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.

Microgeographic variation in recruitment under adult trees: arrival of new genotypes or perpetuation of the existing ones?

Investigating spatial variation in the relative importance of sexual reproduction and clonal propagation is critical to obtain more accurate estimates of future effective population sizes and genetic diversity, as well as to identify ecological correlates of clonality. We combined a stratified sampling scheme with microsatellite genetic analyses to estimate variation in the proportion of sexual versus clonal recruits among saplings in five populations of the tree Pyrus bourgaeana. Using a likelihood framework, we identified clones among the genotypes analysed and examined variation among populations regarding the proportion of saplings coming from clonal propagation. We also examined the relationship between the relative abundance of clonal shoots across the studied populations and their herbivory levels. Our results revealed that one third of the saplings examined (N = 225 saplings) had a probability above 0.9 of being clones of nearby (<10 m) trees, with the ratio between clonal propagation and sexual recruitment varying up to eight-fold among populations. A small portion of these putative clonal shoots reached sexual maturity. Relative abundance of clonal shoots did not significantly relate to the herbivory by ungulates. Our results call into question optimistic expectations of previous studies reporting sufficient levels of recruitment under parental trees without animal seed dispersal services. Nevertheless, given that some of these clonal shoots reach sexual maturity, clonal propagation can ultimately facilitate the long-term persistence of populations during adverse periods (e.g. environmental stress, impoverished pollinator communities, seed dispersal limitation).

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.

Antibodies to Mycobacterium bovis in wild carnivores from Doñana National Park (Spain).

We conducted a retrospective serologic survey for antibodies against the MPB70 protein of Mycobacterium bovis in wild carnivores from Doñana National Park (southwestern Spain). Serum samples from 118 red foxes (Vulpes vulpes), 39 Iberian lynx (Lynx pardinus), 31 Eurasian badgers (Meles meles), five Egyptian mongoose (Herpestes ichneumon), four European genet (Genetta genetta), and one Eurasian otter (Lutra lutra) were analyzed using an indirect competitive enzyme-linked immunoassay. Antibodies against the MPB70 protein of M. bovis were detected in seven badgers, five foxes, and one lynx. The frequency of positive animals was significantly higher in badger (23%) than in lynx (3%) and fox (4%). Antibodies were not detected in other species. Annual antibody frequency peaked at 38% in badgers and 11% for red fox. These species may contribute to persistence of bovine tuberculosis in Doñana.

Effects of an attractive sink leading into maladaptive habitat selection.

Habitat sinks can attract dispersing animals if high mortality or breeding failure are difficult to detect (e.g., when due to human hunting or pollution). Using a simple deterministic model, we explore the dynamics of such source-sink systems considering three scenarios: an avoided sink, no habitat preference, and an attractive sink. In the second two scenarios, there is a threshold proportion of sink habitat above which the whole population decreases to extinction, but this extinction threshold varies with habitat preference and the relative qualities of the two habitat types. Hence, it would be necessary to know the habitat preferences of any species in a source-sink system to interpret data on population increases and declines. In the attractive sink scenario, small changes in the proportion of sink habitat may have disproportionate effects on the population persistence. Also, small changes in growth rates at the source and the sink severely affect the threshold and the time of extinction. For some combinations of demographic parameters and proportion of habitat sink, the decline affects the source first; thus, during some time, it will be hidden to population monitoring at the sink, where numbers can even increase. The extinction threshold is also very sensitive to the initial population sizes relative to carrying capacity. Attractive sinks represent a novel aspect of source-sink dynamics with important conservation and management implications.

A comparative analysis of food-niche relationships and trophic guild structure in two assemblages of vertebrate predators differing in species richness: causes, correlations, and consequences.

We conducted a comparative analysis of foodniche relationships and trophic guild structure in two assemblages of vertebrate predators (including hawks, owls, snakes, and carnivores) living in similar habitats of Chile and Spain, which differed in species richness (11 and 25 predator species, respectively), to explore how the structure of predator assemblages reflects an increase (or decrease) in the number of coexisting species. Our results indicate that the Spanish assemblage appears enriched by the symmetrical "addition" of species with disparately large and small values of body weight, diet breadth, and mean prey size, around the same median value found in Chile. Or, alternatively, the Chilean assemblage appears impoverished by the symmetrical "loss" of peripheral species - those at both tails of the frequency distribution of the above trophic estimators. Spanish predators overlap less extensively among themselves, and with smaller variance, than Chilean predators. Consequently, the higher predator richness in Spain is not ascribable to their narrower diet breadths or more extensive diet overlaps as predicted by some theoretical models. Instead, it is associated with a more spread-out use of the available resources. Trophic guilds can be recognized in the two countries, but in Spain guilds are less tightly packed. Some taxomically unrelated species are close diet analogues between Spain and Chile, whereas some closely related species cannot be matched well between countries. Circumstantial evidence suggests that the presence of some trophic guilds and of some specialist predators in either country is based on the high abundance attained by some particular prey types.

Immobilization of free-ranging red foxes (Vulpes vulpes) with tiletamine hydrochloride and zolazepam hydrochloride.

