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Research Article
Integrative taxonomy revealed a new species of Lefua (Teleostei, Nemacheilidae) from Fukui Prefecture, Japan
expand article infoYuta Katayama‡, Naoto Sawada‡§
‡ Kyoto University, Kyoto, Japan
§ The University of Tokyo, Tokyo, Japan

Corresponding author: Naoto Sawada ( pb3277tcganma@yahoo.co.jp )

Academic editor: Danilo Harms
© 2024 Yuta Katayama, Naoto Sawada.
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: Katayama Y, Sawada N (2024) Integrative taxonomy revealed a new species of Lefua (Teleostei, Nemacheilidae) from Fukui Prefecture, Japan. Evolutionary Systematics 8(2): 247-260. https://doi.org/10.3897/evolsyst.8.131002
ZooBank: urn:lsid:zoobank.org:pub:6ADA06AA-DE1B-4E9B-BB78-CA17732AB226
Open Access

Abstract

Eight-barbel loaches belonging to the genus Lefua have diverged into seven species in freshwaters of East Asia. Recent studies have discovered a new population in the Kuzuryu River drainage system of Fukui Prefecture, central Japan. Based on the results of the genetic analyses and morphological comparisons, we describe this population as a new species, Lefua nishimurai sp. nov. Body width, interorbital width, orbit diameter, preanal length, snout length, and the newly examined head width greatly contributed to the discrimination between L. torrentis, L. tokaiensis and L. nishimurai sp. nov. The new species can be distinguished from other congeners by combining the following characteristics: 1) eyes positioned dorsally on the head; 2) a narrow conspicuous longitudinal mark between the base of the outer rostral barbel and the eye; 3) small dark spots on the body, dorsal, and caudal fins; 4) a small dorsal fin and eye diameter; and 5) black spots above and below the base of the caudal fin. Based on the phylogenetic relationships of the genus, L. nishimurai sp. nov. is estimated to have diverged early from its common ancestor in central Japan. Understanding the natural history of this new species and implementing conservation measures are crucial because of its narrow, fragmented distribution and presence in vulnerable habitats.

Key Words

endangered species, freshwater fish, inland water, nemacheilid loach, nuclear phylogeny, species richness

Introduction

Lefua Herzenstein, 1888, is a genus of eight-barbel loach belonging to the family Nemacheilidae, and its members are widely distributed in Mongolia, Russia, China, Korea, and Japan (Hosoya 2013; Kottelat 2013; Ito et al. 2019). The genus has been characterized by rounded caudal fins, elongated anterior nostrils forming long nasal barbels, and the absence of a postcleithrum and epural (Nichols 1943; Sawada 1982; Prokofiev 2005). The genus Lefua was originally established as a replacement name for the genus Octonema Herzenstein, 1887. Octonema was erected for Diplophysa costata Kessler, 1876, and O. pleskei Herzenstein, 1887, which are indigenous to Korea, China, and Russia, while its name was preoccupied by the piscine and coelenterate genera (Kottelat 2012). Subsequently, in the early 1900s, three species, L. nikkonis (Jordan & Fowler, 1903), L. echigonia Jordan & Richardson, 1907, and L. sayu (Herre & Lin, 1936), were described in China and Japan. After recent descriptions of two new species, L. torrentis Hosoya, Ito & Miyazaki, 2018 and L. tokaiensis Ito, Hosoya & Miyazaki, 2019, from Japan, seven species have been accepted in the genus (Ito et al. 2019).

Lefua species were mainly distinguished in the early 1900s based on a combination of the following characteristics: coloration of the caudal fin base, dorsal fin, and mature male body, as well as vertebral count and head size (Naseka and Bogutskaya 2004; Ito et al. 2019; Ohgita and Matsunuma 2020). Subsequently, Hosoya (1993) examined snout coloration, body elongation, and eye size in L. echigonia, and discovered an unidentified species (currently L. torrentis). Molecular phylogenetic relationships among Japanese Lefua species were clarified in several genetic studies in the 2000s (Sakai et al. 2003; Mihara et al. 2005; Miyazaki et al. 2007, 2011, 2018). These investigations also highlighted the genetic distinctiveness of L. torrentis and another genetic clade, presently identified as L. tokaiensis. These findings substantiated the monophyly of a clade comprising the Japanese L. echigonia, L. torrentis, and L. tokaiensis with both mitochondrial and nuclear loci. These two unidentified species were recently described based on detailed morphological comparisons that emphasized the importance of snout coloration and eye position (Hosoya et al. 2018; Ito et al. 2019). According to integrative analyses conducted in the 2000s, the species richness of Japanese Lefua and genetic relationships within the genus have been well elucidated.

The distribution of the Japanese Lefua has been persistently investigated in sequential studies. Recently, however, a new population of this genus was discovered in the Kuzuryu River drainage system of Fukui Prefecture, central Japan (Natural Environment Division, Department of Safety and Environment, Fukui Prefecture 2016). Subsequent studies employing assessments of morphological features and mitochondrial phylogeny have indicated the morphological and genetic distinctiveness of the population, identifying them as L. torrentis (Nishimura 2020; Nakajima et al. 2021). Katayama (2021) comprehensively surveyed the distribution of the genus in Fukui Prefecture and estimated a considerably fragmented habitat and a small population size of Lefua loaches with morphological features similar to those reported by Nakajima et al. (2021).

Based on the distinctive morphological and mitochondrial phylogenetic traits, the Lefua population present in the Kuzuryu River system may be an independent species with unique evolutionary origins. Considering the mitochondrial introgression observed in the congeners (Miyazaki et al. 2018), however, it is imperative to elucidate the nuclear phylogenetic position of the population to accurately determine its taxonomic account. In the present study, we clarified the nuclear phylogenetic and morphological relationships between the population and congeners, and describe it as a new species.

