Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

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Molecular Phylogenetics and Evolution

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Molecular taxonomy, phylogeny and evolution in the family Stichopodidae(Aspidochirotida: Holothuroidea) based on COI and 16S mitochondrial DNA

Maria Byrne a,*, Frank Rowe b, Sven Uthicke c

a Schools of Medical and Biological Sciences, F13, University of Sydney, NSW 2006, Australiab Australian Museum, 6 College St., Sydney, NSW 2010, Australia and Beechcroft, Norwich Road, Scole, Norfolk IP21 4DY, UKc Australian Institute of Marine Science, PMB No. 3, Townsville MC, Qld 4810, Australia

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 February 2010Revised 6 April 2010Accepted 9 April 2010Available online 23 April 2010

Keywords:PhylogenyEvolutionStichopodidaeHolothuroideaCOI16SMorphology

1055-7903/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.ympev.2010.04.013

* Corresponding author. Fax: +61 2 9351 2813.E-mail address: mbyrne@anatomy.usyd.edu.au (M

The Stichopodidae comprise a diverse assemblage of holothuroids most of which occur in the Indo-Paci-fic. Phylogenetic analyses of mitochondrial gene (COI, 16S rRNA) sequence for 111 individuals (7 genera,17 species) clarified taxonomic uncertainties, species relationships, biogeography and evolution of thefamily. A monophyly of the genus Stichopus was supported with the exception of Stichopus ellipes. Molec-ular analyses confirmed genus level taxonomy based on morphology. Most specimens harvested asS. horrens fell in the S. monotuberculatus clade, a morphologically variable assemblage with others fromthe S. naso clade. Taxonomic clarification of species fished as S. horrens will assist conservation measures.Evolutionary rates based on comparison of sequence from trans-ithmian Isostichopus species estimatedthat Stichopus and Isostichopus diverged ca. 5.5–10.7 Ma (Miocene). More recent splits were estimatedto be younger than 1 Ma.

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1. Introduction

Sea cucumbers (Holothuroidea) in the order Aspidochirotida area conspicuous and diverse group in the world’s oceans. They areparticularly prominent in tropical regions where they inhabit softsediment and reef environments (Rowe and Doty, 1977; Conand,1990; Rowe and Gates, 1995; Massin, 1999, 2007; Rowe and Rich-mond, 2004). Aspidochirotids provide important ecosystem ser-vices enhancing nutrient cycling and local productivity inoligotrophic carbonate sediments through their bioturbation anddeposit feeding activities (Uthicke, 1999, 2001a, b). They are alsofished for bêche-de-mer production (Conand and Byrne, 1993;Uthicke and Benzie, 2000; Conand 2001, 2008; Mangion et al.,2004; Uthicke et al., 2004a; Toral-Granda, 2008). Despite beinglarge and often the dominant mobile invertebrates on reef flatsand lagoons, the taxonomy of many aspidochirotids is uncertain.This is due to the difficulty in application of traditional taxonomiccharacters (e.g. body profile, skeleton morphology) which may nothave been used in sufficient detail to distinguish cryptic speciescurrently included within a single taxon and the difficulty ofextracting taxonomic data from museum specimens (Massin,1999; Massin et al., 2002; Uthicke et al., 2004b). Determination

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. Byrne).

of the correct taxonomy of aspidochirotids such as the Stichopusspecies investigated here is timely, as the fishery for these speciesis expanding and correct identification is critical for managementand conservation (Conand, 2001, 2008; Shepherd et al., 2004;Uthicke et al., 2004a). The family Stichopodidae has long been ataxonomic challenge (Rowe and Gates, 1995; Massin, 1999; Massinet al., 2002; Moraes et al., 2004) and, as noted by Massin (2007),species are often misidentified in field studies.

Revision of the taxonomy of Stichopus Brandt, 1835, resulted inclarification of species imprecisely recognised (Rowe and Gates,1995; Massin, 1999; Massin et al., 2002; Moraes et al., 2004).The Stichopodidae presently comprises nine genera and 32 de-scribed species (Rowe unpubl.). Clark’s (1922) major taxonomicrevision, based on gross morphology and skeletal ossicle form ofpreserved, historic, material divided the family into four genera:Stichopus Brandt, 1835; Thelenota Brandt, 1835; ParastichopusH.L.Clark, 1922; and Astichopus H.L. Clark, 1922. Five more generahave since been described: Neostichopus Deichmann, 1948; Isos-tichopus Deichmann, 1958; Apostichopus Yulin Liao, 1980; Eostich-opus Cutress and Miller, 1982 and Australostichopus Levin, 2004.Recently, two species have been resurrected from synonym(Stichopus herrmanni, S. vastus) (Rowe and Gates, 1995; Rowe andRichmond, 2004); two species understood in greater detail (S. naso,S. monotuberculatus) (Rowe and Gates, 1995; Rowe and Richmond,2004; Massin, 2007) and one species (S. variegatus) reduced to the

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1069

synonymy of S. horrens (Rowe and Gates, 1995; Rowe andRichmond, 2004). Five new species have been added to the genusStichopus since the mid-1960s (Cherbonnier, 1967, 1980; Massin,1999; Massin et al., 2002).

The fishery based on Stichopus, which extends from the Galapa-gos to the Pacific islands, is problematic with several species beingmisidentified under the name ‘S. horrens’. Here we revisited S. hor-rens as a nominal taxon in a molecular and morphological study toclarify the species being harvested as in previous studies of theteatfish and sandfish groups (Uthicke et al., 2004b, 2005; Massinet al., 2009). Clarification of the identity of commercial species pro-vides essential data for fishery management and conservation ofseveral potentially vulnerable species (Bruckner, 2006; Uthickeet al., 2010).

We undertook a phylogenetic analysis of the Stichopodidaeusing sequence data for two mitochondrial genes (COI, 16S rRNA).The aims were to clarify the taxonomic uncertainties of specimensidentified as S. horrens and S. monotuberculatus and to test hypoth-eses on genera and species monophyly and on the evolutionaryrelationships within the Stichopodidae. We place our data in con-text with regard to the cosmopolitan distribution of the familyusing sequence data for specimens collected mainly from theIndo-Pacific (Galapagos to Reunion and from the Philippines toAustralia and New Zealand) and also included sequence for severaltemperate species. Sequence data for 111 individuals across sevengenera and 17 nominal species including voucher registered mu-seum specimens were used. Assisted by traditional morphologicaltaxonomy, we matched where possible DNA from museum speci-mens with those collected for this study. The research coveredthe geographic range of tropical Stichopodidae from shallow waterwith most material from the tropical Pacific.

