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

Molecular Phylogenetics and Evolution 56 (2010) 1068–1081

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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, Australia Australian Museum, 6 College St., Sydney, NSW 2010, Australia and Beechcroft, Norwich Road, Scole, Norfolk IP21 4DY, UK c Australian Institute of Marine Science, PMB No. 3, Townsville MC, Qld 4810, Australia b

a r t i c l e

i n f o

Article history: Received 4 February 2010 Revised 6 April 2010 Accepted 9 April 2010 Available online 23 April 2010 Keywords: Phylogeny Evolution Stichopodidae Holothuroidea COI 16S Morphology

a b s t r a c t The Stichopodidae comprise a diverse assemblage of holothuroids most of which occur in the Indo-Pacific. 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 the family. A monophyly of the genus Stichopus was supported with the exception of Stichopus ellipes. Molecular analyses confirmed genus level taxonomy based on morphology. Most specimens harvested as S. horrens fell in the S. monotuberculatus clade, a morphologically variable assemblage with others from the 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 estimated that Stichopus and Isostichopus diverged ca. 5.5–10.7 Ma (Miocene). More recent splits were estimated to be younger than 1 Ma. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Sea cucumbers (Holothuroidea) in the order Aspidochirotida are a conspicuous and diverse group in the world’s oceans. They are particularly prominent in tropical regions where they inhabit soft sediment and reef environments (Rowe and Doty, 1977; Conand, 1990; Rowe and Gates, 1995; Massin, 1999, 2007; Rowe and Richmond, 2004). Aspidochirotids provide important ecosystem services enhancing nutrient cycling and local productivity in oligotrophic carbonate sediments through their bioturbation and deposit feeding activities (Uthicke, 1999, 2001a, b). They are also fished 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 being large and often the dominant mobile invertebrates on reef flats and lagoons, the taxonomy of many aspidochirotids is uncertain. This is due to the difficulty in application of traditional taxonomic characters (e.g. body profile, skeleton morphology) which may not have been used in sufficient detail to distinguish cryptic species currently included within a single taxon and the difficulty of extracting taxonomic data from museum specimens (Massin, 1999; Massin et al., 2002; Uthicke et al., 2004b). Determination * Corresponding author. Fax: +61 2 9351 2813. E-mail address: [email protected] (M. Byrne). 1055-7903/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2010.04.013

of the correct taxonomy of aspidochirotids such as the Stichopus species investigated here is timely, as the fishery for these species is expanding and correct identification is critical for management and conservation (Conand, 2001, 2008; Shepherd et al., 2004; Uthicke et al., 2004a). The family Stichopodidae has long been a taxonomic challenge (Rowe and Gates, 1995; Massin, 1999; Massin et 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 in clarification 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 described species (Rowe unpubl.). Clark’s (1922) major taxonomic revision, based on gross morphology and skeletal ossicle form of preserved, historic, material divided the family into four genera: Stichopus Brandt, 1835; Thelenota Brandt, 1835; Parastichopus H.L.Clark, 1922; and Astichopus H.L. Clark, 1922. Five more genera have since been described: Neostichopus Deichmann, 1948; Isostichopus Deichmann, 1958; Apostichopus Yulin Liao, 1980; Eostichopus 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 and Richmond, 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

synonymy of S. horrens (Rowe and Gates, 1995; Rowe and Richmond, 2004). Five new species have been added to the genus Stichopus since the mid-1960s (Cherbonnier, 1967, 1980; Massin, 1999; Massin et al., 2002). The fishery based on Stichopus, which extends from the Galapagos to the Pacific islands, is problematic with several species being misidentified under the name ‘S. horrens’. Here we revisited S. horrens as a nominal taxon in a molecular and morphological study to clarify the species being harvested as in previous studies of the teatfish and sandfish groups (Uthicke et al., 2004b, 2005; Massin et al., 2009). Clarification of the identity of commercial species provides essential data for fishery management and conservation of several potentially vulnerable species (Bruckner, 2006; Uthicke et al., 2010). We undertook a phylogenetic analysis of the Stichopodidae using sequence data for two mitochondrial genes (COI, 16S rRNA). The aims were to clarify the taxonomic uncertainties of specimens identified as S. horrens and S. monotuberculatus and to test hypotheses on genera and species monophyly and on the evolutionary relationships within the Stichopodidae. We place our data in context with regard to the cosmopolitan distribution of the family using sequence data for specimens collected mainly from the Indo-Pacific (Galapagos to Reunion and from the Philippines to Australia and New Zealand) and also included sequence for several temperate species. Sequence data for 111 individuals across seven genera and 17 nominal species including voucher registered museum specimens were used. Assisted by traditional morphological taxonomy, we matched where possible DNA from museum specimens with those collected for this study. The research covered the geographic range of tropical Stichopodidae from shallow water with most material from the tropical Pacific. Revisiting the identity and distribution of the Stichopodidae provided insights into the presence of cryptic species and evolution of the family across the Indo-Pacific. The Stichopodidae and holothuroids in general have a poor fossil record (Reich, 2001) and, in the absence of fossil calibrated divergence time, we used the COI phylogeny and sequence data from putative geminate Isostichopus species from either side of the Isthmus of Panama to estimate divergence times for stichopodid species, as in previous studies (Lessios, 2008). The Stichopodidae are unusual in the presence of clonal reproduction by transverse fission, a feature characteristic of several of the most common and abundant members of the family (Uthicke et al., 1998; Conand et al., 2002; Uthicke and Conand, 2005a, b; Massin, 2007). We used the phylogeny to address hypotheses on evolutionary trends in the distribution of asexual reproduction in the stichopodid clades.

