Karyotypes of the Six Species of Frogs (Genus Rana) Endemic to the Ryukyu Islands

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Karyotypes of the Six Species of Frogs (Genus Rana) Endemic to the Ryukyu Islands Mitsuru Kuramoto To cite this article: Mitsuru Kuramoto (1972) Karyotypes of the Six Species of Frogs (Genus Rana) Endemic to the Ryukyu Islands, Caryologia, 25:4, , DOI: / To link to this article: Published online: 30 Jan Submit your article to this journal Article views: 152 View related articles Citing articles: 13 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 30 November 2017, At: 15:49

2 KARYOTYPE$ OF THE SIX SPECIES OF FROGS (GENUS RANA) ENDEMIC TO THE RYUKYU ISLANDS * MITSURU KURAMOTO Department of Biology, Fukuoka University of Education, Munakata, Fukuoka, Japan INTRODUCTION Received: 9 1 h February 1972 With the advance of techniques for observing animal chromosomes, an increasing number of investigations on anuran karyotypes have been reported by many authors. Most of these papers, however, present photographic illustrations of chromosomes, without detailed statistical data. Statistical studies such as the one presented by VoLPE and GEBHARDT (1968) make precise haryotypic comparison possible and promise a fruitful future in this field. KoBAYASHI (1962), SETO (1964, 1965) and others have clarified the karyotypes of some Japanese frogs. On the other hand, karyotypes of the frogs of the Ryukyu Islands, most of which are endemic and interesting from the viewpoint of speciation, remain unknown. Of the seven ranid species inhabiting the Islands, only one, Rana limnocharis, is not endemic; its karyotype was reported by KuRAMOTO ( 1970 ). The present paper deals with the karyotypes of the six species of the genus Rana that are endemic to the Ryukyu Islands: Rana narina, R. ishikawae, R. subaspera, R. holsti, R. okinavana and R. namiyei. Rana narina, R. ishikawae and R. okinavana live in Amami and Okinawa; Rana subaspera occurs only in Amami, and R. holsti and R. namiyei only in Okinawa. Rana subaspera and R. holsti are so specialized among ranid frogs that some authors recognized generic or subgeneric distinctions (OKADA 1966; NAKA MURA and DENO 1963 ). It has been suggested also that R. narina, R. ishi- * This work was supported in part by a research grant from the Ministry of Education of Japan. [Caryologia, Vol. 25, n. 4, 1972

3 548 KURAMOTO kawae and R. namiyei are specialized from the common ranid frogs (NAKA MURA and UENO 1963 ). These species, therefore, constitute favorable materials for the investigation of the relationships between morphological and karyotypic differentiation. MATERIALS AND METHODS Chromosomes were analyzed from 29 frogs which were deposited in the amphibian collection at the Fukuoka University of Education: six males of Rana narina STEJNEGER (FUE ), five females and two males of R. ishikawae (STEJNEGER) (FUE ), four females of R. subaspera BARBOUR (FUE ), and eight males of R. okinavana BoETTGER (FUE ) from Sumiyo, Amami-oshima; a female and a male of R. holsti BouLENGER (FUE 10401, 10402) and two females of R. namiyei STEJNEGER (FUE 10470, 12024) from Yona, Okinawa-honto. Chromosome preparations of bone marrow cells were made by the method of OMURA (1967); 20 hours after subdermal injection of 0.005% colchicine, bone marrow cells of femur and tibio-fibula were squeezed out on slides with 0.6% sodium citrate, fixed with the vapor of ethanol-acetic acid mixture, and colored with Giemsa's stain. For karyotype analysis, chromosome lengths were measured on enlarged photomicrographs of metaphase-spreads (10 or 16 for each species) and relative length (length of each chromosome with the total length of chromosomes being 100) and arm ratio (length of long arm/length of short arm) were calculated. I selected the metaphase-spreads that were easy to measure. Secondary constrictions were included in the measurements. Each chromosome pair was numbered in order of descending mean relative length, but the order of some pairs was reversed to correlate the chromosomes between species. RESULTS Rana narina, R. ishikawae, R. subaspera, R. holsti, and R. okinavana have 13 pairs of chromosomes and R. namiyei has 11 pairs (Figs. 1-2, Table 1). No heteromorphic pairs of chromosomes were found in any species. In the first five species, chromosomes can be divided into two distinct size groups: large chromosomes (Nos. 1-5) and small chromosomes (Nos ). The mean arm ratio of pair No. 1 is below 1.2 in all species and is metacentric. Each species has three subtelocentric chromosomes (arm ratio near and above 2.0); Nos. 3, 8, and 11. All other chromosomes are between 1.2 and 1.9 in arm ratio and regarded as submetacentric. Rana namiyei is unique in having 11 pairs of chromosomes; all other species in the genus Rana reported so far have either 13 or 12 pairs. Chromosomes of R. namiyei cannot be classified into size groups as in the other

