DOI: 10.5061/dryad.6m905qg5g
title: Supporting information for: Integrating morphological, molecular, and cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced chromosomal sets

description: Hybridization could be considered part of the evolutionary history of many
 species. The hybridization among sea turtle species on the Brazilian coast
 is atypical and occurs where nesting areas and reproductive seasons
 overlap. Integrated analysis of morphology and genetics is still scarce,
 and there is no evidence of the parental chromosome set distribution in
 sea turtle interspecific hybrids. In this study, chromosome markers
 previously established for pure sea turtle species were combined with
 morphological and molecular analyses aiming to recognize genetic
 composition and chromosome sets in possible interspecific hybrids
 initially identified by mixed morphology. The data showed that one hybrid
 could be an F2 individual among Caretta caretta × Eretmochelys
 imbricata × Chelonia mydas, and another is resulting from backcross
 between C. caretta × Lepidochelys olivacea. Native alleles of different
 parental lineages were reported in the hybrids, and, despite this, it was
 verified that the hybrid chromosome sets were still balanced. Thus, how
 sea turtle hybridism can affect genetic features in the long term is a
 concern, as the implications of the crossing-over in hybrid chromosomal
 sets and the effects on genetic function are still unpredictable.
description: # Supporting information for: Integrating morphological, molecular, and
 cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced
 chromosomal sets Caroline Regina Dias Machado, Matheus Azambuja, Camila
 Domit, Gabriel Fraga da Fonseca, Larissa Glugoski, Camilla Borges Gazolla,
 Rafael Bonfim de Almeida, Marcela Baer Pucci, Thais Torres Pires, Viviane
 Nogaroto and Marcelo Ricardo Vicari Correspondence: vicarimr@uepg.br This
 supporting information includes: * Table S1 * Table S2 * Table S3 * Table
 S4 * Fig. S1 * Fig. S2 * Fig. S3 * Fig. S4 * Fig. S5 **Table S1** To
 perform this study we used biological samples obtained from four sea
 turtle species that nidificate in different places on the Brazilian coast
 (Chelonia mydas, N = 27: 1 female and 26 juveniles that is, sex
 unidentified; Caretta caretta, N = 12: 2 males, 5 females, and 5
 juveniles; Eretmochelys imbricata, N = 6: 2 females and 4 juveniles; and,
 Lepidochelys olivacea, N = 6: 1 male and 5 juveniles). The blood samples
 from these individuals were used in the methods. The sea turtle samples
 were obtained from the different places that work with sea turtle
 rehabilitation or from wildlife by-catch expeditions and during the
 process of nidification. The place where each sea turtle was sampled was
 summarized in Table S1. **Table S2** Sea turtles species-specific alleles
 (pure lines) corresponding to mitochondrial DNA sequences (mtDNA) and
 nuclear DNA (nucDNA) were mined from the GenBank database. Thus, the
 accession number of the mtDNA 12S and 16S, and nucDNA of brain-derived
 neurotrophic factor - BDNF, oocyte maturation factor - Cmos, and oRNA
 fingerprint protein 35 gene - R35 were provided in Table S2. These
 sequences were used in our molecular data analysis comparatively to the
 gene sequences obtained from the hybrids in the study. **Table S3** Table
 S3 summarizes the diversity indices for each analyzed gene analyzed in
 this study (12S, 16S, BDNF, Cmos, and R35). Thus, the table shows detailed
 data on the number of sequences, haplotypes, polymorphic sites, haplotype
 diversity, and nucleotide diversity. **Table S4** Partial 5S rDNA
 sequences were obtained from the pure species of C. mydas, C. caretta, E.
 imbricata, and L. olivacea. The sequences identity were checked in
 different databases, including BLASTn algorithm and the Rfam. The data
 confirm the sequences belonging to the 5S rDNA gene in each sea turtle
 species. **Fig. S1** In this file, we provide representative idiograms
 from G-band method for Chelonia mydas, Caretta caretta, Eretmochelys
 imbricata, and Lepidochelys olivacea. The readers can use these images to
 verify the interspecific differences in chromosome morphology among
 chromosome pairs 4, 5, 7, and 12. All data used to draw the ideogram were
 according to images published by Machado et al. (2020a). **Fig. S2** In
 this file, we provide representative idiograms from repetitive DNA units
 chromosomal locations for Chelonia mydas, Caretta caretta, Eretmochelys
 imbricata, and Lepidochelys olivacea. The readers can use these images to
 verify the interspecific differences in the number and chromosome location
 of the rDNA and microsatellite sites. All data used to draw the ideogram
 were according to images published by Machado et al. (2020b). **Fig. S3**
 Metaphases used in Figure 4 edition are provided in this file. Here, the
 reader can find the metaphases from *C. mydas* (CMY, images a-f), *C.
 caretta* (CCA, images g-l), *E. imbricata* (EIM, images m-r), and *L.
 olivacea* (LOL, images s-x) pure lineages subjected to FISH using 5S (red
 signals) and 18S rDNAs (green signals) probes. The images are sequentially
 presented, i.e., DAPI, rDNA probe, and merged image. **Fig. S4**
 Metaphases from the Hybrid A used in Figure 5 karyotyping. The images are
 ordered according to what appears in Figure 5. For fluorescence in situ
 images, all layers are provided: DAPI, red channel, green channel
 (exclusively to 18S rDNA probing), and merged images. Excessive background
 may have been deleted in the final image for clearance. **Fig. S5**
 Metaphases from the Hybrid B used in Figure 6 karyotyping. The images are
 ordered according to what appears in Figure 6. For fluorescence in situ
 images, all layers are provided: DAPI, red channel, green channel
 (exclusively to 18S rDNA probing), and merged images. Excessive background
 may have been deleted in the final image for clearance. **References** *
 Machado CRD, Glugoski L, Domit C, Pucci MB, Goldberg DW, Marinho LA et al.
 (2020a) Comparative cytogenetics of four sea turtle species (Cheloniidae):
 G-banding pattern and in situ localization of repetitive DNA units.
 Cytogenet Genome Res 160:531-538 * Machado CRD, Domit C, Pucci MB, Gazolla
 CB, Glugoski L, Nogaroto V et al. (2020b) Heterochromatin and
 microsatellites detection in karyotypes of four sea turtle species:
 Interspecific chromosomal differences. Genet Mol Biol 43:e20200213