Geographical Variations in Morphological Characters of the Fluvial Eight-Barbel Loach, Nagare-Hotoke-Dojo (Cobitidae: Nemacheilinae)

Biogeography 17. 43–52. Sep. 20, 2015

Geographical variations in morphological characters of the fluvial eight-barbel loach, Nagare-hotoke-dojo (Cobitidae: Nemacheilinae)

Taiki Ito*, Kazuhiro Tanaka and Kazumi Hosoya

Program in Environmental Management, Graduate School of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

Abstract. The morphological and color variations of Lefua sp. 1 Nagare-hotoke-dojo individuals from 13 river systems were examined. Analysis of variance revealed highly significant variations in Lefua sp. 1 morphology and coloration among the 13 populations examined, across all 19 measurements and counts. The 13 populations of Lefua sp. 1 were classified into two major clusters (I and II) by using UPGMA cluster analysis. Cluster I comprised fish from the Maruyama, Yura, Muko, Mihara, Yoshino, Hidaka, Kumano, Yoshii, Chikusa, and Ibo river systems. Cluster II comprised fish from the Yoshida, Saita, and Sumoto river systems. Cluster I was further subdivided into sub-clusters: I-i (the Maruyama, Yura, Muko, Mihara, Yoshino, Hidaka, Kumano, and Yoshii river systems) and I-ii (the Chikusa and Ibo river systems). Principal component analysis revealed that populations within cluster II clearly possessed longer caudal peduncles, while populations within cluster I possessed a longer anterior body on average and a deeper body. Populations within sub-cluster I-ii possessed a higher average dorsal fin and a longer average dorsal fin base than those of populations within sub-cluster I-i. A strong correlation was noted between the PC3 score and population latitude (r = 0.621). Observations of body color patterns revealed that individuals from the Yoshino, Mihara, Sumoto, and Hidaka river systems had dark brown mottling on both sides and the dorsal regions of their bodies and many small dark brown spots on the dorsal and caudal fins, while those from the Yura, Muko, and Kumano river systems possessed neither. Individuals from the Yoshii, Chikusa, Ibo, Maruyama, Yoshida, and Saita river systems, no dark brown mottling was present on both sides and the dorsal regions of their body and many small dark brown spots present on the dorsal and caudal fins. Overall, it was determined that Lefua sp. 1 exhibits a high degree of variability in its morphological characters among populations, which may be because of population isolation due to the specify preference for small freshwater bodies, such as streams.

Key words: body color, endangered species, geographical cline, river capture, undescribed species

Introduction were taxonomically separated from L. echigonia as an undescribed species sensu stricto, based on their The fluvial eight-barbel loach, known in Japanese morphological, ecological, and genetic features (Ho- common name as “Nagare-hotoke-dojo” (=Lefua soya, 1993; Hosoya, 2000; Hosoya, 2003; Sakai et sp. 1) is classified as a cobitid in the subfamily al., 2003). Lefua sp. 1 is distributed in freshwaters Nemacheilinae (Hosoya, 2013). Previously, this spe- along the coasts of the Seto Inland Sea, the Ise Bay cies was considered to represent several local popu- and the Sea of Japan that involves Kyoto, Fukui and lations of Lefua echigonia. However, the populations Tottori prefectures, Japan (Hosoya, 2013). ——————————————————————— Some studies have suggested that Lefua sp. 1 *Corresponding author: [email protected] exhibits several morphological and body color

− 43 − Geographical variations of Lefua sp. 1 variations among populations. Takahashi (1999) imens, 38.10–44.91 mm SL, Tatsuno City, Hyogo showed variations in the number of vertebrae among Prefecture. the Lefua sp. 1 populations of the Kinsei, Chigiri, Maruyama River system: KUN-P 44173-44177, 5 and Yoshino river systems in the eastern part of the specimens, 32.25–55.88 mm SL, Toyooka City, Hy- Ehime Prefecture. In addition, Takahashi (1999) ogo Prefecture. showed that the position of the ventral-dorsal fin and Yura River system: KUN-P 44148-44151, 4 spec- the coloration of the dorsal and caudal fins differed imens, 36.53–52.2 mm SL, Tanba City, Hyogo Pre- among river systems. Furthermore, Hosoya (1994) fecture. and Nakatani & Yoshida (1996) reported the obser- Muko River system: KUN-P 44178-44181, 44246- vation of two body coloration morphs in Lefua sp. 1: 44247, 6 specimens, 35.21–47.29 mm SL, Takarazu- one with dark brown mottling on both sides and on ka City, Hyogo Prefecture. the dorsal regions of the body and the other without Kumano River system: KUN-P 44155, 44157- the mottle body coloration. 44160, 5 specimens, 31.33–43.24 mm SL, Yoshino As the studies described above only examined in- District, Nara Prefecture. dividuals from a few river systems, the entire range Saita River system: KUN-P 44596-44616, 21 of intraspecific variation within Lefua sp. 1 is still specimens, 33.71–58.98 mm SL, Mitoyo City, Kaga- unclear. Clarifying the level of variation within Lefua wa Prefecture. sp. 1 and the geographical patterns of this variation Yoshino River system: KUN-P 44562-44573, 12 will contribute not only to the taxonomic description specimens, 35.17–47.69 mm SL, Tokushima City, of Lefua sp. 1 but also to the species’ recognition Tokushima Prefecture. as a conservation unit. In this study, we collected Yoshida River system: KUN-P 42268-42272, specimens of Lefua sp. 1 from a wide distribution of 44165, 6 specimens, 48.2–62.3 mm SL, Shozu Dis- populations and described the geographical variation trict, Kagawa Prefecture. of its morphological characters. Mihara River system: KUN-P 44133-44140, 8 specimens, 37.46–45.26 mm SL, Minamiawaji City, Materials and methods Hyogo Prefecture.

