Phylogeography of the Fiddler Crab Tubuca arcuata (Crustacea: Brachyura: Ocypodidae) in East Asia and Northern Vietnam

© 2022 Academia Sinica, Taiwan Open Access Special issue: Systematics and Biogeography of Fiddler Crabs Phylogeography of the Fiddler Crab Tubuca arcuata (Crustacea: Brachyura: Ocypodidae) in East Asia and Northern Vietnam Hsi-Te Shih1, Min-Yun Liu2,*, Misuzu Aoki3, and Hiroshi Suzuki4 1Department of Life Science and Research Center for Global Change Biology, National Chung Hsing University, Taichung 402, Taiwan. E-mail: [email protected] (Shih) 2Taiwan Ocean Research Institute, National Applied Research Laboratories, Kaohsiung 852, Taiwan. *Correspondence: E-mail: [email protected] (Liu) 3Wetlands International Japan, Tokyo. E-mail: [email protected] (Aoki) 4Department of Fisheries Biology and Oceanography, Kagoshima University, Kagoshima, Japan. E-mail: [email protected] (Suzuki) Received 30 December 2021 / Accepted 11 August 2022 / Published 16 November 2022 Communicated by Benny K.K. Chan Special issue: Systematics and Biogeography of Fiddler Crabs (articles 64–71). Editors: Hsi-Te Shih and Benny K. K. Chan The fiddler crab Tubuca arcuata (Crustacea: Ocypodidae: Gelasiminae) is widely distributed across East Asia and northern Vietnam. Particularly, this species inhabits estuarine mangroves and mudflats of the East Asian continent, as well as the main islands of Japan and the Ryukyus. By comparing sequences of the mitochondrial 16S, cytochrome oxidase subunit I (COI), and control region genes, two main clades of this species were elucidated. The northern (N) clade was mostly restricted to the region north of the Taiwan Strait, whereas the wide (W) clade could be found throughout the entire range inhabited by this species. Based on the distribution of the ancestral haplotypes of COI and the divergence time of the two clades, our findings suggest that the land bridge barrier present in the Taiwan Strait during glaciations might have played an important role in their cladogenesis, approximately 0.93 million years ago. Here, we propose that the distribution of the ancestral haplotype of the W clade in the Ryukyus and the main islands of Japan was determined by the Paleo-Kuroshio Current, whereas the modern distribution pattern is shaped by the modern Kuroshio Current and other warm currents during the crab reproduction season in the summer. Key words: Mitochondrial genes, 16S rDNA, Cytochrome oxidase subunit I, Control region gene, Glaciation, Land bridge, Taiwan Strait. BACKGROUND The Taiwan Strait is a shallow and narrow watergate located in East Asia, close to the Tropic of Cancer (23.5°N) and between the island of Taiwan and the East Asian continent (Fig. 1A). This strait has been hypothesized to play a key role in the dispersal of continental coastal organisms between subtropical and tropical regions by acting as a barrier. During the glacial period, when the sea level was 140 m below the present level (shaded areas in Fig. 1A), the Taiwan Strait became a land bridge (Boggs et al. 1979; Voris 2000; Xu et al. 2009) that presumably allowed some terrestrial animals to migrate from continental China to Taiwan and even to the Ryukyu Islands (e.g., mammals, Ho et al. 1997 2000; Cai 1999; reptiles, Lin et al. 2002; frogs, YH Wang et al. 2017; freshwater crabs, Shih et al. 2007 2011). However, the glacial land bridge of the Taiwan Strait also acted as a geographical barrier that impeded the dispersal of marine coastal organisms between the East China Sea (ECS) and the South China Sea (SCS) (see below). Citation: Shih HT, Liu MY, Aoki M, Suzuki H. 2022. Phylogeography of the fiddler crab Tubuca arcuata (Crustacea: Brachyura: Ocypodidae) in East Asia and northern Vietnam. Zool Stud 61:68. doi:10.6620/ZS.2022.61-68. Zoological Studies 61:68 (2022) doi:10.6620/ZS.2022.61-68 1 © 2022 Academia