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.     

A new cryptic species of pond turtle from southern Italy, the hottest spot in the range of the genus Emys (Reptilia, Testudines, Emydidae)

Geographic variation in the mtDNA haplotypes (cytochrome b gene) of 127 European pond turtles from Italy was investigated. Thirty‐eight of the Italian samples were also studied by nuclear fingerprinting (ISSR PCR) and compared with samples from other parts of the range representing all nine currently known mtDNA lineages of Emys orbicularis. Our genetic findings were compared against morphological data sets (measurements, colour pattern) for 109 adult turtles from southern Italy. Italy is displaying on a small geographical scale the most complicated variation known over the entire distributional area of Emys (North Africa over Europe and Asia Minor to the Caspian and Aral Seas). The Tyrrhenic coast of the Apennine Peninsula, the Mt. Pollino area and Basilicata are inhabited by Emys orbicularis galloitalica, a subspecies harbouring a distinct mtDNA lineage. The same lineage is also found in Sardinia. Along the Adriatic coast of Italy and on the Salentine Peninsula (Apulia, southern Italy), another morphologically distinctive subspecies (Emys orbicularis hellenica) occurs, which also bears a different mtDNA lineage. A higher diversity of mtDNA haplotypes in the south of the Apennine Peninsula suggests that the glacial refugia of E. o. galloitalica and E. o. hellenica were located here. A further refuge of E. o. hellenica probably existed in the southern Balkans. The west coasts of the Balkans and Corfu have probably been colonized from Italy and not from the geographically closer southern Balkanic refuge. In Sicily, a third mtDNA lineage is distributed, which is sister to all other known lineages of Emys. Morphologically, Sicilian pond turtles resemble E. o. galloitalica. However, nuclear fingerprinting revealed a clear distinctiveness of the Sicilian taxon, whereas no significant divergence was detected between representatives of the other eight mtDNA lineages of Emys. Furthermore, nuclear fingerprinting provided no evidence for current or past gene flow between the Sicilian taxon and the mainland subspecies of E. orbicularis. Therefore, Sicilian pond turtles are described here as a species new to science. Some populations in Calabria and on the Salentine Peninsula comprise individuals of different mtDNA lineages. We interpret this as a natural contact. However, we cannot exclude that these syntopic occurrences are the result of human activity. For example, in other parts of Italy, the natural distribution pattern of Emys is obscured by allochthonous turtles. This could also be true for southern Italy. The discovery of the complex taxonomic differentiation in southern Italy requires reconsidering conservation strategies.     

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

Hermann's 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.     

Cryptic diversity of Italian bats and the role of the Apennine refugium in the phylogeography of the western Palaearctic

The Mediterranean Basin is typified by a high degree of species rarity and endemicity that reflects its position, geomorphology, and history. Although the composition and cryptic variation of the bat faunas from the Iberian and Balkan Peninsulas are relatively well studied, data from the Apennine Peninsula are still incomplete. This is a significant shortfall, given the presumed refugial role of this region in the context of Europe's Pleistocene phylogeography. It was thus our aim to supplement the phylogeographical information from the region, generating mitochondrial sequences and reviewing published data, with a focus on the dispersal and diversification patterns characterizing taxa with different life strategies. Site‐specific lineages were ascertained, especially in the genera Myotis and Plecotus and amongst the pipistrelloid bats, representing speciose radiations. It was possible to observe disjunct ranges with patches isolated south of the Alps in several species, corresponding with evolution of elevated genetic distance. The genetic subdivision within the continuous Italian range into northern and southern lineages in several taxa indicated the possible past substructure of the refugium. Several shared lineages between the Apennine and Ibero‐Maghrebian regions were observed, indicating connectivity between the Adriatic and Atlantic?Mediterranean refuges, and raising questions as to which region these clades originated from and what was the direction of faunal exchange between them. In contrast to Europe's other two main refugia, the Apennine Peninsula is a smaller region with simpler phylogeographical patterns. Nevertheless, our results support the idea that the region generated novel lineages. Whereas diversification in sedentary bats may have been driven through the generation of in situ adaptations, specialization, and niche differentiation, the emergence of species with a tramp strategy could have entailed the utilization of faunal drift and the taxon cycle. © 2015 The Linnean Society of London     

