Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

Keywords:
Testudines, Karyotype, Sex determination, CGH, Chromosome banding, Emys orbicularis, FISH
1.
Alföldi J, Di Palma F, Grabherr M, Williams C, Kong L, et al: The genome of the green anole lizard and a comparative analysis with birds and mammals. Nature 477:587-591 (2011).
2.
Araújo NP, Kuhn GCS, Vieira FN, Morcatty TQ, Paglia AP, Svartman M: Comparative genomic in situ hybridization and the possible role of retroelements in the karyotypic evolution of three Akodontini species. Int J Genomics 2017:5935380 (2017).
3.
Bachtrog D, Mank JE, Peichel CL, Kirkpatrick M, Otto SP, et al: Sex determination: why so many ways of doing it? PLoS Biol 12:e1001899 (2014).
4.
Badenhorst D, Stanyon R, Engstrom T, Valenzuela N: A ZZ/ZW microchromosome system in the spiny softshell turtle, Apalone spinifera, reveals an intriguing sex chromosome conservation in Trionychidae. Chromosome Res 21:137-147 (2013).
5.
Badenhorst D, Hillier LW, Literman R, Montiel EE, Radhakrishnan S, et al: Physical mapping and refinement of the painted turtle genome (Chrysemys picta) inform amniote genome evolution and challenge turtle-bird chromosomal conservation. Genome Biol Evol 7:2038-2050 (2015).
6.
Barros RM, Sampaio M, Assis F, Ayres M, Cunha O: General considerations on the karyotypic evolution of Chelonia from the Amazon region of Brazil. Cytologia 41:559-565 (1976).
7.
Bickham JW: A cytosystematic study of turtles in the genera Clemmys, Mauremys and Sacalia. Herpetologica 31:198-204 (1975).
8.
Bickham JW, Baker RJ: Chromosome homology and evolution of emydid turtles. Chromosoma 54:201-219 (1976a).
9.
Bickham JW, Baker RJ: Karyotypes of some neotropical turtles. Copeia 1976:703-708 (1976b).
10.
Bickham JW, Carr JL: Taxonomy and phylogeny of the higher categories of cryptodiran turtles based on a cladistic analysis of chromosomal data. Copeia 1983:918-932 (1983).
11.
Bickham J, Rogers D: Structure and variation of the nucleolus organizer region in turtles. Genetica 67:171 (1985).
12.
Bull JJ, Legler JM: Karyotypes of side-necked turtles (Testudines: Pleurodira). Can J Zool 58:828-841 (1980).
13.
Bull J, Vogt RC, Bulmer M: Heritability of sex ratio in turtles with environmental sex determination. Evolution 36:333-341 (1982).
14.
Ciofi C, Swingland IR: Environmental sex determination in reptiles. Appl Anim Behav Sci 51:251-265 (1997).
15.
Cleiton F, Giuliano-Caetano L: Cytogenetic characterization of two turtle species: Trachemys dorbigni and Trachemys scripta elegans. Caryologia 61:253-257 (2008).
16.
Damas J, O'Connor R, Farré M, Lenis VPE, Martell HJ, et al: Upgrading short-read animal genome assemblies to chromosome level using comparative genomics and a universal probe set. Genome Res 27:875-884 (2016).
17.
Delmas V, Prevot-Julliard AC, Pieau C, Girondot M: A mechanistic model of temperature-dependent sex determination in a chelonian: the European pond turtle. Funct Ecol 22:84-93 (2008).
18.
Demas S, Duronslet M, Wachtel S, Caillouet C, Nakamura D: Sex-specific DNA in reptiles with temperature sex determination. J Exp Zool A 253:319-324 (1990).
19.
De Smet W: Chromosomes of 11 species of Chelonia (Reptilia). Acta Zool Pathol Ant 70:15-34 (1978).
20.
Engel W, Klemme B, Schmid M: H-Y antigen and sex determination in turtles. Differentiation 20:152-156 (1981).
21.
Ezaz T, Quinn AE, Miura I, Sarre SD, Georges A, Graves JAM: The dragon lizard Pogona vitticeps has ZZ/ZW micro-sex chromosomes. Chromosome Res 13:763-776 (2005).
22.
Ezaz T, Valenzuela N, Grützner F, Miura I, Georges A, et al: An XX/XY sex microchromosome system in a freshwater turtle, Chelodina longicollis (Testudines: Chelidae) with genetic sex determination. Chromosome Res 14:139-150 (2006).
23.
Ge C, Ye J, Weber C, Sun W, Zhang H, et al: The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species. Science 360:645-648 (2018).
24.
Girondot M: Statistical description of temperature-dependent sex determination using maximum likelihood. Evol Ecol Res 1:479-486 (1999).
25.
Girondot M, Zaborski P, Servan J, Pieau C: Genetic contribution to sex determination in turtles with environmental sex determination. Genet Res 63:117-127 (1994).
