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.     

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.     

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.     

Combining immunological and molecular data to assess phylogenetic relations of some Greek Podarcis species

Most recent molecular studies revealed the phylogeny of Greek Podarcis species, which for years remained elusive, due to discordant data produced from various chromosomal, complement fixation and protein studies. In this report, we analyzed cellular immune responses of spleen-derived lymphocytes from six allopatric Podarcis species encountered in Greece, by assessing two-way mixed lymphocyte reaction (MLR)-induced proliferation. On the basis of stimulation indices (S.I.) as determined from cultures set up from xenogeneic splenocytes coincubated in pairs, we generated a phylogenetic tree, fully consistent with the phylogenetic relationships of Podarcis as determined by parallel analyses based on partial mitochondrial (mt) DNA sequences. Although the exact mechanisms triggering lymphocyte responses in lizard two-way xenogeneic MLR are not fully understood, our results show the potential use of cell-mediated immune responses as an additional approach to mtDNA analysis, for species delimitation within specific lizard taxa.     

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

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

Genetic Polymorphism of 11 Allozyme Loci in Populations of Wall Lizards (Podarcis sp.) from the Iberian Peninsula and North Africa

The taxonomy of Iberian and North African wall lizards (Podarcis sp.) has been controversial. Recently, morphological and mtDNA sequence data have provided new information on differentiation within these lacertids. To compare these results to those provided by nuclear markers, we investigated variation at 11 polymorphic protein loci using conventional electrophoresis and isoelectric focusing in 11 populations belonging to seven different mtDNA lineages. A total of 62 alleles were found. Populations belonging to the same mtDNA type presented high genetic similarity, whereas strong differentiation was observed between groups. These results are consistent with those previously obtained from morphological and mtDNA analysis and support the idea that Iberian and North African Podarcis are composed of several well-differentiated entities, some of which are already recognized as species, whereas others (belonging to the P. hispanica complex) clearly need taxonomic revision.     

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.     

Evolution and genetic structure of the great tit (Parus major) complex

The great tit complex is divided into four groups, each containing several subspecies. Even though the groups are known to differ markedly on morphological, vocal and behavioural characters, some hybridization occurs in the regions where they meet. The great tit has often been referred to as an example of a ring species, although this has later been questioned. Here, we have studied the genetic structure and phylogenetic relationships of the subspecies groups to clarify the evolutionary history of the complex using control region sequences of the mitochondrial DNA. The subspecies groups were found to be monophyletic and clearly distinct in mitochondrial haplotypes, and therefore must have had long-independent evolutionary histories. This conflicts with the ring species assignment and supports the formation of secondary contact zones of previously temporarily isolated groups. According to the phylogenetic species concept, all the subspecies groups could be considered as separate species, but if the definition of the biological species concept is followed, none of the subspecies groups is a true species because hybridization still occurs.     

Molecular Phylogenetics of Honey Bee Subspecies (Apis melliferaL.) Inferred from Mitochondrial DNA Sequence

A mitochondrial DNA region encompassing part of the NADH dehydrogenase subunit 2 and isoleucine transfer RNA genes was PCR amplified, cloned, and sequenced for 14 morphometrically identifiedApis melliferasubspecies and the New World “Africanized” honeybee. Twenty different haplotypes were detected and phylogenetic analyses supported the existence of 3 or 4 major subspecies groups similar to those based on morphometric measurements. However, some discrepancies are reported concerning the subspecies composition of each group. Based on the sequence divergence ofDrosophila(2% per Myr) we found that the four lineages may have diverged around 0.67 Myr. The variability found in this region enables us to infer phylogenetic relationships and test hypotheses concerning subspecies origin, dispersion, and biogeography.     