Materials examined We examined a total of 110 Lefua sp. 1 individuals from 13 sites, each in one of 13 river systems (Fig. 1), during 2010–2014. The study materials were deposited at Kinki University, Nara, Japan (KUN-P). List of materials examined Yoshii River system: KUN-P 44152-4453, 44584- 44590, 44592-44593, 11 specimens, 36.57–55.72 mm SL, Mimasaka City, Okayama Prefecture. Chikusa River system: KUN-P 44161-44164, 4 Fig. 1. The locations of river systems in this study. C: Chikusa specimens, 43.79–49.31 mm SL, Tatsuno City, Hyo- River, H: Hidaka River, I: Ibo River, K: Kumano River, go Prefecture. Ma: Maruyama River, Mi: Mihara River, Mu: Muko River, Sa: Saita River, Su: Sumoto River, Yd: Yoshida Ibo River system: KUN-P 44129-44132, 4 spec- River, Yi: Yoshii River, Yn: Yoshino River, Yu: Yura River.

− 44 − Taiki Ito, Kazuhiro Tanaka and Kazumi Hosoya

Sumoto River system: KUN-P 44141-44147, Morphological examination 44166-44167, 44574-44583, 19 specimens, 32.27– Measurements and counts were carried out ac- 60.69 mm SL, Minamiawaji City, Hyogo Prefecture. cording to the methods described by Hubbs & Lagler Hidaka River system: KUN-P 44168-44172, 5 (2004), with the exception of the vertebrae counts, specimens, 28.16–47.90 mm SL, Hidaka District, which were conducted according to the methods Wakayama Prefecture. described by Hosoya (1983). The vertebrae were counted on radiographs: we also included the four vertebrae of the Weberian apparatus, and the hypural

Table 1. Measurements and counts of Lefua sp. 1 from each river system

River system Yoshii Chikusa Ibo Maruyama Yura Muko Counts and measurements range mean±SD range mean±SD range mean±SD range mean±SD range mean±SD range mean±SD Measurements Percentage of standard length Head length 20.0-22.3 21.3±0.700 20.1-21.2 20.7±0.415 20.2-21.6 20.8±0.494 19.7-22.3 21.0±1.084 20.0-21.1 20.4±0.426 19.9-21.4 20.6±0.482 Body width 10.9-12.5 11.6±0.561 12.7-13.6 13.2±0.334 11.5-13.7 12.4±0.894 11.6-14.1 12.6±0.884 11.6-13.6 12.8±0.861 11.4-14.2 12.6±0.975 Body depth 11.7-13.9 12.8±0.692 13.8-16.4 15.4±0.981 13.8-14.8 14.3±0.351 13.2-16.2 14.2±1.025 12.7-15.0 13.7±0.934 11.9-14.9 13.5±1.138 Length of caudal peduncle 11.8-16.5 14.3±1.190 11.5-13.6 12.3±0.784 10.8-13.0 11.7±0.787 12.1-15.0 12.8±1.094 11.3-13.3 12.7±0.815 11.6-15.1 12.9±1.248 Depth of caudal peduncle 9.8-11.7 10.8±0.557 11.6-13.1 12.3±0.561 12.0-13.8 12.9±0.769 10.6-12.1 11.3±0.590 10.7-13.0 11.7±0.844 12.0-13.9 12.5±0.650 Predorsal length 63.6-68.4 66.0±1.280 68.6-71.7 70.6±1.167 67.3-70.9 69.2±1.557 65.5-69.0 67.0±1.329 65.7-68.8 67.0±1.151 67.4-68.9 68.1±0.544 Preanal length 77.4-86.4 80.3±2.323 80.4-81.9 81.2±0.549 79.6-84.7 81.9±1.798 80.0-82.2 81.1±0.730 79.7-81.7 80.5±0.815 79.3-83.5 81.8±1.426 Prepelvic length 56.1-59.0 57.6±0.912 59.3-62.1 60.1±1.171 56.9-60.2 58.5±1.170 56.2-61.0 58.5±1.696 57.4-58.5 57.9±0.482 57.0-60.7 58.8±1.147 Pectoral fin to pelvic fin base 35.3-39.2 37.8±1.178 39.4-40.4 40.0±0.377 38.9-39.6 39.2±0.312 36.2-42.2 38.8±2.119 37.7-39.1 38.4±0.497 35.7-41.4 39.2±1.827 Height of dorsal fin 9.2-12.9 11.0±1.005 11.2-15.0 12.9±1.519 12.8-14.7 13.6±0.793 10.5-13.0 12.0±0.912 11.7-13.7 12.8±0.733 9.9-13.4 11.2±1.169 Length of dorsal fin base 6.5-8.8 7.7±0.611 7.9-9.4 8.8±0.561 8.4-9.7 9.1±0.496 7.3-9.1 8.1±0.709 8.0-9.3 8.7±0.461 7.1-9.1 8.1±0.660 Height of anal fin 9.6-12.2 11.2±0.837 11.2-14.1 12.0±1.219 12.1-13.3 12.7±0.570 9.8-13.9 11.8±1.482 11.4-12.8 11.9±0.552 9.7-12.6 11.0±0.898 Pectoral fin length 12.5-15.7 14.1±0.928 12.2-13.2 12.8±0.456 13.8-15.7 14.9±0.686 13.6-15.6 14.6±0.849 13.4-16.1 14.6±1.089 13.4-16.0 14.6±0.936

