A new species of freshwater crab (Decapoda: Brachyura: Potamidae) from Dongyin Island, Matsu, Taiwan, defined by morphological and molecular characters, with notes on its biogeography

A new species of freshwater crab, Nanhaipotamon dongyinense sp. nov. (Decapoda: Brachyura: Potamidae), is described from Dongyin Island, Matsu, Taiwan. Based on morphological and molecular (mitochondrial 16S rRNA) evidence, it can be distinguished from its congeners. The biogeography of the species, found on an island 50 km from mainland China, is also discussed.

(Jhongjhu Bank) and the highest point is 174 m above sea level (Enaishan). Recently, several specimens of the genus Nanhaipotamon were collected in this island with the local name ''shalulu '' or ''halulu'' (''mountain crab''). Based on morphology and molecular data (mitochondrial 16S rRNA), these specimens are clearly distinct from other known species of Nanhaipotamon, and are therefore here described as a new species. The presence of this species on an isolated island 50 km from mainland China is also discussed in relation to past glaciation events.

Materials and methods
Specimens were collected from irrigation ditches beside vegetable gardens, Yansiouwo, Dongyin, Matsu, Taiwan (Figure 1). After collection, specimens were preserved in 75-95% ethanol. Specimens examined are deposited in the National Museum of Natural Science, Taichung, Taiwan (NMNS), Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS), and the Zoological Reference Collection, Raffles Museum, National University of Singapore, Singapore (ZRC).
Genomic DNA was isolated from the muscle tissue of legs by a Sigma mammalian genomic DNA miniprep kit. A region of approximately 550 base pairs (bp) of the 59-end of the 16S rRNA gene was selected for amplification with polymerase chain reaction (PCR) using the primers 1471 (59-CCTGTTTANCAAAAACAT-39) and 1472 (59-AGATAGAAACCAACCTGG-39) (Crandall and Fitzpatrick 1996). The PCR conditions were 35-40 cycles for primers 1471 and 1472 were 50 s at 94uC, 70 s at 45uC, and 60 s at 72uC (denaturation, annealing, and extension), followed by 72uC extension for 10 min. Sequences were obtained by automated sequencing (ABI PRISM 377 Sequencer and MegaBACE DNA Analysis System 500) and were aligned with the aid of CLUSTAL W (version 1.4; Thompson et al. 1994) and BioEdit (version 5.09;Hall 2001), after verification with the complimentary strand. Sequences of the different haplotypes have been deposited in the DDBJ nucleotide sequence databases (accession nos AB212863 to AB212870).
The neighbour-joining (NJ) tree was established by the pairwise distance of nucleotide divergence constructed by the program TREECON for Windows (version 1.3b; Van de Peer and De Wachter 1997) with 2000 bootstrap replications. The distance estimation was based on Kimura's (1980) model with the transition/transversion ratio estimated from the data. A maximum parsimony (MP) tree was constructed using the program PAUP* (version 4.0b8; Swofford 2001), with 2000 bootstrap reiterations of a simple heuristic search, random sequence addition, branch-swapping5TBR (tree bisection-reconnection) and random-addition sequence replications5100. All characters were equally weighted. Gaps in the 16S rRNA alignment were treated as a fifth character state in NJ and MP tree construction (Kambhampati 1995;Miura et al. 2000;Tong et al. 2000).  Table I.