Sinica, Taiwan MONGOLIA MALAYSIA THAILAND Sulawesi RYUKYUS TAIWAN PHILIPPINES LAOS VIETNAM KOREA Yellow Sea    CHINA East China Sea South China Sea Pacific Ocean Taiwan Strait Yangtze River Delta JAPAN 120oE 40oN 110oE 130oE 30oN 20oN 10oN Tropicof Cancer MONGOLIA MALAYSIA THAILAND Sulawesi RYUKYUS TAIWAN LAOS KOREA 1 11 8 3 4 5 6 2 7 9 10 12 13 14 15 16 17 18 JAPAN East China Sea Yellow Sea South China Sea Taiwan Strait CHINA VIETNAM PHILIPPINES Pacific Ocean 120oE 40oN 110oE 130oE 30oN 20oN 10oN Tropicof Cancer AB Several hypotheses for the phylogeographic patterns of marine organisms in East Asia have been proposed (see Fig. 1A). For example, some studies have indicated that (1) there is little divergence along the coastal regions of the East Asian continent (e.g., mudskippers, He et al. 2015; filefish, ZY Wang et al. 2016; flathead fish, Cheng et al. 2019); (2) the Yellow Sea (YS) clades are unique, due to the occurrence of local ocean gyres (e.g., marine snails, Ni et al. 2015; Zhao et al. 2017; swimming crabs, Han et al. 2015); (3) the northern and southern clades in both continental and insular regions are separated by the Yangtze River, due to freshwater discharge (e.g., marine snails, Dong et al. 2012; Yu et al. 2014; Ni et al. 2015 2017; J Wang et al. 2015; algae, Cheang et al. 2010; J Wang et al. 2015); (4) the northern and southern clades in the continental region were separated by the Taiwan Strait during glaciations (e.g., mullets, Liu et al. 2007b; mitten crabs, Xu et al. 2009; bivalves, XX Wang et al. 2017); and (5) the Okinawa (or the Ryukyus) clade is unique (e.g., barnacles, Chang et al. 2017; intertidal crabs including Tmethypocoelis, Mictyris, and Scopimera, Davie and Kosuge 1995; Davie et al. 2010; Wong et al. 2010). Tubuca arcuata (De Haan, 1835), one of the most common species of fiddler crabs in East Asia, is widely distributed across mudflats and estuaries in the subtropical to tropical regions of East Asia, Korea, Japan (including the Ryukyus), Taiwan (including Penghu), China (including Hainan and Hong Kong), and northern Vietnam (Crane 1975; Yoshigou 2001; Shih et al. 2010 2022). This species was grouped among the continental fiddler crabs in East Asia, by exhibiting a similar distribution to Austruca lactea, Gelasimus borealis, and Paraleptuca splendida (see Shih 2012). The general ecology, reproduction, growth and behavior of T. arcuata have been extensively studied (e.g., JT Shih 1990 1992; JT Shih et al. 1991; Murai 1992; JH Shih and Lan 1993; JH Shih and Wang 1993; Jones and Morton 1994; Li 1996; Otani et al. 1997; Wada and Murata 2000; Aoki et al. 2010). During its early development, this species undergoes five zoeal stages within 18 days, followed by the megalopal stage (Ko and Kim 1989). Furthermore, this species has been included in various phylogenetic studies of fiddler crabs (Levinton et al. 1996; Sturmbauer et al. 1996; Shih et al. 2016b) and a study analyzing its full Fig. 1. A, Phylogeographic patterns of coastal organisms in continental and insular East Asia. (1) There is no divergence along the coastal regions of the East Asian continent (not shown); (2) the unique Yellow Sea clade; (3) northern and southern clades of the continental (and insular) regions separated by the Yangtze River; (4) the north and south clades along the continental region separated by the Taiwan Strait; (5) the unique Okinawa or Ryukyus clade. See the BACKGROUND section for more details. B, Collection sites (squares) for specimens of Tubuca arcuata from East Asia and northern Vietnam (the numbers next to the squares correspond to the localities in Table 1). page 2 of 13Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan mitochondrial genome (Karagozlu et al. 2016), as well as two population genetics studies (e.g., allozyme, Huang and JH Shih 1995; restriction fragment length polymorphism (RFLP), Aoki et al. 2008). Tubuca arcuata is widely distributed throughout the continent and islands of East Asia and northern Vietnam, and the Okinawa population was considered to have minimal gene flow with other populations (Aoki et al. 2008). Therefore this species is a good candidate to test the above listed hypotheses regarding the phylogeographic patterns. Our study aimed to: (1) characterize a variety of mitochondrial genetic markers (16S rDNA, cytochrome oxidase subunit I [COI] and control region [CR]) in T. arcuata samples from various locations across their natural habitat range; (2) assess the uniqueness of the Okinawa population; and (3) explain the elucidated phylogeographic patterns in the context of historical glacial events and/or contemporary ocean currents. MATERIALS AND METHODS Tubuca arcuata specimens collected from across East Asia, as well as the outgroup T. forcipata from Malaysia (Table 1; Fig. 1), were preserved in 70–95% ethanol and deposited into the Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL); the Department of Environmental Biology and Fisheries Science, National Taiwan Ocean University, Keelung, Taiwan (NTOU) and the Zoological Reference Collection, Raffles Museum of Biodiversity Research, National University of Singapore (ZRC). Table 1. Specimens and haplotypes of the 16S rRNA, COI, and CR genetic markers of Tubuca arcuata from East Asia and northern Vietnam, as well as the outgroup T. forcipata used in this study. The numbers within brackets following the localities correspond to those in figure 1B. For abbreviations of museums or universities see the MATERIALS AND METHODS Locality Catalogue no. of NCHUZOOL (unless indicated) Haplotype of 16S Access. no. of 16S Haplotype of COI Access. no. of COI Haplotype of control region Access. no. of control region T. arcuata North N clade South Korea: Ganghwa Island, Incheon [1] 13651 S1 LC053353 C1 LC053370 South Korea: Ganghwa Island, Incheon [1] 13651 S1 OP167996 C2 OP153631 Japan: Yoshinogawa R., Tokushima [2] —aS1 OP167997 C3 OP153632 Taiwan: Guandu, Taipei [7] NTOU S1 OP167998 C4 OP153633 D1 OP183984 Taiwan: Guandu, Taipei [7] NTOU S1 OP167999 C5 OP153634 D2 OP183985 Taiwan: Guandu, Taipei [7] NTOU S2 OP168000 C2 OP153635 D3 OP183986 Taiwan: Houlong R., Miaoli [8] 14614 S3 OP168001 C2 OP153636 D4 OP183987 Taiwan: Shengang, Changhua [9] 14615 S1 OP168002 C6 OP153637 D5 OP183988 Taiwan: Dongshih, Chiayi County [9] 14618 S4 OP168003 C7 OP153638 D6 OP183989 Taiwan: Cigu, Tainan [9] 13260 S1 OP168004 C1 OP153639 D7 OP183990 Taiwan: Cigu, Tainan [9] 13260 S5 AB491164 C8 AB491165 D8 OP183991 Taiwan: Cieding, Kaohsiung [9] 15162 S6 OP168005 C9 OP153640 D9 OP183992 Taiwan: Caiyuan, Penghu [10] 14628 S7 OP168006 C2 OP153641 D10 OP183993 Taiwan: Kinmen [13] 14608 S1 OP168007 C10 OP153642 D11 OP183994 China: Qingdao [11] 15163 S1 OP168008 C2 OP153643 D12 OP183995 China: Qingdao [11] 15163 S1 OP168009 C2 OP153644 D13 OP183996 China: Qingdao [11] 15163 S1 OP168010 C11 OP153645 China: Shanghai [12] ZRC 1998.308 S7 OP168011 C2 OP153646 China: Xiamen, Fujian [13] 13350 S1 OP168012 C2 OP153647 D14 OP183997 W clade South Korea: Ganghwa Island, Incheon [1] 13651 S1 OP168013 C12 OP153648 D15 OP183998 South Korea: Ganghwa Island, Incheon [1] 13651 S1 OP168014 C12 OP153649 D16 OP183999 Japan: Yoshinogawa R., Tokushima [2] —aS1 OP168015 C13 OP153650 D17 OP184000 Japan: Yoshinogawa R., Tokushima [2] —aS8 OP168016 C14 OP153651 D18 OP184001 Japan: Yoshinogawa R., Tokushima [2] —aS1 OP168017 C14 AB535432 D19 OP184002 Japan: Yoshinogawa R., Tokushima [2] —aS1 OP168018 C15 OP153652 D20 OP184003 Japan: Kaeda R., Miyazaki [3] 14610 S1 LC097112 C14 AB535432 D21 OP184004 Japan: Kaeda R., Miyazaki [3] 14610 S1 OP168019 C14 AB535432 Japan: Kaeda R., Miyazaki [3] 14610 S1 OP168020 C13 OP153653 D22 OP184005 Japan: Kaeda R., Miyazaki [3] 14610 S1 OP168021 C16 