Materials and methods

Taxon sampling and specimen preparation

A total of 92 Lefua specimens were newly collected from Japan (Table 1). Among them, specimens of L. torrentis and L. tokaiensis were obtained from a wide area, which covers the distribution of the two species and the genetic populations of L. torrentis (Fig. 1; Yamada 2015; Nakajima and Uchiyama 2017; Miyazaki et al. 2018). Moreover, 25 specimens of the new species were collected from the Kuzuryu River system. Following Soto and Burhanuddin (1995), each individual was euthanized using clove oil (NOW Foods, USA). Digital images were captured using a SLT-A55V camera with a SAL1855 lens (Sony Corporation, Japan), and the right pelvic fins of the specimens were cut and preserved in 99.5% ethanol. Subsequently, whole bodies were fixed in 10% neutral formalin solution. The preservation solution was replaced with an 80% ethanol solution after formalin fixation. For morphological comparison, measurements and counts of the holotypes of L. torrentis preserved in the Department of Fisheries, Kindai University (KUN-P 45408) and L. tokaiensis at the National Museum of Natural History (NSMT-P 132821) were obtained from their original descriptions (Hosoya et al. 2018; Ito et al. 2019). Newly collected specimens were deposited at the Department of Fisheries, Faculty of Agriculture, Kyoto University (FAKU) and Lake Biwa Museum (LBM).

Figure 1.

Map of central Japan showing the distribution of three Lefua species. Orange, L. torrentis; red, L. tokaiensis; purple, L. nishimurai sp. nov. The distribution of L. torrentis and L. tokaiensis were in accordance with Yamada (2015) and Nakajima and Uchiyama (2017).

Table 1.
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Specimen list examined in this study with voucher numbers, collection localities, accession numbers of International Nucleotide Sequence Database (INSD), and references.

Voucher # (type status / population) Collection locality INSD Accession # Reference
L. torrentis
KUN-P45408 (holotype) Kasuga, Hyogo Hosoya et al. (2018)
FAKU 211461–211464 (Kii-Shikoku) Aridagawa, Wakayama This study
FAKU 211465–211468 (Kii-Shikoku) Hidaka, Wakayama This study
FAKU 211469–211477 (Kii-Shikoku) Kitanada, Tokushima LC790709 This study
FAKU 211478–211480 (Kii-Shikoku) Gomyo, Kagawa LC790710 This study
FAKU 211481–211484 (Sanyo) Ayuya, Hyogo LC790711 This study
FAKU 211485–211490 (Sanyo) Jindaiurakabe, Hyogo This study
FAKU 211491 (Sanyo) Dojyo, Hyogo LC790712 This study
FAKU 211492 (Sanyo) Nagi, Okayama, LC790713 This study
FAKU 211493–211499 (Sanyo) Santo, Hyogo This study
FAKU 211500 (Unidentified) Yamada, Fukui LC790714 This study
FAKU 211501–211506 (Nihonkai) Taneji, Kyoto LC790715 This study
(Sanyo) Aoi, Kyoto AB586659 Miyazaki et al. (2011)
(Sanyo) Nose, Osaka AB586660 Miyazaki et al. (2011)
(Kii-Shikoku) Hidaka, Wakayama AB586662 Miyazaki et al. (2011)
(Kii-Shikoku) Shioe, Kagawa AB586663 Miyazaki et al. (2011)
(Nihonkai) Natasyo Fukui LC225600 Miyazaki et al. (2018)
(Nihonkai) Oonyu, Kyoto LC225605 Miyazaki et al. (2018)
L. tokaiensis
NSMT-P132821 (holotype) Kadoya, Aichi Ito et al. (2019)
FAKU 211507–211511 Myoken, Aichi LC790716 This study
FAKU 211512–211518 Kadoya, Aichi LC790717, LC790718 This study
FAKU 211519–211526 Hosoe, Shizuoka LC790719 This study
FAKU 211527 Ota, Shizuoka LC790720 This study
Shitara, Aichi AB586682 Miyazaki et al. (2011)
Hourai, Aichi AB586683 Miyazaki et al. (2011)
Gotengawa, Aichi AB586684 Miyazaki et al. (2011)
L. echigonia
(Hokuriku) Ouchi, Akita AB586665 Miyazaki et al. (2011)
(Hokuriku) Nishime, Akita AB586666 Miyazaki et al. (2011)
(Tohoku) Matsushima, Miyagi AB586668 Miyazaki et al. (2011)
(Tohoku) Shionosaki, Fukushima AB586669 Miyazaki et al. (2011)
(Yamagata) Kawanishi, Yamagata AB586686 Miyazaki et al. (2011)
(Yamagata) Shinjo, Yamagata AB586687 Miyazaki et al. (2011)
(Kinki) Kurosaki, Ishikawa AB586671 Miyazaki et al. (2011)
(Kinki) Tsuruga, Fukui AB586672 Miyazaki et al. (2011)
(Tokai) Kakegawa, Shizuoka AB586674 Miyazaki et al. (2011)
(Yokai) Shizuoka, Shizuoka AB586685 Miyazaki et al. (2011)
(North-Kanto) Kakuda, Miyagi AB586676 Miyazaki et al. (2011)
(North-Kanto) Kurobane, Tochigi AB586677 Miyazaki et al. (2011)
(South-Kanto) Kozakuragawa, Ibaraki AB586679 Miyazaki et al. (2011)
(South-Kanto) Maiokagawa, Kanagawa AB586680 Miyazaki et al. (2011)
L. costata Yanggile (Korea) AB586655 Miyazaki et al. (2011)
L. nikkonis Shibecha, Hokkaido AB586654 Miyazaki et al. (2011)
L. nishimurai sp. nov.
LBM 1210059189 (holotype) Fukui, Fukui LC790702 This study
LBM 1210059177–1210059180, 1210059188–1210059195 (paratypes) Fukui, Fukui This study
LBM 1210059181–1210059187 (paratypes) Echizen, Fukui LC790700, LC790701 This study
FAKU 211455–211457 Echizen, Fukui LC790703–LC90705 This study
FAKU 211458–211460 Fukui, Fukui LC790706–LC790708 This study

