Elevation of plasma levels of EC4 in male apolipoprotein E–deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis  Accordingly, reducing apoptosis, and in particular, VSMC apoptosis has been considered as a potential and novel therapeutic strategy to prevent atherosclerotic plaque rupture.A major contributor to atherosclerotic plaque instability and rupture is vascular smooth muscle cell (VSMC) apoptosis. Although VSMC apoptosis is rarely observed in normal blood vessels, in human unstable atherosclerotic plaques, increased apoptosis rates are detected.7 Soluble N-cadherin (SNC), which consists of the extracellular domain, acts as a mimetic to the full-length molecule and also increases VSMC survival. Importantly, SNC was conjugated to the Fc domain to increase half-life in vivo and to ease purification for in vitro and in vivo studies (SNC-Fc).We have previously shown that SNC-Fc activated the fibroblast growth factor receptor, resulting in activation of the prosurvival phosphoinositide 3 kinase/Akt/phospho-Bad pathway.8 SNC-Fc also reduced apoptosis in atherosclerotic plaques and increased features of atherosclerotic plaque stability in apolipoprotein E–deficient mice,8 suggesting that SNC-Fc may have therapeutic potential for retarding plaque instability.Cadherins are homophilic cell–cell adhesion molecules and are involved in prosurvival signaling in various cell types, including VSMCs. Expression of cadherins is cell type specific, VSMCs express N-cadherin, and overexpression of N-cadherin significantly increase VSMC survival.9Our previous work suggests that SNC-Fc can activate the FGFR, as well as phosphoinositide 3 kinase and Akt signaling.8 However, it was unclear whether (i) SNC-Fc could interact with the FGFR directly; (ii) the interaction of SNC-Fc with N-cadherin on the cell surface was required for FGFR activation; or (iii) the interaction with N-cadherin caused additional activation of phosphoinositide 3 kinase and Akt.10–12 To investigate this further, we have mutated the two binding sites to prevent the interaction with either N-cadherin or FGFR.A soluble molecule has obvious advantages therapeutically; however, at ~120 kDa, SNC-Fc is a rather large therapeutic molecule, therefore, we aimed to identify the active antiapoptotic site and evaluate whether a smaller molecule containing this site retained the antiapoptotic and plaque stabilizing properties. SNC consists of five extracellular (EC) domains: EC1–EC5. SNC has two binding domains: the homophilic N-cadherin binding domain (HAV/INPISGQ) in EC1 and the FGFR binding domain (INPDVGQ) in EC4.in vitro and in vivo and has the potential for repressing plaque instability.In this study, we have characterized mutated versions of SNC-Fc to determine the active antiapoptotic site and subsequently truncated the molecule accordingly. We have then investigated whether the truncated form, EC4-Fc, retains prosurvival effects both To determine which part of the SNC molecule was responsible for its antiapoptotic effect, we mutated the two active sites present in the molecule . SNC canInterestingly, we found that when the N-cadherin binding sites were mutated (N-cad mut), this form of SNC-Fc still reduced VSMC apoptosis . In cont8 Immunocytochemistry for phospho-Akt  demonstrated that EC4-Fc increased phospho-Akt to an extent similar to that of SNC-Fc . Additionally, no adverse effects in the mice were observed.Fluorescent immunohistochemistry for cleaved caspase-3 showed that apoptosis within atherosclerotic plaques from the brachiocephalic artery was significantly reduced to a similar extent in the mice infected with RAd EC4-Fc and RAd SNC-Fc . Plaque in vivo and are in line with our previous findings.8 Additionally, we observed a significant increase (approximately threefold) in the ratio of VSMCs:macrophages, using specific cell type markers (The proportion of plaques exhibiting buried fibrous layers (a surrogate marker of plaque instability) were significantly reduced (>35%) in the mice infected with RAd EC4-Fc or RAd SNC-Fc compared with that of the Fc control mice ,c. These markers . This is markers .8 However, the therapeutic potential of SNC-Fc is limited due to the size of the molecule (~120 kDa). In the current study, we therefore aimed to identify the active antiapoptotic site within SNC and generate a smaller molecule, which may have greater therapeutic application. We have identified the active site of SNC-Fc as located within the EC4 domain and evaluated the effects of a truncated form of the molecule (~50 kDa and termed EC4-Fc) on apoptosis and atherosclerotic plaque progression. This study clearly identifies the potential for therapeutic strategies targeted at activating FGFR, in reducing the destabilization of atherosclerotic plaques.VSMC apoptosis is thought to be a major contributor to atherosclerotic plaque instability. Therefore, a therapeutic agent capable of reducing VSMC apoptosis in the fibrous cap of atherosclerotic plaques could be of great clinical benefit. We have previously shown that SNC-Fc significantly reduced apoptosis and increased features of atherosclerotic plaque stability.8 We therefore postulate that a direct interaction between SNC and FGFR is essential for subsequent prosurvival signaling. In support of these observations, N-cadherin and FGFR have also been shown to interact during neurite outgrowth, as reviewed in ref. SNC-Fc consists of five extracellular domains (EC1–EC5). Motifs within EC1 are responsible for interactions with N-cadherin, and another motif in EC4 is responsible for interactions with the FGFR. To investigate whether these sites are required for the previously observed antiapoptotic effect of SNC-Fc, we mutated the two sites independently of each other or in tandem to prevent their interactions. Interestingly, the antiapoptotic effects of SNC-Fc were modulated through the FGFR binding site within the EC4 domain. We have previously shown that SNC-Fc activates the FGFR.in vivo, elevated plasma levels of EC4-Fc reduced intraplaque frequencies of apoptosis, the number of buried layers, and plaque size when compared with the Fc control. In contrast and in accordance with our previous findings,8 elevated levels of SNC-Fc reduced apoptosis and the number of buried layers but had no significant effect on plaque size. Also beneficially for plaque stability, both SNC-Fc and EC4-Fc significantly increased the smooth muscle cell:macrophage ratio. Taken together, these data demonstrate that both SNC and EC4, through interaction with the fibroblast growth factor receptor type 1, can prevent atherosclerotic plaque progression. Although it may appear counter-intuitive that EC4-Fc reduced macrophage apoptosis and the proportion of macrophages in the plaque, we suggest that the lower levels of apoptosis results in less secondary necrosis, inflammation, and proinflammatory cytokine production, leading to reduced influx of macrophages to the plaque.14–17Moreover in vivo study suggested that EC4 possesses greater activity as evidenced by the significant reduction in plaque area that was not observed in SNC-Fc animals. We postulate that the smaller molecular weight of EC4 may increase its permeability and therefore augment its access to intraplaque cells. Consequently in future studies, we will aim to design and test small peptides targeting the FGFR binding site. Indeed, a small peptide encoding the N-cadherin binding site in EC1, producing an HAV peptide which antagonises N-cadherin, is being trialed as a cancer chemotherapeutic,18 demonstrating that this approach has potential for clinical use.Our findings illustrate that in comparison to SNC, EC4 can act as a potent mimetic without any loss in functionality. Indeed, the in vitro, and more importantly, reduced apoptosis, plaque size, and the number of buried layers within murine brachiocephalic artery atherosclerotic plaques, rendering them with an enhanced stable phenotype. Our findings, therefore, suggest that EC4-Fc, or possibly a smaller molecule, may have potential as a therapeutic in the treatment of atherosclerosis.In conclusion, this study demonstrates that site-specific truncation of SNC (EC4) does not diminish its antiapoptotic activity. The ~50 kDa protein EC4-Fc reduced VSMC, endothelial cell, and macrophage apoptosis 4 Ethical permission for this study was gained from the local research ethics committee (NRES 07/H0107/61), and the study was approved by the University of Bristol and National Health Service Review Boards. Each experiment was carried out with VSMCs from at least three different segments of vein.Human saphenous vein VSMCs at passages 4–8 were generated as described.Mouse peripheral blood monocytes were purified using Ficoll-Hypaque gradient , followed by differential adherence and culture in 20 ng/ml of macrophage colony-stimulating factor for 7–10 days to induce differentiation into macrophages.9 and was removed from SNC-Fc using BamHI and HindIII. The second contained the IDPVNGQ motif, which is required for interaction with FGFRs,9 and was removed from SNC-Fc using EcoRV and KpnI. These fragments were cloned into the pDRIVE cloning vector . PCR mutagenesis was used to mutate the binding sites of SNC. This produced the following mutations in the SNC sequence: HAV → HGV, INPISGQ → INPASGQ, IDPVNGQ → IDAVNGQ, which were validated by sequencing. These mutated fragments were then shuttled back into SNC using the same restriction enzyme sites, to produce two plasmids: “N-cadherin mutated” and “FGFR mutated.” The native, mutated SNC constructs were then cloned into pCpG-free mcs  in which a myc-tagged FC domain (FCmyc) had already been cloned. The Fc domain, containing mutations in the immunoglobulin G receptor and complement binding domains, was amplified by PCR from an IL-10-Fc fusion plasmid . This chimeric molecule comprised of SNC and an antibody constant domain, which enables protein A binding, as well as extending plasma half-life19–21 and possibly increasing receptor–ligand interaction through its potential for dimerization. Such immunoadhesins have been used in vivo as potential therapeutic agents.22Two fragments containing the binding sites within EC1 and EC4 were subcloned to facilitate polymerase chain reaction (PCR) mutagenesis. The first contained the HAV and INPISGQ motifs, which are required for interaction with N-cadherin,The EC4 truncation was produced as follows. To facilitate secretion, the signal prepropeptide of SNC was cloned by PCR creating a BamHI site at the 3′ end into FCmyc. This soluble Fc construct was used as a control (sFCmyc). The EC4 domain of SNC was excised by PCR introducing BamHI sites at each end. This product was cut with BamHI and ligated into sFCmyc to create sEC4-FCmyc and was validated by sequencing.8EcoRI fragments were prepared from sFCmyc or sEC4-FCmyc, which included the mouse cytomegalovirus enhancer, EF1α promoter, a short intron, the coding sequence, and a polyA site, and were cloned into pDC511 . Both were recombined with the adenovirus genomic plasmid by cotransfection into 293 cells. The resultant adenoviruses were termed RAd EC4-Fc and RAd Fc (control). RAd SNC-Fc was made as described.Chinese hamster ovary cells were infected with 50 pfu/cell of RAd Fc, RAd SNC-Fc, or RAd EC4-Fc or subjected to AMAXA technology–mediated transfection of plasmids for mutated versions of SNC-Fc. The conditioned media was collected at 66 and 138 hours after infection/transfection. The conditioned media was pooled, and protein purification was achieved with protein A columns (Amersham Biosciences). The protein concentration was determined using the Bradford Protein assay (Sigma) and compared using western blotting.To enable the detection of apoptotic frequencies by immunocytochemistry, all cells were grown on glass coverslips. Human VSMC apoptosis was induced by culturing in serum-free media with 200 ng/ml of Fas-L for 24 hours. Blood-derived macrophage apoptosis was induced by culture in serum-free media for 72 hours. Human umbilical vein endothelial cell apoptosis was induced by culture in serum-free media for 24 hours. Cells were supplemented with 20 pmol/l of the respective purified proteins to assess their antiapoptotic effects.7 Phosphorylated Akt was detected by immunocytochemistry using rabbit anti-pAkt antibody , as described previously.8Apoptosis was assessed by cleaved caspase-3 immunocytochemistry as described previously.