Review of the mudflat varunid crab genus Metaplax (Crustacea, Brachyura, Varunidae) from East Asia and northern Vietnam

Abstract Intertidal mudflat crabs of the genus Metaplax H. Milne Edwards, 1852 (Crustacea: Brachyura: Varunidae) from China, Taiwan, and northern Vietnam are taxonomically revised by morphological and molecular evidence. These crabs show sexual dimorphism and morphological variation of a considerable range in the infraorbital ridge, one of the primary features previously used for species identification. In this study, four species were identified from the region: M. elegans De Man, 1888; M. longipes Stimpson, 1858; M. sheni Gordon, 1930; and M. tredecim Tweedie, 1950. Based on the results of the morphological examination, and as confirmed by molecular evidence from mitochondrial cytochrome oxidase subunit I (COI), taxonomic confusion surrounding M. longipes was resolved, and M. takahasii Sakai, 1939, is considered a junior synonym of M. longipes. The geographical distribution of Metaplax longipes extends along the shores of China, north to Jiangsu, whereas the Southeast Asian M. tredecim was newly recorded from northern Vietnam and Hong Kong.


Introduction
Indo-West Pacific varunid crabs of the genus Metaplax H. Milne Edwards, 1852 commonly inhabit sheltered shores with silty muddy substrate often near or under shades of mangroves in tropical and subtropical regions. While some studies have reported on various biological aspects of selected species (e.g., Macnae 1963;Beinlich and Polivka 1989;Chakraborty and Choudhury 1994), the phylogenetic position of this group within the Thoracotremata remains obscure due to limited taxon sampling (see Kitaura et al. 2002;Chen et al. 2019;Liu et al. 2019).
Species of the genus Metaplax all share a broad, subquadrate, somewhat depressed carapace, which is shallowly marked, broad fronted (approximately 1/3 carapace width), and has lateral margins bearing at most five distinct teeth; slender and elongated ambulatory legs are also shared. One of the frequently used morphological features for species identification remains the number of lobes and tubercles along the infraorbital ridge (Tesch 1918;Tweedie 1950;Dai et al. 1986;Dai and Yang 1991), which are sexually dimorphic as in many varunid groups. The infraorbital tubercles are reported to serve a stridulatory function, which engage with the ridge along the anterior margin of the chelipedal merus (Macnae 1963;Beinlich and Polivka 1989). Metaplax contains around 12 species (Ng et al. 2008; but see Naderloo 2011 on the identity of M. indicus occidentalis Pretzmann, 1971), with an overall distribution extending from the shores of the Persian Gulf, the Arabian Sea, the Bay of Bengal to Southeast and East Asia, and easternmost to western Taiwan. Six species, namely M. elegans De Man, 1888; M. gocongensis Davie & Nguyen, 2003;M. longipes Stimpson, 1858;M. sheni Gordon, 1930;M. takahasii Sakai, 1939;and M. tredecim Tweedie, 1950, have been recorded from East and Southeast Asia, with only M. elegans recorded in the eastern Indian Ocean as well (De Man 1888;Ng and Davie 2002;Dev Roy and Bhadra 2011).
In East Asia, the northern limit of this genus appears to be around Zhejiang, China (Dai et al. 1986;Chen 1991;Dai and Yang 1991). Metaplax is absent from the east coast of Taiwan, the Ryukyus, the main islands of Japan, and Korea (Sakai 1939(Sakai , 1940(Sakai , 1976. Five species were previously reported from the region, including M. elegans De Man, 1888, M. longipes Stimpson, 1858 sheni Gordon, 1930, M. takahasii Sakai, 1939, and M. tredecim Tweedie, 1950. Among these, M. takahasii has been considered a junior synonym of M. longipes (see Davie and Nguyen 2003), whereas tropical M. tredecim had been first listed as part of the fauna of the East China and South China seas by Yang et al. (2008) without any illustration or elaboration.
In the present study, specimens of species of Metaplax were collected from various sites in East Asia (Fig. 1), their morphological features are examined and illustrated, and identifications are confirmed by molecular evidence from mitochondrial cytochrome oxidase subunit I (COI).

