Integrating morphological, molecular and cytogenetic data for F2 sea turtle hybrids diagnosis revealed balanced chromosomal sets.

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.

Comparative cytogenetics among Boana species (Anura, Hylidae): focus on evolutionary variability of repetitive DNA.

Boana comprises a diverse genus of Neotropical treefrogs, currently rearranged into seven taxonomic species groups. Although cytogenetic studies have demonstrated diversity in its representatives, the chromosomal mapping of repetitive DNA sequences is still scarce. In this study, Boana albopunctata, Boana faber, and Boana prasina were subjected to in situ localization of different repetitive DNA units to evaluate trends of chromosomal evolution in this genus. Boana faber and B. prasina had 2n=24 chromosomes, while B. albopunctata has 2n=22 and an intra-individual variation related to the presence/absence of one B chromosome. The location of 45S rDNA sites was different in the analyzed karyotypes, corroborating with what was found in the distinct phylogenetic groups of Boana. We presented the first description of 5S rDNA in a Boana species, which showed markings resulting from transposition/translocation mechanisms. In situ localization of microsatellite loci proved to be a helpful marker for karyotype comparison in Boana, commonly with cis accumulation in the heterochromatin. On the other hand, genomic dispersion of microsatellites may be associated with hitchhiking effects during the spreading of transposable elements. The obtained results corroborated the independent diversification of these lineages of species from three distinct phylogenetic groups of Boana.

A genomic survey of LINE elements in Pipidae aquatic frogs shed light on Rex-elements evolution in these genomes.

The transposable elements (TE) represent a large portion of anuran genomes that act as components of genetic diversification. The LINE order of retrotransposons is among the most representative and diverse TEs and is poorly investigated in anurans. Here we explored the LINE diversity with an emphasis on the elements generically called Rex in Pipidae species, more specifically, in the genomes ofXenopus tropicalis, used as a model genome in the study of anurans,the allotetraploid sister species Xenopus laevis and theAmerican species Pipa carvalhoi. We were able to identify a great diversity of LINEs from five clades, Rex1, L2, CR1, L1 and Tx1, in these three species, and the RTE clade was lost in X. tropicalis. It is clear that elements classified as Rex are distributed in distinct clades. The evolutionary pattern of Rex1 elements denote a complex evolution with independent losses of families and some horizontal transfer events between fishes and amphibians which were supported not only by the phylogenetic inconsistencies but also by the very low Ks values found for the TE sequences. The data obtained here update the knowledge of the LINEs diversity in X. laevis and represent the first study of TEs in P. carvalhoi.

Evolutionary dynamics of DIRS-like and Ngaro-like retrotransposons in Xenopus laevis and Xenopus tropicalis genomes.

Anuran genomes have a large number and diversity of transposable elements, but are little explored, mainly in relation to their molecular structure and evolutionary dynamics. Here, we investigated the retrotransposons containing tyrosine recombinase (YR) (order DIRS) in the genome of Xenopus tropicalis and Xenopus laevis. These anurans show 2n = 20 and the 2n = 36 karyotypes, respectively. They diverged about 48 million years ago (mya) and X. laevis had an allotetraploid origin (around 17-18 mya). Our investigation is based on the analysis of the molecular structure and the phylogenetic relationships of 95 DIRS families of Xenopus belonging to DIRS-like and Ngaro-like superfamilies. We were able to identify molecular signatures in the 5' and 3' noncoding terminal regions, preserved open reading frames, and conserved domains that are specific to distinguish each superfamily. We recognize two ancient amplification waves of DIRS-like elements that occurred in the ancestor of both species and a higher density of the old/degenerate copies detected in both subgenomes of X. laevis. More recent amplification waves are seen in X. tropicalis (less than 3.2 mya) and X. laevis (around 10 mya) corroborating with transcriptional activity evidence. All DIRS-like families were found in both X. laevis subgenomes, while a few were most represented in the L subgenome. Ngaro-like elements presented less diversity and quantity in X. tropicalis and X. laevis genomes, although potentially active copies were found in both species and this is consistent with a recent amplification wave seen in the evolutionary landscape. Our findings highlight a differential diversity-level and evolutionary dynamics of the YR retrotransposons in X. tropicalis and X. laevis species expanding our comprehension of the behavior of these elements in both genomes during the diversification process.

Transposable elements expression in Rhinella marina (cane toad) specimens submitted to immune and stress challenge.

Transposable elements (TEs) are important components of eukaryotic genomes and compose around 30% of the genome of Rhinella marina, an invasive toad species. Considering the possible role of TEs in the adaptation of populations, we have analyzed the expression of TEs in publicly available spleen tissue transcriptomic data generated for this species after immune and stress challenge. By analyzing the transcriptome assembly, we detected a high number of TE segments. Moreover, some distinct TE families were differentially expressed in some conditions. Our result shows that several TEs are capable of being transcribed in R. marina and they could help to generate a rapid response of specimens to the environment. Also, we can suggest that these TEs could be activated in the germinative cells as well producing variability to be selected and shaped by the evolutionary processes behind the success of this invasive species. Thus, the TEs are important targets for investigation in the context of R. marina adaptation.