−/− mice (strain background 71% C57BL/6 and 29% 129) were bred within the Animal Unit of the University of Bristol. Housing, care, and all procedures were performed in accordance with the guidelines and regulations of the University of Bristol and the United Kingdom Home Office, and this study was approved by the University of Bristol Review Board. To induce the formation of complex atherosclerotic lesions, 8-week-old male apolipoprotein E−/− mice were fed a high-fat rodent diet containing 21% (w/w) fat from lard supplemented with 0.15% (w/w) cholesterol  for a period of 8 weeks. Mice were then given an i.v. dose of 8 × 1010 viral particles of an empty adenovirus (RAd66) as a predose 4 hours before administration of 2.25 × 108 viral particles of RAd SNC-Fc (n = 12), RAd EC4-Fc (n = 14), or Fc (n = 15) as previously described.23 This protocol allows efficient hepatic transfection and expression, and subsequent elevated circulating levels of the respective transgene. Mice were then maintained on a high-fat diet for a further 28 days to determine effects on established lesions.Homozygous C57BL/6, 129 male apolipoprotein E4 Blots were detected with a goat anti–mouse horseradish peroxidase secondary antibody diluted 1:1,000 in milk overnight. To detect EC4, immunoprecipitation of c-myc using the ProFound c-myc tag IP/co-IP kit  was performed. Blots were detected with c-myc tag antibody  and a swine anti-rabbit secondary antibody .Plasma samples were taken at 6 and 28 days after RAd administration, and the levels of SNC-Fc and EC4 were analyzed using western blotting. For the SNC-Fc blots, plasmas were pretreated with protein A beads for 30 minutes at room temperature. Beads were centrifuged for 5 minutes at 13,000 rpm, the supernatant was removed, and the beads were resuspended in 30 µl of sodium dodecyl sulfate lysis buffer. These samples were subjected to western blotting as described previously.24Plasma lipid profiles were analyzed in terminal plasma samples as previously described.23 Collagen was identified by PicroSirius red staining observed under polarized light.23 Positive staining was quantified using a computerized image analysis system  and expressed as the percentage of total plaque area.VSMCs, macrophages, and proliferating and apoptotic cells were identified by immunohistochemistry for α-smooth muscle cell actin, Griffonia simplicifolia lectin-1, proliferating cell nuclear antigen, and cleaved caspase-3 as described previously.25Serial sections stained for elastin and α-smooth muscle cell actin were examined for the presence of structures rich in elastin and VSMCs, and these were classed as buried fibrous layers, which we have previously identified as a surrogate marker of previous plaque instability, as previously described.t-test was utilized. Differences were considered significant when P < 0.05.Values are expressed as mean ± SEM. Data were tested for normality. Then normally distributed data were analyzed by analysis of variance for multiple comparisons and the Student–Newman–Keuls posttest, and for experiments with two groups, the paired


Pseudocarcharias kamoharai, a small-sized lamnid shark, is occasionally caught by longline vessels in certain regions of the tropical oceans worldwide. However, the population dynamics of this species, as well as the impact of fishing mortality on its stocks, are still unknown, with the crocodile shark currently one of the least studied of all pelagic sharks. Given this, the present study aimed to assess the population structure of P. kamoharai in several regions of the Atlantic and Indian Oceans using genetic molecular markers. The nucleotide composition of the mitochondrial DNA control region of 255 individuals was analyzed, and 31 haplotypes were found, with an estimated diversity Hd = 0.627, and a nucleotide diversity π = 0.00167. An analysis of molecular variance (AMOVA) revealed a fixation index ΦST = -0.01118, representing an absence of population structure among the sampled regions of the Atlantic Ocean, and between the Atlantic and Indian Oceans. These results show a high degree of gene flow between the studied areas, with a single genetic stock and reduced population variability. In panmictic populations, conservation efforts can be concentrated in more restricted areas, being these representative of the total biodiversity of the species. When necessary, this strategy could be applied to the genetic maintenance of P. kamoharai.Among the various shark species that are captured as bycatch in commercial fishing operations, the group of pelagic sharks is still one of the least studied and known. Within those, the crocodile shark,  Prionace glauca), this group of the pelagic sharks is still one of the least known and studied.Among the various shark species commonly caught as bycatch in commercial fishing operations, the pelagic sharks are one of the groups in more need of attention, management and conservation actions . In receCurrently, there is still a lack of definitive information about the current conservation status of most species , 3; howePseudocarcharias kamoharai, is a small pelagic shark species that inhabits from the sea surface to depths of at least 590 m in tropical and subtropical waters of the Atlantic, Indian and Pacific Oceans. It is usually found in the open sea, although it can sometimes occur in coastal waters [P. kamoharai has been poorly studied and not much is known about its biology [P. kamoharai specimens are usually discarded at the time of fishing gear retrieval, and are not retained for commercialization [P. kamoharai in commercial fisheries are rarely reported, and as such the available data to establish the potential impacts of the fisheries on those populations are also very limited.The crocodile shark,  biology . The cro biology . Due to lization , 9. As aPrionace glauca) that accounted for the large majority of the catches (84% of the elasmobranch catch). In that fishery, and considering the areas and seasons of operation of that particular fleet, the numbers of crocodile sharks caught were in the same order of magnitude of the shortfin mako shark (Isurus oxyrinchus), that represented around 4% of the total elasmobranch catch. This result highlights that in certain areas and seasons, the relative frequency of occurrence of P. kamoharai might be relatively high, but that those catches are probably unaccounted as the specimens are discarded.In a recent study with an overview of pelagic sharks at-haulback mortality in longline fisheries in the Atlantic Ocean,  reportedP. kamoharai opens many questions about the impact of the fisheries on these populations and ultimately about the conservation status of the species. Among the various sources of information needed to develop appropriate management and conservation plans, the study of the population genetics is an essential component, as it provides information regarding the stocks delimitation, biodiversity characterization, and identification of evolutionary units. Therefore, this study aimed to assess the genetic population structure of P. kamoharai, comparing specimens from the Atlantic and the southwestern Indian Oceans, in major areas of operation of pelagic longline fisheries.The current lack of biological and fishery data on P. kamoharai were collected by fishery observers placed onboard commercial pelagic longline vessels from Portuguese, Brazilian and French fleets that operate in several regions of the Atlantic and southwest Indian Oceans (3) which was stored frozen in 96% ethanol. No specific permissions are required for collecting samples of crocodile sharks, a species currently not protected, from those locations/activities.The samples of the n Oceans . For the2) was collected from each specimen and preserved in 96% ethanol.In total of 255 specimens were sampled, with 217 collected from different regions of the Atlantic Ocean, namely 48 from the western equatorial Atlantic, 72 from the northeast tropical Atlantic, 68 from the eastern equatorial Atlantic, 14 from the southeast Atlantic and 15 from the southwest Atlantic . AnotherGenomic DNA was extracted using the NucleoSpin Tissue XS Kit . Partial sequences of the mitochondrial control region were obtained according to the methodology used by . Cycle sh), nucleotide diversity (π), and number of pairwise nucleotide differences among populations were calculated using ARLEQUIN 3.5.1.3 software [P. kamoharai, the diversity measure ΦST was calculated using analysis of molecular variance (AMOVA) [ΦST estimates were non-parametrically tested (1000 bootstrapped replicates) using the ARLEQUIN 3.5.1.3 software, and adjusted for simultaneous pairwise comparisons using the sequential Bonferroni procedure [F test [D [The relative nucleotide composition, number of polymorphic sites, haplotype diversity (software . To esti (AMOVA)  under th (AMOVA)  nucleotirocedure . A minimrocedure . Evidenc [F test  and Taji test [D  in ARLEQ test [D .P. kamoharai resulted in 758 nucleotides that could be analyzed with 26 polymorphic sites, yielding 31 haplotypes (h) and nucleotide (π) diversity were h = 0.627 and π = 0.00167, respectively, with the greatest diversities found in the eastern equatorial Atlantic Ocean , and in the southwest Indian Ocean . Haplotypes 1 and 6, shared by 154 and 36 specimens respectively, were the most common and were found on all the sampled regions, representing 74.5% of crocodile sharks analyzed  and between the eastern and western Atlantic Ocean . Several tests also were made at a finer scale within the five regional groups of the Atlantic Ocean. Likewise, the same analysis to test the hypothesis of structuring between the Atlantic and Indian Oceans also resulted in an index revealing the absence of population genetic differentiation . When the indices of structuring were compared between pairs of regions, on all cases, there was not any evidence of population differentiation, even when