Phylogeography of Balkan wall lizard (Podarcis taurica) and its relatives inferred from mitochondrial DNA sequences

Wall lizards of the genus Podarcis (Sauria, Lacertidae) comprise 17 currently recognized species in southern Europe, where they are the predominant 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 geographical conherence (western island group, southwestern group, Italian group and Balkan group). The Balkan species are divided in two subgroups: the subgroup of Podarcis taurica (P. taurica, P. milensis, P. gaigeae and perhaps P. melisellensis), and the subgroup of Podarcis erhardii (P. erhardii and P. peloponnesiaca). We addressed the question of phylogenetic relations among the species of the P. taurica subgroup encountered in Greece, as they can be inferred from partial mtDNA (cyt b and 16S) sequences. Our data support the monophyly of P. taurica subgroup and suggest that P. gaigeae, P. milensis and P. melisellensis form a clade, which thereinafter connects to P. taurica. Within the previous clade, P. gaigeae is more closely related to P. milensis than to P. melisellensis. However, the specimens of P. taurica were subdivided in two different groups. The first one includes the specimens from northeastern Greece, and the other group includes the specimens from the rest of continental Greece and Ionian islands. Because the molecular clock of the cyt b and 16 rRNA genes was not rejected in our model test, it is possible to estimate times of speciation events. Based on the splitting of the island of Crete from Peloponnisos [c. 5 million years ago (Ma)], the evolutionary rate for the cyt b is 1.55% per million years (Myr) and for the 16S rRNA is 0.46% per Myr. These results suggest that the evolutionary history of P. taurica in Greece is more complex than a single evolutionary invasion. The data analysed, stress the need for a reconsideration of the evolutionary history of Greek Podarcis species and help overcome difficulties that classical taxonomy has encountered at both the species level.     

Cytokeratin cytoskeleton in the differentiating ovarian follicle of the lizard Podarcis sicula Raf

By immunoblotting and immunocytochemical techniques, we characterized the cytokeratins previously localized by us in the previtellogenic ovarian follicle of Podarcis sicula. Our results show that these cytokeratins correspond to those expressed in the monolayered epithelia. In fact, the immunoblotting analysis showed that the NCL-5D3 antibody, specific for human low molecular weight cytokeratins expressed in monolayered epithelia, reacted with the cytokeratins extracted both from the ovary and from the monolayered intestinal mucosa of Podarcis sicula. Furthermore, this antibody, in this reptile as in humans, clearly immunolabeled sections of corresponding tissues. The organization of the cytokeratin cytoskeleton in the main steps of the ovarian follicle differentiation was also clarified. The reported observations suggest that in Podarcis sicula, the cytokeratin cytoskeleton is absent in the early oocytes. It first appears in the growing oocytes as a thin cortical layer in concomitance with its becoming visible also in the enlarging follicle cells. In the larger follicles, this cytoskeleton appears well organized in intermediate cells and in particular in fully differentiated pyriform cells. In both these cells a cytokeratin network connects the cytoplasm to the oocyte cortex through intercellular bridges. At the end of the previtellogenic oocyte growth, the intense immunolabeling of the apex in the regressing pyriform cells suggests that the cytokeratin, as other cytoplasmic components, may be transferred from these follicle cells to the oocyte. At the end of the oocyte growth, in the larger vitellogenic oocytes surrounded by a monolayer of follicle cells, the cytokeratin constitutes a heavily immunolabeled cortical layer thicker than in the previous stages. Mol. Reprod. Dev. 48:536–542, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Phylogeography, genetic structure and diversity in the endangered bearded vulture (Gypaetus barbatus, L) as revealed by mitochondrial DNA

