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Research Article
Two new species add to the diversity of Eoniphargus in subterranean waters of Japan, with molecular phylogeny of the family Mesogammaridae (Crustacea, Amphipoda)
expand article infoAki Shintani‡, Chi-Woo Lee§, Ko Tomikawa‡
‡ Hiroshima University, Hiroshima, Japan
§ Nakdonggang National Institute of Biological Resources, Sangju-si, Republic of Korea

Corresponding author: Ko Tomikawa ( tomikawa@hiroshima-u.ac.jp )

Academic editor: Cene Fišer
© 2022 Aki Shintani, Chi-Woo Lee, Ko Tomikawa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Shintani A, Lee C-W, Tomikawa K (2022) Two new species add to the diversity of Eoniphargus in subterranean waters of Japan, with molecular phylogeny of the family Mesogammaridae (Crustacea, Amphipoda). Subterranean Biology 44: 21-50. https://doi.org/10.3897/subtbiol.44.86914
ZooBank: urn:lsid:zoobank.org:pub:1865B081-1072-4900-BFC7-563C0D29DA7D
Open Access

Abstract

Amphipod crustaceans are a major group of invertebrates that predominantly occur in groundwater ecosystems. Eoniphargus is a mesogammarid genus with only two known species from the groundwater systems of the Japanese archipelago and Korean Peninsula. However, there is a dearth of taxonomic studies on this genus, and the species diversity within Eoniphargus is unclear. Here, we describe two new species, E. iwataorum sp. nov. and E. toriii sp. nov., collected from the interstitial waters in Tochigi and Shizuoka Prefectures in the Japanese archipelago. These two new species are distinguished from their congeners by the following features: head, urosomite 3, first and second antennae, mandibles, and maxilla 1. Eoniphargus kojimai is redescribed here based on material collected near the type locality. Molecular phylogenetic analyses based on the nuclear 28S rRNA and mitochondrial COI genes revealed that E. kojimai is sister to E. iwataorum sp. nov. In this study, we also briefly discuss the phylogenetic relationships of Mesogammaridae based on the molecular phylogenetic analyses.

Keywords

Cryptic species, interstitial water, molecular phylogeny, new species, taxonomy

Introduction

Compared to epigean ecosystems, subterranean environments are generally stable (Badino 2010). Owing to the absence of light, subterranean habitats are almost entirely dependent on external energy, which is mostly scarce (Poulson and Lavoie 2000). Therefore, subsurface habitats usually have low biomass and species diversity (Holsinger 1988; Cardoso 2012). Several species show limited distribution as a result of isolation and adaptation to underground environments (Culver et al. 2000). Crustaceans, segmented worms, flatworms, rotifers, water mites, and larvae of aquatic insects are predominant in subterranean waters, but insects are rarely found in cave waters (Boulton et al. 1998; Sket 1999). Amphipod crustaceans constitute a major portion of the aquatic organisms of groundwater ecosystems and have been reported from a variety of subterranean environments, including caves, interstitial riverbed waters, and springs (Holsinger 1993).

Eoniphargus Uéno, 1955 was established by Uéno (1955) as a new subgenus of Neoniphargus Stebbing, 1899. This subgenus was subsequently elevated to the genus level by Straškraba (1964). The affiliation of this genus to a family is controversial. Straškraba (1964) included Eoniphargus in Gammaridae Leach, 1814. Bousfield (1977) moved this genus to the newly established Neoniphargidae family. However, neither Barnard and Barnard (1983) nor Holsinger (1994) assigned Eoniphargus to a specific family, owing to the unclear nature of its diagnostic traits. A taxonomic revision of Eoniphargus by Tomikawa et al. (2007), based on molecular phylogenetic analyses, included this genus in Mesogammaridae Bousfield, 1977. Mesogammaridae currently include two marine genera, Mesogammarus Tzvetkova, 1965 and Paramesogammarus Bousfield, 1979, and four subterranean genera that occur in freshwaters, Eoniphargus, Indoniphargus Straškraba, 1967, Octopupilla Tomikawa, 2007, Potiberaba Fišer, Zagmajster & Ferreira, 2013; however, the monophyly of the family remains to be tested using molecular methods (see Sidorov et al. 2018 for alternative opinion on Indoniphargus). Eoniphargus currently comprises two species: E. kojimai Uéno, 1955 from the subterranean waters in Tokyo and Shizuoka and E. glandulatus Stock & Jo, 1990 from cave pools in the Korean Peninsula (Uéno 1955; Stock and Jo 1990; Tomikawa et al. 2007). In recent years, molecular phylogenetic analyses using DNA sequences have greatly advanced our understanding of the phylogenetic relationships of amphipods (Hou et al. 2011, 2014; Copilaş-Ciocianu et al. 2020). However, the phylogenetic position of Mesogammaridae and phylogenetic relationships within the family are not fully understood.

The authors recently obtained several specimens of Eoniphargus from interstitial waters in Japan. Detailed morphological observations and molecular phylogenetic analyses of these samples have revealed the presence of two previously undescribed species. Here, we describe these two novel species and redescribe E. kojimai obtained near the type locality.

Methods

Sampling and morphological observation

Specimens of Eoniphargus were collected from four localities in Japan (Fig. 1). The specimens were collected by washing out gravel from the bottom of rivers and springs using fine-mesh hand nets and fixed in 99% ethanol on-site.

Figure 1. 

Map showing the collection localities of the specimens examined in this study. Names of localities are shown in Table 1.

All appendages were dissected by fine needles in 80% ethanol under a stereomicroscope (Olympus SZX7) and mounted in gum-chloral medium on glass slides. Slides were examined using a light microscope (Nikon Eclipse Ni), with appendages illustrated using a camera lucida.

Type specimens are deposited at the National Museum of Nature and Science, Tsukuba (NSMT).

Molecular phylogenetic analyses

Genomic DNA was extracted from the appendage muscle of the specimens following procedures detailed by Tomikawa et al. (2014a, b). The primer sets for PCR and cycle sequencing reactions used in this study were as follows: for 28S rRNA (28S), 28SF and 28SR (Tomikawa et al. 2012); and for cytochrome c oxidase subunit I (COI), LCO1490 and HCO2198 (Folmer et al. 1994). PCR and DNA sequencing were performed using the method detailed by Tomikawa (2015). In total, 15 sequences were newly obtained and deposited in the International Nucleotide Sequence Databases (INSD) through the DNA Data Bank of Japan (DDBJ) (Table 1).

Table 1.
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Samples used for molecular analyses with voucher/isolate number, collection locality, and NCBI GenBank accession number. Sequences marked with an asterisk (*) were obtained for the first time in this study.

