Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-µHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-µHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Landscape Connectivity Limits the Predicted Impact of Fungal Pathogen Invasion.

Infectious diseases are major drivers of biodiversity loss. The risk of fungal diseases to the survival of threatened animals in nature is determined by a complex interplay between host, pathogen and environment. We here predict the risk of invasion of populations of threatened Mediterranean salamanders of the genus Lyciasalamandra by the pathogenic chytrid fungus Batrachochytrium salamandrivorans by combining field sampling and lab trials. In 494 samples across all seven species of Lyciasalamandra, B. salamandrivorans was found to be absent. Single exposure to a low (1000) number of fungal zoospores resulted in fast buildup of lethal infections in three L. helverseni. Thermal preference of the salamanders was well within the thermal envelope of the pathogen and body temperatures never exceeded the fungus' thermal critical maximum, limiting the salamanders' defense opportunities. The relatively low thermal host preference largely invalidates macroclimatic based habitat suitability predictions and, combined with current pathogen absence and high host densities, suggests a high probability of local salamander population declines upon invasion by B. salamandrivorans. However, the unfavorable landscape that shaped intraspecific host genetic diversity, lack of known alternative hosts and rapid host mortality after infection present barriers to further, natural pathogen dispersal between populations and thus species extinction. The risk of anthropogenic spread stresses the importance of biosecurity in amphibian habitats.

Extended Snake Venomics by Top-Down In-Source Decay: Investigating the Newly Discovered Anatolian Meadow Viper Subspecies, Vipera anatolica senliki.

Herein, we report on the venom proteome of Vipera anatolica senliki, a recently discovered and hitherto unexplored subspecies of the critically endangered Anatolian meadow viper endemic to the Antalya Province of Turkey. Integrative venomics, including venom gland transcriptomics as well as complementary bottom-up and top-down proteomics analyses, were applied to fully characterize the venom of V. a. senliki. Furthermore, the classical top-down venomics approach was extended to elucidate the venom proteome by an alternative in-source decay (ISD) proteomics workflow using the reducing matrix 1,5-diaminonaphthalene. Top-down ISD proteomics allows for disulfide bond counting and effective de novo sequencing-based identification of high-molecular-weight venom constituents, both of which are difficult to achieve by commonly established top-down approaches. Venom gland transcriptome analysis identified 96 toxin transcript annotations from 18 toxin families. Relative quantitative snake venomics revealed snake venom metalloproteinases (42.9%) as the most abundant protein family, followed by several less dominant toxin families. Online mass profiling and top-down venomics provide a detailed insight into the venom proteome of V. a. senliki and facilitate a comparative analysis of venom variability for the closely related subspecies, Vipera anatolica anatolica.

Phylogeographic analyses point to long-term survival on the spot in micro-endemic Lycian salamanders.

Lycian salamanders (genus Lyciasalamandra) constitute an exceptional case of micro-endemism of an amphibian species on the Asian Minor mainland. These viviparous salamanders are confined to karstic limestone formations along the southern Anatolian coast and some islands. We here study the genetic differentiation within and among 118 populations of all seven Lyciasalamandra species across the entire genus' distribution. Based on circa 900 base pairs of fragments of the mitochondrial 16SrDNA and ATPase genes, we analysed the spatial haplotype distribution as well as the genetic structure and demographic history of populations. We used 253 geo-referenced populations and CHELSA climate data to infer species distribution models which we projected on climatic conditions of the Last Glacial Maximum (LGM). Within all but one species, distinct phyloclades were identified, which only in parts matched current taxonomy. Most haplotypes (78%) were private to single populations. Sometimes population genetic parameters showed contradicting results, although in several cases they indicated recent population expansion of phyloclades. Climatic suitability of localities currently inhabited by salamanders was significantly lower during the LGM compared to recent climate. All data indicated a strong degree of isolation among Lyciasalamandra populations, even within phyloclades. Given the sometimes high degree of haplotype differentiation between adjacent populations, they must have survived periods of deteriorated climates during the Quaternary on the spot. However, the alternative explanation of male biased dispersal combined with a pronounced female philopatry can only be excluded if independent nuclear data confirm this result.

