Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest,and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae)

Phylogeographic studies using mitochondrial DNA sequence information are frequently used as the principal source of evidence to infer species boundaries. However, a critical analysis of further evidence is essential to test whether different haplotype clades identify different species. We demonstrate a hypothesis‐testing approach, using a combination of phylogeographic methods, multivariate morphometrics and matrix association tests, to investigate species boundaries in eastern Brazilian pitvipers conventionally assigned to the species Bothrops leucurus and B. pradoi. Two basal haplotype clades with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association tests to verify whether generalized morphology, or any of four supposedly diagnostic characters for the two species, show any association with mtDNA variation. Negative results lead to the conclusion that the haplotype clades do not denote independently evolving organismal lineages, and do not constitute separate species under any criterion.     

Phylogeography of the endangered rosewood Dalbergia nigra (Fabaceae): insights into the evolutionary history and conservation of the Brazilian Atlantic Forest

The Brazilian rosewood (Dalbergia nigra) is an endangered tree endemic to the central Brazilian Atlantic Forest, one of the world''s most threatened biomes. The population diversity, phylogeographic structure and demographic history of this species were investigated using the variation in the chloroplast DNA (cpDNA) sequences of 185 individuals from 19 populations along the geographical range of the species. Fifteen haplotypes were detected in the analysis of 1297 bp from two non-coding sequences, trnV-trnM and trnL. We identified a strong genetic structure (F_ST=0.62, P<0.0001), with a latitudinal separation into three phylogeographic groups. The two northernmost groups showed evidence of having maintained historically larger populations than the southernmost group. Estimates of divergence times between these groups pointed to vicariance events in the Middle Pleistocene (ca. 350 000–780 000 years ago). The recurrence of past climatic changes in the central part of the Atlantic forest, with cycles of forest expansion and contraction, may have led to repeated vicariance events, resulting in the genetic differentiation of these groups. Based on comparisons among the populations of large reserves and small, disturbed fragments of the same phylogeographic group, we also found evidence of recent anthropogenic effects on genetic diversity. The results were also analysed with the aim of contributing to the conservation of D. nigra. We suggest that the three phylogeographic groups could be considered as three distinct management units. Based on the genetic diversity and uniqueness of the populations, we also indicate priority areas for conservation.     

Latitudinal Variation in Termite Species Richness and Abundance along the Brazilian Atlantic Forest Hotspot

In this study, we investigated the termites of the Brazilian Atlantic Forest, one of the most threatened biodiversity hotspots in the world, in regularly spaced sites from 7° S to 27° S latitude. To our knowledge, this is the only report of a latitudinal survey of termites at species level performed with a standardized sampling protocol. We evaluate termite diversity and abundance, and describe patterns of species composition based on feeding groups along the latitudinal gradient. We also describe the relative contribution of environmental variables to explain diversity patterns. Termite assemblages were investigated by standardized surveys at 15 Atlantic Forest sites, on six transects divided into five sections of 10 m², with 30 sections per site (or 300 m²/site), which were investigated by one trained person for one hour. Observed species richness and abundance were negatively correlated with latitude. The influence of latitude was explained mainly by variables related to temperature, precipitation and ambient energy (potential evapotranspiration). Our results also suggest that temperature exerts a greater constraint on Atlantic Forest termites than productivity, because ambient productivity increases with latitude in this forest but termite diversity decreases. Termite species richness in the Atlantic Forest showed a different pattern than those described for other organisms, increasing in diversity where the coastal‐forest strip narrows. Overall, our results indicate comparatively high termite species richness at northeastern sites and a significant impoverishment of termite assemblages in the southeastern and southern regions of the Atlantic Forest.     

