Rana susurra, Sekiya, Kunio, Miura, Ikuo & Ogata, Mitsuaki, 2012

Sekiya, Kunio, Miura, Ikuo & Ogata, Mitsuaki, 2012, A new frog species of the genus Rugosa from Sado Island, Japan (Anura, Ranidae), Zootaxa 3575, pp. 49-62 : 51-58

Rugosa susurra sp. nov.

(Japanese name: Sado gaeru) ( Figs. 2–4 )

Rana rugosa (yellow type): Sekiya et al. 2010, p. 71, figs. 2A–B.

Diagnosis. Genetically close to Rugosa rugosa than other species of the genus Rugosa . A medium sized species of Rugosa , with males 33.0– 44.2 mm, females 38.1–49.6 mm in SVL; web rather well developed; dorsum khaki in color with many dermal ridges of varying size, dorso-lateral fold absent, supratympanic fold strong; venter posteriorly deep yellow; anterior ventral less granulated than posterior ventral, especially around jaw granule almost absent. This new species is differentiated from R. rugosa by its yellow ventral coloration and jaw almost without granules and no vocal sac in male.

Holotype: KPM-NFA000078, an adult male from Akitsu, Sado city, Sado Island, Niigata Prefecture, Japan (38°4’N, 138°24’E, 11m a.s.l), Collected on 14 July 2009 by M. Ogata.

Paratypes: Two adult females and an adult male (KPM-NFA000075-77) from the same locality and collected on the same day by M. Ogata and Y. Ogata.

Referred specimens: Is. Sado, Akitsu (10 males, 7 females, collected on 22 June 2008 by M. Ogata IABHU F2462-F2471,F2475-F2481, 3 males collected on 30 May 2011 by M. Ogata IABHU F2472-F2474, one female collected on 11 July 2011 by M. Ogata IABHU F2482) and Suitsu (38°4’N, 138°33’E, 33m a.s.l, 10 males, 3 females, collected on 22 June 2008 by M. Ogata, IABHU F2483-F2495), Nakaoku (38°3’N 138°21’E, 187m a.s.l, 9 tadpoles, collected on 1 July 2010 by K. Sekiya, IABHU F2513-F2521).

Description of holotype (measurements in millimeters). Snout-vent length 37.6; head about as long (14.0) as broad (13.8); snout (5.5) longer than eye (5.0), tip of snout rounded; distance between nostril and eye (3.4) about as same as upper eyelid width (3.3) and internarial distance (3.5), but narrower than interorbital width (4.4), snout dorsally concave and slightly pointed; lore concave; supratympanic fold strong; tympanum conspicuous, diameter (3.8), separated from eye by about one-fifth of tympanum diameter (0.7).

Fingers slender, no webbing, tips of fingers slightly depressed, third finger longer than fourth; subarticular tubercle strong; inner metacarpal tubercle circular; outer metacarpal tubercle elliptical.

Tibia (19.2) about 2.9 times length of arm (6.6); toes slender, tips of toes slightly depressed, web rather well developed, webbing formula: I 1 - 2 II 1 - 2 III 1 - 2 IV 2 – 1 V; excision of membrane between two outer toes reaching near middle subarticular tubercle of fourth toe when toes in contact; inner metatarsal tubercle elliptical, outer metatarsal tubercle weak, tarsal ridge rather strong, skin fold slightly developed on outer margin of fifth toe from near proximal subarticular tubercle to near distal articulation.

Dorsal surface with slightly large dermal ridges and minute tubercles, dorso-lateral fold absent, granules on ventral surface of forelimb, hindlimb and abdomen, but absent on jaw.

Nuptial pad developed on dorso-medial area on first finger from base to subarticular tubercle but weakly elongated to tip.

Dorsum grayish brown, mid-dorsal stripe weak and slender, black transect line on hindlimb.

