Estimation of phylogeographic patterns and divergence times of populations of small mammals from the Iberian Peninsula using genomic data

Abstract

Phylogenetic and phylogeographic methods allow studying the evolutionary relationships between populations and species, patterns of genetic structure, and historical events that have modelled the current distribution of biodiversity. Molecular markers are a fundamental complement of morphological data to accomplish these studies. The divergence time between species and populations is among the most useful parameters to place evolutionary events in the proper historical context as well as to assess population differentiation, evolutionary units and taxonomic categories, but it is difficult to estimate. The emergence of coalescent theory indicated that different genes can reflect different histories and that the coalescence of genes might have occurred much before the actual divergence between populations or species, a phenomenon that is crucial to know divergence times and evidences the importance of multilocus analysis. The Iberian Peninsula played a major role in shaping the distribution of present biodiversity as it harbored different Pleistocene refugia that were the source of recolonizations of other European areas and created internal diversity within this peninsula. In this thesis, two Eurosiberian species, Neomys fodiens and Arvicola scherman, both of which have received little attention despite their biogeographical interest, have been used to advance in this framework of study. To obtain samples of both species, an important effort to optimize laboratory protocols and bioinformatic analysis was made to enable the work with minimally invasive samples. In chapter 1, a subspecies of the Eurasian water shrew, Neomys fodiens niethammeri, which is found in a narrow strip of the northern Iberian Peninsula, was studied. This subspecies presents an abrupt increase in skull size when compared to the rest of the Eurasian population, which has led to the suggestion that it is actually a different species. Skulls obtained from owl pellets collected over the last 50 years allowed us to perform a morphometric analysis in addition to an extensive multilocus analysis based on short intron fragments successfully amplified from these degraded samples. Interestingly, no genetic divergence was detected using either mitochondrial or nuclear data. Additionally, an allele frequency analysis revealed no significant genetic differentiation. The absence of genetic divergence and differentiation revealed here indicated that the large form of N. fodiens does not correspond to a different species and instead represents an extreme case of size increase, of possible adaptive value, which deserves further investigation. In chapter 2, the divergence time between two Iberian populations of the Montane water vole (Arvicola scherman) was estimated using double-digest restriction site-associated DNA (ddRAD) obtained through next-generation sequencing (NGS), including skulls from barn owl pellets among the samples, and applying an isolation-with-migration (IM) analysis. The Cantabrian and Pyrenean populations of A. scherman are geographically isolated and have subtle morphological differences between them. Thousands of single nucleotide polymorphisms (SNPs) derived from the ddRAD were used to study their genetic structure. In addition, a bioinformatic pipeline was developed to find orthologues of the ddRAD loci in other rodents and estimate specific mutation rates using a fossil calibration point (the mouse-rat split). By this means, 85 loci were calibrated. The IMa3 software was then used together with the specific mutation rates of the calibrated loci in order to estimate divergence times and other demographic parameters (population sizes and migration rates). The length of the markers used (145 base pairs) was small compared to other loci generally used with IM models, so the inclusion of only the calibrated loci did not provide enough information. However, it was demonstrated that using a set of 300 ddRAD loci, including the 85 with estimated mutation rates, resulted in good mixing and convergence of the model. The results of the analysis allowed concluding that the two Iberian populations of A. scherman diverged 39 thousand years (Kyr) ago with a 95% highest posterior density interval of 21 – 62 Kyr. A small amount of migration was detected between both populations. Based on this split time and the genetic structure found, it is suggested that the two populations have been present in isolated Iberian refugia at least since the last glaciation and are not the product of a recent colonization of the norther Iberian strip. The methodology developed here based on ddRAD data and coalescent theory allows estimating recent divergence times with high accuracy and comparing the results between different taxa. This can be of great help to understand the generation of diversity in a relatively small geographical area such as the Iberian Peninsula and determine whether the populations have differentiated for enough time to deserve separate taxonomic or conservation status.