Multilocus species-delimitation in the Xerotyphlops vermicularis (Reptilia: Typhlopidae) species complex.

Scolecophidia (worm snakes) are a vertebrate group with high ecomorphological conservatism due to their burrowing lifestyle. The Eurasian or Greek blindsnake Xerotyphlops vermicularis is their only European representative, a species-complex with an old diversification history. However, its systematics and taxonomy has remained untouched. Here, we extend previous work that relied heavily on mitochondrial markers, following a multi-locus approach and applying several species-delimitation methods, including a Bayesian coalescence-based approach (STACEY). Four "species" delimitation analyses based on the mtDNA (ABGD, bGMYC, mPTP, parsimony networks) returned 14, 11, 9 and 10 clusters, respectively. By mitotyping twice as many specimens as before, we have a complete picture of each cluster's distribution. With the exception of the highly-divergent Levantine lineage, the three independent nuclear markers did not help with phylogenetic resolution, as demonstrated in haplotype networks, concatenated and species-trees, a result of incomplete lineage sorting. The prevailing model from the coalescence-based species-delimitation identified two species: the lineage from the Levant and all others. We formally recognize them as distinct species and resurrect Xerotyphlops syriacus (Jan, 1864) to include the Levantine blindsnakes. Finally, X. vermicularis and X. syriacus may represent species-complexes themselves, since they include high levels of cryptic diversity.

Helminth Fauna of Spiny Tailed Lizard, Darevskia Rudis (Bedriaga, 1886) (Sauria: Lacertidae) from Turkey.

The present study investigated the composition of helminth parasites of Darevskia rudis, Spiny Tailed Lizard from Turkey. One hundred and two samples (49♀♀, 53♂♂) from the Tokat, Trabzon, Rize, Gümüshane and Artvin Provinces were collected and examined for helminth parasites. New host and locality records were recorded. As a result of the present study, seven species of Nematoda, Skrjabinodon medinae, Spauligodon sp., Spauligodon carbonelli, Spauligodon aloisei, Skrjabinelazia hoffmanni, Strongyloides darevsky, Oswaldocruzia filiformis; one species of Cestoda, Mesocestoides spp. (tetrathyridium) and one species of Acanthocephala Sphaerirostris scanensis were reported from the lizard samples. Sp. carbonelli and Sphaerirostris scanensis are here recorded for the first time in Turkey. D. rudis is the new host recorded for Sk. medinae, Spauligodon sp., Sp. carbonelli, Sp. aloisei from Nematoda, Mesocestoides spp. from Cestoda and Sphaerirostris scanensis from Acanthocephala. This host has been studied for the first time for the helminth parasites from the Tokat and Gümüshane Provinces. There is, to our knowledge, only one report of helminthes for D. rudis in Turkey.

Helminth Fauna of Valentin's Lizard Darevskia Valentini (Boettger, 1892) (Squamata: Lacertidae) Collected from Central and Eastern Anatolia, Turkey.

In this study, we assessed the helminth fauna of seventy two Valentin's Lizard, Darevskia valentini (32♂♂, 35 ♀♀, 5 subadult). Specimens collected from Kayseri, Ardahan and Van Provinces in Turkey. As a result of the present study, it was detected that forty one hosts are infected with one or more species of helminth. Two species of Cestoda, Oochoristica tuberculata and Mesocestoides spp., and 5 species of Nematoda, Spauligodon aloisei, Skrjabinodon alcaraziensis, Skrjabinodon medinae, Skrjabinelazia hoffmanni and Strongyloides darevsky were found in the hosts. D. valentini represents a new host record for all helminths recorded. Skrjabinodon alcaraziensis is recorded for the first time from Turkey. Van, Kayseri and Ardahan are new locality records for all helminths from D. valentini.

The intestinal helminth community of the spiny-tailed lizard Darevskia rudis (Squamata, Lacertidae) from northern Turkey.