Colors in life. Dorsum almost khaki. Abdomen almost whitish anteriorly, deep yellow posteriorly ( Fig. 2 A and 2B). Similar yellow color is observed on ventral side of arm and ventral side of hindlimb but excluding fifth toe. Some dark brown spots on ventral side of hindlimb.

Variation. Females are larger than males in Akitsu population (Table 1A and 1B, t-test, p<0.001). Male has longer tibia, relative to SVL, than female. Tympanum distance, interorbital width, head width, snout length, distance between nostril and eye, internarial distance and head length of male are larger, all relative to SVL, than female (Mann-Whitney U-test, p<0.05). Males of Akitsu population are smaller than males of Suitsu population (ttest, p<0.001). Males of Akitsu have larger head width, relative to SVL, than males of Suitsu (Mann-Whitney Utest, p<0.01). Some individuals of Akitsu have much less weakly skin granule but others no granule ( Table 2). Ventral colors of some individuals are clear yellow but others less yellow. Mid-dorsal stripe is absent in some individuals (no stripe: Akitsu population=59.1%, Suitsu population=100%).

Comparison. Morphological characters of Rugosa susurra are similar to R. rugosa (Table 1A and 1B). When R. susurra is morphologically compared with R. rugosa from five geographic races, the following differences are detected. In females, head width of R. susurra , relative to SVL, are shorter than those of R. rugosa from the ZW race on Sado Island population (Kruskal-wallis test with nonparametric multiple comparison, p<0.05). In males, R. susurra have longer tibia than R. rugosa from West-Japan race, shorter snout length than the XY race, all relative to SVL (Kruskal-wallis test with nonparametric multiple comparison, p<0.05). Most of R. rugosa (95.6%) have tiny granules especially around ventral surface of jaw, whereas most of R. susurra (94.1%) do not have any there ( Table 2, Chi-square test, p<0.001). A deep yellow coloration of abdomen and ventral surface of hindlimb is observed in R. susurra but not in R. rugosa ( Maeda & Matsui 1999) . Also, R. susurra differs from other species of the genus Rugosa in the following characters ( Chang 1933; Nikolsky 1918; Fei et al.1991 “1990”; 2005): dorsal surface with slightly large dermal ridges and minute tubercles ( R. tientaiensis has oval or elongated narrow warts on dorsal surface, Chang 1933) and smooth ventral skin without granule especially around jaw ( R. emeljanovi and R. tientaiensis have rough skin both on dorsum and ventral side, Chang 1933; Fei et al. 1991 “1990”; 2005). Ventral surface is deep yellow (dirty-yellow in R. emeljanovi, Nikolsky 1918 ). Since the genus Glandirana was considered as related with Rugosa ( Che et al. 2007), we compared R. susurra and G. minima to clarify their morphological difference. R. susurra differs from G. minima in the following characters: webbing on toes is well developed (halfwebbed on toes in G. minima, Fei et al. 1991 “1990”), and ventral surface is deep yellow (gray in G. minima Ting & Ts’ai 1979).

Species Locality Number of frogs with granules Number of frogs without granules

(Geographic race) Male Female Male Female

Rugosa susurra Akitsu 1 1 11 8 Suitsu 0 0 10 3

Rugosa rugosa Akadama (ZW) 8 6 0 0 Nagaoka (ZW) 6 5 0 0 Hitachi-Oomiya (East-Japan) 11 8 4 0 Motosu (XY) 14 2 0 0 Kinomoto (Neo-ZW) 8 3 0 0 Higashi-Hiroshima (West-Japan) 9 6 0 0

Eggs. A female (IABHU F2482 collected from Akitsu on 11 July 2011) contained a total of 710 matured ova in a pair of ovaries. The diameter of twenty ova ranged from 1.2 to 1.5mm (mean±2SE=1.38±0.04). The animal pole is dark brown while the vegetable pole is cream in color.

