Pleistocene fragmentation of the Amazonian rainforest has been hypothesized to be a major cause of Neotropical speciation and diversity. However, the role and even the reality of Pleistocene forest refugia have attracted much scepticism. In Amazonia, previous phylogeographical studies have focused mostly on organisms found in the forests themselves, and generally found speciation events to have predated the Pleistocene. However, molecular studies of open-formation taxa found both north and south of the Amazonian forests, probably because of vicariance resulting from expansion of the rainforests, may provide novel insights into the age of continuous forest cover across the Amazon basin. Here, we analyse three mitochondrial genes to infer the phylogeography of one such trans-Amazonian vicariant, the Neotropical rattlesnake (Crotalus durissus), which occupies primarily seasonal formations from Mexico to Argentina, but avoids the rainforests of Central and tropical South America. The phylogeographical pattern is consistent with gradual dispersal along the Central American Isthmus, followed by more rapid dispersal into and across South America after the uplift of the Isthmus of Panama. Low sequence divergence between populations from north and south of the Amazon rainforest is consistent with mid-Pleistocene divergence, approximately 1.1 million years ago (Ma). This suggests that the Amazonian rainforests must have become fragmented or at least shrunk considerably during that period, lending support to the Pleistocene refugia theory as an important cause of distribution patterns, if not necessarily speciation, in Amazonian forest organisms. These results highlight the potential of nonforest species to contribute to an understanding of the history of the Amazonian rainforests themselves.

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.

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Aim To investigate the phylogeography and execute a historical-demographic analysis of the Neotropical rattlesnake, Crotalus durissus, thereby testing the hypothesis of a Pleistocene central Amazon corridor of dry forest or savanna that partitioned the Amazonian rain forest into western and eastern portions.Location South America.Methods Using sequences of three mitochondrial genes, we estimated the phylogeography, gene and nucleotide diversity across the South American range of C. durissus. Tree topology tests were used to test alternative biogeographical hypotheses, and tests of population genetic structure and statistical parsimony networks and nested clade phylogeographic analysis (NCPA) were used to infer connectivity and historical population processes on both sides of the Amazon basin.Results Tree topology tests rejected the hypothesis of a coastal dispersal in favour of a central corridor scenario. Gene diversity was similar on both sides of the Amazon basin. Nucleotide diversity indicated that the populations from north of the Amazon basin represented ancestral populations. Analysis of molecular variance (amova) showed that intra-population molecular variation was greater than between regions. Historical-demographic statistics showed significant population expansion south of the Amazon, and little differentiation in the north, indicating moderate past gene flow between north and south of the Amazon. The parsimony network connected clades from the Roraima and Guyana populations with Mato Grosso, suggesting an Amazonian central corridor, and NCPA supported allopatric fragmentation between north and south of the Amazon. Main conclusionsThe distribution of C. durissus on both sides of the Amazon basin is evidence of changes in the distribution of rain forest vegetation during the Pleistocene. Our results suggest a formerly continuous distribution of this rattlesnake along a central Amazonian corridor during the middle Pleistocene. Allopatric fragmentation inferred from NCPA is consistent with vicariance resulting from a subsequent closure of this habitat corridor. This study emphasizes the potential of trans-Amazonian open formation species to inform the debate on the past distribution of rain forests in the Amazon Basin.

Morphological differences between two semi-arboreal, bird-eating island pitvipers, Bothrops insularis and Gloydius shedaoensis, and their mainland relatives were investigated. Mitochondrial DNA sequence data for two genes show B. insularis to be rooted within B. jararaca. Bothrops insularis has a more anterior heart, a relatively longer tail, a longer head, and shorter fangs than B. jararaca. The greater head length is paralleled in the ecologically similar G. shedaoensis. Increased head size may represent an adaptation to the abundance of larger food items (migratory passerine birds), providing a selective advantage to snakes able to switch to larger prey at an earlier age. Furthermore, B. insularis and G. shedaoensis have converged on similar body sizes from opposite ancestral states. Other characters, including fang length, tail length and size of neonates do not show parallel variation in G. shedaoensis and B. insularis, suggesting that caution is required when interpreting character state shifts coinciding with ecological shifts in a single species only.