Molecular Analyses in Phycology
Phylogenetics and Evolution
•Who is related to who?
•How have characters evolved?
•What are biogeographic relationships?
•Etc.
Population Genetics
•What is the connectivity (gene flow) among populations?
•How have species dispersed?
•How are invasive species spreading?
•Etc.
Molecular Assisted Identification
•Who is who?
•Who is out there?
•Character analysis?
•Etc.
1
Molecular Analyses in Phycology
DNA based techniques have been around for awhile…
Chromosomes & Nuclear DNA Content
% G+C (melting curves) & Reassociation Kinetics
Restriction Fragment Length Polymorphisms (RFLPs)
•Use restriction enzymes to cut isolated plastid or mitochondrial DNA (e.g.
Misonou et al. 1989, Phycologia 28(4):422-428; Wattier et al. 2001, Amer. J. Bot. 88(7):12091213).
•Use restriction enzymes to cut PCR amplified loci (e.g. Stiller & Waaland 1993, J.
Phycol. 29:506-517).
Misonou et al. 1989
2
Molecular Analyses in Phycology
DNA Sequencing…
Studies used rRNA prior to the spread of Polymerase Chain Reaction
(PCR) methods and thermocyclers (e.g. Buchheim et al. 1990, J. Phycol. 26:689-699;
Zechman et al. 1990, J. Phycol. 26:700-710).
After PCR things became
easier…relatively…and more
loci came into use but initially
two major ones:
18S rDNA - Saunders & Kraft 1994,
Can. J. Bot. 72:1250-1263.
Relatively conserved
“Higher level” relationships
Saunders & Kraft 1994
3
Molecular Analyses in Phycology
rbcL - Freshwater & Rueness 1994, Phycologia 33(3):187-194 (this is an
early example of molecular assisted identification).
•Distinguished previously confused
Gelidium species
•Used samples known to interbreed
from 3 different species
•Set expected intraspecific sequence
divergence based on biological
species concept (this was later done for
N. harveyi by McIvor et al. 2001, Mol. Ecol.
10:911-919)
Relatively less conserved
Wide range of relationships with “higher level” depending upon taxon
sampling
4
Molecular Analyses in Phycology
Other loci with initial or example references…
•Plastid-encoded rbcL-rbcS spacer (Destombe & Douglas 1991, Curr. Genet. 19(5):395-398)
•Mitochondria-encoded cox2-3 spacer (Zuccarello et al. 2002, Mol. Ecol. 8(9):1443-1447)
•Nuclear-encoded rDNA ITS regions (Kooistra 2002, Phycologia 41:453-462)
•Plastid-encoded tufA (Fama et al. 2002, J. Phycol. 38:1040-1050)
•Nuclear -encoded LSU or 28S rDNA (Freshwater & Bailey 1998, J. Appl. Phycol. 10:229-236;
Freshwater et al. 1999, Phycol. Res. 47:33-38; Harper & Saunders 2001, Cah. Biol. Mar. 42:2538)
•Nuclear -encoded EF2 (Le Gall & Saunders 2007, Mol. Phyl. Evol. 43:1118-1130)
•Plastid -encoded 16S rDNA (Olson et al. 2004, J. No. Car. Acad. Sci. 120(4):143-151;
Hommersand et al. 2005, J. Phycol. 42:203-225)
•Mitochondria -encoded cox1 (Saunders 2005, Phil. Trans. R. Soc. B 360:1879-1888; Robba et
al. 2006, Amer. J. Bot. 93(8):1101-1108; McDevit & Saunders 2009, Phycol. Res. 57:131-141)
•Plastid -encoded 23S rDNA (Sherwood & Presting 2007, J. Phycol. 43:605-608)
•atpB, psbA, etc
5
Molecular Assisted Identification
Who is who? - Identifying and distinguishing species
Who is out there? - Identifying which species are present
in mixed assemblage that cannot be separated
Character analysis? - Establishing intraspecific character
state limits
6
Molecular Assisted Identification
Identifying and Distinguishing Species
Example study: The Gelidium isabelae situation
Dr. Alan J. K. Millar
Royal Botanic Gardens Sydney
AJK Millar Scuba Hiking, Lord Howe Is. 2002
© DW Freshwater
RBGS, Sydney, © DW Freshwater
AJK Millar & N Yee, Jervis Bay 2002, © DW Freshwater
7
Identifying and Distinguishing Species: G. isabelae story
Small turfy Gelidium sp.