Molecular phylogeography and genetic differentiation of blue sheep Pseudois nayaurszechuanensis and Pseudois schaeferi in China

Within the genus Pseudois (Bovidae, Artiodactyla), two challenges are faced by taxonomists. First, the designation of the two subspecies of blue sheep, Pseudois nayaur nayaur and Pseudois nayaurszechuanensis, tends to be questionable with the accumulation of morphological and molecular data. Second, the taxonomic status of the dwarf blue sheep (Pseudois schaeferi) has been controversial since its discovery, and molecular analyses with relatively small sample sizes have not painted a clear picture. We collected samples and DNA sequence data from a broad geographic range in China to investigate the population structure of Pseudois nayaurszechuanensis and its phylogenetic relationship with Pseudois schaeferi. Employing mitochondrial cytochrome b and control region sequences, as well as the nuclear gene zona pellucida glycoprotein 3, further investigation was performed to extend upon previous studies. We found that the population of Helan Mountain of Ningxia Hui Autonomous Region has significantly differentiated from the populations in Qinghai, Gansu and Sichuan provinces, and may be classified as a new subspecies according to the 75% rule. The dwarf blue sheep was found to be some individuals of Pseudois nayaurszechuanensis with morphological variations and is not a species or subspecies, since they did not fall into a monophyletic group in the phylogenetic analyses with blue sheep. Gene flow between dwarf blue sheep and common blue sheep might have occurred in different areas of western Sichuan, which would result in the paraphyly of dwarf blue sheep.     

Phylogeography of Iurus dufoureius (Brullé, 1832) (Scorpiones, Iuridae)

Aim  This study uses molecular data in conjunction with palaeogeography to infer the most plausible biogeographical scenario accounting for the current distributional pattern of Iurus dufoureius .
Location  North-eastern Mediterranean region.
Methods  Sequencing of a 441-bp segment of the mitochondrial 16S rRNA gene in seven populations covering the whole distributional range of the species. Phylogenetic analyses performed included neighbour joining, maximum parsimony and Bayesian inference.
Results  The molecular phylogeny showed that two Iurus clades are strongly supported. These clades correspond to the two subspecies Iurus dufoureius dufoureius and Iurus dufoureius asiaticus , currently recognized within the genus. The assumption of a clock-like evolution could not be rejected, and this enabled us to estimate an approximation of the local rate of evolution for the I. dufoureius lineages. Based on the estimated evolutionary rate (0.79 ± 0.17 Myr⁻¹), the split between the two Iurus clades occurred c. 8 Ma.
Main conclusions  Contrary to what was believed in the past, the genus Iurus is an old north-eastern Mediterranean genus that has been differentiating in southern Greece and south-west Turkey at least from the middle Miocene. According to the phylogenetic trees obtained and the dating of the divergence times of lineages, the genus dispersed into the Aegean Archipelago when the Aegean was still a uniform land mass. Although the phylogenetic relationships of I. d. dufoureius populations have been shaped by the most recent vicariant events, the phylogenetic relationships of I. d. asiaticus populations are mostly attributable to older palaeoevents occurring in the area.     

Ancestral polymorphisms in genetic markers obscure detection of evolutionarily distinct populations in the endangered Florida grasshopper sparrow (Ammodramus savannarum floridanus)

Genetic analyses of bird subspecies designated as conservation units can address whether they represent units with independent evolutionary histories and provide insights into the evolutionary processes that determine the degree to which they are genetically distinct. Here we use mitochondrial DNA control region sequence and six microsatellite DNA loci to examine phylogeographical structure and genetic differentiation among five North American grasshopper sparrow (Ammodramus savannarum) populations representing three subspecies, including a population of the endangered Florida subspecies (A. s. floridanus). This federally listed taxon is of particular interest because it differs phenotypically from other subspecies in plumage and behaviour and has also undergone a drastic decline in population size over the past century. Despite this designation, we observed no phylogeographical structure among populations in either marker: mtDNA haplotypes and microsatellite genotypes from floridanus samples did not form clades that were phylogenetically distinct from variants found in other subspecies. However, there was low but significant differentiation between Florida and all other populations combined in both mtDNA (FST = 0.069) and in one measure of microsatellite differentiation (theta = 0.016), while the non-Florida populations were not different from each other. Based on analyses of mtDNA variation using a coalescent-based model, the effective sizes of these populations are large (approximately 80,000 females) and they have only recently diverged from each other (< 26,000 ybp). These populations are probably far from genetic equilibrium and therefore the lack of phylogenetic distinctiveness of the floridanus subspecies and minimal genetic differentiation is due most probably to retained ancestral polymorphism. Finally, levels of variation in Florida were similar to other populations supporting the idea that the drastic reduction in population size which has occurred within the last 100 years has not yet had an impact on levels of variation in floridanus. We argue that despite the lack of phylogenetic distinctiveness of floridanus genotypes the observed genetic differentiation and previously documented phenotypic differences justify continued designation of this subspecies as a protected population segment.     

