A new species of salamander of the genus Hynobius (Amphibia, Caudata, Hynobiidae) from South Korea Author Min, Mi-Sook Author Baek, Hae-Jun Author Song, Jae-Young Author Chang, Min Ho Author Poyarkov Jr, Nikolay A. text Zootaxa 2016 4169 3 475 503 journal article 10.11646/zootaxa.4169.3.4 066ada3e-81a7-4a32-aa33-7621d513edbc 1175-5326 264812 8CA28A0B-4945-40A2-9145-59F6C28D0471 Hynobius unisacculus sp. nov. ( Figs. 3–6 ; Table 3 ) Synonymy: “ Hynobius leechii (partim): Sato 1943 : p. 33; Chang et al. 2011 : p. 20.” “ Hynobius quelpaertensis (partim): Kim et al. 2003 : p. 1166.” “ Hynobius leechii Clade HC2: Baek et al. 2011a : p. 108; Baek et al. 2011b : pp. 25–32.” Holotype. CGRB-15401 (deposited in the Conservation Genome Resources Bank for Korean Wildlife (CGRB), College of Veterinary Medicine, Seoul National University; field number MMS 1962 ), adult male from the Naro Islands (Oenaro-do Island ), from the environs of Oecho-ri , Bongrae-myeon , Goheung-gun , Jeollanam-do , South Korea (127° 28´36.1¨ E; 34° 26´19.6¨ N), collected on March, 10, 2010 by Hae-Jun Baek and Mi-Sook Min. Paratypes. With the exception of the holotype, the type series consists of 68 adult specimens: 55 males and 13 females, all collected during the breeding season. Type series includes: ten specimens (CGRB-15392–400, 402; field numbers: MMS 1951–53 , 1955–58 , 1960- 1961 , 1963; 8 males , 2 females ) with the same collection data as the holotype ; six specimens (CGRB-15386–391; field numbers: MMS 1945–50 ; 3 adult females, 3 subadults) from the vicinity of Singeum-ri, Bongrae-myeon, Goheung-gun, Jeollanam-do , South Korea ( Oenaro-do Island ), collected on March , 10, 2010 by Hae-Jun Baek and Mi-Sook Min ; two specimens (CGRB-15403–404; field numbers: MMS 1968–1969 ; 1 male , 1 female ) from the environs of Bongrim-ri, Podu-myeon, Goheung-gun , Jeollanam-do , South Korea , collected on March , 11, 2010 by Hae-Jun Baek and Mi-Sook Min ; four specimens (CGRB-15442–445; field numbers: MMS 1972–73 , 1975 , 1981; 3 males , 1 female ) from the vicinity of Jinggwang-ri, Beolgyo-eup, Boseoung-gun , Jeollanam-do , South Korea , collected on March , 11, 2010 by Hae-Jun Baek and Mi-Sook Min ; ten specimens (CGRB-15446–455; field numbers: MMS 1989–93 , 1997 , 2114–17 ; 8 males , 1 female ) from the environs of Wangji-dong, Suncheon-si, Jeollanam-dp, South Korea , collected on March, 11 and April 21, 2010 by Hae-Jun Baek and Mi-Sook Min; and thirty four specimens (CGRB-15502-535; field numbers: AR10–15, 17-19, 21-25, 27–30, 71–83, 85–87; 26 males , 8 females ) from Goheung-gun, collected in March 2009 by Jae Young Song and Min Ho Chang. Diagnosis. The species is assigned to the genus Hynobius based on the following character states considered diagnostic for the genus: (1) lungs present; (2) digits in adults lack cornified claw-like structures; (3) dermal flaps on posterior edges of hindlimbs in adults absent; (4) tail relatively short, shorter than body, and distinctly flattened for most of its length; (5) vomerine teeth arranged in a distinctly curved series with inner branches notably longer than outer branches and located posterior to the level of choanae; (6) frontal fontanelle between frontals and parietals absent; (7) light, broad dorsal stripe absent; (8) fifth toe well-developed ( Dunn 1923 ; Sato 1943 ; Zhao et al. 1988 ; Adler & Zhao 1990 ; Fei et al. 2006 ). In external morphology the new species most closely resembles the other Korean species of Hynobius , but is notably smaller. Hynobius unisacculus sp. nov. is distinguished from its congeners by a combination of the following morphological attributes: (1) comparatively small-size (adult SVL up to 61 mm ; range 38.3–60.3 mm in males and 37.5–59.9 mm in females) within the lentic-breeding Hynobius (breeding in shallow still waters, such as ditches or paddy-fields); (2) relatively slender short limbs; large gap (gap of -3.0 to -1.5 costal folds in males and -3.5 to - 1.5 in females) always separating tips of fore- and hindlimbs adpressed on body; (3) comparatively short and flattened tail (TL/SVL ratio in adult males varying from 0.54 to 0.98, in adult females from 0.55 to 0.89), with a low dorsal fin along the posteriormost one-third of its length; (4) usually 11 (occasionally 12) costal grooves (excluding axillary groove); with 13 (occasionally 12) trunk vertebrae; (5) dark brown dorsum in adults, with