Species Voucher or isolate # Locality NCBI GenBank acc. nos.
28S rRNA COI
Acanthogammaridae
Dorogostaiskia parasitica MZH:112037 Lake Baikal, Russia KF586548 KF586540
Anisogammaridae
Jesogammarus hebeiensis 294 Beijing, China EF582998 KT180186
Gammaracanthidae
Gammaracanthus lacustris SLOCHN141 Savonranta Munic, Finland JF965829 JF965997
Gammaracanthu loricatus SLOCHN171 Spitsbergen, Norway JF965830 JF965998
Gammarellus angulosus RBINS-INV.132647 Westkapelle, Netherland KT808715 FJ581638
Gammaridae
Anopogammarus revazi SLOCHN245 Martvili, Georgia KF478431 KF478522
Barnardiorum shadini SLOCHN263 Hodža Obi-Garm, Tajikistan JF965826 JF965994
Chaetogammarus ischnus SLOCHN051 Babadag, Romania KF478441 KF478532
Dikerogammarus villosus SLOCHN052 Babadag, Romania KF478442 KF478533
Echinogammarus acarinatus SLOCHN082 Mostar, Bosnia and Herzegovina KF478458 KF478548
Gammarus lacustris EF582964 EF570317
G. mukudai G857 Katsumoto, Iki, Nagasaki, Japan AB893233 AB893343
G. nipponensis G621 Ukyo, Kyoto, Japan AB893226 AB893336
G. tigrinus 609 Netherland EF582994 EF570348
Jugogammarus kusceri SLOCHN073 Krka, Slovenia KF478462 KF478552
Rhipidogammarus rhipidiophorus SLOCHN162 Dorgali, Sardegna, Italy JF965823 JF965991
Mesogammaridae
Eoniphargus iwataorum sp. nov. NSMT-Cr 30783 Sabi River, Imaizumi, Ohtawara, Tochigi Prefecture, Japan (1) LC709238* LC709248*
E. kojimai G1750 Mamashita Spring, Kunitachi, Tokyo, Japan (2) LC709239* LC709249*
E. kojimai NSMT-Cr 30787 Hinochūōtoshokan Spring, Hino, Tokyo, Japan (3) LC709240* LC709250*
E. kojimai NSMT-Cr 30788 Hinochūōtoshokan Spring, Hino, Tokyo, Japan (3) LC709241* LC709251*
E. toriii sp. nov. G56 Seto River, Terajima, Fujieda, Shizuoka, Japan (4) LC709242* LC709252*
E. toriii sp. nov. G71 Seto River, Terajima, Fujieda, Shizuoka, Japan (4) LC709243* LC709253*
Mesogammarus melitoides G86 Muroran, Hokkaido, Japan (5) LC719002* NA
Octopupilla felix G54 Koza River, Wakayama, Japan (6) LC719003* LC719248*
Pontogammaridae
Obesogammarus crassus SLOCHN055 Babadag, Romania KF478445 KF478536
Paraniphargoides motasi SLOCHN188 Gilan, Iran KF478485 KF478571
Pontogammarus robustoides SLOCHN255 Delta Volgi, Russia JF965822 JF965990
Stenogammarus similis SLOCHN187 Gilan, Iran KF478484 KF478570
Turcogammarus spandli SLOCHN026 Thessaloniki, Greece KF478437 KF478528
Typhlogammaridae
Metohia carinata SLOCHN019 Rijeka Crnojevica, Montenegro KF478498 KF478584
Typhlogammarus mrazeki SLOCHN113 Cetinje, Montenegro KF478504 KF478590
Zenkevitchia admirabilis SLOCHN200 Gudauta, Georgia KF478514 KF478599
Outgroup
Pseudocrangonyx yezonis KUZ Z1969 Daisen, Akita, Japan LC17151 LC171519

In addition to the newly obtained sequences, 50 sequences of 24 gammaroid species and one crangonyctoid Pseudocrangonyx yezonis Akatsuka & Komai, 1922, which was selected as the outgroup, were obtained from the INSD in accordance with the previous studies (Hou et al. 2011, 2014; Copilaş-Ciocianu et al. 2020), and were included in the present phylogenetic analyses (Table 1). The phylogenetic analyses were conducted based on sequences of nuclear 28S and mitochondrial COI. The alignment of COI was trivial, as no indels were observed. The 28S sequences were aligned using the Muscle algorithm implemented in MEGA X (Kumar et al. 2018). The lengths of the 28S and COI were 892 and 658 bp.

Phylogenetic relationships were reconstructed via Maximum Likelihood (ML) and Bayesian Inference (BI). The best evolutionary models were selected based on the corrected Akaike Information Criterion (AIC) for ML using MEGA X (Kumar et al. 2018). ML phylogenies were conducted using MEGA X (Kumar et al. 2018) under the substitution model GTR+G, and 1000 bootstrap replications (Felsenstein 1985) were performed to estimate statistical support for branching patterns. BI and Bayesian posterior probabilities (PPs) were estimated using MrBayes v. 3.2.5 (Ronquist et al. 2012). The best-fit partition scheme and models for each partition were selected with the Bayesian information criterion using PartitionFinder with the “greedy” algorithm: for 28S, SYM+G; for COI 1st position, HKY+G; for COI 2nd position, HKY+I+G; for COI 3rd positions, GTR+I+G. Two independent runs of four Markov chains were conducted for 10 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), and the first 50001 trees were discarded based on the results.

Results

Family Mesogammaridae Bousfield, 1977

Eoniphargus Uéno, 1955

Japanese name: Chikayokoebi-zoku

Neoniphargus (Eoniphargus) Uéno, 1955: 148.

Eoniphargus: Straškraba, 1964, 136, 138; Straškraba 1967, 127; Bousfield 1977, 301; Barnard and Barnard 1983, 581–582; Holsinger 1994, 157; Tomikawa et al. 2007, 646.

Diagnosis

Head without eyes, rostrum short, inferior antennal sinus distinct. Pleonites 1–3 with dorsal setae, lacking processes. Urosomite 2 with dorsal robust setae. Antenna 1 longer than antenna 2; accessory flagellum three- or four-articulate. Male antenna 2 with calceoli. Mandible with triturative molar with seta; left incisors five- or six-dentate; left lacinia mobilis four- or five-dentate; palp article 3 with A-, D- and E-setae. Maxilla 1 with inner plate bearing plumose setae; outer plate with 11 serrate robust setae. Inner plate of maxilla 2 with oblique row of facial setae. Gnathopods subchelate. Coxa of pereopod 4 with posterior concavity. Coxal gills with stalks, on gnathopod 2 and pereopods 3–6. Uropod 1 with basofacial robust setae on peduncle. Uropod 3 with small, scale-like inner ramus; outer ramus one- or two-articulate. Telson cleft with apical robust setae.

Type species

Neoniphargus (Eoniphargus) kojimai Uéno, 1955, original designation.

