D iv e r sity an d E c o sy ste m
F u n c t i o n i n g in E s t u a r i n e
In te r tid a l M ic r o p h y to b e n th o s
ÏP f
Mii
UNIVERSITEIT
GENT
F A C U L T Y O F S C IE N C E S
D e p a r tm e n t B io lo g y
R e s e a r c h u n it P r o tis to lo g y a n d A q u a tic eco lo g y
D iv e r s it y a n d E c o s y s t e m
F u n c t io n in g in E s t u a r in e I n te r t id a l
M ic r o p h y t o b e n t h o s
B art V an elslan d er
P ro m o to r: P ro f. D r. W im V yv erm an
P ro m o to r: P ro f. D r. K oen Sabbe
T h esis s u b m itte d in p a rtia l
fulfillm ent o f th e req u irem en ts
for th e of th e re q u ire m e n ts for
th e degree o f D o cto r (P h .D .) in
Science (Biology)
A cadem ic y ear 2010-2011
E x a m c o m m itte e
M em b ers o f th e reading c o m m itte e
Prof. Dr. G raham J. C. Underwood (U niversity of Essex, UK)
Dr. Jacco C. Kromkamp (NIOO-CEME, T he Netherlands)
Prof. Dr. Tom Moens (Ghent University)
Dr. Frederik De Laender (Ghent University)
O th er m em b ers o f th e exam c o m m itte e
Prof. Dr. Dominique Adriaens (C hairm an, G hent University)
Prof. Dr. Olivier De Clerck (Ghent University)
Prof. Dr. Wim Vyverman (Prom otor, G hent University)
Prof. Dr. Koen Sabbe (Prom otor, G hent University)
C ontent
C h a p ter 1
G en eral In tro d u ctio n
Biodiversity and ecosystem function
Biodiversity
Development of the biodiversity - ecosystem functioning field
M odern B EF experiments
Mechanisms involved
Biodiversity - ecosystem functioning in estuarine intertidal
M icrophytobenthos
Aims and outline
Cited references
1
2
2
3
6
7
8
10
12
C h ap ter 2
E cological D iffe r e n tia tio n b e tw e e n S ym p atric P se u d o c r y p tic
S p ecies in th e E stu a rin e B e n th ic D ia to m N a v ic u la p h y lle p ta
(B a cilla rio p h y cea e)
21
A bstract
Introduction
22
23
M aterials and M ethods
Sam pling sites and clonal cultures
DNA am plification and sequencing
Phylogenetic analyses
Valve morphology
Ecophysiology and natural distribution
Results
Valve morphology
Ecophysiology and distribution pattern
Discussion and Conclusions
Cited References
Supplem entary m aterial
25
25
27
29
32
32
35
37
40
42
46
53
C h a p ter 3
T h erm a l N ic h e E v o lu tio n in th e M arin e D ia to m G en us
C y lin d r o th e c a
55
A bstract
56
Introduction
M aterials and M ethods
57
58
T axon sam pling
Grow th at different tem peratures
Phylogeny
58
61
62
Integration of ecological d a ta and phylogenetic inform ation
63
Sea surface tem perature
Results
Discussion
64
64
71
C ited references
75
C h a p ter 4
H y d ro d y n a m ic D istu r b a n c e G overn s D iv e r sity and B iom ass
P a tte r n s o f E stu a rin e B e n th ic D ia to m s
81
A bstract
Introduction
82
83
M aterials and M ethods
Study site
85
85
Sam pling cam paign
Species composition and diversity
86
87
G ranulom etric analysis
Statistical analysis
89
90
91
Results
Diversity along a disturbance gradient
91
D iversity along a biomass gradient
Species turnover
D iatom cell size
Discussion
96
97
99
100
References
Supplem entary m aterial
104
110
C h a p ter 5
C o m p lem en ta rity E ffects D r iv e P o s itiv e D iv e r sity E ffects on
B io m a ss P ro d u ctio n in E x p e rim en ta l B e n th ic D ia to m B iofilm s
113
A b stract
Introduction
114
115
M aterials and M ethods
117
Biodiversitv-ecosj'stem functioning experim ent
Facilitation
117
119
S tatistical analyses
120
R esults
Discussion
121
125
References
128
C h a p ter 6
D e n s ity -d e p e n d e n t In terferen ce C o m p e titio n b e tw e e n M arine
B e n th ic D ia to m s
133
A b stract
Introduction
134
135
M aterials and M ethods
Strain isolation and culture conditions.
Co-culture experiments.
136
136
137
R esults
Phylogenetic position of N.cf. pellucida
Discussion
138
138
142
References
145
C h a p ter 7
T h e “ M o lecu la r T o o th b r u sh ” o f a M icroalga: D a ily B u r sts o f
B io g e n ic C y a n o g en B ro m id e (B rC N ) C o n tro l B io film F orm ation
a ro u n d a M a rin e B e n th ic D ia to m
151
A b stract
Introduction
152
153
M aterials and Methods
154
Algal strains and culture conditions
154
Bi-algal culture experim ents
155
Effects of spent m edium
Extraction of allelopathic compounds and GC-MS
155
156
Volatile organic compounds
Bioassays
Synthesis of cyanogen iodide
156
157
157
Catalase experiment
Phenol red assay
157
158
Allelopathic effects of N itzschia cf. pellucida
158
158
Extraction and structure elucidation of N. cf. pellucida
allelochemicals
BrCN biosynthesis
161
164
Results
Discussion
References
166
169
C h a p ter 8
G en era l D iscu ssio n and P e r sp e c tiv e s
173
Diversity - ecosystem functioning and organismal traits
173
C ryptic diversity and ecophysiological trait variation
Organismal traits and disturbance
Diversity-ecosystem functioning in m icroalgae communities
173
174
175
Evolutionary history and diversity-ecosystem functioning
Chemically m ediated biotic interactions
176
177
P e r s p e c tiv e s
180
Next generation BEF experim ents
A daptation in marine microalgae
Ecological im portance of microalgal halocarbon chem istry
180
180
181
References
182
S u m m a ry
S a m e n v a ttin g
189
192
A c k n o w led g em e n ts
197
C u rricu lu m v ita e
199
C hap ter 1
G e n e r a l I n t r o d u c t io n
Chapter 1
B io d iv e r s it y a n d E c o s y s t e m
F u n c t io n
H um anity has become a major biogeochemical force and hum an
dom ination of the planet has caused massive destruction and
fragm entation of n atu ral habitats, eutrophication, clim ate change,
acidification, etc (Vitousek et al., 1997, Tilm an et a l, 2001b). Human
activities have resulted in dram atic biodiversity loss and have altered
ecosystems on a global scale (Chapin et ah, 2000, Sala et ah, 2000). The
estim ates and predictions of this ongoing species loss have raised concern
about the consequences of biodiversity loss on ecosystem properties and
the goods and services these ecosystems provide to hum anity (Diaz et ah,
2006, W orm et ah, 2006).
B io d iv e r s it y
Biodiversity is supposed to affect ecosj'stem functioning because the
richness and identity of the species present in an ecosystem determ ine the
functional traits available in an ecosystem. These functional traits may
govern flows of energy and m atter and may influence the response of
ecosystems to abiotic conditions such as disturbance and nutrient
availability. T h e constituents of biodiversity th a t determ ine the presence
and im portance of functional traits in an ecosystem include species
richness, their relative abundance and identity, species interactions and
the tem poral and spatial variation in these properties (Sym stad et a l,
2003).
In order to understand the effects of species loss on ecosystem
functioning, we first need a solid knowledge of the actual biodiversity.
Assessing diversity is not as trivial as it m ay seem a t first, especially in
m icrobial communities. Traditionally, species have been defined on the
basis of discontinuities in morphological and ultrastructural properties
and can therefore be designated as “morphospecies”. A m ultitude of
molecular studies however have dem onstrated th a t traditionally defined
morphospecies often consist of several genetically distinct groups (Darling
et a l, 2004, H ebert et a l, 2004, Slapeta et a l, 2006). Such genetically
distinct b u t morphologically indistinguishable species are called ‘Tryptic
2
Introduction
species”, whereas
the
term
“pseudocryptic
species” refers
to
the
occurrence of morphologically sim ilar species which a t a closer look do
exhibit subtle morphological differences (M ann & Evans, 2007).
(Pseudo)cryptic species appear to be widespread, especially
am ong
microalgae.
Studies
comparing
reproductive,
molecular,
physiological or ecological characters of several strains belonging to the
sam e microalgal morphospecies almost invariably dem onstrated the
occurrence of (pseudo)cryptic species (Knowlton, 1993, Mann, 1999,
Knowlton, 2000, Saez et a l, 2003, Beszteri et a l, 2005, Slapeta et ah,
2006, A m ato et a l, 2007, Vanormelingen et a l, 2008). As a consequence,
our current estim ates of microbial diversity alm ost certainly constitute a
serious underestim ation of actual biodiversity (M ann, 1999).
As mentioned above, biodiversity determ ines th e functional traits
th a t are present in a com m unity. In m ost cases however, it is unknown
whether (pseudo)cryptic species differ in their ecological preferences or
w hether they are functionally equivalent. This notion is not only
im portant for questions relating biodiversity and ecosystem function
(B EF), b u t also to understand mechanisms th a t prom ote cryptic species
coexistence. Niche differentiation is clearly im portant for species
coexistence, but in some cases there is a m arked sim ilarity in coexisting
species. Scheffer & van Nes (2006) used a classical Lotka-V olterra model
to dem onstrate th a t com peting species can evolve to being more similar,
instead of less. According to this model coexisting species are either
sufficiently similar or sufficiently different.
Only a few studies have verified ecological differentiation between
(pseudo)cryptic protist species and suggest possible differentiation in light
requirem ents (Rodriguez et a l, 2005), tem perature preference (Boenigk et
a l, 2007) and susceptibility to parasites (Mann, 1999).
D e v e lo p m e n t o f t h e B io d iv e r s it y E c o s y s t e m F u n c t io n in g F ie ld
Ecology historically treated biodiversity as a corollary of the abiotic
environm ent and ecosystem functioning (Gross k Cardinale, 2007). One
of the m ost im portant ecosystem functions th a t is expected to regulate
biodiversity is productivity. P roductivity is used as a proxy for limiting
3
Chapter 1
resource supply rate which regulates the num ber of competing species
th a t can locall}r coexist (see species-energy theory and resource ratio
theor}' Tilm an, 1977, W right, 1983) During the last two decades this
unidirectional view has been altered and diversity is no loriger considered
as ju st a result of environm ental influences. On th e contrary, it has been
shown th a t diversitj- can regulate h ab itat form ation (Jones et al., 1994,
W right & Jones, 2006), elem ental fluxes and productivity (Naeem, 2002,
Hooper et al., 2005).
This paradigm shift was elicited by the current biodiversity crisis,
b u t the idea th a t diversity can influence ecosystem functioning it is not a
fully novel view. T he first experim ent linking diversity to productivity
dates back to the early 19th century. This experim ent was performed in
1816 by George Sinclair, the head gardener to the Duke of Bedford, in
th e country gardens of W oburn Abbey (Bedfordshire, UK). T he design
comprised 242 plots which were sown w ith different num bers of plant
species. T he aim was to determ ine the role of diversity and under which
conditions diversity m atters (e.g. different soil types). This experiment
was later described by C harles Darwin in his work "The Origin of
Species" and lead him to conclude "it has been experim entally proven
th a t, if a plot of ground be sown w ith one species of grass, and a similar
plot be sown with several distinct genera of grasses, greater num ber of
plants and greater weight of dry herbage can thus be raised" (experiment
described in Hector
Hooper, 2002).
Later on, Elton (1958) and M acA rthur (1955) initiated the
debate about the relation between divershy and stability of ecosystems,
statin g th a t more diverse com m unities are more resilient and stable. The
current focus on biodiversity and ecosystem functioning was instigated by
Ehrlich and Ehrlich (1981) who introduced the ‘‘Rivet hypothesis” which
compared the diversity of an ecosystem w ith the rivets (clinch-nails) of an
airplane. Not devoid of any sense of dram a, the authors suggest th at
some redundant rivets could pop out w ithout the wings being
compromised, but when rivet loss rises, catastrophe is near. In parallel, a
few extinctions in natural com m unities m ay not cause any shift in
functioning, b u t the loss of too many species could result in declining
ecosystem functioning. Several other forms of B EF relations have been
proposed (Naeem, 1998, Schlapfer et al., 1999) such as the proportional
(linear) loss hypothesis which states th a t ecosystem functions decline
linearly w ith species loss (fig. 1).
4
Introduction
T he redundancy hypothesis (W alker, 1992) assumes th a t ecosystem
process rates increase w ith increasing diversity, b u t only to a certain level
at which new species are redundant and do not supply additional
contributions to ecosystem processes. According to this hypothesis, initial
species loss has no effect, but a t a certain level of species loss functioning
diminishes. T he keystone species hypothesis is based on the observation
th a t certain species make a unique and often extrem e contribution to
ecosystem function. Another hypothesis predicts th a t the change in
ecosystem function is context dependent and therefore idiosyncratic (the
idiosyncratic hypothesis, Lawton, 1994). Such p attern could occur when
species m ake different contributions depending on e.g. disturbance level,
n u trien t availability, etc.
Early hvootheses
R edundancy
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iávsl
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Biodiversity
F ig u r e 1. D i f f e r e n t s h a p e s d e s c r i b i n g t h e re la tio n b e t w e e n b i o d iv e r sity a n d e c o s y s t e m
f u n c t i o n i n g ( N a e e m e t al., 2 0 0 2 ) .
Chapter 1
M o d e r n B E F e x p e r im e n ts
More recent experim ental testing of B E F relations started in the 1990s
(Naeem et a l, 1994, Tilm an et a l, 1996, Hector et a l, 1999). These BEF
studies m anipulated biodiversity under controlled conditions and
evaluated the effect of diversity on ecosystem functioning, m ainly primary
productivity. Some of the early B EF experim ents (Naeem et al., 1994,
T ilm an & Downing, 1994) such as the E C O TR O N experim ent (Naeem et
ah, 1994) were criticized by Huston (1997) and Aarssen (1997) because
these experim ents m anipulated diversity indirectly and changes in
richness were confounded with changes in the species composition. Later
on, the experim ental design of most B EF experim ents was optimized so
th a t th e to tal initial biomass of producers was held constant across
several diversity levels. Furtherm ore, com binations of species should be
selected a t random and both species richness and species composition
should be replicated (replicated com binatorial design, Giller et a l, 2004).
Since the initial B EF experim ents, alm ost 200 papers describing
biodiversity ecosystem function experim ents have been published
(C ardinale et a l, 2011). This wealth of d a ta has been extensively
reviewed (Loreau et a l, 2001, Hooper et al., 2005, Srivastava h Vellend,
2005, Reiss et a l, 2009, Loreau, 2010) and several m eta-analyses have
been performed (Balvanera et a l, 2006, C ardinale et a l, 2006, W orm et
al., 2006, Cardinale et a l, 2007, C adotte et a l, 2008). T he compiled
evidence provides persuasive support for a positive relation between
diversity and th e efficiency by which (mainly) plants and algae capture
lim iting resources and convert these into biomass (C ardinale et ah, 2011).
These experim ents also provided support for the diversity-stability
hypothesis which states th a t more diverse com m unities can better resist
disturbance(T ilm an & Downing, 1994, M cCann, 2000). Stability
incorporates b oth the resilience (speed of recovery) and the actual
resistance.
T he
idea behind this
hypothesis
is th a t
more diverse
com m unities harbor more functional traits and th a t
environm ental perturbation some species w ith specific
com pensate for the reduced performance of other species.
6
during an
traits will
Introduction
M e c h a n ism s In v o lv e d
After establishing th e positive biodiversity - ecosystem function relation,
one of th e next goals was (and still is) to elucidate th e mechanisms
responsible for the positive biodiversity effects. Ecological theory suggests
th a t positive B EF relations are caused by three mechanisms: sam pling or
selection probability
effects, niche com plem entarity and
facilitation
(Loreau et ah, 2001). T he sam pling effect refers to th e increased
probability of the presence of species w ith particular, functionally
im portant traits w ith increasing species richness (Huston, 1997). Niche
com plem entarity occurs when species have different
resource
requirem ents, resulting in lower com petition from interspecific neighbours
th an from conspecifics. This m ay lead to a more complete resource use by
more speciose com m unities (Fridle}', 2001). Facilitation occurs when a
species modifies the environm ent in a way favourable for other co­
occurring species (Vanderm eer, 1989). Niche com plem entarity and
facilitation are collectively referred to as ‘com plem entarity’, as it is often
unclear which mechanism prevails.
Although m any researchers claim th a t niche partitioning or
facilitation mechanisms are responsible for positive BEF relations, there is
rarely conclusive proof. C ardinale et al. (2011) recently performed a
m eta-analvsis on BEF experim ents covering 192 peer-reviewed papers and
found th a t not even half of the claims for niche com plem entarity are
backed by an)' direct statistical test or direct experim ental verification.
W ith th e available d ata, they conclude th a t com plem entarity appears to
play the prim ary role in driving positive diversity effects in aquatic
ecosystems, while in terrestrial ecosystems both com plem entarity and
sam pling effects were present.
T he occurrence of com plem entarity is generally inferred indirectly
by com paring m ixture yields w ith the expected yield based on the
m onocultures of the com ponent species (Loreau & Hector, 2001).
However, a t present, we know very little about th e biological mechanisms
th a t are responsible for niche com plem entarity and facilitation. Therefore
we need more direct tests of mechanisms rather th a t merely calculating
the difference between expected and observed yield.
A better understanding of diversity effects will also be gained by getting
better insights in species interactions, both w ith respect to the identity of
these interactions and th e interaction strengths. Species interactions (e.g.
facilitation, m utualism , com petition, and predation) have the potential to
7
Chapter 1
alter biodiversity effects. Recently, a modelling approach (Kirwan et al.,
2009) has been developed to test am ong different hypotheses on how
species interactions can modify diversity effects. By observing patterns in
species interactions, these models suggest which species m echanisms may
occur, b u t they do not produce evidence for specific mechanisms. The
only way to estim ate the im portance of a specific species interaction is to
experim entally m anipulate the factors th a t influence interactions such as
facilitation and competition. ¡Mechanistic evidence for facilitation has
been dem onstrated for N-fixing legumes (Tem perton et al., 2007) and
larvae of suspension-feeding T richoptera (Cardinale et al., 2002), but
further direct evidence for facilitation th a t enhances positive diversity
effects is lacking (Bulleri, 2009).
B io d iv e r s it y - E c o s y s t e m
F u n c t io n in g in E s tu a r in e I n t e r t id a l
M ic r o p h y t o b e n t h o s
Until recently
m ost
BEF
experim ents
were
focused
on
grassland
communities, e.g. the ¡Minnesota Cedar Creek biodiversity experiments
(Tilm an et a i, 2001a), the B IO D EPTH experim ent (Hector et ah, 1999,
Spehn et al., 2005) and the Jena biodiversity experim ent (Roscher et a i,
2004). However, during the last few years researchers have begun to use
microbial systems for B EF experiments. These have a few clear
advantages for B EF research, such as easy control and replication, the
possibility to run experim ents over many generations in a short time
period (Bell et al., 2005, Replansky & Bell, 2009, Striebel et a l, 2009,
Gravel et a l, 2011).
In this thesis I focus on th e biodiversity and ecosystem
functioning of estuarine
intertidal
m icrophytobenthic
(benthic
microalgae) communities. Estuarine and coastal ecosystems provide some
essential ecosystem services such as carbon fixation, food production,
disturbance regulation and coastal protection and nu trien t cycling
(Costanza et a l, 1997). Despite the fact th a t these h abitats cover
relatively small areas on a global scale, their im portance for several
biogeochemical processes is disproportionately large (C ostanza et ah,
1997). In m any m arine and estuarine ecosystems intertidal and subtidal
Introduction
sedim ents support the growth of dense biofilms of benthic microalgae,
which can provide up to 50% of the total prim ary production in estuaries
(Underwood & Kromkamp, 1999). M icrophytobenthos has m any essential
functions for these ecosystems: they are the basis of th e food chain for
m any coastal species (M acIntyre et al., 1996), m ediate nu trien t fluxes
betw een sedim ents and the w ater column (Sundbäck et a/., 2000) and
stabilize sedim ents and as such enhance the resistance of sediment
tow ards erosion (de Brouwer et a l, 2000, Yallop et al., 2000).
W e focused on benthic diatom s as they are often th e dom inant
co nstituent of intertidal mudflats in tem perate regions (Admiraal, 1984).
M any descriptive field studies have assessed the diversity of these benthic
diatom communities and tried to explain species distribution in term s of a
set of abiotic environm ental param eters. O ther studies on the functioning
of these biofilms focused m ainly on their im pact on biogeochemical cycles
(e.g. H erm an et al., 2000, M iddelburg et al., 2000, Risgaard-Petersen,
2003), sedim ent stabilization (de Brouwer et al., 2000, Yallop et al., 2000)
and photophysiology of the microalgae (e.g. Serodio et a l, 1997,
B arranguet et a l, 1998, K rom kam p et a l, 1998, Perkins et a l, 2001).
Very few studies however combined functional studies w ith information
on species identity and richness (but see Nilsson & Sundback, 1996,
Underwood & Provot, 2000, Underwood et a l, 2005, Jesus et a l, 2009).
Therefore we have a limited ability to link diatom biofilm functioning
w ith th e diversity of these diatom communities (Underwood, 2005b).
A recent field study by F orster et al. (2006) on intertidal
m udflats in the W esterschelde estuary (The Netherlands) showed a site
specific relation between prim ary productivity and diversity, w ith either a
positive or a unimodal relation between both. Yet, this field study is
correlative and has no experim ental control on confounding factors which
restricts conclusions about causative relations between diversity and
ecosystem functioning. Moreover, such field studies can not give further
inform ation on the mechanisms
ecosystem functioning.
responsible
for
diversity-m ediated
9
Chapter 1
A im s a n d O u tlin e
T h e general objective of this thesis was to obtain a b etter understanding
of the diversity and ecosystem functioning, and the relationship between
both, in estuarine benthic diatom communities. More specifically, we
w anted to find out (1) w hether cryptic diversity occurred in dom inant
benthic diatom species (Navicula, phyllepta, Cylindrotheca closterium and
C. fusiformis) and how (pseudo)cryptic variation is correlated with
ecological differentiation; (2) w hether the ecological niches of these
crj'ptic species (i.e. Cylindrotheca spp.) are more conserved among more
related lineages th an between more distantly related lineages; (3) how
taxonom ic and functional diversity is regulated by hydrodynamic
disturbance. (4) to w hat degree and how (positive or negative) diversity
affects th e functioning of benthic diatom communities w ith respect to
th eir productivity (overall productivity and growth of individual taxa);
(5) w hat mechanisms are involved in the observed B EF relations
In
C h a p ter
2:
We
investigated
genetic,
morphological
and
ecophvsiological variation in estuarine populations of the widespread
benthic diatom Navicula phyllepta. Genetic variation was assessed by
using sequences of the r&cL, ITS rDNA and 18S rDNA. We assessed
ecophysiological differences by determ ining salinity tolerances of the
different strains.
In C h a p ter 3 we focused on global strains of the diatom genus
Cylindrotheca. We composed a set of strains from a wide range of coastal
h ab itats, from coastal plankton to sea ice and intertidal m udflats. We
determ ined the phylogenetic history of 40 Cylindrotheca strains and
quantified their fundam ental tem perature niches using laboratory
experim ents. W e then assessed if closely related strains tend to be more
sim ilar to each other th an to m ore distantly related ones and therefore if
a phylogenetic signal can be found in clim atic niches.
C h a p ter 4: describes th e relations between th e hydrodynamic
disturbance, species diversity and functional group turnover in estuarine
in tertid al m icrophvtobenthos. We composed an extensive d ata set of
benthic diatom assemblages from the Scheldt estuary covering a wide
range of hydrodynam ic disturbance. W e tested whether diversity -
10
Introduction
disturbance p atterns in estuarine m icrophytobenthos are consistent with
th e Interm ediate Disturbance Hypothesis.
In C h a p ter 5 we experim entally explored the im portance of species
diversity for th e productivity of intertidal benthic diatoms. W e used
naturally co-occurring diatom species to experim entally assemble
communities w ith decreasing diversity. W e measured biodiversity effects
by calculating overyielding (occurring when a m ixture of species performs
better th a n its m onocultures). W e explored the potential mechanisms for
biodiversity effects using the additive partitioning equation of Loreau &
Hector (2001) and by conducting an additional culture experim ent to
elucidate p otential facilitative interactions between different species.
C h a p ter 6: W e dem onstrate th a t cultures of the benthic m arine diatom
Nitzschia cf. pellucida exude m etabolites w ith strong allelopathic effects.
Com peting diatom species were inhibited in their growth and
photosynthetic efficiency. In addition, we show reciprocal, density
dependent allelopathic interactions between N. pellucida and two other
m arine benthic diatom species, Entom oneis paludosa and Stauronella sp.
C h a p ter 7: By chemically characterizing the gas-phase of cultures of the
m arine benthic diatom Nitzschia cf. pellucida using headspace solid phase
m icroextraction (HS-SPME) coupled to GC-MS, we show th a t this
diatom exudes a diverse m ixture of volatile iodinated and brom inated
m etabolites including the new n atu ral product cyanogen brom ide (BrCN)
which
exhibits
pronounced
allelopathic
activity.
We
show
th at
allelopathic effects are H 2 O 2 dependent and link BrCN production to
haloperoxidase activity.
C h a p ter 8: G en eral d isc u ssio n and p ersp ec tiv es
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20
C hap ter 2
E c o lo g ic a l D if f e r e n t ia t io n
b e t w e e n S y m p a t r ic P s e u d o c r y p t ic S p e c ie s in t h e
E s t u a r in e B e n t h i c D i a t o m
N a v ic u la p h yllep ta
( B a c illa r io p h y c e a e )
Modified
from
published
article:
Vanelslander,
B.,
Créach,
V.,
Vanormelingen, P., E rnst, A., Chepurnov, V.A., Sahan, E., Muyzer, G.,
Stal, L.J., Vyverman, W . & Sabbe, K. (2009) Ecological differentiation
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21
Chapter 2
A b str a c t
T he occurrence of cryptic and pseudocryptic species, often living in
svm patry, is widespread am ong microalgae. T his phenomenon raises
im p o rtan t questions about niche partitioning between these closei}'
related species. To date, however, few studies have addressed the
ecological mechanisms underlying sym patry in cryptic and pseudocryptic
species. As a result, we have only a limited understanding of th e factors
th a t govern their distribution along environm ental gradients. Here, we
used the ribosomal internal transcribed spacer (ITS), 18S rRNA gene,
and the RUBISCO large subunit (rbcL) chloroplast gene sequence d ata
together w ith cell wall morphology to show th a t estuarine populations of
th e widespread and common benthic diatom Navicula phyllepta Kiitz.
consist of pseudocryptic species. G row th ra te m easurem ents in function of
salinity showed th a t N. phyllepta strains assigned to the different species
differed in their tolerance to low salinities (< 5 practical salinity units,
psu), which was reflected by their different (but widely overlapping)
distribution in the W esterschelde estuary (the N etherlands). Multiple
regression analyses of the factors determ ining the abundance of the
different species in field samples revealed th a t, in addition to salinity,
sedim ent type and ammonium concentrations were probably equally
im portant. Our results show th a t N. phyllepta sensu lato comprises
different species with specialized ecophysiological characteristics rather
th a n generalists with a broad adaptability to different environm ental
conditions.
22
Pseudocryptic species in N avicula phyllepta
I n tr o d u c t io n
Polyphasic taxonomic studies based on morphology, molecular data, and
reproductive com patibility have led to th e recognition th at
(pseudo)cryptic diversity is widespread am ong microalgae (M ann 1999,
Sáez et a l 2003, Slapeta et al. 2006, Vanormelingen et al. 2008). The
term “ cryptic diversity” refers to the existence of species th a t are
morphologically indistinguishable, but genetically distinct. In man)- cases,
the results of DNA sequence analyses initiated more detailed
morphological studies, which often revealed subtle morphological
differences between species, which are then called “ pseudocryptic species”
(M ann and Evans 2007). In some cases, th e recognition of this hidden
diversity has resulted in the narrowing of the geographic distributions or
ecological am plitudes of species (Rodriguez et al. 2005, Casteleyn et al.
2008, Kooistra et al. 2008). Moreover, an increasing num ber of studies
provide evidence th a t (pseudo)cryptic microalgal species can also occur in
sym patry (M ann et al. 2004, Beszteri et al. 2005). Their coexistence could
be due to fine-tuned niche differentiation, including different preferences
or tolerances w ith respect to small-scale spatial and tem poral
environm ental variation, evolution of different life histories (e.g., in
tim ing of sexual reproduction or the capability of forming resting spores),
or differential susceptibility to predators or parasites (M ann 1999).
Alternatively, cryptic species may lack obvious ecological differentiation,
and random drift and dispersal processes m ay regulate their relative
abundances (Hubbell 2001).
Few studies have addressed the ecological mechanisms underlying
sym patry in (pseudo)cryptic species. As a result, there is a limited
understanding of which factors govern their distribution along
environm ental gradients. Ideall}r, one should combine detailed field
studies on the distribution of cryptic or pseudocryptic species with
ecophysiological experim ents to test whether th e presence of a species at
a specific site is due to its tolerance to certain environm ental conditions
or w hether other factors (such as biotic interactions or neutral
mechanisms) are responsible. However, assessing the distribution of
cryptic or pseudocryptic species in nature has by definition been
ham pered by troublesom e or even impossible identifications using
stan d ard microscopic approaches (M ann et al. 2004).
23
Chapter 2
Recently, it has become possible to use species-specific molecular
probes, which holds great promise to monitor (pseudo)cryptic species in
natural conditions where they are otherwise difficult to detect (Créach et
al. 2006). Laboratory experim ents, on the other hand, have shown
considerable interclonal variation in the physiological traits of m any
microalgal morphospecies (W ood and Leatham 1992). As m ost of these
experim ents have been conducted before DNA identification methods
were available, it is by no means clear w hether the ecophysiological
differences can be attrib u ted to intraspecific variation or to cryptic
species diversity. A study th a t has correlated ecophysiological differences
w ith genetic diversity was conducted using D itylum brigthwellii
(Rynearson and A rm brust 2000). A nother illustrative example is
Skeletonema costatum sensu lato (s. 1.). This diatom has been thoroughly
studied and was considered to be physiologically plastic (Brand 1984),
genotypically diverse (Gallagher 1982), and cosmopolitan. Yet, more
recent phylogenetic and accurate morphological studies showed th a t S.
costatum s. 1. contains several species th a t seem to be geographically
restricted (Sarno et al. 2005, Kooistra et al. 2008). In some cases, the
different distributions suggest ecological differentiation between the
different species (Sarno et al. 2005, Kooistra et al. 2008).
Estuaries are suitable model systems for studying niche
partitioning and changes in species composition along environm ental
gradients because of th e pronounced gradients in, for example, salinity,
turbidity, and nutrients. Sibling species of m any estuarine tax a have
partly overlapping b u t distinct distributions along the salinity gradient
(Bilton et al. 2002). In contrast, some common estuarine diatom s (N .
phyllepta, N. salinarum, N. gregaria) apparently occur along whole
estuarine gradients, and species exhibit a broad adaptability to different
environm ental conditions (K ram m er and Lange-Bertalot 1986).
N. phyllepta is one of the m ost commonly reported diatom s from
brackish and m arine sedim ents worldwide (e.g., Underwood et al. 1998,
Clavero et al. 2000, Sabbe et al. 2003). It is particularly abundant in
intertidal estuarine and littoral sedim ents in northw est Europe, where it
is often the dom inant constituent of m icrophytobenthic biofilms (Sabbe
and Vyverman 1991, Underwood et al. 1998, Haubois et al. 2005). N.
phyllepta has been reported from a broad range of salinities, from
electrolyte-rich freshwaters (Kram m er and Lange-Bertalot 1986) to
hypersaline environm ents w ith salinities up to 75 psu (Clavero et al.
2000). It also shows extensive morphological variability, in particular,
24
Pseudocryptic species in N avicula phyllepta
w ith respect to size and shape (K ram m er and Lange-Bertalot 1986,
W itkowski et al. 2004). Recent sequence analysis of th e ribosomal IT S !
region in a num ber of N. phyllepta clones revealed the presence of two
distinct ITS1 sequence clusters (Créach et al. 2006). A quantitative real­
tim e P C R (qPC R ) approach showed th a t both clades exhibit a distinct
b u t overlapping distribution within the W esterschelde estuary (Créach et
al. 2006). However, because of the covariation of salinity with m any other
environm ental gradients in th e estuary (Ogilvie et al. 1997), it is unclear
which factors govern th e distribution of th e two clades of N. phyllepta.
O ur aim was to further resolve th e species boundaries w ithin N.
phyllepta s. 1. an d to determ ine the ecological preferences of the different
cryptic or pseudocryptic species. We first performed a phylogenetic
analysis by investigating and com paring variation patterns in three
commonly used molecular markers w ith differing rates of sequence
evolution. W e com plem ented this analysis w ith morphological
m easurem ents. W e th en assessed how (pseudo)cryptic variation was
related to ecological characteristics. This was done by using a combined
approach in which we (1) determ ined the grow th of a subset of strains in
response to different salinities and (2) identified the abiotic factors th a t
govern the field distribution of the different species in th e Westerschelde
estuary.
M a t e r ia l a n d M e t h o d s
S a m p lin g s ite s a n d c lo n al c u ltu re s
In addition to the 11 A. phyllepta strains used by Créach et al. (2006)
(strain designations beginning w ith “ C CY ” and “ C O ”) from the
W esterschelde estuary (the Netherlands) (Fig. 1), th e Ems-Dollard
estuary on th e D utch-G erm an border, and the Colne estuary (Essex,
UK), 10 additional clonal cultures were isolated from sedim ent samples
taken in th e W esterschelde estuary (sampling localities and sampling
dates in T able 1).
T he W esterschelde estuary is the 58 km long D utch part of the
160 km long Scheldt estuary. The W esterschelde is well mixed and
characterized by a complex morphology w ith tidal channels surrounding
25
Chapter 2
several large intertidal flats and salt m arshes. T he residence tim e of the
w ater in th e estuary ranges from 1 to 3 m onths, depending on the season,
causing a salinity gradient (ranging from 3 to 32 psu over a distance of 58
km) th a t is prim arily determ ined by th e m agnitude of th e river discharge
and, to a lesser extent, th e tidal oscillation, which is of smaller am plitude
(Meire et al. 2005). T he Ems-Dollard estuary is one of th e larger estuaries
in the W adden Sea (Peletier 1996). T he brackish zone is -3 0 km long and
contains large intertidal mudflats. T he Colne estuary (UK) is a smaller
estuary (-1 6 km long) and is m uddy and highly turbid. T he Colne is
highly eutrophic, and there are pronounced gradients in nutrient
concentrations inversely related to th e salinity gradient (T hornton et al.
2002).
Identification of diatom s (cultures and valves in natural samples)
as N. phyllepta was based on illustrations of the lectotype shown in plate
32 and figure 5 in K ram m er and Lange-Bertalot (1986) and in figure 28 in
Cox (1995). T he salinities (average ± SD, based on m easurem ents
between April 2002 and Septem ber 2003) a t th e four Westerschelde
sam pling sites, Appelzak (51°22’38” N, 4°14’32” E), B ath (51°23’58” N,
4°12’36” E), Biezelingsche Ham (51°26’50” N, 3°55’33” E) and Paulina
Schor (51°20’58” N, 3°43’37” E), were 8.8 ± 4.7 (n = 18), 13.6 ± 3.8 (n =
9), 20.0 ± 3.8 (n = 18), and 22.7 ± 6.3 (n = 18) psu, respectively.
N o rth S e a
WS
■NL
BH
AZ
PS
5 km
F ig u r e
1.
poly­
haline
mesohaline
oligo­
halina
M a p o f t h e W e s t e r s c h e l d e e s t u a r y ( t h e N e t h e r l a n d s ) w i t h t h e d if fe r e n t
s a m p l i n g l o c a t i o n s f r o m w h i c h Navicula p h y lle p ta s t r a i n s w e r e i so la te d . P S , P a u l i n a
S c h o r ; B H , B i e z e l in g s c h e H a m ; B a , B a th ; A Z , A p p e l z a k . T h e o l i g o h a l i n e z o n e r a n g e s
f r o m 0 .5 t o 5 psu ; t h e m e s o h a l i n e z o n e , f r o m 5 t o 18 psu; a n d t h e p o l y h a l i n e zo ne,
f r o m 18 t o 3 0 psu.
26
Pseudocryptic species in Navicula, phyllepta
Clonal cultures were established as described in Chepurnov et al.
(2002) and grown in artificial seaw ater (U ltram arine Sea Salt; W aterlife
Research LTD, Middlesex, UK) w ith a salinity of 30 ± 1 psu, enriched
w ith F / 2 nutrients (Guillard 1975). T he possibility of a bias to clonal
cultures ad apted to 30 psu cannot be fully excluded, but it is considered
unlikely because estuarine benthic diatom s are known for their ability to
grow a t a wide range of salinities. Moreover, we did not find different
m orphotypes of N. phyllepta in the field com pared to th e isolated strains
(see R esults). T he cultures were kept in 24-well plates (Greiner Bio-One,
Frickenhausen, Germany) a t 18 ± 0.3°C w ith a 12:12 light:dark (L:D)
period and 25-30 pmol photons . m 'V 1 from cool-white fluorescent tubes
(Philips, Eindhoven, th e N etherlands, TLD 18W). T he cultures were
transferred every 2 weeks to fresh medium.
