A NEW BEACH-HOPPER, PLATORCHESTIA PARAPACIFICA N. SP. (AMPHIPODA: TALITRIDAE), FROM SOUTH KOREA, WITH MOLECULAR PHYLOGENY OF THE GENUS PLATORCHESTIA

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1 JOURNAL OF CRUSTACEAN BIOLOGY, 33(6), , 2013 A NEW BEACH-HOPPER, PLATORCHESTIA PARAPACIFICA N. SP. (AMPHIPODA: TALITRIDAE), FROM SOUTH KOREA, WITH MOLECULAR PHYLOGENY OF THE GENUS PLATORCHESTIA Min-Seop Kim, Jae-Ho Jung, and Gi-Sik Min Department of Biological Sciences, College of Natural Sciences, Inha University, Incheon , South Korea ABSTRACT To date, the genus Platorchestia Bousfield, 1982 (Crustacea, Amphipoda) includes 17 species, all of which are adapted to terrestrial and supra-littoral habitats. According to a recent study, some species exist as cryptic species complexes. Here, we describe a new beachhopper, Platorchestia parapacifica, collected from beaches in South Korea. This new species can be easily distinguished from other congeners by the morphology of its antennae 1 and 2, gnathopods, coxal plate 6, pereiopod 7, and telson. We detail the morphological characteristics of P. parapacifica, and compare it with related species. In addition, we demonstrate the phylogenetic relationships of the genus Platorchestia based on the mitochondrial cytochrome c oxidase subunit 1. The results of our molecular analyses indicate that the P. platensis-complex is monophyletic and deeply divergent from the P. japonica-complex. Estimation of the genus divergence times using molecular clock analysis suggests that these species shared a most recent common ancestor during the Miocene period ( million years ago). Notably, common ancestry of the P. japonica complex diverged into two species during the Zanclean and Messinian periods ( million years ago), while the ancestral talitrid of the P. platensis complex diverged into three species during the Messinian and Tortonian period (6-8 million years ago). Our results reconfirm the usefulness of molecular techniques for biodiversity studies of talitrids, including phylogenetic relationships, species boundaries, and divergence times. KEY WORDS: amphipod, CO1, Platorchestia parapacifica, South Korea, systematics, Talitridae DOI: / X INTRODUCTION The amphipod Platorchestia includes 17 species distributed worldwide and is one of 52 genera belonging to Talitridae Rafinesque, 1815 (Cheng et al., 2011). This genus can be categorized as beach-hoppers, based on the four systematic ecological units proposed by Bousfield (1982). Species belonging to the genus Platorchestia live in terrestrial and supra-littoral habitats. They are mainly distributed in the Pacific region, especially northeast Asia, where 10 species have been reported to date: P. bousfieldi Hou and Li, 2003; P. humicola (Martens, 1868); P. japonica (Tattersall, 1922); P. joi Stock and Biernbaum, 1994; P. monodi Mateus et al., 1986; P. munmui Jo, 1988; P. pachypus (Derzhavin, 1937); P. pacifica Miyamoto and Morino, 2004; P. paludosus Cheng et al., 2011; and P. zachsi (Derzhavin, 1937). The phylogenetic relationships among the species of Platorchestia are rather controversial, and two cryptic diversities P. japonica and P. platensis (Krøyer, 1845) species complexes were recently proposed (Jo, 1988; Miyamoto and Morino, 2004; Serejo, 2004; Serejo and Lowry, 2008; Cheng et al., 2011). The mitochondrial (mt) cytochrome c oxidase subunit 1 (CO1) gene is frequently used to discriminate species (Hajibabaei et al., 2006; Clare et al., 2007; Elsasser et al., 2009; Zemlak et al., 2009), because it shows high sequence variation between congeneric taxa, but comparatively low sequence variation within species. These features are very useful for providing species-level identification and especially for discriminating species within complexes, which have been demonstrated to occur throughout most animal phyla (Hebert and Gregory, 2005; Hajibabaei et al., 2007). Here, we describe a new species, P. parapacifica, collected from South Korea. Based on mt CO1 gene sequence data, we evaluate the phylogenetic relationships and divergence time of the genus Platorchestia. MATERIALS AND METHODS Materials for Morphological Observation and Molecular Analysis We collected two talitrids (P. parapacifica n. sp. and P. munmui) from five localities in South Korea during (Fig. 1). The talitrids were captured by hand and immediately preserved in 95% ethyl alcohol. The specimens were observed and dissected with a tungsten needle under the stereomicroscope (Model SZX-ILLB2-200; Olympus, Tokyo, Japan) and (Model DM 2500; Leica, Wetzlar, Germany). All dissected appendages were mounted in glycerin and stained with Lignin Pink (Sigma, St. Louis, MO, USA). All drawings were constructed using a drawing tube attached to a microscope. Images were obtained using a microscope digital camera (Model Moticam 2000; Motic, Hong Kong, P.R. China) and edited with Helicon Focus software (Model Helicon Focus ; Helicon Soft, Kharkov, Ukraine). Setae were classified according to the criteria of Garm (2004) and Present address: Marine Ecosystem Management Team, Korea Marine Environment Management Corporation, Seoul , South Korea. Corresponding author; mingisik@inha.ac.kr The Crustacean Society, Published by Brill NV, Leiden DOI: / X

