Ultraconserved Elements Improve the Resolution of Difficult Nodes within the Rapid Radiation of Neotropical Sigmodontine Rodents (Cricetidae: Sigmodontinae).

Sigmodontine rodents (Cricetidae, Sigmodontinae) represent the second largest muroid subfamily and the most species-rich group of New World mammals, encompassing above 410 living species and ca. 87 genera. Even with advances on the clarification of sigmodontine phylogenetic relationships that have been made recently, the phylogenetic relationships among the 12 main groups of genera (i.e., tribes) remain poorly resolved, in particular among those forming the large clade Oryzomyalia. This pattern has been interpreted as consequence of a rapid radiation upon the group entrance into South America. Here, we attempted to resolve phylogenetic relationships within Sigmodontinae using target capture and high-throughput sequencing of ultraconserved elements (UCEs). We enriched and sequenced UCEs for 56 individuals and collected data from four already available genomes. Analyses of distinct data sets, based on the capture of 4634 loci, resulted in a highly resolved phylogeny consistent across different methods. Coalescent species-tree-based approaches, concatenated matrices, and Bayesian analyses recovered similar topologies that were congruent at the resolution of difficult nodes. We recovered good support for the intertribal relationships within Oryzomyalia; for instance, the tribe Oryzomyini appears as the sister taxa of the remaining oryzomyalid tribes. The estimates of divergence times agree with the results of previous studies. We inferred the crown age of the sigmodontine rodents at the end of the Middle Miocene, while the main lineages of Oryzomyalia appear to have radiated in a short interval during the Late Miocene. Thus, the collection of a genomic-scale data set with a wide taxonomic sampling provided resolution for the first time of the relationships among the main lineages of Sigmodontinae. We expect the phylogeny presented here will become the backbone for future systematic and evolutionary studies of the group.[Coalescent; Muroidea; Oryzomyalia; phylogenomics; polytomy; Rodentia; Sigmodontalia; species tree; UCEs.].

On the Origin and Evolution of the Extant System of B Chromosomes in Oryzomyini Radiation (Rodentia, Sigmodontinae).

Heterogeneous supernumerary chromosomes (Bs) are recognized in the oryzomyines Holochilus brasiliensis, Nectomys rattus, N. squamipes, Oligoryzomys flavescens and Sooretamys angouya, representing about 10% of all known B-containing rodent species. They provide an outstanding model for understanding the origin, evolution and diversity of Bs in a phylogenetic context. Therefore, whole chromosome-specific probes were generated from flow-sorted Holochilus brasiliensis (HBR) autosomes 11 and 25+26 and chromosomes X, Y and Bs. Hybridizations were performed on male metaphases of 15 Oryzomyini species of which 3 are B-containing species. The results reveal that among the species sampled, 12 of them, belonging to a monophyletic Oryzomiyini subclade, are positive for an anonymous Oryzomyini shared heterochromatic region (OSHR) on both sex chromosomes. The OSHR is also present on Bs of Holochilus brasiliensis, Nectomys rattus and N. squamipes but not on Bs of O. flavescens and S. angouya. Two distinct additional OSHR/autosome associations are observed on S. angouya. The three species that are OSHR negative belong to an outgroup. Molecular dating suggests that the OSHR originated between 7.8 and 3 Mya on ancestral sex chromosomes. A tentative explanation for the OSHR-positive nature of B regions in three species could be that transposable elements (TEs) from this specific sex chromosome region may have invaded existing B chromosomes. The presence of the OSHR on entire Xp and Yp adjacent to interstitial telomeric sequences at pericentromeric positions, as observed in Drymoreomys albimaculatus, show a similar organization as on B chromosomes in Nectomys squamipes. The diversity of the Oryzomyini Bs in number, size, morphology and genetic content may be explained by the independent origin of B chromosomes in different subgroups of species, with Bs in Holochilus brasiliensis, Nectomys squamipes and N. rattus sharing the OSHR with sex chromosomes, and those in Oligoryzomys flavescens and Sooretamys angouya lacking OSHR in Bs. The species-specific pattern of Bs is probably a consequence of their independent evolutionary origin.

