Camellia taliensis harbors valuable gene resources for tea tree improvement in the future. The knowledge of genetic variation and population structure may provide insights into evolutionary history and germplasm conservation of the species.As one of the most important but seriously endangered wild relatives of the cultivated tea, C. taliensis by using the nuclear PAL gene fragment and chloroplast rpl32-trnL intergenic spacer. Levels of haplotype diversity and nucleotide diversity detected at rpl32-trnL  were almost as high as at PAL . Significant chloroplast DNA population subdivision was detected , suggesting fairly high genetic differentiation and low levels of recurrent gene flow through seeds among populations. Nested clade phylogeographic analysis of chlorotypes suggests that population genetic structure in C. taliensis has been affected by habitat fragmentation in the past. However, the detection of a moderate nrDNA population subdivision  provided the evidence of efficient pollen-mediated gene flow among populations and significant phylogeographical structure . The analysis of PAL haplotypes indicates that phylogeographical pattern of nrDNA haplotypes might be caused by restricted gene flow with isolation by distance, which was also supported by Mantel’s test of nrDNA haplotypes . We found that chlorotype C1 was fixed in seven populations of Lancang River Region, implying that the Lancang River might have provided a corridor for the long-distance dispersal of the species.Here, we sampled 21 natural populations from the species' range in China and performed the phylogeography of C. taliensis showed fairly high genetic differentiation resulting from restricted gene flow and habitat fragmentation. This phylogeographical study gives us deep insights into population structure of the species and conservation strategies for germplasm sampling and developing in situ conservation of natural populations.We found that  Crop wild relatives, which include wild progenitors of cultivated plants as well as other closely related species, are important components of natural habitats and agroecosystem . They maPhylogeographical studies have been emerging as a powerful tool for understanding population structure and evolution of plant species ,5. By syCamellia is composed of over 110 taxa , a measure of the extent of gene flow under an island model at equilibrium [The Mantel test implemented in the program Arlequin 3.1  was applilibrium . StatistAMOVA: Analysis of molecular variance; NCPA: Nested clade phylogeographic analysis; PAL: Phenylalanine ammonia-lyase; SNP: Single nucleotide polymorphisms.YL collected population samples, generated experimental data, performed the whole data analyses, and drafted the earlier versions of the manuscript. SXY and PZJ involved the field sample collection. LZG designed the study, partially analyzed the data, wrote and revised the manuscript. All of the authors have read and approved the final manuscript.Table S1.  Chain of inference from the nested clade analysis of the chlorotype data in C. taliensis using Templeton’s (2004) inference key.Click here for fileTable S2. Chain of inference from the nested clade analysis of the PAL haplotype data in C. taliensis using Templeton’s (2004) inference key.Click here for file


ELISA (Echelle Liégeoise d'Index de Sévérité à l'Admission) is a valid new Emergency Department (ED) triage algorithm including five levels of categorisation , based on vital signs and selected anamnestic data. Previous work has demonstrated that ELISA evidenced a strong inter-rater and intra-rater agreement [From March 2008 to May 2008, we prospectively investigated 545 consecutive admissions to study the potential correlation between ELISA score and impact on resource consumption as well as ED stay. Resources were classified following three categories: complementary examinations , medical treatments , and outcome after ED admission . Each resource was considered a binary variable and was analyzed owing to four statistical tests: chi-square, Wald Wolfowitz, Kolmogorov- Smirnov and Mann-Whitney.Statistical analysis evidenced an effect of ELISA score on the overall need for complementary examinations except for serology, X-rays and Holter ECG. The initial index severity had also related the need for urgent treatments. Outcomes were also significantly correlated with ELISA: the smaller the index, the bigger the number of hospitalisations, ICU admissions and deaths. This study demonstrates ELISA's efficiency; when the initial severity index is close to U1, more complementary examinations are needed and more medical treatments are necessary. Wounds do not have a high emergency degree, which explains why there was no influence of initial index severity on the realisation of sutures. The same reasoning is applied to X-rays that are frequently requested for light traumatic cases with low emergency degree. Finally, hospitalisation, ICU admission and death are more frequent when the ELISA score evidences the highest emergency degree.In addition to previous work demonstrating a strong inter-rater and intra-rater agreement, the present study points out the potent efficiency of ELISA, allowing its further use in the ED.


Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.The distinctness of, and overlap between, pea genotypes held in several The online version of this article (doi:10.1007/s00122-012-1839-1) contains supplementary material, which is available to authorized users. Pisum has received considerable attention  for DNA extraction by the Qiagen DNeasy 96 method.27 RBIP markers were selected from an original set of 45 , each accession has a value of Qrp corresponding to the presumed contribution of each proposed progenitor population (p). The correlation between pairs of runs was calculated using Genstat v13  in DARwin5  + cos(θc)cos(θi)sin2(b/2)]. The values a and b are given as: a = θc − θi and b = φc − φi; all angles were in radians, where θ and φ are latitude and longitude, θc is the latitude of the reference point and θi is the latitude of the sample.Latitude and longitude data were plotted in Google Earth using .kml files (Supplementary Tables 2a and 2b) with the image simplified using Photoshop CS3. Great circle distances were calculated from 35°47′N 72°36′E using the relationship: http://bioinf.scri.ac.uk/germinate_pea/app/. These include the 27 markers used in this study. Corresponding data for the 1,518 extra lines scored with these 27 RBIP markers have been stored in the same database.The original RBIP data of 45 RBIP markers derived from Jing et al. Q corresponding to the STRUCTURE sub-groups of Jing et al. . DNAs from these samples were scored for 27 RBIP markers that were a subset of those 45 previously investigated . The frequency distribution of missing marker scores in the data set is shown in Supplementary Figure 3.A new sample set was assembled from major European g et al. . In totaPisum accessions previously scored, one (D45) with the set of 45 markers and the other (D27) with the set of 27 markers. These two values were strongly correlated .To test correspondence between the old and new data sets, two measures of pairwise genetic distance were made for 394 K, the number of proposed ancestral populations. The value K = 2 partitioned the data set robustly, roughly separating group 2 of Jing et al. . Three of these classes, comprising half of the runs were more closely related to each other than the others and these were taken for further analysis .At Q for the corresponding groups are: QG1, QB 0.32, QG2, QR 0.34 and QG3, QG 0.95, where G1, G2 and G3 are the main STRUCTURE groups of Jing et al. . There were also weaker but significant correspondences between the two other groups (QB and QR) and sub-groups of these analyses  is noticeably distinct and seems to correspond mainly to the components of QR in the new analysis and for high assignments to QR , identified in the present analysis, was examined further by repeating the STRUCTURE analysis on this subset of accessions, using the K = 6 value derived for group 3 by Jing et al.  and a multifactorial analysis (MFA) performed on it “”. The reP. abyssinicum  and one (POZP12) is assigned to P. elatius.A small number of accessions are peripherally located Fig. f. Those The outlying accessions at the lower part of the MFA Fig. f are froP. sativum, and the peripheral germplasm we have analysed mainly derives from the UK (JIC), Dutch (NLD), French (FRA) and Polish (POL) collections. The Dutch accessions sampled , but when this is plotted against distance  no clear relationship between genetic and physical distances is seen.If the accessions marked in green represented a selection from pea genotypes in the Indian sub-continent, then we would expect a measure of genetic distinctness correlated with the distance. The first component of the multifactorial plot clearly separates these accessions Fig. a, x axisnce Fig. c, y axisQ values (Supplementary Table 1). This should correspond to 138 accessions, but the number was 134 because some individuals were selected from both a group and a sub-group. These were augmented with the 7 outliers in the MFA plot discussed above, to maximise the represented diversity, giving 141 accessions. The distributions of these accessions in the STRUCTURE and MFA plots are shown in Fig. Two approaches were taken to identify subsets of accessions that represent the genetic diversity present in the germplasm studied here Fig. . The firhttp://corehunter.org), which identifies subsets of representative accessions on the basis of maximising average genetic distance , reflecting the robust assignment of accessions to the ‘wild’ material of STRUCTURE group 3 and the less reliable assignment for the cultivated germplasm which has lower intrinsic genetic diversity. Although the assignment of accessions to sub-groups within STRUCTURE group 1 is not robust . The pattern of relatedness among these accessions suggests three possibilities: (1) these accessions may represent novel genetic variation within P. abyssinicum, (2) these accessions may be admixed P. abyssinicum, or (3) these accessions may be misclassified. Whichever of these is the case, the accessions are worthy of further analysis and have been included in our representative sets of accessions . We conclude that these accessions represent distinct germplasm and note that samples of these are included in all of the approaches we have taken to identify a representative set of accessions  in our previous analysis and splits into two classes 3A and 3C in the new data  identified to be most distinct from cultivated types by Jing et al.  of markers for the analysis of a relatively large number of accessions  and compared that to the analysis of smaller subsets  with larger numbers of markers  noting that the relationships between accessions were broadly similar. The 45 marker set was noticeably easier to analyse than the 27 marker data set suggesting that something of the order of 50 markers would be sufficient for the analysis of ca. 5000 accessions. Analysis of the data set with the complementary model building (STRUCTURE) and analytical (MFA) approaches was helpful in attracting attention to specific subsets of the data. The marker type we employed (retrotransposon insertion sites) is useful because the insertions are not reversible and occur at a relatively low rate Supplementary material 2 (XLS 2276 kb)Supplementary material 3 (KML 10 kb)Supplementary material 4 (KML 6 kb)Below is the link to the electronic supplementary material.