Populations of the lizard Darevskia rudis (Bedriaga, 1886) from northern Anatolia were examined for intestinal parasites in adult specimens. One cestode, Nematotaenia tarentolae López-Neyra, 1944 and four nematode species, Spauligodon saxicolae Sharpilo, 1962, Skrjabinelazia hoffmanni Li, 1934, Oswaldocruzia filiformis (Goeze, 1782) and Strongyloides darevskyi Sharpilo, 1976, were found. Three of these nematodes, S. saxicolae, S. hoffmanni and S. darevskyi are suggested to be part of a module in the network of Darevskia spp. and their parasites. Only one, S. darevskyi, was identified as a Darevskia spp. specialist. The very low infection and diversity parameters are indicative of the depauperate helminth communities found in this lacertid lizard, falling among the lowest within the Palaearctic saurians. Nevertheless these values are higher than those found in parthenogenetic Darevskia spp. Interpopulation variation in the intensity of S. saxicolae and N. tarentolae is attributable to local changes in ecological conditions. On the other hand, parasite abundance and richness increased in the warmer localities, while the effect of lizard sex and size on infection was negligible. The structure of these helminth communities in D. rudis are compared with those observed in other European lacertid lizards.

Neogene climatic oscillations shape the biogeography and evolutionary history of the Eurasian blindsnake.

Typhlops vermicularis is the only extant scolecophidian representative occurring in Europe. Its main distribution area, the eastern Mediterranean, has a complicated geological and climatic history that has left an imprint on the phylogenies and biogeography of many taxa, especially amphibians and reptiles. Since reptiles are sensitive indicators of palaeogeographical and palaeoclimatic events, we investigated the intraspecific genealogy of T. vermicularis in a phylogeographical framework. A total of 130 specimens were analyzed, while the use of formalin and ethanol as preservatives called for a special treatment of the samples. Partial sequences of two mitochondrial (12S and ND2) and one nuclear (PRLR) marker were targeted and the results of the phylogenetic analyses (NJ, ML and BI) and the parsimony-network revealed the existence of 10 evolutionary significant units within this species. In combination with the results of the dispersal-vicariance analysis, we may conclude that the Eurasian blindsnake has encountered a sequence of extinction events, followed by secondary expansion from refugia. Estimation of divergence times showed that severe climatic changes between significantly wetter and drier conditions in the Late Neogene have played a key role on the evolutionary and biogeographical history of T. vermicularis. Additionally, both markers (mtDNA and nDNA) distinguished a largely-differentiated evolutionary lineage (Jordan and south Syria), which could even be reckoned as a full species. Our study reveals the existence of cryptic evolutionary lineages within T. vermicularis, which calls for further attention both on the protection of intraspecific varieties and the respective geographic areas that hold them.

Phylogeography of the ocellated skink Chalcides ocellatus (Squamata, Scincidae), with the use of mtDNA sequences: a hitch-hiker's guide to the Mediterranean.

We analyze geographic genetic variation in C. ocellatus to evaluate the influences of major climatic, paleogeographic and anthropogenic factors in its biogeographic history. Ninety four specimens from 61 populations were collected across all of its geographical range and analyzed based on partial mitochondrial sequences (cyt b, 12S, and ND1). Our results demonstrate that an ancestral form of C. ocellatus, which expanded in northwestern Africa at the end of Miocene, diverged in at least three separate evolutionary lineages approximately 4.57Ma: C. humilis spread south of the Sahara, while the other two (C. ocellatus sensu stricto) were restricted in the coastal North African region. The complicated history of the ocellated skink is a result of multiple vicariant phenomena followed by multiple active or passive dispersals. The Messinian salinity crisis and the re-flooding of the Mediterranean basin, the climatic transition from Middle to Upper Pliocene, and the hyperarid phase of the Sahara, affected the distribution and diversification of C. ocellatus, while in historical times it was introduced in the central Mediterranean islands and eastern Mediterranean region from Tunisia and Cyrenaica, respectively.

Visualization of sperm by immunohistochemistry: application of alternative counterstains.

The aim of this study was to evaluate the application of hematoxylin, safranin, light green and picric acid as counterstain in sperm immunohistochemistry. This is important to visualize the best staining procedure and to determine the advantage of picric acid as a counterstain in some situations. Picric acid used for counterstaining in the immunohistochemical procedure gives the best image of reaction on sperms when DAB was used as a chromogen.

