The emergence of a new sex-system (XX/XY1Y2) suggests a species complex in the “monotypic” rodent Oecomys auyantepui (Rodentia, Sigmodontinae)

X-autosome translocation (XY1Y2) has been reported in distinct groups of vertebrates suggesting that the rise of a multiple sex system within a species may act as a reproductive barrier and lead to speciation. The viability of this system has been linked with repetitive sequences located between sex and autosomal portions of the translocation. Herein, we investigate Oecomys auyantepui, using chromosome banding and Fluorescence In Situ Hybridization with telomeric and Hylaeamys megacephalus whole-chromosome probes, and phylogenetic reconstruction using mtDNA and nuDNA sequences. We describe an amended karyotype for O. auyantepui (2n = 64♀65♂/FNa = 84) and report for the first time a multiple sex system (XX/XY1Y2) in Oryzomyini rodents. Molecular data recovered O. auyantepui as a monophyletic taxon with high support and cytogenetic data indicate that O. auyantepui may exist in two lineages recognized by distinct sex systems. The Neo-X exhibits repetitive sequences located between sex and autosomal portions, which would act as a boundary between these two segments. The G-banding comparisons of the Neo-X chromosomes of other Sigmodontinae taxa revealed a similar banding pattern, suggesting that the autosomal segment in the Neo-X can be shared among the Sigmodontinae lineages with a XY1Y2 sex system.

Mammals are known to exhibit a stable sex determination system, but distinct sex-autosome translocations may have triggered the separation of Theria and Prototheria (monotremes) (190 MYA) and between Eutheria (placental mammals) and Metatheria (marsupials) (166 MYA) 6 . Although the euchromatic region of the X chromosome is considered conserved among highly rearranged karyotypes of placental mammals 6 , recent investigations on Arvicolinae (Myomorpha) rodents have shown that the X chromosome has undergone several intrachromosomal rearrangements, such as centromere shifts, peri-and paracentric inversions, that were also accompanied by repetitive sequences 7 . Regardless of whether chromosomal rearrangements are the primary cause of speciation 2,8 , or whether karyotypic divergence between closely related species are a casualty of the speciation process 9,10 , the most deleterious among the speciation-linked rearrangements 11,12 are tandem translocations, reciprocal translocations 13,14 and X-autosome translocations 15,16 .
The rise of an X-autosome translocation is subordinated to the same epigenetic mechanism that guarantees dosage compensation between normal females (XX) and males (XY) by silencing one of the Xs in females 17 . In this type of event, the inactivation progress in one of the X chromosomes of females 18 spreads to the autosomal segment translocated to the X, silencing genes in the autosomal portion 19 generating deletion/duplications with deleterious effects 20 .
In rodents from the Brazilian Amazon, the XX/XY 1 Y 2 multiple sex system has been reported only in two genera from the Echimyidae family: Lonchothrix 38 and Proechimys [33][34][35] . In Lonchothrix emiliae, the multiple sex system was identified based on classic banding 38 , while in the Proechimys taxa it was detected by FISH (Fluorescence In Situ Hybridization) with whole chromosome probes (chromosome painting) from P. roberti and P. goeldii 35 . In Sigmodontinae rodents (Rodentia, Cricetidae), the Oryzomyini tribe currently comprises 29 genera and is the most diverse of the 11 tribes within the subfamily 39-41 , but multiple sex systems are acknowledged solely in representatives of the Akodontini, Phyllotini and Reithrodontini tribes: Deltamys kempi (Akodontini) exhibits a X 1 X 1 X 2 X 2 /X 1 X 2 Y sex system due to a translocation involving chromosomes 2 and Y 42 ; Salinomys delicatus (Phyllotini) shows a XY 1 Y 2 system 43 ; and Reithrodon (Reithrodontini) exhibits a XY 1 Y 2 system (Uruguay population) and a Neo-XY system (Brazil population) 44 .
In Oryzomyini, the genus Oecomys has been particularly challenging in taxonomy, distribution patterns and speciation mechanisms. Comprising 19 species to date, Oecomys has been investigated using several approaches, such as morphology, nuclear DNA (nuDNA), mitochondrial DNA (mtDNA), and cytogenetics, which have shown that some lineages correspond to species complexes 39, [45][46][47][48][49][50][51] . Oecomys auyantepui has been recognized as a monophyletic lineage and a monotypic taxon 48,52 . The species is distributed from southeastern Venezuela to north-central Brazil, in the Guiana subregion of Amazonia 39 , and exhibits two sympatric populations with distinct diploid numbers (2n) of 64 and 66 and autosomal fundamental numbers (FNa) of 110 and 114, respectively 52 . A third karyotype of 2n = 72/FNa = 80 was described 53 . In addition, an interstitial telomeric sequence (ITS) was identified at the centromeric region of the bi-armed X chromosomes in karyotypes with 2n = 64 and 66, which suggests that chromosomal rearrangements have driven the evolution of this chromosome in O. auyantepui 52 .