Phylogenetic analysis of the Lacerta agilis subspecies complex

The Sand lizard Lacerta agilis inhabits a huge area across Eurasia with several subspecies. Nine subspecies are generally approved, L. a. agilis, L. a. argus, L. a. bosnica; L. a. chersonensis, L. a. exigua, L. a. grusinica, L. a. iorinensis, L. a. brevicaudata and L. a. boemica, but several more have been described. The emergence of this large number of subspecies is connected with the phylogeographic history of this species, defined by geographic and climatic processes. A study revealing phylogenetic relationships of this species was already conducted with a broad taxon sampling and coverage. However, the latter study was based solely on the cytochrome b gene and, furthermore, the Balkan Peninsula inhabited by the subspecies L. a. bosnica was underrepresented. This peninsula is a centre of European herpetofaunal endemism with high levels of phenotypic and genotypic variation. Therefore, the inclusion of the subspecies L. a. bosnica is important to clarify the overall view of the phylogenetic relations within the Lacerta agilis subspecies complex and to investigate the level of population differentiation within this highly diverse area. Thus, the aim of the present study was a more thorough analysis of the Balkan Peninsula with a broader taxon sampling. Furthermore, we extended the available datasets by adding the mitochondrial d-loop region and by further samples of different areas of the distribution range apart from the Balkan Peninsula. Our study reveals that the Balkan Peninsula is apparently inhabited by several differentiated lineages, whereby the Central Greek lineage might deserve subspecies status. Furthermore, the distribution area of the two subspecies L. a. agilis and L. a. argus should be revised, as the results of our study raise doubt about the commonly assumed distribution of both subspecies. As the most important outcome our results support that L. a. boemica deserves species status.     

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.     

A new cytochrome b phylogroup of the common vole (Microtus arvalis) endemic to the Balkans and its implications for the evolutionary history of the species

The phylogeographic architecture of the common vole, Microtus arvalis, has been well‐studied using mitochondrial DNA and used to test hypotheses relating to glacial refugia. The distribution of the five described cytochrome b (cyt b) lineages in Europe west of Russia has been interpreted as a consequence of postglacial expansion from both southern and central European refugia. A recently proposed competing model suggests that the ‘cradle’ of the M. arvalis lineages is in western central Europe from where they dispersed in different directions after the Last Glacial Maximum. In the present study, we report a new cyt b lineage of the common vole from the Balkans that is not closely related to any other lineage and whose presence might help resolve these issues of glacial refugia. The Balkan phylogroup occurs along the southern distributional border of M. arvalis in central and eastern Bosnia and Herzegovina, Montenegro, and eastern Serbia. Further north and west in Slovenia, Bosnia and Herzegovina, and Serbia, common voles belong to the previously‐described Eastern lineage, whereas both lineages are sympatric in one site in Bosnia and Herzegovina. The Balkan phylogroup most reasonably occupied a glacial refugium already known for various Balkan endemic species, in contrast to the recently proposed model. South‐east Europe is an absolutely crucial area for understanding the postglacial colonization history of small mammals in Europe and the present study adds to the very few previous detailed phylogeographic studies of this region. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 788–796.     

