Diflrerentiation f 70 Populations in the Rana nigromaculata Group by the Method of Electrophoretic Analyses

Seventeen kinds of enzymes and two kinds of blood proteins cxtracted from 1777 frogs of47 populations of Rana nigremacutata, 220 frogs of l2 populations ofR. brevipoda brevipodu and 340 frogs of 11 popuLations ef R. breviPoda Porosa were analyzed by the method ofhorizontal starch-gel electrophoresis. There were 7.6 phcnotypes produced by 4.1 alleles on the average at each of 28 leci. Thc gene frequencies wcre described in 25 ofthc 28 loci other than three which had each a single allele. The fixation index (Fst), average heteTozygosity, proportion of polymorphic loci and mean number of ailclcs per locus wcre calculated in all the populations of R. nigromacalata and R. brevipocla. Genetic distances were estimated by the method of NEi (1975) on the basis of the gene frequencies at 28 loci controlling l7 enzymes and two blood proteins. Thc genetic distances between the 47 popu]ations of R. nigromaculata and the 23 popuLatiuns ofR. brevipoda were O.133-O.768, O.546 on the avcrage. The genetic distanccs among the 45 populations of R. nigromacutata other than the Suwon and Beljing were O.OOO -O.206, while those among the 12 populations ofR. brevipotla brevipoda nd those among the 11 populations of R. brevipoda Porosa were O.O09-O.134 and O.OOO--O.066, respectiyely, Of 10 districts where R. nigromacaiata and R. brevipoda were sympatric, Shibata, Niigata Prefecture, was most peculiar in that the two species were O.133 in genetic distance. In the Matsumoto and Maibara districts, the genetic distances wcre O.417 and O.375, respectively, whi]e in the other seven districts, they were O.509--O.737, A dendrogram was drawn for the 47 populations of R. nigremacutata and the 23 populations of R. hrevipodo n the basis of the genetic distances among them by the UPGMA method. R. brevipoda seems to have invacledJapan earlier than R. nigromacutata, and to have produced R. brevipodu Porosa by frequent hybridization with R. nigromacutata mainly at the Shibata district and introgression of R. nigromacutata genes for a long period. INTRODUCTION The specific name Rana nigromaculata was given by HALLowELL (1860). He reported at the same time on Rana mar?norata collected from Shimoda, Shizuoka Prefecture. CopE (1868) named pond frogs collected from Kanagawa as 7-bmopterna Porosa. STEJNEGER (1907) placed R. mat7norata and 71 porosa as synonyms ofR. Hiroshima University NII-Electronic Library Service iroshimaUniversity 2 M, NISHIOKA, M. SUMIDA and H, OHTANI nigromaculata. According to him, theJapanese pond frogs distributed in Honshu, Shikoku and Kyushu belonged to only one species, R. nigromaculata. ScHMiDT (l927), TEREN[[JEv (1927) and OKADA (1931), moreover, considered that all the pond frogs distributed in Japan belonged to only one subspecies, R. nigromaculata nigromaculata. In 1941, ITo fbund that there were two types of pond frogs in Nagoya district. One type was Rana nigromaculata nigromaculata, while the other was named Rana nigromaculata brevipodo by himsel£ MoRiyA (l951) discovered the sympatric distribution of these two subspecies in Okayama district and found that the R. nigromaculata brevipoda was more unifbrm in color and pattern than that distributed in Nageya district. He observed in detail the differences in morphological, ecological and embryological characters between the two subspecies found in Okayama district. The frogs which were assumed to be natural hybrids between the two subspecies had been seldom found in Okayama district in spite of the easiness of artificial production of hybrids between them. The two subspecies seemed to be isolated from each other mainly by ecological factors. MoRiyA (1960b) confirmed that the males of reciprocal hybrids between the two subspecies were almost completely