Family POTAMIDAE Ortmann, 1896
Nanhaipotamon Bott, 1968 Nanhaipotamon dongyinense sp. nov. ( Description. Carapace distinctly convex longitudinally, surface smooth, finely pitted. Branchial region very swollen, cervical groove wide and deep. H-shaped groove between gastric and cardiac regions deep. Postfrontal lobe prominent, with large pits. Postorbital crest sharp, connected with epibranchial tooth. Front deflexed, anterior border emarginated medially, dorsal orbital border ridged. Exorbital angle triangular, outer border arched. Epibranchial teeth squarish, antero-lateral border carinated, anterior part with indistinct granules, posterior part smooth. Third maxilliped (Figure 2A) with merus about 1.2 times as broad as long, with ischium about 1.5 times as long as broad, exopod reaching to proximal third of merus, with a short flagellum. a The numbers within square brackets correspond to the localities shown in Figure 1; b based on Shih et al. (2004).
Chelipeds strongly unequal, carpus with longitudinal depression on dorsal surface, with rugae on inner border, inner-distal angle with acute spine and spinule; larger manus about 1.3 times as long as high, slightly shorter than movable finger, with large gape when closed. Ambulatory legs slender, last leg with propodus about 2.4 times as long as broad, shorter than dactylus.
Male abdomen ( Figure 2B) triangular, sixth segment about 2.3 times as broad as long, telson about 1.2 times as broad as long. Median longitudinal groove of thoracic sternum  segment resembling an upside down boot in shape, its median line about 2.3 times as broad as long. Inner distal angle blunt triangular, distal margin arched inwards, outer distal angle produced, horn-like, distal end with pore. Male G2 (second pleopod) ( Figure 2F) with subdistal segment about 1.9 times as long as distal segment.
Female abdomen ( Figure 2G) oval, sixth segment about 3.1 times as broad as long, telson about 2.1 times as broad as long. Gonopore ( Figure 2H) with upper part widening gradually, like watermelon seed, opening inwards and downwards.
Coloration ( Figure 3A-D). Carapace is greenish gray and ambulatory legs are yellowish green. Female individuals tend to be more yellowish.
Habitat. The specimens were found in the irrigation ditches next to vegetable gardens ( Figure 3E). The water source is from the springs of this small island. No freshwater shrimps or freshwater snails were found. Brooding females could be found from April to June. A mating pair was found near the entrance within the burrow during July. Sometimes a wall of mud (hood or chimney) around the entrance can be observed ( Figure 3F), especially in habitats far from surface water.
Distribution. The largest population is located in Yansiouwo, eastern part of Dongyin. Some burrows could also be found in Bei-ao, north of Yansiouwo. In the western part of Dongyin (Siyin), local people stated that some crabs were seen in Hou-ao and Rendingshengtian (Figure 1), but none were obtained in the present study.

DNA analysis
A 548 base pair segment (excluding the primer regions) of the 16S mtDNA gene from five species of Nanhaipotamon was amplified and aligned. Out of those, 45 positions were variable and 10 were parsimony informative. Among the total number of sequences, eight different haplotypes were found (Table I). The segment of 16S sequences is AT-rich (72%) (T: 36.4%, A: 35.6%, G: 17.8%, C: 10.3%).
Among the populations of Nanhaipotamon formosanum, the difference in nucleotide number is within 2 bp. N. dongyinense differs from N. formosanum by 6-8 bp, and differs from N. nanriense by 10 bp. There is a 5-6 bp difference between N. nanriense and N. formosanum (Table III). The phylogenetic tree constructed by the Kimura two-parameter model of the NJ analysis, with the bootstrap values larger than 50% from MP analysis, is shown in Figure 4. Both NJ and MP methods support that N. formosanum from the populations of Taiwan form one clade, with N. nanriense forming the sister groups of N. formosanum.
In addition to the morphological difference (Table II), N. dongyinense sp. nov. also differs from congeners at the molecular level. Based on the tree topology (Figure 4), N. dongyinense sp. nov. forms a distinct clade with N. formosanum and N. nanriense. From Table III, there is at least 6 bp difference between N. dongyinense and N. formosanum. In comparison, Shih et al. (2004) have reported that there is no more than 5 bp nucleotide difference within the Geothelphusa tawu clade in southern Taiwan.
It is believed that N. dongyinense was originally distributed throughout Dongyin Island, but anthropogenic factors have affected several populations. The main reasons include the development of military building operations, overuse of pesticides and herbicides,  Table I. cemented trenches, and pollution from a winery. Without appropriate conservation, the endemic species may well be exterminated in the future.