A new species of freshwater crab of the genus Mediapotamon Türkay & Dai, 1997 (Crustacea, Decapoda, Brachyura, Potamidae) from Guizhou, China

Abstract A new species of Mediapotamon Türkay & Dai, 1997 from a karst system in southwest China is described. The new species can be separated from congeners by the combination of a sharp and distinct epibranchial tooth, the anterolateral region lined with few scattered granules, the terminal segment of the male first gonopod distinctly bent with a constant diameter, and the position of the female vulvae. Mitochondrial 16S rDNA genetic data was used to investigate the systematic position of the new species, which is supported as a new taxon.


Introduction
China has the highest number of freshwater crab species in the world, with more than 300 species (Dai 1999, Cumberlidge et al. 2011). There are unique karst landforms in Guizhou, China where numerous caves are distributed (Han et al. 2010), and researchers have discovered a number of new freshwater crab species in these caves (Ng and Trontelj 1996, Ng 2017, Huang et al. 2017. To investigate the species diversity of freshwater crabs in this area, the authors conducted scientific investigations twice in 2010 and 2017 to collect specimens of Chinapotamon Dai & Naiyanetr, 1994, Mediapotamon Türkay & Dai, 1997, Daipotamon Ng & Trontelj, 1996, and Longpotamon Shih, Huang & Ng, 2016, some of which have already been published (Shih et al. 2016). After morphological comparison of the collected specimens, the specimen from Yaozhai village, Dongtang town, Libo County, Qiannan Buyei and Miao Autonomous Prefecture, was found to be a new species of Mediapotamon. This new species is described in this paper, and although also distributed in the karst landforms, is found not in caves but in a hill stream between densely populated mountains. Individuals of this species do not have the characteristics of cave crabs, which determined it as not a karst species but also living in a surrounding karst system. We sequenced the mitochondrial 16S rDNA gene of a specimen and combined the sequence with related reference sequences in GenBank to establish a phylogenetic tree based on Bayesian Inference (BI) and Maximum Likelihood (ML) methods. The molecular data analysis was consistent with the morphological identification results, confirming that it is a new species.

Materials and methods
Specimens were collected from Banzhai Hill, Yaozhai village (25.2128°N, 108.0041°E), Dongtang town, Lino County, Qiannan Buyei and Miao Autonomous Prefecture, Guizhou Province; preserved in 95% ethanol; and deposited at the Department of Parasitology of the Medical College of Nanchang University, Jiangxi, China (NCU MCP). Comparative materials were deposited at the Sun Yat-sen Museum of Biology, Sun Yat-sen University, Guangzhou, China (SYSBM) and the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS CB). Carapace width and length were measured in millimetres. The abbreviations G1 and G2 refer to the first and second gonopods, respectively. The terminology used herein primarily follows that of Dai (1999) and Davie et al. (2015).
Pereiopod muscle tissue was extracted from specimens of the new species with a DP1902 Tissue Kit (BioTeke Inc., Beijing). The mitochondrial 16S rDNA gene was obtained by PCR amplification with the primers 1471 (5'-CCTGTTTAN-CAAAAACAT-3') and 1472 (5'-AGATAGAAACCAACCTGG-3') (Shih et al. 2004). The PCR extension procedure is as follows: denaturation for 50 s at 94 °C, 33 cycles of annealing for 40 s at 52 °C and extension for 1 min at 72 °C and a final extension for 10 min at 72 °C. The PCR products were sequenced on an ABI 3730 automatic sequencer.
Female vulva: large, not reaching sternites 5/6 in situ, with the opening outward and the outer membrane extending outward (Fig. 3B). Reaching approximate threefifths width of sternite 6 and the position generally distantly each other (Fig. 3B). Etymology. The species is named after the type locality, Libo County, Qiannan Bouyei and Miao Autonomous Prefecture, Guizhou Province.
Colour in life. The overall colour is brownish black, which is similar to the colour of the surrounding environment (Fig. 5).
Distribution. The new species is presently known only from the type locality: Libo County, Qiannan Bouyei and Miao Autonomous Prefecture, Guizhou Province.
Ecology. This species lives in karst mountain locations surrounded by low crests and covered with diverse vegetation (Figs 6A, B). The species lives along the stream flowing down the mountain and remains hidden under rocks during the day.
Remarks. The new species fits the characteristics of Mediapotamon Türkay & Dai, 1997, viz., carapace intermediate or small in size (15-20 mm), surface smooth without a conspicuous bulge or depression, anterolateral margin lined with granules, male telson triangular, and G1 slender without any projection and reaching the pleonal locking tubercle in situ (Türkay and Dai 1997). Mediapotamon liboense sp. nov. is similar to M. angustipedum (Dai & Song, 1982), M. leishanense Dai, 1995, andDaipotamon minos, Ng &Trontelj, 1996, but the new species can be differentiated from its congeners by some distinct characters: epibranchial tooth sharp and distinct, anterolateral margin lined with a few scattered granules [versus sharp and distinct in M. angustipedum but blunt and indistinct in M. leishanense, both lined with numerous inseparable granules (cf.  Fig. 7)]. Its differences compared to D. minos can be found in Table 2