Materials and methods
Specimens were collected from China, Taiwan, and Vietnam (Table 1, Fig. 1) and have been deposited in the Kanagawa Prefectural Museum of Natural History, Kanagawa, Japan (KPM-NH); the Zoological Collections of the Department of Life Science, National Chung Hsing University, Taichung, Taiwan (NCHUZOOL), and the Zoological Reference Collection of the Lee Kong Chian Natural History Museum, National University of Singapore, Singapore (ZRC). The abbreviation G1 is used for male first gonopods. Measurement is of the maximum carapace width (CW) in millimeters.
To understand whether the number of infraorbital tubercles and lobes of each species is related to the sex and body size, the numbers on both sides for each specimen were calculated, averaged, and plotted against CWs. Specimens used were 21 males (CW 7.7-15.9 mm) and 19 females (CW 6.3-13.2 mm) for M. elegans, 69 males (CW 6.8-26.6 mm) and 29 females (CW 7.9-23.6 mm) for M. longipes, 7 males (CW 8.6-12.8 mm) for M. sheni (no female specimen), and 23 males  and 13 females (CW 10.3-23.4 mm) for M. tredecim.
Genomic DNA was isolated from the muscle tissue using extraction kits following Shih et al. (2016). A portion of the COI gene was amplified with PCR using the primers LCO1490 and HCO2198 (Folmer et al. 1994 Lanfear et al. 2017) and selected by the Bayesian information criterion (BIC). The obtained best model (GTR + I + G) was subsequently used for a Bayesian inference (BI) analysis. BI analysis was performed with MrBayes (ver. 3.2.3, Ronquist et al. 2012). Phylogenetic analysis was performed with four chains for 10 million generations and four independent runs, 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. 2019), and the first 1000 trees were discarded as the burnin accordingly. Maximum likelihood (ML) analysis was conducted using RAxML (vers. 7.2.6, Stamatakis 2006). The model GTR + G (i.e. GTRGAMMA) was used with 100 runs and finding the best ML tree by comparing the likelihood scores. The robustness of the ML tree was evaluated by 1000 bootstrap pseudoreplicates under the model GTRGAMMA. Base pair (bp) differences and pairwise estimates of Kimura 2-parameter (K2P) distances (Kimura 1980) for genetic diversities between specimens were calculated with MEGA (ver. 10.0.5, Kumar et al. 2018). .45 times as broad as long (N = 127, SD = 0.06), longitudinally convex, broadest between lateral teeth 2 (exorbital angle included), surface sparsely but regularly furnished with short, stiff setae; front broad, divided into two broad lobes, medially concave; lateral margins markedly converging posteriorly, interrupted by four notches, delineating five teeth (including exorbital angle), exorbital angle most distinct, posterior two indistinct; posterolateral facet faintly defined, decorated by two oblique granular ridges. Infraorbital ridge  Gordon, 1930 (G-I NCHUZOOL 15466, male, 9.9 mm), and M. tredecim Tweedie, 1950(J-L paratype, ZRC 1964  Habitat. At Wazihwei Wetland, estuaries of Danshuei River, northwestern Taiwan, this species is found along shores with substantial freshwater influence, on banks with substrates plastic-muddy, somewhat distant from mangrove stands.

Systematics
Remarks. According to previous descriptions (De Man 1888, Dai et al. 1986, Dai and Yang 1991, the infraorbital ridges of M. elegans bear 50-60 tubercles in males and 35-40 tubercles in females. In the present study of 21 males and 19 females, CW ranging from 7.7 to 15.9 mm, and the range of variation is slightly wider than previously reported, being 47-61 in males and 33-42 isomorphic tubercles in females (    (Figs 2D, 4A, E, F) subquadrate, 1.36 times broader than long (N = 98, SD = 0.05), mildly convex longitudinally and laterally, region faintly defined; front medially slightly concave; lateral margin nearly parallel, interrupted by four notches (cutting into five teeth), anterior two lateral teeth pronounced, posterior two very indistinct; posterior facet depressed, decorated by two oblique granular ridges, anterior one extended from second notch. Infraorbital ridge (Figs 2E, 4C) marked sexually dimorphic: males with 7-13 lobes and tubercles, medial 2 broad, decreasing in breadth laterally, innermost four or five decreasing in size, lateral ones small, isomorphic; females with 16-22 small isomorphic tubercles. Chelipeds (Figs 2F, 4D) symmetrical, robust, palm 2.3 times as long as broad, length of palm approximately 1.3 times length of dactyl (N = 16), merus denticulate along anterior and posterior margins; chelae surface smooth, pollex and dactylus unarmed of pronounced molars along cutting edge. Ambulatory legs slender, elongated, meri unarmed along anterior margin, proximal half of meri, and propodi of all furnished with setal mats. G1 (Fig.  7E-H) elongated, relatively stout, almost straight.