Remarks

Eoniphargus is morphologically similar to Octopupilla in the presence of pleonites without dorsal processes, five-dentate incisor of the left mandible, mandibular palp article 3 with A-setae, inner plate of maxilla 2 with oblique row of facial setae, stalked coxal gills, peduncle of uropod 1 with basofacial robust setae, and telson lobes tapering distally. However, Eoniphargus differs from Octopupilla in the following features (features of Octopupilla in parentheses): eyes absent (rudimentary eyes), incisor of right mandible five- to six-dentate (four-dentate), inner lobes of the lower lip absent (vestigial), and inner ramus of uropod 3 shorter than 0.3 × the outer ramus (0.7 ×). Eoniphargus also resembles Indoniphargus Straškraba, 1967, but the familial affiliation of the latter has been controversial. Tomikawa et al. (2007) and Fišer et al. (2013) included Indoniphargus in Mesogammaridae. Sidorov et al. (2018) regarded Indoniphargus as a member of Austroniphargidae Iannilli, Krapp, & Ruffo, 2011. However, since the taxonomic position of Indoniphargus is beyond the scope of this paper, it will not be discussed here further. Eoniphargus is distinguished from Indoniphargus by the presence of facial setae in an oblique row of the inner plate of maxilla 2 (absent in Indoniphargus) and the elongate propodi of gnathopods 1 and 2 (mitten-form in Indoniphargus).

Eoniphargus toriii sp. nov.

https://zoobank.org/2A3DFB47-9FEC-46E4-A003-923CCD93E20D
Figs 2, 3, 4, 5 New Japanese name: Torii-chikayokoebi

Eoniphargus kojimai: Tomikawa et al. 2007, 647, figs 2–6.

Type material

Holotype : ♂ 4.3 mm (NSMT-Cr 16652), Seto River (34.880555°N, 138.218888°E), Terajima, Fujieda, Shizuoka Prefecture, Japan, collected by T. Torii on 3 June 2004. Paratypes:2 ♀♀, 4.6 mm and 4.3 mm (NSMT-Cr 16653 and 16654), data same as for the holotype.

Diagnosis

Urosomite 3 without dorsal setae. Epimeral plates 2–3 without ventral setae. Peduncular article 1 of antenna 1 with robust seta on posterodistal corner. Antenna 2 with peduncular article 2 gland cone not exceeding end of article 3; calceoli present in male. Mandible with 5-dentate left incisor; left lacinia mobilis 4-dentate, right one bifid with three or four teeth. Inner plate of maxilla 1 with six plumose setae. Inner plate of maxilla 2 with seven facial seta in oblique row. Peduncle of pleopod 3 with seta. Uropod 1 with peduncle bearing basofacial robust setae. Uropod 3 with inner ramus 0.3 times as long as outer ramus; outer ramus two-ariticulate, with plumose setae on medial margin. Telson length 1.1 times width, cleft for 67% of length.

Etymology

The new species is named after Dr. Takaaki Torii, who collected the species.

Description

Male holotype, NSMT-Cr 16652. Head (Fig. 2A) as long as pereonites 1 and 2 combined; eyes absent; inferior antennal sinus distinct with rounded angle. Dorsal margins of pleonites 1–3 (Figs 2A, 5A–C) each with four setae. Epimeral plates 1–3 (Figs 2A, 5N–P) with weakly pointed posterodistal corners; ventral margins without setae; posterior margins of plates 1–3 with one, zero, one seta, respectively. Dorsal margins of urosomites 1 and 2 with robust seta, urosomite 3 dorsally bare.

Figure 2. 

Eoniphargus toriii sp. nov., male holotype (NSMT-Cr 16652), female paratype (NSMT-Cr 16653) A habitus, lateral view B upper lip, anterior view C left mandible, medial view D incisor and lacinia mobilis of left mandible, medial view E and F incisor and lacinia mobilis of right mandible, medial view G lower lip, anterior view H maxilla 1, dorsal view I outer plate of maxilla 1, dorsal view J palp of maxilla 1, dorsal view K maxilla 2, dorsal view L maxilliped, dorsal view M palp article 4 of maxilliped, dorsal view N outer plate of maxilliped, dorsal view O, P inner plate of maxilliped, dorsal view F, P female; others, male. Modified from Tomikawa et al. (2007).

Antenna 1 (Figs 2A, 3A) length 0.6 times as long as body; length ratio of peduncular articles 1–3 in 1.0 : 0.8 : 0.4; posterodistal corner of peduncular article 1 with robust seta; accessory flagellum three-articulate (Fig. 3B); primary flagellum with approximately 22 articles, each article with one aesthetasc (Fig. 3C). Antenna 2 (Figs 2A, 3D) length 0.6 times as long as antenna 1; peduncular article 4 length 0.9 times as long as article 5; right peduncular article 5 with calceoli, left one lacking calceoli; flagellum 16-articulate, with calceoli (Fig. 3E).

Figure 3. 

Eoniphargus toriii sp. nov., male holotype (NSMT-Cr 16652), female paratype (NSMT-Cr 16653) A antenna 1, medial view B accessory flagellum of antenna 1, medial view C aesthetasc on flagellar article of antenna 1, medial view D antenna 2 (some flagellar articles are omitted), medial view E calceolus on flagellar article of antenna 2, medial view F antenna 1 (female, some flagellar articles are omitted), medial view G accessory flagellum of antenna 1 (female), medial view H aesthetasc on flagellar article of antenna 1 (female), medial view I antenna 2 (female, some flagellar articles are omitted), medial view J gnathopod 1, lateral view K palmar margin and dactylus of gnathopod 1 (some setae are omitted), lateral view L gnathopod 2, lateral view M palmar margin and dactylus of gnathopod 2 (some setae are omitted), lateral view N gnathopod 1 (female), lateral view O palmar margin and dactylus of gnathopod 1 (female), lateral view P gnathopod 2 (female), lateral view Q palmar margin and dactylus of gnathopod 2 (female, some setae are omitted), lateral view F–H, N–Q female; others, male. Modified from Tomikawa et al. (2007).

Upper lip (Fig. 2B) with rounded apical margin bearing fine setae. Mandibles (Fig. 2C–E) with left and right incisors comprising five and five–six teeth, respectively; left lacinia mobilis comprising four teeth, right lacinia mobilis bifid with three teeth; molar process triturative with plumose seta; length ratio of palp articles 1–3 in 1.0 : 2.4 : 1.9; palp article 1 without setae; palp article 2 with eight marginal setae; palp article 3 with pair of A-setae, many D-setae and E-setae, lateral face with fine setae. Lower lip (Fig. 2G) lacking inner lobes; outer lobes broad, shoulder rounded, with fine setae. Maxilla 1 (Fig. 2H–J) with triangular inner lobe, bearing six plumose setae on medial margin; outer plate rectangular, with 11 serrate robust setae apically; palp 2-articulate, article 1 without setae, article 2 with six robust setae apically. Maxilla 2 (Fig. 2K) with inner plate bearing seven plumose setae in oblique row. Maxilliped (Fig. 2L–O) with inner plate reaching end of palp article 1, subquadrate, bearing three subapical robust setae and medial robust seta; outer plate ovate, reaching middle of palp article 2, with row of robust setae along apical to medial margins; palp 4-articulate, article 2 longest with medial setae, nail of article 4 distinct.