It is noteworthy that cytogenetics studies with Oecomys have shown a substantial diversity in 2n and FNa, ranging from 54 to 86 and from 62 to 140, respectively 39, 45,47,48,50,[52][53][54][55] 47,50 . In addition to elucidating the chromosomal rearrangements that occurred in these species, the chromosome painting analysis helped to delineate taxonomic limits, as the authors 47,50 were able to identify a hidden diversity and proposed that O. catherinae and O. paricola "eastern clade" were composed of two and three species, respectively.
Considering the evolutionary force of chromosomal rearrangements regarding speciation and diversification of species, we set out to investigate if the emergence of a new sex-system triggered the speciation process in the monotypic taxon Oecomys auyantepui.
In order to achieve this goal, we used classic cytogenetics, telomeric and HME whole chromosome probes 54 , mtDNA (mitochondrial DNA) and nuDNA (nuclear DNA) sequences. Here we discuss the chromosomal evolution of the genus, and report for the first time a multiple sex system (XX/XY 1 Y 2 ) in Oryzomyini rodents. We also compared the taxa from the present study with other species analyzed elsewhere using the same set of probes 47,50,[54][55][56][57][58][59] .
The HME 3 probe hybridizes to the short arm of the Neo-X chromosome (OAU Xp), and also hybridizes to OAU 3, and Y 2 ; the HME X chromosome hybridizes to the long arm of the Neo-X (OAU Xq).
FISH with telomeric probes showed hybridization signals at the distal regions of all chromosomes, plus a large interstitial telomeric sequence (ITS) at the centromere of the Neo-X chromosome (Fig. 3).  Table 1). In the COI topology, specimens of O. auyantepui formed a polytomy, with no resolution among most specimens, including those with similar karyotypes (Fig. 4). In the Cytb topology, the specimen N228 was recovered as the most divergent within O. auyantepui, with 5.20% of mean genetic divergence from its conspecifics. The remaining specimens formed a subclade with neither resolution nor support among most of the specimens. As in the COI topology, specimens with similar karyotypes did not nest in a subclade (Fig. 5). Finally, in the concatenated data topology, the specimen ROM 114,316 was the first one to diverge, and the specimen ROM 114,059 was recovered as sister to the 2n = 65♂/FNa = 84 specimens included in this analysis. Although these latter specimens appeared as a subclade, there was no support for that (Fig. 6).  Fig. 1a that corresponded to more than one HME homologue. Single or multiple images are presented to exhibit full coverage with HME probes on OAU chromosomes. OAU chromosomal pairs identification are shown below the chromosomes, while HME probes are shown beside the chromosomes. An asterisk indicates a centromere. HME whole chromosome probes are shown in green (FITC) and red (CY3); the counterstaining is blue (DAPI).   www.nature.com/scientificreports/ translocations, seven pericentric inversions and amplification/deletion of constitutive heterochromatin on two autosomal syntenic blocks plus the X chromosome ( Supplementary Fig. 3), with only seven syntenic blocks conserved without detectable rearrangements. Remarkably, we observed that the rearrangements that differentiate OPA cytotypes (OPA-A, OPA-B, and OPA-C) from each other are different from those responsible for the variability between OCA cytotypes (OCA-PA and OCA-RJ) and OAU. This suggests that the rearrangements mainly occurred in distinct syntenic blocks among these species (Supplementary Fig. 3). Consequently, we propose that each of these three species has evolved independently and has not followed the same path of rearrangements or the same chromosomes. Moreover, by detecting an elevated number of chromosomal rearrangements among three taxa (O. auyantepui, O. catherinae, and O. paricola) with not-so-distant 2n (from 60 to 72), we assume that the chromosomal evolution in Oecomys is more complex than previously thought. In this sense, the use of HME whole chromosome probes  www.nature.com/scientificreports/ in representatives of the main lineages of Oecomys provides a more accurate view of chromosomal evolution, and associated with detailed phylogeographic studies, is a key factor in understanding the speciation processes of this diverse and speciose group of Sigmodontinae rodents. By comparing the OAU karyotype with the other Sigmodontinae species investigated by HME whole chromosome probes (Supplementary Table 2), we identified that OAU exhibited the chromosomal signatures proposed for the Oecomys genus, namely the fragmentation of HME 1 into three blocks and the syntenic block HME (13,22)/21 50 . We also detected an exclusive trait for OAU, the syntenic