Desmognathus fuscus) was once common in the New York City metropolitan area, but has substantially declined throughout the region in recent decades. We used five tetranucleotide microsatellite loci to examine population differentiation, genetic variation, and bottlenecks among five remnant urban populations of dusky salamanders in NYC. These genetic measures provide information on isolation, prevalence of inbreeding, long-term prospects for population persistence, and potential for evolutionary responses to future environmental change. All populations were genetically differentiated from each other, and the most isolated populations in Manhattan have maintained very little genetic variation (i.e. <20% heterozygosity). A majority of the populations also exhibited evidence of genetic bottlenecks. These findings contrast with published estimates of high genetic variation within and lack of structure between populations of other desmognathine salamanders sampled over similar or larger spatial scales. Declines in genetic variation likely resulted from population extirpations and the degradation of stream and terrestrial paths for dispersal in NYC. Loss of genetic variability in populations isolated by human development may be an underappreciated cause and/or consequence of the decline of this species in urbanized areas of the northeast USA.Urbanization is a major cause of amphibian decline. Stream-dwelling plethodontid salamanders are particularly susceptible to urbanization due to declining water quality and hydrological changes, but few studies have examined these taxa in cities. The northern dusky salamander ( Urbanization has emerged as a substantial cause of the decline of amphibian species . IncreasDesmognathus fuscus (D. auriculatus) are inversely correlated with the degree of urbanization  in 2005 based on a note in the herpetological literature  will be included in a future landscape genomics study of urban salamanders (see Discussion).Sampling was conducted in 2010–2011 at five sites known to harbor dusky salamanders : two seeterature . These thttp://www.fws.gov/ventura/species_information/protocols_guidelines/docs/DAFTA.pdf) and the NY State Department of Environmental Conservation’s “Bio-safety Protocols for Reptile and Amphibian Sampling”. Five previously described microsatellite loci were PCR-amplified in 15 µl volumes using Promega PCR master mix and published thermal cycling profiles. PCR included one primer with a CAG or M13R tail and an associated probe with fluorescent WellRED D2, D3, or D4 dye. The total reaction included 7.5 µl master mix, 4.4 µl water, 0.8 µl of the 10 µM untailed primer, and 0.4 µl each of the 10 µM tailed primer and 10 µM fluorescent probe. The amplified loci included Dau3, Dau11, and Dau12 from DOI 10.5061/dryad.q1nc0).We captured salamanders by hand or dipnet after turning over cover objects such as logs, rocks, bottles, and metal/plastic sheeting in or near streams. Tail tips were clipped and stored in 80% ethanol until DNA extraction using the standard protocol for the Qiagen DNEasy tissue kit. All animal handling protocols were approved by the Natural Resources Group of the NYC Department of Parks and Recreation, and followed the recommendations of the Declining Amphibian Task Force’s “Fieldwork Code of Practice” (Each locus was tested for deviations from Hardy–Weinberg (HWE) and linkage equilibrium over the total sample of 141 genotypes and within each of the five sampling sites using GENEPOP 4.0 . We alsoFST between all site pairs using 1,000 random permutations in GenAlex to assess significance. We then used the evolutionary clustering method implemented in STRUCTURE 2.3 to place individual genotypes in clusters that minimized deviations from Hardy–Weinberg and linkage equilibria , and the ΔK method from K. We also used the ‘spatial clustering of groups’ module in BAPS 5.2 to identify the best value of K using predefined sampling sites and spatial coordinates as prior information , but considerably lower for both north (HO = 0.14) and south (HO = 0.29) Highbridge Park in Manhattan , was calculated for K = 6, and the value of ΔK was highest at K = 2. The bar plot for K = 6 indicates substantial admixture between the two Highbridge sites but unique evolutionary clusters present in the three other Staten Island/NJ sites  and further genetic analysis should be conducted on potential source populations before any reintroductions are attempted on Staten Island, as pairwise FST values were relatively high even between populations on the island. The recent discovery of a new species of leopard frog on Staten Island also raises the possibility of previously underappreciated phylogeographic structure or centers of endemism for amphibians in the NYC metropolitan area  genotypes.The only other published study of urban plethodontid genetics found much lower pairwise roadways . P. cineentation . The norFST values than species in more threatened categories  sampled from 14 urban parks in NYC showed a high degree of genetic differentiation between parks, but the FST values were on the lower order of that estimated between the two Manhattan D. fuscus populations in Highbridge park (D. fuscus in NYC (HO = 0.63–0.82), and vegetated urban corridors do allow for weak to moderate gene flow (FST values were in line with those reported for white-footed mice rather than dusky salamanders in NYC (Urban populations of other small vertebrates with limited dispersal ability exhibit significant population structure, but not to the same degree as urban dge park . White-fene flow . Urban ps in NYC . Other ss in NYC .D. fuscus have large genomes (D. fuscus and the northern two-lined salamander, Eurycea bislineata. We will then use landscape genomic approaches to model and compare connectivity between urban and rural streamscapes and landscapes. We will also use new statistical approaches to estimating population history from genetic data (NYC’s dusky salamanders exhibit unusually low genetic variability and substantial genetic structure compared to desmognathines and other species in urban environments. However, this study could not determine the time since population divergence or bottlenecks to rule out the possibility that these genetic phenomena occurred before urbanization of the NYC area. Many salamanders including  genomes , and mor genomes  for hundtic data  to examitic data  and seletic data . SelectiD. fuscus have undoubtedly declined throughout their range in eastern North America where urbanization dominates the landscape (Amphibian responses to urbanization are generally negative , and ourandscape . Loss of


Cardiocrinum giganteum var. yunnanense, an important economic plant in China, were developed with a fast isolation protocol by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphism within each locus was assessed in 24 wild individuals from Gaoligong Mountains in western Yunnan Province, China. The number of alleles per locus ranged from 2 to 4 with a mean of 2.9. The expected and observed levels of heterozygosity ranged from 0.042 to 0.726 and from 0.000 to 1.000, with averages of 0.44 and 0.31, respectively. These polymorphic microsatellite markers should prove useful in population genetics studies and assessments of genetic variation to develop conservation and management strategies for this species.Twenty-one microsatellite markers from the genome of  Cardiocrinum (Endlicher) Lindley is a member of the lily family (Liliaceae) and contains three species and one variety, namely Cardiocrinum cathayanum (E. H. Wilson) Stearn, C. cordatum (Thunb.) Makino, C. giganteum  Makino, and C. giganteum var. yunnanense (Leichtlin ex Elwes) Stearn . PCR amplifications were conducted under the following program: 95 °C for 3 min followed by 30–36 cycles at 94 °C for 30 s, with the annealing temperature optimized for each specific primer , and expected (HE) and observed levels of heterozygosity. We also estimated deviations from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) between pairs of loci using Chi-square tests.Standard genetic diversity parameters of polymorphic loci, e.g., the number of alleles (Cardiocrinum giganteum var. yunnanense. They should prove useful for further investigating the spatial genetic structure, genetic diversity, and levels of gene flow within and among populations of this species, which will help to develop viable strategies for the conservation and management of this important economic plant.The 21 microsatellite markers developed in this study are the first set of such markers for


Chromis margaritifer among the unique and remote atolls in the eastern Indian Ocean. A subtle, but significant genetic discontinuity at 10 microsatellite DNA loci was detected between atoll systems corresponding with a low (≤ 1%) probability of advection across the hundreds of kilometers of open ocean that separates them. Thus, although genetic connections between systems are likely maintained by occasional long-distance dispersal of C. margaritifer larvae, ecological population connectivity at this spatial scale appears to be restricted. Further, within one of these atoll systems, significant spatial differentiation among samples was accompanied by a lack of temporal pairwise differentiation between recruit and adult samples, indicating that restrictions to connectivity also occur at a local scale (tens of kilometers). In contrast, a signal of panmixia was detected at the other atoll system studied. Lastly, greater relatedness and reduced genetic diversity within recruit samples was associated with relatively large differences among them, indicating the presence of sweepstakes reproduction whereby a small proportion of adults contributes to recruitment in the next generation. These results are congruent with earlier work on hard corals, suggesting that local production of larvae drives population replenishment in these atoll systems for a range of coral reef species.We utilized a spatial and temporal analyses of genetic structure, supplemented with ecological and oceanographic analysis, to assess patterns of population connectivity in a coral reef fish  A pelagic larval phase provides the opportunity for demersal marine organisms to disperse long distances by oceanic currents, and thus, contribute demographically and genetically to distant populations. However, how far and in what numbers larvae actually disperse away from their natal area and then survive to reproduce successfully depends on a wide