Molecular survey of morphological subspecies reveals new mitochondrial lineages in Podarcis muralis (Squamata: Lacertidae) from the Tuscan Archipelago (Italy)

Recent analyses of molecular markers have significantly revised the traditional taxonomy of Podarcis species (Squamata: Lacertidae), leading to critically reconsider the taxonomic value of several subspecies described only on morphological bases. In fact, lizards often exhibit high morphological plasticity both at the intra‐specific and the intra‐population level, especially on islands, where phenotypic divergences are mainly due to local adaptation, rather than to evolutionary differentiation. The Common wall lizard Podarcis muralis exhibits high morphological variability in biometry, pholidosis values and colour pattern. Molecular analyses have confirmed the key role played by the Italian Peninsula as a multi‐glacial refuge for P. muralis, pointing out the lack of congruence between mitochondrial lineages and the four peninsular subspecies currently recognized. Here, we analyse a portion of the protein‐encoding cytochrome b gene in the seven subspecies described for the Tuscan Archipelago (Italy), in order to test whether the mitochondrial haplotypes match the morphologically based taxonomy proposed for Common wall lizard. We also compare our haplotypes with all the others from the Italian Peninsula to investigate the presence of unique genetic lineages in insular populations. Our results do not agree completely with the subspecific division based on morphology. In particular, the phylogenetic analyses show that at least four subspecies are characterized by very similar haplotypes and fall into the same monophyletic clade, whereas the other three subspecies are closer to peninsular populations from central Italy. From these results, we conclude that at least some subspecies could be better regarded as simple eco‐phenotypes; in addition, we provide an explanation for the distinctiveness of exclusive lineages found in the archipelago, which constituted a refuge for this species during last glacial periods.     

A phylogenomic resolution for the taxonomy of Aegean green lizards

Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next-generation sequencing and double-digest restriction-site-associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome-wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.     

The impact of nematode parasites on the behaviour of an Australian lizard,the gidgee skink <Emphasis Type="Italic">Egernia stokesii</Emphasis>

The Australian scincid lizard Egernia stokesii lives in social groups and is infected with two nematode species: Pharyngodon tiliquae and Thelandros trachysauri. This study asked whether those nematodes affected levels of lizard activity in field populations. In a laboratory colony, application of a combination of ivermectin and fenbendazole reduced nematode egg count in lizard scats after 12 weeks. In the field, the same doses of those antihelminthic drugs were applied to lizards in six social groups across three populations, and a saline control was given to lizards in six adjacent groups. Observations showed significant changes in behaviour between the two groups developing over 2 months. Drug-treated lizards spent more time basking and moved about for longer times during observation sessions. The results suggest that nematode infection altered host behaviour and reduced fitness. No influence of social group size was detected on the impact of parasitic nematodes.     

Emergence, basking behaviour, mean selected temperature and critical thermal minimum in high and low altitude subspecies of the tropical lizard Mabuya striata

Emergence, basking behaviour, selected temperature, and critical thermal minimum were studied in warm and cold acclimated groups of two subspecies of the lizard Mabuya striata. The high-altitude M. s. punctatissima emerges earlier than the low-altitude M. s. striata. Under conditions of warm acclimation, M. s. punctatissima has a longer initial bask. The two subspecies do not differ in either mean selected temperature or critical thermal minimum, although in both cases values for cold acclimated lizards are lower than for warm acclimated lizards. I have previously demonstrated differences between the subspecies in the pattern of thermal acclimation of rate of oxygen consumption. I thus consider that M. striata is static in some aspects of its thermal biology but labile in others.     