Percentage of head length Snout length 33.6-44.4 39.9±2.668 38.8-44.7 41.0±2.243 36.6-39.2 38.3±0.970 36.0-47.3 42.9±3.923 41.4-45.9 43.4±1.614 37.9-44.5 41.5±2.393 Orbit diameter 11.1-14.7 12.8±1.196 10.5-13.8 12.3±1.350 8.8-13.9 12.0±1.932 8.8-14.4 12.4±1.915 11.0-13.7 12.1±0.983 11.4-13.0 12.1±0.506 Interorbital width 27.8-38.4 34.3±2.550 33.7-40.1 37.6±2.643 35.2-38.9 37.1±1.400 32.9-41.0 35.3±2.958 33.9-38.2 35.2±1.751 34.2-39.2 36.5±1.472

Counts Abdominal vertebral number 20-22 21.0±0.603 21-22 21.5±0.500 22 22.0±0 21-22 21.4±0.490 21 21.0±0 20-23 21.7±1.374 Caudal vertebral number 16-20 18.4±1.226 18-19 18.3±0.433 16-18 17.0±0.707 17-21 19.0±1.265 18-19 18.3±0.433 15-19 17.2±1.462 Total vertebral number 38-41 39.4±1.150 39-41 39.8±0.829 38-40 39.0±0.707 39-42 40.4±1.020 39-40 39.3±0.433 38-40 38.8±0.687

Table 1. Continued River system Kumano Saita Yoshino Yoshida Mihara Sumoto Hidaka Counts and measurements range mean±SD range mean±SD range mean±SD range mean±SD range mean±SD range mean±SD range mean±SD Measurements Percentage of standard length Head length 21.3-22.4 21.7±0.383 18.4-21.8 20.3±0.824 20.0-22.7 21.8±0.724 17.9-21.1 19.4±1.201 19.3-23.2 20.9±1.085 18.0-21.7 19.8±1.017 19.9-22.3 21.1±1.083 Body width 11.1-13.9 12.7±1.098 10.7-12.9 11.9±0.641 11.4-14.7 12.7±0.892 10.9-11.9 11.5±0.382 10.7-13.0 11.5±0.710 11.0-13.2 12.1±0.610 11.2-13.9 12.8±1.221 Body depth 12.0-14.5 13.5±0.848 11.4-14.2 13.1±0.753 13.2-16.6 14.3±0.802 12.3-14.5 13.3±0.709 12.1-14.7 13.2±0.722 12.2-14.6 13.2±0.656 12.2-16.4 14.3±1.554 Length of caudal peduncle 10.7-16.2 12.9±2.027 12.4-18.5 15.3±1.717 10.6-15.5 12.7±1.308 13.9-19.0 17.4±1.692 11.2-13.1 12.1±0.594 9.3-18.7 14.6±2.365 9.4-12.4 11.2±1.009 Depth of caudal peduncle 9.9-13.2 11.1±1.206 9.2-12.2 11.0±0.690 11.2-13.4 12.4±0.663 10.9-12.6 11.8±0.540 11.3-13.4 12.3±0.802 10.1-13.6 11.9±0.915 11.3-12.4 11.8±0.456 Predorsal length 67.6-69.2 68.2±0.637 61.6-67.3 64.5±1.574 66.0-69.2 67.4±0.867 61.8-64.7 63.4±0.877 66.9-69.4 68.5±0.837 62.1-72.0 66.2±2.596 64.7-69.0 66.9±1.461 Preanal length 79.8-82.4 81.0±0.849 72.5-80.6 76.2±2.284 79.3-82.8 81.0±1.171 75.0-79.9 78.3±1.725 80.4-82.9 81.7±0.764 73.9-84.0 78.1±3.121 77.7-82.3 80.6±1.635 Prepelvic length 58.0-61.0 59.6±1.146 53.9-59.3 56.2±1.424 57.4-61.5 58.9±1.032 54.4-56.5 55.4±0.683 57.2-60.5 59.0±1.092 53.4-60.9 56.4±2.105 56.5-58.9 57.9±0.816 Pectoral fin to pelvic fin base 37.4-39.5 38.5±0.751 33.4-39.7 36.7±1.317 35.6-42.9 38.3±1.759 34.8-38.7 36.7±1.478 35.8-39.2 37.4±1.236 33.6-40.5 36.8±1.987 35.5-39.5 37.7±1.537 Height of dorsal fin 10.9-14.6 12.6±1.405 11.4-14.5 12.9±0.952 10.7-13.5 11.5±0.747 11.5-14.1 12.9±0.780 10.0-14.3 12.5±1.153 10.5-14.5 12.2±1.129 12.6-14.5 13.3±0.683 Length of dorsal fin base 7.7-9.1 8.3±0.496 7.7-10.5 9.0±0.763 7.9-9.6 8.5±0.499 7.2-8.7 8.0±0.550 6.4-9.2 8.0±0.767 7.1-10.6 8.5±0.899 7.4-10.1 8.6±0.870 Height of anal fin 10.2-12.7 11.4±0.924 9.8-16.2 13.7±1.780 10.3-12.1 11.5±0.478 12.6-14.8 13.9±0.833 11.8-14.3 13.2±0.806 10.8-15.9 12.2±1.376 11.8-15.5 13.0±1.329 Pectoral fin length 14.0-17.8 15.4±1.399 13.9-17.4 15.9±1.010 12.5-16.0 14.7±0.885 14.1-16.8 15.3±0.943 12.7-16.1 14.8±1.057 10.8-15.8 13.6±1.229 13.0-15.5 14.4±0.802