26.
Goldberg EH, McLaren A, Reilly B: Male antigen defined serologically does not identify a factor responsible for testicular development. J Reprod Immunol 20:305-309 (1991).
27.
Graves JAM: Sex chromosome evolution and the expression of sex-specific genes in the placenta. Placenta 31:S27-S32 (2010).
28.
Graves JAM: Evolution of vertebrate sex chromosomes and dosage compensation. Nat Rev Genet 17:33 (2016).
29.
Howell WM, Black D: Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia 36:1014-1015 (1980).
30.
Ivanov VG: Description of the karyotype of Emys orbicularis L. (Testudines, Emydidade). Tsitologiya 15:1059-1061 (1973).
31.
Janzen FJ: Heritable variation for sex ratio under environmental sex determination in the common snapping turtle (Chelydra serpentina). Genetics 131:155-161 (1992).
32.
Kawai A, Ishijima J, Nishida C, Kosaka A, Ota H, et al: The ZW sex chromosomes of Gekko hokouensis (Gekkonidae, Squamata) represent highly conserved homology with those of avian species. Chromosoma 118:43-51 (2009).
33.
Kichigin IG, Giovannotti M, Makunin AI, Ng BL, Kabilov MR, et al: Evolutionary dynamics of Anolis sex chromosomes revealed by sequencing of flow sorting-derived microchromosome-specific DNA. Mol Genet Genomics 291:1955-1966 (2016).
34.
Killebrew FC: Mitotic chromosomes of turtles: I. the Pelomedusidae. J Herpetol 9:281-285 (1975).
35.
Kretschmer R, de Oliveira TD, de Oliveira Furo I, Oliveira Silva FA, Gunski RJ, et al: Repetitive DNAs and shrink genomes: a chromosomal analysis in nine Columbidae species (Aves, Columbiformes). Genet Mol Biol 41:98-106 (2018).
36.
Literman R, Radhakrishnan S, Tamplin J, Burke R, Dresser C, Valenzuela N: Development of sexing primers in Glyptemys insculpta and Apalone spinifera turtles uncovers an XX/XY sex-determining system in the critically-endangered bog turtle Glyptemys muhlenbergii. Conserv Genet Resour 9:651-658 (2017).
37.
Martinez PA, Ezaz T, Valenzuela N, Georges A, Graves JAM: An XX/XY heteromorphic sex chromosome system in the Australian chelid turtle Emydura macquarii: a new piece in the puzzle of sex chromosome evolution in turtles. Chromosome Res 16:815-825 (2008).
38.
Martinez PA, Boeris JM, Sánchez J, Pastori MC, Bolzán AD, Ledesma MA: Karyotypic characterization of Trachemys dorbigni (Testudines: Emydidae) and Chelonoidis (Geochelone) donosobarrosi (Testudines: Testudinidae), two species of cryptodiran turtles from Argentina. Genetica 137:277-283 (2009).
39.
Matthey R: Chromosomes de tortues. Bull Soc Vaudoise Sci Nat 57:151-152 (1930).
40.
Matthey R: Chromosomes de reptiles sauriens, ophidiens, chéloniens: l'évolution de la formule chromosomiale chez les sauriens. Rev Suisse Zool 38:117-186 (1931).
41.
Matthey R, Van Brink J: Sex chromosomes in Amniota. Evolution 11:163-165 (1957).
42.
McLaren A, Simpson E, Tomonari K, Chandler P, Hogg H: Male sexual differentiation in mice lacking H-Y antigen. Nature 312:552-555 (1984).
43.
Montiel EE, Badenhorst D, Lee LS, Literman R, Trifonov V, Valenzuela N: Cytogenetic insights into the evolution of chromosomes and sex determination reveal striking homology of turtle sex chromosomes to amphibian autosomes. Cytogenet Genome Res 148:292-304 (2016).
44.
Montiel EE, Badenhorst D, Tamplin J, Burke RL, Valenzuela N: Discovery of the youngest sex chromosomes reveals first case of convergent co-option of ancestral autosomes in turtles. Chromosoma 126:105-113 (2017).
45.
Mrosovsky N, Pieau C: Transitional range of temperature, pivotal temperatures and thermosensitive stages for sex determination in reptiles. Amphibia-Reptilia 12:169-179 (1991).
46.
Noleto RB, Kantek DLZ, Swarça AC, Dias AL, Fenocchio AS, Cestari MM: Karyotypic characterization of Hydromedusa tectifera (Testudines, Pleurodira) from the upper Iguaçu River in the Brazilian state of Paraná. Genet Mol Biol 29:263-266 (2006).
47.
Olmo E: Trends in the evolution of reptilian chromosomes. Integr Comp Biol 48:486-493 (2008).
48.
Olmo E, Signorino G: Chromorep: a reptile chromosomes database. Available at http://chromorep.univpm.it/ (2005).