Germination patterns throughout an insular altitudinal gradient: The case of the Macaronesian endemic plant Rubia fruticosa Ait. (Rubiaceae) in El Hierro (Canary Islands)

Seed germination percentage and rate of Rubia fruticosa, an endemic Macaronesian shrub was assessed with seed lots collected at three levels of an altitudinal gradient (200–800 m asl) located in El Hierro Island (Canarian Archipelago). Both seeds collected from control plants and those found in lizard droppings were studied. A significant decrease in the germination percentage was observed with control seeds and those seeds which are previously eaten by lizards if collected at 800 m asl. This indicates that R. fruticosa is a plant whose optimal germination environment is located between 200 and 500 m asl, occurring in higher altitudes only under sub-optimal conditions. The effect of lizard gut on R. fruticosa seeds was variable in the two study years: while germination percentage of seeds having passed through the lizards was not significantly different to that of control seeds in the first year, an increased germination was found for the animal-treated seeds in all three altitudinal zones in the second year. In general, germination rate of control seeds was rather variable between years. However, improvement of R. fruticosa seed germination caused by lizards may be important for its fitness; and a co-evolution may have occurred, since lizards and seeds of this endemic plant have been intensively interacting for millions of years in the lower zones of the Canary Islands.     

Pollination and seed dispersal of Melocactus ernestii Vaupel subsp. ernestii (Cactaceae) by lizards: an example of double mutualism

Recent studies show that the mutualistic role of lizards as pollinators and seed dispersers has been underestimated, with several ecological factors promoting such plant–animal interactions, especially on oceanic islands. Our aim is to provide a quantitative assessment of pollination and seed dispersal mutualisms with lizards in continental xeric habitats. We carried out focal observations of natural populations of Melocactus ernestii (Cactaceae) in the Caatinga, a Brazilian semiarid ecosystem, in order to record the frequency of visits, kind of resource searched and behaviour of visiting animals towards flowers and/or fruits. We made a new record of the lizard Tropidurus semitaeniatus foraging on flowers and fruits of M. ernestii. During the search for nectar, T. semitaeniatus contacted the reproductive structures of the flowers and transported pollen attached to its snout. Nectar production started at 14:00 h, with an average volume of 24.4 μl and an average concentration of solutes of 33%. Approximately 80% of the seeds of M. ernestii found in the faeces of T. semitaeniatus germinated under natural conditions. The roles of T. semitaeniatus as pollinator and seed disperser for M. ernestii show a clear relationship of double mutualism between two endemic species, which may result from the environmental conditions to which both species are subject. Seasonality, low water availability and arthropod supply in the environment, high local lizard densities, continuous nectar production by the flower and fruits with juicy pulp may be influencing the visits and, consequently, pollination and seed dispersal by lizards in this cactus.     

Gene flow across secondary contact zones of the Emys orbicularis complex in the Western Mediterranean and evidence for extinction and re‐introduction of pond turtles on Corsica and Sardinia (Testudines: Emydidae)

European pond turtles represent a phylogeographically deeply structured complex of distinct taxa. Here, we use mitochondrial DNA sequences (cytochrome b gene) and eight polymorphic microsatellite loci to investigate genetic differentiation and gene flow of Sicilian, Corsican and Sardinian pond turtles and of subspecies involved in two secondary contact zones in the Pyrenean region and Southern Italy. Mitochondrial and microsatellite differentiation is largely concordant in populations from the core regions of the distribution ranges of the studied taxa. Both marker systems provide no evidence for gene flow between Sicilian pond turtles (Emys trinacris) and Southern Italian subspecies of E. orbicularis. By contrast, in the contact zones limited gene flow occurs between distinct subspecies of E. orbicularis. Although the Southern Italian contact zone is significantly older than the Pyrenean contact zone of Holocene age, patterns of asymmetric introgression are similar. Introgressive hybridization leads to the exchange of mitochondria, but microsatellite data indicate only a few individuals with mixed ancestry. This suggests that incipient isolating mechanisms maintain largely discrete nuclear genomic gene pools. Furthermore, this implies that Southern Italy acted as a hotspot rather than as a melting pot of genetic diversity during the last glacial. Pond turtles from Corsica and Sardinia are not differentiated from continental populations of the subspecies E. o. galloitalica, neither in the mitochondrial nor in the quickly evolving microsatellite markers. As the fossil record argues for a continuous presence of pond turtles on both islands since the Middle Pleistocene, this suggests that the native island populations became extinct and the extant turtles were later introduced by prehistoric settlers. The lack of genetic differentiation of pond turtles from Corsica and Sardinia supports the view that the subspecies described from these islands are not valid.     

