The second known stygomorphic freshwater crab from China, Phasmon typhlops gen. nov. et sp. nov. (Crustacea, Decapoda, Potamidae), diverged at the beginning of the Late Miocene

Abstract A new genus and new species of blind freshwater cave crab are described from Chongzuo City, Guangxi Zhuang Autonomous Region, China based on morphology and mitochondrial 16S rDNA sequences. The new genus, Phasmongen. nov., is established for P. typhlopssp. nov., which is only the second blind cave crab known from China and East Asia. The combination of a very wide carapace, overall depigmentation, reduced orbits and vestigial unpigmented eyes of Phasmon immediately separates it from all known potamid genera. Molecular divergence estimates based on 16S rDNA suggest that the lineage to which the new genus belongs diverged from other potamids at the beginning of the Late Miocene (10.8 million years ago), much earlier than other Chinese cave crabs.

In 2018, a local collector from Chongzuo City, Guangxi, China, alerted us to an unusual crab trapped from a karst spring. This crab, a female, was apparently a stygobite, lacking body pigmentation and having vestigial, unpigmented eyes. Many further attempts were made to collect more specimens of this unusual species, but they remained elusive. The collector only succeeded once in trapping a second specimen from the karst spring, but only half of the carcass remained when he checked the trap; the other half was apparently consumed by a specimen of the epigean crab Lacunipotamon cymatile Huang, Shih & Ahyong, 2020(Huang et al. 2020c, which was also lured into the trap. It was not until over a year later that the collector discovered the primary habitat of the crab in a nearby cave system in which he successfully trapped more specimens. After we acquired and examined the specimens, it was obvious that these cave crabs were new to science: the combination of highly reduced and unpigmented eyes, long antennules, lack of body pigmentation, unique carapace features, and very wide male anterior thoracic sternum, immediately separate it from all other potamid genera. Molecular data derived from the mitochondrial 16S rDNA gene further support the taxon as new, and we herein describe it as a new genus and new species.