Differential success in northwards range expansion between ecotypes of the marble gallwasp Andricus kollari: a tale of two lifecycles

The Marble gallwasp Andricus kollari has a native range divided into two geographically separated lifecycles. In Eastern Europe and Turkey, the lifecycle involves a sexual generation on Turkey oak, Quercus cerris, while in Iberia and North Africa the sexual generation host is cork oak, Q. suber. Over the last 500 years, A. kollari has expanded its range into northern Europe, following human planting of Q. cerris from Italy and the Balkans. We ask: (i) what is the genetic relationship between eastern and western distributions of Andricus kollari? Can we determine which lifecycle is ancestral, and how long ago they diverged? (ii) To what extent have eastern and western native ranges contributed to northwards range expansion? (iii) Is there any evidence for hybridization between the two life cycle types? We present analyses of allozyme data for 13 polymorphic loci and of sequence variation for a 433 bp fragment of the mitochondrial cytochrome b gene. These show: (i) that four haplotype lineages (one in Spain, two in Hungary/Italy and one in Turkey) diverged more or less simultaneously between 1 and 2 million years ago, suggesting the existence of at least four refuges through recent ice age cycles. Our data cannot resolve which lifecycle type is ancestral. (ii) Populations north of putative refuges are divided into two sets. Populations in south‐west France are allied to Spain, while all remaining populations in northern Europe have been colonized from Italy and the Balkans. (iii) The transition from one race to another in south‐west France is marked by abrupt transitions in the frequency of refuge‐specific private alleles and corresponds closely to the northern limit of the distribution of cork oak. Although hybrids were detected in north‐west France, none were detected where the two lifecycles meet in south‐western France. The biology of oak gallwasps predicts that any hybrid zone will be narrow, and limited to regions where Q. cerris and Q. suber meet. Our data suggest that eastern and western A. kollari are effectively separate species.     

Invaders or endemics? Molecular phylogenetics,biogeography and systematics of Dreissena in the Balkans

1. Zebra mussels and their relatives (Dreissena spp.) have been well studied in eastern, central and western Europe as well as in North America, because of their invasiveness and economic importance. Much less is known about the biology and biogeography of indigenous (endemic) taxa of Dreissena, in the Balkans. A better knowledge of these taxa could help us (i) understand the factors triggering invasiveness in some taxa and (ii) identify other potentially invasive species. 2. Using a phylogenetic approach (2108 base pairs from three gene fragments), Dreissena spp. from natural lakes in the Balkans were studied to test whether invasive Dreissena populations occur in such lakes on the Balkan Peninsula, whether Dreissena stankovici really is endemic to the ancient Lakes Ohrid and Prespa, and to infer the phylogenetic and biogeographical relationships of Balkan dreissenids. 3. No invasive species of Dreissena, such as Dreissena polymorpha, were recorded. The supposedly ‘endemic’D. stankovici is not restricted to the ancient Lakes Ohrid and Prespa, but is the most widespread and dominant species in the west‐central Balkans. Its southern sister taxon, Dreissena blanci, occurs sympatrically with D. stankovici in Lakes Prespa, Mikri Prespa and Pamvotis. Both species are classified into the subgenus Dreissena (Carinodreissena) of which the subgenus Dreissena (Dreissena) (which includes the invasive D. polymorpha) is the sister taxon. Dreissena blanci and D. stankovici are considered to represent distinct species. 4. On a global scale, the two Balkan species have small ranges. An early Pliocene time frame for the divergence of the subgenera Carinodreissena and Dreissena is discussed, as well as potential colonization routes of the most recent common ancestor of Carinodreissena spp. 5. The ambiguous taxonomy of dreissenids in the Balkans is addressed. As nominal D. blanci presbensis from Lake Prespa has nomenclatural priority over D. stankovici, the correct name for the latter taxon should be Dreissena presbensis.     

Phylogeography of western Palaearctic reptiles – Spatial and temporal speciation patterns

A phylogeographic analysis of eight species complexes of European reptiles was performed using different molecular methods. While mitochondrial genes (mainly cytochrome b sequences) enabled conclusions about phylogeography and differentiation, additional application of bisexually inherited markers provided information about speciation stages. As species with similar distribution patterns in southern and Central Europe were selected, matching phylogeographic patterns are useful for drawing general conclusions:

(1) The species complexes are in different stages of speciation. In some cases, cryptic species were detected.