comparing pairs between the Atlantic and Indian Oceans  with significant deviations from the null hypothesis of neutrality, suggesting the occurrence of an event of population expansion. In the evaluations for each sample group, the observed Tajima D ratios were close to zero on all groups, while for the Fu’s FS tests such indices varied between -1.74 and -8.33  shared by the four major areas delimited as the North Atlantic, Equatorial Atlantic, South Atlantic and Indian Ocean. In the two major haplotypes with higher relative frequency , there is a derivation for a number of other rare haplotypes with only one mutation difference between the major haplotype and the derivatives, featuring a star network .In recent years, with the use of molecular tools, there has been an increase in the availability of genetics data for many species, and information such as variability, population structure, gene flow and historical events have became widely considered in the conservation of threatened species and populations, and in the development of more efficient conservation plans.P. kamoharai indexes of nucleotide diversity were similar to reported for other pelagic shark with a single population as Cetorhinus maximus [Rhincodon typus [P. kamoharai constitutes a single genetic stock with high gene flow throughout its distribution range in the Atlantic Ocean. However, there are some features that should be highlighted. While it is clear that there is a sharing of haplotypes by the various sampled regions, a higher nucleotide and haplotype diversity seems to be associated with the eastern equatorial region, closer to the Gulf of Guinea. Considering that there is no population structure among the overall sampled region, the slightly higher diversity in the eastern equatorial sample may just be due to variation among finite samples drawn from a single population. When the samples from the Indian Ocean were included in the AMOVA analysis similar results were obtained, with an absence of genetic structure differentiation between the Indian and all other groups of the Atlantic. Although the sampled area in the Indian Ocean is only representative of a relatively small area in the southwest Indian Ocean, the pattern of a high gene flow observed in almost all areas of occurrence of the species in the Atlantic, as well as the gene flow between the two Oceans, seems to point to the possibility of a single genetic stock of P. kamoharai distributed between these two Oceans basins, configuring a single population.In the present study the  maximus  and Rhinon typus , 23. Wheon typus , 27, 28.D test can mean an excess of low frequency polymorphisms in relation to expectations, observed in the high number of haplotypes with single representative (20), indicating an event of expansion of population size, that probably occurred after a bottleneck event or purification selection. However, the occurrence of shared haplotypes among the Atlantic and Indian Oceans may indicate a recent or contemporary dispersal, suggesting that these sharks cross waters off the southern tip of the African Continent, and support the continuous gene flow between these two Oceans.The negative indices found for the Tajima’s In the haplotype network a configuration related to the absence of population genetic structure with three haplotypes represented in all sampled groups was observed. Considering the distribution of the sampled groups in four major areas, North Atlantic, Equatorial Atlantic, South Atlantic and Indian Ocean, the presence of four shared haplotypes was observed. Other 6 haplotypes are shared by at least two major areas. Haplotype 1, with the highest relative frequency (60.4%) was represented by individuals on all sampled areas, including the Indian Ocean, being found in greater numbers in the North Atlantic (n = 45). However, the frequency of this haplotype was observed in their majority in the South Atlantic (71.4%). The haplotype 1 had many derived less frequent haplotypes, setting a star configuration that suggests a recent process of population expansion after a bottleneck event.In the last years sharks have gained some relative importance in terms of commercial value, mainly due to the increasing value of their meat and fins. In the particular case of the crocodile shark, and even though this species has no commercial value and is therefore usually discarded, there is still fishing mortality taking place and impacting the populations, as part of the catch is captured and discarded already dead . TherefoP. kamoharai suggest the occurrence of a single stock, without a strong differentiation in haplotype frequencies, forming one basic unit of management in the Atlantic and possibly, in the Indian Ocean. With an absence of population genetic structure, genetic diversity similar or higher than that found in many shark species, although still without consideration of conservation measures, attention with a continuing monitoring through time and an evaluation of the effects caused by fishing pressure are recommended.Genetic factors influence the risk of extinction mainly because endangered species usually have small and/or declining populations, and in such populations inbreeding and loss of genetic diversity will inevitably put the populations at risk, with evolutionary potential lost even before they may eventually become effectively extinct. These results for


Ambystoma leorae, an endemic and critically threatened species. The aims of this study were: (1) to assess the demographic parameters of A. leorae and to distinguish and characterize the microhabitats in the river, (2) to determine the number of existing genetic groups or demes of A. leorae and to describe possible relationships between microhabitats types and demes, (3) to determine gene flow between demes, and (4) to search for geographic locations of genetic discontinuities that limit gene flow between demes. We found three types of microhabitats and three genetically differentiated subpopulations with a significant level of genetic structure. In addition, we found slight genetic barriers. Our results suggest that mole salamander’s species are very sensitive to microhabitat features and relatively narrow obstacles in their path. The estimates of bidirectional gene flow are consistent with the pattern of a stepping stone model between demes, where migration occurs between adjacent demes, but there is low gene flow between distant demes. We can also conclude that there is a positive correlation between microhabitats and genetic structure in this population.The reduced immigration and emigration rates resulting from the lack of landscape connectivity of patches and the hospitality of the intervening matrix could favor the loss of alleles through genetic drift and an increased chance of inbreeding. In order for isolated populations to maintain sufficient levels of genetic diversity and adapt to environmental changes, one important conservation goal must be to preserve or reestablish connectivity among patches in a fragmented landscape. We studied the last known population of  Habitat loss is the leading cause of species declines and extinctions worldwide In order for fragmented populations to maintain sufficient levels of genetic diversity, one important conservation goal must be to preserve or reestablish landscape connectivity among patches. This may involve eliminating migration barriers, providing migration corridors between patches, and even manually moving individuals as a last resort Ambystoma leoraeAbies religiosa forest Information about their reproduction is limited and only one pregnant salamander has been found A. leorae and to distinguish and characterize the microhabitats in the river, (2) to determine the number of existing genetic groups or demes of A. leorae and to describe possible relationships between microhabitats types and demes, (3) to determine gene flow between demes, and (4) to search for geographic locations of genetic discontinuities that limit gene flow between demes.The aims of this study are: (1) to assess the demographic parameters of The manipulation of mole salamanders was conducted with permits received for field work from the Mexican government through to the Ministry of Environment and Natural Resources . Our study received the approval of the ethics committee of Universidad Autónoma del Estado de México.Muhlenbergia sp.) surrounded by forest (Pinus hartwegii and Abies religiosa).Iztaccihuatl-Popocatepetl National Park (IPNP) is located in Central Mexico, near Mexico City, The geographical coordinates are 19°21′09″N, 98°40′11″W and 19°35′25″N, 98°66′97″ W, with an altitude of 4,130 masl . SamplinSampling effort per visit was 4 persons for 4.5 hours per day for 2 days per month for 8 months. Each mole salamander encountered was measured (both snout vent length (SVL) and total length (TL)) and weighed. We identified age class based on size: individuals were classified as egg, juvenile (gilled larva), adult or metamorph, according the Sturgess rule 2 at all twelve sampling sites defined a priori based on differences in the percentage of cover vegetation and type of substrates . These characteristics included percentage area covered by grass bushes outside the river (COHAB1), percentage area covered by Pinus-Abies forest outside the river (COHERB2), percentage area covered by vegetation inside the river (COVEGR), percentage area covered by stones in the river (COSTONR), percentage area covered by mud (COMUDR), depth of the river (DEPR), width of river (WIDER), temperature outside of the river (TEMPOUTR), and river water temperature (TEMPR). To reduce the number of microhabitat variables, we performed a principal components analysis (PCA) using the software STATISTICA 8.0 Nine microhabitat characteristics were recorded within a quadrat of 10 mAmbystoma leorae, from six sample sites, using QIAGEN extraction kits  for use as templates in PCR amplification of nine microsatellite loci following published protocols A combination of adult and neotenic larval salamanders were included in this study. We extracted genomic DNA from tissue samples (tail clip) from 96 individuals of et al.is using GENETIC STUDIO. Finally, to determine if some demographic parameters and habitat characteristics were linked to the percentage of alleles and genotypes, we completed a multivariate analysis of Sperman’s correlation We calculated genetic diversity indices  using the software GENALEX 6 et al.st, Rst and Dest with 10000 dememorization steps and 1000 batches of 10000 iterations per batch in order to test the divergence between demes. For the latter we estimated different genetic distances in accordance with a SMM mutation model: ASD 2SWFor population sampling, we defined twelve microhabitat types a priori based on differences in the percentage of cover vegetation and type of substrates .  