OP153654 D23 OP184006 Japan: Ohno R., Kumamoto [4] —aS1 OP168022 C17 OP153655 D24 OP184007 page 3 of 13Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan Locality Catalogue no. of NCHUZOOL (unless indicated) Haplotype of 16S Access. no. of 16S Haplotype of COI Access. no. of COI Haplotype of control region Access. no. of control region Japan: Nakagushuku Bay, Okinawa [5] —aS1 OP168023 C14 AB535432 D25 OP184008 Japan: Nakagushuku Bay, Okinawa [5] —aS9 OP168024 C18 OP153656 D26 OP184009 Japan: Nakagushuku Bay, Okinawa [5] —aS1 OP168025 C14 AB535432 D27 OP184010 Japan: Nakagushuku Bay, Okinawa [5] —aS9 OP168026 C19 OP153657 D28 OP184011 Japan: Nakagushuku Bay, Okinawa [5] —aS9 OP168027 C18 OP153658 D29 OP184012 Taiwan: Yilan [6] 14625 S10 OP168028 C14 AB535432 D30 OP184013 Taiwan: Yilan [6] 14625 S1 OP168029 C20 OP153659 D31 OP184014 Taiwan: Yilan [6] 14620 S11 OP168030 C14 OP153660 D32 OP184015 Taiwan: Jhuwei, New Taipei City [7] 14627 S1 OP168031 C21 OP153661 Taiwan: Jhuwei, New Taipei City [7] 14627 S1 OP168032 C12 OP153662 D33 OP184016 Taiwan: Guandu, Taipei [7] NTOU S1 OP168033 C22 OP153663 D34 OP184017 Taiwan: Guandu, Taipei [7] NTOU S1 OP168034 C23 OP153664 D35 OP184018 Taiwan: Guandu, Taipei [7] NTOU S12 OP168035 C24 OP153665 D36 OP184019 Taiwan: Haishangu, Hsinchu City [8] 13305 S13 AB535406 C14 AB535432 Taiwan: Yuanli, Miaoli [8] 14616 S14 OP168036 C25 OP153666 Taiwan: Wenliao, Taichung [8] 14619 S1 OP168037 C26 OP153667 D37 OP184020 Taiwan: Gaomei, Taichung [8] 14629 S15 OP168038 C27 OP153668 D38 OP184021 Taiwan: Shengang, Changhua [9] 14624 S1 OP168039 C14 AB535432 D39 OP184022 Taiwan: Shengang, Changhua [9] 14615 S16 OP168040 C28 OP153669 D40 OP184023 Taiwan: Shengang, Changhua [9] 14611 S17 OP168041 C29 OP153670 Taiwan: Hanbao, Changhua [9] 14634 S1 OP168042 C30 OP153671 Taiwan: Hanbao, Changhua [9] 15164 S18 OP168043 C31 OP153672 D41 OP184024 Taiwan: Cigu, Tainan [9] 14612 S1 OP168044 C32 OP153673 xxx Taiwan: Cigu, Tainan [9] 14612 S1 OP168045 C33 OP153674 D42 OP184025 Taiwan: Cigu, Tainan [9] 14630 S19 OP168046 C25 OP153675 D43 OP184026 Taiwan: Cieding, Kaohsiung [9] 15162 S1 OP168047 C14 OP153676 D44 OP184027 Taiwan: Linyuan, Kaohsiung [9] 15165 S3 OP168048 C34 OP153677 D45 OP184028 Taiwan: Cingluo, Penghu [10] 14631 S20 OP168049 C35 OP153678 D46 OP184029 Taiwan: Shuangyuan, Penghu [10] 14663 S21 OP168050 C33 OP153679 D47 OP184030 Taiwan: Kinmen [13] 14622 S22 OP168051 C36 OP153680 Taiwan: Kinmen [13] 14747 S1 OP168052 C14 OP153681 China: Shanghai [12] ZRC 1998.308 S23 OP168053 C37 OP153682 D48 OP184031 China: Xiamen, Fujian [13] 13350 S24 OP168054 C14 AB535432 China: Xiamen, Fujian [13] 13350 S1 OP168055 C14 OP153683 D49 OP184032 Hong Kong [14] 13362 S3 OP168056 C38 OP153684 D50 OP184033 Hong Kong [14] 13362 S1 OP168057 C39 OP153685 D51 OP184034 Hong Kong [14] 13362 S1 OP168058 C40 OP153686 D52 OP184035 Hong Kong [14] 13362 S1 OP168059 C41 OP153687 China: Dongzhai, Hainan [15] 13363 S25 OP168060 C42 OP153688 D53 OP184036 China: Dongzhai, Hainan [15] 13363 S25 OP168061 C42 OP153689 D54 OP184037 China: Dongzhai, Hainan [15] 13363 S26 AB813667 C43 AB813684 China: Dongzhai, Hainan [15] 13363 S1 OP168062 C44 OP153690 China: Dongzhai, Hainan [15] 13363 S1 OP168063 C31 OP153691 China: Qinzhou, Guangxi [16] 13362 S3 OP168064 C45 OP153692 D55 OP184038 Vietnam: Cat Ba Island [17] 15140 S1 OP168065 C14 ON193478 Vietnam: Nam Dinh [17] ZRC S3 OP168066 C46 OP153693 Vietnam: Nam Dinh [17] 15144 S1 OP168067 C14 ON193480 D56 OP184039 Vietnam: Nam Dinh [17] 15144 S1 OP168068 C14 ON193481 D57 OP184040 Vietnam: Nam Dinh [17] 15144 S3 OP168069 C14 ON193482 D58 OP184041 Vietnam: Nam Dinh [17] 15100 S1 OP168070 C14 ON193479 D59 OP184042 Vietnam: Ha Tinh [18] 15141 S1 OP168071 C47 ON193483 D60 OP184043 Outgroups T. forcipata Malaysia: Johore NTOU fc-S LC053355 fc-C LC053372 fc-D1 OP184044 Malaysia: Johore NTOU fc-S