Phylogenetic relationships and biogeography of Podarcis species from the Balkan Peninsula, by bayesian and maximum likelihood analyses of mitochondrial DNA sequences

Wall lizards of the genus Podarcis (Sauria, Lacertidae) comprise 17 currently recognized species in southern Europe, where they are the predominant nonavian reptile group. The taxonomy of Podarcis is complex and unstable. Based on DNA sequence data, the species of Podarcis falls into four main groups that have substantial geographic coherence (Western island group, southwestern group, Italian group, and Balkan Peninsula group). The Balkan Peninsula species are divided into two subgroups: the subgroup of P. taurica (P. taurica, P. milensis, P. gaigeae, and perhaps P. melisellensis), and the subgroup of P. erhardii (P. erhardii and P. peloponnesiaca). In the present study, the question of phylogenetic relationships among the species of Podarcis encountered in the Balkan Peninsula was addressed using partial mtDNA sequences for cytochrome b (cyt b) and 16S rRNA (16S). The data support the monophyly of Podarcis and suggest that there are three phylogenetic clades: the clade A (P. taurica, P. gaigeae, P. milensis, and P. melisellensis); the clade B (P. erhardii and P. peloponnesiaca), and the clade C (P. muralis and P. sicula). By examining intraspecific relationships it was found that extant populations of P. erhardii are paraphyletic. Furthermore, subspecies previously defined on the basis of morphological characteristics do not correspond to different molecular phylogenetic clades, suggesting that their status should be reconsidered. The distinct geographic distribution of the major clades of the phylogenetic tree and its topology suggest a spatial and temporal sequence of phylogenetic separations that coincide with some major paleogeographic separations during the geological history of the Aegean Sea. The results stress the need for a reconsideration of the evolutionary history of Balkan Podarcis species and help overcome difficulties that classical taxonomy has encountered at both the species and subspecies level.     

Isolation and characterization of polymorphic microsatellite loci for the Skyros wall lizard Podarcis gaigeae (Squamata: Lacertidae)

Fifteen polymorphic markers were developed from a microsatellite-enriched library for the lizard Podarcis gaigeae. The loci were checked for variability in 68 individuals from a population on the island of Skyros, Greece. The number of alleles ranged from 3 to 23 per locus and expected heterozygosity from 0.29 and 0.94. Most markers were also polymorphic in three closely related Podarcis species, namely P. erhardi, P. taurica and P. milensis. The markers will be used to examine gene flow and differentiation of island and mainland populations of P. gaigeae.     

Variation in the cranium shape of wall lizards (Podarcis spp.): effects of phylogenetic constraints, allometric constraints and ecology

We used geometric morphometrics to explore the influence of phylogenetic and allometric constraints as well as ecology on variation in cranium shape in five species of monophyletic, morphologically similar Podarcis lizards (Podarcis erhardii, Podarcis melisellensis, Podarcis muralis, Podarcis sicula and Podarcis taurica). These species belong to different clades, they differ in their habitat preferences and can be classified into two distinct morphotypes: saxicolous and terrestrial. We found (i) no phylogenetic signal in cranium shape, (ii) diverging allometric slopes among species, and (iii) a significant effect of habitat on cranium shape. The saxicolous species (P. erhardii and P. muralis) had crania with elongated parietals, elongated cranium bases, shortened anterior parts of the dorsal cranium, reduced chambers of the jaw adductor muscles and larger subocular foramina. These cranial features are adaptations that compensate for a flattened cranium, dwelling on vertical surfaces and seeking refuge in crevices. The crania of the terrestrial species (P. melisellensis, P. sicula and P. taurica) tended to be more elongate and robust, with enlarged chambers of the jaw adductor muscle, reduced skull bases and shortened parietals. Terrestrial species exhibited more variation in cranium shape than saxicolous species. Our study suggests that shape variation in Podarcis sp. lizards is largely influenced by ecology, which likely affects species-specific patterns of static allometry.     

