molecular in phylogenetics and evolution · •a partial rbcl sequence from the lectotype of...

Molecular Analyses in PhycologyMolecular Analyses in Phycology

Phylogenetics and EvolutionPhylogenetics and Evolution••Who is related to who?Who is related to who?••How have characters evolved?How have characters evolved?••What are biogeographic relationships?What are biogeographic relationships?••Etc.Etc.

Population GeneticsPopulation GeneticsPopulation GeneticsPopulation Genetics••What is the connectivity (gene flow) among populations?What is the connectivity (gene flow) among populations?••How have species dispersed?How have species dispersed?••How are invasive species spreading?How are invasive species spreading?p p gp p g••Etc.Etc.

Molecular Assisted IdentificationMolecular Assisted Identification••Who is who?Who is who?

1

Who is who?Who is who?••Who is out there?Who is out there?••Character analysis?Character analysis?••Etc.Etc. 1


DNA based techniques have been around for awhile…DNA based techniques have been around for awhile…

Chromosomes & Nuclear DNA ContentChromosomes & Nuclear DNA Content

% G+C (melting curves) & Reassociation Kinetics% G+C (melting curves) & Reassociation Kinetics

Restriction Fragment Length Polymorphisms (RFLPs)Restriction Fragment Length Polymorphisms (RFLPs)••Use restriction enzymes to cut isolated plastid or mitochondrial DNA Use restriction enzymes to cut isolated plastid or mitochondrial DNA (e.g. (e.g. y py p ( g( gMisonou et al. 1989, Misonou et al. 1989, PhycologiaPhycologia 28(4):42228(4):422‐‐428; Wattier et al. 2001, 428; Wattier et al. 2001, Amer. J. BotAmer. J. Bot. 88(7):1209. 88(7):1209‐‐1213).1213).••Use restriction enzymes to cut PCR amplified loci Use restriction enzymes to cut PCR amplified loci (e.g. Stiller & Waaland 1993, (e.g. Stiller & Waaland 1993, J. J. Phycol.Phycol. 29:50629:506‐‐517).517).Phycol.Phycol. 29:50629:506 517).517).

22Misonou et al. 1989Misonou et al. 1989


DNA Sequencing…DNA Sequencing…

Studies used rRNA prior to the spread of Polymerase Chain Reaction Studies used rRNA prior to the spread of Polymerase Chain Reaction (PCR) methods and thermocyclers (PCR) methods and thermocyclers (e.g. Buchheim et al. 1990, (e.g. Buchheim et al. 1990, J. Phycol.J. Phycol. 26:68926:689‐‐699; 699; Zechman et al. 1990, Zechman et al. 1990, J. Phycol.J. Phycol. 26:70026:700‐‐710).710).

After PCR things became After PCR things became i l i l di l i l deasier…relatively…and more easier…relatively…and more

loci came into use but initially loci came into use but initially two major ones:two major ones:18S rDNA18S rDNA Saunders & Kraft 1994Saunders & Kraft 199418S rDNA18S rDNA ‐‐ Saunders & Kraft 1994, Saunders & Kraft 1994, Can. J. Bot.Can. J. Bot. 72:125072:1250‐‐1263.1263.

Relatively conservedRelatively conserved

3

“Higher level” relationships“Higher level” relationships

Saunders & Kraft 1994Saunders & Kraft 1994 3


rbcrbcLL ‐‐ Freshwater & Rueness 1994, Freshwater & Rueness 1994, PhycologiaPhycologia 33(3):18733(3):187‐‐194 (this is an 194 (this is an early example of molecular assisted identification).early example of molecular assisted identification).

••Distinguished previously confused Distinguished previously confused Gelidium Gelidium speciesspecies••Used samples known to interbreed Used samples known to interbreed from 3 different speciesfrom 3 different species••Set expected intraspecific sequence Set expected intraspecific sequence divergence based on biological divergence based on biological species concept species concept (this was later done for (this was later done for N. harveyiN. harveyi by McIvor et al. 2001, by McIvor et al. 2001, Mol. EcolMol. Ecol. . 10:91110:911‐‐919)919)

4

Relatively less conservedRelatively less conservedWide range of relationships with “higher level” depending upon taxon Wide range of relationships with “higher level” depending upon taxon samplingsampling 4samplingsampling


