Trachypithecus leucocephalus) is a Critically Endangered colobine species endemic to the limestone karst forests in southwestern China. We analyzed the mitochondrial DNA (mtDNA) control region sequences of 390 fecal samples from 40 social groups across the main distribution areas, which represented one-third of the total extant population. Only nine haplotypes and 10 polymorphic sites were identified, indicating remarkably low genetic diversity in the species. Using a subset of 77 samples from different individuals, we evaluated genetic variation, population structure, and population demographic history. We found very low values of haplotype diversity (h = 0.570 ± 0.056) and nucleotide diversity (π = 0.00323 ± 0.00044) in the hypervariable region I (HVRI) of the mtDNA control region. Distribution of haplotypes displayed marked geographical pattern, with one population  showing a complete lack of genetic diversity (having only one haplotype), whereas the other population  having all nine haplotypes. We detected strong population genetic structure among habit patches . In addition, the Mantel test showed a significant correlation between the pairwise genetic distances and geographical distances among social groups in FS , indicting isolation-by-distance pattern of genetic divergence in the mtDNA sequences. Analyses of demographic history suggested an overall stable historical population size and modest population expansion in the last 2,000 years. Our results indicate different genetic diversity and possibly distinct population history for different local populations, and suggest that CZ and FS should be considered as one evolutionarily significant unit (ESU) and two management units (MUs) pending further investigation using nuclear markers.Many Asian colobine monkey species are suffering from habitat destruction and population size decline. There is a great need to understand their genetic diversity, population structure and demographic history for effective species conservation. The white-headed langur ( Genetic diversity plays a critical role in ecological adaptation and long-term survival of a species. Loss of genetic variation in wild populations is often associated with increased extinction risk due to reduced individual fitness and weakened resistance to natural and anthropogenic disturbances –3. GenetTrachypithecus leucocephalus, is a flagship species for conservation of the limestone karst forest ecosystem that belongs to the Indo-Burma biodiversity hotspot [2 and in patchy distribution [2 in size [Asian colobines are a morphologically and evolutionarily diverse group of primates including 55 species in seven genera, and mainly found in South and Southeast Asia ,12. Many hotspot . With a  hotspot  and listribution ,16. The  in size –17, rangLittle is known about the species’ historical distribution and population size before the 1980s. The total distribution range for the species shrank by almost 80% during the last two decades of the 20th century, along with extirpation of local populations in small habitat patches ,16. One Trachypithecus francoisi), using mitochondrial DNA (mtDNA) sequences from 54 fecal samples [Genetic data on the white-headed langur are very limited, and large-scale genetic studies have been lacking. One study found very low genetic diversity in the species in comparison with a closely related species, the François' langur ( samples . No data2) in Guangxi Province, China. The landscape is karst topography characterized by limestone peak hills and valleys with evergreen and semi-evergreen forests. The study areas are dominated by agricultural activities. Even within the Nature Reserve, most flatlands and valleys have been exploited for agricultural purposes, and the mean human population density is 220 per km2. White-headed langurs inhabit the hills amid the farmlands, roads, and human settlements.Most of the current distribution range of the white-headed langur is included in the Chongzuo National Nature Reserve  and CZ (area size: 22 km2) areas within the Nature Reserve. A total of 552 individuals in 77 groups and 306 individuals in 33 groups were found in FS and CZ, respectively [The most recent species-wide population census counted a total of 937 individuals in 120 social groups . Over 90ectively . FS is dA reproductive social group of the species usually consists of on average 11.7 individuals (range 3–30), including one adult male, 5.1 adult females (range 1–14), and immatures . All-malSocial groups of wild white-headed langurs use rock ledges and caves on limestone cliffs as sleeping sites at night ,24, and We collected fecal samples in FS and CZ in January-April from 2012 to 2014. Our sampling locations covered most of the species’ current range in FS and CZ. We collected from almost the maximum number of groups that could be sampled in the study areas, since unsampled groups mostly used sleeping sites at inaccessible locations, such as cliffs over 100 meters above the ground level. Fresh fecal samples were selected based on the color, smoothness and moisture level of the surface. A lighter colored, smooth and moist fecal pellet was generally less than three days old and preferable for DNA isolation. Fecal samples were stored in 95–100% ethanol at ambient temperature in the field for up to three weeks, and transferred to -20 or -80°C upon arrival in the laboratory.Extraction of DNA from fecal samples was conducted using a 2CTAB/PCI method , which h2), and ddH2O. The conditions for PCR were predenaturing at 94°C for 3 min; followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 62°C for 40 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min at the end. The PCR products were separated and visualized on 8% polyacrylamide gels. The reliability of this molecular method for sex identification was first verified using fecal samples of known sex that were collected following witnessed defecation of 21 individuals , and by sequencing the PCR products. The amplification patterns were consistent with the prediction based on the sex of the samples, and the sequencing results confirmed that the amplified fragments were of the DEAD-box gene from a nonhuman primate. The X and Y fragment sequences mapped onto human DEAD-box gene on chromosome X and Y (GenBank Accession No. NG_012830.1 and NG_012831.1) with 98% and 96% identity, respectively. Since the white-headed langur was the only primate species that we extracted DNA from in our laboratory, we were confident that the amplified sequences were from this species.Molecular sex identification was done by PCR using a single pair of primers for sex chromosome-linked DEAD-box gene, which produces one fragment (the X fragment) from female DNA, and two fragments (the X and Y fragments) with about 30 bp difference in length from male DNA in human and many nonhuman primate species . PCR werFor sex identification of fecal samples, we followed a procedure modified from the protocol proposed by Eriksson et al.  for deteOther molecular sexing systems including the AMELX-AMELY , amelogetRNAPhe gene, and partial sequences of tRNAPro and 12S rRNA genes. The PCR reagents and conditions were similar as in DEAD-box gene amplification, except with different primers, annealing at 55°C for 30 s, and in a total volume of 50μL. The PCR products were checked on 1.5% agarose gels. If PCR failed to amplify the full length product, we used three primer pairs , and sequenced on an ABI PRISM 3730 Genetic Analyzer with the BigDye Cycle Sequencing Kit (Applied Biosystems) for both strands. Sequencing results were checked by eye, and shorter fragments were manually assembled into full sequences in SeqMan II 5.01 . Negative controls containing no DNA templates were included in PCR to check for contamination.We also amplified and sequenced 10% randomly selected DNA samples from independent extractions to verify sequencing results and exclude contamination. All PCR amplified a single band of predicted size, and no highly divergent sequences were detected. The amplified sequences matched the mtDNA control region sequences of the François' langur (GenBank Accession No. KJ174502.1) with 94% identity. To further exclude the possibility of contamination by nuclear insertions of mtDNA fragments , we conBy direct sequencing of PCR products, we found that all samples amplified a single sequence except samples of one social group from FS-BZ, which yielded two sequences differing at one site. This was most likely to represent a case of mitochondrial heteroplasmy, where more than one mitochondrial genome is present in an organism and can co-transmit . Since ws) and haplotypes (H), haplotype diversity (h), and nucleotide diversity (π) [The mtDNA sequences were aligned and edited using the software CLUSTAL X version 2.1 . We trimsity (π)  of the Hsity (π) . The mossity (π)  and hiersity (π) . This moA median-joining network of the HVRI haplotypes was constructed using the software NETWORK version 4.6.1.2  to show ΦST between the populations were also estimated in ARLEQUIN with 10,000 random permutations. The significance threshold of multiple pairwise ΦST analyses was adjusted using sequential Bonferroni correction [Population genetic structure and differentiation among populations was evaluated by treating the geographic area of sample collection as an a priori defined local population . Because CZ-N and CZ-S samples showed only a single haplotype, we combined their data and considered CZ as a single population in the analyses. Geographical differentiation of the HVRI haplotypes was computed using Analysis of Molecular Variance (AMOVA) as implemented in the software ARLEQUIN version 3.5.1.2 , and therrection .ΦST/(1- ΦST) [ΦST among social groups of the FS population were estimated in ARLEQUIN, and the geographical distances between the sampling sites were calculated online at http://www.gpsvisualizer.com/calculators#distance. The CZ population was excluded from this analysis due to its lack of within-population genetic diversity and a relatively large geographical distance from FS. Statistical significance was calculated by 10,000 iterations at 95% confidence interval (CI).Genetic differentiation due to isolation by distance among social groups of FS (including both FS-JCS and FS-BZ) was assessed by the Mantel test , which a1- ΦST)  and the D [FS tests [g) and mutation-scaled effective population size (θ) using the maximum-likelihood method in the software package LAMARC 2.1.10 [θW [θ, and zero for g. The first 1,000 genealogies were discarded as burn-in. Female effective population size (Nef) can be estimated by Nef = θ/2μ, where μ is the mutation rate per site per generation and can be calculated using a mutation rate of 0.164 per nucleotide per million years estimated for the HVRI sequences in apes and human [Historical changes in the population size of the white-headed langur were assessed by several methods. First, we conducted the neutrality tests including the Tajima’s D  and Fu’sS tests  in ARLEQC 2.1.10  with the.10 [θW  for estind human . Third, nd human  in DNASPnd human  implemennd human . The anand human . We ran We estimated the generation time for female white-headed langurs to be around 10 years, based on the average female age at first birth being 5–6 years  and apprThe research complied with protocols approved by the Chongzuo National Nature Reserve for the White-Headed Langur of Guangxi Province, and adhered to the legal requirements of China.We collected a total of 402 fecal samples from 41 social groups, including 216 samples from 20 groups of FS-JCS, 29 samples from 3 groups of FS-BZ, 132 samples from 14 groups of CZ-N, and 25 samples from 4 groups of CZ-S. Our sampling covered 37% of the known groups in FS and CZ, and one-third of all