Percentage of head length Snout length 39.8-45.9 42.3±2.211 34.7-47.1 40.7±3.292 34.9-44.6 39.1±2.317 40.4-45.7 43.5±1.876 38.2-50.3 42.0±3.805 35.0-50.7 43.3±4.052 35.3-45.6 41.1±3.386 Orbit diameter 11.2-14.7 13.5±1.381 7.3-15.1 10.9±2.096 10.2-16.5 13.6±1.947 10.2-13.6 12.0±1.219 9.6-13.4 11.4±1.196 7.6-13.4 10.6±1.641 12.0-14.5 13.1±0.875 Interorbital width 36.7-41.6 38.8±1.705 31.6-45.1 38.7±3.831 29.7-41.5 36.2±3.506 34.0-41.7 38.7±2.703 31.3-36.1 34.5±1.403 33.3-44.5 37.6±3.824 29.4-42.9 35.7±4.741

Counts Abdominal vertebral number 19-21 20.0±0.632 20-22 21.2±0.499 20-22 20.8±0.722 20-22 20.7±0.745 20-22 20.9±0.599 20-22 21.2±0.614 21 21.0±0 Caudal vertebral number 17-19 18.0±0.632 17-19 18.0±0.617 16-19 17.8±0.924 17-20 18.5±0.957 18-19 18.5±0.500 18-20 18.8±0.670 17-18 17.6±0.490 Total vertebral number 38 38.0±0 39-40 39.2±0.393 38-39 38.5±0.500 38-40 39.2±0.687 39-40 39.1±0.331 39-41 40.1±0.605 38-39 38.6±0.490

− 45 − Geographical variations of Lefua sp. 1 complex. The observation of body color patterns Lefua sp. 1 from each location. The analysis of vari- focused on the lateral sides and dorsal region of the ance indicates that in all 19 variables, there are high- body, as well as the dorsal and caudal fins. ly significant differences among the 13 populations Statistical analysis (Table 3). The results of multiple comparisons for Tests of the homogeneity of variance were carried each character are given in Table 3. out on each character (19 characters, see Table 1) In the cluster analysis of 16 measurements, two using Bartlett’s test in R (Ver. 2.15.2). If the variance major clusters (I and II) were constructed (Fig. 2). were homogeneous, ANOVA (analysis of variance: Cluster I was made up of the populations of the for assessing differences between the means of the Lefua raw measurements and counts for each population) Table 2. Location for sp. 1 sampling sites River system Latitude Longitude Altitude (m) and Tukey–Kramer post hoc test (multiple compar- Yoshii 34.985 134.199 196.0 ison: for pairwise comparisons of means, to deter- Chikusa 34.935 134.457 221.9 Ibo 34.975 134.499 230.7 mine which are significantly different from which Maruyama 35.438 134.934 282.0 others) were used (parametric procedures; R: Ver. Yura 35.135 135.195 206.0 2.15.2). While, variances were heterogeneous, Kru- Muko 34.866 135.308 150.7 Kumano 34.031 135.997 379.0 skal-Wallis test and Steel–Dwass post hoc test were Saita 34.084 133.810 352.0 used (non-parametric procedures; R: Ver. 2.15.2). Yoshino 34.013 134.439 229.0 The morphological similarity among localities Yoshida 34.534 134.316 281.2 Mihara 34.240 134.820 266.0 was analyzed using UPGMA cluster analysis. The Sumoto 34.275 134.846 228.0 means of the measurements (16 characters, see Table Hidaka 33.960 135.160 90.9 1) taken in each population were regarded as a data set. A dendrogram was produced using the Euclidean Table 3. The morphological divergence of 13 populations of Lefua sp. 1. Locality codes refer to Figure 3. Significance distances between populations, using the computa- levels for this table are * = p < .05 and ** = p < .01 ANOVA / tional software program R (Ver. 2.15.2). Variable Bartlett’s Kruskal-Wallis The standardized means (determined using a cor- Measurements relation matrix) of each measurement and count (19 Head length ** Body width ** characters, see Table 1) in each population were an- Body depth ** alyzed using principal component analysis in R (Ver. Length of caudal peduncle * ** 2.15.2). Then, to evaluate the relationships between Depth of caudal peduncle ** Predorsal length ** ** the morphometric values and latitude, longitude, and Preanal length ** ** altitude (Table 2), the coefficients of the correlations Prepelvic length ** Pectoral fin to pelvic fin base * ** (r value) between the component scores of each Height of dorsal fin ** population vs. latitude, longitude, and altitude were Length of dorsal fin base ** Height of anal fin ** ** calculated (Spearman’s rank correlation coefficient) Pectoral fin length ** using Microsoft Excel 2007, in Windows. Snout length * Orbit diameter ** Interorbital width * Results Counts Abdominal vertebral number ** ** Caudal vertebral number * ** Measurements and counts Total vertebral number ** ** Table 1 lists the measurements and counts of

