Neocaridinafonticulata, a new land-locked freshwater shrimp from Hengchun Peninsula, Taiwan (Decapoda, Caridea, Atyidae)

Abstract A new species of land-locked freshwater shrimp, Neocaridinafonticulatasp. n. (Atyidae), is described from Kenting, Hengchun Peninsula, Pingtung County, southern Taiwan. This new species can be distinguished from its congeners by rostrum structure, pereiopods, and male first and second pleopods. The molecular evidence of mitochondrial cytochrome oxidase subunit I (COI) also supports the establishment of a new species. This is the third endemic species of Neocaridina known from Taiwan.

A recent survey of the species diversity of freshwater shrimps of Taiwan showed an undescribed species from southern Taiwan with different morphological characters compared to other known species of Neocaridina, which was supported by molecular evidence. This species is herein described as a new species, endemic to Taiwan Island, which brings the total number of Taiwanese species of Neocaridina to four.

Materials and methods
Specimens of the genus Neocaridina examined in this study were collected from a spring in Sheding, Kenting, Hengchun Peninsula, Pingtung County, Taiwan and preserved in 70%-95% ethanol after collection. Some specimens were selected and illustrated with the help of a drawing tube attached to a Nikon stereo microscope (model SMZ 1000), and deposited in 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 (formerly the Raffles Museum of Biodiversity Research) (ZRC). Carapace length is abbreviated cl, and the mode refers to the most frequently number occurring. The rostral formula was counted based on all specimens available. The egg measurements were based on five eggs each from four ovigerous females (see material examined).
Sequences of mitochondrial cytochrome oxidase subunit I (COI) were obtained following the method described by Shih et al. (2017), with the primers LCO1490 and HCO2198 (Folmer et al. 1994). Sequences were obtained by automated sequencing (Applied Biosystems 3730xl DNA Analyzer), after verification with the complementary strand. Sequences obtained have been deposited in the DNA Data Bank of Japan (DDBJ) and were analyzed with other sequences published in Shih and Cai (2007) and Shih et al. (2017).
The best-fitting model for sequence evolution was determined by MrModeltest (version 2.2, Nylander 2005), selected by the Akaike information criterion (AIC). The obtained best model was HKY + G, and was subsequently used for the Bayesian inference (BI) analysis. The BI analysis was performed with MrBayes (version 3.2.3, Ronquist et al. 2012). The search was run 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. 2005) and the first 1150 trees were discarded as the burnin accordingly. The maximum likelihood (ML) analysis was conducted in RAxML (vers. 7.2.6, Stamatakis 2006). The model GTR + G (i.e., GTRGAMMA) was used with 100 runs, and found 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.
Sixth pleomere in male 0.43cl, 1.40 × as long as 5 th pleomere, slightly shorter than telson; 6 th pleomere in female 0.48cl, 1.38 × as long as 5 th pleomere, slightly shorter than telson. Telson 3.0 × as long as wide, with four or five pairs of dorsal spinules and one pair of dorsolateral spinules; posterior margin rounded, lined with four or five pairs of simple setae, lateral pair distinctly longer than intermediate pairs. Pre-anal carina moderately high, lacking spine.
Eyes well developed, anterior corneal margin reaching to 0.6 × length of basal segment of antennular peduncle. Antennular peduncle 0.6 × as long as carapace; basal segment of antennular peduncle longer than combined length of 2 nd and 3 rd segments, anterolateral angle reaching 0.3 length of 2 nd segment, 2 nd segment distinctly longer than 3 rd segment. Stylocerite reaching 0.7-0.8 length of basal segment of antennular peduncle. Scaphocerite 3.5 × as long as wide, with extension of the distolateral spine reaching end of antennular peduncle.
Mandible with incisor process ending in irregular teeth; molar process truncated. Maxillule lower lacinia broadly rounded; upper lacinia elongate, with a row of 30 distinct spiniform setae on inner margin; palp short. Maxilla distal endite subdivided; palp short; scaphognathite tapering posteriorly with some long, curved setae at posterior end. 1 st maxilliped with stout palp. 2 nd maxilliped typical of genus, endopod with fused dactylus and propodal segments. 3 rd maxilliped reaching to end of antennular peduncle, with ultimate segment slightly longer than penultimate segment.
Endopod of male 1 st pleopod extending to 0.8 × exopod length, inflated at distal ¾, pyriform , 1.7 × as long as wide, with tiny spinules on distal margin of dorsal surface, appendix interna at base of inflated part, short. Appendix masculina of male 2 nd pleopod cylindrical, reaching to about 0.7 length of endopod, inner and distal surface densely lined with long, stout spines, appendix interna reaching to 0.6 length of appendix masculina.