4 KARYOTYPES OF SIX SPECIES OF FROGS 549 TABLE I Relative length and arm ratio (mean ± one standard error) of chromosomes of six ranid species from the Ryukyu Islands. No. Rana narina Rana ishikawae Rana subaspera Relative Relative Relative Arm ratio Arm ratio length length length Arm ratio ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.04 *11.20± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± *4.78± ±0.03 *5.03± ±0.03 *5.07± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.06 (TABLE I cont.) No. Rana holsti Rana okinavana Rana namiyei Relative Relative Relative Arm ratio Arm ratio Arm ratio length length length 15.50± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± *11.36± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± *5.32± ±0.05 ''4.91 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.04 * Chromosome with secondary constriction.

5 550 KURAMOTO species, since the relative lengths decrease rather gradually from one pair to another. Brief remarks on each karyotype follow: 1 ) Rana narina STEJNEGER. Pair No. 1 is easily identified by its distinctly large s1ze and metacen- Fig Karyotypes of R. narina (A, FUE 9722, male), R. ishikawae (B, FUE 9701, male), and R. subaspera (C, FUE 9726, female). Scales equal 10 1-l

6 KARYOTYPES OF SIX SPECIES OF FROGS 551 tric form. Numbers 3 and 4 are similar in length but clearly differ in arm ratio. Although Nos. 2 and 3 as well as Nos. 4 and 5 overlap in the ranges of relative length and arm ratio, it is easy to identify them in the same chromosome spread. Small chromosomes are rather uniform in their relative lengths. Number 9 has a secondary constriction on the long arm (Fig. 1, A). Numbers 8 and 11 can be defined as the larger and smaller subtelocentric chromosomes. Number 13 is the smallest and its range of relative length does not overlap that of No. 12. The most similar chromosomes are Nos. 6 and 7, but they are rarely confused in the same spread. Although No. 11 is significantly longer than No. 10 (t=4.66, P<0.01), this order agrees well with the karyotypes of the other species in view of arm ratio. 2) Rana ishikawae (STEJNEGER). As in R. narina, there are clear disjunctions in relative length between Nos. 1 and 2 and in arm ratio between Nos. 1 and 2 and between Nos. 3 and 4. Identifications of large chromosomes are quite easy in the same chromosome spread. A secondary constriction is always seen on the long arm of No. 9 (Fig. 1, B). Each chromosome of R. ishikawae is very similar in size and shape to that of R. narina. Karyotypes of males and females did not differ in any respect. 3) Rana subaspera BARBOUR. The most characteristic feature of the karyotype of R. subaspera is a secondary constriction on the long arm of No. 4 (Fig. 1, C). Each large chromosome is clearly identifiable by size and shape. Since the difference in relative length of Nos. 3 and 4 is not significant (t=0.74, 0.50>P>0.25), the order is reversed so that the shape of these chromosomes corresponds to those of the other species. Small chromosomes are similar to those of R. ishikawae. Number 9 has a secondary constriction on the long arm as in R. narina and R. ishika:wae. 4) Rana holsti BouLENGER. This species also has two secondary constrictions; one is on the long arm of No. 4 and the other on the long arm of No. 9 (Fig. 2, A). Numbers 3 and 4, Nos. 8 and 10, and Nos. 12 and 13 do not differ in relative length but differ clearly in arm ratio. To correlate each chromosome with that of