Habitat. At Tung Chung Wetland, Hong Kong, where numerous specimens were collected, the habitat of this species is composed of muddy substrates and substantial freshwater influences. Considerable numbers flourish under fringes of mangrove stands, as well as the adjacent more open mudflats.
Remarks. The identity of Metaplax longipes had long remained unclear since the publication of M. takahasii Sakai, 1939. The confusion between the two nominal species was mainly caused by two crucial morphological features used for species identification: the number of tubercles and lobes along the male infraorbital ridge, and the number of teeth on the lateral margin of the carapace.
Interpretations of M. longipes by Dai et al. (1986) and Dai and Yang (1991) brought much confusion. These authors illustrated two forms of infraorbital ridges based on specimens from South China (Guangdong to Zhejiang), one bearing 17 lobes and tubercles, and the other bearing nine ( fig. 288(1) in Dai et al. 1986 andYang 1991). It appeared very likely that their material was composite (also see Remarks under M. tredecim). Anyhow, this "shift" in the number of tubercles was subsequently followed by various authors: "with 15-17 lobules and teeth" (Davie 1992) and "about 9-17 tubercles" (Lee and Leung 1999). Reflecting this confused situation, the dichotomous key to the Metaplax species provided by Davie (1992: 352), which differentiated "M. longipes" (15 to 17 lobules and teeth) from "M. takahasii" (9 teeth), was problematic. Following diagnoses given by Stimpson (1858Stimpson ( , 1907, and authors such as Gordon (1931) and Shen and Dai (1964), only those with around 9 lobes or tubercles, should be considered as the true M. longipes.
Without accessing any material of M. longipes from South China, Sakai (1939) described a similar form named M. takahasii based on one male specimen from Tansui (= Danshuei), northwestern Taiwan, after the naturalist and collector, Sadae Takahashi (or Sadae Takahasi in another translation). The species was subsequently reported elsewhere in China, including Guangdong and Fujian (Dai et al. 1986;Dai and Yang 1991) and Hong Kong (Davie 1992). Regarding the correct spelling of the species epithet, "takahasii" (original as in Sakai 1939), instead of "takahashii" as in Sakai (1976), should be maintained (ICZN 1999: Article 32.2).
Nevertheless, M. takahasii was described with an infraorbital ridge composed of 8 tubercles and the lateral margin of the carapace cut into five teeth. Considering the original descriptions of M. longipes and M. takahasii (Stimpson 1907 andSakai 1939, respectively), holotypes of the two (CW 15.5 mm and 14.2 mm, respectively) differ by the numbers of lateral carapace teeth (four vs. five) of the carapace and the infraorbital lobes and tubercles (seven vs. eight). The number of infraorbital tubercles of both forms overlap might be explained by variation between intraspecific individuals (see "Note on the number of infraorbital tubercles and lobes"; Fig. 8), whereas the posterior-most notch along the lateral margin, however, can be very indistinct and often obscured by a layer of sediment-laden setae and easily omitted (Davie and Nguyen 2003;see Remarks under M. tredecim below). This led Davie and Nguyen (2003: 384) to the view that M. longipes is "almost certain(ly) … a senior synonym of M. takahashii". In enumerating Chinese species of Metaplax, Yang et al. (2008: 803), probably following Davie and Nguyen's (2003) suggestion, listed M. takahasii as a junior synonym of M. longipes without further elaboration. In our material referred to M. longipes, the number of infraorbital tubercles and lobes varies from 7-13 for males and 14-22 in females (Table 2; Fig. 8) In the present study, we compared specimens from Hong Kong (identified as M. longipes) and various lots from Taiwan main island (originally labeled as M. takahasii: see Materials examined above) with morphological and molecular approaches. As noted by Davie and Nguyen (2003; also see above), the number of notches (hence teeth) on the lateral margin of the carapace is easily underestimated unless the surface is carefully denuded. This aspect is well-illustrated in the case of M. tredecim (as discussed below), and also between specimens of M. longipes from Hong Kong (Fig. 4A) and "M. takahasii" from Danshuei, Taiwan (Fig. 4E), the two being identical. Molecular analyses also support only one clade of specimens from various localities of China and Taiwan (Table 1; Fig. 9).  (Figs 2G, 5A, B) subquadrate, 1.45 times broader than long (N = 7, SD = 0.04), longitudinally convex, regions faintly defined; frontal margin sinuous, medially noticeably concave; lateral margin markedly converging posteriorly, furnished with a row of soft setae, interrupted by four notches, cutting into five teeth, anterior two marked, triangular, last two weak, indistinct. Infraorbital ridge (Figs 2H, 5D) of males with 16-20 lobes and tubercles, innermost tubercle more than twice as broad as the adjacent, followed by six broad tubercles, decreasing in size. Chelipeds (Figs 2I, 5E) of males subequal, markedly elongated, palm 2.8 times as long as broad, length of palm approximately 2 times longer than length of dactyl (N = 6), merus and palm subequal in length; both fingers about half-length of palm, deflexed; cutting edges of pollex with low, serrated lobe, dactylus bearing distinct triangular molar. Ambulatory legs slender, elongated, anterior margins of meri finely serrated; anterior margins of carpi and propodi line with thick tomentum. G1 (Fig. 7I-L) elongated, relatively stout almost straight.