D N A a m p lific a tio n a n d se q u e n c in g
T hree DNA regions w ith differing rates of sequence evolution were
selected for sequencing, th a t is, the ITS region (consisting of ITS1, 5.8S
rR N A gene, and ITS2), 18S rRNA gene (which is transcribed into the
ribosom al SSU), and a 457 bp fragm ent of th e chloroplast RUBISCO
large subunit gene (rbcL) (Table 1). T he non-coding ribosomal ITS region
is highly variable and is often used to investigate variation within
populations. T he rbcL and 18S rRNA genes are more slowly evolving. In
raphid diatom s, rbcL evolves slightly faster th a n the 18S rRNA gene
(Evans et al. 2008).
Cells for DNA extraction were harvested from exponentially
growing cultures and pelleted by centrifugation (Sigma Laborzentrifugen
GmbH, Osterode am Harz, Germ any, 4K15 centrifuge). For the
am plification and sequencing of the 18S rRNA gene and the ribosomal
ITS region, DNA was extracted using the bead-beating m ethod with
phenol extraction and ethanol precipitation as described by Zw art et al.
(1998). For rbcL amplification and sequencing, DNA was extracted using
a commercial kit (Mo Bio Lab. Inc., Carlsbad, CA, USA). T he ITS of 11
strains (accession num bers of these strains in Table 1 are not in bold) was
cloned and sequenced earlier (Créach et al. 2006). T he P C R products of
th e ITS were cloned in P C R II-TO PO vector T A cloning kit (Invitrogen,
B reda, th e Netherlands) according to the m anufacturer’s instructions. For
nine strains (Table 1), two to four clones were sequenced and analyzed.
F or the other nine strains (strain designations startin g w ith “ A P ” or
27
Chapter 2
“ B A ” ), th e ribosomal ITS1-5.8S-ITS2 region was amplified by PCR
using the universal prim ers ITS4 (reverse primer located a t the beginning
of th e 28S rRNA gene, W hite et, al. 1990) and 1800F (forward primer
located a t the end of the 18S rRNA gene, Friedl 1996). The P C R reaction
m ixture contained 1 pL of tem plate DNA, dN T P s at 0.2 mM, each
prim er at 0.5 pM, 5 pL 10 X P C R buffer (100 mM Tris-HCl [pH 9], 500
mM KC1), and 2.5 U T aq Polym erase (Qiagen, Hilden, Germ any) and
was adjusted to a final volume of 50 pL w ith sterile w ater (Sigma, St.
Louis, MO, USA). P C R reaction conditions consisted of an initial
preheating step of 3 min a t 94°C followed by 40 cycles of 94°C
dénaturation for 1 min, 55°C annealing for 2 min, and 72°C extension for
1 min. Finally, there was a 72°C extension for 5 min. Sequencing was
perform ed using a Perkin-E lm er ABI Prism 377 autom ated DNA
sequencer (Applied Biosystems, F oster C ity, CA, USA). T he sequencing
prim ers were DITS2 and DITS3 (Zechman et al. 1994), and sequence
coverage was forward and reverse.
For the 18S rRNA gene, forward prim ers were DSSU4 (5’A A C C TG G TT G A TTC TG C C A G TA G -3’), DSSU550 (5’-A A G TC TG G T
GCCAGCAGCC-3’), and DSSU1119 (5’-GGCTG AAACTTAA AGAAA T
T G -3’). Reverse primers were DSSU376 (5’-TC TC A G G C TC C C T C TC
CG -3’), DSSU1180 (S’-TCCA CCA A CTA A G AACGG CC-3’) , DSSU1613
(5’-GTACAAAGGGCAG G G A CG TA -3’), and DSSU1860 (S’-CTGCAG
GTTCA CCTA CG G A A A CC-3’). For the PC R , th e reaction m ixture
contained 1-5 pL of tem plate DNA, dN T Ps a t 0.2 mM, prim ers a t 1 pM
each, 2.5 U T aq poljmierase, and P C R buffer and was adjusted to a total
volume of 50 pL with sterile water. P C R reaction conditions consisted of
an initial 94°C dénaturation during 5 min followed by 40 cycles of 94°C
dén atu ratio n for 30s, 55°C annealing for 2 min, and 72°C extension for 2
min. T he 18S rRNA gene sequences were obtained by direct sequencing
from th e P C R product using the ABI 3100 prism BigDye Term inator
Cycle Sequencing Ready Reaction K it (Applied Biosystems). Sequence
coverage was forward and reverse.
A 554 bp fragm ent of the rbcL gene, which includes the
functional site of the enzyme, was amplified in a P C R with a degenerate
prim er pair as described in W awrik et al. (2002). P C R m ixtures (50 pL)
contained 1 pL of tem plate DNA, prim ers a t 0.5 pM each, dN T Ps a t 0.2
mM, 1.5 mM MgC12, 2.5 U of T aq DNA polymerase, and P C R buffer
(Qiagen). P C R reaction cycles were as described in W awrik et al. (2002).
Sequences were obtained w ith the ABI 3100 prism BigDj'e Term inator
28
Pseudocryptic species in N avicula phyllepta
Cycle Sequencing Ready R eaction Kit. GenBank accession numbers of all
N. phyllepta sequences are listed in Table 1. T he rbcL fragm ents are only
457 bp long, lacking the first ~45 bp a t either end as th e result of
unresolved parts a t
chromatograms.
th e
beginning and
th e
end
of the
sequence
P h y lo g e n e tic a n a ly s e s
Sequences of each m olecular m arker (ribosomal ITS, rbcL, 18S rRNA
gene) were edited separately using BioNumerics version 3.5 (Applied
M aths, Kortrijk, Belgium) and autom atically aligned using ClustalX
(Thompson et al. 1997). T he resulting alignm ent was m anually corrected
where necessary. Phylogenetic analyses were performed using PAUP*
4.0bl0 (Swofford 2001) and, for Bayesian inference (Bí), M rBayes version
3.1.2 (Ronquist and Huelsenbeck 2003). Uncorrected P distances (Nei and
K um ar 2000) were calculated as measures of genetic distance between the
sequences in each alignm ent. Phylogenetic relationships were assessed
separately using m axim um parsim ony (MP) and Bayesian Inference (Bí)
on the ITS1, the ITS2, the com plete ribosomal ITS, the 18S rRNA gene,
and rbcL alignm ents. For th e ITS1 and ITS alignm ents, th e first 36
positions were om itted because this part was lacking for tw o of the
sequences, due to unresolved p arts at the beginning of the sequence
chromatogram s. No outgroup was specified. For MP analysis, the sites
were treated unweighted. G aps were treated as missing data. M P trees
were inferred using heuristic searches with random stepwise additions of
taxa, repeated 100 times, and using the tree bisection-reconnection
branch-swapping algorithm . B ootstrap values were assessed from 1,000
replicates. For th e Bí, we used M rM odeltest 2.3 (Nylander 2004) to
calculate the best-fitting model for the given d ata m atrix using the AIC
(Akaike inform ation criterion). Likelihoods for 24 models of evolution
were calculated using PA U P*, and the command file was provided to
M rM odeltest 2.3. The selected best-fitting models were K80 + I (for the
ITS), K80 (for IT S l), H K Y + l(fo r ITS2), G T R + I + G (for 18S rRNA
gene), and G T R + G (for rbcL). No initial values were assigned to the
model param eters. Two runs of four Markov chains (one cold and three
heated) were run for three million generations and sampled every 100
generations. This yielded a posterior probability distribution of 30,001
trees. After exclusion of 5,000 “ burn-in” trees, posterior probabilities
were calculated by constructing a 50% m ajority-rule consensus tree.
29
Chapter 2
T a b le
1 . N. p h y lle p ta s t r a i n s u se d in t h i s s t u d y t o g e t h e r w i t h p l a c e a n d d a t e o f
iso lation , G e n B a n k a c c e ss io n ( a c c ) n u m b e r s o f t h e c o r r e s p o n d i n g ri b o s o m a l ITS, 18 S
rbcL s e q u e n c e s , m o r p h o m e t r i c m e a s u r e m e n t s ( m e a n ±
rRNA g en e and
SD)
and
a s sig n m e n t to th e different m o r p h o ty p e s, physiotypes a n d phylogenetic clades. S tra in s
f ro m c l a d e B a r e i n d i c a t e d b y g r e y sh a d i n g . A cce ssio n n u m b e r s o f s e q u e n c e s n e w
S tr a in
I s o la tio n
I s o la t io n d a t e
A cc IT S
A c c 18 S
rD N A
rR N A
O r ig in
num ber
A c c rb c L
CC'Y 0221
MOO
A pr '02
Ap[H’l/ak
U Q 19305'.
F.T 624240
E U !)3 8 3 1
C C Y 0230
XIOO
Sep, 02
A p| )i •1/ak
1)0193., 70
E U 938307
F J 6 2 1251
C C Y 0222
N lOO
Apr '02
A p p -U k
DQ193556
A P -0 2 -0 5
p .m .;
16/A p r/0 2
Ap)H'l/,ik
D Q I93559
F J 640068
EU 93831
B A -0 4 - 01
PA F
2 Mar. 01
Bath
D Q 103566
FJ624235
F J 624246
D Q 193557
D Q l93567
B A -0 4 -0 2
PAI
2 / M ur/0 i
Bath
D Q 193563
F J624232
FJ624243
D Q 193564
B A -0 4 -0 4
PM
2 /M a r 01
Balli
F.16 2 4 2 2 6
FJ624233
FJ624244
B A -0 4 -0 5
PA E
2 /M a r /0 1
Bath
D Q 193560
FJ624237
F J 624247
FJ624231
FJ624242
FJ621238
FJ624249
D Q 1935C 1
B A -0 4 -0 6
p a i;
2 /M ar, 0 1
Balli
B A -0 4 -0 7
p a i­
■2. M ar/01
Baili
F J 624228
B A -0 4 -0 8
p a i;
‘2 / Mai 01
Balli
F J624230
FJ624236
FJ624248
B A - 0 1-09
p a i;
‘i /M a r /o -l
Bath
F J 6 2 1220
FJ624234
FJ624245
C O -0 4 -0 1
i
\
.Ian/04
C ..lue (I K)
FJ624225
FJ624239
FJ624250
C C Y 0218
MOO
Apr/02
Bi.-zi'linssflie Hum
DQ10355.S
E U 938308
EU 93831
F J 6 2 1227
M e a n m o r p h o m e tr ic f e a tu r e s c ia d o A
C C Y 0226
M l JO
S ep /02
A p p i-U k
D Q 193.550
C C Y 0201
MOO
■Ian/02
Biezelingsrlie Uam
DQ 103546
E li 938309
EU 93831
C C Y 0212
MOO
A pr/02
Paulina Schor
IK. J103513
F J624241
EU 93831
EU 938310
EU 93831
EU 93832
DQ 193541
C C Y 0213
MOO
Apr /Ö2
Paulina Si hut
DQ 103547
I >Q 193518
C C Y 0227
MOO
S ep /02
Paulina Si lior
1>Q2357S3
FJ624253
EU 93831
C C Y 9804
MOO
A pr/98
Kins I foliar. 1 (N l.)
DQ 19355!
E U 938311
EU 93831
DQ 193552
M e a n m o r p h o m e tr ic f e a tu r e s c l a d e B
30
Pseudocryptic species in N avicu la phyllepta
t o t h i s s t u d y a r e in bold. M o r p h o t y p e : "S ": N a r r o w valves w i t h high s t r i a d e n s i ty ,
“ R ": W i d e v a l v e s a n d low s t r i a d e n s i ty . G r o w t h 2 psu: ability t o g r o w a t 2 psu: " + ”
= y es,
n o a n d cells died, " ± ” =
no g r o w t h b u t cells s u r v iv e d . P A E : R e s e a r c h
G r o u p P r o t i s t o l o g y a n d A q u a t i c E co log y, G h e n t , B e lg i u m . N I O O - C E M E : Y e r s e k e , T h e
N e t h e r l a n d s . E ssex: D e p t . Biol. Univ. Essex, UK. " n m " s t a n d s f o r ' n o t m e a s u r e d ' .
Striae
L en g th
W id th
(p m )
(p m )
n —20
(N o ,10 jim '
M o rp h o -
G ro w th
')
ty p e
2 p su
IT S
1RS
r&cL
n=20
u=20
11 - ± 1 .0
5.0 i 0.3
21.7 ± 0.7
A
A
A
um
mu
lllll
S
A
A
A
mu
mu
um
A
17.0 ± Ü. !
5.1
0.3
20.7 ± ! .0
Ul.2 ! 0.1
5.0 • 0.2
22.2 i 1.0
s
s
ui.-. ± 0.1
5.1 - 0.2
22.3 ± 0.9
s
1-1.1» ± 0.3
LO ± 0.2
21.9 ± 0.7
10.7
5.3
20.S i 0.1
s
s
0.3
0.2
13.0 ± U. !
5.0 ± 0.2
21.7 -± 0,8
11.1 i 0.3
1.0
0.2
22,3 1 0.7
1.1,1 ± 0.3
5.2 ± 0.3
21.5 ± 0.7
13.3 i 0.3
1.0 • 0.2
21.0 1 0.7
1 1.0 ± ¡i.«
1.7 ± 0.2
2.1.3 h 0,7
um
um
um
1 1.2 r 2.2
5.1)
0.2
s
s
s
s
s
+
+
A
A
A
+
A
A
A
+
A
A
A
+
A
A
A
A
A
A
+
A
A
A
-r
A
A
A
+
A
A
A
+
A
A
A
+
A
A
A
A
A
A
21.1, ¡0.0
mu
mu
10.3 ± 1 .1
OR
0 1
■111!
18.3 i 0.9
H
-
B
B
B
B
11.1 ± 0 . 1
7.0
0.3
IS,3 ± O.s
R
-
B
B
B
10.8 ± 0.5
().<S ± 0.1
ls.fi i 0.9
II
-
B
B
B
um
lllll
nui
B
B
B
11.2 ± 0.0
6.3 ± 0.5
13.0 ± 0.9
B
B
B
10.9 ± 0 .2
0 3 =0 1
J S .l ± 0.3
R
B
31
Chapter 2
V a lv e m o rp h o lo g y
For morphological observations of th e siliceous cell wall, all clonal
cultures and three natural samples (Appelzak, Biezelingsche Ham, and
P au lin a Schor) were oxidized using hydrogen peroxide (15% [v / v]) and
acetic acid (50% [v / v]) and repeatedly washed w ith distilled water
before being m ounted in N aphrax (PhycoTech, St. Joseph, MI, USA). LM
was carried out using a Zeiss Axioplan 2 microscope (Zeiss Gruppe, Jena,
Germ any) equipped w ith a digital cam era (Zeiss Axiocam MRm).
M orphom etric m easurem ents on digital images were m ade using Image J
software (Abram off et al. 2004). Length, w idth, and stria density of 20
frustules from each strain and 100 from each natural sample were
m easured. Voucher specimens of cleaned m aterial of th e original natural
sample and the clonal cultures are kept at the Laboratory of Protistology
and A quatic Ecology, G hent University, Belgium (voucher specimens
PAE00101-PAE00115).
E c o p h y sio lo g y a n d n a tu r a l d is tr ib u tio n
T o assess grow th perform ance a t different salinities, eight N. phyllepta
strains (CCY0201 from Biezelingsche Ham; CCY0212 and CCY0213 from
Paulina Schor; and BA-04-01, BA-04-02, BA-04-04, and BA-04-05 from
Bath; and CCY0221 from Appelzak) were grown in triplicate a t eight
different salinities (0, 0.5, 1, 2, 5, 10, 20, and 30 psu). Suspended cells
from stock cultures grown at 30 psu until late exponential phase were
transferred into wells of flat-bottom 96-well m icrotiter plates (Greiner
Bio-One, Frickenhausen, Germ any). W ells were inoculated a t a cell
density of -3,000 cells . mL \ resulting in an initial density of -2 9 cells .
mm 2. One hour after th e inoculation, when m ost of the cells had settled
a t the bottom , the culture medium was gently pipetted off and
im m ediately replaced by fresh medium a t the desired salinity. Growth
was not acclim ated to th e different salinities prior th e experim ents. A
representative growth curve is shown in Figure 2.
32
Pseudocryptic species in N avicula phyllepta,
-
3500
3,000
'*g 2 500
I
/T
r-
I
r/TJ r U 1
Y
2,000
%
1,500
u
1 000
t/
-
I
500
'. *
*
*
2
*
.
.
A
6
.
.
8
10 12
Timt* (d)
F ig u r e 2 . G r o w t h c u r v e o f N avicula p h yllep ta s t r a i n C C Y 0 2 2 1 a t 10 psu. E r r o r bars
r e p r e s e n t s t a n d a r d d eviatio n s.
T here were some clear signs of osmotic stress and cell lysis a t 0,
0.5, and 1 psu, especially in the case of strains belonging to clade B.
C ulture m edia were prepared by adding F / 2 nutrients (Guillard 1975)
and salts (U ltram arine Sea Salt; W aterlife Research LTD, Middlesex,
UK) to distilled w ater up to the required salinity. T he culture m edia were
filtered through glass fiber filters (0.7 pm pore size, W hatm an, M aidstone,
England, G FF) and autoclaved. G row th conditions were identical to
those described above for m aintaining the stock cultures. Cell densities of
each replicate were m onitored daily using an inverted microscope
(Reichert P V 624; Reichert-Jung, Vienna, A ustria) by counting a
minimum of 400 cells per replicate.
G row th rate during th e exponential phase (4-5 d) was calculated
as th e slope of the linear regression of log2-transformed cell densities
versus tim e for individual cultures (Underwood and Provot 2000).
Because th e results of these experim ents indicated th a t the strains
differed mainly in their ability to grow a t low salinities (0.5-5 psu), six
additional strains (AP-02-05 from Appelzak; BA-04-06, BA-04-07, BA-0408, and BA-04-09 from B ath; and CO-04-01 from the Colne estuary [UK])
were grown only a t 2 and 5 psu, and qualitative observations were made
on their survival and growth. C ultures were followed during 2 weeks, a
period during which healthy cultures reached stationary phase.
T he relationship between the environm ental variables and the
distribution of cells belonging to clades A and B in the Westerschelde
estu ary was explored using stepwise forward m ultiple regression as
im plem ented in Statistica version 6.0 for Windows (StatSoft Inc., Tulsa,
OK, USA). T his analysis was performed displaying results a t each step,
w ith an F to enter set a t 1.00 and an F to remove set a t 0; tolerance was
33
Chapter 2
set at 0.0001, and the intercept v/as included in the model. Only
significant variables were included in th e model. T he P earson’s residuals
are given in supplementär}’ figure 1. Standarized residuals were tested for
norm ality using the Shapiro W ilk’s W test. All locations (Appelzak,
Biezelingsche Ham, and Paulina Schor) were sam pled nine times,
spanning two consecutive years. At each location, highshore and midshore
stations were selected on th e exposed m udflats. Samples were taken in
April 2002, May
2002, Septem ber 2002, F ebruary 2003, M arch 2003,
April 2003, M ay
2003, July 2003, and Septem ber 2003. Samples were
taken during low tide. T he strains w ith designation starting with “ CCY ”
were taken during this sam pling campaign. The distribution d ata used for
the stepwise forward m ultiple regression were obtained from Créach et al.
(2006) who conducted a qPC R to determ ine the abundance of clades A
and B in th e samples taken during th e above-m entioned campaign.
T he environm ental param eters m easured during this sampling
cam paign were obtained from Sahan et al. (2007) and included
am m onium , nitrite, nitrate, phosphate, salinity, organic carbon content,
organic nitrogen content, C:N ratio, tem perature, irradiance, water
content, mean grain size, silt content, and height of the sediment. Surface
sedim ent samples (upper 2mm) were collected using a contact corer.
T otal organic carbon (TO C) was m easured using an elem ental analyzer
(Elem entar Analysensysteme, H anau, Germ any). Organic nitrogen
content, nitrite, nitrate, am m onium, and phosphate were measured using
stan d ard colorimetric techniques (Grasshoff 1976) on a SKALAR SA 4000
segm ented
flow
analyzer (Skalar Analytical B.V., Breda, the
Netherlands). Salinity was m easured using a titratio n m ethod with SAC
80 (Radiom eter, Copenhagen, Denmark). Sediment grain size and silt
content of the sediment were determ ined by granulom etric analysis using
a laser diffraction analyzer (M alvern M astersizer 2000; Malvern
Instrum ents Limited, M alvern, UK). T he percentage of w ater was
determ ined by the loss of weight of the sample after 48 h of freeze-drying.
T h e shore heights of the stations were determ ined by reference to a
digital elevation model of the estuary and confirmed by direct observation
of the timing of emersion and immersion periods. Sediment surface
tem peratures were measured w ith an electronic therm om eter a t each
sam pling occasion. T he mean irradiance at all sites was recorded at
hourly intervals during 2002 and 2003 using a Li-Cor Li-192 sensor (LiCor, Lincoln, NE, USA).
34
Pseudocryptic species in N avicula phyllepta
R e s u lt s
Molecular phylogenies. Phylogenetic analyses performed on the ITS
sequences of th e strains identified morphologically as N. phyllepta
revealed two distinct clades (Fig. 3A) possessing intracla.de P distances
not higher th an 0.0148 (12 nucleotide differences) and interclade distances
between 0.0762 and 0.0898 (78 to 92 nucleotide differences). B oth clades
were supported by high to maximal MP bootstrap and B í posterior
probability values. These two clades are referred to as clades A and B
(see Fig. 3A). T he strains collected from the Colne estuary (CO-04-01)
and Ems-Dollard (CCY9804) belonged to clades A and B, respectively.
Intraclonal ITS variation was very low (P distances not higher th a n 0.010
and 12 nucleotide differences) (Fig. 3).
Phylogenetic analyses performed on th e ITS1 and ITS2 regions
generated nearly identical trees supported by high to m axim al MP
bootstrap and B í posterior probability values (not shown). T he ITS1 was
more variable th an IT S 2, w ith interclade uncorrected P distances between
0.119 and 0.141, and 43 to 59 nucleotide differences, com pared to
interclade distances between 0.064 and 0.084, and 25 to 33 nucleotide
differences for the ITS2. T he partial rbcL (which includes the functional
site of the enzyme, W aw rik et al. 2002) and 18S rRNA gene phylogenies
also supported the distinction of clades A and B (Fig. 3, B and C, results
summarized in Table 1). T he num ber of nucleotide differences am ong the
partial rbcL sequences w ithin the clades was 0 or 1 (P distances 0.0000.002), while sequences from different clades (A and B) differed a t 8 to 9
positions (P distances 0.018-0.021). Eight of th e nine differences were in
the third codon position and did not result in am ino-acid differences. One
nucleotide difference was in the first codon position and resulted in a
change of amino acid. In the 18S rRNA gene (the first 129 and last 93
nucleotides are missing), the two clades had two nucleotide differences (P
distance 0.001), while sequences w ithin each clade were identical (except
strain BA-04-07, which differed a t one position from the other strains of
clade A). T he 18S rR N A gene sequence of a Navicula sp. strain (AT145.08) collected in a brackish lagoon of the Baltic Sea by B ruder and
Medlin (2008) belonged to clade A, indicating a wider distribution of this
clade. B oth rbcL and 18S rRNA gene phylogenies suggest th a t Seminavis
cf. robusta and Pseudogomphonema are close relatives of N. phyllepta,
closer th an other Navicula species, such as N. cryptotenella and N.
reinhardtii.
35
Chapter 2
cO'O
CMCoP
§
5 * 1 usjfcsi
"S
> ® o5
<
l i l i
23 § £
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< < - 3
« 0 a5 Q.S
Ul t ü 1/3
~ =t 5
S >- q
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CD
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ro
o
O
^ S o - o r - M j I O n o o o o o o o o N v j
! l |¡ § ? ? l § Í 9 S 3 l s é 3 3 l s g |
Í 5 a§8 8 8 8 B 8 5 8 á á S i á S á á á á 8 8 ö|
h— ¿-a
tfï H
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LU ■
CM CO CM CM CM
O 03 O O O
§ s s |s s s s s s § g s
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ccmcacooj c o c a o oc
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iü < o <
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CM O
par.<
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M r-p
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cM
'rin
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C'4£ÍM- ’’M
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fTitSs
inin
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o c'
«V«;
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F ig u r e 3 . P h y l o g e n y o b t a i n e d f r o m B a y e s i a n i n f e re n c e ( B l ) o f (A ) r i b o s o m a l IT S , (B )
rbcL , a n d ( C ) 1 8 S r R N A g e n e . T h e t r e e s s h o w n a r e t h e 5 0 % m a j o r i t y - r u l e c o n s e n s u s
t r e e b a s e d on Bl an a ly s is. B o o t s t r a p v a l u e s > 5 0 %
( M P ) o r p o s t e r i o r p r o b a b i l it i e s
> 0 . 5 ( B l ) a r e i n d i c a t e d o n t h e r e s p e c ti v e n o d e s . In t ra c l o n a l s e q u e n c e v a r i a n t s a r e
indicated
by a d i f f e r e n t l e t t e r b e h i n d t h e s t r a i n
name.
Scale b ars re p re se n t one
s u b s t i t u t i o n in 10 0 n u c l e o t i d e s ( A ) o r o n e s u b s t i t u t i o n in 10 n u c l e o t i d e s ( B a n d C).
IT S , in t e r n a l t r a n s c r i b e d s p a c e r ; M P , m a x i m u m p a r s i m o n y .
36
Pseudocryptic species in N avicula phyllepta
V a lv e m o rp h o lo g y
LM photographs of N. phyllepta valves are shown in Figure 4. While
there were no obvious differences between th e species in general valve
features (valve outline, striation pattern, etc.), they differed in their
m orphom etry (Table 1 and Fig. 5). Based on a com bination of valve
width and stria density, there was a clear distinction between strains of
th e clades A and B. Strains belonging to clade A were narrow (4.2-5.5
pm wide) and had a high stria density (19.5-24 striae in 10 pm ) (Fig.
5A). S trains belonging to clade B had wider valves (5.5-7.5 pm) and a
lower stria density (16-20 striae in 10 pm) (Fig. 5A). W hen strains with
th e sam e length belonging to the different clades (e.g., CCY0221 and BA04-07 of clade A and CCY0212 and CCY9804 of clade B) were compared,
it was clear th a t the differences in w idth and stria density were
m aintained when cells belonging to different clades had the same length.
F ig u r e 4 . LM p h o t o g r a p h s o f Navicula phyllepta. ( A ) S t r a i n B A - 0 4 - 0 1 ( c l a d e A ). ( B )
S t r a i n C C Y 0 2 1 2 ( c l a d e B ). S c a l e bar, 10 p m .
37
Chapter 2
M orphom etric analysis of N. phyllepta valves from the sam ples from the
W esterschelde (locations Appelzak, Biezelingsche Ham, and Paulina
Schor) showed the presence of the sam e tw o groups based on differences
in stria density and valve width (Fig. 5, B and C). T he natural samples
v/ere composed of a group w ith narrow frustules (4.3-5.5 pm) having
higher stria densities (20-23 striae in 10 pm) and a group w ith broader
frustules (5.4-8 pm) and lower stria densities (16-20 in 10 pm). Both
groups also differed in valve length, w ith the maximal length for the
broader group being 31-32 pm in the field, while th a t of the narrower
group was 21-22 pm. The minim al valve length in the field was 15-16 pm
and 11-12 pm, respectively (Fig. 5C). W ith decreasing length of the cells,
th e w idth also decreased, b u t the length-associated differences were
smaller th an th e differences between the different groups (Fig. 5C).
U nfortunately, we failed to initiate auxosporulation in culture, and
therefore we were unable to determ ine the m axim um cell length for the
two clades. T he minimum size of all strains in culture was more or less
th e same, -9 -1 0 pm. W hen the cell reached this “critical” size, the
cultures ceased to grow and subsequently died.
38
Pseudocryptic species in N avicula phyllepta
23
E
22
O
21
n
O
<5
strains c lade A
strains cla d e B
19
¿0 18
17
5
6
7
W id th ( |jm )
» A p p e lz a k
B ie z e lin g s c h e H a m
o P a u lin a S c h o r
23
E 22
n.
O
21
^
20
0)
ro
C0
8
19
18
17
5
7.5
^
6
7
W id th (p m )
8
C
7
§j_ 6 .5
nt
o»
£
§
^
6
O
55
ítíHÍR*
VS—. «
5
4 .5
' «*
■> &
15
20
.
• Appelzak
Biezelingsche Ham
° Paulina Sch or
25
30
L e n g th ( p m )
F ig u r e 5 . S c a t t e r p lo t s o f (A ) v a l v e w i d t h v e r s u s s t r i a d e n s i ty o f N avicula phyllepta
strains,
(B)
v alv e w i d t h v e r s u s s t r i a
W e s te rs c h e ld e estuary, and
den sity of th r e e
natural
( C ) v alv e l e n g t h v e r s u s v alv e w i d t h
s a m p l e s fro m
o f three
the
natural
s a m p l e s f ro m t h e W e s t e r s c h e l d e e s t u a r y .
39
Chapter 2
E c o p h y sio lo g y a n d d is tr ib u tio n p a t t e r n
Irrespective of clade affinity, all strains tested grew well in a broad range
of salinities but were unable to tolerate freshwater conditions (Fig. 6).
However, strains from th e two clades differed considerably in their
tolerance to low salinities (0.5-2 psu). Strains belonging to clade A were
able to grow a t salinities as low as 0.5 psu, while monoclonal cultures
belonging to clade B showed growth only a t 5 psu or higher and died at
salinities below 5 psu. There was one exception: cells of strain BA-04-05
(belonging to clade A) grown a t 0.5-2 psu did not divide but were still
m otile and contained healthy-looking chloroplasts even after 10 d.
Q ualitative testing of six additional strains for growth a t two salinities (2
and 5 psu) showed th a t all six clonal cultures belonging to clade A were
able to grow a t 2 psu (Table 1). T he strain originating from th e Colne
estuary (UK) showed the sam e response to salinity as the other strains
belonging to clade A.
o?
.Í2 0 .5
CCY 0201
C C Y 0212
C C Y 0213
-♦ — ■8 A -04-Q 1
■■— B A -0 4 -0 2
-B— B A -0 4 -0 4
-A r—
B A -0 4 -0 5
CCY 0221
0.0
0
0 .5
1
2
5
10
20
30
Salinity (p s u )
F ig u r e 6 . G r o w t h r a t e o f e i g h t N avicula p h y lle p ta s t r a i n s g r o w n in t r ip l i c a t e a t e i g h t
d i f f e r e n t sa lin ities. S t r a i n s s h o w n in b lack b e l o n g t o c l a d e A; s t r a i n s in gray , t o cla d e
40
Pseudocryptic species in N avicula phyllepta
The abundances of cells belonging to clade A in the W esterschelde
estuary were negatively correlated w ith salinity (r = -0.33, P = 0.015),
while no significant correlation between salinity and the occurrence of
cells belonging to clade B was found (r = -0.0129, P = 0.926). In a
multiple regression model explaining the abundances of cells belonging to
clades A and B in the W esterschelde estuary, organic nitrogen content,
irradiance, and salinity were selected for clade A (Table 2, Fig. 7).
Together, these variables explained a highly significant [F(3,5) = 14.03, P
value < 0.0001] 42.4% (R2 adj.) of the variation in the abundance of cells
belonging to clade A. Am m onium concentration, silt content, irradiance,
* ♦4
*
0 .2
0 .4
E
CO
O
•s¡i
AM 0 e
<D
O
A
10f -------1------- 1------- 1_
—
10
o j.
o
cv 10
E
-2
tem perature, and n itrite were selected for clade B (Table 2, Fig. 7).
Together, these variables explained a highly significant [F(5,48) = 17.693,
P value < 0.0001] 61.2% (R2 adj.) of th e variation in the abundance of
cells belonging to clade B.
0
o
• •
' * * *
B
io 6 ------ «------- 1------- 1
—
0 .6
10
20
30
S alinity (psu)
O r g N (% )
10
™
CD
O100
20
40
60
80
0
Silt co n te n t (%)
200
400
A m m onium (pM )
F ig u r e 7 . S c a t t e r p l o t s o f t h e l o g - t r a n s f o r m e d a b u n d a n c e s o f Navicula p h yllep ta cla d e
A o r B a g a i n s t s o m e o f t h e e n v i r o n m e n t a l f a c t o r s s e l e c t e d in a s t e p w i s e f o rw a r d
m u l t i p le r e g re ssio n m o d e l. ( A ) O r g a n i c n i t r o g e n ( O r g N ) c o n t e n t v e r s u s a b u n d a n c e s of
cells
belonging t o
clade
A.
(B)
Pore
w ater
sa linity v e r s u s
abundances
o f cells
b e l o n g i n g t o c l a d e A. ( C ) S i l t c o n t e n t v e r s u s a b u n d a n c e s o f cells b e l o n g i n g t o cla d e
B. ( D ) A m m o n i u m c o n c e n t r a t i o n v e r s u s a b u n d a n c e s o f ce lls b e l o n g i n g t o c l a d e B.
S i g n i f i c a n t c o r r e l a t i o n s ( P < 0 . 0 5 ) a r e i n d i c a t e d by a r e g re ssio n line.
41
Chapter 2
T a b le 2 . R e s u l t s o f s t e p w i s e m u l t i p l e r e g re ssio n for t h e a b u n d a n c e s o f cells b e l o n g i n g
t o c l a d e s A a n d B in t h e W e s t e r s c h e l d e e s t u a r y . E s t i m a t e d r e g re ssio n c o e f f ic ie n ts (ß)
a n d t h e i r s t a n d a r d e r r o r s ( S . E . ) a n d a s s o c i a t e d sig n i f i c a n c e level ( P ) for e a c h v ariable.
O n ly s i g n i f i c a n t v a r i a b l e s a r e s h o w n for t h e d if f e r e n t c lad es .
C la d e A
S .E .
C la d e B
P - v a lu e
V a r ia b le
ß
O rg an ic n itro g e n
0.444
0.112
0.0002
0.318
0.110
0.0057
-0.273
0.112
0.0187
Irrad ian ce
ß
S .E .
P - v a lu e
0.546
0.125
0.0001
A m m o n iu m
0.657
0.097
0.0000
Silt c o n te n t
-0.255
0.095
0.0100
T e m p e ra tu re
-0.398
0.129
0.0035
N itrite
-0.199
-0.092
0.0350
S alin ity
D is c u s s io n a n d c o n c lu s io n s
On the basis of three molecular m arkers (viz. ribosomal ITS, 18S rRNA
gene, and rbcL) w ith different rates of sequence evolution, it was shown
th a t N. phyllepta s. 1. consists of at least two clearly distinct clades with
low intraclade sequence divergence compared to the interclade divergence.
These results confirm and strengthen the conclusions drawn by Créach et
al. (2006) who showed the existence of two ITS1 sequence clusters.
Créach et al. (2006) also found th a t the clades A and B differed in the
num ber of copies of the ribosomal operon as well as a 4-fold difference in
the cellular DNA content. T he congruence of the different molecular
markers, the low intraclade variation, the low intraclonal variation, and
the differences in DNA content suggest the presence of intrinsic barriers
to gene flow between these sym patric species, as has been shown in some
other microalgae (e.g., Coleman 2000, Behnke et al. 2004). R epeated
attem p ts to set interclonal m ating tests within and between the clades
were unsuccessful. W e were unable to initiate sexual reproduction despite
using m ethods th a t have been proved successful for m am r other diatom
species (see, e.g., Chepurnov et al. 2004). The average cell size in the N.
phyllepta cultures gradually decreased, and cultures were eventually lost
when they reached the critical m inim um size. W e therefore conclude th at
a mechanism m ust exist th a t restores the cell size, and it seems
42
Pseudocryptic species in N avicula phyllepta
reasonable to assume th a t this is associated w ith sexual reproduction and
auxospore formation, as is the case in m ost diatoms.
T h e differentiation in molecular m arkers was associated with
subtle b u t discrete differences in valve morphology between strains of
clades A and B, which should therefore be referred to as pseudocryptic.
There was a subtle b u t clear morphological distinction based on a
com bination of valve w idth and stria density. B oth w idth and stria
density were dependent on length and change during the life cycle, as is
the case in other diatom s (Mann 1999, M ann et al. 1999). However, these
length-associated differences are smaller th an the differences between the
different clades; hence, there is no overlap in morphology. The
pseudocryptic species are probably sibling as they are more closely related
to each oth er th a n to other Navicula species, although taxon coverage in
this genus is fragm entary. Several m olecular studies have now
d em onstrated th a t protist species traditionally defined on th e basis of
morphology often consist of two or more genetically distinct groups th at
often occur in sym patry (e.g., Slapeta et al. 2006, Vanormelingen et al.
2008). M oreover, evidence of reproductive isolation of such sym patric
cryptic species was found for several protists (Coleman 2000, A m ato et al.