Other loci with initial or example references…Other loci with initial or example references…••PlastidPlastid‐‐encodedencoded rbcrbcLL‐‐rbcrbcS spacer S spacer (Destombe & Douglas 1991, (Destombe & Douglas 1991, Curr. GenetCurr. Genet. 19(5):395. 19(5):395‐‐398)398)••MitochondriaMitochondria‐‐encodedencoded cox2cox2‐‐33 spacer spacer (Zuccarello et al. 2002, (Zuccarello et al. 2002, Mol. Ecol.Mol. Ecol. 8(9):14438(9):1443‐‐1447)1447)pp••NuclearNuclear‐‐encoded rDNA ITS regions encoded rDNA ITS regions (Kooistra 2002, (Kooistra 2002, PhycologiaPhycologia 41:45341:453‐‐462)462)••PlastidPlastid‐‐encodedencoded tufA tufA (Fama et al. 2002, (Fama et al. 2002, J. Phycol.J. Phycol. 38:104038:1040‐‐1050) 1050) ••Nuclear Nuclear ‐‐encoded LSU or 28S rDNA encoded LSU or 28S rDNA (Freshwater & Bailey 1998, (Freshwater & Bailey 1998, J. Appl. Phycol.J. Appl. Phycol. 10:22910:229‐‐236; 236; Freshwater et al. 1999, Freshwater et al. 1999, Phycol. ResPhycol. Res. 47:33. 47:33‐‐38; Harper & Saunders 2001, 38; Harper & Saunders 2001, Cah. Biol. Mar.Cah. Biol. Mar. 42:2542:25‐‐38)38)••Nuclear Nuclear ‐‐encoded EF2 encoded EF2 (Le Gall & Saunders 2007, (Le Gall & Saunders 2007, Mol. Phyl. Evol.Mol. Phyl. Evol. 43:111843:1118‐‐1130)1130)••Plastid Plastid ‐‐encoded 16S rDNA encoded 16S rDNA (Olson et al. 2004, (Olson et al. 2004, J. No. Car. Acad. Sci.J. No. Car. Acad. Sci. 120(4):143120(4):143‐‐151; 151; Hommersand et al. 2005, Hommersand et al. 2005, J. Phycol.J. Phycol. 42:20342:203‐‐225)225)••Mitochondria Mitochondria ‐‐encoded encoded cox1cox1 (Saunders 2005, (Saunders 2005, Phil. Trans. R. Soc. BPhil. Trans. R. Soc. B 360:1879360:1879‐‐1888; Robba et 1888; Robba et al. 2006, al. 2006, Amer. J. Bot.Amer. J. Bot. 93(8):110193(8):1101‐‐1108; McDevit & Saunders 2009, 1108; McDevit & Saunders 2009, Phycol. ResPhycol. Res. 57:131. 57:131‐‐141)141)••Plastid Plastid ‐‐encoded 23S rDNAencoded 23S rDNA (Sherwood & Presting 2007, (Sherwood & Presting 2007, J. Phycol.J. Phycol. 43:60543:605‐‐608)608)••atpB, psbA, atpB, psbA, etcetc

55

Molecular Assisted IdentificationMolecular Assisted Identification

Who is who?Who is who? ‐‐ Identifying and distinguishing speciesIdentifying and distinguishing species

Who is out there?Who is out there? ‐‐ Identifying which species are present Identifying which species are present in mixed assemblage that cannot be separatedin mixed assemblage that cannot be separated

Character analysis?Character analysis? ‐‐ Establishing intraspecific character Establishing intraspecific character state limitsstate limits

66

Molecular Assisted IdentificationMolecular Assisted Identification

Identifying and Distinguishing SpeciesIdentifying and Distinguishing SpeciesExample study: The Example study: The Gelidium isabelaeGelidium isabelae situationsituation

Dr. Alan J. K. MillarDr. Alan J. K. MillarRoyal Botanic Gardens SydneyRoyal Botanic Gardens Sydney

AJK Millar Scuba Hiking, Lord Howe Is. 2002AJK Millar Scuba Hiking, Lord Howe Is. 2002© DW Freshwater© DW Freshwater

RBGS, Sydney, © DW FreshwaterRBGS, Sydney, © DW Freshwater

7

AJK Millar & N Yee, Jervis Bay 2002, © DW FreshwaterAJK Millar & N Yee, Jervis Bay 2002, © DW Freshwater

7

Identifying and Distinguishing Species: Identifying and Distinguishing Species: G. isabelaeG. isabelae storystory

Eastern AustraliaEastern AustraliaSouthwest PacificSouthwest Pacific

Lord Howe IslandLord Howe IslandSouthwest PacificSouthwest Pacific

Small turfy Small turfy GelidiumGelidium sp.sp.