Eastern Australia
Southwest Pacific
Lord Howe Island
Southwest Pacific
New Caledonia Southwest Pacific
Rottnest Island
Western Indian Ocean
South Africa
Eastern Indian Ocean
8
All Photos © DW Freshwater
Identifying and Distinguishing Species: G. isabelae story
9
Identifying and Distinguishing Species: G. isabelae story
•Terete to compressed prostrate branches
•Flattened, stipitate erect branches
•Often decussate lines visible when
observing the blade surface (pointed out by
Taylor in his original description)
Millar & Freshwater 2005
10
Identifying and Distinguishing Species: G. isabelae story
So according to Millar & Freshwater (2005), G. isabelae is a widely
distributed, turfy Gelidium species that may be found on both sides of the
Indian and Pacific Oceans.
End of story, right?
Not quite…
Playa Brasilito, Costa Rica 2000
© DW Freshwater
Playa Guiones, Costa Rica 2000
© DW Freshwater
A number of Gelidium spp. had
been collected from Costa Rica
during collection trips in 1999 and
2000
DT Thomas & RA York, Costa Rica 2000
© DW Freshwater
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Identifying and Distinguishing Species: G. isabelae story
Species B
Species A
Analyses of rbcL sequences from these
samples showed that they:
Species C
Gelidium micropterum South Africa
Gelidium vittatum Namibia
Gelidium isabelae Australia
Gelidium pristoides SouthAfrica
Gelidium pusillum Norway
Gelidium pusillum France
•Represented seven species
•All were Gelidium
Species D
Species E
•Increases the number of Cost Rican
Gelidium pluma Hawaii
Gelidium rex Chile
Gelidum japonicum Taiwan
Gelidium vagum California
Gelidium species by nearly 350%
Some identified but morphological analysis
needed for most
Gelidium floridanum Florida
Gelidium floridanum Costa Rica
Gelidium allanii New Zealand
Gelidium pacificum Taiwan
Gelidium robustum California
Gelidium serrulatum Venezuela
Gelidium pulchellum Spain
Gelidium declerckii South Africa
Gelidium corneum Spain
Species F
Gelidium capense South Africa
Gelidium coulteri California
Gelidium crinale North Carolina
Gelidium bernabei Australia
Capreolia implexa Australia
Gelidium hommersandii Australia
Gelidium caulacantheum New Zealand
Gelidium pusillum v pacificum Hawaii
Gelidium divaricatum JAPAN
Ptilophora scalarimosa Philippines
Ptilophora subcostata Japan
Ptilophora diversifolia South Africa
Pterocladiella capillacea Italy
Pterocladiella caerulescens Hawaii
Pterocladiella bartlettii Costa Rica
Pterocladia lucida New Zealand
Parviphycus tenuissimus Canary Is
Gelidiella acerosa Hawaii
Gelidiella acerosa Costa Rica
0.01 substitutions/site
Species G
12
Identifying and Distinguishing Species: G. isabelae story
Amanda Grusz
“Phylogenetic Assessment of Pacific Costa Rican Gelidium
(Gelidiales, Rhodophyta) using Molecular and
Morphological Analyses”
Amanda Grusz, CMS, © DW Freshwater
Species B
13
Identifying and Distinguishing Species: G. isabelae story
Medullary cells
Obtuse fertile branch tip with
distinct apical cell
Obtuse to acute
sterile branch tip
Inter-medullary
spaces
Rhizines
Cortex
All images, Grusz & Freshwater,unpublished
Terete
stoloniferous
branch
Holdfast
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Identifying and Distinguishing Species: G. isabelae story
Gelidium isabelae
Pacific Costa Rica
Grusz & Freshwater,unpublished
Gelidium isabelae
sensu Millar & Freshwater
Which is the real
Gelidium isabelae?