block HME 26/20/18, which is different from those described for OCA (HME (9,10)/14/5, 23/19/11 and 26/11) and OPA (HME 4/19); OAU also shared and the fragmentation of HME 3 into two blocks with OPA, previously described as exclusive for this species 50 . Among the eleven chromosomal signatures proposed for the Sigmodontinae subfamily (HME 7/(9,10), 8 New sex system in Oecomys auyantepui (XX/XY 1 Y 2 ). In the genus Oecomys, sex chromosomes variation in size and morphology are frequently related to the addition/deletion of CH, which is common in rodents 62 ; the one-armed X chromosome often exhibits CH at the centromeric region, while the bi-armed form presents a heterochromatic block in the short arm and the variation in length of the Y is often due to the size of the heterochromatic block in the long arm 45,47,48,50,52 . However, the bi-armed X chromosome of O. auyantepui from the present study exhibits an euchromatic short arm, with CH at the centromere (Fig. 1b). This indicates that events other than the general addition/deletion of CH were involved in this variation, and this is supported by the HME 3 hybridization signal in the short arm of OAU Neo-X (Xp) and Y 2 (see Supplementary Fig. 1), validating the emergence of a new sex system (XX/XY 1 Y 2 ) in O. auyantepui (2n = 64♀65♂/FNa = 84). Regarding Sigmodontinae rodents, this is the first record of a multiple sex system in Oryzomyini, since this type of sex determination system has previously been reported only in representatives of Akodontini (X 1 X 1 X 2 X 2 /X 1 X 2 Y) 42 , Phyllotini (XY 1 Y 2 ) 43 and Reithrodontini (XY 1 Y 2 and Neo-XY) 44 tribes.

Phylogenetic analysis.
In order to understand the Neo-X origin in O. auyantepui, we constructed a dendrogram that shows a hypothetical scenario with the chromosomes involved. We made a comparative analysis among the other Sigmodontinae taxa studied with the same set of probes (Supplementary Table 2) and considered as outgroup the 16 karyotypes from the genera Akodon, Blarinomys, Necromys, Oxymycterus, Thaptomys (Akodontini), Cerradomys, Hylaeamys, and Neacomys (Oryzomyini), while as ingroup we considered the karyotypes of Oecomys catherinae, O. paricola and O. auyantepui (Oryzomyini). Except for Cerradomys, all karyotypes from the outgroup showed a HME 3 hybridization signal in one large acrocentric chromosome pair. The X was acrocentric, or bi-armed with a heterochromatic block in the short arm. Thus, we propose that: (1) the ancestral forms of the HME 3 and X chromosomes were medium acrocentrics; (2) O. catherinae maintained the autosomal ancestral form, while the X exhibited an addition of CH in the short arm; (3) the HME 3 divided by fission into two blocks of unequal size (one large and one small) before the diversification events that led to the O. paricola and O. auyantepui species; (4) in O. paricola, the HME 3 small block remained as an independent chromosome, and the X exhibited an addition of CH in the short arm; (5) in O. auyantepui, a Robertsonian translocation between the HME 3 small block with the acrocentric X formed the Neo-X chromosome (Fig. 7).
The proposal that intercalary heterochromatic blocks, telomeric repeats and/or rDNA clusters between the ancestral X and the translocated autosome served as a boundary that suppresses the X-inactivation process in the autosomal portion 16,17,[28][29][30] is in accordance with our results, which show a centromeric heterochromatic block (Fig. 1b) and a large block of ITS in the Neo-X chromosome of O. auyantepui (Fig. 3). In the rodent Mus minutoides (Muridae) immunofluorescence techniques demonstrated patterns of histone modification in three types of sex chromosomes (Y, X and a mutant X) that confirmed that the X-inactivation does not spread into the translocated autosomal portion 17 . This feature is of prime importance and guarantees the viability of this multiple sex system. In fact, several studies have described natural populations of vertebrates (e.g., bats, rodents, marsupials and ruminants) that possess this type of rearrangement 16,17,26,[30][31][32][35][36][37]63 ; in cases where such repetitive sequences are absent, deleterious effects such as poor viability and infertility are observed 15,16 . The contribution of repetitive sequences in the evolution of the X chromosome was also proposed 7 . The authors microdissected the Terricola savii X chromosome into five specific-region probes, hybridized in 20 species of Arvicolinae rodents (Myomorpha), and identified multiple intrachromosomal rearrangements, such as centromere shifts, peri-and paracentric inversions, which were related to the amplification and distribution of repetitive sequences among the Xs of distinct Arvicolinae species 7 .