New Caledonia Southwest PacificNew Caledonia Southwest PacificNew Caledonia Southwest PacificNew Caledonia Southwest Pacific

8

Rottnest IslandRottnest IslandWestern Indian OceanWestern Indian Ocean

South AfricaSouth AfricaEastern Indian OceanEastern Indian Ocean 8

All Photos © DW FreshwaterAll Photos © DW Freshwater


99


••Terete to compressed prostrate branchesTerete to compressed prostrate branches

••Flattened, stipitate erect branchesFlattened, stipitate erect branches

f d l bl hf d l bl h••Often decussate lines visible when Often decussate lines visible when

observing the blade surface (pointed out by observing the blade surface (pointed out by

Taylor in his original description)Taylor in his original description)

10Millar & Freshwater 2005Millar & Freshwater 2005 10Millar & Freshwater 2005Millar & Freshwater 2005


So according to Millar & Freshwater (2005), So according to Millar & Freshwater (2005), G. isabelaeG. isabelae is a widely is a widely distributed, turfy distributed, turfy GelidiumGelidium species that may be found on both sides of the species that may be found on both sides of the Indian and Pacific Oceans.Indian and Pacific Oceans.

End of story, right?End of story, right?

Not quite…Not quite…

Playa Brasilito, Costa Rica 2000Playa Brasilito, Costa Rica 2000© DW Freshwater© DW Freshwater

Playa Guiones, Costa Rica 2000Playa Guiones, Costa Rica 2000© DW Freshwater© DW Freshwater

11

A number of A number of GelidiumGelidium spp. had spp. had been collected from Costa Rica been collected from Costa Rica during collection trips in 1999 and during collection trips in 1999 and 20002000 11

DT Thomas & RA York, Costa Rica 2000DT Thomas & RA York, Costa Rica 2000© DW Freshwater© DW Freshwater

20002000


Species BSpecies A

Gelidium floridanum FloridaGelidium floridanum Costa Rica

Gelidium allanii New ZealandGelidium pacificum TaiwanGelidium robustum California

Gelidium serrulatum VenezuelaAnalyses of Analyses of rbcrbcLL sequences from these sequences from these

Species C

Gelidium pulchellum SpainGelidium declerckii South AfricaGelidium corneum Spain

Gelidium micropterum South AfricaGelidium vittatum NamibiaGelidium isabelae Australia

Gelidium pristoides SouthAfricaGelidium pusillum NorwayGelidium pusillum France

samples showed that they:samples showed that they:

••Represented seven speciesRepresented seven species

••All wereAll were GelidiumGelidium Species DSpecies E

Species F

Gelidium pusillum France

Gelidium pluma HawaiiGelidium rex Chile

Gelidum japonicum TaiwanGelidium vagum California

Gelidium capense South Africa

••All were All were GelidiumGelidium

••Increases the number of Cost Rican Increases the number of Cost Rican

GelidiumGelidium species by nearly 350%species by nearly 350%

Species G

Gelidium capense South AfricaGelidium coulteri California

Gelidium crinale North CarolinaGelidium bernabei Australia

Capreolia implexa AustraliaGelidium hommersandii AustraliaGelidium caulacantheum New Zealand

Gelidium pusillum v pacificum Hawaii

Gelidium divaricatum JAPANPtilophora scalarimosa Philippines

Some identified but morphological analysis Some identified but morphological analysis

needed for mostneeded for most

12

Ptilophora scalarimosa PhilippinesPtilophora subcostata JapanPtilophora diversifolia South Africa

Pterocladiella capillacea ItalyPterocladiella caerulescens Hawaii

Pterocladiella bartlettii Costa RicaPterocladia lucida New Zealand

Parviphycus tenuissimus Canary IsGelidiella acerosa Hawaii

Gelidiella acerosa Costa Rica0 01 b tit ti / it

needed for mostneeded for most

120.01 substitutions/site


Amanda GruszAmanda Grusz“Phylogenetic Assessment of Pacific Costa Rican “Phylogenetic Assessment of Pacific Costa Rican GelidiumGelidium

(Gelidiales, Rhodophyta) using Molecular and (Gelidiales, Rhodophyta) using Molecular and Morphological Analyses”Morphological Analyses”

Amanda Grusz, CMS, © DW FreshwaterAmanda Grusz, CMS, © DW Freshwater

Species B

1313


Medullary cells

Obtuse fertile branch tip with distinct apical cell

Obtuse to acute sterile branch tip

Inter-medullary

Terete

yspaces

14

stoloniferous branch

HoldfastCortex

Rhizines

14All images, Grusz & Freshwater,unpublishedAll images, Grusz & Freshwater,unpublished


Gelidium isabelaePacific Costa Rica

Grusz & Freshwater,unpublishedGrusz & Freshwater,unpublished

Gelidium isabelaesensu Millar & Freshwater

Which is the real Which is the real Gelidium isabelae?Gelidium isabelae?