groups of the total extant population. The number of samples per group was 9.8 ± 5.8 .PCR using the primers for DEAD-box gene produced a single fragment of 178 bp (the X fragment) from female DNA, and two fragments of 178 bp and 208 bp  from male DNA in the white-headed langur . We wereWe amplified 1213 bp of the control region sequences of mtDNA from 271 fecal samples, including 178 from FS and 93 from CZ. A total of 10 nucleotide sites were variable, including nine transitions and one transversion, which defined nine different haplotypes  was found in all samples from CZ. Hap C was also the most common haplotype in FS, found in 51% of all samples from FS. Additionally, haplotype distribution showed strong geographical partition within FS, with FS-JCS and FS-BZ patches sharing no haplotypes between them .Using a reduced sample set of 77 sequences representing different individuals from 39 social groups see , we asseThe median-joining network of the mtDNA haplotypes indicated that all haplotypes arose from Hap C after one to three mutational events . The hapΦST = 0.375, P = 0.000 ± 0.000). Additionally, all pairwise comparisons yielded significant ΦST values between the three populations (The AMOVA results showed that large proportions of mtDNA variations occurred both within (62.55%) and among the local populations (37.45%) , and unculations , indicatΦST/(1- ΦST)) and geographical distances among the social groups in FS  were broad and included zero, indicating no or slight population expansion  and 3,162  for the FS and total populations, respectively. The mismatch distribution analyses revealed a rugged shape of distribution for both the FS and total populations, deviating from the unimodal and smooth distribution expected under the assumption of population expansion  used in genetic data analyses included all haplotypes identified in the total samples, increasing sampling from the total dataset can only produce even lower genetic diversity estimations. The very low genetic variation at the mtDNA level might be explained by the species’ relatively short evolutionary history, combined with a small founding population and restricted historical range. Using the mtDNA HVRI sequences, Liu et al. [The white-headed langur exhibited low genetic diversity even when compared with other highly endangered primate species with similar population sizes . In paru et al.  revealedAs a maternally inherited genetic marker, the mtDNA is expected to show pronounced structuring in species characterized by female philopatry and male dispersal . LimitedMaternally inherited mtDNA only provides information for female-mediated genetic processes, whereas male-mediated gene flow is not revealed in our data. Further studies should include nuclear genetic markers to investigate genetic diversity, population structure, and gene flow patterns in this species to fully understand the species’ population genetic dynamics.In contrast to the FS population, which possessed all identified haplotypes, the CZ population only showed one haplotype in all 18 groups. This low genetic variation in mtDNA is puzzling, given the relatively large population size of CZ . Two scenarios might explain such a pattern. One is that the ancestral population first inhabited FS area, and a small number of individuals only recently dispersed to CZ and achieved the current population size in a short period of time. Since the two areas were separated by approximately 50 km of unsuitable habitat and increasing human disturbances in the recent past, further dispersal from FS to CZ was impeded. Therefore, the low genetic variation in the CZ population may be the consequence of a small founding population, restricted gene flow, and a very short population history. An alternative explanation is that the CZ and FS populations were established at a similar time and had comparable genetic diversity in the past, but the former has lost the majority of its genetic variation during severe population bottlenecks in the last century. Of an area size less than one quarter of FS, CZ is expected to have a much smaller historical population size than FS, and its genetic diversity should be more strongly influenced by population bottlenecks and genetic drift .D and Fu’s FS tests, population growth parameter g, and the BSP analyses) suggest an overall stationary historical population size and slight growth in the last 2,000 years. Since the species distribution has been confined to a narrow region with restricted suitable habitat, we expect a moderate size for the historical population, and no large-scale population expansion due to natural limits. Based on the size of the total suitable habitat (360 km2) and the recent census population density at CZ  is estimated to be ~2,500. Under the assumptions of an overall high and even female reproductive success and no population structure, Nef is expected to approach Nf [Nef /Nf ratio close to one in certain human populations [Nef of approximately 3,000 based on genetic variation is within a reasonable range. The most likely explanation for the modest population growth in the last 2,000 years might be attributed to shrinking predator populations as a result of increasing human disturbances and habitat modification following human development in the region. The earliest historical record of the region is the establishment of the administrative division Xiangjun County in Qin Dynasty about 2,200 years ago. Natural predators of the langur such as common leopards (Panthera pardus) and clouded leopards (Neofelis nebulosa) were likely to be common in the region but have declined to near extinction probably since the time of human settlement and exploitation.Analyses of demographic history , we calper km2; , the hisoach Nf , as exemulations . Inferenulations . NonetheAfter drastic declines in both distribution range and population size by the end of last century mainly due to habitat loss and poaching ,19,20, pAccording to the criteria proposed by Moritz , the FS-From a planning perspective, our results emphasize the need to maintain habitat connectivity, as adjacent habitat patches can exhibit strong genetic partitions. Constructing corridors between adjoining patches, such as FS-JCS and FS-BZ, and CZ-N and CZ-S, may promote gene flow and alleviate genetic isolation due to human disturbance.S1 TableTa, annealing temperature.CR, control region; HVRI and HVRII, hypervariable region I and II; (DOCX)Click here for additional data file.S2 TableΦST/(1- ΦST) using the mtDNA HVRI sequences, and geographical distances  between sampling locations are in km.Genetic distances  are expressed as pairwise (XLSX)Click here for additional data file.


Genetic diversity represents the heritable variation both within and among populations of organisms, and in the context of this paper, among bamboo species. Bamboo is an economically important member of the grass family Poaceae, under the subfamily Bambusoideae. India has the second largest bamboo reserve in Asia after China. It is commonly known as “poor man’s timber”, keeping in mind the variety of its end use from cradle to coffin. There is a wide genetic diversity of bamboo around the globe and this pool of genetic variation serves as the base for selection as well as for plant improvement. Thus, the identification, characterization and documentation of genetic diversity of bamboo are essential for this purpose. During recent years, multiple endeavors have been undertaken for characterization of bamboo species with the aid of molecular markers for sustainable utilization of genetic diversity, its conservation and future studies. Genetic diversity assessments among the identified bamboo species, carried out based on the DNA fingerprinting profiles, either independently or in combination with morphological traits by several researchers, are documented in the present review. This review will pave the way to prepare the database of prevalent bamboo species based on their molecular characterization. Bamboo, the fastest growing perennial, evergreen, arborescent plant is a member of the grass family  and constitutes a single subfamily Bambusoideae Kigomo . The Bamhttp://www.eeob.iastate.edu/bamboo/maps.html)  to 47°S latitude in South Argentina. A total number of 1,400 bamboo species are distributed worldwide. The bamboo can grow in an altitudinal range which extends from just above the sea level up to 4,000 m Behari . About 1ml) Fig. .Fig. 1WoArundinaria and Thamnocalamus, whereas, these two genera grow in the temperate region along with Phyllostachys. Arundinaria, Bambusa and Dendrocalamus grow in the subtropical region, the tropical moist region allows to grow Bambusa, Dendrocalamus, Melocanna, Ochlandra and Oxytenanthera; on the other hand, in the dry tropical region Dendrocalamus and Bambusa is predominant Arundinarx = 12), whereas in herbaceous bamboo it is 11 (x = 11) (Grass Phylogeny Working Group 2n = 6x = 72) and the temperate woody bamboo is tetraploid (2n = 4x = 48) , wild cattle (Bos gaurus and B. javanicus) and various species of deer (Cervidae) and primates (including macaques Macaca and leaf monkeys Presbytis), pigs (Suidae), rats and mice (Muridae), porcupines (Hystricidae) and squirrels (Sciuridae) are subsidiary feeders on Southeast Asian bamboos. More than 15 Asian bird species nests exclusively in bamboo; many of these are rare and threatened using bamboo as the significant proportion of their habitat  nests between nodes of mature bamboo (Gigantochloa scortechinii), which it enters through holes created by beetles. The Asian giant panda (Ailuropoda melanoleuca), red panda (Ailurus fulgens) and the Himalayan black bear (Selenarctos thibetanus) are heavily dependent on bamboo for their feed  is recently listed as endangered in the Red Data Book of IUCN. Red Panda mainly lives on bamboo leaves and the destruction of bamboo forest is one of the main reasons for its extinction from the wild . Leaves of Sasa senanesis, S. kurilensis and S. nipponica constitute a major part of the winter diet for Hokkaido voles (Clethrionomys rufocanus), it is when most other plants wither.In addition to the countless direct human uses, bamboo plays an imperative role in many other ways; as per Kratter’s survey, 25 out of 440 bird species living in the Amazon forest are confined to bamboo thickets. Elephants , sporadic or irregular flowering bamboos  and gregarious flowering bamboos   Munro subsp. Spathiflorus , Randomly Amplified Polymorphic DNA (RAPD), Amplified Fragment Length polymorphism (AFLP) or Simple Sequence Repeat (SSR). Nevertheless, it is important to understand that different markers have different properties and will reflect different aspects of genetic diversity  based markers such as RAPD, AFLP, SSR, Inter Simple Sequence Repeats (ISSR), Single Nucleotide Polymorphism (SNP) markers etc. RFLP technique was applied to study the genetic variation and evolution of 20 species of  Kochert . However Kochert . As it i Kochert . It requ Kochert . RAPD an Kochert , many ho Kochert , tea  were generated in this study. In the next year, Zhang et al.  was reported by Peng et al.  of genetic variation between the populations residing in two regions; whereas genetic diversity between populations within the same regions was recorded in a very low level. The ISSR primers showed great amount of variation (49.26 %) between two regions coupled with a low level of variation within population and between populations within region. Ten RAPD primers and ten ISSR primers were utilized for detecting DNA polymorphism, identification and genetic diversity study in 16 barley cultivars , the RAPD technology was employed by Akbar et al. (Curcuma longa) from 10 different agroclimatic zones, utilized both RAPD and ISSR