Genetic analysis

Genomic DNA was extracted from seven specimens of L. torrentis, five specimens of L. tokaiensis, and nine specimens of the new species. DNA was extracted from the right pectoral fin following the procedure outlined by Okamoto et al. (2006) using a protein precipitation solution (Promega Corporation, USA). The primers RPEX1F (5′-TGGCCTCTTCCTTGGCCGTC-3′) and RPEX2R (5′-AACTCGTCTGGCTTTTCGCC-3′) (Miyazaki et al. 2011) were used. Step-down polymerase chain reaction and nucleotide sequencing of approximately 750 bp non-coding sections of the nucleic ribosomal S7 subunit gene were conducted according to the methods described by Miyazaki et al. (2018) using Ex Premier DNA Polymerase (Takara Bio Inc., Japan). The products were sequenced using an ABI 3130xl Genetic Analyzer (Applied Biosystems, USA), and finally, 463 bp alignments were obtained. The sequences were deposited in the International Nucleotide Sequence Database Collaboration through the DNA Databank of Japan (LC790700–LC790720).

The phylogenetic relationships between the 21 newly obtained sequences and the 25 sequences of the five Lefua species provided by Miyazaki et al. (2011, 2018) were estimated (Table 1). Phylogenetic trees were reconstructed based on 452 bp alignments shared by all sequences using maximum-likelihood (ML) with IQ-TREE v1.6.12 (Nguyen et al. 2015) and Bayesian inference (BI) with the MPI version of MrBayes v3.2.7 (Altekar et al. 2004; Ronquist et al. 2012). The best-fit models in the ML phylogenies were calculated as the HKY model based on the Bayesian information criterion and the greedy algorithm using ModelFinder implemented in IQ-TREE (Kalyaanamoorthy et al. 2017). The branch support of the ML phylogenetic tree was assessed using 1,000 non-parametric bootstrap (BS) replicates. The best-fit models in the BI were selected as the HKY+G model under the Bayesian Information Criterion using PartitionFinder v2.1.1 (Lanfear et al. 2016). The tree was sampled every 100 generations from the four Markov chains in eight independent runs. After verifying the parameter estimates and convergence using Tracer v1.7.2 (Rambaut and Drummond 2009), the first million trees were discarded. Based on the phylogenetic relationships within the genus estimated by Miyazaki et al. (2011) using the ribosomal S7 subunit region, L. nikkonis and L. costata were set as outgroups for the present analysis.

The haplotype relationships of ribosomal S7 subunit gene were inferred by statistical parsimony (Clement et al. 2000) using PopART v1.7 (Leigh and Bryant 2015). The mean genetic distances among the Lefua species were estimated using the Kimura two-parameter (K2P) in MEGA v11.0.10 (Tamura et al. 2021) using the same datasets as in the phylogenetic analyses.

Morphological examination and analysis

The external morphological characters and vertebrae of Lefua specimens were examined using the methodologies outlined by Hosoya (1983) and Hubbs and Lagler (2004), respectively. The morphologies of L. torrentis, L. tokaiensis, and the new species were compared based on their morphological similarities indicated previously (Miyazaki et al. 2011; Ito et al. 2019; Nakajima et al. 2021). Moreover, Nishimura (2020) and Nakajima et al. (2021) highlighted that the new species has smaller, more dorsally positioned eyes (i.e. a smaller ratio of interorbital width to body width) compared to L. torrentis and L. tokaiensis. Accordingly, the head width and the ratio of interorbital width to body width were newly measured and calculated, in addition to the following 17 previously examined morphometric characters: standard length, head length, body depth, body width, depth of caudal peduncle, length of caudal peduncle, predorsal length, preanal length, prepelvic length, height of dorsal fin, length of dorsal fin base, height of anal fin, length of anal fin, pectoral fin length, snout length, orbit diameter, and interorbital width. These lengths were measured using a caliper (Popular Caliper 150 mm, Shinwa Rules Co., Ltd., Japan) to the nearest 0.1 mm.

The number of dorsal fin rays, anal fin rays, caudal fin rays, and vertebrae were counted using a CMBW-80 X-ray apparatus box (Softex Co., Ltd., Japan). The first four vertebrae with the Weberian Apparatus and one vertebra fused to the pleurostyle in the hypural complex were also counted. The last two fin rays of the dorsal and anal fins were counted as single rays because they were supported by a shared pterygiophore.

All characters of 19 type specimens of the new species were measured and counted. In non-types of the new species, L. torrentis, and L. tokaiensis, all characters were examined, except for the number of vertebrae and fin rays. In addition, 17 measurements and seven counts of holotypes of L. torrentis and L. tokaiensis were obtained from their original descriptions (Hosoya et al. 2018; Ito et al. 2019).

External morphological relationships among Lefua species were evaluated using principal component analysis (PCA). In addition, discriminant analysis was performed using the Random Forest (RF) algorithm (Breiman 2001). PCA and RF analyses were performed using standardized data for the 18 morphological characters of all newly collected specimens and holotypes of L. torrentis and L. tokaiensis. In the discriminant analysis, three variables were randomly sampled as candidates for each split, and 100,000 trees were generated, given that the out-of-bag error rate fully decreased with a large number of trees. PCA was performed using R v3.6.1 (R Development Core Team 2019). RF analyses were conducted using the package randomForest v4.6–14 (Andy and Matthew 2002) for R.

Results

Genetic analyses

The BI tree (Fig. 2) was identical to the ML tree (not shown). The reconstructed trees based on the 452 bp ribosomal S7 subunit gene supported the monophyly of L. torrentis [BS = 100, Bayesian posterior probabilities (BPP) = 1.00) and L. nishimurai sp. nov. (BS = 100, BPP = 1.00), and the clade consisted of L. tokaiensis and L. echigonia (BS = 95, BPP = 1.00), respectively. Monophyly of the clade formed by these four species was also strongly supported (BS = 100, BPP = 1.00).