Revisiting the identity and distribution of the Stichopodidaeprovided insights into the presence of cryptic species and evolutionof the family across the Indo-Pacific. The Stichopodidae and holo-thuroids in general have a poor fossil record (Reich, 2001) and, inthe absence of fossil calibrated divergence time, we used the COIphylogeny and sequence data from putative geminate Isostichopusspecies from either side of the Isthmus of Panama to estimatedivergence times for stichopodid species, as in previous studies(Lessios, 2008). The Stichopodidae are unusual in the presence ofclonal reproduction by transverse fission, a feature characteristicof several of the most common and abundant members of the fam-ily (Uthicke et al., 1998; Conand et al., 2002; Uthicke and Conand,2005a, b; Massin, 2007). We used the phylogeny to addresshypotheses on evolutionary trends in the distribution of asexualreproduction in the stichopodid clades.

2. Materials and methods

2.1. Sampling of taxa

Tissue samples preserved in ethanol or DMSO were obtained forspecimens of Stichopodidae across the Indo-West Pacific and fromspecimens registered at the Australian Museum, British Museum ofNatural History and Museum of Victoria (Table 1). Specimens col-lected by the seabed biodiversity project in north Queensland(http://www.reef.crc.org.au/resprogram/programC/seabed/in-dex.htm) lodged in the Museum of Tropical Queensland were alsosampled. In tropical Australia we made a concerted effort to locateStichopus horrens in surveys extending from the northern (Raine Is-land, Moulter Cay, Quoin Island, Lizard Island) to the southern (OneTree Island, Heron Island) Great Barrier Reef (GBR) and MoretonBay (Table 1). Several Stichopus species (S. horrens, S. naso, S. mono-tuberculatus) are nocturnal (Rowe and Doty, 1977; Massin, 2007)and night-time searches were conducted. Most specimens col-

lected for this study were sampled at depths ranging between 1and 15 m. Specimens from the seabed biodiversity project werecollected at 15–30 m depth. Geographically we included speci-mens from the Indo-Pacific from the Galapagos to Reunion andfrom the Philippines to Australia and New Zealand (Table 1) andincluded one Caribbean species, Isostichopus badionotus from Cuba.

In total, sequence data were generated for 100 specimens of Sti-chopodidae (Table 1) including 50 identified by fishery scientists asS. horrens. Where possible we sequenced a minimum of three spec-imens per taxon. Sequence data from GenBank were available forseveral temperate and tropical members of the Stichopodidae(Table 1). Additional sequence data were obtained courtesy ofDr. Gustav Paulay (Florida Museum of Natural History). Speciesnames and GenBank accession numbers for all sequences usedare shown in Table 1. Sequence alignments are available fromthe authors on request. In addition, body wall spicule preparationsand live photographs available for Australian Museum specimensprovided reference material to check the identity of specimens col-lected for this study.

Spicule preparations were made from the body wall of speci-mens collected for this project. Small sections of body wall weretreated with diluted bleach to isolate the spicules and preparewhole mounts slides for light microscopic examination and pho-tography (Rowe and Doty, 1977). A photographic record of thespecimens was also made.

2.2. DNA extraction, PCR protocols and sequencing

For DNA extractions approximately 10–20 mg of tissue wasplaced in a 1.5 ml microcentrifuge tube and extracted with aDNeasy tissue extraction kit following the manufacturers’ specifi-cations (Qiagen; DNeasy Blood and Tissue Kit). Sections of thecytochrome oxidase subunit I (COI) and the large subunit 16S ribo-somal DNA (16S rDNA) genes were amplified using primers COIe-Fand COIe-R (Arndt et al., 1996) and 16SA-R and 16SB-R (Palumbiet al., 1991), respectively (Table 2). We used these markers follow-ing previous studies where these have proven useful for taxonomyand phylogeny at the subfamily level (Arndt et al., 1996; Uthickeet al., 2004a, b, 2010).

PCR amplification was conducted as previously described(Uthicke and Benzie, 2003; Uthicke et al., 2004b), using final con-centrations of 1 lM of each primer, 2.5 lM MgCl2, 1 � PCR Buffer,1 � Bovine Serum Albumin, 200 lM of each dNTP 2.5 units Hot-Master Taq DNA polymerase (Eppendorf) and 40–80 ng DNA. PCRreactions involved denaturation for 60 s at 95 �C followed by 40 cy-cles of 30 s denaturation at 95 �C, 30 s annealing at 50 �C, and 80 sextension at 72 �C, and final extension of 10 min. PCR productswere cleaned using QIAquick PCR purification kit (Qiagen) and di-luted to a final concentration of 10–25 lg/ml. For the sequencingreaction, we used Amersham (Dyenamic) sequencing reagents.DNA from each sample was sequenced in both directions, andsequencing products were cleaned using Autoseq50 (Amershan)clean up columns. The PCR amplicons were purified using theGFX PCR DNA Gel Band Purification Kit (GE Healthcare) accordingto manufacturers’ specifications. Some amplicon purification andall sequencing was done by Macrogen (Macrogen DNA SequencingService; Seoul, South Korea). Sequences were aligned using CLUS-TAL W 1.5 (Thompson et al., 1994) under default parameters andchecked by eye.

2.3. Sequence analyses and phylogeny

The COI and 16S sequence data were used for phylogeneticanalyses using Bayesian Markov Chain Monte Carlo (MCMC) asimplemented in MrBayes (v.3.1, Ronquist et al., 2005). Prior tothe analyses we tested for the most appropriate nucleotide

Table 1Stichopodidae analysed for the partial cytochrome oxidase subunit 1 (COI) and 16S genes. Tree ID denotes the abbreviation used in phylogenetic trees. GenBank accessionnumbers for each sample are listed under the respective marker, accession numbers in parenthesis denote sequences deposited on GenBank prior to the present analysis.Sequence data were also generated for specimens obtained from the Australian Museum (AM); British Museum (BM); Museum of Tropical Queensland (MTQ) and, MuseumVictoria (MV); Dr. Gustav Paulay provided sequence for specimens in the University of Florida Museum of Natural History (UF). Museum registration numbers are indicated andsample locations are indicated where known. Specimens used for a FAO funded bêche-de-mer genetic barcoding project (Uthicke et al., 2010) are indicated. Aus, Australia; GBR,Great Barrier Reef; Qld., Queensland; SBD, collected for the seabed biodiversity project; empty fields: no sequence obtained for the respective marker. Holothuria whitmaei wasused as the outgroup.