(2) Highest genetic diversity occurs in southern Europe, the Near East and the Caucasus, regions corresponding with glacial refuges in the Iberian, Apennine and Balkan Peninsulas as well as in Turkey and the Caucasus. Often, several microrefugia must have existed in close neighbourhood. Additional microrefugia were located in southern France and in the Carpathian Basin.

(3) North Africa and the Middle East did not serve as glacial refuges for Central or northern European lineages and are typically inhabited by independent clades.

(4) Evidence for multiple range retractions and expansions, which were postulated for the times of Pleistocene climatic oscillations, could be found in the Balkans, but in Central Europe their traces have been wiped out by the last glacial. Only the Holocene invasion has left imprints in the genomes from this area.

(5) Central and northern Europe were recolonized from Balkan and Pontic refugia in the Holocene.

(6) Groups from the Iberian and Apennine Peninsulas rarely conquered other regions. This limitation can be attributed to the barrier function of the Pyrenees and the Alps.

Italy as a major Ice Age refuge area for the bat Myotis myotis (Chiroptera: Vespertilionidae) in Europe

The distribution of biota from the temperate regions changed considerably during the climatic fluctuations of the Quaternary. This is especially true for many bat species that depend on warm roosts to install their nursery colonies. Surveys of genetic variation among European bats have shown that the southern peninsulas (Iberia and the Balkans) harbour endemic diversity, but to date, no such surveys have been conducted in the third potential glacial refuge area, the Apennine peninsula. We report here the phylogeographical analysis of 115 greater mouse-eared bats ( Myotis myotis ) sampled throughout Italy, and show that 15 of the 18 different haplotypes found in the mitochondrial control region of these bats were unique to the Apennine peninsula. Colonies within this region also showed substantial genetic structure at both mitochondrial ( Φ_ST  = 0.47, P  < 0.001) and nuclear markers ( F _ST = 0.039, P  < 0.001). Based on a comprehensive survey of 575 bats from Europe, these genetic markers further indicate that central Italian populations of M. myotis are more closely related to Greek samples from across the Adriatic Sea, than to other Italian bats. Mouse-eared bat populations from the Apennine peninsula thus represent a complex mixture of several endemic lineages, which evolved in situ , with others that colonized this region more recently along an Adriatic route. Our broad survey also confirms that the Alps represent a relatively impermeable barrier to gene flow for Apennine lineages, even for vagile animals such as bats. These results underline the conservation value of bats from this region and the need to include the Apennine peninsula in phylogeographical surveys in order to provide a more accurate view of the evolution of bats in Europe.     

Phylogeography of the narrow‐headed vole Lasiopodomys (Stenocranius) gregalis (Cricetidae,Rodentia) inferred from mitochondrial cytochrome b sequences: an echo of Pleistocene prosperity

A species‐wide phylogeographic study of the narrow‐headed vole Lasiopodomys (Stenocranius) gregalis was performed using the mitochondrial (mt) cytochrome b gene. We examined 164 specimens from 50 localities throughout the species distribution range. Phylogeographic pattern clearly demonstrates the division into four major mtDNA lineages with further subdivision. The level of genetic differentiation between them was found to be extremely high even for the species level: about 6–11%. The most striking result of our study is extremely high mutation rate of cytb in L. gregalis. Our estimates suggested its value of 3.1 × 10^?5 that is an order of magnitude higher than previous estimates for Microtus species. The mean estimated time of basal differentiation of the narrow‐headed vole is about 0.8 Mya. This time estimate is congruent with the known paleontological record. The greatest mitochondrial diversity is found in Southern Siberia where all four lineages occur; therewith, three of them are distributed exclusively in that area. The lineage that is distributed in south‐eastern Transbaikalia is the earliest derivate and exhibits the highest genetic divergence from all the others (11%). It is quite probable that with further research, this lineage will turn out to represent a cryptic species. Spatial patterns of genetic variation in populations of the narrow‐headed vole within the largest mt lineage indicate the normal or stepping stone model of dispersal to the north and south‐west from the Altay region in Middle Pleistocene. Both paleontological data and genetic diversity estimates suggest that this species was very successful during most of the Pleistocene, and we propose that climate humidification and wide advance of tree vegetation at the Pleistocene–Holocene boundary promoted range decrease and fragmentation for this typical member of tundra‐steppe faunistic complex. However, we still observe high genetic diversity within isolated fragments of the range.     

