Diversity of Taiwanese Brackish Crabs Genus Ptychognathus Stimpson, 1858 (Crustacea: Brachyura: Varunidae) based on DNA Barcodes, with Descriptions of Two New Species

© 2020 Academia Sinica, Taiwan Open Access Diversity of Taiwanese Brackish Crabs Genus Ptychognathus Stimpson, 1858 (Crustacea: Brachyura: Varunidae) based on DNA Barcodes, with Descriptions of Two New Species Jhih-Wei Hsu1 and Hsi-Te Shih1,2,* 1Department of Life Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. E-mail: [email protected] (Hsu) 2Research Center for Global Change Biology, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. *Correspondence: E-mail: [email protected] (Shih) Received 18 August 2020 / Accepted 14 September 2020 / Published 20 November 2020 Communicated by Benny K.K. Chan Species in the brackish crab genus Ptychognathus are common in the seashore and estuary habitats with freshwater input. Due to their similar morphologies and dull colorations, it is always difficult to distinguish the species in this genus. In this study, the DNA barcode gene COI (cytochrome c oxidase subunit I) was used to help identify Ptychognathus from Taiwan. The results showed that the 10 species can be identified successfully based on COI, with intraspecific distances below 1.54% and interspecific distances of 12.2%– 19.57%. In addition, two new species of Ptychognathus are described from Taiwan. Ptychognathus makii sp. nov. from southern Taiwan is similar to P. altimanus (Rathbun, 1914), and P. stimpsoni sp. nov. from southern Taiwan and the southern Philippines resembles P. aff. barbatus (A. Milne-Edwards, 1873) and P. pusillus Heller, 1865. Both species can be distinguished from other congeners by a suite of characters, including the carapace, orbital region, frontal region, telson of male pleon, male first gonopod, and setae on ambulatory legs. Key words: Ptychognathus, P. makii, P. stimpsoni, New species, Morphology, Mitochondrial cytochrome c oxidase subunit I (COI), Barcodes. BACKGROUND The brackish crab genus Ptychognathus Stimpson, 1858 are usually found under stones or the interspaces of pebbles along estuaries or seashores influenced by fresh water, is a diverse genus in the family Varunidae, with 25 reported species (Osawa and N.K. Ng 2006; Ng et al. 2008; N.K. Ng 2010; Sasaki 2019). The first report of this genus in Taiwan was P. barbatus (A. MilneEdwards, 1873) (Maki and Tsuchiya 1923). Sakai (1939) established two new species from Taiwan, P. ishii Sakai, 1939 (type locality: Lanyu, Taitung) and P. takahasii Sakai, 1939 (type locality: Danshuei, New Taipei City). Later, P. hachijoensis Sakai, 1955, P. affinis De Man, 1895, P. altimanus (Rathbun, 1914), P. insolitus Osawa & N.K. Ng, 2006 and P. pilosus De Man, 1892 were reported successively (Fukui et al. 1989; Lee 2001; Naruse et al. 2005; Li 2015; Li et al. 2019). In total, morphological identification suggested that there are eight species in this genus from Taiwan (Ng et al. 2001 2017; Li et al. 2019). Most species of Ptychognathus have minor interspecific morphological differences and dull colorations, so it is always difficult to distinguish species (e.g., Chen 2001; Lee 2001; Nakasone and Irei 2003). Several taxonomic studies of crabs have used COI Citation: Hsu JW, Shih HT. 2020. Diversity of Taiwanese brackish crabs genus Ptychognathus Stimpson, 1858 (Crustacea: Brachyura: Varunidae) based on DNA barcodes, with descriptions of two new