Immunohistochemical localization of epidermal growth factor receptor (EGF-R) in normal and diseased newborn lung tissues.

The distribution of EGF receptors (EGF-R) was examined in normal, hyaline membrane diseased and pneumonic newborn lung tissues by immunohistochemical methods under the light microscope. The PAP technique with polyclonal antibodies was performed to demonstrate the EGF receptor localisation in these tissues. Strong EGF-R reactivity was observed on bronchiolar epithelium and type I and type II alveolar cells in normal newborn lung tissues; whereas, poor reactivity was observed in alveolar macrophages. On the other hand, strong immunoreactivity was detected in type I alveolar cells and alveolar macrophages in hyaline membrane disease, but no reactivity was present in type II alveolar cells. The strongest immunoreactivity was observed in alveolar macrophages of newborn pneumonic lung tissues. In conclusion, the most meaningful form of reactivity was observed in normal newborn lung tissues of airway track and respiration area. This result is related with the maturation of the lungs after birth.

Double staining of skeleton using microwave irradiation.

The fetal skeleton double staining method is used to reveal developmental abnormalities in the skeletal system. We used alizarin red S and alcian blue successfully with microwave irradiation for skeletal double staining. The fixation time was reduced from 4-7 days to 2-2.5 min and the staining time was reduced from 4 days to 23 min.

The expression of VLA integrins in the human tonsilla palatina.

The integrin receptors are a family of transmembrane glycoproteins comprising non-covalent heterodimers. They interact with a wide variety of ligands including extracellular matrix glycoproteins, complement and other cell, while their intracellular domains interact with the cytoskeleton. They participate in cell-matrix and cell-cell adhesion in many physiologically important processes including embryological development, hemostasis, thrombosis, wound healing, immune and nonimmune defense mechanisms, and in oncogenic transformation. This investigation was focused on the histological distribution of the beta 1-integrins in the human tonsil using an indirect immunoperoxidase method. Present data suggest that lymphocyte and antigen presenting cells (FDCs, IDCs, and macrophages) interact with each other following adhesion to extracellular matrix proteins (e.g. fibronectin) through their integrin receptors in order to carry out special immunological functions. In addition, stromal elements and epithelial components were shown to express VLA integrins providing interactions for tissue organization and compartmentalization.

The expression of VLA integrins in the human thymus.

UNLABELLED: The integrin receptors are a family of transmembrane glycoproteins comprising non-covalent heterodimers. They interact with a wide variety of ligands including extracellular matrix glycoproteins, complement and other cells while their intracellular domains interact with the cytoskeleton. They participate in cell-matrix and cell-cell adhesion in many physiologically important processes including embryological development, hemostasis, thrombosis, wound healing, immune and nonimmune defense mechanisms, and oncogenic transformation. This investigation is focused on the histological distribution of the beta 1-integrins in the human thymus, using an indirect immunoperoxidase method. With the exception of VLA-4, none of the beta 1 integrins were expressed on thymocytes which were strongly positive in the cortex and perivascular compartment, somewhat weaker in the medulla. Thymic epithelial cells were positive for VLA-1, VLA-2, VLA-3 and VLA-6, but the distribution pattern of these molecules in epithelial cells at certain locations was quite different. VLA-1 was weakly expressed by both cortical and medullary epithelial cells. VLA-2 was strongly positive in cortical epithelial cells forming a dense framework at the peripheral cortex. VLA-3 and VLA-6 selectively stained a single flattened epithelial cell layer (perilobular epithelial cells) demarcating the peripheral cortex from the surrounding perivascular compartment. VLA-1,3,5,6 were also demonstrated in the endothelial cells and subendothelial layer of the thymic vasculature. IN CONCLUSION: the distribution of integrins in human thymus tissues is of special interest. Such distribution shows that the VLA integrins may have different functions in different areas. The data presented in this study may be important in evaluating the functional role of the VLA integrins in thymocyte maturation in different compartments of the thymus.