primers. Using both RAPD and ISSR markers for 60 genotypes, 62 % correlation between genetic similarity and geographical location were demonstrated. The highest genetic diversity was observed in western central table land.RAPD marker was utilized by Shiran et al.  for deter et al. . RAPD ter et al. , for eva836 for B. balcooa was derived from PW-02 and Bt609 for B. tulda was derived from OPA-08. Bhattacharya et al. . The molecular clustering pattern is in agreement with classification given by Gamble (Bambusa species, the genetic distances between B. bambos, B. ventricosa and B. vulgaris were smaller, while B. atra differed from them for having greater distance. Smaller genetic distance between G. atroviolacea and three Bambusa species indicates that G. atroviolacea has closure affinity with these three species than B. atra. A. hindsii which shows greatest distance from all others. In the year 2009, Bhattacharya et al. utilized random decamer primers for molecular characterization of a gregarious flowering bamboo, T. spathiflorus subsp. spathiflorus. DNA fingerprinting using RAPD markers could not detect any polymorphism either ‘between populations’ or ‘within populations’.Many researchers have employed RAPD and ISSR for genetic diversity analysis and identification of bamboo. Nayak et al.  utilizeda et al.  employedy Gamble , while ty Gamble . The moly Gamble  investigM. baccifera from Mizoram was evaluated using RAPD and ISSR markers by Lalhruaitluanga and Prasad (P. pubescens (Lin et al. P. pubescens. Genetic distance and cluster analysis showed that genetic similarity existed between all the cultivars of P. pubescens under study. In the year 2010, Mukherjee et al. employed 12 ISSR primers and four EST-based random primers for genetic relationship evaluation among 22 taxa of bamboo. The grouping of genotypes based on Jaccard’s similarity matrix using Unweighted Pair Group Method Arithmetic Average, and Principle Coordinate Analysis agreed with earlier reported molecular phylogenetic study only with a few deviations.Genetic diversity among twelve accessions of d Prasad . ClusterPhyllostachys species and for the identification of their hybrid they utilized eight ISSR primers. Using ISSR markers, they identified three hybrids produced by the cross. The fingerprinting pattern and genetic distance measure suggest that two hybrids were authentic, whereas the third one probably an intraspecies offspring. Genetic diversity among 12 natural populations of D. membranaceus was assessed as a preliminary analysis for protection of germplasm resources using ISSR markers (Yang et al. Lin et al.  performe


Src suppress GJIC, through the Ras/Raf/Erk and other effector pathways. High Src activity was found to correlate with high levels of the Src effector, Signal Transducer and Activator of Transcription-3 (Stat3) in its tyrosine-705 phosphorylated, i.e., transcriptionally activated form, in the majority of Non-Small Cell Lung Cancer lines examined. However, Stat3 inhibition did not restore GJIC in lines with high Src activity. In the contrary, Stat3 inhibition in normal cells or in lines with low Src activity and high GJIC eliminated gap junctional communication. Therefore, despite the fact that Stat3 is growth promoting and in an activated form acts like an oncogene, it is actually required for junctional permeability. Gap junctions are channels linking the interiors of neighboring cells. A reduction in gap junctional intercellular communication (GJIC) correlates with high cell proliferation, while oncogene products such as  Gap junctions are channels linking the interiors of neighboring cells that allow the passage of ions and small metabolites. They consist of integral membrane proteins, the connexin gene family, comprising 21 known members in humans [Connexins are composed of four transmembrane domains, two extracellular loops with six conserved cysteine residues, a cytoplasmic loop and cytoplasmic amino- and carboxy-termini. The carboxy-terminus varies in length and provides sites for protein interactions . ConnexiSrc [Src activation [Src decrease GJIC [A number of oncogene products such as v-Src , the midtivation ,10), thetivation , activattivation , v-mos [tivation  and othease GJIC ,15,16. src is the first oncogene to be discovered, as the tumor gene of the Rous sarcoma virus. src encodes a protein with tyrosine kinase activity (Src), which correlates with tyr-418 phosphorylation (Src418). Src is often activated in cancer. As such, Src is one of the best studied targets for cancer therapy. Results from a number of labs indicated that the Src kinase can affect Cx43 phosphorylation either directly on tyrosine, or through its effector pathways on serine or threonine. At first the Src-homology-3 (SH3) domain of Src binds a proline-rich sequence of Cx43, Pro-274 to Pro-280. The proximity between the two proteins facilitates phosphorylation of Cx43 at tyr-265 by Src. This creates a binding site for the Src, Src-homology-2 (SH2) domain that facilitates phosphorylation of tyr-247 of Cx43, triggering channel closure [Src, as opposed to its effectors, upon GJIC in cancer is unclear. The Src effectors, PLCγ  and Erk1/2 also suppress GJIC in their own right, through phosphorylation at serines 368 and 372, or 255, 279 and 282, respectively. In fact, the Ras/Raf/Erk pathway was shown to transduce Src signals leading to GJIC suppression [ closure . Still, pression .Src effector is the Signal Transducer and Activator of Transcription-3 (Stat3) [Src family, a number of growth factor receptors such as the EGF family including Her2/ErbB2 and receptors for cytokines such as the IL6 family phosphorylate Stat3 at tyr-705 (Stat3-705). This triggers Stat3 dimerisation through a reciprocal interaction between the SH2 domain and ptyr-705 of Stat3, migration to the nucleus and activation of transcription of specific genes involved in cell division and survival, such as myc, Bcl-xL, Mcl-1, survivin, HGF [A prominent  (Stat3) , a key c (Stat3) . Besidesvin, HGF  and othevin, HGF ). Recentvin, HGF ,23,24. Tvin, HGF , reviewevin, HGF ,26). Src/Stat3 axis upon GJIC in cells transformed in culture and in lines established from Non-Small Cell Lung Cancer (NSCLC) tumors. Although in the majority of lines high Src418 correlated with high Stat3-705 and the absence of GJIC, Stat3 inhibition did not restore communication in any of the lines examined. This is in sharp contrast to inhibition of the Ras pathway, which did increase GJIC [Despite the extensive literature on the effect of oncogenes upon GJIC, the effect of Stat3 upon GJIC in cancer cells is unclear. Here, we summarise our recent findings on the effect of the ase GJIC . In the ase GJIC , Stat3 iA reliable method to measure gap junctional permeability is a prerequisite for the investigation of the effect of Stat3 upon GJIC. GJIC examination is usually conducted through the introduction of a fluorescent tracking dye such as Lucifer yellow (LY) by microinjection, scrape-loading  or preloin situ was employed to examine the role of Stat3 upon GJIC, both in immortalised rat liver epithelial T51B [Src [Electroporation ial T51B  or mouseial T51B  cells bo51B [Src ,38. The formation of gap junctions depends upon cell to cell contact, and the engagement of cadherins into adherens junctions . Therefoin vitro transformed, T51B cells where Src was activated through mT expression (T51B-Src cells) [Src, a tyrosine kinase with the ability for GJIC suppression, and an oncogene in its own right [Src signals leading to GJIC suppression. However, the results demonstrated that Stat3 downregulation does not restore GJIC, indicating that the high Stat3 activity in T51B-Src cells cannot be responsible for their lack of junctional communication. This is in sharp contrast to Ras, whose inhibition restored GJIC in Src-transformed rodent fibroblasts, and consistent with findings by Ito et al. [We at first examined the effect of Stat3 upon GJIC in c cells) . Stat3 wc cells) ,41 or 23c cells) ,42, or tc cells) . Given twn right , it was o et al. . We next investigated whether Stat3 might, in fact, play a positive role upon GJIC, by examining the effect of Stat3 inhibition in the parental T51B cells. Interestingly, Stat3 downregulation essentially eliminated GJIC in T51B cells . ConversAs noted above, the half-life of Cx43 is 1–3 h, while cell density (which increases Stat3-705) causes a dramatic increase in Cx43 levels in non-transformed cells. Interestingly, Stat3 downregulation through CPA7 treatment or shRNA expression caused a dramatic reduction in Cx43 levels in rat liver epithelial T51B, mouse lung E10 cells or rat F111 fibroblasts at all densities examined, concomitant with GJIC reduction. These findings indicate that Stat3 activity is required for the maintenance of Cx43 protein levels .i.e., activated Src (Src418) in a number of NSCLC biopsies revealed the presence of higher Src activity than the surrounding, non-tumor lung tissue [Src’s contribution to Stat3 activity in NSCLC lines and primary cells which may express other oncogenes in addition to Src is a matter of controversy. Examination of Stat3 levels in certain NSCLC lines demonstrated that Src is a major Stat3 activator [Src inhibition in different NSCLC lines was found to actually increase Stat3-ptyr705. Since cell density causes a dramatic increase in Stat3-705 [Examination of the levels of tyr-418 phosphorylated, g tissue ,45. Howectivator , while ictivator  Src inhitat3-705 , differeSrc is a potent Stat3 activator, Src418 levels did not increase with density in T51B or E10 cells, or in the NSCLC lines SK-LuCi6 . Moreover, Src inhibition with the inhibitors dasatinib , which were found to correlate with their levels of Stat3-705  had low Stat3-705. In sharp contrast however, line LC-T displayed high Stat3-705 despite the fact that Src418 was undetectable. Similarly, SK-MES cells had intermediate Src418 levels (25% of SK-LuCi6-Src), although Stat3-705 was at 90%, and FR-E cells with Src418 of 25% displayed Stat3-705 of 70%, indicating that other, Src-independent factor(s) must also be responsible for the high Stat3-ptyr705 in these lines  dasatinib caused a dramatic reduction in Stat3-705 (vs. 4 and 2 vs. 5), pointing to Src as a significant Stat3 activator. However, in confluent cultures the reduction was only ~50% (lane 3 vs. 6), which further confirms that the density-induced, Stat3 activation is independent of Src. Similar results were obtained with 11 lines with high Src418  had extensive GJIC , or density plus the Stat3 activation triggered by Src and/or other kinases  . HowE, WT-E) . Interesradation ; apparenradation .Src kinase such as Ras/Raf/Erk, PI3k/Akt, the Crk-associated substrate (Cas) and others [Src-mediated GJIC suppression indicated that inhibition of Ras in Src-transformed, rat fibroblasts reinstated gap junctional communication [Src. It was also shown later that Cas is required for the Src-induced, reduction in gap junctional communication [Src-induced, GJIC suppression in these cells is unlikely, despite the fact that constitutively active Stat3 can act as an oncogene and transform established lines [Several signal transducers besides Stat3 are known to be downstream effectors of the d others . Constitd others ,51. Examnication . Conversnication . Taken tnication . In shared lines .The fact that cell density upregulates Stat3 concomitant with an increase in both Cx43 and GJIC prompted us to explore a potential positive role of Stat3 upon GJIC. Interestingly, Stat3 inhibition in non-neoplastic rodent fibroblasts and epithelial cells, as well as two NSCLC lines which exhibit extensive junctional communication  abolished GJIC, indicating that Stat3 is required for the maintenance of gap junction function. Src, tyr-418 