Taxonomic reassessment of salamanders (genus Hynobius) from Tsushima Islands Japan, with a resurrection of Hynobius tagoi Dunn, 1923 (Amphibia: Caudata) Author Niwa, Keita 0009-0009-6442-9555 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu, Sakyo-ku, Kyoto 606 - 8501, JAPAN. Present affiliation: Akita Prefectural Office, Sanno 4 - 1 - 1, Akita 010 - 8570, JAPAN. & tsushimanda 48 @ gmail. com; https: // orcid. org / 0009 - 0009 - 6442 - 9555 tsushimanda48@gmail.com Author Nishikawa, Kanto 0000-0002-6274-4959 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu, Sakyo-ku, Kyoto 606 - 8501, JAPAN. Present affiliation: Akita Prefectural Office, Sanno 4 - 1 - 1, Akita 010 - 8570, JAPAN. & Graduate School of Global Environmental Studies, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606 - 8501, JAPAN. Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori 036 - 8561, JAPAN. kuroo @ hirosaki-u. ac. jp; https: // orcid. org / 0000 - 0003 - 0278 - 2758 & nishikawa. kanto. 8 v @ kyoto-u. ac. jp; https: // orcid. org / 0000 - 0002 - 6274 - 4959 nishikawa.kanto.8v@kyoto-u.ac.jp Author Matsui, Masafumi 0000-0003-2032-2528 Graduate School of Human and Environmental Studies, Kyoto University, Yoshida Nihonmatsu, Sakyo-ku, Kyoto 606 - 8501, JAPAN. Present affiliation: Akita Prefectural Office, Sanno 4 - 1 - 1, Akita 010 - 8570, JAPAN. & fumi @ zoo. zool. kyoto-u. ac. jp; https: // orcid. org / 0000 - 0003 - 2032 - 2528 fumi@zoo.zool.kyoto-u.ac.jp Author Kanamori, Sally 0000-0003-3798-2653 Graduate School of Global Environmental Studies, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606 - 8501, JAPAN. Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho 3, Hirosaki, Aomori 036 - 8561, JAPAN. kuroo @ hirosaki-u. ac. jp; https: // orcid. org / 0000 - 0003 - 0278 - 2758 & s. kanamori. 5328 @ gmail. com; https: // orcid. org / 0000 - 0003 - 3798 - 2653 s.kanamori.5328@gmail.com Author Kuro-O, Masaki 0000-0003-0278-2758 kuroo@hirosaki-u.ac.jp text Zootaxa 2023 2023-08-30 5339 3 201 236 http://dx.doi.org/10.11646/zootaxa.5339.3.1 journal article 265688 10.11646/zootaxa.5339.3.1 ff895335-e448-4de5-a14d-44be257c2306 1175-5326 8308966 6882297C-7C5B-428C-AABF-F671E0440674 Hynobius tagoi Dunn, 1923 (Japanese name: Tago-sanshou-uwo) (English name: Tago’s salamander) ( Figs. 7 , 9B , 13B , 16 , 17D–F , 19 ) Hynobius nebulosus (part): Stejneger 1907 , 31 Hynobius nebulosus (part): Matsui et al. 2019 , 36 Hynobius tsuensis (part): Oyama 1930 , 31 Hynobius tsuensis (part, Group B): Niwa et al. 2021 , 260, Fig. 3B Hynobius tagoi : Dunn 1923a , 29 Hynobius leechii tagoi : Mori 1928 , 48 Holotype : CAS 26563 , an adult male from Tsushima, North Island , collected by Victor Kuhne in October 1910 . Specimens referred in the present paper: All specimens from Tsushima Islands, Nagasaki Prefecture : KUHE 58710 and 59933 ( two females ) from Sumo , Mitsushima ; KUHE 59949–59952 ( four males ) and 59953 ( one female ) from Itose , Toyotama ; KUHE 58864–58865 ( two males ) from Otsuna , Toyotama ; KUHE 58996 ( one female ) from Yoshida , Mine ; KUHE 57664–57665 , 57689–57690 ( four males ), 57666, 60171, and 60180 ( three females ) from Mine , Mine ; KUHE 59937 ( one female ) from Kaidokoro , Kamiagata ; KUHE 58673 ( one female ) from Oshika , Kamitsushima ; KUHE 58701–58703 , 58871 , 58983 , 60550–60562 ( 18 males ), 58704–58705, and 58872 ( three females ) from Kin , Kamitsushima ; KUHE 58709 ( one female ) from Hamakusu , Kamitsushima. All specimens were collected by K. Niwa in 2016–2018 . Etymology: The specific name “ tagoi ” is dedicated to Katsuya Tago who made great contributions to zoology of Japan . Diagnosis: A large species (adult SVL 59.6–71.3 mm in males and 56.4–70.2 mm in females), breeding in lotic water; dorsum brown with numerous dark stipples; fore- hindlimbs medium; tips of fore- and hindlimbs adpressed on body never meeting (overlap of -3.0 to -0.5 costal folds in males and -3.5 to - 0.5 in females); fifth toe long; tail long, low, and narrow; usually distinct yellow stripe(s) absent on the upper side of tail in males, but present in females; usually without distinct yellow stripe(s) on the lower side of tail; lateral color of tail brown with numerous dark stippling; ova large; clutch size small; egg-sacs short and C-shaped (crescent) with distinct whiptail structure on the free end. Phylogenetically, H. tagoi is included within the mtDNA clade of H. nebulosus , but H. tagoi has larger body size (SVL), relatively wider interorbital and upper eyelid, shorter axilla-groin distance, longer and lower tail, narrower tail at middle portion, longer fifth toe, and larger degree of limb overlap than H. nebulosus . Color: In life, dorsum brown to brownish gray, with fine dark stippling ( Fig. 7A, C ). Underside of body lighter than dorsum with ( Fig. 7D ) or without spots ( Fig. 7B ). Throat covered with white nuptial color in males. Tail side not black, but brown to brownish gray, with fine dark stippling (same color as dorsum) ( Fig. 7E, F ). Middorsal line of tail slightly lighter: usually without distinct yellowish stripe in males ( Fig. 7A ), but with narrow yellowish stripe in females ( Fig. 7C ). Variation: Morphometric data and body coloration data are summarized in Tables 3 and 5 , respectively. A significant sexual difference between males (n=28) and females (n=13) was not detected in body size (SVL) (P> 0.05), however, for ratio, males have significantly larger values (P <0.05) than females in snout length (SL, median=6.7%SVL in males vs. 6.4%SVL in females), tail length (TAL, 85.6%SVL vs. 76.4%SVL), basal tail width (BTAW, 10.6%SVL vs. 9.8%SVL), medial tail width (MTAW, 5.5%SVL vs. 4.6%SVL), basal tail height (BTAH, 8.3%SVL vs. 7.5%SVL), medial tail height (MTAH, 9.1%SVL vs. 8.3%SVL), maximum tail height (MXTAH, 9.7%SVL vs. 8.6%SVL), forelimb length (FLL, 23.3%SVL vs. 22.2%SVL), hindlimb length (HLL, 29.0%SVL vs. 27.8%SVL), and vomerine teeth series width (VTW, 5.0%SVL vs. 4.8%SVL). Whereas, males have significantly smaller axilla-groin distance (AGD, median=50.9%SVL) than females (52.9%SVL). Males have significantly larger degree of limb overlap (LO, median= -1.5) than females (-2.5). For body coloration, females tended to have distinct yellow stripe on the upper side of tail (76.9%) compared with males (25.0%). FIGURE 16. Dorsal (A), ventral (B), and lateral (C: right, D: left) views of male holotype of H. tagoi (CAS 26563). A white bar shows 10 mm. Photo by Erica J. Ely. FIGURE 17. Dorsal, lateral, and ventral views of a larva of H. tsuensis (A–C: KUHE 65029) from Izuhara and H. tagoi (D–F; KUHE 65028) from Toyotama. A white bar shows 10 mm. FIGURE 18. An alive female (A: KUHE 60148), egg sacs (B: not captured), a larva (C: not captured), and breeding habitat (D) of H. tsuensis . Egg-sacs and eggs: Morphometric values of egg-sacs, clutch size, and unfertilized egg rate are shown in Table 4 . Egg-sacs of H. tagoi were crescent in shape with thin envelope with some wrinkles and no notable striations ( Figs. 13B , 19B ). The all egg-sacs (n=19) used in the present study had a distinct whiptail structure on the free end, like H. tsuensis and some lotic-breeding salamanders (e.g., H. oni and H. hirosei ). The whiptail structure of H. tagoi ranged 2.5–60 (median=15, n=19) mm in length, which was significantly longer than H. nebulosus but not significantly different from H. tsuensis ( Fig. 12 ; Table 4 ). Egg-sacs of H. tagoi were significantly shorter than those of H. nebulosus ( Fig. 11 ). Clutch size of H. tagoi ranged from 21–62 (mean±SD=45.5±11.4, n=23), which was smaller than those of relatives such as H. nebulosus (population from Fukue Island: range=111–266, mean±SD=175.9±46.9, n=9 [ Fig. 11 ; Table 4 ]; Nagasaki : 77–160, 111.7±23.9, n=12 [ Fig. 11 ; Table 4 ], 89–117, 101.7±14.2, n=3 [ Sato 1943 ]; all Kyushu: 41–333, 135.8±59.0, n=91 [ Matsui et al. 2019 ]), H. dunni (50–200, 95.1±27.5, n=55 [ Sato 1943 ]), and H. bakan sensu lato (56–333, 116.5±58.2, n=32 [ Matsui et al. 2019 ]), but not significantly differed from H. tsuensis (34–74, 52.8±10.8, n=8 [ Fig. 11 ; Table 4 ]). Almost all eggs of H. tagoi were fertilized (unfertilized egg rate: median=2.0%, range=0.0–33.3%, n=24), unlike sympatric occurring H. tsuensis having many unfertilized eggs (unfertilized egg rate: 63.5%, 37.5–80.8%, n=14) in nature ( Fig. 11 ; Table 4 ). Ova from two females ranged from 3.0–3.3 (mean±SD=3.2±0.11, n=10) and 3.3–3.5 (3.4±0.08, n=10) mm in diameter. The animal pole of egg was gray brown and vegetal pole was light in color. Larvae: Views of larvae are shown in Figs. 17D–F and 19C . Larvae after hatching temporarily have a pair of balancers at the side of head. Fully grown larvae (n=7) at Stage 63 of Iwasawa and Yamashita (1991) of the first year collected from Kin, Kamitsushima on 23 June 2017 ranged from 17.6–28.7 (mean±SD=25.1±3.5) mm in SVL and 31.8–54.0 (47.5±7.3) mm in total length, head rounded in dorsal and lateral views ( Fig. 17D, E ); snout short and broadly rounded; eyes slightly protruded, inset from edge of head in dorsal view; labial fold distinct at half of upper jaw; external gills developed; caudal fin higher than head; dorsal fin higher than ventral fin; origin of dorsal fin in the middle portion of trunk; origin of ventral fin in vent; tail tip weakly pointed; limbs slender; claws on fingers and toes absent. In life, dorsum light brown without spots ( Fig. 17D ); venter whitish and transparent without spots ( Fig. 17F ). Metamorphosing larvae (n=4) at Stage 65–67 of Iwasawa and Yamashita (1991) ranged from 