Figure 2.

The Bayesian inference tree of six Lefua species based on 452 bp of the nucleic ribosomal S7 subunit gene. The numbers associated with the nodes represent the bootstrap (BS) values for ML ⁄ and Bayesian posterior probabilities (BPP). BS higher than 50% and ⁄ or BPP higher than 90% are indicated.

The statistical parsimony network calculated based on the 33 haplotypes separated all species, except for L. tokaiensis and L. echigonia by mutational steps (Fig. 3). In this network, the new species was separated from L. torrentis and L. tokaiensis by 31 and 27 steps, respectively. Three haplotypes with a single-base substitution in each other were detected in nine specimens of L. nishimurai sp. nov.

Figure 3.

Statistical parsimony network showing the relationships between the 33 haplotypes detected in a ribosomal S7 subunit gene of six Lefua species. Each connection indicates a single mutation, and hatch marks represent missing intermediate haplotypes.

Lefua nishimurai sp. nov. represented 0.1% intraspecific genetic differences, whereas L. torrentis, L. tokaiensis, and L. echigonia showed broader ranges of 0.8–4.9% (Table 2). The interspecific genetic differences among the four species were estimated to be 4.8–9.9% and the distances of L. torrentis, L. tokaiensis, and L. echigonia to L. nishimurai sp. nov. were 8.2%, 7.8%, and 9.9%, respectively.

Table 2.
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Mean intraspecific (along the diagonal) and interspecific (below the diagonal) genetic distances (%) of six Lefua species calculated by a Kimura two-parameter model.

Species L. torrentis L. tokaiensis L. echigonia L. nishimurai sp. nov. L. nikkonis L. costata
L. torrentis 0.80
L. tokaiensis 4.79 1.08
L. echigonia 7.07 4.82 4.87
L. nishimurai sp. nov. 8.17 7.81 9.90 0.10
L. costata 8.09 7.35 10.06 11.37 –
L. nikkonis 8.57 8.22 10.11 11.34 4.81 –

Morphological analyses

PCA performed on L. torrentis, L. tokaiensis, and L. nishimurai sp. nov. using 18 external morphological characters summarized their morphological relationships. The first two PCs explained 81.8% of the total variation, and PC2 scores primarily separated the three species with large overlaps between L. torrentis and L. tokaiensis, and slight overlaps between L. nishimurai sp. nov. and the other two species (Fig. 4). Among the morphological characters analyzed, standard, predorsal, and prepelvic lengths explained the variation of PC1 more strongly than the others (Suppl. material 1). For PC2, the contributions of the orbit diameter and snout length were greater than those of the other characters.

Figure 4.

Results of the principal component analysis based on the 18 external morphological characters conducted for three Lefua species.

RF classification using the same dataset as PCA correctly classified 79.1% of the specimens into the three species. Bootstrap replicates identified 95.6% L. torrentis, 38.1% L. tokaiensis, and 84.0% L. nishimurai sp. nov. The mean decrease of the Gini coefficients was larger for interorbital width, snout length, orbit diameter, preanal length, body width, and head width (Table 3). The mean decrease in accuracy was greater for snout length, preanal length, interorbital width, body width, head width, and orbit diameter in L. nishimurai sp. nov. The specimens of the new species possessed a smaller snout length, ratio of interorbital width to body width, and interorbital width than those of L. torrentis and L. tokaiensis. The preanal length of the new species was greater than that of L. torrentis and L. tokaiensis. The body and head widths of the new species were larger than those of L. torrentis and smaller than those of L. tokaiensis (Figs 5, 6).

Figure 5.

Comparison of body morphology and coloration of three Lefua species. A. Kii-Shikoku Population of L. torrentis (FAKU 211486); B. Sanyo Population of L. torrentis (FAKU 211492); C. Nihonkai Population of L. torrentis (FAKU 211504); D. L. tokaiensis (FAKU 211510); E. L. nishimurai sp. nov. (LBM 1210059189). Scale bar: 10 mm.

Figure 6.

Comparison of dorsal view of the head of three Lefua species. A. Kii-Shikoku Population of L. torrentis (FAKU 211486); B. Sanyo Population of L. torrentis (FAKU 211492); C. Nihonkai Population of L. torrentis (FAKU 211504); D L. tokaiensis (FAKU 211510); E. L. nishimurai sp. nov. (LBM 1210059179). Scale bars: 5 mm.

Table 3.
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Results of the Random Forest analyses for three Lefua species with specimen numbers, mean decrease in accuracy in each character, and the mean decreases in Gini coefficients.

Character L. torrentis L. tokaiensis L. nishimurai sp. nov. Mean decrease of Gini coefficient
Specimen number 45 21 19
Body depth 0.007 0.020 0.001 2.100
Body width 0.042 0.053 0.004 4.067
Depth of caudal peduncle 0.014 0.011 0.014 2.420
Head length 0.010 0.010 -0.004 1.835
Head width 0.037 0.048 0.020 4.022
Height of anal fin 0.025 0.012 0.012 2.925
Height of dorsal fin 0.016 0.003 -0.002 2.907
Interorbital width 0.026 0.056 0.073 5.539
Length of anal fin base 0.005 0.003 0.003 1.480
Length of caudal peduncle 0.011 0.008 0.011 2.886
Length of dorsal fin base 0.011 0.009 0.001 2.274
Orbit diameter 0.030 0.045 0.013 4.874
Pectral fin length 0.005 0.002 0.004 1.636
Preanal length 0.031 0.057 0.021 4.292
Predosal length 0.017 0.034 0.009 2.915
Prepelvic length 0.016 0.026 0.003 2.549
Snout length 0.033 0.063 0.017 5.026
Standard length 0.014 0.017 0.012 2.659

Systematics

Lefua nishimurai Katayama, sp. nov.

https://zoobank.org/7CD04DBC-DF52-4FD4-AEBC-FBDA719E800C
Table 4, Figs 5E, 6E, 7, 8, 9 New Standard Japanese name: Reihoku-nagare-hotoke-dojyô

Lefua torrentis: Natural Environment Division, Department of Safety and Environment, Fukui Prefecture 2016: 110 (part); Nakajima et al. 2021: 35, fig. 1; Katayama 2021: 54, fig. 5.