Millar & Freshwater 2005
15
Identifying and Distinguishing Species: G. isabelae story
Solution: Generate partial rbcL sequence from the Gelidium
isabelae type specimen
Type Method - When a new seaweed gets a name, a single
specimen is chosen as the name holder (the type specimen) and
consequently for every named seaweed there is a single chosen
reference example of that organism in a herbarium somewhere.
The application of that name is based on that specimen.
The ultimate reference sequence (or other molecular marker) for
any species would be generated from the type specimen.
16
Identifying and Distinguishing Species: G. isabelae story
Sequencing of Seaweed Types:
•A partial rbcL sequence from the lectotype of Gelidium crinale was generated in 1999 and used to establish
that this species is distinct from G. pusillum and is distributed widely (Thomas 2000, UNCW Master’s Thesis).
•Hughey et al. (2001, J. Phycol. 37:1091) published on this method and made numerous changes within the
Gigartinaceae based on the sequencing of type material.
•Rico et al. (2002, Phycologia 41:463) published the first Gelidiales type specimen sequence.
Paul “Gabo” Gabrielson is currently pursuing the
sequencing of type material in earnest and is using it
to resolve taxonomic problems in the Northeast
Pacific (see Gabrielson, 2008, Phycol. Res. 56:105;
Gabrielson, 2008, Phycologia 47:89, and soon to be published
papers on Corallinales)
Paul “Gabo” Gabrielson, CMS Pier, © DW Freshwater
17
Identifying and Distinguishing Species: G. isabelae story
NJ
Gelidium pacificum Taiwan
Gelidium robustum California
Gelidium allanii New Zealand
Gelidium americanum North Carolina
Gelidium isabelae isotype
Gelidium sclerophyllum COSTA RICA
Gelidium floridanum Florida
Gelidium serrulatum Venezuela
Gelidium pteridifolium South Africa
Gelidium "isabelae" COSTA RICA
Gelidium pulchellum Spain
Gelidium latifolium France
Gelidium declerckii South Africa
Gelidium corneum Spain
Gelidium "isabelae" E. AUSTRALIA
Gelidium "isabelae" LORD HOWE IS.
Gelidium "isabelae" W. AUSTRALIA
Gelidium "isabelae" SOUTH AFRICA
Gelidium microdonticum COSTA RICA
Gelidium micropterum South Africa
Real Gelidium isabelae
Gelidium “isabelae”
Costa Rica
Gelidium “isabelae”
sensu Millar & Freshwater
Gelidium vittatum Namibia
Gelidium pristoides SouthAfrica
Gelidium pusillum Norway
Gelidium pusillum France
0.005 substitutions/site
Not one, but two new species!
18
Molecular Assisted Character Analysis
How do we judge the evolutionary and taxonomic
significance of characters (morphological,
developmental, physiological)?
Example: Fixing Fritz’s Frustration - assessment of
morphological characters used to identify Polysiphonia
species.
Good luck figuring
Polysiphonia species
out! How do you know
which characters
define species?
19
Margarita R. Albis Salas
D. “Fritz” Kapraun, 1970s.
Photographer unknown, DFK archives.
Molecular Assisted Character Analysis
Polysiphonia sensu lato (Polysiphonia/Neosiphonia)
•The largest genus of red algae - over 200 currently accepted species names.
•Species exhibit a wide range of morphological variability and which
morphological characters are evolutionarily and taxonomically significant had
not been definitively shown.
Donald “Fritz” Kapraun was the expert on Atlantic
Polysiphonia species, but quite the genus in frustration
despite having a monograph nearly complete!