− 46 − Taiki Ito, Kazuhiro Tanaka and Kazumi Hosoya

Table 3. Continued Variable Tukey-Kramer / Steel-Dwass Measurements Head length Su vs. Yi**, K**, Yn** Yn vs. Sa**, Yd** Yd vs. Yi**, K** Body width C vs. Yi*, Mi* Body depth C vs. Yi**, Sa**, Yd*, Mi**, Su** Yn vs. Yi**, Sa** Length of caudal peduncle Sa vs. Yn*, Mi**, H* Depth of caudal peduncle Sa vs. I**, Mu**, Yn**, Mi**, Su* Yi vs. I**, Yn**, Mi**, Su* I vs. K* Mu vs. Mi** Predorsal length Sa vs. Mu*, K*, Yn**, Mi** Yn vs. Yd* Preanal length Sa vs. Yi*, Mu*, K*, Yn**, Mi** Prepelvic length Yd vs. Ma*, C**, Mu**, K*, Yn**, Mi** Sa vs. C**, Mu*, K**, Yn**, Mi** Su vs. C**, Mu*, K**, Yn**, Mi** Pectoral fin to pelvic fin base Height of dorsal fin I vs. Yi**, Mu*, Yn* Sa vs. Yi**, Mu*, Yn* Yi vs. H* Length of dorsal fin base Yi vs. Sa** Height of anal fin Yn vs. Sa*, Yd*, Mi* Yi vs. Sa*, Yd* Pectoral fin length Sa vs. Yi**, C**, Su** C vs. K*, Yd* Yd vs. Su* Snout length Yn vs. Su* Orbit diameter Yn vs. Sa**, Su** Interorbital width Yi vs. Sa* Counts Abdominal vertebral number Caudal vertebral number Sa vs. Su* Total vertebral number Sa vs. K**, Yn*, Su** Su vs. K*, Yn**

Maruyama, Yura, Muko, Mihara, Yoshino, Hidaka, Kumano, Yoshii, Chikusa, and Ibo river systems. Cluster II was made up of the populations in the Yoshida, Saita, and Sumoto river systems. Cluster I was subdivided into two sub-clusters: I-i, made up of the Maruyama, Yura, Muko, Mihara, Yoshino, Hida- ka, Kumano and Yoshii river systems, and I-ii, made up of the Chikusa and Ibo river systems. The principal component analysis of the 19 morphological variables produced five axes (PC1– PC5) that together explain 85% of the total variation among populations. The principal component analysis was also used to detect correlations among the variables (Table 4). The first axis, PC1, mainly en- compassed head length (-0.662), body width (-0.740), body depth (-0.711), length of caudal peduncle (0.875), predorsal length (-0.904), preanal length (-0.859), prepelvic length (-0.911), and distance from the pectoral to pelvic fin base (-0.895). Because the length and depth of the body loaded strongly on first axis, PC1 was interpreted as body slenderness. The height of the dorsal fin (0.720) and length of the Fig. 2. Dendrogram obtained using UPGMA cluster analysis based on the similarity of the measurements of Lefua sp. 1 dorsal fin base (0.796) loaded on PC2. On the PC3 individuals among river systems. axis, the highest loadings were the pectoral fin length (-0.717), caudal vertebral number (0.630), and total

− 47 − Geographical variations of Lefua sp. 1 vertebral number (0.885). loaded strongly and positively on PC3 (Table 4), the Using the component scores of PC1 and PC2, the coefficients of the correlations between latitude and relationships among the geographical populations of both caudal vertebral number, and between latitude Lefua sp. 1 were displayed in two-dimensional space and total vertebral number were calculated. It was (Fig. 3). In the cluster analysis, PC1 separated clus- found to be a strong correlation between latitude and ters I and II, and PC2 separated sub-clusters I-i and total vertebral number only (r = 0.600; caudal verte- I-ii. Populations contained in cluster II clearly exhib- bral number: r = 0.357). No strong correlations were ited longer caudal peduncles (Fig. 3). While, popula- noted between other component scores and latitude tions contained in cluster I possessed longer anterior (Lat), longitude (Lot), or altitude (Alt) (PC1: Lat r = body sections (hl, pdl, pal, ppl, pec-pel: see Fig. 3) 0, Lot r = -0.324, Alt r = 0.308; PC2: Lat r = 0.044, and deeper bodies (Fig. 3). PC2 represented the vari- Lot r = -0.225, Alt r = -0.022; PC3: Lot r = 0.038, ation in the height of the dorsal fin and length of the Alt r = -0.335; PC4: Lat r = -0.044, Lot r = 0.346, dorsal fin base, with the populations in sub-cluster Alt r = 0.225; and PC5: Lat r = 0.005, Lot r = 0.280, I-ii possessing higher dorsal fins and longer dorsal Alt r = 0.071). fin bases than populations contained in sub-cluster Body color pattern I-i (Fig. 3). On the side and dorsal area of the body, the spec- There was a strong correlation between compo- imens examined were clearly divided into the type nent score of PC3 and latitude (r = 0.621). Because with dark brown mottling and the non-mottled type caudal vertebral number and total vertebral number (Fig. 4A, B). The dark brown mottling color type in-