15Millar & Freshwater 2005Millar & Freshwater 2005

15


Solution: Generate partial Solution: Generate partial rbcrbcL sequence from the L sequence from the Gelidium Gelidium isabelae isabelae type specimentype specimen

Type MethodType Method ‐‐When a new seaweed gets a name, a single When a new seaweed gets a name, a single specimen is chosen as the name holder (the type specimen) and specimen is chosen as the name holder (the type specimen) and

l f d d h i i l hl f d d h i i l hconsequently for every named seaweed there is a single chosen consequently for every named seaweed there is a single chosen reference example of that organism in a herbarium somewhere. reference example of that organism in a herbarium somewhere. The application of that name is based on that specimen.The application of that name is based on that specimen.

The ultimate reference sequence (or other molecular marker) for The ultimate reference sequence (or other molecular marker) for any species would be generated from the type specimen.any species would be generated from the type specimen.

1616


Sequencing of Seaweed Types:Sequencing of Seaweed Types:••A partial A partial rbcrbcL sequence from the lectotype of L sequence from the lectotype of Gelidium crinale Gelidium crinale was generated in 1999 and used to establish was generated in 1999 and used to establish that this species is distinct from that this species is distinct from G. pusillumG. pusillum and is distributed widely (Thomas 2000, UNCW Master’s Thesis).and is distributed widely (Thomas 2000, UNCW Master’s Thesis).H h l (2001H h l (2001 J Ph lJ Ph l 37 1091) bli h d hi h d d d h i hi h37 1091) bli h d hi h d d d h i hi h••Hughey et al. (2001, Hughey et al. (2001, J. Phycol.J. Phycol. 37:1091) published on this method and made numerous changes within the 37:1091) published on this method and made numerous changes within the Gigartinaceae based on the sequencing of type material.Gigartinaceae based on the sequencing of type material.••Rico et al. (2002, Rico et al. (2002, PhycologiaPhycologia 41:463) published the first Gelidiales type specimen sequence.41:463) published the first Gelidiales type specimen sequence.

Paul “Gabo” Gabrielson is currently pursuing the Paul “Gabo” Gabrielson is currently pursuing the sequencing of type material in earnest and is using itsequencing of type material in earnest and is using itsequencing of type material in earnest and is using it sequencing of type material in earnest and is using it to resolve taxonomic problems in the Northeast to resolve taxonomic problems in the Northeast PacificPacific (see Gabrielson, 2008, (see Gabrielson, 2008, Phycol. Res.Phycol. Res. 56:105; 56:105; Gabrielson, 2008, Gabrielson, 2008, PhycologiaPhycologia 47:89, and soon to be published 47:89, and soon to be published papers on Corallinales) papers on Corallinales)

17Paul “Gabo” Gabrielson, CMS Pier, © DW FreshwaterPaul “Gabo” Gabrielson, CMS Pier, © DW Freshwater

p p )p p )

17


Gelidium pacificum TaiwanGelidium robustum California

Gelidium allanii New ZealandGelidium americanum North Carolina

Gelidium isabelae isotype

NJ

Real Gelidium isabelaeGelidium isabelae isotypeGelidium sclerophyllum COSTA RICA

Gelidium floridanum FloridaGelidium serrulatum Venezuela

Gelidium pteridifolium South AfricaGelidium "isabelae" COSTA RICA

Gelidium pulchellum Spain

Gelidium “isabelae”Costa Rica

Real Gelidium isabelae

Gelidium latifolium FranceGelidium declerckii South Africa

Gelidium corneum SpainGelidium "isabelae" E. AUSTRALIAGelidium "isabelae" LORD HOWE IS.Gelidium "isabelae" W. AUSTRALIA

Gelidium "isabelae" SOUTH AFRICA

Gelidium “isabelae”sensu Millar & Freshwater

Gelidium "isabelae" SOUTH AFRICAGelidium microdonticum COSTA RICA

Gelidium micropterum South AfricaGelidium vittatum Namibia

Gelidium pristoides SouthAfricaGelidium pusillum Norway

Gelidium pusillum France

18

0.005 substitutions/site

Not one, but two new species!Not one, but two new species!18

Molecular Assisted Character AnalysisMolecular Assisted Character Analysis

How do we judge the evolutionary and taxonomic How do we judge the evolutionary and taxonomic significance of characters (morphological, significance of characters (morphological, d l l h l l)?d l l h l l)?developmental, physiological)?developmental, physiological)?