indistinct bronze or dark copper spots, lighter greyish-white or pinkish belly; (6) well developed fifth toe; (7) comparatively shallow vomerine tooth series with 13–23 vomerine teeth; (8) small, pigmented ova, located in one, occasionally two, string(s) in small, curved egg sac with folded envelope, lacking distinct mucous stalks or whiptail-like structure on both ends; in the wild usually laid as single egg sacs; the new species is also markedly different from all studied congeners, including other Korean Hynobius , in its sequences of three mitochondrial DNA genes (12S rRNA, COI, cyt b ). FIGURE 3. Holotype of Hynobius unisacculus sp. nov. (CGRB-15401, male; filed number MMS-1962) in preservative: (A) dorsal view, (B) ventral view (C) volar view of the left hand, (D) plantar view of the left foot, (E) ventral view of vent. FIGURE 4. Holotype of Hynobius unisacculus sp. nov. (CGRB-15401, male; filed number MMS-1962) in preservative: (A) head in dorsal view, (B) head in ventral view (C) head in lateral view. Scale bar 2 mm. Description of holotype . An adult male, in a good state of preservation, fixed in and preserved in 70% ethanol, with an SVL of 50.8 mm (measured on the preserved specimen) (see Figs. 3 , 4 ). Trunk. Body elongated, slender and cylindrical ( Fig. 3 , A, B); chest comparatively narrow: the CW/SVL ratio 0.13. The skin of the dorsum and venter is smooth, slimy; diffuse microscopic glands scattered all over the body. Mid-dorsal groove weakly developed on the dorsum, extending from the basis of the head to the base of the tail. Costal grooves well developed, 11 grooves visible on each side of the body (excluding the axillary groove), 11 grooves visible from ventral side; ventral abdominal vein visible through translucent skin on the belly ( Fig. 3 , B). Cloaca. Cloaca noticeably swollen and protuberant in ventral and lateral views ( Fig. 3 , E). Vent longitudinal, elongate Y-shaped with swollen edges and well-developed protuberance on anterior edge of vent ( Fig. 3 , E). Tail. Tail comparatively short, noticeably shorter than the body, ratio of tail length (TL) to body length (SVL) 0.77. Tail laterally compressed for most of its’ length; anterior one-fifth of tail length subcilindrical to oval in transverse section; noticeably flattened for final two-fifths of its length, with a low dorsal tail fin. Tail width / height ratio at the middle of its length (MTW/MTH) is 0.73; tail fin highest point located on the posterior one-third of its length. Final fifth of tail length tapers slightly; tail tip acute. Tail widest near its base. Extremities. Limbs slender and rather short, hindlimbs slightly longer and more robust than forelimbs (FLL/HLL ratio 0.84) ( Fig. 3 ); when forelimb and hind limb adpressed (towards each other) against trunk, digit tips do not meet, separated by a gap equal to three costal segments (folds); forelimb length (FLL) to groin-axilla distance (GA) ratio is 0.36; hind limb length (HLL) to groin-axilla distance (GA) ratio is 0.43. Palmar and tarsal tubercles on palms and feet not developed; digital webbing lacking; digits convex in transverse section. Four fingers and five toes present; finger length in order of increasing length: IV <I <III <II ( Fig. 3 , C); relative length of toes: V <I <II <IV <III ( Fig. 3 , D). Tips of fingers and toes rounded; cornified structures on limbs absent. Head. Head oval, slightly depressed, comparatively small (HL/SVL ratio 0.25), notably longer than wide: head length (HL) / head width (HW) ratio 1.64; head comparatively narrow; head basis not distinct from short neck ( Fig. 4 ). Tongue large, convex-elliptical, adhering to floor of mouth with free lateral margins. Snout wide and rather short, snout length (OR) / head length (HL) ratio 0.38; snout tip rounded in dorsal view ( Fig. 4 , A), bluntly rounded in lateral view ( Fig. 4 , C). Nares small, rounded, with lateral orientation, not protuberant, quite widely separated (IN/HL ratio 0.24), equidistant from eye corner and snout tip (ON/OR ratio 0.49). Eyes comparatively small (EL/HL ratio 0.21), distinctly protuberant in lateral view ( Fig. 4 , C); slightly protuberant in dorsal view ( Fig. 4 , A), eye diameter is notably less than snout length and less than the distance between external nares: eye length (EL) to snout length (OR) ratio 0.71; eye length (EL) / internarial distance (IN) ratio 0.84. Eyes widely