Subaerial Algae in Tropical
Rainforests, part II
Juan M. Lopez-Bautista
The University of Alabama
1
Overview
 Biodiversity and systematics of subaerial algae
 Collection and study of subaerial algae
• Molecular systematics of subaerial microchlorophytes
• Evolution of subaerial microchlorophytes
2
Molecular systematics of subaerial microchlorophytes
3
Klebsormidium
Klebsormidium is one of the most common
genera of terrestrial Charophyta
It consists of few species of green algae
occurring on soil, subaerial surfaces and semiaquatic habitats all around the world
Pisa Italy 2005
The systematics of this genus has been entirely based on traditional morphology.
The species-level relationships in Klebsormidium have not been examined with
molecular tools
Klebsormidium Galway Ireland 2004
Klebsormidium
- One of the most widespread genera of terrestrial and freshwater green algae
- Cosmopolitan distribution
- Uniseriate filaments; no differentiated holdfast; one parietal chloroplast covering a half2/3 of the cell wall, with a single pyrenoid
- 22 species currently described
- Species-level systematics based almost entirely on morphology
- Reproduction by biflagellate zoospores
Principal morphological characters used for species
identification in Klebsormidium
1) Width of filaments
2) Length of filaments
3) Cell shape
4) Presence/absence of
constrictions between
adjacent cells
5) Occasional
presence/absence of
biseriate parts
6) Presence of H-shaped
pieces
7) Shape of the chloroplast
Long
Short
Characters observable in culture
1) Growth habit: filamentous or
fragmented
2) Presence/absence of a superficial
hydrorepellent layer
3) Inducibility of zoospore release
4) Morphology of the zoosporangial
aperture
5) Germination pattern of sporelings
Fragmented
Filamentous
CITIES SAMPLED
- Bergen (Norway)
- Stockholm (Sweden)
- Copenhagen (Denmark)
- Galway (Ireland)
- London, Plymouth and
Manchester (England)
- Hamburg, Rostock and
Konstanz (Germany)
- Bordeaux and Marseilles
(France)
- Porto (Portugal)
- Pisa, Siena and Pavia
(Italy)
- La Valletta (Malta)
- Koper (Slovenia)
- Prague (Czech Republic)
Morphology (field)
Very uniform morphology; virtually no
differences between strains from different
cities
• Filaments mostly long, sometimes mixed with
short fragments
• Filaments unbranched, 6-9 m wide
• Slight constrictions between adjacent cells
present in many strains
• Cells regularly cylindrical
• Chloroplast with smooth margin
• Globular enlargements present in one strain
(Pisa)
• No biseriate parts or pseudo-branches
Morphology (culture)
• All strains growing well in both media used
• Production of superficial hydrorepellent layer
observed in the strains from Bergen, Galway,
Hamburg and Konstanz
• The strains from Marseilles, Pisa, La Valletta,
Hamburg and Porto showed a marked tendency
to fragmentation; after a few weeks the strains
from Pisa and Marseilles consisted entirely of
short fragments (2-5 cells)
• Release of zoospores easily inducible only in
the strain from Galway; release aperture
indistinct
• Sporelings in the Galway strain germinating
with unipolar and bipolar pattern
Although the morphology of the field material was virtually identical to Klebsormidium
flaccidum, preliminary experiments in culture have shown a very large range of
morphological variation
• Most strains produce only 3D filaments that remain submerged
• Others populations produce also a superficial layer of parallel filaments that cover
completely the surface of the medium,
• In other strains, the filaments get fragmented into many short fragments, giving the
cultures the appearance of a green “soup”
Submerged
Submerged
filaments
Filaments +
Superficial
layer
Short
Fragmented
filaments
Samples originally corresponding
to Klebsormidium flaccidum are
spread along the rbcL tree
Samples originally corresponding
to Klebsormidium flaccidum are
spread along the rbcL tree
On the other hand, samples
corresponding to different
morphological species are
grouped together
Samples originally corresponding
to Klebsormidium flaccidum are
spread along the rbcL tree
On the other hand, samples
corresponding to different
morphological species are
grouped together
Furthermore, specimens not
possible to ascribe to any known
taxa, and from distant locations,
are found to be genetically
similar!
