Genetic and morphological variation analyses of Glandirana rugosa with description of a new species (Anura, Ranidae) Author Shimada, Tomohiko Department of Science Education, Aichi University of Education, 1 Hirosawa, Igaya, Kariya, Aichi 448 - 8542, JAPAN Author Matsui, Masafumi 0000-0003-2032-2528 Graduate School of Human and Environmental Studies, Kyoto University, Sakyo, Kyoto 606 - 8501, JAPAN fumi @ zoo. zool. kyoto-u. ac. jp; https: // orcid. org / 0000 - 0003 - 2032 - 2528 fumi@zoo.zool.kyoto-u.ac.jp Author Ogata, Mitsuaki Preservation and Research Center, City of Yokohama, 155 - 1 Asahi Ward, Yokohama 241 - 0804, JAPAN Author Miura, Ikuo 0000-0003-3564-2882 Amphibian Research Center, Hiroshima University, 1 - 3 - 1 Kagamiyama, Higashi-Hiroshima 739 - 8526, JAPAN imiura @ hiroshima-u. ac. jp; https: // orcid. org / 0000 - 0003 - 3564 - 2882 imiura@hiroshima-u.ac.jp Author Tange, Mai Department of Science Education, Aichi University of Education, 1 Hirosawa, Igaya, Kariya, Aichi 448 - 8542, JAPAN & Takahama High School, 1 - 6 - 1 Hongo, Takahama, Aichi 444 - 1311, JAPAN Author Min, Mi-Sook Conservation Genome Resource Bank for Korean Wildlife, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, 1 Gwanak-ro, Seoul 08826, SOUTH KOREA Author Eto, Koshiro 0000-0002-8742-7319 Kitakyushu Museum of Natural History & Human History, Higashida 2 - 4 - 1, Yahatahigashi, Kitakyushu, Fukuoka 805 - 0071, JAPAN koshiro. eto @ gmail. com; https: // orcid. org / 0000 - 0002 - 8742 - 7319 koshiro.eto@gmail.com text Zootaxa 2022 2022-08-08 5174 1 25 45 journal article 177054 10.11646/zootaxa.5174.1.2 36b06def-e42e-44f9-8488-be29f215e3a7 1175-5326 6972938 F72C5C66-5F2F-4DF3-B92F-12D630C306F7 Glandirana reliquia Shimada, Matsui, Ogata et Miura sp. nov. (English name: Proto wrinkled frog) (Japanese name: Mukashi-Tsuchi-gaeru) ( Fig. 6 ) urn:lsid:zoobank.org:act: 2A54EE1B-6A05-4B6D-90C6-8384EB080720 Rana rugosa (part): Stejneger 1907 , p. 123 . Rana ( Rana ) rugosa (part): Nakamura & Uéno 1963 , p. 49 . Rana ( Rugosa ) rugosa (part): Dubois 1992 , p. 322 . Rana rugosa Intermediate subgroup of Eastern group: Nishioka et al . 1993 , p. 126 . Rana rugosa Kanto form: Ogata et al . 2002 , p. 186 . Glandirana rugosa (part): Frost et al . 2006 , p. 368 . Rana rugosa Kanto group: Ogata et al . 2008 , p. 92 . Rugosa rugosa (part): Fei et al . 2010 , p. 37 . Rugosa rugosa East Japan group: Sekiya et al . 2012 , p. 59 . Glandirana rugosa East group: Oike et al . 2017 , p. 446 . Glandirana rugosa East-J group: Ogata et al . 2021 , p. 2 . Holotype . KUHE 64088 (former AUEZ 1131 ), an adult male from Hirata , Kimitsu City , Chiba Prefecture , Japan ( 35 o 13' N , 139 o 59' E , 60 m asl ), collected on 23 March 2013 by Tomohiko Shimada and Ai Sakabe. Paratypes . KUHE 64085–64087 (former AUEZ 0973 , 1129–1130 ) three adult males and KUHE 64089 (former AUEZ 1132 ), an adult female, data same as the holotype . Referred specimens. AUEZ 1305, 1309, 1311–1313, Oshu City, Iwate Prefecture ; KUHE 21569, 21584, Iwaki City, Fukushima Prefecture ; AUEZ 0761–0770, 1168, 2025, 2032, 2033, 2037, 2038, 2047, 2141, 2142, 2324, 2326–2331, 2333–2336, Ichikai Town, Tochigi Prefecture ; KUHE 36529–36530, Utsunomiya City, Tochigi Prefecture ; KUHE 39765–39767, Kanuma City, Tochigi Prefecture ; AUEZ 0379–0381, 0386–0396, 0773, 2368, Higashichichibu Village, Saitama Prefecture ; KUHE 28395–28399, 46177, Ichihara City, Chiba Prefecture ; AUEZ 0843–0844, Kamogawa City, Chiba Prefecture ; AUEZ 0399–0404, KUHE 11254, Akiruno City, Tokyo Metropolis; AUEZ 0782, 0845, 0944–0947, 0949, 0953, Sagamihara City, Kanagawa Prefecture ; KUHE 05117, Ueda City, Nagano Prefecture . Etymology. The specific epithet is from a Latin noun denoting relic, alluding to the facts that the species represents a basic stock of Japanese Glandirana existing far before the western G. rugosa was derived and leaves a part of its own genome within the heteromorphic sex chromosomes of G. rugosa ( Miura et al . 