spaced, interorbital distance relatively great (IO/HL ratio 0.24; EL/ IC ratio 0.57). Eyelid present, well developed; labial folds absent; gular fold distinct ( Fig. 3 , B; Fig. 4 , B). Parotid area slightly swollen, but distinct parotid glands absent. Subparotidal protuberance very distinct, swollen, ovate, extending from angle of jaw to gular fold, separated from the parotid area by a deep longitudinal subparotidal groove ( Fig. 4 , C). Subparotidal groove begins above jaw angle and extends posteriorly, curving ventrally at head basis and intersecting with gular fold at its end. Longitudinal postorbital groove distinct and running from posterior corner of eye toward area located slightly dorsal to angle of jaw. Postorbital groove slightly shorter than eye length, not reaching angle of mouth, terminating ca. 1.0 mm above it, where it joins subparotidal groove and short transverse supraquadratal groove, running ventrally toward mouth angle ( Fig. 4 , C). Dorsal surface of head with two lines of well developed neuromasts extending posteriorly from the snout (area between external nares) to anterior corners of eyes and then further posteriorly along the orbit margins slightly curving laterally posteriorly than the eyes ( Fig. 4 , A) and approaching postorbital grooves on the lateral sides of head ( Fig. 4 , C); over 25 neuromasts discernable in each line. Vomerine teeth. Vomerine teeth in two small, wide, obliquely arched series (VTS), nearly touching at midline, and forming a shallow and longer “”-shaped figure without noticeable gap between branches; outer branch of vomerine tooth series (VTS) short and slightly curved, reaching the level of the outer edge of the internal nares ( Fig. 5 ); VTS inner branches almost straight, only at the very end slightly curved anteriorly. Right VTS with 18 vomerine teeth, left with 17 teeth. Left and right vomerine tooth series in contact with each other with no gap between the medial ends of the inner branches of the tooth series. Branches of outer tooth series less than half the lengths of inner series branches. Vomerine tooth series much wider than long; VTL/ VTW ratio 0.66. Upper jaw with 75 maxillary and premaxillary teeth. Measurements and counts of the holotype . All measurements in mm: SVL: 50.80; TL: 38.96; GA: 29.59; FLL: 10.78; HLL: 12.81; HL: 12.59; HW: 7.68; EL: 2.59; IN: 3.07; ON: 1.78; IO: 3.03; IC: 4.52; OR: 3.64; CW: 6.61; OR: 4.75; 1-FL: 0.84; 2-FL: 2.10; 3-FL: 1.71; 4-FL: 0.75; 1-TL: 1.15; 2-TL: 2.20; 3-TL: 3.17; 4-TL: 2.54; 5- TL: 0.76; MTH: 5.34; MTW: 3.92; MAXTH: 5.67; VTL: 2.15; VTW: 3.28. Counts: VTN (total): 35; VTN (right series): 18; VTN (left series): 17; UJTN: 72; CG: 11 (11 visible from ventral side; 12 if include axillary fold); CGBL: -3.0; TGN: 6. Color of the holotype in life. In life dorsal background dark brown with unclear markings varying in color from tan to dark copper ( Fig. 6 ). Dorsal surfaces of trunk, head and limbs covered with small coppery spots that tend to fuse with each other, becoming larger and lightening to bronze on the dorsal surface of tail. Lateral sides of body dark purplish-grey lightening slightly to purplish or pinkish color ventrally. Posterior half of lateral sides of tail with small, sparse, bluish-white speckling. Throat, chest, ventral surfaces of limbs and anterior third of tail length pinkish; bluish ventral abdominal vein visible through translucent skin of belly. Cloacal area pinkish with indistinct dark markings. Posterior two thirds of tail length brownish ventrally. Iris dark bronze with black markings. FIGURE 5. Mouth cavity of the Hynobius unisacculus sp. nov. holotype (CGRB-15401, male; filed number MMS-1962): (A) photo of mouth cavity in palatal view, (B) schematic drawing of the same mouth showing shape of vomerine tooth series and number of vomerine teeth. Scale bar 1 mm. FIGURE 6. Holotype of Hynobius unisacculus sp. nov. (CGRB-15401, male) showing coloration in life: (A) dorsolateral view; (B) lateral view. Color of the holotype in preservative. After six years in alcohol, the coloration of the holotype is lighter; with dark dorsal background faded to light brown or brownish-grey ( Fig. 3 ); pinkish coloration of ventral surfaces of body disappear and turned yellowish-grey. General coloration pattern without significant change; dark dots of melanophores even more discernible than in life; dorsal surfaces of head, trunk and tail