28.2–30.8 (mean±SD=29.3±0.9) mm in SVL and 52.8–56.2 (54.6±1.5) in total length. The larvae of H. tagoi tended to have larger body size than those of H. tsuensis ( Fig. 17A–C ) (17.7–19.8 [mean±SD=18.4±0.7] mm in SVL and 30.7–35.7 [32.5±1.7] mm in total length, n=5, Stage 63) collected from the same locality (stream) at the same date. In body coloration, H. tagoi tended to do not have any blackish spots (markings) on lateral side of tail ( Fig. 17E ), unlike larvae of H. tsuensis having large blackish or darkish markings on lateral side of tail ( Fig. 17B ). Range: Tsushima Islands (excluding the central to southern parts of Shimojima, former Izuhara-machi), in Nagasaki Prefecture , Western Japan ( Fig. 1 ). The known localities range from 25 m to 125 m (median 65 m , n=17) above sea level. FIGURE 19. An alive male (A: KUHE 63959), a pair of egg-sacs (B: KUHE 60144), a larva (C: not captured), and breeding habitat (D) of H. tagoi . Comparisons: Hynobius tagoi is the closest to H. nebulosus in mtDNA phylogeny, but morphologically distinguished from H. nebulosus by larger body size (SVL: males: mean±SD=64.6± 3.4 mm vs. 61.4± 4.5 mm in H. nebulosus ; females: 63.9± 3.4 mm vs. 59.3± 4.7 mm in H. nebulosus ), relatively wider interorbital (RIOD: males: 6.1%SVL vs. 5.8%SVL in H. nebulosus ; females: 5.9%SVL vs. 5.4%SVL in H. nebulosus ) and upper eyelid (RUEW: males: 3.0%SVL vs. 2.8%SVL in H. nebulosus ; females: 3.1%SVL vs. 2.7%SVL in H. nebulosus ), relatively longer tail (RTAL: males: median 85.6%SVL vs. 77.2%SVL in H. nebulosus ; females: 76.4%SVL vs. 70.7%SVL in H. nebulosus ), relatively narrower tail (RMTAW: males: 5.5%SVL vs. 6.6%SVL in H. nebulosus ; females: 4.6%SVL vs. 6.2%SVL in H. nebulosus ), relatively lower tail at the base (RBTAH: males: 8.3%SVL vs. 9.8%SVL in H. nebulosus ; females: 7.5%SVL vs. 8.9%SVL in H. nebulosus ), relatively lower tail at the middle portion (RMTAH: males: 9.1%SVL vs. 11.2%SVL in H. nebulosus ; females: 8.3%SVL vs. 10.2%SVL in H. nebulosus ), relatively shorter axilla-groin distance (RAGD: males: 50.9%SVL vs. 52.4%SVL in H. nebulosus ; females: 52.9%SVL vs. 54.3%SVL in H. nebulosus ), relatively longer fifth toe (R5TL: males: 3.2%SVL vs. 2.7%SVL in H. nebulosus ; females: 3.1%SVL vs. 2.3%SVL in H. nebulosus ), larger degree of limb overlap (LO: males: -1.5 vs. -2.0 in H. nebulosus ; females: -2.5 vs. - 3.5 in H. nebulosus ). Moreover, H. tagoi differed from H. nebulosus in smaller egg-sacs (MAESL: 139.2± 16.4 mm vs. 277.3± 32.8 mm in Fukue and 278.1± 30.8 mm in topotypic Nagasaki populations of H. nebulosus ) and clutch size (CS: 45.5±11.4 vs. 175.9± 46.9 in Fukue and 111.7± 23.9 in the Nagasaki populations), and longer whiptail structure (WTL: median 15 mm vs. 0 mm in both Fukue and the Nagasaki populations). Hynobius tagoi is distinct from H. tsuensis by the lateral color of the posterior half of tail: brown (not blackish or purplish color) with dark stipples in 96.3% of male and in 76.9% of female H. tagoi ; uniformly blackish [purplish] brown without markings in 96.0% of male and in 69.2% of female H. tsuensis . Also, H. tagoi is distinguishable from H. tsuensis by lacking distinct yellow stripe(s) on the