The influence of ecological and geographical context in the radiation of Neotropical sigmodontine rodents.

BACKGROUND: Much debate has focused on how transitions in life history have influenced the proliferation of some clades. Rodents of the subfamily Sigmodontinae (family Cricetidae) comprise one of the most diverse clades of Neotropical mammals (~400 living species in 86 genera). These rodents occupy a wide range of habitats and lifestyles so that ecological context seems relevant to understand the evolution of this group. Several changes in the landscape of South America through the Neogene might have provided vast resources and opportunity to diversify. The aim of this study was to examine whether transitions between i) lowland and montane habitats, ii) open vegetation and forest, and iii) distinct molar architectures are correlated with shifts in diversification rates and to characterize the general pattern of diversification. RESULTS: Based on a dense taxon sampling of 269 species, we recovered a new phylogeny of Sigmodontinae that is topologically consistent with those of previous studies. It indicates that the subfamily and its major lineages appeared during the Late Miocene. Analyses suggest that vegetation type and elevational range are correlated with diversification rates, but not molar architecture. Tropical lowlands accumulated more lineage diversity than other areas and also supported high speciation rates. Across the radiation the subfamily Sigmodontinae appear to have experienced a decline in diversification rate through time. We detected mixed evidence for lineage-specific diversification rate shifts (e.g., leading to the clades of Akodon, Bibimys, Calomys and Thomasomys). CONCLUSION: We report that the evolution of habitat preference (considering vegetation type and elevational range) was associated with diversification rates among sigmodontine rodents. We propose that the observed diversification slowdown might be the result of ecological or geographical constraints. Our results also highlight the influence of the tropical lowlands -which might have acted as both "a cradle and a museum of species." The tropical lowlands accumulated greater diversity than the remainder of the group's range.

Comparison between Oligoryzomys nigripes and O. flavescens by RAPD and genetic diversity in O. nigripes (Rodentia, Cricetidae) / Comparação entre Oligoryzomys nigripes e O. flavescens e diversidade genética em O. nigripes (Rodentia, Cricetidae) por RAPD

The genus of Oligoryzomys includes species of small size, morphologically similar, which may impede taxonomic identification, mainly between O. flavescens (Waterhouse, 1837) and O. nigripes (Olfers, 1818). The main objective of this work was to investigate whether the RAPD markers are capable of genetically differentiating the specimens O. nigripes and O. flavescens, coming from Rio Grande do Sul (RS) and Santa Catarina (SC) states, and also to estimate the genetic variability among populations of O. nigripes, with the Uruguay River as a geographical barrier. For this purpose, samples were collected in fragments of forests situated in the North of RS, at FLONA (Floresta Nacional de Passo Fundo) and in fragments from SC, close to the Uruguay River. The karyotyping of two samples for each species was carried out and compared using the RAPD technique together with non- karyotyped individuals. Samples of O. nigripes presented 2n = 62; NA = 82, with submetacentric arms on the largest chromosomes, while samples of O. flavescens showed 2n = 64; NA = 66, with the largest chromosomes presenting acrocentric morphology, making such a result the main difference between the species. The analysis was able to detect two distinct groups, being the first one with karyotyped O. flavescens and the second with karyotyped O. nigripes. Identification afforded 211 loci, among them 181 (85.78%) polymorphic. The Jaccard similarity coefficient was in the range of 0.45 to 0.87. The UPGMA and Main Coordinate Analysis techniques demonstrated the existence of heterogeneous genetics among populations, but did not separate them completely in terms of geographical standards, and they are not influenced by the Uruguay River, which did not act as an efficient barrier.