Draft of the famous unpublished Western Atlantic Polysiphonia
monograph. DW Freshwater
DF Kapraun (on right during Chuck Norris phase) with WR
Taylor circa. 1970s. Photographer unknown, DFK archives.
20
Molecular Assisted Character Analysis
Solution: Objectively assign specimens to species using molecular data
and then analyse characters to determine which are consistent within
species.
rbcL was a great locus for this because the level of expected intraspecific
sequence divergence has been established by McIvor et al. (2001, Mol. Ecol.
10:911-919).
Brooke Stuercke
“Consistency of morphological characters used to delimit Polysiphonia
sensu lato species (Ceramiales, Florideophyceae): analyses of North
Carolina, USA specimens”
Phycologia 47:541-559 (2008)
21
B. Stuercke, Sodwana Bay RSA 2005
© DW Freshwater
Molecular Assisted Character Analysis
rbcL sequences generated from multiple
North Carolina specimens
Sequence similarity and position within a
larger Polysiphonia sensu lato phylogeny used
to assign specimens to species
Species specimen tree generated for character
state mapping
Species specimen tree
based on phylogenetic
tree used for character
state mapping
(from Stuercke 2006)
22
Stuercke & Freshwater (2008)
Molecular Assisted Character Analysis
Analyse morphological characters in each molecularly-defiined species
Stuercke & Freshwater (2008)
23
Molecular Assisted Character Analysis
Determine the consistency of characters within species
Poly NC-6
Poly NC-10
Poly NC-13
4 pericentral cells (0)
5-7 pericentral cells (1)
> 8 pericentral cells (2)
Poly NC-16
A
Poly NC-23
Poly NC-17
B
C
Poly NC-10
Poly NC-13
Lanceolate (1)
Poly NC-16
Linear to lanceolate (0/1)
Poly NC-31
Lanceolate to
fractiflexus (1/2)
Linear to
fractiflexus (0/2)
Poly NC-22
No information
Poly NC-2
Poly NC-30
Poly NC-1
Poly NC-3
Poly NC-5
Equivocal
Poly NC-19
Poly NC-6
Linear (0)
A
Poly NC-19
Poly NC-31
Poly NC-22
B
Poly NC-7
Poly NC-14
C
Poly NC-15
D
G
F
H
E
Poly NC-18
Poly NC-20
Poly NC-29
Poly NC-12
Poly NC-21
Poly NC-24
Poly NC-9
Poly NC-33
Poly NC-11
Poly NC-4
Poly NC-26
Poly NC-27
Poly NC-28
Poly NC-32
Poly NC-23
Poly NC-17
Poly NC-2
Poly NC-30
Poly NC-1
Poly NC-3
Poly NC-5
Poly NC-7
Poly NC-14
Poly NC-15
Poly NC-18
D
G
F
H
E
Poly NC-20
Poly NC-29
Poly NC-12
Poly NC-21
Poly NC-24
Poly NC-9
Poly NC-33
Poly NC-11
Poly NC-4
Poly NC-26
Poly NC-27
Poly NC-28
Poly NC-32
24
Molecular Assisted Character Analysis
11 of 22 analysed morphological characters were taxonomically significant in the
study of North Carolina specimens and these were verified in a further study of
New Zealand Polysiphonia species
N. Mamoozadeh
1) Number of pericentral cells
2) Rhizoid-pericentral cell connection
3) Relationship of lateral branches to trichoblasts
4) Presence/absence of trichoblasts
5) Number of segments between trichoblasts
6) Type of holdfast
7) Presence/absence of scar cells
8) Scar cell pattern
9) Presence/absence of cicatrigenous branching
(lateral branches originating from scar cells)
10) Development of spermatangial axes
11) Arrangement of tetrasporangia
Stuercke & Freshwater 2008
J. Kelly
25
Stuercke & Freshwater 2008
Stuercke & Freshwater 2008
Molecular Assisted Identification - DNA Barcoding
DNA Barcoding
“DNA barcoding employs sequence diversity in short, standardized gene
regions to aid speices identification and discovery in large assemblages of
life.” (Ratnasingham & Hebert, 2007, Mol. Ecol. Notes doi:10.1111/j.14718286.2006.01678.x)
“…sequencing a short, diagnostic segment to discriminate between
species.” (Robba et al. 2006, Amer. J. Bot. 93(8):1101-1108)
Initial work with animals, used mitochondria-encoded COI
Hebert et al. 2003, Proc. R. Soc. Lond. B 270:313-321
26
Molecular Assisted Identification - DNA Barcoding
Requirements of a “Barcode” sequence
Need universal primers for amplifications and sequencing (maybe for
marine algae this is impossible).