Exceptions from this proposal are documented in the common ancestor of eutherian mammals, since a sexautosome translocation occurred and it may have triggered the separation between Eutheria (placental mammals) and Metatheria (marsupials) (166 MYA) 6 , with no intercalary heterochromatic block found between the X and autosomal ancestral segments. Distinct processes from the intercalary heterochromatic block would be involved in the regulation of X-autosome viability and in the suppression of deleterious effects 16 .
It is noteworthy that telomeric repeats occur at the ends of chromosomes where they provide stability, while ITS are relics of chromosomal fusions that arose during karyotype evolution 64 . Thus, although we have identified other chromosomes resulting from Robertsonian translocations in O. auyantepui (OAU 21-23, 25; Figs. 2 and 3), the ITS was found only in the Neo-X. An investigation of the effects of these telomeric sequences on gene expression, recombination and rearrangements, was made by introducing 800 bp of the telomere repeat (TTA GGG ) in the adenosine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells 65 . The main result was that gene rearrangements were greatly increased 65 . This type of chromosome instability is in accordance with the proposal that het-ITS (heterochromatic-ITS) seem to be intrinsically prone to breakage 66  www.nature.com/scientificreports/ hotspots for chromosomal rearrangements 64 . Therefore, the elimination of this sequence during chromosomal evolution could be a mechanism that provides karyotypic stability [64][65][66][67] and might explain its absence in the rearranged autosomes of O. auyantepui, while its presence in the Neo-X makes the latter prone to other chromosomal rearrangements, despite providing stability against X-inactivation of autosomal segments 16,17 . We noticed that the other cytotypes found in O. auyantepui (2n = 64, 66 and 72) from the Jatapú and Jari Rivers (Fig. 8, localities 5 and 4, respectively) exhibited distinct morphologies of the X chromosomes (medium submetacentric, large metacentric, and large submetacentric, respectively). Although they are found within a simple sex determination system (XX/XY), the X chromosomes had euchromatic short arms and repetitive sequences at the centromere, such as an ITS in the karyotypes with 2n = 64, 66 52 . Perhaps these differences in size and morphology, plus the presence of ITS, could be a clue to a more complex rearrangement of the X chromosome during its evolution.
Investigations carried out in some groups with multiple sex systems show that the chromosomal evolution in the Neo-X and/or Neo-Y gives rise to other derived systems. This is described in Stenodermatinae bats 68 , in which chromosome painting revealed that a XY 1 Y 2 system originated a Neo-XY system, due to a translocation between Y 1 and Y 2 ; this Neo-XY has then diverged into two more derivate systems: in one branch, a fission in the Neo-X created a X 1 X 1 X 2 X 2 /X 1 X 2 Y; while in the other branch, a fusion between an autosome and the Neo-Y originated a Neo-X 1 X 1 X 2 X 2 /X 1 X 2 Y. In rodents from the genus Reithrodon (Sigmodontinae, Reithrodontini), the Uruguay population exhibits a XY 1 Y 2 system, while the Brazil population shows a Neo-XY system 44 ; a hypothetical intermediate Neo-X/Y 1 Y 2 formula was the ancestor for the Uruguayan form, while the Brazilian form Numbers below idiograms correspond to the identification of the chromosomal pair; numbers beside idiograms correspond to the HME probes. The bottom box encompasses an idiogram of HME karyotype previously elaborated 58 and adapted in the present study. Each HME chromosome is shown with a single color, except the pairs (9,10), (13,22) and (16,17), which have one color each. (H) Indicates large block of constitutive heterochromatin. www.nature.com/scientificreports/ originated through a fusion between the Y 1 and Y 2 , that gave rise to the Neo-XY system 44 . The evolutionary process and specific events responsible for the variation in size and morphology of the X chromosomes in the O. auyantepui cytotypes (2n = 64, 66 and 72) 52,53 will be elucidated only after the employment of HME whole chromosome probes. Taking into consideration the phylogenetic relationships and karyotypic data within O. auyantepui, the COI phylogeny is the only one that includes all karyotyped samples for this species from both the present study (2n = 64♀65♂) and the literature (2n = 64, 66 and 72) 52,53 . Despite being considered as a valuable tool for highlighting cases requiring systematic attention among small mammal species 69 , our COI sequences of O. auyantepui formed a polytomy with only a few subclades mostly with no satisfactory support (Fig. 4). In fact, specimens of O. auyantepui with the multiple sex system grouped in a separated branch only in the concatenated Cytb + FGB-I7 topologies, but with low support in both BI and ML analyses (Fig. 6). Although our molecular analyses do not exhibit a better resolution in the terminal branches, the karyotypic information indicates that we are dealing with at least two distinct lineages. We have three karyotypes with a simple sex system XX/XY (2n = 64, 66 and 72) that could represent variation within a single lineage, while the other lineage corresponds to specimens with a multiple sex system XX/XY 1 Y 2 (2n = 64♀65♂).