Conclusions
• The populations of Klebsormidium occurring in European cities belong to at
least 4 different evolutionary lineages
• In the field material, these show an almost identical morphology and there are
no morphological characters useful to distinguish them
• More differentiation observed in culture, in which different strains show
different growth habits
• The taxonomy of Klebsormidium needs critical reconsideration; none of the
morphological characters used for identification have phylogenetic relevance
• The characterization of Klebsormidium flaccidum, type species of the genus, is
a taxonomic mess that can be solved only sequencing the type specimen;
depending on its identity, the whole genus may require a radical reassessment
Lessons learned
 Overall, these experiments and analyses indicated that a great deal of genetic
diversity is hidden behind a very similar morphology
 Morphology and cytology, even in “simple” and “common” forms, can be
misleading
 Unialgal cultures and phylogenetic analyses are needed to re-evaluate these
so called cosmopolitan species
Spongiochrysis hawaiiensis
Dense yellow-orange coating believed to be Trentepohlia by the
local scientists
Interesting unicellular green alga, reproducing with a budding-like
mechanism known only in two subaerial species of the class
Trebouxiophyceae:
• Marvania geminata Hindák 1976
• Marvania aerophytica Neustupa & Sehjonová 2003/Stichococcus
ampulliformis Handa et al. 2003
•However, sequences of the 18S
rRNA, showed unequivocally that
this alga is a member of the
Cladophorales Siphonocladales
lineage (Ulvophyceae)
•Since this group was so far known
to include basically marine species
with complex thalli, this was a
discovery of exceptional interest,
that shed new light on the
phylogeny of this algal group
•Autosporulation has arisen
separately at least 3 times (twice in
Trebouxiophyceae and once in
Ulvophyceae)
Phylogenetic tree for a 18S rDNA sequence alignment of 1732 characters,
with 55 representatives of the Viridiplantae and Spongiochrysis hawaiiensis.
In a restricted analysis limited to
41 representatives of this group,
our samples were included in
the “Aegagropila-clade” with
high support
*
100/1.00
ML tree 18S rDNA (BS/Bayesian)
Phylogenetic tree for an 18S rDNA sequence alignment of 1595
characters, with 41 representatives of the Siphonocladales
Cladophorales complex, two outgroup taxa, and Spongiochrysis
hawaiiensis
*
*
*
These data suggest that a marine
ancestor gave origin to Spongiochrysis
• Simplification of the thallus
• Carotenoid production
Similar trends during the colonization of
terrestrial habitats have been found in
the evolution of green algae
Phylogenetic tree for an 18S rDNA sequence alignment of 1595
characters, with 41 representatives of the Siphonocladales
Cladophorales complex, two outgroup taxa, and Spongiochrysis
hawaiiensis
*
*
Phylogenetic Considerations
This alga represents a new subaerial lineage from the class Ulvophyceae
It is the first known subaerial member of the Cladophorales
Siphonocladales lineage
For the unicellular habit and for the subaerial habitat this species is well
differentiated from all other members of this lineage and can be considered the
first known successful step of these algae into subaerial habitats
New genus, new species:
Spongiochrysis hawaiiensis
Lessons learned
 The subaerial habit has developed in an algal group that was formerly believed to
be entirely aquatic (the Cladophorales)
 A new subaerial lineage exists in the class Ulvophyceae
 It also showed that identical morphologies and identical mechanisms of
reproduction have developed independently in separated green algal lineages
 Most importantly, great evolutionary surprises can be found in forest
environments, if detailed surveys based on modern methodologies are carried out
The Order Trentepohliales
The group is unusual (to say the least!) in many respects
Unusual features
1.
Exclusively terrestrial and orange-red (unlike the majority of green algae, which
are aquatic and green)
2.
Differentiated sexual and asexual reproductive structures including an absolutely
intriguing, unique zoosporangial abscission process
3.
Remarkable bilaterally keeled flagella and flagellar apparatus (i.e. basal bodies
and associated components), and
4.
Evolutionarily puzzling phragmoplast-mediated cytokinesis found no where else
in the entire chlorophyte lineage and analogous to but distinct from the
phragmoplast-mediated cytokinesis of some green algae and the land plants in
the charophycean lineage
The order Trentepohliales
Five genera currently recognized:
Phycopeltis
Printzina
Trentepohlia
Cephaleuros
Discoid
Epiphytic
Prostrate
Epiphytic or
epilithic
Erect
Epiphytic or
epilithic
Discoid
Obligated
epiphyte
Reduced
Endophytic
Isomorphic
Isomorphic
Isomorphic
Heteromorphic
Heteromorphic
28 species
8 species
38 species
15 species
4 species
Trentepohlia Martius 1817
The genus Trentepohlia is the most diversified of the trentepohliacean algae,
consisting of branched heterotrichous filaments, growing epilithic or epiphytic on the
bark of trees, or in lichenic associations at exposed habitats forming conspicuous
masses, usually yellow to orange in color
As currently redefined, Trentepohlia shows a profused erect system, with a scarce or