Example: Fixing Fritz’s Frustration Example: Fixing Fritz’s Frustration ‐‐ assessment of assessment of p gp gmorphological characters used to identify morphological characters used to identify Polysiphonia Polysiphonia species.species.

Good luck figuring Polysiphonia species

out! How do you know which characters

19

which characters define species?

19Margarita R. Albis Salas

D. “Fritz” Kapraun, 1970s. Photographer unknown, DFK archives.


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 p g p g ymorphological 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!

20DF Kapraun (on right during Chuck Norris phase) with WR Taylor circa. 1970s. Photographer unknown, DFK archives.

Draft of the famous unpublished Western Atlantic Polysiphoniamonograph. DW Freshwater 20


Solution: Objectively assign specimens to species using molecular data Solution: Objectively assign specimens to species using molecular data and then analyse characters to determine which are consistent within and then analyse characters to determine which are consistent within speciesspeciesspecies.species.

rbcrbcL was a great locus for this because the level of expected intraspecific L was a great locus for this because the level of expected intraspecific sequence divergence has been established by McIvor et al. (2001, sequence divergence has been established by McIvor et al. (2001, Mol. Ecol.Mol. Ecol.10:91110:911‐‐919).919).

Brooke Stuercke Brooke Stuercke “Consistency of morphological characters used to delimit “Consistency of morphological characters used to delimit Polysiphonia Polysiphonia

sensu latosensu lato species (Ceramiales, Florideophyceae): analyses of North species (Ceramiales, Florideophyceae): analyses of North

Carolina, USA specimens”Carolina, USA specimens”

21

PhycologiaPhycologia 47:54147:541‐‐559 (2008)559 (2008)

21B. Stuercke, Sodwana Bay RSA 2005B. Stuercke, Sodwana Bay RSA 2005© DW Freshwater© DW Freshwater


rbcrbcL sequences generated from multiple L sequences generated from multiple North Carolina specimensNorth Carolina specimens

S i il it d iti ithiS i il it d iti ithiSequence similarity and position within a Sequence similarity and position within a larger larger Polysiphonia sensu latoPolysiphonia sensu lato phylogeny used phylogeny used to assign specimens to speciesto assign specimens to species

Species specimen tree generated for character Species specimen tree generated for character state mappingstate mapping

Species specimen tree based on phylogenetic

22

p y gtree used for character state mapping(from Stuercke 2006)

22Stuercke & Freshwater (2008)Stuercke & Freshwater (2008)


Analyse morphological characters in each molecularlyAnalyse morphological characters in each molecularly‐‐defiined speciesdefiined species

2323Stuercke & Freshwater (2008)Stuercke & Freshwater (2008)


Poly NC-6Poly NC-10Poly NC-13

4 pericentral cells (0)

5-7 pericentral cells (1)

( )


Linear (0)

Lanceolate (1)

Determine the consistency of characters within speciesDetermine the consistency of characters within species

Poly NC-16Poly NC-23Poly NC-17Poly NC-19

Poly NC-31

Poly NC-22

A

B

> 8 pericentral cells (2) Poly NC-13Poly NC-16Poly NC-23Poly NC-17Poly NC-19Poly NC-31Poly NC-22

A

( )

Linear to lanceolate (0/1)

Equivocal

Lanceolate tofractiflexus (1/2)Linear tofractiflexus (0/2)

No information

Poly NC-2Poly NC-30Poly NC-1Poly NC-3Poly NC-5Poly NC-7Poly NC-14

C

B

C


B

C

Equivocal

Poly NC-15Poly NC-18Poly NC-20Poly NC-29Poly NC-12


DG


P l NC 9

DG

24

Poly NC-9Poly NC-33Poly NC-11Poly NC-4Poly NC-26Poly NC-27Poly NC-28Poly NC-32

HF

E

Poly NC-9Poly NC-33Poly NC-11Poly NC-4Poly NC-26Poly NC-27Poly NC-28Poly NC-32

HF

E

24Poly NC 32 Poly NC 32


11 of 22 analysed morphological characters were taxonomically significant in the 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 study of North Carolina specimens and these were verified in a further study of New Zealand New Zealand PolysiphoniaPolysiphonia speciesspecies

1) Number of pericentral cells2) Rhizoid-pericentral cell connection3) Relationship of lateral branches to trichoblasts4) Presence/absence of trichoblasts4) Presence/absence of trichoblasts5) Number of segments between trichoblasts6) Type of holdfast7) Presence/absence of scar cells8) Scar cell pattern9) Presence/absence of cicatrigenous branching

N. Mamoozadeh

(lateral branches originating from scar cells)10) Development of spermatangial axes11) Arrangement of tetrasporangia