Materials and methods
Specimens were collected by hand, preserved in 75% ethanol, and deposited in the collections of the Sun Yat-sen Museum of Biology, Sun Yat-sen University, Guangzhou, China (SYSBM) and the Australian Museum, Sydney, Australia (AM). The terminology used primarily follows that of Dai (1999) and Davie et al. (2015). Carapace length (CL) was measured along the dorsal midline and carapace width (CW) was the greatest width measured across the branchial margins. The male gonopods 1 and 2 are abbreviated as G1 and G2, respectively. Measurements (mm) are of the carapace width and length, respectively.
A 16S sequence was obtained from the paratype (AM P.105524) following Shih et al. (2009), using the primers 16H10 and 16L29 (Schubart 2009), and aligned with the MUSCLE function of MEGA (vers. 10.0.5; Kumar et al. 2018) after verification with the complimentary strand. The sequence was deposited in NCBI GenBank under the accession number MW289910. A preliminary analysis showed that this genus belongs to the "China-East Asia Islands" Group within the "Eastern-Asia Subclade" of the subfamily Potamiscinae (Shih et al. 2009). Therefore, to confirm the phylogenetic position of the new genus and species, 24 additional 16S sequences from related genera from East Asia, Indochina and Southeast Asia in Shih et al. (2009), Huang et al. (2014, 2017a, b, 2018, 2020d, and Wang et al. (2019) were included for comparison. The variable regions in the loop regions of 16S that could not be aligned adequately for phylogenetic analysis were excluded (Shih et al. 2009).
The best-fitting model for sequence evolution of 16S was determined by Parti-tionFinder (vers. 2.1.1; Lanfear et al. 2017), selected by the Bayesian information criterion (BIC). The best model obtained, GTR+I+G, was subsequently applied for Bayesian inference (BI) and maximum likelihood (ML) analyses. The BI analysis was performed with MrBayes (vers. 3.2.2; Ronquist et al. 2012) using four chains run for 10 million generations, with trees sampled every 1000 generations. The convergence of chains was determined by the average standard deviation of split frequency values below the recommended 0.01 (Ronquist et al. 2005) and the first 1050 trees were discarded as burn-in accordingly. The ML analysis was conducted in RAxML (vers. 7.2.6; Stamatakis 2006). The GTR + G (i.e., GTRGAMMA) model was used for all subsets with 100 runs and found the best ML tree by comparing likelihood scores. The robustness of the ML tree was evaluated by 1000 bootstrap pseudoreplicates under the model GTRGAMMA. The uncorrected p-distances for genetic divergence between haplotypes were calculated by MEGA. Etymology. The genus name is an arbitrary combination of the Latin word "phasma", meaning ghost, which refers to the type species' pale appearance and dark habitat, and the genus name Potamon, which is the type genus of the family. Gender neuter. Description. Carapace broad, about 1.6 times as wide as long; fronto-orbital width about twice width of posterior margin; regions indistinct, dorsal surface slightly convex; surface finely pitted (Fig. 1A). Frontal margin weakly sinuous, continuous  (Fig. 1A). Mesogastric region slightly convex (Fig. 1A). External orbital angle obsolete, outer margin convex, almost indistinguishable from anterolateral margin (Figs 1A, 2A). Epibranchial tooth granular, inconspicuous (Fig. 1A). Anterolateral margin lined with 15-20 small, single or partially fused granules. Posterolateral margin posteriorly convergent (Fig. 1A); posterolateral surface generally smooth (Fig. 1A). Orbits shallow; supraorbital margins weakly cristate, infraorbital margins lined with granules ( Fig. 2A). Eyes almost immobile, greatly reduced, tapering, length about half orbital width; peduncle short, stout; cornea vestigial, surface without facets, unpigmented (Figs 2A, 3F). Suborbital, pterygostomial and sub-hepatic regions generally smooth, pitted ( Fig. 2A). Antennules large, folded within broad fossae; antennae very short ( Fig. 2A). Median lobe of epistome posterior margin broadly triangular, lateral margins sinuous ( Fig. 2A).
Etymology. The species name is derived from the Greek words "typhlos" and "ops", meaning "blind" and "eyes", respectively. It refers to the greatly reduced and non-functional eyes of this species.

