Marc Cheylan

A rangewide phylogeography of Hermann's tortoise, Testudo hermanni (Reptilia: Testudines: Testudinidae): implications for taxonomy

Hermanns tortoise (Testudo hermanni), the best‐known western Palaearctic tortoise species, has a rare natural distribution pattern comprising the Mediterranean areas of the Iberian, Apennine, and Balkan Peninsulas, as well as Sicily, Corsica and Sardinia. The western part of this range is traditionally considered habitat for T. h. hermanni, while T. h. boettgeri occurs in the Balkans. Taxonomy of this tortoise has been challenged in recent years, with the two subspecies being considered full species and the central Dalmatian populations of T. h. boettgeri being considered a third species, T. hercegovinensis. Using an mtDNA fragment approximately 1150 bp long (cytochrome b gene and adjacent portion of tRNA‐Thr gene), we investigated mtDNA diversity with regard to contrasting concepts of two subspecies or three species. Seven closely related haplotypes were identified from the western Mediterranean and 15 different, in part much‐differentiated, haplotypes from the Balkans. Western Mediterranean haplotypes differ from Balkan haplotypes in 16–42 mutation steps. One to seven mutation steps occur within western Mediterranean populations. Balkan haplotypes, differing in 1−37 nucleotides, group in parsimony network analysis into three major assemblages that display, in part, a similar degree of differentiation to that of western Mediterranean haplotypes relative to Balkan haplotypes. Rates of sequence evolution are different in both regions, and low divergence, palaeogeography and the fossil record suggest a slower molecular clock in the western Mediterranean. While monophyly in western Mediterranean haplotypes is well‐supported, conflicting evidence is obtained for Balkan haplotypes; maximum parsimony supports monophyly of Balkan haplotypes, but other phylogenetic analyses (Bayesian, ML, ME) indicate Balkan haplotypes could be paraphyletic with respect to the western Mediterranean clade. These results imply a process of differentiation not yet complete despite allopatry in the western Mediterranean and the Balkans, and suggest all populations of T. hermanni are conspecific. In the western Mediterranean no clear geographical pattern in haplotype distribution is found. Distribution of Balkan haplotypes is more structured. One group of similar haplotypes occurs in the eastern Balkans (Bulgaria, Republic of Macedonia, Romania and the Greek regions Evvia, Macedonia, Peloponnese, Thessaly and Thrace). Two distinct haplotypes, differing in eight to nine mutation steps from the most common haplotype of the first group, are confined to the western slope of the Taygetos Mts. in the Peloponnese. Yet another group, connected over between four and 23 mutation steps with haplotypes of the eastern Balkan group, occurs along the western slope of the Dinarid and Pindos Mts. (Istria, Dalmatia, western Greece). Taygetos haplotypes are nested within other haplotypes in all phylogenetic analyses and support for monophyly of the other Balkan groups is at best weak. We conclude that using the traditional two subspecies model should be continued for T. hermanni. Phylogeographies of T. hermanni and Emys orbicularis, another codistributed chelonian, are markedly different, but share a few similarities. Both were forced to retreat to southern refuges during Pleistocene glaciations. With the advent of Holocene warming, E. orbicularis underwent rapid range expansion and temperate regions of Europe and adjacent Asia were recolonized from refuges in the Balkans and the northern Black Sea Region. By contrast, T. hermanni remained more or less confined to refuges and nearby regions, resulting in a much smaller range, and allopatric and parapatric distribution of haplotype groups and clades. MtDNA lineages are more diverse in E. orbicularis than they are in T. hermanni on southern European peninsulas, indicating several distinct glacial refuges in close proximity and extensive intergradation during Holocene range expansion for E. orbicularis. In T. hermanni it is likely that only on the Balkan Peninsula was more than one refuge located, corresponding to the parapatric ranges of haplotype groups currently there. On the old western Mediterranean islands Corsica and Sardinia no differentiated (E. orbicularis) or only weakly differentiated haplotypes (T. hermanni) occur, even though there is evidence for the presence of both species on Corsica since at least the Middle Pleistocene. High mountain chains constitute major barriers separating distinct mtDNA clades or groups in each species.

