Two new species of freshwater crab of the genus Aparapotamon Dai & Chen, 1985 (Crustacea, Brachyura, Potamidae) from Yunnan, China

Abstract Two new species of freshwater crab of the genus Potamid Aparapotamon Dai & Chen, 1985 are described from Yunnan Province, southwest China. Morphological comparisons were made between the two new species and type materials of other 11 species of Aparapotamon. Aparapotamonbinchuanensesp. nov. and A.huizeensesp. nov. can be separated from their congeners by the shape of the epibranchial tooth, the frontal view of the cephalothorax, the male first gonopod, and the female vulvae. The molecular analyses based on partial mitochondrial 16S rRNA gene are also included. This study brings the number of Aparapotamon species to 13.


Introduction
Crabs of the family Potamidae Ortmann, 1896 (Crustacea, Decapod, Brachyura) spend their whole life history in freshwater or terrestrial environments (Yeo et al. 2008). The juvenile crabs hatch directly from large and yolky eggs, and there is no larval phase in their life history, so they are considered true freshwater crabs (Yeo et al. 2008;Daniels et al. 2015).
Due to their low fecundity and poor dispersal abilities, these crabs are easily blocked by geographical barriers, and their phylogeny often closely reflects relevant historical geological events (Shih et al. 2009Fang et al. 2015;Ji et al. 2016;Jia et al. 2018).
Previous studies have shown that China has the world's highest number of freshwater crab species (Dai 1999;Cumberlidge et al. 2011;Daniels et al. 2015;Chu et at. 2018a). Despite this substantial diversity, the rate of discovery remains high (Chu et al. 2018b;Huang et al. 2018aHuang et al. , b, 2020aNaruse et al. 2018;Zou et al. 2018;Gao et al. 2019;Wang et al. 2019aWang et al. , b, 2020aMao and Huang 2020). With the two new species described in this study, Yunnan has a total of 18 genera and 67 species, highest among all provinces in China (Chu et al. 2018a;Wang et al. 2020). Yunnan is a possible center of origin for the family Potamidae and is located in the southwest of China . It is at the junction of the Asiatic Plate and the Indian Plate (Harrison et al. 1992), the geological movement remains active, the complex geographical features of this area have contributed to the rapid differentiation of crabs (Shih et al. 2009), so species of this area is richer than that in other places at the same latitude such as Guangxi Zhuang Autonomous Region and Guangdong Province .
Aparapotamon was established by Dai and Chen (1985), and eleven species have been reported so far. Since all the species are from Yunnan, Sichuan, Guangxi, Hunan, Hubei, Chongqing, and Shaanxi but with only A. gracilipedum Chen & Chang, 1982 known from Henan Province (Dai 1999). The two new species of Aparapotamon collected from Yunnan Province are herein described. Morphological comparisons were made between the two new species and type materials of other eleven species of Aparapotamon. To analyze the phylogenetic relationship between these species and its congeners, we use the mitochondrial 16S rRNA gene for phylogenetic analysis, which has been proved to be useful in crab taxonomy (Schubart 2000;Bai et al. 2018).
We compared two new species with type materials of other eleven species of Aparapotamon deposited in Chinese Academy of Sciences, Beijing, China (CAS CB). Comparative materials are as follows: The pereiopod muscle tissue was extracted from specimens of the new species with a DP1902 Tissue Kit (BioTeKe Inc. Beijing). Partial mitochondrial 16S rRNA gene sequences were obtained by PCR amplification with the primers 1471 (5'-CCTGTTTAN-CAAAAACAT-3') and 1472 (5'-AGATAGAAACCAACCTGG-3') (Shih et al. 2004). The parameters of the PCR were as follows: denaturation for 50 s at 94 °C, annealing for 40 s at 52 °C, extension for 1 min at 72 °C (33 cycles) and extension for 10 min at 72 °C. The PCR products were examined on an ABI 3730 automatic sequencer to sequence.

NCU MCP
For molecular analysis, 30 partial sequences of 16S rRNA gene were used to construct BI and ML phylogenetic trees, including those of 27 species in 22 genera of potamids (Table 1). Sequences were aligned using MAFFT vers.7.355 (Nakamura et al. 2018) based on the G-INS-I method and the conserved regions were selected with Gblocks 0.91b (Castresana 2000). The best-fitting model for Bayesian Inference (BI) analysis was determined by MrModeltest ver. 2.3 (Nylander 2004), selected by the Akaike information criterion (AIC). The obtained model was GTR+I+G. MrBayes 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 1,000 generations. The first 500,000 generations were discarded as burn-in. The best evolutionary model for Maximum Likelihood (ML) analysis was HKY+G, determined by MEGA X (Kumar et al. 2018) based on the Bayesian information criterion (BIC). A ML tree was built based on 1000 bootstrap replicates in MEGA X (Kumar et al. 2018).
Etymology. The species is named after the type locality, Binchuan County, Dali Bai Autonomous Prefecture, Yunnan Province.
Distribution. The new species is presently known only from the type locality, Binchuan County, Dali Bai Autonomous Prefecture, Yunnan Province.
Remarks. Aparapotamon binchuanense sp. nov. closely resembles congeners in general carapace morphology. However, A. binchuanense sp. nov. can be distinguished from other species by the terminal segment of G1, which is claviform, with distal end tapering and distinctly bent (Fig. 9A) [vs. terminal segment of G1 disc-shaped, straight in A. inflomanum and A. molarum (Fig. 9C, D), terminal segment of G1 of A. emineoforaminum tapering distally but not bent (Fig. 9E), terminal segment of G1 arc-shaped in A. arcuatum and A. muliense (Fig. 9H, I), and terminal segment of G1 of A. tholosum, A. protinum, A. grahami, A. huiliense, A. similium and A.gracilipedum claviform, not bent (Fig. 9F, G, J-M)]. In addition, in A. binchuanense sp. nov., the pterygostomial region is densely covered with round granules, while in the sub-orbital region the granules are sparse. (Fig. 1C). This character can also distinguish A. binchuanense sp. nov. from congeners. For detailed differences between this new species and congeners, see Table 2. Diagnosis. Carapace trapezoidal, dorsal surface slightly convex, regions defined. External orbital angle round, separated from anterolateral margin, postorbital cristae convex, postfrontal lobe prominent. Cervical groove shallow, H-shaped groove distinct, especially in female specimen. Epibranchial tooth distinct, especially in female specimen. Third maxilliped exopod without flagellum. Ambulatory legs slender. Male pleon broad triangular, telson triangular, apex rounded. Vulva ovate, covering anterior half of sternite 6, with the posterior margin distinctly convex. G1 very slender,  dorsal lobe well developed, exceeding suture 4/5 in situ, G2 basal segment ovate, tip of terminal segment round.
Etymology. The species is named after the type locality, Huize County, Qujing City, Yunnan Province.

