University of Wollongong Research Online

Shoalhaven Marine & Freshwater Centre Faculty of Science, Medicine and Health

6-12-2012

A molecular survey of Ulva () in temperate reveals enhanced levels of cosmopolitanism

Lisa Kirkendale University of Wollongong, [email protected]

Gary W. Saunders University of New South Wales

Pia C. Winberg Dr University of Wollongong, [email protected]

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Recommended Citation Kirkendale, Lisa; Saunders, Gary W.; and Winberg, Pia C. Dr, "A molecular survey of Ulva (Chlorophyta) in temperate Australia reveals enhanced levels of cosmopolitanism" (2012). Shoalhaven Marine & Freshwater Centre. 15. https://ro.uow.edu.au/smfc/15

Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] A molecular survey of Ulva (Chlorophyta) in temperate Australia reveals enhanced levels of cosmopolitanism

Abstract The green algal genus Ulva includes a speciose group of marine macroalgae inhabiting shallow seas worldwide. Although algal blooms in Asia highlight the opportunistic nature of several “nuisance” species, recent research clearly reveals important positive benefits of Ulva. Applied research requires accurate, reliable, and rapid identification, however, identification of Ulva spp. has met with con-siderable difficulty. Consequently, many have turned to molecular markers to aid in . Previous studies of plants and algae have relied heavily on ITS and rbcL. Recently, tufA has been presented as a suitable barcoding gene to facilitate species-level identification of green macroalgae and it is used here to explore the diversity of Ulva spp. in temperate Australia. Ninety Ulva specimens collected from 38 sites across five states were sequenced for this gene region with exemplars from each genetic group also sequenced for rbcL to test for congruence. Collections of Australian Ulva spp. were compared to samples from Asia and North America and exhibited trends consistent with recent studies in terms of species relationships. Results support an overwhelmingly cosmopolitan flora in temperate Australia that contrasts with other Australasian surveys of Ulva that report a greater number of endemics and new species. Four new records, as well as numerous range extensions for taxa already known from the country, are documented. Evidence for three nonindigenous Ulva species in temperate Australia is discussed.

Keywords Macroalgae;DNA barcoding;NIS ;tufA ;rbcL

Publication Details Kirkendale, L., Saunders, G. W. & Winberg, P. (2013). A molecular survey of Ulva (Chlorophyta) in temperate Australia reveals enhanced levels of cosmopolitanism. European Journal of Phycology, 49 (1), 69-81.

This journal article is available at Research Online: https://ro.uow.edu.au/smfc/15 © 2012 Phycological Society of America © 2012Phycological ofAmerica Society 10.1111/jpy.12016 version andtheVersi this between differences through the copyediting, typesetting, pagination forpublication accepted This articlehasbeen Centre for Environmental and Molecular Algal Resear Gary W. Saunders University ofWollongong, Shoalhaven Marine andFreshwater Centre, NewSouth Wales, 2541 Australia cosmopolitanism A molecular survey of Article type : Regular Article 04-Sep-2012 : Date Accepted :01-Mar-2012 Date Received Lisa Kirkendale Accepted Running title: MOLEC University ofWollongong, Shoalhaven Marine andFreshwater Centre, NewSouth Wales, 2541 Australia and PiaWinberg Brunswick, Fredericton, Article 2 1

ULAR SURVEY OF ULAR SURVEY Ulva NBE3B5A3Canada (Chlorophyta) in temperate Austra ULVA and undergone full peer review buthasnotbeen review undergone fullpeer and on of Record. Please cite of Record. Please on IN AUSTRALIA and proofreading process, which may lead to ch, Department of Biolo lia reveals enhanced levels of

this article this as doi: gy, University of New New of gy, University (x7522) Abstract the diversity of barcoding gene to facilitate species-level identification of green macroalgae and it is used here to explore plants and algae have relied heavily onITS and difficulty. Consequently, many have turned tomolecular markers to aid in taxonomy. Previous studies of accurate, reliable and rapid identification, however, identification of species, recent research clearly reveals important positive benefits of worldwide. Although algal blooms inAsia highlight genus algal green The © 2012 America Society of Phycological 2 like, Shimodaira-Hasegawa-like; GTR, general-time reversible; BOLD, Barcode of Life Database; NIS, Evidencenon-indigenous forthree records Australasian surveys of Results support an overwhelmingly cosmopolitan flora in temperate Australia that contrasts with other and North America and exhibited trends consistent with recent studies in terms of species relationships. for five states were sequenced for this gene region with exemplars from eachgenetic group also sequenced elongation factor Accepted Article Author forcorrespondence:L. Kirkendale(email: [email protected],61 2 phone: rbc L to test for congruence. Collections of Australian , as well as numerous range extensions for taxa already known from thecountry, are documented. Keywords: macroalgae, DNA barcoding, NIS, DNAbarcoding, macroalgae, Keywords: Abbreviations: , fax:612 4429 1521 Ulva tufA spp. in temperate Australia. Ninety ; ML, maximum likelihood; LRT (aLRT), (approximate) likelihood ratio test; SH- Ulva rbc Ulva L, large subunit of the RUBSICOgene; ITS, internal transcribed spacer; includes aspeciose group of marine macroalgae inhabiting shallow seas that report agreater number of endemics andnew species. Four new Ulva species in temperate Australia is discussed.

rbc L. Recently, the opportunistic nature of several ‘nuisance’ tufA Ulva Ulva , rbc specimens collected from 38sites across spp. were compared tosamples from Asia L tufA has been presented as asuitable Ulva Ulva spp. has met with considerable . Applied research requires 4429 1522 tufA , © 2012 America Society of Phycological coastal zone (Kozhenkova primary trophic producers (Horn 1983) and bio-indicator species in the emerging field ofchemical ecology (Van Alstyne spp. are regarded as key candidates for bioremediation applications (El-Sikaily review of recent publications also points to the diverse range ofpositive applications. For example, negative press owing toblooms inAsia (Hiraoka and locally dominant members of rocky intertidal and subtidal habitats worldwide. They have gained Marine species of the green algal genus Introduction: non-indigenous species. Michael 2009, Paulert related problem withsuch an approach is highlighted by emer properties ofmacroalgae at the generic level, or without robust taxonomic identification. The potential Acceptedespecially for difficult taxa such as those in the genus Taxonomy isfundamental to all biological research, however, it is challenging andtime-consuming work, individuals of a confirmed strain. chemical constituents, physiological requirements and remediation potential directly withtested understanding of ecological processes. Abest practice approach should strive tomarry traits such as implications for both the applied use of environmental gradients and differ in controlled labo present) amongmorphologically similar or closely related species, mayvary seasonally oracross Article Ulva species exhibit unique ecophysiological and chemical characteristics (Eswaran

et al et

et al . 2010,Winberg . 2006). The bulk ofthese studies address chemical analysis or functional Ulva Ulva Ulva

et al. 2011). Key attributes may notbe similar (or even

spp. for specific metabolites and in achieving an Linnaeus in the division Chlorophyta areoften conspicuous

et al ratorysettings versus inthewild. Thispresents . Ulva ging research that has revealed that even closely 2004, Leliaert 2004, s that responds that toeutrophication pollution the in . Previously,

et al et .