Rabbit burrows or artificial refuges are a critical habitat component for the threatened lizard, Timon lepidus (Sauria, Lacertidae)

Refuges are crucial for most animal species as they offer essential protection against predators and provide buffered environmental conditions to their occupants. Our data show that northern populations of the threatened ocellated lizard (Timon lepidus) depend on the availability of the burrows excavated by the European rabbit (Oryctolagus cuniculus). In the last decade, a severe decline in rabbit populations has had a disastrous effect on lizard numbers. To compensate for the lack of refuges, artificial shelters were constructed in autumn 2005 and 2007 and were monitored the following years (2006–2009). Most of the artificial refuges were rapidly occupied by lizards, notably juveniles, suggesting that this technique was successful to improve lizard habitat. Because other factors such as food resources might be also crucial, further assessments are required to determine if artificial refuges are sufficient to stem population decline. These results nonetheless provide an encouraging option to maintain and/or to restore threatened populations, for instance through a buffering of rabbit burrow fluctuations. More generally, the availability of suitable refuges (e.g. natural or artificial) is likely to be a central component for the conservation of many reptile species. The combination of empirical and experimental data further demonstrates that great attention must be paid to the structure and distribution of the refuges and that simple practical actions can effectively improve habitat quality for threatened species.

Heterogeneous responses of temperate-zone amphibian populations to climate change complicates conservation planning

The pervasive and unabated nature of global amphibian declines suggests common demographic responses to a given driver, and quantification of major drivers and responses could inform broad-scale conservation actions. We explored the influence of climate on demographic parameters (i.e., changes in the probabilities of survival and recruitment) using 31 datasets from temperate zone amphibian populations (North America and Europe) with more than a decade of observations each. There was evidence for an influence of climate on population demographic rates, but the direction and magnitude of responses to climate drivers was highly variable among taxa and among populations within taxa. These results reveal that climate drivers interact with variation in life-history traits and population-specific attributes resulting in a diversity of responses. This heterogeneity complicates the identification of conservation ‘rules of thumb’ for these taxa, and supports the notion of local focus as the most effective approach to overcome global-scale conservation challenges.

Differences in gene flow in a twofold secondary contact zone of pond turtles in southern Italy (Testudines: Emydidae:Emys orbicularis galloitalica,E. o. hellenica,E. trinacris)

Using virtually range‐wide sampling for three pond turtle taxa (Emys orbicularis galloitalica, E. o. hellenica, E. trinacris), we analyse gene flow across their southern Italian contact zone. Based on population genetic analyses of 15 highly polymorphic microsatellite loci and a mitochondrial marker, we show that the general genetic pattern matches well with the current taxon delimitation. Yet, single individuals with conflicting genetic identity suggest translocation of turtles by humans. In addition, we identify in south‐western France and the vicinity of Rome populations being heavily impacted by introduced turtles. Cline analyses reveal that the major genetic break between E. o. galloitalica and E. o. hellenica corresponds well with the currently accepted intergradation zone in southern Italy. However, introgression is largely unidirectional from E. o. galloitalica into E. o. hellenica. In the distribution range of the latter subspecies, genetic footprints of E. o. galloitalica are evident along most of the Italian east coast. Our results corroborate that E. o. galloitalica was introduced long ago in Corsica and Sardinia and naturalized there. Gene flow between E. orbicularis and E. trinacris is negligible, with the Strait of Messina matching well with the narrow cline centre between the two species. This contrasts with other Mediterranean freshwater turtle species with extensive transoceanic gene flow. Compared to the two subspecies of E. orbicularis, the Sicilian E. trinacris shows an unexpectedly strong population structuring, a finding also of some relevance for conservation. The differences between the two taxon pairs E. orbicularis/E. trinacris and E. o. galloitalica/E. o. hellenica support their current taxonomic classification and make them attractive objects for follow‐up studies to elucidate the underlying mechanisms of speciation by comparing their properties.

