A new subterranean species of Pseudocrangonyx from China with an identification key to all species of the genus (Crustacea, Amphipoda, Pseudocrangonyctidae)

Abstract A subterranean species of Pseudocrangonyx elegantulus Hou, sp. n. is described from caves of Wulongdong National Forest Park in Henan Province, China. Pseudocrangonyx elegantulus is characterized by both male and female with calceoli on antenna II; urosomite III dorsal margin without armature; uropod III with peduncle 0.30 times as long as outer ramus and terminal article of the outer ramus a little shorter than adjacent spines; telson cleft 0.27 of its length. Phylogenetic analysis based on 28S and COI sequences supported the species distinctness. A key to the genus Pseudocrangonyx with 22 species and a map of their distributions are provided.


Introduction
The genus Pseudocrangonyx was established by Akatsuka and Komai in 1922, includ-ing 21 described species that are widely distributed in subterranean freshwaters or springs of Japan, the Korean peninsula, eastern China, and the Far East of Russia (Labay 2001, Sidorov and Gontcharov 2013, Tomikawa et al. 2016. The genus exhibits typical subterranean adaptive morphology in the loss of eyes and pigmentation, elongated appendages, and vestigialization of dorsal armature on urosomites (Sidorov and Gontcharov 2013).
During a field survey of freshwater amphipods in Henan Province, China, three species were found, including two epigean freshwater gammarids, Gammarus preciosus Wang et al., 2009 andG. monticellus Hou et al., 2014, and one cave Pseudocrangonyx species new to science. In this paper, the fourth species, Pseudocrangonyx elegantulus sp. n., is described and illustrated. In addition, the phylogenetic position of the new species within Pseudocrangonyx was estimated using nuclear 28S rRNA and mitochondrial cytochrome c oxidase subunit I (COI) sequence data. The distributions of all 22 species of the genus Pseudocrangonyx are presented in Figure 1, where only type localities are used for P. elegantulus, P. korkishkoorum, P. febras, P. cavernarius, P. tiunovi, P. holsingeri, P. sympatricus, P. gudariensis, P. elenae, P. kseniae, P. manchuricus, and P. asiaticus, and others are based on the published paper (Sidorov 2006). A key to world species of the genus Pseudocrangonyx is provided.

DNA sequencing and phylogenetic analyses
Genomic DNA was extracted from appendages of the Pseudocrangonyx specimen using a TIANamp Genomic DNA Kit (TIANGEN). The fragments of 28S and COI were amplified and sequenced following published protocols (Hou et al. 2007). The new sequences and reference sequences downloaded from GenBank were aligned using MAFFT v.7.304 (Katoh and Standley 2016). In total, 29 samples of 14 Pseudocrangonyx species were used in molecular phylogenetic analyses (Table 1). There are seven species from Russian Far East including P. febras, P. holsingeri, P. korkishkoorum, P. kseniae, P. susanaensis, P. sympatricus, and P. tiunovi, six species from Japan including P. yezonis and P. gudariensis, and four newly described species (Tomikawa et al. 2016) and Table 1. Samples used for the phylogenetic analyses. The locality information is accompanied by sequence accession numbers. Species names marked with an asterisk were obtained from Tomikawa et al. (2016).  Bousfield, 1963, andCrangonyx pseudogracilis Bousfield, 1958. The best-fit partitioning schemes and nucleotide substitution models were selected using PartitionFinder v.1.1.0 on the Bayesian criterion (Lanfear et al. 2012). The COI data were partitioned into first, second, and third codon positions with TrN+I+G, TrNef+I+G, and TrN+G models, respectively. The best model for 28S was GTR+G. Therefore, a four-partition scheme was used in the following analyses.
Phylogenetic relationships were inferred using maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) on single gene and concatenated sequences. MP analysis and bootstrap evaluation were performed using PAUP* 4.0b10 (Swofford 2002) with tree bisection reconnection swapping algorithm. ML phylogenies were conducted using RAxML v.8.2.7 (Stamatakis 2014) with 1000 rapid bootstrap replicates followed by a thorough tree search. Bayesian analyses were carried out using MrBayes v.3.2.1 (Ronquist et al. 2012), implementing two independent runs of five million generations. The convergence was checked using Tracer v.1.5 (Rambautand Drummond 2009) and the first 25% trees were discarded as burn-in.
