Inbreeding is inevitable in closed populations with a finite number of ancestors and where there is selection. Therefore, management of the rate of inbreeding at sustainable levels is required to avoid the associated detrimental effects of inbreeding. Studies have shown some pedigree dog breeds to have high levels of inbreeding and a high burden of inherited disease unrelated to selection objectives, implying loss of genetic diversity may be a particular problem for pedigree dogs. Pedigree analysis of all 215 breeds currently recognised by the UK Kennel Club over the period 1980–2014 was undertaken to ascertain parameters describing the rate of loss of genetic diversity due to inbreeding, and the presence of any general trend across all breeds.The trend over all breeds was for the rate of inbreeding to be highest in the 1980s and 1990s, tending to decline after 2000. The trend was comparable in very common and rarer breeds, although was more pronounced in rarer breeds. Rates of inbreeding over the entire period 1980–2014 were not correlated with census population size. The existence of popular sires was apparent in all breeds.The trends detected over 1980–2014 imply an initial excessive loss of genetic diversity which has latterly fallen to sustainable levels, even with modest restoration in some cases. The theory of genetic contributions, which demonstrates the fundamental relationship of inbreeding and selection, implies that popular sires are the major contributor to high rate of inbreeding.The online version of this article (doi:10.1186/s40575-015-0027-4) contains supplementary material, which is available to authorized users. Inbreeding’ is widely viewed as being harmful to the wellbeing of individuals and populations. In populations of a limited size, complete avoidance of breeding between individuals with a shared ancestry quickly becomes impossible. Furthermore, selection within dog breeds for desirable traits will inevitably result in the breeding of individuals that resemble each other with respect to the traits under selection. The resemblance of relatives is a fundamental principle of genetics, and means that selected individuals will on average be more closely related than a random pair taken from across the population. Therefore some degree of inbreeding is inevitable in all populations; it is how quickly this changes that is informative. The rate of inbreeding in a population relates to the risk of detrimental effects associated with inbreeding . Therefore the rate of inbreeding is a measure of the sustainability of a population. This study reports the general trends in the rate of inbreeding observed through population analyses of all 215 pedigree dog breeds currently recognised by the UK Kennel Club, over the period 1980 to 2014.http://www.thekennelclub.org.uk/vets-researchers/publications,-statistics-and-health-results/breedpopulation-analyses.For all breeds, the trend was for the rate of inbreeding to be highest in the 1980s and 1990s, representing a major contraction in genetic diversity. Since 2000 however, the general trend has been for the rate of inbreeding to decline to sustainable levels, with some modest restoration of genetic diversity in some cases. It is interesting that this coincides with the relaxation of the UK’s quarantine laws, and is possibly due to the more widespread use of non-UK animals for breeding. The rate of inbreeding (or effective population size) showed no relationship with the actual population size, as judged by mean number of KC registrations. Evidence of popular sires was common in all breeds. Popular sires make large genetic contributions to subsequent generations and are the biggest influence on the rate of inbreeding. There was variation among breeds in the trend of rate of inbreeding over the period 1980-2014. Reports detailing results of the population analysis for each individual breed are publically available at: It is widely believed that pedigree dogs are very inbred, due to closed registries and breeding practices, and that this has had a detrimental effect on the health and welfare of many pedigree breeds. Indeed studies have empirically determined a large depletion in genetic diversity in some pedigree dogs breeds –3, and mn, where n is the generation, i.e. 2 parents, 4 grand-parents, 8 great-grand-parents, and so on). This very quickly leads to an unfeasible number of unrelated ancestors at the nth generation, implying common ancestry and so inbreeding. Where there is common ancestry the probability that both alleles inherited by an individual are copies of a single allele from a common ancestor to both parents is >0. The coefficient of inbreeding (F) is this probability, and F for an individual will be higher if there are more common ancestors and in more recent generations. Given that all individuals carry mutant deleterious alleles, many of which when inherited in duplicate can result in loss of function, F describes the risk to an individual. A high level of inbreeding also has detrimental effects on the wider population, resulting in the loss of genetic variation, loss of heterozygosity, possible effects of inbreeding depression, and a higher rate of spread of deleterious alleles  mean of the coefficient of kinship (expected inbreeding [from random mating]) of all animals born per year. Expected inbreeding is staggered by the breed mean generation interval (n = 121), five had a negative whole period ΔF implying an apparent overall increase in genetic diversity, and consequently no determinable Ne . Of the 116 remaining breeds, the Ne calculated over the period 1980–2014 ranged from 23.8 (Manchester Terrier) to 918.8 (Borzoi). Of these 116 breeds, 68 had Ne of <100, with 29 having Ne of <50. A scatter plot of Ne calculated over the period 1980–2014 on mean annual registrations over the same period for the 116 breeds where Ne was determinable is shown in Fig. e and census population size as measured by mean registrations.Of all breeds with an average of >50 registrations per annum . Fig. Where there are sufficient data a table displaying sire and dam usage statistics and genetic parameters over each of the seven 5-year blocks is included in breed reports. The mean rate of inbreeding  over all breeds with an average of >50 registrations per annum in each of the seven 5-year blocks differed significantly over the blocks (L) from the 1980s to the 1990s onwards was observed . This may partially account for the declining general trend in mean ΔF in the periods 1980–1984 and 1985–1989, where the difference in L is greatest. Mean L is not significantly different over blocks 1990–1994 to 2010–2014 . This observation implies that the continued declining trend in mean ΔF is not solely due to changes in L, and so is in part due to additional changes in breeding practice.A general increase in the generation interval (http://www.thekennelclub.org.uk/getting-a-dog-or-puppy/finding-the-right-dog/vulnerable-native-breeds/) and the 20 most common breeds  for 28 vulnerable native breeds  inform of the recent breed history and of the necessary considerations framing potential breeding strategies in the future. Different breeds with varying dynamics and levels of genetic diversity will have different options available in balancing meeting selection objectives and maintaining a sustainable ΔF. The breed reports are intended to provide a backdrop to discussions over the best approaches.It is hoped that the public availability of population analysis reports for all 215 breeds currently recognised by the Kennel Club will enable breeders and other stakeholders to achieve a better understanding of the unique situation facing each breed. Both the descriptors of population dynamics  and the genetic parameters . Thus, e varied widely across breeds, ranging from 23.8 to 918.8 . Individual breed whole period Ne represents the slope of ‘best fit’ through the ‘observed inbreeding’ plot ; some very numerous breeds had a small whole period Ne  while some much rarer breeds had a relatively high whole period Ne . A possible reason for low Ne in numerically large breeds is underlying population ‘sub-structure’. In common breeds, such as the English Springer Spaniel, several ‘sub-populations’ are likely to exist; for example working, show and pet populations, and even geographically localised populations. The existence of sub-populations (to whatever degree of independence) is indeterminable in this analysis of pedigree data. Breeding practices within each of multiple independent sub-populations giving rise to a positive ΔF in each will lead to a positive breed-wide ΔF. However, this breed-wide figure represents [mean] loss of genetic diversity within each sub-population, but ignores the fact that there is actually an increase in genetic diversity between sub-populations due to drift acting on allele frequencies [e fails to take account of the between sub-population genetic variation, which is easily tapped by migration between sub-populations. Therefore, for some of the numerically larger breeds with show and working ‘types’ , the breed-wide whole period Ne may belie the amount of true within breed genetic diversity.The whole period Nquencies . Thus the for breeds making wide use of imported animals), the principle outlined above also applies to breed populations in different countries. Even where such migrant animals originally trace back to the UK (for example the English Setter), populations which have existed in semi-isolation in different countries will be subject to the effects of drift, potentially increasing genetic diversity between sub-populations while it simultaneously declines within each. However, to what degree the increase in between sub-population diversity counteracts the potential underestimation of ΔF and Ne due to incomplete pedigree information is unknown.Although the lack of complete pedigree data beyond a certain number of generations for migrant animals hampers the ability to fully determine co-ancestry where it does exist  would appear to be related to the effective management of genetic diversity. The plots of observed and expected inbreeding in these breeds show small divergence between the two, implying only a slight departure from random mating  is a single, albeit important, objective among many in the promotion of health and welfare in dog breeds. Many breeds face significant welfare problems due to a high burden of inherited disease  and extrWhile the use of inbreeding coefficients to derive ΔF in a breed or population provides a useful indication of past practice, they remain a retrospective measure of co-ancestry. Well-intentioned but widespread use of unrelated animals in breeding programmes in an attempt to increase genetic diversity, while reducing mean F in the next generation, can lead to such individuals becoming popular sires. Popular sires make a large genetic contribution to future generations of the breed, and are therefore the major contributor to a high ΔF over subsequent generations . BreederThe general trend detected across breeds was that ΔF was highest in the 1980s and 1990s, implying a high rate of loss of genetic diversity within most breeds. However, since 2000 ΔF tends to have decreased, even becoming negative in some cases, indicating a slowing of the rate of loss or even some moderate restoration of genetic variation. This change in trend may have been influenced by the increased availability of migrants since changes to UK quarantine laws in 2000.Most breeds show extensive use of popular sires. The theory of genetic contributions, which demonstrates the fundamental relationship of inbreeding and selection, implies that popular sires are