Madalena Branco

Phylogeography of the European rabbit (Oryctolagus cuniculus) in the Iberian Peninsula inferred from RFLP analysis of the cytochrome b gene

We studied mitochondrial DNA variation in the European rabbit through the examination of restriction fragment length polymorphism in 526 individuals from 20 locations spread across the Iberian Peninsula. Digestion with eight enzymes of a 1120-bp fragment comprising most of the cytochrome b gene resolved 38 different haplotypes. These haplotypes were distributed in two highly divergent clades, with different but overlapping geographical distributions, and with comparable levels of within-clade variation. The overall phylogeographical pattern suggests a history of long-term regional isolation of two groups of rabbit populations, compatible with the recognition of two subspecies within the Iberian Peninsula, followed by recent contact and admixture. The underlying cause is sought in the alternation of glacial and interglacial periods in the late Pleistocene.

POSTGLACIAL DISPERSAL OF THE EUROPEAN RABBIT (ORYCTOLAGUS CUNICULUS) ON THE IBERIAN PENINSULA RECONSTRUCTED FROM NESTED CLADE AND MISMATCH ANALYSES OF MITOCHONDRIAL DNA GENETIC VARIATION

Abstract Nested clade analysis was applied to cytochrome b restriction site data previously obtained on 20 natural populations of the European rabbit across the Iberian Peninsula to test the hypothesis of postglacial dispersal from two main refugia, one in the northeast and the other in the southwest. Apart from historical fragmentation that resulted in geographic discontinuity of two distinct mitochondrial DNA (mtDNA) clades A and B, patterns of haplotype genetic variability have been shaped mostly by restricted gene flow via isolation by distance. The distribution of tip versus interior haplotypes suggests that dispersal occurred from both the southwestern and northeastern groups. Dispersal from the southwest had a north and northwest direction, whereas from the northeast it had mostly a western and southern orientation, with subsequent overlap in a southeastern‐northwestern axis across the Iberian Peninsula. The analysis of the pairwise mismatch distribution of a 179–181‐bp fragment of the mtDNA control region, for seven of those populations, further supports the idea that major patterns of dispersal were in the direction of central Iberia. Additionally, rabbit populations do not show signs of any significant loss of genetic diversity in the recent past, implying that they maintained large population sizes and structure throughout the ice ages. This is congruent with the fact that the Iberian Peninsula was itself a glacial refugium during Quaternary ice ages. Nonetheless, climatic oscillations of this period, although certainly milder than in northern Europe, were sufficient to affect the range distributions of Iberian organisms.

The evolutionary history of the European rabbit (Oryctolagus cuniculus): major patterns of population differentiation and geographic expansion inferred from protein polymorphism

The patterns of population differentiation and geographical expansion of the European rabbit (Oryctolagus cuniculus) remain largely unknown. Using gene frequency data for 20 polymorphic protein loci (102 alleles), we investigated the evolutionary history of the rabbit through the analysis of 13 representative populations and the use of both the neighbor-joining (NJ) and the unweighted pair-group method with arithmetic mean (UPGMA) trees. We also conducted a separate analysis comparing one domestic and one wild population with previously published results. Our data indicate that an ancient split separated southwestern Iberian populations from all others, including domestic breeds, and that this division may have corresponded to the emergence of the subspecies O.c. algirus and O.c. cuniculus. Separation times between the two major groups of populations were estimated with Nei’s genetic distance and were found to be highly discrepant with the mtDNA divergence estimate. The southwestern Iberian populations (algirusgroup) are more polymorphic than northern populations (cuniculusgroup), the latter displaying more than simply a subset of southern alleles. These results are thus compatible with the isolation of a marginal population or with a smaller long-term population size in the north. The high degree of genetic differentiation between the two subspecies allows the reconstruction of rabbit geographical expansion. France, Britain and other European countries, as well as Australia, were colonized by animals belonging to the cuniculus group, from which domestic breeds are exclusively derived. In contrast, Azorean island populations represent an expansion of the algirus group and show evidence of a strong bottleneck effect.

Patterns of colonization, evolution and gene flow in species of the genus Patella in the Macaronesian Islands.