2. Materials and methods 2.1. Sampling of taxa Tissue samples preserved in ethanol or DMSO were obtained for specimens of Stichopodidae across the Indo-West Pacific and from specimens registered at the Australian Museum, British Museum of Natural History and Museum of Victoria (Table 1). Specimens collected by the seabed biodiversity project in north Queensland (http://www.reef.crc.org.au/resprogram/programC/seabed/index.htm) lodged in the Museum of Tropical Queensland were also sampled. In tropical Australia we made a concerted effort to locate Stichopus horrens in surveys extending from the northern (Raine Island, Moulter Cay, Quoin Island, Lizard Island) to the southern (One Tree Island, Heron Island) Great Barrier Reef (GBR) and Moreton Bay (Table 1). Several Stichopus species (S. horrens, S. naso, S. monotuberculatus) are nocturnal (Rowe and Doty, 1977; Massin, 2007) and night-time searches were conducted. Most specimens col-

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lected for this study were sampled at depths ranging between 1 and 15 m. Specimens from the seabed biodiversity project were collected at 15–30 m depth. Geographically we included specimens from the Indo-Pacific from the Galapagos to Reunion and from the Philippines to Australia and New Zealand (Table 1) and included one Caribbean species, Isostichopus badionotus from Cuba. In total, sequence data were generated for 100 specimens of Stichopodidae (Table 1) including 50 identified by fishery scientists as S. horrens. Where possible we sequenced a minimum of three specimens per taxon. Sequence data from GenBank were available for several temperate and tropical members of the Stichopodidae (Table 1). Additional sequence data were obtained courtesy of Dr. Gustav Paulay (Florida Museum of Natural History). Species names and GenBank accession numbers for all sequences used are shown in Table 1. Sequence alignments are available from the authors on request. In addition, body wall spicule preparations and live photographs available for Australian Museum specimens provided reference material to check the identity of specimens collected for this study. Spicule preparations were made from the body wall of specimens collected for this project. Small sections of body wall were treated with diluted bleach to isolate the spicules and prepare whole mounts slides for light microscopic examination and photography (Rowe and Doty, 1977). A photographic record of the specimens was also made. 2.2. DNA extraction, PCR protocols and sequencing For DNA extractions approximately 10–20 mg of tissue was placed in a 1.5 ml microcentrifuge tube and extracted with a DNeasy tissue extraction kit following the manufacturers’ specifications (Qiagen; DNeasy Blood and Tissue Kit). Sections of the cytochrome oxidase subunit I (COI) and the large subunit 16S ribosomal DNA (16S rDNA) genes were amplified using primers COIe-F and COIe-R (Arndt et al., 1996) and 16SA-R and 16SB-R (Palumbi et al., 1991), respectively (Table 2). We used these markers following previous studies where these have proven useful for taxonomy and phylogeny at the subfamily level (Arndt et al., 1996; Uthicke et al., 2004a, b, 2010). PCR amplification was conducted as previously described (Uthicke and Benzie, 2003; Uthicke et al., 2004b), using final concentrations 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 HotMaster Taq DNA polymerase (Eppendorf) and 40–80 ng DNA. PCR reactions involved denaturation for 60 s at 95 °C followed by 40 cycles of 30 s denaturation at 95 °C, 30 s annealing at 50 °C, and 80 s extension at 72 °C, and final extension of 10 min. PCR products were cleaned using QIAquick PCR purification kit (Qiagen) and diluted to a final concentration of 10–25 lg/ml. For the sequencing reaction, we used Amersham (Dyenamic) sequencing reagents. DNA from each sample was sequenced in both directions, and sequencing products were cleaned using Autoseq50 (Amershan) clean up columns. The PCR amplicons were purified using the GFX PCR DNA Gel Band Purification Kit (GE Healthcare) according to manufacturers’ specifications. Some amplicon purification and all sequencing was done by Macrogen (Macrogen DNA Sequencing Service; Seoul, South Korea). Sequences were aligned using CLUSTAL W 1.5 (Thompson et al., 1994) under default parameters and checked by eye. 2.3. Sequence analyses and phylogeny The COI and 16S sequence data were used for phylogenetic analyses using Bayesian Markov Chain Monte Carlo (MCMC) as implemented in MrBayes (v.3.1, Ronquist et al., 2005). Prior to the analyses we tested for the most appropriate nucleotide

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Table 1 Stichopodidae analysed for the partial cytochrome oxidase subunit 1 (COI) and 16S genes. Tree ID denotes the abbreviation used in phylogenetic trees. GenBank accession numbers 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, Museum Victoria (MV); Dr. Gustav Paulay provided sequence for specimens in the University of Florida Museum of Natural History (UF). Museum registration numbers are indicated and sample 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 was used as the outgroup. Species

Location

Tree ID

COI

16S

Stichopus chloronotus

Reef 18–026, Central GBR, Aus Reef 18–026, Central GBR, Aus Reef 18–026, Central GBR, Aus Reef 18–026, Central GBR, Aus Derder Reef, Torres Strait, Aus Derder Reef, Torres Strait, Aus Derder Reef, Torres Strait, Aus Derder Reef, Torres Strait, Aus Etang Sale, Reunion FRA Etang Sale, Reunion FRA Etang Sale, Reunion FRA Etang Sale, Reunion. FRA

Sc1.Cent-GBR Sc2.Cent-GBR Sc3.Cent-GBR Sc4.Cent-GBR Sc1.TS Sc2.TS Sc3.TS Sc4.TS Sc1.RI Sc2.RI Sc3.RI Sc4.RI

EU856610 EU856611 EU856612 EU856613 EU856614 EU856615 EU856616 EU856617 EU856618 EU856619 EU856620 EU856621

EU856682 EU856683 EU856684 EU856685 EU856686 EU856687 EU856688 EU856689 EU856690 EU856691 EU856692 EU856693

S. monotuberculatus

Heron Heron Heron Heron Heron Heron Heron Heron Heron Heron Heron

Sm1.HI-GBR Sm2.HI-GBR Sm3.HI-GBR Sm4.HI-GBR Sm5.HI-GBR Sm6.HI-GBR Sm7.HI-GBR Sm8.HI-GBR Sm9.HI-GBR Sm10.HI-GBR SmHI-GBR (AM)