Comparative postautotomy tail activity in six Mediterranean lacertid lizard species

Tail autotomy, the self-induced tail separation from the body, is a common and effective antipredator mechanism in lizards. In this study, we examine the muscle energetics of tail shedding in six lacertid lizard species (Podarcis erhardii, Podarcis peloponnesiaca, Podarcis muralis, Podarcis gaigeae, Podarcis milensis, and Lacerta graeca) from the northeast Mediterranean region. Very long periods of postautotomy tail movement were demonstrated for all species (range=6-8 min), and differences among species were not statistically significant. Postautotomy tail movement, powered by anaerobic muscle activity, resulted in a strong increase in lactate concentrations and a concomitant depletion of muscle glycogen of exhausted tails relative to resting tails. No significant differences were found in either lactate or glycogen concentrations among the species examined. Duration of movement was negatively correlated with final lactate concentrations. The lack of differentiation in postautotomy energetic physiology in this group of species that have evolved under very different predation environments indicates that postautotomy muscle metabolism involves an overall conservative suite of characters.     

Molecular biogeography of the Mediterranean lizards Podarcis Wagler, 1830 and Teira Gray, 1838 (Reptilia, Lacertidae)

Aim   We discuss biogeographical hypotheses for the Mediterranean lizard species Podarcis and Teira within a phylogenetic framework based on partial mitochondrial DNA sequences.
Methods   We derived the most likely phylogenetic hypothesis from our data set (597 aligned positions from the 12S rDNA and phenyl tRNA) under parsimony, distance and maximum likelihood assumptions.
Results   The species usually included in Teira do not form a strongly monophyletic clade. In contrast, the monophyly of the genus Podarcis is rather well supported. Seven lineages are identified in the genus; in order of appearance within the tree, these are: the Balearic pityusensis and lilfordi pair, the sicula complex, a Tyrrhenian tiliguerta and raffonei pair, muralis , the Siculo-Maltese filfolensis and wagleriana pair, the Balkan group ( erhardi , peloponnesiaca , milensis , melisellensis and taurica ), and the Ibero-Maghrebian group ( bocagei , atrata , hispanica and vaucheri ).
Conclusions   The origin of the three European genera of lacertid assayed ( Lacerta , Teira and Podarcis ) is hypothesized to have occurred in the Oligocene. For Podarcis , a possible scenario of a Miocene diversification is derived from the sequence data, and the zoogeography of the lineages are discussed in relation to the palaeogeography of the Mediterranean. It is hypothesized that in the early history of the genus the main lineages separated by rapid, numerous and close events that produced a starting point very similar to a polytomy, hard to resolve by parsimony analysis of the data set.     

Molecular phylogeny and biogeography of the wall-lizard Podarcis erhardii (Squamata: Lacertidae)

Erhard's wall lizard, Podarcis erhardii (Sauria: Lacertidae), is highly diversified in Greece and especially in the southern Aegean region. Out of the 28 recognized subspecies, 27 are found in Greece from the North Sporades island-complex in the North Aegean (grossly south of the 39th parallel) to the island of Crete in the South. The species exhibits great morphological and ecological plasticity and inhabits many different habitats from rocky islets and sandy shores to mountaintops as high as 2000m. By examining intraspecific variability at a segment of the mitochondrial gene cytochrome b we have found that that extant populations of P. erhardii are paraphyletic. Furthermore, we have found that subspecies previously defined on the basis of morphological characteristics do not correspond to different molecular phylogenetic clades, so that their status should be reconsidered. The DNA based biogeographical and phylogenetic history of Podarcis in Southern Greece is congruent with available paleogeographic data of the region, which supports the view that DNA sequences may be a useful tool for the study of palaeogeography.     

Cross-species testing of 27 pre-existing microsatellites in Podarcis gaigeae and Podarcis hispanica (Squamata: Lacertidae)

We tested 27 microsatellite loci for cross-species amplification in the lacertids Podarcis gaigeae and Podarcis hispanica. We detected 11 and 15 polymorphic loci in the former and the latter species, respectively. In a larger sample of individuals from a single population of each species, the number of alleles ranged from five to 23 in 10 of the polymorphic loci in P. gaigeae, and between four and 13 in nine of polymorphic loci in P. hispanica. Two locus deviated from Hardy-Weinberg equilibrium in P. hispanica. Between 11 and 16 of the 27 loci also amplified successfully in three other Podarcis species.     