Differences in seed rain composition in small and large fragments in the northeast Brazilian Atlantic Forest

Tropical forests are seriously threatened by fragmentation and habitat loss. The impact of fragment size and forest configuration on the composition of seed rain is insufficiently studied. For the present study, seed rain composition of small and large forest fragments (8–388 ha) was assessed in order to identify variations in seed abundance, species richness, seed size and dispersal mode. Seed rain was documented during a 1‐year period in three large and four small Atlantic Forest fragments that are isolated by a sugarcane matrix. Total seed rain included 20,518 seeds of 149 species of trees, shrubs, palms, lianas and herbs. Most species and seeds were animal‐dispersed. A significant difference in the proportion of seeds and species within different categories of seed size was found between small and large fragments. Small fragments received significantly more very small‐sized seeds (<0.3 cm) and less large‐seeded species (>1.5 cm) that were generally very rare, with only one species in small and eight in large fragments. We found a negative correlation between the inflow of small‐sized seeds and the percentage of forest cover. Species richness was lower in small than in large fragments, but the difference was not very pronounced. Given our results, we propose changing plant species pools through logging, tree mortality and a high inflow of pioneer species and lianas, especially in small forest fragments and areas with low forest cover. Connecting forest fragments through corridors and reforestation with local large‐seeded tree species may facilitate the maintenance of species diversity.     

Multilocus tests of Pleistocene refugia and ancient divergence in a pair of Atlantic Forest antbirds (Myrmeciza)

The Atlantic Forest (AF) harbours one of the most diverse vertebrate faunas of the world, including 199 endemic species of birds. Understanding the evolutionary processes behind such diversity has become the focus of many recent, primarily single locus, phylogeographic studies. These studies suggest that isolation in forest refugia may have been a major mechanism promoting diversification, although there is also support for a role of riverine and geotectonic barriers, two sets of hypotheses that can best be tested with multilocus data. Here we combined multilocus data (one mtDNA marker and eight anonymous nuclear loci) from two species of parapatric antbirds, Myrmeciza loricata and M. squamosa, and Approximate Bayesian Computation to determine whether isolation in refugia explains current patterns of genetic variation and their status as independent evolutionary units. Patterns of population structure, differences in intraspecific levels of divergence and coalescent estimates of historical demography fit the predictions of a recently proposed model of refuge isolation in which climatic stability in the northern AF sustains higher diversity and demographic stability than in the southern AF. However, a pre‐Pleistocene divergence associated with their abutting range limits in a region of past tectonic activity also suggests a role for rivers or geotectonic barriers. Little or no gene flow between these species suggests the development of reproductive barriers or competitive exclusion. Our results suggests that limited marker sampling in recent AF studies may compromise estimates of divergence times and historical demography, and we discuss the effects of such sampling on this and other studies.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

Phenotypic evolution of an Atlantic Forest passerine (Xiphorhynchus fuscus): biogeographic and systematic implications

We studied the phenotypic variation of the Atlantic Forest passerine Xiphorhynchus fuscus (Aves: Dendrocolaptidae) with the broad aim of addressing whether the history and type of forest affected the evolution of endemic taxa. We also tested whether the different subspecies and genetic lineages of X. fuscus could be considered full species. We collected plumage and body size measurements and, in combination with genetic data, used multivariate tests to evaluate the working hypotheses. Our results, combined with previous biogeographic analyses, indicate that vicariant events have been important determinants in the evolution of phenotypic characters of X. fuscus, once genetic isolation was complete. Our analysis also suggests that forest heterogeneity and ecotones are important factors in the early evolution of Atlantic Forest taxa, perhaps via divergent selection. Forest instability during the Pleistocene was critical in the evolution of phenotypic traits. We confirm that the subspecies atlanticus should be considered a full species. Other lineages or populations are also phenotypically differentiated but we do not suggest considering them as full species. They share high levels of gene flow and are part of a continuous latitudinal cline of phenotypic variation. Our study suggests that not all the historic events in the Atlantic Forest that affected the evolution of genetic lineages also influenced the evolution of phenotypic characters in the same direction and intensity. Undoubtedly, natural selection played a major role in the evolution of Atlantic Forest organisms. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 1047–1066.     