phosphorylation levels and GJIC in a number of lines. Since Src is known to suppress gap junctional communication it is tempting to speculate that Src may be responsible, at least in part, for gap junction closure in these lines. Therefore, since Stat3 is a positive regulator of GJIC and Cx43 levels, it appears that Src has a dual role upon GJIC; as an inhibitor, through Cx43 phosphorylation, but also as a GJIC activator, through Stat3 stimulation. However, the former prevails, with gap junction closure as a result. Examination of the mechanism of GJIC suppression in NSCLC revealed an inverse relationship between Src or the Large Tumor antigen of Simian Virus 40 led to apoptosis [Src/E2F activity may have accentuated gap junction closure. In lines such as LC-T which has low Src418, other oncogenes that activate Stat3, may also be activating the E2F family and induce apoptosis, with gap junction closure as a result. The fact that another major survival pathway, the PI3k/Akt also has a positive effect upon GJIC [Stat3 downregulation caused apoptosis in all lines examined, which was more pronounced at 3 days post-confluence ,57, the poptosis ,33,63, ppon GJIC  tends topon GJIC ,66,67.Src/E2F. In non-transformed cells with low E2F levels, grown to low densities, Stat3 inhibition was previously shown to cause merely a growth retardation. However, at high densities, such as needed for optimal gap junction formation, Stat3 inhibition leads to apoptosis . This coSrc may have been responsible at least in part for GJIC suppression like the majority of NSCLC lines, but also for lines like LC-T where Src levels were found to be low, that is other oncogenes must be responsible for gap junction closure. In the contrary, Stat3 is required for gap junctional communication and the maintenance of Cx43 levels, both in normal epithelial cells and in certain tumor lines that retain GJIC. In cells such as BH-E the gap junction closure may be independent from the Src/Stat3 axis altogether.Although Stat3 is generally growth promoting, and in an activated form it can act as an oncogene, it does not transmit gap junction suppressing signals in any of the NSCLC cell lines examined. This holds true for cells where The role of connexins in cancer is complex. Numerous models support the idea that connexins are tumor suppressors at the early stages of carcinogenesis. However, recent evidence indicated a positive role for connexins in facilitating metastasis (,69,70, r


In the present study, genetic characterization of Gaddi goat breed, a native to north temperate western Himalayan region of India, was carried out for the purpose of breed characterization and assessing existing intra-population genetic diversity.Totally, 75 blood samples procured at random from genetically unrelated animals of two sexes and different age groups and true to breed type were collected from different locations in the breeding tract of these goats in Himachal Pradesh, of which only 51 samples with desired quantity and quality were subjected to further processing for DNA isolation. The multi-locus genotype data were generated on 51 Gaddi goats sampled across different regions of the breeding tract in Himachal Pradesh using 15 FAO recommended goat specific microsatellite markers, which gave amplification and observed and effective number of alleles, gene frequency, observed and expected heterozygosity were estimated through PopGene software (1.3.1).O) and expected (HE) heterozygosities across all loci were 0.7484±0.02 and 0.8431±0.01 respectively. The polymorphism information content (PIC) value ranged from 0.7148 (SRCPS5) to 0.909 (P19-DYA) with mean PIC of 0.8105±0.01 in the present study. The average heterozygosity was observed to be 0.8347±0.01 ranging from 0.7584 (SRCRSP5) to 0.9156 P19 (DYA) across all loci.A total of 135 distinct alleles were observed with mean observed and effective number of alleles as 9.0000±0.82 and 6.5874±0.56 respectively across all 15 studied loci. The maximum (15) alleles were contributed by loci DRBP1 and P19/DYA and the least (5) by SRCRSP5. The mean heterozygosity was observed to be 0.8347±0.01 ranging from 0.7584 (SRCRSP5) to 0.9156 (P19-DYA) across all loci. The mean observed (HMicrosatellite analysis revealed high level of polymorphism across studied microsatellite markers and informativeness of the markers for genetic diversity analysis studies in Gaddi goats. This high level of polymorphism can be utilized to plan future association studies to exploit the uniqueness and adaptability of indigenous Gaddi goat breed of Western Himalayas. Most studied microsatellite markers had desired neutrality, thus proving to be good candidates for genetic characterization and diversity analysis in Gaddi breed of goats also. Indian goat breeds are recognized as an invaluable component of world’s goat genetic resources. Of the total goat population, 92.76% of goats are found in Asia and Africa. China, India, Pakistan and Bangladesh possess 35.36, 25.46, 10.79 and 7.05% of the total goat of Asia, respectively ) with mean PIC of 0.8105±0.01 . This maThe genetic variability of a population is usually measured as average heterozygosity per locus while the gene differences between two populations may be measured by Nei’s standard genetic distance. The values for these measures varied from 0.7584 (SRCRSP5) to 0.9156 P19 (DYA) with mean genetic distance of 0.8347±0.01. The mean Shannon’s index value was high (1.9504±0.08) showing higher gene diversity in the existing population.In the present investigation, an attempt has been made to genetically characterize “Gaddi” goat population in its breeding tract in HP using 15 FAO recommended goat specific microsatellite markers. Microsatellite analysis revealed high level of polymorphism and informativeness of studied microsatellite markers in genetic diversity analysis in Gaddi goats. The high PIC values as observed in the study are indicative of high informativeness of studied markers for genetic diversity analysis in Gaddi goat breed. Most studied microsatellite markers had desired neutrality, thus proving to be good candidates for genetic characterization and diversity analysis in Gaddi breed of goats also. The information gathered could be utilized to plan breeding, improvement and conservation programs for this valuable goat germplasm resource and future association studies to exploit its unique adaptability traits. The significant level of variability in this population reflects that the Gaddi goat population contains a valuable and substantial amount of genetic diversity among the studied breed and thus there is good scope for bringing effective genetic improvement, conservation and designing future breeding policies for these goats.GS and YT planned and designed the study. GS and AK collected the samples, GS and VS performed test and analyzed the data. All authors participated in draft and revision of the manuscript. All authors read and approved the final manuscript.


Kosciuscola tristis (Sjösted).Mountain landscapes are topographically complex, creating discontinuous ‘islands’ of alpine and sub-alpine habitat with a dynamic history. Changing climatic conditions drive their expansion and contraction, leaving signatures on the genetic structure of their flora and fauna. Australia’s high country covers a small, highly fragmented area. Although the area is thought to have experienced periods of relative continuity during Pleistocene glacial periods, small-scale studies suggest deep lineage divergence across low-elevation gaps. Using both DNA sequence data and microsatellite markers, we tested the hypothesis that genetic partitioning reflects observable geographic structuring across Australia’s mainland high country, in the widespread alpine grasshopper We found broadly congruent patterns of regional structure between the DNA sequence and microsatellite datasets, corresponding to strong divergence among isolated mountain regions. Small and isolated mountains in the south of the range were particularly distinct, with well-supported divergence corresponding to climate cycles during the late Pliocene and Pleistocene. We found mixed support, however, for divergence among other mountain regions. Interestingly, within areas of largely contiguous alpine and sub-alpine habitat around Mt Kosciuszko, microsatellite data suggested significant population structure, accompanied by a strong signature of isolation-by-distance.Consistent patterns of strong lineage divergence among different molecular datasets indicate genetic breaks between populations inhabiting geographically distinct mountain regions. Three primary phylogeographic groups were evident in the highly fragmented Victorian high country, while within-region structure detected with microsatellites may reflect more recent population isolation. Despite the small area of Australia’s alpine and sub-alpine habitats, their low topographic relief and lack of extensive glaciation, divergence among populations was on the same scale as that detected in much more extensive Northern hemisphere mountain systems. The processes driving divergence in the Australian mountains might therefore differ from their Northern hemisphere counterparts.The online version of this article (doi:10.1186/s12862-014-0204-1) contains supplementary material, which is available to authorized users. Mountain landscapes form a matrix of mountain-top ‘sky islands’ , high-elevation ridges and intervening low-elevation habitat. This creates a discontinuous and fragmented landscape with features such as valleys and river drainages forming potential barriers to gene flow for species restricted to high elevations ,2. The s2) area of southeastern Australia, with most of this area within the States of Victoria and New South Wales (‘Kosciuszko region’) .Egernia guthega, an alpine endemic, shows an average 2.2% mitochondrial (ND4) sequence divergence between a population from Kosciuszko and three populations from the Bogong High Plains. However, no genetic structure across this valley was detected among alpine Poa  has shown low genetic structure within this region. Likewise, we found no phylogeographic structure in nisation ,29. We snisation . In addinisation , K. trismisphere ,31,32.K. tristis in the area. Patterns of recent, fine-scale population differentiation (over distances < 50 km) are common across high-elevation taxa – even among species with high dispersal ability  coupled with a strong signature of isolation-by-distance. This could reflect more recent differentiation. In particular, we report a new genetic break, with unambiguous clustering of individuals from Mt Jagungal, which is separated from Kosciuszko proper by 25 km of sub-alpine plains at around ~ 1500 m elevation – also the lower-elevation limit of ty e.g. –36). Hab. HabK. tty  is thought to have promoted the evolution of the cold-adapted sub-alpine and alpine biota, with this species assemblage persisting through subsequent climatic shifts –45. This species . Diverge species ,46,47. A species .Depression of the snowline during glacial periods is thought to have resulted in continuous alpine/sub-alpine conditions from Mt Jagungal into southern Victoria ,16. WhilK. tristis populations from Mt Kosciuszko and the Bogong High Plains. Perhaps more importantly, however, our data suggest that the Murray River valley  may not be the greatest point of divergence among alpine taxa  well-studied phylogeographic break between the Kosciuszko and Bogong High Plains regions, across the Murray River valley. Approximately 100 km separates alpine/sub-alpine habitats on either side of the valley but two of five previous studies ,25 have see also ).For high-elevation taxa in North America and Europe, phylogeographic patterns reflect contrasting population histories of glacial and interglacial expansion and contraction  (Orthoptera: Acrididae) is a small (15–30 mm) grasshopper, endemic to Australia’s alpine region .Second, as there was low variation in the approach  implemen 4.6.1.1 ,61, withCO1 sequences to estimate divergence time among clades. In particular, we were interested in the timing of divergence among Victorian and Kosciuszko populations. No fossil of geological evidence was available for node calibration. Instead, we estimated divergence times using two divergence rates: 3.5% Myr−1, based on a recent insect molecular clock estimate  [The data sets supporting the results of this article are available in the figshare repository [1165613] , or are


Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions. Grapes are used in the production of wine, brandy, or non-fermented drinks and are eaten fresh or dried as raisins Erisyphe necator, was introduced from North America to England in 1845, and onwards to France in 1847. The root-feeding aphid called phylloxera was found in western France about 1865 and rapidly spread throughout Europe. In 1878, resistant rootstocks were introduced to Europe from North America in an effort to control this pest, but the downy mildew pathogen (Plasmopara viticola) was inadvertently introduced into France, probably as oospores on the imported rootstocks, and spread widely throughout Europe Powdery mildew caused by the fungus th century” Cadophora luteo-olivacea was traditionally considered as a minor vascular pathogen associated with Petri disease of grapevine. This fungus has been found in many habitats, such as decaying wood C. luteo-olivacea has been increasingly reported in grapevine nurseries and young vineyards worldwide, causing a major concern in both grape growers and nurseries These past epidemics illustrate the destructive effects of new or introduced pathogens and the diseases they cause on grapevine cultivation. Fungal trunk diseases, namely esca, eutypa and Botryosphaeria diebacks, as well as black-foot and Petri diseases, are currently threatening the viticulture industry worldwide causing significant economic losses due to reduced yields, increased crop management costs for cultural and chemical preventive measures, and shortened life span of the vines C. luteo-olivacea, previously identified as Phialophora sp., from the graft union of apparently healthy plants in commercial nurseries in South Africa. The pathogenicity of one of these isolates was further demonstrated under glasshouse and field conditions C. luteo-olivacea was quite common in esca and Petri-disease affected grapevines and isolated 13 strains throughout Californian vineyards. Gramaje et al. C. luteo-olivacea isolates from grapevines showing black vascular streaking and decline symptoms characteristic of Petri disease, and from environmental samples collected at different growth stages of grapevines in Spanish nurseries. The pathogenicity of representative Spanish isolates was confirmed under field C. luteo-olivacea obtained from nursery planting material showing external and internal symptoms of Petri disease, but results were inconclusive. More recently, Úrbez-Torres et al. C. luteo-olivacea associated with young vine decline in British Columbia (Canada), and reproduced symptoms of dark vascular streaking on inoculated grapevine wood under greenhouse conditions.Halleen et al. C. luteo-olivacea in early stages of grapevine wood disease, the biology, epidemiology and management of this fungus affecting grapevines remain poorly understood. An understanding of its mode of reproduction and the source of inoculum responsible for C. luteo-olivacea dispersal is, however, essential for the efficient management of Petri disease. To date, no sexual fruiting bodies of C. luteo-olivacea have ever been found in vineyards. However, sexual reproduction may be transient in vineyards or may occur on currently unidentified alternative host plants. In Spain, this species was isolated from asymptomatic xylem tissues of weeds collected in commercial vineyards C. luteo-olivacea to hot-water treatments and found that conidial germination of this fungus was inhibited at temperatures above 51°C, while treatments of up to 54°C for 60 min were necessary to inhibit mycelial growth.Despite the clear implication of C. luteo-olivacea in grapevines is problematic. In vineyards, management strategies recommended for prevention and disease management mainly involve the prevention and/or correction of predisposing stress situations Population genetics as applied to plant pathogens hold enormous promise for understanding certain evolutionary forces controlling pathogen populations . Population genetic inference also provides insights into whether populations are sexual or clonal, differentiated or admixed, and whether phenotypes are linked to genotypes Multilocus molecular marker technology has been showed to be highly suited to assess the genetic structure of plant-pathogenic fungi C. luteo-olivacea populations from grapevines was performed by Gramaje et al. C. luteo-olivacea isolates by amplification of the highly conserved ITS region, the partial β-tubulin (BT) or elongation-factor 1α (EF) genes, suggesting that the population of this species was represented by a single clonal lineage. The Spanish and South African C. luteo-olivacea isolates represent the unique collection of this fungus available for its use in population genetic studies. Therefore, the objectives of the present study were (i) to determine the genetic structure of C. luteo-olivacea in Spain at a regional scale by using multilocus ISSR markers, (ii) to compare the genetic structure of C. luteo-olivacea isolates from Spain and South Africa and (iii) to assess the virulence of representative C. luteo-olivacea isolates from the genetic clusters obtained in this study.To date, the only study of the biology and genetic structure of No specific permits were required for the described field studies. Location of grapevine nurseries and vineyard sites were facilitated by regional Plant Health services in Spain and by ARC Infruitec-Nietvoorbij in South Africa. Permission to sample the vineyards and nurseries were granted by the landowner and nurserymen, who are collaborating with the Universidad Politécnica de Valencia . The sites are not protected in any way. The areas studied do not involve any species endangered or protected in Spain and South Africa.C. luteo-olivacea isolates were genotyped. The collection consisted of 65 isolates from Spain and 15 isolates from South Africa  following the manufacturer’s instructions. DNA was quantified using a spectrophotometer  and adjusted to a final concentration of genomic DNA at 15 ng µlC. luteo-olivacea isolates were used in a preliminary screen to determine those ISSR primers that enabled generation of polymorphic, reproducible markers that could be used to generate polymorphic DNA fingerprints for all C. luteo-olivacea isolates. This screen tested 13 ISSR primers . PCR amplifications were performed in a Peltier Thermal Cycler-200. Conditions included an initial step of 5 min at 95°C, followed by 34 cycles of denaturation at 95°C for 1 min, annealing at primer-specific temperature . Only bands  that could be scored consistently for all samples were used, with the assumption that each band with different molecular weight represented a distinct locus and amplicons sharing the same molecular weight were considered to be the same allele at a specific locus. The absence of amplicons was considered as an alternate allele.Amplification profiles of the 80 Amplified DNA fragments were transformed into a binary character matrix . A multilocus genotype (MLG) was constructed for each isolate by combining data for single ISSR fingerprints by using the procedure available in the package POPPR To assess the possible evolutionary relationships among MLGs, minimum spanning networks were constructed, first from genotypes of different provinces or nurseries in Spain (nursery is considered as a single population separate from the vineyard subpopulations by provinces) and also including the genotypes from South Africa. Relative dissimilarity distances were calculated according to the index of association C. luteo-olivacea in Spain using a genetic multivariate analysis C. luteo-olivacea population in Spain, and the global population in Spain and South Africa. PCA is independent of any genetic hypotheses and it is suitable for the analysis of partially clonal species. PCA analysis was performed using the R package ADEGENET We tested for the existence of divergent genetic pools of 2) as implemented in MULTILOCUS pi is the frequency of the ith genotype and n is the number of individuals sampled. D represents the probability that two individuals sampled at random with replacement have the same genotype, and is similar to the diversity measure of Pielou C. luteo-olivacea strains present in each population. Because sample size varied among populations we employed rarefaction to explore the effect of sample size on observed species richness The genetic richness (R) and the evenness index adapted from the Simpson index of genotypic diversity (ED*) were calculated for each population using the R package POPPR gen) was calculated using GENCLONE sex), assuming that the two isolates derive from sexual reproduction IA), and a standardized alternative of the IA (d) IA = 0; the null hypothesis for random mating is rejected where IA>0. These tests were not performed on samples sizes <10 due to lack of statistical power To study the contribution of possible sexual reproduction to the genotypic diversity observed for each population, the probability that a genotype was obtained by chance throughout a sexual event (PST) was also calculated using ARLEQUIN. Significance of FST was tested using 1,000 permutations. Due to low sample sizes, populations from Badajoz, Granada, Mallorca and Zaragoza were excluded from the analysis.Population genetic structure was analyzed by conducting an analysis of molecular variance (AMOVA) C. luteo-olivacea were randomly selected from each of the two genetic clusters (6 isolates representing 4 MLGs from cluster 1 and 8 isolates representing 5 MLGs from cluster 2) inferred by the Bayesian clustering method and the genetic multivariate analysis. Virulence assays were conducted on 1-year-old grapevine cuttings of 110 Richter (R) rootstock. In total 168 dormant cuttings were cut into uniform lengths containing four to five buds, and then hot-water treated at 53°C for 30 min to eliminate the possible incidence of fungal trunk pathogens Representative isolates of −1 streptomycin sulphate (MEAS)  in an attempt to recover the inoculated fungi and confirm Koch’s postulates. Fungi were identified as previously described.Cuttings were collected after 4 months and inspected for lesion development. Extent of vascular discoloration was measured upward and downward from the inoculation point. Additionally, shoot dry weight was evaluated for sprouts formed above the inoculation point. Small pieces (0.5 to 1 cm) of necrotic tissue from the edge of each lesion were cut and plated onto malt extract agar (MEA)  supplemented with 0.5 g L10 transformation was used to improve homogeneity of variance. Treatment means among individuals and genetic clusters were compared using Student’s least significant difference (LSD) test at P<0.05. In all cases the Statistix 10 software  was used.Analyses of variance (ANOVA) were conducted to analyze lesion length and shoot dry weight data. Homogeneity of variance was tested using Levene’s test. Residuals were visually inspected for each treatment, and when necessary a log4 , followed by primer (GAC)5 , primer (ACTG)4 (an anchorless tetra-nucleotide) and primer DDB(CCA)5 (a 5′-anchored tri-nucleotide), both with 11 PBs. The mean percentage of polymorphic loci was 85.65%.In total, 55 polymorphic loci were generated from ISSR-PCR using the four ISSR primers selected from a screen of 13 ISSR primers . The higC. luteo-olivacea isolates  varied across subpopulations defined by different provinces in Spain and South Africa, from very high in Ciudad Real or South Africa (0.64 to 0.78), to high in Valencia or the nursery subpopulation (0.44) . EvennesThe minimum spanning network for the Spanish population distinguished the most frequent MLGs in Spain, MLG13, MLG14, MLG29 and MLG31 . The gloPrincipal component analysis revealed two clusters of MLGs . Axis 1 ST was 0.062 and the permutation P value was 0.03. The AMOVA results based on populations defined by isolates’ country of origin  on all individuals and the clone-corrected data rejected the null hypothesis for recombination (P<0.01) (gen) and that two isolates share these genotypes in the Spanish and South African populations (Psex) were very low (The linkage disequilibrium tests ((P<0.01) . The provery low .P>0.05). All isolates of C. luteo-olivacea used in this study were pathogenic to grapevine cuttings of 110 R rootstock (P<0.05) differences in virulence among treatments. Values of lesion length ranged between 3.6 and 6.0 cm, and the values of shoot dry weight from 1.5 to 4.6 g. All the C. luteo-olivacea isolates caused lesions in the xylem of grapevine rootstock that were significantly longer than in the control. The average shoot dry weight of the isolates CR2, SA11, V23, N5, V13 and CR10 was significantly different compared to the control  and the shoot weight .Data of the two virulence assays were combined because of the lack of significant differences between the two tests and among the studied variables  identified two highly differentiated genetic clusters in the Spanish C. luteo-olivacea population with no intermediate genotypes among these clusters, one including a common MLG, namely MLG13, and the second including the other three dominant genotypes, namely MLG14, MLG29 and MLG31. Movement of MLGs within the Spanish provinces may have occurred repeatedly, given the low level of geographic genetic structure and the frequent retrieval of the same genotype in distant locations. Highly similar, but not identical, clonal genotypes are unlikely to have arisen independently in sexual reproduction and can then be inferred to represent the same clonal lineage This study is the first to employ genetic markers to analyze the population structure of a collection of C. luteo-olivacea genotypes. In fact, AMOVA analyses showed that most genetic diversity (88.1%) was found within populations and only 5.5–6.3% among populations. The two defined clusters would then correspond to an established asexual lineage intermingled with individuals derived from another asexual lineage that were recently admixed. Providing that sexual reproduction is a rare and episodic event in C. luteo-olivaceaPhytophthora ramorumPuccinia striiformisFusarium circinatumPhaeomoniella chlamydosporaThe two genetic groups displayed no specific geographic distribution, thus suggesting clonal divergence and admixture between the Spanish C. luteo-olivacea has been detected in different phases of the propagation process in South Africa and Spain sex values associated with these genotypes also provide evidence for clonal reproduction in the Spanish populations studied. (ii) Linkage disequilibrium tests. The null hypothesis of random mating was rejected for all subpopulations in Spain and South Africa, even when populations were clone corrected. However, caution is required in the interpretation of these findings, since it is difficult to demonstrate the presence of linkage disequilibrium where sample sizes are small. (iii) Finally, sexual structures of C. luteo-olivacea have never been observed under natural conditions and all attempts to induce sexual morph in culture were unsuccessful In our study, the hypothesis of a clonal population structure is supported by several population genetic criteria pointed out by Tibayrenc et al. Phaeomoniella chlamydospora. Given the high haplotype diversity together with linkage disequilibrium, these researchers suggested that P. chlamydospora could be an asexual species possibly undergoing rare recombination or partial recombination due to parasexuality. Continued genotyping of C. luteo-olivacea from vineyards and nurseries will be necessary to confirm the results of genotype diversity, to track the movement and diversification of the lineages and to identify new dominant genotypes or newly introduced lineages.The high levels of genotypic diversity found in this study could be explained by the higher evolutionary rate of change within microsatellite regions compared with other regions. The hypervariable nature of ISSR markers enables detection of a higher level of polymorphism compared with other molecular markers C. luteo-olivacea population using a multivariate approach. Additional sampling of C. luteo-olivacea isolates from South Africa and other countries will be necessary to confirm that the global population of this species consists in two different clonal lineages. MLG13, which was one of the most common MLG in Spain, was found relatively frequently in South Africa. It is worth noting that this MLG was found 8 years apart: namely, in 1999 in Wellington (South Africa) and again, widely distributed in Valencia and Ciudad Real provinces, in 2007–2008. This demonstrates that the fungus can be dispersed on infected plant material for long distance introductions.The South African genotypes also grouped into the two genetic clusters obtained in the analysis of the Spanish C. luteo-olivacea in other grape growing regions to help evaluate how different they may be from the Spanish population. Cadophora luteo-olivacea has been recently associated with Petri disease, one of the most important diseases of young vines C. luteo-olivacea is in accordance with published literature C. luteo-olivacea isolates from other grape growing regions of the world will contribute significantly to better understand the reproductive system of this species, which might be crucial for planning successful control strategies.There is currently no information on populations of C. luteo-olivacea in Spain. Over the past few years this pathogen has been shown to be involved with young grapevine decline in many grape growing regions worldwide. This study revealed the existence of a genetic admixture, with the presence of two divergent and well-differentiated genetic groups corresponding to two sympatric clonal lineages, each composed of closely related clonal variants. The epidemiological importance of these genetic clusters in relation to the different levels of virulence was investigated. All isolates were able to induce typical Petri disease symptoms in xylem vessels of 110 R rootstock. However, no association could be found between virulence phenotype and genetic cluster. Our findings highlight the need to produce healthy and quality planting material in grapevine nurseries to avoid the dispersal of trunk disease pathogens throughout different grape growing regions.The results obtained here provide new insights into the population genetic structure of Table S1Estimates of linkage disequilibrium (IA and d) within Spanish subpopulations, countries and genetic clusters in Spain.(DOC)Click here for additional data file.Table S2Probability of occurrence or a second encounter of multilocus ISSR genotypes observed more than once in the regional populations studied in Spain and South Africa.(DOC)Click here for additional data file.


Macaca silenus) inhabiting the fragmented landscape of Anamalai hills and examined the genetic structure of the species across its distributional range in the Western Ghats. We sequenced around 900 bases of DNA covering two mitochondrial regions–hypervariable region-I and partial mitochondrial cytochrome b–from individuals sampled both from wild and captivity, constructed and dated phylogenetic trees. We found that the lion-tailed macaque troops in the isolated forest patches in Anamalai hills have depleted mitochondrial DNA diversity compared to troops in larger and continuous forests. Our results also revealed an ancient divergence in the lion-tailed macaque into two distinct populations across the Palghat gap, dating to 2.11 million years ago. In light of our findings, we make a few suggestions on the management of wild and captive populations.Genetic isolation of populations is a potent force that helps shape the course of evolution. However, small populations in isolation, especially in fragmented landscapes, are known to lose genetic variability, suffer from inbreeding depression and become genetically differentiated among themselves. In this study, we assessed the genetic diversity of lion-tailed macaques ( Brachyteles hypoxanthus) [Alouatta caraya) [The contemporary spatial distribution of genetic diversity in a species is a function of its life history traits on the one hand and pre-historic and recent vicariance events on the other. Social and dispersal systems have major influences on spatial distribution of genetic diversity . Across xanthus)  and the  caraya) , show li caraya)  which haMacaca fuscata) in Yakushima Island [Macaca mulatta) in India and China and among populations within China [Macaca sylvanus) [The pre-historic vicariance events that influence spatial distribution of genetic diversity include paleo-climatic events, such as glaciations , and geoa Island , geneticin China , and popylvanus)  probablyProcolobus rufomitratus) with male and female dispersal retained more mtDNA haplotypes and had greater nucleotide diversity than the Tana River mangabey  with only male dispersal [Among the major recent vicariance events threatening biodiversity is human-caused habitat fragmentation –12. The ispersal . Both thDermanura watsoni, was greater in the larger forest fragments and in fragments with suitable habitats in the surrounding matrix [Panthera onca) [Ursus americanus floridanus) [While arboreal animals and less mobile terrestrial animals seem to be invariably affected, the genetic impacts of habitat fragmentation on highly mobile animals seem to be more variable –17. Geneg matrix . In mammg matrix . In the ra onca)  and Florridanus) .Macaca silenus) at two temporal levels. We examine the extent of genetic differentiation between two populations on either side of an ecological barrier- the Palghat gap- that predates the arrival of the lion-tailed macaque in the Western Ghats mountain ranges in peninsular India to which it is endemic. Parts of the Western Ghats have undergone extensive habitat loss and fragmentation, starting from 1860’s. Therefore, we also examine the nature and extent of loss of genetic diversity as indicated by mtDNA.Thus, contemporary spatial distribution of genetic diversity is highly variable depending upon so many factors that it might be species and locality specific. In this paper, we examine the spatial distribution of mtDNA in the lion-tailed macaque , long inter-birth interval (30 months), but high survival (80% to adulthood). Population growth rate is, therefore, low. Mean group size in contiguous forests is about 18 individuals and most of these are single-male groups . HoweverThe Western Ghats mountain range extends for about 1600 km from the southern tip of India to the state of Gujarat, running almost parallel to the west coast. The most prominent break in the mountain range is the Palghat gap which is about 40 km wide. This gap is about 500 million years old  and thus2 of tea plantations with more than 20 rainforest