EU856574 EU856577 EU856578 EU856580 EU856581 EU856582 EU856583 EU856584 EU856585 EU856579

Sm1.OTI-GBR Sm2.OTI-GBR Sm3.OTI-GBR Sm4.OTI-GBR Sm1.TSAM Sm2.TSAM Sm3.TSAM Sm4.TSAM Sm1.SSAM Sm2.SSAM Sm3.SSAM Sm4.SSAM Sm5.SSAM Sm6.SSAM Sm.Li-GBR Sm1.MI-QLD Sm2.MI-QLD Sm3.MI- QLD Sm4.MI-QLD Sm5.MI-QLD Sm-PAL Sm-Poh

EU856562 EU856565 EU856568 EU856571 EU856564 EU856567 EU856570 EU856573 EU856563 EU856566 EU856569 EU856572 EU856575 EU856576 EU856556 EU856557 EU856558 EU856559 EU85660 EU85661

EU856645 EU856648 EU856651 EU856654 EU856647 EU856650 EU856653 EU856656 EU856646 EU856649 EU856652 EU856655 EU856657 EU856658 EU856639 EU856640 EU856641 EU856642 EU856643 EU856644 EU856540 EU856543

One Tree Island, GBR, Aus Hawaii (UF#1176) Galapagos (UF – Hickman #97–360) Phillipines Samoa, (BM# 1970.10.8.57)

Sh.OTI-GBR Sh.HAW Sh.GAL

EU856554

EU856638 EU856542 EU856541

Sh.Ph3 Sh.SAM(BM

EU848282 EU856555

EU822434 EU856637

Moreton Bay, Qld, Aus Moreton Bay, Qld, Aus Moreton Bay, Qld, Aus Moreton Bay, Qld, Aus Moreton Bay, Qld, Aus GBR, Aus(MTQ #SBD24743) GBR, Aus (MTQ#SBD24704) GBR, Aus (MTQ#SBD24742) GBR, Aus (MTQ#SBD025742) GBR, Aus (MTQ#SBD01059) New Caledonia (FAO 055) New Caledonia (FAO 056)

Sn1.MOR-QLD Sn2. MOR-QLD Sn3. MOR-QLD Sn4. MOR-QLD Sn5. MOR-QLD Sn1.SBD-GBR Sn2.SBD-GBR Sn3.SBD-GBR Sn4.SBD-GBR Sn5.SBD-GBR Sn1.NC Sn2.NC

EU856586 EU856594 EU856595 EU856596 EU856597 EU856587 EU856588 EU856589 EU856590 EU856591 EU848280 EU848279

EU856666 EU856703 EU856704 EU856705 EU856706 EU856661 EU856662 EU856663

Is, Is, Is, Is, Is, Is, Is, Is, Is, Is, Is,

GBR GBR GBR GBR GBR GBR GBR GBR GBR GBR GBR

Aus Aus Aus Aus Aus Aus Aus Aus Aus Aus Aus

(AM #J17269) One Tree Is, GBR Aus One Tree Is, GBR Aus One Tree Is, GBR Aus One Tree Is, GBR Aus Toamua, Upolu Is Samoa Toamua, Upolu Is Samoa Toamua, Upolu Is Samoa Toamua, Upolu Is Samoa Salelologa, Savaii Is Samoa Salelologa, Savaii Is Samoa Salelologa, Savaii Is Samoa Salelologa, Savaii Is Samoa Salelologa, Savaii Is Samoa Salelologa, Savaii Is Samoa Lizard Is, GBR Aus Magnetic Is, Aus Magnetic Is, Aus Magnetic Is, Aus Magnetic Is, Aus Magnetic Is, Aus Palau (UF# 1589) Pohnpei (UF#2670) S. horrens

S. naso

EU856695 EU856696 EU856697 EU856698 EU856699 EU856700 EU856701 EU856702 EU856694 EU856659

FJ001809 FJ001810

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M. Byrne et al. / Molecular Phylogenetics and Evolution 56 (2010) 1068–1081 Table 1 (continued) Table 1 (continued) Species

Location

Tree ID

COI

16S

S. herrmanni

Heron Is, GBR Aus Heron Is, GBR Aus Heron Is, GBR Aus Heron Is, GBR Aus Heron Is, GBR Aus One Tree Is, GBR Aus Torres Strait Torres Strait Torres Strait Torres Strait New Caledonia (FAO 004) New Caledonia (FAO 005)

Sher1.HI-GBR Sher2.HI-GBR Sher3.HI-GBR Sher4.HI-GBR Sher5.HI-GBR Sher.OTI-GBR Sher1.TS Sher2.TS Sher3.TS Sher4.TS Sher1.NC Sher2.NC

EU856544 EU856546 EU856548 EU856550 EU856552 EU856553 EU856545 EU856547 EU856549 EU856551 EU848281 EU848278

EU856628 EU856630 EU856632 EU856634 EU856636 EU856627 EU856629 EU856631 EU856633 EU856635 EU822451 EU822450

S. vastus

Torres Strait Torres Strait Torres Strait Lizard Island, GBR, Aus (FAO 027)

Sv1.TS Sv2.TS Sv3.TS Sv.Li-GBR

EU856622 EU856623 EU856624 EU848275

EU856707 EU856708 EU856709

S. ocellatus

Torres Strait Torres Strait Torres Strait Bedarra Island, GBR, Aus Central, GBR Long Island, GBR, Aus Lindeman Island, GBR, Aus

So1.TS So2.TS So3.TS SoBI-GBR SoCent-GBR SoLongIs-GBR So.LindIs-GBR

EU856608 EU856609 EU856604 EU856605 EU856606 EU856607

S. ellipes

Batemans Bay, NSW Aus (AM #J14804) Jervis Bay, NSW Aus (AM #J12946)

Se.BB, NSW (AM) Se.JB NSW (AM)

