﻿The systematic position of Cryptopotamonanacoluthon (Kemp, 1918), with the description of a new species of Sinolapotamon Tai & Sung, 1975 (Crustacea, Decapoda, Brachyura, Potamidae) from southern China

﻿Abstract The systematics of the potamid freshwater crab Cryptopotamonanacoluthon (Kemp, 1918) is clarified, and its generic position in Sinolapotamon Tai & Sung, 1975, is confirmed based on morphological comparisons, geographical information and phylogenetic analyses. A new species of Sinolapotamon, Sinolapotamoncirratumsp. nov. is described from the Guangxi Zhuang Autonomous Region of China. Sinolapotamoncirratumsp. nov. is distinguished from its congeners by the combination of characters of its carapace, third maxilliped, anterolateral margin, and unique male first gonopod. Phylogenetic analyses based on partial COX1, 16S rRNA and 28S rRNA genes also support the species as new.


Introduction
Located in the southwest border region of China, with a warm climate, abundant precipitation, and a high percentage of forest coverage and karst landforms, Guangxi (Fig. 1) provides a suitable living environment for freshwater crabs. In China, which has the highest species richness of freshwater crabs globally (Cumberlidge et al. 2011), the species richness in Guangxi (43 species, including S. cirratum sp. nov.) is surpassing that of Taiwan (41 species) and only lower than that of Yunnan (74 species) (Chu et al. 2018;Wang et al. 2019;Cai et al. 2021). Rong County, situated in southeastern Guangxi and adjoining Guangdong Province, is the type locality of Sinolapotamon cirratum sp. nov. (Fig. 1). Hong Kong (Fig. 1), located in the south of China, consists of Hong Kong Island, Kowloon, the New Territories and 262 surrounding islands. The New Territories and Kowloon are connected to mainland China. It is worth noting that the New Territories is connected to Shenzhen, Guangdong Province.

Materials and methods
Specimens were collected from the Duqiaoshan Forest Park and Silaochong, both in Rong County, Yulin City, Guangxi Zhuang Autonomous Region, China. The two sites are so close that they appear as one dot in Fig. 1 (about 5 km). In addition, the sites of the specimens of C. anacoluthon referred to in Ng and Dudgeon (1992) were added to the map (Fig. 1). The two sites are: Tai Po Kau Forest Reserve stream, New Territories, Hong Kong; and the stream at Wu Kwai Sha, New Territories, Hong Kong. The linear distances from the 'Shenzhen' site to the two 'Hong Kong' sites are between 30-40 km (Fig. 1). Ethanol (95%) was used to preserve the collected specimens, which were deposited in the Department of Parasitology of the Medical College of the Nanchang University, Jiangxi, China (NCU MCP). Materials used herein, except for the new species and S. anacoluthon, are as follows: S. patellifer, 1 ♂, Yangshuo County, Guangxi Province, collection date not clear, NCU MCP 407301;S. auriculatum, 2 ♂♂, Shanglin County, Guangxi Province, July 2006, NCU MCP 72301, 72302;S. palmatum, 2 ♂♂, Liuzhou City, Guangxi Province, May 2018, NCU MCP 415301, 415302. Carapace width and length were measured in millimeters. The terminology used herein primarily follows that of Dai (1999) and Davie et al. (2015). The abbreviations used for the male first gonopod and male second gonopod are G1 and G2, respectively.
Approximately 50 mg of muscle tissue was excised from ambulatory legs. Total genomic DNA was extracted using the D3373-01 Mollusc DNA Kit (Omega Biotek, Inc., Norcross, USA). In our study, three fragments of target genes were amplified, including the mitochondrial COX1 and 16S rRNA genes and nuclear 28S rRNA gene. The primers and annealing temperatures used are presented in Table 1. Notably, the COX1 primers used were slightly modified based on the primers LCO1490 and HCO2198. A base T in the primer HCO2198 was replaced with a degenerate base Y (Folmer et al. 1994;Yang 2011). We performed phylogenetic analyses with the single-gene dataset (COX1) and 3-gene combined dataset (COX1, 16S rRNA and 28S rRNA). All molecular data are presented in Table 2. Sequences were aligned using ClustalW (Thompson et al. 2003), and the conserved regions were selected with Gblocks 0.91b (Castresana 2000) using the default settings. The optimal model for Bayesian inference (BI) analysis was determined using MrModeltest v. 2.3 (Nylander 2004) on the basis of the Akaike information criterion (AIC). The best-fitting model was GTR+G+I for both datasets. MrBayes v. 3.2.6 (Ronquist et al. 2012) was employed to perform BI analysis, and four Monte Carlo Markov chains of 2 000 000 generations were run with sampling every 1000 generations. The first 25% of generations were discarded as burn-in. Tracer v. 1.6 (Rambaut et al. 2013) was used to examine the sampling parameter. The optimal model, identified with MEGA X, for maximum likelihood (ML) analysis was also GTR+G+I for both datasets (Kumar et al. 2018). MEGA X was also employed to construct the ML tree based on 1000 bootstrap replicates and to calculate the pairwise distance based on the Kimura 2-parameter (K2P) model (Kumar et al. 2018). The map of the study area was prepared using ArcMap v. 10.2.  Diagnosis. Carapace gently convex, regions indistinct. Cervical groove shallow, indistinct; H-shaped groove depressed and distinct ( Fig. 2A). Epigastric cristae weak, postorbital cristae flat, indistinct. External orbital angle triangular, with about 5 small granules. Epibranchial tooth sharp, distinctly separated with external orbital angle by V-shaped gap. Anterolateral margin of carapace cristate, with about 12 granules ( Fig. 2A). Maxilliped 3 exopod reaching nearly 1/3 of merus length, with long flagellum (Fig. 2C). Chelipeds (pereiopod 1) strongly unequal ( Fig. 2A, B, D). G1 slender, subterminal segment about 1.1 times as long as terminal segment; 2 lobes of terminal segment strongly unequal, dorsal lobe longitudinally extended, oval shaped, ventral lobe sharp and short, reaching 3/7 of terminal segment (Figs 2E, 7C).
Ecology. The species is usually inhabiting the clear hill streams at an altitude below 50 m. Stones could serve as shelter and leaf mould could serve as food (Dai, 1999).