Species Location Tree ID COI 16S

Stichopus chloronotus Reef 18–026, Central GBR, Aus Sc1.Cent-GBR EU856610 EU856682Reef 18–026, Central GBR, Aus Sc2.Cent-GBR EU856611 EU856683Reef 18–026, Central GBR, Aus Sc3.Cent-GBR EU856612 EU856684Reef 18–026, Central GBR, Aus Sc4.Cent-GBR EU856613 EU856685Derder Reef, Torres Strait, Aus Sc1.TS EU856614 EU856686Derder Reef, Torres Strait, Aus Sc2.TS EU856615 EU856687Derder Reef, Torres Strait, Aus Sc3.TS EU856616 EU856688Derder Reef, Torres Strait, Aus Sc4.TS EU856617 EU856689Etang Sale, Reunion FRA Sc1.RI EU856618 EU856690Etang Sale, Reunion FRA Sc2.RI EU856619 EU856691Etang Sale, Reunion FRA Sc3.RI EU856620 EU856692Etang Sale, Reunion. FRA Sc4.RI EU856621 EU856693

S. monotuberculatus Heron Is, GBR Aus Sm1.HI-GBR EU856574Heron Is, GBR Aus Sm2.HI-GBR EU856577 EU856695Heron Is, GBR Aus Sm3.HI-GBR EU856578 EU856696Heron Is, GBR Aus Sm4.HI-GBR EU856580 EU856697Heron Is, GBR Aus Sm5.HI-GBR EU856581 EU856698Heron Is, GBR Aus Sm6.HI-GBR EU856582 EU856699Heron Is, GBR Aus Sm7.HI-GBR EU856583 EU856700Heron Is, GBR Aus Sm8.HI-GBR EU856584 EU856701Heron Is, GBR Aus Sm9.HI-GBR EU856585 EU856702Heron Is, GBR Aus Sm10.HI-GBR EU856579 EU856694Heron Is, GBR Aus SmHI-GBR (AM) EU856659

(AM #J17269)One Tree Is, GBR Aus Sm1.OTI-GBR EU856562 EU856645One Tree Is, GBR Aus Sm2.OTI-GBR EU856565 EU856648One Tree Is, GBR Aus Sm3.OTI-GBR EU856568 EU856651One Tree Is, GBR Aus Sm4.OTI-GBR EU856571 EU856654Toamua, Upolu Is Samoa Sm1.TSAM EU856564 EU856647Toamua, Upolu Is Samoa Sm2.TSAM EU856567 EU856650Toamua, Upolu Is Samoa Sm3.TSAM EU856570 EU856653Toamua, Upolu Is Samoa Sm4.TSAM EU856573 EU856656Salelologa, Savaii Is Samoa Sm1.SSAM EU856563 EU856646Salelologa, Savaii Is Samoa Sm2.SSAM EU856566 EU856649Salelologa, Savaii Is Samoa Sm3.SSAM EU856569 EU856652Salelologa, Savaii Is Samoa Sm4.SSAM EU856572 EU856655Salelologa, Savaii Is Samoa Sm5.SSAM EU856575 EU856657Salelologa, Savaii Is Samoa Sm6.SSAM EU856576 EU856658Lizard Is, GBR Aus Sm.Li-GBR EU856556 EU856639Magnetic Is, Aus Sm1.MI-QLD EU856557 EU856640Magnetic Is, Aus Sm2.MI-QLD EU856558 EU856641Magnetic Is, Aus Sm3.MI- QLD EU856559 EU856642Magnetic Is, Aus Sm4.MI-QLD EU85660 EU856643Magnetic Is, Aus Sm5.MI-QLD EU85661 EU856644Palau (UF# 1589) Sm-PAL EU856540Pohnpei (UF#2670) Sm-Poh EU856543

S. horrens One Tree Island, GBR, Aus Sh.OTI-GBR EU856554 EU856638Hawaii (UF#1176) Sh.HAW EU856542Galapagos Sh.GAL EU856541(UF – Hickman #97–360)Phillipines Sh.Ph3 EU848282 EU822434Samoa, (BM# 1970.10.8.57) Sh.SAM(BM EU856555 EU856637

S. naso Moreton Bay, Qld, Aus Sn1.MOR-QLD EU856586 EU856666Moreton Bay, Qld, Aus Sn2. MOR-QLD EU856594 EU856703Moreton Bay, Qld, Aus Sn3. MOR-QLD EU856595 EU856704Moreton Bay, Qld, Aus Sn4. MOR-QLD EU856596 EU856705Moreton Bay, Qld, Aus Sn5. MOR-QLD EU856597 EU856706GBR, Aus(MTQ #SBD24743) Sn1.SBD-GBR EU856587 EU856661GBR, Aus (MTQ#SBD24704) Sn2.SBD-GBR EU856588 EU856662GBR, Aus (MTQ#SBD24742) Sn3.SBD-GBR EU856589 EU856663GBR, Aus (MTQ#SBD025742) Sn4.SBD-GBR EU856590GBR, Aus (MTQ#SBD01059) Sn5.SBD-GBR EU856591New Caledonia (FAO 055) Sn1.NC EU848280 FJ001809New Caledonia (FAO 056) Sn2.NC EU848279 FJ001810

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Table 2Primers used in this study. COI e primers were developed by Arndt et al. (1996) and16SA-R and 16SB-R by Palumbi et al. (1991).

Primer Sequence 50?30

COIe-F ATA ATG ATA GGA GGR TTT GGCOIe-R GCT CGT GTR TCT ACR TCC AT16SA-R CGC CTG TTT ATC CAG ATC ACG T16SB-R CCG GTC TGA ACT CAG ATC ACG T

Table 1 (continued)

Species Location Tree ID COI 16S

S. herrmanni Heron Is, GBR Aus Sher1.HI-GBR EU856544 EU856628Heron Is, GBR Aus Sher2.HI-GBR EU856546 EU856630Heron Is, GBR Aus Sher3.HI-GBR EU856548 EU856632Heron Is, GBR Aus Sher4.HI-GBR EU856550 EU856634Heron Is, GBR Aus Sher5.HI-GBR EU856552 EU856636One Tree Is, GBR Aus Sher.OTI-GBR EU856553 EU856627Torres Strait Sher1.TS EU856545 EU856629Torres Strait Sher2.TS EU856547 EU856631Torres Strait Sher3.TS EU856549 EU856633Torres Strait Sher4.TS EU856551 EU856635New Caledonia (FAO 004) Sher1.NC EU848281 EU822451New Caledonia (FAO 005) Sher2.NC EU848278 EU822450

S. vastus Torres Strait Sv1.TS EU856622 EU856707Torres Strait Sv2.TS EU856623 EU856708Torres Strait Sv3.TS EU856624 EU856709Lizard Island, GBR, Aus(FAO 027)

Sv.Li-GBR EU848275

S. ocellatus Torres Strait So1.TS EU856679Torres Strait So2.TS EU856608 EU856680Torres Strait So3.TS EU856609 EU856681Bedarra Island, GBR, Aus SoBI-GBR EU856604 EU856675Central, GBR SoCent-GBR EU856605 EU856676Long Island, GBR, Aus SoLongIs-GBR EU856606 EU856677Lindeman Island, GBR, Aus So.LindIs-GBR EU856607 EU856678

S. ellipes Batemans Bay, NSW Aus (AM #J14804) Se.BB, NSW (AM) EU856626Jervis Bay, NSW Aus Se.JB EU856625(AM #J12946) NSW (AM)