Australostichopus mollis

New Zealand New Zealand New Zealand Eliza Pt., Tasmania, Aus Fisher’s Pt, Tasmania, Aus Hope Is, Tasmania, Aus Victoria, Aus (MV#) Port Hacking, NSW AUS (AM #J9224)

Am1.NZ Am2.NZ Am3.NZ Am1.TAS Am2.TAS Am3.TAS Am.VIC Am.NSW (AM)

EU856598 EU856600 EU856602 EU856599 EU856601 EU856603

EU856669 EU856671 EU856673 EU856670 EU856672 EU856674 EU856667 EU856668

Thelenota rubralineata

Phillipines (FAO 20)

TrPh

EU848260

EU822452

T. anax

Lizard Island, GBR (FAO 037) New Caledonia (FAO 046)

Tx-Li Tx-NC

EU848243 EU848292

T. ananas

Central GBR (FAO 070) Central GBR (FAO 071) Central GBR (FAO 072) New Caledonia

Ta1-GBR Ta2-GBR Ta3-GBR Ta1-NC

EU848258 EU848261 EU848259 EU848257

Astichopus multifidus

Cuba

Amu1CUBA

EU848293

EU822453

Isostichopus badionotus

Cuba

Ib1CUBA

EU848264

EU822435

I. fuscus

Mexico, Pacific Mexico, Pacific Mexico, Pacific

If1 If2 If3

(AF486427) (AF486428) (AF486429)

Apostichopus japonicus

Japan Japan

Ij1 Ij2

(AY85220) (AY85221)

Parastichopus californicus

USA, Pacific coast USA, Pacific coast

Pc1 Pc2

(PCU3218) (PPU32199)

Outgroup: Holothuria whitmaei

GBR

H. whitmaei

(AY177134)

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

Sequence 50 ?30

COIe-F COIe-R 16SA-R 16SB-R

ATA ATG ATA GGA GGR TTT GG GCT CGT GTR TCT ACR TCC AT CGC CTG TTT ATC CAG ATC ACG T CCG GTC TGA ACT CAG ATC ACG T

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

EU856679 EU856680 EU856681 EU856675 EU856676 EU856677 EU856678 EU856626 EU856625

(AY509147)

out of 24 models based on the Akaike Information Criterion (AIC). For both genes investigated, AIC suggested the General Time Reversible (Tavaré, 1986) model with gamma distributed variation across sites and a proportion of invariable sites as the most appropriate model. Final model runs were conducted with 107 generations and sampling trees every 100 generations. After 107 generations, the standard deviation of split frequencies for both COI (0.0042) and 16S (0.0064) were well below 0.01, and thus the number of generations run was sufficient (Ronquist et al., 2005). The probability density chosen as priors for all parameters were flat Dirichlets (=values of 1). Trees shown are Bayesian consensus trees of the last 75,000 trees (burnin period = 25,000) and

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Bayesian posterior probabilities for each node. The outgroup sequence was from Holothuria whitmaei, a species representing the closest extant group (Holothuriidae) to the Stichopodidae (Samyn et al., 2005). As alternative analyses we also present trees derived from Maximum Parsimony analyses. These were conducted in Mega 4.0 (Tamura et al., 2007) for both the 16S and COI sequences. CloseNeighbor-Interchange (CNI) with search level 1 and Random tree addition were undertaken with 10 replicates. 1000 bootstrap trees were generated, and the percent of bootstrap replicates supporting each node calculated. Haplotype networks were created using Statistical parsimony analysis as implemented in TCS (v1.13, Clement et al., 2000). To explore phylogenetic relationships in Stichopus and the distribution of asexual reproduction across species, a Neighbor-Joining tree (1000 bootstraps) based on Kimura 2 parameter genetic distances for COI was used just for this genus. Maximum adult size for Stichopus species was obtained from taxonomic studies (Massin, 1996, 1999, 2007; Massin et al., 2002) and field observations made during collections for this study. 2.4. Sequence divergence With the assumption that the two Isostichopus species from either side of the Isthmus of Panama I. fuscus (Pacific) and I. badionotus (Caribbean) are geminate sister species and closure of the Isthmus of Panama at ca. 3.1 Ma (Lessios, 2008) we used the sequence divergence between these species to estimate the Stichopodidaespecific mutation rate for COI. These are the only two species of Isostichopus in this region and so are likely to be closest living relatives, an important consideration for molecular clock calibration (Marco and Moran, 2009). We used the Stichopodidae-specific mutation rate for COI and the maximum rate inferred for evolution rate for echinoderm COI (3.5% Ma1, Lessios, 2008) to estimate a range of clade and species ages. These rates were applied only for the split between the genera Isostichopus and Stichopus, and for the species within the genus Stichopus. To test for similarity in sequence divergence rates in the species involved we applied Tajima’s (1993) test as implemented in Mega 4.0 (Tamura et al., 2007) using H. whitmaei as an outgroup. This test was conducted between all combinations of species involved in the rate calculations for both COI (36 comparisons) and 16S (28 comparisons), using a haphazardly chosen representative sequences for the respective species in case more than one sequences were available. None of the comparisons for both genetic markers indicated significant (v2 test, df = 1, p > 0.05) differences in evolutionary rates between species. 3. Results 3.1. COI and 16S sequence After trimming some base pairs at the beginning and end of the sequences, sequence size for COI was 558 bp and for 16S was 458 bp. Where possible we generated sequence data for both markers (Table 1). For some specimens, particularly the museum material 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 data were similar and Bayesian consensus trees were virtually identical to the Maximum Parsimony consensus tree for both markers (Fig. 1A and B). Monophyly of the genus Stichopus was supported by the COI tree (Fig. 1A). In the 16S tree S. ellipes a warm-temperate

