A palaeobiogeographic model for biotic diversification within Amazonia over the past three million years

Many hypotheses have been proposed to explain high species diversity in Amazonia, but few generalizations have emerged. In part, this has arisen from the scarcity of rigorous tests for mechanisms promoting speciation, and from major uncertainties about palaeogeographic events and their spatial and temporal associations with diversification. Here, we investigate the environmental history of Amazonia using a phylogenetic and biogeographic analysis of trumpeters (Aves: Psophia), which are represented by species in each of the vertebrate areas of endemism. Their relationships reveal an unforeseen 'complete' time-slice of Amazonian diversification over the past 3.0 Myr. We employ this temporally calibrated phylogeny to test competing palaeogeographic hypotheses. Our results are consistent with the establishment of the current Amazonian drainage system at approximately 3.0-2.0 Ma and predict the temporal pattern of major river formation over Plio-Pleistocene times. We propose a palaeobiogeographic model for the last 3.0 Myr of Amazonian history that has implications for understanding patterns of endemism, the temporal history of Amazonian diversification and mechanisms promoting speciation. The history of Psophia, in combination with new geological evidence, provides the strongest direct evidence supporting a role for river dynamics in Amazonian diversification, and the absence of such a role for glacial climate cycles and refugia.

Karyotype characterization reveals an up and down of 45S and 5S rDNA sites in Crotalaria (Leguminosae-Papilionoideae) species of the section Hedriocarpae subsection Macrostachyae

The genus Crotalaria is one of the largest within the family Leguminosae-Papilionoideae, with more than 600 species. However, few karyotypes have been described. In the present paper, five species belonging to the section Hedriocarpae were studied (subsection Machrostachyae), in order to better understand chromosomal evolution in Crotalaria. The results reveals that all species presented 2n = 2x = 16 with symmetrical karyotypes, and slight differences in the chromosome morphology. A secondary constriction was identified at short arm of the pair 1. The 45S rDNA was mapped in the secondary constriction and adjacent heterochromatin (NOR-heterochromatin) and a minor site was identified in C. ochroleuca. The 5S rDNA was mapped linked to 45S rDNA at chromosome 1 short arm in all species. Additional sites for 5S rDNA were identified in C. pallida, C. striata and C. mucronata. Heterochromatin blocks around the centromeres are not CMA(+) neither DAPI(+). The karyotypes of the subsection Macrostachyae are characterized by an inversion at chromosome pair one in relation to previous specialized floral species analyzed. Additional sites of 45S and 5S rDNA were assumed to be a result of transposition events by different ways. The results suggest heterochromatin differentiation and the position of ribosomal genes indicates chromosomal rearrangements during evolution. Karyotype characteristics corroborate the morphological infrageneric classification.

An extraordinary new species of Melanophryniscus (Anura, Bufonidae) from southeastern Brazil

We describe a new species of bufonid from a lowland, sandy soil, restinga habitat in the state of Espirito Santo, southeastern Brazil. Based on the shared occurrence of putative morphological synapomorphies of Melanophryniscus and the results of a phylogenetic analysis of DNA sequences of a broad sample of bufonids, and other anurans, we assign the new species to Melanophryniscus. The new species possesses several peculiar character states that distinguish it from all other Melanophryniscus including, but not limited to: fingers II, III, and V much reduced; nuptial pad with few enlarged, brown-colored spines on medial margin of finger II; seven presacral vertebrae, the last fused with the sacrum; and ventral humeral crest prominent, forming a spinelike projection.