Gnathopod 1 (Fig. 3J, K) with subquadrate coxa bearing setae along anterior to ventral margins; anterior and posterior margins with long setae; carpus length 0.9 times as long as length of propodus and 1.5 times width of carpus; propodus length 1.9 times width, palmar margin weakly serrate, oblique, with three medial and two lateral robust setae; dactylus not reaching posterodistal corner of propodus. Gnathopod 2 (Fig. 3L, M) with coxa expanded proximally bearing setae along anterior to ventral margins; posterior margin of basis with long setae; carpus length 1.2 times as long as length of propodus and 2.8 times width of carpus; propodus length 2.4 times width, palmar margin weakly serrate, almost vertical, with two medial and two lateral robust setae; dactylus not reaching posterodistal corner of propodus.

Pereopod 3 (Fig. 4A, B) with subquadrate coxa, proximally expanded, bearing setae along anterior to ventral margins; posterior margin of basis with long setae. Pereopod 4 (Fig. 4D, E) with coxa bearing setae along anterior to ventral margins, posteroproximally concave; posterior margin of basis with long setae. Pereopod 5 (Fig. 4G, H) with bilobed coxa bearing seta on anterior and posterior lobes; anterior margin of basis with robust setae, posterodistal corner weakly lobate. Pereopod 6 (Fig. 4J, K) with bilobed coxa bearing seta on posterior lobe; basis ovate, with robust setae on anterior margin, posterodistal corner weakly lobate. Pereopod 7 (Fig. 4M) with subtriangular coxa bearing seta on posterior margin; basis elliptical, with robust setae on anterior margin, posterodistal corner weakly lobate.

Figure 4. 

Eoniphargus toriii sp. nov., male holotype (NSMT-Cr 16652), female paratype (NSMT-Cr 16653) A pereopod 3, lateral view B dactylus of pereopod 3, lateral view C coxa of pereopod 3 (female) , lateral view D pereopod 4, lateral view E dactylus of pereopod 4, lateral view F coxa of pereopod 4 (female) , lateral view G pereopod 5, lateral view H dactylus of pereopod 5, lateral view I coxa–merus of pereopod 5 (female), lateral view J pereopod 6, lateral view K dactylus of pereopod 6, lateral view L coxa–merus of pereopod 6 (female) , lateral view M coxa–merus of pereopod 7, lateral view N coxa–merus of pereopod 7 (female), lateral view C, F, I, L, N female; others, male. Modified from Tomikawa et al. (2007).

Coxal gills (Figs 3L, 4A, D, G, J) ovate with stalks on gnathopod 2 and pereopods 3–6.

Pleopods 1–3 (Fig. 5G–K) with peduncles longer than rami, bearing distal seta on pleopod 2 and proximal seta on pleopod 3; retinacula paired (Fig. 5H); mediobasal margin of inner ramus with bifid plumose setae (Fig. 5I); rami well developed.

Figure 5. 

Eoniphargus toriii sp. nov., male holotype (NSMT-Cr 16652), female paratype (NSMT-Cr 16653) A–C dorsal margins of pleonites 1–3, dorsal views D–F dorsal margins of urosomites 1–3, dorsal views G pleopod 1 (plumose setae on rami are omitted), medial view H retinacula of pleopod 1, medial view I bifid setae of pleopod 1, medial view J pleopod 2 (plumose setae on rami are omitted), medial view K pleopod 3 (plumose setae on rami are omitted), medial view L pleopod 1 (female, rami are omitted), medial view M pleopod 2 (female, rami are omitted), lateral view N–P epimeral plates 1–3, lateral views Q epimeral plate 2 (female), lateral view R uropod 1, dorsal view S uropod 2, dorsal view T uropod 2 (female) , dorsal view U uropod 3, ventral view V terminal article of outer ramus of uropod 3, ventral view W uropod 3 (female), ventral view X telson, dorsal view Y telson (female), dorsal view L, M, Q, T, W, Y female; others, male. Modified from Tomikawa et al. (2007).

Uropod 1 (Fig. 5R) with peduncle bearing three basofacial robust setae and dorsal robust setae; inner ramus length 0.7 times as long as peduncle, with two medial and one lateral robust setae, and ventroproximal robust seta; outer ramus length 0.9 times as long as inner ramus, with two robust setae on medial margin. Uropod 2 (Fig. 5S) with peduncle bearing dorsal robust setae; inner ramus length 0.8 times as long as peduncle, bearing two medial robust setae and two ventroproximal slender setae; outer ramus length 0.6 times as long as inner ramus, without marginal setae. Uropod 3 (Fig. 5U, V) with peduncle length 0.3 times as long as outer ramus, bearing bent robust setae on distal edge; inner ramus length 0.3 times as long as outer ramus, with two robust setae on medial margin; outer ramus 2-articulate, proximal article with plumose setae on medial margin and robust setae on medial and lateral margins, terminal article length 0.3 times as long as proximal article with subapical setae.

Telson (Fig. 5X) length 1.1 times width, each lobe with two lateral and three apical robust setae and two dorsolateral penicillate setae, cleft for 67%.

Female paratype, NSMT-Cr 16653. Antenna 1 (Fig. 3F–H) with peduncles 1–3 of which length ratio in 1.0 : 0.8 : 0.5; accessory flagellum 4-articulate (Fig. 3G). Antenna 2 (Fig. 3I) without calceoli.

Incisor of right mandible 6-dentate (Fig. 2F); right lacinia mobilis 4-dentate, weakly bifid. Apical robust setae of inner plate of maxilliped stiffer than those of male (Fig. 2P).

Gnathopod 1 (Fig. 3N, O) with carpus length 1.8 times width; length of propodus 2.0 times width; dactylus reaching posterodistal corner of propodus. Gnathopod 2 (Fig. 3P, Q) with dactylus reaching posterodistal corner of propodus.

Brood plates (Figs 3P, 4I) narrow, lacking setae, on gnathopod 2 and pereopods 3–5.

Peduncles of pleopods 1 and 3 with three and one setae, respectively (Fig. 5L, M).

Uropod 2 (Fig. 5T) with inner ramus bearing two medial and one lateral robust setae; outer ramus with medial robust seta. Uropod 3 (Fig. 5W) with terminal article of outer ramus length 0.2 times as long as proximal article.

Telson (Fig. 5Y) as long as wide, cleft for 71%.

Distribution and environment

The species is known only from its type locality in the Seto River, Shizuoka Prefecture, Japan. Specimens were collected at a depth of 20 cm, from the bank of the river.

Remarks

Eoniphargus toriii sp. nov. is similar to E. kojimai and E. iwataorum sp. nov., with a head bearing deep antennal sinus, antenna 1 peduncular article 1 with a robust seta on the posterodistal corner, antenna 2 peduncular article 2 with a gland cone not exceeding peduncular article 3, uropod 1 peduncle with basofacial robust setae, and uropod 3 with 2-articulate outer ramus. However, E. toriii sp. nov. differs from E. kojimai and E. iwataorum sp. nov. in the following features: right mandible with lacinia mobilis bearing three or four teeth (more than four teeth in E. kojimai and E. iwataorum sp. nov.) and maxilla 1 having six medial setae on the inner plate (eight medial setae in E. kojimai and E. iwataorum sp. nov.).