The best of a harsh lot in a specialized species: breeding habitat use by the yellow-bellied toad (Bombina variegata) on rocky riverbanks

Because of the narrowness of their niches, specialised species are often vulnerable because their populations suffer from fragmentation or low densities. Whatever the causes of their specialization, one might expect micro-habitat selection to be strong within the boundaries of the chosen niche to compensate for the costs linked to habitat constraints (variability of reproductive success due to high larval mortality, habitat fragmentation). We tested this hypothesis by investigating breeding site selection in the yellow-bellied toad (Bombina variegata) in the context of rock pools at riverbanks, which are supposed to be one of the pristine habitats of the species. In this species, breeding habitat use differs markedly from that of other European anurans since spawning takes place in small pools located in the close vicinity of rivers. Such pools experience high risks of drying up and flooding. By measuring 8 habitat variables at 187 rock pools along a 250 m river segment, we show a breeding habitat use based on a preference for pools with large volumes of water provided that they are devoid of alluvia, with a good exposition to sun. By ensuring longer water periods and faster development rates, large volumes and warm water probably reduce the mortality risks due to drying up (within suitable temperature range). This result contributes to understanding the process of niche conservatism (persistence of narrow range on certain dimensions of the niche), which is an important challenge in ecology and conservation biology.

Impacts of a wildfire on the mortality rate and small-scale movements of a Hermann's tortoise Testudo hermanni hermanni population in southeastern France

Because they have high longevity and a weak dispersal capacity, chelonians are known to be highly impacted by fire. We investigated the immediate demographic consequences of a wildfire occurring in 2009 on a population of Hermanns tortoises Testudo hermanni in the Plaine des Maures in southeastern France using the multistate capture–recapture method. Over two years (2009 and 2010), 108 adult individuals were marked. Survival probability was higher in the unburned (0.99) than in the burned area (0.43). The inter-annual transition probability from the burned to the unburned area was high (0.72). These results show that fire affects survival by immediately killing individuals and by inducing small-scale movements from burned to unburned areas.

Breeding traits of Hermann's tortoise Testudo hermanni hermanni in two western populations

Over a period of two years we used radiology to investigate breeding traits in females of two western populations of the threatened Hermanns tortoise Testudo hermanni hermanni. The main purpose of the study was to see if reproductive traits - clutch size, clutch frequency, annual egg production and egg width - varied between populations in Corsica (France) and the Ebro Delta (Spain), and if these traits were affected by female body size and amounts of spring rainfall. All the breeding traits analysed were greater in the Corsican population than in the Ebro Delta population. These differences were also significant when we used female body size as a covariable. In both populations the amount of spring rainfall affected breeding traits but not clutch size. Nevertheless, only in the Corsican population did clutch frequency, annual egg production and egg width increase significantly in the year with a wet spring. Contrary to the predictions of Optimal Propagule Size theory, we found that egg size and clutch size increased with female size, and that both variables were independent. On the other hand, clutch frequency was not related to female body size. Thus, in the Hermanns tortoise, as clutch size did not change between years and clutch frequency was independent of female size, all females are able to increase their reproductive output in years with favourable conditions by increasing their clutch frequency. Nevertheless, other factors besides female size and rainfall may influence in a highly complex way variability in breeding traits among populations.