49.
Pieau C: Modalities of the action of temperature on sexual differentiation in field-developing embryos of the European pond turtle Emys orbicularis (Emydidae). J Exp Zool A 220:353-360 (1982).
50.
Pieau C, Dorizzi M: Determination of temperature sensitive stages for sexual differentiation of the gonads in embryos of the turtle, Emys orbicularis. J Morphol 170:373-382 (1981).
51.
Pieau C, Dorizzi M, Richard-Mercier N, Desvages G: Sexual differentiation of gonads as a function of temperature in the turtle Emys orbicularis: endocrine function, intersexuality and growth. J Exp Zool 281:400-408 (1998).
52.
Pokorna M, Kratochvíl L: Phylogeny of sex-determining mechanisms in squamate reptiles: are sex chromosomes an evolutionary trap? Zool J Linn Soc 156:168-183 (2009).
53.
Pokorna M, Giovannotti M, Kratochvil L, Kasai F, Trifonov VA, et al: Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence. Chromosoma 120:455-468 (2011).
54.
Polli E: The isolated chromosomes from erythrocytes of various species of vertebrates. Chromosoma 4:621-629 (1950).
55.
Quinn AE, Georges A, Sarre SD, Guarino F, Ezaz T, Graves JAM: Temperature sex reversal implies sex gene dosage in a reptile. Science 316:411 (2007).
56.
Rando HM, Farré M, Robson MP, Won NB, Johnson JL, et al: Construction of red fox chromosomal fragments from the short-read genome assembly. Genes 9:308 (2018).
57.
Rhen T, Lang JW: Among-family variation for environmental sex determination in reptiles. Evolution 52:1514-1520 (1998).
58.
Rollinat R: La vie des reptiles de la France centrale (Librairie Delagrave, Paris 1934).
59.
Rovatsos M, Praschag P, Fritz U, Kratochvil L: Stable Cretaceous sex chromosomes enable molecular sexing in softshell turtles (Testudines: Trionychidae). Sci Rep 7:42150 (2017).
60.
Salas AE, Imhof A, Amavet P: Estudios citogenéticos en la tortuga pintada (Trachemys dorbigni: Testudinata, Emydidae). Nat Neotrop 45:9 (2014).
61.
Sarre SD, Georges A, Quinn A: The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. Bioessays 26:639-645 (2004).
62.
Seabright M: A rapid banding technique for human chromosomes. Lancet 298:971-972 (1971).
63.
Shaffer HB, Minx P, Warren DE, Shedlock AM, Thomson RC, et al: The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. Genome Biol 14:R28 (2013).
64.
Sites JW Jr, Bickham JW, Haiduk MW, Iverson JB: Banded karyotypes of six taxa of kinosternid turtles. Copeia 1979:692-698 (1979).
65.
Sumner A: A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75:304-306 (1972).
66.
The Tree of Sex Consortium: Tree of Sex: A database of sexual systems. Sci Data 1:140015 (2014).
67.
Valenzuela N, Adams DC: Chromosome number and sex determination coevolve in turtles. Evolution 65:1808-1813 (2011).
68.
Valenzuela N, Lance V: Temperature-dependent sex determination, in Deeming DC (ed): Reptilian Incubation: Environment, Evolution and Behaviour, pp 211-227 (Nottingham University Press, Nottingham 2004).
69.
Valenzuela N, Adams DC, Janzen FJ: Pattern does not equal process: exactly when is sex environmentally determined? Am Nat 161:676-683 (2003).
70.
Valenzuela N, Badenhorst D, Montiel EE, Literman R: Molecular cytogenetic search for cryptic sex chromosomes in painted turtles Chrysemys picta. Cytogenet Genome Res 144:39-46 (2014).
71.
Van Brink JM: L'expression morphologique de la digamétie chez les sauropsidés et les monotrèmes. Chromosoma 10:1-72 (1959).
72.
Wickbom T: Cytological studies on Dipnoi, Urodela, Anura, and Emys. Hereditas 31:241-346 (1945).
73.
Zaborski P, Dorizzi M, Pieau C: HY antigen expression in temperature sex-reversed turtles (Emys orbicularis). Differentiation 22:73-78 (1982).
74.
Zaborski P, Dorizzi M, Pieau C: Temperature-dependent gonadal differentiation in the turtle Emys orbicularis: concordance between sexual phenotype and serological HY antigen expression at threshold temperature. Differentiation 38:17-20 (1988).
75.
Zachos J, Pagani M, Sloan L, Thomas E, Billups K: Trends, rhythms, and aberrations in global climate 65 Ma to present. Science 292:686-693 (2001).
76.
Zuffi M, Gariboldi A: Sexual dimorphism of the European pond terrapin, Emys orbicularis (L., 1758) from Italy. Sci Herpetol 1995:124-129 (1995).
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