species, from southeast Australia formed a separate clade to other Stichopus species (Fig. 1B). Only 16S data could be obtained for the two S. ellipes specimens. Stichopodid genera Thelenota and Isostichopus formed monophyletic clades and so molecular analyses confirmed current genus level taxonomy based on morphology. Australostichopus is currently known only from one species (A. mollis). The Parastichopus and Apostichopus species cluster together. The two species on either side of Panama I. badionotus (Caribbean) and I. fuscus (Pacific) formed a monopyletic clade positioned basal to all the other Stichopus species (Fig. 1A and B). The two temperate genera from the north Pacific, Parastichopus (represented by P. californicus, North America) and Apostichopus (represented by A. japonicus, Japan) were sister to the temperate genus from the south Pacific, Australostichopus mollis from New Zealand and Southern Australia formed a monophyletic group. 3.3. Stichopus The major Indo-West Pacific species S. chloronotus, the type species for the genus, is a sister taxon to all the other Stichopus species the Indo-West Pacific assemblage (Fig. 1A and B). S. chloronotus has a highly stereotypic morphology throughout its extensive range and is readily identified based on its body profile (Fig. 1A). Throughout the range sampled, including samples from the West Indian Ocean and Pacific, S. chloronotus has surprisingly limited genetic variability (see below). The other major Indo-West Pacific stichopodids divided into two large clades with S. naso forming a monophyletic sister clade to the remaining Stichopus species (Fig. 1A and B). S. naso is readily identified 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 spicule preparations of fresh specimens with those from registered museum specimens (Figs. 1A and 2E). S. naso is also characterised by its prominent and robust papillae, and, to a degree, by its lumpy appearance and colour pattern (Figs. 1A and 2E). The other five nominal Indo-West Pacific species comprised two clades, the S. herrmanni – S. ocellatus – S. vastus and the S. horrens – S. monotuberculatus groups. S. vastus was positioned basal to S. herrmanni and S. ocellatus. These three species are readily identified in the field by their distinctive appearance (Fig. 1A) and their large size. They are larger than all other Stichopus species (Fig. 4). In the other major internal node, S. horrens formed a distinct clade closely related to the S. monotuberculatus assemblage. The three S. horrens specimens sequenced for COI were from a wide distribution including the GBR, Philippines and a specimen from Samoa from British Museum collections. Four specimens sequenced for 16S from Hawaii and the Galapagos were also included. A discrete S. horrens cluster was evident with both COI and 16S analyses. As per taxonomic descriptions and species keys (Rowe and Doty, 1977; Massin et al., 2002), S. horrens sensu stricto is readily identified by the presence of diagnostic tack-like spicules in the body wall (Fig. 3B and C). S. horrens located along the GBR (Raine Is, Quoin Is, One Tree Is.) and elsewhere in the West Pacific (Massin et al., 2002) have distinct conical papillae on the dorsal surface (Fig. 2A–C). Most specimens, identified by field collectors as S. horrens, fell within S. naso or S. monotuberculatus. The latter is a monophyletic group that separated into several sub-clades with high support values. Sequence data were obtained for S. monotuberculatus specimens from the GBR, Samoa, Palau and Pohnpei, from the Pacific distribution of this widely distributed taxon. Unfortunately we could not generate sequence data for museum specimens from the western Indian Ocean type locality of this species (Mauritius). In agreement with the variability indicated by the molecular phylogeny, the S. monotuberculatus specimens sequenced for this

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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 for the Bayesian analysis (left side). The percentage of bootstrap replicates (out of 1000 replicates) is given for the maximum parsimony analysis (right side). Nodes with single values represent Bayesian posterior probability, node not supported by maximum parsimony.

study were morphologically variable in appearance (Fig. 2F-I). This was 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 evident when foraging at night (Fig. 2F), similar to those described for the closely related species, S. rubramaculosa Massin et al., 2002.

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Fig. 1 (continued)

However, those from Samoa had spire-like dorsal papillae similar to those of S. horrens (Fig. 2H) and to those of S. monotuberculatus from 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 juvenile feature.

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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 lumpy appearance. 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 a rock 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. monotuberculatus from 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.

3.4. Sequence divergence, evolutionary rates and asexual reproduction Sequence divergence (expressed as Kimura 2 Parameter distance) between species within the genus Stichopus is between 1.1% and 16.2% for COI and 1.4% and 15.3% for 16S (Table 3). Surprisingly little intra-specific sequence variation existed in the Stichopus species ranging from 0% (S. chloronotus) to 1.2% (S. horrens) for COI, and from 0% (S. vastus) to 1.5% (S. horrens) for 16S. This low intra-specific divergence rates for S. chloronotus were from 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 of the Isthmus of Panama was use to estimate a molecular clock for

evolution of the Stichopodidae. The genetic distance between I. fuscus and I. badionotus is on average 5.6%. With the assumption that I. fuscus and I. badionotus are closest living relatives and closure of the Isthmus of Panama at ca. 3.1 Ma (Lessios, 2008), the rate of evolution for COI was estimated to be 1.81% Ma1(or 1% = 553.000 y). We used this rate and the maximum rate inferred for echinoderm COI (3.5% Ma1, Lessios, 2008) to estimate a range of clade and species ages (Table 4). This analysis suggests that the Isostichopus and Stichopus clades diverged ca. 5.5–10.7 Ma. The split between S. chloronotus and all remaining Stichopus was estimated to be between 4.6 and 8.8 Ma. The youngest splits between S. herrmanni and S. ocellatus and that between S. monotuberculatus and S. horrens were estimated to be younger that 1 Ma.

