The good, the bad and the boa: an unexpected new species of a true boa revealed by morphological and molecular evidence

Snakes of the genus Boa are outstanding elements of the New World biota with a broad sociological influence on pop culture. Historically, several taxa have been recognized in the past 300 years, being mostly described in the early days of binomial nomenclature. As a rule, these taxa were recognized based on a suite of phenotypic characters mainly those from the external morphology. However, there is a huge disagreement with respect to the current taxonomy and available molecular phylogenies. In order to reconcile both lines of evidence, we investigate the phylogenetic reconstruction (using mitochondrial and nuclear genes) of the genus in parallel to the detailed study of some phenotypic systems from a geo graphically representative sample of the cis-Andean mainland Boa constrictor. We used cyt b only (744bp) from 73 samples, and cyt-b, ND4, NTF3, and ODC partial sequences (in a total of 2305 bp) from 35 samples, comprising nine currently recognized taxa (species or subspecies), to infer phylogenetic relationships of boas. Topologies recovered along all the analyses and genetic distances obtained allied to a unique combination of morphological traits (colouration, pholidosis, meristic, morphometric, and male genitalia features) allowed us to recognize B. constrictor lato sensu, B. nebulosa, B. occidentalis, B. orophias and a dis tinct lineage from the eastern coast of Brazil, which we describe here as a new species, diagnosing it from the previously recognized taxa. Finally, we discuss the minimally neces sary changes in the taxonomy of Boa constrictor complex; the value of some usually disre garded phenotypic character system; and we highlight the urgency of continuing environmental policy to preserve one of the most impacted Brazilian hotspots, the Atlantic Forest, which represents an ecoregion full of endemism.

Species limits and recent diversification of Cerradomys (Sigmodontinae: Oryzomyini) during the Pleistocene

Cerradomys is a genus of the tribe Oryzomyini with eight species currently recognized, and a controversial taxonomy. These species are mainly distributed in the South America dry diagonal, but some species extend into Atlantic Forest, reaching the coastal sandy plains known as Restingas. This study aimed to address species limits and patterns of diversification of Cerradomys species. For this purpose, we performed cytogenetic and molecular analyses (phylogeny, coalescent species delimitation, barcoding, and divergence times estimation) using multiple mitochondrial and nuclear markers on a comprehensive sampling, representing all nominal taxa reported so far. Chromosomal information was a robust marker recognizing eight Cerradomys species. Reciprocal monophyly was recovered for all the species, except for C. subflavus. These results together with coalescent analyses recovered eight species as the most congruent species delimitation scenario for the genus (mean Ctax: 0.72). Divergence time estimates revealed that Cerradomys’ diversification occurred about 1.32 million years ago (Mya) during the Pleistocene. Although our results conservatively support the eight Cerradomys species described so far, different lines of evidence suggest that C. langguthi and C. subflavus could potentially be species-complexes. We discussed this scenario in the light of multiple evolutionary processes within and between species and populations, since Cerradomys comprises a species group with recent diversification affected by Pleistocene climatic changes and by the complex biogeographic history of South America dry diagonal. This work supports that the diversity of Cerradomys is underestimated and reiterates that interdisciplinary approaches are mandatory to identify small rodent species properly, and to unhide cryptic species.

Extensive genomic reshuffling involved in the karyotype evolution of genus Cerradomys (Rodentia: Sigmodontinae: Oryzomyini)

Rodents of the genus Cerradomys belong to the tribe Oryzomyini and present high chromosome variability with diploid numbers ranging from 2n=46 to 60. Classical cytogenetics and fluorescence in situ hybridization (FISH) with telomeric and whole chromosome-specific probes of another Oryzomyini, Oligoryzomys moojeni (OMO), were used to assess the karyotype evolution of the genus. Results were integrated into a molecular phylogeny to infer the hypothetical direction of chromosome changes. The telomeric FISH showed signals in telomeres in species that diverged early in the phylogeny, plus interstitial telomeric signals (ITS) in some species from the most derived clades (C. langguthi, C. vivoi, C. goytaca, and C. subflavus). Chromosome painting revealed homology from 23 segments of C. maracajuensis and C. marinhus to 32 of C. vivoi. Extensive chromosome reorganization was responsible for karyotypic differences in closely related species. Major drivers for genomic reshuffling were in tandem and centric fusion, fission, paracentric and pericentric inversions or centromere repositioning. Chromosome evolution was associated with an increase and decrease in diploid number in different lineages and ITS indicate remnants of ancient telomeres. Cytogenetics results corroborates that C. goytaca is not a junior synonym of C. subflavus since the karyotypic differences found may lead to reproductive isolation.