Material examined

Holotype. • LBM 1210059189; 51.2 mm SL; collected from Kuzuryu River system, Fukui, Fukui Prefecture, Japan; 36.0621°N, 136.1304°E; on 6 Sep. 2021 by Yuta Katayama.

Paratypes. Eighteen specimens collected from Kuzuryu River system in Fukui Prefecture, Japan by Y. Katayama. • LBM 1210059177; 44.4 mm SL from Echizen on 17 Jul. 2021; • LBM 1210059178, 58.8 mm SL; • LBM 1210059179, 44.4 mm SL; • LBM 1210059180, 40.0 mm SL from Fukui on 18 Jul. 2021; • LBM 1210059181, 61.5 mm SL; • LBM 1210059182, 52.8 mm SL; • LBM 1210059183, 44.2 mm SL; • LBM 1210059184, 49.6 mm SL from Echizen on 17 Jul. 2021; • LBM 1210059185, 59.9 mm SL; • LBM 1210059186, 43.0 mm SL; • LBM 1210059187, 44.5 mm SL from Echizen on 6 Sep. 2021; • LBM 1210059188, 55.0 mm SL; • LBM 1210059190, 57.0 mm SL; • LBM 1210059191, 44.5 mm SL; • LBM 1210059192, 63.2 mm SL; • LBM 1210059193, 48.7 mm SL; • LBM 1210059194, 62.9 mm SL from Fukui on 6 Sep. 2021; • LBM 1210059195, 57.4 mm SL from Echizen on 13 Nov. 2021.

Additional specimens

Six specimens collected from Kuzuryu River system in Fukui Prefecture, Japan by Y. Katayama. • FAKU 211455, 41.5 mm SL; • FAKU 211456, 61.3 mm SL; • FAKU 211457, 56.1 mm SL from Echi­zen on 1 Oct. 2023; • FAKU 211458, 51.9 mm SL; • FAKU 211459, 47.3 mm SL ; • FAKU 211460, 44.5 mm SL from Fukui on 1 Oct. 2023.

Diagnosis

Lefua nishimurai sp. nov. can distinguished from all other species of Lefua by combing following features: absence of rhomboid or triangular dark blotches on middle of caudal fin base; absence of black longitudinal stripe on both body sides in mature males; absence of dusky cross bars on dorsal area of body; absence of dusky bar beside dorsal fin base; eyes located dorsally on head; narrow conspicuous longitudinal mark between base of outer rostral barbel and eye; small dorsal fin; small orbit diameter (6.3–11.2% of head length); small value of interorbital width relative to body width (28.1–39.4%); dark spots dorsally and ventrally on caudal fin base; small dark brown spots from snout to caudal peduncle; small dark spots on dorsal and caudal fins (approximately same size as eyes).

Description

Measurements and counts listed in Table 4. Body elongated and cylindrical, slightly longitudinally flattened, and caudal peduncle laterally flattened. Dorsal and ventral sides of caudal peduncle keeled, and depth of caudal peduncle, including keel, about same as body depth. Head small (less than one-fourth of SL), slightly dorsoventrally compressed. Snout relatively long, comprising 38.7% in holotype (32.3–46.4% in paratypes and additional specimens) of head length, with tip rounded in dorsal view. Eyes positioned on dorsolateral surface of head (Figs 6–8), very small. Interorbital width comprises 51.5% (42.9–59.0%) of body width, and orbit diameter comprises 6.6% (6.3–11.2%) of head length (Figs 6–8). Mouth subterminal; small and inferior, slightly arched. Upper and lower lips fleshly and smooth (Figs 7, 8). No median incision in upper lip. One median notch in lower lip. Three pairs of barbels, two pairs of rostral barbels, and one pair of maxillary barbels. Inner rostral barbel relatively short, reaching to or slightly past vertical through anterior margin of eye when extended horizontally, outer rostral barbel longest, slightly past vertical through posterior margin of eye when extended horizontally, maxillary barbel past vertical through posterior margin of eye when extended horizontally. Nostrils separated from each other, anterior nostril formed as 1 long nasal barbel, posterior nostril larger than anterior nostril, anterior nostril at anterior side of 1 nasal barbel, close to base. Nasal barbel extended horizontally past posterior border of eye. Dorsal and ventral keels on caudal peduncle; dorsal keel starting slightly posterior to extremity of dorsal fin base, ventral keel starting posterior to extremity of anal fin base. Length of caudal peduncle 1.0 (1.0–1.3) times depth of caudal peduncle (depth including keel). Scales embedded on body. Lateral line absent. Dorsal fin rounded. Origins of dorsal fin nearer to caudal fin base than to tip of snout. Pectoral fin horizontal. Pelvic fin anterior to dorsal fin. Anal fin rounded. Anus positioned slightly anterior to anal fin. Caudal fin rounded. Dorsal fin with 4 (3–4) simple and 5 (5–6) branched rays. Second branched ray longest. Anal fin with 4 (3–4) simple and 5 (5–6) branched rays. Pectoral fin with 1 simple and 11 (10–11) branched rays. Second branched ray longest. Pelvic fin with 1 simple and 5 branched rays. No pelvic axillary lobe. Caudal fin with 2 (1–2) simple, 6 (5–7) branched, 7 (6–7) branched and 1 (1–2) simple principal rays. Total vertebrae 41 (39–43); abdominal vertebrae 23 (21–23), caudal vertebrae 18 (16–21).

Figure 7.