2 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 829 Fig. 1. Sampling localities for Platorchestia species and Orchestia cavimana (used as an outgroup species), examined in this study. Locality codes are shown in Table 2. Zimmer et al. (2009). All specimens were deposited in the National Institute of Biological Resources (NIBR), Incheon, South Korea. CO1 Gene Sequencing Total genomic DNA was extracted from the single pereiopod of each specimen, using a RED-Extract-N-Amp Tissue PCR Kit (Sigma). The CO1 gene was amplified using the primers LCO1490 and HCO2198 (Folmer et al., 1994). Optimized PCR conditions were as follows: denaturation at 94 C for 2 min; followed by 10 cycles of denaturation at 94 C for 10 s, annealing at 37 C for 30 s, and extension at 72 C for 60 s; followed by 25 cycles of denaturation at 94 C for 10 s, annealing at 48 C for 30 s, and extension at 72 C for 60 s; and a final extension step at 72 C for 7 min. The PCR fragments were purified with a QIAquick PCR Purification Kit (Qiagen, Chatsworth, CA, USA). Sequencing was carried out using an ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA). All of the sequences obtained were deposited in GenBank under accession number KF KF Molecular Analysis For phylogenetic analysis, the obtained CO1 sequences were retrieved from GenBank and aligned using ClustalW implemented in BioEdit ver (Hall, 1999). Sequences of four Platorchestia species (P. japonica, P. paludosus, P. monodi and P. platensis) and of Orchestia cavimana Heller, 1865 were retrieved from GenBank; O. cavimana was used as an outgroup. Phylogenetic trees were reconstructed by means of the maximum likelihood (ML) and Bayesian inference (BI) methods, using the bestfit model of DNA substitution estimated by Akaike information criterion (AIC) in jmodeltest ver (Posada, 2008). The selected best-fit model was TIM2 + I + G, with an assumed proportion of invariable sites (0.4320) and a gamma distribution shape parameter (0.4310). ML analysis was generated by PhyML ver. 3.0 (Guindon and Gascuel, 2003). Confidence in the resulting relationship was assessed using the bootstrap procedure with 1000 replications for ML. BI analysis was performed using MrBayes ver (Ronquist and Huelsenbeck, 2003). Markov chain Monte Carlo (MCMC) simulations were run for generations sampled every 100 generations, after a burn-in period of generations. We estimated the divergence time of each clade based on the mt CO1 gene sequence data, using the Test Molecular Clock (ML) option in MEGA 5.05 (Tamura et al., 2011). We used the CO1 clock calibration of % sequence divergence per million years for snapping shrimp (Knowlton et al., 1993; Knowlton and Weigt, 1998). This calibration was used in previous amphipod studies (Cristescu and Hebert, 2005; Kelly et al., 2006; Hou et al., 2007). SYSTEMATICS Talitridae Rafinesque, 1815 Platorchestia Bousfield, 1982 Platorchestia parapacifica n. sp. Figs. 2-7 Type locality. Geumseongcheon Stream, Jeju-do, South Korea. Type material. Holotype (NIBRIV ): adult male (Fig. 2A), 19.2 mm, Geumseongcheon Stream (33 26 N, E), Jeju-do, South Korea, 3 Jun. 2010, coll. M. S. Kim. Paratypes (NIBRIV ): six males and five females, same data holotype. Additional material examined: three males and three females, Sehwa Beach (33 31 N, E), Jeju-do, South Korea, 3 Jun. 2010, coll. M. S. Kim; three males and two females, Seomjin River (34 59 N, E), Jeollanam-do, South Korea, 19 Mar. 2010, coll. M. S. Kim; two males and four females, Osipcheon Stream (36 21 N, E), Gyeongsangbuk-do, South Korea, 10 Jun. 2010, coll. M. S. Kim. Etymology. The species name parapacifica indicates its similarity to P. pacifica. Diagnosis. Antenna 1, peduncular article 3 with four cuspidate setae; antenna 2 ( ), peduncular articles 4, 5 strongly ; gnathopod 1, non-cuspidactylate; gnathopod 2 ( ), propodus marginally bare posteriorly; coxal plate 6, posterior lobe with process ventrally; pereiopod 7 ( ), carpus strongly ; telson, each lobe with apical and three dorsolateral groups of cuspidate setae. Description of male, holotype. Body (Fig. 2A) 12.8 mm. Eyes (Figs. 2A, 3A) sub-round, width about 0.31 times head diameter; interior antennal sinus distinct; buccal mass directed below head. Antenna 1 (Fig. 3D) not reaching endpoint of peduncular article 4 of antenna 2; peduncular articles 1-3 with length

