Three new species of subterranean amphipods (Pseudocrangonyctidae: Pseudocrangonyx) from limestone caves in South Korea

Pseudocrangonyx Akatsuka & Komai, 1922 is the most diverse group of subterranean amphipods in the groundwater communities of Far East Asia. In Korea, the diversity of the group has been underestimated due to the records of morphological variants of Pseudocrangonyx asiaticus Uéno, 1934. To estimate the species diversity, we analyzed the morphological characteristics and conducted molecular analyses of specimens collected from Korean caves that we treated as morphological variants of P. asiaticus. We described three new subterranean pseudocrangonyctid amphipod species, P. deureunensis sp. nov., P. kwangcheonseonensis sp. nov., and P. hwanseonensis sp. nov., from the groundwater of limestone caves in South Korea. Additionally, we determined sequences of the nuclear large subunit ribosomal RNA and the mitochondrial cytochrome c oxidase subunit I gene of the new species for molecular analyses. Molecular phylogenetic analyses revealed that the three new species formed a monophylum together with P. joolaei Lee et al., 2020 and P. wonkimi Lee, Tomikawa & Min, 2020, which are species that are endemic to Korean caves.


INTRODUCTION
Amphipods are the most diverse group of organisms in groundwater communities (Holsinger, 1994), and subterranean amphipods are even more notable from a biogeographic perspective because of their limited dispersal ability and restriction to groundwater aquifers (Holsinger, 1993). Most subterranean amphipods are troglobiont (stygobiont) and are generally characterized by morphological features such as appendage elongation and the loss of eyes and pigment (Holsinger, 1994;Väinölä et al., 2008). These characteristics result in the strikingly convergent morphology of these cave animals (Jones, Culver & Kane, 1992). Classifying subterranean organisms solely on their morphological characteristics leads to several taxonomic problems (Lefébure et al., 2006;Kornobis et al., 2011). Because subterranean and cave amphipod species are particularly difficult to morphologically identify, molecular analyses help in species delimitation (Lefébure et al., 2006;Trontelj et al., 2009;Hou & Li, 2010).
The stygobitic amphipod genus Pseudocrangonyx Akatsuka & Komai, 1922 is the most diverse taxon among the subterranean amphipod genera found in Far East Asia, i.e., the Korean Peninsula, the Japanese Archipelago, eastern China, and the Russian Far East (Sidorov & Holsinger, 2007;Tomikawa & Nakano, 2018). The first record of P. asiaticus Uéno, 1934 on the Korean Peninsula was from North Korea (Uéno, 1940). However, P. asiaticus's type locality is on China's Liaodong Peninsula (Uéno, 1934). Using identification techniques based on morphological characteristics, this species has been found to inhabit several caves in South Korea (Uéno, 1966;Holsinger, 1989). Uéno (1966) mentioned regional morphological variants of the Korean populations, but did not regard them as a distinct species. At that time, there were obvious limitations to correctly identifying subterranean amphipods based solely on morphological characteristics. Recent studies have used molecular analyses and morphological identification to show that the genus' species diversity may be higher than previously believed (Tomikawa et al., 2016;Tomikawa & Nakano, 2018;Lee et al., 2020).
While conducting cave surveys on the Korean Peninsula, we collected Pseudocrangonyx specimens from two caves (Kwangcheonseon Cave and Hwanseon Cave) where Uéno (1966) reported finding one of the P. asiaticus Uéno, 1934 morphological variants. Additionally, specimens were also collected from Deureune Cave, where the genus Pseudocrangonyx had not been previously located. Based on the results of morphological examination of the amphipods, we described and illustrated them as three new Pseudocrangonyx species. Furthermore, we determined the nuclear large subunit ribosomal RNA (28S rRNA) gene and mitochondrial cytochrome c oxidase subunit I (COI) gene sequence data for molecular analyses of the three new species. Additionally, we provided a key to the Korean Pseudocrangonyx species.