patches and the plateau is surrounded by protected areas with contiguous rainforests. The total population of the lion-tailed macaque in Anamalai hills is about 800 animals and nearly half of them are in forest fragments in the tea plantations [The loss and fragmentation of forests in the Western Ghats predates that in most other parts of the tropics, and has its roots in the establishment of tea plantations starting from 1860’s. Even between 1920 and 1990, there was a 40% loss of forest cover and a fourfold increase in the number of forest fragments in the Western Ghats . The raintations . These fntations . This lantations , demograntations ,30, feedntations  and parantations .In this study, we used mtDNA sequences (i) to examine genetic diversity and structure of the lion-tailed macaque across its distribution in the Western Ghats and (ii) to assess the genetic diversity of the population in the fragmented forests of Anamalai hills with reference to a continuous population in the adjacent Nelliyampathy hills. Estimation of genetic diversity in fragmented landscapes will help us employ better management practices and policies in both in-situ and ex-situ conservation efforts . BesidesLion-tailed macaques occur in the wet evergreen forests from the central to the southernmost regions of the Western Ghats. To examine the genetic structure of the lion-tailed macaque population across its distribution in the Western Ghats, fecal samples of individuals were collected from Aghanashini, Karkala, Kundapur and Kudremukh in Karnataka state, Muthanga, Vazhikkadavu, Silent Valley, Nelliyampathy, Kodanad, Vazhachal, Kollam and Amboori in Kerala State, and Anamalai, Meghamalai and Kalakkad in Tamil Nadu State .To understand the impact of habitat fragmentation on the genetic diversity of the lion-tailed macaque populations, a study was undertaken in the rainforest fragments of Anamalai Tiger Reserve, Anamalai hills, Western Ghats, India . Eight fnd September, 2009), the Principal Chief Conservator of Forests (Wildlife), Kerala Forest Department, Thiruvananthapuram 695 014  and the Principal Chief Conservator of Forests, Karnataka Forest Department, Bengaluru 560 003 (Ref. No. B3/WL/CR-8/2008-2009) respectively. Fresh feces were collected by following the troops and the feces were stored in 15 mL or 50 mL falcon tubes containing 90% ethanol at room temperature until DNA isolation.All sample collections were done during the years 2010–2012. Eighty-eight fecal samples were collected from 8 forest fragments in Anamalai hills  and 15 frd January, 2011). Information on the origin of these macaques was collected from studbook records. These animals, part of the Conservation Breeding Programme initiated and coordinated by the Central Zoo Authority of India (CZA), were housed as per CZA norms and fed twice a day. Two to three mL of blood was collected and transported at 4°C to the lab and stored until DNA isolation. All experiments were performed in accordance with the guidelines of the Central Zoo Authority of India, Ministry of Environment, Forests & Climate Change Government of India and the study was also approved by the Institutional Animal Ethics Committee of the Centre for Cellular and Molecular Biology, Hyderabad, India.Twenty-three blood samples from captive lion-tailed macaques were also collected in EDTA vacutainers by qualified veterinarians of Mysore, Thiruvananthapuram and Chennai Zoos by physically restraining the macaques  Vol III, dated 3The ethanol was drained out of the sample collection tubes and the fecal samples were placed on a petriplate for drying at room temperature. After treating the sample with 1X PBS for 4 hours, the resultant mixture was centrifuged and the pellet was lysed by incubating overnight at 50°C in 1.5 mL lysis buffer  containing 2% sodium dodecyl sulfate and 200 μg proteinase K. Genomic DNA was extracted from the lysate using standard phenol-chloroform method , i.e., tM. fascicularis, M. mulatta and M. sylvanus available in GenBank  using the primer designing tool, Primer3 [M. mulatta (NC_005943) and gives an amplicon of ca. 650 bp in the lion-tailed macaque. Universal primers designed by Verma and Singh [Macaque-specific primers, D1 (5’ GTACACTGGCCTTGTAAACC 3’) and D3 (5’ CTTATTTAAGGGGAACGTGTGG 3’), for hypervariable region-I (HVR-I) region were designed from conserved domains of reference sequences of  Primer3 ,36. Thisnd Singh  were use2, 1X BSA, 0.25 μM each of forward and reverse primers and 0.75 units of Taq polymerase . PCRs were performed with initial denaturation at 95°C for 10’, 35 cycles each of denaturation at 95°C for 20”, annealing at 52°C for 20” and extension at 72°C for 30”, followed by a final extension at 72°C for 10’. PCR products were visualized on 2% agarose gel and the DNA extracted with Gel Elution Kit (BioServe India). To prevent cross-sample contamination the workspace was cleaned with bleach and ethanol prior to each extraction. Pre and post-PCR works were carried out in separate, dedicated spaces and all extractions and PCRs were done with a negative control.PCR reactions were performed in 10 μL reaction volumes of 1X PCR buffer, 2.5 mM of MgClSequencing was repeated thrice with both forward and reverse primers. Where the results were contentious, PCR was repeated to resolve the issue. Sequencing was carried out in ABI 3730XL sequencer using BigDye Terminator cycle sequencing kit (Applied Biosystems). Excluding the primer region, we generated sequences of a maximum of 613 bases spanning tRNA-thr , tRNA-pro and partial HVR-I (519 bases) and sequences of a maximum of 521 bases of mitochondrial cytochrome b (cytb) were also generated. The presence of nuclear mitochondrial inserts (numts) is unlikely in our samples as the fecal samples are richer in mitochondrial DNA and no extremely variant sequences were observed. However, we screened cytb sequences for numts by translating into amino acid sequences to detect stop codons.M. nemestrina or M. leonina were deemed to be of lion-tailed macaque origin as no control region sequence of the same was available in the database. Subsequently, after the generation of voucher lion-tailed macaque sequences, the identities were verified by comparing with the voucher. The forward and reverse sequences were aligned and manually edited for base-calling errors using CodonCode Aligner 2.0.6 (http://www.codoncode.com/). A second editing step was performed where all the edited sequences were aligned and the trace sequences were checked for editing-errors at all sites of mismatch. The edited sequences were realigned using CLUSTAL W [The identity of the samples was checked using BLAST and all sequences showing maximum similarity to LUSTAL W  with defLUSTAL W .For assessing genetic diversity, a dataset containing 103 sequences and 519 bases of HVR-I was used. Insertions and deletions were excluded from the analysis. Genetic variability was estimated in terms of haplotype diversity (h) and nucleotide diversity (π). Tajima’s D was also estimated to infer if the data departs from the ideal, neutrally evolving population. All diversity estimations were done in DnaSP 5.10 M. sylvanus (GenBank accession number: KJ567054), M. mulatta (KJ567051 and JQ821843), M. cyclopis (KM023192), M. fuscata (KM401548), M. fascicularis (KM851010 and KM851007), M. thibetana (KJ567056 and EU294187) and M. assamensis (KF990122), cut from complete mitochondrial genome sequences available in GenBank, were used as outgroups. The simplest model of sequence evolution that best explains the pattern of nucleotide variations in the dataset was determined using jModelTest 2.1.3 [For estimating genetic structure, a combined dataset containing 495 bases of HVR-I and 394 bases of cytb was used. Since some of the HVR-I sequences generated low quality bases towards the end, the sequences had to be truncated to 495 bases from 519 bases to accommodate the maximum number of haplotypes in the analysis. Corresponding sequences of st 2.1.3 . Based oBayesian inference trees were reconstructed using Mr.Bayes 3.1.2 . Two parPapio ursinus (accession numbers: JX946204 and JX946205), P. anubis (JX946197 and JX946198) and Theropithecus gelada (KC757412 and FJ785426). To estimate the divergence times, the following fossil-based calibration dates adopted from Liedigk et al. [Papio-Theropithecus split at 5.0 Ma with a 95% credibility interval (CI) of 1.5 Ma and the split between African and Asian macaques at 5.5 Ma with a 95% CI of 1.0 Ma. A relaxed uncorrelated lognormal model of lineage variation and Birth-Death process prior for branching rates were assumed. Four independent MCMC runs with 25 million generations each were performed and trees were sampled every 1,000 generations. Adequacy of 10% burn-in and the stability of the runs were checked using TRACER 1.5 [BEAST 1.7.5  was usedACER 1.5 . The samACER 1.5  and the ACER 1.5 . The treACER 1.5 .One-hundred-and-forty-seven fecal samples of lion-tailed macaques were collected from across the Western Ghats . We amplPhylogenetic trees were reconstructed using Bayesian inference  and maxiPapio-Theropithecus and the African-Asian macaques splits in BEAST 1.7.5  represented by 84 sequences from all the eight fragments and two minor haplotypes (Anm2 and Anm3) from three fragments which are in the periphery of the fragmented Anamalai hills landscape . To compIn the present study, 25.9% of 495 bases of HVR-I region was found to be polymorphic reflecting healthy genetic diversity in the lion-tailed macaque across its range in the Western Ghats.Nyctibatrachus [Nyctibatrachus spp.). Of the 10 species of birds whose divergences across the Palghat gap were dated by Robin et al. [The lion-tailed macaque in the Western Ghats shows an ancient split across the Palghat gap which dates back to 2.11 Ma. Similar observations of divergence across the Palghat gap have been reported in the Asian elephant , the whiatrachus . The estn et al. , 5 have n et al. –53 have n et al. ,55 and in et al. ,49,50.M. fuscata yakui) and the Kyushu haplotypes was 0.047 [Overall genetic diversity of the lion-tailed macaque across its entire distribution is comparable to other macaques. In rhesus macaque, 31% of 544 bases of HVR-I was found to be variable .In rhesuas 0.047 –59, theras 0.047 . Thus, mLion-tailed macaques in the fragmented forests of Anamalai hills have significantly low mtDNA diversity compared to the adjoining large and continuous population of Nelliyampathy hills. Similar reductions in genetic diversity in isolated forest patches as compared to those in continuous forests have been reported in gorillas  and gianOf the three haplotypes found in the Anamalai hills, one haplotype (Anm1) was found in all forest fragments whereas the other two (Anm2 and Anm3) were confined only to the peripheral forest fragments , . One of Lion-tailed macaques of the Anamalai hills, which form almost 20% of the entire wild population, have very low genetic diversity due to habitat fragmentation. Furthermore, lion-tailed macaques in isolated forest fragments have not been observed using non-canopy matrices around them . This prThe present study clearly demonstrates the presence of two genetically distinct populations of lion-tailed macaques across the Palghat gap. These populations can be considered as separate conservation units. Furthermore, we suggest that they should be treated separately in captive breeding programs and any reintroductions to the wild should be done after ascertaining the correct geographic origin of the individuals.S1 FigMaximum Likelihood tree reconstructed from 893 bases (including gaps) of macaque mitochondrial DNA sequences using raxmlGUI 1.3. Leaves are labelled with the samples’ wild origins see  and indi(TIF)Click here for additional data file.