Body coloration of the holotype of Lefua nishimurai sp. nov (LBM 1210059189). A. Lateral view; B. Dorsal view; C. Ventral view. Scale bar: 10 mm.

Figure 8.

Detailed morphology of the holotype of Lefua nishimurai sp. nov (LBM 1210059189). A. Head; B. Lateral view of head; C. Mouth; D. Caudal fin base. An arrow in the lateral view of head (B) indicates narrow conspicuous black stripe between base of outer rostral barbel and eye. Arrows in the caudal fin base (D) indicate black spots on ventral and dorsal surfaces. Scale bars: 5 mm.

Table 4.
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Meristics and morphometrics of Lefua torrentis, L. tokaiensis., and L. nishimurai sp. nov. Measurements indicate minimum–maximum values (mean ± SD). Data of the holotypes of L. torrentis and L. tokaiensis were obtained from Hosoya et al. (2018) and Ito et al. (2019), respectively.

Character L. torrentis L. tokaiensis L. nishimurai sp. nov.
Holotype Non-types (n = 46) Holotype Non-types (n = 21) Holotype Paratypes (n = 18) Non-types (n = 6)
Dorsal fin ⅳ, 6 ⅳ, 6 ⅳ, 5 ⅲ–ⅳ, 5–6
Anal fin ⅳ, 5 ⅳ, 5 ⅳ, 5 ⅲ–ⅳ, 5–6
Pectral fin ⅰ, 9 ⅰ, 7 ⅰ, 11 ⅰ, 10–11
Pelvic fin ⅰ, 5 ⅰ, 5 ⅰ, 5 ⅰ, 5
Caudal fin ⅱ, 6, 8, ⅰ ⅰ, 7, 8, ⅱ ⅱ, 6, 7, ⅰ ⅰ–ⅱ, 5–7, 6–7, ⅰ–ⅱ
Abdominal vertebrae 19 19 23 21–23
Caudal vertebrae 18 18 18 16–21
Total vertebrae 37 37 41 39–43
Standard length (mm) 44.9 44.2 51.2 40.0–63.2
In % of standard length
Head length 20.9 18.4–23.4 (21.0 ± 1.2) 21.3 19.1–26.4 (22.0 ± 1.9) 20.7 19.1–23.6 (20.6 ± 0.9) 19.6–22.0 (20.8 ± 1.0)
Body depth 14.4 8.8–15.2 (12.1 ± 1.2) 14 10.8–17.1 (13.6 ± 1.5) 12.7 10.7–14.2 (12.4 ± 1.0) 11.8–14.3 (13.1 ± 0.9)
Body width 13.9 11.2–14.7 (12.7 ± 0.8) 11.7 12.3–17.2 (14.2 ± 1.4) 13.3 12.5–14.6 (13.6 ± 0.6) 12.9–14.2 (13.6 ± 0.4)
Head width 11.2–15.9 (13.2 ± 1.0) 13.0–17.6 (15.2 ± 1.4) 13.1 12.5–15.2 (13.5 ± 0.6) 12.9–14.4 (13.7 ± 0.5)
Depth of caudal peduncle 12.4 10.8–13.3 (12.1 ± 0.7) 13.2 11.0–17.1 (13.7 ± 1.4) 12.5 10.7–13.5 (12.0 ± 0.7) 11.6–13.0 (12.2 ± 0.5)
Length of caudal peduncle 14.8 11.7–16.8 (14.4 ± 1.1) 16.1 11.3–116.2 (13.7 ± 1.1) 12.7 11.7–15.2 (13.5 ± 0.9) 11.9–14.9 (13.3 ± 0.9)
Predorsal length 67.2 61.5–69.1 (64.8 ± 1.6) 62.9 62.8–76.9 (66.3 ± 3.1) 67.2 63.2–69.3 (66.9 ± 1.3) 65.3–68.1 (67.1 ± 0.9)
Preanal length 76 71.2–80.8 (75.0 ± 1.6) 74.8 72.3–87.0 (75.9 ± 3.1) 77.9 76.6–81.4 (79.3 ± 1.3) 74.3–82.2 (77.1 ± 2.4)
Prepelvic length 56.1 53.3–65.2 (57.0 ± 1.9) 53.6 56.5–74.0 (60.1 ± 4.2) 56.8 53.6–60.9 (57.6 ± 1.7) 56.8–60.5 (58.5 ± 1.1)
Height of dorsal fin 10.9 6.6–11.7 (9.2 ± 1.1) 11.3 6.1–11.8 (8.8 ± 1.3) 8.6 5.6–10.0 (7.9 ± 1.1) 7.0–9.6 (8.5 ± 1.0)
Length of dorsal fin base 8.1 6.7–10.1 (8.6 ± 0.7) 8.6 7.1–9.9 (8.5 ± 0.8) 8.2 7.5–9.7 (8.6 ± 0.6) 7.2–9.0 (8.4 ± 0.6)
Height of anal fin 12.5 5.5–10.7 (7.7 ± 1.1) 10.3 7.0–11.3 (8.7 ± 1.0) 9.4 6.5–9.3 (7.9 ± 0.9) 5.5–9.0 (7.7 ± 1.1)
Length of analfin base 8.1 6.8–9.9 (8.9 ± 0.6) 6.6 6.8–11.9 (8.2 ± 1.0) 7.2 5.8–9.1 (7.8 ± 0.8) 7.0–8.3 (7.6 ± 0.4)
Pectral fin length 13.6 11.6–17.0 (14.1 ± 1.4) 12.4 13.0–18.6 (15.1 ± 1.4) 12.9 11.2–16.5 (13.1 ± 1.4) 12.5–15.1 (13.5 ± 0.9)
In % of head length
Snout length 34 41.6–53.8 (48.9 ± 3.3) 39.7 44.5–55.5 (49.4 ± 3.4) 38.7 32.3–45.7 (40.1 ± 3.4) 42.2–46.4 (44.6 ± 1.5)
Orbit diameter 13 6.9–15.9 (10.5 ± 1.7) 13.6 7.3–13.3 (9.5 ± 1.4) 6.6 6.3–11.2 (8.1 ± 1.5) 7.5–9.4 (8.5 ± 0.6)
Interorbital width 39 32.2–47.3 (39.6 ± 3.8) 36.3 39.3–48.9 (43.8 ± 2.7) 33.0 28.1–39.4 (34.5 ± 3.2) 33.3–39.1 (36.8 ± 1.8)
Ratio of interorbital width to body width 58.6 55.9–82.1 (65.6 ± 6.0) 66.1 56.3–76.4 (68.0 ± 6.2) 51.5 42.9–58.2 (52.2 ± 4.3) 51.6–59.0 (56.1 ± 3.0)

