Inazio Garin

PCR-RFLP identification of mustelid species: European mink (Mustela lutreola), American mink (M. vison) and polecat (M. putorius) by analysis of excremental DNA

Recent advances in molecular scatology have allowed the development of reliable and non-invasive methods that can be applied in monitoring of small carnivores, without disturbance of the animals. Here a method is described that can be used to differentiate European mink Mustela lutreola , polecat M. putorius and American mink M. vison based on the analysis of DNA extracted from faeces. It consists of a nested PCR of a region of the mitochondrial D-loop followed by digestion of the resulting 240 bp amplicons with the restriction enzymes Rsa I and Msp I. The restriction patterns of both enzymes, when used together, are found to detect species-specific sequence variation. Two different haplotypes for European mink (AA, AB), another two for the polecat (AC, AD) and one for American mink (BC) can also be discriminated by this technique. Two new haplotypes for the mitocondrial D-loop of mustelids are described after DNA sequencing.

Detection of alpha and betacoronaviruses in multiple Iberian bat species

Bat coronaviruses (CoV) are putative precursors of the severe acute respiratory syndrome (SARS) CoV and other CoV that crossed the species barrier from zoonotic reservoirs into the human population. To determine the presence and distribution of CoV in Iberian bats, 576 individuals of 26 different bat species were captured in 13 locations in Spain. We report for the first time the presence of 14 coronaviruses in 9 Iberian bat species. Phylogenetic analysis of a conserved CoV genome region (RdRp gene) shows a wide diversity and distribution of alpha and betacoronavirus in Spain. Interestingly, although some of these viruses are related to other European BatCoV, or to Asian CoV, some of the viruses found in Spain cluster in new groups of α and β CoV.

Habitat selection in sympatric Rhinolophus mehelyi and R. euryale (Mammalia: Chiroptera)

Habitat selection was studied in two rhinolophid (Chiroptera) species,Rhinolophusmehelyi andR.euryale, breeding in the same roost in south-west Spain. The first analysis of habitat selection by R. mehelyi is provided, and the hypothesis explored that despite their close relatedness, in sympatry the two bat species showed different habitat preferences. For both species, the least preferred habitats were open habitats such as scrubland, grassland and arable land. As found elsewhere, R. euryale preferred broadleaved woodland. In addition, R. mehelyi also foraged in habitats characterized by loose trees, such as the ‘dehesa’, a semi-natural oak savanna. The progressive disappearance of the dehesa, jeopardized by land-use change, may not harm R. euryale, but could represent a threat to R. mehelyi, which would then be restricted to dense woodland, possibly competing with other bat species, includingR.euryale. To conserve both species, the maintenance of a variety of wooded habitats of differing density is recommended.

Influence of Habitat on the Foraging Behaviour of the Mediterranean Horseshoe Bat, Rhinolophus euryale

In a previous study on habitat use by Rhinolophus euryale in an Atlantic area (Northern Iberian Peninsula), this bat species foraged exclusively in woodland, including both native deciduous woodland and exotic plantations. As the study was carried out in a landscape that was profoundly altered by industrial forestry, we predicted that in a better preserved landscape this species would select smaller feeding areas located at closer distances from the roost, according to the optimal foraging theory, and would use almost exclusively the preferred habitat, i.e., native deciduous woodland. To test these hypotheses, we radiotracked 14 R. euryale from the largest known breeding colony of northern Iberian Peninsula and determined their habitat selection, spatial foraging pattern, and hunting behaviour. Our predictions on habitat selection, as well as on the foraging site size and on commuting range were confirmed. Rhinolophus euryale used almost exclusively native deciduous woodland, and hedgerows were positively selected. We suggest that the richness of tree species in hedgerows provides a high prey availability sustained in time and space. Our findings show that habitat disturbance constitutes a major cause of decline for R. euryale in the study area.

The Foraging Ecology of the Mountain Long-Eared Bat Plecotus macrobullaris Revealed with DNA Mini-Barcodes

Molecular analysis of diet overcomes the considerable limitations of traditional techniques for identifying prey remains in bat faeces. We collected faeces from individual Mountain Long-eared Bats Plecotus macrobullaris trapped using mist nets during the summers of 2009 and 2010 in the Pyrenees. We analysed their diet using DNA mini-barcodes to identify prey species. In addition, we inferred some basic features of the bats foraging ecology that had not yet been addressed. P. macrobullaris fed almost exclusively on moths (97.8%). As prey we detected one dipteran genus (Tipulidae) and 29 moth taxa: 28 were identified at species level (23 Noctuidae, 1 Crambidae, 1 Geometridae, 1 Pyralidae, 1 Sphingidae, 1 Tortricidae), and one at genus level (Rhyacia sp., Noctuidae). Known ecological information about the prey species allowed us to determine that bats had foraged at elevations between 1,500 and 2,500 m amsl (above mean sea level), mostly in subalpine meadows, followed by other open habitats such as orophilous grasslands and alpine meadows. No forest prey species were identified in the diet. As 96.4% of identified prey species were tympanate moths and no evidence of gleaning behaviour was revealed, we suggest P. macrobullaris probably forages by aerial hawking using faint echolocation pulses to avoid detection by hearing moths. As we could identify 87.8% of the analysed sequences (64.1% of the MOTUs, Molecular Operational Taxonomic Units) at species level, we conclude that DNA mini-barcodes are a very useful tool to analyse the diet of moth-specialist bats.