OP168073 fc-C OP153695 fc-D2 OP184045 Malaysia: Johore NTOU fc-S OP168074 fc-C OP153696 fc-D3 OP184046 a Voucher specimen not preserved. Table 1. (Continued) page 4 of 13Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan Genomic DNA was isolated from muscle tissues using commercial kits and the sequences of the mitochondrial 16S rDNA [~560 base pairs (bp)], COI (658 bp), and CR (~905 bp) markers were obtained as described by Shih et al. (2013 2016b) with verification with the complementary strand. The primers used were 1471 (5'-CCTGTTTANCAAAAACAT-3'), 1472 (5'-AGATAGAAACCAACCTGG-3') (Crandall and Fitzpatrick 1996), 16L29 (5'-YGCCTGTTTATCAAAAACAT-3') and 16H10 (5'-AATCCTTTCGTACTAAA-3') (Schubart 2009) for 16S; LCO1490 (5'-GGTCAACAAAT CATAAAGATATTGG-3') and HCO2198 (5'-TA AACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994) for COI; and ILEUCAR3 (5'-GCTAYCCTTTTAAATCAGGCAC-3'; Pie et al. 2008), CR-F4 (5'-TAAGTATAACCGCAGATG-3'; Shih et al. 2013), as well as the three newly designed CR-F2 (5'-TAATAAAACATGCTGAGAACTGAAAC-3'), CRR1 (5'-GATGGGGGCTGATAGGGGCTGTAGTGGT AA-3') and CR-R2 (5'-ATAAATAATAAGGACAAA-3') for CR. The sequences of the different haplotypes were deposited into the NCBI GenBank database (accession numbers are summarized in Table 1). For the combined 16S, COI, and CR dataset, the best fitting models for sequence evolution of individual datasets were determined independently using the jModelTest tool (vers. 2.1.4, Darriba et al. 2012) and selected based on the Bayesian information criterion (BIC). The best models were the HKY, HKY+G, and TIM3+I+G models, respectively, and were subsequently applied to the partitioned Bayesian inference (BI) analysis. BI analysis was performed using the MrBayes package (vers. 3.2.6, Ronquist et al. 2012). Searches were conducted with 4 chains for 10 million generations and 4 independent runs, with trees sampled every 1000 generations. The convergence of the chains was determined by the average standard deviation of the split frequency values below the recommended threshold of 0.01 (Ronquist et al. 2005) and the first 3100 trees were discarded as burnin. A maximum parsimony (MP) consensus tree was constructed using MEGA software (vers. 11, Tamura et al. 2021), with 2000 bootstrap reiterations of a simple heuristic search, TBR branch-swapping (tree bisection-reconnection) (100 random-addition sequence replications; max no. of trees to retain = 10000). The relationships of the 16S and COI haplotypes among the T. arcuata populations were presented separately using the program TCS (vers. 1.23, Clement et al. 2000) with the gaps treated as the fifth state in 16S. As the network of CR haplotypes contains a lot of unsampled sequences (Leigh and Bryant 2015), it was not used in this study. The bp difference, as well as the pairwise estimates of the Kimura 2-parameter (K2P) distance (Kimura 1980) and the uncorrected p-distance for the genetic divergence between haplotypes were calculated using MEGA. RESULTS A 557 bp segment of the 16S gene from 81 specimens, a 658 bp segment of COI from 81 specimens, and a 920 bp segment of CR from 60 specimens of T. arcuata were amplified and aligned. A total of 26 haplotypes of the 16S gene, 47 haplotypes of COI and 60 haplotypes of CR were found (Table 1). Phylogenetic analyses based on the combined 16S, COI, and CR database of 2135 bp from 63 specimens (including the outgroups) elucidated two clades that were highly supported by BI and MP analyses (Fig. 2). One clade was labeled as the “N clade”, because it was mainly distributed in the north region of the habitat range (green line in Fig. 5A), whereas the other clade was labeled as the “W clade” because it was widely distributed across the entire habitat range (red line in Fig. 5A). Two haplotypes ( “S1” and “S3”) of 16S were shared by the N and W clades shown in the 16S haplotype network (Fig. 3?). 