Distribution. China: Shenzhen of Guangdong Province (present record) and
Hong Kong. Remarks. The specimens from Shenzhen, with gently convex dorsal surface of carapace, indistinct postorbital cristae, sharp epibranchial tooth, unequal lobes of the terminal segment of the G1 (Fig. 2), and other characteristics, agree well with the descriptions and illustrations in Ng and Dudgeon (1992) and Dai (1999). The ratio of the subterminal segment to the terminal segment of G1 calculated in this study is 1.1 (Fig. 7C), which is equal to that in Dai (1999) and slightly smaller than that in Ng and Dudgeon (1992) (1.17). Although the specimens are not from Hong Kong, they could still be determined as S. anacoluthon based on morphological examination and the proximity of their collection site to Hong Kong (Fig. 1). Ng and Dudgeon (1992) listed the differences between Cryptopotamon and Sinolapotamon, including carapace, epigastric cristae, postorbital cristae, epibranchial tooth, and the ratio of the subterminal segment to the terminal segment of the G1. We, however, noticed that those differences are interspecific, while two or more species sharing the same character state with the remaining species is not. For instance, S. anacoluthon has a gently convex carapace similar to that of S. cirratum sp. nov. but different from the remaining congeners ( fig. 1a). The different ratios of the subterminal segment to the terminal segment of the G1 could only be regarded as interspecific differences. Most importantly, all five species have accordant fundamental types of G1 (Fig. 7).  (Figs 3A, 5A). Epigastric cristae distinct, separated from postorbital cristae by narrow gap; epibranchial region slightly depressed; mesogastric region gently convex. External orbital angle triangular, distinctly separated from anterolateral margin by wide notch. Anterolateral margin of carapace distinctly cristate, lined with approximately 20 granules (Figs 3A, 5A). Maxilliped 3 exopod reaching nearly 1/2 of merus length, with long flagellum, slightly longer than width of merus (Fig. 4B). Chelipeds (pereiopod 1) strongly unequal in males, subequal in females (Figs 3A, 4A, 5A). G1 slender, subterminal segment about 1.7 times as long as terminal segment; 2 lobes of terminal segment strongly unequal, dorsal lobe longitudinally extended, oval shaped, ventral lobe blunt, reaching 1/2 of terminal segment (Figs 6A-D, 7A). Female vulvae ovate, medium-sized, occupying anterior 2/3 length of sternite 6 (Fig. 5B).
Chelipeds (pereiopod 1) strongly unequal in males, subequal in females. Merus trigonal in cross section. Carpus surface gently depressed, with spine at inner distal angle and spinule at base in both males and females. Palm of lager chela about 1.3-1.5 times as long as high in males, 1.3-1.6 times in females. Dactylus of larger chela 0.6-1.0 times as long as palm in males, practically same proportion in females. Inner margin of fingers lined with granular teeth; fingers of lager chela leaving small gap while smaller one without gap when closed in both males and females (Figs 3A, 4A, 5A).
Remarks. Consistent with the diagnostic characters of Sinolapotamon, Sinolapotamon cirratum sp. nov. has a gently convex dorsal surface, long flagellum of the third maxilliped exopod and unequal lobes of the G1 terminal segment (Figs 3A, 4B, 7A). The dorsal lobe of the G1 terminal segment in S. cirratum sp. nov. is long and oval, which is similar to that of S. anacoluthon. The two species can nevertheless be distinguished by the ratio of the subterminal segment to the terminal segment of G1, which is 1.7 in S. cirratum sp. nov. and 1.1 in S. anacoluthon (Fig. 7A, C). When compared with S. patellifer, S. auriculatum and S. palmatum, the new species could be easily distinguished by the shape of the dorsal lobes and ventral lobes. The ventral lobe of S. cirratum sp. nov. is bluntly angular, while those of the other species in Sinolapotamon are pointed or shortly pointed (Fig. 7A, B, D, E). They also differ in comparative length of the ventral lobe relative to the terminal segment of the G1 (see Table 3). Additional differences among the known species of Sinolapotamon are provided in Table 3.
Etymology. The new species is named Sinolapotamon cirratum sp. nov. because of the curled edges of the dorsal lobe of the G1. In the Latin, 'cirratus' means 'curled'.
Ecology. The specimens were collected from puddles in the Duqiaoshan Forest Park. These crabs live in the shallow water or under the wet stones (Fig. 8A, B). Table 3. Morphological differences between five species of Sinolapotamon.