3 830 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 ratio 1:0.9:1; peduncular article 3 with four cuspidate setae midventrally; flagellum with seven articles, each article with two groups of two or three simple setae distally. Antenna 2 (Fig. 3B, C), peduncular articles 4 and 5 with length ratio 1:1.3, strongly, with many short cuspidate setae; flagellum subequal to peduncular article 5 in length, with 15 articles, six proximal articles incompletely fused, each article flat, all articles with three or four groups of two or three simple setae. Upper lip [labrum] (Fig. 3E) elliptical in shape, apical margin with fine setules. Left mandible (Fig. 3F), incisor process 6-dentate, lacinia mobilis 5-weak teeth, setal row between incisor and molar processes, molar process with one pappose seta; right mandible (Fig. 3G), incisor process 6-dentate, lacinia mobilis bifurcate with many weak dentitions apically. Lower lip (Fig. 3H) bilobed, with small inner lobes; apical and inner margins of outer plate with fine setules. Maxilla 1 (Fig. 4A), inner plate with two pappo-serrate setae distally; outer plate with nine serrate setae distally; palp rudimentary, 2-segmented including tiny distal article. Maxilla 2 (Fig. 4B, C), inner plate with two unequal pappo-serrate setae medio-marginally, distal margin with many simple and serrate setae; outer plate with numerous simple and serrate setae distally. Maxilliped (Fig. 4D), inner plate with pappo-serrate setae on lateral and distal margins, distal margin with three cuspidate setae; outer plate with six pappo-serrate setae on distal margin, inner margin with many simple setae; maxillipedal palp consisting of four articles; article 2 broad and well-developed medio-distal lobe; article 4 projecting beyond article 3, with simple setae distally. Gnathopod 1 (Fig. 4E, F) subchelate; coxal plate 1 with two rows of many simple and cuspidate setae ventrally; basis straight, with seven and five cuspidate setae on anterior and posterior margins; merus with 13 cuspidate setae on posterior margin; carpus with nine cuspidate setae dorsally, posterior margin with tumescent hump, eight lateral setae, and 10 medial setae; propodus 0.63 times as long as carpus, with four groups of one or two cuspidate setae on dorsal margin and tumescent hump posteriorly, lateral margin with 15 setae and medial margin with 15 setae; palmar margin with several setae; dactyl subequal to palm in length, with one simple seta on inner margin, hinge of unguis with three simple setae. Gnathopod 2 (Fig. 4G) powerfully subchelate; coxal plate 2 with many simple setae ventrally and posterior margin with small cusp; anterior margin of basis straight, with nine cuspidate setae, posterior margin with five cuspidate setae; propodus oval in shape, posterior margin marginally bare and palmar margin fringed with many simple and cuspidate setae, with two dome-shaped protuberances, anterior one 2.2 times as long as posterior one, notch situated at about 3/5 length from hinge of dactyl; dactyl strongly curved, inner margin with many simple setae, distal part narrowed. Pereiopods 3-4 (Fig. 5A-F) cuspidactylate; pereiopod 3 longer than pereiopod 4; coxal plate 3 with many simple setae ventrally and posterior cusp triangular; basis straight, with 14 simple and six cuspidate setae on anterior and posterior margins; merus to propodus with several groups of 1-4 cuspidate setae on anterior and posterior margins; dactyl with two setae at hinge of unguis; pereiopod 4 resembling pereiopod 3, but posterior corner of coxal plate 4 slightly expanded and dactyl slightly thickened and pinched posteriorly. Pereiopods 5-7 (Figs. 5G-L, 6A, B) cuspidactylate, with length ratio 1:0.9:0.9 respectively; coxal plates 5-6 bilobate; anterior lobe of coxal plate 5 broader than posterior one, posterior margin with eight simple setae; anterior lobe of coxal plate 6 very small and posterior lobe with process; coxal plate 7 non-lobate; basis of pereiopods 5-7 oval in shape, with several simple setae on posterior margin, anterior margin with several groups of 1-3 cuspidate setae; merus to propodus of pereiopods 5-7 resembling pereiopod 3, but carpus of pereiopod 7. Gills present on pereiopods 2-6 (Figs. 5A, B, D, E, H, K); gill 2 elongated and curved in middle; gills 3-4 similar in size and shape; gill 5 broader than gills 3-4; gill 6 as large as gill 2. Epimeral plates 1-3 (Fig. 6C) with weakly pointed posteroventral corners, ventral margin unarmed and posterior margin bearing 4-6 simple setae. Pleopods 1-3 (Fig. 6D-F) well developed, subequal in length; peduncles of pleopods with two retinaculae distally, Fig. 2. Platorchestia parapacifica n. sp., lateral view. A, male, holotype; B, female, paratype. Scale bar = 2 mm (A, B). This figure is published in colour in the online edition of this journal, which can be accessed via