Australostichopus mollis New Zealand Am1.NZ EU856598 EU856669New Zealand Am2.NZ EU856600 EU856671New Zealand Am3.NZ EU856602 EU856673Eliza Pt., Tasmania, Aus Am1.TAS EU856599 EU856670Fisher’s Pt, Tasmania, Aus Am2.TAS EU856601 EU856672Hope Is, Tasmania, Aus Am3.TAS EU856603 EU856674Victoria, Aus (MV#) Am.VIC EU856667Port Hacking, NSW AUS Am.NSW (AM) EU856668(AM #J9224)

Thelenota rubralineata Phillipines (FAO 20) TrPh EU848260 EU822452

T. anax Lizard Island, GBR (FAO 037) Tx-Li EU848243New Caledonia (FAO 046) Tx-NC EU848292

T. ananas Central GBR (FAO 070) Ta1-GBR EU848258Central GBR (FAO 071) Ta2-GBR EU848261Central GBR (FAO 072) Ta3-GBR EU848259New Caledonia Ta1-NC EU848257

Astichopus multifidus Cuba Amu1CUBA EU848293 EU822453

Isostichopus badionotus Cuba Ib1CUBA EU848264 EU822435

I. fuscus Mexico, Pacific If1 (AF486427)Mexico, Pacific If2 (AF486428)Mexico, Pacific If3 (AF486429)

Apostichopus japonicus Japan Ij1 (AY85220)Japan Ij2 (AY85221)

Parastichopus californicus USA, Pacific coast Pc1 (PCU3218)USA, Pacific coast Pc2 (PPU32199)

Outgroup: Holothuria whitmaei GBR H. whitmaei (AY177134) (AY509147)

Table 1 (continued)

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1071

substitution model using MrModeltest 2.3 (Nylander, J.A.A. 2004.MrModeltest 2.3. Program distributed by the author; www.eb-c.uu.se/systzoo/staff/nylander.htnl). This program chooses the best

out of 24 models based on the Akaike Information Criterion (AIC).For both genes investigated, AIC suggested the General TimeReversible (Tavaré, 1986) model with gamma distributed variationacross sites and a proportion of invariable sites as the most appro-priate model. Final model runs were conducted with 107 genera-tions and sampling trees every 100 generations. After 107

generations, the standard deviation of split frequencies for bothCOI (0.0042) and 16S (0.0064) were well below 0.01, and thusthe number of generations run was sufficient (Ronquist et al.,2005). The probability density chosen as priors for all parameterswere flat Dirichlets (=values of 1). Trees shown are Bayesian con-sensus trees of the last 75,000 trees (burnin period = 25,000) and

1072 M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

Bayesian posterior probabilities for each node. The outgroupsequence was from Holothuria whitmaei, a species representingthe closest extant group (Holothuriidae) to the Stichopodidae(Samyn et al., 2005).

As alternative analyses we also present trees derived from Max-imum Parsimony analyses. These were conducted in Mega 4.0(Tamura et al., 2007) for both the 16S and COI sequences. Close-Neighbor-Interchange (CNI) with search level 1 and Random treeaddition were undertaken with 10 replicates. 1000 bootstrap treeswere generated, and the percent of bootstrap replicates supportingeach node calculated. Haplotype networks were created using Sta-tistical parsimony analysis as implemented in TCS (v1.13, Clementet al., 2000).

To explore phylogenetic relationships in Stichopus and the dis-tribution of asexual reproduction across species, a Neighbor-Join-ing tree (1000 bootstraps) based on Kimura 2 parameter geneticdistances for COI was used just for this genus. Maximum adult sizefor Stichopus species was obtained from taxonomic studies(Massin, 1996, 1999, 2007; Massin et al., 2002) and field observa-tions made during collections for this study.

2.4. Sequence divergence

With the assumption that the two Isostichopus species fromeither side of the Isthmus of Panama I. fuscus (Pacific) and I. badion-otus (Caribbean) are geminate sister species and closure of the Isth-mus of Panama at ca. 3.1 Ma (Lessios, 2008) we used the sequencedivergence between these species to estimate the Stichopodidae-specific mutation rate for COI. These are the only two species ofIsostichopus in this region and so are likely to be closest living rel-atives, an important consideration for molecular clock calibration(Marco and Moran, 2009). We used the Stichopodidae-specificmutation rate for COI and the maximum rate inferred for evolutionrate for echinoderm COI (3.5% Ma�1, Lessios, 2008) to estimate arange of clade and species ages. These rates were applied onlyfor the split between the genera Isostichopus and Stichopus, andfor the species within the genus Stichopus.

To test for similarity in sequence divergence rates in the speciesinvolved we applied Tajima’s (1993) test as implemented in Mega4.0 (Tamura et al., 2007) using H. whitmaei as an outgroup. This testwas conducted between all combinations of species involved in therate calculations for both COI (36 comparisons) and 16S (28 com-parisons), using a haphazardly chosen representative sequencesfor the respective species in case more than one sequences wereavailable. None of the comparisons for both genetic markers indi-cated significant (v2 test, df = 1, p > 0.05) differences in evolution-ary rates between species.

3. Results

3.1. COI and 16S sequence

After trimming some base pairs at the beginning and end of thesequences, sequence size for COI was 558 bp and for 16S was458 bp. Where possible we generated sequence data for bothmarkers (Table 1). For some specimens, particularly the museummaterial we were only able to generate data for one of the markers.

3.2. Higher phylogenetic relationships

The phylogenies generated from COI and 16S sequence datawere similar and Bayesian consensus trees were virtually identicalto the Maximum Parsimony consensus tree for both markers(Fig. 1A and B). Monophyly of the genus Stichopus was supportedby the COI tree (Fig. 1A). In the 16S tree S. ellipes a warm-temperate

species, from southeast Australia formed a separate clade to otherStichopus species (Fig. 1B). Only 16S data could be obtained for thetwo S. ellipes specimens. Stichopodid genera Thelenota and Isostich-opus formed monophyletic clades and so molecular analyses con-firmed current genus level taxonomy based on morphology.Australostichopus is currently known only from one species (A. mol-lis). The Parastichopus and Apostichopus species cluster together.

The two species on either side of Panama I. badionotus (Carib-bean) and I. fuscus (Pacific) formed a monopyletic clade positionedbasal to all the other Stichopus species (Fig. 1A and B). The two tem-perate genera from the north Pacific, Parastichopus (represented byP. californicus, North America) and Apostichopus (represented by A.japonicus, Japan) were sister to the temperate genus from the southPacific, Australostichopus mollis from New Zealand and SouthernAustralia formed a monophyletic group.

3.3. Stichopus

The major Indo-West Pacific species S. chloronotus, the type spe-cies for the genus, is a sister taxon to all the other Stichopus speciesthe Indo-West Pacific assemblage (Fig. 1A and B). S. chloronotus hasa highly stereotypic morphology throughout its extensive rangeand is readily identified based on its body profile (Fig. 1A).Throughout the range sampled, including samples from the WestIndian Ocean and Pacific, S. chloronotus has surprisingly limited ge-netic variability (see below).