Phylogeography of the Lacerta viridis complex: mitochondrial and nuclear markers provide taxonomic insights

Based on broad, nearly rangewide sampling, we reanalysed the phylogeography of the Lacerta viridis complex using the mitochondrial cytochrome b gene and the intron 7 of the nuclear β‐fibrinogen gene. Using the mitochondrial marker, we identified in phylogenetic analyses 10 terminal clades clustering in four deeply divergent main lineages whose relationships are weakly resolved. These lineages correspond to Lacerta bilineata, L. viridis, the previously identified Adriatic or West Balkan lineage and a newly discovered fourth lineage from the Anatolian Black Sea coast and the south‐eastern Balkan Peninsula. Except for the latter lineage, there is considerable phylogeographic structuring in each lineage, with higher diversity in the south of the distribution ranges. This pattern indicates the existence of two distinct microrefugia in the Italian Peninsula and Sicily and of up to seven microrefugia in the Balkan Peninsula, but of only one refugium along the Black Sea coast of Anatolia. We identified secondary contact zones of the main lineages and of terminal clades within these lineages. However, most of the formerly described putative contact zone of L. bilineata and L. viridis turned out to be a contact zone between the Adriatic lineage and L. viridis, but L. bilineata seems to be involved only marginally. Our nuclear marker could not unambiguously resolve whether there is gene flow in contact zones. Thus, further research is necessary to decide whether the four main lineages are conspecific or whether they represent distinct biological species. We restrict the name L. v. meridionalis to the newly identified genetic lineage from Turkey and south‐eastern Europe, synonymize some previously recognized taxa and suggest a tentative nomenclature for the L. viridis complex.     

Taxonomic status of the extinct Canary Islands Oystercatcher Haematopus meadewaldoi

Mitochondrial genes were sequenced from four specimens of the extinct Canary Islands Oystercatcher Haematopus meadewaldoi and compared with African Oystercatcher Haematopus moquini, Eurasian Oystercatcher Haematopus ostralegus and an old unidentified extralimital ‘black’ oystercatcher specimen from The Gambia. At these loci, H. meadewaldoi was approximately 99.65% identical to multiple Eurasian Oystercatcher samples and in phylogenetic trees fell within the range of genetic variation observed in that species. The mystery Gambian bird was resolved as an extralimital H. moquini. We conclude that H. meadewaldoi was most likely a recently diverged melanistic morph or subspecies of H. ostralegus, although further genomic studies will be required to determine whether there has been a period of isolation followed by introgression.     

Refuge sites used by the scincid lizard Tiliqua rugosa

Abstract We examined microhabitat use by the Australian scincid lizard Tiliqua rugosa (the sleepy lizard) in chenopod shrubland. Thirty radiotagged lizards were followed during their spring period of activity (September–November 2000). Characteristics of refuges used by the lizards were compared with the perennial woody plants in 10 10 m × 10 m randomly located quadrats at each of the three sites in the study area. We found that sleepy lizards used multiple refuge sites. Perennial woody bushes constituted an important habitat component for both active and inactive lizards. Use of refuge sites by the lizards was non‐random. They used large bushes with foliage in contact with the ground (‘dome shaped’), and they sheltered under thorny bushes more frequently than expected if choice was random. Bushes that were used repeatedly tended to be larger and were more likely to be dome‐shaped. Under these large and dome‐shaped bushes, daytime temperatures were lower than under smaller bushes. Refuge use was modified as the season progressed, with lizards using larger bushes and a greater proportion of dome‐shaped bushes later in the season. We suggest that sleepy lizards are modifying their refuge site use as ambient temperatures increase, based on microclimatic needs, implying an ability to discriminate among bushes.     