range of interdependent biological and physical processes. Recent evidence suggests that such processes may restrict patterns of realized (or reproductive) population connectivity to smaller scales than those suggested by maximum dispersal potential  current originates near Indonesia and flows polewards along the continental shelf margin, while in the austral spring and summer, seasonal south-west winds induce a weak reversal of the current to the north-east  inhabiting the offshore atolls of north-west Australia. Our primary data are based on a spatial analysis of microsatellite DNA variation among samples of adults collected from the Rowley Shoals and Scott Reef , occupying a variety of reef habitats, including inshore and offshore slopes and lagoons, from the central Pacific Ocean to the east Indian Ocean  following recruitment (pers. obs.). Therefore, migration among geographically separate populations is solely through dispersal of their planktonic larvae. Given that this species has a mean PLD of thirty or thirty-three days depending on location (C. margaritifer is a short-lived (ca. one year) damselfish species .an Ocean . Like molocation , togethelocation . Counts C. margaritifer adults were collected by divers using a combination of hand nets, barrier nets, and clove oil from six sites at Rowley Shoals (n= 276) and seven sites at Scott Reef  were collected from three sites at Scott Reef  and one site at Rowley Shoals (RS3), with average samples sizes of 25 individuals per site  selected from three sites randomly spread across the sampling area.DNA for genotyping was extracted with the high-throughput membrane-based DNA extraction protocol of NA), the unbiased expected heterozygosity (HE), the fixation index (FIS) at each of 10 microsatellite loci, and the number of private alleles at each site averaged across loci are presented in FIS and significance levels were based on 1000 permutations of alleles among individuals within sites and were adjusted with sequential Bonferroni correction for multiple tests when P < 0.05. Micro-Checker v2.2 , among sites relative to variation within each atoll systems (FSR), and among sites relative to overall variation (FST). Additionally, we calculated the variation partitioned among sites at Rowley Shoals and Scott Reef system relative to the variation within that particular system (FSR Rowleys and FSR Scott). To account not only for the high degree of variation within populations of microsatellite markers, but also for the effects that potential differences in effective population sizes might have on subdivision, we also calculated a standardized measure of all the F-statistics  according to the method of DS  and DS estimates are given in To infer the strength of genetic connectivity across a hierarchy of spatial scales among the atolls of north-west Australia, we measured the amount of genetic variation that was geographically structured among adult samples with an analysis of molecular variance (AMOVA) framework in GenAlEx v6.3 . We partod of DS  and FST lEx v6.3 . DS perfistances , and proSR), the degree of relatedness within samples  suggested differences in intensity of sweepstakes reproduction at each system, we tested also differences in allelic richness (RS) and heterozygote deficiency  with the exact tests implemented Genepop v4.0 . Furthermore, to test explicitly for the effects sweepstakes reproduction, we calculated allelic richness  of fish collected for the genetic analyses from Rowley Shoals (n= 98) and Scott Reef (n= 125) to provide relevant dispersion times for the oceanographic modeling. Additionally, we tested whether differences in PLD were associated with differences in genetic structure (see results) at these two atoll systems. The sagittal otoliths were extracted and cleaned, and one from each fish was weighed, mounted on a glass slide, and ground following the method of Because PLD has a pivotal influence on the ions see , and hasC. margaritifer with underwater visual census to infer potential reproductive output and assess whether location-specific densities are associated with patterns of genetic structure. Permanent transects were established at 9-m depth and censused five times at Rowley Shoals (three locations), and 12 times at Scott Reef (six locations), between 1994 and 2008  for the adults  and not within systems , variation among sites within Scott Reef increased when FST was standardized (F′ST Scott= 0.002), but remained zero at the Rowley Shoals. The exact test provided statistical support to these F′ statistics, with significant differences detected among sites at Scott Reef (P < 0.001) but not at Rowley Shoals (P= 0.204).The genetic structure of DS; FST plot, sites from each system tended also to occur on either side of the first axis. Moreover, this PCoA also visually supported the greater local differentiation of genetic structure at Scott reef systems compared with the Rowley Shoals detected by the AMOVA and exact tests, with a much broader spread of sites from Scott Reef contrasting to the tighter clustering of sites from the Rowley Shoals. Finally, the differences in spatial subdivision of adult samples at each atoll system were associated with increased heterozygote deficiency at the Scott Reef system , but no differences in allelic richness .The genetic discontinuity between samples of Rowley Shoals and Scott Reef adults detected by the AMOVA was illustrated in PCoA plots of Nei's standard genetic distance DS; . Sites fC. margaritifer recruits were generally consistent with expectations of sweepstakes reproduction  for the DS plot. However, contrary to expectations of sweepstakes reproduction, recruit samples had a significantly greater proportion of heterozygote deficits , and ranged from twenty-six to forty-two days  than at Scott Reef (35 fish 250 m–2± 9).The mean (±95% CI) PLD of recently recruited two days . There wtwo days . The meaThere was a clear reversal of oceanic currents from predominantly north-east in austral summer . The oceanographic model indicated that the potential for dispersal between the three major systems of the region  over a two-month period was low (≤ 1%) during most years, but that during one of the six years there was a 5% probability that propagules from Scott Reef were transported to within 30 kilometers of Rowley Shoals (C. margaritifer reproduces throughout the year (J. Underwood and M. Travers unpubl. data) and is highly abundant at these offshore atolls, a large number of larvae are likely to be produced across a range of biophysical conditions, and when such oceanographic conditions combine with high reproductive output, occasional dispersal between the Rowley Shoals and the Scott Reef systems appears to be possible. Considering that potential source populations of this damselfish do not exist on the mainland, it therefore appears that a PLD of one to two months, the ability of larvae to locate suitable habitat, and physical connectivity via oceanic currents, has enabled larval exchange over multigenerational time scales to prevent major genetic divergence between these atoll systems.Patterns of genetic structure of y Shoals . Given tC. margaritifer and high mutation rate of microsatellite markers, the genetic signal of potential historical connections is likely to have eroded over the thousands of generations since quaternary sea level and environmental changes  combined with estimates of suitable habitat area (ca 300 km2 at both systems) suggest that population sizes are in the tens of millions on these atolls. Thus, even considering that effective population size (Ne) is likely to be several orders of magnitude smaller than census size (Ne means that a migration rate (m) sufficient to maintain this levels differentiation detected here is far lower than the 10% that is probably necessary for demographically important exchange , and thus larval dispersal is likely to be restricted relative to the simple oval-shaped reefs of the Rowley Shoals  inhabiting hydro-geographically uncomplicated systems is likely to buffer populations against severe and large-scale disturbance events, and in such cases, replicate protected areas at the reef scale (∼30 km) are likely to be adequate. In contrast, for species whose larvae recruit more locally  or in systems where connectivity is restricted due to hydrodynamics , protected areas may need to be replicated over smaller distances (∼10 km) if they are to maintain connections among them and thus enhance resilience to disturbances. If so, then a suitable size and spacing of protected areas for a range of species may reflect the lower end of dispersal distances (10–20 km). General rules of thumb are emerging that are congruent with these conclusions , but rec


Petaurus norfolcensis) provides an excellent case study to examine genetic  connectivity among populations. It has an extensive range that occurs in a wide band along the east coast. However, its forest and woodland habitat has become greatly reduced in area and is severely fragmented within the southern inland part of the species' range, where it is recognised as threatened. Within central and northern coastal regions, habitat is much more intact and we thus hypothesise that genetic connectivity will be greater in this region than in the south. To test this we employed microsatellite analysis in a molecular population biology approach. Most sampling locations in the highly modified south showed signatures of genetic isolation. In contrast, a high level of genetic connectivity was inferred among most sampled populations in the more intact habitat of the coastal region, with samples collected 1400 km apart having similar genetic cluster membership. Nonetheless, some coastal populations associated with urbanisation and agriculture are genetically isolated, suggesting the historic pattern observed in the south is emerging on the coast. Our study demonstrates that massive landscape changes following European settlement have had substantial impacts on levels of connectivity among squirrel glider populations, as predicted on the basis of the species' ecology. This suggests that landscape planning and management in the south should be focused on restoring habitat connectivity where feasible, while along the coast, existing habitat connectivity must be maintained and recent losses restored. Molecular population biology approaches provide a ready means for identifying fragmentation effects on a species at multiple scales. Such studies are