A new species of karst-dwelling freshwater crab of the genus Chinapotamon Dai & Naiyanetr, 1994 (Crustacea: Decapoda: Brachyura: Potamidae), from Guizhou, southwest China

Chinapotamon maolanense sp. n. from Maolan National Nature Reserve, Guizhou, southwest China, is described. C. maolanense sp. n. has diagnostic features of Chinapotamon, such as a slender and sinuous male first gonopod, prominently convex carapace, and one-third ratio of frons to carapace width. This new species can be distinguished from congeners by the combination of the following characters: relatively slender subterminal segment of the first gonopods, nearly oval-shaped carapace, anterolateral margin cristate of carapace and an oval-shaped gap between the fingers of the male major chela. In addition, we used a 16S rRNA gene fragment to explore the relationship between C. maolanense sp. n. and C. glabrum, C. depressum and other freshwater crabs distributed in Guizhou; the results support the new species being assigned to Chinapotamon and clearly different from other species used in the analysis.


INTRODUCTION
Karst is a unique topography formed through the dissolution of soluble rocks such as limestone, dolomite, and gypsum. It is characterized by abundant underground drainage systems with sinkholes and caves (Ruan et al., 2013). The southwestern region of China exhibits the largest continuous distribution of karst topography in the world. The unique topography, water system, vegetation, tropical-subtropical warm-warm humid monsoon climate, and other natural geographical features of this area have created a tropical-subtropical karst ecosystem, that is, both typical and unique. Studies have shown that the ecological environment and biota of the karst terrain in the southwest region of China are extremely fragile, and many plant and animal species are rare or endangered (Liu et al., 2014;Tao, Zhou & Shui, 2011). Hence, detailed and comprehensive scientific research on the biodiversity of this area is urgently required.
Approximately 50 mg of muscle tissue was excised from ambulatory legs and chelipeds. Total genomic DNA was extracted from the tissues using the DP1902 Tissue Kit (BioTeKe Inc., Beijing, China) following the manufacturer's protocol. Then, an ∼550 base pair (bp) region of the 16S rRNA gene was amplified using polymerase chain reaction (PCR) with the primers 1471 (5′-CCTGTTTANCAAAAACAT-3′) and 1472 (5′-AGATAGAAACCAACCTGG-3′) (Shih, Ng & Chang, 2004). The PCR conditions were as follows: denaturation for 50 s at 94 C, annealing for 40 s at 52 C, and extension for 1 min at 72 C (33 cycles), followed by a final extension for 10 min at 72 C. The PCR products were purified and sequenced using an ABI 3730 automatic sequencer.
To further evaluate the validity and interspecies relationships of this new species, we performed molecular analysis with the mitochondrial 16S rRNA gene fragment. Fifteen species of 10 genera were downloaded from GenBank or sequenced, and Portunus trituberculatus was used as the outgroup (Table 1). Sequences were aligned using Muscle in MEGA 6.06 (Tamura et al., 2013), and the conserved regions were selected with Gblocks 0.91b (Castresana, 2000) using the default settings. Maximum likelihood (ML) analysis was conducted using the IQ-TREE web server (Trifinopoulos et al., 2016) with the default settings. MrBayes 3.2.6 (Ronquist & Huelsenbeck, 2003) was employed to perform Bayesian inference (BI) analysis, and four Monte Carlo Markov Chains of 2,000,000 generations were run with sampling every 1,000 generations. The first 500,000 generations were discarded as burn-in, and the consensus tree and Bayesian posterior probability were estimated using the remaining samples.
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:96797C61-B883-4080-B157-4610D5D0CC14. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.  MCP 196102). Others: 5♂ (40.4 Â 30.9, 30.2 Â 22.8, 28.9 Â 21.4, 27.8 Â 20.7, 27.7 Â 19.5) (NCU MCP 196103, 196105, 196106, 196109, 196110), same data as holotype; and 2♀ (38.5 Â 29.7, 31.6 Â 23.5) (NCU MCP 196104, 196108), same data as holotype.