with small dark spots and blotches. Variation. All individuals in the type series appear generally similar in morphology and body proportions; variation of the type series in morphometric characters is shown in Table 3 . All individuals were examined after they had been preserved for 12 months in alcohol. There are 11–12 costal grooves (excluding the axillary groove); no statistically significant differences in number of costal grooves were detected. In all specimens examined limbs and digits are relatively short and adpressed limbs never overlap, leaving two to four intercostal folds uncovered; females tend to have slightly longer bodies and shorter limbs than males. Eyes are comparatively small (horizontal diameter 2.2–3.0 mm) and moderately protuberant. In all specimens all five toes are well-developed. No autotomy has been observed in the new species. Some specimens have very short tails (TL < 25 mm ), possibly due to incomplete regeneration after damage. The combined number of premaxillary and maxillary teeth ranges from 55 to 97 (mean = 70; N = 28). The shape of the vomerine tooth series (VTS) does not show obvious sexual differences and corresponds well to that described for the holotype ; females (19.9 ± 1.42; range 18–23) tend to have slightly more vomerine teeth than do males (18.3 ± 0.98; range 13–20); the number of vomerine teeth ranges from 17 to 23. Coloration. The dorsal color varies from olive to various shades of dark brown or tan. Some specimens have yellowish-brown, bronze or dark-copper speckles and spots from head to tail, while other specimens show no such speckles. In specimens with light speckles or spots on the dorsum, the light markings usually tend to fuse together on the dorsal and lateral sides of the tail, but never form a contrasting light band. Sides of the trunk and belly are always lighter than the dorsum: coloration can vary from pinkish beige to grey. Some specimens show the presence of light silvery-grey or whitish-blue speckles on the lateral sides of tail, trunk and head; such speckles are more abundant on semi-adult and juvenile specimens than on adults. Secondary sexual characteristics. During the breeding period, males have a noticeably swollen cloacal area, with longitudinal vent slit, bifurcating anteriorly in a Y-shaped figure, with two short grooves running from the Yshaped slit in postero-lateral direction; the anterior corner of the vent has a prominent protuberance ( Fig. 3 , E). The cloacal area of females is less swollen and the vent is a simple longitudinal slit. Males usually have a higher tail-fin. Reproduction. Breeding occurs between late February and late March; animals appear to leave water during April, after reproduction. H. unisacculus sp. nov. reproduces in still waters, such as like road-side ditches ( Fig. 7 , A), puddles and trenches on rice paddies ( Fig. 7 , B). During the day time, adult animals usually hide among dead leaves on the waterbody used for breeding. Eggs and clutch. Although information on the ecology of H. unisacculus sp. nov. is limited, to date females have not been observed attaching their egg sacs to substrate. All species of Hynobiidae , including H. unisacculus sp. nov. , are known to lay eggs in a pair of mucous sacs, deposited one per female’s oviduct during spawning; egg sacs are usually within a strong envelope ( Dunn 1923 , Sato 1943 ). The paired sacs as a rule are joined together at the distal end from which two mucous stalks arise; each stalk is used to attach a pair of egg sacs to the substrate (such as leaves, branches or stones); this mucous stalk is very weak and easily breaks during the egg-laying process. Perhaps this is the reason that in the wild we have only observed single egg sacs of H. unisacculus sp. nov. freely deposited in the waterbody, without a gelatinous stalk and not attached to any substrate ( Fig. 8 , A, B). This peculiar reproduction is not typical for other lentic-breeding Hynobius species ( Dunn 1923 , Sato 1943 , Sparreboom 2014 ), and was noticed by Song and Koo (2010) who reported on this unusual reproductive biology (they indicated the gelatinous stalk as a “whiptail-like structure”). However, many details of the reproductive biology of H. unisacculus sp. nov. remain unclear and further studies are necessary to shed light on this question. Egg sacs of the new species are comparatively small ( Fig. 8 , C) and