sterile. On the basis of MoRiyA's observations and expcriments, MooRE (1955, 1960, 1962) rcpeatedly expressed the view that each of Rana nigromaculata nigromaculata and Rana nigromaculata brevipode was a valid species. On the other hand, MoRiyA (1954) collected numerous specimens of the pond frogs from various districts ofJapan, and examined their morphological characters. On the basis of their morphological and distributional diflerences, he divided Japanese pond frogs into the fo11owing five races: 1) nigromaculata common, 2) Niigata intermediate, 3) Tokyo intermediate, 4) Nagoya brevipoda, and 5) Okayama brevipoda, Of these races, the Niigata intermediate was elucidated by KAwAMuRA and NisHioKA (I973, 1977, 1979) to be a mixture of the Tokyo intermediate race, natural hybrids between the latter and the nigromaculata common race, and the offspring of natural hybrids (KAwAMuRA, 1962). They also clarified that natural hybridization was frequently occurring between the nigromaculata common race and the Tokyo intermediate race in Niigata district. According to MoRiyA (1960a, b), the nigromacutata common race diflered from the other four races in many respects of reproduction, ancl the males of reciprocal hybrids between the former race and the latter four races were very poor in reproductive ability, while the females seemed to be quite fertile. Mainly on the basis of MoRiyA's observations, KAwAMuRA (1962) considered it reasonable to divide Japanese pond frogs into the fo11owing two species, Rana nigromaculata HALLowELL and Rana bfevipoda ITo, and one subspecies, Rana brevipodo porosa (CopE). KAwAMuRA and NisHioKA (1978) produced reciprocal hybrids between Rana nigromaculata and Rana brevipodu and their offspring which were repeatedly backcrossed by the paternal and maternal species until B4 offspring were produced. Recently, MATsui and HiKiDA (1985) proposed Rana perosa porosa in place of Rana brevipeda Porosa on the basis ofdescription on only three specimens by CopE (1868). In contrast to KAwAMuRA's consideration that R. brevipoda Perosa was derived from Hiroshima University NII-Electronic Library Service iroshimaUniversity DtLferentiation f ro PopulationJ' in the Rana nigromacutata Greup 3 hybridization between R, nigromaculata and R. brevipoda, MATsui and HiKiDA stated that the hybrid status of Porosa has not been demonstrated, in disregard of the actual existence of natural hybrids between the two species and their offspring. These natural hybrids and their offspring are easily fbund, especially in recent years in most sympatric areas of both species. The present study was designed to clarify the present status and the origin of the Japanese pond frogs by electrophoretic analyses of enzymes and blood proteins, Electrophoretic analyses of enzymes and blood proteins have been perfbrmed to elucidate the intraspecific or interspecific d fllerentiation in Rana tagoi by NisHioKA, OHTA and SuMiDA (1987), Buergeria, Rhacopherus and PoCJ{Pedates by NisHioKA, SuMiDA, OHTA and Suzu(1987), Rana narina by NisHioKA, UEDA and SuMiDA (1987), Btofbjoponicus by KAwAMuRA, NismoKA, SuMiDA and RyuzAKi (1990), 13 Btofb species and subspecies by NisHioKA, SuMiDA, UEDA and Wu (1990), Rana limnocharis by NisHioKA and SuMmA (1990), l!yla by NisHioKA, SuMiDA and BoRKiN (1990), six pond frog species by NisHioKA and SuMmA (1992), and 12 brown frog species by NisHioKA, SuMiDA, BoRKiN and Wu (1992). MATERIALS AND METHODS The materials used in the present study consisted of2337 frogs of 70 populations collected from various districts ofJapan, Korea and China during the 10 years between 1977 and l986. They included 1777 frogs of47 populations belonging to Rana nigromaculata, 340 frogs of 1 1 populations belonging to R. brevipodo porosa and 220 frogs of 12 populations belonging to the Nagoya and Typical races of R. brevipodo