DNA analyses and discussion
We used the mitochondrial 16S rDNA gene sequence for phylogenetic analyses, and 52 species from 41 potamid genera were included (Table 1), using BI and ML analyses to construct phylogenetic trees with support values. The results are shown in Figure 8, and both analysis methods support most of the clades (Shih et al. 2009). The new species clusters with the same species as M. liboense and M. leishanense (specimen collected by Chao Huang in the Maolan Nature Reserve of Libo County in July 2013). After discussion with Huang, we think that the other new species of Mediapotamon with sequence number is LC155165 and M. liboense sp. nov. are the same species, although  Ovitamon artifrons (Bürger, 1894) ZRC Luzon, the Philippines AB428466 Parapotamon spinescens (Calman, 1905) NCUDP Yunnan, China AB428467 Pararanguna semilunatum Dai & Chen, 1985 ZRC Yunnan, China AB428490 Potamiscus yiwuensis Dai & Cai, 1998 ZRC Yunnan, China AB428476 Potamiscus yongshengense Dai & Chen, 1985 NNU150951 Yunnan, China KY963597 Pudaengon sakonnakorn Ng & Naiyanetr, 1995 ZRC Thailand AB428484 Pupamon nayung (Naiyanetr, 1993) ZRC 1995.558 (paratype) Udon Thani, Thailand AB428477 Ryukyum yaeyamense (Minei, 1973) NCHUZOOL 13126 Iriomote, the Ryukyus AB428458 Shanphusa curtobates (Kemp, 1918) NRM 13920 Taunggyi Ng, 1997ZRC 1997 Phitsanulok, Thailand AB428473 Trichopotamon daliense Dai & Chen, 1985 NCHUZOOL 13130 Yunnan, China AB428492 Yarepotamon gracilipa (Dai, Song, Li & Liang, 1980)  the two specimens were collected separately. In the phylogenetic tree, Daipotamon is clustered with Mediapotamon in two separate branches, and the phylogenetic relationships between the new species and Chinapotamon maolanense, which was also collected in Libo County, are distant (Fig. 8) Despite the new species clustering with congeners and Daipotamon in the larger clade, the genetic distance suggests that the congeners are closer, while Daipotamon is farther away. For the habitat, Daipotamon lives in limestone formations and collected from one of karst caves and was determined as a karst species (Ng and Trontelj 1996), while the new species lives in hill streams, which is consistent with congeners, so the new species can be separated from Daipotamon in morphology, phylogenetic analyses, and ecology (Table 2, Fig. 8). Mediapotamon contains M. leishanense and M. angustipedum (Dai 1999), but we were unable to obtain molecular data for the latter, so its phylogenetic relationship with the new species is unclear. From molecular and morphological data, it is distinct from M. leishanense. Although there is no molecular data for M. angustipedum, the distinct morphological differences and more than 400 kilometres geographical distance separate the new species from M. angustipedum clearly. Morphological differences among the three Mediapotamon species, including the new species described in this study, are described in detail (Table 2).