Table 4. Principal component analysis variables from the morphometric values of Lefua sp. 1 and their loadings with descriptive statistics. Loadings in bold are strong (>0.6)

Variable PC1 PC2 PC3 Eigenvalue 7.28 3.36 2.76 % variance explained 38.3 17.7 14.5 Head length -0.662 -0.558 -0.334 Body width -0.740 0.227 0.048 Body depth -0.711 0.426 0.142 Length of caudal peduncle 0.875 -0.034 0.022 Depth of caudal peduncle -0.504 0.497 0.018 Predorsal length -0.904 0.145 0.129 Preanal length -0.859 -0.239 0.080 Prepelvic length -0.911 -0.164 0.027 Pectoral fin to pelvic fin base -0.895 0.078 0.170 Fig. 3. Morphological variation, as revealed by the principal Height of dorsal fin 0.044 0.720 -0.324 component analysis of 13 river systems of Lefua sp. 1. Combinations of variables expressed by the axes are Length of dorsal fin base -0.248 0.796 -0.284 illustrated, and the arrows indicate the direction in which Height of anal fin 0.583 0.541 -0.269 the morphometric values and counts increase along its Pectoral fin length 0.429 -0.113 -0.717 axis. hl: head length, bw: body width, bd: body depth, Snout length 0.490 -0.117 0.400 lcp: length of caudal peduncle, pdl: predorsal length, pal: Orbit diameter -0.537 -0.545 -0.381 preanal length, ppl: prepelvic length, pec-pel: pectoral fin Interorbital width 0.311 0.452 -0.399 to pelvic fin base, hd: height of dorsal fin, ldb: length of dorsal fin base. For the locations and abbreviations of the Abdominal vertebral number -0.293 0.592 0.398 names of the river systems denoted by the black dots, see Caudal vertebral number 0.480 -0.278 0.630 the legend in Fig. 1. Total vertebral number 0.240 0.236 0.885

− 48 − Taiki Ito, Kazuhiro Tanaka and Kazumi Hosoya cludes individuals from the Yoshino, Mihara, Sumo- are also highly variable among the Lefua sp. 1 popu- to, and Hidaka river systems. The non-mottled color lations investigated in this study. type includes individuals from the Yoshii, Chikusa, Ibo, Maruyama, Yura, Muko, Kumano, Saita, and Yoshida river systems. Individuals from the Yoshii, Chikusa, Ibo, Maruyama, Saita, Yoshino, Yoshida, Mihara, Sumo- to, and Hidaka river systems also have many small dark brown spots on their dorsal and caudal fins (Fig. 5A). However, individuals from the Yura, Muko, and Kumano river systems never had spots on their dorsal and caudal fins (Fig. 5B).

Discussion

The variation of measurements and counts among populations The analysis of variance revealed that Lefua sp. 1 has great morphological variations in all 19 variables investigated (Table 3). In these morphological variations within Lefua sp. 1, the diversity of slenderness Fig. 4. The coloration on side and dorsal region of the body of of body and the number of vertebrae had previously Lefua sp. 1. A: type with dark brown mottling, KUN-P 44568, Yoshino River system. B: type without dark brown been reported by Takahashi (1999). In this study, it mottling, KUN-P 44157, Kumano River system. Scales: 5 was newly found that other morphological characters mm.

Fig. 5. The coloration of the dorsal and caudal fins of Lefua sp. 1. A: many small dark brown spots on the fins, KUN-P 44585, Yoshii River system. B: no small dark brown spots on the fins, KUN-P 44157, Kumano River system. Scales: 1 mm.