Speciation in Mediterranean refugia and post‐glacial expansion of Zerynthia polyxena (Lepidoptera,Papilionidae)

Migration of populations to and from glacial refugia is responsible for various cases of speciation and subspeciation in Europe. The pattern of distribution and the degree of diversification between lineages originated by isolation in different glacial refugia usually depends on ecological traits, especially to their dispersal ability. Zerynthia polyxena is a philopatric species, scattered in small populations and rarely colonizing mountain areas. These characteristics probably caused repeated isolation during the Quaternary and may have favoured diversification. Actually two studies, based on both morphological and genetic data, suggest the existence of two highly distinct lineages in Europe having in Northern Italy their contact zone. In this study, I applied geometric morphometrics to male genitalia and demonstrated that (i) two morphotypes exist in Europe approximately facing on the two sides of the Po River; (ii) the two lineages probably survived glaciations in Italy and the Balkan Peninsula, respectively; then the Balkans lineage expanded to Central and Eastern Europe; (iii) no hybrid populations seem to exist in the contact area and, in one locality at least, the two lineages live in sympatry without any evidence of intermediates. These results suggest that (i) two sister species of Zerynthia exist in Europe. Accordingly, Papilio cassandra Geyer, 1828 is reinstated, as Zerynthia cassandra stat. rev., as the species to which the Zerynthia from Italy South of the Po River belong. Male genitalia differences with Zerynthia polyxena are described.     

New insights into the origin and the genetic status of the Balkan donkey from Serbia

The Balkan donkey (Equus asinus L.) is commonly regarded as a large‐sized, unselected, unstructured and traditionally managed donkey breed. We assessed the current genetic status of the three largest E. asinus populations in the central Balkans (Serbia) by analysing the variability of nuclear microsatellites and the mitochondrial (mtDNA) control region of 77 and 49 individuals respectively. We further analysed our mtDNA dataset along with 209 published mtDNA sequences of ancient and modern individuals from 19 European and African populations to provide new insights into the origin and the history of the Balkan donkey. Serbian donkey populations are highly genetically diverse at both the nuclear and mtDNA levels despite severe population decline. Traditional Balkan donkeys in Serbia are rather heterogeneous; we found two groups of individuals with similar phenotypic features, somewhat distinct nuclear backgrounds and different proportions of mtDNA haplotypes belonging to matrilineal Clades 1 and 2. Another group, characterized by larger body size, different coat colour, distinct nuclear gene pool and predominantly Clade 2 haplotypes, was delineated as the Banat donkey breed. The maternal landscape of the large Balkan donkey population is highly heterogeneous and more complex than previously thought. Given the two independent domestication events in donkeys, multiple waves of introductions into the Balkans from Greece are hypothesized. Clade 2 donkeys probably appeared in Greece prior to those belonging to Clade 1, whereas expansion and diversification of Clade 1 donkeys within the Balkans predated that of Clade 2 donkeys.     