The other major Indo-West Pacific stichopodids divided intotwo large clades with S. naso forming a monophyletic sister cladeto the remaining Stichopus species (Fig. 1A and B). S. naso is readilyidentified by the presence of diagnostic large C-ossicles (Fig. 3A)and table spicules possessing spiny, 4-pillared spires (Massin,2007). This was confirmed by comparison of body wall spiculepreparations of fresh specimens with those from registered mu-seum specimens (Figs. 1A and 2E). S. naso is also characterised byits prominent and robust papillae, and, to a degree, by its lumpyappearance and colour pattern (Figs. 1A and 2E).

The other five nominal Indo-West Pacific species comprised twoclades, the S. herrmanni – S. ocellatus – S. vastus and the S. horrens –S. monotuberculatus groups. S. vastus was positioned basal toS. herrmanni and S. ocellatus. These three species are readily identi-fied in the field by their distinctive appearance (Fig. 1A) and theirlarge size. They are larger than all other Stichopus species (Fig. 4).

In the other major internal node, S. horrens formed a distinctclade closely related to the S. monotuberculatus assemblage. Thethree S. horrens specimens sequenced for COI were from a wide dis-tribution including the GBR, Philippines and a specimen from Sa-moa from British Museum collections. Four specimens sequencedfor 16S from Hawaii and the Galapagos were also included. A dis-crete S. horrens cluster was evident with both COI and 16S analy-ses. As per taxonomic descriptions and species keys (Rowe andDoty, 1977; Massin et al., 2002), S. horrens sensu stricto is readilyidentified by the presence of diagnostic tack-like spicules in thebody wall (Fig. 3B and C). S. horrens located along the GBR (RaineIs, Quoin Is, One Tree Is.) and elsewhere in the West Pacific (Massinet al., 2002) have distinct conical papillae on the dorsal surface(Fig. 2A–C).

Most specimens, identified by field collectors as S. horrens, fellwithin S. naso or S. monotuberculatus. The latter is a monophyleticgroup that separated into several sub-clades with high support val-ues. Sequence data were obtained for S. monotuberculatus speci-mens from the GBR, Samoa, Palau and Pohnpei, from the Pacificdistribution of this widely distributed taxon. Unfortunately wecould not generate sequence data for museum specimens fromthe western Indian Ocean type locality of this species (Mauritius).

In agreement with the variability indicated by the molecularphylogeny, the S. monotuberculatus specimens sequenced for this

Fig. 1. Bayesian consensus trees based on the last 75,000 maximum likelihood trees for A. COI and B. 16S. The Bayesian posterior probability (in%) is given near the node forthe Bayesian analysis (left side). The percentage of bootstrap replicates (out of 1000 replicates) is given for the maximum parsimony analysis (right side). Nodes with singlevalues represent Bayesian posterior probability, node not supported by maximum parsimony.

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1073

study were morphologically variable in appearance (Fig. 2F-I). Thiswas particularly evident in the expression of the dorsal papillae.For instance adult S. monotuberculatus from the GBR had low,

wart-like dorsal papillae and prominent lateral papillae evidentwhen foraging at night (Fig. 2F), similar to those described forthe closely related species, S. rubramaculosa Massin et al., 2002.

Fig. 1 (continued)

1074 M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

However, those from Samoa had spire-like dorsal papillae similarto those of S. horrens (Fig. 2H) and to those of S. monotuberculatusfrom the type locality (Mauritius) Cherbonnier (1952). Juvenile S.

monotuberculatus identified from the Northern GBR (Lizard Island)also had large papillae (Fig. 2I), though this may denote a juvenilefeature.

Fig. 2. (A–D) Stichopus horrens from One Tree Reef, GBR. Conical papillae cover the dorsal surface. (E) S. naso from Moreton Bay, Queensland. Note the distinct lumpyappearance. The rectangular profile of this specimen indicates a recent fission event. (F–G) S. monotuberculatus from One Tree Reef at night (F) with erect papillae and under arock during the day (G) with collapsed papillae. Note the distinct lateral papillae and wart-like dorsal papillae on the specimen photographed at night. (H) S. monotuberculatusfrom Samoa with a cover of high-spired papillae. (I) Juvenile S. monotuberculatus from Lizard Island, GBR. scales: (A and D) 4.0 cm; (B and C) 2.0 cm; (E) 1.5 cm; (F) 8.5 cm; (G)10 cm, (H) 3.0 cm; (I) 0.3 cm.

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1075

3.4. Sequence divergence, evolutionary rates and asexual reproduction

Sequence divergence (expressed as Kimura 2 Parameter dis-tance) between species within the genus Stichopus is between1.1% and 16.2% for COI and 1.4% and 15.3% for 16S (Table 3). Sur-prisingly little intra-specific sequence variation existed in theStichopus species ranging from 0% (S. chloronotus) to 1.2% (S. hor-rens) for COI, and from 0% (S. vastus) to 1.5% (S. horrens) for 16S.This low intra-specific divergence rates for S. chloronotus werefrom specimens as distant as Australia and Reunion.

The COI sequence data generated for putative geminate species,I. badionotus (Caribbean) and I. fuscus (Pacific) from either side ofthe Isthmus of Panama was use to estimate a molecular clock for

evolution of the Stichopodidae. The genetic distance between I. fus-cus and I. badionotus is on average 5.6%. With the assumption that I.fuscus and I. badionotus are closest living relatives and closure ofthe Isthmus of Panama at ca. 3.1 Ma (Lessios, 2008), the rate of evo-lution for COI was estimated to be 1.81% Ma�1(or 1% = 553.000 y).We used this rate and the maximum rate inferred for echinodermCOI (3.5% Ma�1, Lessios, 2008) to estimate a range of clade and spe-cies ages (Table 4). This analysis suggests that the Isostichopus andStichopus clades diverged ca. 5.5–10.7 Ma. The split betweenS. chloronotus and all remaining Stichopus was estimated to be be-tween 4.6 and 8.8 Ma. The youngest splits between S. herrmanniand S. ocellatus and that between S. monotuberculatus and S. horrenswere estimated to be younger that 1 Ma.

Fig. 3. Body wall ossicles. (A) Stichopus naso has diagnostic large C-ossicles. (B–D) S. horrens has diagnostic tack-like table ossicles.

1076 M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

Several Stichopus species reproduce asexually splitting theirbody by transverse fission, a feature not observed for Stichopodi-dae outside the genus (Conand et al., 2002; Uthicke and Conand,2005a, b). The distribution of asexual reproduction in Stichopusspecies, maximum adult size and genetic distances are illustratedin a Neighbor-Joining tree (Fig. 4). Fission is characteristic of spe-cies with smaller maximum adult size (ca. <30 cm length) includ-ing S. chloronotus, S. naso and the S. monotuberculatus complex. Onepossible exception is S. horrens, but the reproductive biology of thisspecies has not been documented. Asexual reproduction is absentin the large bodied species, S. herrmanni, S. ocellatus and S. vastus(>40 cm length).