2007, Alstyne 2008), as important et al. 2009, Kong Enteromorpha

et al. 2007),keystoneet al. as (monostromatic

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. et al. 2002, 2011), but Ulva

© 2012 America Society of Phycological sampled Hawaiian sequences provisionally identified as not recover recovered the name in Scopus) and across Australia (235 of 682 studies). However, thevalidity ofsuch abroad application of approximately 30% of the peer-reviewed literature on levels. For example, the reportedly cosmopolitan taxon variation at higher taxonomic levels also confound straightforward identification at lower taxonomic thesebetweenstates two (Tanet al.1999). cha The longer recognized as reliable synapomorphies, apotential morphogenetic switch may explain shifts characters, however, molecular evidence has not supported this distinction (Hayden etal. 2003). No tubes) and Genbankfor individuals identifiedas of genus environmental pollution (El-Sikaily etal. 2007), taxonomic clarification of this and other species in the success and double bands indicative ofdivergent copies for the ITS region (Saunders & Kucera 2010) 2009,al. etal.2010). Kraft mark However,these this trend in marker choice (Woolcott & King 1999,Shimada etal. 2003, Shimada etal. 2008, Heesch et & Waaland 2004, Loughnane et al. 2008, O’Kelly etal. marker among flowering plants (Chase et al. 2005). For the purpose of identifying marine macroalgae, the plastid (Savolainen et al. 2005) with ITS, Molecular tools have proven useful at assisting in the identification process across thediversity of life

AcceptedU. lactuca Article Ulva rbc U. lactuca U. lactuca Ulva L and the nuclear ITS region have proven useful (Coat et al. 1998, Malta et al. 1999, Hayden isimportant. U. lactuca in applied research (Hassan et al. 2011), not least of which are trigger indicators for (distromatic blades) were recognized as separate genera based on morphological is questioned by several recent taxonomic studies. Heesch et al. (2009) only infrequently inNewZealand from disturbed habitats, while Kraft et al. (2010) did from extensive survey work insouthern Australia. O’Kelly etal.’s (2010) newly rbc U. lactuca L and mat

K markers commonly usedtocharacterize relationships from Europe.Considering some ofthe key applications ers are commonly hindered by pooramplification llenges ofecophenotypic or developmentally mediated Ulva U. lactuca 2010) with studies from Australasia reflecting U. lactuca internationally (1499 of 4854 studies searched L. is included as aspecies in donot cluster with sequence data in © 2012 America Society of Phycological al. 2009). Observations of highly cosmopolitan assemblages are often the first line of evidence for non- spp. in other remote Pacific locales aswell asHawa that flora. This shifting balance between cosmopolitanism andendemism hasbeen demonstrated for components of temperate Australian macroalgal communiti notion for (Lopez et al. 2007), as opposed to endemics. However, Kraft et al. (2010) recently challenged the latter Zealand (Heesch et al. 2009). Near identical for diversity genetic of levels low while Acceptedto the notion that regional floras may beincreasingly dominated by widespread or circumglobal (Kong etal. 2011) and high fouling potential (Schaffelke etal. 2006) are all factors that have contributed and distribution of this genus marine macroalgal surveys, have resulted in a relati Difficulties with taxonomic identifications of the speciose genus combination with marker forspecies identification among Ulvaceae (Saunders & Kucera 2010) and is used here in (Kucera & Saunders 2008),Article macroalgae. The plastid marker Armstrong & Ball2005) for red, Rhodophyta (Saunders 2005,Saunders 2009), and brown,Phaeophyceae cryptic species (Saunders & Kucera 2010) and uncover invasive species (Sherwood et al. 2008, Saunders 2009), complete rapid floral surveys atmultiple spatial scales (Sherwood etal. 2008), recognize example, has been used to facilitate difficult taxonomic identification (Saunders 2005, McDevit & (<800bp)short segments of protein-encoding organellar DNA.TheCOI-5P barcode marker, DNA for reliable species-level identifications. Targeted gene regions, often referred to as barcodes, are typically confusion. Recent strides in marker development different species names, as well as distinct sequences often sharing thesame name, only add to the Ulva Ulva spp. providing evidence that, although widespread species are certainly common rbc L data toexplore the diversity of

in Australasia. Rapid growth (Duan et al. 2011), good dispersal capabilities rbc L have hampered species-level resolution amongst New

have, however, provided newoptions for rapid, and vely weak state in our knowledge ofboth the diversity ii (O’Kelly et al. 2010) and New Zealand (Heesch et rbc Ulva L sequences in GenBank bearing anumber of es, anAustralian signature is also apparent in spp. inAustralia. tufA has been tested and proposed as a viable Ulva , coupled with apaucity of recent Ulva Ulva spp.

© 2012 America Society of Phycological track record of taxa (Heesch et al. 2009). Given thehigh priority for biosecurity in Australia, coupled with the invasive indigenous species (NIS), with taxonomic andbiogeo many genetic species groups occur for the genus taxonomy, thefocus of this study was threefold: 1) utilize the Considering theecological, applied and biosecurity contexts and aneed for certainty regarding sample by sample details of voucher locality aswell as other information: www.boldsystems.org) are extractions and photographs detailing keymorphological characters (refer to BOLD project GULVA for Brunswick, Canada. Voucher material of each sample, specifically algal presses, tissue samples, genomic Wales, Australia and the Centre for Environmental and Molecular Algal Research (CEMAR) in New the Shoalhaven Marine and Freshwater Centre (SMF Appendix 1,see supporting information; Fig. 1), aspa Ulva Collection information Materials and Methods: components of the temperate Australian flora. and conduct apreliminary assessment of the potential for cosmopolitan species to be non-indigenous results); and 3) examine the biogeographic context and potential implications of the taxonomic findings in GenBank for data for arepresentative of each of the previous genetic groups for comparison to the wealth of Acceptedhoused attheir respectiveinstitutions. Article samples were collected across five temperate Australian states as well as South Korea (Table 1: Ulva Ulva , such research is critical for identification and management of NIS species. spp. (both to apply species names consistently, and as a secondary check of the

Ulva C) at the University of Wollongong inNewSouth graphic research underpinning recognition of such in the temperate in Australian flora rt of collaborative work between two institutions: tufA barcoding marker to determine how ; 2) generate generate 2) Ulva rbc L data

rbc tufA L

© 2012 America Society of Phycological Acceptedthen visualized. The Australian GenomeFacility Research Westmeade, (AGRF, Sydney) using PCRPurificationKit(Qiagen) QIAquick successful AtSMFC during rounds. subsequent bands double products indicatedby 2010). ThePCRproductwaselectrophoresed, at72°Cfor1min,followed by el final extension min denaturationat94°C;further sterile(sH water with of50µL brought uptoatotalvolume TAQ, primer,0.2µL of 10mMsolutioneach 2010). PCRcocktailsincluded2µL to amplify the used were GGNGCNGCNCAAATGGAYGG-3’ 5’-CCTTCNCGAAT The primersGtufAR 1)followedpub designation inAppendix GWS Articleagaros gel (1-2% via verification was confirmed 250-500 an extra kit (ZR-96Zymo Research) following the manufacturer’srecommendations, withthe addition of extracti weighed for 100-150 mg oftissuewas and scrapedepiphytes toremove ifpresent. werecut Thalli usinga then sterile razor blade and sample ID designation LAK in ethanol-prese at SMFC (fresh, Samples processed Genomic extraction andPCR 2 O). Reactions were run for 38 cycles withtheO). Reactions following for38 were parameters: an run initial 4 μ L of lysis solution during tissue maceration. The presence of genomicDNA L of lysis solutionduring Thepresence of tissuemaceration. Appendix 1) were rinsed with autoclaved seawater, patted dry patted rinsed withautoclavedseawater, 1)were Appendix denaturation at 94°C for 1 min, annealing at 45°Cfor 30sand for1min,annealing at94°C denaturation were subjected toincreased subjected were of template, 5 µL of 10X buffer, 5 µL 10mM dNTPs, 2 µL 10mM dNTPs,2µL 5µL of10Xbuffer, µL 5 of template,

