Two new species of freshwater crabs of the genera Eosamon Yeo & Ng, 2007 and Indochinamon Yeo & Ng, 2007 (Crustacea, Brachyura, Potamidae) from southern Yunnan, China

Abstract Two new species of potamid crabs, Eosamon daiaesp. nov. and Indochinamon malipoensesp. nov. are described from the Sino-Burmese border, southwestern Yunnan and from the Sino-Vietnamese border, southeastern Yunnan, China. The two new species can be distinguished from their closest congeners by several characters, among which is the form of the first gonopod structures. Molecular analyses based on partial mitochondrial 16S rDNA sequences also support the systematic status of these new taxa.


Introduction
China has the most freshwater crab species in the world and Yunnan is the epicenter of this diversity, with over 60 species in 17 genera (Dai 1999;Chu et al. 2018a, b;Naruse et al. 2018). Despite this, the biodiversity of freshwater crabs in this region appears to be still underestimated, especially in the remote areas (Chu et al. 2018b). In this paper we describe two new species belonging to two genera, Eosamon Yeo &Indochinamon Yeo &, from the Sino-Burmese and Sino-Vietnamese border areas in Yunnan Province, China. Eosamon and Indochinamon are widely distributed in the Indochina Peninsula . Including the two new species described in the present study, Eosamon and Indochinamon respectively contain 12 and 40 species Yeo 2010;Naruse et al. 2011Naruse et al. , 2018; Van et al. 2016;Ng and Mar 2018).

Material and methods
Specimens were collected from southwestern and southeastern Yunnan (Fig. 1), preserved in 95% ethanol and identified via a stereo dissection microscope (Nikon SMZ645). Materials examined are deposited in the Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University (NNU), Nanjing, China. Carapace width and length were measured in millimeters. The terminology used here follows Guinot et al. (2013). The following abbreviations are used: G1 for male first gonopod, G2 for male second gonopod, a.s.l. for above sea level. Molecular data. Genomic DNA was extracted from gill tissue using the the Trelief TM Animal Genomic DNA kit (Tsingke). 16S rDNA sequence was selected for amplification with polymerase chain reaction (PCR) using the primers 1471 and 1472 (Crandall and Fitzpatrick 1996). Parameters for PCR were as follows: initial denaturation at 95 °C for 3 min, followed by 35 cycles of 15 sec at 95 °C, 15 sec at 48 °C, 45 sec at 72 °C, and a subsequent 7 min final extension step at 72 °C. Both ends of PCR products were then sequenced using an ABI 3730 automatic sequencer. Sequences were assembled using SEQMAN II 5.05. Sequences of different haplotypes have been deposited in the Genbank (accession numbers listed in Table 1). To confirm the systematic position of newly described taxa, a total of 64 sequences were used in phylogenetic analyses, including 56 downloaded sequences (Table 1).
Phylogenetic analyses. Sequences were aligned using MAFFT 7.310 (Katoh and Standley 2013) based on the G-INS-I method. Gapped positions were treated as missing data. Maximum likelihood (ML) analysis for the dataset was performed using IQ-TREE 1.6.12 (Nguyen et al. 2015). The best substitution model was determined by ModelFinder (Kalyaanamoorthy et al. 2017). Node reliability was obtained through 1000 ultrafast bootstrap replicates (Minh et al. 2013). For Bayesian inference (BI), the best-fitting model was determined by MrModeltest 2.4 (Nylander 2004), selected by the Akaike information criterion (AIC). The best model obtained was GTR+I+G. Bayesian inference was performed using MRBAYES 3.2.6 (Ronquist et al. 2012) with four chains for 20 million generations, with trees sampled every 5000 generations.  The first 25% of MCMC chains were discarded as burn-in. The sampled parameters and convergence of four MCMC chains were investigated using TRACER 1.6 (Rambaut et al. 2014). The effective sampling sizes for all parameters were more than 200. Bootstrap support (BS) and Bayesian posterior probability (BPP) were used to assess statistical support. Diagnosis. Carapace slightly broader than long, dorsal surface strongly convex, densely pitted ( Fig. 2A). Third maxilliped exopod reaching proximal 1/3 of merus length, with long flagellum (Fig. 3A). Male pleon triangular, lateral margin almost straight (Fig. 2C), G1 subterminal segment broad, terminal segment relatively short, clearly sinuous, inferior margin of terminal segment straighter than superior margin, tip of terminal segment gradually tapering to a sharp tip (Fig. 3F), subterminal segment about 3.3 times as long as terminal segment (Fig. 3B, C). G1 strongly curved outwards, not reaching pleonal locking mechanism in situ ( Fig. 3E). Female pleon ovate (Fig. 4A), vulvae on suture between thoracic sternites 5/6, ovate, opening inner upwards, vulvar cover margin slightly arched (Fig. 4B).
