Description of two new species of the genus Heterochelamon Türkay & Dai, 1997 (Crustacea: Decapoda: Brachyura: Potamidae), from southern China

This study describes two new species of freshwater crab of the genus Heterochelamon Türkay & Dai, 1997 from southern China, H. huidongense from Guangdong Province and H. jinxiuense from Guangxi Zhuang Autonomous Region. The two new species can be differentiated from congeners by characters derived from the shape of the epibranchial tooth, external orbital angle, cheliped proportions and structure of the male first gonopod. The present study brings the number of Heterochelamon species to seven. We used the mitochondrial 16S rRNA gene for a molecular analysis and the results are consistent with the morphological features that support the recognition of two new taxa.

In 2011, we collected specimens of a Heterchelamon species from Jinxiu, Guangxi, but the crabs were not identified at the time. It was not until 2018 that our research team identified the specimens as belonging to an undescribed species. In 2019, freshwater crab enthusiast Jia-Ming Tian collected some dark colored freshwater crabs from Huidong, Guangdong. We subsequently collected specimens of this species and found that they belong to another new species of Heterochelamon. We herein describe two new species of Heterochelamon, including the one from Guangdong Province, representing the first member of the genus from outside of Guangxi.
To study the phylogenetic relationships of species within Heterochelamon, we used the mitochondrial 16S rRNA gene data of four species from this genus, including the two new species reported in this paper. Molecular data supports the recognition of the two new species and their assignment to Heterochelamon.

MATERIAL & METHODS
Specimens were collected by Song-Bo Wang, Yi-Yang Xu, Jia-Ming Tian and Xi-Jiao Wei; preserved in 95% ethanol and deposited in the Department of Parasitology of the Medical College of Nanchang University, Jiangxi, China (NCU MCP), National Tropical Disease Research Center, Shanghai, China (TDRC). The abbreviations G1 and G2 are used for the male first gonopod and second gonopod, respectively. Carapace width and length are reported in millimeters. The terminology used primarily follows that of Dai (1999), Naruse, Zhu & Zhou (2013) and Davie, Guinot & Ng (2015).
Muscle tissue was extracted from the ambulatory legs and chelipeds, and total genomic DNA was extracted from the tissues using the Omega Tissue Kit following the manufacturer's protocol. Mitochondrial 16S rRNA gene sequences were obtained by PCR amplification with the primers 1471 (5 -CCTGTTTANCAAAAACAT-3 ) and 1472 (5 -AGATAGAAACCAACCTGG-3 ) (Shih, Ng & Chang, 2004). The PCR procedure was as follows: 33 cycles of denaturation for 50 s at 94 • C, annealing for 40 s at 52 • C, and extension for 1 min at 72 • C, followed by a final extension for 10 min at 72 • C. Sequences were obtained by automated sequencing (ABI3730 automatic sequencer).
Sequences were aligned using MAFFT vers.7.355 (Nakamura et al., 2018) based on the GINS-I method and the selection of conserved regions with Gblocks 0.91b (Castresana, 2000). GTR+I+G was the best fit model for the sequence evolution of the 16S dataset, as determined with MrModeltest vers.2.2 (Nylander, 2005) and then selected based on the Akaike information criterion (AIC). MrBayes vers.3.2. 6 (Ronquist et al., 2012) was used to construct a Bayesian inference (BI) tree. Four Markov chain Monte Carlo (MCMC) chains were run for 2,000,000 generations, with samples stored once every 1000 generations, discarding the first 25% as burnin. Tracer vers.1. 6 (Rambaut et al., 2014) was used to check effective sample size (ESS) values (all of greater than 200). Simultaneously with the BI analysis, we used MEGA vers.X.0 (Kumar et al., 2018) to select the best evolutionary model for maximum likelihood (ML) analysis, which was the HKY+I+G model based on the Bayesian information criterion (BIC). The ML tree was built after 1000 bootstrap replicates by using MEGA vers.X.0 (Kumar et al., 2018).
The electronic version of this article in portable document format will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub: F680C21E-0BED-4724-BA62-1174336F433F. The online version of this work is archived and available from the following digital repositories: Peer J, PubMed Central, and CLOCKSS.     Male chelipeds unequal (Fig. 1A). Merus surface smooth without any pits; carpus with inner angle produced into strong spine, surface sparsely covered with pits ( Fig. 1A). Cutting edges of larger cheliped fingers with row of large blunt teeth, with narrow gap when fingers closed ( Fig. 2A). Ambulatory legs slender; third legs longest when stretched laterally, fourth ambulatory leg propodus about 1.6 times as long as broad, slightly shorter than dactylus, with scattered thorn-like setae (Fig. 2E).