we multiply M by θ. We tested four hypotheses of migration among groups: (1) migration to and from all groups, (2) migration only between adjacent groups, (3) migration between distant groups, and (4) all groups as part of a panmitic population. The same parameters were used for all models. To compare the models and choose the most likely model, we used the logarithm of the Bayes Factor . We also made a skyline plot in order to visualize the changes of the population sizes and migration rates through time Gene flow between demes was estimated using the Bayesian inference implemented in MIGRATE-n 3.0 A. leorae was in seven of the twelve sampling sites. The mean rate of encounter in the seven sites was 2.08 mole salamanders per visit. We found 44 eggs distributed in 11 clutches  from February to June. The clutches were attached to aquatic vegetation and inside caves. The egg shape was almost round and its size was variable (8.5 to 20 mm). One hundred fifty two gilled larva were found. These were classified into eight age classes: (1) 42 to 65 mm, (2) 65.1 to 88 mm, (3) 88.1 to 111 mm, (4) 111.1 to 134 mm, (5) 134.1 to 157 mm, (6) 157.1 to 180 mm, (7) 180.1 to 203 mm, and (8) 203.1 to 226 mm. Class nine included transformed mole salamanders , slow stream (0.3–0.4 m/s), water temperature that ranged from 12 to 18.5°C, water with pH from 6.9 to 7.4, and high water oxygen levels of 78% concentration (6.25 mg/L). The sites had different percentages of aquatic plants ranging from 0% to 40%. Outside the rivers the vegetation was mainly small alpine grassland (Muhlenbergia sp.) surrounded by forest (Pinus hartwegii and Abies religiosa). Air temperature ranged from 10 to 12.5°C  and the effective number of genetically distinct groups according Jost 2 mutation model. The population D1 and D3–D6 were sister groups and D2 was the ancestral deme. However, the other two analyses (ASD and DSW) showed an alternative topology in which D1 and D2 were sister groups and D3–D6 was the ancestral deme.  = –1911.4)  and ΔK men demes . Geneticis increased  assigned by STRUCTURE, GENELAND and TESS . Historical studies have found significantly less individuals. In 1943, only four individuals were found at the type locality Río Frío town A. rivulare with SVL ranging from 60 to 90 mm and 40 to 100 mm for females. Additional studies have found indiviuals of A. dumerilii with SVL of 122 mm A. altamirani with SVL average of 64 mm A. ordinarium with SVL average of 56 mm A. tigrinum tigrinum with average of 94 mm and minimum 80 mm A. leorae becomes sexually mature once they reached the class type 4. Juveniles of the class types 1 to 3 possibly hatched in late January and early February. In other mole salamander, the period of incubation has been found to occur between August and December Most individuals in this study were collected from March to July N = 139,  and the A. leorae have not previously been described in detail. There are only scare reports on water temperature Microhabitat characteristics for Pinus-Abies forest outside the river, percentage area covered by vegetation inside the river and percentage area covered by stones inside the river. The mole salamanders need certain habitat characteristics in order to survive, grow, and reproduce. We believe the percentage area covered by grass bushes outside the river may help to avoid water heating and to decrease predation risk [83–87). The percentage area covered by Pinus-Abies forest outside the river is important because the salamander presence and abundance are highly correlated with forest cover et al.For population sampling, we defined twelve microhabitat types a priori; however, after the principal component analysis and the Ward’s dendogram, we found only three different microhabitats types . The varST values are similar to those detected in other studies of mole salamander’s population genetics in fragmented habitats We found within-population structure; which is not surprising for this species due to its known high breeding site fidelity and low dispersal capability. This within-population structure can also arise due to the existence of temporal reproductive cohorts Figure S1Individuals collected per month.(TIF)Click here for additional data file.Figure S2Individuals per class in each deme.(TIF)Click here for additional data file.Figure S3Classes obtained from Sturges rule (1926) and individuals per class.(TIF)Click here for additional data file.Figure S4Matrix scatter plot of physical distance as a function of genetic distance.(TIF)Click here for additional data file.Figure S5Diversity gradient plot showing observed heterozygosity (HO), expected heterozygosity (HE), expected heterozygosity corrected for small sample sizes (HE), and inbreeding (FIS).(TIF)Click here for additional data file.Figure S6Skyline plot of a population that recently increased strongly, the time is in units of mutation scaled generations.(TIF)Click here for additional data file.Table S1Eigenvalues of each factor and the cumulative score of each factor. In bold are the most important values.(DOCX)Click here for additional data file.Table S2Scores of each microhabitat characteristics of the factor analysis components. In bold are the most important variables.(DOCX)Click here for additional data file.Table S3Table output of the Evanno method results. In bold is the largest value in the Delta K column.(DOCX)Click here for additional data file.Table S4Microenvironmental conditions range.(DOCX)Click here for additional data file.


Peromyscus) is the most abundant mammal in North America, and it occupies almost every type of terrestrial habitat. It is not surprising therefore that the natural history of Peromyscus is among the best studied of any small mammal. For decades, the deer mouse has contributed to our understanding of population genetics, disease ecology, longevity, endocrinology and behavior. Over a century's worth of detailed descriptive studies of Peromyscus in the wild, coupled with emerging genetic and genomic techniques, have now positioned these mice as model organisms for the study of natural variation and adaptation. Recent work, combining field observations and laboratory experiments, has lead to exciting advances in a number of fields—from evolution and genetics, to physiology and neurobiology.The deer mouse (genus DOI:http://dx.doi.org/10.7554/eLife.06813.001 Peromyscus is a genus of small North American rodents known colloquially as deer mice  it is probable that a line, or several lines, could be drawn from Labrador to Alaska and thence to southern Mexico throughout which not a single square mile is not inhabited by some form of this species’  that would wander into his old Kansas City animation studio  do enter human households and partake of their larders. According to legend, Walt Disney drew inspiration for Mickey Mouse from the ‘tame field mice’  is greatest.Much of our understanding of Peromyscus genomes and revising their phylogenySequencing more Peromyscus system. An annotated genome assembly is currently available for P. maniculatus bairdii  and draft sequences are available for P. californicus, P. leucopus and P. polionotus . Several more Peromyscus genomes are being sequenced, but still more are needed.A comprehensive phylogeny based on genome-wide DNA sequences would greatly facilitate the comparative approaches that are the unique advantage of the Peromyscus can complement biomedical studies of other laboratory speciesIdentifying where Peromyscus could be used more widely in biomedical research than previously thought. Indeed, certain aspects of human biology—including aging, epigenetics, retinal development and hematology—have been suitably modeled in Peromyscus  stretches from below sea level in Death Valley to above 4300 meters in the adjacent White and Sierra Nevada mountain ranges  laboratory mouse  and average litter size is 4.6 pups . Such studies have been particularly fruitful for understanding the mechanistic basis of gene by environment interactions. For example, day length can reverse the behavioral action of the hormone estradiol by determining which estrogen receptor pathway is expressed and consequently activated  during development displayed reduced spatial learning and exploratory behavior—traits known to be associated with male–male competition for mates —that we can attribute natural variation in Peromyscus behavior to specific genetic variants. For instance, P. maniculatus and P. polionotus display considerable differences in stereotyped burrowing behavior. P. maniculatus digs short, simple burrows in contrast to the long, complex burrows constructed by P. polionotus that consist of an entrance tunnel, nest chamber and escape tunnel  living on the coastal sand dunes and barrier islands of Florida are considerably paler than their inland counterparts (P. p. subgriseus) that inhabit dark, loamy soils (Mc1r) coding region . These mice served as the founding strains for the Peromyscus Genetic Stock Center (PGSC) established in 1985 by Wallace Dawson at the University of South Carolina, which currently maintains wild-derived stocks of six species, as well as 13 coat-color mutants and four additional mutants on P. maniculatus genetic backgrounds. Additional wild-derived stocks are kept in individual laboratories (http://stkctr.biol.sc.edu) with more than 3000 citations.Francis Sumner, considered the grandfather of logy see , first dratories  and stil10.7554/eLife.06813.010Box 2.Peromyscus biologists (particularly Francis B Sumner) informed influential thinkers in population genetics and evolutionary biology, such as Sewall Wright, Theodosius Dobzhansky and JBS Haldane. Since most early 20th century geneticists came from experimentalist backgrounds, many turned to naturalists for data from wild populations (Peromyscus represented one of the few major studies of evolution in natural populations. As such, Wright closely followed Sumner's analysis of phenotypic intergradation between geographically contiguous P. maniculatus subspecies in California (The work of early ulations . At the lifornia . Wright lifornia . The quePeromyscus, which he then crossed in the laboratory (P. polionotus populations from coastal to inland Florida (Between 1914 and 1930, Sumner carefully measured several quantitative traits—most notably coat color—that varied among geographically distinct subspecies of boratory . Dobzhan Florida . From thPeromyscus also featured in Dobzhansky's studies of reproductive isolation. Certain P. maniculatus subspecies with overlapping geographic distributions are nevertheless separated by habitat, often with one subspecies inhabiting prairie, open fields or sandy lake beaches, and the other being exclusively forest-dwelling (Peromyscus has thus been a cornerstone of evolutionary biology for nearly a century. These and other studies drew the attention of biologists in many fields, launching the many, varied Peromyscus research programs we see today.dwelling . These sdwelling . PeromysDOI:http://dx.doi.org/10.7554/eLife.06813.010Peromyscus colonies are invariably susceptible to inbreeding depression, which necessitates their maintenance as relatively outbred stocks (While the genetic causes and phenotypic consequences differ among strains, d stocks . Thus, ad stocks . Thus, tPeromyscus, this effort was lead by Francis Sumner and continues today. In an era of high-throughput sequencing and expanding transgenic technologies, our concept of the genetic model organism is rapidly changing. We can now widen our focus to include the diverse and naturally evolving species that may further our understanding of life outside the laboratory. The emergence of Peromyscus as a model system has been largely driven by the wealth of natural history information available for the genus. Indeed, deer mice form the foundation of much of our understanding of the biology of small mammals. The multitude of ecological conditions to which deer mice have adapted has contributed to an impressive array of biological diversity within a single, ubiquitous genus. While this radiation is fascinating in its own right, Peromyscus is arguably foremost among nascent model systems that may aptly model the genetic complexity of the human condition, which too has long been shaped by natural selection in the wild. We hope that the continued development—primarily through the growth of genetic and genomic resources—of this model system will galvanize research in all corners of biology.The tradition of dissecting the genetic basis of ecologically relevant traits in the laboratory began in the early 20th century; in