brevipoda (MoRiyA, 1954). These frogs were frozen and preserved in REvco's stocker at -80eC, The collecting stations, dates, numbers of frogs and population names are shown in Tables 1 and 2. Of the 47 populations of R. nigromaculata, the Sakata was collected by Mr. T. IGARAsHi, three of the Matsumoto, Chino and Nagoya were by Mr, R. SHiMoyAMA, the Ina was by Dr. K. TATEism, the Yamaguchi was by Dr. H. SAMBuicHi, the Nangoku was by Dr. K, UTsuMi, the Matsuyama was by Mr. Y. MyoREi, the Sasebo was by Mr. M. ODA, two of the Munakata and Suwon were by Dr. M. KuRAMoTo, the Kageshima was by Dr. S. IsHiKuBo, the Oita was by Mr. S. SATo, and the Beijing was by Dr. Chih-Ye CHANG. Of the 23 populations of R, brevipodo, the Maki of R. b. Porosa was collected by Mr. K. KiNEBucHi, fbur of the Nagano, Iiyama, Shinano and Matsumoto of the same subspecies and the Tsu of the Nagoya race of R. b. brevipotla by Mr. R. SHiMoyAMA and the Ina of the Nagoya race of the same subspecies by Dr. K. TATEISHI. Fourteen kinds of enzymes of skeletal muscles, three kinds of enzymes of livers and two kinds of blood proteins extracted from frogs were analyzed by the method of horizontal starch-gel electrophoresis. The kinds ofenzymes and blood proteins analyzed, their abbreviations, E. C. Nos., samples used fbr electrophoresis and bufler systems are shown in Table3, The method of electrophoresis has been Hiroshima University NII-Electronic Library Service iroshimaUniversity 4 M. NISHIOKA, M. SUMIDA and H. OHTANI TABLE 1 CoL]ecting station, prefecture, area, date, number of Rana nigromacutata and the name ofpopulation Area Prefecture Station Date No.offrogsPopuiation(No.) TehokuAomori Namioka-city Sep.1986 19Namioka <1) Akita Akita-city Aug.1978 6Akita <2) YamagataSakata-city t48Sakata <3) HekurikuNiigata Shibata-city tl 16Shibata (4) Niigata-city t20Niigata <5) Kashiwazaki-city tt 26Kashiwazaki(6) Joetsu-city lt 36Joetsu (7> Toyama Toyama-city tt 37Toyama (8) Ishikawa Kanazawa-city lt S5Kanazawa(9) Fukui Sakai-gun,Mikuni -t 25Mikuni (10) Chubu Nagano Matsumoto-city Oct.1983 3eMatsumoto(11) Okaya-city Aug.1978 64Okaya (12> Chino-city,Miyagawa Sep.1983 3eChino (13) Ina-city June1979 2Ina (14) Iida-city Aug.I978 40Iida (]5) YamanashiKitakoma-gun,Sutama /t 35Sutarna (16) Shizuoka Mishima-city Junel979 67Mishima (17) Aichi Nagoya-city Oct.1981 30Nagoya (18) Inazawa-city,Osato Oct.1979 5Inazawa (19) Kinki Shiga Sakata-gun,Maibara 1978-1986 21Maibara (20) Mie Ueno-city Oct.I979 48Igaueno (21) WakayamaShingu-ci'ty tt 6Shingu (22) Osaka Higashiesaka-city tt 38Higashiesaka(23) Hyogo Himeji-city tt 6Himeji (24) ChugokuTottori Tottori-city Sep.1980 64Tottori (25) Shimane Matsue-city tt 75Matsue (26) Gotsu-city tt 23Gotsu (27) YamaguchiHagi-city Sep.1978 66Hagi <28) Yamaguchi-city,Yahara t52Yamaguchi(29) HiroshimaHiroshima-city l976-1980 60Hiroshima(30) Aki-gun,Kumano 1976-1986 56Kumano (3!) Kure-city,Hiro May1981 19Hiro・ (32) Miyoshi-city Sep.1986 19Miyoshi (33) Shobara-city May1981 19Shobara (34) Okayama Asakuchi-gun,Konko 1976-1978 83Konko (35) ShikokuKagawa Takamatsu-city Sep.1978 42Takamatsu(36) Kochi Nangoku-city June1979 32Nangoku (37) Ehime Uwajima-city tt 39Uwajima (38) Matsuyama-city 1/ 23Matsuyama(39) KyushuFukuoka Munakata-city Sep.1978 58Munakata(40) Nagasaki Sasebo-city tt 58Sasebo (41) KumamotoKumamoto-city Oct1978 49Kumameto(42) KagoshimaKagoshima-city t/ 60Kagoshima(43) Miyazaki Miyazaki-city Sep.1980 56Miyazaki (44) Oita Oita-city Mayl979 55Oita (45) Korea Suwon Junel977 38Suwon {46) China Beljing Sep.1979 21Beijing {47) Total 1777 47populatiens Hiroshima University NII-Electronic Library Service iroshimaUniversity Dijllerentiation of 70 Populations in the Rana nigromaculata GrouP TABLE 2 Collecting station, prefecture, date, number ofRana brevipeda nd the name ef pepulatien 5 Prefecture Station Date No.offrogsPopulation(No,)