Taxonomy and Phylogeography of the Freshwater Crab Geothelphusa tawu Species Complex (Crustacea: Decapoda: Potamidae) from Southern Taiwan and Offshore Islets

© 2023 Academia Sinica, Taiwan Open Access Taxonomy and Phylogeography of the Freshwater Crab Geothelphusa tawu Species Complex (Crustacea: Decapoda: Potamidae) from Southern Taiwan and Offshore Islets Hsi-Te Shih1,2,§,* , Jhih-Wei Hsu1,§ , Kai Chang1,§ , and Min-Wan Chen1 1Department of Life Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. *Correspondence: E-mail: [email protected] (Shih) E-mail: [email protected] (Hsu); [email protected] (Chang); [email protected] (Chen) 2Research Center for Global Change Biology, National Chung Hsing University, 250, Kuo Kuang Road, Taichung 402, Taiwan §HTS, JWH and KC contributed equally to this paper. Received 16 October 2022 / Accepted 4 May 2023 / Published 21 July 2023 Communicated by Benny K.K. Chan The freshwater crabs Geothelphusa tawu Shy, Ng & Yu, 1994, G. lutao Shy, Ng & Yu, 1994 and G. lanyu Shy, Ng & Yu, 1994 from southern Taiwan and the offshore islets, Lyudao (Green I.) and Lanyu (Orchid I.) are closely related in morphology and genetics, and have been proposed to be the same species. Examination of a series of specimens collected from the distributional ranges of the three species indicated that key characters of the ambulatory legs and the male first gonopod (G1) are too variable to support the identity of three species. Based on the mitochondrial 16S rDNA and cytochrome oxidase subunit I (COI) sequences, the phylogenetic analysis did not recover three clades corresponding to the three species, but only a main clade without further clear grouping. The interspecific distances of nucleotides are also too small to support the species delimitation. We conclude that the three species should be treated as a single species. Additionally, unique haplotypes of COI have been found in Lyudao and Lanyu, which are hypothesized as two founder populations that colonized the islets from the Taiwan main island during glacial maxima. Key words: Taxonomy, Phylogeography, 16S rDNA, COI, Founder populations, Morphology, Glaciations Citation: Shih HT, Hsu JW, Chang K, Chen MW. 2023. Taxonomy and phylogeography of the freshwater crab Geothelphusa tawu species complex (Crustacea: Decapoda: Potamidae) from southern Taiwan and offshore islets. Zool Stud 62:37. doi:10.6620/ZS.2023.62-37. BACKGROUND Among the freshwater crabs in East Asia, the species diversity of Geothelphusa Stimpson, 1858 in the East Asian Arc is much higher than any other genera, except Longpotamon Shih, Huang & Ng, 2016 from continental China (Shih and Ng 2011; Shih et al. 2009 2016a). There are 38 species of Geothelphusa from Taiwan and adjacent islets (Shy et al. 1994 2020 2021), but some species complexes have been considered to have unsatisfactory taxonomy (Shih et al. 2004; Shy et al. 2020). The Geothelphusa tawu species complex distributed in southern Taiwan and adjacent islets needs further study, as suggested by Shih et al. (2004). This complex includes G. tawu Shy, Ng & Yu, 1994 from the Hengchun Peninsula, and G. lutao Shy, Ng & Yu, 1994 and G. lanyu Shy, Ng & Yu, 1994 endemic to two offshore islets, Lyudao (= Green Island) and Lanyu (= Orchid Island), respectively (Fig. 1). Other freshwater crabs, including G. ferruginea Shy, Ng & Yu, 1994, G. albogilva Shy, Ng & Yu, 1994 and Candidiopotamon rathbuni (De Man, 1914) are also distributed in southern Taiwan. Populations of freshwater crabs found on offshore islets that are separated from the main island Zoological Studies 62:37 (2023) doi:10.6620/ZS.2023.62-37 1 © 2023 Academia Sinica, Taiwan by more than 30 km might be expected to be different from populations on the main island. In Shih et al. (2004: fig. 2), however, the three recognized species have been suggested to be conspecific, based on an unresolved clade formed by the three species in a phylogenetic tree of mitochondrial 16S rDNA, together with small morphological differences. Additional genetic and morphology studies were referenced to conclusively establish that they are conspecific. In our study, a morphological comparison of the key characters of ambulatory legs and male first gonopod (G1) used in Shy et al. (1994) was conducted on a series of specimens from the distributional ranges of G. tawu, G. lutao and G. lanyu. Our comparison Fig. 1. Collection sites for species of the Geothelphusa tawu species complex from southern Taiwan, and the adjacent islets. Different lines indicate the possible biogeographic boundaries for each species found in southern Taiwan. For locality names, see table 1. Specimens of G. tawu, G. lutao and G. lanyu on the photographs are collected from Dawu R. (9 Jan. 2001), Lyudao (11 Sep. 1999) and Lanyu (8 Jan. 2008), respectively. 0-100 100-500 500-1000 1000-2000 2000-3000 m m m m m Dawu R. Lanyu Lyudao 121°E 22°N GangkouR. G. tawu species complex 8 6 9 11 54 1 2 13 12 CEN RA L RAG N E T 23°N Fenggang R. Sihchong R. 7 Taimali R. G. pingtung G. bicolor G. ferruginea G. albogilva G. lutao G. lanyu Taiwan TAITUNG PINGTUNG3 10 G. tawu N page 2 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan indicated that these characters were not discrete for each of these species, and some were found to be the same in one or more. In addition, the molecular evidence from 16S rDNA and cytochrome oxidase subunit I (COI) did not support the monophyly of the three species. Based on the available evidence in morphology and genetics, our study further proposes the three taxa belong to a single species. MATERIALS AND METHODS Specimens of the G. tawu species complex from around the distributional ranges (including Lyudao and Lanyu; Fig. 1) suggested in Shih et al. (2004 2007) were collected after several surveys. All specimens were preserved in 75–95% ethanol after collection and deposited into the Zoological Collections of the Department of Life Science, National Chung Hsing University (NCHUZOOL; see Table 1 for catalogue numbers). Additional specimens that had been deposited at the Department of Environmental Biology and Fisheries Science, National Taiwan Ocean University, Keelung, Taiwan (NTOU) were also used. Measurements, all in millimeters (mm), are of the maximum carapace width (CW) and carapace length (CL). For the morphological comparison, only adult males (CW ³ 12.0 mm) were selected. The abbreviations P3, P4 and P5 are used for the third, fourth and fifth pereiopods (second, third and fourth ambulatory legs), and G1 for male first gonopods. As different authors may measure different positions from the photograph (Shih and Do 2014; PY Hsu and Shih 2018; Shih et al. 2019; Shy et al. 2020), the