The fact that four potentially karyotypic variants are in a small distribution area (Localities 1-4, Fig. 8) indicates that isolating mechanisms are operating, and the rise of a multiple sex determination system may be acting as a post-zygotic barrier between these apparently sympatric populations, since the difference in sex determination systems will generate aberrations in meiotic synapsis. Taking into consideration the low level of genetic divergence within O. auyantepui (mean p-distance 2.15%; Table 2) and the impossibility of interbreeding between these two sex systems, it is most likely that a speciation process is already on course. Similar results were  www.nature.com/scientificreports/ observed in Deltamys rodents (Sigmodontinae, Akodontini) where the two distinct sex determination systems (XX/XY and X 1 X 1 X 2 X 2 /X 1 X 2 Y) act as a reproductive barrier that is reflected in the phylogenetic divergence of 11.13-12.14% between the two divergent lineages 42 . Despite the low level of genetic divergence in O. auyantepui mentioned above, it is worthy of note that the specimen N228 exhibited 5.20% of mean genetic divergence from other conspecifics included in the Cytb topology (Fig. 5). Further studies are necessary to evaluate if this individual represents a new species, which may be either cytogenetically or morphologically distinct from its closely related O. auyantepui individuals. Some authors have proposed that changes in the sex determination system can alter behavior patterns and contribute to pre-zygotic isolation mechanisms, as described in threespine stickleback fish Gasterosteus aculeatus 70 , in which populations with XX/XY and X 1 X 1 X 2 X 2 /X 1 X 2 Y systems exhibit different courtship behavior. In the rodent Mus minutoides (Muridae), it was observed that XY females have more reproductive success than XX females and are more aggressive and have a stronger bite than XY males 71 . Thus, differences in the X chromosomes of O. auyantepui could lead to behavioral modifications and act in pre-zygotic isolation mechanisms between these two taxonomic entities.
An evaluation of the genetic (Cytb) structure of Oecomys aff. roberti (= O. tapajinus) populations observed isolated and stable populations, but with no influence from the mid-Araguaia River opposite banks 72 . Two sympatric O. paricola "eastern clade" populations (OPA-A and OPA-B) from the Belém area of endemism exhibited distinct karyotypes 50 . Both works reached similar conclusions for their respective analyzed species, proposing that Oecomys taxa can exhibit isolated populations in the absence of strong geographic barriers.
As discussed above, the fact that four potentially karyotypic variants of O. auyantepui are in a small distribution area (Localities 1-4, Fig. 8) could be explained by this type of populational structure for Oecomys taxa 50,72 . In this scenario, the rise of a rearranged chromosomal form within an isolated population could be stablished in a few generations, as rodents exhibit a tendency to be organized in demes 73,74 with low interbreeding among distinct populations 35 . The reproductive barrier would be more intense between the ancestral (XX/XY) and derived (XX/XY 1 Y 2 ) systems, as the hybrid offspring would be infertile 6 . We propose that we are dealing with a case of chromosomal speciation, triggered by the emergence of a new sex system (XX/XY 1 Y 2 ) in isolated O. auyantepui populations. Consequently, O. auyantepui is a species complex with at least two distinct lineages, which disagrees with the literature data that recover this taxon as a monotypic species sensu 48 .