Phylogeography of the Eurasian green woodpecker (Picus viridis)

Aim In this paper we investigate the evolutionary history of the Eurasian green woodpecker (Picus viridis) using molecular markers. We specifically focus on the respective roles of Pleistocene climatic oscillations and geographical barriers in shaping the current population genetics within this species. In addition, we discuss the validity of current species and subspecies limits. Location Western Palaearctic: Europe to western Russia, and Africa north of the Sahara. Methods We sequenced two mitochondrial genes and five nuclear introns for 17 Eurasian green woodpeckers. Multilocus phylogenetic analyses were conducted using maximum likelihood and Bayesian algorithms. In addition, we sequenced a fragment of the cytochrome b gene (cyt b, 427 bp) and of the Z‐linked BRM intron 15 for 113 and 85 individuals, respectively. The latter data set was analysed using population genetic methods. Results Our phylogenetic results support the monophyly of Picus viridis and suggest that this taxon comprises three allopatric/parapatric lineages distributed in North Africa, the Iberian Peninsula and Europe, respectively. The North African lineage split from the Iberian/European clade during the early Pleistocene (1.6–2.2 Ma). The divergence event between the Iberian and the European lineages occurred during the mid‐Pleistocene (0.7–1.2 Ma). Our results also support a post‐glacial range expansion of these two lineages from distinct refugia located in the Iberian Peninsula and possibly in eastern Europe or Anatolia, which led to the establishment of a secondary contact zone in southern France. Main conclusions Our results emphasize the crucial role of both Pleistocene climatic oscillations and geographical barriers (Strait of Gibraltar, Pyrenees chain) in shaping the current genetic structure of the Eurasian green woodpecker. Our molecular data, in combination with diagnosable plumage characters, suggest that the North African green woodpecker (Levaillant’s woodpecker) merits species rank as Picus vaillantii (Malherbe, 1847). The two European lineages could be distinguished by molecular and phenotypic characters over most of their respective geographical ranges, but they locally exchange genes in southern France. Consequently, we prefer to treat them as subspecies (P. viridis viridis, P. viridis sharpei) pending further studies.     

Mitochondrial genomes reveal the global phylogeography and dispersal routes of the migratory locust

The migratory locust, Locusta migratoria, is the most widely distributed grasshopper species in the world. However, its global genetic structure and phylogeographic relationships have not been investigated. In this study, we explored the worldwide genetic structure and phylogeography of the locust populations based on the sequence information of 65 complete mitochondrial genomes and three mitochondrial genes of 263 individuals from 53 sampling sites. Although this locust can migrate over long distances, our results revealed high genetic differentiation among the geographic populations. The populations can be divided into two different lineages: the Northern lineage, which includes individuals from the temperate regions of the Eurasian continent, and the Southern lineage, which includes individuals from Africa, southern Europe, the Arabian region, India, southern China, South‐east Asia and Australia. An analysis of population genetic diversity indicated that the locust species originated from Africa. Ancestral populations likely separated into Northern and Southern lineages 895 000 years ago by vicariance events associated with Pleistocene glaciations. These two lineages evolved in allopatry and occupied their current distributions in the world via distinct southern and northern dispersal routes. Genetic differences, caused by the long‐term independent diversification of the two lineages, along with other factors, such as geographic barriers and temperature limitations, may play important roles in maintaining the present phylogeographic patterns. Our phylogeographic evidence challenged the long‐held view of multiple subspecies in the locust species and tentatively divided it into two subspecies, L. m. migratoria and L. m. migratorioides.     

Diversity and biogeography of Ni‐hyperaccumulators of Alyssum section Odontarrhena (Brassicaceae) in the central western Mediterranean: evidence from karyology,morphology and DNA sequence data