measurements of legs, including the dactyl length (DL) and propodus length (PL) of P3–5 and the merus length (ML) and merus width (MW) of P3 merus; as well as G1, including terminal segment length (TSL), total length of G1 (TLG1), synovial membrane length (SML) and synovial membrane width (SMW) are shown in figure 2. The two ratios of G1s were also measured on the photographs of holotypes of G. tawu, G. lutao and G. lanyu (Shy et al. 2020). Other characters of G1, including the terminal segment curvature and the existence of a tooth on the outer proximal margin of subterminal segment, were also examined, as they were sometimes used to distinguish different species of Geothelphusa (Shy et al. 1994 2020; Shy and Lee 2009). The morphological characters and terminology follow those in WJ Chen et al. (2007) and Shy et al. (2020). Genomic DNA was isolated from muscle tissues using commercial kits, and the sequences of the 16S rDNA [~560 base pairs (bp)] and COI (616–658 bp) genes were obtained as described by Shih et al. (2016b) and verified with the complementary strand. The primers used were 1471 (5'-CCTGTTTANCAAAAACAT-3'), Table 1. Haplotypes of the 16S rDNA and cytochrome c oxidase subunit I (COI) of the Geothelphusa tawu species complex and the related species. Numbers within brackets correspond to the localities in figure 1 Species Localities Sample size Catalogue no. of NCHUZOOL (unless indicated) Haplotype of 16S Access. no. of 16S Haplotype of COI Access. no. of COI G. tawu Taitung: Taimali R. [1] 1 13283 Gtw1 AB535446 Gtw-C1 AB535477 Taitung: Dawu R. [2] 1 (uncatalogued) AB127379 — Taitung: Dawu R. [2] 1 13058 Gtw3 AB127381 Gtw-C2 AB266300 Taitung: Dawu R. [2] 1 13058 Gtw3 OQ822175 — Taitung: Dawu R. [2] 1 13155 Gtw2 OQ822176 Gtw-C3 OQ824907 Taitung: Dawu R. [2] 1 13058 — Gtw-C4 OQ824908 Taitung: Dawu R. [2] 2 13058 Gtw4 OQ822177; OQ822178 — Taitung: Dawu [3] 1 NTOU F10203 (holotype) Gtw5 OQ822179 Gtw-C5 OQ824909 Pingtung: Alangyi, Mudan [4] 1 17155 Gtw6 OQ822180 Gtw-C6 OQ824910 Pingtung: Shuanliou, Danlu [5] 3 17152 Gtw2 OQ822181; OQ822182; OQ822183 Gtw-C7 OQ824911; OQ824912; OQ824913 Pingtung: Shuanliou, Danlu [5] 1 17152 Gtw7 OQ822184 Gtw-C8 OQ824914 Pingtung: (Damei, Mudan [6] 1 14320 Gtw9 AB127374 Gtw-C13 OQ824915 Pingtung: Lilongshan, Shihzih [7] 1 17153 Gtw10 OQ822185 Gtw-C14 OQ824916 Pingtung: Lilongshan, Shihzih [7] 1 17156 Gtw11 OQ822186 Gtw-C15 OQ824917 Pingtung: Fenggang R., Danlu, Shihzih [8] 3 17163 Gtw2 OQ822187; OQ822188; OQ822189 Gtw-C9 OQ824918; OQ824919; OQ824920 page 3 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan Species Localities Sample size Catalogue no. of NCHUZOOL (unless indicated) Haplotype of 16S Access. no. of 16S Haplotype of COI Access. no. of COI Pingtung: Fenggang R., Danlu, Shihzih [8] 1 17163 Gtw8 OQ822190 Gtw-C10 OQ824921 Pingtung: Fenggang R., Danlu, Shihzih [8] 1 13317 Gtw2 OQ822191 Gtw-C11 AB539523 Pingtung: Fenggang R., Danlu, Shihzih [8] 1 13317 Gtw2 OQ822192 Gtw-C12* OQ824922 Pingtung: Shihwen, Chunrih [9] 1 13055 Gtw15 AB127375 Gtw-C20 AB266297 Pingtung: Dahanshan, Chunrih [10] 4 12979 Gtw2 OQ822193; OQ822194; OQ822195; OQ822196 — Pingtung: Cijia, Chunrih [11] 3 13057 Gtw12 OQ822197; OQ822198; AB127376 Gtw-C16 OQ824923; OQ824924; OQ824925 Pingtung: Cijia, Chunrih [11] 1 13056 Gtw13 AB127377 Gtw-C17* AB266298 Pingtung: Cijia, Chunrih [11] 