More than half of the data collected reflect novel genetic variations that were first described in the Omani population, and most disorders with known mutations are inherited in an autosomal recessive fashion. A number of novel Mendelian disease genes have been discovered in Omani nationals, and the corresponding mutations are included here. The current study provides a comprehensive resource of the mutations in the Omani population published in scientific literature or reported through service provision that will be useful for genetic care in Oman and will be a starting point for variation databases as next-generation sequencing technologies are introduced into genetic medicine in Oman.The Sultanate of Oman is a rapidly developing Muslim country with well-organized government-funded health care services, and expanding medical genetic facilities. The preservation of tribal structures within the Omani population coupled with geographical isolation has produced unique patterns of rare mutations. In order to provide diagnosticians and researchers with access to an up-to-date resource that will assist them in their daily practice we collated and analyzed all of the Mendelian disease-associated mutations identified in the Omani population. By the 1 Oman is situated in the South East of the Arabian Peninsula along the East coast of the Arabian Gulf . It has Clinical genetic services were introduced in the Sultanate of Oman in the past decade and they have become an important component of health care. This greatly facilitated the systematic collection of data on genetic diseases and birth defects in the past few decades. With the inauguration of the National Genetic Center in 2013, the existing clinical genetic services were supplemented by sophisticated genetic laboratory services.4 and to link them to specific population groups and geographic locations6, analyze population structure7, and to estimate the impact of genetic disorders and birth defects on the community4 and summarize the genetic services available8. The advances in bioinformatics required to annotate human genomic variants and to place them in public data repositories have not kept pace with their discovery. The deposition of such data in the public domain is essential to maximize both their scientific and clinical utility9.The amount of published data available on genetic disorders in the Sultanate is considerable. There were a few previous attempts to list the genetic diseases reported in OmanHence, in the current study we present a comprehensive compilation of germline mutations in nuclear genes associated with human disease in the Omani population.The wealth of genetic variant data in Omani nationals was collected from multiple sources which form a basis for research into genetic conditions reported from Oman. Multiple sources of data were reviewed to form repository of mutations in Omani nationals introduced in this paper. The sources of data included:(1) 1993–2015 records of patients consulted by clinical geneticists of the Royal Hospital, the largest tertiary hospital in Oman;(2) 2008–2015 publications curated from PUBMED on birth defects and genetic conditions in Omani nationals. The keywords used were: “Oman”, “Genetic disorders”, “Birth defects”, “mutations”;(3) 2012–2014 commercial laboratories referral registry at the Royal Hospital for the samples tested overseas.HTTP://ogvd.net>>;(4) The internal genetic variant repository of the National Genetic Center <<The data presented in this article was manually curated. The OMIM identifiers, Phenotype MIM accession numbers, Phenotype name (OMIM), mutation descriptions, and relevant publications with PMID numbers were all collected from the NCBI database repository. All unavailable through PubMed mutation details were checked with ClinVar, LOVD and CentoMD. The details of unpublished mutations are not included in the present study and feature inIn this study, a wide range of genetic conditions with known mutations collected in Omani nationals were analysed. The disease classifications are comprised of 44 gene variants causing neurodevelopmental disorders, 21 inborn errors of metabolism, 13 endocrinopathies, 15 skeletal dysplasias, nine disorders of the immune system, four hereditary blood disorders as well as other National groups .In total more than 150 rare genetic disorders were listed inFor the majority (85%) of rare disorders presented in10.Soon after the completion of the Human Genome Project in 2003, it was clear that the genetic data collected until then presented only a glimpse of the complexity of the human genome and the significance of genetic variants in human disease. Since then, genetic researchers have unearthed innumerable variants that are not only individual-specific; but also ethnicity-, population- and country-specific. Human genetic variation databases have significant implications for both diagnostic and predictive medicine. Often, the pathogenicity of rare mutations is primarily assessed through multiple reports of occurrence in diseased patients that are documented and routinely updated in mutation databases. Given the fact that gene mutations and their frequencies in many Mendelian disorders differ widely between different ethnic groups, even within a country, national databases are highly valuable resources for studies on disease-gene associations, population diversity and genetic historyThe catalog of Omani mutations presented here will therefore represent a valuable resource that may guide mutation analysis in Omanis suspected of having genetic disease. Unique circumstances in Oman with government-funded comprehensive healthcare throughout the country, and the national coverage for clinical genetics has made the present study possible. Future efforts will be required to extend this database to cover the full spectrum of mutations and population specific variants.The disease-associated mutation data presented ,Table 3The interest in genetic testing is growing among physicians aiming to provide better medical care and genetic disease prevention. The data collected largely represent mutations of rare autosomal recessively inherited disorders in Oman. The mutation data in4, and the interests of individual clinicians in genetic testing.The number of collected mutations among different disease groups  reflect 11. The frequency of hemoglobin disorders in Oman is among the highest in the world, and may reflect natural selection due to advantage for survival, in the heterozygous state, against malaria. Around 10% of Omani nationals are carriers of the allele for sickle cell anemia, 2–3% carry an allele for Beta-thalassemia and 45% are carriers of an alpha-thalassemia allele15.The knowledge of the genetics of Hemoglobin disorders is among the best in Oman due to national preventive programs and research starting from the 1990s. It is not surprising that around a third of all mutations known in Omani population to date are in four genes causing Hemoglobin disorders ,Table 34. The development and use of national mutation data is of importance to Omani medical care because it not only allows the genetic burden of disease to be quantified, but also provides diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research9. National databases for genetic variants are also significant from the perspective of preventive healthcare. There is a significant correlation between the occurrence of rare genetic variants associated with Mendelian disease and the burden of morbidity from complex diseases within a population. Heterozygous carriers for recessive disease genes do not manifest the recessive disease but may be at risk of developing complex trait conditions with some similarity in phenotype. For example, heterozygote carriers of mutations in the ataxia telangiectasia gene locus are reportedly susceptible to breast cancer16, and heterozygote carriers of mutations in the glucocerebrosidase (GBA) gene causing Gaucher disease are at an increased risk for Parkinson disease18. Hence, the collection of genetic variant data in national databases will contribute significantly to the prevention of genetic diseases in the population and might greatly impact the management of complex trait diseases in the future. Genetic scientists and international consortiums studying human genetic variation are increasingly interested in dissecting the interplay between genetic makeup and environmental influences on the pattern of diseases worldwide. Current research is expected to create a foundation for the national data online for the benefit of Oman Healthcare.Genetic disorders causing disabilities and handicap are of great concern. These are different groups of rare disorders leading to intellectual disability or physical handicap requiring detailed clinical classification, genetic testing, research and preventive measures. The high prevalence of birth defects and genetic conditions in Omani communities causes social, psychological and financial difficultiesHTTP://ogvd.net; raw datasets are not available for Royal Hospital laboratory and clinical data, as the registry contains confidential information that could not be deidentified.This article was prepared to introduce the first Omani genetic variation database. This data is available online at Its useful to have region/country specific databases that present a better representation of the prevalence and incidence of region specific diseases and health issues. Putting such data in context of whats known globally shall help in better interpreting whats peculiar to local population and possibly raise some interesting questions on why so? For example, is it the genetic structure or something in the environment?Authors mention existence of tribal structures, consanguineous marriages etc in Omani society. Is it possible to delineate the effects of these factors on the reported mutations? For example, is this reported in the papers that the authors have collated data from and have the authors noted this aspect in their database?How does the