Diagnosis. Female larger than male; eyes absent; lateral cephalic lobe rounded; inferior antennal sinus indistinct; both male and female with calceoli on antenna II; coxal gills present on gnathopod II and pereopods III-VI; sternal gills absent; epimeral plate I without armature on distal margin; urosomite III dorsal margin without armature; uropod I peduncle with one basofacial spine; inner ramus of male uropod II with two serrate and four simple robust terminal spines accompanied by one seta; uropod III peduncle 0.30 times as long as outer ramus and terminal article of the outer ramus a little shorter than adjacent spines.
Head. (  with two articles, subequal to the first article of primary flagellum; both primary and accessory flagella with short distal setae. Antenna II (Fig. 2D, E): peduncle articles 3-5 in length ratio 1.0 : 2.2 : 2.9, with spines accompanied by setae; flagellum with seven articles, with one or two spines and setae on first three articles and with setae on the rest articles; calceoli of crangonyctid type, present on peduncular article 5 and first three flagellum articles; rod-like structures accompanied with setae on first four flagellum articles.
Lower lip ( Fig. 2G): inner lobes absent, outer lobes covered with thin setae. Maxilla I (Fig. 2J, K): asymmetrical, left inner plate with four plumose setae; outer plate with seven serrated apical spines; second article of left palp densely setose, with two simple setae and four slender spines apically; second article of right palp with five spines and two slender setae.
Maxilla II (Fig. 2L): inner plate with four plumose facial setae in an oblique row; inner and outer plates with long setae apically.
Maxilliped (Fig. 2M): inner plate with three stout apical spines, two serrated setae, and five plumose setae; outer plate bearing four setae, four serrated spines and five plumose setae apically; palp 4-articulate, articles 1-2 in length ratio 0.7 : 1, article 2 with a row of simple setae on interior side and one simple seta on exterior side; article 4 hooked, with five setae at hinge of unguis.
Pereon. Gnathopod I (Fig. 3A, B): coxal plate bearing one fine seta on proximal margin and three setae on anterodistal corner, 1.7 times as wide as deep; basis with long setae on posterior margin, anterior margin bare; merus bearing setae on posterodistal corner; carpus as long as wide, approximately 0.5 times as long as propodus, bearing three clusters of setae along posterior margin, two clusters of setae on anterior margin, and three pectinate setae on posterodistal corner; propodus pear-shaped, palm margin with 16 robust spines, some distally notched; dactylus with one seta on anterior margin and two setae at hinge of unguis, posterior margin dentate.
Gnathopod II (Fig. 3C, D): coxal plate bearing one fine seta on proximal margin, three setae on anterodistal corner and one seta on distal margin; basis with setae on posterior margin, anterior margin bare; merus bearing setae on posterodistal corner; carpus 1.5 times as long as wide, approximately 0.7 times as long as propodus, bearing seven clusters of setae along posterior margin and three pectinate setae on posterodistal corner; propodus stout, palm margin with 14 distally notched spines; dactylus with one seta on anterior margin and two setae at hinge of unguis, posterior margin dentate.
Pereopod III (Fig. 4A, B): coxal plate bearing four setae on anterior margin and two setae on distal margin, 1.4 times as wide as deep; basis with seven setae along anterior margin and long setae on posterior margin; merus, carpus, and propodus in length  ratio 1.0 : 0.7 : 0.8; merus with three spines on anterior margin and four clusters of setae on posterior margin, anterodistal corner with one spine; carpus with one fine seta on anterior margin and two setae on posterior margin, anterodistal corner with one seta and posterodistal corner with two spines accompanied with one seta; dactylus with one plumose seta on anterior margin, one seta on posterior margin, and one seta at hinge of unguis.
Pereopod V (Fig. 4E, F): coxal plate irregular, anterior lobe larger than posterior lobe, bearing four setae and one seta on anterior and posterior lobes, respectively; basis with setae on anterior and posterior margins, respectively; merus, carpus, and propodus in length ratio 1.0 : 0.9 : 0.9; merus and carpus with spines accompanied by setae on both margins; dactylus with one plumose seta on posterior margin, one seta on anterior margin, and one seta at hinge of unguis.