3.5. Haplotype network and potential cryptic species inS. monotuberculatus

Because both COI and 16S indicated the presence of sub-cladesin the S. monotuberculatus clade we examined this clade with Sta-tistical parsimony analysis. Haplotype network analyses with COIand 16S for S. monotuberculatus indicated that the sequences fellinto a single 95% confidence network for both markers (Fig. 5).Most individuals grouped into a few larger haplotypes that aregenerally separated by a few mutational steps. However, two spec-imens from Samoa formed a separate haplotype with both markersand were separated from all other common haplotypes by severalmutational steps. Although the sample size is too small for a pop-ulation genetic analysis, the most common haplotype occurs in Sa-moa and the Northern GBR. The museum specimen collected in1961 from Heron Island, Southern GBR only amplified for 16Sand had the same haplotype as specimens collected for this study(in 2005) from Heron Island and Magnetic Island. The presence of

rare haplotypes that are several mutational steps removed fromother haplotypes in Fig. 5 suggests that there may be cryptic spe-cies within S. monotuberculatus.

4. Discussion

4.1. Phylogenetic relationships

Phylogenetic analyses using two mitochondrial markers pro-vided insights into the taxonomic relationships within the familyStichopodidae, and in particular for the genus Stichopus. This wasespecially the case for COI, where more sequence data on a highertaxonomic level were available. Most currently recognised generaformed separate clades supported by high bootstrap values andthe species formed clades that agree with taxonomic revisionsbased on morphology (Rowe and Gates, 1995; Massin, 1999,2007; Massin et al., 2002). The trees generated for both markerswere similar with the major Indo-West Pacific species S. chlorono-tus being a sister taxon to all other Stichopus.

As seen in population genetic and phylogenetic studies ofHolothuria species (Uthicke and Benzie, 2003; Uthicke et al.,2004b), COI is a good marker to document intra-specific relation-ships and evolutionary pathways of closely related stichopodids.Investigation on a higher phylogenetic level required a slower evolv-ing marker such as 16S, as found elsewhere for holothuroid phylog-eny (Kerr et al., 2005). However, 16S, in conjunction with allozymes,was also used as a marker to distinguish closely related sister Holoth-uria species and investigate hybridisation (Uthicke et al., 2005).

The phylogenetic analyses of COI and 16S indicated that Stich-opus is monophyletic with the only exception being the Australian

Fig. 4. Neighbor-Joining tree (1000 bootstraps) on Kimura two parameter genetic distances for COI between Stichopus species and distribution of asexual reproduction byfission and indication of maximum adult size. The collapsed tree shows that the fissiparous (grey) species have smaller maximum size than the non fissiparous (black)species. Stichopus horrens is not known to exhibit fission, but its reproductive biology is poorly documented.

Fig. 5. Ninety-five percent confidence network using sequences (top, COI; bottom16S) for all S. monotuberculatus specimens. The size of the circle is proportional tosample size. Samples from the three major regions are colour coded. Small blackcircles along lines represent substitution of one base pair.

Table 3Kimura 2 parameter distances for Stichopus species, COI distances are above diagonal and 16S distances below. The numbers along the diagonal in bold represent average withinspecies difference (COI/16S).

1 2 3 4 5 6 7

(1) S. chloronotus 0/0.002 0.142 0.138 0.157 0.157 0.162 0.154(2) S. monotuberculatus 0.128 0.006/0.007 0.027 0.072 0.068 0.070 0.127(3) S. horrens 0.134 0.031 0.012/0.015 0.068 0.065 0.072 0.119(4) S. herrmanni 0.143 0.041 0.052 0.002/0.002 0.011 0.030 0.126(5) S. ocellatus 0.143 0.038 0.046 0.014 0.007/0.002 0.024 0.121(6) S. vastus 0.153 0.048 0.056 0.030 0.028 0.004/0 0.117(7) S. naso 0.122 0.079 0.084 0.079 0.074 0.078 0.003/0.002

Table 4Estimated clade ages (expressed as divergence times from the respective sister cladein Ma) of members of the genus Stichopus, based on estimates for COI mutation ratesin geminate Isostichopus spp. (1.81% Ma�1) from this study and the highest value forCOI mutation rates in echinoderms (3.5%) (Lessios, 2008).

1.81% 3.50%

Isostichopus vs. Stichopus 10.67 5.52S. chloronotus vs. S. naso, S. monotuberculatus, S. horrens, S.

herrmanni, S. ocellatus, S. vastus8.84 4.57

S. naso vs. S. monotuberculatus, S. horrens, S. herrmanni, S.ocellatus, S. vastus

6.86 3.55

S. horrens and S. monotuberculatus vs. S. herrmanni, S.ocellatus, S. vastus

3.76 1.95

S. vastus vs. S. herrmanni, S. ocellatus 1.49 0.77S. herrmanni vs. S. ocellatus 0.33 0.17S. monotuberculatus vs. S. horrens 1.00 0.52

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1077

warm-temperate (see Wilson and Allen, 1987) species S. ellipes,from New South Wales, which clustered outside the group with16S. This result was obtained with historic museum material(Rowe and Gates, 1995) from which only one marker was success-ful. Although the specimen used was compared with the holotype,it would be useful to revisit this result with fresh specimens alongwith inclusion of a second temperate species not investigated here,S. ludwigi, from southern Australia. The presence of S. ellipes out-side Stichopus is a surprising result for a species that morphologi-cally appears to be a Stichopus species. This suggests thatmorphological traits considered characteristic for the genus (e.g.C-ossicles, Fig. 3A) may need to be re-evaluated. The presence of‘shield-shaped’ tentacles, abundant tube feet, tentacle ampullaeand two gonad tufts (Haeckel, 1896) and body wall ossicle form(Clark, 1922) has been considered sufficient justification to referspecies to the genus Stichopus. Recent revision of the Australiantemperate species, S. mollis to a new genus, Australostichopus,was largely based on body chemistry and internal anatomy (Moraeset al., 2004). Taxonomic separation of North Pacific stichopodids(Parastichopus californicus and Apostichopus japonicus) from Stich-opus is also supported by data from chemistry (Levin et al., 1986).

Stichopus species appear to be restricted to tropical and sub-tropical regions of the Indo west-Pacific. In our phylogeny, thegenus Stichopus is separated from the east Pacific/Caribbean genusIsostichopus and from temperate genera (e.g. Australostichopus mol-lis: South-west Pacific; P. californicus and A. japonicus: North Paci-fic). Parastichopus is otherwise a North Atlantic genus restrictedto P. tremulus (type species for the genus) and P. regalis. Our resultssupport Australostichopus as a valid genus potentially endemic tothe Tasman region, though we have not investigated its relation-ship with the monotypic genus Neostichopus (South Africa/WesternIndian Ocean). In light of several generic revisions of temperate sti-chopodids (Levin et al., 1986; Moraes et al., 2004) it appears thatthe current inclusion of temperate species in the genus Stichopus,including those not sampled here (e.g. S. ludwigi) warrants exami-nation. In the COI trees, the North Pacific species Parastichopus

1078 M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

californicus and A. japonicus clustered as a monophyletic unit. Thepotential congeneric affinity of these species is supported by bodywall ossicle morphology (Lambert, 1986; Imaoka, 1991). Parastich-opus tremulus and P. regalis, from the North Atlantic differ fromtheir North Pacific congeners in ossicle form, particularly in lacking‘button’-type ossicles (Rowe, unpubl.).