Three in one: fixing marine nematodes for ecological, molecular, and morphological studies

Multidisciplinary benthic studies are still hindered by the lack of a unique fixative that satisfactorily preserves morphology and DNA, and that is simultaneously adequate for ecological surveys. The objective of this study is to test the performance of five fixatives: formalin, ethanol, dimethylsulfoxide with EDTA and NaCl salts (DESS), methanol with acetic acid (METHAC), and ethanol with acetic acid (ETHAC), for the preservation of estuarine and exclusively marine nematode assemblages for morphological, molecular, and ecological studies. The presence of the stain rose bengal in each fixative was also evaluated in the yield of PCR reactions. For molecular analyses, one species of each habitat was considered. Results revealed that fixative performance for morphological studies is habitat-and species-dependent. For studies of estuarine sediment nematodes, we recommend the use of pure ethanol, because it caused little morphological distortion (<10% of the assemblage), preserved all the species for ecological studies, and yielded high quality DNA sequences. For studies of exclusively marine environments, METHAC or DESS are the most adequate. The first performed better for morphological and ecological surveys, whereas the second was more appropriate for molecular research. For ecological studies, DESS should be used in comparison with formalin, in order to cross check the results. Finally, staining of samples with rose bengal is not recommended, because it hindered DNA amplification regardless of the fixative used.

Is a Genome a Codeword of an Error-Correcting Code?

Since a genome is a discrete sequence, the elements of which belong to a set of four letters, the question as to whether or not there is an error-correcting code underlying DNA sequences is unavoidable. The most common approach to answering this question is to propose a methodology to verify the existence of such a code. However, none of the methodologies proposed so far, although quite clever, has achieved that goal. In a recent work, we showed that DNA sequences can be identified as codewords in a class of cyclic error-correcting codes known as Hamming codes. In this paper, we show that a complete intron-exon gene, and even a plasmid genome, can be identified as a Hamming code codeword as well. Although this does not constitute a definitive proof that there is an error-correcting code underlying DNA sequences, it is the first evidence in this direction.

New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?

The Neotropical tribe Trimezieae are taxonomically difficult. They are generally characterized by the absence of the features used to delimit their sister group Tigridieae. Delimiting the four genera that make up Trimezieae is also problematic. Previous family-level phylogenetic analyses have not examined the monophyly of the tribe or relationships within it. Reconstructing the phylogeny of Trimezieae will allow us to evaluate the status of the tribe and genera and to examine the suitability of characters traditionally used in their taxonomy. Maximum parsimony and Bayesian phylogenetic analyses are presented for 37 species representing all four genera of Trimezieae. Analyses were based on nrITS sequences and a combined plastid dataset. Ancestral character state reconstructions were used to investigate the evolution of ten morphological characters previously considered taxonomically useful. Analyses of nrITS and plastid datasets strongly support the monophyly of Trimezieae and recover four principal clades with varying levels of support; these clades do not correspond to the currently recognized genera. Relationships within the four clades are not consistently resolved, although the conflicting resolutions are not strongly supported in individual analyses. Ancestral character state reconstructions suggest considerable homoplasy, especially in the floral characters used to delimit Pseudotrimezia. The results strongly support recognition of Trimezieae as a tribe but suggest that both generic- and species-level taxonomy need revision. Further molecular analyses, with increased sampling of taxa and markers, are needed to support any revision. Such analyses will help determine the causes of discordance between the plastid and nuclear data and provide a framework for identifying potential morphological synapomorphies for infra-tribal groups. The results also suggest Trimezieae provide a promising model for evolutionary research.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Transmission of Human Herpesvirus Type 8 Infection Within Families in American Indigenous Populations From the Brazilian Amazon

Background. The intrafamilial dynamics of endemic infection with human herpesvirus type 8 (HHV-8) in Amerindian populations is unknown. Methods. Serum samples were obtained from 517 Amerindians and tested for HHV-8 anti-latent nuclear antigen (anti-LANA) and antilytic antibodies by immunofluorescence assays. Logistic regression and mixed logistic models were used to estimate the odds of being HHV-8 seropositive among intrafamilial pairs. Results. HHV-8 seroprevalence by either assay was 75.4% (95% confidence interval [CI]: 71.5%-79.1%), and it was age-dependent (P-trend<.001). Familial dependence in HHV-8 seroprevalence by either assay was found between mother-offspring (odds ratio [OR], 5.44; 95% CI: 1.62-18.28) and siblings aged >= 10 years (OR 4.42, 95% CI: 1.70-11.45) or siblings in close age range (<5 years difference) (OR 3.37, 95% CI: 1.21-9.40), or in families with large (>4) number of siblings (OR, 3.20, 95% CI: 1.33-7.67). In separate analyses by serological assay, there was strong dependence in mother-offspring (OR 8.94, 95% CI: 2.94-27.23) and sibling pairs aged >= 10 years (OR, 11.91, 95% CI: 2.23-63.64) measured by LANA but not lytic antibodies. Conclusions. This pattern of familial dependence suggests that, in this endemic population, HHV-8 transmission mainly occurs from mother to offspring and between close siblings during early childhood, probably via saliva. The mother to offspring dependence was derived chiefly from anti-LANA antibodies.