4 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 831 Fig. 3. Platorchestia parapacifica n. sp., male, holotype. A, head; B, antenna 2; C, flagellum of antenna 2; D, antenna 1; E, upper lip; F, left mandible; G, incisor process of right mandible; H, lower lip. Scale bars = 0.25 mm (E-H), 0.5 mm (D), 1 mm (A-C). This figure is published in colour in the online edition of this journal, which can be accessed via

5 832 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 Fig. 4. Platorchestia parapacifica n. sp., male, holotype. A, maxilla 1; B, maxilla 2; C, inner margin of maxilla 2; D, maxilliped; E, gnathopod 1; F, palm and dactylus of gnathopod 1; G, gnathopod 2. Scale bars = 0.25 mm (A-D, F), 0.5 mm (E, G).

6 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 833 Fig. 5. Platorchestia parapacifica n. sp., male, holotype. A, pereiopod 3; B, coxal plate and basis of pereiopod 3; C, dactylus of pereiopod 3; D, pereiopod 4; E, coxal plate and basis of pereiopod 4; F, dactylus of pereiopod 4; G, pereiopod 5; H, coxal plate and basis of pereiopod 5; I, dactylus of pereiopod 5; J, pereiopod 6; K, coxal plate and basis of pereiopod 6; L, dactylus of pereiopod 6. Scale bars = 0.2 mm (C, F), 0.25 mm (I, L), 0.5 mm (B, E, H, K), 1 mm (A, D, G, J). This figure is published in colour in the online edition of this journal, which can be accessed via

7 834 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 Fig. 6. Platorchestia parapacifica n. sp., male, holotype. A, pereiopod 7; B, dactylus of pereiopod 7; C, epimeral plates; D, pleopod 1; E, pleopod 2; F, pleopod 3; G, uropod 1; H, uropod 2; I, uropod 3; J, telson. Scale bars = 0.25mm(B,I,J),0.5mm(D-H),1mm(A,C).