species. Zool Stud 59:59. doi:10.6620/ZS.2020.59-59. Zoological Studies 59:59 (2020) doi:10.6620/ZS.2020.59-59 1 © 2020 Academia Sinica, Taiwan (cytochrome c oxidase subunit I) and other markers to reinstate or recognize new species (cf. Chu et al. 2015), including the taxa within the family Varunidae (e.g., Chu et al. 2003; Shih and Suzuki 2008; Naser et al. 2012; N.K. Ng et al. 2018; Shih et al. 2019a 2020) and Sesarmidae (Li et al. 2019a b; Ng et al. 2020). To help identify species of Ptychognathus from Taiwan, this study used the COI barcodes approach (Hebert et al. 2003a b). Two new species of Ptychognathus collected from southern Taiwan, with morphologies that are distinct from similar species, are described herein based on evidence from the COI gene. MATERIALS AND METHODS Specimens of Ptychognathus collected from Taiwan, the Philippines and Indonesia were examined (Table 1), then deposited into the Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL). The following abbreviations are used: CW = carapace width, CL = carapace length; G1 = male first gonopod; P2–P5 = first to fourth ambulatory legs. Genomic DNA was isolated from muscle tissue using kits (see Shih et al. 2016 for details). A portion of the COI gene was amplified with a polymerase chain reaction (PCR) using the primers LCO1490 and HCO2198 (Folmer et al. 1994). PCR conditions for the above primers were 40 cycles of denaturation for 50 s at 94°C, annealing for 70 s at 45–47°C, and extension for 60 s at 72°C, followed by extension for 10 min at 72°C. Sequences were obtained by automated sequencing (Applied Biosystems 3730, USA) after verification with the complementary strand. Sequences of the different haplotypes were deposited into GenBank (accession numbers given in Table 1). A neighbor-joining (NJ) tree for COI sequences was established using the Kimura (1980) 2-parameter (K2P) model with the complete deletion option using the program MEGA (vers. 10.0.5, Kumar et al. 2018). Basepair (bp) differences and pairwise estimates of K2P distances for genetic diversities between specimens were also calculated in MEGA. RESULTS Molecular analyses of COI Molecular analysis of the COI gene identified 35 specimens and 10 OTUs (operational taxonomic units) with good support (Fig. 1) that correspond to 10 species. Fig. 1. A neighbor-joining tree for species of Ptychognathus from Taiwan, the Philippines and Indonesia, based on the cytochrome c oxidase subunit I (COI) gene. Probability values at the nodes represent support values. Only values > 50% are shown. For haplotype names, see table 1. page 2 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan Among them, the specimens identified as “P. affinis” were previously confirmed to be P. altimanus; two new species (described below) and one unknown species were also identified (Table 1, Fig. 1). The mean pairwise nucleotide divergences of K2P distances and bp differences of haplotypes of the 10 species are shown in table 2. The intraspecific nucleotide divergences (and bp differences) of the 10 species are all ≤ 1.54% (≤ 10 bp), with P. makii possessing the highest intraspecific divergence. The interspecific divergences among the 10 species were 12.2%–19.57%. (73–112 bp), with the lowest interspecific divergence being between P. hachijoensis and P. takahasii, suggesting that the 10 OTUs are distinct species. TANONOMY Family Varunidae H. Milne Edwards, 1853 Subfamily Varuninae H. Milne Edwards, 1853 Genus Ptychognathus Stimpson, 1858 Ptychognathus makii sp. nov. (Figs. 2A, B, 3) urn:lsid:zoobank.org:act:0707FBBA-8465-477F-84C98A25053B45C6 Material examined: Holotype: 1 ♂ (23.5 mm), NCHUZOOL 16062, Jhonggang River (= R.) estuary, Manjhou, Pingtung, coll. P.