The diversity of Alyssum section Odontarrhena in the central–western Mediterranean region was investigated to elucidate relationships and biogeography of Ni‐hyperaccumulators in the group. Karyological, morphometric and molecular phylogenetic analyses were performed on accessions of Ni‐hyperaccumulators from serpentine outcrops and non‐hyperaccumulators from calcareous–dolomitic soils in the region. Alpine and Apennine populations of A. argenteum, Sardinian A. tavolarae and some Tuscan A. bertolonii had a tetraploid chromosome complement and larger silicles, seeds and seed wings than diploid accessions. DNA sequences from the plastid rpoC1 locus corroborated the monophyly of section Odontarrhena but species relationships were poorly resolved. Bayesian analysis of combined ITS‐rpoC1 sequences retrieved three main lineages including hyperaccumulators and non‐hyperaccumulators of contrasting geographical origin. One lineage was mainly continental and included alpine and northern Apennine populations of A. argenteum, the Balkan complex of A. murale and the Iberian group of A. serpyllifolium, sister to Corsican A. robertianum as suggested by their similar diploid karyotype. In this clade no divergence was found between typical A. serpyllifolium and related Ni‐hyperaccumulator races from the Iberian peninsula, supporting their conspecific status. A second lineage was prevalently Mediterranean and included the sister species A. bertolonii and A. tavolarae, and other endemics from Sicily, the southern Balkans and Turkey from dolomite and serpentine habitats. The present data suggest new model systems consisting of hyperaccumulators and non‐hyperaccumulators of proven phylogenetic affinity for further research on the molecular mechanisms of Ni‐hyperaccumulation and serpentine tolerance at the diploid and tetraploid level. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 269–289.     

Testing the efficiency of nested barriers to dispersal in the Mediterranean high mountain plant Edraianthus graminifolius (Campanulaceae)

Due to strong spatial heterogeneity and limited Pleistocene glaciation, the Balkan Peninsula is a major European biodiversity hot spot. Surprisingly little, however, is known about patterns and processes of intraspecific diversification of its biota in general and of high‐altitude species in particular. A well‐suited system to test hypotheses with respect to various isolating factors acting at different geographic scales and to explore full‐range phylogeographic patterns on the Balkan Peninsula is Edraianthus graminifolius (Campanulaceae), distributed in the western Balkan mountain systems, the southwestern Carpathians and the Apennine Peninsula. To this end, we used a dense population sampling and employed amplified fragment length polymorphism (AFLP) markers and plastid DNA sequences supplemented by ecological niche modelling. The strongest splits were inferred to separate southern and northern Balkan populations from the central ones, from where range extension occurred to the Carpathians and, in more recent times, once or twice to the Apennine Peninsula. The three genetic groups in the western Balkan Peninsula were remarkably congruent among molecular markers, suggesting that the barriers to gene flow acted over long time periods facilitating allopatric differentiation. Each main group of Balkan populations contained genetically and geographically distinct subgroups, which likely are the result of local refugia during warmer periods. Evidently, the topographically highly complex and during the Last Glacial Maximum only locally glaciated Balkan Peninsula is a hot spot of species richness and endemism as well as a sanctuary of intraspecific genetic diversity, even if the underlying causes remain insufficiently understood.     

Genetic diversity and invasion history of the European subterranean termite <Emphasis Type="Italic">Reticulitermes urbis</Emphasis> (Blattodea,Termitoidae, Rhinotermitidae)

Biological invasions are among key factors of ecological changes, and social insects appear as highly successful invasive animals. Subterranean termites of the holarctic genus Reticulitermes are present in Europe with six native and one invasive (the nearctic R. flavipes) species. The species R. urbis shows a disjunct distribution in the Western Balkans, Eastern Italy and Southern France. Previous molecular and population genetics data suggested that the taxon originated from the Balkans, and that Italian and French populations are invasive, but it is still unknown how many introduction events occurred and from which Balkan source populations. To address these questions, a population genetics analysis was performed on a larger sampling than previous studies, using mitochondrial cytochrome oxidase II and 6 microsatellite markers on 47 colonies collected across the whole distribution area. Mitochondrial analysis confirmed the presence of two major lineages where colonies from Balkans, Italy, and France intermingle. Similarly, microsatellite loci analysis indicated the presence of two genetic clusters, though not corresponding to the two mitochondrial clades, each including colonies from the three sampled areas and with individuals showing mixed cluster membership. Overall, French and Italian populations showed indications of bottleneck (reduced genetic diversity and change of allele frequencies) and do not appear genetically differentiated from the Balkan population. Results presented here support a history of multiple introductions in Italy and France, in a scenario consistent with continuous exchanges between native and invasive areas, as expected along human trades routes.