7 552 KURAMOTO the other species, the order is reversed between Nos. 3 and 4 and between Nos. 8 and 9; the difference of relative length is not significant in the former pairs (t= 1.62, 0.25>P>0.10), but it is significant in the latter pairs (t=2.87, 0.025>P>O.Ol). I could not find any karyotypic differences between male and female. Fig Karyotypes of R. holsti (A, FUE 10401, female), R. okinavana (B, FUE 10089, male), and R. namiyei (C, FUE 10470, female). Scales equal 10!J..

8 KARYOTYPES OF SIX SPECIES OF FROGS 553 5) Rana okinavana BoETTGER. Although Nos. 3 and 4 do not differ in relative length, the difference in arm ratio is remarkable. Pair No. 9 has a secondary constriction on the long arm (Fig. 2, B). Excepting Nos. 12 and 13, small chromosomes that are similar in relative length are readily distinguishable by the differences in arm ratio. The mean length of No. 10 is longer than those of Nos. 8 and 9, but the differences among the three pairs are not significant (P>0.25). The order is, therefore, determined so as to correlate with the other karyotypes regarding arm ratios. 6) Ran a namiyei STEJNEGER. The karyotype of this species differs significantly from those of the above five species. All chromosomes are submetacentric and none has a secondary constriction (Fig. 2, C). All chromosome pairs can be identified by relative length; differences in relative length between all chromosomes are highly significant (t>2.95, P<0.01). Numbers 1-5 seem to correspond to the same respective pairs of the other species. Numbers 8-11 may correspond respectively to Nos. 6, 7, 10, and 13 of the other species so far as size and shape are concerned. Rana namiyei has unique pairs of intermediate size (Nos. 6 and 7) and, in turn, lacks pairs homologous to Nos. 8, 9, 11, and 12 of the other species. Perhaps Nos. 6 and 7 are composed of the genetic material that occurs in the four smaller pairs of the other five species. The karyotypes of R. narina, R. ishikawae, R. subaspera, and R. holsti remarkably resemble one another, the karyotype of R. okinavana differs somewhat from those of the first four species, and the karyotype of R. namiyei differs remarkably from all the other species. When the relative lengths of chromosomes are plotted against arm ratios, this point can be realized more readily (Fig. 3 ). It is noticed that the corresponding chromosomes of R. narina, R. ishikawae, R. subaspera, and R. holsti occupy relatively narrow areas on the diagram as indicated by the circles. Some chromosomes of R. okinavana and R. namiyei differ considerably from chromosomes of the other four species in relative length, arm ratio, or both. Similarity and dissimilarity of the karyotypes among the six species were checked by Student's t tests. The corresponding chromosomes of different species were regarded to agree with respect to their size and shape when two probability values obtained from t values for relative length and

9 0 I- 0 R. subospero I (!) z w _J 15 w > I- <X _J 10 a:: ~ d G w R. norino 6 R. ishikowoe <> R. holst! 6 5 ~ II ARM RATIO q o R. okinovono I I- 15 o R. nomiye/ (!) z 0 2 w _J ~- ' ', 0. _,- w > 3 :0 I- <X 10 _J w a:: ' ARM RATIO Fig Karyograms of the six ranid species from the Ryukyu Islands. Solid marks are chromosomes with secondary constrictions.