J. Kelly

Stuercke & Freshwater 2008

2525Stuercke & Freshwater 2008 Stuercke & Freshwater 2008

Molecular Assisted Identification Molecular Assisted Identification ‐‐ DNA BarcodingDNA Barcoding

DNA BarcodingDNA Barcoding

“DNA barcoding employs sequence diversity in short, standardized gene“DNA barcoding employs sequence diversity in short, standardized geneDNA barcoding employs sequence diversity in short, standardized gene DNA barcoding employs sequence diversity in short, standardized gene regions to aid speices identification and discovery in large assemblages of regions to aid speices identification and discovery in large assemblages of life.” life.” (Ratnasingham & Hebert, 2007, (Ratnasingham & Hebert, 2007, Mol. Ecol. NotesMol. Ecol. Notes doi:10.1111/j.1471doi:10.1111/j.1471‐‐8286.2006.01678.x)8286.2006.01678.x)

“…sequencing a short, diagnostic segment to discriminate between “…sequencing a short, diagnostic segment to discriminate between species.” species.” (Robba et al. 2006, (Robba et al. 2006, Amer. J. Bot.Amer. J. Bot. 93(8):110193(8):1101‐‐1108) 1108)

Initial work with animals, used mitochondriaInitial work with animals, used mitochondria‐‐encoded COIencoded COIHebert et al. 2003, Hebert et al. 2003, Proc. R. Soc. Lond. Proc. R. Soc. Lond. B 270:313B 270:313‐‐321 321

2626


Requirements of a “Barcode” sequenceRequirements of a “Barcode” sequence

N d i l i f lifi i d i ( b fN d i l i f lifi i d i ( b fNeed universal primers for amplifications and sequencing (maybe for Need universal primers for amplifications and sequencing (maybe for marine algae this is impossible).marine algae this is impossible).

Intraspecific variation must be less than interspecific variation and itIntraspecific variation must be less than interspecific variation and itIntraspecific variation must be less than interspecific variation and it Intraspecific variation must be less than interspecific variation and it helps if there is an appreciable gap (sometimes animal people have used helps if there is an appreciable gap (sometimes animal people have used means to determine the gap but that isn’t really legitimate means to determine the gap but that isn’t really legitimate ‐‐ there must there must be a maximum intraspecificbe a maximum intraspecific ‐‐minimum interspecific gapminimum interspecific gap [see Meier et al[see Meier et albe a maximum intraspecific be a maximum intraspecific minimum interspecific gap minimum interspecific gap [see Meier et al. [see Meier et al.

2008 2008 Syst. BiolSyst. Biol. 57(5):809]. 57(5):809]))

A comprehensive baseline database of sequences for comparison. This is A comprehensive baseline database of sequences for comparison. This is

27

p q pp q preally no different from having a comprehensive understanding of any really no different from having a comprehensive understanding of any character set, such as morphology, etc.character set, such as morphology, etc.

27


Algal DNA Barcoding: Algal DNA Barcoding: AdvantagesAdvantages & & DisadvantagesDisadvantages

DisadvantageDisadvantage Algal lineages are very different in age so a locus that isAlgal lineages are very different in age so a locus that isDisadvantageDisadvantage ‐‐ Algal lineages are very different in age so a locus that is Algal lineages are very different in age so a locus that is best for one might not be the best for anotherbest for one might not be the best for anotherDisadvantageDisadvantage ‐‐ Barcode sequence may not have proper “signal” to Barcode sequence may not have proper “signal” to accurately determine relationshipsaccurately determine relationships ((but that is probably not the purposebut that is probably not the purpose))accurately determine relationships accurately determine relationships ((but that is probably not the purposebut that is probably not the purpose))

DisadvantageDisadvantage ‐‐ Particular genome may not reflect actual species Particular genome may not reflect actual species relationships relationships e.g. mitichondrial or plastid introgression, within population divergence, e.g. mitichondrial or plastid introgression, within population divergence, and adaptive changes may distort the picture. and adaptive changes may distort the picture. (see Zuccarello, (see Zuccarello, PhycologiaPhycologia 48(4) supplement:152 48(4) supplement:152 and I expect future papers)and I expect future papers)

AdvantageAdvantage ‐‐ All data comparable All data comparable (sometimes difficult or impossible when people (sometimes difficult or impossible when people have used multiple markers)have used multiple markers)AdvantageAdvantage ‐‐ Greater universality of primers Greater universality of primers (eventually there will be a set of (eventually there will be a set of

28

gg y py p ( y( yknown primers to throw at a specimen and I can’t wait!).known primers to throw at a specimen and I can’t wait!).