Bearded vulture populations in the Western Palearctic have experienced a severe decline during the last two centuries that has led to the near extinction of the species in Europe. In this study we analyse the sequence variation at the mitochondrial control region throughout the species range to infer its recent evolutionary history and to evaluate the current genetic status of the species. This study became possible through the extensive use of museum specimens to study populations now extinct. Phylogenetic analysis revealed the existence of two divergent mitochondrial lineages, lineage A occurring mainly in Western European populations and lineage B in African, Eastern European and Central Asian populations. The relative frequencies of haplotypes belonging to each lineage in the different populations show a steep East-West clinal distribution with maximal mixture of the two lineages in the Alps and Greece populations. A genealogical signature for population growth was found for lineage B, but not for lineage A; futhermore the Clade B haplotypes in western populations and clade A haplo-types in eastern populations are recently derived, as revealed by their peripheral location in median-joining haplotype networks. This phylogeographical pattern suggests allopatric differentiation of the two lineages in separate Mediterranean and African or Asian glacial refugia, followed by range expansion from the latter leading to two secondary contact suture zones in Central Europe and North Africa. High levels of among-population differentiation were observed, although these were not correlated with geographical distance. Due to the marked genetic structure, extinction of Central European populations in the last century re-sulted in the loss of a major portion of the genetic diversity of the species. We also found direct evidence for the effect of drift altering the genetic composition of the remnant Pyrenean population after the demographic bottleneck of the last century. Our results argue for the management of the species as a single population, given the apparent ecological exchangeability of extant stocks, and support the ongoing reintroduction of mixed ancestry birds in the Alps and planned reintroductions in Southern Spain.     

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).     

Foundations for conservation of intraspecific genetic diversity revealed by analysis of phylogeographical structure in the endangered endemic lizard Podarcis lilfordi

Aim  To describe and analyse phylogeographical patterns in the endangered endemic lizard Podarcis lilfordi from across its remaining range and thereby establish baseline information on genetic diversity that will help determine conservation priorities and assist future reintroduction programs.
Location  Balearic Islands, Spain.
Methods  We analysed mitochondrial DNA (2382 bp sequence from eight genes) from 118 individuals and characterized the relationships among haplotypes using parsimony networks, as well as phylogenetic inference. Analyses of historical gene flow and population growth were used to provide further insights into population histories.
Results  Four unconnected parsimony networks were obtained that mirrored the main clades in the phylogenetic tree: (I) all Menorcan populations, (II) Dragonera, Malgrats and Toro islands (Western Mallorca) (III and IV) and the remaining populations from Cabrera and Mallorca. Two major haplotype groups were detected in Menorca (I) and these provided signatures of a demographic expansion and asymmetrical historical gene flow, respectively, concordant with the expected direction of colonization from south to north of the island. Populations from western Mallorca (II) showed evidence of historical allopatric fragmentation events following isolation around the start of the Pleistocene. In networks III and IV, Cabreran populations appear to have become isolated from north and south Mallorca quite recently, with asymmetric gene flow indicating a northwards dispersal direction.
Main conclusions  P. lilfordi is a genetically diverse species that shows substantial mtDNA structuring both between regions and, at a finer scale, between some islet populations within regions. The precarious state of some islet populations shown here to be quite divergent (e.g. Toro island in western Mallorca) means that conservation of this intraspecific biodiversity requires urgent action.     

Phylogeography and population structure of the golden monkeys (Rhinopithecus roxellana): inferred from mitochondrial DNA sequences

The golden monkey (Rhinopithecus roxellana) is one of the most endangered primate species due to its dramatically shrinking distribution during the past 400 years. Its populations are restricted to three isolated regions, Qinglin (QL), Sichuan/Gansu (SG), and Shennongjia (SNJ) in China. As with other snub-nosed monkeys in China and Vietnam, the biology and evolution of this species is still poorly known. To assess genetic differentiation and explore the relationships among populations of golden monkeys from different geographic locations, 379 bp of mitochondrial DNA control region (CR) hypervariable segment I (HVI) was studied from 60 individuals. Twelve haplotypes were identified from seven populations within the three regions. Haplotype diversity was high (0.845), whereas nucleotide diversity among all haplotypes was low (0.0331). The most recent common ancestor (TMRCA) among mtDNA haplotypes was estimated to have lived approximately 0.48-0.32 million years ago. None of the haplotypes is shared among any of the three regions. Phylogenetic analysis and AMOVA revealed clear and significant phylogeographic structure between the three regions. However, only SG contained haplotypes of the two main clades, indicating either incomplete random sorting of haplotypes or a complex history with phases of population subdivisions and merging of populations. The phylogeographic structure implies that R. roxellana should be regarded as separate management units (MUs) for each of the three regions. It is likely that recent phylogeographic history has shaped the pattern of genetic differentiation observed in the golden monkey and that its populations have suffered significant demographic fluctuation.     