Evidence of facultative parthenogenesis in three Neotropical pitviper species of the Bothrops atrox group

We examined four suspected cases of facultative parthenogenesis in three species of a neotropical lineage of pitvipers of the Bothrops atrox group. Reproduction without mating was observed in captive females of B. atrox, B. moojeni and B. leucurus housed alone for seven years (the two former species) and nine years (the latter one). In addition to the observation of captivity data, we investigated molecularly this phenomenon using heterologous microsatellites. DNA was extracted from the mothers’ scales or liver, from embryo and newborn fragments, and yolked ova. Four of the microsatellites showed good amplification using Polymerase Chain Reaction and informative band segregation patterns among each mother and respective offspring. Captivity information, litter characteristics (comparison of the number of newborns, embryos and yolked ova) and molecular data altogether agreed with facultative parthenogenesis predictions in at least three out of the four mothers studied: B. atrox (ID#933) was heterozygous for three out of the four markers, and the sons S1 and S2 were homozygous; B. moojeni (BUT86) was heterozygous for two out of four markers, offspring S1, S3, E2, and E4, and O1 to O6 were homozygous; and B. leucurus (MJJS503) was heterozygous for three out of four markers, and son E1 and O1 were homozygous. B. moojeni (BUT44) was homozygous for all loci analyzed in the mother and offspring, which although not informative is also consistent with parthenogenesis. This study represents the first molecular confirmation of different pitviper species undergoing facultative parthenogenesis among Neotropical endemic snakes.

A review of Euryoryzomys legatus (Rodentia, Sigmodontinae): morphological redescription, cytogenetics, and molecular phylogeny

The taxonomic history of Euryoryzomys legatus has been complex and controversial, being either included in the synonymy of other oryzomyine species or considered as a valid species, as in the most recent review of the genus. Previous phylogenetic analyses segregated E. legatus from E. russatus, its putative senior synonym, but recovered it nested within E. nitidus. A general lack of authoritative evaluation of morphological attributes, details of the chromosome complement, or other data types has hampered the ability to choose among alternative taxonomic hypotheses, and thus reach a general consensus for the status of the taxon. Herein we revisit the status of E. legatus using an integrated approach that includes: (1) a morphological review, especially centered on specimens from northwestern Argentina not examined previously, (2) comparative cytogenetics, and (3) phylogenetic reconstruction, using mitochondrial genes. Euryoryzomys legatus is morphologically and phylogenetically distinct from all other species-level taxa in the genus, but its 2n=80, FN=86 karyotype is shared with E. emmonsae, E. nitidus, and E. russatus. Several morphological and morphometric characters distinguish E. legatus from other species of Euryoryzomys, and we provide an amended diagnosis for the species. Morphological characters useful in distinguishing E. legatus from E. nitidus, its sister taxon following molecular analyses, include: larger overall size, dorsal fur with a strong yellowish brown to orange brown tinge, flanks and cheeks with an orange lateral line, ventral color grayish-white with pure white hairs present only on the chin, presence of a thin blackish eye-ring, tail bicolored, presence of an alisphenoid strut and a well-developed temporal and lambdoid crests in the skull, and a labial cingulum on M3. Molecular phylogenetic analyses recovered E. legatus as a monophyletic group with high support nested within a paraphyletic E. nitidus; genetic distances segregated members of both species, except for an exemplar of E. nitidus. Our integrated analyses reinforce E. legatus as a full species, but highlight that E. macconnelli, E. emmonsae, and E. nitidus each may be a species complex and worthy of systematic attention. Finally, we also evaluated the chromosome evolution of the genus within a phylogenetic context.