Pliocene and Pleistocene events shaping the genetic diversity within the central corridor of the Brazilian Atlantic Forest

Dinoponera lucida (Formicidae; Ponerinae) is an extinction‐threatened species of ant which is endemic in the central corridor of the Atlantic Forest. We used mitochondrial sequences of the Cox1, Cox2 and Cytb genes in order to infer some aspects of the evolutionary history and phylogeography of this ant. High genetic divergence and population structure were observed for the whole species. The current pattern of D. lucida diversity seems to be shaped during different geological times: middle Pliocene, early Pleistocene and mainly late Pleistocene, when the reduction of populations generated a structure pattern of the genetic variation of this species. Our data show that this structure results from the maintenance of populations of D. lucida within very small putative refuges to the south of the central Bahia refugium. We thus argue that, for some Atlantic forest endemic species, especially those resistant to very small fragments of forest, such as D. lucida, the small putative refuges were as important as, or even more important than, larger and stable refuges for the creation and maintenance of diversity, adding another piece to the puzzle of the mechanisms underlying local endemism. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 949–960.     

Divide to conquer: a complex pattern of biodiversity depicted by vertebrate components in the Brazilian Atlantic Forest

The identification of northern and southern components in different vertebrate species led researchers to accept a two‐component hypothesis for the Brazilian Atlantic forest (BAF). Nevertheless, neither a formal proposal nor a meta‐analysis to confirm this coincidence was ever made. Our main objective here was therefore to systematically test in how many vertebrate components the BAF could be divided by analysing existing empirical data. We used two approaches: (1) mapping and comparing the proposed areas of vertebrate endemism in the BAF and (2) analysing studies mentioning spatial subdivisions in distinct forest‐dependent vertebrates within the biome, by the use of panbiogeography. The four large‐scale endemism area components together with the six small‐scale panbiogeographical ones allowed the definition of three BAF greater regions, subdivided into nine vertebrate components, latitudinally and longitudinally organized. Empirical time estimates of the diversification events within the BAF were also reviewed. Diversification of these vertebrates occurred not only in the Pleistocene but also throughout the Miocene. Our results confirm the BAF's complex history, both in space and time. We propose that future research should be small‐scale and focused in the vertebrate components identified herein. Given the BAF's heterogeneity, studying via sections will be much more useful in identifying the BAF's historical biogeography. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

Phylogeography and genetic differentiation along the distributional range of the orchid Epidendrum fulgens: a Neotropical coastal species not restricted to glacial refugia

Aim Phylogeographical studies in the Brazilian Atlantic Forest (BAF) have mostly included species associated with forest habitats, whereas taxa associated with grassland and sand‐dune plant communities have so far been largely overlooked. This study examines the phylogeography of the orchid Epidendrum fulgens, which occurs on coastal sand dunes and granitic outcrops, in order to identify major genetic divergences or disjunctions across the range of the species and to investigate the genetic signatures of past range contractions and expansions. Location Southern and south‐eastern seashore vegetation along the BAF biome, and granitic and arenitic outcrops that occur in the subtropical grassland plant communities located south of the BAF. Methods Nine nuclear and four plastid microsatellite loci were used to genotype 424 individuals from 16 populations across the distributional range of E. fulgens. For both sets of markers, we estimated genetic diversity and population differentiation, testing for a north–south gradient of genetic diversity. The plastid haplotype network and a Bayesian assignment analysis of nuclear markers were used to infer population structure. Past demographic changes were investigated using a coalescence approach. Results A deep disjunction was found between northern populations within the BAF and southern populations outside the BAF that occur on granitic and arenitic outcrops. Recent demographic reductions were detected in northern populations on coastal sands. Such demographic changes were not expected for those populations, as previous studies with forest species had found evidence of population expansion in the same areas. Higher genetic diversity was found in southern populations on granite, in contrast to patterns observed in previous studies of forest species. Main conclusions The results are consistent with the long‐term persistence of E. fulgens. Bottlenecks were detected in populations from areas where population expansion events have been detected in other plant (and animal) species, suggesting that forest expansion after the Last Glacial Maximum played a role in the population fragmentation and decrease in genetic diversity in E. fulgens. A substantial genetic division in E. fulgens corresponds to the ‘Portal de Torres’, a region that demarcates the northern limits of subtropical grassland plant communities and the southern limits of the BAF.     