MGCRAAWCGC-3’ andtufGF45’- MGCRAAWCGC-3’ on. DNA was extracted using aPlant/SeedDNA extracted on. DNAwas stained, and photodocumented. MultiplePCR stained, andphotodocumented. following manufacturer’s recommendations and recommendations manufacturer’s following lished protocols (Saunders & Kucera 2010). & 2010). Kucera lished protocols(Saunders PCR products were cleaned for cycle sequencing forcycle cleaned productswere PCR e). Samples processed at CEMAR (sample IDat CEMAR (sample Samplesprocessed e). ongation at 72°C for 7 min (Saunders & Kucera, (Saunders ongation at72°Cfor7min rved, frozen or preserved withRNA-later, orpreserved rved, frozen annealing temperatures (50°C) annealing temperatures tufA region (Saunders & Kucera, (Saunders region © 2012 America Society of Phycological Both previously published (Saunders & Kucera 2010) and newly generated Sequence identity 2006). 2006) and initial annotation andediting ofML trees was carried out in TreeDyn v.198.3 (Chevenet et al. assessed using thebootstrap (100) or approximate LRT (aLRT) (SH-like) test (Anisimova & Gascuel in PhyML v 3.0aLRT (Guindon & Gascuel 2003) with likelihood (ML) analyses using the phylogeny.fr online software (Dereeper accession numbers included directly inFig. 3).Both gene alignments were subjected to maximum similarlyandof Saundersadded tothose Kucera representativespecies ofthegenera were added to previously published data (Saunders & Kucera 2010;Appendix 1) and included The Alignment and Molecular analysis 2010). at generated 1)were (Appendix (Saunde aspublished generated data were auto AB3730xl an using sequences generated Accepted(2010) to achieve consistent use of nomenclature (Table 1).Australian collections with blasted in GenBank andsubjected to comparative phylogenetic analysis of Article tufA and rbc L data were easy to align dueto anabsence of indels. Newly generated CEMAR following published protocols (Saunders & Kucera CEMARfollowingKucera & publishedprotocols(Saunders Ulva , Umbraulva

rs & Kucera, 2010). All newly generated Allnewly 2010). & Kucera, rs (2010) with additional data fromGenBank (see matic sequencer. At matic sequencer. and a GTR aGTR + Imodel. +G Tree robustness was Ulvaria . Newly generated CEMAR (Appendix 1) (Appendix CEMAR rbc et al. rbc L data from Kraft et al. L data (Appendix 1) were , 2008) as implemented, 2008)as rbc tufA tufA L data were L data rbc sequences sequences L-3P data tufA

© 2012 America Society of Phycological AcceptedAustralia). The pool of genetic data that exhibited the lower diversity was inferred torepresent evidence included one pool from the test area (within Australia) and one pool globally (within and outside genetic distance data (Kimura 2-parameter) were generated and compared for the recently colonized anarea compared with a population that hadnot recently colonized. pools Two of or harbour. For the second criterion, genetic diversity wasexpected to be lower in apopulation that had suggested if aspecies was found in adisturbed site on artificial substrate in close proximity to a large port Australia (www.boldsystems.org; 2). Table For the data were scored based on collection information for eleven species of with the genetic distance among sequences from overseas. These criteria were followed in this study and where an entity wasobserved; and 2) the genetic distance among sequences from atest area compared NIS (Heeschet al. 2009). Two key criteria for assessmen A number ofcriteria specific to NIS criteria Article(Microsoft Corporation) to facilitate intra- andinterspecific comparisons and estimate barcode gaps. Maddison & Maddison, 2010). Distances were exported to Microsoft Excel for Mac 2008version 12.1.0 Uncorrected p-distances were generated from the Genetic distances species basis). (Saunders & Kucera 2010) were assigned to that species (refer to Table 1 for rationale on species by that fell into well-supported monophyletic groups with previously published North American taxa Ulva have been presented recently to assist in recognition of potential potential of recognition in assist to recently presented been have

tufA first criterion, evidence for an NIS species was alignment using Mesquite v 2.74(build 550; t are: 1) number of collections and type of sites Ulva that had distribution in tufA gene region. This © 2012 America Society of Phycological orinsularAustra focused on41sitesatmainland The tufA id illustrated taxonomic Final 1). GenBank (Table genetic matching our theaim of with analysis toML 744bpthatwasalso subjected alignment and of108taxa determined of45taxa analysis for final retained were Identical co our toassign analyses was subjectedtoML the genus of from NorthAmerica and tennew One hundred Molecular analyses of genetic distance data for each species tovisualize trends. As available, of a recent colonization, suggestive of a NIS. Neighbor-joining trees were constructed fromglobal pools Accepted Article Results: website (www.boldsystems.org).two criteria These thwere diversity bothwithin Australia andglobally. Genetic analyses were carried out through theBOLD results tufA gene region resolved 19 lineages from the genus 19lineages fromthe resolved generegion tufA Umbraulva rbc sequences were culled and representative andrepresentative culled were sequences L sequences (Appendix 1) were similarly 1)were L (Appendix sequences groups to names currently recorded in Kraft et al. (2010) aswell (2010) etal. inKraft recorded tonamescurrently groups (Appendix 1).Thefulldatasetof (Appendix entity and demonstrated Ulva tufA rbc , Lanalysesconducted ona were smaller that with83taxa dataset Ulvaria sequences were added tosixty- added were sequences

obscura

(Fig. 2 and supporting Eightand (Fig. information). newly 2 llections togeneticllections sp as an outgroup, as well as representatives from representatives anoutgroup, aswell as corroborationwith lia (Table 1; Fig. 1). These lineages were were lineages 1).These Fig. 1; (Table lia en considered together to assess NIS status. 179 specimens (Appendix 1)and807bp 179 specimens(Appendix sequences highlighting genetic highlightingsequences diversity added to published data for a final topublisheddatafor added Ulva nine previously published records publishedrecords nine previously withbiogeographic sampling tufA rbc ecies groups (Table 1). groups (Table1). ecies L data were also queried for (Fig. 3). © 2012 America Society of Phycological in Island(both NSW) Lord Howe and from Bendalong geographic distributionto (Fig. mostdiversetaxon 2B). of next dive thehighest levelofintraspecific exhibited intheUII subgroup. data geneticdistance characterized again divergence thesamelevelsofw exhibited UI. in distance interspecific highest the exhibited fasciata and alsoha (0.78%) within speciesdivergence of 2B).theUIgroup Within (Fig. potentially newtaxa generate were Genetic distances 2A). Umbraulva outgroup Australian representatives ofeither + linza, U.stenophylla gr representatives 2A). NoAustralian U. gigantea U. procera groups, UI(Fig.and UIIdistributed acrosstwo 2A U. compressa

AcceptedU. compressa Article , U. Ulvaria obscura Ulvaria , lineages, sp.5GWS and sp.5GWS U. prolifera, U. linza U. prolifera, sister to sisterto . or U. fasciata Umbraulva U U. intestinalis . sp. 5GWS. UII tw . sp.5GWS. included was distantly related all sampled to U. intestinalis, U. sp.10GWS,showednogenetic vari ithin species genetic divergence (0-0.13%). Little withinspecies genetic ithin species divergence(0-0.13%). hadthehighest ( size sample , d for 15 lineages that had Australian representation or were orwere hadAustralianrepresentation that d for15lineages sp.1AUSand U. ohnoi , U. stenophylla, U. lobata of samples North American ouped with and2)

but possessed almost eightthe times diversity. Samples , unidentified or d the largest sample size. Three lineages, lineages, Three largest samplesize. d the U. lobata rsity observed (1.03%), more thanfourtimesthat more (1.03%), observed rsity Umbraulva japonica Umbraulva U. lactuca U. Ulva flexuosa, U. laetevirens Ulva flexuosa,U.laetevirens