Habitat. In Hong Kong, in comparison to M. longipes, M. tredecim tends to occur in habitats of coarser, grittier substrates, with less freshwater input, and frequently on open sandflats rather unsheltered by mangroves.
Remarks. Identification of the Metaplax tredecim had been confusing, particularly based on the number of teeth along the lateral margin of the carapace. Tweedie (1950: fig. 6) showed merely three conspicuous lobes, the posterior one occupying more than half of carapace length. However, as noted by Davie and Nguyen (2003), members of the genus often have the structures around the posterolateral facet obscured by setae-trapped sediments, and not visible unless carefully denuded. Reexamination of a paratype male (16.2 mm;ZRC 1964.7.14.4-18), after denudation, showed the lateral margin to be interrupted by 4 notches (hence 5 teeth) (Figs 2J, 6A, E), the posterior two being inconspicuously defined by the last notch.  Gordon, 1930 (I-L NCHUZOOL 15466, male, 9.9 mm), right G1; and M. tredecim Tweedie, 1950(M-P paratype, ZRC 1964.2 mm), left G1. Scale bars: 0.5 mm (A-H, M-P); 0.2 mm (I-L).
As mentioned above, two forms, differing in the number of tubercles or lobes on the infraorbital ridge, were recognized in specimens identified with M. longipes by Dai et al. (1986) and Dai and Yang (1991). We confirmed that specimens from the study area, characterized by the possession of about 17 tubercles or lobes closely represent M. tredecim. There is little doubt that the material studied by Dai et al. (1986) and Dai and Yang (1991) included two species, M. longipes and M. tredecim. Chertoprud et al. (2012) recorded "M. longipes" from Nha Phu, southeastern Vietnam. However, given the infraorbital ridge with 17 tubercles and the chelipeds with the length of palm/the length of dactyl ratio about 1.5 (estimated from their plate 47F on page 295), this record is suspected to represent M. tredecim instead (see Table 2).

Note on the number of infraorbital tubercles and lobes
As one of the major morphological features for the identification of species of Metaplax species, the number of lobes and granules along both infraorbital ridges, which are in all cases sexually dimorphic, differs substantially among species. The following range indicate the number of these lobes and tubercles of both sexes (with the exception of M. sheni for which only males were collected), with differences between left and right ridges placed in brackets: in M. elegans 46-61 (0-5) for males and 33-42 (0-3) for females, M. longipes 7-13 (0-2) for males and 14-22 (0-2) for females, M. sheni 16-20 (0-2) for males, and M. tredecim 13-20 (0-3) for males and 20-27 (0-3) for females (Table 2 (Table 2; Fig. 8).

Molecular analyses
The molecular analysis of the COI marker included 22 specimens of Metaplax, with 13 haplotypes (Table 1). The phylogenetic reconstruction (Fig. 9) shows four wellsupported clades, which could correspond to the four species treated in this study. It is obvious that only one clade is represented by specimens of M. longipes from South China and specimens from the type locality of M. takahasii (Danshuei, Taiwan). Metaplax longipes and M. tredecim are in sister-relation, and the two species and M. sheni form a main clade. Metaplax elegans is distant from other species of Metaplax.