Phylogeography of the canyon treefrog, Hyla arenicolor (Cope) based on mitochondrial DNA sequence data

Patterns of phylogeography and gene flow were examined in the canyon treefrog, Hyla arenicolor. A total of 973 bp of mitochondrial cytochrome b sequence data were obtained for 65 individuals from 53 populations, yielding 50 unique haplotypes. Interpopulation sequence variation ranged from 0 to 13.7%. Phylogenetic analysis revealed three deeply divergent mtDNA lineages. These three Clades were mapped onto geography and found to represent completely concordant, nonoverlapping, geographical regions. Levels of sequence divergence between the three Clades were equal to or greater than published levels of divergence found in other vertebrate species and genera. Furthermore, one Clade of H. arenicolor was found to be more closely related to the outgroup H. eximia than to other H. arenicolor, suggesting that the taxonomy of this species may require revision.     

Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.     

Phylogeography of the New Zealand cicada Maoricicada campbelli based on mitochondrial DNA sequences: ancient clades associated with cenozoic environmental change

New Zealand's isolation, its well-studied rapidly changing landscape, and its many examples of rampant speciation make it an excellent location for studying the process of genetic differentiation. Using 1520 base pairs of mitochondrial DNA from the cytochrome oxidase subunit I, ATPase subunits 6 and 8 and tRNA(Asp) genes, we detected two well-differentiated, parapatrically distributed clades within the widespread New Zealand cicada species Maoricicada campbelli that may prove to represent two species. The situation that we uncovered is unusual in that an ancient lineage with low genetic diversity is surrounded on three sides by two recently diverged lineages. Using a relaxed molecular clock model coupled with Bayesian statistics, we dated the earliest divergence within M. campbelli at 2.3 +/- 0.55 million years. Our data suggest that geological and climatological events of the late Pliocene divided a once-widespread species into northern and southern components and that near the middle of the Pleistocene the northern lineage began moving south eventually reaching the southern clade. The southern clade seems to have moved northward to only a limited extent. We discovered five potential zones of secondary contact through mountain passes that will be examined in future work. We predict that, as in North American periodical cicadas, contact between these highly differentiated lineages will exist but will not involve gene flow.     

Phylogeography of the Turkish brown trout Salmo trutta L.: mitochondrial DNA PCR‐RFLP variation

In the present study, mitochondrial DNA polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) assay was used to assess the phylogenetic and phylogeographic relationships among 27 brown trout Salmo trutta populations from Turkey. The complete NADH 5/6 region and a second segment comprising the cytochrome b gene and D‐loop of mtDNA amplified by PCR were digested with six and five restriction enzymes, respectively. A total of 27 haplotypes were observed and divided into three major phylogenetic assemblages, namely Danubian (DA), Adriatic (AD) and a newly proposed Tigris (TI) lineage. The timing of the net nucleotide divergence between the major lineages along with the geological history of Turkey suggested pre‐Pleistocene isolation of the Turkish brown trout and provided evidence that Turkey could be considered as a centre of diversification for these lineages. The average haplotype diversity (0·1397) and the nucleotide diversity (0·000416) within populations were low in comparison to the observed interpopulation nucleotide diversity (0·021266). PCR‐RFLP analysis showed that most of the mtDNA sequence variation found in the Turkish brown trout populations was imputable to differences among lineages. On the other hand, there was also an obvious relationship between geographical distribution of the populations and their clustering. The present study showed that brown trout populations from Turkey are highly divergent and mainly have a unique genetic profile that could be used for conservation and management purposes.     