Biological and immunological characteristics of the poison of Bothrops cotiara (Serpentes: Viperidae)

Bothrops cotiara is a venomous snake sporadically found in the province of Misiones in Argentina, South of Brazil and Paraguay. Data on the clinics of the envenomation produced by its bite and on its venom are scarce. There is no information on the neutralizing capacity of the antivenoms available. In this study, the lethal potency, hemorrhagic, necrotizing, coagulant and thrombin-like, defibrinogenating, indirect hemolytic and fibrinolytic activities of the venom of B. cotiara specimens from the province of Misiones were determined. The toxic activities were within the range of those described for the other Bothrops species from Argentina, and the electrophoretic and chromatographic studies showed similarities with those described for the other bothropic venoms. The immunochemical reactivity of six South American anti Viper antivenoms (ELISA) have a strong reactivity with all the antivenoms studied. The neutralizing capacity of three of these therapeutic antivenoms against the lethal potency and hemorrhagic, necrotizing, coagulant, thrombin-like and hemolytic activities showed a very close neutralizing capacity. Our data strongly suggest that the antivenoms for therapeutic use available in this area of South America are useful to neutralize the toxic and enzymatic activities of the venom of this uncommon specie of Bothrops.     

Ethnobotany of Atlantic Forest Coastal Communities: II. Diversity of Plant Uses at Sepetiba Bay (SE Brazil)

This is an ethnobotanical study of Atlantic Forest coastal communities located at Sepetiba Bay, Rio de Janeiro State, Brazil. Atlantic Forest remnants are top priority conservation areas, and include native communities that depend on fish and small-scale agriculture. We conducted fieldwork in the community of Calhaus (Jaguanum Island) from 1989 to 1991, and interviewed adults on their use of plants. We examined the diversity of medicinal plants used among communities of different islands and found results similar to previous research at Gamboa (Itacuruçá Island); communities living in smaller islands and on islands further from the coast use a lower diversity of plants. Also, older islanders show a deeper knowledge of medicinal plants than younger islanders.     

A complex biogeographic history of diversification in Neotropical lancehead pitvipers (Serpentes,Viperidae)

Based on the literature, we had predicted that the diversification within the Neotropical snake genus Bothrops occurred along a latitudinal gradient from north to south, with diversification into unoccupied niches through ecological opportunity, not correlated with geoclimatic events. Using a dated phylogeny and estimating likelihoods of ancestral states at cladogenesis events, we reconstructed ancestral areas and assessed major events of the diversification of Bothrops clades, and we also discuss systematic implications for this group. Based on the phylogeny we produced, B. lojanus was not considered as part of the genus Bothrops since the results recovered this species nested within the Bothrocophias clade. We infer that the diversification of the Miocene Bothrops pictus and Bothrops alternatus clades may be related to the uplift of the western slopes of the Andes and the Argentinian Patagonian Andes, respectively. The Pliocene Bothrops taeniatus and Bothrops osbornei clades may be related to the uplift of the eastern and northern Andes, respectively. The Plio-Pleistocene Bothrops neuwiedi clade may be related to the habitat transitions from a warmer and forested environment to a cooler and open landscape; the Bothrops jararaca (i.e. island endemic species) and Bothrops lanceolatus clades to over-water dispersal with island speciation; and Bothrops atrox clade to the appearance of the Panamanian land bridge. We found that a multitemporal and multidirectional history of diversification may be correlated with geoclimatic and dispersalist events. We argue that the vacant niche hypothesis by itself does not explain Bothrops diversification.     