O gênero Oligoryzomys inclui espécies de tamanho pequeno, morfologicamente semelhantes, com difícil identificação taxonômica, principalmente entre O. flavescens e O. nigripes. O objetivo principal deste trabalho foi investigar se os marcadores RAPD são capazes de diferenciar geneticamente as amostras de O. nigripes e O. flavescens, oriundas do Rio Grande do Sul (RS) e Santa Catarina (SC) e também para estimar o variabilidade genética entre populações de O. nigripes, tendo o rio Uruguai como uma barreira geográfica. Para este fim, as amostras foram coletadas em fragmentos florestais situados no Norte do RS, na FLONA (Floresta Nacional de Passo Fundo) e em fragmentos florestais de SC, próximos ao Rio Uruguai. O cariótipo de duas amostras de cada espécie foi realizado e comparado com a técnica RAPD em conjunto com indivíduos não cariotipados. As amostras de O. nigripes apresentaram 2n = 62, NA = 82, com braços submetacêntricos nos cromossomos maiores, enquanto que as amostras de O. flavescens mostraram 2n = 64, NA = 66, com os maiores cromossomos apresentando morfologia acrocêntrica, tornando tal resultado a principal diferença entre as espécies. As análises por RAPD foram capazes de detectar dois grupos distintos, sendo o primeiro com O.flavescens cariotipados e o segundo com O. nigripes cariotipados. Foram avaliados 211 loci, e entre eles 181 (85,78%) foram polimórficos. O coeficiente de similaridade de Jaccard variou de 0,45 a 0,87. A análise por UPGMA e análise de coordenadas principais demonstraram a existência de heterogeneidade genética entre as populações, mas não foi possível separá-las completamente em termos de padrões geográficos, e estas não são influenciadas pelo rio Uruguai, o qual não agiu como uma barreira eficiente.

In the wake of invasion: tracing the historical biogeography of the South American cricetid radiation (Rodentia, Sigmodontinae).

The Great American Biotic Interchange (GABI) was greatly influenced by the completion of the Isthmus of Panama and impacted the composition of modern faunal assemblages in the Americas. However, the contribution of preceding events has been comparatively less explored, even though early immigrants in the fossil records are evidence for waif dispersals. The cricetid rodents of the subfamily Sigmodontinae are a classic example of a species-rich South American radiation resulting from an early episode of North American invasion. Here, we provide a temporal and spatial framework to address key aspects of the historical biogeography and diversification of this diverse mammal group by using mitochondrial and nuclear DNA datasets coupled with methods of divergence time estimation, ancestral area reconstruction and comparative phylogenetics. Relaxed-clock time estimates indicate that divergence of the Sigmodontinae began in the middle-late Miocene (ca. 12-9 Ma). Dispersal-vicariance analyses point to the arrival of a single lineage of northern invaders with a widespread ancestral distribution and imply that the initial differentiation between Central and South America gave rise to the most basal groups within the subfamily. These two major clades diversified in the late Miocene followed by the radiation of main tribes until the early Pliocene. Within the Oryzomyalia, tribes diverged initially in eastern South America whereas multiple dispersals into the Andes promoted further diversification of the majority of modern genera. A comparatively uniform background tempo of diversification explains the species richness of sigmodontines across most nodes, except for two akodontine genera with recent increases in diversification rates. The bridging of the Central American seaway and episodes of low sea levels likely facilitated the invasion of South America long before the onset of the post-Isthmian phase of the GABI.

Dating an impressive Neotropical radiation: Molecular time estimates for the Sigmodontinae (Rodentia) provide insights into its historical biogeography.

With about 400 living species and 82 genera, rodents of the subfamily Sigmodontinae comprise one of the most diverse and more broadly distributed Neotropical mammalian clades. There has been much debate on the origin of the lineage or the lineages of sigmodontines that entered South America, the timing of entrance and different aspects of further diversification within South America. The ages of divergence of the main lineages and the crown age of the subfamily were estimated by using sequences of the interphotoreceptor retinoid binding protein and cytochrome b genes for a dense sigmodontine and muroid sampling. Bayesian inference using three fossil calibration points and a relaxed molecular clock estimated a middle Miocene origin for Sigmodontinae (∼12Ma), with most tribes diversifying throughout the Late Miocene (6.9-9.4Ma). These estimates together results of analyses of ancestral area reconstructions suggest a distribution for the most recent common ancestor of Sigmodontinae in Central-South America and a South American distribution for the most recent common ancestor of Oryzomyalia.