− 49 − Geographical variations of Lefua sp. 1

One of the causes of this extensive diversification the populations of the above river systems, those of among populations of Lefua sp. 1, is supposed to be the Yoshii, Chikusa, Ibo, Maruyama, Saita, Mihara, the strict isolation of the populations of each river Sumoto, Yoshida, and Hidaka river systems also had system. Generally, the dispersal of freshwater fishes many small dark brown spots on their dorsal and (especially fishes of primary division: sensu Dar- caudal fins. lington, 1957) relies on marine regressions, when Because individuals with dark brown mottling on the sea level drops, allowing for the formation of their bodies were limited to so-called the Kii-Shi- downstream connections between freshwater ba- koku populations, which were classified using sins (Lindberg, 1981; Nishimura, 1974; Mizuno & genetic techniques (Miyazaki et al., 2007), this char- Gotoh, 1987). These events allow the different fish acteristic might reflect the phylogeny of Lefua sp. populations to come into genetic contact; however, 1 populations. On the other hand, individuals with Lefua sp. 1 has a low chance of experiencing such small dark brown spots on their dorsal and caudal type of migration opportunity as described above, fins were observed in both the Sanyo and Kii-Shi- because this species characteristically persists to the koku populations; thus, fin coloration might reflect upper reaches of mountain streams (Hosoya, 1997). an adaptation to the environmental conditions of In fact, Nakano (1992) used isozyme analysis to each river system, and/or random genetic drift. show that some populations of Lefua sp. 1 (treated The relationship between morphological variation as L.echigonia by the author) from the Minato River and geography system in the Kagawa Prefecture and the Yoshino In the vertebral counts, geographical cline was River system in the Tokushima Prefecture, were found to be significant: Lefua sp. 1 from higher lati- strongly isolated in particular river systems and/or tudes had more vertebrae, because there was a strong streams. Over a long period of time, the adaptation correlation between latitude and total vertebral of Lefua sp. 1 populations to the disparate environ- number. Similar geographic clines, called “Jordan’s mental conditions of each river system may have led rule” (Jordan, 1891), were reported to apply to a to increases in the differentiation of morphological few of Japanese freshwater fish species Misgurnus( characteristics among different populations. In addi- anguillicaudatus: Kubota & Ono, 1965; Kubota, tion, small sizes of the populations inhabiting only 1967; Oryzias latipes: Yamahira & Nishida, 2009). the upper reaches of mountain streams (Suzuki et Generally, this cline is mainly caused by differences al., 1997; Ito & Hosoya, 2011), may have accelerat- in water temperature: i.e., the greater numbers of ed this evolutionary trend via the founder effect and vertebrae seen at higher latitudes are the result of genetic drift (see Wright, 1931; 1932; 1942; Mayr, lower habitat temperatures. Like wise, in Lefua sp. 1, 1954). variations in vertebral counts may also be influenced Variations of body color pattern by the water temperatures in each river system. We confirmed that Lefua sp. 1 individuals exhibit Similarity of measurements among populations is two different patterns of body and fin coloration (Ho- not correlated with the geographic locations of each soya, 1994; Nakatani & Yoshida, 1996; Takahashi, river system. According to the cluster analysis, the 1999). Takahashi (1999) reported that individuals similarity of measurements among some populations from the Kinsei, Chigiri, and Yoshino river systems such those from the Maruyama, Yura, and Muko have many small dark brown spots on their dorsal river systems, and the Ibo and Chikusa river systems and caudal fins. In our observation, in addition to reflect the populations’ geographic locations (Fig. 1,

− 50 − Taiki Ito, Kazuhiro Tanaka and Kazumi Hosoya

2). Meanwhile, other populations, especially the pop- 9: 43–48. (in Japanese) ulations from the Yoshino and Saita river systems, Hosoya, K., 1993. Cobitidae. In Nakabo, T. (Ed), and the Sumoto and Mihara river systems had very Fishes of Japan with pictorial keys to the species: different characteristics, in spite of the fact that these 231–235. Tokai Univ. Press, Tokyo. (in Japanese) river systems are in geographically small areas (Fig. Hosoya, K., 1994. Lefua echigonia. In The Fisheries 1, 2). The cause of this disparity between physically Agency (Ed), Basic data on threatened wild close populations is estimated to be due to differ- aquatic organism in Japan-I: 386–391. The Fish- ences in the degree of isolation among populations. eries Agency, Tokyo. (in Japanese) Lefua sp. 1 might well mainly disperse when river Hosoya, K., 1997. Protection of freshwater fishes in captures occurred from one side to opposite (Mizuno terms of biodiversity. In Nagata, Y. and Hosoya, & Gotoh, 1987; Yamashina et al., 1994; Kato, 2000; K. (Eds), Circumstances in endangered Japanese Higashiyama, 2002). Because river captures take freshwater fishes and their protection: 315–329. place locally and at low frequency (Mizuno & Got- Midori Shobo, Tokyo. (in Japanese) oh, 1987), profound differences in the degree of iso- Hosoya, K., 2000. Cobitidae. In Nakabo, T. (Ed), lation among populations might arise. Therefore, in Fishes of Japan with pictorial keys to the species populations strongly isolated from nearby river sys- second edition: 272–277. Tokai Univ. Press, To- tems, characteristics differ greatly among adjacent kyo. (in Japanese) populations. In the future, further work is needed to Hosoya, K., 2003. Lefua sp. In Ministry of the envi- clarify the relationship between the morphological ronment (Ed), Threatened wildlife of Japan: red and genetic differentiations among populations of data book, 2nd edition, vol. 4. Pisces: Brackish Lefua sp. 1. and freshwater fishes: 108–109. Japan Wildlife Research Center, Tokyo. (in Japanese) Acknowledgments Hosoya, K., 2013. Cobitidae. In Nakabo, T. (Ed), Fishes of Japan with pictorial keys to the species We are deeply grateful to Dr. S. Aoyama, Mr. A. third edition: 328–334. Tokai Univ. Press, Tokyo. Inotsuka, and Ms. Y. Yamashina for assisting in the (in Japanese) collection of specimens. Special gratitude is con- Hubbs, C. L. & Lagler, K. F., 2004. Fishes of the veyed to Dr. S. Kawase for the advice and comments Great Laks Region. Revised edition. University of on this manuscript. Michigan Press, Bloomfield Hills, xxxii+276 pp. Ito, T. & Hosoya, K., 2011. Preliminary report on the References current environment and status endangered loach, Lefua sp., “Nagare-hotokedojou” in Shodoshima Darlington, P. J. Jr., 1957. Zoogeography: the geo- Island. Bull. Biogeogr. Soc. Jpn., 66: 235–241. (in graphical distribution of animals. Wiley &Sons, Japanese with English abstract) New York, 675 pp. Jordan, D. S., 1891. Relations of temperature to ver- Higashiyama, N., 2002. Distributions of Lefua sp. in tebrae among fishes. Proc. Natl. Acad. Sci. USA., Kyoto Prefecture, Japan. Nankiseibutu, 44: 42–47. 14: 107–120. (in Japanese) Kato, F., 2000. Notes on the morphology and the Hosoya, K., 1983. Geographic variation of number distribution of two species of the genus Lefua, the of vertebrae in Squalidus. The Freshwater Fishes, genus Tridentiger and three species of the genus