Coleodactylus meridionalis, which was used to illustrate the model in the original paper. We incorporate data on inferred historic habitat dynamics, phylogeography and thermal physiology to test for divergence between coastal and inland populations in the Atlantic Forest of Brazil. Environmental and genetic analyses are concordant with divergence through the VRM, yet physiological data are not. We emphasize the importance of multidisciplinary approaches to test this and alternative speciation models while seeking to explain the extraordinarily high genetic and phenotypic diversity of tropical biomes.Much of the debate around speciation and historical biogeography has focused on the role of stabilizing selection on the physiological (abiotic) niche, emphasizing how isolation and vicariance, when associated with niche conservatism, may drive tropical speciation. Yet, recent re-emphasis on the ecological dimensions of speciation points to a more prominent role of divergent selection in driving genetic, phenotypic, and niche divergence. The vanishing refuge model (VRM), first described by  Speciation is a dynamic, multifaceted, and continuous process . Much of“Some populations of forest-restricted species may be pre-adapted to life in open formations. If, during the dry part of a climatic cycle, they happen to be confined to a refuge that eventually vanishes, they may, in the process, become completely adapted to open formation conditions and constitute a full ecological variant.”Revisiting and refining the VRM is relevant for present-day discussions about the drivers of diversification, particularly given the renaissance of ecological and biogeographic thinking in speciation studies, and the new methods used to infer population and biogeographic history. This often neglected model mirrors recent emphasis on the role of ecology and adaptive divergence on speciation processes . Though As originally illustrated by In this paper, we (1) articulate why and how the VRM contributes to ongoing discussions about the links between diversification and niche evolution; (2) make clear predictions about the expected patterns of genetic structure, phylogenetic relationships, realized niches, and phenotypic disparity resulting from the processes described by the VRM; (3) consider alternative historical processes that can result in diversity patterns similar to those expected under the VRM; (4) discuss multidisciplinary, integrative approaches to test the model; and (5) illustrate an initial test of the predictions of the VRM using new data on the distribution, genetics and physiology of lizards in the Brazilian Atlantic Forest and adjacent dry biomes – the same habitats and taxa used by In general, theory on speciation processes considers the interaction of suppression of gene flow with opportunity for divergent selection and/or genetic drift . The VRMWhether the opportunity for speciation under the VRM will be realized depends on the remnant, initially forest-associated, populations remaining viable under divergent selection. Given potentially rapid environmental change, persistence of populations will be enhanced by (i) the presence of standing genetic variation on which selection can act, (ii) plasticity in key traits to buffer the demographic costs of selection, or (iii) a high rate of intrinsic growth relative to the rate of environmental change ; all “prTable 1A). Yet we exemplify the predictions with the original conditions illustrated by Here, we extend the original formulation of the VRM to include specific predictions regarding habitat structure, phylogeography and historical demography that are pertinent to any time period and habitat type  habitat types , yet show evidence for “pre-adaptation”  to broader environmental conditions, possibly inferred through natural history observations, for instance through species records in edge or anthropogenic habitats.Populations of taxa undergoing the initial stages of diversification as described by the VRM will inhabit distinct isolates of suitable habitat. While some populations will remain in more climatically stable (core) areas and under stabilizing selection, others populations will occur in patches (the vanishing refugia) which are being replaced by the surrounding, ecologically distinct matrix. The later will hence be subject to strong directional selection.initial stage, we expect to find evidence of:At this (1)Recent habitat connections between core areas and vanishing refugia, followed by habitat fragmentation and near or complete loss of the original preferred habitats; and(2)Table 1A, steps 2–6).Genetic differentiation among populations in core areas and vanishing refugia, due to the cessation of gene flow, and increasing with time since isolation Broad physiological tolerances relative to populations in core (stable) areas. This can be detected through:(a)Table 1A, steps 1 and 2);Inter-individual trait variation (polymorphism), where some individuals in vanishing refugia are more tolerant of the matrix conditions ((b)Table 1A, steps 1 and 2; Greater capacity for plasticity, in terms of either developmental plasticity or  reversible acclimation ((c)Table 1A, step 3).Adaptation to ecotone conditions, where all individuals are able to tolerate both original habitat and the matrix types of habitat: lineages in core areas occupy ancestral habitat types, whereas lineages evolving in vanishing refugia will have different climatic niches and occupy a distinct (matrix-like) habitat type . In this instance and hereafter, we use the term “lineage” to refer to lineages of individuals (organisms) rather than lineages of genes.Sister lineages occupy distinct As individuals of the lineage undergoing climatic niche evolution  colonize the matrix, we expect to uncover:(5)Table 1A, steps 5–6).Genetic signatures of population expansion in the newly occupied habitat (Multiple mechanisms may then contribute to:(6)Pre- or post-mating reproductive isolation between individuals of the ancestral lineages in core habitats relative to those of the newly evolved lineage occupying the matrix, including for instance divergent sexual selection and reinforcement.In isolation and under the new regime of selective pressures:(7)The lineage occupying the matrix becomes further differentiated, phenotypically, from those in ancestral habitats. Because the VRM stresses the role of divergent selection, at least some of these phenotypic differences will be adaptive. Whether the physiological tolerances of the diverging lineage will remain broader than that of the ancestral population (or just shift to a different optimum) will depend on the direction of selective pressures in the matrix and hence cannot be predicted.Table 2).The diversification process described by the VRM results in the pattern that originally motivated The VRM was initially proposed to avoid the assumption of divergence with gene flow, yet a parapatric mode of speciation, in which two populations diverge across an environmental gradient in the presence of continuous gene flow  could rePeripatric speciation, for instance caused by founder events , may alsTable 2). Given a suitable test system, we suggest integrative testing that combines habitat modeling over climatic fluctuations, genetic analyses, and phenotypic comparisons (Table 1B).As with any discussion of refugial dynamics  or of spRegions of high climatic heterogeneity and steep environmental gradients are good candidates for operation of the VRM process because climatic changes, such as those of the Quaternary, can readily lead to habitat deterioration and fragmentation and hence to the isolation and diversification of populations. Extreme differences across habitats can also prevent organisms from experiencing similar microhabitats through behavioral thermoregulation that may otherwise shield them from divergent selection pressures . Local eAs Generally, we expect that low dispersal organisms will more likely diversify through vanishing refuge processes than will high dispersal species. Because the former may be unable to track habitats through movement or migration , therefoTable 2).A key approach to validating the VRM is through paleo-habitat modeling. By using bioclimatic distribution models to infer the distribution of the inferred ancestral habitat rather than individual species, this approach can reveal past habitat fragmentation  and highTable 1, step 2). Population genetic methods  mentioned in the original VRM paper. Coleodactylus meridionalis’ potential exposure to divergent selection, they hypothesized that populations of this species could be in the initial stages of speciation under the VRM.We illustrate an initial test of the VRM within one lizard species  the dactyloid Norops brasiliensis (at the time Anolis chrysolepis), and (3) the tropidurid Plica plica. The authors suggested that all of them could possibly be “pre-adapted” to tolerate non-forest habitat. P. plica is an arboreal species, typical of primary forest and rarely found at forest edges, which shows occasional basking behavior. G. humeralis and N. brasiliensis show inter-population variation in habitat use, being found in primary forests as well as highly disturbed habitats. Based on distribution and morphological distinctiveness, Copeoglossum arajara (at the time Mabuya arajara) has completed the speciation process according to the VRM , its putative sister species, was restricted to forested environments (despite being able to actively thermoregulate). Our recent field observations , however, do not support the presumed differences in habitat use between these two species. the VRM . This suColeodactylus meridionalis are diverging according to the VRM, we combined novel occurrence data, preliminary phylogeographic analyses, and physiological assays with existing hypotheses about the historical climatic stability of the Northern AF. Because related species are primarily distributed in forest habitats  suggests limited dispersal capacity, which may prevent it from tracking shifting forests in periods of rapid climatic and habitat change.To test whether geographically isolated lineages of habitats , we suggFigure 1). To test for the VRM predictions with Coleodactylus meridionalis, we sampled populations in the climatically stable and currently continuous forested area along the Brazilian coast (mostly in Bahia), as well as inland populations in forested areas that have been climatically unstable over the last 120 ky and are currently isolated from coastal AF habitats by the surrounding by Caatinga biome . If VRM mechanisms are in progress, we should find evidence of: (1) recent shifts in the distribution of the AF, (2) recent AF fragmentation, (3) genetic, and (4) phenotypic differentiation between populations in core (stable) forest areas and more unstable, isolated forest patches. We also expect (5) greater acclimation capacity and broader thermal tolerances in lineages occupying historically unstable areas. Although one can expect more within-populations variation (polymorphism) in tolerance traits in unstable areas than in stable areas, our limited sample sizes in some key areas prevents us from testing it statistically.Our distribution data reveals that this species is indeed restricted to the leaf litter and occurs primarily in the Northern AF, yet is also found in several localities within the much drier Caatinga and Cerrado biomes of Northern and Northeastern Brazil, in addition to the site Coleodactylus meridionalis is found in large forested areas along the coast as well as in small, isolated forest patches further inland, with an overall gradient of decreasing historical stability of forest habitat from the coast to the inland regions .Two lines of environmental evidence are concordant with a VRM of diversification in this system. Based on correlative paleo-modeling of the Northern AF, developed at 4 ky intervals through a full glacial cycle (120 ky), historical climatic stability of forest habitats has varied throughout this species’ range ; some poColeodactylus meridionalis. Such low genetic differentiation suggests that, if diverging under the VRM, this system must be in its very early diversification stages . The data nonetheless indicate the existence of a few differentiated clades within this species (bootstrap support >85): (a) a large clade including samples from the climatically stable coastal Bahia, (b) a north coastal clade within a region with low-medium stability, (c) a clade including inland, relatively unstable sites , yet covering most of the species distribution . We also assessed short-term reversible acclimation capacity in three localities with varying degrees of climatic stability by conducting experiments immediately after capture as well as after two different acclimation treatments (details in supplementary methods). We nonetheless found no evidence of acclimation capacity in CTmax, CTmin, thermal tolerance (CTmin–CTmax), or thermal preferences , except for a significant shift in CTmin in Chapada . Because this result probably reflects the small sample size of the 30°C treatment , we avoid over-interpreting it. Nevertheless, if CTmin is actually plastic in Chapada, this would be in opposition to the VRM prediction given the moderate to high stability score of this locality . Tolerance did vary across populations , yet the greatest difference (∼5°C) was observed between individuals collected in Salvador (narrower tolerance) relative to those in Chapada . These two sites have high historical climatic stability, yet Salvador is a coastal, lowland site while the Chapada is an inland and higher elevation region where climate is more seasonal  and current annual temperature range . Together, these results suggest that thermal physiology in Coleodactylus meridionalis is labile. Whether such correlation represents local adaptation or developmental plasticity is yet to be determined. In contrast to predictions .In contrast to predictions of VRM, we did not detect a correlation between historical habitat stability and thermal tolerance  may be one such candidate. This lineage is nested within Coleodactylus meridionalis, and may have diverged quite recently Coleodactylus meridionalis lineages. Coleodactylus natalensis is known from only one forested area on a coastal dune system , and is morphologically distinct from sister lineages (Coleodactylus natalensis lineage (It is possible that other phenotypic traits, such as eco-morphology, may have diverged in lineages . Some da lineage , though maintenance of diversity in space and over time (generating adaptive diversity (Our case study illustrates how multiple lines of evidence can be combined to identify lineages potentially diverging under the VRM. Recent integrative studies support the view that historic climatic stability promotes the accumulation and ver time . The vaniversity . Importaiversity . We arguDNA sequences: GenBank accessions KM852739-KM852829.Roberta Damasceno and Maria L. Strangas contributed equally to this study. Roberta Damasceno, Maria L. Strangas, Ana C. Carnaval, Craig Moritz, and Miguel T. Rodrigues designed this study. Roberta Damasceno and Miguel T. Rodrigues collected data. Roberta Damasceno performed analyses. Roberta Damasceno, Maria L. Strangas, Ana C. Carnaval, Craig Moritz, and Miguel T. Rodrigues interpreted the data. Roberta Damasceno, Maria L. Strangas, Ana C. Carnaval, and Craig Moritz wrote the manuscript. Miguel T. Rodrigues revised it critically for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.