More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus
Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n = 52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n = 22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.
KeywordsMeiotic multivalents Multiple sex chromosomes Translocations Amphibia Leptodactylidae
We thank Natalia Maria Espíndola Salles, Juliano Mafra Neves, and Karll Cavalcante Pinto for help in field work, and to João Rodrigo Cabeza as well as Companhia Hidrelétrica Teles Pires for allowing collecting activities in their study areas. We are grateful to Heidi Horn, for a revision in the final version of this manuscript. TG thanks Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio – SISBIO) for providing a collection permit (no. 30202-2). The authors are also grateful to the Centro de Estudos de Insetos Sociais, UNESP – Univ. Estadual Paulista, Rio Claro, Brazil, for allowing the utilization of facilities for molecular analyses.
T. G., P. P. P. M., and C. F. B. H. designed the study. T. G. performed the cytogenetic analyses. D. C. C. M. assisted on microsatellite and CGH experiments. M. L. L. performed DNA sequencing and similarity analyses. T. G. and H. N. collected the specimens. T. G. and P. P. P. M. wrote the main manuscript, and all authors contributed to improve it. All authors approved the final version of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
- Amaro-Ghilardi RC, Rodrigues MT, Yonenaga-Yassuda YY (2004) Chromosomal studies after differential staining and fluorescence in situ hybridization using telomeric probe in three Leptodactylus species (Leptodactylidae, Anura). Caryologia 57(1):53–65. https://doi.org/10.1080/00087114.2004.10589372 CrossRefGoogle Scholar
- Bachtrog D, Mank JE, Peichel CL, Kirkpatrick M, Otto SP, Ashman T-L, Hahn MW, Kitano J, Mayrose I, Ming R, Perrin N, Ross L, Valenzuela N, Vamosi JC (2014) Sex determination: why so many ways of doing it? PLoS Biol 12(7):e1001899. https://doi.org/10.1371/journal.pbio.1001899 PubMedPubMedCentralCrossRefGoogle Scholar
- Baldissera FA Jr, Oliveira PSL, Kasahara S (1993) Cytogenetics of four Brazilian Hyla species (Amphibia-Anura) and description of a case with a supernumerary chromosome. Rev Bras Genet 16:335–345Google Scholar
- Cnaani A, Lee BY, Zilberman N, Ozouf-Costaz C, Hulata G, Ron M, D’Hont A, Baroiller JF, D’Cotta H, Penman DJ, Tomasino E, Coutanceau JP, Pepey E, Shirak A, Kocher TD (2008) Genetics of sex determination in Tilapiine species. Sex Dev 2(1):43–54. https://doi.org/10.1159/000117718 PubMedCrossRefGoogle Scholar
- Coelho AC, de Mattos TL, Viana P, Terencio ML, Schneider CH, Menin M, Gross MC (2016) Intra-generic and interspecific karyotype patterns of Leptodactylus and Adenomera (Anura, Leptodactylidae) with inclusion of five species from Central Amazonia. Genetica 144(1):37–46. https://doi.org/10.1007/s10709-015-9876-8 PubMedCrossRefGoogle Scholar
- Frost DR (2017) Amphibian species of the world: an online reference (Version 6.0) http://research.amnh.org/vz/herpetology/amphibia/Amphibia/Anura/Leptodactylidae/Leptodactylinae/Leptodactylus/Leptodactylus-pentadactylus. Accessed 10 March 2017
- Gazoni T, Gruber SL, Silva APZ, Araújo OGS, Narimatsu H, Strüssmann C, Haddad CFB, Kasahara S (2012) Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae), including a new diploid number and a karyotype with multiple translocations. BMC Genet 13(1):109. https://doi.org/10.1186/1471-2156-13-109 PubMedPubMedCentralCrossRefGoogle Scholar
- Gross MC, Feldberg E, Cella DM, Schneider MC, Schneider CH, Porto JI, Martins C (2009) Intriguing evidence of translocations in discus fish (Symphysodon, Cichlidae) and a report of the largest meiotic chromosomal chain observed in vertebrates. Heredity 102(5):435–441. https://doi.org/10.1038/hdy.2009.3 PubMedCrossRefGoogle Scholar
- Grützner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O’Brien PCM, Jones RC, Ferguson-Smith MA, Graves JAM (2004) In the platypus a meiotic chain of ten sex chromosomes shares genes with the bird Z and mammal X chromosomes. Nature 432(7019):913–917. https://doi.org/10.1038/nature03021 PubMedCrossRefGoogle Scholar
- Heyer WR, Diment MJ (1974) The karyotype of Vanzolinius discodactylus and comments on usefulness of karyotypes in determining relationships in the Leptodactylus-Complex (Amphibia, Leptodactylidae). Proc Biol Soc Wash 87:327–336Google Scholar
- Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
- Matsubara K, Knopp T, Sarre SD, Georges A, Ezaz T (2013) Karyotypic analysis and FISH mapping of microsatellite motifs reveal highly differentiated XX/XY sex chromosomes in the pink-tailed wormlizard (Aprasia parapulchella, Pygopodidae, Squamata). Mol Cytogenet 6(1):60. https://doi.org/10.1186/1755-8166-6-60 PubMedPubMedCentralCrossRefGoogle Scholar
- Matsubara K, O’Meally D, Azad B, Georges A, Sarre SD, Graves JA, Matsuda Y, Ezaz T (2016) Amplification of microsatellite repeat motifs is associated with the evolutionary differentiation and heterochromatinization of sex chromosomes in Sauropsida. Chromosoma 125(1):111–123. https://doi.org/10.1007/s00412-015-0531-z PubMedCrossRefGoogle Scholar
- Palumbi SR, Martin AP, Romano SL, Mcmillan WO, Stice L, Grabowski G (1991) The simple fool’s guide to PCR. Honolulu, Dept. of Zoology, University of HawaiiGoogle Scholar
- Rens W, O’Brien PCM, Grützner F, Clarke O, Graphodatskaya D, Tsend-Ayush E, Trifonov VA, Skelton H, Wallis MC, Johnston S, Veyrunes F, Graves JAM, Ferguson-Smith MA (2007) The multiple sex chromosomes of platypus and echidna are not completely identical and several share homology with the avian Z. Genome Biol 8(11):R243. https://doi.org/10.1186/gb-2007-8-11-r243 PubMedPubMedCentralCrossRefGoogle Scholar
- Sarre SD, Ezaz T, Georges A (2011) Transitions between sex-determining systems in reptiles and amphibians. Annu Rev Genom Human Genet 12(1):391–406. https://doi.org/10.1146/annurev-genom-082410-101518 CrossRefGoogle Scholar
- Schmid M, Feichtinger W, Steilein C, Visbal García R, Fernandez Badillo A (2003) Chromosome banding in Amphibia XXVIII. Homomorphic XY sex chromosomes and derived Y-autosome translocation in Eleutherodactylus riveroi (Anura, Leptodactylidae). Cytogenet Genome Res 101(1):62–73. https://doi.org/10.1159/000073420 PubMedCrossRefGoogle Scholar
- Šíchová J, Ohno M, Dincă V, Watanabe M, Sahara K, Marec F (2016) Fissions, fusions, and translocations shaped the karyotype and multiple sex chromosome constitution in the northeast-Asian wood white butterfly, Leptidea amurensis. Biol J Linnean Soc 118(3):457–471. https://doi.org/10.1111/bij.12756 CrossRefGoogle Scholar
- Siqueira S, Ananias F, Recco-Pimentel SM (2004) Cytogenetics of three Brazilian species of Eleutherodactylus (Anura, Leptodactylidae) with 22 chromosomes and re-analysis of multiple translocations in E. binotatus. Genet Mol Biol 27(3):363–372. https://doi.org/10.1590/S1415-47572004000300010 CrossRefGoogle Scholar
- Stöck M, Horn A, Grossen C, Lindtke D, Sermier R, Betto-Colliard C, Dufresnes C, Bonjour E, Dumas Z, Luquet E, Maddalena T, Sousa HC, Martinez-Solano I, Perrin N (2011b) Ever-young sex-chromosomes in European tree frogs. PLoS Biol 9(5):e1001062. https://doi.org/10.1371/journal.pbio.1001062 PubMedPubMedCentralCrossRefGoogle Scholar
- Stöck M, Savary R, Betto-Colliard C, Biollay S, Jourdan-Pineau H, Perrin N (2013a) Low rates of X-Y recombination, not turnovers, account for homomorphic sex chromosomes in several diploid species of Palearctic green toads (Bufo viridis subgroup). J Evol Biol 26(3):674–682. https://doi.org/10.1111/jeb.12086 PubMedCrossRefGoogle Scholar
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30 (12):2725–2729Google Scholar
- Tanaka K, Takehana Y, Naruse K, Hamaguchi S, Sakaizumi M (2007) Evidence for different origins of sex chromosomes in closely related Oryzias fishes: substitution of the master sex-determining gene. Genetics 177(4):2075–2081. https://doi.org/10.1534/genetics.107.075598 PubMedPubMedCentralCrossRefGoogle Scholar
- Uno Y, Nishida C, Yoshimoto S, Ito M, Oshima Y (2008) Diversity in the origins of sex chromosomes in anurans inferred from comparative mapping of sexual differentiation genes for three species of the Raninae and Xenopodinae. Chromosom Res 16(7):999–1011. https://doi.org/10.1007/s10577-008-1257-z CrossRefGoogle Scholar
- Van der Meijden A, Vences M, Hoegg S, Boistel R, Channing A, Meyer A (2007) Nuclear gene phylogeny of narrow-mouthed toads (family: Microhylidae) and a discussion of competing hypotheses concerning their biogeographical origins. Mol Phylogenet Evol 44(3):1017–1030. https://doi.org/10.1016/j.ympev.2007.02.008 PubMedCrossRefGoogle Scholar