Ambulatory legs relatively stout, dactylus slender with spine-like setae ( Fig. 2A); second ambulatory leg merus about 1.3 times as long as dactylus; last leg with propodus about 1.7 times as long as broad, slightly shorter than dactylus ( Fig. 2A).
Male pleon triangular, third somite widest; sixth somite about 2.2 times broader than long; telson triangular, with about 1.3 times as broad as long; the lateral margin of pleon almost straight (Fig. 2C); sterno-pleonal cavity reaching anteriorly to level of mid-length of cheliped coxae bases, broad, deep, median longitudinal groove between sternites 7, 8 long (Fig. 3E). Female pleon ovate, surface pitted; sixth somite about 2.8 times as broad as long; telson semicircular, terminal gently protuberant, about 2.3 times as broad as long (Fig. 4A).
G1 stout, tip of terminal segment not reaching pleonal locking mechanism in situ ( Fig. 3E); subterminal segment stout, about 3.3 times as long as terminal segment (Fig. 3B, C); G1 terminal segment cone-shape, bent outwards, inferior margin of terminal segment straighter than superior margin, tip of G1 terminal segment gradually tapering to sharp tip (Fig. 3F). G2 slightly longer than G1, basal segment about 2.1 times as long as distal segment (Fig. 3D). Female vulvae on suture between thoracic sternites 5/6, ovate, opening inwards towards the median of the cavity, vulvar cover slightly arched (Fig. 4B).  Live coloration. Carapace is usually dark brown, while chelipeds and ambulatory legs are usually light brown in life.
Etymology. The species is named after the late Prof. Aiyun Dai, who made a huge contribution to freshwater crab studies in China during her lifetime.
Remarks. Eosamon daiae sp. nov. can be distinguished from other Eosamon species by the combination of male abdomen with straight lateral margins, relatively broad G1 subterminal segment, conical and straight G1 terminal segment, the superior margin of G1 terminal segment curved and the inferior margin of G1 terminal segment comparatively straight.
Eosamon daiae sp. nov. is morphologically and geographically closest to E. tumidum (Wood-Mason, 1871), E. tengchongense (Dai & Chen, 1985) and E. lushuiense (Dai & Chen, 1985). These species are characterized by a male abdomen with straight lateral margins and superficially similar G1 structure (Fig. 5). But Eosamon daiae sp. nov. can be distinguished by the fact that the superior margin of G1 terminal segment is curved and the inferior margin is comparatively straight (Fig. 3F, 5A) (versus superior margin comparatively straight and inferior margin slightly curved in both E. tumidum and E. lushuiense, Fig. 5B, C; outer and inner margins all comparatively curved in E. tengchongense, Fig. 5D); the distal part of G1 subterminal segment slightly sunken (Fig. 5A) (versus barely sunken in E. tumidum, Fig. 5B, prominently sunken in E. tengchongense, Fig. 5D). Other characters as shown in Table 2.