Environmental temperatures shape thermal physiology as well as diversification and genome-wide substitution rates in lizards.

Climatic conditions changing over time and space shape the evolution of organisms at multiple levels, including temperate lizards in the family Lacertidae. Here we reconstruct a dated phylogenetic tree of 262 lacertid species based on a supermatrix relying on novel phylogenomic datasets and fossil calibrations. Diversification of lacertids was accompanied by an increasing disparity among occupied bioclimatic niches, especially in the last 10 Ma, during a period of progressive global cooling. Temperate species also underwent a genome-wide slowdown in molecular substitution rates compared to tropical and desert-adapted lacertids. Evaporative water loss and preferred temperature are correlated with bioclimatic parameters, indicating physiological adaptations to climate. Tropical, but also some populations of cool-adapted species experience maximum temperatures close to their preferred temperatures. We hypothesize these species-specific physiological preferences may constitute a handicap to prevail under rapid global warming, and contribute to explaining local lizard extinctions in cool and humid climates.

New data of spiders (Arachnida, Araneae) of Cyprus. 1. Dysderidae found in caves.

This paper is the first in a series describing the previously unstudied cave spiders from Cyprus. Two new species, Dysderocrateskibrisensis sp. n. and Harpacteakalavachiana sp. n., are described. Detailed morphological descriptions and diagnostic characteristics are presented. This is the first report of the genus Dysderocrates Deeleman-Reinhold & Deeleman, 1988 from Cyprus.

Investigating the cytotoxic effects of the venom proteome of two species of the Viperidae family (Cerastes cerastes and Cryptelytrops purpureomaculatus) from various habitats.

Animal secretions are of great interest in terms of drug development due to their complex protein and peptide composition. Especially, in the field of therapeutic medications such as anti-cancer drugs snake venoms receive attention. In this study, we address two Viperidae species from various habitats with a particular focus on the cytotoxic potential along with the decomplexation of the venom proteome: the horned desert viper (Cerastes cerastes), native to desert regions of North Africa and the mangrove pit viper (Cryptelytrops purpureomaculatus), found in coastal forests of Southeast Asia. Initial cytotoxic screenings of the crude venoms revealed diverse activity, with the highest effect against SHSY5Y human glioblastoma carcinoma cells compared to other cancerous and non-cancerous cell lines. In-depth cytotoxicity studies of SHSY5Y cells with purified venom fractions revealed heterodimeric disintegrins from C. cerastes venom, which exerted a high cytotoxic activity with IC50 values from 0.11 to 0.58 µM and a disintegrin-like effect on SHSY5Y morphology was observed due to cell detachment. Furthermore, two polyproline BPP-related peptides, one PLA2 and a peptide-rich fraction were determined for C. purpureomaculatus with moderate IC50 values between 3 and 51 µM. Additionally, the decryption of the venom proteomes by snake venomic mass spectrometry and comparison of the same species from different habitats revealed slight differences in the composition.

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey.

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. BIOLOGICAL SIGNIFICANCE: Population level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.

Ancient diversification, biogeography, and the role of climatic niche evolution in the Old World cat snakes (Colubridae, Telescopus).

The process of species diversification is often associated with niche shifts in the newly arising lineages so that interspecific competition is minimized. However, an opposing force known as niche conservatism causes that related species tend to resemble each other in their niche requirements. Due to the inherent multidimensionality of niche space, some niche components may be subject to divergent evolution while others remain conserved in the process of speciation. One such possible component is the species' climatic niche. Here, we test the role of climatic niche evolution on the diversification of the Old World cat snakes of the genus Telescopus. These slender, nocturnal snakes are distributed in arid and semiarid areas throughout Africa, southwest Asia and adjoining parts of Europe. Because phylogenetic relationships among the Telescopus species are virtually unknown, we generated sequence data for eight genetic markers from ten of the 14 described species and reconstructed a time-calibrated phylogeny of the genus. Phylogenetic analysesindicate that the genus is of considerably old origin that dates back to the Eocene/Oligocene boundary. Biogeographical analyses place the ancestor of the genus in Africa, where it diversified into the species observed today and from where it colonized Arabia and the Levant twice independently. The colonization of Arabia occurred in the Miocene, that of the Levant either in the Late Oligocene or Early Miocene. We then identified temperature and precipitation niche space and breadth of the species included in the phylogeny and examined whether there is phylogenetic signal in these climatic niche characteristics. Despite the vast range of the genus and its complex biogeographic history, most Telescopus species have similar environmental requirements with preference for arid to semiarid conditions. One may thus expect that the genus' climatic niche will be conserved. However, our results suggest that most of the climatic niche axes examined show no phylogenetic signal, being indicative of no evolutionary constraints on the climatic niche position and niche breadth in Telescopus. The only two variables with positive phylogenetic signal (temperature niche position and precipitation niche breadth) evolved under the Brownian motion model, also indicating no directional selection on these traits. As a result, climatic niche evolution does not seem to be the major driver for the diversification in Telescopus.