FORAGING BY MEDITERRANEAN HORSESHOE BATS (RHINOLOPHUS EURYALE) IN RELATION TO PREY DISTRIBUTION AND EDGE HABITAT

Abstract We studied the effect of habitat type and prey availability on the foraging decisions of the Mediterranean horseshoe bat (Rhinolophus euryale), a species specialized for cluttered environments. We modeled seasonal habitat selection using radiotelemetry in relation to prey availability in a heterogeneous landscape, determined seasonal diet and prey selection, and used geographic information system data to characterize the landscape surrounding 10 breeding colonies in order to assess the radiotracking results at the population level. Although R. euryale typically has been associated with woodland, our results suggest that the existence of edge habitat, created by semicluttered structures such as hedgerows and woodland edges, was a significant factor in the choice of foraging areas by these bats. Edge habitat was associated with meadows and pastures, creating a landscape highly suited to moths, the preferred prey of R. euryale. In the study area, however, moths were evenly distributed among habitat types; therefore, distribution of moths cannot explain the preference of these bats for semicluttered habitats. The results of our study are consistent with the presumed origin of R. euryale in an edge-rich ecosystem (i.e., the savannahs of northern Africa) and establish a new paradigm for how this species uses habitat. This new paradigm, which might also apply to other members of the genus in Europe, should prompt reconsideration of the presumed habitat requirements for this species, and should be incorporated into the conservation policies for the Mediterranean horseshoe bat.

Novel papillomaviruses in free-ranging Iberian bats: no virus-host co-evolution, no strict host specificity, and hints for recombination.

Papillomaviruses (PVs) are widespread pathogens. However, the extent of PV infections in bats remains largely unknown. This work represents the first comprehensive study of PVs in Iberian bats. We identified four novel PVs in the mucosa of free-ranging Eptesicus serotinus (EserPV1, EserPV2, and EserPV3) and Rhinolophus ferrumequinum (RferPV1) individuals and analyzed their phylogenetic relationships within the viral family. We further assessed their prevalence in different populations of E. serotinus and its close relative E. isabellinus. Although it is frequent to read that PVs co-evolve with their host, that PVs are highly species-specific, and that PVs do not usually recombine, our results suggest otherwise. First, strict virus–host co-evolution is rejected by the existence of five, distantly related bat PV lineages and by the lack of congruence between bats and bat PVs phylogenies. Second, the ability of EserPV2 and EserPV3 to infect two different bat species (E. serotinus and E. isabellinus) argues against strict host specificity. Finally, the description of a second noncoding region in the RferPV1 genome reinforces the view of an increased susceptibility to recombination in the E2-L2 genomic region. These findings prompt the question of whether the prevailing paradigms regarding PVs evolution should be reconsidered.

Diet and prey selection in the Mediterranean horseshoe bat Rhinolophus euryale (Chiroptera, Rhinolophidae) during the pre-breeding season

In May 2001 we collected droppings of Rhinolophus euryale from a colony located in the Basque Country (souhtwestern Europe). Simultaneously, we used Malaise traps to determine prey availability in different habitats, weighting it according to the habitat use of bats. Prey categories found in droppings were then compared to availability to perform the selection analysis. The main consumed prey were by far small lepidopterans (5-11 mm long), followed by tipulids, and scarabaeid beetles (entirely Rhizotrogus sp.). Moths and beetles were selected positively, whereas the other categories were underrepresented in the diet. These results suggest the importance of small moths in the pre-breeding diet of this species, although other items might seasonally play an important role.

Landscape features in the habitat selection of European mink ( Mustela lutreola ) in south-western Europe

Habitat change is one of the main factors influencing the decline of the western population of European mink Mustela lutreola, but data on habitat selection are scarce. Landscape features influencing selection of habitat and resting sites of male European mink were studied using radio-tracking. None of the habitat descriptors accounted for the habitat selection of European mink during their activity periods. On the other hand, resting site selection was correlated with the presence of bramble patches. Intensive use of bramble patches is explained as a consequence of the need of mink for protection against predators. Moreover, the high availability of bramble patches provides the mink with easy resting sites.

Seasonal foraging by Rhinolophus euryale (Rhinolophidae) in an Atlantic rural landscape in northern Iberian Peninsula

ABSTRACT We investigated the foraging behaviour of the Mediterranean horseshoe bat (Rhinolophus euryale) in an Atlantic mosaic-like landscape consisting predominantly of meadows and broadleaved tree structures, and containing the largest known breeding colony in northern Iberian Peninsula. We radio-tracked a total of 46 individuals during pre-breeding, lactation and post-lactation periods. Bats were divided into different classes based on season, sex, reproductive condition or age. Significant differences between classes were apparent in the distances travelled to foraging areas. During pre-breeding foraging occurred on average within 1.3 km, and at most 4.2 km from the roost. Contrary to our predictions, lactating females extended mean foraging distances to 4.3 km, and covered the widest range with a maximum individual distance from the roost of 9.2 km. Males in the same period foraged closer (mean 1.9 km), but with lower fidelity to the main roost. The foraging distances of adult bats during post-lactation did not differ from that of lactating females (mean 4.6 km). Newly volant juveniles flew on average 2.6 km, though showed a varied behaviour. No seasonal effect was found on the size of individual foraging home ranges, as great variation was recorded within all groups. We conclude that the increase in foraging distances is the consequence of higher density as colony size increased by 55% from pre-breeding into the lactation period on. Our results show another aspect of the scale of foraging movements of R. euryale and highlight the need to take these into account when formulating conservation policy, especially during such crucial periods as lactation and juvenile dispersal.