Phylogeography, population structure and dispersal patterns of the beluga whale Delphinapterus leucas in the western Nearctic revealed by mitochondrial DNA

The recent evolutionary history, population structure and movement patterns of beluga whales in the western Nearctic were inferred from an analysis of mitochondrial DNA control region sequence variation of 324 whales from 32 locations representing five summer concentration areas in Alaska and north-west Canada. Phylogenetic relationships among haplotypes were inferred from parsimonious networks, and genetic subdivision was examined using haplotypic frequency-based indices and an analysis of variance method modified for use with interhaplotypic distance data. MtDNA relationships were characterized by a series of star-like phylogenies which, when viewed in conjunction with information on haplotype frequency and distribution, suggested a rapid radiation of beluga whales into the western Nearctic following the Pleistocene, and an early divergence of the Beaufort Sea from the Chukchi and Bering Seas subpopulations. Overall nucleotide diversity was low (0.51%) yet all major summering concentrations were significantly differentiated (Φ_ST= 0.33) from one another. Stratification of samples by gender and age from the three northernmost subpopulations suggested that female cohorts from neighbouring subpopulations were more differentiated than males. Further stratification of adult animals by age revealed that older adults were substantially less subdivided among locations than younger adults, particularly for males, suggesting that dispersal, although limited, is biased toward older adult males. Overall, the patterns of mtDNA variation in beluga whales indicated that the summering concentrations are demographically, if not phyletically distinct. Population structure appears to be maintained primarily by natal homing behaviour, while asymmetries in dispersal may be associated with the type of mating system.     

Evidence for biogeographic patterning of mitochondrial DNA sequences in Eastern horse populations

Equine mitochondrial DNA (mtDNA) phylogeny reconstruction reveals a complex pattern of variation unlike that seen in other large domesticates. It is likely that this pattern reflects a process of multiple and repeated, although not necessarily independent, domestication events. Until now, no clear geographic affiliation of clades has been apparent. In this study, amova analyses have revealed a significant non-random distribution of the diversity among equine populations when seven newly sequenced Eurasian populations were examined in the context of previously published sequences. The association of Eastern mtDNA types in haplogroup F was highly significant using Fisher's exact test of independence (P = 0.00000). For the first time, clear biogeographic partitioning has been detected in equine mtDNA sequence.     

Phylogenetic relationships among the subfamily Coregoninae as revealed by mitochondrial DNA restriction analysis

Mitochondria1 DNA (mtDNA) restriction analysis was used to assess phylogenetic patterns among 21 taxa of the subfamily Coregoninae. The genus Prosopium formed a very distinct group differing by 10% (sequence divergence estimate) from other species. Coregonus and Stenodus species were closely related, diverging by sequence divergence estimates of less than 5.6%. These species split into two major sister groups. One comprised all 'true whitefish' (subgenus Coregonus ) and four cisco species (subgenus Leucichrhys ). The most distant species within this assemblage was the Acadian whitefish ( C. huntsmani ). The other group included all other cisco species and also the Inconnu ( Stenodus leucichthys ). These results supported a polyphyletic origin of the ciscoes, and did not support Stenodus as a sister taxon of the genus Coregonus . The levels of sequence divergence observed suggested that most extant coregonines radiated during the Pleistocene.     