Vicariance divergence and gene flow among islet populations of an endemic lizard

Allopatry and allopatric speciation can arise through two different mechanisms: vicariance or colonization through dispersal. Distinguishing between these different allopatric mechanisms is difficult and one of the major challenges in biogeographical research. Here, we address whether allopatric isolation in an endemic island lizard is the result of vicariance or dispersal. We estimated the amount and direction of gene flow during the divergence of isolated islet populations and subspecies of the endemic Skyros wall lizard Podarcis gaigeae, a phenotypically variable species that inhabits a major island and small islets in the Greek archipelago. We applied isolation-with-migration models to estimate population divergence times, population sizes and gene flow between islet-mainland population pairs. Divergence times were significantly correlated with independently estimated geological divergence times. This correlation strongly supports a vicariance scenario where islet populations have sequentially become isolated from the major island. We did not find evidence for significant gene flow within P. g. gaigeae. However, gene-flow estimates from the islet to the mainland populations were positively affected by islet area and negatively by distance between the islet and mainland. We also found evidence for gene flow from one subspecies (P. g. weigandi) into another (P. g. gaigeae), but not in the other direction. Ongoing gene flow between the subspecies suggests that even in this geographically allopatric scenario with the sea posing a strong barrier to dispersal, divergence with some gene flow is still feasible.     

Characterization of polymorphic microsatellite markers in the Dalmatian wall lizard Podarcis melisellensis (Squamata: Lacertidae)

We describe polymerase chain reaction primers and amplification conditions for 13 highly polymorphic microsatellite DNA loci isolated from the Dalmatian wall lizard, Podarcis melisellensis. The number of alleles per locus ranged from 12 to 41, with levels of observed heterozygosity between 0.62 and 0.94. Most of these loci were successfully cross-amplified in the closely related species P. sicula, but levels of polymorphism were always lower.     

Molecular phylogenetics of Iberian wall lizards (Podarcis): is Podarcis hispanica a species complex?

Phylogenetic relationships between species and morphotypes of Podarcis wall lizards from the Iberian Peninsula and north Africa were estimated using partial 12S rRNA and cytochrome b mitochondrial DNA sequences. All species except Podarcis hispanica form monophyletic units. P. hispanica is paraphyletic, although all identified morphotypes are monophyletic. These morphotypes represent highly divergent lineages showing 10-15% pairwise sequence divergence with the cytochrome b gene. The data suggest that P. hispanica is a species complex. We recommend using P. hispanica* until additional sampling delimits the number and ranges of species currently referred to P. hispanica. P. carbonelli, which has recently been raised to species status, is confirmed as a genetically distinct form. P. atrata is genetically distinct, but much more closely related to some populations of P. hispanica than previously thought.     

Digestive performance in five Mediterranean lizard species: effects of temperature and insularity

Temperature sensitivity of digestive processes has important ramifications for digestive performance in ectothermic vertebrates. We conducted a comparative analysis of temperature effects on digestive processes [gut passage times (GPTs) and apparent digestive efficiencies (ADEs)] in five lacertid lizards occurring in insular (Podarcis erhardii, P. gaigeae), and mainland (P. muralis, P. peloponnesiaca, Lacerta graeca) Mediterranean environments. GPTs were negatively correlated to temperature with mainland taxa having 10–20% longer GPTs than island taxa. In contrast to previous studies that estimate ADEs using bomb calorimetry, we compare ADEs by analyzing discrete efficiencies for lipids, sugars and proteins at three temperature regimes (20, 25, and 30°C); each of these categories produces different results. ADEs for lipids and sugars showed a monotonic increase with temperature whereas ADEs for proteins decreased with temperature. Island taxa had consistently higher ADEs than their mainland counterparts for lipids and for proteins but not for sugars. They are characterized by superior energy acquisition abilities despite significantly shorter GPTs. Their increased digestive performance relative to the mainland species appears to allow them to maximize energy acquisition in unproductive island environments where food availability is spatially and seasonally clustered.     

