First Record of the Genus Pseudohelice Sakai, Türkay & Yang, 2006 from India and Description of a New Pseudocryptic Species (Crustacea: Brachyura: Varunidae)

© 2022 Academia Sinica, Taiwan Open Access First Record of the Genus Pseudohelice Sakai, Türkay & Yang, 2006 from India and Description of a New Pseudocryptic Species (Crustacea: Brachyura: Varunidae) Mani Prema1,§ , Jhih-Wei Hsu2,§ , Hsi-Te Shih2,3,* , and Samuthirapandian Ravichandran1,4,* 1Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai – 608 502, Tamil Nadu, India. E-mail: [email protected] (Prema) 2Department of Life Science, National Chung Hsing National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. E-mail: [email protected] (Hsu) 3Research Center for Global Change Biology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. *Correspondence: E-mail: [email protected] (Shih) 4Government Thirumagal Mills College, Gudiyatham, 635 803, Tamil Nadu, India. *Correspondence: E-mail: [email protected] (Ravichandran) §MP and JWH contributed equally to this paper. Received 4 March 2022 / Accepted 26 July 2022 / Published 29 September 2022 Communicated by Benny K.K. Chan A new pseudocryptic species of the varunid crab genus Pseudohelice Sakai, Türkay & Yang, 2006, is described from India based on morphological and molecular evidence. The new species was collected from higher intertidal zones in the Vellar River estuary, Tamil Nadu, southeastern India, in a habitat composed of muddy and sandy sediment. Pseudohelice annamalai n. sp. is similar to P. subquadrata (Dana, 1851) and P. latreillii (H. Milne Edwards, 1837) in general form, but can be distinguished from the congeners by the characters of the infraorbital ridges, male first gonopod, and female vulvae. In addition, the mitochondrial cytochrome oxidase subunit I sequences also support the new species. The occurrence of Pseudohelice from India links the distribution gap between the western Indian Ocean and western Pacific Ocean. The new species provides additional evidence for the geographic isolation of the eastern Indian Ocean for some marine organisms. Key words: Mitochondrial cytochrome oxidase subunit I, Morphology, Pseudohelice annamalai, Taxonomy, Tamil Nadu. BACKGROUND Species of the genus Pseudohelice Sakai, Türkay & Yang, 2006 (family Varunidae) are small-sized and distributed in subtropical and tropical regions of the Indo-West Pacific (Sakai et al. 2006). To date only two species have been confirmed within this genus. Pseudohelice subquadrata (Dana, 1851) was considered widely distributed in Indo-West Pacific (Sakai et al. 2006), but a recent study showed the range of this species to be from the eastern margin of the Indian Ocean to French Polynesia. Pseudohelice latreillii (H. Milne Edwards, 1837) is distributed in eastern Africa, western Indian Ocean (WIO) (Hsu et al. 2022a). Based on the distributional range of the two species of Pseudohelice (Hsu et al. 2022a: fig. 4), species of this genus seems not distributed around the Indian subcontinent. In the region of the Vellar River estuary, Tamil Nadu, southeastern India, 17 species of crabs have been recorded, including sesarmids and fiddler crabs (Austruca annulipes (A. Milne-Edwards, 1873) and A. variegata (Heller, 1862)) as the dominant species in mangroves (Khan et al. 2005; Fredrick and Citation: Prema M, Hsu JW, Shih HT, Ravichandran S. 2022. First record of the genus Pseudohelice Sakai, Türkay & Yang, 2006 from India and