Investigation of Vipera Anatolica Venom Disintegrin via Intracellular Uptake with Radiolabeling Study and Cell-Based Electrochemical Biosensing Assay.

Snake venoms are a natural biological source that has potential therapeutic value with various protein compounds. Disintegrins originally were discovered as a family of proteins from snake venoms composed of cysteine rich low molecular weight polypeptides. Disintegrins exhibit specific binding and higher affinity toward integrin with potential inhibition of function. Trans-membrane receptors of the integrin family may involve in many pathological conditions such as inflammation and tumor progression with important processes related to invasion and migration. Since disintegrins have the ability to bind to integrins, they could be used for cancer detection and treatment, and in monitoring of therapy in select cancer types. The main purpose of the study is to investigate disintegrin containing Vipera anatolica (VAT) crude venom potential for radiolabeling and intracellular uptake as well as electrochemical biosensing assay against U87MG human brain glioblastoma cells. For this purpose, VAT crude venom containing U87MG cell-specific disintegrin was investigated in terms of radiolabeling and intracellular uptake as well as electrochemical biosensing assay in comparison with echistatin (ECT) disintegrin in cells. The interaction between VAT crude venom and ECT with HEK293 human non-tumorigenic embryonic kidney cells and glioblastoma U87MG cells was electrochemically investigated using pencil graphite electrodes (PGEs). The interaction of the VAT crude venom and ECT with HEK293 and U87MG cells was detected according to the changes in oxidation signals. Then, VAT crude venom and echistatin were labeled with 131I via iodogen method. Intracellular uptakes of radiolabeled molecules were investigated in U87MG cell line. 131I-VAT can be an agent for imaging of glioblastoma cancer. Further work will focus on the production of large quantities of pure VAT disintegrin with a biotechnological approach to improving imaging agent.

A new subspecies of Zamenis hohenackeri (Strauch, 1873) (Serpentes: Colubridae) based on morphological and molecular data.

Based on morphological characteristics, two subspecies of the Transcaucasian rat snake (Zamenis hohenackeri) are currently recognized, namely Z. h. tauricus and Z. h. hohenackeri. Both subspecies are repeatedly considered to be conspecific colour morphs, or have even been confused with Z. situla. Although, few studies involved the Transcaucasian rat snake in a phylogenetic approach, none has so far led to any taxonomic changes. We assessed the intraspecific morphological variation and phylogeographic relationships among specimens from different locations across its updated distribution. Our molecular (1191 bp mtDNA, 565 bp nuDNA) and morphological data provide sufficient evidence to support three distinct lineages within the Z. hohenackeri complex with a different arrangement compared to a previous study. These represent the subspecies Z. h. hohenackeri, Z. h. tauricus, and a lineage from southwestern Turkey which is described as a new subspecies. Aspects of historical biogeography and conservation status are briefly discussed.

Anti-inflammatory properties of Centaurea calolepis Boiss. and cnicin against Macrovipera lebetina obtusa (Dwigubsky, 1832) and Montivipera xanthina (Gray, 1849) venoms in rat.