-Y. Hsu et al., 7 Nov. 2018. Paratype: 2 ♂♂ (13.2–13.6 mm), 1 ♀ (11.2 mm), Table 1. The haplotypes of the COI gene from the genus Ptychognathus used in this study. See MATERIALS AND METHODS for abbreviations of museums and universities. *, species identified in Lee (2001) Species Locality Sample size Catalogue no. of NCHUZOOL (unless indicated) Haplotype Access. no. P. altimanus Taiwan: Gangkou R. estuary, Pingtung 1 16052 Pa1 MW000763 Taiwan: Gangkou R. estuary, Pingtung 1 16052 Pa2 MW000764 (id as “P. affinis”)* Taiwan: Linbian R. estuary, Pingtung 1 16055 Pa3 MW000765 Taiwan: Linbian R. estuary, Pingtung 1 16055 Pa4 MW000766 P. aff. barbatus Taiwan: Fubao, Changhua 1 16075 Pb1 MW000767 Taiwan: Baoli R. estuary, Pingtung 1 16072 Pb2 MW000768 Taiwan: Watong, Baisha, Penghu 1 16076 Pb2 MW000768 Taiwan: Dasi R. estuary, Yilan 1 16063 Pb1 MW000767 Taiwan: Yanliao, Hualien 1 16064 Pb3 MW000769 Taiwan: Jihuei, Taitung 1 16067 Pb1 MW000767 Taiwan: Dulanwan, Taitung 1 16065 Pb1 MW000767 Taiwan: Shanyuan, Taitung 1 16066 Pb2 MW000768 Taiwan: Shanyuan, Taitung 1 16505 Pb1 MW000767 Indonesia: Bali 1 16504 Pb4 MW000770 P. hachijoensis Taiwan: Chaishan, Kaohsiung 1 15807 Ph1 MW000771 Taiwan: Houwan, Pingtung 1 15818 Ph2 MW000772 Taiwan: Jioupeng, Pingtung 1 15821 Ph3 MW000773 Taiwan: Dasi, Yilan 1 15820 Ph3 MW000773 Taiwan: Yanliao, Hualien 2 15809, 15810 Ph3 MW000773 P. insolitus Taiwan: Houwan, Pingtung 1 16044 Pin1 MW000774 Taiwan: Houwan, Pingtung 1 16045 Pin2 MW000775 P. ishii Taiwan: Gangkou R. estuary, Pingtung 1 16039 Pis1 MW000776 Taiwan: Gangkou R. estuary, Pingtung 1 16506 Pis2 MW000777 Taiwan: Dulanwan, Taitung 116035 Pis3 MW000778 P. makii Taiwan: Gangkou R. estuary, Pingtung 1 16049 Pm1 MW000779 Taiwan: Gangkou R. estuary, Pingtung 1 16050 Pm2 MW000780 P. pilosus Taiwan: Gangkou R. estuary, Pingtung 1 15395 Pp MW000781 P. stimpsoni Taiwan: Wanlitong, Pingtung 1 16501 Ps1 MW000782 Philippines: Camiguin 1 16502 Ps2 MW000783 Philippines: Camiguin 1 16502 Ps3 MW000784 Philippines: Camiguin 1 16502 Ps4 MW000785 P. takahasii Taiwan: Jihuei, Taitung 1 16058 Pt MW000786 Ptychognathus sp. Taiwan: Dingtanzih 2 16503 Psp MW000787 Total 35 page 3 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan NCHUZOOL 16049, Gangkou R. estuary, Hengchun, Pingtung, coll. P.-Y. Hsu and C.-Y. Chi, 4 Dec. 2016; 1 ♂ (20.3 mm), NCHUZOOL 16050, Gangkou R. estuary, Hengchun, Pingtung, coll. P.-Y. Hsu et al., 4 Sep. 2017; 4 ♂♂ (12.8–21.0 mm), 1 ♀ (16.3 mm), NCHUZOOL 16052, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-J. Li, 19 Mar. 2018; 5 ♂♂ (7.9–19.8 mm), 3 ♀ (6.6–10.6 mm), NCHUZOOL 16053, Gangkou R. estuary, Hengchun, Pingtung, coll. P.-Y. Hsu et al., 4 Sep. 2017. Comparative material: Ptychognathus pilosus: 1 ♂ (20.2 mm), NCHUZOOL 15395, Gangkou R. estuary, Pingtung, coll. J.-J. Li, 10 Aug. 2015. P. altimanus: 2 ♂♂ (9.5–19.1 mm), NCHUZOOL 16055, Linbian R. estuary, Checheng, Pingtung, coll. J.