8 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 835 both rami with about 8-10 articles, fringed with plumose setae; peduncle of pleopod 1 marginally bare; peduncle of pleopod 2 with seven cuspidate setae in middle part of outer margin; peduncle of pleopod 3 with nine cuspidate setae on outer margin, inner margin with five simple spines. Uropod 1 (Fig. 6G), peduncle with two rows of 6-9 marginal cuspidate setae and distolateral cuspidate seta; outer ramus 0.65 times of peduncle, marginally bare, bearing four distal cuspidate setae; inner ramus with four, five, and four cuspidate setae on inner, outer, and distal margins respectively. Uropod 2 (Fig. 6H), peduncle with four cuspidate setae on outer margin, inner margin with three simple and nine cuspidate setae; outer ramus with two marginal and three distal cuspidate setae, inner ramus with three, two, and five cuspidate setae on inner, outer, and distal margins respectively. Uropod 3 (Fig. 6I), peduncle slightly broadened dorsally, with three dorsal cuspidate setae; ramus slightly shorter than peduncle, with three marginal cuspidate setae, distal margin with two long simple, two smooth simple, two cuspidate setae. Telson (Fig. 6J) notched distally; each lobe with three groups of 1-3 cuspidate setae on dorsal margin, distal margin with four cuspidate setae. Description of female, paratype (NIBRIV ). Body 13.5 mm long. General appearance is similar to male. Differsasfollows: Antenna 2 (Fig. 2B), peduncular articles 4 and 5 with length ratio 1:1.4, narrow, with many short cuspidate setae. Gnathopod 1 (Fig. 7A-C), carpus and propodus without tumescent hump, parallel-sided; dactylus much longer than palmar margin, hinge of unguis with two simple setae. Gnathopod 2 (Fig. 7D-F), basis anterior margin expanded, with 18 simple setae, posterior margin straight and marginally bare; merus with 13 cuspidate setae and small tumescent hump posteriorly; carpus with tumescent hump posteriorly posterodistally; propodus tumescent posteriorly, lateral margin with 19 serrate setae and medial margin with 26 serrate setae; dactylus longer than palm, with three simple setae on inner margin, hinge of unguis with two simple setae. Pereiopod 7 (Fig. 2B), carpus not. Oöstegites present on pereiopods 2 to 5 (Fig. 7D, G-I); oöstegites 2-4 with many bristles; elongate and parallelsided; distal margin of oöstegite 5 triangular in shape, with nine simple setae distally. Habitat. This new species lives mainly on the sandy shores around river mouths (salinity ranged between 5-20 psu). It is usually found under rotting hay or in silt beside the water (Fig. 8). Remarks. Miyamoto and Morino (2004) divided Platorchestia into three subgroups based on development of sexual dimorphism in antenna 2 and pereiopod 7. Platorchestia parapacifica n. sp. belongs to group 1, which includes six species with strong sexual dimorphism in antenna 2 and pereiopods 6, 7: P. joi; P. munmui; P. pacifica; P. pachypus; P. paraplatensis (Serejo and Lowry, 2008); and P. platensis. This new species morphologically resembles P. pacifica, P. paraplatensis, and P. platensis in the following characters: (1) antenna 2 ( ), peduncular articles 4, 5 strongly ; (2) gnathopod 2 ( ), propodus without marginal cuspidate setae posteriorly; (3) coxal plate 6 with process ventrally; (4) pereiopod 7 ( ), carpus strongly. However, the new species differs from congeners in the following features: (1) antenna 1, peduncular article 3 with four cuspidate setae ventrally (vs. one cuspidate seta); (2) gnathopod 1 ( ) non-cuspidactylate (vs. cuspidactylate); and (3) telson, each lobe with apical and three dorsolateral groups of cuspidate setae (vs. apical and two dorsolateral groups of cuspidate setae). A comparison between P. parapacifica and P. platensis complex is given in Table 1. Molecular Data. 596 bp CO1 gene sequences (GenBank accession numbers KF KF016047) were obtained from four specimens of the new species. Additionally, three sequences (GenBank accession numbers KF KF016043) were determined from P. munmui, and 12 sequences from five species retrieved from National Center for Biotechnology Information (NCBI) (Table 2). Sequence alignment was straightforward without any insertion and deletion. We also verified that 388 sites were conserved and 209 sites were variable, while 177 sites were parsimonyinformative. Intraspecific variation of the CO1 sequence of the new species ranged from 0.2% to 0.3%, while interspecific variation ranged from 14.7% (P. parapacifica n. sp. and P. paludosus) to 21.3% (P. parapacifica n. sp. and P. munmui). DISCUSSION Platorchestia platensis was first reported as Orchestia platensis by Krøyer (1845), but Bousfield (1982) subsequently transferred the species to Platorchestia. Until recently, P. platensis has been regarded as a cosmopolitan species (Morino, 1975; Bousfield, 1982; Serejo, 2004). However, Jo (1988) compared several local species of P. platensis s.s., which were previously identified as P. platensis s.l., in the Western Pacific region. After examination of local species collected from Denmark, Netherlands, and Florida, he suggested that P. platensis s.l. was not a single species but rather made up of several morphologically similar species, including P. joi and P. munmui. Since then, four more species have been reported in the P. platensis-complex. Currently, the P. platensis-complex comprises six sibling species: P. munmui, P. joi, P. ashmoleorum Stock, 1996, P. pacifica, P. paraplatensis and P. platensis. To verify the phylogenetic relationships among species in the genus Platorchestia, including P. parapacifica, we performed phylogenetic analyses based on the CO1 sequences of seven newly determined sequences from two species of Platorchestia (P. parapacifica and P. munmui) and 12 sequences from five species retrieved from NCBI (Table 2). Monophyly for all of the traditional species based on morphological criteria was well supported by ML and BI analyses, with high support values (Fig. 9). Despite the morphological similarity, the species showed high levels of interspecific variation ( %); by contrast, genetic divergences within the species were very low ( 0.7%) (Table 3).