mutation data reported here compare with mutation patterns or frequency seen in other populations? Do the authors provide information on this or link out to other similar resources from other countries? If a physician in Oman is looking up some mutation from this database and wants to know if this is something specific to Oman or is a more general mutation found in other populations too, would this information appear automatically in the database; or could they perform a manual search of the database?Do the authors want to comment about any data privacy issues that maybe associated with such a database, if any and to what extent?Are there any attributes on quality of data in the database? perhaps based on the technique used in the original paper or some other measure some quality metric can be assigned to the mutation information recorded in the database?It would be helpful if authors were able to illustrate out a case or two on how they expect a doctor in Oman to be able to use this database in a real clinical setting; to illustrate the usefulness of the database from a simple collection of data to something that can be used on a more regular basis by doctors in clinical setting.Some specific comments follow:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The paper is well-written and though it does not present any new findings, it can be used as a reference database for Omani mutations.There are a few suggestions for the authors:a. Standard HGVS nomenclature should be followed for all the mutations and the authors should preferably stick to one format i.e. either DNA or protein notation. There is no uniformity in the present nomenclature followed in Table 2.b. Number of patients in whom each mutation was found, should be indicated – this would give an idea about any preponderance of specific mutations in this population.c. Functional validation studies, if available, or at least the mutation prediction scores should be mentioned for the novel sequence variants identified, which will help create a comprehensive database of new likely-pathogenic variants. Again, if the number of patients in whom each of these novel variants were identified is mentioned, we will get an idea as to whether these novel variants were present in more than one case and we can get further proof of the pathogenicity of these mutations.d. It would also be interesting to see if there is any ethnic group-wise preponderance of genetic diseases or mutations in the various subsets/ tribes/ regional groups that constitute the Omani population.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Among the European countries, Italy counts the largest number of local goat breeds. Thanks to the recent availability of a medium-density SNP (single nucleotide polymorphism) chip for goat, the genetic diversity of Italian goat populations was characterized by genotyping samples from 14 Italian goat breeds that originate from different geographical areas with more than 50 000 SNPs evenly distributed on the genome.ST values between breeds ranging from 0.013 to 0.164 and 7.49 % of the total variance assigned to the between-breed level. Only 2.11 % of the variance explained the clustering of breeds into geographical groups .Analysis of the genotyping data revealed high levels of genetic polymorphism and an underlying North–south geographic pattern of genetic diversity that was highlighted by both the first dimension of the multi-dimensional scaling plot and the Neighbour network reconstruction. We observed a moderate and weak population structure in Northern and Central-Southern breeds, respectively, with pairwise FOur results indicate that the present-day genetic diversity of Italian goat populations was shaped by the combined effects of drift, presence or lack of gene flow and, to some extent, by the consequences of traditional management systems and recent demographic history. Our findings may constitute the starting point for the development of marker-assisted approaches, to better address future breeding and management policies in a species that is particularly relevant for the medium- and long-term sustainability of marginal regions.The online version of this article (doi:10.1186/s12711-015-0140-6) contains supplementary material, which is available to authorized users. According to archeozoological and genetic data, goats were domesticated some 10 000 years ago in the geographical region that spans from Eastern Anatolia to the Zagros Mountains in Northern Iran . After dIn modern European agriculture, the economic role of goats is mainly linked to the products of either high-yielding dairy breeds that developed in the central Alps  or of local stocks that were often improved by crossing with more productive dairy or meat breeds . In Europe, 96 % of the 2.8 millions of tons of goat products are dairy products and only 4 % are meat products (see FAOSTAT at faostat.fao.org).www.assonapa.it). Diversity in orography and climate together with historical factors and traditions, led to the development of a large variety of livestock populations, which were later standardized in modern breeds. Although, since the early 1950s the genetic diversity of farm species has suffered from steady erosion, an opposite trend is observed for goats. A number of local goat breeds still populate rural environments where harsh climate and pastures challenge the diffusion of more productive species and cosmopolite breeds. According to the FAO classification, 58.3 % of Italian goat breeds are considered not to be at risk, each with more than 1200 heads, but there is a declining trend. These breeds include three cosmopolite or exotic breeds , which represent 24 % of recorded Italian goat heads. Among the remaining 41.7 % of breeds classified as at risk, 11 are endangered (number of heads less than 1200 with a declining trend) including Orobica, Valdostana and Ciociara Grigia breeds, and four are in a critical status (number of heads less than 100) as for example the Di Teramo breed.Even if the main hotspots of the world goat diversity are probably in Africa and Asia, among European countries, Italy can be considered as a reservoir of genetic resources for the caprine species with 36 breeds recorded by the National Goat and Sheep Breeder Association (www.assonapa.com). These data from the national goat and sheep breeders association refer only to registered animals that, according to FAOSTAT, represent only 20 % of the goats reared in Italy.Italian goats are mainly reared in the Alps and in the Mediterranean environments that are typical of Southern Italy and of the islands. In 2013, the regions that counted the highest proportion of goats were Calabria , the two major islands Sardinia (18 %) and Sicily (10 %) in Southern Italy, and Lombardy (10 %) in the north  traditional farming, with an indoor system in the winter, natural grazing in the spring and autumn, and vertical transhumance in the summer; (2) intensive and semi-intensive indoor farming, with animals kept in flocks of medium to large size and reared under controlled feeding that includes hay and concentrate. The farming system for breeds in Central Italy is characterized by small sedentary herds that practice transhumance from spring to fall while that for breeds in Southern Italy is mainly characterized by small- to large-sized farms with either semi-sedentary farming based on natural pasture or free-ranging farming. In Sardinia, goat farming systems differ from the traditional ones and range from small infrastructures with low management costs to a semi-intensive system as for Maltese goats .Several molecular studies, which often included limited numbers of breeds and loci, have been carried out on local Italian goat breeds –7 in attRecently, the availability of SNP panels –17 allown = 24; Camosciata delle Alpi, CAM, n = 31; Orobica, ORO, n = 24; Saanen, SAA, n = 24; Valpassiria or Passeirer Gebirgziege, VPS, n = 24; Valdostana, VAL, n = 24; Ciociara Grigia, CGI, n = 19; Dell’Aspromonte, ASP, n = 24; Nicastrese, NIC, n = 25; Girgentana, GIR, n = 24; Argentata dell’Etna, ARG, n = 25; Maltese sampled in Sicily, MAL, n = 16; Di Teramo, TER, n = 23; Sarda, SAR, n = 32; Maltese sampled in Sardinia, SAM, n = 15 . The geographical origin of the breeds is shown in Fig. A total of 354 animals from 14 Italian goat breeds were sampled (Table Blood samples were collected according to the recommendations of the European Council (1986) concerning animal care. Whole blood was collected in Vacutainer tubes with K-EDTA as anticoagulant and stored at −20 °C until genomic DNA was extracted using a commercial kit  according to the manufacturer’s instructions.http://www.lgscr.it) and Porto Conte Ricerche s.r.l.  facilities. Raw signal intensities of the 53 347 SNPs were converted into genotype calls with GenomeStudio software v2011.1 and by using the SNP genomic locations and cluster files made available by the IGGC. GenABEL ver. 1.7-6 [DNA samples were genotyped using the GoatSNP50 BeadChip  developed by the International Goat Genome Consortium (IGGC) . SNP typE) and observed heterozygosities (HO) were calculated with an in-house script. Arlequin software ver. 3.5.1.3 [http://cmpg.unibe.ch/software/arlequin3) was used to (i) calculate population specific inbreeding coefficients (FIS); (ii) compute the FST [Data parsing, recoding and formatting were performed using PLINK ver. 1.07 suite . Expecte 3.5.1.3   at diffST distances was obtained with the software SplitsTree ver. 4.10 [A Neighbor-network graph based on between-breeds Fer. 4.10 . The R per. 4.10  was useder. 