required to examine the generality of our findings for other tree-dependent species.Forests and woodlands are under continuing pressure from urban and agricultural development. Tree-dependent mammals that rarely venture to the ground are likely to be highly sensitive to forest fragmentation. The Australian squirrel glider ( Loss and fragmentation of habitat are recognised worldwide as substantial threats to biodiversity Population genetic approaches provide a direct way of determining functional connectivity for a given species at the landscape scale. This is because the degree of genetic similarity among populations is determined largely by gene flow, which in turn reflects success in both dispersal among habitat remnants, and breeding Among the most heavily human-impacted ecosystems worldwide are forests and woodlands, which are not only exploited for timber, but are cleared for agriculture because they typically occur on high fertility soils Petaurus norfolcensis) typifies this pattern, being listed as endangered in the south of its geographic range Some hollow-using species with wide latitudinal ranges in eastern Australia are listed as threatened in only part of their range, promoting some indifference to a range-wide concern about their conservation. The squirrel glider , after Bonferroni correction for multiple tests (Rice 1989): Pet9 in the Cape Hillsborough and Karawatha samples. This locus also showed heterozygous deficits in two other coastal populations based on pre-correction probability scores, suggesting the possible presence of null alleles at this locus in the coastal region. There was no indication of concerted linkage disequilibrium for any locus pair, and of 130 population/locus pair tests none were significant after correction for multiple tests.FST values .All pairwise T values  differed59–0.119 . The popl region . The popr region . OverallThe lowest level of genetic diversity was seen in the western Victorian population of Deep Lead . The ThuKnowledge of functional (genetic) connectivity among populations is essential for a complete understanding of the threats posed to biodiversity by human activities. This is particularly the case for species with broad geographic ranges because threats may vary geographically or may be obscured by geographic barriers or historical events e.g. . Our anaFST values were significantly higher overall among southern compared to coastal populations (with the exception of Padaminka). In addition, populations identified as ‘isolates’ by STRUCTURE showed significantly elevated levels of pairwise individual relatedness, consistent with them experiencing little or no immigration. The extensive clearing for grazing (more than a century earlier) of the species' preferred habitat in the south has meant that habitat is largely restricted to linear strips of remnant vegetation associated with roads, travelling stock reserves and waterways Antechinus flavipes) Squirrel glider populations in the inland woodlands of Victoria and southern NSW typically showed high levels of genetic differentiation, even over small distances (e.g. ∼25 km between Murraguldrie and Mate's Gully). This general pattern was indicated by the STRUCTURE analysis, and the fact that Phascolarctos cinereus) populations in urbanised areas of southeast Queensland has also recently been demonstrated Two patterns were observed in the coastal range. The first was that samples from central NSW and central Queensland, separated by at least 1400 km, had similar genetic cluster membership, and the second, that two sample locations showed a high level of genetic isolation. The large extent of forest and woodland cover persisting in the coastal part of the species' range appears to have facilitated broad-scale connectivity, at least until recently: the extent to which the genetic signatures examined here will lag behind cessation of gene flow is not clear. The five locations with similar genetic cluster membership are embedded within, or closely connected to, broader areas of suitable habitat We identified two populations in the coastal and four in the southern region showing signs of isolation, most likely as a consequence of urban and agricultural development. These six populations exhibit the loss of genetic diversity expected for small isolates, having significantly lower diversity than those populations that showed signs of connectivity with a broader population in their region. Loss of neutral genetic diversity as a result of habitat fragmentation was a consistent pattern observed in a meta-analysis performed by DiBattista (2008)The neutral genetic markers used in this study are unlikely to have a direct effect on fitness of individuals or populations. Whether genetic diversity at neutral markers reflects that in quantitative traits, and thus, their evolutionary potential, is unclear We have identified two important characteristics of contemporary squirrel glider populations: i) genetic connectivity has persisted over large spatial scales where habitat is relatively continuous, and ii) fragmentation of habitat leads to genetic isolation. The latter was found for populations across the geographic range, suggesting that this reflects local impacts and is a general phenomenon. Our data are thus consistent with the suggestion that habitat fragmentation is associated with the regional decline of the species The vegetation-cover threshold at which squirrel glider population connectivity will be lost is not yet understood, but may become clear following fine-scale landscape genetics analyses that will be undertaken as our sample coverage improves e.g. see . The resThe samples were collected with institutional animal ethics approval, namely: Southern Cross University 02/2, 03/4, 03/10, 04/7 and 04/28; Central Queensland University 03/06–141; Deakin University (A05/96); and The University of Melbourne 02119, 05141, and 0810924.Genetic samples from squirrel gliders were collected opportunistically as part of other studies on the species from 14 sites across eastern Australia, ranging from south-western Victoria, to around Mackay in central Queensland see . GlidersSquirrel gliders were caught in traps placed on the trunks of trees  among sampled individuals within that population was calculated using GENALEX. The probability that mean observed PSA for a population was greater than that expected under the null hypothesis of no difference among populations, was assessed by comparison with upper and lower 95% confidence intervals generated from 1000 random reshufflings of values among populations.Deviations of genotype frequencies from those expected under Hardy-Weinberg equilibrium were tested for each locus in each population sample using the exact test as implemented in GENEPOP 3.2d FST values as a measure of genetic differentiation among populations were calculated, and their significance assessed by 1000 permutations, using FSTAT. These were used along with Euclidean distances among sampling sites to examine isolation-by-distance for the coastal and southern regions separately, via Mantel tests performed in GENEPOP using 100 permutations. The difference between the coastal and southern regions in pairwise intra-region FST values was assessed in a two-sided permutation test using 10000 resamplings in Resampling Stats 5.0.2, (http://www.resample.comPairwise Table S1Genetic diversity parameters per locus and population for 13 STRUCTURE-defined squirrel glider populations (sample sizes in parentheses).(DOC)Click here for additional data file.


Petaurus norfolcensis), a gliding mammal that is commonly subjected to a loss of habitat connectivity. We identified genetically differentiated local populations over distances as little as 3 km and within 30 years of landscape change. Genetically isolated local populations experienced the loss of genetic diversity, and significantly increased mean relatedness, which suggests increased inbreeding. Where tree cover remained, genetic differentiation was less evident. This pattern was repeated in two landscapes located 750 km apart. These results lend support to other recent studies that suggest the loss of habitat connectivity can produce fine-scale population genetic change in a range of taxa. This gives rise to the prediction that many other vertebrates will experience similar genetic changes. Our results suggest the future collapse of local populations of this gliding mammal is likely unless habitat connectivity is maintained or restored. Landscape management must occur on a fine-scale to avert the erosion of biodiversity. Understanding how populations respond to habitat loss is central to conserving biodiversity. Population genetic approaches enable the identification of the symptoms of population disruption in advance of population collapse. However, the spatio-temporal scales at which population disruption occurs are still too poorly known to effectively conserve biodiversity in the face of human-induced landscape change. We employed microsatellite analysis to examine genetic structure and diversity over small spatial (mostly 1-50 km) and temporal scales (20-50 years) in the squirrel glider ( Habitat loss and fragmentation are viewed as two of the most serious threats to biodiversity worldwide –3. MitigIsolation and loss of available habitat lead to reduced gene flow and small populations. The extent that populations become functionally isolated  depends on a number of factors, including the availability of habitat corridors, the composition of the landscape matrix in which remnants are embedded and a species’ vagility (i.e. its ability to use or cross the matrix)  -11. The The ability to cross the matrix is unknown for most species . HoweverGenetic theory predicts that small, genetically-isolated populations will be subject to inbreeding and loss of genetic variation through drift. Increased levels of inbreeding can give rise to inbreeding depression, leading to reduced fitness and an increased extinction risk . ErosionPetaurus norfolcensis), an Australian gliding mammal dependent on tree cover for movement . Studies on additional species are required to test these generalities. Further confirmation would allow land managers to target those species with life history attributes that predispose them to fine-scale effects of urbanisation. Species identified as vulnerable could then be used to guide efforts to restore and maintain landscape-level habitat connectivity for broader components of biodiversity.