1 13057 Gtw12 OQ822199 Gtw-C18* AB266299 Pingtung: Cijia, Chunrih [11] 1 13057 Gtw13 OQ822200 Gtw-C19 OQ824926 Pingtung: Cijia, Chunrih [11] 1 13057 Gtw14 OQ822201 — G. lutao Taitung: Lyudao [12] 1 13060 Gtw16 AB127382 Glt-C1 AB266302 Taitung: Lyudao [12] 1 13061 Gtw16 OQ822202 Glt-C2 AB266303 Taitung: Haisenping, Lyudao [12] 1 14992 Gtw16 OQ822203 Glt-C3 OQ824927 Taitung: Guanyin Cave, Lyudao [12] 2 17145 Gtw16 OQ822204; OQ822205 Glt-C1 OQ824928; OQ824929 Taitung: Haishenping, Lyudao [12] 2 14993 Gtw16 OQ822206; OQ822207 Glt-C4 OQ824930; OQ824931 Taitung: Haishenping, Lyudao [12] 1 14992 Gtw16 OQ822208 Glt-C1 OQ824932 Taitung: Haishenping, Lyudao [12] 2 14992 Gtw16 OQ822209; OQ822210 Glt-C5 OQ824933; OQ824934 Taitung: Lyudao [12] 1 NTOU F10200 (holotype) Gtw16 OQ822211 Glt-C6* OQ824935 G. lanyu Taitung: Longmen R., Lanyu [13] 1 13059 Gtw2 AB127380 Gly-C1 AB266301 Taitung: Longmen R., Lanyu [13] 1 13059 Gtw17 OQ822212 Gly-C1 OQ824936 Taitung: Hongtou, Lanyu [13] 1 14326 Gtw17 OQ822213 Gly-C1 OQ824937 Taitung: Hongtou, Lanyu [13] 1 14326 Gtw17 OQ822214 Gly-C1 OQ824938 Taitung: Hongtou, Lanyu [13] 1 14326 Gtw17 OQ822215 Gly-C1 OQ824939 Taitung: Hongtou, Lanyu [13] 1 14326 Gtw2 OQ822216 Gly-C1 OQ824940 Taitung: Yeyou R., Lanyu [13] 1 17146 Gtw2 OQ822217 Gly-C3 OQ824941 Taitung: Yeyou South R., Lanyu [13] 1 17147 Gtw2 OQ822218 Gly-C4 OQ824942 Taitung: Yuren R., Lanyu [13] 1 17150 Gtw2 OQ822219 Gly-C5 OQ824943 Taitung: Hongtou R., Lanyu [13] 1 17149 Gtw2 OQ822220 Gly-C6* OQ824944 Taitung: Longmen R., Lanyu [13] 1 17148 Gtw17 OQ822221 Gly-C6* OQ824945 Taitung: Langdao R., Lanyu [13] 1 17151 Gtw2 OQ822222 Gly-C7 OQ824946 Taitung: Longmen R., Lanyu [13] 1 17142 — Gly-C1 OQ824947 Taitung: Longmen R., Lanyu [13] 1 17142 Gtw17 OQ822223 Gly-C1 OQ824948 Taitung: Yeyou R., Lanyu [13] 1 17154 Gtw2 OQ822224 — Taitung: Yeyou R., Lanyu [13] 1 17154 Gtw18 OQ822225 — Taitung: Yuren R., Lanyu [13] 3 17141 Gtw2 OQ822226; OQ822227; OQ822228 Gly-C1 OQ824949; OQ824950; OQ824951 Taitung: Langdao R., Lanyu [13] 1 17143 Gtw2 OQ822229 Gly-C1 OQ824952 Taitung: Langdao R., Lanyu [13] 1 17143 Gtw2 OQ822230 Gly-C2 OQ824953 Taitung: Langdao R., Lanyu [13] 1 17143 Gtw19 OQ822231 Gly-C1 OQ824954 Taitung: Lanyu [13] (holotype) 1 NTOU F10100 (holotype) Gtw3 OQ822232 — total 69 G. ferruginea Pingtung: Maozaikengnei, Hengchun 1 13062 Gf-1 AB127383 Gf-C1 AB266304 Pingtung: Nanrenshan, Manjhou 1 13282 Gf-2 OQ822233 Gf-C2 OQ824955 G. albogilva Pingtung: Maozaikengnei, Hengchun 1 13053 Ga AB127366 Ga-C AB266295 *, sequence is shorter. See MATERIALS AND METHODS for abbreviations of universities. Table 1. (Continued) page 4 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan 1472 (5'-AGATAGAAACCAACCTGG-3') (Crandall and Fitzpatrick 1996), 16L29 (5'-YGCCTGTTTATCAAAAACAT-3'), 16H10 (5'-AATCCTTTCGTACTAAA-3'), and 16H11 (5'-AGATAGAAACCRACCTGG-3') (Schubart 2009) for 16S; and LCO1490 (5'-GGTCAACAAATCATAA AGATATTGG-3'), HCO2198 (5'-TAAACTTCAGGGT GACCAAAAAATCA-3') (Folmer et al. 1994), COL14 (5'-GCTTGAGCTGGCATAGTAGG-3') (Roman and Palumbi 2004), jgLCO (5'-TITCIACIAAYCAYAA RGAYATTGG-3'), jgHCO (5'-TAIACYTCIGGRTG ICCRAARAAYCA-3') (Geller et al. 2013), LCOB (5'-CAAAYCATAAAGAYATYGG-3') and HCOex3 (5'-GCTCANACTACRAATCCTA-3') (Shih et al. 2022b), as well as the newly designed primers HCOex0 (5'-GAYTCTTTTTTDCCDGAYTC-3') for COI. The sequences of the different haplotypes have been deposited in the NCBI GenBank database (accession numbers are summarized in Table 1). As the sequences of COI are shorter than others by using the internal primer COL14, the missing data were designated as a ‘?’ in the alignment of the Bayesian inference (BI) and maximum likelihood (ML) analyses, but the segments with missing data were excluded in network analysis and nucleotide pairwise comparison. Sequences of the related species, G. ferruginea and G. albogilva distributed in southern Taiwan and sometimes sympatric with G. tawu, were included in the phylogenetic analyses. For the combined 16S and COI dataset, the best-fitting models for sequence evolution of individual datasets were determined by PartitionFinder (ver. 2.1.1, Lanfear et al. 2017) and selected by the Bayesian information criterion (BIC). The obtained best models for the two individual datasets were HKY+I, and were subsequently used for the partitioned BI and ML analyses. The BI was performed with the program BEAST (vers. 2.6.7, Bouckaert et al. 2019) and the divergence times among taxa were estimated by using a strict clock (Yule Model) with the substitution rates of 0.44% and 1.165% per million years for 16S and COI, respectively (Schubart et al. 1998). A Yule speciation process was conducted for the divergence within the G. tawu species complex. An HKY+I model with the parameters obtained from PartitionFinder was used for each gene. Two independent MCMC chains were run for 10 TSL TLG1 SML SMW DL PL (A) (B) Fig. 2. Schematic drawings of the ambulatory leg (A) and the G1 (B) measurements used in this study. DL, dactyl length; PL, propodus length; ML, merus length; MW, merus width; TLG1, total length of G1, TSL, terminal segment length; SML, synovial membrane length; SMW, synovial membrane width. page 5 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan million generations sampled every 1000 generations. The convergence of the two combined chains was determined by the ESS (> 200 as recommended) for each parameter in Tracer (vers. 1.7.2, Rambaut et al. 2018). Trees in the two chains were combined using LogCombiner (vers. 2.6.7, distributed as part of the BEAST package) and were assessed using TreeAnnotator (vers. 2.6.7, distributed as part of the BEAST package) with the default burnin cutoff (10% of sampled trees). A chronogram was constructed by FigTree (vers. 1.4.4, Rambaut 2018). A ML analysis was conducted in IQ-TREE (vers. 2.2.0, Minh et al. 2020) with the best models and 30,000 ultrafast bootstrap replicates (Hoang et al. 2017). A TCS haplotype network of the 16S+COI haplotypes was generated using the program PopART (vers. 1.7, Leigh and Bryant 2015). Bp differences and the pairwise estimates of Kimura 2-parameter (K2P) distances (Kimura 1980) for genetic diversities between COI haplotypes were calculated with MEGA (vers. 11.0, Tamura et al. 2021). Specimens examined: G. tawu: 1 ♂ (13.3 × 10.6 mm), NCHUZOOL 13283, Taimali R., Taitung, 30 Jan. 2007; 1 ♂ (14.5 × 11.5 mm), NCHUZOOL 13155, Dawu R., Taitung, coll. H.-T. Shih, 9 Jan. 2001; 1 ♂ (17.3 × 13.5 mm), 3 ♀♀ (17.3 × 13.4, 16.9 × 13.4, 13.4 × 10.3 mm), NCHUZOOL 13058, Dawu R., Taitung, coll. H.-T. Shih, 9 Jan. 2001; 1 ♀ (16.6 × 13.1 mm), NCHUZOOL 17155, Alangyi, Mudan, Pingtung, coll. Y.