U. torta,flexuosa ). UI splitintotwo was major subgroups:1) were the most divergent populationstested werethemostdivergent showed comparable levels ofsampleand levels showedcomparable o well-supported lineages: 1) o well-supportedlineages: Ulva U. , U. lactuca sp. 5GWS and sp.5GWS were identifiedin thisstudy. The , U. procera U. procera 41) and a huge geographic spread, spread, geographic 41) andahuge Ulva ability, however the latter taxon taxon thelatter ability, however as well asthetwoincluded aswell and that fell sister to and U. australis exhibited the most Ulva compressa U. laetevirens U. californica U. prolifera, U. and U. ohnoi U. howensis . No Ulva (Fig. (Fig. Ulva and2)

(Fig. all

© 2012 America Society of Phycological Accepted limitedinmost was Inference sp subgroup; 2). Table introduced toAustralia fromoverseas: establish potential NIS inAustralia. status Thre ofAustralian species Eleven NIS assessment etal.2010). (Kraft stenophylloides example, For latter. the with compared region gene former for the between arose discrepancies agreem 2).Although Fig. 3 withFig. (compare Figs.inconsistencies (Table1; Articlepermitted identification of The purposeofincluding criterion exhibiting wastaxa met withall less intraspecific diversity than(Fig. interspecific 2B). Umbraulva U.ohnoi U. flexuosa,laetevirens, divergent australis with the relatively high ofnearest level ne (1/4 or25%)tothatof ofthediversity afraction yet exhibited only anditsclosestsister. Umbraulva sp.1AUSshowedhigh ofinters levels , U. tanneri sp. 1AUS exhibited thesame1AUS exhibited levelof sp. rbc Ulva and rbc Ulva L data from Genbank wastwofold: L Genbank from data Ulva 2 and 3); and 2) itservedto and 3);2) 2 species, which in some instances pointed to taxonomic some instancespointedtotaxonomic whichin species, U. brisbanensis L and were scored for two published criteria (Heesch et al. 2009)to et two publishedcriteria(Heesch for scored were

howensis and tufA U. procera

U.intestinalis ighbor interspecific distance observed between between observed interspecificdistance ighbor trees as a result of the inclusion ofadditionalspecies aresultoftheinclusion treesas exhibited no within species divergence. The nowithinspeciesdivergence. exhibited ent between the two topologies was high, slight washigh, between thetwotopologies ent e species are considered likely to have been been have to likely considered are e species have been sequenced for havebeensequenced pecific genetic distances. The barcode gap Thebarcode distances. genetic pecific , U. torta ecies due to low sample sizeand/or missingecies duetolowsample . Like corroborate lineages resolved by by resolved lineages corroborate (UI subgroup) and (UI subgroup)and within species divergence (0.13%) as (0.13%) divergence withinspecies Ulva 1)itprovidedscaffolding that U. rotundata sp. 10GWS and sp.10GWS U. compressa rbc L, butnot U. australis , U. . Thiscontrasts U. australis tufA U. (UII (UII tufA ,

© 2012 America Society of Phycological Accepted 034)identifiedas code EU933957 Voucher discussed by Couceiro (Tab former the to going priority nomenclatural 100% sequencesimilarity( morphological hold.S indications clathratioides U. torta 2010)and & Kucera (Saunders Canadian waters endemic taxonomic reconsideratio al. (2010)i Aligning withKraftet ourresults onco able toassign based names Articlenew, species.All ofthe remaining Australian collections fell into11groups forwhich were we tufA remaining lineages, 1).Twoofthe Appendix U. stenophylla viz., Australian representatives, The Distribution Diversity, Taxonomyand Discussion: data. or tufA rbc . It geneticgroup is . thatthis isclear gene region delineated nineteen lineages of lineages delineatednineteen generegion U. clathratioides L thattheyGenbank indicating in matches with the reportedly globally distributed globally withthereportedly distributed (based on

et al. (2011) with the two sequences (EU933954 Voucher code 031and code Voucher (EU933954 al. (2011)withthetwosequences et n of two species (Table 1, Appendi 1, n oftwospecies(Table tufA was genetically assignable to aspeci to assignable wasgenetically rbc sequences from non-Australianco sequencesfrom L) to the newly described L) described tothenewly Ulva gigantea,U.lactuca,linza,lobata,prolifera mparisons to data in Genbank. inGenbank. mparisons todata imilarly, as plantsidentified

U. australis not endemic to Australia and synonymy of toAustraliaandsynonymy not endemic ndicates general congruence but alsotheneedfor but congruence general ndicates Ulva Ulva le 1). We didnotfindsequencemisidentification 1). We le loosely identified on morphological groundsas identified onmorphological loosely sp. 5GWS and sp. 5GWS Ulva represent previously unsampled, possibly previously represent U. torta by Kraft et al. (2010). byet al.(2010). Kraft U. proliferoides (Fig. 2). Of these, six didnot include six (Fig. Ofthese, 2). x 1). The newly described Australian Australian described 1).Thenewly x will be necessary ifthe willbenecessary U. howensis es that was widely distributedin es thatwas llections available in GenBank, inGenBank, available llections Ulva sp. 10GWS, didnothave sp.10GWS, (Kraft et al. 2010) et (Kraft in ourstudy had U. and

© 2012 America Society of Phycological Ulva tufA U. torta and tentatively (Table 1) assigned to the species: 1) (Appendix2, seesupporting information). nationalAt the level,genetic fournew identifiedwere groups Several new records for on effortto inventory floras (Heeschetet al.2009,Kraft Recent molecular surveys of Shifting the balance from endemism to cosmopolitanism here simply toestablish consistency in the literature for Australian records of this genus). matching that of Kraft et al. (2010), but in conflict with that ofO’Kelly etal. (2010), the former selected critical to acknowledge that the identity of reinforces the notion that reports of absence of published records from Victoria with the synonymy of was collected not only from mainland NSW, but al uncovered among Australian states (Appendi only from Australia (Lord Howe Island, mainland NSW and WA). Sixteen range extensions were genus from outside Australia (Table 1; Appendix 2). There is little evidence in this study ofendemicity for the Australia, 10/11 taxa (10/11 or91%) showed high sequence similarity torepresentative species sampled

AcceptedUlva Article data inGenbank and may represent new species, fornow they are certainly newrecords toAustralia. sp. 10GWSwas also sampled from eastern Canada, while Ulva . Two additional . Two taxa, spp. that were collected and sequenced for Ulva lactuca in the Australian flora excepting from oursurvey, anobservation consistent with Kraft et al. (2010), and one that Ulva Ulva Ulva spp. in Australia were uncovered at the national, state and local levels U. sp.10GWS and in remote regions of the Pacific Ocean have been conducted in an U. lactuca U. lactuca

U. x 2). The Lord Howe IslandHowe (NSW)endemic2). TheLord x inAustralia are based on misidentifications (although it is

howensis Umbraulva so Western Australia (Appendix 1) and includes tufA Ulva californica requires further investigation,usageour here al. 2010, O’Kelly etal. 2010). Focusing exclusively U. proliferoides with representation both within and outside of . Even sp.1AUS, were unique relative to Umbraulva U. howensis ;

2) (discussednote above).Of isthe U.ohnoi; sp.1AUS isthus far known , previously considered 3) U.procera; Ulva howensis rbc and 4) L and