Tadpole. Developmental stages of the nine tadpoles (IABHU F2513-F2521 collected from Nakaoku) varied from St.29 to St.42. In preserved specimens, dorsum almost grayish brown, abdomen white ( Fig. 5 ). Tail and tail fin fine and grayish brown network. A lot of small white spots were distributed on entire body including tail fin. Head and body oval shaped, body slightly rounded below. Dermal ridges strongly on the dorsum of tadpoles at St.41 and St.42. Head-body length of two tadpoles at St.29 was 16.5mm and 18.0mm and that of tadpoles at St. 31 and St. 32 was 19.1 mm and 19.9 mm. Head-body length of four tadpoles at St.41 and St.42 varied from 22.6 mm to 27.0 mm whereas that of tadpole at St. 39 is 25.0 mm. Tail fin slightly develop around the center of tail.

Oral slightly below. Dental formula of six tadpoles (St.29-St.32 and St.39-St.42) was 1:1+1/1+1:2 having relatively large interrupted portion on upper dentition, while that of the remaining three tadpoles was 1:1+1/3 (St.41) or 1/1+1:2 (St.29).

Range. Rugosa susurra is mainly distributed in the Kuninaka Plain of Sado Island including the type locality ( Fig. 1 ), not in any areas outside Sado Island, Japan. Five localities are known other than the three as mentioned above, Umetsu (38°6’N, 138°24’E, 75m a.s.l.), Nagae (38°5’N, 138°23’E, 54m a.s.l), Tassya (38°4’N, 138°15’E, 77m a.s.l.), Ogawa (38°3’N, 138°15’E, 29m a.s.l.), Niibo-Iuchi (38°1’N, 138°27’E, 102m a.s.l.). The elevations of the eight localities range from 11m to 187m (in a.s.l.). Five of the eight localities are on the Kuninaka Plain ( Fig.1 , No.6–No.8 and No.12–No.13)

Calls. A total of five calls from one male of Rugosa susurra were recorded in Akitsu at air and water temperatures of 18.5°C and 21.5°C, respectively. Call duration ranged from 1112 to 1646 ms (mean±SD = 1341.7±226.9 ms), whereas number of pulse in a single call ranged from 67 to 93 (mean±SD = 78.8±11.6) ( Fig. 6 ). When the male frog was calling, we could not observe any apparent expansion of vocal sac as R. rugosa is doing. Recording at laboratory was conducted at air temperatures of 24.6–26.8°C for R. susurra collected from Akitsu on 30 May 2011 (IABHU F 2472-2474) and R. rugosa from Akadama on Sado Island on 30 May 2011 (ZW race, IABHU F 2504-2506). A total of 16 calls ( R. susurra ) and 19 calls ( R. rugosa ) were recorded. Call duration of R. susurra is much longer than that of R. rugosa ( R. susurra , mean±SD = 1333.5±382.4ms; R. rugosa , mean±SD = 111.7±9.1ms, t-test p<0.05). Also, number of pulse in a single call is completely different between them (R.

susurra , mean±SD =63.0±6.4; R. rugosa , 6.8±0.7, p<0.005, t-test). In the three male specimens of R. susurra , any vocal sac and vocal opening could not be identified inside the mouth by dissection. Therefore, R. susurra has no vocal sac.

Natural history. Adult frogs of Rugosa susurra are always observed near the water, such as in rims of rice field, small streams and ponds. Egg masses laid are attached to short withered branches or water grass ( Fig. 7 ). Breeding seems to occur from middle May to early August, because egg masses are observed in ponds during the season. In winter, both adult frogs and tadpoles are observed in the mud under water. Thus, the tadpoles of R. susurra can hibernate successfully like those of R. rugosa ( Maeda & Matsui 1999) , although we have no information whether all tadpoles that are produced in the season hibernate or not.

Karyotype. 2n= 26 (males and females) with no heteromorphic pair of sex chromosomes, and the chromosome 7 is very similar in morphology and late replicating banding pattern to that of East-Japan (Kanto) race of R. rugosa , suggesting their genetically close relationship (cf. Fig. 3 in Sekiya et al. 2010).