-J. Yang, 26 Mar. 2020; 2 ♂♂ (17.9 × 14.6, 17.8 × 14.7 mm), 2 ♀♀ (18.6 × 14.3, 17.3 × 13.3 mm), NCHUZOOL 17152, Shuanliou, Danlu, Pingtung, 30 Mar. 2021; 1 ♀ (19.9 × 15.7 mm), NCHUZOOL 14320, Damei, Mudan, Pingtung, 9 Mar. 2000; 1 ♀ (18.5 × 14.3 mm), NCHUZOOL 17153, Lilongshan, Shihzih, Pingtung, 16 Jul. 2017; 1 ♂ (16.5 × 13.4 mm), NCHUZOOL 17156, Lilongshan, Shihzih, Pingtung, coll. S.-P. Wu, 7 Jul. 2020; 1 ♂ (19.5 × 15.8 mm), 2 ♀♀ (21.9 × 16.1, 16.3 × 13.2 mm), NCHUZOOL 13317, Fenggang R., Danlu, Shihzih, Pingtung, coll. H.-T. Shih, 11 Mar. 1999; 3 ♂♂ (19.3 × 15.6, 19.0 × 15.0, 17.5 × 13.6 mm), 3 ♀♀ (17.9 × 14.1, 17.5 × 14.3, 13.8 × 11.2 mm), NCHUZOOL 17163, Fenggang R., Danlu, Shihzih, Pingtung, coll. H.-T. Shih, 11 Mar. 1999; 1 ♂ (12.7 × 10.1 mm), NCHUZOOL 13055, Shihwen, Chunrih, Pingtung, 10 Sep. 2002; 1 ♂ (18.2 × 14.4 mm), 1 ♀ (17.8 × 14.4 mm), NCHUZOOL 17165, Shihwen, Chunrih, Pingtung, 10 Sep. 2002; 1 ♂ (18.1 × 13.5 mm), 2 broken ♂♂, 1 ♀ (19.2 × 14.7 mm), NCHUZOOL 12979, Dahanshan, Chunrih, Pingtung, 2 Mar. 1997; 1 ♂ (16.4 × 13.3 mm), NCHUZOOL 13056, Cijia, Chunrih, Pingtung, 10 Sep. 2002; 4 ♂♂ (18.8 × 14.7, 16.7 × 13.2, 16.6 × 13.1, 9.4 × 7.3 mm), 2 ♀♀ (22.1 × 16.8, 12.9 × 10.5 mm), 2 ovig. ♀♀ (20.3 × 15.4, 18.8 × 14.7 mm), NCHUZOOL 13057, Cijia, Chunrih, Pingtung, 10 Sep. 2002; 1 ♂ (15.6 × 12.1 mm), NCHUZOOL 17164, Cijia, Chunrih, Pingtung, 10 Sep. 2002; 1 ♂ (17.5 × 13.7 mm), 1 ♀ (21.7 × 16.6 mm), NCHUZOOL 17144, Cijia, Chunrih, Pingtung, 10 Sep. 2002. G. lutao: 1 ♂ (18.5 × 14.2 mm), NCHUZOOL 13060, Lyudao, coll. H.-C. Liu, 11 Sep. 1999; 1 ♂ (18.2 × 14.3 mm), NCHUZOOL 13061, Lyudao, coll. H.-C. Liu, 11 Sep. 1999; 2 ♂♂ (12.6 × 9.8, 11.8 × 9.1 mm), NCHUZOOL 17145, Guanyin Cave, Lyudao, coll. Y.-H. Huang, 26 Sep. 2021; 7 ♂♂ (21.4 × 16.9, 19.2 × 14.5, 18.9 × 14.3, 16.9 × 13.0, 15.7 × 11.8, 14.8 × 11.2, 14.5 × 11.1 mm), 2 ♀♀ (21.2 × 16.2, 18.7 × 14.3 mm), NCHUZOOL 14992, Haishenping, Lyudao, 21 Apr. 2012; 4 ♂♂ (15.0 × 11.5, 13.8 × 11.0, 13.7 × 10.8, 13.0 × 9.9 mm), 1 ♀ (13.5 × 10.5 mm), NCHUZOOL 14993, Haishenping, Lyudao, 21 Apr. 2012. G. lanyu: 1 ♂ (12.5 × 9.7 mm), 3 ♀♀ (17.9 × 14.1, 17.0 × 13.1, 16.5 × 12.7 mm), 1 ovig. ♀ (17.9 × 14.0 mm), NCHUZOOL 17160, Lanyu, coll. H.-C. Liu & C.-H. Wang, 21–22 Mar. 1996; 1 ♂ (11.1 × 8.7 mm), NCHUZOOL 17151, Langdao R., Lanyu, 1 Apr. 2008; 1 ♂ (12.8 × 9.5 mm), 1 ♀ (20.6 × 15.5 mm), NCHUZOOL 14326, Hongtou, Lanyu, 4 Apr. 2015; 1 ♂ (19.7 × 15.1 mm), NCHUZOOL 17162; Hongtou, Lanyu, 4 Apr. 2015; 1 ♀ (9.3 × 7.0 mm), NCHUZOOL 17146, Yeyou R., Lanyu, 8 Jan. 2008; 2 ♀♀ (9.0 × 7.4, 5.3 × 3.9 mm), NCHUZOOL 17154, Yeyou R., Lanyu, 8 Jan. 2008; 1 broken ♀ (12.1 mm), NCHUZOOL 17147, Yeyou South R., Lanyu, 8 Jan. 2008; 1 ♀ (15.2 × 11.7 mm), NCHUZOOL 17150, Yuren R., Lanyu, 11 Jul. 2003; 2 ♀♀ (15.9 × 12.7, 13.0 × 10.3 mm), NCHUZOOL 17161, Yuren R., Lanyu, coll. T.-P. Tseng, 11 Jul. 2003; 2 ♂♂ (18.7 × 14.5, 12.0 × 9.1 mm), 1 ♀ (18.0 × 14.1 mm), NCHUZOOL 17141, Yuren R., Lanyu, coll. K. Chang et al., 28 Sep. 2022; 1 ♂ (16.3 × 12.7 mm), NCHUZOOL 17149, Hongtou R., Lanyu, 13 Jul. 2003; 1 ♀ (14.1 × 10.9 mm), NCHUZOOL 17148, Longmen R., Lanyu, 2 Apr. 2008; 2 ♂♂ (15.1 × 12.1, 13.6 × 10.9 mm), 1 ovig. ♀ (12.9 × 10.2 mm), NCHUZOOL 17158, Longmen R., Lanyu, 2 Apr. 2008; 2 ♂♂ (17.2 × 13.2, 16.9 × 13.2 mm), 2 ♀♀ (13.5 × 10.7, 12.9 × 9.8 mm), NCHUZOOL 14327, Longmen R., Lanyu, 3 Apr. 2015; 1 ♂ (13.5 × 10.5 mm), 2 ♀♀ (17.4 × 13.2, 16.3 × 12.6 mm), NCHUZOOL 17142, Longmen R., Lanyu, coll. K. Chang et al., 2 Oct. 2022; 1 ♂ (16.3 × 12.3 mm), 2 ♀♀ (16.5 × 13.0, 11.3 × 9.0 mm), 1 ovig. ♀ (16.4 × 12.6 mm), NCHUZOOL 13059, Longmen R., Lanyu, 5 Jun. 1993; 5 ♂♂ (17.2 × 13.2, 15.2 × 11.7, 14.7 × 11.3, 14.4 × 11.1, 7.7 × 5.9 mm), 1 ♀ (14.0 × 11.0 mm), NCHUZOOL 17143, Langdao R., Lanyu, 1 Apr. 2008. page 6 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan 1.0 1.1 1.2 1.3 10 12 14 16 18 20 22 1.0 1.1 1.2 1.3 10 12 14 16 18 20 22 1.0 1.1 1.2 1.3 10 12 14 16 18 20 22 2.0 3.0 4.0 5.0 10 12 14 16 18 20 22 (A) (B) (C) (D) CW (mm) CW (mm)CW (mm) CW (mm) G. tawu G. lanyu G. lutao G. tawu G. lanyu G. lutao G. tawu G. lanyu G. lutao G. tawu G. lanyu G. lutao DL/PL of P3 DL/PL of P4 DL/PL of P5 ML/MW of P3 RESULTS Taxonomic identity of the G. tawu species complex The CWs of male adult specimens used for comparison of legs and G1s (13.3–19.3 mm for G. tawu; 12.6–21.4 mm for G. lutao; and 12.0–19.7 mm for G. lanyu), as well as the ratios of DL/PL of P3–P5, ML/MW of P3, TLG1/TSL and SML/SMW of G1 are shown in table 2. From the scatter plots of the DL/PL ratios of P3–P5, as well as the TLG1/TSL (Fig. 3A–C) and SML/SMW ratios of G1 (Fig. 4), no clear patterns can be found among the three species. However, the ML/MW ratios of P3 in G. lanyu tend to be higher (i.e., more slender) than the other two species (Fig. 3D), but some specimens of the three species have very close values. With regard to the terminal segment curvature of G1, all three species have specimens with straighter (Fig. 5A, C, E) and slightly curved (Fig. 5B, D, F) forms. Regarding the tooth on the outer proximal margin of the subterminal segment, both G. tawu and G. lutao have specimens with the tooth or without (Fig. 6A–D), but all specimens of G. lanyu have the tooth (Fig. 6E). Fig. 3. (A–C) Ratios of dactyl length (DL) to propodus length (PL) of P3 (A), P4 (B) and P5 (C) plotted as a function of carapace width (CW) in male G. tawu (gray triangles), G. lutao (green squares) and G. lanyu (blue diamonds). (D) Ratios of merus length (ML) to merus width (MW) of P3 as a function of CW in males of three species. page 7 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan 4.5 5.5 6.5 7.5 12 14 16 18 20 22 2.0 3.0 4.0 12 14 16 18 20 22 (A) (B) CW (mm) CW (mm) G. tawu G. lanyu (holotype) G. lutao G. tawu (holotype) G. lutao (holotype) G. lanyu TLG1/TSL SML/SMW Molecular analyses A 550 bp segment of the 16S rDNA from 67 specimens and a 616–658 bp segment of COI from 57 specimens of G. tawu, G. lutao and G. lanyu were amplified and aligned. A total of 19 haplotypes of 16S gene were found for the three species, with one haplotype (“Gtw2”) shared by G. tawu and G. lanyu; and only one haplotype (“Gtw16”) found in G. lutao (Table 1). There were 33 haplotypes of COI, with 20 for G. tawu, six for G. lutao and seven for G. lanyu. Five shorter segments of COI using the primer COL14 Fig. 4. (A) Ratios of total length of G1 (TLG1) to terminal segment length of G1 (TSL) as a function of carapace width (CW) in G. tawu (gray triangles), G. lutao (green squares) and G. lanyu (blue diamonds). (B) Ratios of synovial membrane length (SML) to synovial membrane width (SMW) as a function of CW in three species. Empty symbols are for holotypes. Fig. 5. Right G1s showing the degree of terminal segment