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Chaenogobius found in Fukui Prefecture, Japan. His., 14: 29–30. (in Japanese) Bull. Fukui City Mus. Nat. His., 47: 33–45. (in Nishimura, S., 1974. Origin and history of the Sea Japanese) of Japan: An approach from biogeographic stand- Kubota, Z. & Ono, T., 1965. Morphology of the point. Tsukiji Shokan, Tokyo, 227 pp. (in Japa- Japanese loach, Misgurnus anguillicaudatus nese) (Cantor)-V. Geographical variation of the number Sakai, T., Mihara, M., Shitara, H., Yonekawa, H., of vertebrae. Contribution from the Shimonoseki Hosoya, K. & Miyazaki, J., 2003. Phylogenet- University of Fisheries, 14: 41–52. (in Japanese ic relationships and intraspecific variations of with English abstract) loaches of the genus Lefua (Balitoridae, Cyprini- Kubota, Z., 1967. Morphology of the Japanese loach, formes). Zool. Sci., 20: 501–514. Misgurnus anguillicaudatus (Cantor)-VI. Verte- Suzuki, T., Masuda, O., Yamashina, Y. & Yuasa, bral number of the loach hatched at various water Y., 1997. Lefua echigonia and L. sp. in Hyogo temperature. Contribution from the Shimonoseki Prefecture - distribution and habitat environment. University of Fisheries, 15: 17–22. (in Japanese Hyogo Freshwater Biology, 48: 1–4. (in Japanese) with English abstract) Takahashi, H., 1999. Distribution of the Lefua sp. Lindberg, G. U., 1981. Large-scale fluctuations of in the eastern part of Ehime Prefecture, Shikoku, sea level in the quaternary period: Hypothesis Japan. Bull. Tokushima Pref. Mus., 9: 39–47. (in based on biogeographical evidence (Japanese Japanese with English abstract) translation). Tokai Univ. Press, Tokyo, 366 pp. (in Wright, S., 1931. Evolution in Mendelian popula- Japanese) tions. Genetics, 16: 97–159. Mayr, E., 1954. Change of genetic environment and Wright, S., 1932. The roles of mutation, inbreeding, evolution. In Huxley, J. A., Hardy, A. C. & Ford, E. crossbreeding, and selection in evolution. Proc. B. (Eds), Evolution as a Process: 157–180. Allen 6th Internat. Congr. Genet., 1: 356–366. & Unwin, London. Wright, S., 1942. Statistical genetics and evolution. Miyazaki, J., Nakao, K., Mihara, M., Sakai, T., Bull. Amer. Math. Soc., 48: 223–246. Gunji, Y., Tojo, K., Muraoka, K & Hosoya, K., Yamahira, K. & Nishida, T., 2009. Latitudinal varia- 2007. Incongruence between mtDNA phylogeny tion in axial patterning of the medaka (Actinopte- and morphological and ecological characters in rygii: Adrianichthyidae): Jordan’s rule is substan- loaches of the genus Lefua (Balitoridae, Cyprini- tiated by genetic variation in abdominal vertebral formes). Zool. Sci., 24: 666–675. number. Bio. J. Linn. Soc., 96: 856–866. Mizuno, N. & Gotoh, A., 1987. Freshwater fishes of Yamashina, Y., Kamei, T. & Hosoya, K., 1994. Pre- Japan. Tokai Univ. Press, Tokyo, ix+244+33 pp. liminary report on two Lefua species obtained from (in Japanese) Hikami district. Hyogo Freshwater Biology, 45: Nakano, H., 1992. Teaching materialization of in- 5–11. (in Japanese) traspecific differentiations of Lefua echigonia in- habit in Shikoku Island. Education of Biology, 32: (Received June 4, 2015; Accepted August 4, 2015) 44–45. (in Japanese) Nakatani, Y. & Yoshida, M., 1996. The current dis- tributional state of Lefua sp. in Wakayama Prefec- ture. Report of Wakayama Prefectural Mus. Nat.

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