description of a new pseudocryptic species (Crustacea: Brachyura: Varunidae). Zool Stud 61:56. doi:10.6620/ZS.2022.61-56. Zoological Studies 61:56 (2022) doi:10.6620/ZS.2022.61-56 1 © 2022 Academia Sinica, Taiwan Ravichandran 2013; Shih et al. 2019a). In recent surveys in this region, specimens of the genus Pseudohelice were collected, representing the first record of this genus from India. Using detailed comparison of the morphological characters with the other two congeners, and with support from analysis of mitochondrial cytochrome oxidase subunit I (COI), the specimens are confirmed to be a different species and the new species described herein. MATERIALS AND METHODS Specimens were collected from high intertidal areas in the Vellar River estuary, Parangipettai, Tamil Nadu, southeastern India, with sediments composed of mud and sand (Fig. 3). The mangroves in the habitats were artificially planted and covered an area of about 5 ha along the northern bank of the Vellar River, with two distinct zones including Rhizophora spp. toward the estuary and Avicennia spp. toward land in the intertidal area of estuary (Fig. 3). The examined specimens were deposited into the reference collections of the crustacean research laboratory, Centre of Advanced Study in Marine Biology, Annamalai University (CASAU). However, the holotype of the new species will be moved to the national repository of the Zoological Survey of India (ZSI), Kolkata; the Museo Zoologico dell’Università di Firenze, Italy (MZUF); Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL); the Queensland Museum, Brisbane, Australia (QM); and the Zoological Reference Collection of the Lee Kong Chian Natural History Museum, National University of Singapore (ZRC). Morphological characters were illustrated with the aid of a drawing tube attached to a stereomicroscope. The morphological characters and terminology follow those of Sakai et al. (2006), Guinot et al. (2013), and Davie et al. (2015). The abbreviation G1 is used for male first gonopod. Measurements are of the maximum carapace width (CW), and carapace length (CL) in millimeters. Genomic DNA was isolated from the muscle tissue of legs or gills using the GeneMark tissue and cell genomic DNA purification kit (Taichung, Taiwan). A portion of the COI gene was amplified with PCR using the primers LCO1490 (5'-GGTCAACAAAT CATAAAGATATTGG-3'), HCO2198 (5'-TAAACT TCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994), LCOB (5’-CAAAYCATAAAGAYATYGG-3'), HCOex (5'-GCTCATACTACAAATCCTAAA-3'), HCOex2 (5'-GCTCANACTACAAATCCTAA-3') and HCOex3 (5'-GCTCANACTACRAATCCTA-3') (Shih et al. 2022b). PCR conditions for the above primers were denaturation for 50 s at 94°C, annealing for 70 s at 45–47°C, and extension for 60 s at 72°C (40 cycles), followed by another extension for 10 min at 72°C. Sequences were obtained by automated sequencing (Applied Biosystems 3730) after verification with the complementary strand. Sequences of different haplotypes have been deposited into GenBank, with other sequences published in Shih and Suzuki (2008), Shih et al. (2020), and Hsu et al. (2022a) (accession numbers given in Table 1). Parahelice daviei (Sakai, Türkay & Yang, 2006), Par. pilimana (A. MilneEdwards, 1873), and Par. pilosa (Sakai, Türkay & Yang, 2006) were selected as outgroups following Hsu et al. (2022a). The best-fitting model for sequence evolution was determined by jModeltest (vers. 2.1.4; Darriba et al. 2012), selected by