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

Several Stichopus species reproduce asexually splitting their body by transverse fission, a feature not observed for Stichopodidae outside the genus (Conand et al., 2002; Uthicke and Conand, 2005a, b). The distribution of asexual reproduction in Stichopus species, maximum adult size and genetic distances are illustrated in a Neighbor-Joining tree (Fig. 4). Fission is characteristic of species with smaller maximum adult size (ca. <30 cm length) including S. chloronotus, S. naso and the S. monotuberculatus complex. One possible exception is S. horrens, but the reproductive biology of this species has not been documented. Asexual reproduction is absent in the large bodied species, S. herrmanni, S. ocellatus and S. vastus (>40 cm length). 3.5. Haplotype network and potential cryptic species in S. monotuberculatus Because both COI and 16S indicated the presence of sub-clades in the S. monotuberculatus clade we examined this clade with Statistical parsimony analysis. Haplotype network analyses with COI and 16S for S. monotuberculatus indicated that the sequences fell into a single 95% confidence network for both markers (Fig. 5). Most individuals grouped into a few larger haplotypes that are generally separated by a few mutational steps. However, two specimens from Samoa formed a separate haplotype with both markers and were separated from all other common haplotypes by several mutational steps. Although the sample size is too small for a population genetic analysis, the most common haplotype occurs in Samoa and the Northern GBR. The museum specimen collected in 1961 from Heron Island, Southern GBR only amplified for 16S and 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 from other haplotypes in Fig. 5 suggests that there may be cryptic species within S. monotuberculatus.

4. Discussion 4.1. Phylogenetic relationships Phylogenetic analyses using two mitochondrial markers provided insights into the taxonomic relationships within the family Stichopodidae, and in particular for the genus Stichopus. This was especially the case for COI, where more sequence data on a higher taxonomic level were available. Most currently recognised genera formed separate clades supported by high bootstrap values and the species formed clades that agree with taxonomic revisions based on morphology (Rowe and Gates, 1995; Massin, 1999, 2007; Massin et al., 2002). The trees generated for both markers were similar with the major Indo-West Pacific species S. chloronotus being a sister taxon to all other Stichopus. As seen in population genetic and phylogenetic studies of Holothuria species (Uthicke and Benzie, 2003; Uthicke et al., 2004b), COI is a good marker to document intra-specific relationships and evolutionary pathways of closely related stichopodids. Investigation on a higher phylogenetic level required a slower evolving marker such as 16S, as found elsewhere for holothuroid phylogeny (Kerr et al., 2005). However, 16S, in conjunction with allozymes, was also used as a marker to distinguish closely related sister Holothuria species and investigate hybridisation (Uthicke et al., 2005). The phylogenetic analyses of COI and 16S indicated that Stichopus is monophyletic with the only exception being the Australian

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Fig. 4. Neighbor-Joining tree (1000 bootstraps) on Kimura two parameter genetic distances for COI between Stichopus species and distribution of asexual reproduction by fission 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.

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

(1) (2) (3) (4) (5) (6) (7)

S. S. S. S. S. S. S.

chloronotus monotuberculatus horrens herrmanni ocellatus vastus naso

1

2

3

4

5

6

7

0/0.002 0.128 0.134 0.143 0.143 0.153 0.122

0.142 0.006/0.007 0.031 0.041 0.038 0.048 0.079

0.138 0.027 0.012/0.015 0.052 0.046 0.056 0.084

0.157 0.072 0.068 0.002/0.002 0.014 0.030 0.079

0.157 0.068 0.065 0.011 0.007/0.002 0.028 0.074

0.162 0.070 0.072 0.030 0.024 0.004/0 0.078

0.154 0.127 0.119 0.126 0.121 0.117 0.003/0.002

Table 4 Estimated clade ages (expressed as divergence times from the respective sister clade in Ma) of members of the genus Stichopus, based on estimates for COI mutation rates in geminate Isostichopus spp. (1.81% Ma1) from this study and the highest value for COI mutation rates in echinoderms (3.5%) (Lessios, 2008).

Isostichopus vs. Stichopus S. chloronotus vs. S. naso, S. monotuberculatus, S. horrens, S. herrmanni, S. ocellatus, S. vastus S. naso vs. S. monotuberculatus, S. horrens, S. herrmanni, S. ocellatus, S. vastus S. horrens and S. monotuberculatus vs. S. herrmanni, S. ocellatus, S. vastus S. vastus vs. S. herrmanni, S. ocellatus S. herrmanni vs. S. ocellatus S. monotuberculatus vs. S. horrens

1.81%

3.50%

10.67 8.84

5.52 4.57

6.86

3.55

3.76

1.95

1.49 0.33 1.00

0.77 0.17 0.52

Fig. 5. Ninety-five percent confidence network using sequences (top, COI; bottom 16S) for all S. monotuberculatus specimens. The size of the circle is proportional to sample size. Samples from the three major regions are colour coded. Small black circles along lines represent substitution of one base pair.

warm-temperate (see Wilson and Allen, 1987) species S. ellipes, from New South Wales, which clustered outside the group with 16S. This result was obtained with historic museum material (Rowe and Gates, 1995) from which only one marker was successful. Although the specimen used was compared with the holotype, it would be useful to revisit this result with fresh specimens along with inclusion of a second temperate species not investigated here, S. ludwigi, from southern Australia. The presence of S. ellipes outside Stichopus is a surprising result for a species that morphologically appears to be a Stichopus species. This suggests that morphological 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 ampullae and two gonad tufts (Haeckel, 1896) and body wall ossicle form (Clark, 1922) has been considered sufficient justification to refer species to the genus Stichopus. Recent revision of the Australian temperate species, S. mollis to a new genus, Australostichopus, was largely based on body chemistry and internal anatomy (Moraes et al., 2004). Taxonomic separation of North Pacific stichopodids (Parastichopus californicus and Apostichopus japonicus) from Stichopus is also supported by data from chemistry (Levin et al., 1986). Stichopus species appear to be restricted to tropical and subtropical regions of the Indo west-Pacific. In our phylogeny, the genus Stichopus is separated from the east Pacific/Caribbean genus Isostichopus and from temperate genera (e.g. Australostichopus mollis: South-west Pacific; P. californicus and A. japonicus: North Pacific). Parastichopus is otherwise a North Atlantic genus restricted to P. tremulus (type species for the genus) and P. regalis. Our results support Australostichopus as a valid genus potentially endemic to the Tasman region, though we have not investigated its relationship with the monotypic genus Neostichopus (South Africa/Western Indian Ocean). In light of several generic revisions of temperate stichopodids (Levin et al., 1986; Moraes et al., 2004) it appears that the current inclusion of temperate species in the genus Stichopus, including those not sampled here (e.g. S. ludwigi) warrants examination. In the COI trees, the North Pacific species Parastichopus