Lateralization When Monitoring Predators in the Wild: A Left Eye Control in the Common Wall Lizard (Podarcis muralis)

Lateralization is the function specialization between left and right brain hemispheres. It is now ascertained in ectotherms too, where bias in eye use for different tasks, i.e., visual lateralization, is widespread. The lateral eye position on the head of ectotherm animals, in fact, allows them to observe left/right stimuli independently and allows lateralized individuals to carry out left and right perceived tasks at the same time. A recent study conducted on common wall lizards, Podarcis muralis, showed that lizards predominantly monitor a predator with the left eye while escaping. However, this work was conducted in a controlled laboratory setting owing to the difficulty of carrying out lateralization experiments under natural conditions. Nevertheless, field studies could provide important information to support what was previously found in the laboratory and demonstrate that these traits occur in nature. In this study, we conducted a field study on the antipredatory behavior of P. muralis lizards. We simulated predatory attacks on lizards in their natural environment. We found no lateralization in the measure of eye used by the lizard to monitor the predator before escaping from it, but the eye used was probably determined by the relative position of the lizard and the predator just before the attack. This first eye used did not affect escape decisions; lizards chose to escape toward the nearest refuge irrespective of whether it was located to the lizard’s left or right side. However, once they had escaped to a refuge, lizards had a left eye–mediated bias to monitor the predator when first emerging from the refuge, and this bias was likely independent of other environmental variables. Hence, these field findings support a left eye–mediated observation of the predator in P. muralis lizards, which confirms previous findings in this and other species.     

Fine‐scale genetic structuring in a group‐living lizard,the gidgee skink (Egernia stokesii)

By constraining gene flow, group living and natal philopatry can result in fine‐scale genetic structure. Although the genetic structure of some group‐living lizards has been characterised, studies are few compared with those for group‐living bird and mammal species. The Egerniinae group of lizards exhibits a high diversity of social structures, making it a useful group for comparative studies of genetic structure across a broader range of social taxa. A well‐studied member of Egerniinae is Egernia stokesii, a lizard that forms long‐term pair bonds and stable social groups and exhibits natal philopatry and limited dispersal. Evidence exists for consistent E. stokesii social structure across seven close but disconnected rocky outcrops within a 40 × 10 km area. We used summary statistics, analysis of molecular variance, Bayesian clustering, and discriminant analysis of principal components to assess if E. stokesii exhibit a consistent pattern of fine‐scale genetic structure across the same seven outcrops. Due to E. stokesii social structure and constrained dispersal, we predicted significant genetic structuring – based on microsatellite markers – among outcrops. We found significant fine‐scale genetic structuring and evidence for two genetic clusters. We discuss features of E. stokesii biology and ecology that may explain our findings. Some rocky outcrops, and some social groups, contained lizards from both genetic clusters. An examination of the composition of mixed cluster social groups did not detect any notable patterns. Therefore, further work is necessary to identify how the observed patterns may have arisen. Future investigations in E. stokesii and other group‐living lizard species are likely to contribute greatly to our understanding of the genetic consequences of group living.     

Asymmetric Response to Heterotypic Distress Calls in the Lizard Liolaemus chiliensis