Colour in life.
Pale yellowish-white all over (Fig. 3F). Habitat. Phasmon typhlops gen. nov. et sp. nov. occurs in subterranean karst streams, but little is currently known about its precise habitat. According to the collector, subterranean streams in the dark zone of caves appear to be the primary habitat of P. typhlops sp. nov., where it has been found in shallow and still water as well as flowing streamways. However, some specimens have also been captured at night from a karstic spring that is immediately connected to the more extensive subterranean karst system. We only examined the two type specimens, of which the holotype was collected from the former habitat and the paratype from the latter. An epigean species, Lacunipotamon cymatile, inhabits the areas immediately adjacent to the spring and has been observed to prey on Phasmon typhlops gen. nov. et sp. nov. (Huang et al. 2020c).
Distribution. Chongzuo City, Guangxi Province, China. Remarks. Phasmon typhlops gen. nov. et sp. nov. can be considered a true stygobite owing to its stygomorphic features, in particular the strong reduction of the eyes, body depigmentation and slightly elongated appendages, which are consistent with its subterranean lifestyle (Holthuis 1986;Ng and Goh 1987). Apart from P. typhlops gen. nov. et sp. nov., Diyutamon cereum and Cerberusa caeca are the only other apparently blind stygomorphic potamid crabs known. We have not directly examined the eyes of C. caeca, but those of D. cereum and P. typhlops are unpigmented and the cornea is vestigial and without facets. Although we cannot exclude the possibility that the eyes of D. cereum and P. typhlops are capable of light detection, the absence of pigmentation or ommatidial facets indicates that the eyes are incapable of image formation. The enlarged antennules as present in Phasmon gen. nov. are otherwise seen in only a few cavernicolous freshwater crabs such as the gecarcinucids Sundathelphusa waray Husana, Naruse & Kase, 2009, and S. lobo Husana, Naruse & Kase, 2009(Husana et al. 2009, and are likely a sensory compensation for the loss of vision (Culver et al. 1995). Other than these two species, there are other stygomorphic gecarcinucids from Asia, but these can be separated from the new species by obvious family-level characters.
Sexual dimorphism is evident in our two specimens of P. typhlops: the smaller male holotype has proportionally longer but stouter legs in comparison to the larger female. The anterior carapace of the larger female is also proportionately wider than the posterior than in the male. Although the differences in leg proportions follow the pattern of sexual dimorphism observed in other potamids (e.g., Huang et al. 2020b), whether this carapace difference is due to size, sex or general variation remains to be determined.
Phasmon typhlops gen. nov. et sp. nov. is similar to Cerberusa caeca in general physiognomy and size. However, the new species can immediately be distinguished by its proportionally wider carapace (CW/CL=1.6 vs. 1. 3-1.4 in C. caeca;Holthuis 1979: pl. 8); almost indiscernible postorbital cristae (Fig. 1)  The G1 characteristics of Phasmon gen. nov. are rather unremarkable and particularly similar to those of Chinapotamon and Diyutamon. Chinapotamon is also found in Guangxi and includes two cavernicolous species, C. dashiwei Ng, 2017 andC. clarkei Ng, 2017, of which the latter displays evidence of stygomorphism in reduced body pigmentation and well-developed, albeit proportionally smaller eyes than epigean congeners (Ng 2017). Phasmon gen. nov. is readily distinguished from Chinapotamon in: the proportionally wider carapace (CW/CL=1.

DNA analyses and discussion
A 512-basepair segment of the 16S rDNA gene, excluding the variable regions, was amplified and aligned from 25 potamid genera. The phylogenetic tree of the 16S sequences reconstructed using BI analysis is shown with support values from ML analysis (Fig. 5). The phylogenetic results place Phasmon gen. nov., albeit with weak support, in a basal position of the "China-East Asia Islands" Group. Although the sister group to Phasmon could not be robustly determined, Phasmon gen. nov. is clearly phylogenetically distant from the stygobitic Diyutamon, indicating that the two lineages independently colonized subterranean habitats. Treating Phasmon gen. nov. as a basal clade and applying the substitution rate of 0.88% for 16S rDNA for terrestrial Sesarma and other freshwater crabs (Schubart et al. 1998;Shih et al. 2006Shih et al. , 2009Huang et al. 2017b), the divergence time between Phasmon gen. nov. and other genera in the "China-East Asia Islands" Group is estimated at 10.8 ± 1.0 mya (= million years ago) (with uncorrected p-distance of 9.49% ± 0.91%).
The divergence time of 10.8 mya estimated for Phasmon gen. nov. is much older than the 5.7 mya estimated for another Chinese cave crab genus, Diyutamon (Huang et al. 2017b). The karst caves of this region are estimated to have formed in the Pleistocene (2.58-0.0117 mya) based on animal and plant fossils (Mead et al. 2014;Yan et al. 2014;Li et al. 2020). If ancestral P. typhlops gen. nov. et sp. nov. entered subterranean karst caves at the beginning of the Late Miocene (11.63-5.333 mya), then previous age estimates of the regional karsts would be substantially too young. However, it seems also more likely that although the lineage to which Phasmon gen. nov. belongs diverged in the Late Miocene, the ancestors of P. typhlops gen. nov. et sp. nov. probably entered the subterranean environment after it was formed later in the Pleistocene. The present-day distribution of Phasmon gen. nov. is probably relictual, and given its isolated phylogenetic position in the "China-East Asia Islands" Group, P. typhlops possibly represents the last of an otherwise extinct lineage.