Coloration

Live specimens, body and head light brown with strong yellowish tinge except for whitish gray ventral surface (Fig. 7). One narrow conspicuous black stripe between base of outer rostral barbel and eye (Fig. 8). Entire body from snout to caudal peduncle scattered dark brown spots. Largest of spots slightly larger than orbit diameter. Both lips white, with one pair of small brown spots inside lower lip tissue (Fig. 8). All fins pale to translucent. Pectoral fin yellowish brown, with dark spots at the base and anterior half. Dorsal fin yellowish brown with dark spots from base to middle. Anal fin with a few dark spots on basal half (sometimes absent). No spots on pelvic fin. Caudal fin yellowish brown with dark spots from base to a little closer to tip of middle, but translucent and without spots on margins. Dorsal and ventral surfaces of caudal fin base with dark brown spots (rarely absent) (Figs 8, 9). No obscure dark band in skin of caudal fin base (rarely present) (Figs 5, 7, 9).

Figure 9.

Body coloration of atypical specimen of Lefua nishimurai sp. nov. (LBM 1210059188), showing no distinct dark brown spots scattered over the body and obscure dark band in the skin of the caudal fin base. Scale bar: 10 mm.

Etymology

The specific name is dedicated to Toshiaki Nishimura, who first morphologically distinguished this new species.

Distribution

This new species was recorded in the Kuzuryu River drainage system of the Reihoku Region in Fukui Prefecture, central Japan (Katayama 2021; Fig. 1).

Habitat and biology

Lefua nishimurai sp. nov. inhabits small mountain streams surrounded by natural or planted forests, and prefers sandy or gravelly bottoms (Fig. 10). The new species usually hides under rocks and stones at the river edge or under sediments, such as fallen leaves. It has been estimated that spawning of this species occurs from late April to mid-June (Katayama 2021).

Figure 10.

Typical habitat of Lefua nishimurai sp. nov. in the Kuzuryu River drainage system in Fukui Prefecture, Japan.

Remarks

The new species is similar to L. torrentis and L. tokaiensis with a narrow conspicuous longitudinal mark between the base of the outer rostral barbel and eye. However, L. nishimurai sp. nov. can be distinguished from L. torrentis based on the following characteristics: eyes located dorsally on head (more lateral in L. torrentis), small orbit diameter (larger in L. torrentis), dark spots on the caudal fin base (rarely absent in the new species; usually absent in L. torrentis), small dark spots on dorsal and caudal fins, and distinct dark brown spots scattered over the body (rarely absent in the new species; entirely absent in L. torrentis). The new species and L. tokaiensis can also be discriminated by eyes located dorsally on the head (more lateral in L. tokaiensis), small orbit diameter (larger in L. tokaiensis), dark spots dorsally and ventrally on the caudal fin base (rarely absent in the new species; entirely absent in L. tokaiensis), distinct dark brown spots scattered over the body (rarely absent in the new species; entirely absent in L. tokaiensis), and fan-shaped caudal fin of L. nishimurai (squared-off apex in L. tokaiensis). Both the new species and L. torrentis inhabiting Wakayama Prefecture, and Shikoku and Awaji Islands possess dark brown spots of varying sizes on the body, dorsal side, and caudal peduncle (Hosoya et al. 2018). However, L. nishimurai sp. nov. represents smaller spots than the other species on the entire body and dorsal surfaces as well as on the dorsal and caudal fins (approximately the same size as the eyes) (Fig. 5).

Comparative material examined

Lefua torrentis, 46 specimens (33.2–66.9 mm SL). Holotype: • KUN-P 45408; 44.9 mm SL collected from Yura River system, Kasuga, Tanba, Hyogo; • FAKU 211461–211464, 4 specimens, 33.2–49.4 mm SL from Arida River system, Aridagawa, Wakayama; • FAKU 211465–211468, 4 specimens, 43.4–55.5 mm SL, from Hidaka River system, Hidaka, Wakayama; • FAKU 211469–211477, 9 specimens, 38.4–49.9 mm SL from Orino River system, Kitanada, Tokushima; • FAKU 211478–211480, 3 specimens, 42.9–55.5 mm SL from Yoshino River system, Gomyo, Kagawa; • FAKU 211481–211484, 4 specimens, 40.4–63.6 mm SL from Sumoto River system, Ayuya, Hyogo; • FAKU 211485–211490, 6 specimens, 44.2–69.9 mm SL from Dainichi River system, Jindaiurakabe, Hyogo; • FAKU 211491, 1 specimen, 49.6 mm SL from Muko River system, Dojyo, Hyogo; • FAKU 211492, 1 specimen, 40.0 mm SL from Yoshii River system, Nagi, Okayama; • FAKU 211493–211499, 7 specimens, 39.0–60.0 mm SL from Maruyama River system, Santo, Hyogo; • FAKU 211500, 1 specimen, 44.9 mm SL from Saburi River system, Yamada, Fukui; • FAKU 211501–211506, 6 specimens, 35.6–57.4 mm SL from Onyu River system, Taneji, Kyoto.