the Bayesian information criterion (BIC). The best model obtained was HKY+I+G, which was subsequently used for Bayesian inference (BI) and maximum likelihood (ML) analyses. The BI analysis was performed with MrBayes (ver. 3.2.3, Ronquist et al. 2012). Phylogenetic analyses were run with four chains for 10 million generations and four independent runs, with trees sampled every 1,000 generations. The convergence of chains was determined by the average standard deviation of split frequency values below the recommended 0.01 (Ronquist et al. 2020), and the first 2,650 trees were accordingly discarded as “burnin”. A ML analysis was conducted in MEGA (vers. 11.0, Tamura et al. 2021) with the HKY+I+G model and 2,000 replicates and the options of the Nearest-NeighborInterchange (NNI) in the ML heuristic method, NJ/ BioNJ in the initial tree for ML, and moderate in the branch swap filter. A maximum parsimony (MP) consensus tree was also constructed using MEGA, with 2,000 bootstrap reiterations of a simple heuristic search, TBR branch-swapping (tree bisection-reconnection) (100 random-addition sequence replications; max no. of trees to retain = 10,000). Basepair (bp) differences and pairwise estimates of Kimura 2-parameter (K2P) distances (Kimura 1980) for genetic diversities between specimens were calculated with MEGA. RESULTS TAXONOMY Family Varunidae H. Milne Edwards, 1853 Subfamily Cyclograpsinae H. Milne Edwards, 1853 Genus Pseudohelice Sakai, Türkay & Yang, 2006 page 2 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan Pseudohelice annamalai n. sp. (Figs. 1, 2, 4) urn:lsid:zoobank.org:act:A5E686EC-C98A-48CF-81DF47F1EBB3DF32 Material examined: Holotype: 1 male (17.3 × 14.6 mm) (CASAU CR-1011), Vellar River estuary, Tamil Nadu, India, coll. M. Prema and S. Ravichandran, 28 Feb.–5 Mar. 2022. Paratypes (same locality as holotype): 1 male (18.9 × 15.2 mm) (CASAU CR-1013), 21 Sep. 2020; 2 males (20.1 ×16.8, 16.7 × 14.0 mm) (CASAU CR-1014), 17 Dec. 2021; 2 males (16.4 × 13.1, 15.2 × 13.9 mm) (CASAU CR-1015), 25 Feb. 2022; 2 males (18.7 × 15.4, 16.7 × 14.5 mm) (CASAU CR-1016), 2 Mar. 2022; 2 females (18.3 × 15.4, 17.7 × 14.2 mm) (CASAU CR-1017), 5 Jun. 2021; 2 females (17.5 × 16.0, 10.4 × 9.5 mm) (CASAU CR-1018), 3 Mar. 2022, coll. M. Prema; 3 males (18.2 × 15.3, 16.7 × 14.3, 14.3 × 11.9 mm) (NCHUZOOL 17048), coll. M. Prema and S. Ravichandran, 12 Dec. 2020; 5 males (16.6 ×14.4, 18.2 × 15.9, 16.6 × 14.2, 17.1 × 14.3, 17.1 × 14.4 mm), 2 females (14.3 × 12.5, 13.9 × 11.6 mm), 1 ovig. female (18.8 × 15.3 mm) (NCHUZOOL 17049), coll. M. Prema and S. Ravichandran, 28 Feb.–5 Mar. 2022; 2 males (16.9 × 14.2, 17.2 × 14.5 mm), 2 females Table 1. Haplotypes of the cytochrome c oxidase subunit I (COI) gene of specimens of Pseudohelice species from the Indo-West Pacific and the outgroups of Parahelice species Species Locality Catalogue no. Sample size Haplotype Access. no. of COI Pseudohelice annamalai India: Vellar R., Tamil Nadu NCHUZOOL 17048 1 PA1 ON971913 India: Vellar R., Tamil Nadu NCHUZOOL 17049 1 PA2 ON971914 India: Vellar R., Tamil Nadu NCHUZOOL 17048 2 PA3 ON971915, ON971916 India: Vellar R., Tamil Nadu NCHUZOOL 17049 2 PA3 ON971917, ON971918 India: Vellar R., Tamil Nadu CASAU CR-1011 1 PA3 ON971919 India: Vellar R., Tamil Nadu ZRC 2022.0189 2 PA3 ON971920, ON971921 India: Vellar R., Tamil Nadu NCHUZOOL 17049 1 PA4 ON971922 India: Vellar R., Tamil Nadu ZRC 2022.0189 1 PA5 ON971923 India: Vellar R., Tamil Nadu NCHUZOOL 