the major contributor to high ΔF. Breeders should guard against the over-use of sires as a strategy to maintain sustainable ΔF in the breed.th February 2015).All electronically recorded pedigree data held by the Kennel Club was used to determine population statistics per year from 1980–2014 for each of the 215 recognised breeds  regressed on year of birth, the regression coefficient describing the overall trend in breed registrations over the 35 year period.Over each year 1980–2014 the total number of unique sires used was determined, and the maximum, mean, median, mode and standard deviation in number of progeny per sire computed. The percentage of registered animals born per year to the most prolific 50 %, 25 %, 10 % and 5 % of sires was calculated to describe the extent of ‘popular sire’ usage in each breed.L) was computed as the mean of generation intervals calculated per year from 1980–2014, and is quoted in years.The generation interval is the mean age of parents at the birth of progeny which themselves go on to reproduce . The meat) on year of birth, where Ft is the mean inbreeding coefficient of animals born in year t. The rate of inbreeding per annum was multiplied by the breed mean generation interval (L) to obtain the rate of inbreeding per generation (ΔF). The effective population size (Ne) was calculated for each breed over the period 1980–2014 as (2 ΔF)−1 [Inbreeding coefficients for all animals in each breed pedigree were calculated using the algorithm of Meuwissen and Luo . Mean in(2 ΔF)−1 .L, since animals born in year t would on average produce breeding progeny at t + L in the future). Calculation of coefficient of kinship per year of birth is computationally intensive for large populations, since the number of calculations is ½(n2-n)  and so the method used was dependent on the number of animals born in a particular year. Where ≤500 animals were born in a particular year, all kinships were calculated and the mean computed. Where >500 and ≤2000 animals were born in a year, kinships of a single, random sample of 500 animals were calculated and the mean computed. Where >2000 animals were born in a year, kinships were calculated for 10 samples of 50 randomly selected animals. The overall mean and standard deviation of the 10 sample means were calculated. In this final scenario, the standard deviation of samples mean kinships is indicated by the ‘bleeding’ of the line denoting expected inbreeding.The coefficient of kinship between two animals is equal to the inbreeding coefficient of offspring of those two animals . Thus, mL between the 5-year blocks were tested for statistical significance using ANOVA, which would indicate a change in general (across breed) trends.The statistics described above were also calculated over seven 5-year blocks  to provide more detail of changing trends within the overall 35 year period. Where the numerical size of the breed sufficed (mean of >50 registrations per annum in each of the seven 5-year blocks), differences in across breeds mean ΔF and


Rangifer tarandus). In central Canada, the ranges of two caribou subspecies (barren-ground/woodland caribou) and two woodland caribou ecotypes  overlap. Our objectives were to reconstruct the evolutionary history of the eastern migratory ecotype and to assess the potential role of introgression in ecotype evolution. STRUCTURE analyses identified five higher order groups . The evolutionary history of the eastern migratory ecotype was best explained by an early genetic introgression from barren-ground into a woodland caribou lineage during the Late Pleistocene and subsequent divergence of the eastern migratory ecotype during the Holocene. These results are consistent with the retreat of the Laurentide ice sheet and the colonization of the Hudson Bay coastal areas subsequent to the establishment of forest tundra vegetation approximately 7000 years ago. This historical reconstruction of the eastern migratory ecotype further supports its current classification as a conservation unit, specifically a Designatable Unit, under Canada’s Species at Risk Act. These findings have implications for other sub-specific contact zones for caribou and other North American species in conservation unit delineation.Understanding the evolutionary history of contemporary animal groups is essential for conservation and management of endangered species like caribou ( Similarly, the two sub-specific lineages . The assessment of the goodness-of-fit of a model parameter posterior combination was tested via the model-checking option in DIYABC v. 2.0.4 [a–c).We ran approximately 3 million simulations and compared scenarios by estimating posterior probabilities using the logistic regression . For modv. 2.0.4 . Runs we3.3.1In total, 1360 unique genotypes  were identified. Samples with at least eight out of 10 successfully scored loci were used in subsequent analysis (missing data in the microsatellite dataset was 2.9%). However, one locus (BMS1788) was taken out of further analysis because it showed mutational patterns that are inconsistent with 1 bp shifts.icro-Checker tests nor tests for deviations from HWE and LD revealed any significant evidence that specific populations showed null alleles, deviated from HWE (9/333 cases significant after Bonferroni correction), or showed signs of LD (none significant after Bonferroni correction). Therefore, we retained nine loci for the subsequent analyses to examine phylogeographical structuring rather than delineate fine-scale local structure that might also exist but is beyond the scope of this work.Neither MK=2, corresponded to the subspecies level distinguishing barren-ground from all other populations . The second level, at K=6 , revealed population genetic structure at the ecotype and within-ecotype level, identifying barren-ground, the eastern migratory ecotype, including the Pen Island herd and coastal region in Ontario, as structured groups. Further, the boreal caribou ecotype consisted of four populations : (1) and (2) two populations in western Manitoba, (3) one population in southwestern Manitoba and (4) one population including eastern Manitoba and Ontario mainland. For further analysis, the two populations found in western Manitoba were combined because only a single sampling site with fairly low sampling size (N=23) showed high membership coefficients for that cluster with additional individuals being highly admixed. It is currently unknown whether that cluster is biologically meaningful and the pattern requires further investigation with more local sampling. Since the focus here was to understand the evolutionary origin of the eastern migratory ecotype, western Manitoba was considered one population with potential substructure. All subsequent analyses were carried out with the five major groups identified in STRUCTURE. Further, a population mean q bar plot  revealed that individuals of the eastern migratory ecotype had higher average membership assignments to their focal population. Likewise, Ontario/eastern Manitoba showed a different genetic signature including higher average membership coefficients to a different population cluster. Hence, the eastern migratory ecotype and the Ontario/eastern Manitoba population were treated as different populations in succeeding analyses. Additionally, a sorted q bar plot  confirmed the presence of highly assigned individuals to each of the identified populations indicating that, albeit high admixture levels in Ontario, two distinct clusters exist there (i.e. eastern migratory ecotype and Ontario/eastern Manitoba)  .Figure Lastly, the phylogeographical pattern in BMS1788 is consistent with results in that a preliminary assessment of allele variation supports a phylogeographical pattern separating western Manitoba and Ontario. An AMOVA on microsatellite data revealed lower differentiation values that nevertheless were highly significant; therefore, supporting the results found in the STRUCTURE analysis.Since barren-ground (B haplotypes) and woodland caribou (A haplotypes) clearly differed in mtDNA control region sequences ,12,13, iA hierarchical AMOVA (mtDNA) revealed that approximately 22% of the genetic variation was found between the STRUCTURE groups indicating clear genetic structure  and 3. Wa–c). As would be expected for the rates of change at each type of loci and the time frame under investigation, these results suggest that mtDNA is more reliable in estimating the more ancient admixture event, whereas the microsatellite data are superior in estimating the more recent divergence event . Since mtDNA has a lower mutation rate than microsatellites, haplotype resolution may simply be insufficient to fully capture the divergence of eastern migratory and Ontario/eastern Manitoba boreal caribou, and therefore microsatellite data are likely more suitable for the analysis of the more recent divergence event. Cornuet et al. [ABC  identified scenario 3 as the most likely evolutionary scenario based on posterior probability values, credible intervals  and logit et al.  pointed 4.Increased understanding of the evolutionary processes that lead to contemporary spatial genetic partitioning is critical for delineating units for conservation assessment and subsequent recovery plans and may enhance our ability to protect important adaptive potential of species at risk ,65,66. IUnder this scenario, barren-ground caribou likely migrated along the retreating ice sheet allowing for lineage mixing in northern Ontario and Manitoba. The eastern migratory ecotype diverged from this ancient introgressed population, leaving residual barren-ground-specific haplotypes in both the eastern migratory ecotype and Ontario/eastern Manitoba boreal caribou population. The lack of B haplotypes in western and southwestern Manitoba might be best explained by later arriving western (i.e. A2 lineage) caribou populations that migrated eastwards and consequently did not readily interbreed with barren-ground. The occurrence of B haplotypes in eastern migratory and the boreal ecotypes in Ontario and eastern Manitoba is possibly the result of the ancient introgression event identified in the ABC analysis. Alternatively, gene flow between the eastern migratory ecotype and boreal caribou populations led to distributions of a few B haplotypes.In western Canada, several studies ,13 detecet al. [McDevitt et al.  also suget al. ) may be Migratory behaviour has been shown to increase fitness through exposure to seasonal changes in predator avoidance ,72 and rRegardless of the source of migratory behaviour, lineage-specific diversity is important, both biologically and for conservation ranking because genetically, different populations in widespread species often follow independent evolutionary trajectories and therefore may respond differently to new or changing environments ,75. The The maintenance of significant phylogeographical structure found in this study also points to the potential importance of additional environmental factors and landscape features  in shaping of genetic diversity. Since environmental factors are likely to be diverse across the distribution range of widespread species, locally adapted populations will be spatially distributed as well. It has been proposed  that pro