2007, Vanorm elingen et al. 2008). In m ost cases, it is unknown whether
these seemingl}r similar species differ in their biology or whether they are
functionally equivalent. As a consequence, we know little about the
mechanisms th a t prom ote the coexistence of cryptic protist species. The
lim ited available data suggest or show differentiation between cryptic
species for susceptibility to different parasitic fungi (M ann 1999) and
light requirem ents (Rodriguez et al. 2005), which m ight help to explain
their sym patric occurrence. There is evidence th a t different tolerances to
salinity m ay contribute to niche differentiation between allopatric cryptic
species (Koch and Ekelund 2005, Lowe et al. 2005).
F o r N. phyllepta, the sibling species were differentiated in terms
of grow th a t different salinities, and this corresponded w ith their
distribution in the Schelde estuary. As previously shown for other
estuarine diatom s (Admiraal 1977, B rand 1984, Clavero et al. 2000), all
strains grew well over a wide range of salinities, including th e average
salinities a t the different sampling stations (8.8-22.7 psu), seemingly
suggesting th a t th e distribution is not the result of physiological
constraints. However, strains of clade B were not able to grow a t low
(0.5-5 psu) salinities, in contrast to the strains belonging to clade A.
Such small differences in physiological tolerances may influence species
43
Chapter 2
interactions and consequently their distribution in th e field (De Jong and
Adm iraal 1984). In particular, the different tolerances to low salinities
could be im portant during heavy rainfall when the salinities of the
sedim ent top la}’er often drop to alm ost freshwater conditions (Admiraal
1977, Coull 1999). This tolerance difference would give clade A organisms
a com petitive advantage in the oligohaline and mesohaline p arts of the
estuary. T hus, despite the wide salinity range in which the species were
able to grow, their tolerance to low salinities may nevertheless determ ine
their abundance. In line with this, the abundance of clade A organisms in
th e W esterschelde estuary was indeed positively associated with low
salinities, while clade B organisms reached their highest abundances at
higher salinities (>15 psu). However, this difference was not significant
because of the large interannual variation (clade B only bloomed in spring
of the first sampling year). From Figure 6, it is clear th a t strains of clade
A have higher growth rates in culture th a n those of clade B a t any given
salinity and th a t their optimal salinity is ~20 psu. T his finding suggests
th a t clade A should be able to outcom pete clade B. However, making
predictions ab out growth rates and com petitive ability in th e field based
on absolute growth rates obtained in the laboratory is difficult. For
example, N. salinarum displa 3 's optim al growth rates a t 30 psu
(Underwood and Provot 2000) but occurs only in brackish environments
where it outcom petes other species because of its tolerance to low
salinities and high am m onium and sulfide concentrations (Peletier 1996,
Underwood and Provot 2000). This phenom enon suggests th a t tolerance,
rath er th a n th e growth rate, determ ines the ecological success of an
organism and would explain species distribution.
Salinity is an im portant factor influencing the distribution of the
two clades w ithin N. phyllepta (and of other diatom s, Sabbe and
V yverm an 1991, Underwood et al. 1998). However, other environm ental
variables m ay be equally im portant, as is suggested by the multiple
regression model. T he model shows th a t, independent of each other,
organic nitrogen content, irradiance, and low salinity positively influenced
th e abundance of clade A. T he organic nitrogen content had the strongest
contribution to the model and is significantly correlated w ith silt content
(r = 0.6521, P = 0.000), ammonium concentrations (r = 0.5225, P =
0.000), phosphate concentrations (r = 0.6479, P = 0.000), w ater content
(r = 0.8114, P = 0.000), and organic carbon content (r = 0.9095, P =
0.000) (All correlations are given in supplem entary table 1). T hus, clade
A typically blooms in spring and summ er in the mesohaline parts of the
44
Pseudocryptic species in N avicula phyllepta
estuary on silty sediments (70%-90% silt) w ith high organic m atter and
w ater content and high nutrient concentrations. Clade B was positively
influenced by high ammonium concentrations and lower silt content. This
clade reached its highest abundances a t sedim ents w ith lower silt content
(30%-70%) and with high am m onium concentrations (200-450 phi).
N itrite, which was generali}' higher in silty sediments, also
negatively influenced its occurrence. Furtherm ore, clade B was positively
influenced by irradiance, but negatively by tem perature, suggesting th a t
this species peaks in spring, which could be due to tem perature tolerances
but m ight also be due to high grazing pressures in late spring and
sum m er (Sahan et al. 2007). Salinity, sedim ent type, organic m atter
content, ammonium concentrations, and w eather conditions thus
differently influence the spatial and tem poral distribution of both clades
of N. phyllepta. Several studies show th a t these variables strongly affect
the species composition of microalgal biofilms on intertidal mudflats
(Peletier 1996, Underwood et al. 1998, T hornton et al. 2002). Ammonium
can have positive effects on th e growth and distribution of benthic algae
as it is an easily assim ilated source of nitrogen (Underwood and Provot
2000). However, high am m onium concentrations may have detrim ental
effects on benthic algae, especially on N. phyllepta (Peletier 1996,
Underwood and Provot 2000). T he very high am m onium concentrations
(400-1,000 pM) necessary to cause detrim ental effects were (almost) not
encountered in the W esterschelde estuary, and a negative effect was
therefore not expected.
Our conclusion
th a t
N.
phyllepta
consists
of
different
pseudocryptic species possessing different preferences for salinity and
other environm ental factors m ay explain th e puzzling p attern of field
observations of N. phyllepta. T his “ morphospecies” was reported in
different estuaries (Underwood and B arnett 2006) at salinities ranging
from 6 to 35 psu (Cox 1995, Snoeijs and P otapova 1995, Peletier 1996,
T h ornton et al. 2002). These observations can probably be attrib u ted to
the occurrence of different pseudocryptic species w ith distinct
environm ental preferences, rather th a n a single generalist species th a t
exhibits a broad adaptability to different salinity conditions. This
phenom enon is found for several coastal and estuarine macroscopic
eukar 3 'ote morphospecies displaying substantial morphological and
physiological variation. Detailed stu d y revealed th a t these morphospecies
consist of several (pseudo) cryptic species w ith different habitat
requirem ents (Knowlton 1993). This finding has been shown, for instance,
45
Chapter 2
in nem atodes (Derycke et al. 200-5), in copepods, and for m any other
invertebrates (Bilton et al. 2002). Finlay (2002) argues th a t because
m icrobial eukaryotes are able to tolerate or ad a p t to a wide range of
ecologically im portant factors, there will be few physiological “species”
w ithin microbial morphospecies. O ur findings oppose th e view th a t
m icrobial species are ecological generalists. We show th a t there may be a
strong discrepancy between th e measured theoretical niche (which can be
very broad) and the realized niche in the field. W e further dem onstrated
th a t th e morphospecies N. phyllepta consists of a t least two distinct
clades, and th a t despite their genetically and evolutionary relatedness,
these clades display im portant physiological differences and are influenced
differently by environm ental gradients, resulting in distinct distributions
along the W esterschelde estuary.
A c k n o w le d g e m e n ts
T his research was supported by grants from the F und for Scientific
Research-Flanders (Belgium) in the framework of th e Flem ish-Dutch
collaboration on m arine research (VLANEZO) grants ALW 832.11.003
and G .0630.05, and by the BOF project GOA 01GZ0705 (Ghent
University). This is Publication 4623 Netherlands In stitu te of Ecology
(NIOO-KNAW ). W e thank three anonym ous reviewers for valuable
comments.
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52
Pseudocryptic species in N avicula phyllepta
S u p p le m e n t a r y m a te r ia l
P e a r s o n ’s r e s i d u a l s f o r C l a d e A
O r g a n ic N
I r r a d ia t io n
S a lin ity
-M* * .* 5 0
.
. *
P e a r s o n 's r e s i d u a ls fo r C l a d e B
I r r a d ia t io n
NH
of
te m p e ra tu re
NO
S u p p le m e n ta r y fig u r e 1 . P e a r s o n ' s r e s i d u a l s f o r t h e s t e p w i s e m u l t i p le reg re s s i o n s
s h o w n in t a b l e 2.
53
Chapter 3
A b s tr a c t
How sim ilar are closely related lineages of microalgae in their ecological
niches? C an therm al niches evolve fast on an evolutionary timescale
resulting in regular switches between climates or are they instead highly
conserved? To address these questions we composed a set of closely
related strains of th e globally distributed diatom genus Cylindrotheca,.
W e collected strains from a wide range of m arine h abitats, from coastal
plankton to sea ice and intertidal mudflats. We first inferred the
evolutionary relationships of these strains using a multi-locus DNA
dataset and obtained a well-resolved phylogeny. W e then determined
tem perature preferences of closely related lineages in laboratory
experiments. Combining the m olecular phylogeny with the therm al niches
of lineages revealed a very weak phylogenetic signal in therm al niche
characteristics. This indicates th a t closely related species tend to differ
more in therm al niche th a n expected by a random walk model. This
seems to be caused by a com bination of adaptive evolution and frequent
shifts in environm ents in related lineages.
56
Thermal niche evolution in C ylindrotheca
I n t r o d u c t io n
T o w hat ex tent closely related species are similar in their ecological
niches is a m ajor and recurring them e in evolutionary biology and ecology
(Harvey & Pagei, 1991, Losos, 2008, W iens et a i, 2010). Two extremes
along a continuum of niche evolution can be distinguished: phylogenetic
niche conservatism and adaptive radiation (Ackerly, 2009). Phylogenetic
niche conservatism (PNC) is th e tendency of species to retain aspects of
their fundam ental niche over time (Wiens
G raham , 2005).
Conservative evolution of ecological traits can arise from different
processes such as stabilizing selection (Ackerly, 2003), insufficient genetic
variation (Bradshaw, 1991) and homogenizing gene flow counteracting
local niche ad aptation (Holt & Gomulkiewicz, 1997). W hile several
studies have dem onstrated PNC in a broad range of organisms (e.g.
Ackerly, 2004, Kozak & Wiens, 2006, W iens et al., 2006, Verbruggen et
a l, 2009, Bahl et a l, 2011), others find no or only weak evidence for
PN C and instead report no or only little relation between phylogenetic
relatedness and niche sim ilarity (Rice et a l, 2003, Cavender-Bares et a l,
2004, G raham et a l, 2004), for instance in the case of adaptive radiation
(Losos et ah, 2003). A daptive radiation can be defined as “the
evolutionary divergence of members of a single phylogenetic line into a
variety of different adaptive forms”; usually this divergence occurs over a
relatively short geological time span (Futuym a, 1998). Given the paucity
of d ata, it is a t present not known how im portant PN C is, and more
im portantly to w hat degree
influence the incidence of PNC.
organism al or
environm ental
features
Ideally, th e com bination of robust phylogenetic inform ation and
inform ation on niche preferences allows estim ating how fast ecological
divergence evolves between related species (Cooper et ah, 2010). Niche
inform ation can be obtained either by ecological niche modelling or by
ecophysiological experim ents. Niche modelling techniques are based on
d ata on the current distribution of a species and th e range of
environm ental conditions it occupies (the realized niche), which is
typically sm aller th a n the suit of environm ents a species can potentially
inhabit (the fundam ental niche) (Colwell & Rangel, 2009). This, together
w ith the choice of modelling techniques can influence the patterns
inferred from a phylogenetic com parative analysis. Ecophysiological
57
Chapter 3
experim ents allow defining th e fundam ental niche of organisms for the
environm ental variable of interest.
O ur views on evolutionary niche dynamics are biased towards
macroscopic and often eye-catching species m ainly from terrestrial
communities; a selection often guided by natural history observations on
striking ecological differentiation (Ackerly, 2009). This clade selection
disregards th e microbial world. Little is known about evolutionary niche
dynam ics in m arine microbial species (Palum bi, 1994). M arine microbial
populations are often believed to lack geographical barriers to gene flow
(Cermeño h Falkowski, 2009, b u t see Casteleyn et al., 2010) which would
frequently lead to the dispersal of large num bers of cells into
unfavourable environments. It has been hypothesized th a t marine
com m unities are characterized by a widespread occurrence of ecological
spéciation, i.e. reproductive isolation caused by divergent natural
selection (e.g. Ingram , 2011), but the im portance of this m echanism is
unknown in m arine microbial communities.
In this study we focus on the globally distributed diatom genus
Cylindrotheca. Species of this genus are common in a wide range of
coastal h ab itats, from coastal plankton to sea ice and intertidal m udflats.
O ur goal was to determ ine the evolutionary therm al niche dynam ics of
m arine Cylindrotheca species. Therefore, we first determ ined the
phylogenetic
relationships
between
Cylindrotheca
lineages
and
experim entally quantified their fundam ental tem perature niches. W e then
integrated th e fundam ental therm al niche and th e phylogenetic
inform ation to infer tem perature niche evolution by calculating the
phylogenetic signal in the available data.
M a t e r ia ls a n d M e t h o d s
T a x o n s a m p lin g
W e collected a set of 40 strains belonging to Cylindrotheca cf closterium
and C. cf. fusiform is (Table 1). Tw enty-tw o strains were newly isolated
from m arine and brackish sediment and plankton sam ples (sampling
localities in T able 1 and Fig. 1). Diatoms were harvested from sediments
using th e lens tissue technique (Eaton & Moss, 1966); m igratory
58
Thermal niche evolution in C ylindrotheca
microalgae were collected by placing a piece of lens tissue on top of the
sedim ent followed by a coverslip on the tissue, which was transferred to
autoclaved seawater after 3 hours of incubation a t low light. We
established monoclonal, non-axenic cultures by isolating single cells by
m icropipette and subsequent culturing in sterile filtered (0.2 pm)
seaw ater (33 psu) enriched with f/2 nutrients (Guillard, 1975). T he other
18 strains were obtained from the Provasoli-Guillard N ational Centre for
C ulture of M arine Phytoplankton, U.S. (CCM P); th e Commonwealth
Scientific and Industrial Research O rganization (CSIRO) collection of
living microalgae (Australia), and th e culture collection of algae and
protozoa (C C A P), UK. Strains PT01, SP01, GB01 and CIM222 were
kindly provided by J. Serodio, I. Moreno G arrido and J. Taylor and hi.
Pfannkuchen.
F ig u r e 1 . G e o g r a p h i c d i s t r i b u t i o n o f C ylindrotheca s t r a i n s .
59
Chapter 3
T ab le
1. List o f C ylindrotheca s t r a i n s u se d in t h i s s t u d y .
C.f. =
C ylindrotheca
fusiform is, C.c. = C ylindrotheca closterium .
S tra in
I s o l a to r
I s o l a ti o n
O rig in
L a titu d e /L o n g itu d e
d a te
num ber
S p e c ie s
id e n t i t y
A N T 1001
B. V anelslander
2010
King G eorge, A n ta rc tic
62“ 13' S, 58=40' W
C.f.
A N T 902
B. V anelslander
2010
King G eorge, A n tarctic
62“ 13’ S, 58 40' W
C.f.
A N T105
B. V anelslander
2010
King George, A n tarctic
62 13’ S, 58 4 0 ' W
C.c.
A N T304
B. Vanelslander
2010
King George, A n ta rc tic
62 13' S, 5 8 4 0 ' W
C.c.
A N T401
B. V anelslander
2010
King George, A n tarctic
62“13’ S, 58 40' W
C.c.
A N T 6 04
B. Vanelslander
2010
King George, A n tarctic
62 13' S, 58 40' W
C.f.
A N T903
B. V anelslander
2010
King George, A n tarctic
62“ 13' S, 58°40' W
C.c.
CC A P101708
B res nan
2004
Buckie, S co tlan d , UK
57“ 40 N , 2“ 58’ W
C.c.
CC A P101709
Bresnan
2004
Cove Bay, S co tlan d , UK
57“ 06’ N, 2' 04’ W
C.c.
C C A P101711
B rosnan
2004
Stonehaven, Scotland, UK
56“ 58’ N, 2“ 12’ W
C.c.
C C M P 2086
C. Lovejoy
1998
Baffin Bay
78“ 36' N , 74" 29’ W
C.f.
CC M P1554
D . .laeobson
1993
Boothbay H arbor, M aine
43' 50’N, 69“ 38' W
C.c.
C C M P1725
J . S tirn
1995
G ulf o f O m an , A rab ian Sea
'23° 34' N , 58“ 51’ E
C.c.
C C M P1989
N. Rolde
1997
M idw ay Islands
28“ 12' N, 177° 21’ W
C.c.
C C M P340
B. Palenik
1988
Open ocean, Sargasso Sea
2 8 ' 59’ N, 64“ 22’ W
C.f.
C C M P343
S. W atso n
1958
W oods Hole, M assachusetts
41“ 31’ N, 70° 40’ W
C.f.
C C M P 344
W . M addox
1960
S an d y Hook, New Jersey
40“ 27’ N, 74 W
C.f.
C C Y 9601
H. Peletier
2007
Em s-D ollard, Holland
53“ 15' N, 7 ’ 05’ E
C.f.
C IM 2 2 2
M. Pfannkuchen
2009
A driatic, C ro a tia
44“ 0 2 ’ N, 1 3 ' 14’ E
C.c.
C S -1 1 4
J.L . Stauber
1980
Coral Sea, A u stra lia
17“ S, 149“ E
C.c.
C S -5
M. Wot.ten
1962
P o rt H acking-A ustralia
34" 04’ S, 151 08’ E
C.c.
G B 01
.1. T aylo r
2008
Colne, UK
51“ 50’ N, 0“ 59’ E
C.c.
H3
B. Vanelslander
2007
W estern S ch eld t, Holland
51 “21' N , 3°43' E
C.c.
I ID 0 2
B. V anelslander
2006
W estern S cheldt, Holland
51“19' N , 4° 16' E
C.f.
I I I D 13
B. V anelslander
2006
W estern S cheldt, Holland
51 “19' N, 4“ 16' E
C.f.
IIP 0 3
B. V anelslander
2006
W estern S cheldt, Holland
5 1 '2 1 ' N, 3 43' E
C.f.
IIP 1 4
B. Vanelslander
2006
W e ste rn S cheldt, Holland
51 “21 ' N, 3“ 43’ E
C.f.
K D 10
.1. W ölfel
2005
N y-Â lcsund, Spilbergen
78“ 55’ N , 11 56’ E
C.c.
M ID 2 2
B. Vanelslander
2007
Veerse M eer, Holland
51-33'N , 3“47’ E
C.c.
OS1
B. V anelslander
2007
E a ste rn S cheldt, Holland
51 32’N, 3“44’ E
C.c.
OS13
B. Vanelslander
2007
E astern S cheldt, Holland
5 1 '3 2 'N , 3‘44’ E
C.c.
O S9B
B. V anelslander
2007
E a ste rn S cheldt, Holland
51°32’N, 3°44’ E
C.c.
PS10
B. Vanelslander
2008
W e ste rn S cheldt, Holland
5 T 2 1 ’ N , 3“43’ E
C.c.
PS2
B. Vanelslander
2008
W estern S cheldt, Holland
51*21’ N, 3°43’ E
C.c.
PT01
.1. Serodio
2004
Rio d e A veiro, P o rtug al
40“ 39’ N, 8 40’ E
C.c.
SP01
I. M oreno-G arrido
2000
P u e rto Real, Spain
36“ 36’ N, 6“ 12’ E
C.c.
W 0214
B. Vanelslander
2010
N o rth Soa, Belgium
51 13’ N, 2“ 51’ E
C.f.
W 0222
B. Vanelslander
2010
N o rth Sea, Belgium
51°13’ N , 2“ 51’ E
C.f.
W 0224
B. Vanelslander
2010
N o rth Sea, Belgium
51“ 13’ N , 2° 51’ E
C.f.
60
Thermal niche evolution in Cylindrotheca
All cultures were kept in 24-well plates (Greiner Bio-One,
Frickenhausen, Germany) a t 18 ± 0.3°C w ith a 16:8 lightidark period and
50 pmol photons m 'V from cool-white fluorescent tubes. T he strains with
strain designation beginning w ith “A N T ’, strain KD10 and CCM P 2086
were isolated and m aintained a t 6°C. The cultures were transferred every
2 weeks to fresh medium.
G r o w th a t d iffe re n t t e m p e r a tu r e s
W e assessed the growth perform ance of the 40 strains at 7 different
tem peratures (0.2, 5, 10, 15, 20, 25 and 33°C). Each treatm ent was
replicated four times. Cells for growth experiments were harvested from
exponentially growing cultures and inoculated in 24-well plates a t a cell
density of -3,000 cells mL h Light conditions and culture m edium were
identical to those described above for the stock cultures. W e monitored
growth by pulse am plitude m odulated (PAM) fluorescence (MAXI
Im aging PAM fluorometer, Walz, Germ any). We used the minimum
fluorescence yield Fo as a proxy for biomass (Honeywill et al., 2002). We
determ ined the growth rate during the exponential grow th phase (4-5 d)
as the slope of the linear regression of log2-transformed F0 fluorescence
versus time for individual cultures. T he relation between growth ra te and
tem perature was modeled as in R innan et al. (2009) using the following
function:
p = a* (T -T min) * ( l - e(b‘(T-Tm"x)))
Tnuw and T m¡„ are the m aximum and m inimum tem perature
permissible for growth. T he param eter “a ” is the slope and b is a fitted
param eter related to the decrease in growth above T opt. T he equation was
fitted to the growth a t different tem peratures for each replicate using
S tatistica 6.0 (StatSoft, Tulsa, OK, USA). O ptim al tem perature and the
tem peratures (both low and high) at which the growth was 20% of the
m axim al grow th rate were th en calculated using a function calculator.
61
Chapter 3
P h y lo g e n y
W e inferred the evolutionary history of the 40 Cylindrotheca strains from
a multi-locus DNA dataset. W e selected five DNA regions, i.e. the nuclear
ITS region (consisting of ITS1, 5.8S rRNA gene, and ITS2) and D1/D 2
LSU rDNA, th e chloroplast RUBISCO large subunit gene (r6cL) and
psbA and th e m itochondrial gene coxi. Cells for DNA extraction were
harvested from exponentially growing cultures and pelleted by
centrifugation. W e extracted DNA using the bead-beating m ethod with
phenol extraction and ethanol precipitation as described by Zwart (1998).
Sequence d ata were obtained using previously published P C R primers
and protocols (rbcL and ITS: Vanelslander et al. (2009), co x i: Evans et
al. (2007), LSU rDNA: Vanelslander et al. (subm itted), psbA: Souffreau
et al. (subm itted). All sequences generated during this study were
deposited in Genbank (accession num bers XXX-XXX, will be added later
on).
W e autom atically aligned the sequences using M A FFT v6.843b
(Katoh et al., 2005) and removed ambiguous positions from th e ITS
alignm ent with Gblocks 0.9'lb (Talavera & C astresana, 2007) allowing a
m aximum of 8 nonconserved contiguous positions. D ata for some DNA
regions were missing for several species (mainly due to erratic
performance of the coxi prim ers), but the concatenated d a ta m atrix was
84.9 % filled.
W e performed Bayesian phylogenetic inference (Bí) on the
concatenated dataset using MrBayes version 3.1.2 (Ronquist &
Huelsenbeck, 2003). W e used a G T R + I+ G model in which each proteincoding gene (coxi, psbA and rbcL) was partitioned into three codon
positions and LSU rDNA and ITS rDNA were treated as separate
partitions resulting in 11 partitions. Pseudo-nitzschia pungens was chosen
as outgroup. All param eters were unlinked between partitions. Two
independent runs of four increm entally heated Metropolis-coupled MonteCarlo M arkov Chains (MCMC) were run for 20 million generations using
default settings. Runs were sampled every 1,000th generation, and
convergence and stationarity of the log-likelihood and param eter values
was assessed using T racer v.1.5 (R am baut and Drum m ond, 2007). The
first 4 million generations were discarded as burn-in and a m ajority rule
consensus tree was generated.
W e estim ated divergence times under a relaxed m olecular clock
using an uncorrelated lognormal model in BEAST vl.4.6 following
62
Thermal niche evolution in C ylindrotheca
Souffreau et al. (subm itted). We used one single calibration point, being
the split of C. closterium and C. fusiform is estim ated a t 9 Ma
(Sorhanrms, 2007). T he resulting chronogram displayed such a large
uncertainty of divergence times th a t we did not include it in further
analyses and it is not shown in this m anuscript.
I n te g r a tio n o f e co lo g ical d a t a a n d p h y lo g e n e tic
in fo r m a tio n
Most analyses of tra it evolution require ultram etric trees in which branch
lengths are proportional w ith time, and not th e estim ated am ount of
molecular evolution inferred by our Bayesian analysis. Therefore, we
converted our consensus phylogram into a chronogram using penalized
likelihood rate sm oothing (Sanderson, 2002) in th e R package Ape
(Paradis et al., 2004) w ith a lam bda value of 0.1. C urrent traits were
plotted on this chronogram to visualize trait diversity. W e further
visualized tra it diversity by plotting traitgram s (Ackerly, 2009) which
draw phylogenies in which the y position of nodes and tips corresponds to
the value of a continuous tra it variable, and x position corresponds to
node depth using the R package Picante (Kembel et al., 2010).
W e quantified th e degree to which phylogenetic relatedness
predicts tra it sim ilarity by calculating the K statistic presented by
Blomberg et al. (2003) using the R package Picante. This statistic
compares th e observed phylogenetic signal in biological traits to the
signal expected under a Brownian m otion model of tra it evolution using
the same topology and branch length of the phylogeny. This Brownian
m otion model calculates evolution of traits as a random walk along
lineages in which th e change in traits at each tim e unit is sam pled from a
norm al d istribution w ith a m ean of zero and a certain variance. To test
for statistical significance of this K value, we performed 1,000 sim ulations
to compare th e observed signal with the distribution of randomized
values. K values range between zero and infinity. K > 1 mean th a t traits
of related lineages are more similar th an expected on the basis of
Brownian m otion, which is often used as an indication of phylogenetic
niche conservatism (Losos, 2008). K values = 1 correspond to a Brownian
m otion process and also suggest a certain degree of conservatism in trait
evolution. K values < 1 suggest lower th an expected ecological sim ilarity
between closely related lineages and may result from divergent selection.
63
Chapter 3
Because th e K statistic is a standardized value, it can be used to compare
p attern s of tra it evolution in different clades.
W e com pared the distribution of tra it disparity w ithin and
among clades by constructing disparity-through-tim e plots (Harm on et
a l , 2003) in the R package Geiger (Harm on et al., 2008). For each
interior tree node, m ean relative tra it disparity is calculated among all
subclades present a t th a t time. This m ethod thus provides a running
average tra it disparity through tim e and compares this to the expected
tra it disparity under a Brownian motion model.
T h e phylogenetic com parative m ethods used here are based on
species-specific tra it means and do not incorporate within-species
variation. Therefore, we pruned the phylogenetic tree by removing strains
w ith identical sequences and assigning their mean tra it value to the single
sequence retained. T his is not ideal because it removes p a rt of the
observed tra it variation, but otherwise there would be an artificial
increase in th e num ber of clades (each strain is seen as a different lineage)
which would bias the results in the case of lineages w ith more strains.
S ea s u rfa c e te m p e r a tu r e
T em perature traits were plotted against the yearly average sea surface
tem perature (SST) from their sampling origin. SST values were the
average SST between 1971 and 2000 and were obtained from th e National
Oceanic
&
Atmospheric
A dm inistration
h ttp ://w w w .esrl.noaa.gov/psd/ accessed on M arch 10, 2011).
(NO A A,
R e s u lt s
Bayesian phylogenetic analysis of the concatenated alignm ent of rbcL,
psbA, LSU rDNA, ITS and coxi sequences (a to tal of 3780 characters)
resulted in a well-resolved Cylindrotheca phylogeny (fig. 2). We
morphologically assigned strains to C. cf closterium and C. fusiformis.
Our phylogeny clearly indicates th a t these morphospecies consist of a
diverse set of genetic lineages, possibly representing cryptic species. There
appears to be no pronounced geographic pattern in the phylogenetic tree,
especially when we added rbcL and ITS sequence d a ta of Chinese strains
64
Thermal niche evolution in C ylindrotheca
from Qingdao (Fig. 3) (Li et al., 2007), a strain from Maine (CCMP339).
Lineages containing Chinese strains d id n ’t group together but were
dispersed over the phylogeny, even though no lineages were shared
betw een tem perate A tlantic and Chinese strains.
W e examined growth and survival of the Cylindrotheca strains
across a tem perature range from 0.2 to 33 °C. Strains from different
clim ate zones strongly differed in their ability to cope w ith low and high
tem peratures (fig. 2). Strains sam pled from areas w ith cold clim ates (i.e.
Spitsbergen, Baffin Bay, A ntarctica) showed a lim ited ability to grow at
tem peratures >20°C, while strains from tropical and subtropical regions
(Coral Sea, Gulf of Oman) had strongly reduced growth rates at
tem peratures <10°C. Strains from tem perate regions had a broader
tem p eratu re range. Yearly average sea surface tem perature from the
sam pling location was significantly related w ith optim al growth
tem p eratu re (Spearm an R = 0.625, P — 0.00001), low tem perature
threshold for 20% growth (R = 0.590, P = 0.00006) and high tem perature
threshold for 20% growth (R = 0.700, P < 0.00001) (Fig. 4). We also
observed a strong correlation between growth a t high and low
tem peratures (R = 0.652, P < 0.0001) (Fig. 4), suggesting th a t species
th a t are able to grow a t low tem peratures are on average less able to
grow a t high tem peratures and vice versa.
65
Chapter 3
ío
zS
So o
H S'?
F ig u r e 2 . C o n s e n s u s t r e e f r o m a B a y e s i a n an a l y s i s o f five m o l e c u l a r loci. N u m b e r s a t
n o d e s a r e p o s t e r i o r p r o b a b ilitie s i n d i c a t i n g t h e s u p p o r t f o r e a c h n o d e ; b r a n c h l e n g t h s
a r e pro p o rtio n al to se q u en ce ch a n g e . F o r each strain t h e g ro w th r a te as a function of
7 d i f f e r e n t t e m p e r a t u r e s is s h o w n .
66
Thermal niehe evolution in C ylindrotheca
—
P se u d o n itz s c h ia p u n g e n s
P se u d o n itzsc h ia m ultiseries
A N T604
A n ta r c tic a
CC M P340
S a rg a sso
W 0222
W 0 2 2 4 N o rth S e a
W 0214
P A N TS02
C C M P2086
« n la r c llc a
B a ffin B a y
C C M P 3 4 3 M a s s a c h u s e tts
CC Y 9601
H o lla n d
C y lin d r o th e c a
C C M P 3 4 4 N ew J e rse y
cf. fu s ifo r m is
IID 02
T 1 IID 0 3 \
H o lla n d
IIP 0 3
IIP 1 4
C C M P 1 7 2 5 G u lf o f O m a n
C S 1 14
C o r a l S e a , A u s tra lia
,r — C I M 7 2 2 A d ria tic , C r o a tia
C C M P 1 9 8 9 M id w a y I s la n d s
C S 5 P o r t H a c k in g , A u s tra lia
i 1 O S 9 B H o lla n d
PS10
H o lla n d
C y lin d r o th e c a
i— C C M P 1 5 5 4
M a in e
cf. c lo s te r iu m
Jj ANT105
A N T 3 0 4 A n ta rc tic a
AN T401
A N T903
'C C A P 1 0 1 7 1 1 S c o tla n d
K D 10 S p its b e rg e n
S P 0 1 S p a in
G B 01
E n g la n d
C C A P 1 0 1 7 0 8 S c o tla n d
C C A P 1 0 1 7 0 9 S c o tla n d
H3
H o lla n d
M id 2 2
o m
PT01
I O S1
O S13
P o r tu g a l
H o lla n d
_QJ
F ig u r e 3 . C o n s e n s u s t r e e f r o m a B a y e s i a n an aly s is o f five m o l e c u l a r loci u s i n g t h e
s e q u e n c e s f r o m fig 2, b u t w i t h a d d i t i o n a l s t r a i n s a d d e d (o n ly rbcL a n d I T S d a t a for
t h e s e a d d e d s t r a i n s ) . S t r a i n s i n d i c a t e d in g r e y w e r e n o t used f o r t h e e c o p h y s io lo g ic a l
e x p e r i m e n t s a n d t h e r m a l n i c h e a n a l y s e s . T h e origin o f s t r a i n M 8 7 3 2 6 is u n k n o w n .
67
Chapter 3
u 30 A
Q)
3 25
fö
S
a
20
I
15
*-•
♦ ♦
t
*
£ io
I
¡2
5
0
-5
a.
0
5
10
15
20
25
30
A n n u a l M e a n S e a S u r f a c e T e m p e r a t u r e ("C)
u
45
J
40
<N
30
0
25
0
5
10
15
20
25
30
A n n u a l M e a n S e a S u rfa c e T e m p e ra tu re fC )
D
I 35
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-5
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t*
**
♦
.
♦
1 20
♦
♦
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-5
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A n n u a l M e a n S e a S u r f a c e T e m p e r a t u r e (°C)
30
0
5
10
L o w e r lim it f o r pó g r o w t h (°C)
F ig u r e 4 . P l o t s o f n ich e t r a i t s v e r s u s a v e r a g e a n n u a l m e a n s e a s u r f a c e t e m p e r a t u r e
( S S T ) A: O p t i m a l g r o w t h t e m p e r a t u r e in f u n c t i o n o f a n n u a l m e a n S S T . B: L ower
l im it for 2 0 % g r o w t h v e r s u s m e a n a n n u a l S S T . C: U p p e r limit f o r 2 0 % g r o w t h v e r su s
m e a n a n n u a l S S T . D: U p p e r lim it f o r 2 0 % g r o w t h v e r s u s L o w e r lim it f o r 2 0 % g r o w t h .
Thermal niche evolution in C ylindrotheca
F ig u r e 5 .
Consensus
chronogram
th e
tem p eratu re
n O O
L1- r*-»
w ith
current
r
z o o o o z z o o q o i£ û û (o o lœ u w ü )ü z zû O z zû .iD ü ü ^ h W w
< U 5 S ^ < < Ü Ü Ü Ü r = = = O O O £ L U 0 ü ü « í ¿ U < < W Ü U ü 5 lú .0 0
tr
TO ÇTT
traits
p l o t t e d on t h e t r e e
tips.
i
•T- (\J T-
u“j 5 ^
T
P
s y g g P P iI^ i? 8 8 S ^ s|® s S » s p P 5 < ^ p 5 5 < < § u , .
zügÇ gzzüUOün.iQQiooim om M UzzQOzzam üüSnhm w
< U S S S « Ü U O U = = = = Ü O O I1 Ü O O O C < VO <<C 0O O O S X D.OO
tr
T O I^ T T
T
c
I
05
COTf
S
O O
¡??89?2gg;
f— - > CNC\J C P P ss^ sjg g g ;:!^ ® :
ZQOOÇ z i z 0 u b 0 í g Q Q ¡ 0 0 1 w ü m m O z z ü O 2 z ° m o o 5 n h r o ( ñ
<o£->: «ouou====oüOû.üOüo«ïu«rooou5iD.oo
CD
O O
69
Chapter 3
T o deduce p attern s in niche evolution we first plotted th e tem perature
tra it values of th e individual strains on the chronogram (Fig. 5) and
m ade traitg ram s to visualise the distribution of tra its along th e phylogeny
(Fig. 6). Using th e K estim ate of Blomberg et al. (2003), we found very
weak phylogenetic signals in optim al tem perature (K = 0.261,
P = 0.0009), low tem perature thresholds for 20% grow th (K — 0.221,
P = 0.0019) and high tem perature thresholds for 20 % growth
(K = 0.353, P = 0.0019).
T he disparity-through-tim e plots (Fig. 7) confirmed the findings
from the K statistic and showed th a t tra it diversification is higher than
expected under a Brownian motion model of evolution. T he plots further
show th a t th ere is m ainly a trait diversification in low tem perature
thresholds and th a t tra it disparity starts already at th e origin in the
clade and is alm ost constantly higher th a n expected under a Brownian
motion model.
L O W T E M P E R A T U R E T h R E S H O L D M =20<P, <°C)
H IG H T E M P E R A T U R E T h R E S H O L D M = 2 0 '!.
F ig u r e 6 . P h y l o g e n e t i c t r a i t g r a m s w i t h
C
T E M P E R A T U R E O P T IM U M C C )
lin e a g e s a r r a n g e d a l o n g a c o n t i n u o u s t r a i t
ax is (lo w a n d h ig h t e m p e r a t u r e t h r e s h o l d a n d t e m p e r a t u r e o p t i m u m ) a n d c o n n e c t e d
by t h e u n d e r l y i n g p h y l o g e n y .
70
Thermal niche evolution in C ylindrotheca
Low t e m p e r a t u r e tre sh o ld
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show
th e
observed
relative
trait
d i s p a r i t y v e r s u s re la tiv e t i m e f o r C ylindrotheca l i n e a g e s c o m p a r e d w i t h m e a n e x p e c t e d
t r a i t d i s p a r i t y b a s e d o n 1 ,0 0 0 s i m u l a t i o n s o f B r o w n i a n m o t i o n e v o l u t i o n ( d a s h e d line).