1998 ; Miura 2007 ; Mawaribuchi et al . 2016 ). Diagnosis. A moderate-sized species of the genus Glandirana , with adult SVL 31–54 mm in females and 29–43 mm in males. From G. rugosa , this new species is differentiated morphologically in only in several morphometric characters relative to SVL in metamorphs, but is fairly different in development of larval skin glands, and definitely differs from them in nuclear genome characters. It differs from G. susurra and G. emeljanovi , in ratios of morphometric characters and ventral coloration, from G. tientaiensis by less flat dorsal ridges and more granulated ventral skin, and from G. minima by larger body and much well developed toe webbing. Diploid chromosomes are homomorphic, i.e., not sexually dimorphic unlike most of G. rugosa . Description of holotype (in millimeters). Snout-vent length (SVL) 40.7; body robust; head large, slightly wider (HW 17.3, 42.5%SVL) than long (HL 16.6, 40.7%SVL); snout triangular, tip slightly pointed in dorsal outline; projecting beyond lower jaw, slightly rounded in lateral profile; canthus very distinct; lore vertical, concave; nostril below canthus, midway between tip of snout (S-NL 3.8, 9.3%SVL) and anterior margin of upper eyelid; internarial distance (IND 3.4, 8.2%SVL) shorter than distance from nostril to eye (N-EL 3.8, 9.4%SVL); eye large, length (EL 5.4, 13.3%SVL) one and half times eye-nostril distance but smaller than snout length (SL 6.9, 17.0%SVL); interorbital (IOD 3.9, 9.5%SVL) subequal to width of upper eyelid (UEW 3.9, 9.6%SVL) and wider than internarial distance; pineal spot present; tympanum large and very distinct, nearly circular (TD 4.7, 11.6%SVL), about fivesixth eye diameter; vomerine teeth in indistinctly oval, small, and slightly oblique raised series (each of 2 teeth), the center posterior to line connecting posterior margins of choanae, narrowly separated from each other, but widely separated from choanae; tongue narrow anteriorly, moderately notched, without papilla; a pair of internal vocal sacs and vocal openings on corners of mouth. Forelimb stout (FLL 26.9, 66.1%SVL; LAL 20.4, 50.0%SVL; FAW 3.9, 9.6%SVL); fingers slender unwebbed, but with fringes of skin; finger length formula: II<I<IV<III ( Fig. 6C ), first finger as long as second; finger tips blunt, without disk; three large palmar tubercles, and indistinct supernumerary tubercles; subarticular tubercles prominent, circular; distinct gray nuptial pads on dorsal, medial, and ventral surfaces of first finger extending from its base to distal phalanx, covered with minute asperities. Hindlimb long (HLL 71.4, 175.4%SVL), about 2.6 times the length of forelimb; tibia (TL 21.5, 52.8%SVL) shorter than foot (FL 23.7, 58.3%SVL); heels overlapping when limbs are held at right angles to body; tibiotarsal articulation of adpressed limb reaching anterior corner of eye; toe tips blunt, without disk; toe length formula I<II<III<V<IV; third toe shorter than fifth; toes moderately webbed, formula I 1/2– 11 / 2 II 1–2 III 1/2–2 IV 11 / 2 –0 V ( Fig. 6D ); excision of membrane between two outer toes reaching the point between distal and middle subarticular tubercles of fourth when toes in contact; webs thick, not crenulate; subarticular tubercles prominent, rounded; inner metatarsal tubercle distinct, oblong (IMTL 2.1, 5.2%SVL), less than half length of first toe (5.0, 12.2%SVL); outer metatarsal tubercle small but distinct; tarsus with strong tarsal fold. FIGURE 6. Dorsal (A) and ventral (B) views of the whole body and ventral view