And Identity of Triploids of Rana
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Bijdragen tot de Dierkunde, 60 (3/4) 199-207 (1990) SPB Academie Publishing bv, The Hague kl. esculenta Linnaeus On the occurrence and identity of triploids of Rana and R. lessonae Camerano in The Netherlands (Anura: Ranidae) Rose M.A. Blommers-Schlösser Institute of Taxonomie Zoology, University of Amsterdam, P.O. Box 4766, 1009 AT Amsterdam, The Netherlands Keywords: Ranidae, Rana lessonae, R. ridibunda, R. kl. esculenta, erythrocyte size, triploidy, genotype, biometry, distribution Abstract tage de R. kl. esculenta triploide est d’environ 1% dans des populations de R. lessonae — R. kl. esculenta des zones orientales. According to electrophoresis and erythrocyte size the genotypes On n’a pas trouvé des différences biométriquesentre R. kl. es- of 756 waterfrogs, collected during 1986—1988 in 54 localities in culenta triploïdeet diploïde, ou bien entre R. lessonae triploïde The Netherlands, were classified as belonging to 5 different et diploïde. lessonae genotypes: 331 diploid R. lessonae (LL), 5 triploid R. (LLL), 250 diploidR. kl. esculenta (LR), 133 triploidR. kl. es- culenta (LLR), and 37 diploid R. ridibunda (RR). The occurrenceof triploidR. kl. esculenta in The Netherlands Introduction is reported for the first time and triploidR. lessonae has not yet been LL reportedpreviously. There are indications that gametes The edible frog Rana klepton (thief in Greek) es- could be produced by LLR triploids and LL diploids. R. kl. es- culenta Linnaeus, 1758 is hybridogenetic, its ge- culenta in R. kl. esculenta and R. ridibunda —R. kl. esculenta nome containing parental chromosome sets of both populations of the western regions seems to be exclusively triploid, whereas the percentage oftriploidR. kl. esculenta in R. the little waterfrog Rana lessonae Camerano, 1882 lessonae — R. kl. esculenta populations of the eastern regions is and the lake frog Rana ridibunda Pallas, 1771 (cf. about 1%. Günther, 1987). Both parental species and the Biometrical differences were neither found between R. kl. es- hybrid form occur in The Netherlands (Wijnands & culenta triploid and diploid,nor between R. lessonae triploid Van 1976; and diploid. Gelder, Wijnands, 1977). In most cases R. kl. esculenta coexists with one of the parental species and is maintained by back- Résumé crossing with the syntopic parental species, clonally the of the En se basant sur l’électrophorèse et sur les dimensions des transmitting genome allotopic parental érythrocytes, les génotypes de 756 grenouilles vertes, collectées species (Günther & Plötner, 1988). de 1986 à 1988 dans 54 localités des Pays-Bas, sont rangés dans Mixed diploid R. lessonae - R. kl. esculenta 5 génotypes différents: 331 exemplaires de R. lessonae (LL) populations are the most widespread within the ge- diploïde, 5 de R. lessonae (LLL) triploide, 250 de R. kl. esculen- ographical range of R. kl. esculenta (western and ta (LR) diploïde, 133 de R. kl. esculenta (LLR) triploïdeet 37 de central In these populations R. kl. es- R. ridibunda (RR) diploïde. Europe). L’existence de R. kl. esculenta démontrée la culenta ridibunda and is triploïdeest pour generates (R) gametes première fois dans les Pays-Bas, et des R. lessonae triploïdes maintained by backcrossing with R. lessonae, n’ont pas encore été décrites. Il y a des indications que des ga- which produces lessonae (L) gametes. Crosses of R. LLR LL mètes LL peuvent être produits par des triploïdes et des kl. esculenta x R. kl. esculenta are usually lethal diploïdes. Il paraît que tout R. kl. esculenta est triploïde dans des and R. ridibunda is extremely rare in such popula- populations de R. kl. esculenta ou de R. ridibunda —R. kl. es- culenta des zones occidentales du pays, tandis que le pourcen- tions. 200 R.M.A. Blommers-Schlosser Triploids of Rana kl. esculenta and R. lessonae 1. Fig. Distribution of collecting sites. The encircled numbers refer to localities in Table I. Coordinates according to the Amersfoort grid (quadrats are 25 km²). - 201 Bijdragen tot de Dierkunde, 60 (3/4) 1990 - localities Both mixed R. ridibunda R. kl. esculenta study focussed onthis part of the country. Sample are shown in Fig. 1 and listed in Table I. populations and R. lessonae - R. kl. esculenta The ratios tibia length/callus internus length (index 1) and from The populations are known only Netherlands, digitusprimus length/callusinternus length (index 2) are consid- and Poland East and West Germany (Günther, characteristics ered by most authors the most useful biometrical 1975; Berger, 1977; Wijnands, 1977; Rahmel, to distinguishdifferent genotypes of waterfrogs from each other For captured (sub)adults 1988). (cf. Günther, 1975; Berger, 1977). all the following measurements were taken using a vernier caliper: Populations, containing only R. kl. esculenta in- body length, tibia length, length of digitusprimus, length of cal- dividuals, comprise always a large fraction of lus internus, and height ofcallus internus. Biometrical compari- and have been found in the northern triploids parts sons are restricted to animals larger than 36 mm. and in the fourth of East and West Germany and Poland Blood was taken by severing ablood vessel between the and fifth toe. Aftermeasuring, bleeding, and sex determination southern part of Sweden (Günther, 1975; Berger, the frogs were released. 