The study of phylogeographical patterns may contribute to a better understanding of factors affecting the dispersal of organisms in ecological and historical times. For intertidal organisms, islands are particularly suitable models allowing the test of predictions related to the efficacy of pelagic larvae dispersal. Here, we study the phylogeographical patterns and gene flow within three groups of species of the genus Patella present in the Macaronesian Islands that have been previously shown to be monophyletic. The genetic variability of around 600 bp of the mitochondrial gene cytochrome c oxidase subunit I was studied by single strand conformation polymorphism and/or sequencing for seven species of limpets. A total of 420 samples were analysed from the Macaronesian archipelagos, North Africa, and Atlantic and Mediterranean shores of the Iberian Peninsula. No clear geographical pattern or temporal congruence was found between the three groups of species, pointing to independent histories and colonization events. However, for the three groups, the split between the Macaronesian and the mainland forms most probably occurred before 3.9 million years ago, predating the establishment of the current circulation patterns. The presence of pelagic larvae in these species is shown to be insufficient to ensure gene flow between continental and Macaronesian populations and between the Macaronesian archipelagos. In the endangered Azorean populations of Patella candei, there is restricted gene flow to Flores and Graciosa.

Absence of a genetic bottleneck in a wild rabbit (Oryctolagus cuniculus) population exposed to a severe viral epizootic.

Infectious diseases and their demographic consequences are thought to influence the genetic diversity of populations. In Europe, during the last 50 years, the European rabbit (Oryctolagus cuniculus) has suffered two important viral epizootics: myxomatosis and rabbit viral haemorraghic disease (RVHD). Although mortality rates were very high, the impact of these diseases on genetic diversity has never been assessed directly. The subject of this paper is a wild rabbit population in France, which has been studied since the beginning of the 1980s. The first outbreak of RVHD occurred in 1995 and provoked a demographic crash. The population, sampled for the first time in 1982 and 1994, was sampled again at the end of 1996 to examine the impact of the epizootic on genetic diversity. In spite of the observed high mortality rate (≈ 90%), analysis of 14 polymorphic loci (allozymes and microsatellites) showed no loss in genetic diversity after the epizootic. Determination of temporal changes in allele frequencies indicated that the population evolved under genetic drift. The temporal method of Waples demonstrated a significant decrease in the effective population size (Ne) correlated with the demographic crash due to the epizootic. However, the population had only been studied for two generations after the epizootic and the remnant population size probably stayed high enough (≈ 50 individuals) to keep its genetic diversity at the precrash level. These results suggest that, contrary to what is usually thought and in spite of the subsequent high mortality rates, past epizootics (especially myxomatosis) may have had little effect on the genetic diversity of wild rabbit populations in Europe.

tcsBU: a tool to extend TCS network layout and visualization

MOTIVATION TCS is a widely used haplotype network reconstruction software, but lacks the capability of overlapping genetic with geographic structure, which is often a first step in phylogeographic analysis. RESULTS tcsBU is a web-based program that extends the capabilities of TCS, by implementing haplotype classification into an arbitrary user-defined scheme, which is displayed as pie-chart like graphs embedded into the network. Taking advantage of modern graphic libraries, tcsBU also improves the speed at which the final network layout is reached. Networks can be saved as a Scalable Vector Graphics format. AVAILABILITY AND IMPLEMENTATION tcsBU is available on-line at http://cibio.up.pt/software/tcsBU/. The source code is freely available from https://github.com/sairum/tcsbu/ under a standard MIT license.

Invasion or invisibility: using genetic and distributional data to investigate the alien or indigenous status of the Atlantic populations of the peracarid isopod, Stenosoma nadejda (Rezig 1989).

The peracarid isopod, Stenosoma nadejda (Rezig 1989), until recently considered to be endemic of the Mediterranean region, was first reported in the Atlantic coast of southern Spain in 2001, and in 2006 abundant populations were discovered throughout the southwestern Portuguese coast. This fast expansion was intriguing because, as a direct brooder, this species has limited mechanisms for dispersal, such as rafting on seaweeds. Did S. nadejda recently extend its range into the Atlantic or was it overlooked in the past? We examined the patterns of genetic diversity and population differentiation accordingly by sequencing the cytochrome c oxidase subunit I mitochondrial gene from 75 individuals collected in five locations in Atlantic Iberia and one in the Mediterranean. Our results indicate that the newly discovered Atlantic populations of S. nadejda appear to be old and have long persisted on Atlantic shores rather than being a recent introduction. High levels of genetic diversity and geographic structure were uncovered in what was initially suspected to be an ‘invasive’ species. Recent changes in population dynamics may have made S. nadejda more conspicuous in the Atlantic shores, or a more comprehensive survey led to the recognition of this species where it was not expected.