A comparative analysis of the G-banding patterns among the Sigmodontinae Neo-X chromosomes of O. auyantepui (present work), Reithrodon (Reithrodontini 44 ), and Salinomys delicatus (Phyllotini 43 ) was performed and this revealed that the autosomal portion translocated in the Neo-X exhibits similar G-banding patterns in the three taxa. This suggests that the same autosomal segment is shared among these distinct lineages. Literature data show that distinct genera within groups that have multiple sex systems may also share the same autosome in the sex-autosome translocation, as in primates from genera Alouatta 75 and Aotus 76 and in bats from genera Artibeus, and Uroderma 77 , Chiroderma, Mesophylla, and Vampyriscus 68 . However, it has been shown that, in rodents from genera Proechimys (Echimyidae) and Nannomys (Muridae), species within a genus can have different autosomes translocated to the X chromosome 26,35 .
We suggest that the employment of HME whole chromosome probes in Reithrodon, Salinomys delicatus and Oecomys auyantepui with 2n = 64, 66 and 72 will elucidate the origin of Neo-X chromosomes in Sigmodontinae rodents; also, this will shed light on the evolutionary relationships among the four karyotypes of O. auyantepui, and clarify if we are dealing with simple (XX/XY) and multiple (XX/XY 1 Y 2 ) sex determination systems; or with derived lineages from the XY 1 Y 2 system.

Material and methods
Ethics. The specimens were captured using pitfall traps 78 and kept stress-free with full access to food and water until their necessary euthanasia that was performed in accordance with animal welfare guidelines established by Brazilian resolution CFMV n.1000/2012. The necessary euthanasia occurred in accordance with animal welfare guidelines established by the Animal Ethics Committee ( Cytogenetics. The metaphase chromosomal preparations were obtained from bone marrow extraction 79 . The slides containing chromosomal preparations underwent C-Banding 80 , G-Banding 81  www.nature.com/scientificreports/ HME whole chromosome probes, 21 correspond to one chromosome each (including the X chromosome), while three corresponded to two pairs of chromosomes each (HME (9,10), (13,22) and (16,17).
Image capture and analysis. We used an Olympus BX41 microscope and a CCD 1300QDS digital camera to obtain digital images from G-banded and C-banded karyotypes, which were analyzed using the GenA-SIs software v. 7.2.7.34276. The Nikon H550S microscope with a DS-Qi1Mc digital camera captured the FISH images, which were analyzed using the Nis-Elements software. The karyotypes were organized according to the literature 83 . The final images were edited using Adobe Photoshop CS6 software.

Molecular analysis.
We obtained sequences from Oecomys auyantepui samples UFPAM 2027 (Field number LTO05) and MPEG 40,457 (field number CN285). We used partial nucleotide sequences of the mitochondrial genes Cytochrome b (Cytb; 801 base pairs) and Cytochrome C Oxidase Subunit I (COI; 657 base pairs), and sequence data from nuclear beta-fibrinogen intron 7 (FGB-I7; 727 base pairs). We followed the protocols described in 49 for the extraction, amplification, and sequencing of Cytb and FGB-I7 genes; we also followed the same protocols for the COI gene, with the primers Fish F2 (TCG ACT AAT CAT AAA GAT ATC GGC AC) and Fish R2 (ACT TCA GGG TGA CCG AAG AAT CAG AA) 84 Table 1).
The alignment and editing of the Cytb, COI and concatenated (Cytb + FGB-I7) sequences were conducted using the BioEdit Sequence Alignment Editor program, version 7.0.5.3 85 with ClustalW tool. A search for the best nucleotide substitution model was made using jModeltest 2.1.6 software 86 on CIPRES platform 87 , which selected TIM2 + I + G for Cytb, GTR + I + G for COI, and TIM2 + I + G and TPM2uf + G for the concatenated Cytb and FGB-I7, respectively.
The phylogenetic reconstructions were made using Bayesian Inference (BI) and Maximum Likelihood (ML) methods. BI run in MrBayes 3.2.7 88 with four chains. The algorithm was based on 50 million generations, sampled every 1,000 generations. ML reconstruction was obtained using Garli-2.0 89 with 1,000 bootstrap replicates and majority consensus tree. The tree was displayed and edited in Figtree v. 1.4.3 90 . Branches supports were evaluated on Bayesian posterior probability in BI and by bootstrap in ML. Cytb genetic divergence values (p-distances) for O. auyantepui clades (obtained on Cytb analysis) were calculated with MEGA7 91 .

Data availability
The datasets generated and/or analysed during the current study are available in the GenBank repository (https:// www. ncbi. nlm. nih. gov/ genba nk/). All accession numbers supporting the results reported in this article are found in the main text and in the supplementary files.