Complex longitudinal diversification across South China and Vietnam in Stejneger's pit viper,Viridovipera stejnegeri (Schmidt, 1925) (Reptilia: Serpentes: Viperidae)

Viridovipera stejnegeri is one of the most common pit vipers in Asia, with a wide distribution in southern China and Vietnam. We investigated historical demography and explored how the environment and climatic factors have shaped genetic diversity and the evolutionary history of this venomous snake. A total of 171 samples from 47 localities were sequenced and analysed for two mitochondrial gene fragments and three nuclear genes. Gene trees reveal the existence of two well‐supported clades (Southwest China and Southeast China) with seven distinct and strongly supported, geographically structured subclades within V. stejnegeri. Estimation of divergence time and ancestral area suggests that V. stejnegeri originated at ~6.0 Ma in the late Miocene on the Yunnan–Guizhou Plateau. The estimated date of origin and divergence of the island populations of Taiwan and Hainan closely matches the geological origin of the both islands. The mtDNA gene tree reveals the presence of west–east diversification in V. stejnegeri populations. Complex orogenesis and heterogeneous habitats, as well as climate‐mediated habitat differentiation including glacial cycles, all have influenced population structure and the distribution of this taxon. The validity of V. stejnegeri chenbihuii is questionable, and this subspecies most probably represents an invalid taxon.     

Phenotypic plasticity to light of two congeneric trees from contrasting habitats: Brazilian Atlantic Forest versus cerrado (savanna)

The Brazilian Atlantic Forest is a typically multi-layer tropical forest, while cerrado (savanna) is a patchy habitat with different physiognomy. Despite these differences, both habitats have high light heterogeneity. Functional traits of Dalbergia nigra and D. miscolobium from the Atlantic Forest and cerrado, respectively, were evaluated under shade (25% of full sunlight) and full sunlight in a nursery experiment. We hypothesised that both species should benefit from high phenotypic plasticity in relation to light. Plasticity was estimated using the relative distance phenotypic index (RDPI). D. miscolobium had lower shoot growth under both light conditions, suggesting it has low competitive capacity in the forest environment, which could explain its limited ability to expand over areas of Atlantic Forest. The studied species exhibited photoprotection strategies under high light and improved light capture under low light. Stomatal conductance, ETR(max) (maximum electron transport rate), PPFD(sat) (saturating photosynthetically active photon flux density), chlorophyll and carotenoid content had higher RDPI than stem morphological traits. Although both species showed considerable phenotypic plasticity, D. miscolobium had higher RDPI for eight of 11 evaluated traits. This high plasticity could be one of the factors that explain the occurrence of this species in a wide range of environmental conditions, from open grassland to dense woodlands, and it could also reflect its adaptation to high light. D. nigra also had considerable plasticity and good growth performance in both shade and full sunlight, but its absence in areas of cerrado suggests that factors other than light limit its occurrence in these habitats.     

The development of the pit organ of Bothrops jararaca and Crotalus durissus terrificus (Serpentes, Viperidae): support for the monophyly of the subfamily Crotalinae

The development of the pit organ and the course of the lateral branch of the ethmoidal nerve of the two South American crotalines Bothrops jararaca and Crotalus durissus terrificus are described in detail using serially sectioned material from fetal stages. Serial sections of the Asian pit vipers Agkistrodon halys halys and Trimeresurus albolabris were included in this work. The viperines, as the sister‐group of the Crotalinae, and the Colubridae, Elapidae and Boidae as their outgroups were used for comparisons. New synapomorphies of the advanced subfamily Crotalinae were found: namely, absence of the foramen epiphaniale and an extracapsular course of the lateral branch of ethmoidal nerve. These results support the monophyly of the crotalines.