© 2012 America Society of Phycological sampled taxa such as U. intestinalis solely Australian individuals (Fig. 2). Cosmopolitanism characterizes trends in the bulk of Australian taxa falling with included representatives from outsideAustralia and not in discrete groups of Lord HoweIsland (94% support in phylogenetic analyses; Fig. 2). More pervasive is the trend of signature is evident only in one strongly supported lineage within samples from outside Australia, this potentially is evidence of incipient species. Anemerging Australian signature. If Australian samples exhibit reciprocal monophyly withother Australian samples and not with Geographic structuring within species offers the potential for additional insight into apossible Australian than was previously thought. endemic toLord HoweIsland, has been shown through ourwork tohave awider distribution in Australia U. australis, U. fasciata sampled taxa with minor genetic differentiation (Fig. 3). Indeed, conservative and thus less variable marker than species. This finding isonly strengthened by consideration of the thatnotion thebulkof sampledAustralian taxascreen included al. 2010, O’Kelly et al. 2010). In particular is the remote Pacific that report higher incidences of endemism inthegenus These results contrast with the findings of other recent molecular studies of regional floras from the Acceptedcosmopolitan/widely distributed taxa and 1endemic while Kraft et al overlapping geography betweenthe two studies in temperate Australia. Our study yields 11 Article rbc L sequence data. and even U. torta U.australis, and U. tanneri and U. procera where sample sizes are relatively high, butas well for moderately show very little variation across large geographic distances for

. This intermingling ofgeographic samples supports the tufA contrast with Kraft et al., of interest given the , and still points to multiple instances of widely ed in this study are members ofwidely distributed U. stenophylla, U. prolifera. U. lactuca, rbc U. compressa L genetic data, which is amore . (2010) reported six cosmopolitan Ulva (Heesch et al. 2009, Kraft etet al.2009,Kraft (Heesch from NSW, including U. compressa, U. compressa, © 2012 America Society of Phycological suggest that stenophylloides brisbanensis species, two subspecies and described four new species endemic toAustralia, including: 1) represent gathering evidence of the cosmopolitan nature of growing a to contributing of instead verification, endemicity outlined by Kraft et al synonym of the putative introduced or test range (= Australia), then this was considered an indication ofNIS. global genetic diversity ‘pools’ where possible. If agiven taxon exhibited higher global diversity than in (Cheang et al. 2010). In regards toCriterion 2, test introduced to British Columbia, Canadabut is found in awide range of habitats, including pristine areas and introduced species can occur widely, including atsuch sites. For example, sites pristine inhabit to taxa only the not are species Native hold. necessarily not does reciprocal the NIS, modified site or to areas with frequent vessel traffic is a solid indicator for geographical recognition of with thefollowingclarifi exhibiting high genetic similarity with samples from overseas. These criteria were adhered to in this study given toataxon both found inhighlymodified environments and/or areas with frequent vessel traffic and a number ofcriteria specific to transport, anthropogenic transport (and therefore NIS) orboth. distinguishTo between these alternatives, The presence of anumber of wide-ranging Accepted ArticleIndications of putative NIS? U. howensis U. clathratioides , 2) exhibits close match to U. stenophylloides, (Table 1; Fig. 3). These proposed amendments cast doubt onthe level of cations. In regards to Criterion 1,although restriction of a taxon to ahighly

could be widely distributed Ulva . (2010). If these proposed amendments holdup following additional have beenproposed by Heesch et al. (2009), with an NIS designation 3) U.clathratioides U . sp1CHEAY255871 from Chile,while results from this study Ulva Ulva

spp. in temperate Australia could be the result of natural Australian signature, these records could instead area genetic diversity ‘pools’ were compared with and 4) U.torta Ulva U. proliferoides. spp. and U. proliferoides Sargassum muticum However, However, islikelya U. U. is © 2012 America Society of Phycological (n=1) negated a test area genetic diversity assessment at this time. Status of Zealand (Heesch et al. 2009), it was only given possible open coast areas on natural substrates. Although andTAS NSW) compared to 68 overseas samples and that both Australian samples were collected from observed diversity within Australia is based ononly twosamples (albeit over wide geographic distances, sampling sites in good proximity toport towns. However, the argument against NIS status is that the intestinalis overseas or within Australia, would clarify preliminary patterns indicated here. For example, U. intestinalis Conflicting trends complicated straightforward NIS inference for several species of Australian Australia in comparison to a global diversity pool (Table 2). collected from adiversity ofhabitat types and settings andexhibited zero or less genetic diversity within and Application of these criteria toeleven et al. 2010 for Australian Atlantic seaboards of Canada. Querying additional studies that utilize other molecular markers (e.g. Kraft trends for this species and exhibited nogenetic diversity betweensamples collected from thePacific and suggestive ofintroduction from Australia to rest of the world. Data from Ulva compressa as this species has arestricted distribution to southern Australia and is most likely anAustralian endemic. Acceptedcomparisons (Table 1). others, a solid taxonomic foundation needs to be in place for would improve the preliminary NIS assessments discussed here. However, as evident from this study and Article U. australis, exhibits nointra-Australian diversity, higher global diversity forboth , U. fasciata, U. flexuosa, U. laetevirens was found broadly andexhibited higher diversity within than outside Australia, considered likely NIS introduced to Australia from overseas. These species were Ulva using using rbc Ulva L and ITS), as well as increasing sample sizes for all species, spp. in this study resulted in three taxa,

Ulva californica and NIS status in this study, asthe small sample size U.ohnoi Ulva was identified as a likely NIS toNew in order to facilitate meaningful additional sampling, either from rbc Ulva howensis L were consistent with tufA U.procera, U. torta and rbc was clearcut, U. L and Ulva tufA . For

© 2012 America Society of Phycological The recently identified green algal barcode marker Conclusions species. In contrast with recent work on congruent except for twonew species designations that wereinterpret as likely members ofwidespread work andthen analyzed together with nomenclatural consistency, individual differentiation between tested Australian species and overseas representatives. Inan effort to achieve discriminated between closely related Australian species and indicated low levels of genetic AcceptedAdditional support to GWS wasprovided by theCanada Research Chair Program,as well as Engineering Research Council ofCanada, aswell Network from Genome Canada through theOntario Genomics Institute, the Natural Sciences and McDevit and Dixon.K. This work hasbeen supported by grants from theCanadian Barcode of Life technical contributions from numerous personnel in th deeply indebted to the collectors listed in Appendix 1for samples from Western Australia and for analysis of Government RuralIndustries Research and Development Corporation (RIRDC) forcollection and This research project is collaboration across two research teams andis in part funded by theAustralian Acknowledgments putative NIS inAustralia. Australian Articlefor NIS, analyses based on ecological and genetic data were conducted to ascertain ifany ofthe tested endemism inthegenus Ulva Ulva species were also potential NIS. Three widespread taxa from NewSouth Wales, Victoria, SouthAustralia and Tasmania. In addition, weare Ulva Ulva in temperate Australia. As widespread species are the most likely candidates rbc rbc Ulva L data from Kraft et al. (2010). Identifications were broadly L sequencesgenerated were forspecies delineated byour

spp. in the region, our findings provide little evidence of asother sponsors listed at www.BOLNET.ca. tufA e Saunders laboratory, most notably T. Moore, D. delineated twenty-two lineages of Ulva species were found to be Ulva , reliably tufA

generated from this study (JN GenBank prefix) Fig. 3.MLphylogram of above branches. B. Table of intraspecific and interspecific distances. data. Genbank number reported aswell as total sequences in brackets. Bootstrap (100) support values prefix). Names have been retained on branches if not in agreement with bulk ofidentified taxa. © 2012 America Society of Phycological Accepted ArticleFig. 2.A.MLphylogram ofrepresentative Australian and Appendix 1). Fig. 1.Collection localities forAustralian taxa included in this study (numbers cross reference to Table 1 Foundation. infrastructure support from theCanada Foundation for Innovation, and NewBrunswickInnovation