17049 1 PA6 ON971924 India: Vellar R., Tamil Nadu NCHUZOOL 17049 2 PA7 ON971925, ON971926 India: Vellar R., Tamil Nadu ZRC 2022.0189 1 PA7 ON971927 India: Vellar R., Tamil Nadu NCHUZOOL 17049 1 PA8 ON971928 Pseudohelice subquadrata Japan: Miyako, Ryukyus NCHUZOOL 13102 1 PS1 AB334557 Taiwan: Gangnan, Hsinchu City NCHUZOOL 15724 1 PS2 LC511075 Taiwan: Sinfeng, Hsinchu County NCHUZOOL 15723 1 PS3 LC511076 Taiwan: Dingtouer Shoal, Tainan NCHUZOOL 15731 1 PS4 LC511077 Taiwan: Yanshuei R., Tainan NCHUZOOL 15726 1 PS5 LC511078 Taiwan: Wanlitong, Pingtung NCHUZOOL 15733 2 PS6 LC511079 Taiwan: Dongsha Island NCHUZOOL 15725 1 PS7 LC511080 Guam ZRC 2013.1034 1 PS8 LC511081 Indonesia: Bali NCHUZOOL 15722 1 PS2 LC511082 Indonesia: Bali NCHUZOOL 15727 1 PS9 LC511083 Australia: SE Queensland QM W19922 1 PS10 LC511084 Australia: SE Queensland QM W19922 1 PS11 LC511085 Pseudohelice latreillii Egypt: Nabq-El Arwashie, Sinai MZUF 3790 1 PL1 MZ400781 Kenya: Mida Creek MZUF 4985 1 PL2 MZ400782 Kenya: Mida Creek MZUF 4993 1 PL2 MZ400783 Kenya: Gazi MZUF 4987 1 PL4 MZ400784 Kenya: Mida Creek NCHUZOOL 1 PL3 MZ400785 Seychelles: Mahé, Port Launay MZUF 2935 1 PL4 MZ400786 Mauritius: Poste Lafayette MZUF 4994 2 PL2 MZ400787, MZ400788 Mauritius: Melville MZUF 4997 1 PL2 MZ400789 Mauritius: Rodrigues I. MZUF 4991 1 PL2 MZ400790 Mauritius: Rodrigues I. MZUF 4999 1 PL2 MZ400791 outgroups Parahelice daviei Taiwan: Gangkou R., Pingtung NCHUZOOL 15717 1 LC511052 Parahelice pilimana Taiwan: Baoli R., Pingtung NCHUZOOL 15681 1 LC511064 Parahelice pilosa Taiwan: Gangkou R., Pingtung NCHUZOOL 15700 1 LC511071 page 3 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan (15.6 × 13.2, 18.1 × 15.4 mm) (ZRC 2022.0189), coll. M. Prema and S. Ravichandran, 28 Feb.–5 Mar. 2022. Comparative material: Pseudohelice subquadrata and P. latreillii (see material examined in Hsu et al. 2022a). Description: Carapace (Fig. 2A) quadrate, slightly broader than long, 1.08–1.15 times as broad as long; surface convex, irregularly punctated and finely granulated; mesogastric and protogastric regions deeply low with noticeable epigastric groove. Frontal margin slightly concave. Anterolateral margins with 3 teeth including larger orbital tooth, second tooth slightly narrower than preceding, last tooth very small, distinct. Infraorbital ridge (Fig. 2C–E) heteromorphic in both sexes; in male, mesial part with 4 or 5 rounded, smooth, less interspaced small tubercles, followed by several large, elongated and less convex tubercles; lateral part with 1 significantly largest, very convex and elliptical tubercle, followed by 1 less convex tubercle, and 2 or 3 large convex tubercles (Fig. 2C); in female form I, mesial part with several (5 or 6) dense, small rounded tubercles, followed by several larger, less convex elongated tubercles; lateral part with well-spaced 3 or 4 elliptical, more convex, larger tubercles, ending with 1 or 2 small rounded tubercles (Fig. 2D); in female form II, mesial part with several (5 or 6) dense, small rounded tubercles, followed by well-spaced several elongated and less convex tubercles, lateral part with 1 significantly largest elongated tubercle, and 2–5 closely spaced, larger tubercles (Fig. 2E). Chelipeds (Fig. 2B) usually unequal in adult male and equal in some adult male (similar size) and all female; palm bulky, inner palm meagerly granulated, outer surface smooth, line of short setae present at base of anterior margin of palm. Ambulatory