Phylogeographic Structure and Karyotypic Diversity of the Brazilian Shrew Mouse (Blarinomys breviceps, Sigmodontinae) in the Atlantic Forest

Blarinomys breviceps possesses cryptic and burrowing habits with poorly documented genetics and life history traits. Due to its rarity, only a few specimens and DNA sequences have been deposited in collections worldwide. Here, we present the most comprehensive cytogenetic and molecular characterization of this rare genus. Phylogenetic analyses based on partial cytochrome b sequences were performed, attempting to establish the relationships among individuals with distinct karyotypes along the geographic distribution of the genus in the Atlantic Forest. Classical and molecular cytogenetics, using banding patterns and FISH of telomeric and whole chromosome X-specific painting probes (obtained from the Akodontini Akodon cursor) were used to characterize and compare the chromosomal complements. Molecular phylogenetic analyses recovered 2 main geographically structured clades, northeastern and southeastern with pair-wise sequence divergences among specimens varying between 4.9 and 8.4%. Eight distinct karyomorphs are described: (A) 2n = 52 (50A, XX), (B) 2n = 52 (48A, XY+2Bs), (C) 2n = 45 (42A, XY+1B), (D) 2n = 43 (37A, XX+4Bs), (E) 2n = 37 (34A, XY+1B), (F) 2n = 34 (32A, XX), (G) 2n = 31 (27A, XX+2Bs), (H) 2n = 28 (26A, XY), all with the same number of autosomal arms (FNA = 50). Variation of 0-4 supernumerary chromosomes (Bs) presenting heterogeneity in morphology and distribution of interstitial telomeric sequences (ITSs) is reported. ITSs are also found in some metacentric autosomes. The phylogeographic separation between 2 major lineages with high levels of genetic divergence, and the wide karyotypic diversity indicate that B. breviceps is a diverse group that warrants taxonomic re-evaluation. Copyright (C) 2012 S. Karger AG, Basel

Decreasing the number of false positives in sequence classification

Abstract Background A large number of probabilistic models used in sequence analysis assign non-zero probability values to most input sequences. To decide when a given probability is sufficient the most common way is bayesian binary classification, where the probability of the model characterizing the sequence family of interest is compared to that of an alternative probability model. We can use as alternative model a null model. This is the scoring technique used by sequence analysis tools such as HMMER, SAM and INFERNAL. The most prevalent null models are position-independent residue distributions that include: the uniform distribution, genomic distribution, family-specific distribution and the target sequence distribution. This paper presents a study to evaluate the impact of the choice of a null model in the final result of classifications. In particular, we are interested in minimizing the number of false predictions in a classification. This is a crucial issue to reduce costs of biological validation. Results For all the tests, the target null model presented the lowest number of false positives, when using random sequences as a test. The study was performed in DNA sequences using GC content as the measure of content bias, but the results should be valid also for protein sequences. To broaden the application of the results, the study was performed using randomly generated sequences. Previous studies were performed on aminoacid sequences, using only one probabilistic model (HMM) and on a specific benchmark, and lack more general conclusions about the performance of null models. Finally, a benchmark test with P. falciparum confirmed these results. Conclusions Of the evaluated models the best suited for classification are the uniform model and the target model. However, the use of the uniform model presents a GC bias that can cause more false positives for candidate sequences with extreme compositional bias, a characteristic not described in previous studies. In these cases the target model is more dependable for biological validation due to its higher specificity.