Ulva rbc Ulva L sequence data. SH-like supportvalues above branches. Data

and from Saunders& Kucera (2010) (HQ GenBank Ulva to highlight genetic diversity of of diversity genetic highlight to tufA sequence © 2012 America Society of Phycological B A 1803: xxii, 1 xxii, 1803: Ulva flexuosa Ulva Ulva Ulva Ulva (Kützing) Bliding Bliding (Kützing) Ulva gigantea Linnaeus 1163 1753: Ulva compressa 1-25 figs 17, 2004: Shimada Ulva ohnoi Harvey 265 1855: Ulva lobata 1163 1753: Ulva linza 5, 1826] fig. 58: 297[pl. 1813: Ulva fasciata Linnaeus 1163 1753: Ulva intestinalis Areschoug 1854: 370 Areschoug 1854: Ulva laetevirens 1163 1753: Ulva lactuca Umbraulva Umbraulva I.K.Lee 230 2001: (Holmes) Bae & japonica Umbraulva 574 1969: C.Bliding (Kützing) P.Gayral ex obscura Ulvaria 290 2003: J.R.Waaland & M.J.Stanhope P.C.Silva, Maggs, Blomster, (K.Ahlner) Hayden, Ulva procera 611 no. 1899: & Setchell in F.S. Collins,Holden Ulva californica DCCLXIII: fig. 1 fig. DCCLXIII: 7, pl. 1778: O.F.Müller Ulva prolifera Trevisan 1841: 480 1841: Trevisan torta Ulva A-I fig. 37, 319, 2007: 14 Kraft (A.H.S.Lucas) Ulva howensis 29 558 1969 N Distribution Global Rationale 370 Areschoug 1854: Ulva australis Name used for Kraft et al. (2010) notes are except as indicated. Distribution on based own our collection records, as as (98% 5 well top hits Table1. Name usage, speciesauthority, rationale, distributionAustralian and collection informationfor speciesstudyin this 282, pl. 26: fig. 2; pl. pl. 2; fig. 26: pl. 282, N.L.Gardner 1920: Setchell & Ulva stenophylla Numbers cross-reference 1 to and FigureAppendix 1. AcceptedN/Arefers taxa to from outside Australia. Article rbc s.5W Unique genetic group relative to all available data in Unique genetic group relative to all available data in sp. 5GWS sp. 1GWS p 0U Uniquegenetic group relativeall to available data in sp.10AUS L data1). A, I, (Appendix Indian Pindicate Atlantic, and Pacific ocean, respectively and N refers to number total of sequence Linnaeus (Mertens) Hiraoka & p ASUniquegenetic group relativeall to available data in sp.1AUS (Kützing) Linnaeus Delile Wulfen Wulfen Wille name(s) necessary. species true of confirmation - Genbank in names identifications, which likely explains the multiple morphologyuse gross Impossible for to tubes. to blades this group in plants range from blades, to full linza-like actually 3-5 genetic groups cluster. in Further, this our geneshere,there are used both Based(2010). on al. Included thein Kraft al. et (2010) Genbank. Genbank. Genbank. Genbank. species name necessary. allied sequences Genbank in confirmation - trueof to applied nameshavebeen Considerable complex. (2010) al. Kraft et the to Equivalent ossetwt sg nKate l 21) NrhAeia() N/A 1 America North (P). laetevirens with data Genbankin Consistent and with falls with usageConsistent in Kraft et al. (2010). speciestrue namenecessary. type locality is reported as England - confirmation of the only, Pacific are but group this in collections our of with Consistent Australia (P), North America Consistent with usage in Kraft et al. (2010). ossetwt sg nKate l 21) Australia (P), North America Consistent with usage in Kraft et al. (2010). ossetwt sg nKate l 21) NrhAeia(,) N/A 1 (I,P), Australia North America North (A,P). with usageConsistent in Kraft et al. (2010). with usageConsistent in Kraft et al. (2010). ossetwt opooyaddt nGnak SuhKra() N/A 3 match Genbank. Closest N/A Korea (P). South 1 with morphologyConsistent and data in Genbank. America North (A,P). with morphologyConsistent and data in Genbank. The The Included thein Kraft al. et (2010) Matches Australia?) have be to subsumed into the global (introduced to our identificationiscorrect, the new 'endemic' would L.G.Kraft, Kraft & R.F.Waller in Kraft et al. (2010). If locations. Genetic match to the new weEurope, but have collected this species from many Identification loosely based onmorphology. Type from collections to originally species). keyed GWS manytheseof linzoides and we apply the former name to this group (also see (also group this applyformerand we name to the incorrect latter are The group. this match (AY255874) and Data Genbank forin both applied. name, olderthe species which for America North and Europe of Kraft et al. These (2010). likely represent a single 100% with usageConsistent in Kraft et al. (2010). necessary name species ttl2010) l t ft K on data in on data in Genbank. species). This name appears to be the best match based (which is clearly composed of multiple closely related linzoides well. ranunculata U. priority.NewSouth Wales collectionsfield identified as on our genetic data. australis with usageConsistent in Kraft et al. (2010) to to sequencesallied in Genbank - of confirmation true U. taeniata U. procera rbc U. prolifera and L.G.Kraft,Kraft &R.F.Waller complex complex (clearly multiple closely related L match to newly described group of Kraft et al. 2010. U. torta U. U. prolifera U. pertusa FIN/ U. linza Kraft & A. Millar fall this groupin as (Setchell) Setchell &N.L.Gardner . Ulva australis GRi rf ta.(00. ot mrc AP.1N/A 1 America (A,P). North (2010). Kraft al. et in GBR U. linza Umbraulva Kjellm.are conspecific based USA in Kraft et al. (2010). All U. stenophylla U. stenophylla . Considerable namesapplied Considerable .

JPN clusterJPN from Kraftet U. howensis U. U. stipitata U. U. stipitata U. , the older name, has name, older the , U. compressa U. clathratioides spp. spp. U. proliferoides U. proliferoides (AY255874) (AY255874) Ulva Ulva var var. , is U. (A,P). America North (P), Australia ot mrc P.3N/A 3 (A). N/A ( America North (P), Australia 1 North America (P). North America (P). and Asia (A) and Asia Europe (P), (A,P). America North (P), Australia utai IP n aa P WA: 8 (P) Japan and (I,P) Australia N/A 1 North America (P). (A,P). utai IP.1 NSW: 13 (I,P). Australia match). (100% Japan (P) as and (A) Europe (A), America America (A,P), South Korea.America (A,P), South North (I, P), Australia (A,P). America North (P), N/A Australia 1 North America (P). utai IP NSW: 9 (I,P) Australia (A). America (A,P) North (P), Australian and South Korea. and South (A,P) Europe and America North (I,P), Australia U. armoricana U. armoricana 0TAS: 10 8TAS: 28 6TAS: 26 1TAS: 41 2TAS: 8NSW 8TAS: 4NSW: 7TAS 1N/A 2VIC: 40 26, 33-6, 38-9 33-6, 38-9 26, SA: 37, 39 37, 12,13,17,18 12,13,17,18 SA: 22, 33-35 11, WA: WA: 14, 19 14, NSW: Australian Collection sites : 2 NSW: : 2 13 17 15, 16 NSW 41 5NSW: 2, 4 VIC: 1-7 VIC: 4 4 VIC: 6, 8 VIC: : 22, 24, 28, 24, : 31, 26, 22, 38, rbc 22, 35, 36, 37 SA: 36, 35, 22, WA: 35 20, 22, 25, 22, 27 23, 20, . Name. usage was bas or greater) in BLAS L data only) 41 36 36, 36, 37 SA: 29 18 9,10 NSW: NSW: 9,10 10 WA: 41 NSW: s. 32 WA: WA: 32 : 22-23, : 22-23, 30, 32 13, 13, 32 21, 21, 32 A,B T e © 2012 America Society of Phycological C B A Ulva howensis Ulva australis UII Subgroup Ulva torta Ulva compressa Ulva procera Ulva ohnoi laetevirens Ulva Ulva flexuosa Tabl Ulva intestinalis pce Types Sites of Species to Australia. introduced was species that forlikelihood are Predictions estimates. distance 2-parameter Kimura using website Ulva fasciata Ulva californica UI Subgroup Number ofNumber individua ofNumber individua AcceptedOpencoast (OC) or harbour/ Article e 2. Like l ihood of introduction to to ihoodAustra of introduction l l s sa s sa OC and HE, N and A 30 0 44; 8 0.259; 0.136 0.259; 8 44; 0 0.136 0.259; 2 19; 30 N and andA HE, OC 0.129 4 N and andA HE, OC CadH,NadA701990.129 9 0.129 7 and N OC and HE, A OC and HE, N and A 15 1.043 38; 13 1.043; 0 1.043; 38; 13 1.043 15 N and andA HE, OC CadH,NadA2070.13 7 0 2 and N A HE, OC and m m pl pl e e e OC, N 9 0 ; 4 ; 0 ; 4 ; 0 9 N OC, HE, A 2 0.78 44; 8 1.185; 0.136 1.185; 8 44; 0.78 No data 2 Nodata HE, A 0.129 8 OC, N OC, N 2 0 70; 23 0.134; 0.411 0.134; 70; 23 0 2 OC, N OC, N 8 0 No data No data No data No 0 0.27 0.389; 8 5 14; No data N OC, 1 HE, A m d globa d from Austra baym e l l nt (HE) and natura and (HE) nt y inc A l l l ao aacol ia oftaxa uding Austra ia N B genetic diversity diversity genetic A l l l ia for e (N) orartificia (N) u ct