Eoniphargus iwataorum sp. nov.

https://zoobank.org/4234A07E-026A-4DB1-A2E8-2DE845E6295E
Figs 6, 7, 8 New Japanese name: Iwata-chikayokoebi

Type material

Holotype : ♀ 5.6 mm (NSMT-Cr 30782), Sabi River (36.898181°N, 140.012153°E), Imaizumi, Ohtawara, Tochigi Prefecture, Japan, collected by Y. Iwata on 27 January 2019. Paratype: ♀ 5.3 mm (NSMT-Cr 30783; G1751), ♀ 6.0 mm (NSMT-Cr 30784), data same as for the holotype.

Diagnosis

Urosomite 3 without dorsal setae. Epimeral plates 2–3 each with ventral robust seta. Peduncular article 1 of antenna 1 with robust seta on posterodistal corner. Antenna 2 with peduncular article 2 gland cone not exceeding end of article 3. Mandible with 5-dentate left incisor; left lacinia mobilis 4-dentate, right one bifid with many teeth. Inner plate of maxilla 1 with eight plumose setae. Inner plate of maxilla 2 with seven facial seta in oblique row. Peduncle of pleopod 3 with seta. Uropod 1 with peduncle bearing basofacial robust setae. Uropod 3 with inner ramus 0.25 times as long as outer ramus; outer ramus 2-ariticulate, with plumose setae on medial margin. Telson almost as long as width, cleft for 67% of length.

Etymology

The specific epithet was derived from the names of Mr. Yasuyuki Iwata and Mr. Tomofumi Iwata, who collected the specimens of this new species.

Description

Female holotype, NSMT-Cr 30782. Head (Fig. 6A) as long as pereonites 1 and 2 combined; eyes absent; inferior antennal sinus distinct with rounded angle. Dorsal margins of pleonites 1–3 (Fig. 6B–D) each with four setae. Posterodistal corners of epimeral plates 1–3 (Fig. 6E–G) weakly produced; ventral margin of epimeral plate 1 without setae, epimeral plates 2 and 3 each with ventral robust seta; posterior margins of epimeral plates 1–3 each with two setae. Dorsal margin of urosomite 1 (Fig. 6H) with slender setae; urosomite 2 (Fig. 6I) with dorsal robust setae; urosomite 3 (Fig. 6J) without dorsal setae.

Figure 6. 

Eoniphargus iwataorum sp. nov., female holotype (NSMT-Cr 30782) A head, lateral view B–D dorsal margins of pleonites 1–3, dorsal views E–G epimeral plates 1–3, lateral views H–J dorsal margins of urosomites 1–3, dorsal views K antenna 1 (some flagellar articles are omitted), lateral view L antenna 2 (some flagellar articles are omitted), lateral view M upper lip, anterior view N right mandible, medial view O incisor and lacinia mobilis of right mandible, medial view P incisor and lacinia mobilis of left mandible, lateral view Q lower lip, anterior view R maxilla 1, dorsal view S apical robust setae on outer plate of maxilla 1, dorsal view T maxilla 2, dorsal view.

Antenna 1 (Fig. 6K) length 0.25 times as long as body; length ratio of peduncular articles 1–3 in 1.0 : 0.8 : 0.4; posterodistal corner of peduncular article 1 with robust seta; accessory flagellum 4-articulate; primary flagellum 27-articulate. Antenna 2 (Fig. 6L) length 0.7 times as long as antenna 1; peduncular article 4 slightly shorter than article 5; flagellum 22-articulate; calceoli absent.

Upper lip (Fig. 6M) with rounded apical margin bearing fine setae. Mandibles (Fig. 6N–P) with left and right incisors comprising five and six teeth, respectively; left lacinia mobilis comprising four teeth, right lacinia mobilis bifid with many teeth; molar process triturative with plumose seta; length ratio of palp articles 1–3 in 1.0 : 2.0 : 1.5; palp article 1 without setae; palp article 2 with eight marginal setae; palp article 3 with pair of A-setae, several D-setae and E-setae, lateral face with fine setae. Lower lip (Fig. 6Q) lacking inner lobes; outer lobes broad, shoulder rounded, with fine setae. Maxilla 1 (Fig. 6R, S) with elliptical inner lobe, bearing eight plumose setae on medial margin; outer plate rectangular, with 11 serrate robust setae apically; palp 2-articulate, article 1 without setae, article 2 with five robust setae and slender plumose seta apically. Maxilla 2 (Fig. 6T) with inner plate bearing seven plumose setae in oblique row. Maxilliped (Fig. 7A–C) with inner plate exceeding end of palp article 1, subquadrate, bearing three subapical robust setae and medial robust seta; outer plate ovate, reaching middle of palp article 2, with row of robust setae along apical to medial margins; palp 4-articulate, article 2 longest with medial setae, nail of article 4 distinct.

Figure 7. 

Eoniphargus iwataorum sp. nov., female holotype (NSMT-Cr 30782) A maxilliped, dorsal view B inner plate of maxilliped, dorsal view C outer plate of maxilliped, dorsal view D gnathopod 1, lateral view E palmar margin and dactylus of gnathopod 1, medial view F gnathopod 2, lateral view G palmar margin and dactylus of gnathopod 2, medial view H pereopod 3, lateral view I dactylus of pereopod 3, lateral view J pereopod 4, lateral view K pereopod 5, lateral view L pereopod 6, lateral view.

Gnathopod 1 (Fig. 7D, E) with subquadrate coxa bearing setae along anterior to ventral margins; anterior and posterior margins of basis with long setae; carpus length 0.9 times as long as length of propodus and 1.4 times width of carpus; propodus length 1.7 times width, palmar margin weakly serrate, oblique, with three medial and one lateral robust setae; dactylus reaching posterodistal corner of propodus. Gnathopod 2 (Fig. 7F, G) with coxa expanded proximally bearing setae along anterior to ventral margins; posterior margin of basis with long setae; carpus length 1.2 times as long as length of propodus and 2.8 times width of carpus; propodus length 2.2 times width, palmar margin weakly serrate, almost vertical, with two medial and two lateral robust setae; dactylus reaching posterodistal corner of propodus.

Pereopod 3 (Fig. 7H, I) with subquadrate coxa, proximally expanded, bearing setae along anterior to ventral margins; posterior margin of basis with long setae. Pereopod 4 (Fig. 7J) with coxa bearing setae along anterior to ventral margins, posteroproximally concave; posterior margin of basis with long setae. Pereopod 5 (Fig. 7K) with bilobed coxa bearing seta on posterior lobe; anterior margin of basis with robust setae, posterodistal corner weakly lobate. Pereopod 6 (Fig. 7L) with bilobed coxa bearing seta on anterior and posterior lobes; basis with robust setae on anterior margin, posterodistal corner weakly lobate. Pereopod 7 (Fig. 8A–C) with elliptical basis bearing robust setae on anterior margin, posterodistal corner weakly lobate.