Separation of S. herrmanni, S. ocellatus and S. vastus in one cladewith high support values corroborates recent taxonomic revisions(Rowe and Gates, 1995; Massin et al., 2002). These three speciescomprise the so-called commercial ‘curry fish’ group; readily iden-tified in the field by their distinctive appearance and large size.

4.2. S. horrens and S. monotuberculatus

In both COI and 16S trees S. horrens formed a discrete cluster, asister taxon to the variable S. monotuberculatus complex. Of the 50specimens identified by field collectors as S. horrens only two ofthese were revealed to be S. horrens. The others were placed inthe S. naso or S. monotuberculatus clades. In total we obtained fivespecimens of S. horrens (field and museum collections) from dispa-rate localities across the tropical Pacific range of this species, Gala-pagos, Philippines, Hawaii, Samoa and Australia. Molecular supportfor a distinct S. horrens clade corroborates morphological taxon-omy based on spicule structure (Rowe and Doty, 1977; Massinet al., 2002). Examination of body wall spicules from museumand freshly collected specimens revealed the distinct tack-like ta-ble ossicles, a diagnostic character used to identify S. horrens.

Despite the presence of distinct morphological characters of S.horrens sensu stricto, this taxon name is often incorrectly used infield studies and by fishers (e.g. Harriott, 1982; Young and Ryan,2004; Eriksson et al., 2007; Kohtsuka et al., 2005). S. horrens how-ever did have about twice the within species sequence variationcompared with other species, a feature indicating the presence ofcryptic diversity. Our analyses only included five specimens, andgiven the vast distribution of S. horrens in the tropical Pacific, itseems likely that more variability will be discovered. Specimensof S. horrens from near the type locality of this taxon (Society Is-lands, French Polynesia) need to be examined.

The two species often mistakenly called S. horrens, that is S. nasoand S. monotuberculatus, are common in the tropical waters of east-ern Australia. Extensive night-time searches (GBR, Samoa) indi-cated that S. monotuberculatus is a common and conspicuousnocturnal species on reefs (Byrne and Eriksson, pers obs). Theabundance of S. naso and S. monotuberculatus may be due to theirfissiparous reproduction which can result in high local densitieson coral reefs (Uthicke, 2001b). Thus far fissiparity has not been re-ported for S. horrens sensu stricto. Earlier descriptions of asexualreproduction in this species (Harriott, 1982; Kohtsuka et al.,2005) referred to S. monotuberculatus and S. naso. Although S. hor-rens was found at a number of locations along the GBR, it appearsto be uncommon in the shallow tropical waters of eastern Austra-lia. Concerted effort is needed to find this species in night searches.

According to both mitochondrial markers S. monotuberculatus isa variable assemblage including several sub-clades. S. quadrifascia-tus Massin, 1999 and S. rubramaculosus from Malaysian waters ap-pear to fall in this clade based on morphology, but we have notused molecular data to examine these taxa. The variability of theS. monotuberculatus group is reflected by their morphologicaldiversity. For instance the S. monotuberculatus from tropical NEAustralia have low wart-like papillae and prominent lateral papil-lae (Fig. 2). They accord in body and spicule form with S. rubrama-culosus, but lack the red colour associated with this species (Massinet al., 2002). The specimens from Samoa had taller dorsal papillae,similar to those of S. horrens and are also similar to S. monotuber-culatus from its type locality (Mauritius) (Cherbonnier, 1952).Massin et al. (2002) indicate that the presence of prominent

papillae is diagnostic for S. monotuberculatus, but this feature isnot characteristic of the morphs on the GBR. Regional differencesin body wall spicules, external morphology and colour indicatethe potential for taxonomic diversity in S. monotuberculatus acrossits wide Indo-Pacific distribution (Red Sea and Madagascar to Eas-ter Island; Massin, 1996; Massin et al., 2002). The figure of S. var-iegatus from the Philippines (drawn in Semper, 1867) should bereferred to S. monotuberculatus (see Rowe and Gates, 1995; Massin,1999). Molecular analysis of specimens from the type locality ofmonotuberculatus (Mauritius) is required to determine the identityof S. monotuberculatus sensu stricto. There is an urgent need to char-acterise the species composition of the developing East AfricanIndian Ocean bêche-de-mer fishery that appears to includeS. monotuberculatus and other cryptic species (Eriksson and Byrne,pers obs).

Further research is required to determine if the variable mor-phology and molecular diversity within the Pacific S. monotubercul-atus investigated here is linked to different species or potentialhybridisation between this species and the closely related S. hor-rens. Hybridization has been documented in molecular studies ofother bêche-de-mer species (Uthicke et al., 2005, 2010). In additionstudies of widely distributed echinoderm species (Dartnall et al.,2003; Hart et al., 2003, 2006; O’Loughlin and Rowe, 2006; Uthickeet al., 2010) have revealed the presence of cryptic species. Revisionof S. monotuberculatus remains a challenge. The molecular phylog-eny established here for Stichopus provides a framework to assistwith this.

Haplotype-network based approaches to recognising speciesboundaries have been used in several recent studies where theanalyses have indicated the presence of cryptic species withinnominal taxa (e.g. Hart et al., 2006; Hunter and Halanych, 2008).In these studies multiple haplotype networks are interpreted asindication of multiple species. Although multiple networks werenot present in S. monotuberculatus, the presence of several haplo-types removed by several mutational steps from the most commontypes indicate that cryptic species may exist in the S. monotubercul-atus analysed here.

4.3. Clarification of commercially exploited Stichopus

Comparison of sequence data from species being fished or listedas commercial species indicates that at least three species are cur-rently being fished under the name S. horrens. The species listed inthe south Queensland (East Australia) fishery as S. horrens is S. nasoand the species fished as S. horrens in Samoa is S. monotuberculatus(Young and Ryan, 2004; Eriksson et al., 2007). In the Galapagos S.horrens is cited as forming the basis of a fishery (Toral-Granda,2008), but the photograph of a specimen from this fishery (inHearn and Pinnillos, 2006) indicates that it differs from S. horrens.Unfortunately we were not able to obtain a sample from the Gala-pagos fishery. It is difficult to manage a fishery without knowingwhat species are harvested. For the ‘S. horrens fishery’ several spe-cies, potentially differing ecologically are being fished under thisname. This study and ongoing investigations on the identity of glo-bal bêche-de-mer species (Uthicke et al., 2005, 2010) will contrib-ute to the conservation and sustainable use of these resources.