Genetic variation and differentiation in the lizard, Podarcis wagleriana (Reptilia: Lacertidae)

The electrophoretic variation at 26 presumptive gene loci was investigated in populations of Podarcis ivagleriana from Sicily, the Aegadian Islands, and the Aeolian Islands. For interspecific comparison, samples of the closely related lizard P. sicula from the same geographic area were also used. Population heterogeneity analyses carried out by the estimation of F-statistics and Nei's standard genetic distance, showed a high genetic homogeneity within P. sicula, but a noticeable genetic differentiation within P. wagleriana. In the latter species, Nei's D ranged from 0 to 0.212, and this is because the Aeolian populations were quite distinct from those inhabiting Sicily and the Aegadian Islands. Fixed differences identified at three loci (Ck, Ada, Gp-4) contributed to a relatively high value of Nei's standard genetic distance between the two population groups (D= 0.147). This value is very similar to those found comparing pairs of well-recognized biological species included in the genera Podarcis and Lacerta. Estimation of the time of evolutionary divergence shows that the Aeolian and Sicilian populations of P. wagleriana have been isolated geographically for a long time (0.7 Myr according to Nei's formula; 2 Myr according to Sarich's calibration), indicating evolutionary divergence at the species level. Based on genetic and biogeographic data, it is suggested the recognition of full specific status for the Aeolian populations, for which the name P. rajfonei comb. nova (Aeolian wall lizard) is proposed. Electrophoretic data and comparative species distributions suggest that (1) Podarcis sicula recently colonized the Aeolian Islands, and (2) it has competed successfully with P. rajfonei in this area, greatly reducing the range of the latter and causing the extinction of most of its populations. In fact, P. sicula is widespread in the Aeolian Archipelago, while P. rajfonei is confined at present to one large island (Vulcano) and three tiny islands (Strombolicchio, Scoglio Faraglione, La Canna). This can be considered a classic example of competitive exclusion of a native form (P. rajfonei) by a species accidentally introduced by man (P. sicula).     

Phylogeography of the Italian wall lizard, Podarcis sicula, as revealed by mitochondrial DNA sequences

In a phylogeographical survey of the Italian wall lizard, Podarcis sicula, DNA sequence variation along an 887-bp segment of the cytochrome b gene was examined in 96 specimens from 86 localities covering the distribution range of the species. In addition, parts of the 12S rRNA and 16S rRNA genes from 12 selected specimens as representatives of more divergent cytochrome b haploclades were sequenced (together about 950 bp). Six phylogeographical main groups were found, three representing samples of the nominate subspecies Podarcis sicula sicula and closely related subspecies and the other three comprising Podarcis sicula campestris as well as all subspecies described from northern and eastern Adriatic islands. In southern Italy a population group with morphological characters of P. s. sicula but with the mitochondrial DNA features of P. s. campestris was detected indicating a probably recent hybridization zone. The present distribution patterns were interpreted as the consequence of natural events like retreats to glacial refuges and postglacial area expansions, but also as the results of multiple introductions by man.     

Cytochrome b sequence divergence in the European sea bass (Dicentrarchus labrax) and phylogenetic relationships among some Perciformes species

The entire cytochrome b (cyt b ) gene has been sequenced in eight Mediterranean populations of the European sea bass, Dicentrarchus labrax and in one of D. punctatus . Our data indicate that both eastern (Greece and Egypt) and northwestern (French coast) populations of D. labrax were genetically differentiated from the Tyrrhenian ones, which were nearly indistinguishable from each other at the cyt b level. The D. labrax population from the Atlantic coast (Portugal) was genetically quite distinct from all the Mediterranean ones. These results strongly confirm the conclusions from previous studies where the same populations were screened for allozymes, random amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) d -loop variation. Seven other species of Perciformes belonging to five different families (Sparidae, Serranidae, Carangidae, Pomatomidae, Sciaenidae) were sequenced to explore the usefulness of the cyt b gene for inferring evolutionary relationships at different hierarchical levels. The data were analysed together with other published cyt b sequences from Perciformes fishes. Our data suggest that the superfamily Percoidea is not monophyletic. At the family level, the Sparidae and Moronidae seem to be monophyletic. The evolutionary relationships among families were not resolved. Possible causes for this lack of resolution are discussed.     