Intraspecific variation must be less than interspecific variation and it
helps if there is an appreciable gap (sometimes animal people have used
means to determine the gap but that isn’t really legitimate - there must
be a maximum intraspecific - minimum interspecific gap [see Meier et al.
2008 Syst. Biol. 57(5):809])
A comprehensive baseline database of sequences for comparison. This is
really no different from having a comprehensive understanding of any
character set, such as morphology, etc.
27
Molecular Assisted Identification - DNA Barcoding
Algal DNA Barcoding: Advantages & Disadvantages
Disadvantage - Algal lineages are very different in age so a locus that is
best for one might not be the best for another
Disadvantage - Barcode sequence may not have proper “signal” to
accurately determine relationships (but that is probably not the purpose)
Disadvantage - Particular genome may not reflect actual species
relationships e.g. mitichondrial or plastid introgression, within population divergence,
and adaptive changes may distort the picture. (see Zuccarello, Phycologia 48(4) supplement:152
and I expect future papers)
Advantage - All data comparable (sometimes difficult or impossible when people
have used multiple markers)
Advantage - Greater universality of primers (eventually there will be a set of
known primers to throw at a specimen and I can’t wait!).
28
Molecular Assisted Identification - DNA Barcoding
Algal DNA Barcoding - initial and example studies:
•Saunders 2005 “Applying DNA barcoding to red macroalgae: a preliminary appraisal holds
promis for future application” Phil. Trans. R. Soc. B 360:1879-1888. COI, red algal species
identification (Mazzaella; Dilsea/Neodilsea; Asteromenia)
•Robba et al. 2006 “Assessing the use of the mitochondrial cox1 marker for use in DNA
barcoding of red algae (Rhodophyta)” Amer. J. Bot. 93(8):1101-1108. COI, red algal species
identification (Saunders specimens + Porphyra; Corallina; Calliblepharis; Mastocarpus; Gracilaria)
•Sherwood & Presting 2007 “Universal primers amplify a 23S rDNA plastid marker in eukaryotic
algae and cyanobacteria” J. Phycol. 43:605-608. 23S, multiple algal groups, higher level taxonomic
designations, maybe species identification? (Rhodophyta, Phaeophyta, Chlorophyta, Euglenophyta?,
Xanthophyta, Bacillariophyta?)
•McDevit & Saunders 2009 “On the utility of DNA barcoding for species differentiation among
brown macroalgae (Phaeophyceae) including a novel extraction protocol” Phycol. Res. 57(2):131141. COI, brown algal species identification
•Sherwood et al. 2008 “Contrasting intra versus interspecies DNA sequence variation for
representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding
approach” Phycol. Res. 56:269-279. COI and 23S, red algal species identification (Batrachospermales)
29
Molecular Assisted Identification - DNA Barcoding
Prior studies with COI have shown it to be a good barcoding locus in red algae, but
two things are yet to be explicitly shown…
1)
2)
Resolution of “biological species” (ones that fit the biological species concept).
Verification of the maximum intraspecific - minimum interspecific gap
Requirements of a “Barcode” sequence
Need universal primers for amplifications and sequencing (maybe for marine algae this is impossible).
Intraspecific variation must be less than interspecific variation and it helps if there is
an appreciable gap. There must be a maximum intraspecific - minimum
interspecific gap.
A comprehensive baseline database of sequences for comparison. This is really no different from having a comprehensive understanding of any
character set, such as morphology, etc.