L. tokaiensis, 22 specimens (32.8–60.9 mm SL). Holotype: • NSMT-P 132821, 44.2 mm SL from Toyo River, Kadoya, Aichi; • FAKU 211507–211511, 5 specimens, 32.8–51.3 mm SL from Yahagi River system, Myoken, Aichi; • FAKU 211512–211518, 7 specimens, 33.8–54.4 mm SL from Toyo River system, Kadoya, Aichi; • FAKU 211519–211526, 8 specimens, 40.7–60.0 mm SL from Miyakoda River system, Hosoe, Shizuoka; • FAKU 211527, 1 specimen, 36.8 mm SL from Ota River system, Ota, Shizuoka.

Discussion

The genetic and morphological distinctiveness of L. nishimurai sp. nov. in the Kuzuryu River system was clarified through integrative analyses. The phylogenetic relationships among the Lefua species corresponded to those estimated in previous studies (Miyazaki et al. 2011, 2018). The reconstructed tree strongly supported the monophyly of L. nishimurai sp. nov. In addition, the statistical parsimony network showed that the new species possesses unique haplotypes of the ribosomal S7 subunit gene in the Japanese Lefua species.

The long-branched clade consisting of the new species and the large interspecific genetic distances between the new species and other congeners were estimated in this study. Although only one nucleic locus was analyzed in this study, these results suggest that the new species was genetically isolated at an early stage of diversification of the common ancestors of L. torrentis, L. tokaiensis, L. echigonia, and L. nishimurai sp. nov. In addition, the observed low haplotypic diversity may indicate past population size declines in this new species. In Fukui Prefecture, populations of freshwater fishes, such as Pungtungia herzi Herzenstein and Tanakia limbata (Temminck & Schlegel), have been geographically isolated between the northern Reihoku and the southern Reinan Regions (Fig. 1; Kato 1906; Igarashi and Kato 1966; Kato 1985, 1998; Matsumiya 2001). Therefore, the restricted distribution of the new species in the Reihoku Region might be due to the high altitudes surrounding the region, with patterns similar to those of other fish populations.

The incongruence between mitochondrial and nuclear phylogenies caused by historical mitochondrial introgression has been estimated in Japanese Lefua species (Miyazaki et al. 2018). The Nihonkai Population of L. torrentis in the Tango, Tajima, and Reinan Regions represents mitochondrial haplotypes closely related to L. echigonia and L. tokaiensis, suggesting past hybridization among them or their ancestors (Miyazaki et al. 2018). A hybrid population of Oryzias species was also found in this area, and their hybridization has been attributed to the topographic features of the watershed boundary between the Paleo-Yura River and the Paleo-Kako River (Iguchi et al. 2018).

Given that the distribution of L. echigonia extends along the Sea of Japan (Nihonkai) (Miyazaki et al. 2018), the mitochondrial phylogeny of Lefua in Fukui Prefecture can be incorrectly estimated because of populations influenced by introgression. On the other hand, the genetic distinctiveness of L. nishimurai sp. nov. was clarified with mitochondrial and nuclear genes by Nakajima et al. (2021) and this study. Based on the genetic and morphological distinctiveness of the Lefua population in the Kuzuryu River system of the Reihoku Region, we consider L. nishimurai sp. nov. to be an independent species.

The characters of standard, predorsal, prepelvic, and head lengths contributed more strongly to explaining the morphological variation among specimens in the PCA. In contrast, interorbital width, snout length, and orbit diameter were more important in explaining interspecific variations in PCA and/or discriminant analysis. These results show that both previous and newly obtained characters are useful for discriminating the three Lefua species. The snout length, preanal length, interorbital width, body width, head width, and orbit diameter were particularly important for the identification of L. nishimurai sp. nov. in the RF classification. According to this analysis, the specimens of this new species can be discriminated by head morphology, which is characterized by more dorsally positioned small eyes and shorter snouts. The preanal length, and body and head widths of the new species are also larger than those of L. torrentis and smaller than those of L. tokaiensis.

According to prefectural surveys, a decrease in the number of freshwater fishes and salamanders, such as Cottus pollux Günther, Hynobius abei Sato, and H. kimurae Dunn, whose habitats potentially overlap with those of L. nishimurai sp. nov., has been progressing due to recent habitat degradation in the Reihoku Region (Natural Environment Division, Department of Safety and Environment, Fukui Prefecture 2016). Given the restricted distribution and vulnerable habitats of this new species, it is important to accumulate further knowledge on its distribution, population size, and natural history, as well as investigate its conservation status.

Acknowledgments

We are grateful to Katsutoshi Watanabe (Kyoto University; KU) and Yusuke Fuke (National Institute of Genetics) for their supports in taking photographs and preparing specimens. We also thank Seigo Kawase (LBM) and Ryoichi Tabata (LBM) for providing radiographs and registering specimens. We are grateful to Mizuki Matsunuma (FAKU) for registering the specimens. We appreciate supports by Tomoyuki Takahashi (KU), Taro Jonishi (KU), and Chiharu Endo (KU) in the genetic analyses. We thank Danilo Harms (Leibniz Institute for the Analysis of Biodiversity Change), Artem M. Prokofiev (Severtsov Institute of Ecology and Evolution), Apinun Suvarnaraksha (Maejo University), Jun Nakajima (Fukuoka Institute of Health and Environmental Sciences), Toshiaki Nishimura, Takafumi Nakano (KU), and Yoshiharu Sasaki for their constructive suggestions to our study. We thank Keigo Kitao (KU), Jo Hamahashi (KU), and Yusei Takaishi (KU) for assisting our taxon samplings. We are grateful to Daichi Kajiwara (Keio University) for collecting literatures. We thank Editage (www.editage.jp) for English language editing.

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Supplementary material

Supplementary material 1 

Loadings of the 18 external morphological characters, proportion of variance, and cumulative proportion for the 18 principal components (PC) caluculated by the principal component analysis

Yuta Katayama, Naoto Sawada

Data type: docx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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