Material and methods
Chromosome analyses were carried out in males and females of L. flavifrenatus, L. meridionalis, L. dilepis and L. anomalus (see details in the Appendix). Voucher specimens are deposited in the Colección Herpetológica de la Universidad Nacional del Nordeste (UNNEC), Corrientes, Argentina.
Four hours prior to animal dissection, specimens were injected intraperitoneally with 0.1% colchicine (1 ml/100 g body weight). The euthanasia method proposed by Beaupre et al. (2004) was used. Chromosomes were obtained from intestinal epithelium by dispersion of cells on the hot stage. Chromosome preparations were stained conventionally using a 10% Giemsa solution at pH 6.8. The NORs were detected using the silver staining (Ag-NOR) technique applied by Howell and Black (1980). The Ag-NORs banding staining was performed only for species which provided a sufficient number and quality of metaphase plate.
To calculate the centromeric index (CI), the arms of macrochromosomes were measured on ten metaphase plates of each specimen using the software MicroMeasure version 3.3 (Reeves and Tear, 2000). The chromosomal formula was determined following Peccinini-Seale (1981): (2n = I + II + III), with I = metacentric or submetacentric macrochromosomes, II = telocentric or subtelocentric macrochromosomes and III = microchromosomes. Chromosomes that measured around 1 micron (µm) were classified as microchromosomes.
To reconstruct karyotype evolution within the Xenodontini clade, the diploid number (from our results and from the literature) of Xenodontini species was optimized on the most recent phylogenetic hypothesis of Squamata by Pyron et al. (2013) using the parsimony criterion with TNT software (Goloboff et al., 2008).