Phylogenetic analyses
Thirty 529 bp 16S rRNA gene sequences were used to construct BI and ML trees. The phylogenetic tree in this study included five species of Aparapotamon, and the results showed that they were clustered into one clade (Fig. 10). Eight sequences of Figure 10. Bayesian inference (BI) phylogenetic tree based on 16S rRNA gene for two new species with their sequence accession numbers see Table 1. Probability values at nodes represent support values for BI and ML. Only values > 50% are shown. five species were clustered into one branch, including the two new species reported in this paper, along with A. huiliense, A. similium, and A. grahami. The pairwise distance based on the K2P model showed that the pairwise genetic distances among five species of Aparapotamon range from 0.0019 to 0.0095 (Table 3). The minimum genetic distance is between A. grahami and A. binchuanense sp. nov., indicating that they have a close relationship. The minimum genetic distances between A. huizeense sp. nov. and other four species is 0.0038, which is same as the genetic distance between A. grahami and A. huiliense. Densely covered with round granules (Fig. 1C) Sparely covered with round granules (Fig. 1C) Exceeding pleonal locking tubercle but not suture 4/5 (Fig. 2F) Slender, distal end tapering, distinctly bent ( Fig. 9A) Ovate, posterior margin not convex (Fig. 3C) A. huizeense sp. nov. Sharp (Fig. 5A) Sparely covered with round granules (Fig. 5C) Smooth (Fig. 5C) Exceeding suture 4/5 (Fig. 6F) Very slender, distal end slightly bent, dorsal lobe well developed inward (Fig. 9B) Ovate, posterior margin distinctly convex (Fig. 7C) A. inflomanum (cf. Dai 1999: fig. 196

Discussion
There are currently 13 species in this genus including those described in this study. The original eleven species of Aparapotamon are morphologically diverse, with the distal end of G1s of A. inflomanum and A. molarum being disc-shaped but that of A. emineoforaminum tapering distally, and the three G1s extend to suture 4/5, while the other eight species have G1s that are claviform in terminal segment and distal ends do not extend to suture 4/5 (Dai 1999). A. binchuanense sp. nov. and A. huizeense sp. nov. can be distinguished from above eleven species by their G1s, which distinctly bent in distal end and very slender, dorsal lobe distinctly developed, distal end exceeds suture 4/5 respectively. In this study, 30 sequences of 16S rRNA gene from 27 species of 22 genera were used to performed phylogenetic analyses. Since the two new species cluster with other Aparapotamon species form a separate branch in clade (Fig. 10), the phylogenetic tree supports the assignment of these two new species to Aparapotamon. However, the genetic distances between species of Aparapotamon are small, ranging from 0.0019 to 0.0095 (Table 3). A. grahami and A. binchuanense sp. nov. are close at molecular level, with the genetic distance 0.0019, but they are significantly different in morphology (Table 2). Most importantly, A. binchuanense sp. nov. can be distinguished from A. grahami by the terminal segment of G1, which is claviform, with distal end tapering and distinctly bent (Fig. 9A) [vs. terminal segment of G1 A. grahami claviform, not bent ( Fig. 9J)]. In this study, the molecular results of 16S rRNA gene were not sufficient for species identification in Aparapotamon. Therefore, it is recommended to use other markers (e.g. COI and nuclear genes) for further phylogenetic studies of this genus. If the results of other makers indicate that the genetic distance between Aparapotamon is also too small compared to other freshwater crab genera, revision of Aparapotamon is necessary.
The present molecular results show five species of Aparapotamon were clustered into one clade. And Aparapotamon cluster with other genera from Yunnan form 'Yunnan' clade. The genera in the branch of 'Yunnan' have many similarities in terms of morphological structure, such as the G1 slender, the terminal segment is longer than the half of subterminal segment, third maxilliped exopod without flagellum, and the ability to live at an altitude of 1500-2900 meters (Dai and Chen 1985;Dai 1999;Chu et al. 2017). Specimens of A. molarum were collected at Baishui River, Yulong Naxi Autonomous County, Lijiang City, Yunnan Province at an altitude of 2910 meters, which is the highest altitude at which freshwater crab specimens have been discovered in China so far (Dai 1999).