Pereopod VI (Fig. 4G, H): coxal plate similar to that of pereopod V, with smaller anterior lobe, bearing one seta on posterior lobe; basis with setae on anterior and posterior margins; merus, carpus, and propodus in length ratio 1.0 : 1.0 : 0.9; merus and carpus with spines accompanied by setae on both margins; dactylus with one plumose seta on posterior margin, one seta on anterior margin, and one seta at hinge of unguis.
Pereopod VII (Fig. 4I, J): coxal plate subtriangular, with two setae on posteroproximal corner; basis with setae on anterior and posterior margins; merus, carpus, and propodus in length ratio 1.0 : 1.1 : 1.1; merus and carpus with spines accompanied by setae on both margins; dactylus with one plumose seta on posterior margin, one seta on anterior margin, and one seta at hinge of unguis.
Pleon. Epimeral plates ( Fig. 5A-C): plate I distally rounded, bearing three fine setae on posterior margin and one seta on posterodistal corner, distal margin without armature; plate II with two spines on distal margin and three fine setae on posterior margin, posterodistal corner rounded with one seta; plate III with two spines on distal margin and two fine setae on posterior margin, posterodistal corner rounded with one seta.
Pleopods I-III (Fig. 5D-F): similar, peduncle with two retinacula on interior side and one fine seta on exterior distal corner; outer ramus shorter than inner ramus, both inner and outer rami fringed with plumose setae.
Urosome. Urosomites (Fig. 2N): urosomite I with two setae on dorsal margin; urosomite II with two spines on left side and two spines accompanied by one seta on right side; urosomite III dorsal margin without armature.
Uropods I-III (Fig. 5G-I): uropod I peduncle with one basofacial spine, with three spines on exterior side, interior and exterior distal corners with one spine respectively; inner ramus approximately 0.77 times as long as peduncle, with three spines on interior side, one seta and one spine on exterior side, and six terminal spines accompanied by one seta; outer ramus approximately 0.76 times of inner ramus, with two spines on exterior side and five terminal spines. Uropod II half shorter, peduncle bearing two spines on exterior side and one spine on each distal corner; inner ramus with two spines on interior side, one spine on exterior side, and six terminal spines accompanied by one seta; outer ramus approximately 0.71 times of inner ramus, with two spines on exterior side and five terminal spines. Uropod III with peduncle 0.30 times as long as outer ramus, with one dorsal and three ventral robust spines; inner ramus absent; outer ramus 2-articulate, first article of outer ramus with stiff spines on interior and exterior sides, terminal article 0.19 times of the first article, with three distal setae, a little shorter than adjacent spines.
Telson (Fig. 5J): 1.2 times as long as wide, cleft 0.27 of its length, each lobe with two setae on surface and two distal spines.
Description of paratype male (IZCAS-I-A1602-2), 6.3 mm. Head. Antenna II (Fig. 7A): peduncle articles 3-5 in length ratio 1.0 : 2.6 : 3.0, with setae along anterior and posterior margins; flagellum with six articles, with spines and setae on first article and with setae on the rest articles; calceoli of crangonyctid type present on peduncular article 5 and first two flagellum articles; rod-like structures accompanied with setae on flagellum articles.
Pereon. Gnathopod I (Fig. 6A, B): coxal plate bearing three setae on anterodistal corner, 1.6 times as wide as deep; basis with long setae on posterior margin, anterior margin bare; merus bearing setae on posterodistal corner; carpus 0.8 times as long as wide, approximately 0.5 times as long as propodus, bearing five clusters of setae along posterior margin and two pectinate setae on posterodistal corner; propodus pear-shaped, palm margin with 14 distally notched spines; dactylus with one seta on anterior margin.
Gnathopod II (Fig. 6C, D): coxal plate bearing five setae on distal margin; basis with long setae on posterior margin, anterior margin bare; merus bearing setae on posterodistal corner; carpus 1.5 times as long as wide, approximately 0.7 times as long as propodus, bearing seven clusters of setae along posterior margin; propodus stout, palm margin with 12 distally notched spines; dactylus with one seta on anterior margin. Uropods I-III (Fig. 5K-M): uropod I peduncle with one basofacial spine, with three spines on exterior side, interior and exterior distal corners with one spine respectively; inner ramus approximately 0.73 times as long as peduncle, with two spines on interior side, two simple setae on exterior side, and six terminal spines accompanied by one seta; outer ramus approximately 0.8 times of inner ramus, with two spines on exterior side and five terminal spines. Uropod II shorter, peduncle bearing one spine on exterior side and one spine on each distal corner; inner ramus with two spines on each side, distal part with two serrated and four simple robust spines accompanied by one  seta; outer ramus approximately 0.72 times of inner ramus, with one spine on exterior side and five terminal spines. Uropod III peduncle 0.31 times as long as outer ramus, with one dorsal and three ventral robust spines; inner ramus absent; outer ramus biarticulate, first article of outer ramus with three groups of stiff spines on interior and exterior sides, terminal article 0.18 times of the first article, with three distal setae, a little shorter than adjacent spines.