tail, having brown dorsum with dark stipples (vs. usually, distinct yellow stripe(s) on the tail; blackish [purplish] brown dorsum with yellowish markings [spots] or yellowish dorsum with blackish [purplish] brown markings [spots] or dark brownish dorsum with dark [blackish] markings [spots] in H. tsuensis ). Hynobius tagoi morphologically differed from H. tsuensis by relatively narrower and shallower vomerine teeth series (RVTW: males: 5.0%SVL vs. 5.2%SVL in H. tsuensis ; females: 4.8%SVL vs. 5.2%SVL in H. tsuensis ; RVTL: males: 5.1%SVL vs. 5.5%SVL in H. tsuensis ; females: 5.0%SVL vs. 5.4%SVL in H. tsuensis ), and larger degree of limb overlap (LO: males: -1.5 vs. -2.0 in H. tsuensis ; females: -2.5 vs. -3.0 in H. tsuensis ). Clutch size and morphology of egg-sacs of H. tagoi are similar to H. tsuensis , but H. tagoi is distinct from H. tsuensis by having lower unfertilized egg rate (2.0% vs. 63.5% in H. tsuensis ). The lotic-breeding H. tagoi is also genetically close to lentic-breeding H. dunni and H. bakan sensu lato (containing H. nagatoensis and H. nihoensis ), but distinguished from H. dunni by smaller degree of limb overlap (LO: males: -1.5 vs. +2.0 in H. dunni ; females: -2.5 vs. +2.0 in H. dunni [ Sato 1943 ]) and smaller clutch size (CS: mean±SD=45.5±11.4 vs. 95.1± 27.5 in H. dunni [ Sato 1943 ]); from H. bakan sensu lato by larger body size (SVL: mean±SD=64.6± 3.4 mm vs. 51.8± 5.7 mm in H. bakan sensu lato [ Matsui et al. 2019 ]), relatively longer tail (RTAL: median 85.6%SVL vs. 74.4%SVL in H. bakan sensu lato [ Matsui et al. 2019 ]), relatively lower tail (RMXTAH: 9.7%SVL vs. 12.4%SVL in H. bakan sensu lato [ Matsui et al. 2019 ]), relatively longer fifth toe (R5TL: 3.2%SVL vs. 2.0%SVL in H. bakan sensu lato [ Matsui et al. 2019 ]), and smaller clutch size (CS: 45.5±11.4 vs. 116.5± 58.2 in H. bakan sensu lato [ Matsui et al. 2019 ]). Natural history: Hynobius tagoi breeds in lotic water such as mountain streams ( Fig. 19D ), unlike the lentic-breeding H. nebulosus . Sometimes, H. tagoi breeds syntopically with H. tsuensis ( Niwa et al. 2021 ) . Breeding occurs from early February to early March, which is earlier than that of H. tsuensis (late March to mid-April). Females of H. tagoi oviposit a pair of egg-sacs to stones or debris under the water in streams ( Fig. 19B ). Hatching occurs in late March to mid-April and larvae metamorphose in June to August of the first year. Overwintered larvae have not been discovered, unlike syntopic H. tsuensis , in which some overwintered larvae are found in mountain streams. Conservation: Hynobius tagoi is conserved as specified class II nationally rare species of wild fauna/flora on the Environment Government of Japan , under the name of H. nebulosus . Hynobius tagoi has been listed as vulnerable (VU) on the Red List of Ministry of the Environment Government of Japan , 2020 (Ministry of the Environment Government of Japan 2020 ), under the name of H. nebulosus . Also, it is listed as VU on the Prefectural Red List of Nagasaki , 2022 ( Nagasaki Prefecture 2022 ), under the name of H. nebulosus . These ranks should be reconsidered because the distribution range of H. tagoi is smaller than 400 km 2 .