A new species of the genus Pseudocrangonyx (Crustacea, Amphipoda, Pseudocrangonyctidae) from Korea

Abstract A new subterranean species of pseudocrangonyctid amphipod, Pseudocrangonyx daejeonensis sp. n. is described from the interstitial waters in Daejeon, Korea. Pseudocrangonyx daejeonensis sp. n. is distinguished from three morphologically similar congeners, P. coreanus Uéno, 1966, P. febras Sidorov, 2009, and P. gudariensis Tomikawa & Sato, 2016, by the characteristics of antenna 1, antenna 2, mandible, gnathopod 2, pleopods, uropods 1–2, and telson. Molecular phylogenetic analyses based on nuclear 28S rRNA and histone H3, and mitochondrial cytochrome c oxidase subunit I and 16S rRNA genes revealed that P. daejeonensis is a sister species of the unnamed Pseudocrangonyx sp. 3 inhabiting central Japan.


Introduction
Amphipod species of the genus Pseudocrangonyx Akatsuka & Komai, 1922 have been known from subterranean waters and springs in Japan, the Korean Peninsula, Eastern China, and the Far East of Russia (Sidorov and Holsinger 2007;Tomikawa et al. 2016;Zhao and Hou 2017). Among the 22 known species of Pseudocrangonyx, only two rRNA, histone H3, COI and 16S rRNA from three Korean Pseudocrangonyx specimens were newly obtained in this study, and deposited into the International Nucleotide Sequence Database Collaboration (INSDC) through the DNA Data Bank of Japan (Table 1).
The OTU set for phylogenetic analyses was almost identical to that used in the previous phylogenetic analyses in Tomikawa et al. (2016) with the DNA sequences of Table 1. Samples used for the phylogenetic analyses. The information on the vouchers is accompanied by the collection locality and the INSDC accession numbers. Sequences marked with an asterisk were obtained for the first time in the present study. Acronyms: IZCAS, Institute of Zoology, Chinese Academy of Sciences; NNIBR, Nakdonggang National Institute of Biological Resources; NSMT, National Museum of Nature and Science, Tokyo.  (Katoh and Standley 2013). The lengths of the 28S, H3, COI and 16S were 1357, 328, 658, and 430 bp, respectively. The concatenated sequences thus yielded 2773 bp of alignment positions. Phylogenetic trees were constructed using maximum likelihood (ML) and Bayesian inference (BI). The ML phylogeny was constructed using RAxML v. 8.2.8 (Stamatakis 2014) with the substitution model set as GTRCAT, immediately after nonparametric bootstrapping (BS) conducted with 1000 replicates. The best-fit partition scheme was identified with Akaike information criterion using PartitionFinder v. 2.1.1 (Lanfear et al. 2017) with the "greedy" algorithm (Lanfear et al. 2012): 28S/1st and 2nd positions of H3/H3 3rd position/COI 1st position/COI 2nd position/COI 3rd position/16S. BI and Bayesian posterior probabilities (PPs) were estimated using MrBayes v. 3.2.6 (Ronquist et al. 2012). The best-fit partition scheme and models for each partition were selected based on the Bayesian information criterion using PartitionFinder with the "greedy" algorithm: for 28S, GTR+G; for H3 1st and 2nd position, JC+I; for H3 3rd position, K80+G; for COI 1st position, SYM+I+G; for COI 2nd position, F81+I; for COI 3rd position, GTR+I+G; and GTR+I+G for 16S. Two independent runs of four Markov chains were conducted for 20 million generations, and the tree was sampled every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.6.0 (Rambaut and Drummond 2013), and the first 50001 trees were discarded based on the results.  2) with short dorsal setae; rostrum reduced; lateral cephalic lobe rounded; antennal sinus shallow with rounded angle; eyes absent. Dorsal margin of pleonites 1-3 and urosomites 1-2 with setae (Fig. 2). Ventral margin of urosomite 1 without setae (Fig. 2). Ventral margin of epimeral plate 1 with seta, posteroventral corner rounded with seta ( Upper lip (Fig. 3D) with rounded anterior margin, bearing fine setae. Mandibles ( Fig. 3F, G, H) with left and right incisors with 5-and 4-dentate, respectively; left lacinia mobilis 4-dentate, right lacinia bifid, bearing many teeth; molar process triturative; accessory setal rows of left and right mandibles with 3-and 2-pectinate setae, respectively; palp 3-articulate, article 3 with 1 A-, 7 D-, and 3 E-setae. Lower lip ( Fig. 3I) with broad outer lobes with fine setae, mandibular process of outer lobe rounded apically; inner lobes indistinct. Maxilla 1 (Fig. 3J) with inner and outer plates, and palp; inner plate subovate with 2 plumose setae; outer plate subrectangular with 7 serrate teeth apically; palp 2-articulate, longer than outer plate, article 2 with 2 apical robust setae. Maxilla 2 ( Fig. 3K) with oblique inner row of 2 setae on inner plate. Maxilliped (Fig. 4A) with inner and outer plates, and palp; inner plate reaching base of palp article 1, with 2 apical robust setae; outer plate not exceeding end of palp article 1, with 2 plumose setae and some medial setae; palp 4-articulate, medial margin of article 2 lined with setae, article 4 with nail.