9 836 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 Fig. 7. Platorchestia parapacifica n. sp., female, paratype. A, gnathopod 1; B, palm and dactylus of gnathopod 1; C, medial view of carpus and propodus in gnathopod 1; D, gnathopod 2; E, palm and dactylus of gnathopod 2; F, medial view of propodus in gnathopod 2; G, oostegite 3; H, oostegite 4; I, oostegite 5. Scale bars = 0.25 mm (B, E), 0.5 mm (A, C, D, F-I).

10 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 837 Fig. 8. Habitat of Platorchestia parapacifica n. sp. A, type locality; B, estuarine shoreline in Geumseongcheon Stream; C, hiding place (rotting seaweed). This figure is published in colour in the online edition of this journal, which can be accessed via Table 1. Morphological differences among P. parapacifica n. sp. and congeners. Species/Distribution Antennae 1 and 2 (male) Platorchestia parapacifica n. sp./south Korea P. ashmoleorum Stock, 1996/Saint Helena Island P. monody Mateus et al., 1986/Mid-Atlantic Islands, Brazilian coast, Israel and South Korea P. munmui Jo, 1988/South Korea P. joi Stock and Biernbaum, 1994/Soviet coast, Japan, Taiwan and South Korea P. pacifica Miyamoto and Morino, 2004/Soviet coast, Japan and Taiwan Antenna 1, peduncular article 3 with four cuspidate setae ventrally; antenna 2, strongly Antenna 1, peduncular article 3 with two cuspidate setae ventrally; antenna 2, weakly Antenna 1, peduncular article 3 with one cuspidate seta ventrally; antenna 2, weakly Antenna 1, peduncular article 3 with one cuspidate seta ventrally; antenna 2, strongly Antenna 1, peduncular article 3 with two cuspidate setae ventrally; antenna 2, strongly Antenna 1, peduncular article 3 with one cuspidate seta ventrally; antenna 2, strongly Gnathopods 1 and 2 (male) non-cuspidactylate; propodus marginally bare posteriorly non-cuspidactylate; propodus marginally bare posteriorly cuspidactylate; propodus marginally bare posteriorly cuspidactylate; propodus marginally bare posteriorly non-cuspidactylate; propodus with cuspidate setae posteriorly cuspidactylate; propodus marginally bare posteriorly Pereiopods 6 and 7 (male) coxal plate 6 with process ventrally; pereiopod 7, carpus strongly coxal plate 6 with weakly process ventrally; pereiopod 7, carpus very weakly coxal plate 6 lacking process ventrally; pereiopod 7, carpus very weakly coxal plate 6 lacking process ventrally; pereiopod 7, carpus strongly coxal plate 6 with weakly process ventrally; pereiopod 7, carpus very weakly coxal plate 6 with process ventrally; pereiopod 7, carpus strongly Pleopods 1-3 (male) with seven marginal median setae; pleopod 3, peduncle with nine marginal and five facial setae with six marginal median setae; pleopod 3 (N/A) with one marginal median seta; pleopod 3, peduncle with two marginal distal setae with five marginal median setae; pleopod 3, peduncle with nine marginal and eight facial setae with 13 marginal setae; pleopod 3, peduncle with 11 marginal and seven facial setae with three marginal proximal setae; pleopod 3, peduncle with nine marginal and six facial setae Telson (male) apical and three dorsolateral groups of cuspidate setae apical and one dorsolateral group of cuspidate setae apical and two dorsolateral groups of cuspidate setae apical and two dorsolateral groups of cuspidate setae apical and three dorsolateral groups of cuspidate setae apical and two dorsolateral groups of cuspidate setae