T h e d i s p a r i t y a t a given t i m e p o i n t is t h e m e a n d i s p a r i t y o f t h e s u b c l a d e s w h o s e
a n c e s t r a l l i n e a g e s w e r e p r e s e n t a t t h a t t i m e r e l a t i v e t o t h e t o t a l d i s p a r i t y o f all t a x a
i n c o r p o r a t e d ( H a r m o n e t al., 2 0 0 3 ) .
D is c u s s io n
T he well-resolved, five-gene phylogeny combined with inform ation on the
fundam ental tem perature niche enabled us to infer therm al niche
evolution w ithin m arine lineages of the diatom genus Cylindrotheca. We
only observed a weak phylogenetic signal in therm al niche evolution,
which suggests th a t closely related lineages tend to differ more in their
71
Chapter 3
tem perature niche th an expected on th e basis of a Brownian motion
model. Phylogenetic signals can also be reduced when there is
convergence in traits in distantly related lineages, like we observed for the
cold adapted strains ANT105, ANT604, ANT1001 and CCMP2086.
However, despite the overall low phylogenetic signal, conservatism in
tra its does appear to exist in some clades. For example, in the case of the
three Scottish strains which originate from the sam e clim ate conditions
b u t belong to different lineages (CCAP 101708, C CA P 101709 and CCAP
101711), we observed th a t th e strain which has cold adapted sister
lineages in th e A ntarctic and Spitsbergen (CCAP 101711) is unable to
grow a t 25°C, while the two other strains do show higher tem perature
thresholds.
T h e K statistic is only a measure of a p attern and the
in terp retatio n of the K statistic in term s of niche conservatism and
adaptive radiation should be done w ith care (Ackerly, 2009). K values
should not be light-heartedly attrib u ted to a certain evolutionary process
because very different evolutionary processes can produce similar results
in term s of phylogenetic signal (Reveil et al., 2008). Low K values could
be caused by a combination of adaptive evolution and frequent habitat
shifts between closely related lineages. Low K values however can also
result from constant stabilizing selection tow ards a single peak (Reveil et
al., 2008) in which some lineages will adopt extrem e niches, while most
will be draw n back towards traits optim al for more common conditions
(Ackerly, 2009). Low K values can also arise by a random walk pattern
w ith constant evolutionary bounds such as a certain physiological
constraint on th e occurrence in particular conditions. Bounded evolution
however should lead to a uniform distribution of tra it values (Ackerly,
2009), which is not observed in our data.
T he disparitv-through-tim e plots show th a t there is higher trait
diversification th a n expected under a Brownian m otion model of
evolution, especially for growth a t low tem peratures and a t interm ediary
phylogenetic distances. It is im portant to recognize th a t our molecular
plrylogeny is far from complete. An extended sam pling effort would
certainly increase the num ber of extant clades we detected so far. In
addition, extinct Cylindrotheca species are very hard to detect, given
th eir very weakly silicified cell wall and thus no fossil records. In an
incom plete molecular phylogeny, the internal nodes near th e root will be
on average more often included than nodes near the tips because deeper
nodes have more descendants (Pybus & Harvey, 2000). T his can lead to
72
Thermal niche evolution in C ylindrotheca
incorrect conclusions about rates of spéciation and tra it variation through
tim e. So the interpretation of disparity-tlirough-tim e plots should be done
w ith caution.
O ur results are sim ilar to those of Ackerly et al. (2006), Evans et
al. (2009), Knouft et al. (2006) and G raham et al. (2004) who found th a t
clim atic tolerances are evolutionary highly labile and observed strong
variation in climatic tolerances between closely related lineages in a range
of terrestrial organisms (the woody plant group Caenothus, evening
primroses, Anolis lizards and dendrobatid frogs).
A comparison w ith other algae is difficult, since to our knowledge
only three other studies have performed an analysis of therm al niche
evolution in algae using a com bination of phylogenetic information and
d a ta on niche preferences. Verbruggen et al. (2009) studied the climatic
niche evolution in the seaweed genus Halimeda and concluded th a t there
was a strong phylogenetic signal in th e niches for four out of five sections,
w ith most lineages displaying a conserved preference for high average
tem peratures and low nu trien t levels. Several lineages in a fifth clade
however dic! show several independent adaptations to colder nutrient-rich
waters.
In a second study, B reem an et al. (2002) determ ined the
evolution of therm al niches of related lineages in the green alga
Cladophora and found differentiation in cold tolerance. Interestingly, they
also found a negative relation between the capacities to grow a t high and
low tem peratures. Such correlation can be caused by two different
mechanisms. One is antagonistic pleiotropy (A ngilletta et a l, 2003, Zhan
& M cDonald, 2011) in which m utations in a single gene result in
ad ap tatio n to one tem perature regime but negatively affects growth at
other tem peratures. T his would constitute a true trade-off between
abilities to grow a t different tem peratures. A nother possibility is
m u tatio n accum ulation (Cooper et al., 2001) in which different sets of
genes are responsible for a good perform ance in different environments.
D uring ad aptation in one environm ent, those genes th a t are no longer
under selective pressure (e.g. those inferring heat tolerance in polar
environm ents) become non-functional by the gradual accum ulation of
m utations through genetic drift. A t the m om ent we can not distinguish
between these two possibilities because we do not know the genes
responsible for th e adaptation to high or low tem peratures.
therm al
In a third study, Souffreau et al. (unpublished) recently compared
ad ap tation of different lineages within the cosmopolitan
73
Chapter 3
freshwater diatom Pinnularia borealis. Although th e num ber of sampled
locations is limited, they observed a clear separation between a group of
cold-adapted lineages from Chili, A ntarctica, Mongolia and the French
Alps/Belgium and a group of tem perate clim ate adapted lineages from
C anada/C zech Republic, Belgium and C anada/B elgium . T he divergence
between both groups of lineages is estim ated at ca 22 million years ago.
Some other studies on microalgae th a t were initially not m eant to
compare niche characteristics and phylogenetic relations can nevertheless
provide further information. Several studies on biogeographic distribution
p attern s and phylogenetic relationships in marine microalgae hint th at
there can be a high capacity for adaptation to new environments.
Casteleyn et al. (2010) dem onstrated th e form ation of distinct lineages
within the marine planktonic diatom species Pseudo-nitzschia pungens
during the Pleistocene, w ith a lineage typical for tropical to warmtem perate waters, an antitropical (tem perate) lineage and a third lineage
in the northeastern Pacific. Logares et al. (2008) dem onstrated the rapid
evolution of a set of closely related m arine dinoflagellate lineages which
inhabit a broad range of habitats from freshwater to marine, vet they all
were typical for low tem perature conditions.
Several laboratory experim ents have dem onstrated th a t microbial
populations can rapidly ad ap t to stressful and selective environm ents
(Vanormelingen (unpublished), Bell k. R,eboud, 1997, Bell & Gonzalez,
2009) and th a t adaptation depends on population sizes and the extent of
genetic variation, next to m utation rates and th e frequency of sexual
reproduction and the strength of the selection pressure. Micro-algae often
have extremely large population sizes, especially in m arine environments,
and can harbor pronounced genetic variation w ithin species (De B ruin et
ah, 2004, Vanormelingen et ah, 2009). In com bination w ith a high
dispersal potential in th e m arine environm ent (Cermeño & Falkowski,
2009) this could set th e stage for rapid adaptation to new environments.
To which extent divergent adaptation to a variety of environm ental
conditions is responsible for th e strong diversification in the
Cylindrotheca lineages is difficult to deduce from correlative analyses as
ours. Did ecological adaptation spur spéciation or vice versa? Several
studies on diverse tax a (but not micro-algae) indicate th a t niche shifts
due to natural selection trigger rapid divergence and even reproductive
isolation (Losos et al., 1997, Funk, 1998, Filchak et a l, 2000, Jiggins et
a l, 2001, Rundle & Nosil, 2005, Funk et a l, 2006). On an evolutionary
74
Thermal niche evolution in C ylindrotheca
tim e scale, this coulcl result in th e here observed repeated tem perature
niche shifts.
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C h a p te r 4
H y d r o d y n a m ic D is tu r b a n c e
G o v e r n s D iv e r s it y a n d
B io m a s s P a t t e r n s o f
E stu a r in e B e n th ic D ia to m s
Vanelslander B., V yverm an W ., Verleyen E. & K. Sabbe
Laboratory of Protistology & A quatic Ecology, D epartm ent of Biology,
Ghent University, Krijgslaan 281-S8, 9000 G hent, Belgium.
Unpublished m anuscript
Chapter 4
A b s tr a c t
We examined the relations between hydrodynam ic disturbance, biomass,
species diversity and functional group turnover in estuarine intertidal
m icrophytobenthos (M PB). T o this end we analysed an extensive diatom
d ata set representative of the variation in salinity, hydrodynam ic
disturbance and standing stock biomass of intertidal flats of the
W esterschelde estuary (The N etherlands). T otal MPB species richness
displayed a unim odal relationship w ith hydrodynamic disturbance and
standing stock biomass. In contrast, evenness increased w ith increasing
hydrodynam ic disturbance and decreasing biomass. Changes in diversity
reflected a strong turnover in functional group com position along the
biomass and hydrodynam ic disturbance gradients, and were visible at
different hierarchical levels of functional group classification. Maximal
functional diversity was observed a t interm ediate levels of habitat
disturbance.
82
Diversity - disturbance in benthic diatoms
I n t r o d u c t io n
How
species
diversity
and
coexistence
change
along
environm ental gradients has been a long-standing question in ecology.
One of the m ost influential hypotheses accounting for spatial variation in
diversity invokes disturbance (Grime, 1973, Connell, 1978). T he effect of
disturbance on diversity, formalized in the Interm ediate Disturbance
Hypothesis (IDH, Grime, 1973, Connell, 1978), predicts th a t interm ediate
disturbance promotes maxim al diversity. T he basic idea behind the IDH
is th a t exposure to low disturbance results in com petitive species
excluding inferior com petitors resulting in low diversity. Increasing
disturbance will diminish com petition intensity by killing or removing
cells and m aking space available for settling of inferior competitors
resulting in th e coexistence of com petitive and disturbance tolerant
species leading to higher species richness. W ith further increasing
disturbance, com petitive species become unable to cope w ith disturbance
and only disturbance resistant species remain. Many empirical studies
confirmed this hypothesis (Sousa, 1979, Molino & Sabatier, 2001,
Svensson et a l, 2007), though there rem ains considerable debate about
th e im portance of com petition along disturbance gradients (Violle et al.,
2010) and the necessity of patchiness in disturbance events (Roxburgh et
al., 2004). Reviews by Mackey & Currie (2001) and Hughes et al. (2007)
revealed th a t in less th a n 25 % of the studies on disturbance - species
richness (SR) relations a unimodal relation was dem onstrated while only
% of studies provided evidence for such a relationship when evenness
was used as a m easure of diversity (Mackey & Currie, 2001). One reason
why so m any observations are inconsistent w ith the IDH is th a t this
11
hypothesis relies on a num ber of assum ptions/prerequisites, which are
seldom accounted for, including the occurrence of com petitive exclusion,
the need of a large regional species pool, a. trade-off between competitive
and disturbance resistance related traits and the occurrence of multiple
succession stages (Svensson et al., 2007).
In ad d ition to disturbance, productivity (PR) - or its often used
surrogate stan d ing stock biomass (M ittelbach et a l, 2001, Irigoien et al,
2004) - is an o ther main driver of diversity patterns. M any studies have
explored th e shape of this relation and also often found unim odal or
“hum p-shaped” relations, especially in plant communities (Grime, 1973,
M ittelbach et al., 2001). However, biomass can be a ver}' poor surrogate
for productivity (W hittaker, 2010). This notion is im portant, as SR-PR
83
Chapter 4
studies focus on productivity because it can be used as a proxy for
limiting resource supply rate. Ecological theories predict th a t species
richness should be driven by the supply rate (species-energy theory,
W right, 1983) and the relative ratios of limiting resources (resource ratio
theory, Tilm an, 1977) th a t regulate the num ber of com peting species th a t
can locally coexist (Gross & Cardinale, 2007, C ardinale et al., 2009).
Based on hum ped biomass diversity relations, Grime (1973) developed his
classical com petitive exclusion theory as an explanation for these
unimodal relationships. He stated th a t m axim al diversity a t interm ediate
biomass values was due to the coexistence of species belonging to
different p lan t strategies. Com munities w ith high biomass are species
poor because they are dom inated by com petitive species and low biomass
com m unities are again species poor because they lack the competitive
species.
M ittelbach (2001) pointed to the fact th a t our knowledge of
species richness - biomass and productivity relationships is m ainly based
on particular taxonom ic groups (vascular plants, mammals, aquatic
invertebrates) and therefore may be biased. Since 2001, several studies
investigating th e SR-PR. relation focused on other taxonom ic groups, but
aquatic microbial communities for instance are still underrepresented (but
see Li, 2002, Horner-Devine et al., 2003, Irigoien et al., 2004, C ardinale et
a l, 2006).
In
this
paper,
we
examine
the
relationships
between
the
hydrodynam ic disturbance, biomass an d species diversity in estuarine
intertidal m icrophytobenthos (M PB). M PB plays an im portant role in
th e ecology of estuarine systems. They provide up to 50% of the prim ary
production (Underwood & Krom kamp, 1999), contribute to sediment
stabilization through the production of extracellular polymer secretions
(Decho, 2000) and influence nutrient fluxes between sedim ent and water
(Sundbäck et a l, 2000). In our stud}r area, the W esterschelde estuary,
M PB is largely made up of benthic diatom s.
E stuarine M PB communities are subject to strong species sorting
effects due to th e structuring of com m unities by salinity, light exposure
and hydrodynam ic forces (Admiraal, 1984, Sabbe & Vyverm an, 1991,
Underwood, 1994, Paterson Sz Hagerthey, 2001). Dispersal is high due to
erosion/sedim entation dynamics and by im port of propagules from
upstream and from the sea. There is a large gradient in salinity spanning
more th a n 60 km. A second im portant gradient is the degree of
hydrodynam ic disturbance. Intertidal areas experience strong wave action
Diversitj’ - disturbance in benthic diatoms
th a t carries away fine sedim ent particles and leave coarser, well sorted
sand, while finer sediment like silt and clay particles are deposited in
more sheltered areas (Oh <k Koh, 199-5). T he degree in wave action thus
produces a continuous gradient in sedim ent grain size which strongly
influences other environm ental factors such as w ater content (Paterson et
al., 2000) and nutrient concentrations (Paterson & Hagerthey, 2001). At
the microscale, there are steep gradients in nutrients, light and oxygen
(Adm iraal, 1984, Paterson & Hagerthey, 2001). All these gradients result
in a very heterogeneous intertidal environm ent a t various spatial and
tem poral scales. This heterogeneity creates various different niches for
estuarine benthic diatoms. Benthic diatom diversity in estuaries is
therefore high, not only from a taxonom ic point of view (> 2 0 0 species
commonly present in the W esterschelde estuary) b u t also from a
functional point of view. Benthic diatom s can be classified in three large
functional groups, namely th e motile epipelic diatom s, th e sand grainattached epipsammon and tychoplankton (Admiraal, 1984).
In the past, species diversity and distribution p attern s have
m ainly been assessed for epipelic assemblages (Underwood, 1994). We
explored this relationship using a large dataset comprising m onthly
sam pling of 32 stations in th e W esterschelde estuary during one year
(>360 samples) covering a wide range of hydrodynam ic disturbance,
salinity and MPB biomass. O ur m ain questions were: 1) w hat is the
relationship between hydrodynam ic disturbance and diatom species
richness and evenness; 2 ) w hat is the relation between diatom diversity
and diatom biomass; and 3) how do the different functional groups
respond tow ards disturbance and biomass gradients?
M a t e r ia l a n d M e t h o d s
S tu d y s ite
T h e W esterschelde is th e meso- to euhaline p a rt of the Schelde estuary
(B elgium /T he Netherlands). It is a m acrotidal coastal plain estuary
subject to considerable organic pollution and eutrophication. The
W esterschelde is well mixed and characterized by a complex morphology
w ith tidal channels surrounding several large intertidal flats and salt
m arshes. Residence time of the w ater in the W esterschelde estuary ranges
85
Chapter 4
from one to three m onths (depending on the season) resulting in a rather
stable and gradual salinity gradient (ranging from 3 to 32 psu in the
Westerschelde) th a t is prim arily determ ined by th e m agnitude of the
river discharge and to a lesser extent to the tidal oscillation, which is of
smaller am plitude (Meire et al.7 2005).
S a m p lin g c a m p a ig n
T hirty-tw o sampling stations (F ig.l) were sam pled m onthly during one
year (Oct. 1991-Oct. 1992). Eu-, poly- and mesohaline stations were
selected in order to cover the entire range in salinity and sedim ent types
in the W esterschelde estuary. Sedim ent cores (diam eter 22mm) from the
upper 1 cm of the sedim ent were taken a t low tide and fixed w ith 4%
formalin. Five cores from each locality (within lm 2) were combined to
com pensate
for
microscale
differences
in
diatom
composition
and
abundance.
P o lyhaline
E uhaline
M outh
M p4
M p3
M p2
M pl
Mp6
M p5
M esohaline
N b3
N b1
Snl
Sn2
A z3
A zi
H p4
H p3
H p2
H p1
P s3
P s2
Ps1
T e3
T e2
T el
Ph3
Ph2
Phi
B h3
B h2
Bh 1
K s2
K sl
5 km
F ig u r e 1 . M a p o f t h e W e s t e r s c h e l d e e s t u a r y s h o w i n g t h e p o si t i o n o f t h e s a l in i t y z o n e s
( M c L u s k y , 1 9 9 3 ) a n d t h e l o c a t i o n o f t h e s a m p l i n g s t a t i o n s . I n te rtid a l a r e a s below
m e a n high w a t e r a r e s h o w n in l i g h t grey , in t e r ti d a l a n d s u p r a - t i d a l a r e a s a b o v e m e a n
h ig h w a t e r ( p r e d o m i n a n t l y s a l t m a r s h e s ) in d a r k grey . Az: A p p e l z a k , Bh: B a a lh o e k ,
Hp:
H o o g e p l a t e n , Ks: K o n i j n e n s c h o r , M p : M o l e n p l a a t ,
P laten
van
T ern eu zen .
H ulst,
Ps:P aulinaschor,
Ra :
Nb:
Ram m ekenshoek,
N a u w v a n B a t h , Ph:
Sn:
S taartse
Nol,
Te:
Diversity - disturbance in benthic diatoms
S p ecies c o m p o s itio n a n d d iv e r s ity
P a rt of the homogenized, combined samples was oven-dried a t 60 °C. One
gram sedim ent dry weight (SDW) of each sample was then oxidized with
hydrogen peroxide (27 %) and acetic acid (99 %) (Sabbe, 1993). The
sediment was th en washed 3 to 4 tim es with distilled w ater and diluted to
a final volume of 50 ml. A fter thorough homogenization a subsam ple of
50 pi was taken w ith a m icropipette, transferred to a coverslip and airdried. P erm anent preparations were made w ith N aphrax m ounting
medium. On each coverslip all diatom valves in a transect of known
surface area were identified and counted. Allochtonous species (planktonic
and freshwater) were om itted from the analyses. Cell counts of 400 cells
per sample can not adequately estim ate the occurrence of rare species
(Alverson et al., 2003), therefore we also om itted species which occurred
less th an three times in th e dataset. The original dataset contained 294
tax a and after omission of these species it contained 193 species. The
counted surface wms then extrapolated to the whole surface of th e dried
drop, th u s allowing us to determ ine the total num ber of diatom s in one
gram SDW. For each slide a minimum area of 5000 by 100 pm was
counted. Counts were continued beyond this m inimum area until a t least
400 valves were counted. In some samples, diatom densities were very
low; a m axim um of ten transects of 5000 by 100 pm was then scanned for­
di atoms.
W e assigned species to functional groups based on the following
traits: cell-size and shape, hold-fast mechanisms and m otility. These
traits are all valuable for characterizing functional traits in benthic
inicroalgae. Cell size determ ines specific physiological activities such as
grow th (Weithoff, 2003). Furtherm ore, cell-size and shape jointly
determ ine the surface to volume ratio which in tu rn influences nutrient
uptake. Hold-fast mechanisms evidently determ ine to which extent cells
are able to cope with hydrodynam ic disturbance. M otility is undeniably
an im portant tra it in in tertidal m udflats as these environm ents are
characterized by steep gradients in light and nutrients and enables cells
to position themselves in favorable patches. Inhibiting and limiting light
conditions are often only a few hundred pm away from each other.
Moreover, n utrients such as silica can become limiting in the upper part
of the sedim ent when production is high (Jesus et al., 2009). It has
recently been shown th a t epipelic diatom s display the ability to sense
87
Chapter 4
silica concentration gradients and m igrate to favourable patches (Gillard
et al. unpublished).
Based on these groups we classified benthic diatom species into
the motile epipelic diatom s, the sand grain-attached epipsamm on and
tychoplankton, functional groups which are well established in
m icrophytobenthos research (Paterson & Hagerthey, 2001). W ithin the
epipsammon, we further distinguished between adnate, stalked and small
(<10pm ) motile species (Hamels et a l, 1998). T he epipelon was divided
into small-celled ( 1 0 - 2 0 pm) and big-celled ( > 2 0 pm) diatom s (fig. 2 ) will
explore if these autccological traits are able to predict th e effect of
disturbance on functional group species richness.
Cell biovolumes of each taxon was calculated using linear
dimensions (length, w idth and height) and a standardized set of equations
for biovolume calculations for the different diatom genera (Hillebrand et
al., 1999). T he to tal biovolume per sample was calculated based on the
cell densities and the m ean biovolume for each diatom species.
W hile the oxidation m ethod ensures th a t all diatom cells are
quantitatively separated from the sediment and also allows accurate
identification, it does not allow to distinguish between dead and living
cells, which could lead to an overestim ation of th e to tal diatom biomass.
W e therefore estim ated the relative proportion of living cells in the
different functional groups in about 30 fixed samples stained w ith Bengal
Rose, which allowed us to distinguish between em pty frustules and those
w ith a cell content (the latter were referred to as living diatom s) (Hamels
et al., 1998). These countings dem onstrated th a t the oxidized counts do
supply realistic estim ations of the absolute biomass values since th e ratios
of the living to dead cells of the epipelon closely follow trends in cell
abundance (Hamels et a l, 1998). On average, th e living:dead ratio was
relatively stable, between 40 to t 60% of the cells was alive. Using these
living/dead ratios, the original quantitative counts on th e perm anent
preparations were then corrected to yield the total num ber of living
diatom s per gram SDW.
Diversity - disturbance in benthic diatoms
P la n k to n
T y c h o p la n k to n
M o tile
M o tile
2 0|im
M o tile
A d n a te
F ig u r e
2. T h e differen t fu n ctio n al
g r o u p s o f d i a t o m s t h a t o c c u r s in t h e S c h e l d t
estu ary . T h e ep ip s a m m o n (co m p risin g t h e stalked, a d n a te a n d m otile < 1 0 p m groups),
w hich
is t h e s m a l l e s t g r o u p s ,
can
grow
attac h ed
to
in d iv id u al s a n d g r a i n s . T h e
e p i p e l o n ( c o m p r i s i n g t h e m o t i l e 1 0 - 2 0 p m a n d t h e > 2 0 p m g r o u p s ) c o n s i s t s o f larger,
m o t i l e d i a t o m s t h a t f o rm d e n s e biofilm s. T h e t y c h o p l a n k t o n h a s a m o r e a m p h i b i o u s
life style.
G r a n u lo m e tric a n a ly s is
Granulom etric analyses of the sediment (top 1 cm) were
performed every two m onths a t each station; for the intervening sampling
m onths, values were obtained by linear extrapolation. Size analysis was
performed w ith a Coulter C ounter (LS100, Coulter Electronics, UK). By
adopting a grade scale (the W entw orth scale, B uchanan & Kain, 1971),
we then assessed w hat sedim ent volume percentage was present in each
size class. T he following particle size classes were distinguished: clay (<
3.9 pm), silt (3.9 -63 pm ), very fine sand (63-125 pm ), fine sand (125-250
pm ), m edium sand (250 pm-500 pm) and coarse sand (500 pm- 1000 pm).
89
Chapter 4
It is well known th a t hydrodynam ic disturbance strongly determines the
sediment composition of intertidal sedim ents (Oh & Koh, 1995). We
therefore conducted a principal com ponent analysis (PCA) based on the
standardized abundances of the particle size classes and m edian grain size
and used the first principal com ponent axis sample scores as a proxy for
hydrodynamic disturbance of the sediment (Van Colen et al., 2010) . The
first axis explained 64.1% of the variation in granulom etry and was
strongly correlated w ith th e silt fraction (r=0.963, pCO.OOl).
S ta tis tic a l an a ly sis
W e calculated both species richness and Pielou’s evenness index (based
on cell abundance) for each of the 360 samples. Trends in to tal species
richness, functional group species richness and to ta l evenness along log
transform ed biomass and hydrodynam ic disturbance were explored using
linear and quadratic regressions for which we checked the homogeneity
and homoscedasticity. We used the Akaike inform ation criterion (AIC) to
select the m ost reliable model. If the quadratic regression was selected, we
used the Mitchell-Olds and Shaw (MOS) test (M itchell-Olds & Shaw,
1987) to determ ine w hether there was a significantly unimodal (or so
called ‘hum ped’) shape and to estim ate the peak of the unimodal curve.
W e further verified if the estim ated peak diversity is significantly greater
th an the diversity at th e m inim um biom ass/disturbance and less th an the
diversity at th e observed m axim um biom ass/disturbance, i.e. if the
m axim um richness is reached w ithin the observed range of
biom ass/disturbance. T he MOS test is a classical m ethod to look for
hum p-shaped species richness p attern s (M ittelbach et al., 2001, Chase &
Leibold, 2002) and was conducted using the VEGAN package in R
(Oksanen et ah, 2008).
Salinity gradients in estuaries strongly affect diatom species
distributions and richness (Sabbe &; Vyverman, 1991). As our sampling
campaign covered a broad range of salinities (between 8.5 and 35 psu for
the interstitial w ater), we tested for the effect of salinity on species
richness. As we found a weak b u t significant trend in species richness
along the salinity gradient (supplem entary table 3, to tal species richness
= 41.30 + 0.22 X salinity, F = 11.44, P = 0.0007, R 2 = 0.028), we
recalculated
the
above
regressions
between diversity
and
biom ass/disturbance, b u t now using the residual species richness after
accounting for the salinity trend (Cardinale et a l, 2006).
90
Diversity - disturbance in benthic diatoms
B eta diversity was quantified using the B ray-C urtis dissimilarity
index (based on standardized quantitative abundance data) and the
Jaccard’s index (based on presence/absence data).
W e compared th e diversity in samples from different silt content
classes (0-1%, 1-5%, 5-10%, 10-20%, 20-40%, >40% silt) using Analysis
Of Sim ilarity (ANOSIM, (Clarke, 1993), implemented in the software
package P R IM E R 6 (Clarke & Gorley, 2006) based on the B ray-Curtis
and on the Jaccard index. ANOSIM produces a test statistic R which lies
between 0 and 1 depending on th e sim ilarities between groups. An R
value of 1 indicates maxim al dissimilarity, while 0 stands for full
similarity. C ontrasting q u antitative and qualitative m etrics enables one
to assess w hether dissimilarities between groups are m ainly due to
compositional change or change in relative abundances (Schaefer et a l,
2005).
R e s u lt s
D iv e r s ity a lo n g a d is tu r b a n c e g r a d ie n t
Total diatom species richness showed a significant quadratic relation with
hydrodynam ic disturbance (Table 1, Fig. 3A) and the MOS test indicates
th a t the relation is hum p-shaped. Samples from stations associated with
low and high disturbance displayed low diversity, while samples from
interm ediately disturbed stations had the highest diversity. T he Pielou’s
evenness index (Fig. 5A) showed a significant negative correlation with
increasing hydrodynam ic disturbance (Spearman R ank R = - 0.636 , P —
0 . 0001 ) .
T he species richness of the epipsammon showed a significant
linearly b u t not unim odally decreasing relation w ith decreasing
hydrodynam ic disturbance (Table 1, Fig. 3A). T he species richness of the
epipelic and tychoplanktonic functional groups displayed a significant
quadratic relation with hydrodynam ic disturbance (Table 1, Fig. 3A).
T he MOS test showed th a t both relations were unim odal and th a t the
estim ated peak richness of both groups was located w ithin the observed
range of disturbance values and is expected a t 0.419 disturbance and
91
Chapter 4
1.559 (PC A axis 1 values) disturbance for the epipelon and tychoplankton
respectively.
Species richness of the adnate epipsammic diatom s linearly
decreased w ith decreasing hydrodynam ic disturbance (Table 1, Fig. 3C).
T he small motile (<10 p m ) epipsammic diatom s showed a quadratic
relation w ith hydrodynam ic disturbance (Table 1, Fig. 3C). T he MOS
test indicated th a t the relation was unim odal, b u t the estim ated peak
diversity was not significantly greater th an the diversity a t the maximal
hydrodynam ic disturbance. T he stalked epipsammic diatom s showed a
quadratic (Table 1, Fig. 3A), unim odal (MOS test) relation with
disturbance w ith estim ated peak diversity a t -1.075. T he epipelic diatoms
between 1 0 and 2 0 p m significantly decreased along decreasing
hydrodynam ic disturbance (Table 1, Fig. 3A). For the epipelic diatom s >
20
p m species richness, a linear regression was not possible as the
assum ption of hom oscedasticitv was not fulfilled. Therefore we used a non
param etric Spearm an rank correlation and found a positive correlation
between decreasing disturbance and species richness of m otile diatom s >
20 p m (R=0.2373; p=0.000005).
92
Diversity - disturbance in benthic diatoms
.
T o ta l
ty ch e
» e p ip s a m
* e p ip e l o n
T o ta l
ty c h o
e p ip s a m
e p ip e l o n
1
H y d ro d y n a m ic d is tu rb a n c e
!0
to o
1OO0
B i o v o l u m o ( p m '1 x 10J i / m 2 )
20
■ M o tile < 1 0 p m
• A d n a te
. S t.i l k e d
18
16
14
12
10
6
4
7
0
1.0
0
1.0
2.0
10
H y d r o d y n a m ic d is tu rb a n c e 20
• M o t il e > 2 0 p m
• M o t il e I O - » p m
18
)
M o t il e > 2 0 p m
M o t il e 10-20 p m
lo
1614
14
xr
; 10
ÿ
100
B io v o lu m e t p m - x 1 0 : / m
a
I
12
*
1
«0
s
-6 •"
■1 •
2
-
0-1
• 2 .0
0
10
1.0
H y d ro d y n a m ic d is tu r b a n c e
100
f iio v o ltim e ( p m
luoo
x 10 !- / m ! )
F ig u r e 3 . S p e c i e s r i c h n e s s ( S R ) a l o n g d i s t u r b a n c e a n d t o t a l b i o m a s s g r a d i e n t s . (A-
B): T o t a l S R , t y c h o p l a n k t o n i c , e p i p s a m m i c a n d ep ip elic S R a l o n g t h e d i s t u r b a n c e (A)
and
b io m ass ( B ) g ra d ie n t (low est d istu r b a n c e v a lu e =
m a x i m a l d i s t u r b a n c e ) . O n ly
s i g n i f i c a n t r e g r e s s i o n s a r e p l o t t e d . U p p e r b l a c k line: r e g re ssio n for t o t a l SR, d a s h e d
b l a c k line: e p i p s a m m i c SR, g r e y line: t y c h o p l a n k t o n S R , lo w er b lack line: ep ip elic SR.
(C -D ):
SR
o f 3 epipsam m ic gro w th
m o r p h o lo g i e s
(adnate,
<
10pm
m otile and
s t a l k e d ) a l o n g d i s t u r b a n c e ( C ) a n d b i o m a s s ( D ) g r a d i e n t . B lac k line: reg re ssio n for
m o t i l e < 10 p m S R , g r e y line: a d n a t e SR, d a s h e d b l a c k line: s t a l k e d d i a t o m S R . ( E F ): S R o f 2 ep ip elic g r o w t h
m o r p h o lo g i e s ( m o ti l e 1 0 - 2 0 p m a n d m o t i l e >
20pm)
a l o n g t h e d i s t u r b a n c e ( E ) a n d b i o m a s s ( F ) g r a d i e n t . R e g re s s i o n e q u a t i o n s a r e giv en in
T a b l e s 1 a n d 2.
93
Chapter 4
1 . R e g re s sio n
T a b le
hydrodynam ic
e q u a t i o n s f o r t o t a l a n d f u n c t i o n a l g r o u p s p e c i e s ric h n e s s vs.
d isturbance
(lin ear:
Y =a+bX
or
Q uadratic:
Y=
a+
bX
+
cX 2).
R e g r e s s i o n s a r e s h o w n in Fig. 3. S i g n i fi c a n c e c o d e s : 'N . S . ' P > 0 . 0 5 ; ‘* ’ : 0 . 0 1 < P
<
0 . 0 5 ; '* * ': 0 .0 1 <
C r i t e r io n .
N.A.
P <
0 . 0 0 1 ; ' * * * ’: P < 0 .0 0 1 . AIC: A k a i k e ’s in f o rm a t i o n
n o t ap p licab le. S E : S t a n d a r d
E rror. T h e AIC v a l u e f o r t h e m o s t
r e lia b le m o d e l is s h o w n in bold.
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Diversity - disturbance in benthic diatoms
T a b l e 2. T h e reg re ssio n e q u a t i o n s for t h e t o t a l s p e c i e s ric h n e s s a n d f u n c t i o n a l g r o u p
s p e c i e s ric h n e s s vs. t o t a l s t a n d i n g b i o v o l u m e s t o c k (lin e ar: Y = a + b X o r Q u a d r a t i c :
Y = a + b X + c X 2). R e g re s s io n s a r e s h o w n in Fig. 3. S i g n i fi c a n c e c o d e s : ‘N . S . ' P >
0.05 ;
: 0 .0 1 <
P <
0.05 ;
0.01 <
A k a i k e ’s i n f o r m a t i o n C riterio n . N.A.
P <
0 . 0 0 1 ; ' * * * ’: P < 0 .0 0 1 . AIC:
n o t a p p lic a b le . SE: S t a n d a r d
E rro r. T h e AIC
v a l u e f o r t h e m o s t reliable m o d e l is s h o w n in bold.
/
x
Çf
z
s
r_,
-i
§
CD
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t?
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<33
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cm
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95
Chapter 4
T h e hydrodynamic disturbance is also strongly related to total diatom
biomass (biomass = 0.498*disturbance + 4.755, R 2=0.654, P = 2.2 . IO'16)
(Fig. 4). The strong collinearity between biomass and disturbance makes
it impossible to discern th e relative im portance of both on species
richness using a multiple regression.
1000
o
-
100
X
£
n.
■~U
£
■
10
o>
m
-
2 .0
0
1 .0
2.0
10
Hydrodynamic d i s t u r b a n c e
F ig u r e
4.
R e la ti o n
betw een
biom ass
and
hydrodynam ic
disturbance.
(Lowest
d istu r b a n c e value = m axim al d istu rb a n c e ).
D iv e r s ity a lo n g a b io m a ss g r a d ie n t
T o tal diatom species richness showed a significant quadratic relation with
to tal cell biovolume (used as a proxy for biomass) (Table 2, Fig. 3B). The
MOS test revealed th a t the relation is unimodal and hum p-shaped with
th e estim ated peak diversity w ithin the observed biomass ranges
(estim ated a t 23.70 . IO' 2 pin 3 bio volum e/m 2). Pielou’s evenness (Fig. 5B)
showed a negative correlation w ith increasing biomass (Spearm an R ank R
= -0.542 , P = 0.00000).
A
A
•*
-1.0
o
1.0
H y d ro d y n a m ic d is tu r b a n c e
*
*
,
10
E io v o lu n v I p m
100
» I O 1’- ' / m - )
F ig u r e 5 . P i l o u 's e v e n n e s s a l o n g t h e d i s t u r b a n c e a n d b i o m a s s g r a d i e n t .
96
*
Diversity - disturbance in benthic diatoms
T he species richness of th e epipsammic and epipelic functional
groups also displayed a significant unim odal, hum ped-shaped relation
w ith biomass (Table 2, Fig. 3B). T he estim ated peak epipelic diversity
(using the MOS test) was located w ithin the observed range of biomass
values and is expected a t 59.53 . IO12 pm 3 biovolum e/m 2. The estim ated
peak epipsammic diversity was also within the observed range of biomass
values, but it was not significantly greater th a n the epipsammic diversity
at the m inim al observed biomass. T he species richness of the
tychoplankton showed a significant linear positive relation w ith increasing
biomass. T hus th e different functional groups show peak diversity at
distinct biomass values.