We evaluated Zoletil on free-ranging red foxes (Vulpes vulpes) in Spain. Twenty-two pup and 49 adult wild-caught red foxes (Vulpes vulpes) were immobilized with a combination of tiletamine hydrochloride and zolazepam hydrochloride in a 1:1 proportion (Zoletil). Mean (+/- SE) Zoletil doses were 10.57 (+/- 0.41) mg/kg (range = 7.58-15.39 mg/kg, n = 22) for pups and 10.51 (+/- 0.33) mg/kg (range = 5.88-16.67 mg/kg, n = 45) for adults. Mean induction and first recovery times for pups were 2.3 (+/- 0.2) minutes (range = 1 to 5 minutes) and 35.5 (+/- 3.28) minutes (range = 18 to 78 minutes), respectively. Mean induction and first recovery times for adults were 3.7 (+/- 0.21) minutes (range = 2 to 8 minutes) and 35.4 (+/- 2.22) minutes (range = 13 to 90 minutes), respectively. We recommend Zoletil doses of 10 mg/kg for red foxes. For wild adult red foxes of unknown weight, an initial dose of 60 to 70 mg Zoletil should be administered. This dose should allow about 40 minutes of handling time.

Immobilization of the endangered Iberian lynx with xylazine- and ketamine-hydrochloride.

A combination of the dissociative anesthetic ketamine hydrochloride (KH) and the sedative xylazine hydrochloride (XH) was used to immobilize 31 wild Iberian lynx (Felis pardina) 45 times at Doñana National Park, Spain. A mean (+/- SE) dose of 4.6 (+/- 0.2) mg/kg KH and 4.0 (+/- 0.2) mg/kg XH resulted in mean (+/- SE) induction time of 5.6 (+/- 0.3) min and mean (+/- SE) first reaction time of 59.3 (+/- 6.5) min. Convulsions occurred four times (9%), but with no noteworthy consequences.

Xylazine hydrochloride-ketamine hydrochloride immobilization of free-living red foxes (Vulpes vulpes) in Spain.

A combination of xylazine hydrochloride-ketamine hydrochloride was used to immobilize 83 wild red foxes (Vulpes vulpes) (15 pups and 68 adults) at Doñana National Park (Spain). Mean ketamine hydrochloride doses were 17.1 mg/kg (SE = 1.53) and 12.3 mg/kg (SE = 0.4) for pups and adults, respectively, and mean xylazine hydrochloride doses for the same groups were 6.2 mg/kg (SE = 0.63) and 4.7 mg/kg (SE = 0.14), respectively. Mean induction times and first reaction times were 1.6 minutes and 22.5 minutes for pups and 3.8 minutes and 39.4 minutes for adults, respectively. Recommended doses for wild adult foxes of unknown weight are 75 mg of ketamine hydrochloride and 20 mg of xylazine hydrochloride.

Co-existence between Iberian lynx and Egyptian mongooses: estimating interaction strength by structural equation modelling and testing by an observational study.

We used path analysis to investigate the causal relationships between Iberian lynx and Egyptian mongoose track numbers, and to estimate the direct effect of the former on the latter in the Doñana area (2750 km(2) ), south-western Spain. Relative abundance of rabbits, shrub cover and protection level were also considered in the path analysis. An observational study consisting of a repeated track survey with a split-plot design was also carried out between 1990 and 1996 to independently test the results obtained by path analysis. Maximum likelihood ratio tests of the goodness-of-fit of the model to the data and other indices used all indicated that the proposed theoretical model depicting the relationships among variables was adequate. Egyptian mongooses suffered a significant direct negative effect from lynx, and rabbits and protection level positively, and significantly affected lynx track abundance; rabbits were also significantly affected by shrub cover (all these standardized partial correlation coefficients ranged from 0.34 to 0.41). Shrub cover did not directly affect either lynx or mongoose tracks. Relatively important indirect effects were found between shrubs and lynx (0.12), and between rabbits and mongooses (-0.14), and protection and mongooses (-0.17). The two latter non-intuitive indirect effects were through the lynx path, as a consequence of the negative direct influence of lynx on mongooses. The repeated track censuses confirmed that lynx presence significantly affects number of mongoose tracks. In areas without lynx, mean number of mongoose tracks were 3.6 times higher than in areas with lynx. It remains an interesting question which of several proposed hypotheses explains co-existence of both species in south-western Spain.

Phylogenetic and phylogeographic analysis of Iberian lynx populations.

The Iberian lynx (Lynx pardinus), one of the world's most endangered cat species, is vulnerable due to habitat loss, increased fragmentation of populations, and precipitous demographic reductions. An understanding of Iberian lynx evolutionary history is necessary to develop rational management plans for the species. Our objectives were to assess Iberian lynx genetic diversity at three evolutionary timescales. First we analyzed mitochondrial DNA (mtDNA) sequence variation to position the Iberian lynx relative to other species of the genus LYNX: We then assessed the pattern of mtDNA variation of isolated populations across the Iberian Peninsula. Finally we estimated levels of gene flow between two of the most important remaining lynx populations (Doñana National Park and the Sierra Morena Mountains) and characterized the extent of microsatellite locus variation in these populations. Phylogenetic analyses of 1613 bp of mtDNA sequence variation supports the hypothesis that the Iberian lynx, Eurasian lynx, and Canadian lynx diverged within a short time period around 1.53-1.68 million years ago, and that the Iberian lynx and Eurasian lynx are sister taxa. Relative to most other felid species, genetic variation in mtDNA genes and nuclear microsatellites were reduced in Iberian lynx, suggesting that they experienced a fairly severe demographic bottleneck. In addition, the effects of more recent reductions in gene flow and population size are being manifested in local patterns of molecular genetic variation. These data, combined with recent studies modeling the viability of Iberian lynx populations, should provide greater urgency for the development and implementation of rational in situ and ex situ conservation plans.