of left hand (C) and foot (D) of the male holotype of Glandirana reliquia sp. nov. (KUHE 64088). Scale bar = 20 mm (A, B)/5 mm (C, D). A lateral view of a nearly topotypic male in life (AUEZ 0843: Kamogawa City, Chiba Prefecture) is shown as well (E). Head dorsally covered with small tubercles. Dorsum from posterior to upper eyelid to anterior to vent, covered with longitudinal skin folds, with pustular warts and granules in between; no dorsolateral fold; a supratympanic fold from eye, but not curved to axilla; side of trunk coarsely granular; longitudinal skin folds dorsally on forearm and tibia; ventral side weakly rugose. Color in life. Dorsum dark brown with faint dark blotches, but without interorbital bar; no vertebral line medially; lores with light dark markings below canthus; upper lip with dark bars; upper half of tympanum surrounded by a weak brown band; limbs marked dorsally with dark brown crossbars; ventrum light brown scattered with minute white spots; blackish brown bar on lower lip not clear; ventral surfaces of thigh and tibia with irregular dark spots. Variation. A summary of morphometric data is shown in Table 2 together with those on mitochondrial groups of G. rugosa . Females are significantly larger in SVL (mean ± SD = 47.9 ± 1.4 mm , n = 35) than males (36.8 ± 1.0 mm, n = 41; t-test, P <0.01). Size of all characters relative to SVL, except for FFL, tended to be greater in males than females (Mann-Whitney U-test, two-tailed, P <0.05). When the hindlimb was bent forward along the body, tibiotarsal joint reached the point between middle of upper eyelid and nostril in males, and between posterior and anterior corners of upper eyelid in females, but the medians had no sexual difference, both to the point anterior to upper eyelid. Males tended to have more developed toe webbing than females, and the incision of outer web in males varied from distal to same level of middle subarticular tubercles on fourth toe, with the median same level of middle subarticular tubercles. In contrast, females had the incision from same level to proximal to middle subarticular tubercles on fourth toe, with the median proximal to middle tubercles. Degree of development of larval ventral glands varied among populations, and medians ranged from state B (Kimitsu, 41.7%; Sendai, 33.3%) through state C (Hiratsuka, 16.7%) to state D (Higashichichibu, 72.7%), with the ground median state C (see below). Eggs and larvae. In a population from Matsudo City, Chiba Prefecture , eggs laid at a time ranged from 892–1452 (mean ± SD = 1245 ± 246, n = 4) ( Okada 1930 ). In the paratypic female, left ovary contained 627 mature eggs, each 1.3 mm in diameter and light brown in animal hemisphere. Eggs are laid in small clumps of about 30 to 60 eggs . A total of nine overwintered tadpoles in stages 31–35 (total length [TOTL] = 37.8–53.6 [mean ± SD = 45.2 ± 14.5] mm, head body length [HBL] = 13.0–20.3 [mean ± SD = 16.4 ± 1.6] mm), and two in stages 36–41 (TOTL = 46.9–55.9 [mean = 51.4] mm, HBL=18.1–18.6 [mean = 18.3] mm), from the type locality were closely examined. Head and body slightly flattened above, spheroidal below; head body width (HBW) maximum slightly anterior to level of spiracle 59–69% (median = 63%) of HBL; head body depth (HBD) 74–90% (median = 83%) of HBW; snout rounded; eyes dorsolateral, not visible from below; nostril open, dorsolateral, rim raised, midway between tip of snout and eye; internarial 100–101% (median = 101%) of interorbital. Oral disk anteroventral, emarginate, width 33–34% (median = 33%) of HBW; marginal papillae on upper labium with wide gap; lower labium with a continuous row of papillae, submarginal papillae present near corners; denticles 2(2)/3(1) ( Fig. 7D ); beaks with black outer