Figure 8. 

Eoniphargus iwataorum sp. nov., female holotype (NSMT-Cr 30782) A, B pereopod 7, lateral views C dactylus of pereopod 3, lateral view D coxal gill 4, lateral view E coxal gill 6, lateral view F peduncle of pleopod 1, lateral view G pleopod 2, medial view H peduncle of pleopod 3, lateral view I retinacula of pleopod 2, medial view J uropod 1, dorsal view K uropod 2, dorsal view L uropod 3, ventral view M telson, dorsal view.

Coxal gills (Fig. 8D, E) ovate with stalks on gnathopod 2 and pereopods 3–6.

Pleopods 1–3 (Fig. 8F–I) with peduncles longer than rami, bearing distal seta on pleopods 1 and 2 and proximal seta on pleopod 3; retinacula paired (Fig. 8I); mediobasal margin of inner ramus with bifid plumose setae; rami well developed.

Uropod 1 (Fig. 8J) with peduncle bearing three basofacial robust setae and dorsal robust setae; inner ramus length 0.7 times as long as peduncle, with two medial and one lateral robust setae, and ventroproximal seta; outer ramus length 0.9 times as long as inner ramus, with two robust setae on medial margin. Uropod 2 (Fig. 8K) with peduncle bearing dorsal robust setae; inner ramus as long as peduncle, bearing two medial robust setae and ventroproximal slender seta; outer ramus length 0.7 times as long as inner ramus, without marginal setae. Uropod 3 (Fig. 8L) with peduncle length 0.3 times as long as outer ramus, bearing robust setae on distal edge; inner ramus length 0.2 times as long as outer ramus, with seta on medial margin; outer ramus 2-articulate, proximal article with plumose setae on medial margin and robust setae on medial and lateral margins, terminal article length 0.2 times as long as proximal article with subapical setae.

Telson (Fig. 8M) length slightly shorter than wide, each lobe with two lateral and three apical robust setae and two dorsolateral penicillate setae, cleft for 67%.

Distribution and environment

The species is known only from its type locality in the Sabi River, Tochigi Prefecture, Japan.

Remarks

Eoniphargus iwataorum sp. nov. is similar to E. kojimai but differs from the latter in the following features (features of E. kojimai in parentheses): urosomite 3 without robust setae on dorsal margin (bearing robust setae), maxilla 2 with inner plate bearing seven setae in oblique row (nine setae), and uropod 2 without robust seta on lateral margin of outer ramus (bearing robust seta).

Eoniphargus kojimai Uéno, 1955

Figs 9, 10, 11, 12 Japanese name: Kojima-chikayokoebi

Neoniphargus (Eoniphargus) kojimai Uéno, 1955: 148, figs 1–3.

Eoniphargus kojimai: Straškraba, 1964, 138; Straškraba 1967, 127; Bousfield 1977, 301; Barnard and Barnard 1983, 581.

Material examined

♀ 6.3 mm (NSMT-Cr 30785, G1905), ♂ 4.8 mm (NSMT-Cr 30786), Mamashita Spring (35.680066°N, 139.428283°E), Kunitachi, Tokyo, Japan, collected by K. Tomikawa on 25 June 2016. ♀ 5.0 mm (NSMT-Cr 30787, G 1930), ♀ 4.5 mm (NSMT-Cr 30788, G 1931), Hinochūōtoshokan Spring (35.655783°N, 139.382133°E), Hino, Tokyo, Japan, collected by K. Tomikawa on 25 June 2016.

Diagnosis

Urosomite 3 with dorsal robust setae. Epimeral plates 2–3 each with ventral robust seta. Peduncular article 1 of antenna 1 with robust seta on posterodistal corner. Antenna 2 with peduncular article 2 gland cone not exceeding end of article 3; calceoli present in male. Mandible with 5- or 6-dentate left incisor; 4- or 5-dentate left lacinia mobilis, right one bifid with many teeth. Inner plate of maxilla 1 with eight plumose setae. Inner plate of maxilla 2 with nine facial seta in oblique row. Peduncle of pleopod 3 without seta. Uropod 1 with peduncle bearing basofacial robust setae. Uropod 3 with inner ramus 0.25 times as long as outer ramus in female and 0.27 times in male; outer ramus 2-ariticulate, with plumose setae on medial margin. Telson length 0.9 times width, cleft for 69% of length.

Description

Female (NSMT-Cr 30785). Head (Fig. 9A) as long as pereonites 1 and 2 combined; eyes absent; inferior antennal sinus distinct with rounded angle. Dorsal margins of pleonites 1–3 (Fig. 9B–D) each with 4 setae. Posterodistal corners of epimeral plates 1–3 (Fig. 9E–G) weakly produced; ventral margin of epimeral plate 1 without setae, epimeral plates 2 and 3 each with ventral robust seta; posterior margins of epimeral plates 1–3 each with two setae. Dorsal margin of urosomite 1 (Fig. 9H) with slender setae and robust seta; urosomites 2 and 3 (Fig. 9I, J) with pair of dorsal robust setae.

Figure 9. 

Eoniphargus kojimai Uéno, 1955, female (NSMT-Cr 30785) A head, lateral view B–D dorsal margins of pleonites 1–3, dorsal views E–G epimeral plates 1–3, lateral views H–J dorsal margins of urosomites 1–3, dorsal views K antenna 1 (some flagellar articles are omitted), lateral view L antenna 2 (some flagellar articles are omitted), medial view M upper lip, posterior view N right mandible, medial view O incisor and lacinia mobilis of left mandible, medial view P incisor and lacinia mobilis of right mandible, medial view Q lower lip, anterior view R maxilla 1, dorsal view S apical robust setae on outer plate of maxilla 1, dorsal view T maxilla 2, dorsal view.

Antenna 1 (Fig. 9K) length 0.22 times as long as body; length ratio of peduncular articles 1–3 in 1.0 : 0.8 : 0.4; posterodistal corner of peduncular article 1 with robust seta; accessory flagellum 4-articulate; primary flagellum 26-articulate. Antenna 2 (Fig. 9L) length 0.6 times as long as antenna 1; peduncular article 4 length 0.9 times as long as article 5; flagellum 13-articulate; calceoli absent.

Upper lip (Fig. 9M) with rounded apical margin bearing fine setae. Mandibles (Fig. 9N–P) with left and right incisors comprising five and six teeth, respectively; left lacinia mobilis comprising four teeth, right lacinia mobilis bifid with many teeth; molar process triturative with plumose seta; length ratio of palp articles 1–3 in 1.0 : 2.3 : 1.7; palp article 1 without setae; palp article 2 with nine marginal setae; palp article 3 with pair of A-setae, several D-setae and three E-setae, lateral face with fine setae. Lower lip (Fig. 9Q) lacking inner lobes; outer lobes broad, shoulder rounded, with fine setae. Maxilla 1 (Fig. 9R, S) with subtriangular inner lobe, bearing eight plumose setae on medial margin; outer plate rectangular, with 11 serrate robust setae apically; palp 2-articulate, article 1 without setae, article 2 with three robust setae and slender plumose seta along apically to medial margins. Maxilla 2 (Fig. 9T) with inner plate bearing nine plumose setae in oblique row. Maxilliped (Fig. 10A–C) with inner plate exceeding end of palp article 1, subquadrate, bearing three subapical robust setae and medial robust seta; outer plate ovate, reaching middle of palp article 2, with row of robust setae along apical to medial margins; palp 4-articulate, article 2 longest with medial setae, nail of article 4 distinct.