Since early 2013, H7N9-subtype avian influenza virus (AIV) has caused human infection in eastern China. To evaluate AIV contamination and the public risk of infection, we systematically implemented environmental sampling from live poultry markets in Guangdong Province. Through real-time polymerase chain reaction assays and next-generation sequencing, we generated full nucleotide sequences of all 10 H6N6 AIVs isolated during sampling. Focusing on sequence analyses of hemagglutinin genes of the 10 H6N6 AIVs revealed that the viruses were low pathogenic AIVs with the typical hemagglutinin cleavage site of P-Q-I-E-T-R-G. The hemagglutinin, neuraminidase, and nucleocapsid genes of nine AIVs were of ST2853-like (H6-subtype) lineage, ST192-like (N6-subtype) lineage, and HN573-like (H6-subtype) lineage, respectively; whereas the other five genes were of ST339-like (H6-subtype) lineage. However, the polymerase PB2 and nucleocapsid genes of one strain (HZ057) were of GS/GD-like (H5N1-subtype) and ST339-like lineages. Phylogenic analysis revealed that all eight genes of the 10 viruses belonged to Eurasian avian lineage. Altogether, the 10 AIVs were reassortants of different genetic groups of exchanges with the same virus subtype, thus illustrating the genetic diversity and complexity of H6N6-subtype AIVs in Guangdong Province. Avian influenza viruses (AIVs) are single-stranded, negative-sense RNA viruses of the family Orthomyxoviridae that contain eight gene segments  have long been regarded as potential hotbeds for AIVs, whose transmission can be facilitated there via humans' direct and indirect contact with poultry. It has been reported that LPMs have become an important source for human infection with H5N1 highly pathogenic AIV  and thereafter analyzed the molecular evolution of 10 H6N6-subtype AIVs. Ultimately, with this study we have contributed fundamental data regarding controlling AIVs as well as epidemiological and molecular evidence of their evolution in Guangdong's LPMs.This study was approved by the Guangdong Provincial Center for Disease Control and Prevention (CDC) Experimental Animal Welfare Ethics Committee (permit number 2015–09).Surveillance of AIVs was conducted in LPMs in Guangdong for 12 months beginning in January 2013. Samples including poultry manure and material swabbed from defeathering machines, sewage, and chopping tools were randomly taken from LPM environments weekly. Samples were initially tested for influenza A virus with real-time polymerase chain reaction (PCR) in the local CDC. Positive influenza A samples were differentiated by HA subtype (H1–H18) via real-time PCR in local CDC laboratories and further verified by Guangdong Provincial CDC laboratories. Positive samples of H6-subtype were further tested by NA subtype (N1–N11), also with real-time PCR. In all, 10 positive samples of H6N6-subtype were purified and propagated in embryonated, 10-day-old specific-pathogen–free (SPF) chicken eggs and stored at −70°C . Reverse transcription and PCR amplification of all eight gene segments was performed using PathAmp™ FluA Preamplification Reagents . Products of PCR were purified with the Ampure XP purification kit  and quantified using an Ultrospec 2000 mass spectrophotometer . Full genome sequences of the isolated AIVs were sequenced using the Personal Genome Machine Sequencing 200 Kit v2 (American Life Technologies) and the Ion 316 Chip V2 on an Ion PGM System to sequence.Full sequences of the isolated AIVs were obtained by aligning the resulting sequences against representative sequences downloaded from GenBank , which included ST339-like and ST2853-like AIVs in internal gene trees. Unlike surface gene trees, AIVs in Group II rarely formed a monophyletic clade in internal gene trees. Meanwhile, AIVs circulating in natural hosts could also form the monophyletic Group III in all internal gene trees Figure .Phylogenetic analyses of internal genes showed that polymerase PB1 (PB1), polymerase PA (PA), matrix protein (M), and non-structural protein (NS) genes of all 10 viruses were of Eurasian lineage and ST339-like viruses, as well as shared 94.2–96.4, 95.3–95.5, 95.4–97.7, and 95.6–96.4% nucleotide identity with A/duck/Shantou/339/2000 (H6N2), respectively Figures . The polTen H6N6-subtype AIVs contained multiple basic amino acids (PQIETR↓G) at the cleavage site between HA1 and HA2, thereby indicating that the 10 AIVs were of low pathogenicity in domestic poultry. Amino acid residues T160, Q226, and G228 (H3 numbering) were retained in the receptor-binding pocket of HA1, thus indicating that the 10 H6N6-subtype AIVs preferred to bind to α-2,3-linked sialic acid AIV receptor , whereas the other nine AIVs had eight potential N-linked glycosylation sites in NA . Position 274 of the NA protein (NA of GS/GD number) was Y, which suggested sensitivity to the neuraminidase inhibitor oseltamivir  and their transmission among humans.RY, YK, ML, and CK conceived and designed the experiments. RY, YS, LZ, JW, and XZ performed the experiments. RY analyzed the data. RY, LL, XZ, HN, JYL, JL, ML, and CK contributed reagents/materials/analysis tools. RY wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bifurcaria bifurcata. ENMs for B. bifurcata were developed using marine and terrestrial climatic variables, and its range projected for 2040-50 and 2090-2100 under two greenhouse emission scenarios. Geographical patterns of genetic diversity were assessed by screening 18 populations spawning the entire distribution for two organelle genes and 6 microsatellite markers. The southern limit of B. bifurcata was predicted to shift northwards to central Morocco by the mid-century. By 2090-2100, depending on the emission scenario, it could either retreat further north to western Iberia or be relocated back to Western Sahara. At the opposing margin, B. bifurcata was predicted to expand its range to Scotland or even Norway. Microsatellite diversity and endemism were highest in Morocco, where a unique and very restricted lineage was also identified. Our results imply that B. bifurcata will maintain a relatively broad latitudinal distribution. Although its persistence is not threatened, the predicted extirpation of a unique southern lineage or even the entire Moroccan diversity hotspot will erase a rich evolutionary legacy and shrink global diversity to current (low) European levels. NW Africa and similarly understudied southern regions should receive added attention if expected range changes and diversity loss of warm-temperate species is not to occur unnoticed.The global redistribution of biodiversity will intensify in the coming decades of climate change, making projections of species range shifts and of associated genetic losses important components of conservation planning. Highly-structured marine species, notably brown seaweeds, often harbor unique genetic variation at warmer low-latitude rear edges and thus are of particular concern. Here, a combination of Ecological Niche Models (ENMs) and molecular data is used to forecast the potential near-future impacts of climate change for a warm-temperate, canopy forming seaweed,  Global warming constitutes, together with land-use change and pollution, a major global anthropogenic threat to biodiversity in both marine and terrestrial systems –3. TempeRange shifts involve regional extirpations and/or colonization of newly suitable areas, often but not restricted to peripheral range edges, as species track their climatic niches . Ecologica. 20000 years BP] colonized areas [The use of genetic assessments extends ENMs by allowing potential losses of cryptic diversity  associated with climate change to be predicted . Geneticed areas ,15. Thised areas ,17.Global warming is anticipated to trigger similarly diverse demographic responses across species ranges , and thuBifurcaria bifurcata.Rocky-shore organisms, especially the conspicuous structural species, are regarded as early-warning indicators for the impacts of climate change ,27. IsotB. bifurcata R. Ross 1958 (Brown Forking Weed) is a perennial and hermaphroditic brown alga endemic to the warm-temperate eastern Atlantic. It occurs from southern Morocco (27.5° N) to north-western Ireland (55° N) on moderately exposed rocky shores in the mid/low intertidal and in rock-pools. The range of B. bifurcata has expanded and contracted along the leading English Channel edge in response to decadal-scale variations in climatic conditions [B. bifurcata is overwhelmingly concentrated along this overlooked southern range. Our models predict a poleward range shift associated with future climate change that is likely to result, depending on greenhouse gas emissions, in the loss of a distinct and spatially restricted phylogeographic lineage located at the current trailing edge, or even in the complete extirpation of the Moroccan diversity hotspot.nditions , and othnditions . In contnditions , distribB. bifurcata were collated from field observations and the available literature . Multivet al.  for machCross-validation was performed by randomly dividing the distribution records (presences and pseudo-absences) into two datasets. The first was generated with 70% of the records and was used to train the models with all possible combinations of non-correlated predictors  when a predictor was modeled alone and the mean gain in accuracy when added to a given model. The combinations of predictors with highest potential for transferability per modelling technic (BRT and MARS) were determined by sorting the combinations by decreasing accuracy scores and by running successive Kruskal-Wallis rank tests. In this process, the raw TSS values resulting from the 50 interactions were sequentially added to independent tests until the level of significance  was reached. This allowed pinpointing a threshold in accuracy separating the top-ranked combinations from those retrieving significantly lower accuracy scores.The full set of distribution records were used to train the final distribution maps. However, because several combinations of predictors could retrieve equally high accuracy scores (on average), the final predictions were produced by ensembling  the reshttp://www.r-project.org/) using the packages: adehabitat, dismo, gbm, gstat, mda, parallel, raster and SDMTools.All modeling analyses were performed in R  were sampled from 18 locations covering the entire known distribution of the species .Individuals of  species . No specin silico by screening with MSATCOMMANDER [cox3 (618bp) and the chloroplast rbc operon (527–537 bp), the latter spanning the intergenic spacer between the large (rbcL) and small (rbcS) sub-unit genes. Primer sequences were developed using published sequences available in Genbank database (cox3: EU681436 [rbcL: AY590500 [rbcS: FM957154 [http://www.vgl.ucdavis.edu/STRand) using the 500 LIZ size standard (Applied Biosystems). Sequences were aligned, proofread and edited in GENEIOUS 4.8 .A geographically comprehensive panel of individuals was used to assess the polymorphism of 25 microsatellite loci identified OMMANDER  an EST lEU681436 ; rbcL: AAY590500 ; rbcS: FER [cox3 8bp and tFM957154 ). Amplifhttp://kimura.univ-montp2.fr/genetix), including allele frequencies, mean allelic richness (A), Nei’s gene diversity (HE), observed heterozygosity (HO) and inbreeding coefficients (FIS). Pairwise FST and D  was run with all individuals combined and with no prior population assignment. Each number of assumed populations  was run five times using a burn-in of 200000 iterations and a run-length of 1000000 iterations. The “best” number of genetic clusters was inferred following the ΔK choice criterion of [Summary statistics of microsatellite genetic diversity were calculated with GENETIX 4.05  were es . Haplotype and nucleotide diversities were calculated for each population and selected geographical regions using DNASP 5.10 [The geographic distribution of the ASP 5.10 .Bifurcaria bifurcata was mostly explained by air and sea surface temperatures . This and the ensemble based on the AOGCMs showed that presently the niche of f Biscay . By 2040f Biscay . In the  Channel . In the  Channel . A simil