Paulo C. Alves

Advancing ecological understandings through technological transformations in noninvasive genetics

Noninvasive genetic approaches continue to improve studies in molecular ecology, conservation genetics and related disciplines such as forensics and epidemiology. Noninvasive sampling allows genetic studies without disturbing or even seeing the target individuals. Although noninvasive genetic sampling has been used for wildlife studies since the 1990s, technological advances continue to make noninvasive approaches among the most used and rapidly advancing areas in genetics. Here, we review recent advances in noninvasive genetics and how they allow us to address important research and management questions thanks to improved techniques for DNA extraction, preservation, amplification and data analysis. We show that many advances come from the fields of forensics, human health and domestic animal health science, and suggest that molecular ecologists explore literature from these fields. Finally, we discuss how the combination of advances in each step of a noninvasive genetics study, along with fruitful areas for future research, will continually increase the power and role of noninvasive genetics in molecular ecology and conservation genetics.

Invasion from the cold past: extensive introgression of mountain hare (Lepus timidus) mitochondrial DNA into three other hare species in northern Iberia

Mitochondrial DNA introgression from Lepus timidus into Lepus granatensis and Lepus europaeus was recently reported in Iberia, although L. timidus presumably retreated from this region at the end of the last ice age. Here we assess the extent of this ancient mtDNA introgression by RFLP analysis of 695 specimens representing the three hare species present in Iberia. The introgressed L. timidus lineage was found in 23 of the 37 populations sampled. It is almost fixed in L. europaeus across its Iberian range in the Pyrenean foothills, and in L. granatensis, which occupies the rest of the peninsula, it is predominant in the north and gradually disappears further south. We also found it in Lepus castroviejoi, a species endemic to Cantabria. Multiple hybridizations and, potentially, a selective advantage for the L. timidus lineage can explain the remarkable taxonomic and geographical range of this mitochondrial introgression.

Genomics and the challenging translation into conservation practice

The global loss of biodiversity continues at an alarming rate. Genomic approaches have been suggested as a promising tool for conservation practice as scaling up to genome-wide data can improve traditional conservation genetic inferences and provide qualitatively novel insights. However, the generation of genomic data and subsequent analyses and interpretations remain challenging and largely confined to academic research in ecology and evolution. This generates a gap between basic research and applicable solutions for conservation managers faced with multifaceted problems. Before the real-world conservation potential of genomic research can be realized, we suggest that current infrastructures need to be modified, methods must mature, analytical pipelines need to be developed, and successful case studies must be disseminated to practitioners.

Ancient introgression of Lepus timidus mtDNA into L. granatensis and L. europaeus in the Iberian Peninsula.

A 587 bp fragment of cytochrome b sequences from 90 individuals of 15 hare (Lepus) species and two outgroups were phylogenetically analysed and compared to an analysis derived from 474 bp sequences of the nuclear transferrin gene. Mountain hare (Lepus timidus) type mtDNA was observed in L. granatensis and L. europaeus from the Iberian Peninsula, far away from the extant distributional range of L. timidus. In addition to these two hare species, other hare species may also contain mtDNA from L. timidus. This species may have introgressed with other species of Lepus that occur within its present range, or where fossils indicate its historical presence during glacial periods. L. timidus mtDNA is common in the northern part of the L. granatensis range. Finally, we reassessed the phylogenetic relationships of the five European hare species based on both mitochondrial and nuclear DNA sequences.

Reference-Free Population Genomics from Next-Generation Transcriptome Data and the Vertebrate–Invertebrate Gap

In animals, the population genomic literature is dominated by two taxa, namely mammals and drosophilids, in which fully sequenced, well-annotated genomes have been available for years. Data from other metazoan phyla are scarce, probably because the vast majority of living species still lack a closely related reference genome. Here we achieve de novo, reference-free population genomic analysis from wild samples in five non-model animal species, based on next-generation sequencing transcriptome data. We introduce a pipe-line for cDNA assembly, read mapping, SNP/genotype calling, and data cleaning, with specific focus on the issue of hidden paralogy detection. In two species for which a reference genome is available, similar results were obtained whether the reference was used or not, demonstrating the robustness of our de novo inferences. The population genomic profile of a hare, a turtle, an oyster, a tunicate, and a termite were found to be intermediate between those of human and Drosophila, indicating that the discordant genomic diversity patterns that have been reported between these two species do not reflect a generalized vertebrate versus invertebrate gap. The genomic average diversity was generally higher in invertebrates than in vertebrates (with the notable exception of termite), in agreement with the notion that population size tends to be larger in the former than in the latter. The non-synonymous to synonymous ratio, however, did not differ significantly between vertebrates and invertebrates, even though it was negatively correlated with genetic diversity within each of the two groups. This study opens promising perspective regarding genome-wide population analyses of non-model organisms and the influence of population size on non-synonymous versus synonymous diversity.