Figure 10. 

Eoniphargus kojimai Uéno, 1955, female (NSMT-Cr 30785) A maxilliped, dorsal view B inner plate of maxilliped, dorsal view C outer plate of maxilliped, dorsal view D gnathopod 1, lateral view E palmar margin and dactylus of gnathopod 1, medial view F gnathopod 2, lateral view G palmar margin and dactylus of gnathopod 2, medial view H pereopod 3, lateral view I dactylus of pereopod 3, lateral view J pereopod 4, lateral view K pereopod 5, lateral view.

Gnathopod 1 (Fig. 10D, E) with subquadrate coxa bearing setae along anterior to ventral margins; anterior and posterior margins of basis with long setae; carpus length 1.1 times as long as length of propodus and 1.7 times width of carpus; propodus length 1.9 times width, palmar margin weakly serrate, oblique, with three medial and two lateral robust setae; dactylus almost reaching posterodistal corner of propodus. Gnathopod 2 (Fig. 10F, G) with coxa expanded proximally bearing setae along anterior to ventral margins; posterior margin of basis with long setae; carpus length 1.2 times as long as length of propodus and 3.3 times width of carpus; propodus length 2.8 times width, palmar margin weakly serrate, almost vertical, with two medial and one lateral robust setae; dactylus reaching posterodistal corner of propodus.

Pereopod 3 (Fig. 10H, I) with subquadrate coxa, proximally expanded, bearing setae along anterior to ventral margins; anteroproximal and posterior margins of basis with long setae. Pereopod 4 (Fig. 10J) with coxa bearing setae along anterior to ventral margins, posteroproximally concave; anteroproximal and posterior margins of basis with long setae. Pereopod 5 (Fig. 10K) with bilobed coxa bearing seta on anterior and posterior lobes; anterior margin of basis with robust setae, posterodistal corner weakly lobate. Pereopod 6 (Fig. 11A) with bilobed coxa bearing two setae on posterior lobe; basis with robust setae on anterior margin, posterodistal corner weakly lobate. Pereopod 7 (Fig. 11B) with elliptical basis bearing robust setae on anterior margin, posterodistal corner weakly lobate.

Figure 11. 

Eoniphargus kojimai Uéno, 1955, female (NSMT-Cr 30785) A pereopod 6, lateral view B pereopod 7, lateral views C pleopod 1, lateral view D retinacula of pleopod 1, lateral view E peduncle of pleopod 2, medial view F peduncle of pleopod 3 G coxal gill 4, lateral view H oostegite of pereopod 4, medial view I uropod 1, dorsal view J uropod 2, dorsal view K uropod 3, dorsal view L telson, dorsal view.

Coxal gills (Fig. 11G) ovate with stalks on gnathopod 2 and pereopods 3–6.

Pleopods 1–3 (Fig. 11C–F) with peduncles longer than rami, bearing distal seta on pleopod 1; retinacula paired (Fig. 11D); mediobasal margin of inner ramus with bifid plumose setae; rami well developed.

Uropod 1 (Fig. 11I) with peduncle bearing three basofacial robust setae and dorsal robust setae; inner ramus length 0.6 times as long as peduncle, with two medial robust setae and two ventroproximal setae; outer ramus length 0.9 times as long as inner ramus, with two medial and one lateral robust setae. Uropod 2 (Fig. 11J) with peduncle bearing dorsal robust setae; inner ramus as long as peduncle, bearing two medial and one lateral robust setae; outer ramus length 0.7 times as long as inner ramus, with lateral robust seta. Uropod 3 (Fig. 11K) with peduncle length 0.3 times as long as outer ramus, bearing robust setae on distal edge; inner ramus length 0.3 times as long as outer ramus, with seta on medial margin; outer ramus 2-articulate, proximal article with plumose setae on medial margin and robust setae on medial and lateral margins, terminal article length 0.2 times as long as proximal article with subapical setae.

Telson (Fig. 11L) length slightly shorter than width, each lobe with two lateral and three apical robust setae, and two dorsolateral and 1 subapical penicillate setae, cleft for 69%.

Male (NSMT-Cr 30786). Antenna 1 (Fig. 12A) length 0.24 times as long as body length; accessory flagellum 3-articulate; primary flagellum 25-articulate. Antenna 2 (Fig. 12B) length 0.6 times as long as antenna 1; peduncular article 4 length 0.8 times as long as peduncular article 5; peduncular article 5 and flagellum with calceoli (Fig. 12C); flagellum 16-articulate. Gnathopod 1 (Fig. 12D, E) with carpus length 1.2 times as long as length of propodus and 1.6 times width of carpus; propodus length 1.5 times width. Gnathopod 2 (Fig. 12F, G) with carpus length 1.1 times as long as length of propodus and 2.3 times width of carpus; propodus length 2.2 times width, palmar margin with two medial and two lateral robust setae. Uropod 3 (Fig. 12H) with peduncle length 0.4 times as long as outer ramus, peduncle bearing bent robust setae on distal edge.

Figure 12. 

Eoniphargus kojimai Uéno, 1955, male (NSMT-Cr 30786) A antenna 1 (some flagellar articles are omitted), lateral view B antenna 2 (some flagellar articles are omitted), lateral view C calceolus on flagellar article of antenna 2, medial view D gnathopod 1, lateral view E palmar margin and dactylus of gnathopod 1, medial view F gnathopod 2, lateral view G palmar margin and dactylus of gnathopod 2, medial view H uropod 3, dorsal view.

Distribution and environment

This species has been found in interstitial waters in Tokyo: the sand-filter bed of the Komae Purification Plant near the Tama River (Uéno 1955); two springs — Mamashita Spring, Kunitachi and Hinochūōtoshokan Spring, Hino (this study).

Remarks

The present specimens conform with the original description of E. kojimai by Uéno (1955). However, our specimens differed from the original description in the left mandible with 4-dentate lacinia mobilis and maxilla 1 with 11 serrate robust setae on the outer plate, which was 6-dentate incisor and 5-dentate lacinia mobilis of the left mandible, and 10 serrate robust setae in Uéno’s (1955) description. The exact number of these setae is difficult to ascertain because they are minute and overlap. This suggests that he may have misstated the number of setae. Unfortunately, the type specimen of this species is believed to be lost (Tomikawa et al. 2007), so the character could not be verified. Examination of the present material from Kunitachi and Hino, Tokyo revealed some features that were not mentioned in the original description: maxilla 2 with inner plate bearing nine plumose setae in oblique row, uropod 1 peduncle with facial robust setae, and maxilliped inner plate with three subapical robust setae and medial robust seta.