Channel . By compcox3 (Genbank accession nos. KR002845-48) and 7 rbc (accession nos. KR002849-55) haplotypes were identified in the 144 sequenced individuals of Bifurcaria bifurcata. Both markers exhibited low haplotypic diversity and sequence divergence, but they were concordant in delimiting two phylogeographic groups , and B3 and B4 (rbc), were found in high frequencies in specific populations of northwest Iberia. Additional diversity was recovered within the rbc spacer, where variations in the repeat number of a pentanucleotide microsatellite distinguished the haplotypes B1, B2 and B5  than in Europe .A total of 4 c groups . The firps cox3,  or two (2 and B5 . These wFIS). Within populations, genetic diversity (A and HE) was higher throughout Morocco  than in Iberia, Brittany or the British Isles , with very few exceptions  and central  populations . Most populations exhibited heterozygote deficiency  temperatures. The coldest winter temperatures and hottest summer temperatures (both air and SST) should be particularly relevant in setting the northern and southern limits respectively. Predictors nevertheless interact in complex ways, and critical air and sea thresholds do not coincide spatially. At its northern limits in the British Isles, B. bifurcata is essentially restricted to rock-pools, presumably due to excessively cold air winter temperatures [A wealth of empirical, biogeographical, and niche modelling studies have demonstrated the key importance of marine and terrestrial thermal environments in setting the general distributions (and range shifts) of intertidal sessile species ,35,69–71eratures  as suppoB. bifurcata and the observed records. Weighting different sources of data allowed to partially capture the inherent uncertainty of predicting ranges using niche based models [B. bifurcata, although the course and extent of shifts differed between emission scenarios. By 2040–50, and irrespective of the emission scenario, the severe deterioration of environmental conditions throughout southern Morocco and the Western Sahara was predicted to lead to a northward repositioning of the trailing edge to central Morocco. Local probabilities of occurrence there were nevertheless much lower in the RCP8.5 than in the RCP2.6 scenario, indicating a more marginal and spatially restricted presence in the former.Our modelling approach, weighting a set of best candidate models, showed excellent transferability and high consistency between the modelled distribution of d models . The simB. bifurcata to reoccupy its lost southern Moroccan range, their aggravation in the RCP8.5 scenario predicts the elimination of the species from NW Africa and southwest Iberia. The potential southern limit of B. bifurcata by 2100 may thus be located either along the coast of the Western Sahara or central Iberia, depending on emission levels. At the leading edge B. bifurcata was predicted to experience a significant poleward expansion along western Scotland. In the RCP8.5 scenario, it may even reach Norway, although in this case large distributional gaps are predicted in northern Iberia and the English Channel.The forecast southern ranges differed much more significantly between emission scenarios by the end of the century. While the improvement of environmental conditions in the RCP2.6 scenario could allow B. bifurcata will be able to swiftly track its moving niche envelope, the species as a whole is not expected to be facing any significant risk of near-future extinction. However, as shown below, these shifts are likely to have negative impacts on its global genetic diversity. Diversity loss across climatic shifts largely depends on the geographical distribution of genetic diversity. In B. bifurcata, diversity is rather skewed along the distributional range, concentrated within the vanishing area most at risk and low at its leading expanding edge; the potential biodiversity loss from climate change may thus be severe [Our results show that climate change will affect both the species range and its gene pool. There will be positive and negative population-level effects, dependent on local shifts in conditions across the distribution. Even if range centers and southern edges varied between emission scenarios, globally this species is also predicted to expand northwards and maintain, as presently, a relatively ample latitudinal distribution. In the RCP2.6 scenario, it could even increase its latitudinal span. Assuming that e severe .Bifurcaria bifurcata is composed of two divergent lineages with contrasting distributions and range sizes, as shown by the mitochondrial and chloroplast sequences. Phylogroup A was only detected in a ca. 170km stretch of shoreline at its modern rear-edge in southern Morocco (0.5 degrees in latitude), whereas phylogroup B occupied the remaining Moroccan and European distribution (> 20 degrees). Cytoplasmic genomes are inherited maternally in fucoid algae (reviewed in [iewed in ), but thIt remains difficult, with the data at hand, to establish the historical demographic contexts promoting (and maintaining) the genetic divergence  of these two main phylogroups. Given their differentiation, the observed pattern may result from the recent secondary contact of former allopatric groups. However, the high genetic diversity found in these groups  supports long term regional persistence of large populations rather than recent expansions, which are often associated with loss of diversity in fucoids . We furtB. bifurcata has its extant diversity hotspots located in northwest Africa, much beyond other known northeast Atlantic (European) seaweed refugia.The pattern of high and distinct diversity at the lower latitudes is likely to reflect the effects of the last glaciation(s), as the advance of the ice-sheets and permafrost belts eliminated most temperate species from colder, high-latitude areas. The lower levels of genetic diversity and differentiation found in Europe are a common signature of consecutive bottlenecks due to founder events associated with recent , stepping stone colonizations originating in a single or limited set of source populations ,77. The B. bifurcata.It is generally assumed that range dynamics, and particularly population extinctions, will have the highest impact for global patterns of genetic diversity in the next century of climate change. Indeed, given the short evolutionary time frame (<100 years) considered here, the impacts of mutation and lineage sorting will be negligible. Also, at the warm range edge local alleles are more likely to go extinct than to disperse to other populations. Effective gene flow is generally very low in direct-developing structural seaweeds, as they have limited dispersal abilities  and B. bifurcata may recover its lost southern range by the end of the century, but potential colonizers will likely originate from the nearest surviving sites, which will be central Moroccan populations belonging to lineage B. The CPR8.5 scenario is even more negative. By 2090–2100, B. bifurcata could be completely eliminated from Morocco, where its presently high diversity and divergence requires large persistent populations, indicating that it has had a relatively stable presence during the past glacial-interglacial cycles. In addition to lineage A, this refugial region harbors the highest regional and intra-population microsatellite diversity, including 18 private alleles (only two in Europe); its loss would shrink the global genetic diversity of B. bifurcata to the levels presently found in the rather homogenous European genetic cluster. The potential loss of the endemic lineage A is particularly relevant due to its distinctiveness. Individual lineages have partially independent evolutionary trajectories in unique environmental spaces, and thus are also likely to harbor unique and potentially adaptive genetic variability [By 2040–2050, the elimination of this species from southern Morocco will predictably result in the extinction of the unique lineage A. Such loss would be irreversible and independent of future range changes. In the CPR2.6 scenario iability ,81,82. TB. bifurcata itself has a local habitat refugium in the northern range where it only occurs in tide pools never exposed to critically cold winter air temperatures.Estimates of genetic loss, here and elsewhere, depend on the accuracy of the data  and of the predictions of range loss. It is possible that the distribution of phylogroup A extends further north along the transitional region between El Ouatia (phylogroup A) and Essaouira (phylogroup B), increasing survival chances (in the best scenario CRP2.6) despite the anticipated extinction of its two presently known populations. The complete extirpation of the entire Moroccan hotspot by the end of the century (CRP8.5) may also be overestimated if some populations unpredictably persist in suitable habitat islands. Local habitat refugia in the warm edge has been documented for another intertidal fucoid alga  and B. bsuch as Himanthalia elongata and Fucus serratus to occur in isolated pockets in northwest Iberia [Laminaria ochroleuca and Saccorhiza polyschides also occur in small isolated areas in central Morocco where upwelling is stronger. However, a recent SST-warming trend in Morocco was shown by [Fucus vesiculosus from extinction. These observations imply that other variables, namely related to the terrestrial climate, are as relevant as oceanographic conditions in setting range limits of southern intertidal species [B. bifurcata to escape the effects of projected climatic changes.Meteorological (e.g. cloudiness), oceanographic (e.g. upwelling) or tidal (e.g. time of low tides) eccentricities can also create atypical thermal environments over spatial scales of biogeographic significance, that may regionally superimpose onto latitudinal temperature clines ,84. Regut Iberia ,86, wellshown by  who note species ,83. It r species ,88, but In the northeast Atlantic, most records of range changes in the shallow marine realm involve limits located along the European coast e.g. ,89,90). ,90. 89,9B. bifurcata in European countries is well known and based on abundant and geographically precise occurrence records . The. TheB. bnditions . There, project, ). By comara (see ), but ouB. bifurcata to be promptly detected, and their genetic consequences assessed.The role of rear edges as important and vulnerable areas of diversification and endemism has long been recognized, particularly for terrestrial organisms but also for cold-temperate marine taxa; as our study shows, the same should apply to warm-temperate coastal species. Understudied low-latitude regions at the rear-edge of many species should thus receive added attention to allow range changes of warm-temperate species like S1 FigThe upper matrix shows the coefficient of correlation between predictors , the lower matrix shows the scatter plot of pairs of predictors, and the diagonal shows the histogram and name of individual predictors.(DOCX)Click here for additional data file.S2 Figet al.  and Evanno et al. . Five iterations were run for each K.The “best” number  was inspected using the choice criteria of Pritchard (DOCX)Click here for additional data file.S1 TableCoordinates , countries, approximate locations, and original sources are reported for each site.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableModel accuracy was assessed using True Skill Statistics (TSS). Note the similarity between MARS and BRT models.(DOCX)Click here for additional data file.S4 TableFST (θ) values are given above diagonal, in bold if non-significant. Jost’s Dest are given below diagonal. Population codes are as in (DOCX)Click here for additional data file.