-H. Lee, 6 Feb. 2000; 1 ♂ (17.4 mm), 1 ♀ (19.7 mm), NCHUZOOL 16051, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-J. Li, 10 Aug. 2015. Description: Carapace (Figs. 2A, 3A) quadrate, slightly broader than long, 1.1 times as broad as long, flat; dorsal surface smooth, glabrous, regions weakly defined, with noticeable groove between epigastric regions. Front broad, flat, not sloping forward, part near orbital regions slightly convex; frontal margin straight, without lobes; postfrontal region indistinct, separated into two obscure lobes, without distinct shallow grooves. Anterolateral margins each with conspicuous three teeth including orbital tooth, first tooth largest and most distinct, third tooth smallest and most acute. Posterolateral margins divergent posteriorly, moderately sloping outwards; lateral and posterolateral margins regularly furnished with short, soft setae. Infraorbital ridge consisting of several small, rounded granules, decreasing in size laterally. Epistome broad, median part triangular, margin with tiny granules. Third maxillipeds (Figs. 2B, 3B) broad, surface with short setae sparsely, exopod distinctly broader than ischium, exopod 1.7 times as broad as ischium; mesial part of merus with oblique groove, anteroexternal angle very broad, slightly sloping outwards; ischium with distinct vertical shallow groove. Chelipeds (Fig. 3D) symmetrical in male. Merus without spines, margins lined with small, rounded granules, dorsal margins with long soft setae, ventral margins glabrous. Surface of carpus with several tiny granules, part near inner distal angle with single ridge consisting of large granules; inner surface sometimes with short setae sparsely; inner distal angle with single long blunt spine, slightly curving forward, and single smaller spine on margin in male (Fig. 3C); inner distal angle with single small spine in female. Outer surface of palm in male (Fig. 3D) smooth, without distinct granules; inner surface glabrous, middle part convex. Movable finger distinctly longer than palm; immovable finger with obscure horizontal ridge toward palm; cutting edges of both fingers with numerous blunt teeth, teeth near base of fingers larger; margins between movable finger and palm with short soft setae, without Table 2. Matrix of percentage pairwise nucleotide divergence with Kimura 2-parameter (K2P) distances (lower left) and mean numbers of differences (upper right) based on cytochrome c oxidase subunit I (COI) within and between 10 species of Ptychognathus from Taiwan, the Philippines and Indonesia (see Table 1). Values of the range are shown in parentheses Intraspecific Interspecific Nucleotide divergence Mean nucleotide difference P. altimanus P. aff. barbatus P. hachijoensis P. insolitus P. makii P. ishii P. pilosus P. stimpsoni P. takahasii Ptychognathus sp. P. altimanus 0.31 (0–0.46) 2 (0–3) 101.9 (100–103) 104.67 (102–106) 104 (102–106) 99.33 (97–101) 84 (82–86) 95 (95) 101 (100–102) 100 (99–101) 99 (98–100) P. aff. barbatus 0.15 (0–0.77) 1 (0–5) 17.49 (17.11–17.71) 86.43 (83–87) 91.3 (90–92) 96.63 (94–98) 101.4 (100–102) 104.9 (103–106) 77.85 (76–81) 83.8 (83–84) 76.9 (76–77) P. hachijoensis 0.2 (0–0.61) 1.33 (0–4) 18.02 (17.5–18.29) 14.86 (14.16–14.98) 83.67 (81–87) 93.83 (91–96) 107.5 (105–110) 106.83 (105–108) 81.42 (79–83) 74.17 (73–76) 80.17 (79–82) P. insolitus 0.46 (0.46–0.46) 3 (3) 17.95 (17.55–18.36) 15.67 (15.41–15.81) 14.26 (13.72–14.92) 99.83 (99–101) 111 (110–112) 105.5 (104–107) 90.25 (89–92) 86.5 (86–87) 79.5 (79–80) P. makii 1.03 (0.15–1.54) 6.67 (1–10) 17.05 (16.59–17.37) 16.61 (16.09–16.89) 16.02 (15.48–16.44) 17.24 (17.06–17.48) 101.83 (99–106) 97.67 (97–98) 