Macrovipera lebetina obtusa (Dwigubsky, 1832) and Montivipera xanthina (Gray, 1849) (Ottoman Viper) are viper snakes from Viperidae family and found in various locations in Anatolia. Both snakes are responsible for major snake bite cases in Turkey Their venoms cause necrosis, hemorrhage, pain and local edema. Centaurea L. (Asteraceae) species draw attention as potential anti-inflammatory sources due to their traditional uses and accomplished studies on this field. C. calolepis Boiss. is an endemic taxon distributed in Aegean and Antalya regions in Turkey. Chloroform extract of C. calolepis and its major compound cnicin, a sesquiterpene lactone, are reported to have strong anti-inflammatory activities in-vitro, by previous studies. In the present study, in-vivo anti-inflammatory activities of C. calolepis chloroform extract and the sesquiterpenoid cnicin against edema induced by Macrovipera lebetina obtusa and Montivipera xanthina venoms were evaluated in the rat model. Protein contents and induction doses of the venoms were determined. Carrageenan and snake venoms were used as inducing agents in paw edema tests. Extract demonstrated strong inhibition on edema at all doses and hours against M. xanthina venom and carrageenan. Inhibition ratio of extract at 25 mg/kg dose (84.13% inhibition) after 0.5 h M. xanthina venom injection was more than indomethacin's value (45.4% inhibition). The extract also showed significant effect also on inflammation caused by M. lebetina obtusa venom at all doses. However, 2.5 mg/kg cnicin was more effective than total extract of C. calolepis against rat paw edema induced by (27.31%) M. lebetina obtusa venom. This is the first study reported therapeutic potential of C. calolepis, an endemic plant of Turkey, in case of snake-bites cause inflammation by venomous species in natural fauna of Anatolia.

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, Vipera ammodytes transcaucasiana, Venom on U87MG and SHSY5Y Neuronal Cancer Cells.

Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC50 values of nanoparticles with snake venom were calculated as 37.24 and 44.64 µg/ml for U87MG and SHSY5Y cells, respectively. The IC50 values of nanoparticles with snake venom were calculated as 10.07 and 7.9 µg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA™ protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.

Comparative Venomics of the Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni from Turkey Provides Insights into Kinship.

The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytestranscaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

Insecticide Susceptibility Status of Wild-Caught Sand Fly Populations Collected from Two Leishmaniasis Endemic Areas in Western Turkey.

BACKGROUND: In Turkey, vector control programs are mainly based on indoor residual spraying with pyrethroids against mosquitoes. No special control program is available for sand flies. Most insecticide susceptibility tests were done for mosquitoes but not for sand flies. We therefore aimed to determine the insecticide susceptibility against two commonly used insecticides; deltamethrin and permethrin, on wild-caught sand fly populations collected in two geographically separated leishmaniasis endemic areas. METHODS: Insecticide susceptibility of wild-caught sand flies to deltamethrin (0.05%) and permethrin (0.75%) using ready-to use impregnated insecticide papers of WHO was investigated in 2010 based on knockdown time using standard WHO tube-test kit and procedures. Sand flies used in this study were collected from villages of Aydin (Bascayir) and Mugla (Tepecik). RESULTS: The resistance and early resistance were detected on the sand fly population from Mugla province against deltamethrin and permethrin, respectively. However, populations from Aydin Province were sensitive to both insecticides. CONCLUSION: The resistance against deltamethrin and permethrin was detected on sand fly population in Mugla Province where both insecticides have been applied for long time while no resistance was found in the insecticide free area, Aydin Province. These findings can be an indicator for showing the ability for developing the insecticide resistance in sand flies. Because of the presence and dominancy of vector sand fly species of Leishmania infantum (Phlebotomus neglectus, P. tobbi) in both study areas, the systematic monitoring for resistance of sand fly populations and more attention are needed by the authorities involved in control programs for sand fly-borne diseases.

Inferring the shallow phylogeny of true salamanders (Salamandra) by multiple phylogenomic approaches.