Telson (Fig. 7G): 1.2 times as long as wide, cleft 0.24 of its length, each lobe with two setae on surface and two distal spines accompanied by one short seta.
Habitat. This species was collected from groundwater flowing through a cave of the Wulongdong National Forest Park.
Remarks. Pseudocrangonyx elegantulus sp. n. is clustered with P. yezonis Akatsuka & Komai, 1922 supported by high statistical supports in the molecular phylogenetic tree. Unfortunately, the original description of the latter species is poor and no redescription has been published. The following comparisons are based on recent observations (Ko Tomikawa pers. comm.). The new species is morphologically similar to P. yezonis in antenna II with calceoli; the armature of gnathopods I and II and pereopods III-VII; both rami of pleopods with more than five articles; urosomite III dorsal margin without armature. It can be distinguished from P. yezonis Akatsuka & Komai, 1922 by the following characters (P. yezonisin parentheses): pereonites I-VI without armature on dorsal margin, only pereonite VII with dorsal setae (with long setae on dorsal margins of pereonites I-VII); uropod III terminal article of outer ramus a little shorter than adjacent spines (subequal).
The new species is most similar to P. cavernarius Hou & Li, 2003 in the armature of gnathopods I and II and pereopods III-VII; epimeral plate I without armature on distal margin; both rami of pleopods with more than five articles. The new species can be distinguished from P. cavernarius Hou & Li, 2003 by the following characters (P. cavernarius in parentheses): antenna II with calceoli (absent); inner plate of maxilla II with four plumose facial setae in an oblique row (with five plumose setae); urosomite I with two setae on dorsal margin (with four clusters of setae); urosomite III dorsal margin without armature (with one pair of fine spines); outer ramus of uropod I with five terminal spines (with four terminal spines); uropod II inner ramus with six terminal spines accompanied by one seta (with five terminal spines) and outer ramus with five terminal spines (with three terminal spines); uropod III peduncle with one dorsal and three ventral robust spines (with three distal spines), terminal article of outer ramus a little shorter than adjacent spines (longer); each lobe of both male and female telson with two setae on surface (with no armature).
The new species is similar to P. asiaticus Uéno, 1934, which was redescribed by Uéno (1966), in the accessory flagellum being subequal to the first article of primary flagellum; the armature of gnathopods I and II and pereopods III-VII. It can be distinguished from P. asiaticus Uéno, 1934 by the following characters (P. asiaticus in parentheses): antenna II with calceoli (absent); incisor of mandible with five teeth (with 5-6 teeth); mandible spine row with five serrated spines (with 8-10 serrated setae); maxilliped inner plate with three stout apical spines, two serrated setae, and five plumose setae (with five serrated spines and seven plumose setae); sternal gills absent (present on gnathopod II and pereopods III-IV); each lobe of both male and female telson with two setae on surface (with no armature).
The new species is similar to P. elenae Sidorov, 2011 in body length (longer than 6.0 mm); the armature of gnathopod I and II and pereopods III-VII; epimeral plate I without armature on distal margin; both rami of pleopods with more than five articles; urosomite III dorsal margin without armature; terminal article of outer ramus of uropod III shorter than adjacent spines. It can be distinguished from P. elenae Sidorov, 2011 by the following characters (P. elenae in parentheses): accessory flagellum of antenna I subequal to the first article of primary flagellum (shorter than accompanying flagellar article); antenna II of female with calceoli (absent); mandible spine row with five serrated spines (with six serrated setae); maxilla I with four plumose setae on inner plate (with five plumose setae); inner plate of maxilla II with four plumose facial setae in an oblique row (with five plumose setae); inner plate of maxilliped with three stout apical spines, two serrated setae, and five plumose setae (with five simple strong apical setae and nine plumose setae); epimeral plate II with two spines on distal margin (with one seta).