Recently, the development of biobanks linked to electronic medical records has presented new opportunities for genetic and epidemiological research. Studies based on these resources, however, present unique challenges, including the accurate assignment of individual-level population ancestry. In this work we examine the accuracy of administratively-assigned race in diverse populations by comparing assigned races to genetically-defined ancestry estimates. Using 220 ancestry informative markers, we generated principal components for patients in our dataset, which were used to cluster patients into groups based on genetic ancestry. Consistent with other studies, we find a strong overall agreement (Kappa  = 0.872) between genetic ancestry and assigned race, with higher rates of agreement for African-descent and European-descent assignments, and reduced agreement for Hispanic, East Asian-descent, and South Asian-descent assignments. These results suggest caution when selecting study samples of non-African and non-European backgrounds when administratively-assigned race from biobanks is used. Hospital-based biobanks linked to electronic medical records (EMRs) are a growing and cost-effective way to ascertain large segments of a population for biomedical research studies. Genetic and clinical studies increasingly require larger numbers of samples to provide statistical power to discover genetic variation associated with complex human diseases; using existing surveyed clinical populations is a way to meet this demand quickly. Multiple studies have been published illustrating the basic utility of biobanks for validating existing association studies The Vanderbilt DNA biobank (BioVU) contains nearly 160,000 DNA samples linked to electronic medical records at Vanderbilt University and continues to accrue additional patient samples. DNA is extracted from discarded blood samples collected during routine patient care. EMR data is drawn from administrative databases and scrubbed of identifying information to generate a resource for researchers known as the Synthetic Derivative (SD) With increased emphasis on the use of DNA biobanks, it is important to note the critical role of race in genetic association studies. A sample drawn from multiple underlying populations is subject to population stratification, where each population has a slightly different genetic architecture. If not properly accounted for, these differences in allele frequency can result in false associations. As such, it is common practice in genetic studies to correct for underlying population sub-structure by estimating global genetic ancestry for each sample In lieu of genotyping AIMs, genetic studies sometimes use self-reported race as a covariate, either as a surrogate for genetic ancestry or to capture social and demographic components Dumitrescu et al. The accuracy of third-party racial assignments is especially critical for biobank-based studies. Should an investigator seek to perform a genetic study within a diverse population, sample selection is likely dependent on the third-party racial assignment within the EMR. As a result, samples of a different ethnicity may be selected and genotyped, only to be excluded from analysis after ancestry is determined using genetic data, resulting in a waste of research funds. Additionally, genetic ancestry can influence some clinical decision-making processes, including automated decision support, which is being integrated into some EMRs BioVU, Vanderbilt University's biobank, uses de-identified patient electronic medical records. This study is considered non-human subjects research by the Vanderbilt institutional review board.A total of 7,252 individuals were selected from BioVU, specifically to over-represent diverse populations and individuals with “unknown” administrative race assignments. Within the synthetic derivative (SD) and BioVU, race is administratively assigned to one of eight predefined categories: White (W), Black (B), Asian/Pacific (A), Native American (N), Indian (I), Hispanic (H), other (O), or unknown (U) . Based oAll 7,252 BioVU samples were genotyped using the Illumina VeraCode GoldenGate assay in the Center for Human Genetics Research (CHGR) DNA Resources Core at Vanderbilt University for 308 ancestry informative markers (AIMs) and scanned on the Illumina BeadXpress reader. AIMs genotypes were merged with existing data for 805 individuals from the International HapMap Project , including 165 CEU, 203 YRI, 137 CHB, 113 JPT, 101 GIH samples, and 86 MXL, as reference populations to assist in determining genetic ancestry . The genmclustmclust should define five clusters in order to differentiate the five ancestry groups known to be present in the dataset . By plotting a 10 by 10 matrix of all pairs of PCs, colored by the defined clusters, we visually determined that PCs 1, 2, 3, 7, 9, and 10 optimally captured separation of the five clusters. These six PCs were used to perform clustering. Genetic variance within the European-descent cluster was captured in the unused principal components, and may reflect a bias toward European-descent components within this set of AIMs.We performed principal components analysis (PCA) for 220 SNPs using the EIGENSTRAT package Administratively-assigned race was compared to cluster-based ancestry assignment  through The distribution of administratively-assigned race across the sample used in this study, within BioVU, and within the entire synthetic derivative (SD)—as well as population-level counts for Davidson County Tennessee—are shown in Genetic and epidemiological studies routinely use self-reported race or genetic ancestry to adjust for confounding factors and/or to tailor genetic effects to specific population subgroups. Global genetic ancestry is often used to correct for population stratification in genetic analyses, because it roughly reflects differences in allele frequencies between continental populations. The social construct of race is often used to capture other demographic factors, such as access to care, dietary and environmental exposures, and socioeconomic status. Self-reported race has been shown to be highly correlated to genetic ancestry and is often used as a surrogate for continental ancestry. In many clinical datasets, self-reported ancestry is not available and various administrative procedures are used to assign race status. While it is unknown to what degree administratively-assigned race captures the various social and cultural aspects of an individual, in this work we show that it has only moderate agreement with genetic ancestry for certain populations. We observed strong agreement between administrative race assignment and genetically determined ancestry for European-descent and African-descent individuals; there was less agreement between assigned race and genetic ancestry for East Asian-descent, South Asian-descent, and Hispanic-descent individuals. Given this fact, investigators should use caution when using administratively-assigned race as a proxy for genetic ancestry, and expect some misappropriation of racial categories by third party assignment.Interestingly, East Asian-descent, South Asian-descent, and Hispanic-descent individuals all have slightly different agreement statistics by sex, with females tending to have slightly higher agreement between administrative assignment and genetic ancestry. Previous studies have reported subjective misclassification of Hispanic individuals by sex, causing non-Hispanic females to be classified as Hispanic because of adopted spousal surnames Approximately 18% of the individuals in our dataset had an administratively-assigned race specified as “unknown” . The disIn conclusion, administratively assigned race is an accurate predictor of genetic ancestry for the ascertainment of European-descent and African-descent individuals, but is less accurate for other diverse populations. Investigators accessing Asian-descent or Hispanic-descent populations should expect a moderate number of samples to have administrative race labels inconsistent with genetic ancestry. When race is an important factor in a study, we recommend, when possible, that a low-cost genotyping array, such as a fixed content Illumina BeadChip (i.e. Illumina HumanCore array) be used to genotype ancestry-informative markers (AIMs) to determine genetic ethnicity.Table S1Race/ethnicity terminology usage.(DOC)Click here for additional data file.Table S2Percentages of each administratively-assigned race assigned to each genetic ancestry based on PCA clustering group.(DOC)Click here for additional data file.Table S3Kappa agreement statistics, including percent agreement and expected agreement, by genetic ancestry, as determined by clustering.(DOC)Click here for additional data file.Table S4Genetic ancestry for samples with administratively-assigned race listed as 'unknown'.(DOC)Click here for additional data file.


Lentil is a self-pollinated annual diploid (2n = 2× = 14) crop with a restricted history of genetic improvement through breeding, particularly when compared to cereal crops. This limited breeding has probably contributed to the narrow genetic base of local cultivars, and a corresponding potential to continue yield increases and stability. Therefore, knowledge of genetic variation and relationships between populations is important for understanding of available genetic variability and its potential for use in breeding programs. Single nucleotide polymorphism (SNP) markers provide a method for rapid automated genotyping and subsequent data analysis over large numbers of samples, allowing assessment of genetic relationships between genotypes.In order to investigate levels of genetic diversity within lentil germplasm, 505 cultivars and landraces were genotyped with 384 genome-wide distributed SNP markers, of which 266 (69.2%) obtained successful amplification and detected polymorphisms. Gene diversity and PIC values varied between 0.108-0.5 and 0.102-0.375, with averages of 0.419 and 0.328, respectively. On the basis of clarity and interest to lentil breeders, the genetic structure of the germplasm collection was analysed separately for cultivars and landraces. A neighbour-joining (NJ) dendrogram was constructed for commercial cultivars, in which lentil cultivars were sorted into three major groups . These results were further supported by principal coordinate analysis (PCoA) and STRUCTURE, from which three clear clusters were defined based on differences in geographical location. In the case of landraces, a weak correlation between geographical origin and genetic relationships was observed. The landraces from the Mediterranean region, predominantly Greece and Turkey, revealed very high levels of genetic diversity.Lentil cultivars revealed clear clustering based on geographical origin, but much more limited correlation between geographic origin and genetic diversity was observed for landraces. These results suggest that selection of divergent parental genotypes for breeding should be made actively on the basis of systematic assessment of genetic distance between genotypes, rather than passively based on geographical distance.The online version of this article (doi:10.1186/s12863-014-0150-3) contains supplementary material, which is available to authorized users. Lens culinaris Medik.) is a self-pollinating, diploid (2n = 2× = 14) grain legume crop with a large genome size (c. 4 Gbp) [Lentil . 4 Gbp) . Contemp Gbp . Co. 4 Gbp) . Cultiva. 4 Gbp) ,7, follo. 4 Gbp) . Lentil . 4 Gbp) -11. Worl. 4 Gbp) .Numerous landraces of lentil have been sampled from different geographical regions world-wide, and are now preserved within the Australian Grains Genebank (AGG), Horsham, Victoria, Australia. Many of these landraces are yet to be exploited for breeding activities. The key to increases in lentil yield is the conservation and surveillance of existing genetic diversity for broadening the use of available genetics . One priGenetic diversity in both cultivated and wild lentil has been explored using several approaches, including morphological and physiological markers, isoenzymes, DNA-based markers such as randomly amplified polymorphic DNAs (RAPDs), inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs) ,11,15-17A number of studies have reported the use of SSR markers for germplasm characterisation in a multiplicity of crop species ,22, but Assessment of genetic diversity in lentil is desirable for prospective future breeding activities, in terms of broadening and maintaining the diversity of the genetic base, improving opportunities for selection of improved genetics and cultivar identification. In the present study, the genetic diversity of 505 lentil cultivars and landraces obtained from different geographical regions and preserved within the AGG has been determined through the use of a genotyping tool based on 384 SNP markers.Lens culinaris Medik.), including cultivars (111) and landraces (394), were obtained from the AGG, Horsham, Victoria, Australia. All available passport data from these accessions is summarised in Additional file A total of 505 accessions of lentil , allele frequency, minor allele frequency, heterozygosity, gene diversity (GD), and polymorphism information content (PIC). Genetic similarities between each pair of accessions were measured by using an in-house customised program Genomic Relationship Matrix (genomicRelMatp). A heat map was generated using the R package. The NJ dendrogram from cultivar data was generated using the DARwin package based on genetic distance calculated using NTSYS v2.1.The genetic structure of the germplasm collection was first analysed by performing PCoA implemented in the program GenAlex 6.41. Basic statistics were calculated using the genetic analysis package PowerMarker  for divFor analysis of population structure, a Bayesian model-based analysis was performed with STRUCTURE v2.3.4 . The posA sub-set of 384 genome-wide distributed SNPs was used to assess genetic diversity within lentil germplasm, of which 192 were assigned to locations on the lentil genetic map . The estimation of true value of K was based on the observation that L(K) reached a plateau (or continued to increase slightly) and displayed high variance between runs. This analysis showed an optimum value of K = 3 for commercial cultivars Figure  and K = L. culinaris) and 0.31 per kb (L. ervoides) [Medicago truncatula Gaertn.  