Caracterización molecular de poblaciones venezolanas de Sigmodon hirsutus (Rodentia: Cricetidae)

Molecular characterization of Sigmodon hirsutus (Rodentia: Cricetidae) populations in Venezuela. Recent phylogenetic studies based on cytochrome b gene sequence, have determined that the species historically known as Sigmodon hispidus (Rodentia) from South America comprises a species S. hirsutus of paraphyletic origin. The aim of this study was to test the hypothesis that populations from Venezuela, represent the sensu strict form, ancestral haplotypes, and monophyletic subspecies. For this, 12 individual sequences from three localities of different biogeographic regions in Venezuela were evaluated and sequenced based on cyto b. Additionally, the sequences were used to develop a cladistic analysis and genetic distance calculations, and to compare this information with two individual sequences of Sigmodon specimens available in Genbank. Phylogenetic analyses show that the three populations of S. hirsutus of Venezuela form an ancestral and monophyletic subclade supported by high bootstrap values and significant genetic distance between subclade within the S. hirsutus. Besides, the existence of two lineages suggests two subspecies, S. hirsutus hirsutus from Venezuela, and S. hirsutus mexicanus from Mexico-Central America, but, both species need formal description. Rev. Biol. Trop. 59 (2): 795-807. Epub 2011 June 01.

Recientes estudios filogenéticos basados en la secuencia del gen del citocromo b, han determinado que la especie conocida históricamente como Sigmodon hispidus (Rodentia) en Suramérica incluye una especie S. hirsutus de origen parafilético. El objetivo de este estudio fue probar la hipótesis de que las poblaciones de Venezuela, representan la forma sensu estricto, los haplotipos ancestrales y una subespecie monofilética. La metodología consistió en un análisis cladístico y cálculos de distancia genética, a partir de secuencias de citocromo b en 12 individuos de tres localidades en Venezuela que pertenecen a diferentes regiones biogeográficas, y a su vez compararlas con las dos secuencias disponibles en Genbank de especies del género Sigmodon. Los análisis filogenéticos indican que las tres poblaciones de S. hirustus de Venezuela forman un subclado ancestral y monofilético con el apoyo de valores de bootstrap altos y con significativa distancia genética, entre subclados dentro de S. hirsutus. La existencia de dos subclados dentro de S. hirsutus sugiere dos subespecies, S. hirsutus hirsutus en Venezuela y S. hirsutus mexicanus en México y Centroamérica. Sin embargo, ambas subespecies necesitan una descripción formal.

Genetic characterization of hantaviruses associated with sigmodontine rodents in an endemic area for hantavirus pulmonary syndrome in southern Brazil.

An ecological assessment of reservoir species was conducted in a rural area (Jaborá) in the mid-west of the state of Santa Catarina in southern Brazil, where hantavirus pulmonary syndrome is endemic, to evaluate the prevalence of hantavirus infection in wild rodents. Blood and tissue samples were collected from 507 rodents during seven field trips from March 2004 to April 2006. Some of the animals were karyotyped to confirm morphological identification. Phylogenetic reconstructions of rodent specimens, based on the mitochondrial DNA cytochrome b gene sequences, were also obtained. Hantavirus antibody was found in 22 (4.3%) of the 507 rodents: 5 Akodon montensis, 2 Akodon paranaensis, 14 Oligoryzomys nigripes, and 1 Sooretamys angouya. Viral RNAs detected in O. nigripes and A. montensis were amplified and sequenced. O. nigripes virus genome was 97.5% (nt) and 98.4% (nt) identical to sequences published for Araucaria (Juquitiba-like) virus based on N and G2 fragment sequences. Viral sequences from A. montensis strain showed 89% and 88% nucleotide identities in a 905-nt fragment of the nucleocapsid (N) protein-coding region of the S segment when it was compared with two other Akodontine rodent-associated viruses from Paraguay, A. montensis and Akodon cursor, respectively. Phylogenetic analysis showed the cocirculation of two genetic hantavirus lineages in the state of Santa Catarina, one from O. nigripes and the other from A. montensis, previously characterized in Brazil and Paraguay, respectively. The hantavirus associated with A. montensis, designed Jaborá virus, represents a distinct phylogenetic lineage among the Brazilian hantaviruses.