T he species richness of the three different epipsammic growth
forms all had a significant unimodal, hum ped-shaped relation with
biomass (Table 2, Fig. 3D). The estim ated peak adnate diversity was
located a t lower th an observed biomass values (0.51. IO12 pm 3
biovolum e/m 2), while th e small motile and stalked diatom s showed peak
diversities a t 5.69 . IO12 pm :i biovolum e/m 2 (1.30 . IO12 pm 3 biovolum e/m 2
- 10.4 . IO12 pm 3 biovolum e/m 2, 95% confidence interval) and 14.21 . IO12
pm 3 biovolum e/m 2 (4.14 . IO12 pm 3 biovolum e/m 2 - 23.4 . IO12 pm 3
biovolum e/m 2, 95% confidence interval) respectively. The epipelic
diatom s between 1 0 and 2 0 pm showed a unimodal, hum ped-shaped
relation w ith biomass (Table 2, Fig. 3F). T he estim ated peak diversity
was estim ated a t 2.97 . IO12 pm 3 biovolum e/m 2 (no confidence interval
could be determ ined). For the epipelic diatom s > 20pm species richness, a
linear regression was again not possible as the assum ption of
homoscedasticity was not fulfilled. W e performed a non param etric
Spearman rank correlation and found a positive correlation between
increasing biomass and species richness of motile diatom s > 2 0 pm
(Spearm an R 0.1815 p = 0 .00053). So again the different growth
morphologies show peak diversity a t different biomass values.
We repeated th e above described regressions for disturbance and
biomass using th e residual species richness after accounting for the
salinity tren d and found largely the same results (supplem entary table 1
and
2
).
S p ecies tu r n o v e r
Analysis of Sim ilarity (ANOSI3VI) based on quantitative and qualitative
species d ata was used to assess differences in species composition and
97
Chapter 4
species turnover along the disturbance gradient (Tables 3 and 4). As siltcontent of sedim ent samples is strongly related to th e hydrodynamic
disturbance proxy used during this study (logsilt = 0.534*disturbance +
1.02, R2=0.912, P = 2.2 . IO'12), we classified the sam pling stations in
different silt classes and then analyzed their sim ilarity. T he most
disturbed sam pling stations w ith a silt content between 0 and 1 % had a
very specific species composition which showed relatively low sim ilarity
with the 1-5% silt class and almost no sim ilarity w ith th e least disturbed
sites (20 % silt or more) (Table 3 and 4). The sam pling stations w ith silt
content > 1 0 % showed high sim ilarity in species composition.
C ontrasting the quantitative (ANOSIM based on the B ray-C urtis index,
Table 4) and qualitative (ANOSIM based on the Jaccard index, Table 3)
m etrics showed th a t both compositional change and change in relative
abundances occurred. For instance, the sampling stations w ith 1-5% silt
content and w ith a 10-20% silt showed a R value of 0.39 based on
qualitative d ata, but a R value of 0.598 based on qualitative data,
showing th e im portance of changes in relative abundances. Overall, the
strongest turnover in species occurred a t low silt content ( 0 - 1 0 % silt), but
species turnover continued also a t higher silt content.
T a b l e 3. O n e - w a y a n a ly s is o f sim ilarities ( A N O S I M ) u sin g t h e J a c c a r d d iss im ila rity
in d ex
based
on
p resence/absence
data
of
sp e c ie s
alo n g
different
silt
content
p e r c e n t a g e cl a s s e s . V a l u e s g iv en a r e t h e R s t a t i s t i c a n d t h e p r o b a b i l i t y level ( P ) .
0-1 90 silt
1- 5 % silt
5-10 % silt
10-20 % silt
20-40 % silt
0 - 1 % silt
1 - 5 % silt
0.523 (0.001)
5 - 10 % silt
0.733 (0.001)
0.288 (0.001)
10 - 20 % silt
0.811 (0.001)
0.390 (0.001)
0.084 (0.003)
20 - 40 % silt
0 .945 (0.001)
0.673 (0.001)
0 .286 (0.001)
0.124 (0.0 0 1 )
> 40 % silt
0.999 (0.001)
0.946 (0.001)
0 .615 (0.001)
0 .2 7 8 (0.003)
T a b le
4.
O n e - w a y an a l y s is
of
si m ilarities ( A N O S I M ) u s i n g t h e
0.008 (0.418)
B r a y - C u r t i s in d ex
b a s e d o n s t a n d a r d i z e d s p e c i e s a b u n d a n c e d a t a a l o n g d i f f e r e n t silt c o n t e n t p e r c e n t a g e
classes. V a l u e s giv en a r e t h e R s t a t i s t i c a n d t h e p r o b a b i l it y level ( P ) .
0 -1 % s ilt
1- 5 % s ilt
5 -1 0 % s i l t
1 0 -2 0 % s i l t
2 0 -4 0 % s i l t
0 - 1 % s ilt
1 - 5 % s ilt
0 .6 6 8 (0 .0 0 1 )
5 - 10 % s i l t
0 .6 9 9 (0 .0 0 1 )
0 .3 7 1 (0 .0 0 1 )
10 - 2 0 % s ilt
0 .8 3 8 (0 .0 0 1 )
0 .5 9 7 (0 .0 0 1 )
0 .1 7 8 ( 0 .0 0 3 )
2 0 - 4 0 % s ilt
0 .9 5 8 (0 .0 0 1 )
0 .8 1 2 (0 .0 0 1 )
0 .4 0 7 ( 0 .0 0 1 )
0 .0 7 3 ( 0 .0 0 1 )
> 4 0 % s ilt
1 .0 0 0 (0 .0 0 1 )
0 .9 7 5 (0 .0 0 1 )
0 .4 7 3 ( 0 .0 0 1 )
0 .0 1 0 (0 .4 1 2 )
98
- 0 .0 8 9 (0 .8 4 8 )
Diversity - disturbance in benthic diatoms
D ia to m cell size
Differences in species richness, functional groups and species composition
along the biomass and disturbance gradients coincided with changes in
average cell size and cell size distribution within samples. T here w~as a
significant increase in average cell size w ith increasing biomass values
(Spearm an R ank R = 0.502, P = 0.0000 ; Fig. 6 B) and a significant
increase in average cell size w ith decreasing hydrodynam ic disturbance
(Spearm an R ank R = 0.496, P = 0.0000 , Fig. 6 A).
T h e m ost disturbed sampling sites with silt content between 0
and
1
% were dom inated
by very small diatom s
(< 1 0 0
pm 3
cell
biovolume) (Fig. 7). Sampling sites w ith 1 - 5 % silt also had a significant
portion of the smallest diatom s ( < 1 0 0 pm 3), b u t also a second group of
diatom s w ith cell biovolumes between 100 and 240pm3. A further increase
in silt content and thus lower disturbance resulted in a gradual decrease
of the < 1 0 0 pm 3.
10000
s
A
IOC-0
1000
i #
100
100
- 1 .0
?0
1
H y d r o d y n a m ic d is iu r b a n c e
10
8 ¡o v o lu ir o i p m » x 1 0 '-’ / n v )
F ig u r e 6 . A A v e r a g e cell b i o v o l u m e v e r su s t o t a l b i o m a s s . B: A v e r a g e cell b i o v o l u m e
versus d is tu r b a n c e (low est value = m axim al d istu rb an ce)
99
Chapter 4
0 .7
0.6
,
Jo.,
0
° 0 1
o
C 0 .3
0 .4
0 .3
01 0.2
0.2
0.1
-r fi- . rFí
o
o
f i , B
jj
§
0 • 2 0 °i> Silt
on.
1
3R
4 0 0 -5 5 0
1 0 0 -2 4 0
2 4 0 -4 0 0
<50
6 0 -1 0 0
lí
0 - 4 0 % Silt
0 7
■a 0.5
■fi 0.3
o
n 02
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rü
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ffi
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7 ° ? ™
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8
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05
0.6
0 5
os
04
0.4
0.3
OJ
0.2
0.2
[
o
*
3
o
5
ó
C
o
S
ó
O
^
_ i § ... r k
(Ti f i S
o
o
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8
o
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o
ó
7
5
^
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-
04
t
O
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8
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a
A
8
°
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> 4 0 “a s m
ñ
o
*
ffi
o
i f ) , rfi
ñ
m
8
S
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8
8
!
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, S
c
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ó
tO
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c
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O
Diatom cell b i o v o lu m e classes (pim3)
F ig u r e 7 . A v e r a g e f r e q u e n c y o f d i a t o m cell b i o v o l u m e - c la s s d i s t r i b u t i o n a l o n g
d i f f e r e n t silt c o n t e n t s . E r r o r b a r s a r e s t a n d a r d d e v i a t i o n s .
D is c u s s io n
Species richness in intertidal m icrophytobenthic diatom communities of
the W esterschelde estuary shows a significant hum p-shaped relation with
disturbance (Table 2), which is in agreem ent with the interm ediate
disturbance hypothesis (IDH, Grime, 1973, Connell, 1978). As most
studies to date have been focused on epipelic communities of the less
disturbed, silt rich intertidal sedim ents, it was not known whether
in tertid al m icrophytobenthos would respond to th e IDH, although
(Paterson & Hagerthey, 2001) hypothesized th a t it would. T he IDH has
previously been applied to phytoplanktonic communities, in both
experim ental and field investigations (Reynolds et al., 1993, Sommer,
1995, Floder & Sommer, 1999), most of which also comply w ith the IDH.
T he IDH assumes th a t there is a trade-off in traits th at
determ ine com petitive ability and disturbance tolerance (H addad et a l,
2008), resulting in m axim al diversity where com petitive and disturbance
tolerant species co-occur (Haddad et ah, 2008). Here we assigned species
100
Diversity - disturbance in benthic diatoms
to functional groups based on th e following traits: cell-size and shape,
hold-fast mechanisms and m otility. These functional traits were able to
explain the effect of disturbance on functional group species richness.
High energy conditions with strong wave action and tidal currents create
strong selection gradients allowing only a small num ber of erosionresistant, species w ith hold-fast mechanisms to ad a p t to these conditions.
Highly disturbed, coarse sedim ents were therefore characterized by the
occurrence of m ainly epipsammic diatom species and a relatively high
evenness. W ithin the epipsammic communities we observed a turnover
from adnate and small ( < 1 0 pm) motile species tow ards stalked species
along decreasing disturbance gradients.
T he presence of th e small (<10 pm) m otile forms in the most
dynam ic sedim ents may appear contradictory at first, as it would increase
th e risk of resuspension. However, their m otility m ight actually be a good
ad aptation to life on these larger sand grains as it enables them to go for
shelter (in crevices) when circum stances are m ost dynamic (e.g. at high
tide), while they can move across th e sand grains to optimize e.g. light
exposure when circumstances are less dynamic (low tide). Stalked forms
were more dom inant in less dynam ic sedim ents which was also noted by
(Sundbäck, 1983), as they are more prone to abrasion when the sediments
become too dynamic.
W ith decreasing disturbance, sedim ents are often poorly sorted
(fig. 8 ), containing both silt and sand fractions. Epipelic diatom s are able
to colonize these sediments, m ainly small motile ( 1 0 - 2 0 pm) species, while
larger motile ( > 2 0 pm ) tax a have higher species richness at lower
disturbance. The least disturbed, silt rich, nutrient rich sedim ents
harbour few species which can reach high dominance resulting in low
evenness, suggesting strong com petitive interactions. These sedim ents
harboured m ainly bigger celled epipelic ( > 2 0 pm ) and tychoplanktonic
species.
The turnover in functional groups coincides w ith strong species
turnover along the disturbance gradient with a complete disparity
between the least and m ost disturbed communities. Species turnover was
especially strong at the high disturbance end. T he functional and
taxonom ical turnover also results in a changing diatom cell size
distribution along the disturbance gradient. T he m ost disturbed
sedim ents are dom inated by very sm all diatom s (< 1 0 0 pm3), while mixed
sand and m uddy stations harbour several size classes and average cell size
increasing w ith decreasing disturbance. A greater variety in cell sizes in
101
Chapter 4
less disturbed sediments was also observed by Jesus et al. (2009) who
hypothesized th a t m uddier stations may provide more potential niche
spaces prom oting greater size variation.
Irigoien et al. (2004) and Li (2002) also observed an increasing
cell size with increasing biomass in phytoplankton communities. They
a ttrib u ted this to size-governed protection against Zooplankton predation.
Here we do not have d a ta on grazing intensity, so it is difficult to draw
any conclusions about this. T he occurrence of large species in the most
competitive communities seemingly contradicts w ith their lower surface
area to volume ratio causing them to have a less efficient nutrient
acquisition compared to small species (Raven, 1998). However, analyses
by Grover (1989) on microalgae revealed
cells are indeed better com petitors, but
shape are often better com petitors than
potential mechanism is th a t larger cells
th a t for spherical cells, smaller
large cells w ith an elongated
elongated small ones. Another
have a better nutrient storage
ability which is im portant in fluctuating n utrient environm ents (Grover,
1991, Litchm an et a l, 2009) such as intertidal m udflats which typically
have strong nutrient concentration gradients.
80
♦ < 63 pm
□ 1 2 5 - 2 5 0 pm
2 5 0 - 5 0 0 pm
"a'
70 60 50 40 30 -
uWiÎ
»
20
-
10
-
0
O
-
*
-
2.0
-
1.0
0
1.0
2.0
H ydrodynam ic d istu rb an ce
F ig u r e
8.
Sedim ent
g r a in
size
classes
against
th e
disturbance
gradient.
The
d i s t u r b a n c e g r a d i e n t larg ely c o i n c i d e s w i t h a g r a d i e n t in s e d i m e n t c o m p o s i t i o n . T h e
m o s t d i s t u r b e d s i t e s ( n e g a t i v e v a l u e s ) c o n t a i n m a i n l y m e d i u m siz ed s a n d , w h ile t h e
least
disturbed
sites
are
d om inated
by
silt
and
clay
particles.
At
d i s t u r b a n c e , fine s a n d ( 1 2 5 - 2 5 0 p m ) is t h e m o s t i m p o r t a n t s e d i m e n t ty p e .
102
interm ediate
Diversity - disturbance in bentliic diatoms
T he classical explanation of the IDH relies heavily on competitive
exclusion and environm ental filtering based on species specific tolerances
towards disturbance. M ichalet et al. (2006) recently suggested to include
facilitation into models explaining the IDH. They argued th a t facilitation
can prom ote diversity a t m edium to high disturbance intensities by
expanding th e realized niche of disturbance-intolerant com petitive species
tow ards stressful conditions. Typical examples for intertidal communities
are the large macroalgal canopy-forming species th a t protect understory
algae against physical stressors (Molina-Montenegro et al., 2005). Though
on another scale, facilitation through alleviation of stressful conditions
could apply to m icrophytobenthos as well. Laboratory experiments
dem onstrated th a t single cells of epipelic diatom species have limited
resistance tow ards resuspension by w ater currents, which is in contrast to
epipsammic species (Harper 1977). However, n atu ral epipelic diatom
assemblages exude copious am ounts of extracellular polymeric secretions
(EPS), thereby creating stick)- biofilms th a t have pronounced influence
on erosion thresholds of sedim ents (Paterson, 1989). Thus intertidal
diatom biofilms can trap and retain sedim ents a t current velocities th a t
would norm ally lead to sedim ent and MFB resuspension (Paterson &
Hagerthey, 2001, Van De Koppei et a l, 2001). Yet, different epipelic
diatom species seem to have varying abilities to produce EPS and
stabilize the sedim ent (Holland et a l, 1974, Underwood, 1994, Paterson
& Hagerthey, 2001). Field observations and culture experim ents show
th a t the ab u d an t diatom s species Navicula flanatica and Cylindrotheca
closterium presum ably have little contribution to sedim ent stabilization
(Holland et al., 1974, Underwood, 1994, P aterson & Hagerthey, 2001) but
likely take advantage of this h a b ita t amelioration.
Next to modifying hydrodynam ic disturbance, benthic microalgae
can also facilitate other benthic microalgae through the alleviation of
lim iting conditions. Benthic diatom s often face light lim itation due to
wave induced burial or by self shading in thick diatom biofilms. Some
M PB species are m ixotrophic and have the ability to take up dissolved
organic substrates as a carbon or nitrogen source (Linares & Sundback
2006). W e recently showed th a t a mixotrophic strain of Cylindrotheca
closterium was able to enhance its growth by recycling organic exudates
of other benthic diatom s and was therefore facilitated in its growth by
naturally co-occurring diatom species (Vanelslander et a l, 2009). To
which extent these facilitative interactions influence th e diversity of
n atu ral com m unities is difficult to deduce from our field d ata and thus
103
Chapter 4
field and lab experim ents are needed to determ ine if facilitation actually
prom otes diversity in interm ediate to highly disturbed sediments.
In addition to th e disturbance gradient, we also assessed diversity
p attern s along biomass gradients. Generally th e same p attern s show up,
which is not surprising given th a t disturbance and biomass are strongly
correlated. Biomass is often used as proxy for productivity to stud}'
productivity-diversity relationships. This m ight be reasonable in some
cases, but quite often there is no correlation between biomass and
productivity, as shown for intertidal benthic diatom s (F orster et al.,
2006). This im proper use of productivity surrogates lead to the
misconception th a t productivity-diversity relations were m ost frequently
hum p shaped (M ittelbach et al., 2001). So caution is required when
choosing a m easure for productivity (and thus the availability of
resources th a t limit production).
In sum m ary, we show th a t diversity of intertidal benthic diatom
communities is m axim al a t interm ediate hydrodynam ic disturbance. We
further show a decreasing evenness tow ards the least disturbed sites,
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109
Chapter 4
S u p p le m e n t a r y m a te r ia l
T a b l e 1. T h e r e g re s s i o n e q u a t i o n s for t h e s p e c i e s r i c h n e s s / f u n c t i o n a l g r o u p ric h n e s s
vs. h y d r o d y n a m i c d i s t u r b a n c e (lin ear: Y = a + b X
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sp e c ie s
richness
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accounting
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c X 2)
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s i g n i f i c a n t r e l a t i o n b e t w e e n d i v e r sity a n d sa lin ity . S i g n i f i c a n c e c o d e s : 'N . S . ' P > 0.0 5
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: 0.01 <
P < 0.05 ;
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vs. t o t a l s t a n d i n g b i o v o l u m e s t o c k (lin ear: Y = a + b X o r Q u a d r a t i c : Y = a + b X + c X 2)
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s i g n i f i c a n t re l a t i o n b e t w e e n d i v e r sity a n d salin ity . S i g n i f i c a n c e c o d e s : 'N . S . ' P > 0 .0 5
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r i c h n e s s vs. s a lin ity ( Y —a + b X ) . S ig n ific a n c e c o d e s : 'N . S . ' P > 0 . 0 5 ;
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: 0.01 < P
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112
C h a p te r 5
C o m p le m e n t a r it y E ffe c ts
D r iv e P o s it iv e D iv e r s it y
E ffe c ts o n B io m a s s
P r o d u c t io n in E x p e r im e n ta l
B e n t h ic D ia t o m B io film s
B art Vanelslander1, Aaike De W ever1, Nicolas Van Oostende1, P a tta ra tjit
Kaew nuratchadasorn2, P ieter Vanormelingen1, Frederik H endrickxi4,
Koen Sabbe 1 and W im V 3 'ver m an 1
la b o r a to r y of Protistology & Aquatic Ecology, D epartm ent of Biology,
G hent University, Krijgslaan 281-S8, B-9000 G hent, Belgium
S o u th e ast Asian Fisheries Development Center, Training D epartm ent,
Phrasam utchedi S am utprakan 10290, Thailand
3T errestrial Ecology U nit, D epartm ent of Biology, Ghent University, K.
L. Ledeganckstraat 35, B-9000 Ghent, Belgium
4Royal Belgian Institute of N atural Sciences, V autierstraat 29, B-1000
Brussels, Belgium
Published as: Vanelslander, B., De Wever, A., V an Oostende, N.,
K aew nuratchadasorn, P., Vanormelingen, P., Hendrickx, F., Sabbe, K. &
Vyverman, W. (2009) Com plem entarity effects drive positive diversity
effects on biomass production in experim ental benthic diatom biofilms.
Journal o f Ecology, 97, 1075-1082.
113
Chapter 5
A b s tr a c t
Positive effects of species diversity on ecosystem functioning have often
been dem onstrated in ‘m acrobiaP communities. This relation and the
responsible mechanisms are far less clear for microbial communities. Most
experim ental studies on microorganisms have used randomly assembled
communities th a t do not resemble natural communities. It is therefore
difficult to predict th e consequences of realistic, non-random diversity
loss.
In this study, we used naturally co-occurring diatom species from
intertidal mudflats to assemble communities w ith realistically decreasing
diversity and analysed th e effect of non-random species loss on biomass
production. O ur results dem onstrate a highly positive biodiversity effect
on production, w ith m ixtures outperform ing the most productive
component species in more th a n half of the combinations. These strong
positive diversity effects could largely be attrib u ted to positive
com plem entarity effects (including both niche com plem entarity and
facilitation), despite the occurrence of negative selection effects which
partly counteracted th e positive com plem entarity effects a t higher
diversities.
Facilitative interactions were, a t least in p art, responsible for the
higher biomass production. For one of the species, Cylindrotheca
closterium,, we show its ability to significantly increase its biomass
production in response to substances leaked into the culture m edium by
other diatom species. In these conditions, the species drastically reduced
its pigment concentration, which is typical for mixotrophic growth.
We show th a t both species richness and identity have strong
effects on the biomass production of benthic diatom biofilms and th a t
transgressive overyielding is common in these communities. In addition,
we show mechanistic evidence for facilitation which is partly responsible
for enhanced production. U nderstanding th e mechanisms by which
diversity enhances th e perform ance of ecosystems is crucial for predicting
the consequences of species loss for ecosystem functioning.
114
Diversity-Productivity in benthic diatom biofilms
I n t r o d u c t io n
T he effects of biodiversity and changes in com m unity composition on the
functioning of ecosystems have been a central topic in ecological research
during th e past decade. T he m ain research question has been whether
ecosystems w ith decreasing species num bers and altered species
compositions are able to m aintain their functional properties and process
rates. T h e m ajority of these studies dem onstrate th a t a decline in species
richness and functional diversity can adversely affect central ecosystem
processes such as productivity (Hooper et al. 2005; S riv astav i & Vellend
2005; C ardinale et al. 2006).
Theory suggests th a t positive relations between biodiversity and
ecosystem functioning arise through three prim ary mechanisms: sampling
or selection probability effects, niche com plem entarity and facilitation.
T he sam pling effect refers to the increased probability of selecting species
w ith a specific property in random ly assembled experim ental treatm ents
w ith a higher num ber of species (Huston 1997). Niche com plem entarity
occurs when species differ in their resource requirem ents, resulting in
lower com petition from interspecific neighbours th an from conspecifics.
This m ay lead to a more complete resource use by more speciose
com m unities (Fridley 2001). Facilitation occurs when a species modifies
the environm ent in a way th a t benefits a co-occuring species (Vandermeer
1989). Niche com plem entarity and facilitation are collectively referred to
as ‘com plem entarity’, as it is often unclear which mechanism prevails.
A lthough niche com plem entarity and facilitation are often
considered to enhance ecosystem functioning w ith increased species
richness, direct functional and ecophvsiological evidence is scarce.
C om plem entarity is generally dem onstrated indirectly by comparing
m ixture yields w ith th e expected yield based on the m onocultures of the
com ponent species (Loreau 1998; Loreau k Hector 2001).W hile there are
numerous examples of niche com plem entarity (e.g. com plem entary N
uptake strategies in plants: Jum pponen et al. 2 0 0 2 ), there is little direct
evidence showing how it can lead to increased resource use and thus serve
as a functional m echanism in positive biodiversity ecosystem functioning
(BEF) relationships (Kahm en et al. 2006). M echanistic evidence for
facilitation has been dem onstrated for N-fixing legumes (Tem perton et al.
2007) and larvae of suspension-feeding T richoptera (Cardinale et al.
2 0 0 2 ), b u t further direct evidence for facilitation is lacking.
115
Chapter 5
T he m ajority of the studies linking com m unity diversity with
ecosystem functioning have focused on terrestrial plant communities,
whereas microbial communities have received less attention, especially in
m arine system s (Petchey et al. 2002; Giller et al. 2004).Microbial systems
seem extrem ely attractive for testing B EF hypotheses because they
provide high levels of experim ental control and replication, and the
possibility to ru n experiments over m any generations. However, microbial
B EF experim ents th a t are relevant to natural situations are difficult to
realize due to the fact th a t the m ajority of microorganisms resist
cultivation in th e laboratory (up to 99% of prokaryotes, Kaeberlein et al.
2002). As a result, most BEF experim ents w ith microbial communities
used artificial communities of easily cultivable taxa, which usually bear
little resem blance to natural assemblages in term s of both species
com position and species richness. In addition, these artificial communities
are generally randomly assembled, while natural species loss is typically
non-random due to differences in population size, im m igration rate and
specific resistance to stressors (Giller et al. 2004; Srivastava & Vellend
2005). T his raises concerns about the relevance of previously observed
diversity effects for natural microbial communities and renders it more
difficult to predict the consequences of realistic diversity loss. T he few
studies th a t have used non-random diversity changes (Solan et al. 2004;
Z avaleta & Hulvey 2004; Bracken et al. 2008) found more rapid and
disproportionate loss of function th a n expected from random ized loss
experim ents.
W e focused
on
estuarine
benthic
diatom
(Bacillariophyta)
com m unities which provide a useful microbial system to address the link
between biodiversity and ecosystem functioning because these
com m unities are relatively species-poor (local diversity m ostly ranging
from two to nine species, Forster et al. 2006). In addition, m any species
are cultivable (M ann & Chepurnov 2004) and readily identifiable using
light microscopy (Forster et al. 2006). Benthic diatom s play a pivotal role
in the functioning of estuarine ecosystems; they provide up to 50% of the
prim ary production of estuaries (Underwood & Krom kam p 1999),
contribute to sedim ent stabilization through th e production of
extracellular polymer secretions (Decho 2000) and influence nutrient
fluxes between sedim ent and w ater (Sundback et al. 2000).
A field study by Forster et al. (2006) on intertidal mudflats in
th e
W esterschelde
estuary
(The
Netherlands)
showed
a
positive
relationship between net prim ary production and diversity of natural
116
Diversity-Productivity in benthic diatom biofilms
diatom communities. We further explored this relationship by composing
experim ental assemblages of naturally co-occurring diatom species with
nonrandom ly decreasing diversity and analysed the effect of species
richness and composition on biomass production. Biodiversity effects were
assessed by m easuring overyielding (occurring when a m ixture of species
perform s better than its m onocultures). T he mechanisms behind the
biodiversity effects were explored by partitioning the biodiversity effects
using the additive partitioning equation of Loreau A Hector (2001) and
by conducting an additional culture experim ent to elucidate potential
facilitative interactions between different species.
M a t e r ia l a n d m e t h o d s
B io d iv e rs ity -e c o s y s te m fu n c tio n in g e x p e r im e n t
T h e effect of diatom species richness on com m unity yield was assessed
using eight benthic diatom species (Table 1). These species were isolated
from th e ‘P au lina’ intertidal m udflat in th e W esterscheldc E stuary, The
N etherlands (51°21’ N, 3°43’ E) where they are dom inant components of
th e m icrophytobenthos (Sabbe & V yverm an 1991; Forster et al. 2006).
Voucher slides of the cultures used for th e experim ents were kept in the
L aboratory of Protistology and A quatic Ecology, G hent University,
Belgium.
In total, we assembled 19 species combinations spanning four
species richness levels (one, two, four and eight species, combinations
shown in Table 1) in a replicated com binatorial design (Giller et al. 2004)
which replicated both species richness and species composition, with five
replicates per treatm ent. W e replicated three species levels regarding
species composition (one, two and four species); th e eighth species
com m unity was only the pool of species used here. Therefore, we cannot
separate the effect of species num ber and identity on the production for
th e eighth species level. Although this is a serious drawback, we included
th is m ixture because it can shed some light on the interspecific
interactions when all species are combined.
117
Chapter 5
S ta tis tic a l a n a ly se s
W e used the additive partitioning m ethod (Loreau & Hector 2001) to
calculate the n et biodiversity, selection and com plem entarity effects. The
net biodiversity effect of a m ixture was calculated as the difference
between the observed yield and th e expected yield (weighted average of
the monocultures), under the null hypothesis th a t there was no selection
or com plem entarity effect. T he net biodiversity effect could be
partitioned into two additive com ponents: the selection effect and the
com plem entarity effect. T he com plem entarity effect for a specific number
of species N was N A R Y M , where À R Y
is th e average change in
relative yield for all species in th e m ixture and
M
is the average
m onoculture yield. T he selection effect N • cov (ARY, M) was calculated
as the covariance between the m onoculture yield of species (hi) and their
change in relative yield in th e m ixture (ARY) m ultiplied by the number
of species in th e m ixture (N). Positive selection occurred when species
w ith high monoculture yield dom inated the m ixtures, while negative
selection arose when species w ith low m onoculture yield dom inated or
when species w ith high m onoculture yield performed worse in mixtures.
Negative selection effects were docum ented in seagrass communities
where plant m ortality in m ixtures was reduced in genotypes th at were
weak in m onoculture (Reusch et al. 2005). A nother example is found in
serpentine grasslands where perennial bunchgrass w ith high monoculture
yields produced substantially less biomass in m ixtures with other
functional groups (Hooper k Vitousek 1997). As our d ata were not
normally distributed, we used th e Spearm an rank m ethod for calculating
the correlation (rho) of species richness with biovolume yield, net
biodiversity
effects,
com plem entarity
and
selection
effects
(R
Development Core Team 2005).
W e evaluated the effects of species composition (identity effect)
and species richness (diversity effect) on biomass yield using a generalized
linear mixed model (GLMM) w ith species richness as a fixed factor and
species composition as a random factor nested w ithin species richness. We
treated species composition as a random factor because replicate species
compositions were only a sm all sample of all possible species
combinations. Species richness was treated as a fixed factor because its
levels are deliberately selected and form a larger fraction of the possible
range. As the standard deviations of the different combinations were
120
Diversity-Productivity in benthic diatom biofilms
correlated w ith the average yield, we log-transformed the yield values.
T he variances between the different com binations differed between
species richness levels. Therefore, a GLMM w ith the variances estim ated
for each species richness level was used. T he link function was “proc
m ixed” and the distribution family was normal. T he variance structure
was t-variance com ponents”.
W e verified which m ixtures outperform ed their most productive
component m onoculture by calculating the transgressive overyielding
measure D„mx (Loreau 1998). We assessed the performance of individual
species in each m ixture by considering th e proportional deviation (D¡) of
species i’s yield from its expected value based on its m onoculture yield
(Loreau 1998).
R e s u lt s
After 25 days, th e initial biovolume of 4.78 • x IO7 pm 3 algal cells added to
the experim ental microcosms significantly increased in all treatm ents.
Analysis of diatom biovolume (Fig. la ) of the different species
assemblages revealed an increase in yield with species richness (Spearm an
rank correlation rho of 0.54, P = 0.016). T he transgressive overyielding
measure Dmax, which indicates when a m ixture outperform s its most
productive com ponent
treatm ents.
m onoculture,
was positive in
6
out
of
11
The GLMM (Table 2) confirmed th a t species richness influenced
biomass yield (F = 4.59, P = 0.018) and showed th a t the identity of the
m onocultures significantly influenced their production (Z = 1.86, P
=0.0312) b u t was unable to show significant differences in yield between
different com binations w ithin higher species richness levels. A TukeyK ram er post hoc test showed significant differences in yield between
communities of four species and m onocultures (adjusted P — 0.0422).
T he net biodiversity effect (Loreau & Hector 2001) increased
w ith species richness (Fig. lb , Spearm an rank correlation of 0.82, P =
0.002). T he biovolume d a ta for the individual species in the assemblages
allowed us to separate th e net biodiversity effect into a selection and a
com plem entarity effect (sensu Loreau & Hector 2001). The selection effect
showed a decreasing trend and became negative w ith increasing species
121
Chapter 5
richness (Fig. Id), which indicated th a t assemblages w ith higher species
richness were dom inated by species w ith low m onoculture yields. The
negative selection effects in treatm ents one, three, four, five and nine
were mainly caused by th e dominance of C. closterium and / or Nitzschia
sp., wherein th e yields in the above m ixtures exceeded their monoculture
yields. T h e com plem entarity effect was positive for all except one
treatm ent (seven) and increased w ith species richness (Fig. le).
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F ig u r e 1 . E f f e c t s o f d i a t o m d i v e r s i t y o n ( a ) b i o m a s s yield, ( b ) n e t b i o d iv e r sity effect
( t h e d i f fe r e n c e b e t w e e n t h e o b s e r v e d yield o f t h e m i x t u r e a n d t h e e x p e c t e d yield,
L o reau
L o reau
H e c t o r 2 0 0 1 ) , ( c ) c o m p l e m e n t a r i t y e f f e c t s a n d ( d ) se l e c t i o n e f f e c t s ( sensu
&
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A rrow s
indicate
th e
treatm en ts
w here
transgressive
o v er y ield in g ( D ma*) o c c u r r e d . T h e n u m b e r s d e n o t e t h e d i f f e r e n t t r e a t m e n t s as d efined
in T a b l e 1. O p e n c ircles r e p r e s e n t t r e a t m e n t s w i t h o u t C ylindrotheca closterium , filled
circles r e p r e s e n t t r e a t m e n t s w h i c h c o n t a i n C. closterium .
122
Diversity-Productivity in benthic diatom biofilms
T a b l e 2 . R e s u l t s o f a g e n e r a l i z e d lin ear m ix ed m o d e l ( G L M M ) w i t h s p e c i e s rich n ess
a s a fixed f a c t o r a n d s p e c i e s c o m p o s i t i o n as a r a n d o m f a c t o r n e s t e d w i t h i n sp e cies
richness. V a r ia n c e s w ere e s tim a te d
com b:
f o r e a c h s p e c i e s r i c h n e s s level. A b b r e v i a t i o n s :
s p e c i e s c o m b i n a t i o n s , SRL: sp e c i e s r i c h n e s s level, S E : s t a n d a r d
erro r, d.f.:
d e g r e e s o f f r e e d o m , n.a.: n o t a p p lic a b le . Diff.: d if fer en ce.
R a n d o m effec ts
E stim a te (SE)
Z -value
P -value
S pecies C om b in ation s in SR L 1
1.2378 (0.6053)
1.86
0 .0 3 1 2
S p ecies C om b in ation s in SR L 2
0.06049 (0.04137)
1.46
0.0718
S pecies C om b in ation s in SRL 4
0.03607 (0.03347)
1.08
0.1406
S pecies C om b in ation s in SR L 8
0 (n .a.)
n.a.
n.a.
F-value
F ix e d e ffe c ts
S pecies richness
P -value
4 .5 9
0 .0 1 8
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T u k e y -K r a m e r p o s t h o c te s t
Effect
E stim ate (S E )
d.f.
t-v a lu e
D iff SR L 1 - 2
-0.7734 (0.3905)
15
-1.98
0.2384
D iff SR L 1 - 4
-1.1581 (0.3897)
15
-2.97
0 .0 4 2 2
D iff SR L 1 - 8
-0.9959 (0.3828)
15
-2.60
0.0838
D iff SR L 2 - 4
-0.3846 (0.1454)
15
-2.64
0.0775
D iff SR L 2 - 8
-0.2225 (0.1258)
15
-1.77
0.3254
D iff SRL 4 - 8
0.1621 (0.1232)
15
1.32
0.5672
T h e relation between the performance of individual species (D¡),
and the num ber of species in the assemblage is shown in Fig. 2. A
positive correlation was observed for C. closterium and N. gregaria (Fig.
2a,b), while N. arenaria, N. flanatica, N. cincta and H. crucigera showed
a neutral to negative relation (Fig. 2e-h). The D¡ of Nitzschia sp. and of
N. phyllepta (Fig. 2c,d) showed a nonlinear relation w ith species richness,
indicating th a t th e species composition rather than its richness influenced
the perform ance of Nitzschia sp. T he proportional deviation of the other
species was not significantly correlated w ith species richness.
T o test w hether facilitation between different diatom species was
the m echanism th a t prom oted the grow th of C. closterium in multispecies
assemblages (see D¡ of this species in Fig. 2a), we conducted an additional
experim ent assessing th e growth of a C. closterium m onoculture in an
environm ent where there was no influence of other diatom species and in
an environm ent where we added spent medium of different Navicula
123
Chapter 5
species. W e observed a disproportionate threefold increase in cell numbers
when we added spent medium and, surprisingly, a drastic six fold
decrease in chlorophyll a content per cell (Fig. 3).
1 2 3 4 5 6 7
N u m b e r of s p e c i e s
F ig u r e 2 . P r o p o r t i o n a l d e v i a t i o n Di ( L o r e a u 1 9 9 8 ) o f sp e c i e s yield f r o m its e x p e c t e d
v alue
(a):
C ylindrotheca
( d ):N a v ic u la phyllep ta , ( e ):
c losterium ,
( b ):
N avicula
gregaria,
( c ):N itzsch ia
N avicula arenaria, (f): N avicula flanatica, (g):
sp.,
Haslea
crucigera, ( h ) : N avicula cincta.
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(a)
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C ylindrotheca clo ste riu m w h e n g r o w n in i n o r g a n i c ' f /
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'f/2'
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in 'f /
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m e d i u m e n r i c h e d w ith s p e n t m e d i u m o f a m i x t u r e o f Navicula s p e c ie s, ( c ): b o x p l o t o f
t h e c h l o r o p h y ll a t o b i o v o l u m e r a t i o in b o t h t r e a t m e n t s .
124
Diversity-Productivity in benthic diatom biofilms
D is c u s s io n
O ur results dem onstrated strong effects of non-random diversity changes
on biomass production in experim entally assembled benthic diatom
comm unities. Positive diversity effects could largely be attrib u ted to
strong positive com plem entarity effects (incorporating both niche
com plem entarity and facilitation). A t the highest diversity (eight species)
the shape of the species richness-net biodiversity effect curve levelled off.