Genetic variability, management, and conservation implications of the critically endangered Brazilian pitviper Bothrops insularis

Information on demographic, genetic, and environmental parameters of wild and captive animal populations has proven to be crucial to conservation programs and strategies. Genetic approaches in conservation programs of Brazilian snakes remain scarce despite their importance for critically endangered species, such as Bothrops insularis, the golden lancehead, which is endemic to Ilha da Queimada Grande, coast of São Paulo State, Brazil. This study aims to (a) characterize the genetic diversity of ex situ and in situ populations of B. insularis using heterologous microsatellites; (b) investigate genetic structure among and within these populations; and (c) provide data for the conservation program of the species. Twelve informative microsatellites obtained from three species of the B. neuwiedi group were used to access genetic diversity indexes of ex situ and in situ populations. Low‐to‐medium genetic diversity parameters were found. Both populations showed low—albeit significant—values of system of mating inbreeding coefficient, whereas only the in situ population showed a significant value of pedigree inbreeding coefficient. Significant values of genetic differentiation indexes suggest a small differentiation between the two populations. Discriminant analysis of principal components (DAPC) recovered five clusters. No geographic relationship was found in the island, suggesting the occurrence of gene flow. Also, our data allowed the establishment of six preferential breeding couples, aiming to minimize inbreeding and elucidate uncertain parental relationships in the captive population. In a conservation perspective, continuous monitoring of both populations is demanded: it involves the incorporation of new individuals from the island into the captive population to avoid inbreeding and to achieve the recommended allelic similarity between the two populations. At last, we recommend that the genetic data support researches as a base to maintain a viable and healthy captive population, highly genetically similar to the in situ one, which is crucial for considering a reintroduction process into the island.

Caracterização da diversidade genética de bothrops insularis baseada em sequências de microssatélites

Bothrops insularis é uma espécie endêmica da Ilha da Queimada Grande, São Paulo, considerada ameaçada de extinção. Em decorrência da sua vulnerabilidade, são mantidas cinco populações de B. insularis em plantéis de quatro institutos de ensino e pesquisa para fins de reprodução e conservação ex situ. Nos últimos anos, essa espécie tem sido alvo de estudos relacionados à história natural, evolução, ecologia e composição de seu veneno. Entretanto, existem poucos relatos sobre a diversidade genética das populações existentes, informação crucial para a condução e manutenção de programas de conservação. Neste contexto, o presente estudo objetivou caracterizar e comparar a diversidade da população ex situ mantida no Laboratório Especial de Ecologia e Evolução do Instituto Butantan com alguns exemplares da população in situ, por meio de 12 marcadores microssatélites heteroespecíficos dese nvolvidos para três espécies de Bothrops do grupo neuwiedi, bem como testar a hipótese de estruturação e divergência genética inter e intrapopulacional. Os resultados mostraram que as populações apresentaram baixos/médios índices de diversidade genética, sendo a maior diversidade genética observada na população ex situ. Ambas as populações apresentam baixos, porém significativos, graus de endogamia de grau de parentesco, como esperado para populações de espécies ameaçadas de extinção e com restrita distribuição geográfica. Adicionalmente, foram recuperados valores negativos de endogamia de sistema de acasalamento, possivelmente relacionado a existência de processos que evitem a endogamia nas populações. As divergências interpopulacionais das frequências alélicas e a existência de alelos privados observados podem estar relacionados a efeitos de deriva genética, eventos demográficos recentes e/ou a ausência de amostras de uma região da ilha. Apesar dessas divergências, nenhum dos índices de diferenciação populacional apresentou valores significativos e, portanto, não há indícios de diferenciação genética entre as populações. Quando as amostras são estudadas em conjunto, as análises de distância genética e de componentes discriminantes principais (DAPC) sugerem a existência de cinco agrupamentos genéticos, porém tais agrupamentos não recuperam as relações geográficas dos espécimes da população in situ, e não são totalmente explicados por relações de consanguinidade. Além das informações inéditas apresentadas no presente estudo, consideramos importante que abordagens futuras utilizem novos marcadores moleculares (como SNPs e/ou novos microssatélites) para a melhor compreensão da diversidade genética e para investigar a estrutura genética populacional de Bothrops insularis.