curved forming a C-shaped or O-shaped figure, with a transparent envelope, covered by numerous irregular folds ( Fig. 8 , A, C). Eggs are pigmented, located in two rows or in a single row within the egg-sac ( Fig. 8 , C). Variation in the morphology of egg sacs in the H. leechii complex in southern Korea is discussed in detail by Song and Koo (2010) ; their study also includes data on the egg sacs of H. unisacculus sp. nov. (as Narodo and Boseong populations of H. leechii ). According to Song and Koo (2010) , clutch size (CS) in the new species varies from 17 to 88 eggs ; median values were 34.9 ± 9.0 (N = 101) for the Narodo and 47.1±15.4 (N = 39) in the Boseong population. H. unisacculus sp. nov. has comparatively small egg sacs; egg sac length (ESL) is less than 150 mm : 130.5 ± 20.9 mm ( 86 mm − 209 mm ) in the Narodo and 134.1 ± 28.8 mm ( 85 mm − 187 mm ) in the Boseong populations. Egg sac width (ESW) varies from 13.9 ± 1.5 mm ( 10 mm − 18 mm ) in the Narodo to 12.9 ± 1.8 mm ( 10 mm − 17 mm ) in the Boseong populations. The ESL/ESW ratio for H. unisacculus sp. nov. (mean 9.1±1.5 for the Narodo and 9.9±1.8 for the Boseong populations) was significantly different from several populations of H. leechii (11 <ESL/ESW<13) and H. quelpaertensis (ESL/ ESW>14) ( Song & Koo 2010 ). Larval morphology and metamorphosis. To date we lack any data on the development and morphology of the larval stage of the new species. Metamorphosis takes place during the second half of summer. Etymology. The specific name “ unisacculus ” is a noun in masculine gender, used in apposition, based on the Latin words “ unicus ” (single) and “ saccus ” (sac), or, more precisely, “ sacculus ” (small sac), meaning “a single small sac”. The species name refers to the unusual egg structure and breeding biology of the new species. The suggested vernacular name in English is “ Korean small salamander ”; the suggested common name in Korean: “ Kkoma– Dorongnyong ”. Distribution, habitats and natural history. To date, the new species has been found in four localities within the southeastern part of Jeollanam-do , South Korea : Goheung, Suncheon, Bosoeung and Yeosu areas ( Fig. 9 , localities 1–6). Specimens from these localities were used in the genetic study of Baek et al. (2011a , 2011b ) and their taxonomic assignation to Hynobius unisacculus sp. nov. was confirmed by mtDNA sequence analysis. However, Chang et al. (2011) reported several other populations of the H. leechii species complex on adjacent offshore islands along the southeastern coast of Jeollanam-do ( Fig. 9 , localities 7–12); the taxonomic status of these populations requires further study. The new species reproduces in still waters of ditches or rice paddies, after the breeding season, adults move onto surrounding land to hide in forest litter or other shelters (see Fig. 7 ). According to the data of Chang et al. (2011) , on Oenaro-do Island , Hynobius unisacculus sp. nov. is sympatric with Bufo gargarizans Cantor , Hyla japonica Günther and Pelophylax nigromaculatus (Hallowell) . On Gogeum-do Island , what is assumed to be the new species (identified as H. leechii ) was recorded in sympatry with the above mentioned anuran species, and also with Bombina orientalis (Boulenger) , Rana coreana (Okada) , and the introduced Lithobates catesbeianus (Shaw) . On Geomun-do Islands (Dong-do and Seo-do) the population tentatively assigned to the new species represents the only amphibian species recorded ( Chang et al. 2011 ). Comparisons. Among the 35 currently recognized species of the genus Hynobius (see Frost 2016 ), Hynobius turkestanicus Nikolskii appears to be an enigmatic taxon (see Kuzmin 1999) and is likely not a member of this genus. The rest of the species are grouped into three discrete groups, differing in morphology, phylogenetic position, chromosome structure, breeding ecology, and natural history. The stream-breeding or lotic Hynobius found in Taiwan and Japan , as well as H. ( Satobius ) retardatus , found in Hokkaido , are markedly distinct from the lentic-breeding species of Hynobius , reproducing mostly in still waters and inhabiting eastern and northeastern China , the Korean Peninsula and Japan . The new species, H. unisacculus sp. nov. , is both most morphologically and most molecularly similar to other lentic-breeding species of Hynobius from the Korean Peninsula: H. leechii , H. quelpaertensis