The accelerating development of biochemical and DNA-based diagnostic tests for human genetic conditions in the last decade has engendered a revolution in genetic diagnosis. Both genetic testing and genetic screening involve the same testing processes to examine an individual’s chromosomes, DNA, or the biochemical product of a gene, typically a protein to confirm or refute a suspected chromosomal, DNA, or gene product change. The identification of genetic disorders, and the potential for developing a therapy, is a powerful force in genetics and medicine. Genetic testing is the use of laboratory tests to determine the genetic status of individuals already suspected to be at high risk for a particular genetic disorder based on family history or a positive screening test, and genetic testing and screening are similar in that both involve the use of laboratory tests to reveal the presence of disease-causing genes. The accelerating development of biochemical and DNA-based diagnostic tests for human genetic conditions in the last decade has engendered a revolution in genetic diagnosis. Finally, the pace of development and application of DNA and biochemical genetic tests and their acceptance by the public may be accelerated by the recent widespread media coverage of the work of human geneticists. A genetiIn gene tests, scientists scan a patient’s DNA sample for mutated sequences. A DNA sample can be obtained from any tissue, including blood. For some types of gene tests, researchers design short pieces of DNA called probes, whose sequences are complementary to the mutated sequences. These probes will seek their complement among the 3 billion base pairs of an individual’s genome. If the mutated sequence is present in the patient’s genome, the probe will bind to it and flag the mutation. Another type of DNA testing involves comparing the sequence of DNA bases in a patient’s gene to a normal version of the gene. Cost of testing depends on the sizes of the genes and the numbers of mutations tested. Both genetic testing and genetic screening involve the same testing processes to examine an individual’s chromosomes, DNA, or the biochemical product of a gene, typically a protein to confirm or refute a suspected chromosomal, DNA, or gene product change. Genetic screening is done for a particular condition in individuals, groups, or populations without family history of the condition, and genetic testing is done for a particular condition where an individual is suspected of being at increased risk due to their family history or the result of a genetic screening test.Advances in genome technology and other fruits of the Human Genome Project are playing a growing role in the delivery of health care. With the development of new technologies and opportunities for large-scale analysis of the genome, transcriptome, proteome, and metabolome, the genome sciences are poised to have a profound impact on clinical medicine. Cancer prognostics will be among the first major test cases for a genomic medicine paradigm, given that all cancer is caused by genomic instability, and microarrays allow assessment of patients’ entire expressed genomes. Genomic The diagnostic techniques outlined briefly above are a powerful new tool in all genetics, but most especially in the arena of human genetics. By application of these tools of biotechnology, and other techniques, molecular biologists and geneticists are providing a basis on which to make a genetic diagnosis. The identification of genetic disorders, and the potential for developing a therapy, is a powerful force in genetics and medicine. Diagnostic techniques can be used to identify specific proteins or fragments of DNA. These techniques can be used to detect viruses of animal and plant diseases, to detect harmful substances in the environment, or to match the DNA left at a crime scene to a possible criminal. Diagnostic techniques often utilize DNA probes, RFLP (restriction fragment length polymorphism) analysis, and mono- or polyclonal antibodies. Advances in genomics have led to mounting expectations in regard to their impact on health care and disease prevention. In light of this fact, a comprehensive research agenda is needed to move human genome discoveries into health practice in a way that maximizes health benefits and minimizes harm to individuals and populations. We present a framework for the continuum of multidisciplinary translation research that builds on previous characterization efforts in genomics and other areas in health care and prevention. The continuum includes four phases of translation research that revolve around the development of evidence-based guidelines. Phase 1 translation (T1) research seeks to move a basic genome-based discovery into a candidate health application . Phase 2 translation (T2) research assesses the value of a genomic application for health practice leading to the development of evidence-based guidelines. Phase 3 translation (T3) research attempts to move evidence-based guidelines into health practice, through delivery, dissemination, and diffusion research. Phase 4 translation (T4) research seeks to evaluate the “real world” health outcomes of a genomic application in practice.Genetic testing is a complex process, and the results depend both on reliable laboratory procedures and accurate interpretation of results. Tests also vary in sensitivity, that is, their ability to detect mutations or to detect all patients who have or will get the disease. Interpretation of test results is often complex even for trained physicians and other health care specialists. When interpreting the results of any genetic test, one must take into account the probability of false positive or false negative test results. Special training is required to be able to analyze and convey information about genetic testing to affected individuals and their families. Available types of genetic testing are listed in Table 1A comprehensive study of the significance and varieties of genetic discrimination is critical to design strategies to ensure the ethical and appropriate use of genetic testing in the future. The dominant theme noted in the responses in this study is that genetic conditions are regarded by many social institutions as extremely serious, disabling, or even lethal conditions without regard to the fact that many individuals with “abnormal” genotypes will either be perfectly healthy, have medical conditions which can be controlled by treatment, or experience only mild forms of a disease. As a result of this misconception, decisions by such institutions as insurance companies and employers are made solely on the basis of an associated diagnostic label rather than on the actual health status of the individual or family. The appropriate use of genetic testing information to restrict or limit access to public entitlements such as health care or employment has not been established and may not exist. The cost of such labeling is magnified by the fact that errors in testing and interpretation do occur. The threGiven this situation – powerful and attractive new techniques, social and economic forces pressing for their application, and an incomplete understanding of the potential negative social and personal consequences of genetic testing – concern about the burdens engendered by widespread utilization of genetic tests seems justified. Genetic


Anopheles gambiae s.s. We found no evidence of isolation between the Bijagós archipelago and mainland Guinea-Bissau, despite separation by distances beyond the known dispersal capabilities of this taxon. Conversely, the Comoros Islands appear to be genetically isolated from the East African mainland, and thus represent a location worthy of further investigation for field trials. Based on assessments of gene flow within and between the Comoros islands, the island of Grande Comore was found to be genetically isolated from adjacent islands and also exhibited local population structure, indicating that it may be the most suitable site for trials with existing genetic modification technologies.Considerable technological advances have been made towards the generation of genetically modified mosquitoes for vector control. In contrast, less progress has been made towards field evaluations of transformed mosquitoes which are critical for evaluating the success of, and hazards associated with, genetic modification. Oceanic islands have been highlighted as potentially the best locations for such trials. However, population genetic studies are necessary to verify isolation. Here, we used a panel of genetic markers to assess for evidence of genetic isolation of two oceanic island populations of the African malaria vector,  Aedes aegypti L. mosquitoes  are the most significant vectors of human malaria. However, in many regions, the majority of malaria transmission is attributable to the highly anthropophilic species, An. gambiae s.s.. Genetic data has shown that An. gambiae s.s. is comprised of two incipient species, known as the M and S molecular forms. Where M and S co-occur in West and Central Africa they have been shown to exhibit strong reproductive isolation with rates of cross-matings typically <1%  An. gambiae is the primary vector of malaria and is responsible for active malaria transmission and (ii) where the island is located sufficiently far away from the mainland to be potentially isolated. Moreover, studies of both oceanic , with >100 malaria cases/1000 people (WHO 2) and the furthest from the mainland. We also assessed the islands of Bubaque (85 km2) and Formosa (140 km2). These three islands have an isolation index of 10–17 according to the United Nations Environment Programme (UNEP) classification, which assesses the isolation of an island from potential colonization sources by calculating the sum of the square roots of the distances to the nearest equivalent or larger island, the nearest island group or archipelago and the nearest continent  that runs north to south. The highly permeable volcanic substrate results in an absence of surface water on the island. As such, An. gambiae s.s. larval sites are largely restricted to outdoor cisterns, which are widely used across the island to store rainwater for domestic use. In fact, according to Mouchet et al. (2), with a population of ∼40 000. The island is largely forested and consists of wide valleys with multiple rivers. The majority of Moheli is low elevation (<500 m), with the exception of a ridge reaching 600 m which dissects the centre of the island. The third island, Anjouan, is the most densely populated (∼280 000 people across 424 km2). The topography is severe, consisting of a number of steep ridges which reach up to 1500 m in elevation dissected by rivers and mountain streams. Across the islands, malaria is classified as mesoendemic  to hyperendemic . The number of malaria cases is highest on Grande Comore where most parts of the island report >100 cases/1000 population compared with 1–50/1000 on Moheli and Anjouan , along with a site on mainland Tanzania, which is an important trading partner due to a free trade agreement between the two countries  and locally reared  mosquitoes at all sites visited for Anopheline sampling and identified specimens using morphological keys when possible. Rearing of larvae to adult stages was also used to aid identification of the Anopheline species present. Representative preserved specimens were deposited in the Bohart Museum at the University of California-Davis. Due to the limited spatial and temporal scale of sampling, these collections should be viewed as representing only a subset of the mosquito community present.An. gambiae s.l. species complex we used the Scott et al.  were used to extract DNA from Anopheline samples using the Qiagen Biosprint 96 system. To distinguish between the different members of the morphologically indistinguishable t et al.  