Ischium of third maxilliped elongate rectangular, about 1.3 times longer than broad, with distinct, longitudinal median sulcus; merus trapezoidal, about 1.1 times broader than long; exopod reaching beyond base of merus slightly, with short flagellum, about half the width of the merus (Fig. 8A).
Ambulatory legs relatively slender, dactylus slender, with spine-like setae (Fig. 7A); second ambulatory leg merus about 1.8 times as long as dactylus; last leg with propodus about 2.7 times as long as broad, slightly shorter than dactylus (Fig. 7A).
Live coloration. The crabs usually have two colors: brownish-red (Fig. 11A) and yellowish-cyan (Fig. 11B). From the type locality, Tianbao Town, both brownish-red and yellowish-cyan crabs have been found, while from Babu Town, only yellowishcyan crabs have been found. Morphologically, there is no distinct difference between individuals with different colors. Similar color variation also can be seen in another potamid crab, Geothelphusa pingtung Tan & Liu, 1998(Shy et al. 2019.
Etymology. This species is named after the type locality, Malipo County, Yunnan Province, China.
All Indochinamon species have a well-developed flagellum on the exopod of the third maxilliped. The length of the flagellum varies among species. In some species, the flagellum does not exceed the width of the merus, e.g., I. tannanti, I. changpoense, I. gengmaense (Dai, 1995), I. guttus (Yeo & Ng, 1998), I. hispidum (Wood-Mason, 1871), I. jinpingense, I. mieni (Dang, 1967 and I. yunlongense. In I. malipoense sp. nov., the flagellum is about half the width of the merus, which is shorter than that in other species. The G1 of I. malipoense sp. nov. is very similar to I. tannanti, I. changpoense, I. ahkense, and I. daweishanense. They are also geographically close. But I. malipoense sp. nov. can be distinguished from the similar I. tannanti and I. changpoense by several characters (Table 3) . 85) carapace gently convex, regions indistinctly defined (Fig. 7A) flat, regions distinctly defined gently convex, regions distinctly defined G1 terminal segment obviously curved, unciform ( Fig. 10A) slightly curved, conical, with short, conspicious setae,tip tapering ( Fig. 10B) slightly curved, conical, with few very short setae, dorsal lobe of pleopod opening visible (Fig. 10C) base of G1 terminal segment slightly inflated (Fig. 10A) nearly straight (Fig. 10B) nearly straight (Fig. 10C (versus distinctly defined in I. tannanti and I. changpoense (Dai 1999)), the G1 terminal segment is obviously curved, unciform (Fig. 10A) (versus slightly curved, conical in both I. tannanti and I. changpoense, Fig. 10B, C)), the base of the G1 terminal segment is slightly inflated (Fig. 8F) (versus nearly straight in both I. tannanti and I. changpoense, Fig. 10B, C). The G1 structure of I. malipoense sp. nov. is also similar to I. ahkense (Naruse et al. 2018: fig. 4) and I. daweishanense (Dai 1999: fig. 87) by relatively slender terminal segment. However, the G1 terminal segment is more curved in I. malipoense sp. nov. and stronger bent outward in I. daweishanense. The carapace of I. malipoense sp. nov. is superficially similar to I. ahkense by smooth and shallow grooves of the dorsal surface. In I. ahkense, the carapace is subquadrate (versus subtrapezoidal in I. malipoense sp. nov.) and flatter (versus slightly convex in I. malipoense sp. nov.).
In I. khinpyae, the carapace and G1 show considerable variations (Ng and Mar 2018). In smaller individuals, the carapace is less sculptured and the G1 terminal segment is shorter and straighter (Ng and Mar 2018). In I. malipoense sp. nov., the morphology of the carapace is relatively stable while the ratio of G1 subterminal segment to terminal segment varies in sampled individuals.