4.4. Evolution of fission in Stichopus

Asexual reproduction through transverse fission is characteris-tic of several small-bodied Stichopus species including S. chlorono-tus, the most abundant shallow water Stichopus species across theIndo-Pacific (Uthicke, 1999, 2001a). The high density and successof this species with respect to local abundance has been attributedin part to its propensity for asexual reproduction (Conand et al.,1998, 2002; Uthicke, 2001a; Uthicke and Conand, 2005a, b).

M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 1079

Asexual reproduction is also common in S. naso and S. monotuber-culatus, which are also locally abundant (Kohtsuka et al., 2005;Massin, 2007). The original description of S. naso (Semper, 1867)was based on a regenerating specimen with an anterior ‘nose-like’protrusion – the regenerating anterior region of the body. Largebodied stichopodids (e.g. S. herrmanni) are not known to exhibitfission. They may not be able to sustain respiration following lossor damage of respiratory organs due to fission, whereas small indi-viduals can acquire sufficient oxygen through the body wall(Uthicke, 1998). Asexual reproduction is not known for S. horrens,but the reproductive biology of this species has not been investi-gated. Although they do not exhibit fissiparity, large bodied sticho-podids use the break down of mutable body wall connective tissue,the mechanism that underlies fission, for defence.

Both Bayesian and Parsimony phylogenetic methods identifiedS. chloronotus as the basal taxon in the genus Stichopus. The pres-ence of fission in this and other clades (S. naso, S. monotuberculatus)with smaller maximum body size suggests that asexual reproduc-tion may have been lost in evolution of larger body size (e.g. S. herr-manni, S. vastus, S. ocellatus), representing a single loss withinStichopus. However, the alternative hypothesis that asexual repro-duction has evolved multiple times within the genus Stichopus isequally parsimonious. Asexual reproduction is not known for theother stichopodid genera. Fission also occurs in Holothuria species(Conand, 1996; Uthicke, 1998) and so may be a basal character ofthe Aspidochirotida or evolved independently in Holothuria andStichopus.

The within stichopodid species COI sequence variation arewithin the range observed in other holothuroid genera (e.g. Boh-adschia sp ca. 0.6–0.7%; Holothuria nobilis, H. whitmaei 0.5–0.5%)(Uthicke et al., 2004b, 2010; Clouse et al., 2005). In other echino-derms (e.g. Echinometra sp., Linckia sp.) within species differencesup to 3% are reported (Williams, 2000; Landry et al., 2003). SomeStichopus species however had a very low within species sequencedivergence. The lack of sequence divergence within S. chloronotusappears paradoxical given the large geographic distance betweensamples (Australia to Reunion). One possible hypothesis for thelow intra-specific diversity in some Stichopus species may beslower evolutionary rates due to asexual reproduction. However,genetic divergence described here for asexual (COI: 0–0.6%; 16S:0.2–0.7%) and sexual species (COI: 0.2–1.2%, 16S: 0–1.5%) overlapand provide no support for this hypothesis. This analysis is some-what confounded by sampling on different geographic scales.However, since asexual species also reproduce sexually (broadcastspawners), fission could simply be seen as an amplification of thegenome to enhance success of density dependent sexual reproduc-tion (Uthicke et al., 1998). It appears that asexual reproductioncannot explain the low within species sequence divergence in mostof the Stichopus species.

4.5. Evolution of Stichopus

Evolutionary rates estimated for the COI region (�1.8% Mya�1)are on the lower end of the range estimated for other echinoderms(1.6–3.5% Mya�1, reviewed by Lessios, 2008). Using our rates andthe upper end of previous estimates the age of the split betweenStichopus and Isostichopus, ca. 5.5–10.7 Ma suggests a Miocene evo-lution of these genera. The most derived taxa and most recentsplits between species were estimated to be younger than 1 Main the Pleistocene. This range of evolutionary ages is similar to thatreported for other echinoderms based on use of transithian gemi-nate species. Several species of the echinoid genus Diadema di-verged in the Pleistocene although the genus is known from theMiocene (12 Ma) (Lessios et al., 2001). Similarly, several speciesof the echinoid genus Echinometra and asteroid genus Cryptasterinadiverged only 0.5–1.6 Ma (McCartney et al., 2000; Hart et al.,

2003). Estimates of divergence times based on comparison of se-quence data from putative geminate species is however fraughtwith difficulty (Lessios, 2008; Marco and Moran, 2009). We donot know for instance if the Isostichopus species include undetectedcryptic species raising the possibility that the sequences used maynot be from closest living relatives. This problem was recentlyidentified in a study of geminate bivalve species (Marco and Mor-an, 2009).

The order Aspidochirotida is evolutionarily old with the oldestrecorded family (Synallactidae) described from the mid Triassic(Amesian: 225–220 Ma, Gilliland, 1993). The Stichopodidae is con-sidered to be geologically young because it has not been identifiedin fossil deposits (Gilliland, 1993). Our genetic data generally sup-port Gilliland’s (1993) notion that Stichopodidae are evolutionarilyrelatively young, but our estimates indicate that they should bedetectable in fossil deposits. An earlier evolution of the Stichopodi-dae is indicated by the recent discovery of fossil ‘table-form’ spic-ules from the late Cretaceous (ca. 70 Ma) (Reich, 2001). In light ofshared characters between recent Stichopodidae and some recentsynallactid taxa (Rowe, unpubl.) however, the family Synallactidae,sensu extenso appears to comprise a heterogeneous assemblage ofgenera, posing the possibility that some fossil representativesmay be incorrectly ascribed to that family instead of theStichopodidae.

In conclusion, genetic analyses of two mitochondrial markerslargely supported current taxonomy of the Stichopodidae on high-er level and species scales. However, the need for a reassessment ofdiagnostic morphological characters was suggested by geneticanalyses (e.g. S. ellipes). In addition, we clarified the species statusof several commercial species and highlighted difficult groups (e.g.S. monotuberculatus, S. horrens) that may harbour cryptic speciesand warrant further investigation with species being harvestedfor bêche-de-mer of particular concern.

Acknowledgments

The research was funded by Australian Biological ResourcesSurvey and a grant from the FAO. Thanks to many colleagues thatprovided specimens, photographs and other assistance, particu-larly Hampus Eriksson, Tim O’Hara, Steve Purcell and MaraWolkenhauer. We are grateful to Stephen Keable, AustralianMuseum; Andrew Cabrinovic, British Museum; Chris Bartlett,Museum of Tropical Queensland; Mark O’Loughlin, MuseumVictoria and Gustav Paulay, Florida Museum of Natural Historyfor assisting us with access to museum specimens or sequencedata. Anne Hoggett and Thierry Rakotoarivelo provided photographs.Assistance from Zoran Ilic, Paula Cisternas, Natalie Soars and ErikaWoosley is gratefully acknowledged. We also thank the reviews forhelpful comments on the manuscript. This represents contributionno. 34 from the Sydney Institute of Marine Science and 1292 LizardIsland Research Station.

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