Phylogenetic relationships
A single-gene dataset (COX1) and a 3-gene combined dataset (COX1, 16S rRNA, and 28S rRNA) were used to reconstruct the ML tree and BI tree, respectively. The topologies of the ML tree and BI tree based on the single-gene dataset and the 3-gene combined dataset were analogous. Both evolutionary trees based on the single-gene and 3-gene datasets offer strong evidence for the recognition of the new species as Sinolapotamon cirratum sp. nov., since it is clustered with the species of Sinolapotamon as a monophyletic clade. Sinolapotamon patellifer and S. auriculatum form a sister group. Notably, S. anacoluthon (previously C. anacoluthon) is in 'Clade Sinolapotamon', which provides supporting evidence for recognizing the species in Sinolapotamon (Figs 9, 10). The results show that pairwise genetic distances range from 0.0600-0.1106 within the genus Sinolapotamon, and the genetic distances between Sinolapotamon cirratum sp. nov. and its congeners range from 0.0728-0.0947 (Table 4). Phylogenetic analyses, therefore, provided evidence for the identification of Sinolapotamon cirratum sp. nov. as a new species.

Discussion
Previous studies on Sinolapotamon focused on morphological descriptions and lacked molecular evidence (Tai and Sung 1975;Ng and Dudgeon 1992;Zhu et al. 2010). For our study, we obtained sequences of the partial COX1, 16S rRNA and 28S rRNA genes of all the members of Sinolapotamon, thus compensating for this gap. Moreover, the taxonomic statuses of the new species and S. anacoluthon are demonstrated based on morphology, molecular phylogeny and geographical distribution. Ng and Dudgeon (1992) listed the morphological differences between Cryptopotamon and Sinolapotamon, including a gently convex carapace against a strongly inflated carapace, and the extent of prominence of the epigastric and postorbital cristae. Dai (1999) stated that these differences could only be regarded as interspecific and that the fundamental types of G1 are accordant, thus considering Cryptopotamon as a synonym of Sinolapotamon. Ng et al. (2008), however, listed S. anacoluthon as belong to Cryptopotamon. We assessed the morphological differences among the five known species of Sinolapotamon (see Remarks above) (Table 3) and reconstructed the phylogenetic relationships in Sinolapotamon, in turn providing molecular evidence for transferring C. anacoluthon to Sinolapotamon. Sinolapotamon anacoluthon was previously recorded only from Hong Kong (Ng and Dudgeon 1992;Stanton et al. 2017), but we also collected this species in Shenzhen of the Guangdong Province. There is some geographical distance between S. anacoluthon (from Guangdong) and its congeners (from Guangxi). We, however, noticed that all the species of Sinolapotamon are distributed near the Pearl River Basin. We speculate that the Pearl River contributed to the spread of Sinolapotamon, but further surveys will be needed to validate this hypothesis.

Conclusion
In this study, a new species of Sinolapotamon is described from the Guangxi Zhuang Autonomous Region of China, based on its morphological characteristics, especially its unique G1 among congeners, and the results of phylogenetic analyses (phylogenetic tree based on COX1 and 3-gene combined datasets). In addition, the generic position of Cryptopotamon anacoluthon in Sinolapotamon is confirmed largely on the basis of its morphology, with further evidence from the genetic data. Sinolapotamon is now known by five species. Based on the geographical distributions of Sinolapotamon, there is still possibility to discover new species in Guangxi or Guangdong.