Taxonomy
Description. Carapace nearly oval, widest at anterior one-third, 1.3-1.4 times as broad as long (mean = 1.34, specimens in the Materials examined sections were measured);  Ischium of third maxilliped (Figs. 2C and 2D) subtrapezoidal, approximately 1.4 times as long as broad (mid-length), with longitudinal depressions; merus trapezoidal, approximately 0.7 times as long as broad; exopod reaching proximal two-fifths of the merus, with slender flagellum exceeding the distal end of the merus when stretched anteriorly.
Male major chelipeds (Figs. 2A and 3E) unequal. Merus with inner-lower margin crenulated. Carpus surfaces smooth, with a long sharp inner angle, followed inferiorly by two small teeth at base; chela palm smooth in large males, approximately 1.2 times as long as broad; movable finger approximately 1.2 times as long as the immovable finger; both fingers slender, movable finger gradually curving downward, bending outward at base, immovable finger curving upward and outward at distal 2/3, forming a large oval gap, not crossing, occlusal margins of both fingers irregularly lined with short, sharp teeth distally. Minor chela shorter than major chela, and two fingers relatively straight, with a smaller gap when closed. Ambulatory legs (Figs. 2A, 3D and 4A) long and slender. Outer surface of merus with shallow, irregular pits, length-to-width ratio of merus of the fourth leg 3.7; Surface of carpus with shallow, irregular pits, length-to-width ratio of the fourth leg 2.3; propodus subrectangular, with short sharp spines on the distal inner margins, length-to-width ratio of the fourth leg 3.0; dactylus gently curved, with sharp spines on both the inner and outer margins.
Male thoracic sternites (Figs. 3A, 3B and 5) 1 and 2 fused to form a triangular structure; sternites 2 and 3 demarcated by horizontal groove; sternites 3 and 4 fused, forming subtrapezoidal, superficially demarcated by oblique depressions; median longitudinal suture of sternites 7, 8 deep. Female vulvae (Fig. 4B) oval, deep, opening directed anteromesially. Male pleon (Figs. 3A-3C) narrowly triangular; lateral margins of somites 3-6 forming slightly concave curvature; proximal margin of telson significantly wider than the distal margin of somite 6; somite 6 approximately 1.8 times as broad as long; telson approximately 1.3 times as broad as long. G1 (Figs. 5 and 6A-6C) generally slender, sinuous, reaching or slightly not reaching the press button on thoracic sternite 5, but not reaching the suture between sternite 4/5; subterminal article directed inward proximally but slightly bent outward distally, approximately 2.2 times as long as the terminal article; terminal article tapering to subacute, slightly recurved tip. G2 (Fig. 6D) slightly shorter than Living color. The dorsal surfaces of the carapace and pereopods are dark purple-red, and the joints of the cheliped merus and carpus and the ambulatory legs are bright red. The inner surface of the immovable finger and distal part of the movable finger are almost milky (Fig. 7).
Etymology. The species is named after the type locality: the Maolan National Nature Reserve.
Ecological note. Karst terrain usually lacks soil and water, but in karst forests such as the Maolan National Nature Reserve, water could be conserved by dead branches and deciduous layers, and groundwater is another source. C. maolanense sp. n. crabs are locally known as "mountain crabs" because the species is generally distributed in low-altitude mountain forests. This species inhabits small mountain streams with low water flow and even moist soil where the surface has no flow; in contrast, most other Chinapotamon species live under stones in streams, while C. dashiwei and C. clarkei live in streams in caves. This species inhabits environments with dead leaves, dead branches, and humus (Fig. 8).
Remarks. Chinapotamon is characterized by the sinuous and slender G1, prominently convex dorsal surface of the carapace, and frons approximately one-third as wide as the carapace (Dai, 1999;Ng, 2017). C. maolanense sp. n. has all these features. Compared with the other Chinapotamon species, the shape of the G1 of C. maolanense sp. n. is similar to that of C. anlongense, C. depressum, C. dashiwei, and C. clarkei, but the terminal segment of the G1 of the new species is fairly straight, whereas those of the other four species are clearly curved. The subterminal segment of the G1 of C. maolanense sp. n. is almost as slender as the terminal segment, but the subterminal segment of the other four species are clearly stouter than the terminal segment. Additionally, the nearly ovate and prominent convex carapace, the large, oval-shaped gap between the fingers of the male major chela, and the uncountable granular teeth along the anterolateral margin of C. maolanense sp. n. are all distinguishable features (Table 2).

DNA analysis
In this research, 25 16S rRNA sequences of 461 bp were obtained for subsequent analysis, including 15 species in 10 genera, most of which are distributed in southwestern China. The pairwise distance based on the K-2-P model showed that most of the pairwise genetic distances between the 15 species reached a threshold of 0.02 (Table 3); some were slightly below this value. The genetic distances between C. maolanense sp. n. and the other two Chinapotamon species ranged from 0.027 to 0.040. The minimum value between Chinapotamon species and the other genera was 0.095, supporting the new species being assigned to Chinapotamon but still maintaining species-level differences. The phylogenetic trees constructed by the BI and ML methods showed a similar topology, with credible support values (Fig. 9). The phylogenetic trees strongly supported  the monophyly of Chinapotamon species used in the analysis. The seven specimens of C. maolanense sp. n. exhibited minimal intraspecific genetic variation; C. depressum and C. glabrum form a sister clade to C. maolanense sp. n. Chinapotamon is the next sister to the genus Tiwaripotamon, which is mostly distributed in Guangxi Province, unlike the other genera in Guizhou Province. Tenuilapotamon, Longpotamon, Parapotamon, Table 3 Mean genetic distance among the 15 species based on the K-2-P model. and Mediapotamon form another clade sister to Chinapotamon and Tiwaripotamon, Tenuipotamon, Artopotamon, Trichopotamon, and Pararanguna form a clade at the base. This result indicates that there are at least three lineages of freshwater crabs in the mountainous terrain of southwestern China, and they may have different origins.

GENERAL DISCUSSION
China has the largest area of karst in the world, most of which is located in the subtropical climate zone in southern China (Liu et al., 2014;Zhou, 1987). Based on theoretical inference or speculation due to existing forest fragments, these karst landforms are believed to have been covered with dense forest vegetation before human influence, but these forests are gradually disappearing and are nearly destroyed (Han, Tang & Wu, 2010). Thus, the discovery of the Maolan karst forest and the primitiveness and richness of the forest vegetation in this area have attracted attention from researchers (Han, Tang & Wu, 2010), but biodiversity surveys in this region have mainly focused on plants (Zhou, 1987). Among the collections of freshwater crab specimens in this area, we found a new species described herein as C. maolanense sp. n. Most known Chinapotamon species are distributed in Guangxi and Guangdong Provinces, China, except for C. anlongense and C. xingrenense from southwest Guizhou Province, where the average altitude is 1,000-2,000 m. Dai (1999) also collected observations at altitudes above 1,000 m. However, the average altitude of the type locality of C. maolanense in southern Guizhou Province is approximately 500 m, and the main terrain of this area is a low karst peak cluster instead of high mountains. In contrast to the other karst-dwelling crabs, the specimens of C. maolanense were not collected from caves but rather from the humus layer in the forest with small water flow or even just a wet environment; this is a rare habitat for freshwater crabs. In this case, we speculate that this species may have more athletic ability.