was slightly lower than that obtained for other crops such as soybean , field rvoides) . These vrvoides) ), field rvoides) ) and Med per kb; ), supporInformation content of markers was assessed on the basis of a number of different criteria, the most fundamental being number of alleles, higher values of which are likely to lead to higher polymorphisms in any given germplasm set. However, this criterion is most relevant to SSR markers, which are capable of displaying multiallelic structure. For SNPs, in contrast, for which biallelic patterns are standard (using Goldengate assays), expected heterozygosity is a more accurate measure of polymorphism as this parameter measures distribution of alleles across the germplasm under examination. In general, the level of genetic diversity quantified as heterozygosity based on SNP markers was approximately half that estimated through use of SSR markers . This poEstimation of the degree of differentiation between accessions that are included in a crossing program is useful for selection of parental genotypes. The maximum distance Figure  was calcBased on knowledge of the pedigrees of Australian cultivars and breeding lines that were included in this study, the PCoA obtained a number of consistent relationships. For example, variety Nipper, which was derived from a three-way cross between Indianhead and (twice-over) Northfield, is located mid-way between these two lines. Similarly, CIPAL0715 and CIPAL0714 (released as variety Grampians), lie between the parental lines Nugget and French Green, while the widely adopted variety PBA Flash is positioned close to mid-way between its parents, Nugget and ILL7685. Many of the breeding lines in the largest cluster of Australian varieties  were located at intermediate positions between their parents.In contrast, some other varieties appear in positions inconsistent with recorded ancestry. For instance, CIPAL0801 (syn. PBA Bolt) appears close to Aldinga, distant from the parents, ILL7685, Nugget, and Matador. In the same way, Boomer and CIPAL0501 are sister progeny of a Digger × Palouse cross, but are located in a separate cluster. Anomalous placements were also observed for PBA Blitz, PBA Bounty, CIPAL0803 (syn. PBA Ace), 01-068 L-04H014 and CIPAL0901, all being elite Australian cultivars or breeding lines. The explanation of such anomalous results is not clear, although errors in pedigree record-keeping, or labelling of seed samples, are more probable. In general, however, PCoA confirmed many of the known relationships between cultivars. Given the relatively short history of Australian lentil breeding, such affinities may be attributable to the contributions of original source germplasm, predominately landraces or cultivars obtained from ICARDA.Lens species are endemic. Following the initial domestication event, cultivation of lentil spread to Europe, Central Asia, Pakistan, India and South America [The analysis of the landraces did not reveal a strong correlation between geographical origin and genetic diversity. It is generally accepted that landraces may consist of highly diverse mixtures of different genotypes, and may hence require substantial within-accession sampling for a meaningful analysis of genomic diversity . The lan America , and the America ,38-40.Knowledge of genetic variation and genetic relationships between lentil landraces is important for efficient germplasm preservation, characterisation and subsequent use by lentil breeders. The narrow genetic base of the cultivars compared to the landraces, as shown by genetic distance estimates, reveals a relatively untapped pool of genetic diversity that could be highly valuable for further advances in yield potential, along with resistance to biotic and abiotic stresses. In addition to this, information on regional differentiation has practical significance for the management of germplasm and to assist selection of parental genotypes for breeding activities. Selection of genetically diverse landraces as parents should contribute to genetic gain through identification of superior progeny combinations from within breeding populations. This will also lead to cultivars with superior local adaptation, if the populations are evaluated and analysed carefully. Furthermore, information on regional differentiation should provide evidence for identification of parents with enhanced abiotic stress tolerance or resistance to biotic challenges.The indicative number of clusters obtained from use of STRUCTURE was K = 5, but substantial overlaps were observed between different clusters. The result of the present study for lentil accessions, revealing limited correspondence between geographical origin and genetic diversity, is similar to that obtained in previous studies of other crops such as field pea  and saffOf the 384 SNPs used in the current study, genetic map positions were known for 192 (50%). Such information could contribute to future detailed genome wide-association studies (GWAS) studies  for lentAssessment of genetic variation among a global collection of lentil cultivars and landraces was performed using a set of genome-wide distributed SNP markers. Genetic diversity analysis revealed clear grouping within cultivars based on geographical origin, but no such correspondence was observed within landraces collection. This result indicates that assessment of genetic diversity is critical for choice of germplasm suitable for breeding activities, and the data presented in the present study will highly assist such efforts.


Eudyptula minor) in southeast Australia documented low spatial structuring of genetic variation with the exception of colonies at the western limit of sampling, and this distinction was attributed to an intervening oceanographic feature (Bonney Upwelling), differences in breeding phenology, or sea level change. Here, we conducted sampling across the entire Australian range, employing additional markers . The zone of elevated genetic structuring previously observed actually represents the eastern half of a genetic cline, within which structuring exists over much shorter spatial scales than elsewhere. Colonies separated by as little as 27 km in the zone are genetically distinguishable, while outside the zone, homogeneity cannot be rejected at scales of up to 1400 km. Given a lack of additional physical or environmental barriers to gene flow, the zone of elevated genetic structuring may reflect secondary contact of lineages (with or without selection against interbreeding), or recent colonization and expansion from this region. This study highlights the importance of sampling scale to reveal the cause of genetic structuring.Factors responsible for spatial structuring of population genetic variation are varied, and in many instances there may be no obvious explanations for genetic structuring observed, or those invoked may reflect spurious correlations. A study of little penguins ( Knowledge of the factors influencing gene flow and genetic structuring among populations is essential for our understanding of evolutionary processes , and alsIn an effort to understand the factors most influential for genetic structuring among seabird colonies, Eudyptula minor (Spheniscidae), is a flightless marine bird that breeds in colonies irregularly distributed throughout southern Australia, New Zealand, and associated islands  among 7 southeast Australian colonies  approach described in G2-2 discontinued owing to consistent deviation from Hardy–Weinberg expectations). An additional 8 loci were employed from A total of 12 microsatellite loci were employed for analysis. These comprise 4 of the 5 loci employed by P value) with Arlequin , an estimate of genetic distance between populations (θ (θ /1 − θ) and the shortest marine distance  between 1 and 6. Selection of the optimum number of clusters followed the ΔK method of K, assessed using STRUCTURE HARVESTER  of loci per colony ranged from 0.05 to 0.85  to 10 (Granite Island) with a mean of 6.47 per colony . Microsa to 0.85 .P > 0.05), and there was no evidence of scoring errors due to stuttering, large allele dropout, or null alleles at any of the loci as assessed by Microchecker. Independence of genotypes among microsatellite loci was rejected for only 9 tests following Benjami–Yekutieli correction—none of which involved the same comparison of loci among populations. When ignoring correction for simultaneous tests, no more than 5 out of 17 populations exhibited P < 0.05 for the same comparison of loci. Therefore, genotypes among loci were considered independent.The Ewens–Watterson–Slatkin exact test only rejected selective neutrality of mtDNA variation for the Spencer Gulf composite sample, which may reflect the low sample size and pooling of data from proximate colonies. Genotype frequencies at each microsatellite locus were consistent with Hardy–Weinberg equilibrium (θ (Supplementary Table 1 online) failed to reject genetic homogeneity of most colonies in southeast Australia, while homogeneity was rejected elsewhere, often over much smaller spatial scales. Samples collected between West Island and Lion Island, including the 2 Tasmanian colonies, were homogeneous for microsatellite allele frequencies in the majority of pairwise tests (θ than the microsatellite loci (Supplementary Table 1 online). The small Spencer Gulf sample was infrequently distinguished from other colonies based on mtDNA haplotype frequencies.Pairwise estimates of θ  and resuse tests , with thK method suggested that K = 2 was the optimum number of clusters for the data based on STRUCTURE analysis , but there is no suggestion of a hard genetic break among samples; a continuous transition in coancestry coefficient occurs from a western group to an eastern group , but not for mtDNA . We also tested whether the IBD relationship differed regionally, based on the spatially variable scales of genetic differences observed. Significant IBD relationships for microsatellites were obtained from analysis of colonies between Troubridge and Granite Island . A significant relationship was also observed based on all colonies from outside this region  was weaker than that for the colonies between Troubridge and Granite Island  , and also west of Troubridge Island , and the slopes of these relationships  were similar to that obtained from all colonies outside the Troubridge–Granite zone, and shallower in comparison to that for the Troubridge–Granite subset of colonies A significant isolation-by-distance (IBD) relationship was evident throughout the entire study range for microsatellites (03×10−3) . Significolonies . Analysecolonies  and coulZ = 103.79, r = 0.59, P = 0.04), but not for comparisons of all colonies outside this region  , but not west of Troubridge Island . As for microsatellites, the slope of the relationship inside the Troubridge–Granite subset  was steeper than that outside  .A significant IBD relationship was observed for mtDNA within the Troubridge–Granite subset ( = 0.14) . A signi14×10−4) . ExclusiSupplementary Figure 3 online). However, the estimated cline center was similar to that obtained from microsatellites . A likelihood ratio test revealed that analysis with both microsatellite and mtDNA clines constrained to have the same center  was not significantly worse than that where clines were allowed to have different centers . Analysis of microsatellite loci individually revealed a geographic spread of cline centers . Clines could not be adequately fitted to variation at loci Emm4, AM13, and G3-11. Constraining clines fitted for individual microsatellite and mtDNA loci to have the same center  was not significantly worse than that where clines were allowed to have different centers .Analysis of multilocus microsatellite data revealed a cline centered 17 km west of West Island . The disThe most striking observation from this study was the different geographic scales at which significant population genetic structuring was observed. Spatial structuring of genetic variation was weak among colonies in southeast Australia, matching previous observations of The presence of finer spatial structuring of genetic variation in the Troubridge–Granite Island region and different IBD relationships among regions can be more readily explained by nonequilibrium population genetic structuring . There aUria aalge , traditional interpretations of the genetic structuring observed herein should be avoided ; Phillip Island Nature Park.