T his was due to negative selection effects which partly counteracted the
positive com plem entarity effects and was m ainly caused by the
dom inance of species w ith low m onoculture yields (particularly C.
closterium and Nitzschia sp.). Negative selection effects seem to be
relatively common in B EF experim ents (Fargione et al. 2007; Jiang 2007)
and can significantly decouple effects of com plem entarity from net effects
on production.
T he overall effects of biodiversity on biomass production were
highly positive, especially when m ixture yields were com pared w ith
m onoculture yields. Six out of 11 com binations outperform ed their most
productive
com ponent m onoculture.
This
phenomenon, called
transgressive
overyielding,
is a
strong
indication
for
niche
com plem entarity and /o r facilitation, since sam pling effects alone cannot
cause this type of overyielding (Drake 2003). T he frequent occurrence of
transgressive overyielding (in 54% of our m ixtures) was rem arkable as
most. B EF studies dem onstrated very low frequencies or absence of
transgressive overyielding (Hector et al.2002; Drake 2003; Hooper &
Dukes 2004; Cardinale et
al. 2006). T he few studies th a t did observe
transgressive overyielding were long-term experim ents (covering 1 0
generations, Fargione et al. 2007), showing th a t positive diversity effects
m ight increase over time. Hector et al. (2002) suggest th a t the conditions
where resource partitioning and positive feedback mechanisms become
ap p aren t were not yet fully developed in m any short-term experiments
(in term s of num ber of generations) using higher plants. O ur experiment
was conducted w ith rapidly dividing microbial species (covering five to
six generations), which may explain the observed high frequency of
transgressive overyielding. In addition, the fact th a t we sim ulated nonrandom species loss, which often results in more rapid and
disproportionate loss of function due to faster disappearance of functional
125
Chapter 5
groups (Solan et al. 2004; Z avaleta & Hulvey 2004; Bracken et al. 2008)
m ight also have led to more pronounced biodiversity effects.
T he high frequency of transgressive overyielding in our
experim ents (in 54%of the m ixtures) can be further explained by the
effects of species richness on production and the underlying mechanisms
m ediated by specific environm ental conditions (C ardinale et al. 2000).
Facilitation, one of the mechanisms potentially leading to overyielding, is
considered to be more im portant under harsh environm ental conditions
(Bertness & Leonard 1997; M ulder et a,I. 2001). H arsh lim iting conditions,
such as desiccation, highly variable salinity and tem perature and regular
physical disturbance are constantly encountered by diatom s of intertidal
m udflats and could be alleviated through the intim ate association of cells
in biofilms which offers shelter against bottom shear stress from wave
action. This physical protection is strengthened by positive feedback
interactions between diatom growth and decreased sedim ent erosion (Van
de Koppei et al. 2001). Here we dem onstrated th a t diversity per se (and
th e increasing probability of positive interactions) could thus maximize
n et production of biofilms which m ight reinforce the reported positive
feedback mechanisms between biofilm production and decreased sediment
erosion. F uture research should be conducted on th e effects of species
diversity on biofilm stability.
T he observed positive diversity effects were largely attributable
to strong positive com plem entarity effects which could be caused by niche
com plem entarity and /o r facilitation. A t the m om ent we do not have
insights into mechanisms of niche com plem entarity b u t it is conceivable
th a t there is com plem entary use of light and /o r nutrients. We did find
facilitative interactions when we calculated th e difference between the
yield of a particular species in the presence of other species and as a
m onoculture. We found th a t the yield of C. closterium strain VD18 and
Nitzschia sp. was alm ost always strongly prom oted in the presence of
other diatom species, indicating facilitation (Hodgson et al. 2002).While
th is suggests th a t facilitation is an im portant com ponent of the observed
com plem entarity, we cannot infer exactly to w hat extent facilitation
contributed to the positive diversity effect in our experiment. Although
Nitzschia sp. generally benefited from th e presence of other diatom
species, its performance decreased in the eight species m ixtures (Fig. 2c).
In this case, interspecific com petition was probably stronger than the
facilitative interactions. Some caution is w arranted, however, when
drawing conclusions from the eight species level, because the species
126
Diversity-Productivity in benthic diatom biofilms
composition a t this diversity level is not replicated. Because of this,
species id entity effects cannot be separated from species diversity effects
for this species richness level. T he results of additional experim ents w ith
C. closterium strain VD18 confirmed the occurrence of facilitation. The
growth (in term s of cell num bers) of C. closterium m onocultures was
strongly prom oted when spent medium of a different Navicula species was
added, while the cellular chlorophyll a content showed a sixfold decrease.
Since the Navicula species modified the environm ent in a way favourable
to C. closterium cells, we can conclude th a t facilitation occurred
(Vanderm eer 1989; Fridley 2001). Previous experim ents w ith C.
closterium strain VD18 showed th a t th e culture medium (f / 2 ) was
sufficiently n u trient rich th a t even half strength did not depress growth
rate over sim ilar periods of tim e and under identical culture conditions,
implying th a t inorganic nutrients should never be lim iting to C.
closterium strain VD18 in th e additional experim ent (B. Vanelslander,
unpublished data). O n the other hand, we previously found th a t the
growth of this strain im proved when galactose was added to the culture
medium, showing the m ixotrophic capacities of this strain. Mixotrophic
growth of microalgae is known to cause a decrease in specific chlorophyll
a content and reduction or even loss of photosynthetic capacity (T ittel et
al. 200-5). Considering th e m ixotrophic capacities of this strain and the
absence of inorganic n u trien t shortage, we concluded th a t th e improved
growth and concomitant decrease in chlorophyll a content was caused by
a shift from photoautotrophic to mixotrophic growth on exudates of other
diatom species, potentially modified by bacteria. In contrast, two other C.
closterium strains (VD05 and VD06) incapable of using galactose to
improve their growth did not show significantly differing growth rates
and had an unaltered chlorophyll a content when the same spent medium
of different Navicula species was added (B. Vanelslander, unpublished
data). These observations strengthened our conclusion th a t the grow th of
C. closterium strain VD18 was prom oted by the availability of organic
exudates in th e spent medium. Benthic environm ents are often
characterized by frequent light lim itation due to burial and shading by
thick algal m ats and by relatively high concentrations of dissolved organic
compounds (Cook et al. 2004). These conditions provide a competitive
advantage to phototrophic cells which have the ability to take up
dissolved organic substrates as a carbon or nitrogen source (Linares &
Sundback 2006). L aboratory studies have shown th a t the growth of a
num ber of benthic microalgae (including C. closterium) was enhanced by
127
Chapter 5
addition of dissolved organic substrates, ranging from monomeric sugars
to complex organic molecules (Hellebust & Lewin 1977; T uchm an et al.
2006). T he im portance of m ixotrophic grow th in the nutritional ecology
of benthic microalgae is alm ost unexplored. O ur results indicate th at
mixotrophic capacities of microalgae can cause facilitative interactions,
and th a t these interactions can contribute to the observed positive effects
of biodiversity on biomass production in intertidal m icrophytobenthic
communities.
A c k n o w le d g e m e n t s
This research was supported by grants from th e F und for Scientific
Research, Flanders (Belgium) in the framework of the Flem ish-Dutch
C ollaboration on M arine Research (VLANEZO; G .0630.05), by the BOF
project GOA 01GZ0705 (G hent University) and by FW O project
G.0222.09N. W e wish to th an k the two anonymous referees for their
helpful comments.
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Diversity-Productivity in benthic diatom biofilms
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132
C h a p te r 6
I n te r fe r e n c e C o m p e t it io n
b e t w e e n M a r in e B e n t h ic
D ia t o m s
B art Vanelslander, Koen Sabbe and Wirn Vyverm an
Laboratory of Protistology & Aquatic Ecology, D epartm ent of Biology,
Ghent University, Krijgslaan 281-S8, 9000 Ghent, Belgium.
Unpublished M anuscript
Chapter 6
A b s tr a c t
Spatial
variation
in
species
composition
of
benthic
microalgae
communities is poorly understood. Variation in com m unity composition
over large spatial scales is typically related to gradients in environm ental
param eters. Y et, a t smaller spatial scales biofilms of microalgae often
show patchy distribution p attern s even when there is little or no obvious
abiotic heterogeneity. W hile it can be envisaged th a t colonisation
dynamics and local biotic interactions are able to generate such
m icropatchiness, little is know about the nature and im portance of these
mechanisms. In this study we use co-culture experim ents to dem onstrate
th a t estuarine benthic diatom s differ strongly in their capacity for
interference com petition. W e show th a t cell cultures of the marine
benthic diatom N. cf.pellucida exude m etabolites w ith strong allelopathic
effects. All nine com petitor species tested were inhibited in their growth
and some in their photosynthetic efficiency as well. In addition, we show
reciprocal, density dependent allelopathic interactions between N.
cf.pellucida and two other m arine benthic diatom species, Entomoneis
paludosa and Stauronella sp. O ur results suggest th a t allelopathic
interactions m ight be common in benthic microalgal communities and
m ay explain small -scale spatial distribution patterns observed in nature.
134
Interference competition in benthic diatoms
I n t r o d u c t io n
E stuarine intertidal m udflats are characterized by steep gradients in
light, n u trien ts and oxygen and hence there is strong com petition for
lim iting resources among m icrophytobenthic algae (Adm iraal, 1984). In
tem perate regions, the intertidal m icrophytobenthos is dom inated by
diverse diatom communities. These diatom assemblages display
pronounced spatial and tem poral variation in species composition.
Environm ental param eters such as sedim ent type, tem perature, light,
salinity, am m onium concentration, etc explain m uch of the large-scale
rates of com m unity turn-over in m icrophytobenthos (T hornton et al.,
2002, Du et al., 2009, Underwood, 1994). However, a t centim etre scales,
th e biom ass and species composition of intertidal benthic diatoms
sometimes display spatial p attern s (Shaffer & Onuf, 1985, de Brouwer et
al., 1999), w ith different species showing a com plem entary spatial
arrangem ent w ith non-overlapping areas of maxim al density (Saburova et
a l, 1995). T h e negative correlation between the abundances of different
species could be due to microscale h ab itat heterogeneity a n d /o r biological
interactions, including species specific grazing (Sm ith et al., '1996,
H agerthey et al., 2002, Hamels et a l, 2004) and interspecific competition.
Evidence is accum ulating th a t m icroalgae are able to engage in
interference com petition as a means to acquire resources (Gross, 2003,
Legrand et a l, 2003).
In phytoplankton, density dependent allelopathy, the release of
chemicals th a t inhibit the growth or survival of com peting species, has
been shown to be an im portant determ inant of species composition and
succession in phytoplankton (Keating, 1977, V ardi et a l, 2002, Kubanek
et a l, 2005), and affects the initiation a n d /o r continuation of plankton
blooms (Sm avda, 1997, Legrand et al., 2003, Prince et a l, 2008a). Such
chemically m ediated interference com petition has been dem onstrated for
phytoplankton tax a from groups as divergent as dinoflagellates,
chlorophytes, diatom s and cyanobacteria (Gross, 2003, Legrand et al.,
2003, Poulson et a l, 2009). In m any cases, the chemical nature of
allelopathic compounds is unknown, b u t several modes of action of
allelochemicals on susceptible target cells have been dem onstrated. These
include inhibition of Photosystem II efficiency (Gross, 2003, Prince et ah,
2008a), m em brane damage (Legrand et al., 2003), inhibition of enzymes
(Sukenik et a l, 2002), reduced m otility (Uchida et a l, 1999) and
135
Chapter 6
oxidative dam age (Legrand et al., 2003, Prince et al., 2008a, Poulson et
al., 2009).
In benthic phototrophic biofilms, the intim ate association and
high density of cells enables strong chemical com m unication and cell-cell
interactions between microbial cells (Decho, 2000). Allelopathic
interactions have been dem onstrated for several benthic cyanobacteria
(Mason et a l, 1982a, Gross et a l, 1991, Ju ttn e r, 1999, Gross, 2003), but
there is less evidence for eukaryotic benthic microalgae. De Jong and
Adm iraal (1984) observed th a t cells of the estuarine benthic diatom
Cylindrotheca closterium died in m ixted cultures w ith Entomoneis cf.
paludosa. A nother m arine diatom , Haslea ostrearia, produces a blue-green
polyphenolic pigment, marennine, th a t inhibits the growth of naturally
co-occurring microalgae (Pouvreau et a l, 2007). Leflaivc and Ten-Hage
(2009) showed th a t menthanolic cell extracts (so not only exuded
m etabolites) of Uronema confervicolum and Desmodesmus quadrispina
inhibited the growth of several microalgae.
In this study we docum ent th a t the diatom N itzschia cf. pellucida
inhibits th e growth of several key epipsam m ic and epipelic diatom species
of intertidal m icrophytobenthos of the Scheldt E stuary. W e further show
th e occurrence of dependent m utual allelopathic interactions between N.
pellucida and two other marine benthic diatom species, Entomoneis
paludosa and Stauronella sp.
M a t e r ia l a n d M e t h o d s
S tr a in is o la tio n a n d c u ltu r e c o n d itio n s
All diatom strains were isolated from the ‘•Rammekenshoek” intertidal
m udflat in the Westerschelde E stuary, T he Netherlands (51°26’50” N,
3°38’38” E) on M arch 03, 2009 except CCY 9601and IID03 which
originate from Ems Dollard (Holland) and Doei (Belgium). Voucher slides
of the cultures used for the experim ents are kept in the L aboratory of
Protistology and A quatic Ecology, G hent University, Belgium. The
identification of the diatom strains was based on K ram m er and LangeB ertalot (1986-1991).
136
Interference competition in benthic diatoms
Clonal cultures were established as described in Chepurnov et al.
(2002). C ulture medium was composed of filtered and autoclaved North
Sea seaw ater enriched w ith f/2 nutrients (Guillard, 1975). Clonal cultures
were m aintained in a clim ate room a t 18 ± 1 °C and illum inated by coolwhite fluorescent lamps at a rate of 50 pmol photons m ' 2 s' 1 w ith a
lig h t/d ark cycle of 12/12 hours. T he experiments described below were
performed under the sam e conditions.
C o - c u ltu re e x p e r im e n ts
Prelim inar)' experiments w ith co-cultures of diatom species revealed th at
the diatom Nitzschia cf. pellucida exerted strong adverse effects on other
diatom species. We therefore focused on the grow th interactions between
N. cf. pellucida and 9 benthic diatom species belonging to various genera
( Gyrosigma sp, Navicula arenaria Donkin, Nitzschia sp., Stauronella sp.,
Cylindrotheca fusiform is Reim ann & Lewin strain CCY 9601,
Cylindrotheca fusiform is (Ehrenberg) Reimann &: Lewin strain IID03,
Opephora sp, Amphora sp, and Leyanella arenaria Hasle, von Stosch &
Syvertsen). Com petition experim ents were performed in polystyrene 24well cell culture plates (Greiner Bio-One, Frickenhausen, Germany)
containing 2.0 mL f/2 culture medium. Each species was inoculated a t a
cell density of ~ 5,000 cells mL
1
resulting in an initial density of ~ 50
cells m m '2. Uni-algal treatm ents were included as control and inoculated
a t 10,000 cells mL ‘. Cells for inoculations were obtained from
monoclonal, exponentially growing cultures. Each treatm ent was
replicated four times. T he culture medium was renewed daily (1.0 mL of
culture medium was replaced w ith 1.0 mL of double strength f/2
medium) to minimise nutrient lim itation and hence resource competition
for nutrients. The cell density of each species in all replicates was
m onitored daily using an inverted microscope (Axiovert 135 Zeiss
microscope, Jena, Germany) by counting a m inimum of 300 cells per
replicate. G row th rate during the exponential phase (4-5 days) was
calculated as the slope of the linear regression of log2 transform ed cell
densities versus tim e for individual cultures.
T o assess the im portance of direct cell contact for allelopathic
interactions, we co-cultured Navicula arenaria, Cylindrotheca fusiform is
IID 0 3 , Gyrosigma sp. (three of the m ost susceptible species, see below)
and Stauronella sp. together w ith N. cf. pellucida b u t separated from each
other by a. 1 pm pore size m em brane filter using cell culture inserts (Thin
137
Chapter 6
C ert 12 well plates, Greiner Bio-One, Frickenhausen, Germ any). We
inoculated 20,000 cells of each species (~ 50 cells/m m 2 in the outer
cham ber and ~ 177 cells/m m 2 in the inner cham ber). N. cf. pellucida cells
were cultured in the outer cham ber, th e second species in the inner
chamber. As a control, we replaced th e N. cf. pellucida culture with fresh
F /2 medium. Each treatm ent was replicated four times. T he culture
m edium was renewed daily (2.0 mL of double strength f/2 medium). We
m onitored growth and photosynthetic efficiency by pulse am plitude
m odulated (PAM ) fluorescence (MAXI Im aging PAM fluorometer, Walz,
Germ any). We used the m axim um quantum yield of Photosystem II
(PSII) as a proxy for photosynthetic efficiency (Krom kam p et al., 1998).
T his was measured as F v : Fm, w ith F v= F,„- Fo (Parkhill et al., 2001). F,„
is the maximum fluorescence emission level in the dark measured with a
satu ratin g pulse of light (emission peak a t 450 nm, 2700 pmol photons.nr
2s '\ 800 ms). The initial fluorescence, Fo was used as a proxy for biomass
(Honeywill et al., 2002).
From prelim inary experim ents and from th e experim ent shown in
Fig. 1, we suspected reciprocal allelopathic interactions for the
com binations of N. cf. pellucida w ith E. paludosa and Stauronella sp.
Therefore we conducted bi-algal culture experim ents with different cell
densities and ratio’s of these com peting species. W e inoculated these
species with total initial cell densities of 10,000 and 30,000 cells mL ' 1 and
w ith the initial ratio’s between the two species of 1:4; 1:1, 4:1.
R e s u lt s
Between 1 and 4 days, th e growth of the 9 tested benthic diatom species
was first suppressed and then arrested in the presence of Nitzschia cf.
pellucida and eventually all cells of these species suffered cell lysis and
death (Fig. 1). The cell density a t which Nitzschia cf. pellucida was
inhibiting and lethal was species specific. Gyrosigma sp., Navicula
arenaria and Cylindrotheca fusiform is strain IID 03 were the most
sensitive strains as they were inhibited and died after 1 day of exposure
to relatively low densities of N. cf. pellucida (between ~ 7500 to 9500 cells
m L '1). Entomoneis paludosa, Cylindrotheca fusiform is strain CCY 9601,
Stauronella sp., and Am phora sp. were inhibited after 2 to 4 days
138
Interference competition in benthic diatoms
exposure (at N. cf. pellucida densities of between ~ 30,000 ariel 150,000
cells mL-1) and died soon after, while Leyanella arenaria and Opephora
spp. were more resistant and died gradually w ith an almost complete
extinction after approxim ately 1 0 days.
T he growth ra te of N. cf. pellucida did not differ significantly in
the presence or absence of other species (paired two-tailed t-test, p >
0.1), except when grown together w ith Stauronella sp. (p = 0.008), which
significantly decreased th e grow th of N. cf. pellucida b u t still died after 4
days.
Gyrosigma sp.
Nitzschia+ s e c o n d s p e c i e s
Nitzschiam o n o c u l t u r e
S p e c ie s X + N
itzschia
S p e c i e s Xm o n o c u l t u r e
Stauronella sp.
. C. fusiformis CCY9601
p C. fusiformis IID03
A
Ampliora sp.
o
i
2
3
-!
s
Tim e (d)
F i g u r e 1. G r o w t h i n t e r a c t i o n s b e t w e e n
d ia to m species belonging t o
t r ia n g le s ,
dashed
lines)
and
8
N itzschia cf. pellucida a n d 9 o t h e r b e n t h i c
different genera. S pecies w ere cu ltu red
tog eth er
(grey
open
circle,
solid
lines)
a l o n e (g re y
w ith
N. cf.
pellucida (filled b l a c k s q u a r e , solid lines). N. cf. pellucida m o n o c u l t u r e s w e r e in c lu d e d
( o p e n b lack s q u a r e s , d a s h e d lines). T a r g e t sp e c ie s w e r e G yrosigm a sp . (A ), N avicula
arenaria ( B ) , E n to m o n e is paludosa ( C ) , Stauronella sp . ( D ) , C ylindrotheca fusiform is
CCY 9601 (E),
C. fu sifo rm is IID03 ( F ) ,
O pephora s p ( G ), A m p h o ra s p ( H ) a n d
Leyanella arenaria (I) E rro r b a r s a r e s t a n d a r d d ev iatio n s.
139
Chapter 6
T hin C ert experim ents w ith selected species com binations showed
broadi}' similar, b u t on average less pronounced effects of N. cf. pellucida.
T h e growth of N. arenaria was significantly affected after 4 dat's; after 6
days biomass estim ated by fluorescence was 39.2% lower th a n in
monocultures, a t a N. cf. pellucida cell density of -180,000 cells/mL. The
photosynthetic efficiency (Fv : F,„) showed only a weak decrease (-17 %).
Cells of C. fusiform is strain IID03 were affected after 4 days and after 5
days biomass was 56 % lower than in m onocultures (a t N. cf. pellucida
cell densities of - 160,000 cells mL’1) T he photosynthetic efficiency
decreased w ith 52.0 % after 5 days com pared to the m onocultures. The
growth of Stauronella sp. was already affected after 2 days and after 5
days biomass was 36.9% lower th a n in m onocultures (a t a N. cf. pellucida
cell density of - 160,000 cells m L'1). Its photosynthetic efficiency however
was unaffected. T he growth and photosvnthetic efficiency of Gyrosigma
sp. were not affected a t all after
6
days indirect exposure to N. cf.
pellucida cells.
C. clo steriu m
S ta u ro n ella
sp.
p
G y ro sig m a
sp.
Time (d)
F i g u r e 2. C o - c u l t i v a t i o n o f N. cf. pellucida w i t h 4 o t h e r d i a t o m s p e c i e s b u t s e p a r a t e d
by a 1 p m m e m b r a n e filter . B i o m a s s ( A - D ) ( m e a s u r e d a s initial f l u o r e s c e n c e FO) a n d
p h o t o s y n t h e t i c efficien c y ( E - H ) ( F v : F m m e a s u r e d by P A M f l u o r e s c e n c e ) o f Navicula
arenaria ( A , E ) , C ylindrotheca fusiform is I I D 0 3 ( B , F ), Stauronella sp . (C , G) a n d
Gyrosigm a sp . (D , H) in m o n o c u l t u r e s ( t r i a n g l e s a n d d a s h e d lines) a n d t o g e t h e r w i t h
N. cf. pellucida b u t s e p a r a t e d by a 1 p m p o r e size m e m b r a n e ( s q u a r e s a n d solid lines).
Error bars a r e s ta n d a r d deviations.
140
Interference competition in benthic diatoms
Based on the N. cf. pellucida vs Stauronella sp. co-culture
experim ent shown in Fig. 1 and on prelim inary experim ents w ith E.
paludosa and N. cf. pellucida, we suspected the occurrence of reciprocal,
density-dependent allelopathic interactions. W hen we varied the (relative)
startin g densities of N. cf. pellucida an d Stauronella sp., we indeed
observed reciprocal inhibition between both species and this depended
strongly on th eir respective cell concentrations (Fig. 3 and 4). W hen
Stauronella sp. cell num bers reached ~ 100,000 cells m L'1, N. cf. pellucida
grow th was suppressed. Yet, when N. cf. pellucida reached ~ 80.000 cells
mL'1, Stauronella sp. cell num bers declined. Similar reciprocal densitydependent effects were seen between N. cf. pellucida and E. paludosa. E.
paludosa cell num bers declined when N. cf. pellucida reached cell densities
of ~ 50,000 - 70,000 cells m L'1. On the other hand, when E. paludosa
reached a cell density of ~ 100,000 cells m L'1, growth of N. cf. pellucida
was suppressed, again in the presence of an adequate nutrient supply.
F ig u re
3.
G row th
interactions
betw een
N itzsch ia cf.
pellucida a n d
E n to m o n eis
p a lu d o sa a t d i f f e r e n t initial cell d e n s itie s . S p e c i e s w e r e c u l t u r e d a l o n e ( d a s h e d lines)
a n d t o g e t h e r (solid lines). Cell d e n s i ti e s o f N. cf. p e llu c id a r e p r e s e n t e d by b l a c k lines,
E. p aludosa cell d e n s i t i e s r e p r e s e n t e d by g r e y lines. ( A - C ) : t o t a l initial cell d e n s i t y w a s
1 0 , 0 0 0 cells m L '1 w i t h N. cf. pellucida : E. paludosa initial cell d e n s i t y r a t i o ’s o f 1:4
(A ) ; 1:1 ( B ) , 4 : 1 ( C ) . ( D - E ) t o t a l initial cell d e n s i t y a t 3 0 , 0 0 0 cells m L '1 w i t h N. cf.
pellucida : E. p a lu d o sa initial cell d e n s i t y r a t i o ’s o f 1 : 4 ( D ) ; 1:1 ( E ) , 4:1 ( F ) . E r r o r b ars
a re sta n d a rd deviations.
141
Chapter 6
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T im e (d)
F i g u r e 4. G r o w t h i n t e r a c t i o n s b e t w e e n N itzsch ia cf. pellucida a n d Sta u ro n ella sp. a t
d i f f e r e n t initial cell d en s ities . S p e c i e s w e r e c u l t u r e d a l o n e ( d a s h e d lines) a n d t o g e t h e r
(solid lines). Cell d e n s i ti e s o f N. cf. pellucida r e p r e s e n t e d by b lack lines, Stauronella
sp . cell d e n s i t i e s r e p r e s e n t e d b y g r e y lines. ( A - C ) : t o t a l initial cell d e n s i t y w a s 1 0 ,0 0 0
ce lls m L - 1 w i t h N. cf. pellucida : S tauronella s p . initial cell d e n s i t y r a t i o 's o f 1:4 (A);
1:1 ( B ) , 4 :1 ( C ) . E rro r b a r s a r e s t a n d a r d d e v i a t i o n s .
D is c u s s io n
W e show th a t the diatom Nitzschia cf. pellucida inhibits the growth of
and ultim ately kills all nine tested benthic diatom species. These
com petitors naturally co-occur w ith N. cf. pellucida and m ost of them are
common in European intertidal m udflats. Several of the diatom species
tested ( Gyrosigma sp., N. arenaria, C. fusiform is, E. paludosa) were
inhibited a t relatively low cell densities (~ 8 , 0 0 0 - 1 0 , 0 0 0 cells m l / 1
equivalent w ith ~ 8,000 - 10,000 cells.cm'2) of N. cf.pellucida. It seems
unlikely th a t nutrient depletion would cause the observed growth
inhibition as m ost inhibition and cell death occurs already in th e first 3
days (fig. 1). In contrast, cells of N. cf. pellucida in the co-cultures display
exponential growth during the first 5 days, implying nu trien t replete
conditions during the first days of the co-culture experiments.
T he observed p attern s of allelopathic interactions suggest th a t in
natu ral epipelic diatom communities these negative interactions may
operate and influence com m unity composition, given th a t diatoms can
reach to tal cell densities of 1.IO6 cells.cm ' 2 in estuarine mudflats
(A dm iraal et al., 1982). W hereas N. pellucida is a cosmopolitan species
inhabiting m arine coasts and is commonly found worldwide (Kram m er &
Lange-Bertalot, 1986-1991), it is currently unknown whether all strains
142
Interference competition in benthic diatoms
(or lineages) of this morphospecies produce allelochemicals and thus how
widespread this mechanism occurs.
However, effects of co-cultivation in different com partm ents
separated by a ly m pore size m em brane are less pronounced th an in co­
cultures where cells of the two species were completely mixed. This may
indicate th a t close cell-cell contact or contact with the extracellular
polymeric saccharides (EPS) m atrix enhances th e allelopathic interactions
a n d /o r th a t the allelopathic compound is volatile an d /o r little soluble in
w ater. Close proxim ity of com peting algae and cell-cell contact may have
a large contribution to the effectiveness of allelochemicals since toxin
concentration in close proxim ity of a cell can be m any times higher than
further away from th e cell (Jonsson et a l, 2009). Transfer of more
hydrophobic allelochemicals by direct cell-cell contact is highly
conceivable in microalgal biofilms where cells are densely packed in a
polymeric m atrix (Decho, 2000). Several surface-associated allelopathic
interactions have been dem onstrated for benthic cyanobacteria such as
Fischerella sp. (Gross, 1999) and Scytonema hofm anni (Mason et a l,
1982b), b u t only very few for eukaryotic benthic microalgae.
Interestingly, three of th e five m ost resistant diatom species were nonmotile (Leyanella arenaria, Am phora sp. and Opephora sp.) epipsammic
diatom s which live attached to sand grains. In contrast, th e more
susceptible diatom species were all epipelic diatom s which exhibit a
motile lifestyle and thus have higher probabilities for physical encounters
w ith cells of N. cf. pellucida but are also able to escape them.
N. cf. pellucida exudates can diminish the photosynthetic
efficiency of competing algae, as was shown in our T hin C ert co­
cultivation experiments. Several studies already dem onstrated decreased
PSII efficiency due to allelopathy (Legrand et al., 2003, Prince et al.,
2008a), but whether PSII is really the target of allelopathic compounds or
if decreased efficiency is merely a sym ptom of deteriorating cell
functioning is usually unknown. Sukenik et al. (2002) for instance showed
th a t M icrocystis exudates inhibited photosynthesis of Peridinium
gatunense. This was not the result of direct effects on the photosystems,
but due to a strong suppression of internal carbonic anhydrase which
caused a reduced availability of CO 2 and thus a reduced photosynthesis.
Allelochemicals can be very effective, b u t th e target cells are not
necessarily suffering passively and m ay try to escape through the
form ation of tem porary cysts (Uchida et a l, 1999, Fistarol et a l, 2004).
C om petitors
m ight
also
counteract
allelopathy
b}-
degrading
143
Chapter 6
allelochemicals (Prince et a l, 2008b) or can be constitutively resistant
tow ards specific allelochemicals (Kubanek et al., 2005). Still others can
produce their own allelopathic compounds as a response on
allelochemicals (Yardi et ah, 2002). Furtherm ore, relative abundance can
determ ine the outcome of allelopathic interactions as suggested by our
experim ents using different initial densities of N. cf.pellucida and E.
paludosa or Stauronella sp. in co-culture experiments. The species which
first reached a certain cell density threshold could suppress growth of the
other species resulting in considerable m ortality, even with ample
n u trient supply. Basically, this phenomenon can be described as a
priority effect which is a lasting im pact of the order of arrival of species
on the community development (Van Grem berghe et a l, 2009). Priority
effects can be caused by two different mechanisms, firstly, a numerical
effect in which the first species can reach carrying capacity before other
species arrive. Secondly, species can alter their environm ent in a
favourable or detrim ental way for later colonizing species (Van
Gremberghe et a l, 2009), which is w hat we observed in our culture
experiments.
O ur observation th a t E. paludosa is able to exude growth
inhibiting m etabolites confirms th e preliminary observations by De Jong
and Adm iraal (1984) on a E. cf.paludosa strain. Reciprocal, density
dependent allelopathic interactions between the planktonic dinoflagellate
Peridinium gatunense and th e cyanobacterium M icrocystis sp. in the Sea
of Galilee were proposed to prom ote the observed patchy distribution
patterns of these species in th e lake (Vardi et al., 2002). Similarly, in
m icrophytobenthos such chemical cross-talk may also help to understand
observations of patchy spatial occurrence of m icrophytobenthos species at
small (cm) spatial scales, and are in agreem ent w ith the findings of
Saburova et al (1995) who showed th a t different species showed
com plem entary spatial arrangem ent w ith non-overlapping areas of
maximal densities, b u t only when threshold densities of interacting
species were reached.
Overall, we dem onstrate th a t the m arine benthic diatom N.
cf.pellucida produces strong allelochemicals th a t kill all tested diatom
species. W e further show reciprocal, density dependent allelopathic
interactions between N. pellucida and two other m arine benthic diatom
species, Entom oneis paludosa and Stauronella sp. O ur results suggest th a t
allelopathic
144
interactions
m ight
be
common
in
benthic
microalgal
Interference competition in benthic diatoms
communities and m ay influence their sm all - scale spatial distribution
patterns.
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148
Interference competition in benthic diatoms
Vardi, A., Schatz, D., Beeri, K., M otro, U., Sukenik, A., Levine, A. h
K aplan, A. 2002. Dinoflagellate-cyanobacterium com munication
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149
150
C h a p te r 7
T h e “ M o le c u la r T o o th b r u s h ”
o f a M icro a lg a : D a ily B u r s ts
o f B io g e n ic C y a n o g e n
B r o m id e (B r C N ) C o n tr o l
B io film F o r m a tio n a r o u n d a
M a r in e B e n th ic D ia to m
B art V anelslander1, Georg P ohnert2, C arsten P aul2, Jan Grueneberg2,
Emily K. Prince2, Jeroen G illard1, Koen Sabbe1 and W im Vyverm an1
1 L aboratory of Protistology & Aquatic Ecology, D epartm ent of Biology,
Ghent University, Krijgslaan 281-S8, 9000 G hent, Belgium
2 In stitu te for Inorganic and Analytical Chem istry, Bioorganic Analytics,
Friedrich Schiller U niversity of Jena, Lessingstrasse 8, 07743 Jena,
Germ any
Subm itted m anuscript.
Chapter 7
A b s tr a c t
T he spatial organization of biofilms is strongly regulated by chemical cues
released by settling organisms. Yet, the exact nature of these interactions
and the repertoire of chemical cues and signals th a t micro-organisms
produce and exude in response to the presence of com petitors remain
largely unexplored. Biofilms dom inated by microalgae often show
rem arkable, yet unexplained fine-scale patchy variation in species
composition. Since this occurs even in absence of abiotic heterogeneity,
antagonistic interactions m ight play a key role. Here we show th a t the
m arine benthic diatom Nitzschia, cf. pellucida exudes a diverse m ixture of
volatile iodinated and brom inated m etabolites including the new natural
product cyanogen bromide (BrCN) which exhibits pronounced
allelopathic activity. Toxin production is light-dependent w ith a short
toxin b urst after sunrise. This labile compound acts as a short-term
signal, leading to daily “cleaning'’ events around the algae. W e show th a t
allelopathic effects are H 2 O 2 dependent and link BrCN production to
haloperoxida.se activity. This novel strategy is a highly effective means of
biofilm control and provides an explanation for the poorly understood
role of volatile halocarbons from marine algae which contribute
significantly to the atm ospheric halocarbon budget.
152
The molecular toothbrush of a microalga
I n t r o d u c t io n
Biofilm form ation in m arine habitats is a rapid and ubiquitous process
and m ost submerged surfaces, natural or man-m ade, are covered with
complex microbial communities. Intense efforts are m ade to control
biofilm form ation on industrial surfaces such as ship hulls because this
biofouling can result in severe economic loss (Yebra et al., 2004). Among
the early settlers, microalgae play a key role in the biofilm development
and diatom s, especially, are able to settle on even the m ost fouling
resistant surfaces (Molino & W etherbee, 2008). In this context it is
interesting to observe th a t certain microalgae can obviously control their
m icroenvironm ent since the patchy variation in species composition
observed around these algae (Shaffer & Onuf, 1985, Saburova et a i, 1995,
de Brouwer et a l, 2000) cannot be explained by abiotic heterogeneity or
by grazers because the production of m icroalgae in dense biofilms far
exceeds th e total consumption, making the effect of grazing limited
(Saburova et a l, 1995). This spatial organization of species is
characterized by com plementary distribution patterns and negative
correlation of species densities (Saburova et a l, 1995). Allelopathic
interactions have been suggested as a possible explanation for such
observed patchiness (Saburova et a l, 1995). Because biofilms are
composed of densely packed cells embedded w ithin a m atrix of exuded
polymeric compounds, metabolites produced by any cell can efficiently
targ et its neighbours rather th an diffusing into the surrounding water
column (Decho, 2000).
Studies th a t focused on interspecific interactions between biofilmforming diatom s revealed th a t synergistic (Vanelslander et a l, 2009) and
antagonistic (De Jong & Admiraal, 1984, C hapter 6) interactions are
common an d can have a strong influence on biofilm performance
(Vanelslander et a l, 2009). The underlying chem istry of these
interactions is unknown, but several modes of action of allelochemicals on
susceptible targ et cells have been dem onstrated, including th e inhibition
of photosynthesis (Gross, 2003, Prince et a l, 2008), m em brane damage
(Legrand et a l, 2003), inhibition of enzymes (Sukenik et a l, 2002),
reduced m otility (Uchida et a l, 1999) and oxidative damage (Legrand et,
a l, 2003, Prince et a l, 2008, Poulson et a l, 2009). In this study we
selected th e common biofilm forming diatom Nitzschia cf. pellucida due to
153
Chapter 7
th e high allelopathic activity observed in chapter 6 of this PhD . Several
N itzschia species are known for their production of volatile halocarbons
(Sturges et al., 1993, Moore et a l, 1996, Hill & Manley, 2009) and a first
screening revealed th a t the selected alga is also a rich source of such
compounds.