28


Algal DNA Barcoding Algal DNA Barcoding ‐‐ initial and example studies:initial and example studies:••Saunders 2005 “Applying DNA barcoding to red macroalgae: a preliminary appraisal holds Saunders 2005 “Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promis for future application” promis for future application” Phil. Trans. R. Soc.Phil. Trans. R. Soc. BB 360:1879360:1879‐‐1888. 1888. COI, red algal species COI, red algal species id tifi ti (id tifi ti (M ll Dil /N dil A t iM ll Dil /N dil A t i ))identification (identification (Mazzaella; Dilsea/Neodilsea; AsteromeniaMazzaella; Dilsea/Neodilsea; Asteromenia))••Robba et al. 2006 “Assessing the use of the mitochondrial Robba et al. 2006 “Assessing the use of the mitochondrial cox1cox1marker for use in DNA marker for use in DNA barcoding of red algae (Rhodophyta)” barcoding of red algae (Rhodophyta)” Amer. J. Bot.Amer. J. Bot. 93(8):110193(8):1101‐‐1108. 1108. COI, red algal species COI, red algal species identification (Saunders specimens + identification (Saunders specimens + Porphyra; Corallina; Calliblepharis; Mastocarpus; GracilariaPorphyra; Corallina; Calliblepharis; Mastocarpus; Gracilaria))••Sherwood & Presting 2007 “Universal primers amplify a 23S rDNA plastid marker in eukaryoticSherwood & Presting 2007 “Universal primers amplify a 23S rDNA plastid marker in eukaryotic••Sherwood & Presting 2007 Universal primers amplify a 23S rDNA plastid marker in eukaryotic Sherwood & Presting 2007 Universal primers amplify a 23S rDNA plastid marker in eukaryotic algae and cyanobacteria” algae and cyanobacteria” J. Phycol.J. Phycol. 43:60543:605‐‐608. 608. 23S, multiple algal groups, higher level taxonomic 23S, multiple algal groups, higher level taxonomic designations, maybe species identification? (Rhodophyta, Phaeophyta, Chlorophyta, Euglenophyta?, designations, maybe species identification? (Rhodophyta, Phaeophyta, Chlorophyta, Euglenophyta?, Xanthophyta, Bacillariophyta?)Xanthophyta, Bacillariophyta?)••McDevit & Saunders 2009 “On the utility of DNA barcoding for species differentiation among McDevit & Saunders 2009 “On the utility of DNA barcoding for species differentiation among y g p gy g p gbrown macroalgae (Phaeophyceae) including a novel extraction protocol” brown macroalgae (Phaeophyceae) including a novel extraction protocol” Phycol. Res.Phycol. Res. 57(2):13157(2):131‐‐141. 141. COI, brown algal species identificationCOI, brown algal species identification••Sherwood et al. 2008 “Contrasting intra versus interspecies DNA sequence variation for Sherwood et al. 2008 “Contrasting intra versus interspecies DNA sequence variation for representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding representatives of the Batrachospermales (Rhodophyta): Insights from a DNA barcoding

29

p p ( p y ) g gp p ( p y ) g gapproach” approach” Phycol. Res.Phycol. Res. 56:26956:269‐‐279. 279. COI and 23S, red algal species identification (Batrachospermales)COI and 23S, red algal species identification (Batrachospermales)

29


1)1) Resolution of “biological species” (ones that fit the biological species concept)Resolution of “biological species” (ones that fit the biological species concept)

Prior studies with COI have shown it to be a good barcoding locus in red algae, but 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…two things are yet to be explicitly shown…

1)1) Resolution of “biological species” (ones that fit the biological species concept).Resolution of “biological species” (ones that fit the biological species concept).2)2) Verification of the maximum intraspecific Verification of the maximum intraspecific ‐‐minimum interspecific gapminimum interspecific gap

Requirements of a “Barcode” sequenceRequirements of a “Barcode” sequence

Need universal primers for amplifications and sequencing (maybe for marine algae this is impossible).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 Intraspecific variation must be less than interspecific variation and it helps if there is an appreciable gap. There must be a maximum intraspecific an appreciable gap. There must be a maximum intraspecific ‐‐minimum minimum i t ifii t ifiinterspecific gap.interspecific gap.

A comprehensive baseline database of sequences for comparison. This is really no different from having a comprehensive undersA comprehensive baseline database of sequences for comparison. This is really no different from having a comprehensive understantanding of any ding of any character set, such as morphology, etc.character set, such as morphology, etc.