stralian e tufA d and identifi d and . l (A) report rbcL). (tufA; (tufA; e N nti study. Maxi d in this C diversity ( G e hr known. d where oa ge lobal rbc L). n tufA NIS assessment eti m c a ; l eei distanc genetic Levels for 2 overseas sequences of sequences for2overseas Levels Likely diversity within 5 samples from overseas (B.C. Canada), Canada), (B.C. overseas from 5samples within diversity Genetic data consistent with introduction from from Australia introduction with consistent data Genetic Likely TAS, to one VICand to one but overseas, from Australia present trends suggestive of introduction from from Australia. ofintroduction suggestive trends present reference pool lacking. Habitat data do not suggest NIS. suggest do not data Habitat lacking. pool reference and Coledale, NSW 20 km north of Port Kembla and 64 Kembla Port ofnorth 20km NSW and Coledale, likely. Genetic data consistent with dual introduction to introduction with dual likely. consistent data Genetic Unlikely. diversity between Australia and Canada + South Korea. Korea. + South and Canada between Australia diversity globally for More samples needed. Habitat data are not inconsistent inconsistent not are data Habitat needed. samples More Only Korea. and South Canada and between of Canada WA (between Australia coasts two between and NSW), Possible Possible necessary. Habitat data inconsistent with NIS, but both both but NIS, with inconsistent data necessary. Habitat genetic diversity from Australia also supportive of NIS ofNIS supportive from also diversity Australia genetic Uncertain (Pacific) and Bay of Fundy (Atlantic) for (Atlantic) ofFundy and Bay (Pacific) (samples from mainland NSW and Lord Howe Island) Island) Howe Lord and NSW mainland from (samples sample sites (Devonport, TAS(Devonport, sites sample ofTasmania Spirit dock Possible. Two solely (TASAustralian one groups, WA) and and other Canada, NSW and WA.NSW and Canada, other diversity, intragroup No Unlikely Unlikely for km south of Sydney) in somewhat close proximity to proximity close in kmsomewhat of south Sydney) only intergroup diversity. More samples needed, but but needed, samples intergroup More diversity.only to rest of world. No diversity between B.C. Canada B.C. Canada between No diversity ofworld. to rest more Australian samples needed to confirm pattern. Possible instead all diversity between Australia and Canada. Canada. and between Australia diversity all instead both both status. Habitat data arenot inconsistentwith NIS. likely. Habitat data are not inconsistent with NIS. NIS. with inconsistent not are data Habitat likely. test area diversity for comparison to overseas. No No overseas. to comparison for diversity area test Likely for both for both makes introduction plausible, but global genetic genetic global but plausible, introduction makes with NIS and one site (Swan River, (Swan WA) site one and with NIS highly tufA NIS. suggest not do data Habitat comparison. Habitat data from TASVIC and from data NIS. suggest Habitat . Higher diversity in global pool than test pool for pool test than pool global in diversity . Higher . Higher diversity in global pool than test pool for pool test than pool global in diversity . Higher tufA tufA . Higher diversity in global pool than test pool pool test than pool global in diversity Higher . . Genetic sampling. (n=1)assess to insufficient No genetic diversity from Australian samples samples from Australian diversity Nogenetic or . Diversity within Australia half that found that half within Australia . Diversity . Test area diversity driving global diversity. global . driving Test diversity area Complete lack of diversity within Australia . Test area diversity driving global diversity. global . Test driving diversity area . Complete lack of genetic diversity within within diversity ofgenetic lack . Complete . No genetic data from overseasgenetic samples data from. No for and and rbc tufA tufA e data w data Habitat data suggest NIS. L. Not found outside Australia. Likely Likely foundAustralia. outside Not L. rbc and and rbc makes introduction to likely.to Australia introduction makes Australian endemic. endemic. Australian L makes introductionto Australia L makes introduction to to LAustralia introduction makes Found broadly. rbc e re generat re disturbed. disturbed. broadly. L. More Australiansamples ports. e tteBOLD the d at rbc L than higher rbc L. Found Found L. tufA

alga Couceiro, L.,Cremades, J. & Barreiro, R.2011.Evidence for multiple introductions of the Pacific green analysis. ITS sequence rDNA Goer, S.1998. Coat, G.,Dion, P.,Noailles, M.C.,deReviers, B., annotations for analyses of trees. Chevenet, F.,Brun, C.,Baculs, A., Jacq, B. & Chri © 2012 America Society of Phycological by aglobal analysis of native and introduced populations. Serisawa, Y.& Ang, P.O.2010.Lowgenetic variabi Cheang, C.C.,Chu,K. H., Fujita, D.,Yoshida, G., Sci. 2005.V. Land plants andDNA barcodes Chase, M. W., Salamin, N.,Wilkinson, M., Dunwell, J. Trans. R.Soc. B: Biol. Sci. S.Armstrong, F.&Ball, L.2005.DNAbarcodes K. for biosecurity: invasive species identification. marine macroalgae. Alstyne, L.2008.K. Ecological and physiological role References Accepted Articlepowerful alternative. Anisimova, M.& Gascuel, O. 2006.Approximate likelihood-ratio test for branches: Afast, accurate, and pp. 173-94.

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s ofdimethylsulfoniopropionate and its products in 7 M., Kesanakurthi, R.P.,Haidar, N.& Savolainen, :439-9. J. Phycol.

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54 :391-402. Phil. Phil. © 2012 America Society of Phycological molecular characterization of free-floating and attached green macroalgae Duan, W.,Guo, L.,Sun,D., Zhu, S.,Chen,X., Zhu, W., Xu, T. & Chen,C.2011. Morphological and the non-specialist. Lefort, Lescot,V., M., Claverie, J.-M. & Gascuel, O.2008. Phylogeny.fr: robust phylogenetic analysis for Dereeper, A.,Guignon, V., Blanc, G.,Audic, S., Bu wastewater using green alga alga green using wastewater El-Sikaily, A.,Nemr, A.E.,Khaled, A.& Abde China. biochemical composition of three Eswaran, K., Ganesan, M., Periyasamy, C.& Rao,P. Acceptednortheast Pacific. Hayden, H.S.& Waaland, R. J. 2004.Amolecular systematic study of along: all Hayden, H. S.,Blomster, J., Maggs, C.,Silva, P., Linnaeus. of hypercholesterolemic albino rats by polysaccharides extracted from thegreen alga Hassan, S., El-Twab, S.A., Hetta, M. & Mahmoud, B. maximum likelihood. Guindon, S.& Gascuel, O. 2003. Asimple, fast, and accurate algorithm toestimate large phylogenies by Mar. Sci. Article J. Appl. Phycol.