10 KARYOTYPES OF SIX SPECIES OF FROGS 555 arm ratio were larger than When the two probabil.ity values were smaller than 0.01, the difference between the two chromosomes was regarded as significant. The reason for adopting the confidence level of 0.01 instead of 0.05 is that some factors, such as differential coiling and artifacts in preparation, will probably make the identification of each chromosome uncertain, especially those of closely resembled pairs, and it seems pertinent to take a higher level of confidence for drawing some conclusions on karyotypic differences. Karyotypic similarities, on the other hand, would be greater than they actually are using this procedure. The results of t tests are summarized in Table II. Numbers 8, 9, 10, and 11 of R. namiyei are correlated respectively with Nos. 6, 7, 10, and 13 of the other species. For the discussion below, the results of R. limnocharis from Amami (karyological data are from KuRAMOTO 1970) are included in Table II. TABLE II Numbers of chromosome pairs that agree in both relative length and arm ratio (upper right) and those that differ in both (lower left). R. narina R. ishi- R. sub- R okina- R.limno- R. holsti kawae as per a van a charts R. namiyei R. narina R. ishikawae R. subaspera R. holsti R. okinavana R. limnocharis R. namiyei The karyotypes of R. subaspera and R. holsti are very similar to each other; 9 pairs agree both in relative length and in arm ratio and there are no pairs which differ together in both. It is noticed also that the karyotype of R. ishikawae resembles those of R. narina, R. subaspera, and R. holsti; 7 pairs between R. ishikawae and R. narina, 8 pairs between R. ishikawae and R. subaspera and 8 pairs between R. ishikawae and R. holsti show nearly the same values of relative length and arm ratio and only one pair of R. ishikawae and R. holsti differs in both (No. 13, t = 7.31 for relative length and t=4.92 for arm ratio). In all other combinations, less than half of the chromosome pairs agree in both relative length and arm ratio. None of No. 4 differs between the seven species both in relative length and in arm ratio at the confidence level of 0.01, and it agrees in both in

11 556 KURAMOTO 18 of 21 combinations. Number 1 is also similar between species, differing only between R. subaspera and R. namiyei (t=2.83 for relative length and t=5.72 for arm ratio) and agreeing in 12 combinations. Number 8 does not differ between species and agrees in 10 of 15 combinations (since R. namiyei has no chromosome corresponding to No. 8 of the other species, comparisons were made between the other six species). The most dissimilar chromosome is No. 13; it differs in 9 and agrees only in 2 of 21 combinations. DISCUSSION The ranids of the Ryukyu Islands differ remarkably in appearance from one another except R. subaspera and R. holsti. Rana narina, about 70mm in snout-vent length, resembles common ranid frogs but the tips of its digits dilate into small disks, which allow it to leap actively on smooth rocks in running waters without difficulty. Rana ishikawae, often over 100mm in snout-vent length, is a brilliant species with coarse skin and large sucking disks on the digits. NAKAMURA and UENO ( 1963) suggested that this species might well be classified as a different subgenus. Rana subaspera and R. holsti, toad-like in shape and size, are distinct in having five fingers; the first finger which is lost in most frog species is short but sharply pointed and often injures a collector's hands to the point of bleeding. Considering this additional digit to be diagnostic, OKADA ( 1966) treated both species as members of the genus Babina and NAKAMURA and UENO ( 1963) recognized Babina as a subgenus in the genus Rana. Rana namiyei, over 100mm in snout-vent length and aquatic in habit, is also considered to be worthy of subgeneric separation although any subgeneric name has not yet been postulated (NAKAMURA and UENO 1963 ). Rana okinavana is a member of the complex of brown frogs and its close relatives occur in Japan and Taiwan. Differing from these six species, R. limnocharis is not endemic to the Islands but instead occurs from southeastern Asia to Japan. In view of karyotypic characteristics, phylogenetic relationships of the ranids in the Ryukyu Islands may be expressed as in Fig. 4. All the species have apparently been derived from southeastern Asia through Taiwan (INGER 1950). As discussed previously, R. limnocharis seem to have extended its range rather recently, probably in the Pleistocene (KuRA MOTO 1968 ). It is obvious that R. limnocharis had differentiated before it moved into the Islands. Detailed comparisons of the karyotype of R. okinavana with those of the brown frogs occurring in the adjacent areas are insufficient at present since the numerical data on the latter species are not available. But the preliminary data obtained in my laboratory show that the karyotypes of