absent prostrate system; sporangia are ovoid, sporangiate-laterals solitary or grouped,
borne terminally or on an enlarged terminal head-cell of a branched sporangiophore;
gametangia are terminal only
Described species: 35
Physolinum H. Printz 1921
The species type for the genus Physolinum was originally described as Trentepohlia
monile, later the specific epithet was changed to T. moniliformis
The genus Physolinum was based on the discovery of aplanospores as the sole
reproductive structures found in this alga, and concluding that was markedly different
from Trentepohlia
Described species: 1
Printzina Thompson et Wujek 1992
Printzina is remarkably similar to Trentepohlia, except
•
•
the presence of globular to reniform sporangia, and
an almost pseudoparenchymatic thallus with few or any upright filaments
Described species: 9
Phycopeltis Millardet 1870
The filaments of Phycopeltis grow openly or form a pseudoparenchymatous thallus,
but always superficial upon the plant host or other surfaces
Another feature distinguishing Phycopeltis from other superficial trentepohlialean
genera is the terminal position of the papilla-pore or ostiole on the sporangium,
which is opposite to the end of attachment whereas, in the other genera it is basal
and adjacent to the area of attachment
Described species: 25
Cephaleuros Kunze in Fries 1829
This genus is reported as an obligate epiphyte and subcuticular, that may be parasitic
The prostrate portion can be open-filamentous to pseudoparenchymatous
Sporangiophores bear one or more head cells subtending sporangiate-laterals
Cephaleuros is one of the most studied genera among the Trentepohliales, in part for
its worldwide distribution, its obvious presence and economic damage to their host
Described species: 15
Stomatochroon Palm emend. Thompson and Wujek 1997
This alga grows as branching filaments endophytically in the substomatal chamber
and protruding its sporangiophores through the stomata
The reduced morphology of this alga is extraordinary, one species has become
reduced to a single massive and lobed anchoring cell
Described species: 4
Life cycles
They have been described as either an isomorphic or heteromorphic alternation of
generations
Trentepohlia and Phycopeltis
M
n
n
n
2n
1n
n
F
Isomorphic generations occur in taxa where the vegetative morphology is simpler
(Trentepohlia & Phycopeltis),
Cephaleuros and Stomatochroon
n
n
M
n
2n
1n
n
F
Whereas a more complicated heteromorphic alternation is said to occur in taxa with
a complex vegetative morphology (Cephaleuros & Stomatochroon) and possibly
secondarily reduced morphology
Reproductive Cells: sporangium is unique to this order
Trentepohliales displays a characteristic abscission between the sporangium and the
suffultory cell (or stalk cell)
The sporangium and suffultory cell form together the sporangiate-lateral, which
representa an important taxonomic character for the circumscription of the order
Abscission
The abscission involves a central area rich in plasmodesmata surrounded by a
thickened area or internal ring
In the periphery of this abscission septum there is a second area of thickened wall
material, the external ring; the region between the rings lacks plasmodesmata
Flagella
The flagellate cells are compressed in a dorsiventral fashion with either two (gametes)
or four (zoospores) flagella
The overlapping configuration of the basal bodies in Trentepohliales is CCW and it
has been cited as an evidence for an affinity with the Ulvophyceae
Cytokinesis
A phragmoplast-type cytokinesis has
been reported for C. parasiticus and
Trentepohlia odorata
Chapman and Henk 1986
Ecology and Distribution
•The geographic distribution of the Trentepohliales is basically pantropical
•All Trentepohliales are subaerial, none having ever been found in aquatic habitats,
freshwater or marine
•The presence of sporopollenin-like substances in the cell walls, as well as a special
pattern of carbohydrates and alcohols, probably are adaptative features against
desiccation in the subaerial habitat
•Trentepohliales are well known to form lichenic associations with fungi
•Trentepohlia commonly occurs upon rocks or tree bark
•Phycopeltis is an epiphyllous alga, growing on the surface of leaves
•Cephaleuros is a strict epiphyte living beneath the cuticle of leaves, twigs, and fruits
•Stomatochroon is found in the air chambers and stomata of leaves
Questions on the evolution of the Trentepohliales
Where does the Trentepohliales belong in the various proposed classification
schemes of green algal classes?
•Phragmoplast cytokinesis suggested an affinity with Charophyceae
•The CCW flagella can be cited as evidence for an affinity with the Ulvophyceae
•Based on biochemical and physiological features they have been classified as
Pleurastrophyceae
•Moreover, Trentepohliales share a rare ultrastructural feature (presumptive
mating structure in the gametes) with members of a fourth class, the
Chlorophyceae
Molecular studies in green algae
VIRIDIPLANTAE (CHLOROBIONTA, GREEN PLANTS)
CHLOROPHYTA
STREPTOPHYTA
Chlorophycean lineage
CHLOROPHYCEAE
Charophycean lineage
TREBOUXIOPHYCEAE
LAND PLANTS
CHAROPHYCEAE
Chlorokybales
Klebsormidiales
Coleochaetales
Zygnematales
Charales
ULVOPHYCEAE
Mesostigma
PRASINOPHYCEAE
PRASINOPHYCEAE
PRASINOPHYCEAE
PRASINOPHYCEAE
ANCESTRAL
GREEN
FLAGELLATE