蜂猴线粒体细胞色素b基因变异特点及系统发育分析

测定了蜂猴属（Nycticebus)1个蜂猴（N.coucang)和2个矮蜂猴（N,pygmaseus)个体的线粒体细胞色素b(cyt-b)基因全序列,比较现有的司猴科其他种序列,分析了核苷酸序列差异和碱基替换特点,以指猴为外群重建了系统发育树,结果表明,在所研究的个体中,2个蜂猴物种碱基组成具有哺乳动物的共同特点,它们之间转换比（特别是密码子第3位）是颠换比的6倍多,大于其他种间比较;低的Ka/Ks值（＜0．1）,说明懒猴科cyt-b基因的异义突位点受到强的选择压力作用。由cyt-b基因构建的系统发育树符合懒猴科化石记录和形态学分类观点,根据化石记录和与分化时间有一定线性关系的第3位颠换和同义突变速率,估算蜂猴与倭蜂猴种间,蜂猴与蜂属间可能的分化时间分别为300和600万年。     

Microevolution and taxonomy of European reptiles with particular reference to the grass snake Natrix natrix and the wall lizards Podarcis sicula and P. melisellensis

The grass snake Natrix natrix and the wall lizards Podarcis sicula and P. melisellensis are used as examples to compare the procedure and achievements of the conventional approach to naming subspecies with the use of multivariate morphometries to investigate racial differentiation.
The conventional procedure, which has changed little over the last 50 years, fails to take into account the appropriate evolutionary facts or refer to any abstracted levels of divergence necessary for subspecific recognition. Consequently, the patterns of population differentiation are obscured by the recognition of a large number of rather meaningless subspecies. There is a tendency to section clines into artificial categories and arbitrarily delimit subspecies by physiographic features.
On the other hand, the use of multivariate morphometries reveals the patterns of population differentiation which can be related to geological events and patterns of differentiation in other species and species groups. The nature of 'hybrid' zones and population differentiation enables the relative importance of evolutionary forces such as gene flow, selection and genetic drift to be discussed and provides evidence concerning speciation mechanisms. These techniques also contribute to the discussion regarding the nature of species and provide abstracted and operational criteria for taxonomic decisions.
The difference between the results of multivariate analysis and the conventional approach cannot be explained solely on the basis of choice of characters. Some of the advantages and disadvantages of using multivariate morphometries, as opposed to other modern techniques, for investigating racial affinities are discussed.     

Molecular survey of Apicomplexa in Podarcis wall lizards detects Hepatozoon, Sarcocystis, and Eimeria species

The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from 3 genera, Hepatozoon , Sarcocystis , and Eimeria , were found. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis gallotiae , known only from endemic Gallotia sp. lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae . Eimeria sp. is reported for the first time from Podarcis spp. lizards. This study shows the power of detecting multiple different apicomplexan parasites through screening of tail tissue samples and blood drops that are often collected in reptiles for other purposes.     

Cytochrome b sequences reveal Acomys minous (Rodentia, Muridae) paraphyly and answer the question about the ancestral karyotype of Acomys dimidiatus

Sequences of the cytochrome b (cyt b) mitochondrial gene show that the spiny mouse Acomys from Crete, known as the endemic species A. minous, is composed of two distinct maternal lineages ("A" and "B"). Group "A" sequences cluster with A. nesiotes (Cyprus) and group "B" sequences cluster with A. cilicicus (Turkey), which is evidence of paraphyly of A. minous in regard to these two species. From cyt b sequences, the three taxa are very closely related to A. cahirinus (Egypt): the maximum divergence found among these sequences is 1.6%, which is equivalent to the intraspecific diversity observed in other Acomys species. Paleozoology evidenced that man unintentionally introduced Acomys into Crete and Cyprus during antiquity. The divergence time between Acomys cyt b sequences found in Crete was estimated at 0.4 Myr, which means that the diversity observed did not appear after the introduction but reflects a much more ancient polymorphism. Cytochrome b phylogeny and cytogenetic data together comprise evidence that, within the species A. dimidiatus (Saudi Arabia, Israel, Egypt), it is the karyotypic form with 36 chromosomes that derives from the form with 38 chromosomes, due to a single acrocentric fusion.