Cytogenetic characterization and mapping of the repetitive DNAs in Cycloramphus bolitoglossus (Werner, 1897): More clues for the chromosome evolution in the genus Cycloramphus (Anura, Cycloramphidae).

Cycloramphus bolitoglossus (Werner, 1897) is a rare species with a low population density in the Serra do Mar region of Paraná and Santa Catarina, in southern Brazil. Currently, it has been assigned to the Near Threatened (NT) category in the Brazilian List of Endangered Animal Species. Here, we described the karyotype of this species for the first time and investigated the patterns of some repetitive DNA classes in the chromosomes using molecular cytogenetic approaches. We isolated, sequenced and mapped the 5S rDNA and the satellite DNA PcP190 of C. bolitoglossus, as well as mapped the telomeric sequences and seven microsatellites motifies [(GA)15, (CA)15, (GACA)4, (GATA)8, (CAG)10, (CGC)10, and (GAA)]10. Cycloramphus bolitoglossus has 2n = 26 chromosomes and a fundamental number (FN) equal to 52, with a highly conserved karyotype compared to other genus members. Comparative cytogenetic under the phylogenetic context of genus allowed evolutionary interpretations of the morphological changes in the homologs of pairs 1, 3, and 6 along with the evolutionary history of Cycloramphus. Two subtypes of 5S rDNA type II were isolated in C. bolitoglossus genome, and several comparative analysis suggests mixed effects of concerted and birth-and-death evolution acting in this repetitive DNA. The 5S rDNA II subtype "a" and "b" was mapped on chromosome 1. However, their different position along chromosome 1 provide an excellent chromosome marker for future studies. PcP190 satellite DNA, already reported for species of the families Hylidae, Hylodidae, Leptodactylidae, and Odontophrynidae, is scattered throughout the C. bolitoglossus genome, and even non-heterochromatic regions showed hybridization signals using the PcP190 probe. Molecular analysis suggests that PcP190 satellite DNA exhibit a high-level of homogenization of this sequence in the genome of C. bolitoglossus. The PcP190 satDNA from C. bolitoglossus represents a novel sequence group, compared to other anurans, based on its hypervariable region. Overall, the present data on repetitive DNA sequences showed pseudogenization evidence and corroborated the hypothesis of the emergence of satDNA from rDNA 5S clusters. These two arguments that reinforced the importance of the birth-and-death evolutionary model to explain 5S rDNA patterns found in anuran genomes.

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences.

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)n, (GA)n, (CAG)n, (GATA)n, (GAA)n, (CGC)n and (GACA)n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)n, (GA)n, (CAG)n and (GATA)n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.

Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae).

The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.

Recurrent variation in the active NOR sites in the monkey frogs of the genus Pithecopus Cope, 1866 (Phyllomedusidae, Anura).

Treefrogs of the genus Pithecopus Cope, 1866 exhibit expressive chromosomal homogeneity which contrasts with a high variation frequency of the nucleolus organizer region (NOR) related to the group. Currently, the genus contains eleven species and no chromosomal data are available on P. palliatus Peters, 1873, P. ayeaye Lutz, 1966 and P. megacephalus Miranda-Ribeiro, 1926. Here, we describe the karyotypes of these three species based on Giemsa staining, C-banding, silver impregnation and in situ hybridization (FISH). We were also analyze the evolutionary dynamic of the NOR-bearing chromosome in species of genus under a phylogenetic view. The results indicate that P. palliatus, P. ayeaye, and P. megacephalus have similar karyotypes, which are typical of the genus Pithecopus. In P. palliatus the NOR was detected in the pericentromeric region of pair 9p whereas in P. ayeaye and P. megacephalus we report cases of the multiple NOR sites in karyotypes. In P. ayeaye the NOR was detected in the pericentromeric region of pair 9p in both homologues and additional sites was detected in pairs 3q, 4p, and 8q, all confirmed by FISH experiments. Already in P. megacephalus the NOR sites were detected in pericentromeric region homologues of pair 8q and additionally in one chromosome of pair 13q. A comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs and the position of the NORs sites on these chromosomes. We hypothesized that this feature is a result of a polymorphic condition present in the common ancestor of Pithecopus. In such case, the lineages derived from polymorphic ancestor have reached fixation independently after divergence of lineages, resulting in a high level of homoplasy observed in this marker. Our findings help to fill the gaps in the understanding of the karyotype of the genus Pithecopus and reinforce the role of the evolutionary dynamics of the rDNA genes in karyotype diversification in this group.

Chromosome Spreading of the Retrotransposable Rex-3 Element and Microsatellite Repeats in Karyotypes of the Ancistrus Populations.