PCR assaSNP discovery was conducted by assessing published sequences representing both the M and S forms from multiple locations in Western (M & S forms), Eastern (S forms) and Southern Africa (S forms)  using GenALEX6.3  as estimated by FSTAT 2.9.3.2  to 1  gene, which have been evaluated in h) and sequence diversity (π) were calculated using DnaSP v5 . These haplotypes were then cross-referenced with published sequences from East African populations in order to detect novel haplotypes and to resolve the genetic relationship between the islands and mainland Africa  were collected in the habitats from which we sampled. Full taxonomic records are detailed in Anopheles pretoriensis Theobald samples consistently varied from those on continental Africa  sunyaniensis Edwards and Cx. (Eum.) wigglesworthi Edwards, neither of which have been recorded in the Comoros Islands (Aedes (Steg.) albopictus Skuse, providing the first record of this important arbovirus vector on the island of Anjouan , and the salt water tolerant species, Anopheles melas, which was relatively rare . The 418 An. gambiae s.s. samples consisted of both the M (n = 57) and S (n = 244) molecular forms, as well as hybrids  which was common, and the saltwater tolerant, Anopheles merus, which was rare   were not identified by PCR to be members of either the An. gambiae s.l. complex or An. funestus species group, particularly on Anjouan , which has previously been recorded on all of the islands  found at Miringoni on Moheli which also has previously been recorded on the Comoros  as previous assessments showed the hybrids to consist of a range of backcrosses, and thus could not be classed as a ‘population’ that could be assessed separately. The final data set consisted of 52 loci for 213 samples including 141 samples from the Bijagós archipelago , and 72 samples from the coastal region of mainland Guinea-Bissau . Genetic diversity metrics for the M form (RS and HE) were found to be slightly higher on the island of Formosa than on the mainland, whereas they were similar between mainland and island populations of the S form , so we could not assess FST among M form island populations. S form samples were collected from all three islands, and genetic differentiation was found to be low among these sites , Anjouan (n = 66) and Moheli (n = 149) and mainland Tanzania (n = 49). It is noteworthy that all samples from the site of Malé on Grande Comore had to be excluded due to DNA degradation.We assayed 75 SNPs in female , 3 on X . We exclHE estimates were similar. Pairwise comparisons detected significant genetic differentiation between each of the Comoros Islands and mainland Tanzania , and highest between Anjouan and Grande Comore which are separated by the greatest distance . No differentiation was observed amongst the six sites within Moheli island , whereas significant FST was found between the site of Bouni (BOU) and the three other sites on Grande Comore  Mainland Tanzania; (ii) Grande Comore; (iii) Moheli and Anjouan B. We fouAn. gambiae population by sequencing the ITS and ND5 genes. Previous studies detected limited variation at the ITS : 31 from Bouni and 31 from Bouenindi (Grand Comore); 29 from Wala and 30 from Wanani (Moheli); 30 from Moya (Anjouan); and 32 from Dar es Salaam (Tanzania). The ND5 sequences generated in this study were ∼150 bp longer (811 bp) than those deposited in Genbank (665 bp), which yielded three extra variable sites. To utilize this additional information as well as published sequence data, we conducted analyses on two ND5 data sets; the first using the full-length sequences (811 bp) generated for the samples screened in this study; and the second using the sequences from this study trimmed to 665 bp and combined with published sequences from East Africa.Due to the higher levels of variation in the ND5 gene in comparison to the ITS  relative to the mainland  which may be explained by stronger genetic drift in isolated populations due to bottlenecks and/or smaller Ne. The lower ND5 diversity may also reflect that there were few colonization events from the mainland. We detected two to three of the six haplotypes on each island. No haplotypes were shared across all three islands, but the two most common haplotypes were each detected on more than one island; ISL02 was detected on Grande Comore and Anjouan, and ISL03 between Anjouan and Moheli  and Moheli (n = 23/30), and H11 which was found on Anjouan (n = 56/59) and Moheli (n = 6), have previously been detected in Malawi, Kenya and Tanzania . These private haplotypes were similar to the two common island haplotypes (ISL04 differed by one base pair from H11 and ISL06 by a single base pair H32) indicating that they may represent mutation events that occurred on the Comoros islands post colonization, which is consistent with isolation of the islands from mainland Africa. However, more widespread sampling of Africa is required to verify this.Trimming of the ND5 sequences condensed the six Comoros haplotypes into four, but did not affect the number of haplotypes in Dar es Salaam 8; . Two of Tanzania . Due to Considerable advances have been made in recent years with regards to the development of transgenic Anopheline mosquitoes refractory to the malaria parasite Alphey . HoweverAnopheles gambiae s.s. was found to be present alongside An. melas, at all sites in Guinea-Bissau , we found no evidence of genetic sub-division between the island and mainland sites in either the M or S forms, suggesting there is considerable gene flow . However, ongoing malaria transmission presents the highest health burden on the islands  to be derived from different genetic populations of mosquitoes from mainland Tanzania, and found no evidence of recent migrants between the mainland and islands amongst our samples  resulting in a higher isolation index 49, UNEP . We foun samples , which iHE estimates which were similar on the mainland and islands despite lower allelic richness on the islands, which in itself is suggestive of a recent bottleneck  was also suggestive of isolation between the islands and mainland. Furthermore, as expected for isolated populations, genetic diversity estimates based on SNP data  and ND5 ttleneck . Lastly,FST values were not indicative of complete genetic isolation between the Comoros islands and mainland (FST = 1). This could be the result of FST not reflecting the true level of isolation. For example, if the Comoros islands were colonized relatively recently, there may have been insufficient time for differentiation to occur despite the absence of any ongoing gene flow (non-equilibrium population). This is consistent with ND5 haplotype data which shows the presence of both unique and shared haplotypes. Or, levels of divergence may have been underestimated due to the absence of Comoros samples in the SNP ascertainment panel, which would have resulted in under-representation of polymorphisms unique to the Comoros. Another explanation is that an FST value of <1 may reflect that there is ongoing gene flow between the mainland and islands sites. Indeed, population bottlenecks can cause rapid divergence in allele frequencies resulting in elevated FST values despite high gene flow as the population is not at equilibrium. In these situations, methods that do not assume mutation-equilibrium, such as STRUCTURE, are more appropriate. As stated above, STRUCTURE analyses and the distribution of ND5 haplotypes showed strong genetic structuring between mainland and island sites, indicating gene flow is restricted. Moreover, amongst our samples we found no evidence of recent migrants. As such, our data do not appear to be consistent with a hypothesis of elevated FST despite high gene flow due to a bottleneck. However, there may well be a low level ongoing migration which our sampling regime failed to detect. Additional sampling from the Comoros, sites along coastal East Africa  and Madagascar (400–450 km), as well as sampling from transport locations (airports and ports) would be useful to quantify any migration, particularly from the Comoros islands.Despite this evidence of restricted movement, An. gambiae is always found alongside human populations which may inadvertently transport individuals, there will always be some risk of migration and thus complete isolation of field site is unrealistic. General guidelines concerning acceptable levels of risk have not been developed as these will be specific to the species, trial conditions (e.g. fitness of the GMM) and technology being applied: A low level of migration would pose a smaller risk for self-limiting approaches (versus self-propagating see Introduction), such as are available for An. gambiae. For this reason, mathematical models would be useful in assessing the potential risk posed by migration on a case by case basis , indicating that human-assisted movement is lowest between the islands which are located the furthest apart , which were shown to constitute a single genetic population  will be necessary , but samples were degraded and unsuitable for SNP analysis, and therefore genetic evaluations of mosquitoes from this area would be useful to assess isolation.Population structure within a release site, that is, an island, has the potential to inhibit GMM programmes as genetic sub-division may impede the spread of genes across the target population if a genetic drive system is linked to an effector gene so to facilitate the rapid spread of modified genes through the population. However, in taxa such as Benedict . In thes2) and relatively flat terrain of Moheli, make this island an attractive prospect if trying to conduct an island wide implementation.In addition to genetic considerations, other island characteristics, such as island size, political stability and logistical considerations, are important. Whilst a full evaluation of these factors is outside of the scope of this study and thus not specifically investigated here, it is noteworthy that the highly mountainous terrain and political instability of Anjouan, make this island less appealing as a release site. In contrast, the smaller size  conduct additional sampling from the Comoros, mainland Africa and Madagascar, including some transport locations (airports and ports), to further evaluate the risk of escapees; (ii) evaluate the distribution of populations, and their genetic isolation, in the most southern region of Grande Comore.We evaluated


