Joana Paupério

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia.

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.

Genetic identification of Iberian rodent species using both mitochondrial and nuclear loci: application to noninvasive sampling.

Species identification through noninvasive sampling is increasingly used in animal conservation genetics, given that it obviates the need to handle free‐living individuals. Noninvasive sampling is particularly valuable for elusive and small species such as rodents. Although rodents are not usually assumed to be the most obvious target for conservation, of the 21 species or near‐species present in Iberia, three are considered endangered and declining, while several others are poorly studied. Here, we develop a genetic tool for identifying all rodent species in Iberia by noninvasive genetic sampling. To achieve this purpose, we selected one mitochondrial gene [cytochrome b (cyt‐b)] and one nuclear gene [interphotoreceptor retinoid‐binding protein (IRBP)], which we first sequenced using tissue samples. Both genes allow for the phylogenetic distinction of all species except the sibling species Microtus lusitanicus and Microtus duodecimcostatus. Overall, cyt‐b showed higher resolution than IRBP, revealing a clear barcoding gap. To allow these markers to be applied to noninvasive samples, we selected a short highly diagnostic fragment from each gene, which we used to obtain sequences from faeces and bones from owl pellets. Amplification success for the cyt‐b and IRBP fragment was 85% and 43% in faecal and 88% and 64% in owl‐pellet DNA extractions, respectively. The method allows the unambiguous identification of the great majority of Iberian rodent species from noninvasive samples, with application in studies of distribution, spatial ecology and population dynamics, and for conservation.

The usefulness of field data and hunting statistics in the assessment of wild rabbit (Oryctolagus cuniculus) conservation status in Portugal

Context. The wild rabbit (Oryctolagus cuniculus) is a keystone species from the Iberian Peninsula that has suffered a strong decline in Spain during the past decades. Data on historical and current population trends in Portugal are virtually non-existent. Aims. To investigate changes in rabbit abundance at the national level so as to inform conservation status assessments, and to evaluate the usefulness of hunting bags as a rabbit abundance index. Methods. Field surveys based on latrine counts in linear transects were performed in two periods (1995 and 2002) to assess variation in population abundance. Hunting bags were also analysed for the same period to verify whether these data showed the same trends. General trends of rabbit abundance were estimated using TRIM software. Key results. Field data revealed that most of the sampling units across Portugal have low abundances, despite the observation of local high-density spots. A population reduction of 27% was estimated. Although some fluctuations were observed in hunting bags, global results obtained from these data suggest a slightly increasing trend in rabbit abundance. Conclusions. A discrepancy between field data and hunting statistics was observed. Because hunting bags may be influenced by sporadic management operations undertaken by hunters and the lack of systematic procedures in data collection, we believe that hunting statistics are not representative of real changes in rabbit populations. Thus, observed reduction in rabbit abundance estimated by field data, combined with the high initial morbidity due to rabbit haemorrhagic disease (RHD) after 1988 and the potential for continuing decline in population trends because of other factors, led to an inference of a reduction of >30% in rabbit abundance in Portugal during the past decade. Implications. Taking into account the estimated reduction, the species’ biology and socioeconomic implications, wild rabbit in Portugal was listed in 2005 in the Near Threatened category under IUCN criteria. Because of its ecological and economic importance, this classification prompted the definition of several conservation actions aimed at the recovery of the species in Portugal.

Diet of the Iberian hare (Lepus granatensis) in a mountain ecosystem

The diet of the Iberian hare (Lepus granatensis) was studied through microhistological pellet analysis in two areas from a mountain ecosystem in Central Portugal. Fecal pellets were collected monthly in 24 plots spatially distributed throughout the two study areas. For each period, a sample of 15 to 20 pellets was milled and 400 epidermal fragments were identified, by comparison with a reference collection. A wide range of plant species was observed in hare’s diet. Grasses represent the basis of the Iberian hare diet, with frequencies always higher than 50% in both study areas (annual average = 69.98%). Most of the 35 species of grasses assembled for the reference collection (91.43%) were identified in the pellets. Nevertheless, only six of these were consumed in proportions greater than 5%, being Anthoxanthum odoratum, Secale cereale and Agrostis spp. the species ingested in higher frequencies. The rate of grasses consumption reached 80.69% in winter but decreased in summer to around 55%. In this season, a concurrent rise in the ingestion of other plant groups, like herbs and shrubs, and of plant inflorescences was observed. This work provides the first results on the Iberian hare’s diet on mountain ecosystems, and suggests that the Iberian hare diet in a mountain ecosystem is similar to the observed in L. europaeus and L. timidus.

Endemic species may have complex histories: within-refugium phylogeography of an endangered Iberian vole

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near‐threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast‐disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex‐linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well‐supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera voles detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

Molecular and morphological insights into the origin of the invasive greater white-toothed shrew (Crocidura russula) in Ireland

Identifying routes of invasion is a critical management strategy in controlling the spread of invasive species. This is challenging however in the absence of direct evidence. Therefore, indirect methodologies are used to infer possible invasion sources and routes, such as comparisons of genetic and morphological data from populations from invasive ranges and putative source areas. The greater white-toothed shrew (Crocidura russula) was first discovered in Ireland from skeletal remains in the pellets of birds of prey collected in 2007 and is it is now sufficiently established that the species has a detrimental impact on Ireland’s small mammal community. In this study, we address the uncertain origin(s) of the Irish population of C. russula. The cytochrome b gene of mitochondrial DNA was analysed from 143 individuals from throughout its range within a phylogenetic and approximate Bayesian computation framework. These analyses revealed that the Irish population stemmed from Europe as opposed to North Africa. Additionally, mandibles from 523 individuals from Ireland and 28 other European populations were subjected to multivariate and distance-based analyses, which demonstrated an association between the Irish population and those in France, Switzerland and Belgium. When the genetic and morphological analyses were considered together, an origin stemming from France was deemed the most likely scenario for the source of the invasive Irish population. This study has demonstrated the importance of utilising a multidisciplinary approach when attempting to identify the origins and invasion routes of invasive species.