legs (Fig. 1A‒D) slender, anterior margins of merus, carpus, and propodus covered with short setae, posterior margins with sparse short setae. Male G1 (Fig. 2F‒I) slender, tapering, slightly curved towards lateral end in distal part, chitinous Fig. 1. Pseudohelice annamalai n. sp. with preserved coloration. A, dorsal view of male; B, ventral view of male; C, dorsal view of female; D, ventral view of female. A, B, holotype, CASAU CR-1011 (17.3 × 14.6 mm); C, D, NCHUZOOL 17049 (14.3 × 12.5 mm). page 4 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan Fig. 2. Pseudohelice annamalai n. sp. A, carapace; B, outer view of male left cheliped; C, left male infraorbital ridge; D, E, left female infraorbital ridge (D, form I; E, form II); F, G, dorsal view of right G1; H, I, ventral view of right G1; J, K, right vulva (J, form I; K, form II). A, B, male, CASAU CR-1013 (18.9 × 15.2 mm); C, F–I, holotype male, CASAU CR 1011 (17.3 × 14.6 mm); D, female, NCHUZOOL 17049 (18.8 × 16.4 mm); E, J, female, ZRC 2022.0189 (18.1 × 15.4 mm); K, female, NCHUZOOL 17049 (13.9 × 11.6 mm). page 5 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan Fig. 3. Habitats of Pseudohelice annamalai n. sp. A, higher intertidal zone of the Vellar River estuary, in front of the Centre of Advanced Study in Marine Biology, Tamil Nadu, southeastern India; B, artificial mangroves of Rhizophora sp. and Avicennia sp., in the margin of Vellar River. page 6 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan endpiece shorter, wider and thicker, bilobed and rounded end; female vulvae (Fig. 2J, K) with a semicircular sternal vulvar cover; sunken on inner part. Size: Largest male specimen is CW 20.1 mm (CASAU CR-1014); largest female is (ovigerous) CW 18.8 mm (NCHUZOOL 17049). Color in life: Varied from dark purple to dark gray, with irregular light brown, yellowish brown, or white patches on posterior carapace and some individuals on entire carapace as white dots or patches; some young individuals yellowish orange-brown. Chelipeds usually lighter brown (most of surface) and upper regions of palm lighter purple in adult male (Fig. 4A–G). Ecological notes: In the southeastern coast of India, this species inhabits sand-muddy banks of mangroves (Fig. 3) and it is sympatric with A. annulipes (H. Milne Edwards, 1837). Some burrows (Fig. 4H) were located near the pneumatophores of Avicennia mangroves. Burrows have a depth of 25–30 cm and are branched, with larger-sized pellets around the burrow entrance. Etymology: This species is named after Annamalai University, in honor of 100 years’ service in education and research as a state university of India. In addition, the present specimens were collected from the intertidal areas in front of the Faculty of Marine Science, Research Centre (Centre of Advanced Study in Marine Biology, Annamalai University), Vellar River, Tamil Nadu. The name is used as a noun in apposition. Distribution: Currently known only from the type locality, the Vellar River estuary, southeastern India. Remarks: Morphologically, this new species is similar to P. subquadrata and P. latreillii, but can be distinguished by the infraorbital ridges, male G1s, and female vulvae. Both infraorbital ridges and male G1s (or female vulvae) should be compared to identify the species of Pseudohelice, because male infraorbital ridges and female vulvae are similar in P. annamalai and P. subquadrata, and male G1s and female infraorbital ridges are similar