11 838 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 Table 1. (Continued.) Species/Distribution Antennae 1 and 2 (male) P. paraplatensis Serejo and Lowry, 2008/Australia P. platensis (Krøyer, 1845)/Nearly cosmopolitan Antenna 1 (N/A); antenna 2, strongly Antenna 1, peduncular article 3 with one cuspidate seta ventrally; antenna 2, strongly Gnathopods 1 and 2 (male) cuspidactylate; propodus marginally bare posteriorly cuspidactylate; propodus marginally bare posteriorly Pereiopods 6 and 7 (male) coxal plate 6 with process ventrally; pereiopod 7, carpus strongly coxal plate 6 with process ventrally; pereiopod 7, carpus strongly Pleopods 1-3 (male) with three marginal median setae; pleopod 3, peduncle with three marginal distal setae with four marginal median setae; pleopod 3, peduncle with four marginal distal setae Telson (male) apical and two dorsolateral groups of cuspidate setae apical and two dorsolateral groups of cuspidate setae Fig. 9. Mitochondrial (mt) cytochrome c oxidase subunit 1 (CO1) phylogenetic tree of Platorchestia species based on maximum likelihood (ML) analysis and Bayesian inference (BI). Orchestia cavimana was used as an outgroup. The numbers above the nodes indicate the ML bootstrap values (BV, left) and BI posterior probability values (PPV, right). We also revealed that monophyly of the two cryptic species complexes was supported by ML and BI analyses, although the ML support values were low (P. japonicacomplex, ML bootstrap values BV:62; BI posterior probability values PPV:98; P. platensis-complex, BV:90; PPV:100). Interspecific variation between P. parapacifica and other species in the P. platensis-complex ranged from 14.7 to 21.3%. We detected no overlap between distribution divergences at the interspecific level. This pattern of genetic variation is typical of mt CO1 gene sequence data. Previous amphipod studies showed similar levels of sequence variation. Meyran et al. (1997) determined 36.4% CO1 gene variation between Gammarus pulex and G. marinus; Cristescu and Hebert (2005) reported variation values ranging from

12 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 839 Table 2. Sample information used in this study, including the number of samples examined, sampling dates, sampling localities and GenBank accession numbers. Species Number of samples examined Date Sample locality Locality code Longitude/latitude Accession number Source Platorchestia japonica Biwa Lake, Japan (Type locality) PJaJapB N/A HQ Cheng et al., 2011 P. monodi 1 23 March 2010 Yeonangyo Bridge, Incheon-si, South Korea PMoKorS N/ E JN Kim and Min, June 2010 Dadaepo Beach, Busan-si, South Korea PMoKorD N/ E JN May 2009 Wangsan Beach, Incheon-si, South Korea PMoKorY N/ E JN September 2009 Onsucheon Stream, Incheon-si, South Korea PMoKorG N/ E JN712923, 4 P. munmui 3 20 November 2010 Bonggiil Beach, Gyeongsangbuk-do, South Korea (Type locality) PMuKorB N/ E KF This study P. paludosus Taipei, Taiwan (Type locality) PPalTaiT N/A HQ HQ P. parapacifica n. sp. 1 3 June 2010 Geumseongcheon Stream, Jeju-do, South Korea (Type locality) Cheng et al., 2011 PPaKorG N/ E KF This study 1 19 March 2010 Seomjin River, Jeollanam-do, South Korea PPaKorS N/ E 1 3 June 2010 Sehwa Beach, Jeju-do, South Korea PPaKorJ N/ E 1 10 June 2010 Osipcheon Stream, Gyeongsangbuk-do, South PPaKorO N/ E Korea P. platensis Estuary and Gulf of the St Lawrence River, PPlCan N/A FJ581856, Canada FJ Outgroup Orchestia cavimana Radulovici et al., Tegeler See, Germany OCaGer N/13 9 E EF Browne et al., 2007

13 840 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 6, 2013 Table 3. K2P differences (%) of the CO1 gene among Platorchestia species and Orchestia cavimana PMuKorB1 02 PMuKorB2 0.3 P. munmui ( %) 03 PMuKorB PMuKorS PMuKorD PMuKorG P. monodi ( %) 07 PMuKorG PMuKorY PP1Can PP1Can P. platensis (0.2%) 11 PPaKorJ PPaKorO P. parapacifica n. sp. ( %) 13 PPaKorS PPaKorG PJaJapB PPalTaiT PPalTaiT P. paludosus ( %) 18 PPalTaiT OCaGer