curvature. (A) G. tawu (CW 17.9 mm, NCHUZOOL 17152). (B) G. tawu (CW 19.0 mm, NCHUZOOL 17163). (C) G. lutao (CW 18.2 mm, NCHUZOOL 13061). (D) G. lutao (CW 18.9 mm, NCHUZOOL 14992). (E) G. lanyu (CW 17.2 mm, NCHUZOOL 14327). (F) G. lanyu (CW 17.2 mm, NCHUZOOL 17143). Scale bars = 1 mm. page 8 of 15Zoological Studies 62:37 (2023) © 2023 Academia Sinica, Taiwan shared the same sequences with longer segments, viz. Gtw-C12=Gtw-C9, Gtw-C18=Gtw-C16, GtwC17=Gtw-C19, Glt-C6=Glt-C1 and Gly-C6=Gly-C1 (Table 1). Phylogenetic analysis based on the combined 16S and COI database with 1208 bp from 70 specimens showed one main clade with high supports by BI and ML methods (Fig. 7). However, even though the clades of G. lutao and G. lanyu are supported, haplotypes of G. tawu from the Taiwan main island do not form a clade or any clear groupings, even when excluding the more different Taimali haplotype (“Gtw1+Gtw-C1” in Figs. 7, 8; Fig. 1: no. 1). The haplotypes of G. lutao and those from Alangyi (Fig. 1: no. 4) and Shuanliou (Fig. 1: no. 5) from the Taiwan main island are very closely related and form another larger clade, named as “large G. lutao clade”. Based on the substitution rates of 16S and COI, the divergence time estimation for the main nodes (Fig. 7) showed the G. tawu species complex separated from related species about 1.16 mya (million years ago), and the divergence time between the Taimali haplotype and others is about 0.7 mya. The divergence times of G. lutao and G. lanyu from others are estimated as 0.2 and 0.33 mya, respectively. In the network of 16S+COI haplotypes (Fig. 8), haplotypes of G. lutao and G. lanyu are only separated from those from the Taiwan main island by ≥ 2 bp and ≥ 3 bp , respectively. From the pairwise nucleotide divergences of K2P distances and bp differences among COI haplotypes of the G. tawu species complex (Table 3), the maximum intraspecific nucleotide divergences (and bp differences) of G. lanyu, G. lutao, G. tawu and G. tawu excluding distinct haplotypes (from Shihwen and Taimali; Fig. 1: no. 9, 1, respectively; Figs. 7, 8) are ≤ 0.49% (≤ 3 bp), ≤ 0.65% (≤ 4 bp), ≤ 2.5% (≤ 15 bp) and ≤ 1.48% (≤ 9 bp), respectively. The minimum interspecific divergence (and bp difference) of the species complex are as small as 0.16–0.65% (1–4 bp). DISCUSSION Morphological comparison According to Shy et al. (1994 2020) and Shy and Lee (2009), the main characters of ambulatory legs and G1 were useful to distinguish G. tawu, G. lutao and G. lanyu. For example, the merus of ambulatory legs are slender in G. lanyu (vs. stout in other two species) (Shy et al. 2020: 16). In our study, most G. lanyu specimens tend to have more slender ambulatory merus, but some are still close to the other two species (Table 2; Fig. 3D), which shows that this character is not very reliable. Geothelphusa lanyu also was thought to have longer dactyl to propodus ratios than that in G. lutao (Shy et al. 2020: 95), but the ratios of DL/PL of P3–P5 in the three Fig. 6. Right G1s showing the outer proximal margin of subterminal segment with or without a tooth. (A) G. tawu (CW 17.8 mm, NCHUZOOL 17152). (B) G. tawu (CW 19.3 mm, NCHUZOOL 17163). (C) G. lutao (CW 15.7 mm, NCHUZOOL 14992). (D) G. lutao (CW 18.2 mm, NCHUZOOL 13061). (E) G. lanyu (CW 17.2 mm, NCHUZOOL 14327). Scale bars = 1 mm. page 9 of 15Zoological Studies 62:37 (2023)