This minimum interspecific - maximum intraspecific variation needs to
be assessed based on known sister species.
30
Molecular Assisted Identification - DNA Barcoding
Verification of the maximum intraspecific - minimum interspecific gap
with Gelidiales sister species and comparisons with rbcL.
Enrico Tronchin, road to Mozambique 2005 © DW Freshwater
Pterocladiella psammophila, Sodwana Bay RSA 2005 © DW Freshwater
31
Pterocladiella caerulescens, Sodwana Bay RSA 2005 © DW Freshwater
Molecular Assisted Identification - DNA Barcoding
Gelidium crinale
vs.
Gelidium coulteri
Freshwater et al. 1995, J. Phycol. 31:614-630;
Millar & Freshwater 2005, Aust. Syst. Bot. 18:215-263
G. crin. (rbcL)
(COI)
G. coul. (rbcL)
(COI)
G. crin.
G. coul.
0.00-1.23
0.00-2.62
2.82-5.65
12.48-13.41
2.29x
4.76x
0.00-0.83
0.00-0.73
COI – rbcL comparison for specimens of
G. crinale (G.crin.) and G.coulteri
(G.coul.). Minimum “tra-ter-var” values
are shown in red above diagonal
32
Molecular Assisted Identification - DNA Barcoding
Gelidium pristoides PORT EDWARD, RSA
Gelidium pristoides
vs.
Gelidium foliaceum
Tronchin et al. 2002, Bot. Mar. 45:548-558
COI – rbcL comparison for specimens of
G. pristoides (G. prist.) and G. foliaceum
(G. fol.). Minimum “tra-ter-var” values
are shown in red above diagonal
L100
D100
P100
L98
D100
P100
Gelidium pristoides KIDDS BEACH, RSA
Gelidium pristoides FALSE BAY, RSA
Gelidium foliaceum PORT EDWARD, RSA
L94
D100
P99
Gelidium foliaceum EAST LONDON, RSA
Gelidium foliaceum BREEZY POINT, RSA
G. prist. (rbcL)
(COI)
G. fol. (rbcL)
(COI)
G. prist.
G. fol.
0.00-0.49
0.29-1.31
1.84-2.10
7.60-8.61
3.68x
5.80x
0.00-0.21
0.15-0.44
33
Molecular Assisted Identification - DNA Barcoding
Pterocladiella caerulescens
vs.
Pterocladiella psammophila
Tronchin & Freshwater 2007, Phycologia 46:325-348
COI – rbcL comparison for specimens
of P. caerulescens (P. caer.) and P.
psammophila (P. psam.). Minimum
“tra-ter-var” values are shown in red
above diagonal
P. caer. (rbcL)
(COI)
P. psam. (rbcL)
(COI)
P. caer.
P. psam.
1.18-3.15
0.00-5.33
2.04-3.71
8.10-10.56
<1.00x
1.52x
0.00-0.11
0.00-0.15
34
Molecular Assisted Identification - DNA Barcoding
P. caer. SA (rbcL)
(COI)
P. caer. HI (rbcL)
(COI)
P. caer. CR (rbcL)
(COI)
P. psam. (rbcL)
(COI)
P. caer. SA
0.07-0.42
0.44-1.33
2.14-2.13
4.69-5.33
1.18-3.15
4.27-5.33
2.04-3.36
9.37-10.56
P. caer. HI
5.10x
3.53x
0.29
0.15
2.15-3.15
4.42-4.56
2.28-3.71
8.93-9.32
P. caer. CR
2.81x
3.21x
7.41x
29.47x
NA*
NA*
2.04-2.48
8.10-8.28
P. psam.
4.86x
7.05x
7.86x
59.53x
18.55x
54.00x
0.00-0.11
0.00-0.15
COI – rbcL comparison for geographic specimens of P. caerulescens
(P. caer.) and P. psammophila (P. psam.). Minimum “tra-ter-var”
values are shown in red above diagonal
35