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.     

Molecular phylogeny and historical biogeography of the Anatolian lizard Apathya (Squamata,Lacertidae)

Apathya is a lacertid genus occurring mainly in south-east Turkey and its adjacent regions (part of Iran and Iraq). So far two morphological species have been attributed to the genus; A. cappadocica (with five subspecies, A. c. cappadocica, A. c. muhtari, A. c. schmidtlerorum, A. c. urmiana and A. c. wolteri) and A. yassujica. The first species occupies most of the genus’ distribution range, while A. yassujica is endemic of the Zagros Mountains. Here, we explored Apathya’s taxonomy and investigated the evolutionary history of the species by employing phylogenetic and phylogeographic approaches and using both mitochondrial (mtDNA) and nuclear markers. The phylogenetic relationships and the genetic distances retrieved, revealed that Apathya is a highly variable genus, which parallels its high morphological variation. Such levels of morphological and genetic differentiation often exceed those between species of other Lacertini genera that are already treated as full species, suggesting the necessity for a taxonomic revision of Apathya. The phylogeographical scenario emerging from the genetic data suggests that the present distribution of the genus was determined by a combination of dispersal and vicariance events between Anatolia and Southwest Asia dating back to the Miocene and continuing up to the Pleistocene. Key geological events for the understanding of the phylogeography of the genus are the movement of the Arabian plate that led to the configuration of Middle East (orogenesis of the mountain ranges of Turkey and Iran) and the formation of Anatolian Diagonal.     

Calcium hypochlorite on mouse embryonic fibroblast cells (NIH3T3) in vitro cytotoxicity and genotoxicity: MTT and comet assay

Molecular Biology Reports - Antimicrobial irrigation solutions are widely used under clinical settings. Their effect on dental tissue is a subject of recent research, which aims for a safer...     

Comparative phylogeography of six herpetofauna species in Cyprus: late Miocene to Pleistocene colonization routes

The colonization patterns of oceanic islands are often interpreted through transmarine dispersal. However, in islands with intense human activities and unclear geological history, this inference may be inappropriate. Cyprus is such an island, whose geotectonic evolution has not been clarified yet to the desired level for biogeographical reconstructions, leaving the questions of ‘how the Cypriote biota arrived’ and ‘does the dispersal have the formative role in patterns of its diversification’ unanswered. Here, we address these issues through a reconstruction of the evolutionary history of six herptiles (Ablepharus budaki, Ophisops elegans, Acanthodactylus schreiberi, Telescopus fallax, Pelophylax cf. bedriagae, and Hyla savignyi) by means of mitochondrial DNA (cytochrome b and 16S rRNA), applying a Bayesian phylogenetic, biogeographical, and chronophylogenetic analyses. The phylogeographical analyses show that the colonization history of those species in Cyprus started in the late Miocene and extended into the Pliocene and Pleistocene, with geodispersal, transmarine dispersal, and human‐mediated dispersal having their share in shaping the diversification of Cypriote herptiles. The revealed patterns could be divided into three biogeographical categories: old colonizers that arrived in Cyprus during the late Miocene or early Pliocene either by a land bridge (geodispersal) which connected Cyprus with the mainland or by transmarine dispersal, younger colonizers that reached the island through transmarine dispersal from the Middle East, and new settlers that arrived through human‐induced (voluntary or not) introductions. This work advances our knowledge of the biogeography of Cyprus and highlights the need to consider both geo‐ and transmarine dispersal when dealing with islands whose associations do not have a straightforward interpretation. © 2013 The Linnean Society of London     

Phylogenetic relationships of the populations of Iranolacerta brandtii (de Filippi, 1863) (Squamata: Lacertidae) recently found in Eastern Anatolia,Turkey

The Persian Lizard, Iranolacerta brandtii, was until recently considered to be restricted to north-western Iran (Azerbaijan and Esfahan provinces). However, two recent studies have revealed the existence of populations in Eastern Anatolia, extending the range of this species for about 230?km westwards. The fragmented distribution of this species has been considered to be a consequence of the climatic oscillations during the Pleistocene and Holocene, which created events of alternating contact and isolation of populations in distinct glacial refugia. According to our obtained genealogy derived from three mitochondrial fragments (12S rRNA, 16S rRNA and cytb), the Turkish specimens cluster together but form an independent clade, sister to the individuals from Tabriz in Iran. The separation of these two clades is concurrent with the cladogenesis between the Esfahan and Ardabil clades, estimated to have taken place during the late Holocene.     

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.     

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: Antioxidants protect DNA integrity against cryodamage

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze–thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20 s in a water bath for the evaluation.The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9 ± 1.3% and 51.3 ± 1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5 mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6 ± 2.9% and 54.2 ± 4.9%) and inositol (34.9 ± 2.0% and 47.3 ± 2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06 ± 0.38 mM) than that of control (0.96 ± 0.29 mM) following the freeze–thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.     

Multiple dispersal out of Anatolia: biogeography and evolution of oriental green lizards

The oriental green lizards of the Lacerta trilineata group are widely distributed in Greece, Anatolia, the eastern Mediterranean, the southern Caucasus, and the Zagros mountains in Iran. We studied their phylogeography using three mitochondrial markers with comprehensive sampling from most representatives of the group. Their phylogeny and divergence times (implementing fossil‐based molecular clock calibrations) were inferred using Bayesian methods, and haplotype networks were reconstructed to assess how genetic diversity and current distributional patterns were shaped. According to our phylogenetic analyses, the group constitutes a well‐supported monophylum containing several distinct evolutionary lineages with high haplotype diversity. Vicariance might explain the divergences within most lineages that have accumulated by range restriction and expansion of populations as a result of Quaternary climate oscillations and glacial refugia. However, niche divergence appears to be a major force promoting speciation, and large scale distributional patterns between lineages were shaped earlier by multiple, independent dispersals out of Anatolia during the Pliocene and early Pleistocene. The results of the present study also suggest that the group is in need of a taxonomical revision because the identified lineages and genetic diversity are not congruent with the currently recognized subspecies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 398–408.     

Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide–lomeguatrib combination

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ–lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ–lomeguatrib (50 μM) was used in AA cell lines, IC₅₀ values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib–TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.