Giant sex chromosomes retained within the Portuguese lineage of the field vole (Microtus agrestis)

The field vole (Microtus agrestis) is characterised by extremely large blocks of heterochromatin on both the X and Y chromosome. Some other Microtus also have blocks of heterochromatin on their sex chromosomes but not as extensive and always of independent origin from the heterochromatic expansion found in M. agrestis. Coupled with evidence of geographic variation in large heterochromatic blocks within other species (e.g. in the western hedgehog Erinaceus europaeus), it might be expected that field voles would show substantial variation in size and disposition of the sex chromosome heterochromatin. In fact, only minor variation has been described up to now. Those studies conducted previously were largely on field voles from central and northern Europe. Here, we describe the karyotype of field voles from Portugal, of interest because recent molecular studies have shown field voles from western Iberia to be a separate evolutionary unit that might be considered a cryptic species, distinct from populations further to the east. The two Portuguese field voles (one female, one male) that we examined also had essentially the same karyotype as seen in other field voles, including the giant sex chromosomes, but with small differences in the structure of the Y chromosome from that described previously. The finding that field voles throughout Europe show relatively little variation in their giant sex chromosomes is consistent with molecular data which suggest a recent origin for this complex of species/near-species.

Genetic non-invasive sampling (gNIS) as a cost-effective tool for monitoring elusive small mammals

Genetic non-invasive sampling (gNIS) may provide valuable information for population monitoring, as it allows inferences of population density and key behavioural traits such as dispersal, kinship and reproduction. Despite its enormous potential, gNIS has rarely been applied to small mammals, for which live-trapping is still the most commonly used sampling method. Here we evaluated the applicability and cost-effectiveness of gNIS compared with live-trapping, to monitor a metapopulation of an Iberian endemic and elusive rodent: the Cabrera vole (Microtus cabrerae). We compared the genetic diversity, kinship and dispersal movements inferred using both methods. For that, we optimised microsatellite markers for individual identification of M. cabrerae, using both tissue (n = 31) and faecal samples (n = 323) collected from a metapopulation in south-western Iberia. An initial set of 20 loci was optimised for tissue samples, from which 11 were selected to amplify in faecal samples. Overall, gNIS revealed a higher number of identified individuals (65) than live-trapping (31), and the estimated genetic diversity was similar using data from tissues and gNIS. Kinship analysis showed a higher number of inferred relationships and dispersal events when including gNIS, and indicated absence of sex-biased dispersal. The total cost (fieldwork and genetic analysis) of each genotype obtained through live-trapping was three times greater than for gNIS. Our data strongly supports the high potential and cost-effectiveness of gNIS for monitoring populations of elusive and/or threatened small mammals. We also illustrate how this genetic tool can be logistically feasible in conservation.

The Microtus voles: Resolving the phylogeny of one of the most speciose mammalian genera using genomics

Sequential rapid radiations pose some of the greatest difficulties in phylogenetics, especially when analysing only a small number of genetic markers. Given that most of the speciation events occur in quick succession at various points in time, this creates particular challenges in determining phylogenetic relationships, i.e. branching order and divergence times. With the development of high throughput sequencing, thousands of markers can now readily be used to tackle these issues. Microtus is a speciose genus currently composed of 65 species that evolved over the last 2 million years. Although it is a well-studied group, there is still phylogenetic uncertainty at various divergence levels. Building upon previous studies that generally used small numbers of mitochondrial and/or nuclear loci, in this genomic-scale study we used both mitochondrial and nuclear data to study the rapid radiation within Microtus, using partial mitogenomes and genotyping-by-sequencing (GBS) on seven species representing five Microtus subgenera and the main biogeographic ranges where this group occurs. Both types of genome (mitochondrial and nuclear) generated similar tree topologies, with a basal split of the Nearctic (M. ochrogaster) and Holarctic (M. oeconomus) species, and then a subdivision of the five Palearctic species into two subgroups. These data support the occurrence of two European radiations, one North American radiation, and a later expansion of M. oeconomus from Asia to both Europe and North America. We further resolved the positioning of M. cabrerae as sister group of M. agrestis and refute the claim that M. cabrerae should be elevated to its own genus (Iberomys). Finally, the data support ongoing speciation events, especially within M. agrestis, with high levels of genetic divergence between the three Evolutionarily Significant Units (ESUs) previously identified. Similar high levels of divergence were also found among ESUs within M. oeconomus and M. arvalis.

Coccidiosis in European rabbit (Oryctolagus cuniculus algirus) populations in the Iberian Peninsula

The European rabbit Oryctolagus cuniculus is a keystone species from the Iberian Peninsula where viral diseases have played a prominent role in regulating their populations. Coccidiosis, a parasitic disease caused primarily by Eimeria spp., is also thought to have important negative effects. However, few studies have investigated the impact of coccidia on wild European rabbit populations on the Iberian Peninsula. Here we estimate coccidian prevalence in rabbit faecal samples collected along transects established in two ecological regions. Six Eimeria species, with different pathogenicity, were identified (E. coecicola, E. perforans, E media, E. magna, E. irresidua and E. flavescens). Species diversity varied significantly between regions although mean oocyst excretion levels were generally low in both areas (57.61 s.d.±78.07 and 17.03 s.d.±27.72, oocyst per gram of rabbit faeces). This study is the first to describe the composition of the Eimeria spp. assemblage for wild rabbit populations on the Iberian Peninsula and provides fundamental information for future studies on the potential interaction of viral and parasitic diseases.