Uropod 1 (Fig. 9A) with robust seta on inner margin of inner ramus; outer ramus 0.62 times as long as inner. Uropod 2 (Fig. 9B) with 2 serrate and 4 simple robust setae and slender seta at distal part. Uropod 3 (Fig. 9C) with peduncle 0.32 times as long as outer ramus; terminal article of outer ramus 0.5 times as long as proximal article.
Variation. Peduncle of pleopod 1 with or without seta on outer margin. Distribution. This species is known only from the type locality. Remarks. Pseudocrangonyx daejeonensis is morphologically similar to P. coreanus in having 1) relatively small body size (smaller than 6 mm), 2) eyes completely absent, 3) carpus of gnathopod 2 without serrate robust setae on posterodistal corner, 4) outer margin or outer distal corner of pleopods 1 and 2 with setae, 5) inner basal margin of inner ramus of pleopods without bifid setae, and 6) small number of articles (less than 5) of rami of pleopods. However, the former is distinguished from the latter by the following features (features of P. coreanus in parentheses): 1) antenna 1 shorter (longer) than 0.4 times as long as body length, 2) antenna 2 of female without calceoli (with calceoli), 3) uropod 1 not exceeding (slightly exceeding) tip of uropod 2, and 4) outer ramus of uropod 2 without robust seta (with robust seta).
Although the phylogenetic position of P. coreanus remains uncertain, the results of the previous molecular phylogenetic studies (Tomikawa et al. 2016;Zhao and Hou 2017) and our phylogenetic analyses showed that P. daejeonensis and the two morphologically similar species, P. febras and P. gudariensis, did not form a monophyletic lineage with large genetic divergences. Because these three species inhabit interstitial waters, not subterranean habitats, morphological similarities observed among them may reflect their similar habitat preferences.
The phylogenetic position of P. daejeonensis also sheds light onto the complex faunistic relationships between the Pseudocrangonyx species inhabiting the Japanese Archipelago and those inhabiting the Far Eastern continental area. Common ancestors of the Japanese Pseudocrangonyx species were considered to have migrated from the continental part to the Japanese Archipelago (Sidorov and Holsinger 2007). Previous systematic studies revealed that the Pseudocrangonyx amphipods distributed in northern Japan and the western tip of Honshu, Japan, i.e., P. yezonis and Pseudocrangonyx sp. 2, are phylogenetically close to the continental species (Sidorov and Holsinger 2007;Tomikawa et al. 2016;Zhao and Hou 2017). As P. daejeonensis formed a wellsupported clade with Pseudocrangonyx sp. 3, which is indigenous to the central part of Honshu, their phylogenetic relationship suggested that the species diversity of the Japanese Pseudocrangonyx has been increased as a result of multiple continental-origins. It is also feasible that P. daejeonensis diverged from a common ancestor indigenous to the Japanese Archipelago. To clarify the biogeographical history of Pseudocrangonyx amphipods, further faunistic surveys along with molecular phylogenetic analyses are essentially needed.
The uncorrected p-distance of 15.0 % for COI, calculated using MEGA7.0.16 (Kumar et al. 2016) between P. daejeonensis and Pseudocrangonyx sp. 3 is equivalent to sequence divergence thresholds for discriminating amphipod species (Witt et al. 2006;Rock et al. 2007;Hou et al. 2009). The former is distinguished from the latter in having the following features (features of Pseudocrangonyx sp. 3 in parentheses): 1) outerdistal corner of peduncle of pleopod 3 without seta (with seta), 2) each of inner and outer ramus of pleopod 3 2-articulate (3-articulate), 3) outer rami of uropods 1 and 2 without marginal robust setae (with marginal seta), and 4) robust setae on distal part of proximal article of uropod 3 short, not reaching tip of terminal article (long, exceeding tip of terminal article) (Tomikawa pers. observation).