Sample collection and morphological examination
Pseudocrangonyx specimens were collected from the groundwater of three Korean caves: Deureune Cave (Fig. 1A), Kwangcheonseon Cave (Fig. 1B), and Hwanseon Cave (Fig. 1C). We fixed and preserved the specimens in 99% ethanol. All specimen appendages were dissected in 80% ethanol and mounted with gum-chloral medium on glass slides under an Olympus SZX7 stereomicroscope (Tokyo, Japan). The specimens were examined using a Nikon Eclipse Ni light microscope (Tokyo, Japan) and were illustrated with the aid of a drawing tube. The body length from the tip of the rostrum to the base of the telson was measured along the dorsal curvature to the nearest 0.1 mm. The nomenclature of the setal patterns on the mandibular palp followed the method described by Stock (1974). The specimens examined in this study have been deposited in the collection at the Nakdonggang National Institute of Biological Resources, Korea (NNIBR).

Molecular analyses
We extracted genomic DNA from the muscles of the specimen appendages using the LaboPass Tissue Mini Kit (Cosmo GENETECH, Seoul, South Korea), according to the manufacturer's instructions. We used the following primer sets for the PCR reaction used in this study: 28F and 28R for 28S rDNA (Hou, Fu & Li, 2007), and LCO1490 and HCO2198 for COI (Folmer et al., 1994). The sequences of 28S rDNA were aligned using MAFFT v. 7.388 L- INS-i (Katoh & Standley, 2013), and COI was aligned using Geneious 8.1.9 (Biomatters,Auckland,New Zealand). For phylogenetic analysis, these two alignments were combined. All data used in molecular analyses were provided, including the newly obtained sequences (Table 1). Pairwise comparisons of uncorrected p-distances for COI sequences were calculated using MEGA X (Kumar et al., 2018). Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). We performed ML analysis using RAxML v. 8.2.10 (Stamatakis, 2014) with the substitution model immediately set as GTRCAT after nonparametric bootstrapping was 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 10 million generations, and the tree was sampled at every 100 generations. Parameter estimates and convergence were checked using Tracer v. 1. 7.1 (Rambaut et al., 2018), and the first 50001 trees were discarded based on results.

