A new cave amphipod, Pseudocrangonyx wonkimi sp. nov. (Crustacea, Amphipoda, Pseudocrangonyctidae), from the Korean Peninsula

Abstract A new species of pseudocrangonyctid amphipod, Pseudocrangonyx wonkimisp. nov., was found in the groundwater of a cave in the southwestern Korean Peninsula. Pseudocrangonyx wonkimisp. nov. is morphologically most closely related to P. joolaeiLee et al., 2020. However, P. wonkimi is clearly distinguished from P. joolaei by lacking sternal gills, fewer setae on maxilla 1 inner plate, fewer serrate robust setae on the carpus of the gnathopods, lacking bifid setae on the inner ramus of pleopod 3, and fewer articles of rami on pleopod 3. We also determined sequences of mitochondrial cytochrome c oxidase subunit I (COI) of P. wonkimisp. nov. for molecular diagnosis. From the molecular analysis based on COI sequences, P. wonkimi showed the closest relationship with P. joolaei with 15.1% genetic distance.


Introduction
The genus Pseudocrangonyx Akatsuka & Komai, 1922 is one of the stygobitic groups of groundwater environments in Eastern Asia (Holsinger 1994). Species of the genus Pseudocrangonyx are known from subterranean waters and springs in the Korean Peninsula, Japan, Eastern China, and the Far East of Russia (Sidorov and Holsinger 2007;Tomikawa et al. 2016;Zhao and Hou 2017). So far, the genus contains 27 species (Lee et al. 2020), four of which have been recorded in the Korean Peninsula: P. asiaticus Uéno, 1934;P. coreanus Uéno, 1966; P. daejeonensis Lee et al., 2018;and P. joolaei Lee et al., 2020. Although only four species have been recorded in Korea, it is possible that the specific diversity of the genus Pseudocrangonyx in the Korean Peninsula may have been underestimated. This is because there are about 1,000 natural caves in South Korea (Kim et al. 2004), many of which are known to be inhabited by unidentified species of Pseudocrangonyx. In addition, as mentioned in previous studies (Uéno 1966;Lee et al. 2020), P. asiaticus, which is distributed in various regions of Korea, includes cryptic species.
Recently, we found an unidentified species of Pseudocrangonyx collected from a cave in the southwestern part of the Korean Peninsula. Based on the results of the morphological examination of these specimens, we herein describe and illustrate them as representing a new species. These specimens were also confirmed to represent a distinct new species through molecular analysis using the mitochondrial cytochrome c oxidase subunit I (COI) gene.

Sample collection and morphological examination
Pseudocrangonyx specimens were collected from the groundwater of Jungchangjin Cave, Yongseong-ri, Daedong-myeon, Hampyeong-gun, Jeollanam-do, South Korea ( Fig. 1), using a fine-meshed hand net. A small pool where specimens were collected is 10 m from the entrance to the cave. Specimens were fixed and preserved in 99% ethanol. All appendages of the specimens were dissected in 80% ethanol and mounted in gum-chloral medium on glass slides under a stereomicroscope (Olympus SZX7). The specimens were examined using a light microscope (Nikon Eclipse Ni) and illustrated with the aid of a drawing tube. Body length (BL, to the nearest 0.1 mm) was measured from the tip of the rostrum to the base of the telson, along the curvature of the dorsal surface. The nomenclature of the setal patterns on the mandibular palp follows Stock (1974). The specimens examined in this study have been deposited in the collection of the Nakdonggang National Institute of Biological Resources, South Korea (NNIBR).

Molecular analysis
Genomic DNA was extracted from the muscles of the appendages of two Korean Pseudocrangonyx specimens using LaboPass Tissue Mini (Cosmo GENETECH, Seoul, South Korea), according to the manufacturer's instructions. The primer sets used for polymerase chain reaction (PCR) followed Tomikawa et al. (2016). Molecular analyses were performed using the COI sequences aligned by Geneious 8.1.9 (Biomatters, Auckland, New Zealand). Phylogenetic tree was constructed using maximum likeli-hood (ML) and Bayesian inference (BI). ML analysis was performed using RAxML v. 8.2.10 (Stamatakis 2014) with the substitution model set as GTRCAT, immediately after nonparametric bootstrapping conducted with 1,000 replicates. The best fit-partitioning scheme for the ML analysis was identified with the Akaike information criterion using PartitionFinder v. 2.1.1 (Lanfear et al. 2017) with the "greedy" algorithm. BI and posterior probabilities were estimated using MrBayes v. 3.2.6 (Ronquist et al. 2012). Two independent runs of four Markov chains were conducted for one million generations, and the tree was sampled every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.7.1 (Rambaut et al. 2018). Paratypes: 1 male (NNIBRIV36158, BL = 8.3 mm), 1 female (NNIBRIV36157, BL = 8.9 mm), collection data same as for the holotype.
Uropod 1 (Fig. 6G) with basofacial seta on peduncle; inner ramus 0.7 times as long as peduncle, inner and outer margins with 3 and 2 robust setae, respectively, basal part with 4 slender setae; outer ramus 0.8 times as long as inner ramus, with 2 outer marginal robust setae. Uropod 2 (Fig. 6H) with inner ramus 1.1 times as long as peduncle, inner and outer margins each with 2 robust setae; outer ramus 0.7 times as long as inner ramus, with 2 outer marginal robust setae, respectively. Uropod 3 (Fig. 6I, J) with peduncle 0.3 times as long as outer ramus; inner ramus absent; outer ramus 2-articulate, proximal article with robust setae, terminal article 0.1 times as long as proximal article, with 3 distal setae.
Telson (Fig. 6K) length 1.57 times as long as wide, cleft for 25.0% of its length, each telson lobe apical with penicillate seta and 2 robust setae.
Telson (Fig. 8E) length 1.38 times as long as wide, cleft for 27.4% of its length. Distribution. Known only from the type locality. Etymology. The name of the new species is dedicated to Prof. Won Kim (Seoul National University, South Korea), who has significantly contributed to our knowledge of crustaceans in South Korea.
DNA sequences. Sequences of COI gene (MT316534 and MT316535) were determined from two specimens (NNIBRIV35119 and NNIBRIV36158).
Molecular analyses. The topologies of the BI and ML trees were identical (Fig.  9). Pseudocrangonyx wonkimi sp. nov. and P. joolaei Lee et al., 2020 showed the closest relationship.  Remarks. We revealed that Pseudocrangonyx wonkimi sp. nov. is most closely related to P. joolaei Lee et al., 2020 based on molecular analyses. The genetic distance between these two species was 15.1% for the COI gene, and this distance is larger than that between members of two distinct species among the other congeners examined. Pseudocrangonyx wonkimi sp. nov. is distinguished from P. joolaei in having the following features (features of P. joolaei in parentheses): 1) sternal gill absent (present), 2) maxilla 1 inner plate with 4 (6) plumose setae, 3) carpus of gnathopods 1 and 2 each with serrate robust seta (with 2-3) on the posterodistal corner, 4) inner basal margin of inner rami of pleopod 3 without (with) bifid seta, and 5) less than 10 (more than 10) articles on the rami of pleopods.