31 Ulva Saudi J. Biol. Sci. :334-36. and Phycologia Nucleic Acids Res. Acids Nucleic Enteromorpha Syst. Biol. :1-12.

18 Ulva lactuca

43 :333-40.

:364-82. 52 Ulva are not distinct genera. genera. distinct not are :696-704.

36 species (Chlorophyta) from southeast coast ofIndia. :W465-W69. and its activated carbon.

lwehab, O.2007.Removal oftoxic chromium from Stanhope, M. & Waaland, R.2003. Linnaeus was right ffet, S.,Chevenet, F., Dufayard, J.-F., Guindon,S., V. S.2002.V. Effect of ultraviolet-B radiation on 2011. Improvement oflipid profile and antioxidant Eur. J.Phycol. J. Hazard. Mater. Ulva

38 Ulva :277-94. (Ulvaceae, Ulvales) from the spp. in the Yellow Seaof

Ulva lactuca 148 :216-28. Indian J.

pertusa Lopez, S.B.,Fernandez, I.B., Lozano, R.B.& Ugarte, J.C.2007.Is the cryptic alien O. 2009.Research note: Identity ofthe Qingdao algal bloom. Leliaert, F., Xiaowen, Z., Ye,N.,Malta, E. EngeJ., Phaeophyceae) Canadianin torecognized waters species using DNAbarcoding. Kucera, H.& Saunders, G. W. S.2008. Assigning morphological variants of endemic cryptic species. (Ulvophyaceae, Chlorophyta) taxonomy andmolecular phylogeny indicate both cosmopolitanism and Kraft, L.G. K., Kraft, G. T. & Waller, R.F.2010. Investigations into southern Australian fenestrata from Peter the Great Bay, Sea of Japan. Kozhenkova, S.,Chernova, E.& Shulkin, 2006.V. Mi forming green tides in Qingdao, China. Kong, F.,Mao, Y.,Cui, F., Zhang, & X. Gao, Z. 2011.Morphology and molecular identification of © 2012 America Society of Phycological from atemperate rocky intertidal zone. Horn, M.H. 1983. Optimal diets in complex environments: feeding strategies of two herbivorous fishes ohnoi Hiraoka, M., Shimada, S., Uenosono, M. & Masuda, M. 2004.A new green-tide-forming alga, 44 and Heesch, S., Broom, J.E.S., Neill, F.,Farr,K. Dalen, L.&T. J., Nelson,J. W.A.2009.

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46 :1257-77. Oecologia Journal of Ocean University ofChina (English Edition)

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Russ. J.Mar. Biol. 58 len, A.H.,Mineur, F., Ve :345-50. croelement composition of the green alga Ulva Phycol. Res.

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(Fucales, Bot. Mar. Bot. 52 :17-29.

Ulva, Umbraulva Umbraulva Ulva, 86 Ulva :1065-79. Eur. J. Phycol.

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invasive species Saunders, G. W. S.2009. Routine DNAbarcoding of promise for future applications. Saunders, G. W. S.2005. Applying DNAbarcoding macroalgae: to red a preliminary appraisal holds against powdery mildew in wheat and barley plants. rice andwheat cell cultures by ulvan, apolysaccharide from green macroalgae, and enhanced resistance Paulert, R., Ebbinghaus, D., Urlass, C.& Moerschbacher, B.M. 2010.Priming of the oxidative burst in © 2012 America Society of Phycological (Ulvophyceae, Chlorophyta) in the Hawaiian Islands. O’Kelly, C. Kurihara,J., A., Shipley, C.& T. Sherwood, A.R.2010. Molecular assessment of Michael, S.2009.T. Glycoconjugate organization of among brownmacroalgae (Phaeophyceae) including anovel extraction protocol. D.C.&Saunders,McDevit, G.W.S.2009. Onthe 54. species or morphotypes? A morphological Malta, E. J., Draisma, S.G. A.& Kamermans, P.1999. Free-floating Version 2.74 Maddison, D.R.& Maddison, W.P.2010. Mesquite: a modular system forevolutionary analysis. Britain. phylogeny anddistribution of distromatic Loughnane, C. McIvor,J., L.M.,Rindi, F., Stenge Accepted(Chlorophyta) zoospores. Article Phycologia http://mesquiteproject.org Gracilaria vermiculophylla

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45 :660-77. . Ulva , molecular and ecological comparison.

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9 :140-50. Ulva fasciata

57 Ulva :131-41.

34 spp :443-

Schaffelke, B.,Smith, & J. Hewitt, C.2006.Introduced macroalgae-a growing concern. Insights from a DNA barcoding approach. Insights approach. from DNAbarcoding a versus interspecies DNAsequence variation for representatives of Batrachospermalesthe (Rhodophyta): Sherwood, A.R., Vis, M. L., Entwisle, Necch T. J., 18 encyclopaedia an oflife: introduction to DNAbarcoding. Savolainen, Cowan,R. V., S., Vogler, A.P.,Roderick, & G.K. Lane, R.2005. Towards writing the © 2012 America Society of Phycological free-floating analysesof theJapanese Shimada, S.,Masanori, H., Shinichi, N.,Masafu markers for the marine green macroalgae. G. Saunders, W. S.&Kucera, H.2010. Anevaluation rbcL, UPA,LSUand tufA, of ITS asDNAbarcode (DMSP) production in Alstyne,Van K., Koellermeier, L.& Nelson, T. 2007. Spatial variation in dimethylsulfoniopropionate Evol. the gross morphology oftwo common genera ofgreen , Stanhope, M. 1999.J. Molecular phylogenetic evidence for areversible morphogenetic switch controlling Tan, I. H.,Blomster, Hansen, G.,Leskinen,J., E. Phycol. (Ulvophyceae, Chlorophyta), with special reference to the Japanese freshwater and brackish taxa. Shimada, S.,Yokoyama, Arai, N., S.& Hiraoka, M.2008. Phylogeography ofthe genus

Accepted Article:529-41.

16

20 :1011-18. :979-89. Ulva . Phycol. Res. Ulva lactuca Ulva Ulva and

51 Enteromorpha :99-108. (Chlorophyta) from theNortheast Pacific. Cryptogam Algol Phycol. Res.

mi, I.& Michio, M. 2003. Molecular phylogenetic , Maggs, C.A.,Mann,D.G.,Sluimam, & H.J. (Ulvales, Ulvophyceae), with special reference to the i Jr, O.& Presting, G.2008. Contrasting intra

56 Phil. Trans. R.Soc. B:Biol. Sci. :269-79.

31 :487-528. Ulva and Enteromorpha Mar. Biol. Mar. J. Appl. Phycol. Ulva

. 150

360 Mol. Biol. :1127-35.

:1805-11. J. Appl.

Bot. Comparison Australia: of morphological features and analyses nuclear of rDNA sequence data. Woolcott, G.& R.1999.King, Canberra. PRJ-000162 © 2012 America Society of Phycological Accepted Articleand marketable products. Winberg, P.,Skropeta, D. & Ullrich, A.2011. Seaweed cultivation pilot trials: towards culture systems 19 16

18 15 12 17 14 :709-25. 12 13 . Rural Industries Research& Devel Ulva and Enteromorpha

32 (Ulvales, Ulvophyceae, Chlorophyta) in eastern opment Corporation Technical Report. 9 41 10 4 8 5 1 6 7 2 3 40 37 39 38 35 36 33 30

34 31 29 Aust. Syst. 20-8

© 2012 America Society of Phycological Accepted Article

© 2012 America Society of Phycological Accepted Article