The rise of high-throughput sequencing techniques provides the unprecedented opportunity to analyse controversial phylogenetic relationships in great depth, but also introduces a risk of being misinterpreted by high node support values influenced by unevenly distributed missing data or unrealistic model assumptions. Here, we use three largely independent phylogenomic data sets to reconstruct the controversial phylogeny of true salamanders of the genus Salamandra, a group of amphibians providing an intriguing model to study the evolution of aposematism and viviparity. For all six species of the genus Salamandra, and two outgroup species from its sister genus Lyciasalamandra, we used RNA sequencing (RNAseq) and restriction site associated DNA sequencing (RADseq) to obtain data for: (1) 3070 nuclear protein-coding genes from RNAseq; (2) 7440 loci obtained by RADseq; and (3) full mitochondrial genomes. The RNAseq and RADseq data sets retrieved fully congruent topologies when each of them was analyzed in a concatenation approach, with high support for: (1) S. infraimmaculata being sister group to all other Salamandra species; (2) S. algira being sister to S. salamandra; (3) these two species being the sister group to a clade containing S. atra, S. corsica and S. lanzai; and (4) the alpine species S. atra and S. lanzai being sister taxa. The phylogeny inferred from the mitochondrial genome sequences differed from these results, most notably by strongly supporting a clade containing S. atra and S. corsica as sister taxa. A different placement of S. corsica was also retrieved when analysing the RNAseq and RADseq data under species tree approaches. Closer examination of gene trees derived from RNAseq revealed that only a low number of them supported each of the alternative placements of S. atra. Furthermore, gene jackknife support for the S. atra - S. lanzai node stabilized only with very large concatenated data sets. The phylogeny of true salamanders thus provides a compelling example of how classical node support metrics such as bootstrap and Bayesian posterior probability can provide high confidence values in a phylogenomic topology even if the phylogenetic signal for some nodes is spurious, highlighting the importance of complementary approaches such as gene jackknifing. Yet, the general congruence among the topologies recovered from the RNAseq and RADseq data sets increases our confidence in the results, and validates the use of phylotranscriptomic approaches for reconstructing shallow relationships among closely related taxa. We hypothesize that the evolution of Salamandra has been characterized by episodes of introgressive hybridization, which would explain the difficulties of fully reconstructing their evolutionary relationships.

Combined venom profiling and cytotoxicity screening of the Radde's mountain viper (Montivipera raddei) and Mount Bulgar Viper (Montivipera bulgardaghica) with potent cytotoxicity against human A549 lung carcinoma cells.

Here we report the first characterization of the endemic Mount Bulgar Viper (Montivipera bulgardaghica) and Radde's mountain viper (Montivipera raddei) venom by a combined approach using intact mass profiling and bottom-up proteomics. The cytotoxicity screening of crude venom as well as isolated serine proteases revealed a high activity against A549 human lung carcinoma cells. By means of intact mass profiling of native and reduced venom we observed basic and acidic phospholipases type A2. Moreover, the analysis revealed snake venom metalloproteases, cysteine-rich secretory proteins, disintegrins, snake venom serine proteases, C-type lectins, a vascular endothelial growth factor and an L-amino acid oxidase.

An integrative systematic revision and biogeography of Rhynchocalamus snakes (Reptilia, Colubridae) with a description of a new species from Israel.