Systematics
Male thoracic sternites 2/3 demarcated by distinct suture, sternites 3/4 demarcated by shallow groove (Fig. 2C). Male sterno-pleonal cavity deep, reaching anteriorly to mid-length of thoracic sternite 4; median groove between sternites 7/8 long (Fig. 2E). Male pleon triangular, somites 3-6 gradually decreasing in width, increasing in length in males; somite 3 widest; somite 6 trapezoidal, margins slightly oblique (Fig. 2D). Telson triangular with rounded apex, lateral margin slightly concave (Fig. 2C). Female pleon broadly ovate (Fig. 3B); vulva small, ovate, located at middle of sternite 6, opening inwards, posterior margin slightly bulged (Fig. 3C). G1 slender; terminal segment flat, bent medially, not tapered distally, distal part rounded; exceeding pleonal locking tubercle on mid length of sternite 5, not reaching suture between sternites 4/5 (Figs. 4A-4D, 2F). Distinct boundary between terminal segment and subterminal segment, latter length about 2.5 times as former, groove for G2 in ventral surface; G2 basal segment triangular, length about 2.1 times distal segment (Fig. 4). Etymology. The species is named after the type locality, Huidong County, Huizhou City, Guangdong Province. Distribution. The new species is known only from the type locality presently, Huidong County, Huizhou City, Guangdong Province, southern China. Ecology. Specimens of the new species were collected at three sites along a river using fish cages or by hand (Figs. 5B, 5C). The water of the river is relatively clear, and the water flow is slow. The width of the river is approximately 1-3 m and the depth is approximately 0.2-0.3 m. The river bed is mainly sand and soil (Fig. 5E). There are many shrubs and low hills around the river, many households located dozens of meters away from the river and some vegetable gardens along the river (Fig. 5A). At one of the collection points, we also collected specimens of the gecarcinucid Somanniathelphusa sinensis H. Milne-Edwards, 1853 (Fig. 5D), which we believe coexists with the new species in the river. Remarks. Compared to congeners, Heterochelamon huidongense n. sp. can easily be separated by its G1 not reaching sternites 4/5 suture in situ and terminal segment being medially bent (Figs. 2E and 4A, Table 1) (versus reaching sternites 4/5 suture and straight in congeners, except H. tessellatum which has G1 that also does not reach the sternites 4/5 suture but is strongly bent proximally), as well as a combination of characters: very sharp epibranchial tooth , triangular external orbital angle, slightly convex anterolateral margins (Fig. 1A), triangular male telson (Fig. 2C), and inward opening female vulva (Fig. 3C). For detailed differences between this new species and congeners, see Table 1.     Epistome posterior margin distinctly cristate, median lobe triangular, distinctly protruding, lateral margins almost straight (Fig. 6B). Third maxilliped exopod exceeding posterior margin of merus, with slender flagellum; merus about 1.2 times as broad as long, with median depression; ischium about 1.4 times as long as broad, longitudinal median sulcus distinct (Fig. 7B).
Chelipeds strongly unequal in adult males (Fig. 7A). Merus surface smooth without any pits; carpus inner angle forming stout spine, surface not covered with pits (Fig. 1A). Cutting edge of larger cheliped fixed finger with large rounded teeth, movable finger cutting edge lined with several small teeth, with medium gap when fingers closed (Fig. 7A). Ambulatory legs slender; third legs longest, the fourth ambulatory leg propodus about 2.0 times as long as broad, equal to dactylus in length (Figs. 6A, 7F).

Distribution.
The new species is known only from the type locality presently, Jinxiu Yao Autonomous County, Laibin City, Guangxi Zhuang Autonomous Region, southern China. Ecology. The collection site is a small river running behind Tongfu village. The river is mostly surrounded by dwarf mountains and many kinds of fruit trees. The river is approximately 2 m in width, the water depth is approximately 0.4 m, the riverbed mainly consists of sand and gravel and the water quality is good.

Remarks
Heterochelamon jinxiuense n. sp. can easily be differentiated from its congeners by its distally tapered G1 (Fig. 9, Table 1) (versus widening distally or not tapered distally in congeners), blunt external orbital angle (Fig. 6A, Table 1) (versus triangular or acutely triangular in congeners, except that of H. guangxiense which is also blunt), strongly unequal chelipeds in adult males and medium gap when male major chela fingers closed (Fig. 7A, Table 1) (versus strongly unequal in H. tessellatum, H. purpureomanualis and H. yangshuoense, but fingers with very broad and oblong gap when closed; other species unequal with narrow gap when closed). The detailed differences between this new species and congeners are also presented in Table 1.
The new species was collected in 2011, of which the material consisted of five males (one adult and four immature) and one female. The G2 of the only mature male had been destroyed by the time we examined the specimens. We were also unable to find this species in a subsequent collection trip. As of such, the G2 is not described here.

