Validation of loop-mediated isothermal amplification for fast and portable sex determination across the phylogeny of birds.

PCR is a universal tool for the multiplication of specific DNA sequences. For example, PCR-based sex determination is widely used, and a diversity of primer sets is available. However, this protocol requires thermal cycling and electrophoresis, so results are typically obtained in laboratories and several days after sampling. Loop-mediated isothermal amplification (LAMP) is an alternative to PCR that can take molecular ecology outside the laboratory. Although its application has been successfully probed for sex determination in three species of a single avian Family (raptors, Accipitridae), its generality remains untested and suitable primers across taxa are lacking. We designed and tested the first LAMP-based primer set for sex determination across the modern birds (NEO-W) based on a fragment of the gene chromo-helicase-DNA-binding protein located on the female-specific W chromosome. As nucleotide identity is expected to increase among more related taxa, taxonomically targeted primers were also developed for the Order Falconiformes and Families Psittacidae, Ciconiidae, Estrildidae and Icteridae as examples. NEO-W successfully determined sex in a subset of 21 species within 17 Families and 10 Orders and is therefore a candidate primer for all modern birds. Primer sets designed specifically for the selected taxa correctly assigned sex to the evaluated species. A short troubleshooting guide for new LAMP users is provided to identify false negatives and optimize LAMP reactions. This study represents the crucial next step towards the use of LAMP for molecular sex determination in birds and other applications in molecular ecology.

Habitat use, but not gene flow, is influenced by human activities in two ecotypes of Egyptian fruit bat (Rousettus aegyptiacus).

Understanding the ecological, behavioural and evolutionary response of organisms to changing environments is of primary importance in a human-altered world. It is crucial to elucidate how human activities alter gene flow and what are the consequences for the genetic structure of a species. We studied two lineages of the Egyptian fruit bat (Rousettus aegyptiacus) throughout the contact zone between mesic and arid Ecozones in the Middle East to evaluate the species' response to the growing proportion of human-altered habitats in the desert. We integrated population genetics, morphometrics and movement ecology to analyse population structure, morphological variation and habitat use from GPS- or radio-tagged individuals from both desert and Mediterranean areas. We classified the spatial distribution and environmental stratification by describing physical-geographical conditions and land cover. We analysed this information to estimate patch occupancy and used an isolation-by-resistance approach to model gene flow patterns. Our results suggest that lineages from desert and Mediterranean habitats, despite their admixture, are isolated by environment and by adaptation supporting their classification as ecotypes. We found a positive effect of human-altered habitats on patch occupancy and habitat use of fruit bats by increasing the availability of roosting and foraging areas. While this commensalism promotes the distribution of fruit bats throughout the Middle East, gene flow between colonies has not been altered by human activities. This discrepancy between habitat use and gene flow patterns may, therefore, be explained by the breeding system of the species and modifications of natal dispersal patterns.

Sex determination in the wild: a field application of loop-mediated isothermal amplification successfully determines sex across three raptor species.

PCR-based methods are the most common technique for sex determination of birds. Although these methods are fast, easy and accurate, they still require special facilities that preclude their application outdoors. Consequently, there is a time lag between sampling and obtaining results that impedes researchers to take decisions in situ and in real time considering individuals' sex. We present an outdoor technique for sex determination of birds based on the amplification of the duplicated sex-chromosome-specific gene Chromo-Helicase-DNA binding protein using a loop-mediated isothermal amplification (LAMP). We tested our method on Griffon Vulture (Gyps fulvus), Egyptian Vulture (Neophron percnopterus) and Black Kite (Milvus migrans) (family Accipitridae). We introduce the first fieldwork procedure for sex determination of animals in the wild, successfully applied to raptor species of three different subfamilies using the same specific LAMP primers. This molecular technique can be deployed directly in sampling areas because it only needs a voltage inverter to adapt a thermo-block to a car lighter and results can be obtained by the unaided eye based on colour change within the reaction tubes. Primers and reagents are prepared in advance to facilitate their storage at room temperature. We provide detailed guidelines how to implement this procedure, which is simpler (no electrophoresis required), cheaper and faster (results in c. 90 min) than PCR-based laboratory methods. Our successful cross-species application across three different raptor subfamilies posits our set of markers as a promising tool for molecular sexing of other raptor families and our field protocol extensible to all bird species.

Phylogeography of Southern Water Vole (Arvicola sapidus): evidence for refugia within the Iberian glacial refugium?

The role of Southern European peninsulas as glacial refugia for temperate species has been widely established, but phylogeographic patterns within refugia are being only recently addressed. Here we describe the phylogeographic patterns for Southern water vole (Arvicola sapidus) in its whole distribution across Iberia and France. Control region and cytochrome b sequences were obtained for 228 samples from 130 localities across Iberia and France. Eighty-five haplotypes were found in total yielding a high overall mitochondrial diversity (pi = 0.027; H = 0.974). Phylogeographic structure was relatively shallow (3.1% average intraspecific divergence) with few supported clades and 95% and 90% maximum parsimony unconnected networks, but significant, as reflected in increased pairwise nucleotide divergences with distance (r = 0.197, P = 0.03) and significant autocorrelation up to approximately 500 km. Spatial analysis of molecular variance analysis detected seven geographical groups explaining 43.73% of the total mitochondrial variation. We detected demographic expansions in three of these groups. A recent colonization of France from Iberia was suggested and estimated around 62 000 years bp by an isolation-with-migration model. Our results suggest the contribution of episodes of isolation in glacial subrefugia in Iberia, but seem to exclude a long-term isolation over successive glacial cycles. Phylogeographic divergence was probably tempered by relatively large population sizes and rapid and extensive mixing among subrefugia during interglacials, that might have eroded the phylogeographic structure accumulated at glacial peaks. Phenotypic differences in A. sapidus do not delineate historically isolated intraspecific divisions and do not warrant subspecific delimitations. Our results do support the existence of subrefugia within Iberia and their role in promoting intraspecific divergences.