Molecular phylogeny. The bootstrap consensus tree of mitochondrial genes was constructed by maximum likelihood method ( Fig. 8 ). Rugosa rugosa comprises two main lineages: one contains East-Japan (Kanto), XY and Neo-ZW races, while the other contains West-Japan and ZW races. R. susurra was always clustered with the lineage comprising East-Japan (Kanto), XY and Neo-ZW races of R. rugosa in the three topologies with high bootstrap probability. Topologies of the other two methods were almost the same as that of Fig. 6 , although two sequences of R. emeljanovi were clustered with the linage (East-Japan +XY+Neo-ZW + R. susurra ) in maximum parsimony methods with low bootstrap probability (<50%). DNA distances between R. susurra and R. rugosa are 0.041 to 0.062 whereas those of R. susurra and the remaining three species are 0.068–0.075 ( R. emeljanovi ), 0.071 ( R. tientaiensis ) and 0.096 ( Glandirana minima ).

Etymology. The specific epithet is derived from "susurrus" in Latin meaning “whispering”. The advertisement call of Rugosa susurra is much quieter than those of other anuran species in the same locality ( Hyla japonica Günther , Rhacophorus arboreus Okada and Kawano ), and sounds like whispering among them.

Figures

FIGURE 1. Distribution map of the five geographic races of Rugosa rugosa and the localities of specimens (closed circle) used in this study including those (yellow circle) of R. susurra in Sado Island. Circles without number are the localities of R. rugosa on Sado Island referred from Sekiya et al. (2010). Chromosomes 7 are given for the groups except Neo-ZW. Names of localities are as follows: 1, Nagaoka; 2, Hitachi-Oomiya; 3, Motosu; 4, Kinomoto; 5, Higashi-Hiroshima; 6, Umetsu; 7, Nagae; 8, Akitsu; 9, Suitsu; 10, Tassya; 11, Ogawa; 12, Nakaoku; 13, Niibo-Iuchi; 14, Akadama; 15, Hamochi-Ohashi.

FIGURE 2. Holotype of Rugosa susurra (KPM-NFA 000078) in life. Dorsal side (A) and ventral side (B).

FIGURE 3. Holotype of Rugosa susurra (KPM-NFA 000078) in alcohol. Dorsal side (A) and ventral side (B). Scale bars represent 1 mm.

FIGURE 4. Ventral view of left foot of holotype of Rugosa susurra in alcohol.

FIGURE 5. Tadpole of Rugosa susurra (IABHU F 2521). Dorsal (A), ventral (B) and lateral (C) views. Developmental stage is 41 (Gosner 1961). Scale bars represent 1 mm.

FIGURE 6. Spectrogram of an advertisement call of Rugosa susurra. Air temperature and water temperature are 18.5 ° C and 21.5 ° C, respectively

FIGURE 7. Egg masses of Rugosa susurra from Tassya on Sado Island.

FIGURE 8. Maximum-likelihood tree based on the 867 bp sequences of the mitochondrial 12 S rRNA and 16 S rRNA genes of genus Rugosa. Numeral at each node denotes the bootstrap support (% in 1000 replicates). R. susurra indicates a specimen from Akitsu in Sado Island (AB 430349). East-Japan (AB 430340), West-Japan (AB 430341), XY (AB 430342), Neo-ZW (AB 430343), ZW 1 (AB 430345) and ZW 2 (AB 430344) indicate specimens from Isehara City in Kanagawa Prefecture, Miyoshi City in Hiroshima Prefecture, Ohtsu City in Shiga Prefecture, Kanazawa City in Ishikawa Prefecture and Niigata City in Niigata Prefecture, respectively. ZW-Sado (AB 430352) indicates a specimen from Hamochi-Ohashi in Sado Island. See other abbreviations in the text.