Research Article |
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
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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.
Cryptic species, interstitial water, molecular phylogeny, new species, taxonomy
Compared to epigean ecosystems, subterranean environments are generally stable (
Eoniphargus Uéno, 1955 was established by
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.
Specimens of Eoniphargus were collected from four localities in Japan (Fig.
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 (
Genomic DNA was extracted from the appendage muscle of the specimens following procedures detailed by
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. |
|
Sabi River, Imaizumi, Ohtawara, Tochigi Prefecture, Japan (1) | LC709238* | LC709248* |
E. kojimai | G1750 | Mamashita Spring, Kunitachi, Tokyo, Japan (2) | LC709239* | LC709249* |
E. kojimai |
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Hinochūōtoshokan Spring, Hino, Tokyo, Japan (3) | LC709240* | LC709250* |
E. kojimai |
|
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 (
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 (
Neoniphargus (Eoniphargus) Uéno, 1955: 148.
Eoniphargus: Straškraba, 1964, 136, 138;
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.
Neoniphargus (Eoniphargus) kojimai Uéno, 1955, original designation.
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.
Eoniphargus kojimai:
Holotype
: ♂ 4.3 mm (
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.
The new species is named after Dr. Takaaki Torii, who collected the species.
Male holotype,
Eoniphargus toriii sp. nov., male holotype (
Antenna 1 (Figs
Eoniphargus toriii sp. nov., male holotype (
Upper lip (Fig.
Gnathopod 1 (Fig.
Pereopod 3 (Fig.
Eoniphargus toriii sp. nov., male holotype (
Coxal gills (Figs
Pleopods 1–3 (Fig.
Eoniphargus toriii sp. nov., male holotype (
Uropod 1 (Fig.
Telson (Fig.
Female paratype,
Incisor of right mandible 6-dentate (Fig.
Gnathopod 1 (Fig.
Brood plates (Figs
Peduncles of pleopods 1 and 3 with three and one setae, respectively (Fig.
Uropod 2 (Fig.
Telson (Fig.
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.
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.).
Holotype
: ♀ 5.6 mm (
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.
The specific epithet was derived from the names of Mr. Yasuyuki Iwata and Mr. Tomofumi Iwata, who collected the specimens of this new species.
Female holotype,
Eoniphargus iwataorum sp. nov., female holotype (
Antenna 1 (Fig.
Upper lip (Fig.
Eoniphargus iwataorum sp. nov., female holotype (
Gnathopod 1 (Fig.
Pereopod 3 (Fig.
Eoniphargus iwataorum sp. nov., female holotype (
Coxal gills (Fig.
Pleopods 1–3 (Fig.
Uropod 1 (Fig.
Telson (Fig.
The species is known only from its type locality in the Sabi River, Tochigi Prefecture, Japan.
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).
Neoniphargus (Eoniphargus) kojimai Uéno, 1955: 148, figs 1–3.
Eoniphargus kojimai: Straškraba, 1964, 138;
♀ 6.3 mm (
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.
Female (
Eoniphargus kojimai Uéno, 1955, female (
Antenna 1 (Fig.
Upper lip (Fig.
Eoniphargus kojimai Uéno, 1955, female (
Gnathopod 1 (Fig.
Pereopod 3 (Fig.
Eoniphargus kojimai Uéno, 1955, female (
Coxal gills (Fig.
Pleopods 1–3 (Fig.
Uropod 1 (Fig.
Telson (Fig.
Male (
Eoniphargus kojimai Uéno, 1955, male (
This species has been found in interstitial waters in Tokyo: the sand-filter bed of the Komae Purification Plant near the Tama River (
The present specimens conform with the original description of E. kojimai by
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. |
The obtained ML tree showed a topology identical to that of the BI tree (Fig.
Although there are many taxonomic studies on the amphipods that occur in East Asian groundwaters (
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.
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 (
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 (
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.