93.25 (92–95) 82.33 (82–83) 89 (88–91) P. ishii 0.15 (0.15–0.15) 1 (1) 14.28 (13.89–14.67) 17.39 (17.11–17.51) 18.62 (18.13–19.14) 19.36 (19.15–19.57) 17.47 (16.91–18.3) 109.5 (109–110) 94.25 (93–96) 100.5 (100–101) 93.5 (93–94) P. pilosus — — 16.17 (16.17) 18.05 (17.67–18.28) 18.47 (18.11–18.71) 18.27 (17.97–18.58) 16.73 (16.59–16.8) 18.98 (18.87–19.08) 91 (91) 105 (105–105) 91 (91) P. stimpsoni 0.28 (0–0.46) 1.83 (0–3) 17.32 (17.12–17.52) 13.05 (12.7–13.65) 13.73 (13.28–14.03) 15.44 (15.19–15.79) 15.89 (15.65–16.23) 15.97 (15.73–16.31) 15.37 (15.37) 83.75 (83–85) 75.25 (74–77) P. takahasii — — 17.12 (16.92–17.32) 14.19 (14.03–14.23) 12.43 (12.2–12.78) 14.77 (14.67–14.86) 13.8 (13.74–13.92) 17.24 (17.14–17.34) 18.11 (18.11) 14.14 (13.99–14.38) 80 (80) Ptychognathus sp. 0 0 16.86 (16.67–17.06) 12.95 (12.78–12.97) 13.58 (13.34–13.93) 13.4 (13.31–13.5) 15.09 (14.89–15.47) 15.82 (15.73–15.92) 15.37 (15.37) 12.52 (12.29–12.85) 13.47 (13.47) page 4 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan pulvinus at base of fingers. Female with outer surface of palm almost glabrous, inner surface glabrous and middle part convex; immovable finger with single prominent horizontal ridge toward palm; part near cutting edges with sparse short setae. Ambulatory legs (Fig. 3F, G) slender, P3 and P4 longest, P4 and P5 obviously more flat than P2 and P3; anterior margins of each merus with long soft setae, posterior margins with short setae densely and long setae sparsely, with dense black short setae on margins near carpus; merus with single small spine on anterior margins near carpus in P2–P4, spine indistinct or absent in P5; propodus of P2–P4 narrower, propodus of P5 wider. Carpus and propodus of P2 covered with dense short setae on anterior margins, posterior margins of carpus without dense short setae, with dense black short Fig. 2. A–B: Ptychognathus makii sp. nov., holotype (male, CW 23.5 mm; NCHUZOOL 16062). C–F: Ptychognathus stimpsoni sp. nov.; C, D: holotype (male, CW 7.9 mm; NCHUZOOL 16501). E, F: paratype (male, CW 10.9 mm; NCHUZOOL 16502). page 5 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan Fig. 3. Ptychognathus makii sp. nov., holotype (male, CW 23.5 mm; NCHUZOOL 16062). A, carapace; B, left third maxilliped; C, left carpus of cheliped (dorsal view); D, outer view of left cheliped; E, pleon; F, left third ambulatory leg; G, left forth ambulatory leg; H–I, right G1; H, I, dorsal view; J, K, lateral view. page 6 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan setae on posterior margins of propodus, ventral surfaces of carpus and propodus with rows of short setae. P3 with anterior margins of propodus and carpus covered with dense short setae, ventral surface of propodus with rows of short setae, posterior margins of carpus without dense short setae, posterior margins of propodus with dense short setae and sparse long setae. P4 (Fig. 3F) shorter; carpus with anterior margins covered with dense short setae, posterior margins often glabrous; propodus two times as wide as long, anterior margins covered with dense short setae, posterior margins with dense long setae, denser on distal part. P5 (Fig. 3G) relatively shortest, anterior margins of carpus covered with dense short setae (denser on distal part); propodus oval, width as 1.5 times as length, anterior margins covered with dense short setae, posterior margins with dense long setae, denser on distal part. Male pleon (Fig. 3E) narrow, surface smooth, without any granules, margins covered with short setae, setae on margins of telson longer; telson tongue-shaped, approximately as 1.2 times long as sixth segment, distal margin of telson not concave, without tuft of setae. Male G1 (Fig. 3H–K) slender, slightly curving dorsally; tip blunt, with short chitinous structure, semicircle-shaped lobes in lateral view (Fig. 3J, K), opened laterally and dorsally; G2 shorter than 1/4 length of G1. Coloration: Preserved specimens with carapace and ambulatory legs gray, brown or dark brown; ventral surface of carapace and chelipedal palm light brown; setae dark brown or black. Habitat: This species inhabits estuarine areas with sandy mud sediment. Individuals always hid under stones or objects and were sometimes sympatric with P. altimanus and Varuna litterata in southern Taiwan. Etymology: This species is named for the Japanese zoologist Moichiro Maki, who published the first monograph of Taiwanese decapods (Maki and Tsuchiya 1923; Ng et al. 2009), which was also the first record of Ptychognathus in Taiwan. Distribution: Southern Taiwan. Size: Large; largest male CW 23.5 mm, largest female CW 16.3 mm. Remarks: Among the species of Ptychognathus from Taiwan, this species is similar to P. altimanus (Rathbun, 1914) and P. pilosus De Man, 1892 in morphology , but can be distinguished by the characters of its frontal region, supraorbital margins, posterolateral margins of carapace, male chelipedal palms and G1s. Ptychognathus makii differs from P. altimanus by having a shorter frontal region with lateral margins not vertical (vs. longer with lateral margins vertical), supraorbital margins gently sinuous (vs. margins strongly sinuous) (Figs. 2A, 3A; Tesch 1918: pl. 4(5a), Naruse et al. 2005: fig. 1A); posterolateral margins of carapace distinctly divergent posteriorly (vs. almost parallel, not divergent posteriorly) (Figs. 2A, 3A; Tesch 1918: pl. 4(5a), Naruse et al. 2005: fig. 1A ); male palms proximally almost smooth, without distinguishable granules (vs. with several tiny granules) (Fig. 3D; Naruse et al. 2005: fig. 1C), but the character is indistinct in smaller specimens; the margins of the chitinous structure of G1 without obvious notch (vs. with a notch) (Fig. 3H–K; Naruse et al. 2005: fig. 1E). Ptychognathus makii and P. pilosus can be distinguished by the setae on chelipeds and G1. In P. makii, male and female with short soft setae on the margin between palm and movable finger (Fig. 3D), and cutting edge with sparse short setae in females. In P. pilosus, both male and female have a small tuft of shorter setae on tip of fixed finger, and longer setae on both fingers only in males (Li et al. 2019: fig. 7B). The tips of male G1 is blunt, and the chitinous structure is large semicircle-shaped in lateral view in P. makii (vs. tapering, and the chitinous structure is small pyramidshaped pyramid-liked in P. pilosus) (Fig. 3H–K; Li et al. 2019: fig. 7C, D). Ptychognathus stimpsoni sp. nov. (Figs. 2C–F, 4) urn:lsid:zoobank.org:act:DF3F6955-235A-4588-A984793595D6FDC0 Material examined: Holotype: 1 ♂ (7.9 mm), NCHUZOOL 16501, Wanlitong, Hengchun, Pingtung, Taiwan, coll. J.-W. Hsu, 15 Aug. 2016. Paratype: 16 ♂♂ (7.3-10.9 mm), 4 ♀♀ (7.5-8.8 mm), NCHUZOOL 16502, Camiguin, the Philippines, 31 Aug. 2003. Comparative material: Ptychognathus aff. barbatus (see Remarks): 10 ♂♂ (7.7–12.4 mm), 5 ♀♀ (8.9–13.9 mm), NCHUZOOL 16063, Dasi, Toucheng, Yilan, coll. J.