The new species resembles P. gudariensis Tomikawa & Sato, 2016 in epimeral plate I without armature on distal margin; urosomite III dorsal margin without armature. It can be distinguished from P. gudariensis Tomikawa & Sato, 2016 by the following characters (P. gudariensis in parentheses): accessory flagellum of antenna I subequal to the first article of primary flagellum (longer); antenna II of female with calceoli (absent); mandible spine row with five serrated spines (with 2-3 weakly pectinate setae); maxilla I with four plumose setae on inner plate (with three plumose setae); inner plate of maxilla II with four plumose facial setae in an oblique row (with three plumose setae); inner plate of maxilliped with three stout apical spines, two serrated setae, and five plumose setae (with three apical and two subapical robust setae); terminal article of uropod III outer ramus a little shorter than adjacent spines (longer); epimeral plates II and III with two spines on distal margins (with one seta); telson of male cleft 0.24 of its length (0.08).
The new species is also similar to P. holsingeri Sidorov & Gontcharov, 2013 in the armature of gnathopod I and II and pereopods III-VII; epimeral plate I without armature on distal margin; both rami of pleopods with more than five articles. It differs from P. holsingeri Sidorov & Gontcharov, 2013 by the following characters (P. holsingeri in parentheses): accessory flagellum of antenna I subequal to the first article of primary flagellum (longer); inner plate of maxilliped with three stout apical spines, two serrated setae, and five plumose setae (with two apical and three sub-apical setae); epimeral plate III with two spines on distal margin (with three setae); uropod I peduncle with one basofacial spine (with two basofacial spines in female); uropod III peduncle with one dorsal and three ventral robust spines (with two sets of stiff setae on distal margins).
Distinguishing features of all the 22 species of genus Pseudocrangonyx can be found in the key below.

Molecular phylogeny
The final alignment contained 32 taxa with 2123 bp, including 1465 bp for 28S and 658 bp for COI. MP, ML and BI yielded a congruent topology (Fig. 8). The monophyly of the genus Pseudocrangonyx was well supported, but the relationships within the genus Pseudocrangoyx remained unresolved, as found in the previous molecular study (Tomikawa et al. 2016). The new species P. elegantulus was clustered with P. yezonis with high support value. The uncorrected p-distance between P. elegantulus and P. yezonis was 12-15% for COI, which was comparable to distances found between Russian Pseudocrangonyx species (Sidorov and Gontcharov 2013).
As mentioned in the Remarks, the new species P. elegantulus is morphologically similar to P. cavernarius. Unfortunately, we were unable to obtain fresh material for P. cavernarius, because of tourist development in the type locality. Our phylogenetic results revealed that P. elegantulus was grouped with P. yezonis from the northern part of Japan. However, the divergences of 12-15% for COI confirmed the distinctness of new species, in comparison with the various COI distances used for amphipod delimitation (Rock et al. 2007). In addition, P. elegantulus and P. yezonis are mutually allopatric, as P. elegantulus is located at the inner land of China and separated from P. yezonis by sea. Therefore, morphological examination, molecular phylogenetic analyses, and distribution data support P. elegantulus being a new species.

Discussion
Four Pseudocrangonyx species are recorded from subterranean freshwaters of China. Pseudocrangonyx asiaticus and P. manchuricus are known from interstitial water strata Figure 8. The ML tree derived from concatenated data set of 28S and COI. Support values greater than 70% are shown above branches in order for MP, ML, and BI analyses. Names of terminal taxa include voucher numbers for ingroups according to literature (Tomikawa et al. 2016).
approximately 10 meters under the surface of the earth, while P. cavernarius and P. elegantulus inhabit caves. Because the subterranean habitats are imperiled by drought and tourism, conservation plans should be strengthened.
Our molecular analyses revealed significant COI differentiation (12-20%) for species of the genus Pseudocrangonyx. Molecular evidences help us to discover new species, especially for subterranean or cave species which are morphologically indistinguishable (Hou and Li 2010). Phylogenetic results supported a single origin of the genus Pseudocrangonyx, however the diversification pattern across the Asia-Pacific margins was uncertain. Extensive sampling and detailed genetic data are needed to clarify the evolutionary history of Pseudocrangonyx amphipods.