Ornithodoros peropteryx (Acari: Argasidae) in Bolivia: an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guaporé River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Rickettsial infection in Amblyomma cajennense ticks and capybaras (Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area

Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras (Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF. In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen. A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192. A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.

Ornithodoros peropteryx (Acari Argasidae) in Bolivia an argasid tick with a single nymphal stage

By the end of the 1960s, the argasid tick Ornithodoros peropteryx was described from larval specimens collected from the bat Peropteryx macrotis in Colombia. Since its original description, no additional record of O. peropteryx has been reported, and its post-larval stages have remained unknown. During July 2010, 18 larvae were collected from 9 bats (Centronycteris maximiliani), resulting in a mean infestation of 2.0 ± 2.2 ticks per bat (range 1–8). These bats were captured in a farm in northeastern Bolivia close to Guapore´ River in the border with Brazil. Morphological examinations of the larvae revealed them to represent the species O. peropteryx. One engorged larva that was kept alive in the laboratory moulted to a nymph after 9 days. Fourteen days after the larval moulting, the nymph moulted to an adult female without taking any blood meal during the nymphal period. This adult female was used for a morphological description of the female stage of O. peropteryx. In addition, the larvae were used for a morphological redescription of this stage. One larva and two legs extirpated from the adult female were submitted to DNA extraction and PCR targeting a fragment of the mitochondrial 16S rDNA gene, which yielded DNA sequences at least 11 % divergent from any available argasid sequence in Genbank. We show that O. peropteryx ontogeny is characterized by a single, non-feeding, nymphal stage. This condition has never been reported for ticks.

Adaptation and Constraint in the Plant Reproductive Phase

Conservatism is a central theme of organismic evolution. Related species share characteristics due to their common ancestry. Some concern have been raised among evolutionary biologists, whether such conservatism is an expression of natural selection or of a constrained ability to adapt. This thesis explores adaptations and constraints within the plant reproductive phase, particularly in relation to the evolution of fleshy fruit types (berries, drupes, etc.) and the seasonal timing of flowering and fruiting. The different studies were arranged along a hierarchy of scale, with general data sets sampled among seed plants at the global scale, through more specific analyses of character evolution within the genus Rhamnus s.l. L. (Rhamnaceae), to descriptive and experimental field studies in a local population of Frangula alnus (Rhamnaceae). Apart from the field study, this thesis is mainly based on comparative methods explicitly incorporating phylogenetic relationships. The comparative study of Rhamnus s.l. species included the reconstruction of phylogenetic hypotheses based on DNA sequences. Among geographically overlapping sister clades, biotic pollination was not correlated with higher species richness when compared to wind pollinated plants. Among woody plants, clades characterized by fleshy fruit types were more species rich than their dry-fruited sister clades, suggesting that the fleshy fruit is a key innovation in woody habitats. Moreover, evolution of fleshy fruits was correlated with a change to more closed (darker) habitats. An independent contrast study within Rhamnus s.l. documented allometric relations between plant and fruit size. As a phylogenetic constraint, allometric effects must be considered weak or non-existent, though, as they did not prevail among different subclades within Rhamnus s.l. Fruit size was correlated with seed size and seed number in F. alnus. This thesis suggests that frugivore selection on fleshy fruit may be important by constraining the upper limits of fruit size, when a plant lineage is colonizing (darker) habitats where larger seed size is adaptive. Phenological correlations with fruit set, dispersal, and seed size in F. alnus, suggested that the evolution of reproductive phenology is constrained by trade-offs and partial interdependences between flowering, fruiting, dispersal, and recruitment phases. Phylogenetic constraints on the evolution of phenology were indicated by a lack of correlation between flowering time and seasonal length within Rhamnus cathartica and F. alnus, respectively. On the other hand, flowering time was correlated with seasonal length among Rhamnus s.l. species. Phenological differences between biotically and wind pollinated angiosperms also suggested adaptive change in reproductive phenology.