Dalbergia miscolobium, a widespread tree from the Cerrado, and to verify its concordance with plant phylogeographical and biogeographical patterns so far described. A total of 287 individuals from 32 populations were analyzed by sequencing the trnL intron of the chloroplast DNA and the internal transcribed spacer of the nuclear ribosomal DNA. Analysis of population structure and tests of population expansion were performed and the time of divergence of haplotypes was estimated. Twelve and 27 haplotypes were identified in the cpDNA and nrDNA data, respectively. The star-like network configuration and the mismatch distributions indicated a recent spatial and demographic expansion of the species. Consistent with previous tree phylogeographical studies of Cerrado trees, the cpDNA also suggested a recent expansion towards the southern Cerrado. The diversity of D. miscolobium was widespread but high levels of genetic diversity were found in the Central Eastern and in the southern portion of Central Western Cerrado. The combined analysis of cpDNA and nrDNA supported a phylogeographic structure into seven groups. The phylogeographical pattern showed many concordances with biogeographical and phylogeographical studies in the Cerrado, mainly with the Cerrado phytogeographic provinces superimposed to our sampling area. The data reinforced the uniqueness of Northeastern and Southeastern Cerrados and the differentiation between Eastern and Western Central Cerrados. The recent diversification of the species (estimated between the Pliocene and the Pleistocene) and the ‘genealogical concordances’ suggest that a shared and persistent pattern of species diversification might have been present in the Cerrado over time. This is the first time that an extensive ‘genealogical concordance’ between phylogeographic and phytogeographic patterns is shown for the Cerrado biome.Few studies have addressed the phylogeography of species of the Cerrado, the largest savanna biome of South America. Here we aimed to investigate the phylogeographical structure of  On the other hand, the species was encountered in only 37% and 28% of the north-eastern and central-western Cerrado provinces, respectively. It is a tree pollinated by bees and its seeds are wind-dispersed The species chosen for this study,  Cerrado . It is aD. miscolobium populations encompassing a large portion of the Cerrado biome in order to investigate the species' phylogeographical structure and to verify its congruence with phylogeographical and biogeographical patterns reported to the Cerrado biome so far. More specifically, we aimed to answer the following questions: 1) is the phylogeographical structure of this wide range species congruent with the Cerrado biogeographical provinces proposed by Ratter et al.D miscolobium we predicted some congruency between them; 2) in what extension is the phylogeographical structure of D. miscolobium similar to other Cerrado tree species studied so far? Based on previous studies Here, we undertook an extensive sampling of Dalbergia miscolobium is not included in the Brazilian Official List of Threatened Plants. The collection of samples of leaves in conservation units was realized with the legal authorization of the ‘Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis’  and of the ‘Instituto Estadual de Florestas – MG’ (population RPE – license 018/05). The collection of the samples in localities out of conservation units had authorization for collection and transport of IBAMA  and of the owners of the lands. Also we had authorization for the access to sampling of component of genetic patrimony of the Brazilian ‘Conselho de Gestão do Patrimônio Genético’ (CGEN/MMA) number 03/2004.D. miscolobium was estimated through field trips, databases of floristic checklists  and herbarium records  and the internal transcribed spacer (ITS) region of the 18S–26S nuclear ribosomal DNA (nrDNA) genes (which includes the ITS1 and ITS2 intergenic spacers and the 5.8S gene). The trnL intron and ITS region were amplified and sequenced using primers as described by Taberlet et al.et al.2, 200 µM of each dNTP, 0.2 ng bovine serum albumin (BSA), 0.5 µM of each primer, 1 U Taq DNA polymerase , and autoclaved deionized water. The nrDNA region reaction mixture also contained 2% dimethyl sulfoxide (DMSO) and 1 M Betaine (N3-trimethyglycine). Thermal conditions of the reaction were as follows: initial denaturation at 94°C for 2 min, followed by 35 cycles at 94°C for 1 min, 56°C (cpDNA) or 60°C (nrDNA) for 1 min, and 72°C for 1 min, and a final elongation at 72°C for 7 min. Each PCR product was then double-strand sequenced using the DYEnamic ET dye terminator sequencing Kit . Sequencing reactions were analyzed on a MegaBACE 1000 automated sequencer  following the manufacturer's instructions.The following regions were chosen for DNA sequencing: the Sequences were assembled using the package PHRED/PHRAP/CONSED to generate consensus sequences. The cpDNA and nrDNA sequences were deposited in GenBank under accession numbers JQ612719 to JQ612730 and JQ582850 to JQ582876, respectively. The consensus sequences were aligned using CLUSTAL-W et al.For nrDNA sequences, all variable sites were carefully checked in the original electropherograms and, when heterozygous nucleotide positions identified by double peaks were present, samples were re-sequenced independently in order to confirm the genotype. To reconstruct the nrDNA haplotypes, we used PHASE 2.1 software DSFD. miscolobium based on cpDNA and nrDNA data.Genetic diversity indices were estimated in ARLEQUIN 3.5 The phylogenetic relationships among haplotypes were inferred using the Median-Joining (MJ) network method et al.'s consensus analysis and k = 5 to Ratter et al.'s Twinspan analysis; see Results for details). The location of potential geographic barriers was explored through the Monmonier's algorithm in BARRIER 2.2 et al.CT, FSC and FST were computed considering the pairwise distances between haplotypes. The level of significance was computed by 1000 nonparametric permutations.The phylogeographic structure of populations was evaluated through a Bayesian Analysis of genetic Population Structure in BAPS 5.3 D. miscolobium were estimated through a Bayesian coalescent approach implemented in BEAST 1.6 8 generations. Substitution rates for the trnL-trnF and ITS regions of Inga species, estimated to be 1.3×10−9 and 2.34×10−9 substitutions per site per year, respectively Inga shares many features with Dalbergia. In particular, both species belong to the Leguminosae family, are composed of trees, and occur in the neotropics. Thus, the estimated rates for sequence evolution of Inga are suitable for use in our study.The divergence times among the cpDNA and nrDNA haplotypes identified in D. miscolobium for the cpDNA and nrDNA data, respectively. For cpDNA, the alignment produced a total of 606 bp with 13 detected polymorphic sites, which included 11 base substitutions and two indels , resulting in 27 haplotypes  and nrDNA   pointed to the most probable existence of five and six clusters of populations for the cpDNA and the nrDNA datasets, respectively . With thctively) , confirmet al.We named each one of the seven clusters using as a basis the biogeographic units described by Ratter et al.et al's phytogeographical Cerrado provinces. Those priors correspond to the number of Cerrado's provinces that are superimposed to our sampling area in two ways: Ratter et al's Consensus results  and Twinspan results  and nrDNA (FCT = 0.68), which was higher than with the four clusters division , and we show only the consensus barriers among the matrices . With thExclusive haplotypes could be found in six of the seven groups  showing D and Fu's SF), on the other hand, did not have significant values for all populations together or for any of the seven groups (data not shown), and thus do not support any population expansions. The neutrality tests can be more sensitive to sequence variability Population expansion, both demographic and spatial, of the species was corroborated by the mismatch distributions for the cpDNA and nrDNA data, where the observed values did not differ from the expected values under a sudden-expansion model. For the phylogeographic groups, only the CW2 and CE1 for nrDNA and CW1 for cpDNA did not exhibit population expansion pattern by mismatch distributions, as well as the groups of CE1+CE2 and CW+CW2 for both cpDNA and nrDNA . The neu6 (0.49–3.88) and 2.94×106 (0.75–5.71) years before the present (YBP), respectively. These dates place the beginning of diversification of D. miscolobium lineages at the transition between Pliocene and Pleistocene, with confidence intervals spanning from Late Miocene to Middle Pleistocene. This estimated recent origin might be the cause of the low resolution of the networks. The results are very concordant between the cpDNA and nrDNA analyses, with the estimated dates being very close to each other and having overlapping confidence intervals.The times to the most recent common ancestor (TMRCA) for cpDNA and nrDNA haplotypes were estimated in 2.03×10D. miscolobium , revealed a phylogeographic structure in colobium  largely et al.Some other longstanding phytogeographical hypotheses also are corroborated by our data. Several different authors have proposed Southern Cerrados and Northeastern Cerrados as completely different units from the remaining Cerrado D. miscolobium, estimated at the Pliocene/Pleistocene (see results), these ‘genealogical concordances’ suggest that a shared and persistent pattern of species diversification might have been present on the Cerrado over time. Ecological as well as historical biogeographic factors have acted collectively in shaping species diversification and distribution in the biome and the relative importance of each of these factors has been the object of considerable debate et al.et al. D. miscolobium in a similar way to the events of speciation that are behind the current phytogeographic patterns. Future study on genetic, morphological, and physiological diversity of widespread Cerrado species might provide the basis for better understanding the contribution of each of these factors to the observed patterns of diversification.Such ‘genealogical concordances’ D. miscolobium in the southern Cerrado (SC group) is also very similar to that reported for three other tree species studied in the Cerrado, Caryocar brasilienseHymenaea stigonocarpaPlathymenia reticulataD. miscolobium presents populations with low genetic diversity, probably descent from northernmost sources, a star-like network and mismatch distributions not significantly different from the demographic and expansion model distribution. Together, these patterns point to a recent range expansion D. miscolobium seems to better tolerate lower temperatures, it seems to have been similarly affected by the extremely arid and cold conditions of the LGM. It is suggestive that maybe, the entirely Cerrado biota, including its more cold-tolerant species, were unable to resist to the coldest conditions of LGM times in the southern portion of the Cerrado. This same pattern was not observed for nrDNA data, whose variation, in SC group, has contrasting patterns, that is, high genetic diversity and genetic uniqueness. Rapid expansion of those populations coupled with the high rate of mutation of nrDNA, nrDNA paralogue evolution, seed/pollen bias due to cpDNA gene flow only by seeds and even hybridization/introgression are among the factors that could have been responsible for those results D. miscolobium.The pattern of cpDNA diversity of D. miscolobium is in accordance to the commonly reported heterogeneity of Cerrado biome in terms of species distribution D. miscolobium has been reported to three other species Hymenaea stigonocarpaPlathymenia reticulataThe heterogeneous distribution of genetic diversity of These many patterns found rise novel questions for future consideration. Do these biogeographic patterns persist in other widespread Cerrado species? What would have occurred to species endemic to these provinces, especially those found commonly in the southern Cerrado? How did the different ecological and historical factors lead to the diversification of the Cerrado biota? Phylogeography of the Cerrado is in its infancy, and many studies are still needed to improve our understanding of the historical biogeography of this rich and threatened biome to help in its conservation and sustainable use.Figure S1Results from Bayesian Analysis of Population Structure (BAPS) of Dalbergia miscolobium populations with a prior of k = 4. See text and  for details.(TIF)Click here for additional data file.Table S1Variable sites of aligned cpDNA sequences.(XLS)Click here for additional data file.Table S2Variable sites of aligned nrDNA sequences.(XLS)Click here for additional data file.


