Ichthyological Research https://doi.org/10.1007/s10228-020-00783-1 FULL PAPER Morphologies and population genetic structures of the eight‑barbel loach of the genus Lefua on southern Sakhalin Yoshiyasu Machida1 · Minoru Kanaiwa2 · Sergey V. Shedko3 · Hajime Matsubara4 · Hirozumi Kobayashi5 · Ixchel F. Mandagi5,6 · Akira Ooyagi7 · Kazunori Yamahira5 Received: 7 December 2019 / Revised: 5 August 2020 / Accepted: 6 September 2020 © The Ichthyological Society of Japan 2020 Abstract Coldwater, primary freshwater, fsh such as the eight-barbel loach, Lefua nikkonis, are thought to have colonised Hokkaido from the continental Far East via Sakhalin Island during the Late Pleistocene. Lefua populations have been reported on southern Sakhalin, but detailed morphological and population structure analyses have not yet been completed. This infor- mation is important for reconstructing the colonisation history of L. nikkonis to Hokkaido. In this study, morphological analysis revealed that L. nikkonis and two continental congeners, Lefua pleskei and Lefua costata, are distinguishable from each other, and Lefua collected from southern Sakhalin is morphologically more similar to L. nikkonis. Random forest analysis, a machine learning classifcation method, classifed all Sakhalin individuals as L. nikkonis. Haplotype analysis of mitochondrial DNA sequences revealed that all but one Sakhalin haplotype are shared with L. nikkonis and none is shared with the two continental congeners, which supports the hypothesis that Sakhalin Lefua are L. nikkonis. However, none of the Sakhalin haplotypes was distributed on northern Hokkaido. This discontinuous distribution of haplotypes across Sakhalin and Hokkaido suggests that the Sakhalin Lefua populations are not native. Some of the Sakhalin haplotypes were found only on Hokkaido’s Ishikari River system or the Tokachi River system, suggesting that they originated from these regions. Because previous feld surveys reported wild Lefua only from northwestern Sakhalin, we concluded that native Lefua on southern Sakhalin may have gone extinct after they colonised Hokkaido in the Middle Pleistocene. Keywords Random forest analysis · MtDNA · Siberian primary freshwater fsh · Alien species Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s1022 8-020-00783 -1) contains Introduction supplementary material, which is available to authorized users. One dispersal mechanism used by coldwater, primarily * Yoshiyasu Machida [email protected] freshwater, fsh to move between islands was land bridges that appeared during glacial periods (Goto and Nakano 1 Bihoro Museum, Bihoro, Hokkaido 092-0002, Japan 1993; Avise 2000). In the Japanese archipelago, these fsh 2 Mie University Graduate School, Faculty of Bioresources, likely moved from the continental Far East to the island of Tsu, Mie 514-8507, Japan Hokkaido via one of the land bridges that repeatedly formed 3 Federal Scientifc Center of the East Asia Terrestrial between the islands of Sakhalin and Hokkaido (Goto and Biodiversity FEB RAS, Vladivostok 690022, Russia Nakano 1993). Consistent with this view, coldwater, primary 4 Faculty of Biological Science and Technology, Institute freshwater, fsh on Hokkaido have conspecifc populations, of Science and Engineering, Kanazawa University, Noto or closely related (sub)species, on both Sakhalin and the Center for Fisheries Science and Technology, Kanazawa continental mainland (Lindberg 1972; Watanabe et al. 2006). University, Noto, Ishikawa 927-0552, Japan Population structure analyses also support this hypothesis. 5 Tropical Biosphere Research Center, University For example, populations of the lake minnow, Rhynchocy- of the Ryukyus, Okinawa 903-0213, Japan pris perenurus sachalinensis, on northern and eastern Hok- 6 Faculty of Fisheries and Marine Science, Sam Ratulangi kaido share mitochondrial DNA (mtDNA) haplotypes with University, Manado 95115, Indonesia those on southern Sakhalin (Sakai et al. 2014). 7 Shimokita Field Science Nest, Mutsu, Aomori 035-0077, Japan Vol.:(0123456789)1 3 Y. Machida et al. The eight-barbel loach Lefua nikkonis is another cold- for reconstructing the colonisation history of L. nikkonis on water, primary freshwater, fish distributed throughout Hokkaido. Hokkaido and parts of northern Honshu (the Shimokita In this study, we examine the morphologies of Sakhalin Peninsula) (Takeuchi and Ohta 1993; Ooyagi 2013; Naka- Lefua and compare them with L. nikkonis, collected from jima and Uchiyama 2017). Recently, Ooyagi et al. (2018) Hokkaido and Honshu, and with the two continental con- examined the population structures of L. nikkonis using geners. We also investigate mtDNA haplotypes within the mtDNA haplotypes and demonstrated that they dispersed Sakhalin populations