Spermatocyte apoptosis, which involves both intrinsic and extrinsic pathways, explains the sterility of Graomys griseoflavus x Graomys centralis male hybrids.

Spermatogenic impairment and the apoptotic pathways involved in establishing sterility of male hybrids obtained from crossing Graomys griseoflavus females with Graomys centralis males were studied. Testes from G. centralis, G. griseoflavus and hybrids were compared at different ages. Terminal transferase-mediated dUTP nick-end labelling assay (TUNEL), Fas, Bax and cytochrome c labelling were used for apoptosis evaluation, and calbindin D(28k) staining as an anti-apoptotic molecule. In 1-month-old animals, spermatocytes were positive for all apoptotic markers, but moderate TUNEL (+) spermatocyte frequency was only found in G. centralis. At subsequent ages, the apoptotic markers were downregulated in testes from parental cytotypes, but not in hybrid testes. TUNEL (+) spermatocytes were present at 78% and 44% per tubule cross-section in 2- and 3-month-old hybrid animals, respectively. Pachytene spermatocyte death in adult hybrids occurs via apoptosis, as revealed by high caspase-3 expression. Calbindin was highly expressed in spermatocytes of adult hybrids, in which massive cell death occurs via apoptosis. Calbindin co-localisation with TUNEL or Fas, Bax and cytochrome c was very limited, suggesting an inverse regulation of calbindin and apoptotic markers. Hybrid sterility is due to breakdown of spermatogenesis at the pachytene spermatocyte stage. Both extrinsic and intrinsic pathways are involved in apoptosis of spermatocytes, which are the most sensitive cell type to apoptotic stimuli.

Nitric oxide production and nitric oxide synthase type 2 expression by cotton rat (Sigmodon hispidus) macrophages reflect the same pattern as human macrophages.

Our knowledge of the antibacterial role of nitric oxide (NO) during infection is based on studies of murine macrophages, which secrete large amounts of NO. In contrast, human macrophages produce very little NO and its relevance as an antibacterial mediator during infection of humans is uncertain. We have defined bone marrow-derived macrophages from cotton rats (Sigmodon hispidus). These macrophages display phenotypical and functional characteristics similar to other rodent and human macrophages. The most interesting finding was the low level of NO production which is in contrast to findings for murine macrophages, but consistent with those of humans. In spite of these low levels, inhibition of NO production led to a decrease in killing of bacteria. Cotton rats are highly susceptible to a variety of human pathogens and therefore offer a rodent model of infectious diseases with similar characteristics to humans in terms of NO production.

Genetic diversity and relatedness within and between species of the genus Oligoryzomys (Rodentia; Sigmodontinae)

A RAPD analysis on six species of the rodent genus Oligoryzomys trapped in a wide area (ranging from 01° N to 32° S) of Brazilian territory was performed in order to determine the levels of genetic variability within and between its populations and species. One-hundred and ninety-three animals were collected in 13 different sites (corresponding to 17 samples) located at Pampas, Atlantic Rain Forest, Cerrado, and Amazon domains. Oligoryzomys sp., O. nigripes (8 populations), O. flavescens (4 populations), O. moojeni, O. stramineus, and O. fornesi were the taxa analyzed. Of the 20 primers tested, 4 generated a total of 75 polymorphic products simultaneously amplified in 151 specimens. Various diversity estimators analyzed showed considerable differences between species and populations, indicating a great genetic variation occurring in the Oligoryzomys taxa investigated. A cluster analysis was made using Nei's standard genetic distances, however, it did not correlate the genetic heterogeneity of the species and populations with the geographical areas.