BACKGROUND: The colubrid snakes of the genus Rhynchocalamus are seldom studied and knowledge of their ecology and life history is scarce. Three species of Rhynchocalamus are currently recognized, R. satunini (from Turkey eastwards to Iran), R. arabicus (Yemen and Oman), and R. melanocephalus (from the Sinai Peninsula northwards to Turkey). All are slender, secretive, mainly nocturnal and rare fossorial snakes. This comprehensive study is the first to sample all known Rhynchocalamus species in order to review the intra-generic phylogenetic relationships and historical biogeography of the genus. METHODS: We revised the systematics of Rhynchocalamus using an integrative approach and evaluated its phylogeography. The phylogenetic position within the Colubridae and the phylogenetic relationships within the genus were inferred using 29 individuals belonging to the three known species, with additional sampling of two other closely-related genera, Muhtarophis and Lytorhynchus. We analysed three mitochondrial (12S, 16S, cytb) and one nuclear (c-mos) gene fragments. Phylogenetic trees were reconstructed using maximum likelihood and Bayesian inference methods; the latter method also used to provide the first time-calibrated molecular phylogeny of the genus. We generated a nuclear network and carried out a topology test and species delimitation analysis. Morphological comparisons were used to differentiate among species and to describe a new species from Israel. The studied material was comprised of 108 alcohol-preserved specimens, 15 photographs, and data from the literature for the examination of 17 mensural, 14 meristic, and two categorical characters. RESULTS: The molecular results support Rhynchocalamus as monophyletic, and as having split from its sister genus Lytorhynchus during the Late Oligocene. The three recognized species of Rhynchocalamus comprise four independently evolving groups. The molecular results reveal that the genus began to diverge during the Middle Miocene. We revealed that the best-studied species, R. melanocephalus, is paraphyletic. A population, formally ascribed to this species, from the Negev Mountain area in southern Israel is phylogenetically closer to R. arabicus from Oman than to the northern populations of the species from Israel, Syria and Turkey. Herein we describe this population as a new species: Rhynchocalamus dayanaesp. nov. DISCUSSION: We identify four species within Rhynchocalamus: R. satunini, R. arabicus, R. melanocephalus, and R. dayanaesp. nov., the latter, to the best of our knowledge, is endemic to southern Israel. The onset of Rhynchocalamus diversification is very old and estimated to have occurred during the Middle Miocene, possibly originating in the Levant region. Radiation probably resulted from vicariance and dispersal events caused by continuous geological instability, sea-level fluctuations and climatic changes within the Levant region.

Biological activities of skin and parotoid gland secretions of bufonid toads (Bufo bufo, Bufo verrucosissimus and Bufotes variabilis) from Turkey.

Toad glandular secretions and skin extractions contain numerous natural agents which may provide unique resources for novel drug development. Especially the skin-parotoid gland secretions of toads from genus Bufo contain as many as 86 different types of active compounds, each with the potential of becoming a potent drug. In the present study, crude skin-parotoid gland secretions from Bufo bufo, Bufo verrucosissimus and Bufotes variabilis from Turkey were screened against various cancer cells together with normal cells using MTT assay. Furthermore, the antimicrobial properties of skin secretions were tested on selected bacterial and fungal species for assessing the possible medical applications. Antimicrobial activity of skin secretions was studied by determining minimal inhibitory concentration (MIC) in broth dilution method. Hemolytic activity of each skin-secretion was also estimated for evaluating pharmaceutical potential. Both skin-parotoid gland secretions showed high cytotoxic effect on all cancerous and non-cancerous cell lines with IC50 values varying between <0.1µg/ml and 6.02µg/ml. MIC results of antimicrobial activity tests were found to be between 3.9µg/ml and 250µg/ml. No hemolytic activities on rabbit red blood cells at concentrations between 0.5µg/ml and 50µg/ml were observed. In conclusion, skin-parotoid secretions of bufonid toads might be remarkable candidates for anti-cancer and antimicrobial agents without hemolytic activities.

Phylogeny of the genus Rhynchocalamus (Reptilia; Colubridae) with a first record from the Sultanate of Oman.

The genus Rhynchocalamus comprises three species distributed in Southwest Asia. Little is known about them, most probably because of their secretive fossorial lifestyle. The poor knowledge of the genus is even underscored by the fact that its phylogenetic affinities remained unclear until very recently. The least known of the species, Rhynchocalamus arabicus, is known only from the holotype collected in Aden, Yemen, and it has not been observed since its description in 1933. Here we provide a second record for this species, which represents the first record of this genus for Oman. This extends its range in southern Arabia by more than 1000 km. The observed specimen was determined as R. arabicus on the basis of its similarity in size, color, and scalation with the holotype. Furthermore, we sequenced three mitochondrial (12S, 16S, cytb) and one nuclear (cmos) genes for R. arabicus and for two individuals of R. melanocephalus and one R. satunini and inferred the phylogenetic relationships of all currently recognized species of the genus for the first time. The results of our phylogenetic analyses indicate that Rhynchocalamus is a member of the Western Palearctic clade of Colubrinae and is sister to Lytorhynchus, with which it forms a very well supported clade and shares some morphological characters. As our results show, R. satunini is the basal lineage of the genus and R. melanocephalus is sister to R. arabicus.