margins; outer surface smooth; margin finely serrate; upper beak weekly convex medially; neither beak divided. Spiracle sinistral, tube pointing upward and backward, free of body wall slightly. Anal tube dextral, attached to ventral fin; loops of gut visible ventrally only in young larvae. Tail moderately long and lanceolate, both margins weakly convex, tapering gradually to slightly rounded tip; tail length 164–199% (median = 175%) of HBL, maximum depth 26–44% (median = 31%) of length; dorsal fin origin at posterior end of body, deeper than ventral fin except near tail tip; ventral fin origin continuous to vent; caudal muscle moderately strong, maximum tail width 26–40% (median = 31%) of HBW; muscle depth at anterior one-third of tail 45–59% (median = 50%) of tail depth, steadily narrowed posteriorly, shallower than either fin in distal half of tail. Indistinct supranaso-orbital, infranaso-orbital, mental, pregular, and lateral neuromasts discernible. Larval skin glands variously developed but overall very few on dorsum ( Yamamoto & Shimada 2021 ). Ventral glands also variable, but the median was state C (glands partly absent between throat and abdomen, center of abdomen without glands). In life dorsal and lateral body brown, spotted with black and covered with silver; venter dirty white, scattered with dark gray on throat; tail scattered with black and densely covered by silver spots ( Fig. 7A–C ). Karyotype. Diploid chromosome 2n = 26, with five large and eight small pairs, that are homomorphic and lacking sexual difference ( Nishioka et al ., 1994 ). Chromosome Nos. 1, 2, 4, and 5 in the larger group and Nos. 6, 8 and 10 in the smaller group are metacentric, while Nos. 3 in the larger group and Nos. 9, 11, 12, and 13 in the smaller group are submetacentric. The small chromosome No. 7 is subtelocentric, of which short arm is shorter than that of subtelocentric chromosome 7 in G, rugosa ( Miura et al ., 1998 ; Ogata et al ., 2002 ). The small chromosome No. 11 has a distinct secondary constriction in the longer arm. Call. We analyzed mating calls of a single male, recorded at a paddy field at Osho, Itsukaichi, Akiruno City, Tokyo at an air temperature of 20.1°C on 4 June 2013 by N. Maeda. Calls (15 notes were analyzed) consisted of a series of notes each emitted at an interval (between the beginnings of two successive notes) of 0.56 ± 0.08 (0.48– 0.77) s ( Fig. 8 ). Each note was composed of 22.4 ± 6.2 (17–37) short pulses and lasted for 0.39 ± 0.12 (0.29–0.65) s. Frequency bands spread over the 0.56–2.5 kHz range, and the dominant frequency was 0.84 ± 0.04 (0.76–0.91) kHz. Frequency and intensity modulations were only slight. Comparisons. The new species tends to be different from the groups of G. rugosa in much less developed larval dorsal skin glands ( Yamamoto & Shimada 2021 ). Compared with the Central group of G. rugosa , G. reliquia sp. nov. differs by relatively longer limbs (forelimb, lower arm, outer palmar tubercle, hand, hindlimb, tibia, and foot) in both sexes. Also, the new species has wider upper eyelid margin and longer first toe in females, and third finger in males, but has smaller inner metatarsal tubercle in females. From the North group of G. rugosa , the new species is distinguished by having relatively wider upper eyelid margins and longer limbs (forelimb, lower limb, outer palmar tubercle, hand, foot, and first toe) in both sexes, and in third finger length in males. Compared with the Western group of G. rugosa , the new species has relatively longer limb parts (forelimb, hindlimb, tibia, and foot) in both sexes, and in longer snout-nostril and larger tympanum in females. FIGURE 7. Dorsal (A), lateral (B), and ventral (C) views and the oral disc (D) of a tadpole of Glandirana