and compared them with those of L. southward, from Hokkaido to Honshu, via a land bridge(s) nikkonis collected throughout their geographic range. Based that emerged at the Tsugaru Strait during the MIS 5e (i.e. on the results, we discuss the origin of the Sakhalin Lefua 130,000–200,000 years ago). However the origins of the populations and the colonisation history of L. nikkonis on Hokkaido populations of this primary freshwater species Hokkaido. remain unclear. Although Lefua populations have been reported on southern Sakhalin (Shedko and Shedko 2003; Shedko et al. 2008; Pietsch et al. 2012; Labai et al. 2014; Materials and methods Dyldin and Orlov 2016; Nakajima and Uchiyama 2017), detailed morphological analysis and population structure Field collections. Twenty-eight and 39 Lefua individuals analysis, using molecular markers, have not been com- were collected from Lake Bol’shoye Vavayskoye and Lake pleted. Since L. nikkonis has two continental congeners, Maloye-Chibisanskoye, respectively, on southern Sakhalin Lefua pleskei and Lefua costata, (Fig. 1; Bogutskaya and (Fig. 1; Table S1). Each individual was euthanised using Naseka 1996; Novikov et al. 2002; Bogutskaya et al. 2008; clove oil (NOW Foods, USA) (Soto and Burhanuddin 1995). Shedko et al. 2008; Pietsch et al. 2012; Dyldin and Orlov The right pelvic fn was cut and preserved in 99.5% ethanol 2016), the Sakhalin Lefua populations are very important for mitochondrial analysis, described below. The remaining Fig. 1 Schema of geographic distribution and phylogenetic relationships of Lefua nikkonis and two related continental congeners, Lefua pleskei and Lefua constata [the phylogeny was modifed from Ooyagi et al. (2018)]. Sites from which sam- ples for morphological analysis was obtained are also shown 1 3 Morphologies and population structures of Lefua body was fxed in 10% formalin and then preserved in 70% Honshu L. costata. In addition, 13 individuals of L. costata, ethanol. which were collected from the Korean Peninsula and stored Additionally, one to 10 individuals of Lefua nikkonis in the Natural History Museum and Institute in Chiba, Japan were collected from each of the 17 locations on the islands (CBM), and in the Hokkaido University Museum in Hako- of Hokkaido and Honshu (65 individuals in total), where date, Japan (HUMZ), were examined in a similar manner. Ooyagi et al. (2018) examined mitochondrial haplotypes of Each morphometric measurement was divided by SL and this species (Fig. 1; Table S1). Furthermore, 10 and fve log-transformed, and morphometric variations among the individuals of Lefua pleskei were collected from the Illistaya samples were described using principal component analysis River (St. LP1) and the Kraskino area (St. LP2), respec- (PCA). Because sexual diferences in fn lengths were evi- tively, in Far East continental Russia (Fig. 1; Table S1). dent (Fig. S1, see also Results), the PCAs were conducted The Illistaya River is just the type locality of L. pleskei. separately for fn length measurements (fve total) and body- Twelve Lefua costata individuals were also collected from shape measurements (17 total). Midoriko, Honshu Island (St. LC4; Fig. 1; Table S1), which We also classifed the Sakhalin Lefua into one of the three is known to be an introduction site for this species from the Lefua species using random forest analysis (Breiman 2001; continental Far East (Nakajima and Uchiyama 2017). All Cutler et al. 2007). The morphometric measurements and collected individuals of L. nikkonis, L. pleskei and L. costata VN of L. nikkonis from Hokkaido and Honshu, L. pleskei were euthanised, fxed in 10% formalin and transferred to a and L. costata were used as ‘training data’. Random forest 70% ethanol solution for preservation. analysis is a machine learning classifcation method, which Morphological analysis. Among the Sakhalin Lefua 1) can be used when the number of variables is larger than individuals preserved, 10 specimens (fve males and fve the number of samples; 2) does not overft; 3) performs well, females) taken from Lake Bol’shoye Vavayskoye and Lake even if many variables do not contribute to the results; 4) Maloye-Chibisanskoye were examined to assess their mor- incorporates variable interactions and 5) estimates each phology. Twenty-three morphometric measurements (Fig. 2; variable’s importance (Díaz-Uriarte and De Andrés 2006). Table S2) were obtained from each individual using a dial In this study, we constructed 10,000 decision tree, using caliper (D15F, Mitutoyo) to the nearest 0.01 mm. Vertebrae the 23 morphometric measurements (each divided by SL numbers without Weberian apparatus (VN) were counted and log-transformed), VN (log-transformed) and the sex using radiographs (CMB-2, Softex). Each