Foram realizadas análises com RAPD em seis espécies de roedores do gênero Oligoryzomys capturados em uma ampla área (estendendo-se de 01° N a 32° S) do território brasileiro com o objetivo de determinar os níveis de variabilidade genética dentro e entre as populações e espécies. Cento e noventa e três animais foram coletados em 13 locais diferentes (correspondendo a 17 amostras) localizados nos Pampas, Floresta Atlântica, Cerrado e Amazônia. Oligoryzomys sp., O. nigripes (8 populações), O. flavescens (4 populações), O. moojeni, O. stramineus e O. fornesi foram as espécies analisadas. Vinte primers foram testados, sendo que quatro deles geraram um total de 75 produtos polimórficos amplificados simultaneamente em 151 exemplares. Várias estimativas de diversidade apresentaram diferenças consideráveis entre as espécies e as populações, indicando uma grande variação genética entre os taxa de Oligoryzomys investigados. As análises de agrupamento utilizando a distância genética de Nei, entretanto, não correlacionaram a heterogeneidade genética das espécies e populações com as áreas geográficas.

(T2AG3)n telomeric sequence hybridization indicating centric fusion rearrangements in the karyotype of the rodent oryzomys subflavus.

Chromosome preparations of 30 specimens of Oryzomys subflavus trapped in eight Brazilian localities were C-, and G-banded and analyzed by fluorescence in situ hybridization (FISH). Two karyotypes were found, 2n = 50/FN = 64, at three coastal localities of the Atlantic Forest domain, and 2n = 58/FN = 70 at two sites located in the Cerrado biome, Brazil Central. Two fluorescence in situ hybridization (FISH) patterns of the telomeric sequence (T2AG3)n were observed: in both karyotypes the probes hybridized to the telomeres of all chromosomes and also a hybridization signal in the centromeric regions of two autosome pairs was seen in the 2n = 50 karyotype. These results, together with the occurrence of other diploid numbers described in the literature, suggest that 0. subflavus is a complex species, bearing fusion/fission rearrangements proper to the different biomes which it inhabits.

Genetic diversity of the major histocompatibility complex of cotton rats (Sigmodon hispidus) inhabiting an oil refinery complex.

We examined genetic diversity of an immune-response gene within the major histocompatibility complex in cotton rats (Sigmodon hispidus) inhabiting an oil refinery complex. Genetic diversity of MHC-DQA exon-2 was examined within and among contaminated and noncontaminated reference grids. The level of gene diversity within contaminated grids (0.748) was lower than within reference grids (0.818), but the difference was not statistically significant (p > 0.5). Analysis of molecular variance, pairwise FST values, and hierarchical clustering failed to reveal population genetic structure related to contamination. Results of this study indicate either that the level of contaminant-induced selection is insignificant at this major histocompatibility complex (MHC) locus or that gene flow from surrounding areas has obliterated the effects of selection.

Cloning, expression and purification of recombinant cotton rat interleukin-5.

The coding sequence of the hispid cotton rat (Sigmodon hispidus) interleukin-5 (IL-5) was isolated by a combination of reverse transcription (RT)-PCR and RACE protocols from concanavalin A stimulated spleen cells. The open reading frame of 399 bp encodes a polypeptide of 132 amino acids. Comparison with the rat, mouse, gerbil and human counterparts revealed 88, 88, 87 and 75% identity at the nucleotide level and 88, 90, 89 and 70% at the amino acid level, respectively. The entire coding sequence, minus the putative signal peptide sequence, was inserted into an inducible Escherichia coli expression vector. The recombinant protein possessed an expected molecular mass of 14kDa and was located in bacterial inclusion bodies. A purification scheme under reducing and denaturing conditions followed by subsequent successive dialysis steps led to the recovery of a recombinant dimeric cotton rat IL-5. The biological activity of the recombinant protein was demonstrated in a murine cell line proliferation assay. This activity was specifically inhibited by rat monoclonal antibodies directed against mouse IL-5. Together with specific antibodies that can be generated easily, cotton rat IL-5 constitutes a useful tool for extending the use of the cotton rat animal model in the study of various human pathogens.