Discussion
In this study, based on morphological and molecular evidences, we resolve the taxonomic confusions and updated the distribution of Metaplax species from East Asia and northern Vietnam. The presence of four species, viz., M. elegans, M. longipes, M. sheni, and M. tredecim are confirmed, and it is verified that M. takahasii is conspecific with M. longipes, and thus synonymized.
With regard to the number of infraorbital tubercles and lobes, despite elaborate sexual dimorphism among varunid species, serve as a reliable morphological feature in identifying Metaplax species (cf. Table 2), whereas the numbers of M. elegans substantially exceed those of congeners (Fig. 8). The numbers of infraorbital tubercles of the Helice/ Chasmagnathus complex (Varunidae) are also used for species identification (K. Sakai et al. 2006), and likewise for species of Helicana K. Sakai & Yatsuzuka, 1980, all supported by genetic evidences. However, three species belong to the "Helice latimera complex" under Helice De Haan, 1833, with varying ranges of tubercle count, were shown to be otherwise (Shih and Suzuki 2008;Ng et al. 2018). The latter case implied H. latimera Parisi, 1918;H. formosensis Rathbun, 1931;and H. tientsinensis Rathbun, 1931 might well belong to a single species, as discussed in Ng et al. (2018). This ambiguity of specific delimitation requires further morphological and developmental investigations.
Phylogenetic relationships in the genus Metaplax or among genera of the Varunidae are far from settled. Monophyly of Metaplax has not yet been confirmed. Moreover, despite various recent research effort employing even complete mitochondrial sequences, the sister group of Metaplax remains unclear (Kitaura et al. 2002;Chen et al. 2019;, which is probably due to the limited genera sampled in phylogenetic analyses. In our study, the four species of Metaplax can be separated by the COI marker with a minimum interspecific distance of 17.5 %, which is higher than that of most other crab species (see Chu et al. 2015). The phylogenetic tree based on COI (Fig. 9) showed M. longipes and M. tredecim as sister species, and both species form a clade that is sister to M. sheni, whereas M. elegans is sister to the three as a whole. The phylogenetic relationships of the four species are also consistent with the number of infraorbital tubercles and lobes, i.e., M. longipes and M. tredecim, have the fewest number of these structures; M. sheni has moderate number; and M. elegans has the greatest number (Table 2; Fig. 8). This implies the number of infraorbital tubercles is possibly higher in Table 3. Matrix of percentage pairwise nucleotide divergence with K2P distance (lower left) and mean number of differences (upper right) based on COI within and between species of Metaplax from East Asia and northern Vietnam (see Table 1). Values of range are shown in parentheses. the ancestral form, becoming reduced in successive clades. More species of this genus, however, should be included in the future to test this hypothesis. Species of Metaplax are mainly distributed in the tropical and subtropical continental regions, in muddy and muddy sand habitats, always accompanied by mangroves (Sakai 1939(Sakai , 1976Dai et al. 1986;Dai and Yang 1991). It has been suggested that the pattern of geographical distributions agrees with the "continental type" of fiddler crabs, in contrast with the "oceanic type" mainly inhabited on islands (cf. Shih et al. 2010Shih et al. , 2016Shih 2012). This also explains why no East Asian species of Metaplax are recorded from Korea, the main islands of Japan, the Ryukyu islands, and eastern Taiwan. The habitat preferred by species of Metaplax is suggested to be related to physiological constraints (e.g., food, temperature, salinity, etc.; Curtis and McGaw 2012;Theuerkauff et al. 2018). Understanding of the population structure may help reveal larval dispersal in the region of East Asia and northern South China Sea (Chan et al. 2007;Shih et al. 2015;Wang et al. 2015;Chai et al. 2017).
The results of this study clarify the biogeographic distribution of three species (Fig. 1). With M. takahasii synonymized with M. longipes, the distribution of M. longipes stretches from western Taiwan (including Matsu and Kinmen) to China (from Jiangsu to Guangxi) and northern Vietnam. Metaplax sheni is found in China (Zhejiang and Fujian), Taiwan (Kinmen), Vietnam, and Malay Peninsula (including Singapore); and the known range of M. tredecim include South China (Hainan and Hong Kong), Vietnam, and northern Borneo.