Colour in life.
Body colour varying from translucent to light blue, with darker red-brown spots on dorsal surface and lighter red-brown spots on lateral surface of carapace; pleon usually with several dark red-brown vertical stripes on lower lateral surface, and white star-shaped pigment scattered on whole body ( Figure 4A-D). Appendages mostly transparent.
Etymology. Neocaridina fonticulata is named after its known habitat, from the Latin root, fonticulus, for little spring.
Ecological notes. Specimens of the new species were collected from leaf litter layer of a small stream ( Figure 4E, F) next to a spring outlet at a limestone hill. The collection site consists of concretized substrate and banks, representing the headwater of the stream. The water flow is slow, cool temperature (about 25 °C), neutral (pH 7.06-7.16), and with moderately high dissolved oxygen (7.33-7.70 mg/L). The freshwater crabs, Candidiopotamon rathbuni (De Man, 1914) and Geothelphusa ferruginea Shy, Ng & Yu, 1994, were found to be sympatric with this new species. Ovigerous females were found in July.
Neocaridina fonticulata sp. n. can be separated from N. ketagalan (cf. Shih and Cai 2007) by its shorter rostrum (reaching from slightly short of to slightly beyond end of 1 st segment of antennular peduncle vs. reaching the middle or end of the 2 nd segment of the antennular peduncle; Figures 1A, 3A vs. figs 5A, 6A in Shih and Cai 2007). It also differs from N. ketagalan (cf. Shih and Cai 2007) by the slender male 1 st pleopod (1.7 × as long as broad vs. 1.4 × in N. ketagalan; Figure 2G vs. fig. 5J in Shih and Cai 2007); the male 2 nd pleopod appendix masculina being half the endopod length (vs. 0.7 × in N. ketagalan; Figure 2H vs. fig. 5K in Shih and Cai 2007); and the appendix interna of the male 2 nd pleopod being relatively longer, reaching to 0.7 × length of appendix masculine (vs. 0.6 × in N. ketagalan; Figure 2H vs. fig. 5K in Shih and Cai 2007).
With its relatively short rostrum, Neocaridina fonticulata sp. n. morphologically also resembles the recently described Japanese species Neocaridina ikiensis Shih, Cai, Niwa & Nakahara, 2017. It can be differentiated from the latter by its shorter rostrum (reaching from slightly short of to slightly beyond the end of the 1 st segment of antennular peduncle vs. reaching slightly short of to distinctly beyond the end of the 2 nd segment of antennular peduncle; cf. Figures 1A, 3A vs. figs 2A, 4A in Shih et al. 2017). The propodus and dactylus of the 3 rd pereiopod of the new species displays sexual dimorphism (vs. no sexual dimorphism in N. ikiensis); the male 2 nd pleopod appendix masculina is 0.7 × endopod length (vs. 0.5 × in N. ikiensis; Figure 2H vs. fig.  3I in Shih et al. 2017); and the appendix interna of the male 2 nd pleopod is relatively shorter (reaching to 0.6 length of appendix masculina vs. 0.8 in N. ikiensis; Figure 2H vs. fig. 3J in Shih et al. 2017).
With the relatively slender endopod of the male 1 st pleopod, the new species is similar to N. koreana Kubo, 1938. It can be separated from the latter by the relatively shorter rostrum, which mostly reaches to or slightly beyond the end of the 1 st segment of antennular peduncle vs. almost reaching to or slightly beyond antennular peduncle in N. koreana (cf. Kubo 1938); and the fewer ventral rostral teeth (2-6 teeth, mode 2-4 vs. 4-6, average 5.6 in N. koreana (cf. Kubo 1938)).

DNA analyses and discussion
A total of four specimens from Sheding, Kenting, were used in the molecular phylogenetic analysis. A 658-bp segment of COI was amplified, resulting in one haplotype (accession number LC427866). Based on the COI haplotypes, the phylogenetic tree was reconstructed using BI analysis, with the support values from the BI and ML analyses shown in Figure 5. Specimens assigned to Neocaridina fonticulata sp. n. formed a clade distinct from other species. The pairwise nucleotide divergences with the K2P distance and bp differences of haplotypes are shown in Table 1. The minimum K2P interspecific divergences between N. fonticulata sp. n. and N. ketagalan and N. saccam are 5.42% and 5.43% respectively, which are close to or larger than the values between species of N. davidi, N. denticulata, N. koreana, and N. palmata (from 2.17% to 5.56%; Table 1). Consequently, the establishment of the new taxon is warranted.
The discovery of the new species increases the number of Neocaridina species in Taiwan to four, i.e., N. davidi, N. saccam, N. ketagalan, andN. fonticulata sp. n. (Shih andCai 2007, Shih et al. 2017). While the common species, N. davidi, is distributed in both western and eastern sides of Taiwan Island, as well as the offshore islands, Penghu, Siaoliouciou and Kinmen Cai 2007, Shih et al. 2017;unpublished data), the distributional range of the other three endemic species is narrower. Neocaridina ketagalan is distributed in northern Taiwan, N. saccam is limited in southwestern Taiwan, and N. fonticulata sp. n. is presently known only from Kenting. Previous molecular studies on aquatic organisms, including the freshwater crab Candidiopotamon rathbuni (De Man, 1914) and frog Hylarana latouchii (Boulenger, 1899) (= Sylvirana latouchii)