in P. annamalai and P. latreillii (Table 2). Pseudohelice annamalai can be discerned from P. subquadrata by the infraorbital ridges in both sexes and male G1s. In P. annamalai, the lateral part of male infraorbital ridge has 1 less convex but obvious tubercle between the largest tubercle and 2 or 3 large convex tubercles (vs. this tubercle absent or not obvious in P. subquadrata) (Fig. 2C; Hsu et al. 2022a: fig. 1E); the lateral part of the female infraorbital ridge lacks the largest elongated tubercle (form I) or is present but smaller and less convex (form II) (vs. largest tubercle relatively larger and more convex in P. subquadrata) (Fig. 2D, E; Hsu et al. 2022a: fig. 1F). Pseudohelice annamalai has the G1 slender, the upper part tubular, the chitinous endpiece with distal part shorter and thicker (vs. G1 stouter, upper part slightly flatter, chitinous endpiece with distal part relatively longer and thinner in P. subquadrata) (Fig. 2F–I; Hsu et al. 2022a: fig. 1G–J). Pseudohelice annamalai can be differentiated from P. latreillii by the male infraorbital ridges and the female vulvae. In P. annamalai, the lateral part of male infraorbital ridge has 1 less convex but obvious tubercle between the largest elliptical tubercle and 2 or 3 large convex tubercles (vs. tubercle absent between the largest rounded tubercle and 2 or 3 large convex tubercles in P. latreillii) (Fig. 2C; Hsu et al. 2022a: fig. 3E). In females, the sternal vulvar cover is often longer (but shorter in some individuals) in P. annamalai (vs. shorter in P. latreillii) (Fig. 2J–K; Hsu et al. 2022a: fig. 1L, M). Molecular analyses We used 16 specimens from the Vellar River estuary for molecular study, with 8 haplotypes of COI (Table 1). The pairwise nucleotide divergences of K2P distances and bp differences among haplotypes of the species of Pseudohelice are shown in table 3. The intraspecific nucleotide divergences (and bp differences) of P. subquadrata, P. annamalai n. sp., and P. latreillii are ≤ 1.39% (≤ 9 bp), ≤ 0.77% (≤ 5 bp), and ≤ 1.86% (≤ 12 bp), respectively. Pseudohelice annamalai has interspecific divergences ≥ 1.54% (≥ 10 bp) with P. subquadrata and ≥ 3.45% (≥ 22 bp) with P. latreillii. The phylogenetic tree (Fig. 5) based on COI shows three clades within the genus Pseudohelice, corresponding to P. subquadrata, P. annamalai, and P. latreillii, although P. annamalai is not highly supported by BI and MP methods. The relationship between P. subquadrata and P. annamalai is closer, but the support values are not high in BI and MP methods. DISCUSSION Biogeographic implication The occurrence of Pseudohelice from India links the distribution gap between the WIO and western Pacific Ocean. Currently the geographical distributions of the three species of Pseudohelice are different and do not overlap. From west to east, P. latreillii is distributed in the WIO (including the Red Sea, Arabian Sea, eastern Africa, Mauritius Island, and Rodrigues Island); P. annamalai is found from southeastern India (Vellar River estuary); and P. subquadrata is distributed widely from the eastern margin of the Indian Ocean (Koh Surin page 7 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan Fig. 4. Color in life of Pseudohelice annamalai n. sp. (A–G) from higher intertidal zone of the Vellar River estuary, Tamil Nadu, India. A, B, dorsal view of male; C, D, frontal and dorsal views of female, respectively; E, F, dorsal and frontal views of an ovigerous