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californicus and A. japonicus clustered as a monophyletic unit. The potential congeneric affinity of these species is supported by body wall ossicle morphology (Lambert, 1986; Imaoka, 1991). Parastichopus tremulus and P. regalis, from the North Atlantic differ from their 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 clade with high support values corroborates recent taxonomic revisions (Rowe and Gates, 1995; Massin et al., 2002). These three species comprise the so-called commercial ‘curry fish’ group; readily identified 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, a sister taxon to the variable S. monotuberculatus complex. Of the 50 specimens identified by field collectors as S. horrens only two of these were revealed to be S. horrens. The others were placed in the S. naso or S. monotuberculatus clades. In total we obtained five specimens of S. horrens (field and museum collections) from disparate localities across the tropical Pacific range of this species, Galapagos, Philippines, Hawaii, Samoa and Australia. Molecular support for a distinct S. horrens clade corroborates morphological taxonomy based on spicule structure (Rowe and Doty, 1977; Massin et al., 2002). Examination of body wall spicules from museum and freshly collected specimens revealed the distinct tack-like table 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 in field studies and by fishers (e.g. Harriott, 1982; Young and Ryan, 2004; Eriksson et al., 2007; Kohtsuka et al., 2005). S. horrens however did have about twice the within species sequence variation compared with other species, a feature indicating the presence of cryptic diversity. Our analyses only included five specimens, and given the vast distribution of S. horrens in the tropical Pacific, it seems likely that more variability will be discovered. Specimens of S. horrens from near the type locality of this taxon (Society Islands, French Polynesia) need to be examined. The two species often mistakenly called S. horrens, that is S. naso and S. monotuberculatus, are common in the tropical waters of eastern Australia. Extensive night-time searches (GBR, Samoa) indicated that S. monotuberculatus is a common and conspicuous nocturnal species on reefs (Byrne and Eriksson, pers obs). The abundance of S. naso and S. monotuberculatus may be due to their fissiparous reproduction which can result in high local densities on coral reefs (Uthicke, 2001b). Thus far fissiparity has not been reported for S. horrens sensu stricto. Earlier descriptions of asexual reproduction in this species (Harriott, 1982; Kohtsuka et al., 2005) referred to S. monotuberculatus and S. naso. Although S. horrens was found at a number of locations along the GBR, it appears to be uncommon in the shallow tropical waters of eastern Australia. Concerted effort is needed to find this species in night searches. According to both mitochondrial markers S. monotuberculatus is a variable assemblage including several sub-clades. S. quadrifasciatus Massin, 1999 and S. rubramaculosus from Malaysian waters appear to fall in this clade based on morphology, but we have not used molecular data to examine these taxa. The variability of the S. monotuberculatus group is reflected by their morphological diversity. For instance the S. monotuberculatus from tropical NE Australia have low wart-like papillae and prominent lateral papillae (Fig. 2). They accord in body and spicule form with S. rubramaculosus, but lack the red colour associated with this species (Massin et al., 2002). The specimens from Samoa had taller dorsal papillae, similar to those of S. horrens and are also similar to S. monotuberculatus 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 is not characteristic of the morphs on the GBR. Regional differences in body wall spicules, external morphology and colour indicate the potential for taxonomic diversity in S. monotuberculatus across its wide Indo-Pacific distribution (Red Sea and Madagascar to Easter Island; Massin, 1996; Massin et al., 2002). The figure of S. variegatus from the Philippines (drawn in Semper, 1867) should be referred to S. monotuberculatus (see Rowe and Gates, 1995; Massin, 1999). Molecular analysis of specimens from the type locality of monotuberculatus (Mauritius) is required to determine the identity of S. monotuberculatus sensu stricto. There is an urgent need to characterise the species composition of the developing East African Indian Ocean bêche-de-mer fishery that appears to include S. monotuberculatus and other cryptic species (Eriksson and Byrne, pers obs). Further research is required to determine if the variable morphology and molecular diversity within the Pacific S. monotuberculatus investigated here is linked to different species or potential hybridisation between this species and the closely related S. horrens. Hybridization has been documented in molecular studies of other bêche-de-mer species (Uthicke et al., 2005, 2010). In addition studies of widely distributed echinoderm species (Dartnall et al., 2003; Hart et al., 2003, 2006; O’Loughlin and Rowe, 2006; Uthicke et al., 2010) have revealed the presence of cryptic species. Revision of S. monotuberculatus remains a challenge. The molecular phylogeny established here for Stichopus provides a framework to assist with this. Haplotype-network based approaches to recognising species boundaries have been used in several recent studies where the analyses have indicated the presence of cryptic species within nominal taxa (e.g. Hart et al., 2006; Hunter and Halanych, 2008). In these studies multiple haplotype networks are interpreted as indication of multiple species. Although multiple networks were not present in S. monotuberculatus, the presence of several haplotypes removed by several mutational steps from the most common types indicate that cryptic species may exist in the S. monotuberculatus analysed here. 4.3. Clarification of commercially exploited Stichopus Comparison of sequence data from species being fished or listed as commercial species indicates that at least three species are currently being fished under the name S. horrens. The species listed in the south Queensland (East Australia) fishery as S. horrens is S. naso and 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 (in Hearn and Pinnillos, 2006) indicates that it differs from S. horrens. Unfortunately we were not able to obtain a sample from the Galapagos fishery. It is difficult to manage a fishery without knowing what species are harvested. For the ‘S. horrens fishery’ several species, potentially differing ecologically are being fished under this name. This study and ongoing investigations on the identity of global bêche-de-mer species (Uthicke et al., 2005, 2010) will contribute to the conservation and sustainable use of these resources. 4.4. Evolution of fission in Stichopus Asexual reproduction through transverse fission is characteristic of several small-bodied Stichopus species including S. chloronotus, the most abundant shallow water Stichopus species across the Indo-Pacific (Uthicke, 1999, 2001a). The high density and success of this species with respect to local abundance has been attributed in part to its propensity for asexual reproduction (Conand et al., 1998, 2002; Uthicke, 2001a; Uthicke and Conand, 2005a, b).