Martes foina) and red foxes (Vulpes vulpes) share a widespread Palearctic distribution and are considered habitat generalists, but in the Iberian Peninsula stone martens tend to occur in higher quality habitats. We compared their genetic structure in Portugal to see if they are consistent with their differences in ecological plasticity, and also to illustrate an approach to explicitly delineate the spatial boundaries of consistently identified genetic units. We analysed microsatellite data using spatial Bayesian clustering methods , a progressive partitioning approach and a multivariate technique . Three consensus Bayesian clusters were identified for the stone marten. No consensus was achieved for the red fox, but one cluster was the most probable clustering solution. Progressive partitioning and sPCA suggested additional clusters in the stone marten but they were not consistent among methods and were geographically incoherent. The contrasting results between the two species are consistent with the literature reporting stricter ecological requirements of the stone marten in the Iberian Peninsula. The observed genetic structure in the stone marten may have been influenced by landscape features, particularly rivers, and fragmentation. We suggest that an approach based on a consensus clustering solution of multiple different algorithms may provide an objective and effective means to delineate potential boundaries of inferred subpopulations. sPCA and progressive partitioning offer further verification of possible population structure and may be useful for revealing cryptic spatial genetic patterns worth further investigation.The identification of populations and spatial genetic patterns is important for ecological and conservation research, and spatially explicit individual-based methods have been recognised as powerful tools in this context. Mammalian carnivores are intrinsically vulnerable to habitat fragmentation but not much is known about the genetic consequences of fragmentation in common species. Stone martens ( In conservation biology, interest is growing on the population genetic status and viability of species traditionally classed as ‘common’ ,2,3 sincMuch remains unknown about the genetic consequences of habitat fragmentation and heterogeneity on common, vagile and apparently continuously distributed species. These species are generally assumed to have absent or negligible population structure or an isolation-by-distance pattern (IBD). However, an increasing number of studies are showing that unexpected patterns of genetic differentiation can be observed in mobile and widespread habitat generalists ,10,11,12Microsatellites  provide Studies on large carnivores e.g. ,11,13) h,13 h11,1Martes foina, which in Portugal seems to be forest-adapted . . Capreols strong .When building the consensus clustering for the stone marten, we only included individuals assigned to the same cluster by no-admixture models using a threshold of 0.90 , 103 forTo obtain a consensus pattern for further analysis, it is not required that all of the Bayesian clustering methods used yield the same optimal number of clusters K (as exemplified by the case of the stone marten). A consensus solution can be derived from the results of the methods supporting the modal K, and subsequently examined using multiple analytical tools . Again, IS value  and related measures . Despite the short distance, this differentiation was detected in all Bayesian analyses and in the sPCA Figs . The subth century and in the 1930s [Rivers and other hydrologic features have been recognised as obstacles to dispersal in martens ,23,47. Hhe 1930s .In contrast to the stone marten, here there was no clear consensus among the Bayesian clustering algorithms, but most of them  strongly supported the lack of population structure in Portugal. This might be expected given the high ecological plasticity of the red fox, and the adaptability and dispersal ability of the species have been invoked to explain the weak genetic structuring found in other studies ,49.ST < 0.03 [However, the uncorrelated frequency model in GENELAND inferred the presence of a north-south division, which was also suggested by the admixture models in TESS, albeit only when using a low posterior probability threshold of 0.5, and by the sPCA . DiscordT < 0.03 ,28. The T < 0.03 . It is nT < 0.03 .ST estimate for the north-south separation was low (≈ 0.01), although this may be, at least in part, due to the high heterozygosity in the data (Table D in ST and DEST (= 0.06–0.08). The BAYESASS estimates of migration rates between the two clusters identified by the uncorrelated frequency model in GENELAND were relatively low . However, migration rate estimates from BAYESASS can be inaccurate when FST < 0.03 [The north-south split could be an artefact due to an underlying IBD pattern, but the Mantel test showed a very weak correlation between genetic and geographic distances across Portugal . The FSTT < 0.03 : the posT < 0.03 . GENECLAT < 0.03 . The indT < 0.03 ,121.ST could result from the high heterozygosity of the markers used, but the G”ST and DEST values for the subdivision in the red fox were still lower than in the stone marten. Although a large effective population size may contribute to the low genetic differentiation in the red fox, the results of the Bayesian clustering models with admixture and of the BAYESASS analysis indicate recurrent gene flow.The north-south discontinuity is intriguing given the similar patterns observed here in the stone marten and reported previously in the otter and wild boar ,52, evenThus, the north-south division may be the legacy of past landscape fragmentation associated with the flood history of the Tagus River  123,129129, and The identification of hidden genetic structure is important for the management of species, even those that are currently abundant and widespread so as to guide management actions that can prevent them from becoming threatened in the future. In this study, we used a combination of Bayesian and multivariate methods to assess and compare population structure and spatial genetic patterns in two common mesocarnivores, the stone marten and the red fox, across the fragmented and heterogeneous landscape of Portugal. Our results are compatible with the known differences in ecological plasticity between the two species in the Iberian Peninsula, and are relevant for their conservation management in Portugal. Further work is needed to examine the role and influence of the Tagus and Sado rivers, as long-standing barriers with spatially and temporally variable permeability to gene flow, and of the highly modified contemporary landscapes adjacent to these rivers in shaping the observed spatial genetic patterns.From a methodological point of view, we wanted to draw attention to an approach to delimit the spatial boundaries of consistently identified genetic units, based on a consensus clustering solution of different Bayesian algorithms. As far as we know, the explicit use of a consensus solution among Bayesian clustering methods to spatially delineate and characterise population genetic structure was introduced by , but hasS1 FileMartes foina samples analysed in this study (Table A). Information on the Vulpes vulpes samples analysed in this study (Table B). Total and within-subpopulation microsatellite genetic diversity in Portuguese stone martens (Table C). Summary statistics for microsatellite loci in Portuguese red foxes (Table D). Cluster membership of stone marten individuals in the best run of, respectively from left to right, STRUCTURE assuming admixture and correlated allele frequencies (K = 3), TESS using the BYM admixture model (K = 3), TESS using the CAR admixture model (K = 3), and GENELAND for the correlated frequency model (K = 6) . Progressive partitioning results for the northern cluster of stone martens inferred by the uncorrelated frequency model in GENELAND . sPCA for stone martens . sPCA for red foxes .Information on the (PDF)Click here for additional data file.S2 FileTable A). Microsatellite genotypes of the red fox samples (Table B).Microsatellite genotypes of the stone marten samples ((XLS)Click here for additional data file.