-W. Hsu, 15 Aug. 2016; 2 ♂♂ (10.4–14.7 mm), 1 ♀ (13.0 mm), 1 ♀ (ovig.) (13.2 mm), NCHUZOOL 16064, Yanliao, Shoufeng, Hualien, coll. J.-W. Hsu, 29 June 2016; 2 ♀♀ (6.7– 7.4 mm), NCHUZOOL 16065, Dulanwan, Donghe, Taitung, coll. P.-Y. Hsu et al., 9 Aug. 2017; 1 ♂ (13.0 mm), NCHUZOOL 16066, Shanyuan, Taitung, 27 July 2014; 2 ♂♂ (10.9–11.0 mm), 1 ♀ (9.4 mm), NCHUZOOL 16067, Gihui, Chenggong, Taitung, coll. J.-W. Hsu, 28 Apr. 2017; 3 ♂♂ (12.1–14.9 mm), NCHUZOOL 16075, Fubao, Changhua, 16 Jan. 2017; 2 ♂♂ (18.9–20.2 mm), 1 ♀ (11.5 mm), NCHUZOOL 16074, Gaoping R. estuary, Linyuan, Kaohsiung City, 29 Apr. 2009; 4 ♂♂ (9.9–16.9 mm), 1 ♀ (8.6 mm), NCHUZOOL 16073, Houwan, Hengchun, Pingtung, 11 July 2017; 1 ♂ (13.0 mm), NCHUZOOL 16072, page 7 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan Fig. 4. Ptychognathus stimpsoni sp. nov., holotype (male, CW 7.9 mm; NCHUZOOL 16501). A, carapace; B, left infraorbital ridge; C, left third maxilliped; D, left carpus of cheliped (dorsal view); E, outer view of left cheliped; F, pleon; G, left third ambulatory leg; H, left forth ambulatory leg; I–N, right G1; I, J, dorsal view; K, L, lateral view; M, N, ventral view). page 8 of 13Zoological Studies 59:59 (2020) © 2020 Academia Sinica, Taiwan Baoli R. estuary, Checheng, Pingtung, 23 June 2014; 8 ♂♂ (9.3–12.3 mm), 3 ♀♀ (8.8–11.9 mm), 2 ♀♀ (ovig.) (8.2–9.7 mm), NCHUZOOL 16070, Wanlitong, Hengchun, Pingtung, coll. J.-W. Hsu et al., 11 Dec. 2018; 5 ♂♂ (7.8–11.4 mm), 1 ♀ (9.4 mm), NCHUZOOL 16071, Dingtanzih, Hengchun, Pingtung, coll. P.-Y. Hsu et al., 19 Mar. 2018; 3 ♂♂ (6.5–10.5 mm), 1 ♀ (8.9 mm), NCHUZOOL 16069, Gangkou R. estuary, Hengchun, Pingtung, 18 Aug. 2016; 1 ♂ (14.7 mm), NCHUZOOL 16068, Jhonggang R. estuary, Manjhou, Pingtung, 7 Nov. 2018; 2 ♂♂ (6.3–12.3 mm), 1 ♀ (10.8 mm), NCHUZOOL 16076, Watong, Baisha, Penghu, 2 Sep. 2014. Ptychognathus ishii Sakai, 1939: 3 ♂♂ (5.3–7.4 mm), 1 ♀ (5.8 mm), NCHUZOOL 16033, Dasi, Toucheng, Yilan, coll. J.- W. Hsu, 15 Aug. 2016; 2 ♂♂ (8.9–11.4 mm), 2 ♀♀ (8.9-9.1 mm), NCHUZOOL 16034, Dasi, Toucheng, Yilan, coll. J.-W. Hsu, 16 Aug. 2016; 1 ♂ (10.8 mm), NCHUZOOL 16035, Dulanwan, Donghe, Taitung, coll. P.-Y. Hsu, 29 June 2016; 1 ♂ (9.2 mm), 1 ♀ (8.4 mm), NCHUZOOL 16036, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-J. Li, 5 June 2015; 6 ♂♂ (11.0–13.2 mm), 7 ♀♀ (9.1-13.7 mm), NCHUZOOL 16037, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-J. Li, 10 Aug. 2015; 1 ♂ (8.2 mm), 1 ♀ (6.9 mm), NCHUZOOL 16038, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-W. Hsu et al., 19 Mar. 2018; 1 ♀ (ovig.) (10.27 mm), NCHUZOOL 16039, Gangkou R. estuary, Hengchun, Pingtung, coll. J.-W. Hsu et al., 18 Aug. 2016. Description: Carapace (Figs. 2C–F, 4A) subquadrate, slightly broader than long, 1.2 times as broad as long, flat; dorsal surface smooth, glabrous, regions weakly defined, with noticeable groove between epigastric regions. Front broad, slightly sloping forward, part near orbital regions slightly convex; frontal margin slightly concave, weakly divided into indistinct two lobes; anterior margin lined with small, rounded granules, with row of small granules behind frontal margin, granulated ridge contiguous to anterior margin in lateral part, separated from anterior margin in middle part; postfrontal region indistinct, separated into obscure 2 lobes by shallow grooves. Anterolateral margins and supraorbital margins lined with small