Phylogenetic analyses
We obtained the mitochondrial 16S rRNA gene molecular data from four species of Heterochelamon for this study. Other sequences used were downloaded from GenBank. To show where these two new species fit within the Asian freshwater crab phylogeny, 60 species of 47 genera in the family Potamidae Ortmann, 1896, were used ( Table 2). The trees were constructed via the BI and ML methods and showed a high degree of consistency, and the topological structure was determined from BI with support values from both methods (Fig. 10). The phylogenetic results support the recognition of the two new Heterochelamon species, H. tessellatum and H. castanea cluster with the two new species to form an independent branch in the ''China-East Asia Islands'' clade (Shih, Yeo & Ng, 2009). It is possible that the two new species are in sister relationship. H. castanea is most closely related to the two new species in the two known congeneric species available for the molecular phylogenetic analysis.

DISCUSSION
The genetic analysis supports the recognition of four species of Heterochelamon which can also be differentiated morphologically. These four species form a monophyletic group with relatively high support values. Due to the lack of specimens of the other three species of Heterochelamon, we were unable to obtain their molecular data for analysis and therefore unable to determine the phylogenetic relationships of these species with the above four species.  Phylogenetically, it is possible that Heterochelamon huidongense n. sp. is more closely related to Heterochelamon jinxiuense n. sp. than any other congener (Fig. 10)  Türkay & Dai (1997) established Heterochelamon with Potamon (Geothelphusa) pupureomanualis (Wu, 1934) as a type species. However, the real identity of the specimens examined by Türkay & Dai (1997) is questionable, because their specimens are different from the original description by Wu (1934) in the following particulars, although we could not examine type material or topotypic material from Luocheng Country, Guangxi Zhuang Autonomous Region: the epibranchial tooth is very sharp in the type material (Wu, 1934) versus blunt in the specimens examined by Türkay & Dai (1997), and the G1 terminal segment has a bent tip in the type material (Wu, 1934) versus straight terminal segment in the specimens examined by Türkay & Dai (1997) (see also Table 1). Furthermore, the specimens examined by Türkay & Dai (1997) were said to be collected from Xiushui County, Guangxi. The locality record seems to be incorrect, because Xiushui County is in Jiangxi, not in Guangxi. Dai (1999) corrected the locality of the specimens studied by Türkay & Dai (1997) to Xiuren County, Guangxi, and we believe that this is correct locality. We concur with Naruse, Zhu & Zhou (2013) that the specimens referred to H. pupureomanualis by Türkay & Dai (1997) could actually represent an undescribed species. This matter can only to be addressed when the specimens can be re-examined.
With a few exceptions, most Chinese freshwater crab genera have restricted distributional ranges. The geographical distribution pattern of many freshwater crabs is caused by geographical isolation, which is due to the limited dispersal abilities of freshwater crabs and low fertility (Yeo et al., 2008;Ji et al., 2016;Jia et al., 2018). The distribution pattern of freshwater crabs in mainland China is closely related to the regional differentiation of China's natural geographical environment. Heterochelamon has been reported to be mainly distributed in northern Guangxi (Türkay & Dai, 1997;Naruse, Zhu & Zhou, 2013). Noteworthy in this paper is that we describe a new species assigned to the genus from southern Guangdong, which is not in the known range of the genus, suggesting that it has a wider distributional pattern than previously thought. That being said, the distribution of Heterochelamon is still within the Huanan freshwater zoogeographical province, which mainly consists of Guangxi, Guangdong and Hainan (Huang, Ebach & Ahyong, 2020). Within this zoogeographical province, there are relatively few abiotic factors such has high mountains and rivers that may obstruct the dispersal of freshwater crabs. We therefore believe that there are other new species of Heterochelamon in this zoogeographical province.

CONCLUSIONS
This study describes two new species of freshwater potamid crabs, referred to the genus Heterochelamon. We obtained sequence data of the mitochondrial 16S rRNA gene from the two new species and two other congeneric species for genetic analysis. The analysis supports the recognition of the two new species and the monophyly of the genus. The known distribution of this genus has been heretofore restricted to northern Guangxi, but our study now shows that the genus also occurs in southern Guangdong. (GenBank: MN823645 to MN823652).