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Asexual reproduction is also common in S. naso and S. monotuberculatus, 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. Large bodied stichopodids (e.g. S. herrmanni) are not known to exhibit fission. They may not be able to sustain respiration following loss or damage of respiratory organs due to fission, whereas small individuals 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 investigated. Although they do not exhibit fissiparity, large bodied stichopodids use the break down of mutable body wall connective tissue, the mechanism that underlies fission, for defence. Both Bayesian and Parsimony phylogenetic methods identified S. chloronotus as the basal taxon in the genus Stichopus. The presence of fission in this and other clades (S. naso, S. monotuberculatus) with smaller maximum body size suggests that asexual reproduction may have been lost in evolution of larger body size (e.g. S. herrmanni, S. vastus, S. ocellatus), representing a single loss within Stichopus. However, the alternative hypothesis that asexual reproduction has evolved multiple times within the genus Stichopus is equally parsimonious. Asexual reproduction is not known for the other stichopodid genera. Fission also occurs in Holothuria species (Conand, 1996; Uthicke, 1998) and so may be a basal character of the Aspidochirotida or evolved independently in Holothuria and Stichopus. The within stichopodid species COI sequence variation are within the range observed in other holothuroid genera (e.g. Bohadschia 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 echinoderms (e.g. Echinometra sp., Linckia sp.) within species differences up to 3% are reported (Williams, 2000; Landry et al., 2003). Some Stichopus species however had a very low within species sequence divergence. The lack of sequence divergence within S. chloronotus appears paradoxical given the large geographic distance between samples (Australia to Reunion). One possible hypothesis for the low intra-specific diversity in some Stichopus species may be slower 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%) overlap and provide no support for this hypothesis. This analysis is somewhat confounded by sampling on different geographic scales. However, since asexual species also reproduce sexually (broadcast spawners), fission could simply be seen as an amplification of the genome to enhance success of density dependent sexual reproduction (Uthicke et al., 1998). It appears that asexual reproduction cannot explain the low within species sequence divergence in most of the Stichopus species. 4.5. Evolution of Stichopus Evolutionary rates estimated for the COI region (1.8% Mya1) are on the lower end of the range estimated for other echinoderms (1.6–3.5% Mya1, reviewed by Lessios, 2008). Using our rates and the upper end of previous estimates the age of the split between Stichopus and Isostichopus, ca. 5.5–10.7 Ma suggests a Miocene evolution of these genera. The most derived taxa and most recent splits between species were estimated to be younger than 1 Ma in the Pleistocene. This range of evolutionary ages is similar to that reported for other echinoderms based on use of transithian geminate species. Several species of the echinoid genus Diadema diverged in the Pleistocene although the genus is known from the Miocene (12 Ma) (Lessios et al., 2001). Similarly, several species of the echinoid genus Echinometra and asteroid genus Cryptasterina diverged only 0.5–1.6 Ma (McCartney et al., 2000; Hart et al.,

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2003). Estimates of divergence times based on comparison of sequence data from putative geminate species is however fraught with difficulty (Lessios, 2008; Marco and Moran, 2009). We do not know for instance if the Isostichopus species include undetected cryptic species raising the possibility that the sequences used may not be from closest living relatives. This problem was recently identified in a study of geminate bivalve species (Marco and Moran, 2009). The order Aspidochirotida is evolutionarily old with the oldest recorded family (Synallactidae) described from the mid Triassic (Amesian: 225–220 Ma, Gilliland, 1993). The Stichopodidae is considered to be geologically young because it has not been identified in fossil deposits (Gilliland, 1993). Our genetic data generally support Gilliland’s (1993) notion that Stichopodidae are evolutionarily relatively young, but our estimates indicate that they should be detectable in fossil deposits. An earlier evolution of the Stichopodidae is indicated by the recent discovery of fossil ‘table-form’ spicules from the late Cretaceous (ca. 70 Ma) (Reich, 2001). In light of shared characters between recent Stichopodidae and some recent synallactid taxa (Rowe, unpubl.) however, the family Synallactidae, sensu extenso appears to comprise a heterogeneous assemblage of genera, posing the possibility that some fossil representatives may be incorrectly ascribed to that family instead of the Stichopodidae. In conclusion, genetic analyses of two mitochondrial markers largely supported current taxonomy of the Stichopodidae on higher level and species scales. However, the need for a reassessment of diagnostic morphological characters was suggested by genetic analyses (e.g. S. ellipes). In addition, we clarified the species status of several commercial species and highlighted difficult groups (e.g. S. monotuberculatus, S. horrens) that may harbour cryptic species and warrant further investigation with species being harvested for bêche-de-mer of particular concern.

Acknowledgments The research was funded by Australian Biological Resources Survey and a grant from the FAO. Thanks to many colleagues that provided specimens, photographs and other assistance, particularly Hampus Eriksson, Tim O’Hara, Steve Purcell and Mara Wolkenhauer. We are grateful to Stephen Keable, Australian Museum; Andrew Cabrinovic, British Museum; Chris Bartlett, Museum of Tropical Queensland; Mark O’Loughlin, Museum Victoria and Gustav Paulay, Florida Museum of Natural History for assisting us with access to museum specimens or sequence data. Anne Hoggett and Thierry Rakotoarivelo provided photographs. Assistance from Zoran Ilic, Paula Cisternas, Natalie Soars and Erika Woosley is gratefully acknowledged. We also thank the reviews for helpful comments on the manuscript. This represents contribution no. 34 from the Sydney Institute of Marine Science and 1292 Lizard Island Research Station.

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