granules, anterolateral margin with three teeth including orbital tooth, first tooth most distinct and acute, slightly sloping forward; second and third teeth blunt, third tooth indistinct or notch. Posterolateral margins divergent posteriorly, moderately sloping outwards; lateral and posterolateral margins regularly furnished with short, soft setae. Infraorbital ridge (Fig. 4B) consisting of 12– 15 small, rounded granules. Surface of pterygostome with sparse soft setae. Epistome broad, median part triangular, margin with tiny granules. Third maxillipeds (Figs. 2D, F, 4C) broad, surface with short setae sparsely, exopod slightly broader than or equal to ischium; mesial part of merus with oblique groove, anteroexternal angle broad, slightly sloping outwards; ischium with obscure vertical shallow groove. Chelipeds (Fig. 4E) symmetrical in male. Merus without spines, dorsal margins with long soft setae, ventral margins glabrous. Surface of carpus smooth, without distinct granules, inner surface sometimes with short setae sparsely, inner distal angle blunt or obtuse triangular in male (Fig. 4D); inner distal angle with single spines in female. Outer surface of palm in male (Fig. 4E) smooth, without distinct granules or horizontal ridge; inner surface glabrous, middle part convex. Movable finger approximately as long as palm, inner surface glabrous and middle part convex; cutting edges of both fingers with numerous small blunt teeth, immovable finger with 2–3 larger blunt teeth; proximal half of fingers with long dense soft setae in male (absent in female), margin between movable finger and palm with short soft setae, with single small pulvinus at base of fingers. Female with outer surface of palm glabrous and granulated, inner surface glabrous, middle part convex; immovable finger with ridge consisting of large granules toward palm, fingers with sparse short setae at tips; movable finger slightly longer than palm. Ambulatory legs (Fig. 4G, H) slender, P3 and P4 longest; mesial half of anterior margins of each merus with long soft setae, without spines, posterior margins without setae; propodus as long as dactylus in P2–P4 (Fig. 4G), propodus about 2 times length of dactylus in P5 (Fig. 4H). Carpi of P2 and P3 with dense short setae on anterior margins of distal half, anterior margins of propodus and dactylus all covered with dense short setae, posterior margins of propodus sparsely setose, ventral surfaces of carpus and propodus with rows of short setae. Carpi of P4 and P5 with dense short setae on anterior margins of distal 1/3–1/4, posterior margins of carpus glabrous. P4 (Fig. 4G) relatively long, anterior margins of propodus covered with dense short setae, posterior margins with sparse long setae; posterior margins of distal 1/2–1/3 propodus covered with dense short setae, ventral surface with rows of long setae sparsely. P5 (Fig. 4H) relatively short, anterior margins of propodus covered with dense short setae (denser on distal part), posterior margins of distal 1/3 propodus covered with dense short setae. Male pleon (Fig. 4F) narrow, surface smooth, without any granules, lateral margins covered with short setae; telson tongue-shaped, approximately as long as sixth segment, distal margin of telson not concave, without tuft of setae. Male G1 (Fig. 4I–N) slender, almost straight, part near tip more slender and slightly curving outwards; tip page 9 of 13Zoological Studies 59:59 (2020)