Terminology
Pseudocrangonyx asiaticus sensu stricto refers to the species that was originally described by Uéno (1934).
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: [urn:lsid:zoobank.org:pub:A60F095A-2A50-4D87-876C-6D8E3D8539CE]. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.  gill; antenna 1 being 0.51 times as long as body length; antenna 2 with calceoli in both sexes; mandible palp article 3 longer than article 2; maxilla 1 outer plate with 7 serrate teeth; maxilla 2 inner plate with oblique inner row of 6 setae; gnathopods 1 and 2, carpi with serrate setae on posterodistal corners in both sexes; palmar margins of propodi of gnathopods 1 and 2 with 11-15 and 14-18 robust setae, respectively; pleopod peduncles lacking marginal setae, inner margin of inner rami with bifid setae; uropod 1 inner ramus 0.9 times as long as peduncle, inner and outer margins of inner ramus with 3 and 2 robust setae, respectively, basal part of inner ramus with 3 slender setae, outer ramus with 2 marginal robust setae; uropod 2 inner ramus 1.2 times as long as peduncle, outer ramus with 2 marginal robust setae; uropod 3 terminal article longer than adjacent robust setae; telson each lobe with 2 apical robust setae and 1 penicillate seta. Description. Female holotype (NNIBRIV39838). Head ( Fig. 3A) with short dorsal seta; rostrum short; lateral cephalic lobe rounded; antennal sinus with rounded angle; eyes absent. Pereonites 1-6 with short dorsal setae; dorsal margin of pereonite 7 with long setae. Dorsal margins of pleonites 1-3 with long setae (Fig. 3B). Posterior margin of epimeral plate 1 with 6 setae, posteroventral corner with seta; ventral and posterior margins of plate 2 with 3 and 4 setae, respectively, posteroventral corner with seta; ventral and posterior margins of plate 3 with 3 setae, respectively, posteroventral corner subquadrate with seta ( Fig. 3B). Dorsal margin of urosomites 1-3 with setae. Anteroventral corner of urosomite 1 with seta, posteroventral corner of urosomite 3 with setae ( Fig. 3B). Antenna 1 (Fig. 3C) 0.51 times as long as body length, peduncular articles 1-3 in length ratio of 1.0: 0.7: 0.4; accessory flagellum ( Fig. 3D) 2-articulate, more than shorter primary flagellar article 1, terminal article with 3 setae and 1 aesthetasc; primary flagellum 18-articulate, 1 aesthetasc on some articles. Antenna 2 (Figs. 3E, 3F) 0.59 times as long as antenna 1; peduncular article 5 with 2 calceoli; flagellum 0.65 times as long as peduncular articles 4 and 5 combined, consisting of 9 articles, first 5 each with calceolus.
Uropod 1 (Fig. 6H) with basofacial seta on peduncle; inner ramus 0.9 times as long as peduncle, inner and outer margins with 3 and 2 robust setae, respectively, basal part with 3 slender setae; outer ramus 0.7 times as long as inner, with 2 outer margin robust setae, inner margin bare. Uropod 2 ( Fig. 6I) with inner ramus 1.2 times as long as peduncle, outer margin and marginal with 2 robust setae, respectively; outer ramus 0.7 times as long as inner ramus, inner margin bare and outer margin with 2 robust setae. Uropod 3 ( Fig. 6J) with peduncle 0.24 times as long as outer ramus; inner ramus absent; outer ramus 2-articulate, proximal article with robust setae, terminal article 0.2 times as long as proximal article, with 3 distal setae.
Uropod 1 (Fig. 12H) with basofacial seta on peduncle; inner ramus 0.76 times as long as peduncle, inner and outer margins with 4 and 3 robust setae, respectively, basal part with 3 slender setae; outer ramus 0.6 times as long as inner, with 3 outer marginal robust setae, inner margin bare. Uropod 2 (Fig. 12I) with inner ramus 1.1 times as long as peduncle, outer margin and marginal with 3 and 2 robust setae, respectively; outer ramus 0.7 times as long as inner ramus, inner margin bare and outer margin with 2 robust setae. Uropod 3 (Figs. 12J, 12K) with peduncle 0.29 times as long as outer ramus; inner ramus absent; outer ramus 2-articulate, proximal article with robust setae, terminal article 0.07 times as long as proximal article, with 3 distal setae.
Telson (Fig. 12L) base laterally concave and shallowly at the top, length 1.33 times as long as wide, cleft for 40.2% of length, each telson lobe with lateral penicillate setae, apical with 3 robust setae and 1 seta.
Telson (Fig. 18L) length 1.31 times as long as wide, cleft for 36.8% of length, each telson lobe with 2 lateral penicillate setae, apical robust setae and one short penicillate seta.
Telson (Fig. 20E) length 1.25 times as long as wide, cleft for 40.0% of length. Distribution. Known only from the type locality. Etymology. The specific name is an adjective derived from the name of the cave where the new species was found. Remarks. Pseudocrangonyx hwanseonensis sp. nov. is morphologically similar to P. asiaticus Uéno, 1934 in having (1) eyes completely absent, (2) pereonites 1-6 without short dorsal setae, (3) urosomite 1 with ventral robust seta, (4) maxilla 1 inner plate with 4 plumose setae, (5) antenna 2 longer than half of antenna 1 length, and (6) carpi of gnathopods 1 and 2 with serrate robust setae on posterodistal corner. The new species can be clearly distinguished from P. asiaticus by the following characters (features of P. asiaticus in parentheses): (1) sternal gills of 1 pair (single) present on each pereonites 2-4 (pereonites 2-5), (2) maxilla 1 outer plate with 7 (with 5) serrate teeth, (3) antenna 1 longer (shorter) than as long as body length half, and (4) uropod 3 terminal article shorter (longer) than adjacent robust setae. Molecular Analyses. The uncorrected COI p-distance among the species of the genus Pseudocrangonyx in Korean caves is shown in Table 2; this divergence was calculated based on the 657 aligned positions from the data set. The range of interspecific variation was 11.7-17.0%. However, the maximum intraspecific variation was 0.2% within each species. In the phylogenetic analyses (Fig. 21), the topologies of the BI and ML trees were almost identical. Results of the present analyses showed that the species of the genus Pseudocrangonyx, inhabiting individual caves, were distinct new species.