The rise and fall of the mountain hare ( Lepus timidus ) during Pleistocene glaciations: expansion and retreat with hybridization in the Iberian Peninsula

The climatic fluctuations during glaciations have affected differently arctic and temperate species. In the northern hemisphere, cooling periods induced the expansion of many arctic species to the south, while temperate species were forced to retract in southern refugia. Consequently, in some areas the alternation of these species set the conditions for competition and eventually hybridization. Hares in the Iberian Peninsula appear to illustrate this phenomenon. Populations of Iberian hare (Lepus granatensis), brown hare (Lepus europaeus) and broom hare (Lepus castroviejoi) in Northern Iberia harbour mitochondrial haplotypes from the mountain hare (Lepus timidus), a mainly boreal and arctic species presently absent from the peninsula. To understand the history of this past introgression we analysed sequence variation and geographical distribution of mitochondrial control region and cytochrome b haplotypes of L. timidus origin found in 378 specimens of these four species. Among 124 L. timidus from the Northern Palaearctic and the Alps we found substantial nucleotide diversity (2.3%) but little differentiation between populations. Based on the mismatch distribution of the L. timidus sequences, this could result from an expansion at a time of temperature decrease favourable to this arctic species. The nucleotide diversity of L. timidus mtDNA found in Iberian L. granatensis, L. europaeus and L. castroviejoi (183, 70 and 1 specimens, respectively) was of the same order as that in L. timidus over its range (1.9%), suggesting repeated introgression of multiple lineages. The structure of the coalescent of L. granatensis sequences indicates that hybridization with L. timidus was followed by expansion of the introgressed haplotypes, as expected during a replacement with competition, and occurred when temperatures started to rise, favouring the temperate species. Whether a similar scenario explains the introgression into Iberian L. europaeus remains unclear but it is possible that it hybridized with already introgressed L. granatensis.

The ubiquitous mountain hare mitochondria: multiple introgressive hybridization in hares, genus Lepus

Climatic oscillations during the glaciations forced dramatic changes in species distributions, such that some presently temperate regions were alternately occupied by temperate and arctic species. These species could have met and hybridized during climatic transitions. This phenomenon happened for three hare species present in Iberia (Lepus granatensis, Lepus europaeus and Lepus castroviejoi), which display high frequencies of mitochondrial DNA (mtDNA) from Lepus timidus, an arctic/boreal species presently extinct in Iberia. Here, we extend our previous geographical survey to determine whether the distribution of this mtDNA lineage extends beyond the northern half of the Iberian Peninsula, where it is found at high frequencies. We also review the taxonomy, distribution and molecular phylogeny of the genus Lepus. The phylogenetic inference reveals the presence of L. timidus-like mtDNA in several other hare species in Asia and North America, suggesting that the mitochondrial introgression observed in Iberia might be generalized. Comparison with the available nuclear gene phylogenies suggests that introgression could have happened repeatedly, possibly during different climatic transitions. We discuss demographic and adaptive scenarios that could account for the repetition in time and space of this spectacular phenomenon and suggest ways to improve our understanding of its determinants and consequences. Such high levels of introgressive hybridization should discourage attempts to revise hare taxonomy based solely on mtDNA.

Recurrent Introgression of Mitochondrial DNA Among Hares (Lepus spp.) Revealed by Species-Tree Inference and Coalescent Simulations

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.

Hybridization versus conservation: are domestic cats threatening the genetic integrity of wildcats (Felis silvestris silvestris) in Iberian Peninsula?

Cross-breeding between wild and free-ranging domestic species is one of the main conservation problems for some threatened species. The situation of wildcats (Felis silvestris silvestris) in Europe is a good example of this critical phenomenon. Extensive hybridization was described in Hungary and Scotland, contrasting with occasional interbreeding in Italy and Germany. First analyses in Portugal revealed a clear genetic differentiation between wild and domestic cats; however, four hybrids were detected. Here, we extended the approach to Iberian Peninsula using multivariate and Bayesian analyses of multilocus genotypes for 44 Portuguese wildcats, 31 Spanish wildcats and 109 domestic cats. Globally, wild and domestic cats were significantly differentiated (FST=0.20, p<0.001) and clustered into two discrete groups. Diverse clustering methods and assignment criteria identified an additional hybrid in Portugal, performing a total of five admixed individuals. The power of admixture analyses was assessed by simulating hybrid genotypes, which revealed that used microsatellites were able to detect 100, 91 and 85% of first-generation hybrids, second-generation genotypes and backcrosses, respectively. These findings suggest that the true proportion of admixture can be higher than the value estimated in this study and that the improvement of genetic tools for hybrids detection is crucial for wildcat conservation.

Phylogeography of roe deer ( Capreolus capreolus ) populations: the effects of historical genetic subdivisions and recent nonequilibrium dynamics

We sequenced 704 mitochondrial DNA (mtDNA) control‐region nucleotides and genotyped 11 autosomal microsatellites (STR) in 617 European roe deer (Capreolus capreolus) samples, aiming to infer the species’ phylogeographical structure. The mtDNA sequences were split in three distinct haplogroups, respectively, named: Clade West, sampled mainly in Iberia; Clade East, sampled mainly in Greece and in the Balkans; and Clade Central, which was widespread throughout Europe, including the eastern countries and Iberia, but not Greece. These clades might have originated in distinct Iberian and Balkanic refuges during the penultimate or the last glaciations. Clades East and West contributed little to the current postglacial mtDNA diversity in central Europe, which apparently was recolonized mainly by haplotypes belonging to Clade Central. A unique subclade within Clade Central grouped all the haplotypes sampled from populations of the Italian subspecies C. c. italicus. In contrast, haplotypes sampled in central and southern Spain joined both Clade Central and Clade West, suggesting that subspecies C. c. garganta has admixed origin. STR data support a genetic distinction of peripheral populations in north Iberia and southern Italy, and show the effects of anthropogenic disturbance in fragmented populations, which were recently reintroduced or restocked and not may be in mutation–drift equilibrium. Roe deer in central Europe are mainly admixed, while peripheral populations in north Portugal, the southern Italian Apennines and Greece represent the remains of refugial populations and should be managed accordingly.