Testing phylogenetic hypotheses for reconstructing the evolutionary history of Dolichopoda cave crickets in the eastern Mediterranean

Aim To investigate the molecular phylogenetic divergence and historical biogeography of cave crickets belonging to the genus Dolichopoda (Orthoptera, Rhaphidophoridae). Location Caves in continental and insular Greece. Methods We sequenced 1967 bp of mitochondrial DNA, corresponding to three fragments of the small and large subunit of the ribosomal RNA (16S and 12S rRNA, respectively) and to the subunit I of cytochrome oxidase (COI), to reconstruct phylogenetic relationships among all 30 known Greek species of Dolichopoda. Alternative hypotheses about the colonization of the Hellenic Peninsula by Dolichopoda species were tested by comparing the degree of discordance between species trees and gene trees under four plausible biogeographical scenarios. Results The present study revealed a rather well resolved phylogeny at species level, identifying a number of clades that represent long‐separated lineages and diverse evolutionary histories within the genus Dolichopoda. Two main clades were revealed within Hellenic–Aegean species, identifying a north‐western and a south‐eastern species group. Based on Bayesian analysis, we applied a relaxed molecular clock to estimate the divergence times between the lineages. The results revealed that the origins of eastern Mediterranean lineages are much older than those of previously studied western Mediterranean Dolichopoda. Tests of alternative biogeographical hypotheses showed that a double colonization of the Hellenic Peninsula, following separate continental and trans‐Aegean routes during the Messinian stage, best accounts for the present distribution of Greek Dolichopoda species. Main conclusions Reconstruction and biogeographical hypothesis testing indicated that the colonization of Greece by Dolichopoda species comprised two episodes and two different routes. The southern lineage probably arose from a trans‐Aegean colonization during the Messinian salinity crisis (5.96–5.33 Ma). The northern lineage could be the result of dispersal from the north through the Balkan Peninsula. The opening of the Mid‐Aegean Trench could have promoted an initial diversification within the uprising Anatolian Plateau, while the Messinian marine regression offered the conditions for a rapid dispersal through the whole Aegean–Hellenic region. In addition, climatic events during the Plio‐Pleistocene may have been responsible for the speciation within each of the two different phylogeographical units, principally attributable to vicariance events.     

A molecular phylogeny of New Zealand's Petroica (Aves: Petroicidae) species based on mitochondrial DNA sequences

The New Zealand robin (Petroica australis), tomtit (P. macrocephala), and Chatham Island black robin (P. traversi) are members of the Petroicidae family of Australo-Papuan robins, found throughout Australasia and the western Pacific. In the nearly 200 years since the New Zealand members of Petroicidae were first described, the division of species, subspecies, and even genera has undergone many changes. In this study, we investigate whether molecular phylogenies based on mitochondrial DNA sequences support current taxonomic classifications based on morphology. Petroica traversi, P. australis, and P. macrocephala form distinct clades in phylogenetic trees constructed from Cytochrome b and control region sequences, however the position of the black robin is at odds with the morphological and behavioral data. The black robin does not appear to be a derivative of the New Zealand robin, instead it groups strongly with the tomtit, indicating that lineage sorting and/or introgressive hybridization may have occurred. There is some evidence to support the hypothesis that two invasions of Petroica from Australia have occurred, however additional data from Australian Petroica taxa are required to confirm this. Control region sequences confirm a deep split between the North and South Island P. australis lineages, but suggest a recent radiation of P. macrocephala.     