1977; Ebendal & Uzzell, 1982; Eikhorst, 1984). In As tadpoles from homotypic R. kl. esculenta crosses mostly and R. kl. esculenta LLR (2n 3n) populations (two die before metamorphosis or produce clumsy froglets (Berger, lessonae and one ridibunda genomes) triploids 1970; Blankenhorn, 1977) tadpoles were reared until they Just the animals hatched asvital froglets. after metamorphosis, largely outnumber the LRR (one lessonae and two were anaesthetized in a solution of MS 222 (Sandoz, Basel) and ridibunda genomes) ones. Studies on these pure R. blood was taken by heart puncture. kl. esculenta indicate that LLR populations small amount of The blood was collected in glass capillaries, a fertile L triploids generate mainly haploid gametes, a 4% Na-citrate or heparinefrog saline solution being added to A blood smear was prepared in most cases and formed after normal genetic recombination pro- prevent clotting. the plasma of the remainingblood, after centrifugation with a the R. cesses, thus replacing parental lessonae, 40% hand centrifuge, was mixed with anequal volume of a su- while diploid LR males produce R gametes and LR crose solution and stored at -20°C. females LR R of vertical produce or ova. Reproduction seems The plasma protein pattern was studied by means of LR Polyacrylamide slab-gel electrophoresis (Maurer, 1971). After to depend essentially on the mating diploid staining with Coomassie Blue up to three bands of different females with triploid LLR males, which results in anodical mobilities were expected to show up on the gels. A fast both LLR and LR progeny. Other crosses seem to moving band "Type A" is typical of R. lessonae while a slower be much less successful. The R. kl. esculenta pure moving band "Type B" and/or a slightly slower moving band is modified lessonae/esculenta R. kl. population thus a (than"Type B") "Type C" are typical of ridibunda. R. esculenta is heterozygous, having both the lessonae and a system (Günther et aí., 1979; Günther, 1983;Berger ridibunda band (Tunner, 1973; Wijnands, 1977). & Günther, 1988; Eikhorst, 1988b; Borkin et al., Triploid R. kl. esculenta can be distinguished from diploid 1989). animals in two ways: (1) from the gene dosage effectthe albumin The on the of the present paper reports genotypes band, representing double genome, is darker and broader than the albumin band, representing the single (Eik- waterfrogs and occurrence of triploid R. kl. es- genome horst, 1984) and (2) on account of their (about 20%) longer culenta in The Netherlands. Data were collected erythrocytes (Günther, 1977). Therefore, the length ofat least 10 and during a study on the distribution, ecology, erythrocytes was measured from the dried smear and scored need for of R. lessonae. more adequate protection blind. For sizes Student's As the LLR triploid R. kl. esculenta can take the both the biometrical data and erythrocyte r-test was used to test the differences found, after application of place of R. lessonae functionally in the main- the normality test. tenance of R. kl. esculenta, it is potentially threatening the survival of R. lessonae. Ecological Results and conservational aspects will be treated elsewhere. With electrophoresis the following albumin pat- A terns were found in 756 specimens: ( lessonae), B Material and methods and BC (ridibunda), and AB and AC (esculenta). In darker and some of theesculentapatterns, a slightly & According to the distribution map provided by Bergmans broader A band, combined with a lighter and slight- Zuiderwijk (1986), in which waterfrog identifications are based narrower B or C band was observed (Fig. This mostly on biometry, R. lessonae occurs mainly in the eastern ly 2). part (above sea level) of The Netherlands. Hence, the present was interpreted as a gene dosage effect, indicating 202 R.M.A. Blommers-Schlosser - Triploids of Rana kl. esculenta and R. lessonae Table I. Localities and genotypes of waterfrogs, grouped by province. Coordinates according to the Amersfoort grid. Genotypes: L lessonae R = ridibunda LL = R. lessonae LLL = lessonae LR = = genome; genome; 2n; R. 3n; R. kl. esculenta 2n; LLR R. kl. * ** esculenta 3n; RR = R. ridibunda 2n. N = number of specimens. Presence observed. Leg. P. Bellink, University of Nijmegen. Localities Coordinates Year Water bodies Genotypes (N) LL LLL LR LLR RR Province Drenthe 1. Vledder "Vledderesch" 211.4-543.0 87 Fen in moorland 5 2 1 2. De Wijk "Havixhorst" 213.8-521 87 Pool in deciduous forest 4 8 Province Flevoland 3. Lelystad "Natuurpark" 164 -499 87 Small lakes 15 Province Zuid-Holland 4. Leiden "Knotterpolder" 93.5-461.2 88 Ditch in meadow 8 1 5. Zoeterwoude "Weipoort" 96.5-458.4 88 Ditch in meadow 1 * Province Zeeland 6.