Key to species of Eoniphargus

1 Antennal sinus shallow; antenna 1 peduncular article 1 with slender seta on posterodistal corner; antenna 2 peduncular article 2 with elongate gland cone exceeding peduncular article 3; uropod 1 peduncle without basofacial robust setae; uropod 3 with uniarticulate outer ramus E. glandulatus Stock & Jo, 1990
– Antennal sinus deep; antenna 1 peduncular article 1 with robust seta on posterodistal corner; antenna 2 peduncular article 2 with gland cone reaching distal end of peduncular article 3; uropod 1 peduncle with basofacial robust setae; uropod 3 with 2-articulate outer ramus 2
2 Right mandible with lacinia mobilis bearing 3 or 4 teeth; maxilla 1 with 6 medial setae on inner plate E. toriii sp. nov.
– Right mandible with bifid lacinia mobilis bearing many teeth; maxilla 1 with 8 medial setae on inner plate 3
3 Urosomite 3 with robust setae on dorsal margin; maxilla 2 with inner plate bearing 9 setae in oblique row; uropod 2 with robust seta on lateral margin of outer ramus E. kojimai (Uéno, 1955)
– Urosomite 3 without robust setae on dorsal margin; maxilla 2 with inner plate bearing 7 setae in oblique row; uropod 2 without robust seta on lateral margin of outer ramus E. iwataorum sp. nov.

Molecular phylogenetic analyses

The obtained ML tree showed a topology identical to that of the BI tree (Fig. 13). The monophyly of Mesogammaridae was shown with the highest support (100% bootstrap support [BS] and 1.0 Bayesian posterior probability [BPP] in the ML and BI trees, respectively). Mesogammaridae formed a monophyletic group with two gammarids genera, Barnardiorum Iwan & Löbl, 2007 and Rhipidogammarus Stock, 1971 (BS = 99%, BPP = 1.0). Within Mesogammaridae, the first split formed Mesogammarus and (Eoniphargus + Octopupilla) (BS = 69%, BPP = 1.0). Eoniphargus kojimai is sister to E. iwataorum sp. nov. (BS = 76%, BPP = 1.0). The uncorrected pairwise distances among the three Eoniphargus species are 16.9–19.8% for COI and 1.3% for 28S.

Figure 13. 

Maximum likelihood tree for 1550 bp of nuclear 28S rRNA and mitochondrial cytochrome c oxidase subunit I markers. Numbers at nodes represent maximum likelihood bootstrap values and Bayesian posterior probabilities (Values below 60% for the former and 0.8 for the latter are omitted).

Discussion

Although there are many taxonomic studies on the amphipods that occur in East Asian groundwaters (Akatsuka and Komai 1922; Derzhavin 1927; Uéno 1927, 1934), their species diversity is not fully understood. To date, studies on the amphipods in groundwater have been based on specimens from small streams in caves that are relatively easily accessible and incidentally collected from dug wells (Tomikawa et al 2008; Tomikawa and Nakano 2018). Recently, a variety of groundwater amphipods have been reported to occur in the interstitial waters of rivers and springs (e.g. Tomikawa et al. 2007), but little is known about their species diversity and evolutionary history.

In this study, we conducted a molecular phylogenetic analysis of Mesogammaridae, including one marine genus, Mesogammarus, and two subterranean genera, Eoniphargus and Octopupilla, and showed that they form a monophyletic group (Fig. 13). Our phylogenetic trees show that Mesogammarus was the first to diverge within Mesogammaridae, followed by Octopupilla and Eoniphargus. Mesogammarus occurs under coastal cobbles, Octopupilla in brackish and freshwater interstitial environments, and Eoniphargus only in freshwater groundwater (Tzvetkova 1965; Tomikawa et al. 2007). This suggests that species diversification in Mesogammaridae likely occurred through the adaptive dispersal of epigean marine ancestor into brackish and freshwater groundwater environments.

Mesogammaridae formed a monophyletic group with gammarid genera Barnardiorum and Rhipidogammarus. Barnardiorum occurs in epigean freshwaters in Tajikistan and Afghanistan, and Rhipidogammarus in brackish and freshwater groundwaters (interstitial waters) in the Mediterranean belt (Barnard and Barnard 1983; Özbek and Sket 2020). Hou et al. (2014) showed the Tethys Sea origin of Gammaridae and that geohistorical and environmental changes in the Tethys Sea facilitated the diversification of this group. Although the details of the evolutionary history of Mesogammaridae and related groups are not clear in this study, judging from the fact that their distribution extends from the North Pacific coast to Central Asia and the Mediterranean Sea, ancestral species that inhabited the coastal surface waters of the Tethys Sea may have diversified by adapting to different salinity and subterranean environments. Barnardiorum and Rhipidogammarus are presently treated as members of Gammaridae. Our results challenge their family affiliation, but the low statistical support for the results of the phylogenetic analyses in this study precluded further discussion of the monophyly of Gammaridae.

Prior to this study, two species of Eoniphargus, E. kojimai and E. glandulatus, were found in the interstitial waters in Japan and cave pools in the Korean Peninsula, respectively. In this study, we described two new species, E. iwataorum and E. toriii, found in the Kanto region of Japan, indicating that the species diversity of amphipods in interstitial water is higher than previously thought. Interestingly, Eoniphargus mainly occurs in interstitial waters and is rarely reported from the groundwater in caves. In contrast, the groundwater amphipod genus Pseudocrangonyx Akatsuka & Komai, 1922 is widely distributed in East Asian groundwater systems, and species of this genus often occur in cave groundwater (Lee and Min 2020). Speciation in subterranean environments is commonly a consequence of geographic isolation, as subterranean environments are always dark, oligotrophic, and ecological niches are narrow (Mammola and Isaia 2016; Mammola et al. 2018). Recently, however, the potential for diversification through niche differentiation has been demonstrated in Niphargus amphipods and Troglohyphantes spiders, even in energy-poor environments such as underground habitats (Fišer et al. 2013; Mammola et al. 2018). In Eoniphargus and Pseudocrangonyx, the choice between different groundwater environments, relatively open cave groundwater, and interstitial water may have played a role in the differentiation of the two genera.

Acknowledgements

We thank Takaaki Torii (Idea Consultants Inc.), Tomofumi Iwata (Toyama Science Museum), and Yasuyuki Iwata (Japan Institute of Insect and Fungal Damage to Cultural Properties) for providing materials. Thanks are also due to Drs Denis Copilaş-Ciocianu (Nature Research Centre, Vilnius, Lithuania) and Cene Fišer (University of Ljubljana), and an anonymous reviewer for their critical reading and valuable comments on our manuscript. This work was partly supported by the Japan Society for the Promotion of Science KAKENHI grants JP 21H00919, JP22H01011, and JP22K06373 to KT, and a grant from the Nakdonggang National Institute of Biological Resources (NNIBR) funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR202201101) to CL. We would like to thank Editage (www.editage.com) for English language editing.

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