DISCUSSION
The three new species described in this paper are similar to P. asiaticus Uéno, 1934 in morphology, and they share the following characteristics: relatively large body size (about 8.0-10.0 mm), completely absent eyes, presence of basal setae on urosomite 1, present sternal gills, and carpi of gnathopods 1 and 2 with serrate robust setae on the posterodistal  corner. However, the three new species have following characteristics that distinguished them as distinct new species: (1) P. deureunensis sp. nov., urosomite 3 with dorsal setae; (2) P. kwangcheonseonensis sp. nov., maxilla 1 inner plate with 8 plumose setae, telson base laterally concave, and shallow at the top; and (3) P. hwanseonensis sp. nov., sternal gills of 1 pair present on each pereonites 2-4. Furthermore, the COI genetic distance among the three species showed significant differentiation (12.5-13.4%) that was sufficient to designate the species as distinct , which was confirmed by a previous study (12-20%) on the genus Pseudocrangonyx (Zhao & Hou, 2017). Most geographically separated subterranean species are likely to be independent in origin due to their poor dispersal and small ranges (Trontelj et al., 2009;Trontelj, Blejec & Fišer, 2012). Likewise, our molecular phylogenetic analyses revealed that the species within the genus Pseudocrangonyx endemic to the Korean Peninsula caves formed a monophyletic clade (Fig. 21), suggesting that the genus Pseudocrangonyx inhabits groundwater environments where dispersal is limited, such as a cave, and that they may have an independent origin in each habitat. A previous study (Lee et al., 2020) found that P. joolaei Lee et al., 2020 andP. akatsukai Tomikawa &Nakano, 2018 formed a clade. However, our results showed that P. joolaei has a closer relationship with the Korean cave Pseudocrangonyx species. Additionally, our phylogenetic analyses showed that all Korean cave Pseudocrangonyx species were a same clade, but the Japanese P. akatsukai was in a different clade. This means that rather than forming a clade with the Japanese species, the Korean cave Pseudocrangonyx species may share a single lineage with P. asiaticus sensu stricto, which is geographically adjacent and morphologically similar. Unfortunately, we could not obtain molecular data for P. asiaticus Uéno, 1934, and it is unclear whether P. asiaticus sensu stricto inhabit the Korean Peninsula. Additional molecular data for P. asiaticus should be examined in order to confirm the presence of P. asiaticus sensu stricto and explore the true species diversity of Pseudocrangonyx amphipods inhabiting the Korean Peninsula.
Ultimately, it is important to study the biogeography of pseudocrangonyctids in order to better understand the origin and evolution of subterranean amphipod fauna in the Far East (Sidorov & Holsinger, 2007). Further molecular phylogenetic analyses of Pseudocrangonyx are essential for enhancing the understanding of subterranean Crangonyctoidea species diversity and evolutionary history in Far East Asia.

CONCLUSIONS
This study described three new species described found in caves in Korea. Two new species of them were found from caves treated as the morphological variants of P. asiaticus Uéno, 1934. The other new species was found from a cave with no former records of the genus Pseudocrangonyx. These new species may receive a unique species status within the genus Pseudocrangonyx based on our morphological examination and molecular analyses. These results suggest that the genus Pseudocrangonyx may have greater species diversity in the Korean Peninsula than previously believed. Although we failed to obtain molecular data of the originally described P. asiaticus, obtaining those data in future studies may make it possible to determine the true species diversity of the subterranean amphipod Pseudocrangonyx in Far East Asia including the Korean Peninsula.