Systematics of the genus Oecomys (Sigmodontinae: Oryzomyini): molecular phylogenetic, cytogenetic and morphological approaches reveal cryptic species

Oecomys is a genus of Neotropical arboreal rodents composed of 17 species with diploid number ranging from 2n = 54 to 86. Despite this high taxonomic and karyotypic diversity, the species-level systematics remains uncertain. We investigated the phylogenetic relationships and species delimitation of Oecomys using multiple approaches based on cytogenetic, molecular (mtDNA and nuDNA sequences) and morphological data sets. Sampling included 73 individuals from 25 localities in Amazonia, Cerrado, Pantanal and the Atlantic Forest, as well as 128 DNA sequences from GenBank. Molecular species boundaries associated with karyotype, morphological characters and geographic distribution led us to recognize 15 distinct lineages in Oecomys. These include five major well-supported clades composed of O. bicolor, O. catherinae, O. cleberi, O. mamorae, O. paricola and O. roberti, which were hypothesized as species complexes with at least eight putative new taxa. Three new karyotypes are also reported for the genus: 2n = 54 (FN = 54), 2n = 62 (FN = 62) and 2n = 70 (FN = 74). Sympatry of up to four species with different diploid numbers recovered in distinct clades illustrates the complex evolutionary history in Oecomys. These data highlight the importance of combining cytogenetic, morphological and geographic information along with molecular coalescent analyses in developing species delimitation scenarios.

Systematics of the genus Oecomys (Sigmodontinae: Oryzomyini): molecular phylogenetic, cytogenetic and morphological approaches reveal cryptic species

Oecomys is a genus of Neotropical arboreal rodents composed of 17 species with diploid number ranging from 2n = 54 to 86. Despite this high taxonomic and karyotypic diversity, the species-level systematics remains uncertain. We investigated the phylogenetic relationships and species delimitation of Oecomys using multiple approaches based on cytogenetic, molecular (mtDNA and nuDNA sequences) and morphological data sets. Sampling included 73 individuals from 25 localities in Amazonia, Cerrado, Pantanal and the Atlantic Forest, as well as 128 DNA sequences from GenBank. Molecular species boundaries associated with karyotype, morphological characters and geographic distribution led us to recognize 15 distinct lineages in Oecomys. These include five major well-supported clades composed of O. bicolor, O. catherinae, O. cleberi, O. mamorae, O. paricola and O. roberti, which were hypothesized as species complexes with at least eight putative new taxa. Three new karyotypes are also reported for the genus: 2n = 54 (FN = 54), 2n = 62 (FN = 62) and 2n = 70 (FN = 74). Sympatry of up to four species with different diploid numbers recovered in distinct clades illustrates the complex evolutionary history in Oecomys. These data highlight the importance of combining cytogenetic, morphological and geographic information along with molecular coalescent analyses in developing species delimitation scenarios.

Advances in cytogenetics of Brazilian rodents: cytotaxonomy, chromosome evolution and new karyotypic data

Rodents constitute one of the most diversified mammalian orders. Due to the morphological similarity in many of the groups, their taxonomy is controversial. Karyotype information proved to be an important tool for distinguishing some species because some of them are species-specific. Additionally, rodents can be an excellent model for chromosome evolution studies since many rearrangements have been described in this group. This work brings a review of cytogenetic data of Brazilian rodents, with information about diploid and fundamental numbers, polymorphisms, and geographical distribution. We point out that, even with the recent efforts on cytogenetic studies in this group, many species lack karyotypic data. Moreover, we describe for the first time the karyotype of Carterodon sulcidens (Lund, 1838) (Family Echimyidae), a new fundamental number for an undescribed species of Neacomys Thomas, 1900 (Family Cricetidae, Subfamily Sigmodontinae), and illustrate the karyotype of a Brazilian specimen of Mus musculus Linnaeus, 1758 (Family Muridae). This review compiles the cytogenetic data on Brazilian rodents reported in the last three decades, after the last revision published in 1984, including synonyms, chromosomal variations, and geographic distribution. Additionally, it also reinforces that Brazilian biodiversity is still poorly known, considering the new data reported here.

Noteworthy records of Ixodes schulzei (Acari: Ixodidae) on rodents from the State of Parana, southern Brazil.

Ixodes schulzei Aragão & Fonseca is an endemic tick to Brazil and has already been reported in the northern (State of Rondonia), southeastern (States of Minas Gerais, Rio de Janeiro and Sao Paulo) and southern (State of Santa Catarina) regions, always found in association with the water rat Nectomys squamipes (Brandt). This communication records the first occurrence of this tick species in the State of Parana, southern region of Brazil, and it constitutes its first report on Akodon montensis (Thomas) and Oryzomys russatus (Wagner) hosts.

First cytogenetic information for Drymoreomys albimaculatus (Rodentia, Cricetidae), a recently described genus from Brazilian Atlantic Forest.