14 KIM ET AL.: PHYLOGENY OF PLATORCHESTIA 841 Fig. 10. Divergence time estimates among Platorchestia species based on the mt CO1 gene sequence data, using the Test Molecular Clock (ML) option in MEGA 5.05 (Tamura et al., 2011). Gray bars represent the 95% confidence intervals of the age of each node. This figure is published in colour in the online edition of this journal, which can be accessed via 17 to 28% (Tamura-Nei s distance) for Ponto Caspian amphipods; Witt et al. (2006) recorded a divergence value of 35.2% (K2P distance) for Hyalella spp.; Hou et al. (2007) reported a divergence value of 36.9% for the CO1 gene (K2P distance) from freshwater Gammarus spp.; Kim et al. (2010) revealed 39-44% CO1 gene variation among G. gageoensis, G. electrus, G. pulex and G. sinuolatus (K2P distance); and Cheng et al. (2011) determined % CO1 gene variation among P. japonica, P. paludosus and P. platensis (K2P distance). Based on the results of these previous studies and our present sequence divergence data, P. parapacifica is considered to be a new talitrid species. Morphologically, Platorchestia parapacifica resembles the P. platensis complex. This is evident in the development of sexual dimorphism of antenna 2 and pereiopod 7. However, although this new species was clustered with the P. platensis-complex as a sister group, the relationship between them was weakly supported by our ML (BV: 48) and BI analyses (PPV: 49). We also estimated the divergence time of the genus Platorchestia using the molecular clock (Knowlton et al., 1993; Knowlton and Weigt, 1998; Tamura et al., 2011). Our results suggest that the genus shared a most recent common ancestor during the Miocene period ( million years ago, Mya). The ancestral talitrid appears to have diverged into six species during the Zanclean and Tortonian periods ( Mya). Furthermore, common ancestry of the P. japonica-complex diverged into two species sometime during the Zanclean and Messinian periods ( Mya), while the ancestral talitrid of the P. platensis-complex diverged into three species during the Messinian and Tortonian period (6-8 Mya) (Fig. 10). The results of our present study demonstrate the phylogenetic relationships and divergence times of the P. japonica and P. platensis complexes. Despite their morphological similarity, the two complexes show extensive genetic divergence. Our findings reconfirm the usefulness of molecular techniques for species identification, and also for description of new cryptic diversity. Our study was based solely on the mitochondrial CO1 gene sequence. Additional studies, using other mitochondrial/nuclear sequence data and more species, are required to further elucidate the phylogenetic relationships and evolutionary history of the P. japonica- and P. platensis-complexes. ACKNOWLEDGEMENTS The first two authors contributed equally to this work. This work was supported by the Invasive Species Management Program in Marine Ecosystem (2012), the Ministry of Land, Transport and Maritime Affairs of the Korean Government, and the National Institute of Biological Resources of Korea as a part of the Discovery of Korean Indigenous Species Project REFERENCES Bousfield, E. L The amphipod superfamily Talitroidea in the northeastern Pacific Region. 1. Family Talitridae: systematics and distributional ecology. Publications in Biological Oceanography 11: Browne, W. E., S. H. D. Haddock, and M. Q. Martindale Phylogenetic analysis of lineage relationships among hyperiid amphipods as revealed by examination of the mitochondrial gene, cytochrome oxidase I (COI). Integrative and Comparative Biology 47: Cheng, Y. T., K. Nakazono, Y. K. Lin, and B. K. K. Chan Cryptic diversity of the semi-terrestrial amphipod Platorchestia japonica (Tattersall, 1922) (Amphipoda: Talitrida: Talitridae) in Japan and Taiwan, with description of a new species. Zootaxa 2787: Clare,E.L.,B.K.Lim,M.D.Engstrom,J.L.Eger,andP.D.N.Hebert DNA barcoding of Neotropical bats: species identification and discovery within Guyana. Molecular Ecology Notes 7: Cristescu, M. E. A., and P. D. N. Hebert The Crustacean Seas an evolutionary perspective on the Ponto-Caspian peracarids. Canadian Journal of Fisheries and Aquatic Sciences 62: Elsasser, S. C., R. Floyd, P. D. N. Hebert, and A. I. Schulte-Hostedde Species identification of North American guinea worms (Nematoda: Dracunculus) with DNA barcoding. Molecular Ecology Resources 9: Folmer, O., M. Black, W. Hoeh, and R. Vrijenhoek DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I

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