The weeping lizard, Liolaemus chiliensis, emits distress calls when trapped by a predator. Conspecific lizards respond to such calls with prolonged immobility, which may increase their probability of remaining undetected by a predator. This benefit, however, depends on the ability to react to the alert message of the call, which may be impaired by natural variation in the calls. The distress calls of L. chiliensis show geographic variation, and here we tested the response of two geographically distant populations (>700 km apart) to local (homotypic) and non‐local (heterotypic) distress calls; if populations are finely tuned to their local calls, they may not be able to respond to heterotypic calls. We found that geographic variation in calls affects the lizards’ response, but this effect was population dependent; whereas southern lizards responded to calls of both populations, the northern lizards only reacted to homotypic distress calls. The factors that determine this asymmetric response to heterotypic calls are unclear and we discuss three hypotheses that have a common component in the difference in body size between the tested populations, which seems to play a key role in determining the response to distress calls in this species.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Extended gestation with late‐autumn births in a cool‐climate viviparous gecko from southern New Zealand (Reptilia: Naultinus gemmeus)

Abstract Female viviparous lizards from temperate locations in the Southern Hemisphere (New Zealand, Tasmania (Australia), South Africa and South America) often have reproductive activity spanning many months of the year. In contrast, vitellogenesis and pregnancy are often confined to the spring/summer months in viviparous species from temperate zones of the Northern Hemisphere. An extreme Southern Hemisphere example is the nocturnal common gecko from New Zealand, Hoplodactylus maculatus (Gray 1845), in which females exhibit biennial reproduction with pregnancy lasting up to 14 months in a cool‐climate population. Here, we examined whether such an extended reproductive cycle also occurs in a diurnal species, the jewelled gecko Naultinus gemmeus (McCann 1955), at a similar latitude. Palpation was used to assess reproductive condition non‐invasively. In contrast to the nearby higher‐altitude population of H. maculatus, N. gemmeus reproduces annually. Vitellogenesis occurs from autumn to spring in both species, but pregnancy ends after about 7 months in N. gemmeus. Birth occurs in the seemingly unpropitious season of mid‐ to late autumn, a pattern that may be unique for lizards from cool‐temperate zones. We hypothesize that there are major differences between populations of N. gemmeus and H. maculatus with respect to survival of autumn‐born neonates and/or costs to females from remaining pregnant over winter. Museum specimens of N. gemmeus support anatomical inferences from palpation; they also suggest that vitellogenesis may begin before the end of pregnancy (which may be essential to completing each reproductive cycle within a year) and that some populations may show gestation in utero over winter, as in H. maculatus. Extended gestation appears to be a common response to cool climates for Southern Hemisphere lizards that have independently evolved viviparity.     

Predatory lizards perceive plant‐derived volatile odorants

Many lizards are olfactory foragers and prey upon herbivorous arthropods, yet their responses to common herbivore‐associated plant volatiles remain unknown. As such, their role in mediating plant indirect defenses also remains largely obscured. In this paper, we use a cotton‐swab odor presentation assay to ask whether lizards respond to two arthropod‐associated plant‐derived volatile compounds: 2‐(E)‐hexenal and hexanoic acid. We studied the response of two lizard species, Sceloporus virgatusand Aspidoscelis exsanguis, because they differ substantially in their foraging behavior. We found that the actively foraging A. exsanguisresponded strongly to hexanoic acid, whereas the ambush foraging S. virgatus responded to 2‐(E)‐hexenal—an herbivore‐associated plant volatile involved in indirect defense against herbivores. These findings indicate that S. virgatus may contribute to plant indirect defense and that a species' response to specific odorants is linked with foraging mode. Future studies can elucidate how lizards use various compounds to locate prey and how these responses impact plant‐herbivore interactions.     

The visual perceptual range of a lizard, <Emphasis Type="Italic">Tiliqua rugosa</Emphasis>

The fragmentation of landscapes produces habitat gaps where the distance between visual landmarks may exceed the perceptual range of a species and impose navigational constraints. We estimated the visual perceptual range of the Australian sleepy lizard, Tiliqua rugosa, by releasing individuals in the centre of a cleared arena in high temperature conditions, with a 0.5-m-high bush placed either 10, 20 or 30 m from the release site. Lizards were more likely to locate those bushes and shelter under them when they were closer, and no lizards found a bush at 30 m. In addition, lizards were less likely to move from the release point when bushes were at 30 m than when they were at the two closer distances. These data suggest that for sleepy lizards the perceptual range for a 0.5-m-high bush is about 20 m. In the uncleared chenopod shrub-land where these lizards live, suitable shelter bushes are an average of 10.5 m from any point in their home range, well within their perceptual range.     