Ambystoma tigrinum melanostictum) in northern regions of Yellowstone National Park are declining due to climate-related habitat changes. In this study, we used ancient and modern mitochondrial haplotype diversity to model the effective size of this amphibian population through recent geological time and to assess past responses to climatic changes in the region. Using subfossils collected from a cave in northern Yellowstone, we analyzed >700 base pairs of mitochondrial sequence from 16 samples ranging in age from 100 to 3300 years old and found that all shared an identical haplotype. Although mitochondrial diversity was extremely low within the living population, we still were able to detect geographic subdivision within the local area. Using serial coalescent modelling with Bayesian priors from both modern and ancient genetic data we simulated a range of probable population sizes and mutation rates through time. Our simulations suggest that regional mitochondrial diversity has remained relatively constant even through climatic fluctuations of recent millennia.Recent data indicates that blotched tiger salamanders ( Detailed contemporary genetic characteristics of a population can be analyzed to reveal aspects of its history. Nonetheless, the modern genetics of a population serve only as a snapshot of complex and dynamic processes averaged over time and biased by recent events. Incorporating ancient information into genetic studies can place a population into its historical context, and can reveal details of a population's history that might otherwise be lost with time Ambystoma spp.) suggest complex colonization, migration and bottleneck patterns through their histories that could be explored in greater detail by anchoring modern genetic variation in ancient variation. Nonetheless, no previous study has included assessments of ancient amphibian DNA, either in a phylogeographic or a population genetic context. To our knowledge, the oldest analyzed amphibian genetic material was extracted from museum specimens accessioned in 1920 Amphibian populations are sensitive environmental indicators, tracking both aquatic and terrestrial environments, and their responses to historical environmental changes may lend insight into their prospects in rapidly changing modern ecosystems. Genetic variation in modern populations of tiger salamanders  midden deposit chronicling over 3000 years of vertebrate community history Ambystoma tigrinum melanostictum is the most abundant non-mammalian vertebrate present in the deposit, and Bruzgul et al.Lamar Cave in northern Yellowstone National Park, Wyoming, USA, is a rich wood rat  was extracted from samples in a designated aDNA facility physically isolated from any area in which amplification or analyses were performed. Personnel were not permitted to enter the aDNA area for 24 h after contact with PCR products or facilities. Prior to extracting aDNA, all the surfaces in the aDNA facility were cleaned with 25% bleach. The room, the inside of the flow hood, and all reagents and materials were irradiated with UV light for a minimum of 12 hours. Extractions and PCR setup were performed in a sterilized Class II laminar flow hood within the sterilized aDNA facility.We adhered to strict extraction and amplification protocols, and numerous precautions were taken to prevent contamination see . NeitherExtractions of ancient material were performed in small batches of 5 or fewer samples. Weight of sampled cervical vertebrae was measured and ranged from 1 to 12 mg (average = 5.4 mg). After crushing the vertebra inside of sterilized aluminum foil using a sterilized mortar and pestle, powdered material was digested in 275 µl 0.5 M EDTA pH 8.0 with 0.5% sodium dodecyl sulfate and 100 µg/ml proteinase K for 24 h at 55°C, then purified using Qiaquick® DNA purification silica columns . We included two dummy negative controls in this process, the first control added with the samples as they were weighed and powdered, and the second added at the addition of buffer during the extraction phase. Each of these controls was used alongside the samples through the amplification process to monitor for cross-contamination. A sample of the sequences were cloned and sequenced to verify sequence fidelity see .We designed five sets of overlapping primers, altogether 729 base pairs (bp) of the mitochondrial genome:AAACATCGATCTTGTAAGTCG 1R: ATTAAAAAAGCTCCTACGCTT (to amplify a 184 bp product); 2F: CTTAAAATCAGTAAACTGC 2R: CAAGAGGGGAGGATTTTCAC (201 bp product); 3F: CAACTCGAATTTTCTATTGC 3R: CATCCAACTATCTGCCACACG (214 bp product); 4F GTAACGTGGCAACATATTATG 4R: CTGGTTAAAATCTATGGAC (201 bp product); 5F: GAGTGCCTTACTTCCCTTG 5R: GTAATATGACTGGTACTAC (205 bp product).1F: AmpliTaq® Gold polymerase , 1× Taq Gold buffer, 5 mM MgCl2, 1 mM each dNTP, 1.3 mg/ml spermidine  and 0.25 µM of each primer. A third control was added during PCR preparation.Amplifications were performed in 50 µL reactions containing 1 U Samples were transferred into the physically isolated PCR room and run on a 9700 thermocycler  for 10 min at 95°C, 45 cycles of 95°C for 30 sec, 45°C for 30 sec, 72°C for 30 sec, followed by 10 min at 72°C. Every amplification reaction included a negative control with no added DNA. We tested each of the dummy negative controls collected during the extraction process, of which there were eight in total (two for each of the four extraction batches performed). These were amplified along with the actual DNA samples, and none of the dummy negative controls ever yielded product visible on an agarose gel stained with ethidium bromide. Likewise, the negative control amplification reactions containing no DNA never yielded detectable products. Our PCRs were successful for 51% of positive (DNA-containing) reactions. We found no correlation between amplification success and fragment size or sample age. Amplified fragments were purified with the Qiaquick® PCR purification kit and sequenced in both forward and reverse directions by Genaissance Pharmaceuticals .A. t. melanostictum were collected from 13 ponds in northern Yellowstone National Park , 1× PCR buffer, 2 mM MgCl2, 0.2 mM each dNTP and 0.25 µM of primers 1F and 5R (see sequences in previous section). PCRs were run at 95°C for 2 min, 35 cycles of 95°C for 30 sec, 55°C for 30 sec, 72°C for 1 min, followed by 5 min at 72°C. PCR samples were sequenced in both forward and reverse directions by Cogenics, Inc. . In order authenticate our sequences  or both the observed ancient segregating sites (0 sites) as well as the number of modern segregating sites for the Lamar Valley population (i.e. 1 site). After running Bayesian models, we ultimately performed simulations based on fixed parameters. In these simulations, we fixed both the mutation rate and the population size, then ran 1000 independent simulations and evaluated the percentage of successful simulations. A simulation was deemed successful when it produced the appropriate numbers of segregating sites in the ancient (Ancient Data) or in both the modern and ancient populations (Ancient+Modern Data). We calculated the probability of any potential population size as the percent success in these simulations.Sequence reads were aligned with Sequencher software . Mantel tests and AMOVA, both with 9999 permutations, were performed for the modern population using GenAlEx 6.2 A. t. melanostictum from northern Yellowstone (We sampled 143 modern and 16 ancient lowstone  and ampllowstone . All 16 xy = 0.23 (p<0.001) with distance explaining 5.1% variation between ponds. The amount of subdivision within the population was significant . We calculated Tajima's D for individual ponds, for the ponds in Lamar Valley and for all ponds; in all scenarios, there was no significant deviation from 0.Multiple AMOVAs of the modern genetic data indicated that the population exhibited significant isolation by distance . A ManteWe used Bayesian serial coalescent modeling A. t. melanostictum is comparable to the low mtDNA diversity described in other Ambystoma populations The low mitochondrial diversity that we observed in modern A. t. melanostictum collected from other geographic regions in the Great Plains and the Pacific Northwest A. t. melanostictum collected from the eastern Great Plains. The greater Yellowstone ecosystem was covered by ice until ∼14,000 ybp, and the region was colonized by amphibians soon after this ice cap melted, and this genetic similarity is consistent with a recolonization westward from the Great Plains. The low diversity throughout the region suggests that there has been relatively little gene exchange with outside populations since this initial colonization, however the mutation rate and overall diversity is so low that moderate levels of migration and gene exchange cannot be excluded, especially from populations with identical haplotypes present. Although diversity was low, the distribution of the four haplotypes revealed significant spatial differentiation (H1 shares more than 99% identity with mitochondrial sequences of ntiation . We founntiation . The amo16 = 0.52 or 52%. This relatively large probability suggests that the spatial distribution of modern haplotypes reflects patterns that have likely persisted for millennia, and that the haplotype distribution seen in the current Lamar Valley is similar to the distribution that has existed for several thousand years.Our analysis of aDNA indicated that H1 was historically prevalent in this population as well; indeed H1 was the only haplotype identified in the ancient samples. Rodríguez and colleagues Ambystoma may be as high as 1.5% mutations per base pair per million years in the D-loop, but is probably closer to 0.75% in coding regions such as the cytochrome-b gene A. t. melanostictum was simulated using the number of mitochondrial segregating sites identified in ancient (Of course, haplotype frequencies do change through time and even if populations remain geographically stable, new haplotypes are introduced by mutation. To construct models representing the dynamic nature of population genetics, we simulated coalescence based on a range of population sizes and mutation rates  ancient  or ident ancient  to evalu ancient . Conside ancient . We descA. t. melanostictum ranging in age from 100 to 3300 years, sequencing a total of more than 10,000 ancient base pairs with a minimum of 2× coverage. PCR misincorporations were not detected in any of the sequences, showing that DNA from all these samples could be faithfully amplified. While our laboratory has performed numerous aDNA-based studies (i.e. Base pair misincorporations, specifically cytosine-deamination transitions are shown to be pervasive in amplifications of ancient and historic DNA, even from samples as young as 50 years ies i.e. , all theThe vertebrate specimens used in the present study, like other samples from Lamar Cave A. t. melanostictum population sizes, we conclude that the bottlenecks caused by fish stocking and disease are not the primary cause of the low mitochondrial diversity in this region.Signals of population bottlenecks are evident across northern Yellowstone, and these have been attributed to the bottlenecks induced by recent events such as fish stocking in the 1950s