Evolution and phylogenetic information content of the ITS-1 region in the tiger beetle Cicindela dorsalis

Sequence divergence in the internal transcribed spacer region 1 (ITS-1) of the ribosomal DNA locus was assessed in subspecies of the coastal North American tiger beetle, Cicindela dorsalis. The spacer region was amplified using the polymerase chain reaction and cloned for sequencing. Of a total of 50 clones obtained from 12 specimens, 42 clones were different in at least one nucleotide position. In a parsimony analysis of these sequences, the main phylogenetic distinction was found to separate sequences from the Gulf of Mexico and the Atlantic Ocean. Within these two assemblages phylogenetic resolution was low, and the variation within individuals was almost as high as the variation within the entire lineage. The pattern of sequence variation suggests the existence of two forms of the ITS-1 that are maintained on different chromosomes. Polymorphisms of limited geographical distribution could be detected, and 41 additional clones were partly sequenced, to assess the geographic distribution of these polymorphisms in more detail. In a population aggregation analysis, the geographic pattern of ITS-1 distribution was basically congruent with that obtained in earlier studies from mitochondrial DNA in the same C. dorsalis populations.      

Multilocus phylogenetic analysis of true morels (Morchella) reveals high levels of endemics in Turkey relative to other regions of Europe

The present study was conducted to better understand how the phylogenetic diversity of true morels (Morchella) in Turkey compares with species found in other regions of the world. The current research builds on our recently published surveys of 10 Turkish provinces and the northern hemisphere in which DNA sequence data from 247 and 562 collections respectively were analyzed phylogenetically. Herein we report on phylogenetic analyses of 243 additional collections made in spring 2009 and 2010 from eight additional provinces in the Aegean, Black Sea, central Anatolia, eastern Anatolia and Marmara regions of Turkey. Our analysis revealed that five species within the Esculenta clade (yellow morels) and 15 species within the Elata clade (black morels) were present in Turkey. Our preliminary results also indicate that M. anatolica, recently described from a collection in Mu?la province in the Aegean region of Turkey, is a closely related sister of M. rufobrunnea; these two species comprise a separate evolutionary lineage from the Esculenta and Elata clades. Nine species of Morchella currently are known only from Turkey, four species were present in Turkey and other European countries and seven species might have been introduced to Turkey anthropogenically. Three of the putatively exotic species in Turkey appear to be endemic to western North America; they are nested within a clade of fire-adapted morels that dates to the late Oligocene, 25 000 000 y ago. Our results indicate that there are roughly twice as many Morchella species in Turkey compared with the other regions of Europe sampled. Knowledge of Morchella species diversity and their biogeographic distribution are crucial for formulating informed conservation policies directed at preventing species loss and ensuring that annual morel harvests are sustainable and ecologically sound.     

A new subspecies of Linum gyaricum (Linaceae) from Greece

Christodoulakis, D. 1995. A new subspecies of Linum gyaricum (Linaceae) from Greece. - Nord. J. Bot. 15: 145–147. Copenhagen. ISSN 0107–055X.
A new subspecies, Linum gyaricum ssp. icaricum from Ikaria island (E Aegean, Greece), is described and illustrated.     

Are subspecies useful in evolutionary and conservation biology?

The taxonomic rank of subspecies remains highly contentious, largely because traditional subspecies boundaries have sometimes been contradicted by molecular phylogenetic data. The most complete meta-analysis to date, for instance, found that only 3% of traditional avian subspecies represented distinct phylogenetic lineages. However, the global generality of this phenomenon remains unclear due to this previous study's narrow geographic focus on continental Nearctic and Palearctic subspecies. Here, we present a new global analysis of avian subspecies and show that 36% of avian subspecies are, in fact, phylogenetically distinct. Among biogeographic realms we find significant differences in the proportion of subspecies that are phylogenetically distinct, with Nearctic/Palearctic subspecies showing significantly reduced levels of differentiation. Additionally, there are differences between island and continental subspecies, with continental subspecies significantly less likely to be genetically distinct. These results indicate that the overall level of congruence between taxonomic subspecies and molecular phylogenetic data is greater than previously thought. We suggest that the widespread impression that avian subspecies are not real arises from a predominance of studies focusing on continental subspecies in North America and Eurasia, regions which show unusually low levels of genetic differentiation. The broader picture is that avian subspecies often provide an effective short-cut for estimating patterns of intraspecific genetic diversity, thereby providing a useful tool for the study of evolutionary divergence and conservation.