reliquia sp. nov. in stage 35 of Gosner (1960) , collected on 9 May 2013 at Sendai City, Miyagi Pref., Japan. Scale bar = 10 mm. FIGURE 8. Advertisement call of Glandirana reliquia sp. nov. from Tokyo, Japan, recorded at an air temperature of 20.1°C, showing sonogram (top) and wave form (bottom). From G. susurra , G. reliquia sp. nov. is distinct in having relatively longer (head, snout, tympanum) and wider head parts (head width, upper eyelid, upper eyelid margin), and longer finger (third finger and first finger) in both sexes, and larger nostril-eye, intercanthal, and hand, and smaller inner palmar tubercle in females. Compared with G. emeljanovi , the new species has relatively larger head parts (tympanum, head width, upper eyelid margin) and limbs (forelimb, lower arm, third finger, first finger, hand, hindlimb, and foot) in both sexes, and larger eye, upper eyelid, and first toe in females. Of the two remaining members of wrinkled frogs, G. tientaiensis ( Chang 1933 ) resembles G. reliquia sp. nov. , but has much flatter dorsal ridges and less granulated ventral skin than the latter. Glandirana minima (Ting & Tsai 1979) has significantly smaller body and much less developed toe webbing than G. reliquia sp. nov. ( Fei et al . 2012 ). Range. Eastern half of southern Tohoku to Kanto and Chubu regions ( Fig. 9 ). Eastern Tohoku region: Iwate Prefecture (Oshu City, Kitakami City, Ichinoseki City [former Higashiyama Town], Hiraizumi Town, Tono City, Hanamaki City), Miyagi Prefecture (Minamisanriku Town, Sendai City, Kawasaki Town), Fukushima Prefecture (Iwaki City). Kanto region: Ibaraki Prefecture (Tsukuba City, Hitachiomiya City), Tochigi Prefecture (Ichikai Town, Ashikaga City, Nasushiobara City, Utsunomiya City, Kanuma City), Gunma Prefecture (Shibukawa City [former Akagi Village], Katashina Village), Saitama Prefecture (Ogawa Town, Higashichichibu Village), Chiba Prefecture (Mobara City, Kamogawa City, Kimitsu City, Ichihara City), Tokyo Metropolis (Akiruno City [former Itsukaichi Town]), Kanagawa Prefecture (Yamakita Town, Odawara City, Sagamihara City, Minamiashigara City, Isehara City). Chubu region: Yamanashi Prefecture (Kofu City, Uenohara City), Nagano Prefecture (Nakano City, Yamanouchi Town, Nagano City [former Togakushi Village], Chikuma City, Ueda City, Tomi City, Komoro City, Saku City, Matsumoto City, Shiojiri City, Hakuba Village, Minamimaki Village). Natural History. Glandirana reliquia sp. nov. inhabits widely plains and low mountains, near various water bodies from artificial ponds in urban area to paddy fields, rivers, montane streams, and wetlands. Breeds during late May and late August in still waters in rice fields, ponds, ditches, sometimes in pools of dry riverbeds, but also in slowly flowing waters. Multiple clutches spawned by some females in a year. Eggs are laid in small batches on vegetations in the shallow water of very slowly flowing interceptor connecting water canals and paddies. Ashizawa et al. (2013) reported flow velocity to have the most impact on the egg batch density, in Ichikai Town, Tochigi Prefecture . Larvae hatched from eggs laid at the end of breeding season usually overwinter and metamorphose in the following year. Conservation status. Glandirana rugosa including G. reliquia sp. nov. is listed as Least Concern (LC) in IUCN category ( Matsui et al . 2021 ). It is not listed in Japanese Red List by Ministry of Environment, but populations assigned to G. reliquia sp. nov. is variously treated to levels of Critically Endangered ( Chiba Prefecture ), Endangered ( Saitama Prefecture ), Vulnerable ( Tochigi , Gunma , and Nagano Prefectures and Tokyo Metropolis), and near threatened ( Miyagi Prefecture ) by local governments in the range of its distribution.