The recently described taxon Drymoreomys albimaculatus is endemic to the Brazilian Atlantic Forest and its biology and genetics are still poorly known. Herein, we present, for the first time, the karyotype of the species using classical and molecular cytogenetics, which showed 2n=62, FN=62, and interstitial telomeric signals at the sex chromosomes. Nuclear and mitochondrial DNA sequences from the two karyotyped individuals verify the taxonomic identity as the recently described Drymoreomys albimaculatus and confirm the relationship of the species with other Oryzomyini. Additionally, external morphological information is provided.

Noteworthy records of Ixodes schulzei (Acari: Ixodidae) on rodents from the State of Parana, southern Brazil / Relatos de Ixodes schulzei (Acari: Ixodidae) em roedores do estado do Paraná, sul do Brasil

Ixodes schulzei Aragão & Fonseca is an endemic tick to Brazil and has already been reported in the northern (State of Rondonia), southeastern (States of Minas Gerais, Rio de Janeiro and São Paulo) and southern (State of Santa Catarina) regions, always found in association with the water rat Nectomys squamipes (Brandt). This communication records the first occurrence of this tick species in the State of Parana, southern region of Brazil, and it constitutes its first report on Akodon montensis (Thomas) and Oryzomys russatus (Wagner) hosts.

Ixodes schulzei Aragão & Fonseca é um carrapato endêmico do Brasil e já foi relatado nas regiões Norte (estado de Rondônia), Sudeste (estados de Minas Gerais, Rio de Janeiro e São Paulo) e Sul (estado de Santa Catarina), tendo sido encontrado sempre em associação com o rato d'água Nectomys squamipes (Brandt). Esta comunicação registra a primeira ocorrência dessa espécie de carrapato no estado do Paraná, região Sul do Brasil e constitui o primeiro relato de Akodon montensis (Thomas) e Oryzomys russatus (Wagner) como hospedeiros.

Thaptomys Thomas 1915 (Rodentia, Sigmodontinae, Akodontini) with karyotypes 2n = 50, FN = 48, and 2n = 52, FN = 52: two monophyletic lineages recovered by molecular phylogeny

A novel karyotype with 2n = 50, FN = 48, was described for specimens of Thaptomys collected at Una, State of Bahia, Brazil, which are morphologically indistinguishable from Thaptomys nigrita, 2n = 52, FN = 52, found in other localities. It was hence proposed that the 2n = 50 karyotype could belong to a distinct species, cryptic of Thaptomys nigrita, once chromosomal rearrangements observed, along with the geographic distance, might represent a reproductive barrier between both forms. Phylogenetic analyses using maximum parsimony and maximum likelihood based on partial cytochrome b sequences with 1077 bp were performed, attempting to establish the relationships among the individuals with distinct karyotypes along the geographic distribution of the genus; the sample comprised 18 karyotyped specimens of Thaptomys, encompassing 15 haplotypes, from eight different localities of the Atlantic Rainforest. The intra-generic relationships corroborated the distinct diploid numbers, once both phylogenetic reconstructions recovered two monophyletic lineages, a northeastern clade grouping the 2n = 50 and a southeastern clade with three subclades, grouping the 2n = 52 karyotype. The sequence divergence observed between their individuals ranged from 1.9 percent to 3.5 percent.

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.

Karyological geographic variation of Oligoryzomys nigripes Olfers, 1818 (Rodentia, Cricetidae) from Brazil

The karyotypes of 85 specimens of Oligoryzomys nigripes (Rodentia, Sigmodontinae) collected in the Cerrado and Atlantic Forest of seven states of Brazil were analyzed. Eighty four specimens presented a karyotype with 2n = 62 and one individual had 2n = 61 due to a monosomy of the X chromosome. High levels of intra- and inter-population karyotypic variability, due to sex chromosomes heteromorphisms and pericentric inversions in four autosomes (pairs 2, 3, 4 and 8), led to a variation of the autosomal arm numbers (fundamental number, FN) from 78 to 82. Synaptonemal complex analyses revealed normal meiosis in males heterozygous for pericentric inversions. We found 39 different cytotypes, 27 of which are herein described for the first time. A literature survey revealed 46 described karyotypes for O. nigripes. We tested the hypothesis that chromosomal variants frequencies are dependent on geographical distribution and we propose a model for the karyotypical evolution of Oligoryzomys nigripes with 2n = 62/FN = 78-82.