4.10  softwareAfter quality control: (i) 551 markers with a MAF less than 0.01 and (ii) 1660 SNPs and four animals with call rates less than 0.95 were excluded. The final working dataset included 350 animals and 51 136 SNPs of which only 0.46 % carried rare alleles (0.01 ≤ MAF ≤ 0.05).IS) of 0.074 (P < 0.05) revealed a slight excess of homozygotes. The pairwise FST values between breeds ranged from 0.013 to 0.164 [See Additional file ST values, while ARG, NIC, CGI and ASP breeds from Southern and Central Italy showed the lowest FST values.The percentage of within-breed polymorphic SNPs ranged from 95.13 % to 99.70 % , a large number of polymorphic SNPs were detected for the Italian breeds , which suggests that the impact of ascertainment bias is small in our dataset.Although the 50 K SNP panel was developed from sequence data for goat breeds such as Saanen, Alpine, Creole, Boer, Kacang, and Savanna  high levels of genetic polymorphism, (ii) a limited amount of inbreeding, (iii) a geographical pattern that underlies the distribution of genomic diversity, and (iv) a moderate and weak population structure in the Northern and Central-Southern breeds, respectively. The latter two results can also be influenced by traditional management practices, recent demographical events and adaptation to different climatic conditions. However, a more extensive sampling of Italian goat breeds that would cover more evenly the geographical range is necessary to assess the impact of these factors.The presence of a clear North–south geographical distribution of genetic diversity Fig.  along thProbably, isolation by geographical distance played a major role in shaping the differentiation of Italian goats, although it may have acted in synergy with other factors. Introgression from gene pools of animals that are native to foreign countries  may have also played a role, together with adaptation to local environmental conditions, as already suggested by a genome-wide analysis based on AFLP markers . The effE values were in line with those calculated for other breeds that are included in the SNP discovery panel [O and HE was not significant for any of the investigated breeds, with the sole exception of NIC for which a slightly positive FIS was found. It is likely that pedigree records and occasional DNA-based controls helped to design mating plans that enabled farmers to control inbreeding. The institution of National Registers and Herd Books  further contributed to preserve goat breeds from indiscriminate crossbreeding. This is particularly the case for breeds with distinctive phenotypic traits, such as horn shape in VAL and GIR, and horns and coat colours in the ORO breed.Although the sizes of the populations analyzed were small, Hry panel , 32. In K = 2, this breed is clearly assigned to one of the two gene pools . Nevertheless, an excess of TER heterozygotes was observed, which, together with the large number of rare SNPs identified for this breed and the wide area covered by TER individuals on the MDS plot, suggest that crossbreeding with bucks from other breeds occurred. However, since admixture analysis assigned TER individuals to a well-defined cluster at a K value as low as 3, which suggests a low level of admixture, if a recent episode of introgression occurred, it probably involved a donor gene pool from a breed that is not included in our dataset.Among the central Italian breeds investigated, TER had the smallest population size, with only 58 animals recorded by the National Breeders Association (www.assonapa.it). The distribution of individual points on the MDS plot and an HE of 0.402 indicate that there is a certain level of genetic variability within this breed. According to admixture analysis, this breed shares a common genomic background with three Southern Italian populations . Although these three breeds are bred in non-contiguous geographical areas - i.e., Southern Lazio for CGI, Calabria for NIC and ASP and Sicily for ARG –, their pairwise FST distances were the lowest, which can be partly explained by the impact of transhumance in Central and Southern Italy [CGI from Central Italy is also at risk of extinction; in 2008, when the national Register was created, this breed counted only 181 animals that were distributed across a few farms in the Lazio region. Thanks to local (Regolamento (European Council EC) 1698/2005 - Programma di Sviluppo Rurale 2007–2013) and EU economic support, after five years, the population size had increased by 3.7 folds and reached 674 animals in 2013  No 1698/2005 to support rural development that includes the presence of autochthonous breeds in danger of extinction; this is funded by the European Agricultural Fund for Rural Development (EAFRD). The absence of male rotational schemes between farms has probably caused inbreeding at the farm level (www.goatit.eu), which shows that conservation strategies that are based exclusively on remuneration of farmers are not efficient when proper breeding schemes and extension services are lacking [NIC is the only breed that showed a significant F lacking .Besides demographic factors, historical factors are likely to have an impact on the genetic makeup of Southern Italian and insular breeds. In fact, since the Neolithic age, the area that covers North Africa, Malta, Sicily, Sardinia, and Southern Italy has been deeply interconnected by routes of migration, trade and conquest. The tightly interconnected history of these areas was favoured by their role of crossroads of the trading and warring routes through the land-locked Mediterranean Sea .The differentiation between Maltese goats that were sampled in Sardinia and Sicily as revealed by admixture and Neighbour-network analyses, is probably the consequence of the combined effects of genetic drift, small population size, founder effects and reproductive isolation (since the Southern Tyrrhenian Sea acted as a strong genetic barrier). However, the two populations were not separated on the MDS plot based on a genomic kinship distance matrix. In fact, this measure may be showing that these two populations share a common origin since it can account for older evolutionary relationships and is less affected by recent population dynamics . Anyway,According to our results, the axis of the main source of genetic variation for Italian goat populations stretches along the longitudinal axis of the Italian peninsula. Among the major factors which could have acted on the goat genome in Italy, reproductive isolation due to geographical distance, adaptation to local conditions and breeders’ management may all have a role, although the identification of their relative impacts and contribution is not straightforward. To assess the importance of any of these factors, a larger and more comprehensive set of breeds is necessary, in particular to have a more uniform distribution of sampling locations. Nevertheless, some breeds displayed a clear genetic identity that confirms previous findings , 28, 35,A thorough genomic characterization of breeds represents a key point to develop efficient conservation strategies, which, to become effective, should also take into account population viability. This variable, in turns, depends on budgetary limitations, management practices and on the existence of services to support shepherds.Based on the use of a standardized genotyping array, such as the GoatSNP50 BeadChip, it will be possible to combine various datasets and to provide a global picture of goat genetic diversity both at a local and global scale. This will help to understand the origins of genetic diversity and to manage biodiversity of these animal genetic resources that are particularly relevant for poor and marginal rural areas of the world.


Pandion haliaetus) is one of only six bird species with an almost world-wide distribution. We aimed at clarifying its phylogeographic structure and elucidating its taxonomic status (as it is currently separated into four subspecies). We tested six biogeographical scenarios to explain how the species’ distribution and differentiation took place in the past and how such a specialized raptor was able to colonize most of the globe.The Osprey (b and ND2), the Osprey appeared structured into four genetic groups representing quasi non-overlapping geographical regions. The group Indo-Australasia corresponds to the cristatus ssp, as well as the group Europe-Africa to the haliaetus ssp. In the Americas, we found a single lineage for both carolinensis and ridgwayi ssp, whereas in north-east Asia (Siberia and Japan), we discovered a fourth new lineage. The four lineages are well differentiated, contrasting with the low genetic variability observed within each clade. Historical demographic reconstructions suggested that three of the four lineages experienced stable trends or slight demographic increases. Molecular dating estimates the initial split between lineages at about 1.16 Ma ago, in the Early Pleistocene.Using two mitochondrial genes  contains supplementary material, which is available to authorized users. The modern distribution of living organisms has been shaped by multiple processes that had profound effects on the dispersal, genetic structure and evolutionary histories of plant and animal populations. Movements of land-masses and successive multiple glacial events that occurred during the Pleistocene caused severe habitat changes which confined many species to warmer refugia and led other taxa to experience demographic reductions or complete extinction . FavouraArdea alba, the Cattle Egret Bubulcus ibis, the Glossy Ibis Plegadis falcinellus, the Barn Owl Tyto alba, the Peregrine Falcon Falco peregrinus and the Osprey Pandion haliaetus.Despite the high potential dispersive power of flying birds, it is striking that only a few taxa did colonize most of the world. Excluding seabirds, for which the distribution pattern depends more on ocean basins than on the major land-masses , only siHaliaeetus albicilla: [Falco peregrinus: [The Osprey is a medium-sized raptor with flexible breeding habitat requirements across its range. Despite its specialization as a piscivore, it is an opportunistic forager that can feed in both freshwater and marine environments . Northerbicilla: ; Falco pegrinus: ). Howeveegrinus:  and suchP. h. haliaetus  in the Palearctic from Europe, northwest Africa, and Asia north of the Himalayas, P. h. carolinensis  in North America, P. h. ridgwayi  in Caribbean Islands, and P. h. cristatus  in the Indo-Pacific and Oceania  to merib and ND2 genes) from samples covering the worldwide distribution of the Osprey. This exhaustive dataset allowed addressing specific questions. a) Does the Osprey show any phylogeographic structure in mitochondrial DNA at the continental level? b) How was such a specialized raptor able to colonize the entire world? We propose a hypothetical evolutionary scenario explaining how the species’ distribution and differentiation took place in the past. In the light of these new genetic clarifications, we discuss the potential implications for revisions of the taxonomy and for conservation  to desiSampling covered the whole species’ distributional range, with 204 individuals (114 fresh samples + 90 museum specimens) from 31 countries across five continents  were obtained from wild ospreys at the nest during ringing activities in different breeding sites. For each individual, about 0.5 ml of blood was taken by venepuncture from the wing and stored either on filter blotting papers or in 70 % ethanol in Eppendorf tubes. In some cases, growing body feathers containing traces of blood within the calamus were collected and stored in envelopes.Fresh samples  from museum study Osprey-skins following the procedure described by Mundy et al. . From eab (cyt b) and NADH dehydrogenase subunit 2 (ND2). For the cyt b, five sequences from the GenBank database were also included, leading to a total of 209 sequences , Tawny eagle (Aquila rapax), Black-breasted buzzard (Hamirostra melanosternon) and Gray-headed kite (Leptodon cayanensis). The choice of these outgroups was motivated because 1) both cyt b and ND2 sequences were available in GenBank  for the mitochondrial Cytochrome ank . The nuTotal genomic DNA was extracted from fresh and historical samples using a Qiagen DNeasy Tissue kit, following the manufacturer’s instructions . To avoid contamination with contemporary DNA , all extb and ND2 were amplified by PCR. Specific external and internal mitochondrial cyt b and ND2 primers were designed in this study for Pandion haliaetus  and maximum likelihood (ML). Bayesian analyses were performed with MrBayes 3.2  using thb on 146 samples.Relationships between haplotypes were also visualized as a minimum spanning network, using the Median-Joining (MJ) network algorithm implemented in the program network 4.1.1.0 . In ordep-distance and a pairwise deletion for the gaps/missing data treatment, as implemented in the Mega 5.10 software [We used Dnasp 5.10  to compusoftware .b dataset using the skyline plot method  separately with the cyt b partitioned according to codon position and using the HKY + G model as sequence evolution. The likelihood-ratio test performed with tree-puzzle 5.2 [p < 0.05). BSP analyses were thus conducted using a lognormal-relaxed molecular clock with a substitution rate of 0.01973 per lineage per million years as estimated by Nabholz et al. [b. Although this rate has been obtained for the third codon position we apply it because most substitutions (83.3 %) are located at this position in our dataset. Analyses were run for 50 million generations, sampled every 1000 generations, after discarding the first 10 % as burn-in. We used Tracer 1.5 to analyse the results and draw the BSPs.Demographic history of the haplogroups and the whole dataset was determined with different methods. Firstly, R2 , Fu’s Fsod . The whole alignment was partitioned according to the three codon positions using a HKY + G model of sequence evolution. As previously, analyses were performed using a lognormal-relaxed molecular clock using a substitution rate (ucld. mean) following a normal prior distribution of mean 0.01973 [Time of the most recent common ancestor (TMRCA) was estimated with Beast 1.8.0 based on 23 sequences (19 Osprey haplotypes and 4 outgroups) of the cytochrome  0.01973  and a stProbabilistic inference of ancestral range was performed using BioGeoBEARS  as impleb gene. We implemented a model including four broad geographic regions: America (A), Indo-Australasia (I), Asia (J) and Europe-Africa (E). The maximal number of areas that could be occupied by one terminal grouping  was set to two. Analyses were conducted using a non-time-stratified approach under default settings [As input tree, we used the ultrametric tree obtained from the MrBayes Fig.  analysissettings . These sb dataset , the best-fit scheme was a partitioning according to codon position with the models HKY + I for position 1 (367 nucleotides), TrN for position 2 (368) and K80 + G for position 3 (368). As the TrN substitution model was not available in MrBayes, the parameter Nst was set to 6. The partitioned ML analysis was performed with 1000 bootstrap replicates using a GTR + G substitution model for each codon position in RaxMl software. The average Bayesian posterior probabilities (pp) and bootstrap values (BP) for supported clades are shown on the tree in Fig. n = 209) is reported in supplementary information  returned 19 mtDNA haplotypes that were used to represent phylogenetic relationships  belong to two haplotypes which are scattered with other carolinensis samples  is composed of 3 haplotypes including 16 individuals retrieved from the Indo-Australasian area: 6 from various islands of Indonesia and 10 from Australia.A third group was composed of 2 haplotypes and 11 ospreys from Asia : 6 from the Pacific coast of Siberia  and five from Japan Fig. . In addiFinally, the largest clade  was formed by 102 individuals representing 9 haplotypes, mainly belonging to the Western Palearctic area, with a few exceptions. 93 of these samples were collected in Europe along a latitudinal gradient scattered from northern Europe (Fenno-Scandia and western Russia), central Europe , to southern localities in the Mediterranean area . Samples from North African coasts (e.g. Morocco) and Atlantic islands (e.g. Canary and Cape Verde) were included in this haplogroup, together with ospreys from the Red Sea and Persian Gulf areas. Three single haplotypes identified particular geographical regions  one p-distances within groups (Table p = 0.001 - 0.002), indicating limited genetic variability internal to each lineage. On the other hand, the greatest genetic differences between groups (Table p = 0.026), whereas lowest values were obtained between IND-AUS and ASIA (p = 0.015) and between IND-AUS and EUR-AFR (p = 0.017). At the same time, distance between AMER and IND-AUS was smaller (p = 0.020) than those between AMER and ASIA (p = 0.025).Despite the different sample sizes, mean genetic ps Table  were lowb phylogenetic tree , one for the cyt b-position 2 and ND2-position 3 (model HKY + I), one for the cyt b-position 3 and the ND2-position1 (model TrN) and one for the ND2-position 2 (model K81uf + G). As previously stated, the TrN model was approximated with Nst = 6 in MrBayes. The analysis carried out on the two genes  resulted in nine haplotypes differing by only one nucleotide change. Two out of the nine haplotypes were most frequent, and shared by the majority of the individuals . Despite remarkable differences in breeding and movement ecology, Osprey populations of lower latitudes within the EUR-AFR did not show notable haplotypic differences when compared to the northern and central European birds. The four samples from the Canary Islands shared a single haplotype  only three haplotypes were found, differing by only one nucleotide position. Five haplotypes were observed within the AMER group and 13 samples out of 17 showed the same haplotype, which was shared by ospreys from both western and eastern USA and from the Caribbean. Finally, within the ASIA group (n = 11) two haplotypes were recorded. The five samples from Japan were characterized by a single haplotype, which was shared with 2 samples from East Siberia.The cyt ork Fig.  confirmeype Fig. . Within Despite slight variation within each group (haplotypes were mainly distant by only one or two positions), a larger number of nucleotide differences were recorded between clades. The AMER group recorded the greatest genetic distance with EUR-AFR (a minimum of 15 nucleotides changed), whereas the minimum number of changes is 11 positions between IND-AUS and ASIA Fig. .b fragment. The haplotype diversity (H), nucleotide diversity (π) and other statistics were computed for the four recognized haplogroups and the whole dataset combined  because “j” was estimated to be greater than zero (j = 0.108) whereas the estimated rate of both dispersal and extinction was essentially zero ; the ancestor (AI) first diversified in the Indo-Australasian area by vicariance and then underwent a range expansion to occupy the Eastern Asia (J) and Western Palearctic (E).Among the six models tested in biogeographic inference, the ancestral area reconstruction method using the likelihood dispersal-vicariance analysis with the founder parameter (DIVAj) had a higher probability of being the best model for each of the three scenarios tested Table . With thOur study revealed that the Osprey is structured into four main genetic groups, representing quasi non-overlapping geographical regions. Each lineage, though including birds from distant geographic areas, showed low internal genetic variability as revealed by the very low values  is 1.2 %, which is higher than the values recorded for the Red kite Milvus milvus  and Hal3-9.8 %; ).Populations within each group were poorly differentiated, suggesting that they might have experienced a reduction of genetic variation. Such low values are usually related to populations that experienced demographic crashes or remained isolated in fragmented habitats , 39. Howth century. Then, despite declines during the 1960-70s, populations were able to recover, showing positive demographic trends in most areas [For the EUR-AFR clade, Bayesian skyline curves Fig.  suggest st areas . On the n = 102). We found that for 10 oldest museum specimens  despite a population bottleneck in the 20th century [Why then does each clade show such low genetic variability? Low levels of genetic variation can be the consequence of recent population declines, or represent an ancestral condition, inherited from an ancient evolutionary history . To brin century . In our b phylogenetic tree  than by the ND2 alone , patterns of colonization phases should be considered as tentative.The major result yielded by phylogeographic inferences is a split of Pandion  than those recorded between AMER and EUR-AFR (0.026). This result indicates that the EUR-AFR group did not originate directly from the AMER group, as also suggested by the lowest likelihood values yielded by the alternative biogeographical reconstruction  that are undistinguishable morphologically. This reflects the poor knowledge of the species in Asia where detailed information about biology and distribution are needed [The four genetic groups that we found do not entirely correspond to the four subspecies described, based on morphological characters , 13. Theristatus . Contrare needed . More saGypaetus barbatus, 2.9 % for the control region; [Haliaetus albicilla, 0.7 %). It might be objected that more variability would potentially have been found by including more geographic samples for the different clades. However, if we consider the EUR-AFR clade , the variability was as weak as for other groups that are represented by a much smaller number of birds (15 to 37). The decision for splitting ospreys into different species  should  Osprey; .n = 15) from only a few areas; 3) the majority of the samples analysed were museums specimens, so the current presence of this lineage in East Russia and Indonesia needs to be confirmed; 4) it is the only one clade showing signals of demographic decline; and 5) no clear morphological characteristics can presently help identification.A first step towards a sound global management and conservation plan is to define Management Units (MUs) and Evolutionary Significant Units . As a mOur study revealed that, at the global scale, the particular evolutionary history of the Osprey has partitioned the species into 4 distinct clades with clear genetic differentiation. In addition, our findings indicate that broad habitat requirements and high mobility of Osprey were more important factors than philopatry in shaping genetic diversity at the intra-clade level. Further genetic study using microsatellite markers are ongoing  in order to reveal more recent differences and to quantify gene flow among populations that show differences in migratory or reproductive behaviours .Even though the Osprey is currently globally listed as “Least Concern” according to IUCN criterions , it is cA specific ethical committee was not required for this study. Bird handling was performed under animal experimentation permits 34–369 (David Grémillet) delivered by the French ‘Direction Départementale de la Protection des Populations’ and under the licence of Olivier Duriez from the Centre for Bird Population Studies (CRBPO) of the Natural History Museum : according to the French law of 22 September 2008, the CRBPO has the delegation by the Ministry of Ecology, Energy, Sustainable Development and Land Settlement for allowing the owners of a general bird ringing licence to capture and handle birds from protected species, and collect samples or mark them (with rings or any other device like GPS units).The data set supporting the results of this article is included within the article and its additional files “Additional files