The repetitive DNAs are the expressive substrate to genomic evolution and directly related to chromosomal diversification in eukaryote, including fishes. Ancistrus is an interesting group for studies about interplay between repetitive DNA and karyotype evolution, given its extensive chromosomal variation. In this study, we aimed to understand the evolutionary dynamics in genome of the distinct Ancistrus populations of the Paraná basin to the contribution of three classes of repetitive DNA sequences. Nucleotide sequence was isolated, characterized the nonlong terminal repeat (non-LTR) retrotransposable Rex-3, and evaluated the chromosomal organization in the Ancistrus populations. In addition, we also mapped microsatellite repeats on chromosomes. A high conserved level of the Rex-3 element was presented in Ancistrus genome sequences to record in other fish genomes. We recognized also five domains conserved in the amino acid sequence presumed from nucleotide sequence of the reverse transcriptase fragment, which indicates that it is potentially active in the genome. The physical mapping using the Rex-3 as probe revealed signals scattered throughout the chromosomes of all the Ancistrus specimens, while the microsatellite probes hybridized preferentially in the subterminal and interstitial regions. Physical mapping also reveals interplay between these two classes of repetitive DNA in some chromosome pairs. Besides, the spreading of Rex-3 signals in adjacencies of the 5S recombinant DNA (rDNA) sites could reflect their role in the dispersion of these regions. Our findings provide important insights into the mechanisms of karyotype diversification in the genus Ancistrus, which involve these repetitive sequences.

Frequência da Mutação Mitocondrial C1494T, do Gene MT-RNR1, em Coortes de Ouvintes e Surdos Brasileiros / Frequency of Mitochondrial Mutation C1494T, of the Gene MT-RNR1, in Cohorts of Brazilian Hearing and Deaf People

Fatores ambientais e genéticos, ou mesmo ainteração destes, podem ser causadores da surdez sendo amesma o déficit sensorial mais frequente em humanos, sendoestimado que um a cada 500 recém-nascidos tenha perdaauditiva bilateral permanente, e que esta incidência aumentepara 3,5:1000 em indivíduos na adolescência. Determinadosfatores ambientais podem ser potencializados, quandodeterminadas mutações estão presentes no genoma. Éamplamente conhecida que a utilização de antibióticosaminoglicosídeos pode originar a surdez; especialmentequando o sujeito possuir mutações mitocondriaispredisponentes. A mutação C1494T do gene mitocondrial MTRNR1,o qual é responsável pela formação da subunidade12S do RNA ribossômico (12S rRNA), tem sido descritaassociada à perda auditiva pelo uso de antibióticosaminoglicosídeos. Objetivo: É o de investigar a prevalênciada mutação mitocondrial C1494T do gene MT-RNR1 em coortesde sujeitos ouvintes e surdos, das regiões Norte e Nordeste doEstado do Paraná, Brasil. Material e Método: Foraminvestigados 80 sujeitos ouvintes saudáveis e 80 surdos queapresentavam surdez pré-lingual, não sindrômica, de etiologiadesconhecida, sendo casos isolados dentro das famílias. Foiutilizada a técnica de PCR para amplificação da região, deum fragmento de 936 pb, na posição 1494, do gene MT-RNR1seguido de digestão pela enzima de restrição Hph I. Resultados:A mutação C1494T não foi encontrada nas amostras de sujeitosouvintes e de surdos. Conclusões: A mutação C1494T do geneMT-RNR1 pode ser considerada ausente ou rara nas populaçõesde sujeitos ouvintes e surdos das regiões Norte e Nordeste doestado do Paraná, Brasil...

Deafness is the most common sensory deficit inhumans, which may be caused by environmental and geneticfactors or even by a combination of both. It has been estimatedthat one in every 500 newborns has bilateral permanent hearingloss, and this incidence increases to 3.5:1,000 individuals inadolescence. Certain environmental factors may be enhancedwhen specific mutations are present in the genome. It is widelyknown that the use aminoglycoside antibiotics may lead tohearing loss, especially if the individual has predisposingmitochondrial mutations. Mutation C1494T of themitochondrial gene MT-RNR1 which causes the formation ofthe subunit 12S of the ribosome RNA (12S rRNA), has beenassociated with hearing loss due to the use of aminoglycosideantibiotics. Objective: This study investigates the occurrenceof mitochondrial mutation C1494T in the gene MT-RNR1 incohorts of hearing and deaf people in northern and northwesternregions of Paraná state, Brazil. Material and Methods: Eightyhearing people and eighty deaf people were analyzed. Thelatter were characterized as pre-lingual, non-syndromic, ofunknown etiology, being isolated cases within the family. PCRtechnique was used for amplification of the fragment 936 bpof the gene MT-RNR1 at position 1494, followed by digestionwith restriction enzyme Hph I. Results: Mutation C1494T wasnot detected in the samples of hearing and deaf people.Conclusions: Mutation C1494T of the gene MT-RNR1 may beabsent or rare in populations of hearing and deaf people innorthern and northwestern regions of Paraná state, Brazil...