Population history of European mountain pines Pinus mugo and Pinus uncinata revealed by mitochondrial DNA markers

The dwarf mountain pine (Pinus mugo) and the Pyrenean pine (P. uncinata) constitute a pair of closely related coniferous taxa of poorly resolved evolutionary history and affinity, which inhabit numerous stands scattered over subalpine environments of European mountain ranges. The aim of the study was to investigate their phylogeography and mutual relationships, shedding new light on their taxonomy and the past of the alpine flora. Previous evolutionary reconstructions of the mountain pines relied mainly on bi‐parentally or paternally inherited markers that quickly homogenize between populations, showing rather shallow and recent differentiation of gene pools. Therefore, to contrast these pictures, we analyzed diversity and differentiation within a large set of new mitochondrial loci, inherited in maternal line and distributed by seeds at short geographical distances. Samples of the taxa were taken from 27 natural populations representing their range‐wide distributions—17 populations of P. mugo and 10 of P. uncinata. All markers appeared polymorphic, providing a total of 31 multilocus haplotypes. Two of the loci proved to be species‐diagnostic and nearly fixed between analyzed samples. Distribution of mitotypes indicate that allopatric populations of the taxa constitute separate mitochondrial haplogroups, and the two mountain pines have independent evolutionary history. However, introgression of P. mugo mitotypes by P. uncinata specimens revealed in the species contact zone in Western Alps shows that their speciation is not fully completed.     

Isolation and characterization of polymorphic microsatellite markers in the wall lizard Podarcis erhardii (Squamata: Lacertidae)

Five polymorphic microsatellite loci (containing di‐ and penta‐nucleotide repeats) were developed for the Erhard's wall lizard, Podarcis erhardii, an endemic species of southern Balkan Peninsula. The number of alleles ranged from five to 17. Observed and expected heterozygosities ranged from 0.28 to 0.92 and 0.57 to 0.91, respectively. These markers will provide a valuable tool for population genetics analyses and can contribute to the evaluation of conservation programs.     

Genetic variability and phylogeography of the cyprinid Telestes muticellus within the Italian peninsula as revealed by mitochondrial DNA

A phylogeographic analysis of 12 populations of Telestes muticellus was performed on the basis of mitochondrial DNA sequences of the cytochrome b gene. The main aims were to determine phylogenetic relationships from congener species and to highlight the patterns of genetic differentiation within the Italian peninsula. The results obtained showed a significant divergence with the congener species, indicating a possible Miocene origin for the genus. Divergence was found to be low for T. muticellus , yet distinct lineages resulted as being allopatric within the two main Italian biogeographic districts, the Padano-Venetian and the Tuscano-Latium, suggesting the existence of recent vicariance coupled with past demographic regression. The high level of genetic differentiation found between populations highlighted the low dispersal ability of this rheophilic Cyprinid, whose migration was mainly related to connections between the upper parts of river basins. The distribution of sequence characteristics found suggests that particular attention should be paid to the conservation management of this species.     

Isolation,characterization and molecular cloning of cathepsin D from lizard ovary: Changes in enzyme activity and mRNA expression throughout ovarian cycle

During vitellogenesis, the oocytes of oviparous species accumulate in the cytoplasm a large amount of proteic nutrients synthetized in the liver. Once incorporated into the oocytes, these nutrients, especially represented by vitellogenin (VTG) and very low‐density lipoprotein (VLDL), are cleaved into a characteristic set of polypeptides forming yolk platelets. We have studied the molecular mechanisms involved in yolk formation in a reptilian species Podarcis sicula, a lizard characterized by a seasonal reproductive cycle. Our results demonstrate the existence in the lizard ovary of an aspartic proteinase having a maximal activity at acidic pH and a molecular mass of 40 kDa. The full‐length aspartic proteinase cDNA produced from total RNA by RT‐PCR is 1,442 base pairs long and encodes a protein of 403 amino acids. A comparison of the proteic sequence with aspartic proteinases from various sources demonstrates that the lizard enzyme is a cathepsin D. Lizard ovarian cathepsin D activity is maximal in June, in coincidence with vitellogenesis and ovulation, and is especially abundant in vitellogenic follicles and in eggs. Ovarian cathepsin D activity can be enhanced during the resting period by treatment with FSH in vivo. Northern blot analysis shows that cathepsin D mRNA is exceedingly abundant during the reproductive period, and accumulates preferentially in previtellogenic oocytes. Mol. Reprod. Dev. 52:126–134, 1999. © 1999 Wiley‐Liss, Inc.