and H. yangi , and comparisons with these taxa and other lentic-breeding Hynobius species of the region appear to be the most pertinent. Morphological comparison of H. unisacculus sp. nov. with three other species of Korean Hynobius are given in Tables 2 and 3 . The new species is a small-sized Hynobius (up to 61 mm SVL; mean SVL 49.0 mm in males and 51.4 mm in females), with short limbs and slender body. In SVL, post hoc analyses of one-way ANOVA revealed that H. unisacculus sp. nov. was not significantly different from H. yangi from Busan , but was significantly much smaller than H. quelpaertensis from Jeju Island and H. leechii from mainland Korea (one-way ANOVA, df = 3, F = 42.49, P <0.05; Duncan test, P <0.05). The new species can be further differentiated from H. quelpaertensis by its much shorter tail with mean TL/SVL ratio for males being 0.79 ± 0.01 and for females 0.73 ± 0.02 ( versus 0.90 ± 0.02 in males and 0.79 ± 0.02 in females of H. quelpaertensis ); these differences are statistically significant (TL, one-way ANOVA, df = 3, F = 9.360, P <0.01). H. unisacculus sp. nov. has relatively shorter limbs and longer body than H. quelpaertensis : between adpressed limbs in the new species there is always a wide gap of -2.4 costal folds in males and -2.5 costal folds in females (ranging from -3.5–-1.5), whereas in H. quelpaertensis the adpressed limbs often touch or overlap and the CGBL value is 0 in males and - 1.3 in females (ranging from -1.5–1.0). H. unisacculus sp. nov. has a lower number of vomerine teeth than does H. quelpaertensis (18.3 ± 0.98 (13–20) in males and 19.9 ± 1.42 (18–23) in females of the new species versus 23.8 ± 3.06 (18–30) in males and 22.8 ± 1.25 (21–24) in females of H. quelpaertensis ). FIGURE 7. Breeding habitats of Hynobius unisacculus sp. nov. , salamanders and egg sacs were found in water-filled ditches and puddles on the fields: (A) typical breeding habitat in environs of the type locality, Naro Islands, Goheung-gun, Jeollanamdo, environs of Dongil-myeon village; (B) breeding habitat at environs of Podu-myeon village, Naro Islands, Goheung-gun, Jeollanam-do. FIGURE 8. Egg sacs of Hynobius unisacculus sp. nov. in environs of the type locality (Naro Islands, Goheung-gun, Jeollanam-do): (A) a single egg sac in situ showing the folded texture of the envelope; (B) a group of egg sacs in situ laid in a roadside ditch; (C) a single egg sac compared with a 500 Korean won coin showing two rows of eggs within the egg sac. The new species can be further diagnosed from H. leechii from mainland Korea by its relatively shorter tail length: TL/SVL ratio is 0.79 ± 0.01 (0.54–0.98) in males and 0.73 ± 0.02 (0.55–0.89) in females of the new species versus 0.89 ± 0.05 (0.76–0.99) in males and 0.86 ± 0.03 (0.74–1.06) in females of H. leechii (TL, one-way ANOVA, df = 3, F = 9.360, P <0.01). H. unisacculus sp. nov. has a significantly shorter head, too: HL/SVL ratio is 0.24 ± 0.00 (0.21–0.26) in males and 0.23 ± 0.00 (0.21–0.25) in females of the new species versus 0.27 ± 0.01 (0.26–0.29) in males and 0.25 ± 0.00 (0.22–0.28) in females of H. leechii (HL, one-way ANOVA, df = 3, F = 9.356, P <0.01). The new species has a significantly longer trunk than does H. leechii : TRL/SVL ratio is 0.77 ± 0.00 (0.74–0.82) in males and 0.78 ± 0.00 (0.75–0.81) in females of H. unisacculus sp. nov. versus 0.73 ± 0.01 (0.71– 0.74) in males and 0.75 ± 0.00 (0.72–0.78) in females of H. leechii (TRL, one-way ANOVA, df = 3, F = 7.514, P <0.01); in the new species when limbs are adpressed to the body, digit tips are always separated by a large gap comprising -2.4 ± 0.42 (-3–-1.5) of costal folds in males of H. unisacculus sp. nov. whereas there is only a small gap—less than one costal fold—in H. leechii (-0.4 ± 0.35 (-1–0)). FIGURE 9. Known distribution of Hynobius unisacculus sp. nov. (genetically confirmed localities—red dots) and other known localities of H. leechii complex (species assignation requires further studies—grey dots) in offshore islands of southern part of Jeollanam-do, southern Korea. Locality information (all in Jeollanam-do), confirmed localities (this study): 1—Oenarodo Island, Goheung-gun (type locality); 2—Naenaro-do Island, Goheung-gun; 3—Podu-myeon, Goheung-gun; 4—Boseunggun [Boseong]; 5—Suncheon-si; 6—Yeosu-si; unconfirmed localities (data from Chang et al. 2011): 7—Geomun-do (Dongdo) Island; 8—Geomun-do (Seodo) Island; 9—Gogeum-do Island; 10—Saengil-do Island; 11—Sinji-do Island; 12—Cheongsando Island. The new species is not significantly different in size and most body ratios from H. yangi , but H. unisacculus sp. nov. has slightly deeper vomerine tooth series than H. yangi (VTW/VTL in males of the new species 1.53 (1.00–1.78) versus 1.62 (1.24–1.89) in males of H. yangi ); in the latter species vomerine series are slightly less curved than in the new species. Finally, H. unisacculus sp. nov. has relatively shorter limbs and a comparatively longer body than H. yangi : between adpressed limbs in the new species there is always a wide gap of -2.4 costal folds in males and -2.5 costal folds in females (ranging from -3.5–-1.5), whereas in H. yangi males the adpressed limbs often in touch or overlap and the CGBL value is -0.9 ± 0.32 (ranging from -1.5–-0.5). The new species is also morphologically distinct from the undescribed sister lineage Hynobius clade 1 (HC1) from coastal and lowland areas in the southern part of Gyeongsangnam-do Province in number of morphological traits: in having 11–12 costal folds (11.1 ± 0.18) in males ( versus 10 costal folds in males of Hynobius clade 1); in having much shorter tail, TL/SVL ratio is 0.74 ± 0.18 in males ( versus longer tail in males of Hynobius clade 1, TL/ SVL ratio is 0.92 ± 0.10); in having relatively shorter hindlimbs, HLL/SVL ratio in males of the new species is 0.26 ± 0.01 ( versus comparatively longer hindlimbs, HLL/SVL ratio is 0.26 ± 0.01 in males of Hynobius clade 1); by comparatively smaller head, in the males of the new species HL/SVL ratio is 0.24 ± 0.01 and HW/SVL ratio is 0.16 ± 0.00 (vs. comparatively larger head in males of Hynobius clade 1: HL/SVL ratio is 0.27 ± 0.01 and HW/ SVL ratio is 0.18 ± 0.00); by comparatively shorter eye to snout distance, OR/SVL ratio is 0.07 ± 0.00 in males of the new species ( versus OR/SVL ratio is 0.09 ± 0.00 in males of Hynobius clade 1); finally, between adpressed limbs in the new species there is always a wide gap of -2.4 costal folds in males (ranging from -3.0–-1.5), whereas in Hynobius clade 1 males the adpressed limbs usually in touch or overlap and the CGBL value is 0.0 ± 0.2 (ranging from -1.0–1.0). Other lentic-breeding Hynobius species found in East and Northeast Asia can be distinguished from H. unisacculus sp. nov. by a combination of several morphological characters. H. tsuensis Abé , inhabiting the Tsushima Islands in the Korea Strait, can be distinguished from the new species by much longer vomerine tooth series (VTW/VTL ratio 1.13 (0.98–1.27) versus 1.53 (1.00–1.82) in the new species; data for males), by a longer tail (TL/SVL ratio 0.82 (0.71–0.86) versus 0.79 ± 0.01 (0.54–0.98) in the new species; data for males) and by a bright yellow stripe on the dorsal surface of the tail ( versus absent in the new species). H. nebulosus (Temminck et Schlegel) , inhabiting Kyushu and the western part of Honshu Island in Japan , also has bright-yellow stripe on the dorsal edge of the tail ( versus absent in H. unisacculus sp. nov. ), deeper vomerine tooth series (VTW/VTL ratio 1.04 (0.85–1.32) versus 1.53 (1.00–1.82) in the new species; data for males), and a longer tail (TL/SVL ratio 70.5 (62.2–80.4) versus 0.79 ± 0.01 (0.54–0.98) in the new species; data for males). H. dunni Tago , from the western part of Kyushu and the easternmost Shikoku, can be distinguished from H. unisacculus sp. nov. by deeper vomerine series; longer limbs, overlapping or in touch when adpressed to body; uniform brown-olive coloration ( versus shallower vomerine series; smaller limbs, separated by a wide gap of 3 costal folds; bronze-spotted darkbrown coloration in the new species). Several Japanese lentic-breeding species can be distinguished from the new species by coloration; they all have numerous silvery, whitish or bluish spots and speckles on the dorsum and body flanks: H. lichenatus Boulenger from northeastern Japan (Tohoku, Honshu); H. takedai Matsui et Miyazaki from the Hokuriku region of central Honshu, along the coast of the Sea of Japan (Eastern Sea); and H. hidamontanus Matsui from mountain areas in central Honshu. H. tokyoensis Tago from the Kanto area of eastern Honshu usually lacks bluish spots, but has more costal grooves than does the new species: from 12 to 14 costal grooves (median—13 costal grooves) in males. H. abei Sato from the Sea of Japan (Eastern Sea) coast of western Honshu differs from H. unisacculus sp. nov. in its longer and broader head, shorter trunk, and much higher tail. The above-mentioned species can be further diagnosed from the new species by its comparatively longer limbs with digits overlapping or separated by a small gap when limbs are adpressed to the body; in males, the CGBL value varies from 0 to 2.5 in H. lichenatus , from -1 to 1 in H. takedai , from -2 to 1 in H. tokyoensis , from -2 to -0.5 (mean -2) in H. abei and from -2 to -0.5 (mean -1) in H. hidamontanus . H. nigrescens Stejneger inhabiting northeastern Japan (Tohoku, Honshu), can be easily diagnosed from H. unisacculus sp. nov. by its larger body size (total length up to 190 mm ; SVL = 66.4 ± 0.94 ( 60.5–73.4 mm ) in males), longer tail and longer limbs, which overlap on 1-3 costal segments when adpressed to the body ( versus smaller size and shorter limbs, separated when adpressed to body by never less than 3 costal segments in the new species). H. unisacculus sp. nov. can be diagnosed from the group of Chinese lentic-breeding Hynobius , which inhabit eastern and central parts of China, by its smaller body size (SVL up to 61 mm ; mean SVL 49.0 mm in males and 51.3 mm in females): versus H. amjiensis Gu (SVL in males 83.6, from 79.5 to 86.5), H. guabangshanensis Shen (SVL in males 81.4, from 72.8 to 87.5), H. maoershanensis Zhou, Jiang et Jiang (SVL in males 86.1, from 83.1 to 91.1), and H. yiwuensis Cai (SVL in males 63.8, from 47.0 to 74.0). H. chinensis Günther , H. amjiensis and H. maoershanensis also have tails longer than the new species (mean TL/SVL values 0.85, 0.92 and 0.84, respectively). The new species markedly differs from all other Hynobius in its breeding biology; to date in all types of waterbodies used for breeding that were examined by us we observed only single egg sacs, which lacked the gelatinous stalk-like structure typical for other lentic-breeding Hynobius ( Song & Koo 2010 ) . Finally, the new species is markedly distinct from all other congeners for which comparable sequences are available, including its closest relatives from the Korean Peninsula in its large genetic distances in the 12S rRNA mtDNA (genetic distance from p = 9.40%) and COI gene (genetic distance from p = 9.10%) fragments (see Table 4 ). This degree of pairwise divergence in the 12S rRNA and COI gene is greater than that usually representing differentiation at the species level in amphibians ( Smith et al. 2008 ; Xia et al. 2012 ; Murphy et al. 2013 ). To date, despite intensive fieldwork in the southern part of the Korean Peninsula we have not found any signs of sympatry of the new species with other Korean Hynobius (see Baek et al. 2011a , b ). Phylogenetic position. Baek et al. (2011a , b ) compared mitochondrial DNA sequences between the new species and the Korean hynobiids Hynobius leechii , H. quelpaertensis , and H . yangi . The analysis of the partial cytochrome b gene (907 bp), COI (1,097 bp), and the 12S rRNA (788 bp) gene showed profound differences between the new species and other Korean Hynobius (see Table 5 and Results for details). The observed level of genetic divergence between the new species and other Korean Hynobius clearly corresponds to a species level of differentiation ( Lee et al. 1998 ; Lai & Lue 2008 ). Based on the data of Baek et al. (2011a , b ), H. unisacculus sp. nov. is recovered as a sister species of lineage HC1, both lineages forming a clade with H. quelpaertensis . Conservation status. All species of Hynobius in South Korea are confronted with severe habitat destruction and fragmentation due to anthropogenic activities: economic growth, construction of roads, and further development, disappearance of agricultural areas and fragmentation of forests (see Baek et al. 2011a ). Moreover, Yang et al. (2009) revealed that chytridiomycosis is a growing threat to Korean amphibian populations, indicating the need to pay more attention to amphibian conservation and protection. Although our knowledge of H. unisacculus sp. nov. distribution is still incomplete, it appears that the new species has a very narrow range, restricted to several offshore islands and a short segment of coastal area in Jeollanam-do, South Korea. Given the available information, we suggest that H. unisacculus sp. nov. be considered Vulnerable (Vu2a), following IUCN’s Red List categories ( IUCN 2001 ).