T he
formation
of low
m olecular
weight
halogenated
m etabolites is widely distributed in macro- and microalgae which
contribute significantly to the atm ospheric halocarbon budget (Sturges et
al., 1992, C arpenter et a l, 2003, P aul & P ohnert, 2011). Local m axima of
volatile halogenated m etabolites are often observed in coastal regions but
th e function of these metabolites is poorly understood. Here we directly
link th e halocarbon chem istry of microalgae to an allelopathic activity by
establishing th a t the novel natural product cyanogen brom ide BrCN is
highly inhibitory against com petitors. T his m etabolite is released during a
short period after the onset of light in quantities sufficient to kill or
inhibit th e growth of competing microalgae.
M e th o d s
A lg a l s tr a in s a n d c u ltu r e c o n d itio n s
W e isolated diatom strains from the “Ram m ekenshoek’' intertidal m udflat
in the W esterschelde Estuary, T he N etherlands (51°26’50” N, 3°38’38r E)
on M arch 03, 2009. C ultures and perm anent slides of the cultures used for
th e experim ents are kept in the Laboratory of Protistology and Aquatic
Ecology, G hent University, Belgium. W e established clonal cultures as
described in Chepurnov et al. (2002). W e prepared culture medium by
filtering and autoclaving North Sea seaw ater enriched w ith f/2 nutrients
(Guillard, 1975). W e m aintained clonal cultures in a clim ate room a t 19
± 1 °C and illum inated by cool-white fluorescent lamps a t a rate of 50
pmol photons m'2 s '1 w ith a lig h t/d ark cycle of 12/12 hours. Iodine
enriched culture medium was composed of ESAW artificial seawater
(Berges et a l, 2001) supplem ented w ith 45 pM KI. C ulture medium
enriched w ith 13C was based on ESAW artificial seawater w ith 2.07 mM
N aH 13C 0 3.
154
The molecular toothbrush of a microalga
B i-a lg a l c u ltu re e x p e r im e n ts
W e examined the growth interactions between N itzschia cf. pellucida and
3 benthic diatom species belonging to various genera (Navicula arenaria
Donkin, Cylindrotheca closterium (Ehrenberg) Reim an & Lewin, and
E ntom oneis paludosa (W. Smith) Reimer using bialgal cultures. We
performed growth experim ents in polystyrene 24-well cell culture plates
(Greiner Bio-One, Frickenhausen, Germany) containing 2.0 inL f/2
culture medium. We inoculated each species a t a cell density of ~ 5,000
cells m l / 1 resulting in an initial density of ~ 50 cells m nr2. We included
m onoculture treatm ents (inoculated at 10,000 cells mL ’) as a control. We
harvested cells for inoculations from monoclonal, exponentially growing
cultures. W e replicated each treatm ent four times. We daily renewed the
culture medium (1.0 mL of double strength f/2 medium) to avoid
n u trien t lim itation and hence resource com petition for nutrients. We
daily m onitored the cell density of each replicate using an inverted
microscope (Axiovert 135 Zeiss microscope, Jena, Germany) by counting
a m inim um of 300 cells per replicate.
E ffe c ts o f s p e n t m e d iu m
W e prepared N. cf. pellucida spent medium by filtering exponentially
growing cultures (200,000 - 250,000 cells m L'1) on G F /F filters
(W hatm an, M aidstone, UK) and subsequently on 0.2 pm membrane
filters (Acrodisc, Pali Life Sciences, Ann Arbor, MI, USA). W e enriched
this filtered spent medium w ith f/2 nutrients and then applied it to cells
of C. closterium, Stauronella sp. and E. paludosa. In parallel, we cultured
cells of these species w ith f/2 enriched seaw ater as a control. We
m onitored the effect of N. cf. pellucida spent m edium by m easuring the
biomass and photosynthetic efficiency using Pulse-am plitude-m odulated
(PAM ) fluorescence.
We used PAM fluorescence (MAXI Imaging PAM fluorometer,
Walz, Germany) to determ ine the m axim um quantum yield of
photosystem II (PSII), which is frequently applied as a proxy for
photosynthetic efficiency (Krom kam p et a l, 1998). W e determined
photosynthetic efficiency as F v : F m, where F. = F m-Fo. Fm is the
m axim um fluorescence emission level in the dark m easured w ith a
satu ratin g pulse of light (emission peak at 450 nm, 2700 pinoi photons nr
155
Chapter 7
V , 800 ms). W e used th e initial fluorescence F0 as a proxy for biomass
(Honeywill et al., 2002).
L iq u id -L iq u id e x tr a c tio n o f a lle lo p a th ic c o m p o u n d s
a n d G C -M S a n a ly sis
W e filtered 150 mL N. cf. pellucida spent m edium (G F /F , W hatm an,
M aidstone, England) and extracted it 3 times w ith 50 mL ethylacetate.
W e dried the extract w ith anhydrous sodium sulfate and concentrated it
a t reduced pressure. W e perform ed GC-EI-MS m easurem ents of the
concentrated extracts w ith a W aters G C T premier (W aters, M anchester,
UK) time of flight mass spectrom eter (MS) coupled to an Agilent 6890N
gas chrom atograph (GC) equipped w ith a DB-5ms column (30 m x 0.25
m m internal diam eter, 0.25 pm film thickness and 10 m D uraguard pre­
column, Agilent, W aldbronn, Germ any). T he carrier gas was Helium 5.0
w ith a constant gas flow of 1.0 mL m in 1. T he source tem perature was at
300 °C w ith an electron energy of 70 eV. T he column was held a t 40 °C
for 2 min, heated up from 40 °C to 150 °C with 5 °C m in'1, from 150 °C
to 280 °C with a ra te of 20 °C m in 1 and held for 4.5 min. T he samples
were injected in splitless mode.
V o la tile o rg a n ic c o m p o u n d s
We
used
solid
phase
m icroextraction
(SPM E,
C arboxen/
Polydimethylsiloxane, Supelco, Bellefonte, PA) to measure the volatile
com pounds em itted by N. cf. pellucida cultures. W e exposed the SPME
fiber for 30 min to the headspace of 82 mL m agnetically stirred filtrate
(G F /F filters) of N. cf. pellucida. We used C DCh (Eurisotop, Gif-surY vette, France) (at 1.24 pM) as an internal standard to enable
quantification. We analyzed the extracted compounds using the GC-MS
described above. We calibrated the SPM E extracting by m easuring a
dilution series of commercially available BrCN (Sigma Aldrich, Germany)
v.’ith CDCI3 as an internal standard. We determ ined BrCN concentrations
by calculating th e GC peak area using stand ard program peak detection.
W e normalized the BrCN peak area to the CDCh peak area and used the
calibration curve to calculate th e BrCN concentrations in the N. cf.
pellucida cultures.
156
The molecular toothbrush of a microalga
B io a ssa y s
W e used bioassays to detect the presence of allelochemicals in N. cf.
pellucida spent medium and used E. paludosa as the susceptible strain.
W e inoculated cells of exponentially growing E. paludosa in N. cf.
pellucida spent medium a t a final density of 2,000 cells mL-1. After two
hours exposure to spent medium, we checked for the occurrence of resting
cell formation and cell lysis using an inverted microscope (counting min
300 cells per replicate).
To check the occurrence of allelochemicals in the ethylacetate
extracts, we concentrated the extracts using reduced pressure and finally
dried the ex tract under a nitrogen enriched atm osphere. W e dissolved the
residue in acetone and added to an E. paludosa culture w ith 2,000 cells
mL'1 a t a final concentration of 1% acetone. This acetone concentration
did not affect cell integrity itself within the tim efram e of the bioassay.
We tested the toxicity of nine halogenated compounds on the
diatom E. paludosa a t concentrations of 0.1, 1, 5 and 10 pM (Fig. 4). We
first dissolved the halogenated compounds in acetone and added to an E.
paludosa culture w ith 2.103 cells m L'1 a t a final concentration of 1%
acetone. We microscopically checked for lysed cells, resting cells, and
healthy cells 3h after application. W e used the same approach to check
the toxicity of NaCN on E. paludosa cells at 2, 10, 20 and 40 pM Na.CN.
S y n th e s is o f c y a n o g e n io d id e
W e performed ICN synthesis as previously described (Bak, 1952). We
dissolved 0.25 mmol NaCN (Sigma Aldrich) in 0.5 mL w ater and cooled it
to 0 °C. W e gradually added 0.25 mmol iodine (Fluka, G erm any), waiting
until the last portion has reacted. W e extracted the w aten- solution 3
times w ith diethyl ether, dried th e ether extract w ith sodium sulfate and
removed it by a stream of argon. ICN was received as colourless crystals.
C a ta la s e e x p e r im e n t
We assessed the effect of the H^CL-decomposing enzyme catalase (600
units bovine liver catalase m L'1 dissolved in water, Sigma Aldrich) on the
toxicity of N. cf. pellucida cultures. We added catalase to the N. cf.
pellucida cultures one hour before the onset of light and we tested the
presence of allelochemicals three hours after the onset of light using the
157
Chapter 7
E. paludosa bioassay. W e included a control treatm ent in which we
stirred N. cf. pellucida cultures (analogous to th e catalase treatm ent) one
hour before the onset of light. We replicated treatm ents 4 times.
P h e n o l r e d a ssa y
W e used the brom ination of phenol red (phenolsulfonphthalein) into
brom inated phenol blue (3',3",5',5n-tetrabrom ophenolsulfonphthalein) as
an indicator for halogenating activity (Hill & Manley, 2009). The
conversion of phenol red into brom inated phenol blue indicates the
involvement of haloperoxidase enzymes. These enzymes catalyze the
oxidation of halide ions to hypohalous acid by H'iCL- Hypohalous acid (or
a similar oxidized interm ediate) can then react w ith organic substrates
th a t are susceptible to electrophilic halogénation (Butler & Sandy, 2009).
W e added phenol red (30 pM final concentration) to N. cf. pellucida
cultures (200,000 - 250,000 cells mL'1) three hours after daybreak. Two
hours later, we m easured phenol red and brom inated phenol blue
spectrophotom etrically a t 433 run and 592 nm respectively (Hill &
Manley, 2009). P rior to th e m easurem ents, we removed cells by filtering
on a 0.2 pm filter and adjusted the pH to 6.5 w ith acetic acid.
R e s u lt s
A lle lo p a th ic e ffe c ts o f N itzsc h ia cf. pellucida
Experim ents w ith co-cultures of biofilm forming diatom species revealed
th a t the diatom N. cf. pellucida (Fig. la ) exerted strong allelopathic
effects on other diatom species. W e observed th a t the naturally co­
occurring diatom s N. arenaria (Fig. Id), C. closterium. and E. paludosa
(Fig. lb) were inhibited and killed after 24h exposure to relatively low
cell densities (7,000-10,000 cells m L'1) of N. cf. pellucida. A nother diatom ,
Stauronella sp. (Fig. le) was more resistant b u t was killed w ithin 24h
when exposed to circa 80,000 cells m L'1 of N. cf. pellucida. The
mechanism of inhibition appeared to be th e same for all species
investigated: exposure to N. cf. pellucida cells resulted in loss of
pigm entation, shrivelling of chloroplasts and finally cell death (Fig. 1).
158
The molecular toothbrush of a microalga
b 0 min
10 min
120 min
s < e
d 0 min
F i g u r e 1. A n t a g o n i s t i c e f f e c t s o f N. cf. pellucida o n a n a t u r a l l y c o - o c c u r i n g d i a t o m
s p e c i e s , a:
N itzsch ia cf. pellucida, b: E. paludosa, h e a l t h y cell, cell a f t e r 10 min
e x p o s u r e t o N. cf. pellucida cells, d e a d cell a f t e r 1 20 m in e x p o s u re , c: S tauronella sp.,
h e a l t h y cell, cell a f t e r 3 0 h e x p o s u r e t o N. cf. pellucida cells, cell a f t e r 4 8 h e x p o s u re ,
d: N avicula arenaria, h e a l t h y cell, cell a f t e r 3 0 h e x p o s u r e t o N. cf. pellucida cells, cell
a f t e r 4 8 h e x p o s u r e . S c a l e b a r = 2 0 pm .
Application of nutrient-enriched spent N. cf. pellucida culture
m edium on cells of different diatom species induced a collapse of
photosynthetic efficiency (Fig. 2) and massive cell lysis w ithin 2h for E.
paludosa (Fig. 3) and C. closterium. T he diatom Stauronella sp. was
again more resistant and was able to m aintain its photosvnthetic
efficiency, b u t its growth was suppressed for two days (measured as the
initial fluorescence, F(), a proxy for algal biomass) after which cell growth
a t rates sim ilar to the control was restored.
159
Chapter 7
C closterium
500
S ta u ro n ella sp.
500
E. p a lu d o s a
800
c
400
400
300
300
200
200
400
100
100
200
o
o
c
a>
o
¡E
1
2
3
4
600
1
*
*
2
3
4
d
0 .6
Ht- C
O
o
0 .6
0 .5
0 .5
- - i
0 .5
a> 0 . 4
0 .4 1
o
m
sz
c
>.
in
O
o
JZ
CL
0 .3
0 .3 •
0.2
0 .2
0 .2 .
0 .1
0 .1
•
0 •%
± = fc
0
L
L
O
1
2
3
4
T im e (d)
F ig u re
pellucida
2.
E ffects
on
the
of n u trie n t
biom ass
enriched
( A -C )
spent
(m easured
cell-free
as
m edium
initial
of
N itzschia cf.
fluorescence
Fo)
and
p h o t o s y n t h e t i c efficien cy ( D - F ) ( F v:F m usin g P A M f l u o r e s c e n c e ) o f 3 n a t u r a l l y c o ­
o c c u r r i n g d i a t o m spe cies. W e c u l t u r e d s p e c i e s w i t h f / 2 n u t r i e n t e n r i c h e d s e a w a t e r
( b l a c k t r i a n g l e s w i t h d a s h e d lines) a n d w i t h f / 2 e n r i c h e d s p e n t m e d i u m o f N. cf.
pellucida (filled s q u a r e s a n d solid lines). ( A ) a n d ( D ) : C ylindrotheca closterium , (B)
a n d ( E ) Stauronella sp ., (C ) a n d ( F ) E n to m o n eis paludosa. ( M e a n s ± S .D .) .
An additional response was detected in E. paludosa since N. cf.
pellucida spent medium induced E. paludosa cells to lose m otility and
cells displayed a strong condensation of the protoplast within 10 min
after exposure (Fig. lb). Prolonged exposure to the N. cf. pellucida
medium caused massive cell lysis w ithin 2-3h (Fig. 3). As this species is
highly sensitive tow ards N. cf. pellucida, we selected it as a model for
further bio-assay experiments.
T he allelochemical potential of N. cf. pellucida varied
dram atically w ith tim e of day. Using a bioassay with E. paludosa and cell
free spent m edium from N. cf. pellucida cultures sampled in lh to lh.30
tim e intervals, we showed th a t allelopathic activity is highest between
two to four hours after daybreak when application of spent medium
resulted in nearly complete eradication of E. paludosa cells (Fig. 3). Five
hours after th e onset of light, the activity diminished and th e effect was
reduced to th e induction of protoplast shrinkage in >90 % of E. paludosa
cells (Fig. 3). Six hours after daybreak, m ost of th e cells showed no effect
160
The molecular toothbrush of a microalga
of spent medium, with > 50 % healthy cells, increasing to nearly 100 %
tow ards the end of the night. This striking p attern suggests th a t labile,
reactive or volatile m etabolites are responsible for th e allelopathic
activity. F u rth er studies therefore focused on the characterization of
m etabolites present 3h after the onset of light when toxicity was
maximal.
D ead cells
41
H ealthy cells
S hrunken p ro to p la st
-á t
100
cu
u
s
«„2
m
a
LU
80
60
40
20
0
1
0
1
2
3
4
5
6
7
8
T im e a fte r d a y b re a k (h)
D a yb re a k
F i g u r e 3. T i m e d e p e n d e n t a lle lo c h e m ic a l p o t e n t i a l o f N. cf. pellucida t h r o u g h o u t t h e
d a y . G r a p h s h o w s t h e e f f e c t o f N. cf. pellucida cell fre e s p e n t m e d i u m h a r v e s t e d a t
d i f f e r e n t t i m e p o i n t s on E. paludosa cells. W e m e a s u r e d t h e e f f e c t s 2 h a f t e r e x p o s u r e
t o N. cf. pellucida f i ltr a te . ( M e a n s ± S . D . )
E x tr a c tio n a n d s tr u c t u r e e lu c id a tio n o f N . cf.
p ellu cid a a lle lo c h e m ic a ls
W e performed GC-MS analyses of ethylacetate extracts (EAe) of cell-free
spent culture medium and of volatile organic compounds collected by
headspace solid phase m icroextraction (HS-SPME) and revealed the
161
Chapter 7
occurrence of 18 different brom inated and iodinated volatiles in N. cf.
pellucida cultures. These compounds were a m ixture of methylhalogens
(CH:iBr, CH 3I), dihalom ethanes (CH2Br2, CH2I2, CH2C1I*, CH2BrI*) as
well as trihalom ethanes (CHBr:i, CH I3) CHBr2I*, CHBr2Cl*, CHClIo*)
(EAe and SPM E), 1-iodopropane (EAe), di and tri-halogenated
acetaldehydes
(dibrom oacetaldehyde*,
brom ochloroacetaldehyde*,
chlorodibromo-acetaldehyde*, EAe), an d l,2-dichloroethane(EAe). We
identified these compounds based on their retention tim e and mass
spectra using GC-MS and, if not indicated otherwise, compared with
commercially available or synthetic (Bak, 1952) standards (* — identified
only by mass spectrom etry and retention tim e). In addition to these
m etabolites th a t are known from m arine algae (Paul Sz P ohnert, 2011),
we could also detect the volatile and highly toxic cyanogen bromide,
BrCN using SPME. A commercially available synthetic standard of this
novel natu ral product provided m aterial for the confirmation of the
structure, quantification and a dose response assessment in bioassays with
E. paludosa.
T o check if these halocarbons caused the observed allelopathic
interactions, we assessed the toxicity of different concentrations of the
nine m ost abundant halogenated com pounds and NaCN on the diatom E.
paludosa (Fig 4). In these bioassays, cyanogen brom ide turned out to be
by far the most toxic halogenated m etabolite produced by the algae. The
minim al lethal concentration was 2 pM (causing 96% of the E. paludosa
cells to die within 3h). BrCN is by far more potent com pared to NaCN,
which is active only in concentrations above 40 phi. BrCN concentrations
in the Nitzschia cultures (320,000 cells mL-1) (determ ined by SPM E GCMS with CDCI 3 as an internal standard) reached on average 5.18 pM (±
3.10; n= 4). T he concentration of BrCN can thus fully explain the lethal
effect of th e spent N. cf. pellucida medium. No other tested halogenated
m etabolites were active in the concentration range reached in cultures.
Cells of N. cf. pellucida are more resistant to BrCN and could cope with
concentrations of up to 16 pM w ith m inor or no growth reduction, and
onljr displayed a reduced grow th and photosynthetic efficiency a t 32 phi
BrCN (Fig. 5).
162
The molecular toothbrush of a microalga
B rC N
C H B r3
—
100
•
80
.
C H i3 ^
r
C H 2B r2
cu
o
co
«
"c5
a
c h 2 i2
CFhBr
—
60
.
40
.
- o - c h 3i
C2 H , Cl¿
ui
■a
20
.
cu
Q
0
:
to
—
c
—
NaCN
3 h 7i
Nitzschia B rC N
1
0 1
10
C o n c e n tra tio n (pM)
F i g u r e 4. D o s e - r e s p o n s e c u r v e f o r E. paludosa for 9 h a l o g e n a t e d c o m p o u n d s d e t e c t e d
in
N.
pellucida
cf.
cultures.
The
grey
square
represents
the
average
B rCN
c o n c e n t r a t i o n in N. cf. pellucida c u l t u r e s ( t h r e e h o u r s a f t e r o n s e t o f lig ht) a n d t h e
a v e r a g e r e s p o n s e o f E. paludosa t o t h e N. cf. pellucida f i lt r a t e s . ( M e a n s ± S . D . )
600
d)
500
s 400
— •—
«—
c o n tro l
2 pM B rCN
— »—
4 pM BrCN
-
0 .4
a - - 8 pM B rC N
¿rs
1 6 pM BrCN
VJ
.
• 3 2 p M B rC N
S? 300
_2
200
o
-
^
LX-
'
100
c o n tro l
2 pM B rC N
?
4 pM B rC N
- o- - 8 pM B rC N
_ _
1 6 pM B rC N
-,
3 2 pM B rC N
o
0
20
40
10
60
H o u r s a f t e r BrCN e x p o s u r e
F ig u re
5.
E ff e c t
of
BrCN
20
30
40
H o u rs a f t e r BrCN e x p o s u r e
on
(A )
grow th
(m easured
as
FO)
and
(B )
the
p h o t o s y n t h e t i c efficiency o f N. cf. pellucida. ( M e a n s ± S . D .)
To further validate th a t BrCN is the m ajor m etabolite
responsible for th e allelopathic activity, we deprived N. cf. pellucida
cultures of brom ide and iodide. O m itting these halogens from the culture
medium alm ost completely elim inated the allelochemical activity (Fig. 6).
W hen we altered the I- : Br- ratio in the culture m edium from ca. 1:2,000
in natural seawater to 1:16, production of brom inated hydrocarbons
ceased and was replaced by the form ation of iodinated compounds.
Likewise, BrCN production was reduced and instead there was formation
163
Chapter 7
of cyanogen iodide (ICN), a second new natural product. Spent medium
of N. cf. pellucida grown at high I:Br ratios was even more toxic than
spent m edium derived from natural seawater and caused massive cell lysis
of E. paludosa cells within 45 min after application (Fig. 6).
100
o80
<
L>
O
<U 60
■§
-5
CD
Q.
UJ
T5
(0
0)
Cs
40
I“: Br "= 1:16
I": Br "= 1 :2 0 0 0
20
W ith o u t I a n d Br
0
0
0.5
1.5
2
2.5
3
T i m e (h)
F ig u re
6.
Cell d e a t h in E. p aludosa f o llo w in g e x p o s u r e t o f i l t r a t e o f N. cf. pellucida
g r o w n in m e d i a w ith d i f f e r e n t I' : B r r a t i o s ( 1 : 1 6 a n d 1 : 2 ,0 0 0 ) a n d in a b s e n c e o f I'
a n d B r . ( M e a n s ± S .D .) .
B rC N b io s y n th e s is
Since BrCN and ICN are not known as natural products and since their
production evidently relies on the availability of external halides, we
aimed to verify their biogenic origin by further addressing their
biosynthesis. Therefore we incubated N. cf. pellucida cells in culture
medium enriched with i:iC labeled bicarbonate for 5 days. Analysis of the
isotope distribution of the cyanides provided proof for a biosynthetic
origin. BrCN and ICN exhibited a 12C : 13C ratio of 1 : 1.13 and 1 : 1.18
in BrCN and ICN respectively while the cyanides from cells in natural
seawater exhibited the natural ratio of 1 : 0.012. (Fig 7).
164
The molecular toothbrush of a microalga
104.9 106.9
100
C
100
152.Í
78.9
126.9
138.9
76.5
100
90
80
110
100
120
140
m/z
100
104.9
107.9
153.9
100
152.9
126.9
c
78.9 80.9
76.5
138.9
100
110
80
100
120
140
m/z
F i g u r e 7 . G C - E I - M S s p e c t r a o f B rC N (A a n d C) a n d ICN ( B a n d D ). A a n d B w ere
harvested
from
N. c f pellucida c u l t u r e s g r o w n
in n a t u r a l s e a w a t e r b a s e d
culture
m e d i u m a n d C a n d D f r o m c u l t u r e s g r o w n in N a H 13C 0 3 e n r i c h e d m e d i u m .
100
80
60
•§
,3
"c5
a 40
hi
-a
A
3
V
Q
20
0
Filtrate
Filtrate
+ cata lase
N itzschia
Control
+ catalase
F i g u r e 8 . Cell d e a t h in E. paludosa follo w in g e x p o s u r e t o f i l t r a t e o f N. cf. pellucida,
to filtrate a n d
catalase,
to
f i lt r a t e f r o m
N. cf. pellucida c u l t u r e s i n c u b a t e d w ith
c a t a l a s e ( “N itzsc h ia + c a t a l a s e ” ) a n d t o a filtered s e a w a t e r c o n t r o l . ( M e a n s ± S .D .)
165
Chapter 7
Biosynthetic considerations of BrCN or ICN suggested the
presence of an oxidized halogen species. Known enzymes which could be
involved are H 2 O 2 consuming haloperoxidases (H PO ). T o test if
allelochemical production is linked w ith cellular H20 2 production, we
assessed th e effect of the H20 2-decomposing enzyme catalase incubated
w ith N. cf. pellucida cultures. C atalase application indeed largely
suppressed the allelochemical potential in the N. cf. pellucida cultures.
T h e activity cannot be attrib u ted to th e catalase enzyme itself since it
did not inhibit the allelopathic activity of spent medium in control
experim ents (Fig 8).
In a second assay for haloperoxidase activity we added phenol red
(phenolsulfonphthalein) a t 36 pM to N. cf pellucida cultures and
spectrophotom etrically checked for conversion into brom inated phenol
blue
(3',3",5',5''-tetrabrom ophenolsulfonphthalein).
Halogénation
of
phenol red occurred shortly after daybreak and phenol red concentration
decreased to 26.38 ±2.26 (mean ± s.d., n=5) pM and 8.60 ± 1.86 (n=5)
pM bromophenol blue was formed w ithin three hours.
D is c u s s io n
In this stu d y we show th a t the benthic diatom Nitzschia cf. pellucida
produces allelochemicals th a t cause chloroplast bleaching and a reduced
photosynthetic efficiency leading to growth inhibition and massive cell
lysis in n aturally co-occurring com peting microalgae. T he allelopathic
com pounds are effective w ith low threshold concentrations which is
dem onstrated by the fact th a t medium from cultures w ith relatively low
cell densities (8,000 cells m L'1) was active against com petitors. The
threshold concentrations relevant for cell-cell interactions in our
experim ents are probably overestim ated given th e recent finding (Jonsson
et al., 2009) th a t local concentrations of allelochemicals (near the
chemical envelope of cells) can be orders of m agnitude higher th an those
in the well mixed cell-free filtrates which we used in our experiments.
O ur work adds to a very limited num ber of studies dem onstrating
the m olecular basis for chemically m ediated interactions between biofilm
forming microalgae. Using a com bination of chemical analyses and
bioassays we identified the highly reactive m etabolite BrCN as the
166
The molecular toothbrush of a microalga
causative agent of the observed activity. This compound has not been
previously detected as a natural product. BrCN is highly toxic and has
been applied as fumigant and pesticide and was even briefly used as a
chemical weapon during W orld W ar I (Iiosch, 2009). BrCN is currently
used to fragm ent proteins by hydrolyzing peptide bonds a t the Cterm inus of methionine residues (M ortvedt et al., 1991). Besides this
effective m etabolite, N. cf. pellucida also produces a diverse m ixture of
iodo- and bromocarbons w ith com paratively lower allelopathic properties.
T he halom ethanes, halogenated acetaldehvdes and iodopropane detected
have been reported previously from micro- and macroalgae (Giese et al.,
1999, K am enarska et al., 2007, B utler &¿ Sandy, 2009). Cyanogen bromide
is hydrolyzed by water to release hydrogen cyanide (HCN) b u t we can
exclude th a t the allelopathic effects were caused by cyanide alone since
NaCN caused only minor effects when applied to our bio-assay species E.
paludosa. C oncentrations of 40 pM NaCN (20 tim es higher th an the
active BrCN concentration) caused no short term (8 h) effect on E.
paludosa cells, which is mild com pared to the toxicity of BrCN. Since
BrCN is not a common natural product we verified if it m ight result from
abiotic transform ations in the m edium or if it is a true natural product
biosynthesized by the alga. 13C labeled bicarbonate was incorporated into
BrCN and ICN in high yields confirming unam biguously a biogenic
origin.
A pplication of the ITOs-decomposing enzyme catalase suppressed
the allelochemical potential in the N. cf. pellucida cultures which suggests
th a t haloperoxidase (HPO) activity (B utler & Sandy, 2009) is involved in
th e BrCN synthesis. Furtherm ore, we observed a preference for iodide
over brom ide incorporation, which corresponds w ith the halide selectivity
for haloperoxidases (Verhaeghe et al., 2008). Also the absence of C1CN in
Br and I depleted cultures m atches with the halide preference of algal
HPO. Lastly, the conversion of phenol red into brom inated phenol blue in
the N itzschia cultures points to the involvement of haloperoxidase
enzymes. These enzymes catalyze the oxidation of halide ions to
Irypohalous acid by H 2 O 2 . Hypohalous acid (or a sim ilar oxidized
interm ediate) can then react w ith organic substrates th a t are susceptible
to electrophilic halogénation (B utler & Sandy, 2009). W e can not,
however, conclude whether biogenic CN~ (or equivalent) reacts w ith “Br+”
from th e haloperoxidase reaction or if the transform ation of halomethanes
or other precursors to BrCN is involved in the biosynthetic pathw ay. CN'
production is known from a broad range of organisms, including bacteria,
167
Chapter 7
fungi, insects, algae, and plants, as a m eans to avoid predation or
com petition but the production and several pathw ays to this m etabolite
are described (Knowles & Bunch, 1986).
Haloperoxidase enzymes are distributed in m arine organisms
including Rhodophyta, P haeophyta, C hlorophyta (Paul k P ohnert, 2011)
and Bacillariophyta (Moore et al., 1996). An im portant function of HPO
is to scavenge harmful H 2 O 2 produced during photosynthesis,
photorespiration and other m etabolic processes (Manley, 2002). It has
also been suggested th a t HPO of m arine organisms are involved in
defense mechanisms such as th e m ediation and prevention of bacterial
biofilm formation, but evidence for the involved m etabolites was not
given till now (B rochardt et al., 2001, Cosse et al., 2009). Here we provide
a link between HPO activity and allelopathic potential supporting an
ecological role for this enzyme in diatoms.
It is interesting to note th a t we only detected BrCN during the
m orning hours in the culture and only if we quickly extracted this
reactive metabolite. Given th e methodological difficulties in detecting
highly reactive m etabolites, it would be w orth verifying th e potential of
oth er microalgae to produce this reactive m etabolite, which would most
likely not have been picked up in other determ inations of halogenated
volatiles. The mechanism introduced here could thus have a broader
occurrence.
T he production of BrCN only w ithin few hours after daybreak
coincides w ith the time span of hydrogen peroxide production in algal
cells due to the Mehler reaction (Collen et al., 1995). A short term release
of a toxic m etabolite to suppress grow th of com petitors m ight be a highly
efficient allelopathic strategy. Like a “molecular toothbrush” BrCN could
elim inate the surrounding flora daily after sunrise, leading to increased
access to nutrients present in the environm ent and even elevated
concentrations resulting from nutrients leaking from killed cells. The
clean and n u t rient-rich area could then be used by N. cf. pellucida for
effective proliferation in the absence of toxins. This diatom species is
more resistant to BrCN com pared to its com petitors (Fig. 5) but at
elevated concentrations still sensitive to the toxin. T hus th e short term
toxin b urst is an effective means of reducing the risk of auto-toxicity and
represents a novel strategy for allelopathic interactions.
In this study we illustrate th a t a highly active simple m etabolite
from diatom s has the potential to prom ote daily cleaning events around a
biofilm forming diatom . Our results provide a novel m echanism by which
168
The molecular toothbrush of a microalga
diatom s can generate microscale chemical territoria in which com petitors
are deterred or killed. Obviously, such strategy contributes to complex
micro-landscapes m aintained by interacting species and may boost the
small-scale patchy growth habits of biofilm forming species. Our results
also suggest a potential link between the globally significant emissions of
volatile halocarbons released by m arine algae and allelopathic activity.
A c k n o w le d g e m e n t s
This research was supported by the BOF-GOA projects 01GZ0705 and
01G01911 (G hent University), by the FW O project G.0374.11 and by
VLANEZO project G.0630.05. W e thank th e Jena School for Microbial
Com munication (JSMC) for a grant to C P and JG r and the Volkswagen
Foundation for a Lichtenberg Professorship to GP.
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172
231870
C h a p te r 8
G e n e r a l D is c u s s io n
D iv e r s ity - E c o sy s te m F u n c tio n in g a n d O rg a n ism a l
T r a its
T he m ain aim of this thesis was to gain a better understanding of the
diversity and functioning of estuarine benthic diatom communities. The
relationship between biodiversity and ecosystem functioning (BEF) has
now been docum ented for a wide range of ecosystems. These studies
generally found positive effects of diversity on different measures of
ecosystem functioning (reviewed by B alvanera et a l, 2006, W orm et a l,
2006, Stachowicz et a l, 2007, C adotte et a l, 2008, C ardinale et a l,
2011). M any studies suggest th a t organismal tra it differentiation is
required for positive diversity effects, but actual information on trait
differentiation is mostly lacking (Hillebrand & M atthiessen, 2009, Reiss et
a l, 2009). These organismal traits govern energy and m aterial flows and
m ay also m ediate environm ental conditions such as disturbance and
n u trien t availability.
C r y p tic d iv e r s ity a n d e c o p h y sio lo g ic a l t r a i t v a r ia tio n
An even more fundam ental requirem ent to gain insights in BEF
relationships is a solid knowledge of th e diversity before we can begin to
assign organism al trait characteristics to species. Assessing microalgal
diversity has traditionally been based on the morphology of species, but
species delineation and hence diversity measures based on morphology
can be deceiving. M any recent studies using m ultiple approaches
including morphology, molecular d ata and breeding experim ents to assess
species boundaries have shown high levels of cryptic and pseudocryptic
variation in microalgae (Saez et a l, 2003, Beszteri et a l, 2005, Slapeta et
173
Chapter 8
al., 2006, Evans et a l, 2007) and therefore suggest th a t diversity has been
severely underestim ated (Mann, 1999). In this thesis I add two more
examples (the dom inant epipelic diatom species Navicula phyllepta
(chapter 2) and Cylindrotheca closterium, chapter 3) to the expanding
list of (pseudo)cryptic species. More im portantly, I show how closely
related and morphologically similar lineages in both tax a differ in their
ecophysiological traits (salinity and therm al niche respectively). Our
results show th a t these microbial m arine species are not th e ecological
generalists we once thought they were, b u t consist of different lineages
w ith distinct ecological preferences. These insights are not only im portant
when assessing effects of diversity on ecosystem function, but also help
understanding the mechanisms th a t prom ote cryptic species coexistence.
It is likely th a t many other currently recognized estuarine benthic
diatom species are actually species complexes, consisting of several closely
related lineages w ith different ecological preferences. For example, several
common estuarine diatoms are known for their presence along broad
estuarine gradients (Kramm er & Lange-Bertalot, 1986-1991) and it is
unclear w hether these morphospccies represent cryptic species with
com plem entary distributional ranges and physiological properties or
single species with broad ecological tolerances. Navicula gregaria for
instance, another dom inant benthic diatom in many estuaries, seems to
consist of different genetic lineages; Sabbe (1997) and Cox (1987)
reported the occurrence of two different morphological groups within N.
gregaria which display distinct ecological preferences, and experiments
showed th a t different N. gregaria genotypes differ in their salt tolerance
(VaneIslander, 2004).
O rg a n is m a l T r a its a n d D is tu r b a n c e
Species’ traits arc not only im portant for species coexistence and
biodiversitv-ecosystem function relations (see below), they may also
determ ine how species can cope w ith disturbance. Based on an extensive
dataset of benthic diatom assemblages covering a wide range of
hydrodynam ic disturbance, we showed th a t growth form and cell size are
im p o rtan t traits to predict th e effect of disturbance on species richness
(chapter 4). O ur results are consistent w ith the interm ediate disturbance
hypothesis which implicitly assum es th a t there is a trade-off in functional
traits determ ining com petitive ability and disturbance tolerance (Haddad
174
General Discussion
et a l, 2008). These trade-offs lead to maximal diversity when competitive
and disturbance tolerant species are able to coexist (Haddad et al., 2008).
D iv e rs ity -E c o s y s te m F u n c tio n in g in M ic ro a lg a e
C o m m u n itie s
Field studies on benthic and planktonic microalgae suggest th a t diversity
can enhance ecosystem functions such as resource use efficiency and
productivity (Forster et a l, 2006, Ptacnik et a l, 2008). Yet, as is true for
m ost other BEF studies, th e underlying mechanisms are unclear. By
experim entally assembling diatom communities w ith decreasing diversity,
we dem onstrated a highly positive biodiversity effect on biomass
production (chapter 5). Based on the additive partition m ethods designed
by Loreau k Hector (2001), we inferred th a t com plem entarity effects
(sensu Loreau & H ector (2001), thus incorporating both niche
com plem entarity and facilitation) were responsible for positive diversity
effects.
However,
these
m athem atically
derived
measures
of
com plem entarity do not full}' correspond with the ecological meaning of
niche com plem entarity and facilitation (Cardinale et ah, 2011). Several
studies have shown th a t com plem entarity effects (sensu Loreau k Hector
2001) can be the balance of both negative and positive interactions
between species (Hooper k Dukes, 2004, Cardinale et a l, 2007).
