Juan L. García-Mudarra

The Iberian contribution to cryptic diversity in European bats

ABSTRACT We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples from the rest of Europe. In this general screening, almost 20% of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other European samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16%, indicating that these taxa represent a complex of several biological species. Other well-differentiated lineages (K2P distances between 5–10%) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at least, Switzerland.

Bat predation on nocturnally migrating birds

Bat predation on birds is a very rare phenomenon in nature. Most documented reports of bird-eating bats refer to tropical bats that occasionally capture resting birds. Millions of small birds concentrate and cross over the worlds temperate regions during migration, mainly at night, but no nocturnal predators are known to benefit from this enormous food resource. An analysis of 14,000 fecal pellets of the greater noctule bat (Nyctalus lasiopterus) reveals that this species captures and eats large numbers of migrating passerines, making it the only bat species so far known that regularly preys on birds. The echolocation characteristics and wing morphology of this species strongly suggest that it captures birds in flight.

Phylogeny and systematics of Old World serotine bats (genus Eptesicus , Vespertilionidae, Chiroptera): an integrative approach

Integrative taxonomy aims to document biodiversity by incorporating all useful characters to increase confidence in hypotheses about phylogenetic relationships. In this study, we combine data obtained independently from morphology, two maternally inherited mtDNA genes and two biparentally inherited nuDNA genes to make phylogenetic and taxonomic hypotheses about the Palaearctic members of the bat genus Eptesicus (Vespertilionidae). This genus is distributed worldwide (except for Antarctica) and is highly diversified, presenting one of the most entangled taxonomic puzzles among all mammals. Our results support restoring the genus Rhyneptesicus and separating E. isabellinus and E. pachyomus from E. serotinus and E. ognevi and E. anatolicus from E. bottae. Differences in the phylogenetic hypotheses from mtDNA and nuDNA data suggest the occurrence within E. serotinus of evolutionary processes such as mtDNA capture and secondary contacts between partially differentiated ecomorphs. These two evolutionary processes deserve more in‐depth studies within the group.

Presence of Plecotus macrobullaris (Chiroptera: Vespertilionidae) in the Pyrenees

In July 2002, several bats of the genus Plecotus (Geoffroy, 1818) were captured at two localities of ‘Ordesa y Monte Perdido’ National Park (Central Pyrenees, Spain). They showed external characters that appeared intermediate between those of P. auritus and P. austriacus. Morphometric and genetic analysis have revealed that these long-eared bats should be ascribed to the recently recognized species P. macrobullaris. This study extends the distribution of this new species, whose western limit was considered, until now, to lie in the Alps, and adds a new mammal species to the Iberian fauna.

Presence of Myotis alcathoe Helversen & Heller, 2001 (Chiroptera: Vespertilionidae) in the Iberian Peninsula

In a survey of bats from La Rioja (Spain), several specimens of the mystacinus group were captured at different mountain localities. Genetic and morphologic analyses have revealed the presence of two lineages within this group in La Rioja. The lineages have been identified as corresponding to two different species: Myotis mystacinus sensu stricto and the recently described M. alcathoe. Both species were found using the same nocturnal refugia (caves) and the same forest habitats. This study extends the distribution of M. alcathoe west and southwards and adds a new mammal species to the Iberian fauna.

Single-tube HotSHOT technique for the collection, preservation and PCR-ready DNA preparation of faecal samples: the threatened Cabrera's vole as a model

A cost-effective, reliable and efficient method of obtaining DNA samples is essential in large-scale genetic analyses. This study examines the possibility of using a threatened vole species, Microtus cabrerae, as a model for the collection and preservation of faecal samples for subsequent DNA extraction with a protocol based on the HotSHOT technique. Through the examination of the probability of multi-copies (mitochondrial) and single copy (microsatellite) loci amplification (including the genotype error) and of the DNA yield (estimated by real-time qPCR), the new protocol was compared with both the frequently employed methods that successfully use ethanol to preserve faecal samples and with commercial kit-based DNA extraction. The single-tube HotSHOT-based protocol is a user-friendly, non-polluting, time-saving and inexpensive method of faeces sample collection, preservation and PCR-quality gDNA preparation. This technique therefore provides researchers with a new approach that can be employed in high-throughput, noninvasive genetic analyses of wild animal populations.

Concealed by darkness: interactions between predatory bats and nocturnally migrating songbirds illuminated by DNA sequencing

Recently, several species of aerial‐hawking bats have been found to prey on migrating songbirds, but details on this behaviour and its relevance for bird migration are still unclear. We sequenced avian DNA in feather‐containing scats of the bird‐feeding bat Nyctalus lasiopterus from Spain collected during bird migration seasons. We found very high prey diversity, with 31 bird species from eight families of Passeriformes, almost all of which were nocturnally flying sub‐Saharan migrants. Moreover, species using tree hollows or nest boxes in the study area during migration periods were not present in the bats’ diet, indicating that birds are solely captured on the wing during night‐time passage. Additional to a generalist feeding strategy, we found that bats selected medium‐sized bird species, thereby assumingly optimizing their energetic cost‐benefit balance and injury risk. Surprisingly, bats preyed upon birds half their own body mass. This shows that the 5% prey to predator body mass ratio traditionally assumed for aerial hunting bats does not apply to this hunting strategy or even underestimates these animals’ behavioural and mechanical abilities. Considering the bats’ generalist feeding strategy and their large prey size range, we suggest that nocturnal bat predation may have influenced the evolution of bird migration strategies and behaviour.

A molecular approach to the study of avian DNA in bat faeces

The molecular identification of prey in faeces is an efficient non-invasive technique to study diet which requires both a satisfactory method of DNA extraction and the design of specific primers to selectively amplify preys DNA. In this study we evaluated and compared the efficiency of two total DNA extraction methods and five primer pairs for the molecular identification of birds from scats, in particular from the giant noctule bat (Nyctalus lasiopterus). A modified DNA stool Mini Kit of Qiagen was tested against a modified silica method with a guanidinium thiocianate (GuSCN) applied after freezing and pulverizing the samples. We also checked two published vertebrate- and bird-generalist primer pairs and three bird-specific primer pairs designed by us (two pairs targeting the cytochrome b and one the cytochrome oxidase subunit I genes) that amplified shorter DNA fragments. The results show that pulverizing the scat remains before extraction was a very important step, presumably facilitating access to the well preserved DNA located inside the rachis of the feathers. The combination of our bird-specific designed primers showed a higher amplification rate than the generalist primers and allowed successful bird identification from the feathers excreted by the giant noctule bat in all the scat samples analyzed, independent of the preservation method used (dried and frozen). These methodological improvements will allow not only the study of the avian diet composition of the enigmatic giant noctule, but the extension of this methodology to other bird predators such as raptors.