Evidence for genetic similarity of two allopatric European hares (Lepus corsicanus and L. castroviejoi) inferred from nuclear DNA sequences

During the Pleistocene, the severe climatic changes induced major shifts in species distributions, forcing them to retract, expand, displace and/or fragment their ranges (Hewitt, 1996). In Europe, these continuous oscillations led to the production of greater subspecific and specific diversity in the southern peninsulas since they were the major ice age refugial areas (Hewitt, 1999). Hares (genus Lepus) seem to perfectly demonstrate these phenomena. Despite earlier controversies, the latest taxonomic view accepts five species of genus Lepus occurring naturally in Europe: L. europaeus, L. timidus, L. granatensis, L. castroviejoi, and L. corsicanus (Mitchell-Jones et al., 1999). Of these species, the latter two have allopatric and restricted ranges, L. castroviejoi in the Cantabrian Mountains of the Iberian Peninsula and L. corsicanus in the Apennines from central and southern Italy and also in Sicily (Fig. 1). These two southern European endemisms occupy highly specialized patches of scarce habitat and thus the establishment of suitable conservation mechanisms is a major concern (e.g., Temple and Terry, 2007). Several molecular studies have tried to clarify the evolutionary relationships between the European hare species (e.g., Perez-Suarez et al., 1994; Thulin et al., 1997a; Pierpaoli et al., 1999; Suchentrunk et al., 1999; Alves et al., 2000; Estonba et al., 2006; Ben Slimen et al., 2005). However,

New genetic variation in European hares, Lepus granatensis and L. europaeus.

Six genetic polymorphisms for the Iberian hare (Lepus granatensis) and four for the brown hare (L. europaeus) are newly described. The genetic variation of peptidases B (PEPB) and C (PEPC), hemoglobin CHKαCHK chain (HBA), hemopexin (HPX), vitamin D binding protein (GC), and properdin factor B (BF) was assessed by conventional electrophoresis and isoelectric focusing in carrier ampholytes and hybrid pH gradients. Six alleles were detected in PEPB, three in PEPC, four in HBA, six in GC, five in HPX, and six in BF. At least one allele was shared between species at all loci except HBA. The allelic overlap between the two species was medium to high in PEPB, GC, and HPX and small in PEPC and BF.

Invasion history of Caprella scaura Templeton, 1836 (Amphipoda: Caprellidae) in the Iberian Peninsula: multiple introductions revealed by mitochondrial sequence data

The caprellid Caprella scaura, native to the western Indian Ocean, is one striking example of a successful invader. It was first recorded in the Iberian Peninsula in Gerona (north-eastern coast of Spain), in 2005, and has recently been reported throughout the Mediterranean and Atlantic coasts of this region. The most likely vector of introduction and distribution is within the hull-fouling community on recreational craft. However, beyond the dates of detection, the introduction history of C. scaura remains unclear. Direct sequencing of mitochondrial DNA (cytochrome c oxidase subunit I) was used to compare genetic composition in native and introduced populations in order to infer the invasion history of this species. In addition, 18S rDNA sequences were used to resolve phylogenetic relationships within this species and with the morphologically closest species Caprella californica and Caprella scauroides. The high genetic divergence and population subdivision found between non-native Iberian populations together with a high level of genetic diversity in some populations indicate multiple geographical sources and introduction points for this caprellid. Our data suggest that Iberian populations may derive from at least two sources: (1) Pacific Australian, and (2) Indian Ocean, either directly, or, more likely, through stepping-stone events from central Mediterranean population(s). Atlantic Iberian populations seem to be the most recently established populations. On the other hand, this is the first study providing molecular evidence confirming C. scaura, C. californica and C. scauroides as distinct species. It also provides strong molecular evidence that C. scaura typica and C. scaura scaura correspond to the same subspecies, and the Japanese C. scaura diceros and the Chilean C. scaura spinirostris could merit specific rank.