In our study, we showed th a t growth of a strain of Cylindrotheca
closterium was significantly prom oted by the availability of organic
exudates of neighbouring diatom species, suggesting th a t m ixotrophy may
constitute an im portant m echanism m ediating facilitative interactions
between benthic diatom species. Again, different strains of C. closterium
showed different capabilities to grow mixotrophicallv, confirming th at
there can be considerable cryptic variation in organism al traits in
morphologically sim ilar strains.
As yet, we do not have further evidence for other mechanisms of
niche com plem entarity th a t caused the observed positive diversity effects
in our experim ents, b u t it is likely th a t com plem entary use of light and
/o r nutrients is also involved. Com plem entary nutrient uptake leading to
higher resource uptake in more diverse communities has been shown for
different macroalgae species which are able to use different nitrogen forms
(Bracken k
Stachowicz, 2006). In the case of benthic microalgae,
Underwood and Provot (2000) showed th a t different m icrophytobenthic
175
Chapter 8
species and strains display distinct preferences and tolerances for nitrate
and ammonium. In addition, Nilsson and Sundback (1996) showed species
specific differences in the ability to take up amino acids.
Com plem entarity in light use has recently been shown for
phvtoplankton communities. More diverse phytoplankton communities
harbour on average a more diverse arra}' of photosynthetically active
pigments which results in a more efficient absorbance of th e available
light spectrum leading to a higher biomass accrual (Striebel et a l, 2009).
Moreover, planktonic cvanobacteria display com plem entary chromatic
adaptation which allows them to tune their pigm ent com position to the
prevailing light spectrum and adjust to the presence of other
cyanobacteria with specific spectral light requirem ents (Stom p et a l,
2004, Kehoe &¿ G utu, 2006).
In contrast to phytoplankton living in relatively deep mixed
layers, m icrophytobenthos often faces a very steep gradient in light with
cells in biofilms at a depth of 300-400 pm experiencing alm ost complete
darkness (Paterson et al., 2003). In such conditions, light limiting and
inhibiting conditions are separated by only a few hundred pm. Epipelic
diatom s can cope w ith such harsh conditions by exhibiting vertical
m igration p atterns which are synchronized with daily emersion (Paterson,
1989). Kromkamp et al. (1998) suggested th a t there is a further
differentiation possible w ithin these m igration cycles. He suggested th at
species “m icro-m igrate” w ithin the upper layers of th e biofilm, with
different species surfacing a t different timings. This was confirmed in field
experim ents by Underwood et al. (2005) who dem onstrated th a t different
m icrophytobenthos species do display differentiation in m igration timing
and photophysiological adaptation to inhibiting light conditions
(especially w ith respect to non photochemical quenching). T he niche
differentiation in light use could thus also contribute to the overall higher
resource use efficiency and a higher productivity of more diverse biofilms.
E v o lu tio n a r y H is to r y a n d D iv e rs ity -E c o s y s te m
F u n c tio n in g
T he evolutionary history of species determ ines current species traits,
therefore it has been proposed th a t evolutionary history influences
biodiversity and ecosystem functioning relations (Kinzig et al., 2002,
Loreau et a l, 2002, Gravel et a l, 2011). Gravel et al. (2011) showed th a t
176
General Discussion
bacterial communities of experim entally evolved specialists have a
stronger B E F relation th an bacterial communities of experim entally
evolved generalists. Maherali and Klironomos (2007) showed th a t
phylogeneticall}' more diverse communities of mycorrhizal fungi caused a
higher associated plant productivity. They reasoned th a t there is a lot of
functional redundancy of mycorrhizal species w ithin families, and thus
th a t traits are conserved within families, corresponding w ith the idea of
phylogenetic niche conservatism. To which extent this phylogenetic niche
conservatism applies to higher clades of microalgae is currently unknown,
b u t we show th a t, at least for climatic niches, niche conservatism is weak
w ithin the diatom genus Cylindrotheca (chapter 3). To which extent this
fast adaptive n atu re is specific for Cylindrotheca or if it can be broadened
to m arine microalgae is open for further discussion.
C h e m ic a lly M e d ia te d B io tic I n te r a c tio n s
In addition to these examples of niche differentiation leading to higher
resource efficiency use through com plem entary resource use, there is a
growing awareness th a t biotic interactions m ediated by chemical cues can
also strongly influence ecosystem function (Hay, 2009). Microalgae
produce and exude a wide array of different molecules th a t are involved
in selection of m ates (Tsuchikane et ah, 2008), defence against grazing
(M iralto et ah, 1999), repelling com petitors (Gross, 2003), etc. These
secondary m etabolites are supposed to be om nipresent, b u t their identity
is often unknown (Hay, 2009). These chemical cues have been described
as the “language of life in the sea” and a better understanding of this
language will evidently increase our understanding of biotic interactions
and their im portance for ecosystem functioning (Hay, 2009). The
com bination of chemical characterization m ethods and advanced
ecological assays has only recently led to th e identification of a relatively
small set of chemical cues th a t affect the behaviour an d /o r physiology of
m arine microorganisms (Pohnert et al., 2007, Hay, 2009, Poulson et a l,
2009), b u t th eir precise effects on ecosystem functioning and com m unity
stru ctu re are still virtually unknown.
How chemical cues affect the development and spatial
organization of biofilms is poorly understood. Phototrophic biofilms,
dom inated by eukaryotic micoalgae, are known to display complex micro­
scale patchiness which cannot readily be explained by abiotic
heterogeneity
or
bioturbation
by
grazers.
It
has
therefore
been
177
Chapter 8
hypothesized th a t allelopathic interactions between biofilm eukaryotic
microalgae m ay be invoked to explain this micro-scale patchiness
(Saburova et al., 1995). In C hapter 6, I show th a t three different benthic
diatom species produce allelopathic substances in a density-dependent
way. These findings suggest th a t allelopathic interactions m ight be more
common in benthic microalga.1 com munities th an previously thought.
F urtherm ore, I dem onstrate the physiological and molecular basis of
allelopathic interactions caused by Nitzschia cf. pellucida. I show th a t this
m arine benthic diatom exudes a diverse m ixture of volatile halogenated
hydrocarbons including the highly toxic and new natural product
cyanogen brom ide (BrCN). Biosynthesis of th e toxin is light-dependent,
w ith a short burst of production shortly after the onset of light.
Inhibiting cellular H 2 O 2 production prevented th e form ation of
allelochemicals, strongly suggesting th a t haloperoxidase enzyme activity
(which is H 2 O 2 dependent) is involved in BrCN synthesis. These
antagonistic interactions have the potential to generate micro-scale
chemical landscapes which may result in the observed small-scale patchy
grow th habits of biofilm forming species.
T his work adds to a very limited num ber of studies
dem onstrating the m olecular basis for chemically m ediated interactions
between microalgae. Production of volatile halogenated m etabolites is
widespread am ong micro- and macroalgae and contributes significantly to
th e global atm ospheric halocarbon budget, b u t their biological role has
always been poorly understood (Sturges et a l, 1992, C arpenter & Liss,
2000, Salawitch, 2006, Paul & P ohnert, 2011). Furtherm ore, our results
suggest a potential function for haloperoxidase enzymes, which are
present in m arine red, green and brown algae, diatom s, fungi and
bacteria, b u t whose role to date remained elusive. Several studies have
dem onstrated allelopathic activity in algae possessing these enzymes, but
a direct link between these enzymes an d allelopathic activity has never
been suggested. In addition, production of th e toxin BrCN is coupled
w ith photosynthetic activity, and raises interesting questions regarding
photosynthesis related H 2 O 2 production (e.g. in th e Mehler reaction,
Collen et
production.
a l,
1995),
haloperoxidase
activity
and
allelochemical
M any other allelopathic interactions have been described
(reviewed by Gross, 2003, Legrand et a l, 2003), b u t only a few studies
actually show th e chemical nature of th e interaction (Leao et a l, 2010).
Chemical cues may also m ediate access to nutrients and thus influence
178
General Discussion
resource use efficiency. For instance, when it comes to phosphate
acquisition, certain cyanobacteria have a cunning plan. Bar-Yosef et al.
(2010) showed th a t Aphanizom enon ovalisporum produces the cyanotoxin
cylindrosperm opsin which causes th e production of extracellular
phosphatases in other phytoplankton species. These enzymes break down
external phosphate esters (which can not be readily assimilated) w ith the
production of inorganic phosphate which can then be (partly) used by
Aphanizomenon. O ther chemical cues th a t govern resource use efficiency
are exchanged between certain bacteria and phytoplankton species.
C ertain sym biotic bacteria can help microalgae by increasing the
solubility of boron and iron by producing non-specific boron and iron
binding siderophores which can be shared with microalgae (Amin et al,
2007, Amin et al., 2009). B acteria m ay also enhance microalgal growth by
suppling microalgae w ith essential compounds such as cobalam in (vitam in
B 12) (Croft et a l, 2005, King et a l, 2011).
C o n c lu s io n
Overall, our results contribute to the understanding of the species
com position and functional organization of complex phototrophic biofilms
and its consequences for ecosystem functioning. W e suggest th a t cryptic
diversity is widespread am ong estuarine diatom s and th a t this diversity is
linked w ith ecophysiological differentiation and h ab itat partitioning by
closely related species. Over large environm ental gradients it appears th at
lineages behave in a conservative fashion, in the sense th a t they are
lim ited in their distribution to suitable hydrodynamieal conditions. At
the local scale, strong positive and negative interactions (facilitation,
allelopathy) appear to be im portant in these diatom communities.
179
Chapter 8
P e r s p e c t iv e s
T he results in this thesis invite further research in several research areas.
I highlight some of the specific topics th a t, in my opinion, deserve further
research.
N e x t G e n e r a tio n B E F E x p e rim e n ts
O ur laboratory B EF experim ent yielded insights into the potential
mechanisms responsible for enhanced performance of more speciose
biofilms. Yet, this experim ent largely ignored the natural complexity,
th u s its applicability to real natural systems is unclear. Future
experim ents should include more environm ental realism such as the
inclusion of natural environm ental variation and trophic complexity and
should explore the im pact of diversity loss a t greater spatial and tem poral
scales. T he effect size of diversity should be com pared w ith the effect
sizes of environm ental variation and heterogeneity (see also Cardinale et
ah, 2011).
Next to these larger scale experim ents, there is a long-lasting
need to gain more insights into th e m echanisms and functional traits
responsible for BEF relations. Ideally, B EF experim ental designs could
combine measurem ents of m ultiple biogeochemical processes, a
m etabolomics approach th a t scans th e extracellular chemical cues, and a
m onitoring of key cell functions by profiling the expression of selected
genes. O r more simply: linking the ‘omics’ to com m unity ecology to
b etter understand how organismal traits affect species interactions and
com m unity functioning.
A d a p ta tio n in M a rin e M ic ro a lg a e
On the basis of the results presented in chapter 3 in which we focused on
th e therm al niche of closely related lineages w ithin the m arine diatom
genus Cylindrotheca, we can draw some further questions th a t merit
study: How fast can a strain ad a p t to another tem perature regime?
W hich genes are involved in tem perature adaptation? C an this
ad ap tatio n potentially influence spéciation? Is ecological spéciation
im portant for diversification in m arine phytoplankton? Is the negative
correlation between cold and heat tolerance caused by antagonistic
180
General Discussion
pleiotropy or m utation accum ulation? P a rt of these questions could be
resolved by conducting evolution experim ents w ith e.g. cold adapted
strains and let them adapt gradually to sublethal tem peratures (similar to
Bell & Gonzalez, 2009). Differences in gene expression between adapted
and ancestral strains could then be assessed to gain more knowledge in
th e genes responsible for therm al adaptation.
E c o lo g ica l I m p o r ta n c e o f M ic ro a lg a l H a lo c a rb o n
C h e m is try
Many
marine micro- and
macroalgae produce volatile halogenated
metabolites, an emission which is of global significance, but why they do
this is not understood (Paul & P ohnert, 2011). Several macroalgae such
as the green alga Ulva sp. and the red algae Gracilaria sp. and Corallina
sp. are known to possess haloperoxidases (Sheffield et al., 1995, Manley &
Barbero, 2001, Ohsawa et a i, 2001, W einberger et a l, 2007) and other
reports have shown allelopathic interactions for these species (Jeong et
a l, 2000, W ang et a l, 2009), b u t no reliable mechanistic link nor
halogenated allelochemicals have been identified so far for these species.
Next to these algae w ith known halogenating activities, several
dinoflagellates produce unknown toxins th a t are associated w ith hydrogen
peroxide and superoxide (M arshall et a l, 2005). In at least one case,
toxin production is H 2 O 2 dependent (Kim et a l, 1999) b u t the identity of
the active compound remains unknown (Kim et a l, 2002). An enhanced
effort w ith the m ethods described in this thesis m ight yield new insights
in the ecology of halocarbon chemistry.
Haloperoxidase enzymes also have th e potential to
disrupt
bacterial quorum sensing (a cell-cell com m unication system th a t enables a
single cell to sense the total cell num ber of its population, governed by
the accum ulation of signaling molecules). Quorum sensing (QS)
disruption has already been shown for the brown algae Lam inaria digitata
(B rochardt et a l, 2001). It m ight be interesting to test if the
halogenating enzymes in the diatom Nitzschia cf pellucida are also able to
enzymatically alter or degrade bacterial QS signals such as Nacylhomoserine lactone (AHL) which can be easily checked using
stan d ard bacterial bioassavs (Dobretsov et a l, 2009).
181
Chapter 8
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188
Sum m ary
H um anity has become a m ajor biogeochemical force and hum an
dom ination of the planet has caused massive destruction and
fragm entation of natural habitats, eutrophication, clim ate change,
acidification, etc which caused a dram atic biodiversity loss. This
biodiversity crisis raised concern about the consequences of biodiversity
loss on ecosystem properties and th e goods and services these ecosystems
provide to hum anity.
T he m ain aim of this thesis was to gain a better understanding of
the diversity and functioning of estuarine benthic microalgae
com m unities. Estuarine ecosystems provide some essential ecosystem
services to hum ans such as carbon fixation, sustaining fisheries, coastal
protection and nitrogen cycling. Although these estuaries cover relatively
small areas on a global scale, their im portance for several biogeochemical
processes is disproportionately large. We focused on benthic microalgae
inhabiting estuarine sediments; these algae have m any essential functions
for estuarine and coastal ecosystems: e.g. they are the basis of the food
chain for m any coastal species, m ediate nutrient fluxes and stabilize
sediments.
A fundam ental requirem ent to gain insights in biodiversity and
ecosystem function relationships is a solid knowledge of the diversity.
Assessing diversity is not as trivial as it may seem a t first, especially in
microbial communities. Traditionally, species have been defined based on
morphological properties. A m ultitude of molecular studies however have
dem onstrated th a t morphologically defined species often consist of several
genetically distinct lineages. Such genetically distinct b u t morphologically
indistinguishable species are called “cryptic species”, whereas the term
“pseudocr 3 rptic species” refers to th e occurrence of morphologically similar
species which a t a closer look do exhibit subtle morphological differences
between species. In m ost cases it is unknown w hether (pseudo)cryptic
species differ in their ecological preferences or w hether they are
functionally equivalent. This notion is not only im portant for questions
relating diversity and ecosystem function, b u t also to understand
m echanisms th a t prom ote cryptic species coexistence.
In C h a p ter 2, we used sequence d a ta of 3 different genetic
m arkers (ITS rDNA, th e 18S rRNA gene and rbcL) together w ith cell
189
Summary
wall morphology to show th a t estuarine populations of th e widespread
and common benthic diatom Navicula phyllepta K ütz. consist of
pseudocryptic species. ¡Moreover, we show th a t these pseudocrvptic
species differed in their tolerance to low salinities (< 5 practical salinity
units, psu), which was reflected by their different (but widely
overlapping) distribution in the W esterschelde estuary (the Netherlands).
O ur results show th a t N. phyllepta sensu lato comprises different species
w ith specialized ecophysiological characteristics rather th an generalists
w ith a broad adaptability to different environm ental conditions.
In C h a p ter 3 we further investigated th e ecological divergence
in closely related (pseudo)cryptic microalgae and assessed p attern in
phylogenetic relatedness and ecological niche sim ilarity. We explored if
therm al niches can evolve fast on an evolutionary timescale resulting in
regular switches between climates or if these niches are instead highly
conserved. W e composed a set of closely related strains of the globally
distributed diatom genus Cylindrotheca. We collected strains from a v/ide
range of m arine habitats, from coastal plankton to sea ice and intertidal
m udflats. W e first inferred the evolutionary relationships of these strains
using a m ulti-locus DNA dataset and obtained a well-resolved phylogeny.
W e th en determ ined tem perature preferences of closely related lineages in
laboratory experim ents. Combining th e molecular phylogeny w ith the
therm al niches of lineages revealed a very weak phylogenetic signal in
therm al niche characteristics. This indicates th a t closely related species
tend to differ more in therm al niche th a n expected b}' a random walk
model. This seems to be caused by a com bination of adaptive evolution
and frequent shifts in environm ents in related lineages.
Species specific niches and functional traits are not only
im p o rtan t for species coexistence and biodiversity-ecosvstem function
relations (see below), they may also determ ine how species can cope with
disturbance. In C h a p ter 4, we examined th e relations between the
hydrodynam ic disturbance, biomass, species diversity and functional
group turnover in estuarine intertidal m icrophytobenthos. W e used an
extensive dataset of benthic diatom assemblages covering a wide range of
hydrodynam ic disturbance to show th a t growth form and cell size are
im p o rtan t tra its to predict the effect of disturbance on functional group
species richness. Total m icrophytobenthos species richness displayed a
unim odal relationship w ith hydrodynam ic disturbance and standing stock
190
Summary
biomass. O ur results are consistent with the interm ediate disturbance
hypothesis which implicitly assumes th a t there is a trade-off in functional
traits determ ining competitive ability and disturbance tolerance. These
trade-offs lead to maximal diversity when com petitive and disturbance
tolerant species are able to coexist.
In C h ap ter 5 we assessed the effects of species diversity on
productivity of intertidal m icrophytobenthos. W e used naturally co­
occurring diatom species from intertidal m udflats to experimentally
assemble communities with decreasing diversity. O ur results dem onstrate
a highly positive biodiversity effect on production, w ith mixtures
outperform ing th e most productive com ponent species in more th an half
of the combinations. These strong positive diversity effects could largely
be a ttrib u ted to facilitation and com plem entarity effects. In addition, we
show m echanistic evidence for facilitation which is partly responsible for
enhanced production. We show th a t a strain of Cylindrotheca closterium
has th e ability to significantly increase its biomass production in response
to substances leaked into the culture medium by other diatom species. In
these conditions,
the species drastically reduced its
concentration, which is typical for m ixotrophic growth.
pigment
In C h a p ter 6, I show th at, next to positive, facilitative
interactions, direct negative interactions (allelopathy) can strongly
influence species composition in m icrophytobenthos. I show th a t cell
cultures of the m arine benthic diatom N. cf pellucida exude metabolites
w ith strong allelopathic effects. All nine com petitor species tested were
inhibited in their growth and some in their photosvnthetic efficiency as
well. In addition, I show the occurrence of reciprocal, density dependent
allelopathic interactions between N. pellucida and two other marine
benthic diatom species, Entom oneis paludosa and Stauronella sp. These
results suggest th a t allelopathic interactions m ight be common in benthic
microalgal communities and m ay explain small -scale spatial distribution
p attern s observed in nature.
In C h a p ter 7, I dem onstrate the physiological and molecular
basis of allelopathic interactions caused by N itzschia cf. pellucida. I show
this m arine benthic diatom produces chemical cues th a t cause chloroplast
bleaching and a reduced photosynthetic efficiency leading to growth
inhibition and massive cell lysis in naturally co-occurring competing
191
Summary
microalgae. Using headspace solid phase m icroextraction (HS-SPME) GC-MS, I dem onstrate th a t this diatom exudes a diverse m ixture of
volatile iodinated and brom inated m etabolites including the new natural
products cyanogen bromide (BrCN) and cyanogen iodide (ICN) which
exhibits pronounced allelopathic activity. Besides these effective
m etabolites, this diatom also produces a diverse m ixture of mono- di- and
trihalom ethanes, halogenated acetaldehydes and iodopropane with
com paratively lower allelopathic properties. Production of these toxins is
light-dependent with a short toxin burst after sunrise. This labile
compound thus acts as a short-term signal, leading to dailj- “cleaning”
events around the algae. W e show th a t th e allelopathic effects are H 2 O2
dependent and therefore link BrCN production to haloperoxidase activity.
T his novel strategy of chemical warfare is a highly effective means of
biofilm control. Obviously, such strategy contributes to complex micro­
landscapes m aintained by interacting species and m ay boost the smallscale patchy growth habits of biofilm forming species. Our results also
provide a potential explanation for the poorly understood role of volatile
halocarbons from m arine algae which contribute significantly to the
atm ospheric halocarbon budget.
Overall, our results contribute to th e understanding of the species
com position and functional organization of complex phototrophic biofilms
and its consequences for ecosystem functioning. W e suggest th a t cryptic
diversity is widespread am ong estuarine diatom s and th a t this diversity is
linked with ecophvsiological differentiation and h ab itat partitioning bv
closely related species. Over large environm ental gradients it appears th a t
lineages behave in a conservative fashion, in the sense th a t they are
lim ited in their distribution to suitable hydrodynam ical conditions. At
th e local scale, strong positive and negative interactions (facilitation,
allelopathy) seem to be im portant in these diatom communities.
192
Summary
S a m e n v a ttin g
De menselijke overheersing van de planeet veroorzaakt een massale
vernietiging en versnippering van
natuurlijke habitats, eutrofiëring,
klim aatverandering, verzuring, etc die allen leiden to t een drastisch
verlies aan biodiversiteit. Deze biodiversiteitscrisis leidde to t ongerustheid
over de gevolgen van biodiversiteitverlies voor het functioneren van
ecosystemen en de goederen en diensten deze ecosystemen leveren aan de
mensheid.
Het belangrijkste doei van dit proefschrift was om een beter
begrip van de diversiteit en het functioneren van estuariene benthische
microalgen gem eenschappen te verkrijgen. Estuariene ecosystemen leveren
een aantal essentiële ecosysteemdiensten zoals koolstoffixatie, het
ondersteunen van de visserij en de bescherming van de kustgebieden.
Ondanks d a t deze estuaria slechts een relatief klein oppervlak innemen op
wereldschaal,
is het
belang van
estuaria voor
verschillende
biogeochemische processen onevenredig groot. Deze scriptie focuust op
estuariene benthische microalgen; deze algen hebben vele essentiële
functies voor estuariene en m ariene ecosystemen. Ze vormen de basis van
de voedselketen voor vele kustsoorten, beinvloeden nutriëntencycli en
zorgen voor sedim ent stabilisatie.
Een fundam entele vereiste om inzicht te verkrijgen in de relatie
tussen biodiversiteit en ecosysteemfuncties is een gedegen kennis van de
diversiteit. Het
schatten
van
de
diversiteit
van
microbiële
gemeenschappen is niet zo triviaal ais het lijkt. Traditioneel zijn soorten
gedefinieerd op basis van morfologische eigenschappen. Een veelheid van
moleculaire studies hebben echter aangetoond d a t morfologisch
omschreven soorten echter vaak bestaan
verschillende groepen. Dergelijke genetisch
uit meerdere genetisch
te onderscheiden, m aar
morfologisch niet te onderscheiden soorten worden "cryptische soorten"
genoemd, terwijl de term "pseudocryptische soorten" verwijst naar
morfologisch verw ante soorten die subtiele morfologische verschillen
vertonen. In de m eeste gevallen is het onbekend of (pseudo)cryptische
soorten verschillen in hun ecologische voorkeuren of d at ze functioneel
gelijkwaardig zijn. Inzicht in de ecologische niche van nauwverwante
(pseudo) cryptische soorten is niet alleen belangrijk voor vragen
betreffende diversiteit en het functioneren van ecosystemen, m aar ook
193
Summary
voor een beter begrip van de mechanismen die sam en voorkomen van
cryptische soorten bevorderen.
In H o o fd stu k 2 gebruikten we DNA sequentie gegevens van 3
verschillende genetische merkers (ITS rDNA, het 18S rRNA-gen en rbcL)
samen m et morfologische kenmerken van de celwand om aan te tonen dat
estuariene populaties van de wijdverspreide benthische diatom ee No,ricula
phyllepta bestaan uit verschillende pseudocryptische soorten. Bovendien
tonen we aan d at deze pseudocryptische soorten verschillen in hun
tolerantie voor lage zoutconcentraties (< 5 PSU), w at to t uiting kwam
door hun verschillende (m aar grotendeels overlappende) verspreiding in
de W esterschelde (Nederland). Onze resultaten tonen aan d at N.
phyllepta sensu lato bestaat uit verschillende soorten m et specicifieke
ecofysiologische karakteristieken, het is dus geen generalistische soort met
een breed aanpassingsvermogen aan verschillende milieu-omstandigheden.
In H o o fd stu k
3 onderzochten we verder of er ecologische
verschillen tussen de nauw verwante (pseudo)cryptische microalgen
bestaan en onderzochten we de relatie tussen fylogenetische verwantschap
en
gelijkenis
in
ecologische
niches.
We
onderzochten
of
tem peratuursniches snel kunnen evolueren op een evolutionaire tijdschaal.
We verzamelden hiervoor een reeks van nauw verwante stam m en van het
wereldwijd verspreide diatom eeëngeslacht Cylindrotheca. We verzamelden
stam m en uit een breed scala van mariene habitats, van kust plankton to t
zee-ijs en intertidale slikken. We onderzochten de evolutionaire relaties
van deze stam m en m et behulp van een multi-locus D N A -dataset en
verkregen een goed ondersteunde fylogenetische boom. D aarna bepaalden
we de tem peratuurpreferenties van nauw verwante stam m en in
ecofysiologische experim enten. Het combineren van de moleculaire
fylogenie m et de tem peratuursniche van deze stam m en toonde aan d at er
een zeer zwak fylogenetisch signaal in de therm ische niche was. D it geeft
aan dat nauw verwante soorten de neiging hebben om meer in therm ische
niche af te wijken dan verw acht door een random walk model. Dit lijkt te
worden veroorzaakt door een combinatie van adaptieve evolutie en
frequente verschuivingen in de leefomgeving van deze stam m en.
Soortsspecifieke niches en functionele kenmerken zijn niet alleen
belangrijk voor soorten samenleven en biodiversiteit-ecosysteern functie
relaties (zie hieronder), zij kunnen ook bepalen hoe soorten kunnen
194
Summary
om gaan m et verstoringen. In H o o fd stu k 4 onderzochten we de relaties
tussen hydrodynam ische verstoring, biomassa, diversiteit en functionele
kenm erken van estuariene bodembewonende microalgen. W e gebruikten
een
uitgebreide
dataset
van
bodembewonende
diatomeeëngemeenschappen. W e toonden aan d at het effect van
verstoring op functionele groep soortenrijkdom deels kan verklaard
worden door de groeivorm en celgrootte van de bodembewonende
microalgen. De totale soortenrijkdom vertoonde een unimodale relatie met
hydrodynam ische verstoring. Onze resultaten zijn in overeenstemming
m et de interm ediate disturbance hypothese.
In H o o fd s tu k 5 onderzochten we de effecten van de
soortenrijkdom op de productiviteit van estuariene bodembewonende
microalgen. We gebruikten diatomeeënsoorten die sam en in intergetijde
m odderplaten voorkomen om experimentele gemeenschappen met
afnemende diversiteit sam en te stellen. Onze resultaten wezen op een
positief effect van diversiteit op de biomassa productie, waarbij
soortenmengsels meer biomassa produceerden dan de m eest productieve
soorten in m onocultuur. Deze sterk positieve effecten kunnen grotendeels
v/orden toegeschreven aan facilitatie en niche com plem entariteit.
Bovendien tonen we een mechanisme voor facilitatie. We toonden aan dat
een stam van de diatomeeënsoort Cylindrotheca closterium de potentie
heeft om zijn groei aanzienlijk te versnellen door suikers op te nemen die
door andere diatom eeënsoorten uitgescheiden werden.
In H o o fd stu k 6, toon ik aan dat, naast positieve, faciliterende
interacties ook direct negatieve interacties (allelopathie) sterk de
soortensam enstelling van bodembewonende microalgen kan beïnvloeden.
Ik toon aan d at celculturen van de m ariene benthische diatom ee N. cf
pellucida m etabolieten m et sterke allelopathic effecten uitscheiden. Alle
geteste diatom eeënsoorten werden geremd in hun groei en stierven
uiteindelijk n a blootstelling aan N. cf pellucida. D aarnaast toon ik d a t er
wederkerig, dichtheidsafhankelijke allelopathische interacties bestaan
tussen N. pellucida en twee andere mariene benthische diatomeeën
soorten, Entom oneis paludosa en Stauronella sp. Deze resultaten
suggereren
d at
allelopathic
interacties
algemener
kunnen
zijn
dan
gebruikelijk verwacht w ordt en deze interacties helpen meer inzicht te
verkrijgen in kleinschalige ruim telijke variatie in het voorkomen van
soorten.
195
Summary
In H o o fd stu k 7, toon ik de fysiologische en moleculaire basis
van allelopathic interacties veroorzaakt door Nitzschia cf pellucida. Ik
toon aan d a t deze mariene benthische diatom ee m etabolieten uitscheidt
die chloroplasten van concurrerende soorten afbleken w at leidt to t een
verlaagde fotosynthetische efficiëntie en uiteindelijk massale celsterfte.
M et behulp van vaste fase m icro-extractie (SPME) - gaschromatografie
gekoppeld aan een m assaspectrom eter (GC-MS), toon ik aan d at deze
diatom eeën een diverse mix van vluchtige jodium houdende en
gebromeerde m etabolieten produceert, inclusief de nieuwe natuurlijke
producten broomcyanide (BrCN) en iodiumcyanide (ICN). N aast deze
extrem e giftige m etabolieten produceert deze diatom eeënsoort ook een
diverse mix van mono- di- en trihalom ethanen, gehalogeneerde
acetaldehydes en iodopropaan. De productie van deze gehalogeneerde
producten is lichtafhankelijk, m et een korte puls van BrCN kort na
zonsopgang. Deze labiele verbinding w erkt dus ais een korte-term ijnsignaal, w at leidde to t de dagelijkse "reiniging" rond de algen. W e tonen
aan d at de vorming van allelopathische stoffen waterstofperoxide (H 2 O 2 )
afhankelijk is en linken dus BrCN productie aan haloperoxidase activiteit.
Deze nieuwe strategie van chemische oorlogsvoering is een zeer effectief
m iddel van biofilm controle. Dergelijke strategie draagt bij to t het
o n tstaan van complexe m icro-landschappen door biotische interacties.
Onze resultaten bieden ook een mogelijke verklaring voor weinig
begrepen, m aar globaal belangrijke uitstoot van vluchtige gehalogeneerde
koolwaterstoffen door mariene algen.
Ais conclusie kunnen we stellen d at onze resultaten bijdragen tot
een beter begrip van de soortensam enstelling en functionele organisatie
van complexe fototrofe biofilms en de gevolgen daarvan voor het
functioneren van deze ecosystemen. Wij suggereren d a t cryptische
diversiteit wijdverbreid is bij estuariene diatom eeën en d at deze
diversiteit verbonden is met ecofysiologische differentiatie tussen nauw
verw ante soorten. Over grote m ilieu-gradiënten blijkt d a t soorten zich op
een conservatieve wijze gedragen, in de zin d at ze beperkt zijn in hun
verspreidingsgebied
worden
om
geschikte
hydrodynamische
om standigheden. Op de lokale schaal zijn wellicht sterk positieve en
negatieve interacties (facilitatie, allelopat.hie) belangrijk voor deze
diatom eeën gemeenschappen.
196
A c k n o w le d g m e n ts
F irst of all I would like to thank Prof. Wim Vyverman who gave me the
chance to work in his lab. I ’m very grateful for his enduring support,
advice and ideas, patience, for the m uch appreciated freedom he gave me,
for the m anuscript editing and m uch more.
Prof. Koen Sabbe shared me his fascination w ith th e wonderful and weird
world of microphytobenthos. I’m thankful for his enthusiasm , support,
ideas, constructive criticism, for providing th e lab with th e superb PAM
fluorometer (which saved me a lot of time), m anuscript editing and much
more. Koen and Wim: a big thanks to both of you for developing the lab
into w hat it is now!
I th ank Prof. Georg P ohnert for inviting me into his lab. It was one of
th e m ost exciting times of my PhD! Those nine weeks a t his ‘Bioorganic
A nalytics’ research group opened m y eyes to the emerging and thrilling
field of m arine chemical ecology. Also thanks to his lab members for their
sweet kindness and helpfulness which made m y stay one to remember.
M any thanks to Pieter Vanormelingen (“m anusje van alles” in the lab)
for the m any interesting discussions, helpful advice and for supporting irie
when I was writing my first paper. I would also like to thanks Aaike De
W ever for introducing me into R, again nice discussions and support
when w riting my second paper.
I would also like to thank Victor Chepurnov v/ho introduced me into the
world of diatom culturing and more broadly into scientific research. The
skills he learned me were essential for the PhD I completed now. Also
thanks for supplying me some very nice pictures of intertidal diatoms
such as the one on the frontpage of this PhD.
M any thanks to Sofie D ’hont and Tine V erstraete for the superb
molecular work which was done a t an impressive speed!
197
T hanks to all the other members of the PA E lab for th e nice atmosphere,
entertainm ent and distraction, nice lab excursions and so on. A special
thanks to the “support staff” to keep the lab clean and running!
Mijn liefste ouders verdienen uiteraard een ongelooflijk dikke merci voor
alle zorg, aanmoediging en liefde. Heel erg bedankt voor jullie
on voorwaardelijke steun en om er altijd te zijn voor mij!!
Mijn vriendin Ellen wil ik danken voor alle mooie momenten, ondanks de
weinige tijd die er was. Voor alle zorg en steun, om er te zijn, voor alles.
Bedankt!
198
C u r r ic u lu m v ita e
B art Vanelslander (°1981, Bruges, Belgium) graduated as a biologist at
G hent U niversity in 2004. In 2005 he started a PhD in the Laboratory of
Protistology and A quatic Ecology in the Biologj' departm ent of G hent
University. As a research assistant he organised and supervised many
laboratory classes and field courses. During his PhD , B art supervised
m any Bachelor and blaster students during their thesis.
B art is the author of several publications in international peerreviewed academ ic journals. He also presented his research a t several
European conferences. B art acted as a referee for several international
journals such as PLoS ONE, Phycological Research and Journal of
Applied Phycolog}-. He also refereed a research proposal for the U.S.
National Science Foundation (N.S.F.).
B art visited the Friedrich Schiller University of Jena (Germany)
for two m onths where he worked a t the chemical ecology lab of Prof.
Georg Pohnert. B art also visited the lab of Prof. Ulf K arsten (Rostock
University, Germ any) and th e sedim ent ecology research group of Prof.
David P aterson (St Andrews University, U.K.) for a study visit and a
training course.
A r tic le s in in te r n a tio n a l jou rn als in clu d ed in th e IS I W eb o f
S cien ce index:
Creach, V., E rnst, A., Sabbe, K., V a n elsla n d er, B ., Vyverm an, W. &
Stal, L.J. (2006) Using q uantitative P C R to determ ine the distribution of
a semicryptic benthic diatom , Navicula phyllepta (Bacillariophyceae).
Journal of Phycology, 42, 1I42-1I54.
Van Colen, C., Lenoir, J., De Backer, A., V a n elsla n d er, B ., Vincx, bí.,
Degraer, S. k Y sebaert, T. (2009) Settlem ent of Macoma balthica larvae
in response to benthic diatom films. Marine Biology, 156, 2161-2171.
van Grem berghe, I., Vanormelingen, P ., V a n elsla n d er, B ., Van der
G ucht, K., D 'H ondt, S., De bleester, L. k
Vyverm an, W . (2009)
G enotype-dependent interactions among S3 'm patric M icrocystis strains
m ediated by Daphnia grazing. Oikos, 118, 1647-1658.
199
V a n elsla n d er,
B .,
Creach,
V.,
Vanormelingen,
P .,
Ernst,
A.,
Chepiirnov, V. A., Sahan, E., Muyzer, G., Stal, L. J., Vyverman, W. &
Sabbe, K. 2009a. Ecological differentiation betw een sym patric
pseudocryptic species in the estuarine benthic diatom Navicula phyllepta
(Bacillariophyceae). Journal o f Phye oiogy 45:1278-89.
V a n elsla n d er,
B .,
De
Wever,
A.,
Van
Oostende,
N.,
K aew nuratchadasorn, P., Vanormelingen, P ., Hendrickx, F., Sabbe, K. &
Vyverman, W. 2009b. Com plem entarity effects drive positive diversity
effects on biomass production in experim ental benthic diatom biofilms.
Journal o f Ecology 97:1075-82.
Sabbe, K.; V a n elsla n d er, B.; Ribeiro, L.; W itkowski, A.; M uylaert, K.
& Vvyerman, W. (2010) A new genus, Pierrecomperia gen. nov., a new
species and two new combinations in the m arine diatom family
Cymatosiraceae. Life and Environm ent 60(3): 243-256.
200
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GENT