Phylogeography and cryptic variation within the Lacerta viridis complex (Lacertidae, Reptilia)

It is well known that the current genetic pattern of many European species has been highly influenced by climatic changes during the Pleistocene. While there are many well known vertebrate examples, knowledge about squamate reptiles is sparse. To obtain more data, a range‐wide sampling of Lacerta viridis was conducted and phylogenetic relations within the L. viridis complex were analysed using an mtDNA fragment encompassing part of cytochrome b, the adjacent tRNA genes and the noncoding control region. Most genetic divergence was found in the south of the distribution range. The Carpathian Basin and the regions north of the Carpathians and Alps are inhabited by the same mitochondrial lineage, corresponding to Lacerta viridis viridis. Three distinct lineages occurred in the south‐eastern Balkans — corresponding to L. v. viridis, L. v. meridionalis, L. v. guentherpetersi— as well as a fourth lineage for which no subspecies name is available. This distribution pattern suggests a rapid range expansion of L. v. viridis after the Holocene warming, leading to a colonization of the northern part of the species range. An unexpected finding was that a highly distinct genetic lineage occurs along the western Balkan coast. Phylogenetic analyses (Bayesian, maximum likelihood, maximum parsimony) suggested that this west Balkan lineage could represent the sister taxon of Lacerta bilineata. Due to the morphological similarity of taxa within the L. viridis complex this cryptic taxon was previously assigned to L. v. viridis. The distribution pattern of several parapatric, in part highly, distinct genetic lineages suggested the existence of several refuges in close proximity on the southern Balkans. Within L. bilineata sensu stricto a generally similar pattern emerged, with a high genetic diversity on the Apennine peninsula, arguing for two distinct refuges there, and a low genetic diversity in the northern part of the range. Close to the south‐eastern Alps, three distinct lineages (L. b. bilineata, L. v. viridis, west Balkan taxon) occurred within close proximity. We suggest that the west Balkan lineage represents an early offshoot of L. bilineata that was isolated during a previous Pleistocene glacial from the more western L. bilineata populations, which survived in refuges on the Apennine peninsula.     

Genetic analysis of a contact zone between two lineages of the ocellated lizard (Lacerta lepida Daudin 1802) in south‐eastern Iberia reveal a steep and narrow hybrid zone

Measuring the diffusion of genes between diverging taxa through zones of secondary contact is an essential step to understand the extent and nature of the reproductive isolation that has been achieved. Previous studies have shown that the ocellated lizard (Lacerta lepida Daudin, 1802) has endured repeated range fragmentation associated with the climatic oscillations of the Plio‐Pleistocene that promoted diversification of many different evolutionary units within the species. However, the oldest divergence within the group is estimated to have occurred much earlier, during the Miocene, around 9 Ma and corresponds to the split between the subspecies Lacerta lepida nevadensis Buchholz (1963) and Lacerta lepida lepida Daudin (1802). Although these two evolutionary units have documented genetic and morphological differentiation, most probably accumulated during periods of allopatry, little is known about patterns of gene flow between them. In this study, we performed a population genetic analysis of a putative area of secondary contact between these two taxa, using mtDNA and microsatellite data. We assessed levels of gene flow across the contact zone to clarify to what extent gene flow may be occurring. Hybridization between the subspecies was observed by the presence of genetically introgressed individuals. However, the overall coincidence of mitochondrial and multilocus nuclear clines and generally steep clines support the idea that this contact zone is acting as a barrier to gene flow. Taken together, these results suggest that L. l. lepida and L. l. nevadensis are in independent evolutionary trajectories and should be considered as two different species.