11 and 36 haplotypes of COI were present in N and W clades, respectively, without a shared haplotype (Table 1). For the CR marker, there were 14 and 46 haplotypes for the N and W clades, respectively, without a shared haplotype (Table 1). For the COI haplotype network analysis based on 81 specimens, the network (Fig. 4) indicated that there were two clades that were separated by at least eight steps, which is consistent with the clades revealed by the combined 16S, COI, and CR phylogeny (Fig. 2). The ancestral haplotype of the N clade is “C2,” which was represented by eight specimens distributed in Korea, South and North China, and Taiwan. In contrast, the ancestral haplotype of the W clade is “C14”, which was represented by 19 specimens distributed in Japan (including Okinawa), Taiwan, South China, and Vietnam (Table 1; Figs. 4, 5B). The nucleotide divergence with the K2P distance and bp difference between the COI haplotypes of the N and W clades are shown in table 2. The average nucleotide divergences and bp differences within the N and W clades were 0.23% (1.53 bp) and 0.32% (2.09 bp), respectively, whereas the divergence between clades was 1.44% (9.32 bp). DISCUSSION The distribution of fiddler crabs is known to page 5 of 13Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan be greatly influenced by the dispersal of planktonic larvae via ocean current transport (Barnwell and Thurman 1984; Hopkins and Thurman 2010; LópezDuarte et al. 2011). However, according to the study of López-Duarte et al. (2011), species in mesohaline or oligohaline habitats (e.g., the estuaries) exhibit narrower distributional ranges than species in euhaline habitats (e.g., the open coasts) because larvae migrate vertically in estuaries to avoid currents and ensure their retention (Shih et al. 2015 2019). The distribution of T. arcuata is consistent with the above described estuarine pattern, with a narrower distribution compared to other open coastal species (e.g., Austruca annulipes, A. perplexa, Gelasimus tetragonon, Paraleptuca crassipes; Crane 1975; Shih et al. 2009 2013; Shih and Poupin 2020). Our study identified two clades within T. arcuata: the N clade distributed in the northern region and the W clade distributed widely throughout the entire habitat range (Figs. 2, 5A). However, only one unresolved clade in the network of 16S haplotypes (Fig. 3) and some shared 16S haplotypes in N and W clades were found, which implies a lower resolution of 16S compared with COI (Chu et al. 2015). Based on the pairwise divergence rates of 1.66% per million years for COI in marine coastal crabs of the family Sesarmidae (Schubart et al. 1998), the N and W clades of T. arcuata possibly diverged approximately 0.93 ± 0.3 million years ago (mya) (with p-distance divergences of 1.54% ± 0.47%) during the Pleistocene. The divergence between these clades was probably caused by glaciation events, with fluctuations in the global sea levels occurring in cycles of 100,000 years during the Pleistocene (Haq et al. 1987; Woodruff 2003; Shih et al. 2011). During these glaciation periods, the main PaleoKuroshio Current was hypothesized to flow along the Fig. 2. Bayesian inference (BI) tree for Tubuca arcuata based on the combined mitochondrial 16S, COI, and control region markers. The probability values at the nodes represent support values (> 50%) for BI and maximum parsimony (MP), respectively. See table 1 for haplotype names. W N S3+C34+D45 (Taiwan) S22+C33+D47 (Penghu) S9+C14+D18 (Japan) S3+C38+D50 (Hong Kong) S10+C19+D28 (Ryukyus) S1+C14+D39 (Taiwan) S1+C14+D59 (Vietnam) S1+C22+D34 (Taiwan) S10+C18+D29 (Ryukyus) S10+C18+D26 (Ryukyus) S3+C14+D58 (Vietnam) S1+C20+D31 (Taiwan) S20+C25+D43 (Taiwan) S1+C14+D25 (Ryukyus) S1+C13+D17 (Japan) S1+C26+D37 (Taiwan) S1+C14+D27 (Ryukyus) S17+C28+D40 (Taiwan) S13+C24+D36 (Taiwan) S1+C15+D20 (Japan) S26+C42+D54 (Hainan) S1+C39+D51 (Hong Kong) S1+C17+D24 (Japan) S1+C33+D42 (Taiwan) S26+C42+D53 (Hainan) S1+C23+D35 (Taiwan) S1+C13+D22 (Japan) S1+C12+D16 (Korea) S19+C31+D41 (Taiwan) S11+C14+D30 (Taiwan) S1+C14+D44 (Taiwan) S1+C14+D21 (Japan) S1+C47+D60 (Vietnam) S1+C12+D15 (Korea) S1+C14+D19 (Japan) S1+C14+D49 (S China) S16+C27+D38 (Taiwan) S12+C14+D32 (Taiwan) S1+C40+D52 (Hong Kong) S1+C14+D56 (Vietnam) S24+C37+D48 (N China) S3+C45+D55 (S China) S1+C14+D57 (Vietnam) S1+C12+D33 (Taiwan) S21+C35+D46 (Penghu) S1+C6+D5 (Taiwan) S8+C2+D12 (N China) S7+C2+D10 (Penghu) S6+C9+D9 (Taiwan) S1+C2+D7 (Taiwan) S1+C4+D1 (Taiwan) S3+C2+D4 (Taiwan) S5+C8+D8 (Taiwan) S1+C2+D14 (S China) S1+C5+D2 (Taiwan) S2+C2+D3 (Taiwan) S1+C10+D11 (Kinmen) S8+C2+D13 (N China) S4+C7+D6 (Taiwan) S1+C16+D23 (Japan) 1/88 0.94/97 1/100 0.005 BI / MP support values Tubuca arcuata fc-S+fc-C+fc-D3 (Malaysia: Johore) fc-S+fc-C+fc-D1 (Malaysia: Johore) fc-S+fc-C+fc-D2 (Malaysia: Johore) 1T. forcipata page 6 of 13 Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan eastern side of the Ryukyus (Ujiié et al. 2003), with branches extending to areas further north (Ujiié et al. 1991) (Fig. 5B). This differs from the modern pattern of the Kuroshio Current and its branches (Fig. 5A). We hypothesize that the ancestral distributions of the N and W clades could be determined from the current distributions based on their ancestral haplotypes. That is, the ancestral N haplotype was likely distributed across the coastal regions of the ECS and the YS (green line in Fig. 5B). In contrast, the distribution of the ancestral W haplotype includes the northern coastal region of the SCS, Taiwan, Okinawa, and the main islands of Japan. Therefore, we hypothesize that the original ancestral distribution of the latter during the glaciation period was the northern SCS, but was further dispersed northwardly to Okinawa and the main islands of Japan by the PaleoKuroshio Current (Fig. 5B). After the glaciation period, due to the emergence of modern warm currents (including the Kuroshio Current and its branches, the South China Sea Warm Current and the Taiwan Worm Current; blue arrows in Fig. 5A), the ancestral haplotypes of both clades were dispersed to their current habitat range in coastal regions (green and red arrows in Fig. 5B), and their descendant haplotypes also dispersed to more distant areas (green and red arrows in Fig. 5A). Based on the distribution of descendent W haplotypes in the coastal regions of the ECS and the YS as well as the presence of the descendent N haplotype in the main islands of Japan (Fig. 5A), we propose that the crabs’ larvae were dispersed northwardly by warm currents during the reproduction seasons in the summer (blue solid arrows in Fig. 5A). However, the N clade does not extend its range to the SCS (only some extension in southwestern Taiwan). This may have been because the only southward current is the cold China Coastal Current in winter (blue dotted arrow in Fig. 5A; Kang et al. 2021), which would have impeded the southward dispersion of larvae. In other studies, the mixing between the N and W clades separated by the Taiwan Strait was more limited due to the low larvae dispersal (e.g., mullets, Liu et al. 2007b; mitten crabs, Xu et al. 2009; bivalves, XX Wang et al. 2017). In our study, the Okinawa population did not form an isolated clade and even shared an ancestral COI haplotype with others (Figs. 2, 4), which is different from a previous conclusion that the Okinawa population of T. arcuata was genetically distinct based on RFLP Fig. 3. Genealogical network for the 557-bp 16S haplotypes observed within Tubuca arcuata, constructed with TCS. The ancestral haplotype is indicated by square. Green and red circles mean the haplotypes of the N and W clades, respectively; and the square and circle in purple represent the haplotypes