female, respectively; G, male at the burrow entrance near mangrove pneumatophores; H, burrow in the habitat. A, CASAU CR-1012 (18.2 × 15.5 mm); B, CASAU CR-1016 (18.7 × 15.4 mm), C–F, specimens not collected; G, NCHUZOOL 17049 (18.2 × 16.8 mm). page 8 of 13Zoological Studies 61:56 (2022) © 2022 Academia Sinica, Taiwan in western Thailand) to the eastern region of the West Pacific (French Polynesia) (Sakai et al. 2006; Hsu et al. 2022a: fig. 4; this study). In addition to the endemic P. latreillii, other species of coastal crabs are known to be endemic in the WIO, e.g., Metopograpsus cannicci Innocenti, Schubart & Fratini, 2020 and M. messor (Forskål, 1775) (Grapsidae) (Fratini et al. 2018; Innocenti et al. 2020); Neosarmatium africanum Ragionieri, Fratini & Schubart, 2012 and N. meinerti (De Man, 1887) (Sesarmidae) (Ragionieri et al. 2009 2012); Leptodius exaratus (H. Milne Edwards, 1834) (Xanthidae) (Lee et al. 2013); seven species of fiddler crabs (Shih et al. 2022b); and several species or clades of ghost crabs (genus Ocypode) (Ocypodidae) (Sakai and Türkay 2013; Ma et al. 2018). Some marine barriers to larval dispersal in the WIO have been proposed, including the hydrochemical front found at approximately 10°S, the upwelling off the coast of Somalia and Arabia, and high salinity at the surface in the Arabian Sea (Ma et al. 2018). Several crab studies examined the differentiation within the eastern Indian Ocean (EIO). Lai et al. (2010) reported that Portunus reticulatus (Herbst, 1799) distributed in the EIO is different genetically from Por. pelagicus (Linnaeus, 1758) from the western Pacific, although the Bay of Bengal may be a zone of hybridization of the two species. Shih et al. (2019a) confirmed two species of fiddler crabs in the EIO with Austruca variegata (Heller, 1862) in the Bay of Bengal and A. bengali (Crane, 1975) in the Andaman Sea, which differ genetically from A. triangularis (A. Milne-Edwards, 1873) in the western Pacific. Similarly, Lai et al. (2006) showed that Calappa guerini BritoCapello, 1871 is only distributed in the Indian Ocean, which differ from Calappa quadrimaculata Takeda & Shikatani, 1990 from the western Pacific. It is suggested that some kinds of geographic isolation (e.g., ocean currents) in the EIO may promote speciation for some marine organisms. Within the Helice/Chasmagnathus complex, species of Parahelice also have wide distributions, but less wide than species of Pseudohelice and the Fig. 5. A Bayesian inference (BI) tree for species of Pseudohelice and the outgroups, based on the cytochrome c oxidase subunit I (COI) gene. Probability values at the nodes represent support values for BI, maximum likelihood (ML) and maximum parsimony (MP). For haplotype names, see table 1. 0.02 PL3 (Kenya) PA4 PS5 (Taiwan: Tainan) PS1 (Japan: Ryukyus) PA7 (x3) Parahelice daviei (Taiwan) PS6 (Taiwan: Pingtung) (x2) PA5 PA8 PS10(Australia: Queensland PL4(Kenya; Seychelles) (x2) PL1(Egypt: Nabq) Parahelice pilimana (Taiwan) PS2 (Taiwan; Bali) (x2) PS8 (Guam) PS3 (Taiwan: Hsinchu) PA1 PS9 (Indonesia: Bali) PS7 (Taiwan: Dongsha) PA6 PS4 (Taiwan: Tainan) Parahelice pilosa (Taiwan) PA2 PL2(Kenya; Mauritius; Rodrigues) (x7) PA3 (x7) PS11(Australia: Queensland) 1/99/99 1/100/100 0.64/80/83 0.8/92/64 0.53/96/80 0.99/93/90 BI / ML / MP support values Pseudohelice subquadrata Pseudohelice latreillii Pseudohelice annamalai n. sp. (Vellar R. estuary, SE India) page 9 of 13Zoological Studies 61:56 (2022)