High Genetic Differentiation Among French Populations of the Orsini’s Viper (Vipera ursinii ursinii) Based on Mitochondrial and Microsatellite Data: Implications for Conservation Management

The Orsinis viper (Vipera ursinii) is one of the most threatened snakes in Europe due to its highly fragmented distribution and specific open environment (steppic habitat) requirement. French populations are isolated on top of mountain massifs of the southern Prealps/Alps. Mitochondrial sequences (cytochrome b) and 6 microsatellite loci have been used to estimate the levels of genetic diversity and isolation within and among 11 French fragmented populations (a total of 157 individuals). Eleven cytochrome b haplotypes with a limited divergence were observed (mean divergence between haplotypes: 0.31%). However, we detected considerable genetic differentiation among populations (global F(ST) = 0.76 and 0.26 for mitochondrial and nuclear DNA, respectively). Results indicate that 3 populations possibly went through a bottleneck and 1 population showed low genetic diversity compared with the others. Although a significant isolation by distance was detected for both markers, strong differentiation was also observed between geographically close populations, probably due to the ragged landscape that constitutes a serious barrier to gene flow owing to the limited dispersal capability of the viper. Despite some discrepancies between the 2 markers, 8 Management Units have been identified and should be considered for future management projects.

Assessing spatial and temporal population dynamics of cryptic species: an example with the European pond turtle

Within the current context of biodiversity loss a number of biodiversity indicators have been developed to help measure the state of nature and how it is changing. However, most indicators are derived from bird populations. Reptiles and amphibians could be useful ecosystem indicators, but this requires obtaining precise and unbiased population parameters. This is a particularly challenging task for these two groups of species, because individuals are extremely difficult to detect for various reasons. We illustrate the use of a sampling and analytical method that explicitly takes into account imperfect detection to assess the population dynamics of a reptile species and its temporal and spatial variation. European pond turtles (Emys orbicularis) were sampled at two different locations during a 10-year study. The two sites differed by their water management regimes and number of livestock. At each site and for each sex, the data were modeled using a robust design capture-mark-recapture framework to obtain and compare estimates of survival, temporary emigration, time-specific abundance, density, and population growth rate, while taking into account effects of individual heterogeneity, trap-response, and time on capture probabilities. Temporary emigration was higher in males (0.344 +/- 0.046) (mean +/- SE) than in females (0.228 +/- 0.071), did not differ between sites for each sex, and was mainly Markovian. Apparent adult survival was higher in females (0.966 +/- 0.031) than in males (0.864 +/- 0.024), and was negatively related to the proportion of individuals with repaired shell fractures in the population. Average density was 63.7 +/- 6.6 turtles/km2 but was higher for females (39.2 +/- 14.0 females/km2) than for males (23.5 +/- 5.7 males/km2). The population sizes of males and females decreased where long periods of artificial drought and the highest density of livestock occurred. This research highlights differences in demographic parameters according to sex and site in a turtle species, and it provides evidence that significant human-induced disturbance represents a potential risk to European pond turtle populations. The sampling and analytical approaches illustrated in this study are applicable to many other species of reptiles and amphibians, and estimated population parameters could be combined to produce population indicators useful for conservation and management.

Congruent signals of population history but radically different patterns of genetic diversity between mitochondrial and nuclear markers in a mountain lizard

Historical factors, current population size, population connectivity and selective processes at linked loci contribute to shaping contemporary patterns of neutral genetic diversity. It is now widely acknowledged that nuclear and mitochondrial markers react differently to current demography as well as to past history, so the use of both types of markers is often advocated to gain insight on both historical and contemporary processes. We used 12 microsatellite loci genotyped in 13 populations of a mountain lizard (Iberolacerta bonnali) to test whether the historical scenario favoured by a previous mitochondrial study was also supported by nuclear markers and thereby evaluated the consequences of postglacial range movements on nuclear diversity. Congruent signals of recent history were revealed by nuclear and mitochondrial markers using an Approximate Bayesian computation approach, but contemporary patterns of mtDNA and nuclear DNA diversity were radically different. Although dispersal in this species is probably highly restricted at all spatial scales, colonization abilities have been historically good, suggesting capability for reestablishment of locally extinct populations except in fully disconnected habitats.