Ambystoma maculatum from five ponds in Missouri, and analyzed them at 15 microsatellite loci. We calculated allelic richness, heterozygosity and effective population sizes for each life stage at each pond and tested for genetic differentiation (FST and DC) and isolation-by-distance (IBD) among ponds. We tested for differences in each of these measures between life stages, and in a pooled population of all life stages. All calculations were done with and without sibling pairs to assess the effect of sibling removal. We also assessed the effect of reducing the number of microsatellites used to make inference. No statistically significant differences were found among ponds or life stages for any of the population genetic measures, but patterns of IBD differed among life stages. There was significant IBD when using adult samples, but tests using embryos, larvae, or a combination of the three life stages were not significant. We found that increasing the ratio of larval or embryo samples in the analysis of genetic distance weakened the IBD relationship, and when using DC, the IBD was no longer significant when larvae and embryos exceeded 60% of the population sample. Further, power to detect an IBD relationship was reduced when fewer microsatellites were used in the analysis.In population or landscape genetics studies, an unbiased sampling scheme is essential for generating accurate results, but logistics may lead to deviations from the sample design. Such deviations may come in the form of sampling multiple life stages. Presently, it is largely unknown what effect sampling different life stages can have on population or landscape genetic inference, or how mixing life stages can affect the parameters being measured. Additionally, the removal of siblings from a data set is considered best-practice, but direct comparisons of inferences made with and without siblings are limited. In this study, we sampled embryos, larvae, and adult  An overarching goal of any study is to obtain accurate, unbiased estimates of the parameters of interest. In population and landscape genetics, it is often recommended that 25–30 individuals be sampled from each population . For manAmbystoma annulatum), A. maculatum), and Rana sylvatica). The drastic decline in abundance can also be seen in fish and insects. Nezara viridula) found mortality rates to be as high as 96% .https://dx.doi.org/10.6084/m9.figshare.1621318.v2.All data and code used in this study can be accessed from Figshare at To determine how researchers are currently collecting tissue samples from amphibians with complex life cycles, we conducted a literature search of the Scopus database of population and landscape genetic studies of amphibians. We used the search terms “amphibia*” , “microsatellite*” , and NOT “reptil*”  and limited the search to findings from Molecular Ecology, Conservation Genetics, Heredity, Biological Journal of the Linnean Society, Amphibia-Reptilia, Animal Conservation, Molecular Ecology Resources, Evolution, Plos One, or Journal of Zoology published through December 2012. For each study, we determined if different life stages were sampled and if the study gave an indication as to whether sampling multiple life stages influenced analysis or inferences made from the data.Quercus spp.) and hickory (Carya spp.), with varying amounts of sugar maple (Acer saccharum) and red cedar (Juniperus virginiana) in the understory  from five ponds at DBCA (2), depth (<1.2 m), age (27–47 yrs), and permanent hydroperiod. We sought to collect 25 adult and embryo samples and 30 larval samples from each pond. Adult salamanders were captured in mesh funnel traps placed in breeding ponds in March 2013, and tissue samples were obtained by removing 0.5 cm of tail tissue. Following oviposition, we sampled embryos by collecting a single embryo per clutch in April 2013. In June 2013 larvae were captured with dip nets, and to minimize the sampling of siblings, we collected larvae from the entire perimeter of each pond. Upon collection in the field, each tissue sample was placed in 95% ethanol and stored at −20 °C until DNA extraction.Our study was conducted at Daniel Boone Conservation Area (DBCA), in Warren County, Missouri, USA . This 1,derstory . There aderstory . Only ma at DBCA . Each of at DBCA , and havDNA was extracted from tissue using chelex-based resin . Approximately 2.5 mm × 2.5 mm of tissue was finely chopped with a sterile razor and was incubated at 60 °C for 2 hrs in 250 µL of InstaGene, vortexed, incubated for 20 min at 100 °C, then vortexed again. Following centrifugation, a 100 µL aliquot was removed and used as template DNA and the remainder was kept at −20 °C . NineteeFST and allelic richness  were calculated with the R package hierfstat (DC) were calculated with the R package adegenet (Ne) were made using the linkage disequilibrium method implemented in COLONY  for each life stage with siblings present. All population genetic measures were compared among life stages and between estimates made with and without siblings using analysis of variance (ANOVA) and paired t-tests. Due to small sample sizes, we bootstrapped our ANOVA analyses and conducted permutation t-tests to more robustly assess differences among life stages and removal of siblings.Before proceeding with analyses we created a data set free of full sibling pairs using COLONY . For ourierfstat , observeadegenet , and effn COLONY . The prohierfstat , we further assessed how the IBD relationship changed with the inclusion of larval and embryo samples. For this analysis, we varied the proportion of larval and embryo samples included with our adult samples. This was assessed at proportions ranging from 0  to 1  at increments of 0.05. At each increment, we assessed the mean and 95% confidence intervals of the Mantel r and the corresponding P-value based on 1,000 bootstrap samples of the data. We used the data set without siblings for this analysis and sampled each population to the minimum adult sample size (n = 18).For the isolation-by-distance (IBD) analysis we conducted simple Mantel tests correlating genetic distance with Euclidean distance between ponds. This test was repeated for all life stages, with and without siblings, and significance was assessed using 100,000 permutations using the R package ecodist . We testr and the corresponding P-value. We also calculated the observed heterozygosity and allelic richness at each mixture proportion and for each level of microsatellite subsampling. These estimates were averaged over all populations sampled.Concurrent with assessing the effects of mixing life stages, we also assessed the effects of reducing the number of microsatellites used in an analysis. Within the bootstrapping procedure for assessing the proportion of larval and embryo samples described above, we sub-sampled our microsatellite data set to include either 5, 10, or all 15 of the microsatellites. At each bootstrap iteration at each mixture proportion, microsatellites were randomly chosen to calculate Mantel We found that 20 out of 95 (21%) of studies meeting our search criteria on Scopus conducted population or landscape genetic analyses of amphibian species using mixed tissue sampling (searched on 13 February 2016). Five of these studies stated that one life stage was sampled only when the target life stage was not available . None ofAm_13, Am_60) were not polymorphic, and two loci  showed very little polymorphism and deviated significantly from expected heterozygosity values under Hardy Weinberg equilibrium (HWE). These four loci were removed from the dataset and all population genetic statistics were calculated using the remaining 15 loci  or within ponds and among life stages  in adults, 0.021 (±0.007) in larvae, and 0.019 (±0.014) in embryos, while DC averaged 0.226 (±0.025) in adults, 0.237 (±0.011) in larvae, and 0.240 (±0.030) in embryos  for adult-only samples, and increased to 0.231 (0.223–0.239) for larvae/embryo-only samples  when only adults were included and increased to 0.146 (0.140–0.152) without adult samples . When 10 microsatellites are used, the 0.05 threshold is exceeded when the proportion of larvae and embryos reaches 0.30–0.35 . The IBD relationship was not significant at any mixture proportion when only five microsatellites were used.Our assessment of increasing the proportion of larval or embryo tissue samples clearly demonstrated that the calculated Mantel ncreases . This pa P-value . When us samples . In cont samples . The 0.0There was a trend for the mean heterozygosity to decrease and mean allelic richness to increase as the proportion of larvae and embryos increased, but we note that these trends occur over a very limited parameter space . Adult-oOur literature search revealed that mixed tissues have been used in about one fifth of amphibian population genetic studies using microsatellites, despite the lack of knowledge concerning the effects that this may have on population or landscape genetic inferences. Collection of genetic samples from the field is likely influenced by numerous factors, such as the timing of life stage development and accessibility to sampling sites. Many of the studies that mixed tissues did so when the target life stage was not found and minimum sample requirements had to be met. Decisions made during sampling can undoubtedly influence the inferences made, as genetic variation within structured populations can vary spatially, temporally, or as a result of life history .FST differed substantially between life stages as well as from the mixed-tissue sample, but this difference was not statistically significant. Additionally, this pattern was not observed when pairwise genetic distance was measured using DC. The greatest impact of mixing life stages was evident in our tests of IBD among pond pairs. Only the tests using adults were significant, while larvae-only, embryo-only, and mixed-tissue samples showed little correlation with distance. The IBD relationship was strongest when genetic distance was measured using DC, and we found that the IBD relationship seen in adult-only samples decreased as the proportion of larvae and embryos included in the population sample increased. In our dataset, the IBD relationship, when assessed with DC, remained significant until the frequency of larvae and embryos in the population sample exceeded 60%. However, our power to detect a significant IBD relationship was substantially affected by the number of microsatellites used. When 10 of the 15 microsatellites were used, the IBD relationship became non-significant when the frequency of larvae and embryos exceeded 30%, and no significant IBD relationship was evident when only five microsatellites were used. As such, our results suggest the greatest effects of mixing different life stages may be evident in landscape genetic analyses assessing pairwise distances among populations, with different conclusions potentially being drawn from adult-only samples as compared to larvae, embryo, or mixed-tissue samples. Further, such relationships may be sensitive to the genetic distance statistic used as well as the number or polymorphism of the microsatellites used.In our study, however, we found little evidence for adverse effects of including siblings or with mixing tissue samples collected from different life stages when assessing levels of allelic richness, heterozygosity, or effective population size. Contrary to FST, which increased (insignificantly) after the removal of full siblings from the data, as well as from adults to embryos, to larvae. These increases in genetic differentiation in the embryonic and larval stages, as compared to the adults is predicted by population genetic theory (Rana luteiventris)  .The clearest result from our study was the proportion of samples that had to be removed from each life stage due to redundancy of siblings. It is actually quite unlikely that we sampled full sibling adults given their life history and longevity , but we In our study, we assessed IBD through the use of simple Mantel tests. We readily acknowledge the limitations and criticisms of the Mantel test for making robust inference e.g., . HoweverR. luteiventris and four populations of A. macrodactylum. We do note that the power to infer differences is likely greater in our study due to the large number of polymorphic microsatellites used, which was reinforced by our results of subsampling the number of genetic markers used. Perhaps of greater importance is the fact that our populations are relatively close together  and situated within continuous forest habitat. In contrast, To our knowledge, our study is the first attempt to determine how the sampling and mixing of different life stages affects genetic parameter estimates. It is unclear how differences in life history  alter the effects of sampling different life stages, as we currently do not have a mechanistic explanation for the patterns we observed. We found that mixed-tissue samples can lead to different conclusions when conducting spatial analyses, such as IBD, and these results would likely extend to more complex landscape genetic analyses as well. As such, we caution researchers to carefully consider the implications of mixing samples collected from multiple life stages. Our finding that population genetic parameters differed little with the removal of siblings or mixing of life stages was surprising and contrary to our predictions. In our study, we had a relatively small sample size from which to draw inference, although we note that it is not much smaller than that used by Like 10.7717/peerj.1813/supp-1Supplemental Information 1Click here for additional data file.