that are shared by N and W clades. Unlabelled hatches and node indicate inferred haplotypes not found in the sampled population. S3 (x6) S4 S7 (x2) S9 (x3) S11 S20 S10 S2 S5 S12 S14 S15 S16 S21 S17 S25 (x2) S26 S24 S23 S19 S18 S13 S22 S8 S6 S1 (x47) page 7 of 13 Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan Fig. 4. Genealogical network for the 658-bp COI haplotypes observed within Tubuca arcuata, constructed with TCS. The ancestral haplotypes of the N and W clades are indicated by squares. Unlabelled hatches and nodes indicate inferred haplotypes not found in the sampled population. ancestral haplotype of N clade: C2 (Korea;S&N China; Taiwan) x8 C4 C1 (x2) C3 C6 C5 C7 C8 C11 C10 C9 C17 C13 (x2) C15 C18 (x2) C19 C21 C12 (x3) C23 C24 C26 C27 C34 C16 C37 C20 C22 C29 C30 C35 C36 C38 C39 C40 C41 C42 (x2) C43 C31 (x2) C47 C44 C45 C46 C32 C33 (x2) C25 (x2) C28 ancestral haplotype of W clade: C14 (Japan; Taiwan; Kinmen; S China; Vietnam) x19 C2 b C14 page 8 of 13Zoological Studies 61:68 (2022) © 2022 Academia Sinica, Taiwan MONGOLIA MALAYSIA THAILAND Sulawesi LAOS Kuroshio Branch Tsushima Current South China Sea WarmCurrent Kuroshio Current ChinaCoastalCurrent TaiwanWarmCurrent 120oE 40oN 110oE 130oE 30oN 20oN 10oN TropicofCancer cold current & direction warm current & direction range & haplotypes of N clade range & haplotypes of W clade haplotypes of N & W clades MONGOLIA MALAYSIA THAILAND Sulawesi LAOS Paleo-Kuroshio Current Taiwan Strait 120oE 40oN 110oE 130oE 30oN 20oN 10oN Tropicof Cancer AB analysis (Aoki et al. 2008). In fact, although one RFLP haplotype is dominant in the Okinawa group, all three haplotypes are shared with other populations (Aoki et al. 2008: table 2) and an alternative conclusion might be obtained by using a larger sample size. Another similar study on the phylogeography of the Parasesarma bidens complex (Sesarmidae) using 16S and COI (Shahdadi et al. 2022) showed one clade mainly distributed in the main islands of Japan and the Ryukyus, which might belong to the pattern of the unique Ryukyus clade (see BACKGROUND) (no. 5 in Fig. 1A), although the range also extended to the main islands of Japan and Taiwan. Fig. 5. A, Distributions of the N haplotypes (green squares) and the W haplotypes (red squares). The green and red lines and arrows indicate the possible biogeographical boundary and possible dispersal direction of the N and W clades after glaciation, respectively. The square with both red and green represents the locality with both N and W haplotypes. The blue solid and dotted lines and arrows represent the warm and cold currents in summer and winter, respectively. B, Distribution of the ancestral haplotypes of the N clade (green squares) and the W clade (red squares). The green and red lines and arrows indicate the possible biogeographical boundary and the assumed dispersal direction of the ancestral N and W clades after glaciation, respectively. The shaded sea areas represent continental shelves (< 120 m) that would have been dry during periods of low sea level (after Voris 2000; Xu et al. 2009). The blue solid line indicates the Paleo-Kuroshio Current (after Ujiié et al. 2003) and the dotted lines are the possible extended branches of the Paleo-Kuroshio Current (after Ujiié et al. 1991). Table 2. Matrix of the percentage pairwise nucleotide divergences with K2P distance and mean number of bp differences based on COI within and between clades of Tubuca arcuata from across East Asia and northern Vietnam (see Table 1). In the right half, the lower-left values are K2P distance and the upper-right ones are mean bp differences. Ranges are indicated in parentheses Within clade Between clades nucleotide divergence mean bp difference N clade W clade N clade 0.23 (0–0.76) 1.53 (0–5) 9.32 (6–13) W clade 0.32 (0–0.92) 2.09 (0–6) 1.44 (0.92–2.01) page 9 of 13Zoological Studies 61:68 (2022)