30This minimum interspecific This minimum interspecific ‐‐maximum intraspecific variation needs to maximum intraspecific variation needs to be assessed based on known sister species.be assessed based on known sister species. 30


Verification of the maximum intraspecific Verification of the maximum intraspecific ‐‐minimum interspecific gap minimum interspecific gap with Gelidiales sister species and comparisons with with Gelidiales sister species and comparisons with rbcrbcL.L.

Enrico Tronchin, road to Mozambique 2005 © DW FreshwaterEnrico Tronchin, road to Mozambique 2005 © DW Freshwater

31

Pterocladiella psammophilaPterocladiella psammophila, Sodwana Bay RSA 2005 © DW Freshwater, Sodwana Bay RSA 2005 © DW Freshwater

31Pterocladiella caerulescensPterocladiella caerulescens, Sodwana Bay RSA 2005 © DW Freshwater, Sodwana Bay RSA 2005 © DW Freshwater


Gelidium crinaleGelidium crinaleGelidium crinaleGelidium crinalevs.vs.

Gelidium coulteriGelidium coulteriFreshwater et al. 1995, Freshwater et al. 1995, J. Phycol.J. Phycol. 31:61431:614‐‐630;630;

Millar & Freshwater 2005Millar & Freshwater 2005 Aust Syst BotAust Syst Bot 18:21518:215 263263Millar & Freshwater 2005, Millar & Freshwater 2005, Aust. Syst. Bot.Aust. Syst. Bot. 18:21518:215‐‐263263

COI – rbcL comparison for specimens of G i l (G i ) d G lt i

G. crin. G. coul.G crin (rbcL) 0 00 1 23 2 29x

32

G. crinale (G.crin.) and G.coulteri(G.coul.). Minimum “tra-ter-var” values are shown in red above diagonal

G. crin. (rbcL) 0.00-1.23 2.29x(COI) 0.00-2.62 4.76x

G. coul. (rbcL) 2.82-5.65 0.00-0.83(COI) 12.48-13.41 0.00-0.73 32


Gelidium pristoides PORT EDWARD, RSAGelidium pristoides KIDDS BEACH, RSA

Gelidium pristoides FALSE BAY, RSAD100L98

P100D100L100

Gelidium pristoidesGelidium pristoidesvsvs Gelidium foliaceum PORT EDWARD, RSA

Gelidium foliaceum EAST LONDON, RSA

Gelidium foliaceum BREEZY POINT, RSA

D100P100

D100L94

P99

vs.vs.Gelidium foliaceumGelidium foliaceum

Tronchin et al. 2002, Tronchin et al. 2002, Bot. Mar.Bot. Mar. 45:54845:548‐‐558558

G. prist. G. fol.pG. prist. (rbcL) 0.00-0.49 3.68x

(COI) 0.29-1.31 5.80xG. fol. (rbcL) 1.84-2.10 0.00-0.21

(COI) 7.60-8.61 0.15-0.44

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

3333


Pterocladiella caerulescensPterocladiella caerulescensvsvsvs.vs.

Pterocladiella psammophilaPterocladiella psammophilaTronchin & Freshwater 2007, Tronchin & Freshwater 2007, PhycologiaPhycologia 46:32546:325‐‐348348

P. caer. P. psam.COI – rbcL comparison for specimens P. caer. P. psam.P. caer. (rbcL) 1.18-3.15 <1.00x

(COI) 0.00-5.33 1.52xP. psam. (rbcL) 2.04-3.71 0.00-0.11

(COI) 8.10-10.56 0.00-0.15

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

34

( )above diagonal

34


P. caer. SA P. caer. HI P. caer. CR P. psam.P. caer. SA (rbcL) 0.07-0.42 5.10x 2.81x 4.86x

(COI) 0 44 1 33 3 53x 3 21x 7 05x(COI) 0.44-1.33 3.53x 3.21x 7.05xP. caer. HI (rbcL) 2.14-2.13 0.29 7.41x 7.86x

(COI) 4.69-5.33 0.15 29.47x 59.53xP. caer. CR (rbcL) 1.18-3.15 2.15-3.15 NA* 18.55x

(COI) 4.27-5.33 4.42-4.56 NA* 54.00xP. psam. (rbcL) 2.04-3.36 2.28-3.71 2.04-2.48 0.00-0.11

(COI) 9.37-10.56 8.93-9.32 8.10-8.28 0.00-0.15

COI – rbcL comparison for geographic specimens of P. caerulescens(P. caer.) and P. psammophila (P. psam.). Minimum “tra-ter-var”

l h i d b di l

35

values are shown in red above diagonal

35

molecular in phylogenetics and evolution · •a partial rbcl sequence from the lectotype of...

Documents