12 KARYOTYPES OF SIX SPECIES OF FROGS 557 the brown frogs differ from one another. For example, among the eight pairs of small chromosomes of R. longicrus, the only brown frog occurring in Taiwan, three are below 1.4 and two are above 3.1 in arm ratio (KURA MOTO, unpubl.), while all the small chromosomes of R. okinavana are between 1.6 and 2.4. Probably the brown frogs of eastern Asia (Japan, Ryukyu, and Taiwan) were derived from the stock of continental China and the karyotype of each species differentiated independently. ANCESTRAL FORMS (SE ASIA 8 TAIWAN) R. limnochoris R. okinovono R. norino R. ishikowoe R. subospero R. ho/sti R. nomiyei.cryukyu ISL.) Fig Hypothetical phylogeny of the seven ranid species in the Ryukyu Islands based on karyotypic similarity. Rana narina, R. ishikawae, R. subaspera, and R. holsti differ greatly in their external morphology except for the last two species belonging to the subgenus Babina. Nevertheless, the karyotypes of these four species closely resemble one another. In fact, interspecific differences in the karyotypes of these species are as small as intraspecific differences in R. limnocharis (KURAMOTO 1970). The ranid frogs for which relative length and arm ratio of chromosomes were given by previous authors are R. temporaria, R. dalmatina (GUIL LEMIN 1967), R. limnocharis (KURAMOTO 1970), and R. guentheri (KURA MOTO and TESHIMA 1970). From the comparison of these karyotypes, we conclude that the large chromosomes are similar in size and shape but the small chromosomes differ significantly to such an extent that they cannot be properly correlated (KuRAMOTO and TESHIMA 1970). Considering that R. na-

13 558 KURAMOTO rina, R. ishikawae, R. subaspera, and R. holsti seem to be much diverged and specialized forms which can be classified in different subgenera, the karyotypic resemblance of the four species is rather surprising. A clear conclusion from these comparisons is that the similarity in karyotypes of R. narina, R. ishikawae, R. subaspera, and R. holsti cannot be ascribed to the basic karyotype of the genus Rana. To explain the similarity by convergence seems improbable as well. The only explanation is that these four species are related to one another in spite of their morphological divergence. They must have differentiated genetically without accompanying modifications in their karyotypes. They retain the ancestral karyotype unchanged except for pair No.4 of R. subaspera and R. holsti, which has a distinctive secondary constriction. Thus, the subgenus Babina can be diagnosed not only on the basis of external characters but also on karyotypes. Differing from R. okinavana and R. namiyei, these four species have no relatives in Taiwan and it is probable that they have differentiated within the Islands as a result of long geographic isolation. The chromosome number of 2n = 22 is the basic number of the families Bufonidae, Leptodactylidae, and Atelopodidae and not of the Ranidae (see the list of RABELLO 1970). Including the present results, chromosome numbers of about 40 species in the genus Rana have been known up to now. Among these, only one, R. namiyei here reported, has 2n=22, four (R. arvalis, R. chensinensis, R. dybowskii, and R. ornativentris, all belonging to the complex of brown frogs) have 2n=24, and all the others have 2n=26 chromosomes. This suggests strongly that R. namiyei is karyotypically the most diverged stock in the genus and may well be separated from other ranid species by giving it a distinctive recognition at the subgeneric rank. This species is allied to R. kuhlii of Taiwan and continental China (NAKAMURA and DENO 1963 ), and it is highly probable that R. namiyei and R. kuhlii are derived from the common ancester. A diagnostic external feature of R. namiyei and R. kuhlii is long bony protuberances on the lower jaw (OKADA 1966; Lm and Hu 1961). To investigate the karyotype of R. kuhlii seems critical for the proper taxonomic treatment of R. namiyei. Acknowledgements. - I thank Mr. FuMIHIRO NAKAMURA, Mr. MASAO TAKEGAMI, and Miss REIKO TESHIMA for their assistance in collecting materials and in making chromosome preparations. Special thanks are due to Dr. CHARLES ]. CoLE of the American Museum of Natural History who kindly read the manuscript ar:.d offered me stimulating suggestions. REFERENCES GurLLEMIN C., Caryotypc:s de Rana temporaria (L.) et de Rana dalmatina (Bonaparte). Chromosoma, 21: INGER R. F., Distribution and speciation of the amphibians of the Riu Kiu Islands. Amer. Nat., 84:

14 KARYOTYPES OF SIX SPECIES OF FROGS 559 KoBAYASHI M., Studies on reproductive isolation mechanisms in brown frogs. II. Hybrid sterility. J. Sci. Hiroshima Univ., Ser. B, Div. 1, 20: KuRAMOTO M., Studies on Rana limnocharis Boie. II. Geographic variation in external characters. Bull. Fukuoka Univ. Educ., Part III, 18: , Studies on Rana limnocharis Boie. IV. Karyotypic differentiation of subspecies. Ibid., 20: KuRAMOTO M. and TESHIMA R., Karyotype of Rana guentheri Boulenger. Ibid., 20: Lru C. C. and Hu S.C., Chinese tailless batrachians. Ka-siie-chu-ban sha, Peiching, China. NAKAMURA K. and UENO S., Japanese reptiles and amphibians in colour. Hoiku-sha, Osaka, Japan. OKADA Y., Fauna japonica: Anura (Amphibia). Tokyo Electrical Engineering College Press, Tokyo, Japan. OMURA T., A method for chromosome preparations from amphibian bone marrow cells without in vitro culture and centrifugation. Zoo!. Mag. (Tokyo). 76: RABELLO M. N., Chromosomal studies in Brazilian anurans. Caryologia, 23: SETo T., The karyotype of Hyla arborea japonica with some remarks on heteromorphism of the sex chromosome. ]. Fac. Sci. Hokkaido Univ., Ser. VI, Zoo!., 15: , Cytogenetic studies in lower vertebrates. II. Karyological studies of several species of frogs (Ranidae). Cytologia, 30: VoLPE E. P. and GEBHARDT B. M., Somatic chromosomes of the marine toad, Bufo marinus (Linne). Copeia, 1968: SUMMARY Karyotypes of Rana narina, R. ishikawae, R. subaspera, R. holsti, R. okinavana, and R. namiyei were analyzed by making use of chromosomes from bone marrow cells. The first five species have 13 pairs of chromosomes, of which five pairs (Nos. 1-5) are large and eight pairs (Nos. 6-13) are small. Number 1 is metacentric, Nos. 3, 8 and 11 are subtelocentric, and the others are submetacentric. Number 9 has a secondary constriction on the long arm. Number 4 of R. subaspera and R. holsti has an additional secondary constriction on the long arm. Differing from these species, R. namiyei has 11 pairs of chromosomes that are not readily separated into different size groups, which is unique in the family Ranidae. All pairs of chromosomes of R. namiyei are submetacentric and none has a secondary constriction. The karyotypes of R. narina, R. ishikawae, R. subaspera, and R. holsti resemble one another, suggesting a common origin. Comparisons with karyotypes of other species of Rana suggest that these species have become differentiated in external morphology without large changes in the ancestral karyotype. The karyotype of R. okinavana differs from those of the above four species and probably from those of the allied forms of Japan and Taiwan. The karyotype of R. namiyei is decidedly different from that of all other species of ranids and thus R. namiyei should be separated taxonomically from all other ranids at the subgeneric rank.