Molecular results
In the present phylogenetic analyses, 60 species from 48 genera were included (Table 1). Phylogenetic trees reconstructed using BI and ML resulted in similar topologies. The phylogenetic trees indicate that two new species were placed in the 'Indochina -SW China' clade (Shih et al. 2009) with strong support (Fig. 12). Eosamon daiae sp. nov. clusters with E. tengchongense and E. lushuiense and Indochinamon malipoense sp. nov. clusters with I. tannanti (Fig. 12).

Discussion
The two new species cluster with several congeneric taxa (but not all), which tentatively supports recognition of the two genera, Eosamon and Indochinamon, following the systematic revision of . However, based on our molecular analyses, Eosamon and Indochinamon are not monophyletic (Fig. 12). Eosamon boonyaratae (Naiyanetr, 1987), E. smithianum (Kemp, 1923) and E. yotdomense (Naiyanetr, 1984) were placed in the 'Indochina' clade instead of the 'Indochina -SW China' clade, suggesting a polyphyletic topological structure for the current composition of Eosamon sampled to date. Morphologically, some characters, e.g., carapace dorsally convex and male pleon with straight lateral margins, in E. daiae sp. nov., E. tumidum, E. lushuiense and E. tengchongense, distributed in China, also differ from the description of Eosamon that was proposed based on specimens of the species distributed in Thailand, Laos and Vietnam . Several relatives, Potamiscus yiwuensis Dai & Cai, 1998, Pupamon nayung (Naiyanetr, 1993 and Beccumon jarujini (Ng & Naiyanetr, 1993), are nested within the Indochinamon clade suggesting that Indochinamon is paraphyletic (Fig. 12). Ng and Mar (2018) separated Indochinamon into several groups on the basis of their G1 structures. Although only few Indochinamon species were included, our molecular results indicate that their classification is still problematic. Indochinamon tannanti (Rathbun, 1904) is genetically closer to Beccumon Yeo &Pupamon Yeo &, rather than I. ou (Yeo & Ng, 1998). Due to the lack of taxa and sampling of molecular markers, we could not delve deeper into these questions in the present study. Further studies are needed to clarify the systematic treatments of Eosamon and Indochinamon.
Eosamon daiae sp. nov. and Indochinamon malipoense sp. nov. are not threatened by human activity. Eosamon daiae sp. nov. is distributed in the vicinity of the Tongbiguan Nature Reserve and Indochinamon malipoense sp. nov. is distributed in the vicinity of the Laoshan Nature Reserve. In these areas, large-scale developments are strictly regulated.
Yunnan is a global biodiversity hotspot (Myers et al. 2000), and also an important center for global biodiversity and endemism of primary freshwater crabs (Cumberlidge et al. 2011). Generations of scientists have done plenty of species discovery of freshwater crabs in this area (reviewed by Dai 1999;Chu et al. 2018b). However, investigations of freshwater crabs on the Sino-Burmese border, Sino-Vietnamese border and Sino-Lao border have rarely been carried out, because of the proximity of the 'Golden Triangle'. With constant efforts by the governments, conducting field surveys in these areas became possible. Many species have been newly described (e.g., Yu et al. 2019;Zhao et al. 2019;Lin and Li 2020;Zhang et al. 2020). In addition, some old type localities of freshwater crabs from Myanmar, e.g., Indochinamon andersonianum (Wood-Mason, 1871), I. edwardsii (Wood-Mason, 1871) and I. hispidum (Wood-Mason, 1871), are within Yunnan Province, China nowadays due to changes of national boundaries over one hundred years ago (Ng and Mar 2018). To fully understand the biodiversity of freshwater crabs in Yunnan, further investigations are expected in the poorly sampled frontier zones of China. This work was supported by the National Natural Science Foundation of China (No. 31772427) and Ocean Park Conservation Foundation, Hong Kong (No. OT02.1920) to SHY. This work was also supported by Biodiversity Survey Observation and Assessment Program (2019-2023) of the Ministry of Ecology and Environment of China.