Scutellaria species in Taiwan. Scutellaria taipeiensis, which was originally assumed to be an endemic species of Taiwan Island, is shown to be part of the widespread species S. barbata based on the overlapping ranges of genetic variation and climatic niches as well as their morphological similarity. Rejection of the scenario of “early divergence with secondary contact” and the support for multiple origins of populations of S. taipeiensis from S. barbata provide strong evolutionary evidence for a taxonomic revision of the species combination. Further tests of a climatic effect on genetic variation were conducted. Regression analyses show nonlinear correlations among any pair of geographic, climatic, and genetic distances. However, significantly, the bioclimatic variables that represent the precipitation from late summer to early autumn explain roughly 13% of the genetic variation of our sampled populations. These results indicate that spatial differences of precipitation in the typhoon season may influence the regeneration rate and colonization rate of local populations. The periodic typhoon episodes explain the significant but nonlinear influence of climatic variables on population genetic differentiation. Although, the climatic difference does not lead to species divergence, the local climate variability indeed impacts the spatial genetic distribution at the population level.Spatial climate heterogeneity may not only affect adaptive gene frequencies but could also indirectly shape the genetic structure of neutral loci by impacting demographic dynamics. In this study, the effect of local climate on population genetic variation was tested in two phylogenetically close  Species delimitation is a subjective judgment of the level of divergence between organisms despite using objective evidences, while exploring the mechanisms causing genetic divergence is a more objective approach to describing the process of species divergence. In the genic species concept Wu, , introgrInterspecific gene flow between two genetically distinct species could be the result of two contrasting evolutionary hypotheses. The first hypothesis is ecologically based speciation with continuous gene flow during the speciation process. When species occur in sympatry, ecological selection should act to maintain their divergence. An alternative hypothesis addresses the introgression following secondary contact after the completion of speciation. Such ecologically divergent species might be able to interbreed with each other during the onset of divergence and secondary contact  and the recency of island formation  are two morphologically similar species with overlapping distributional ranges . In addition, the climate's effect on genetic divergence at the population level was further examined to understand the local adaption of these two allied species. Based on our genetic evidence, we suggest that the amount of genetic divergence between these two morphologically divergent organisms may not be sufficient to grant them the level of species.In this study, the genetic variation and genetic differentiation between Scutellaria barbata is distributed in northern Taiwan. Some populations were recorded from western and northeastern Taiwan. This species is usually scattered in the sallow hills, on farmland ridges and along roadsides, and is usually distributed sporadically, making it hard to form a dense population. Scutellaria taipeiensis is very rare and restricted to the Taipei Basin of northern Taiwan. It grows attached to bare rock or the surrounding soil, usually in sunny environments along trails . Identification of other tpe populations was based on the macromorphology and growth habit of the MK population. In total, 80 and 74 individuals of bar and tpe were sampled, respectively, for genotyping and sequencing. The seed coat patterns of all populations were further checked by Scanning Electron Microscopy (SEM).ly Table . BecauseSeeds were collected from all studied populations for SEM observation. An accelerating voltage of 15 kV was applied in order to obtain high image resolution signals. SEM was conducted using the tabletop SEM TM3000 .ndhF-rpl32 and rpl32-trnL were chosen for sequencing. The PCR amplification and sequencing conditions followed Chiang et al.'s  was used for testing the degrees of genetic variation between species, among, and within populations. We conducted the AMOVA using Arlequin 3.5  was estimated by 10 independent runs using one-million steps Markov chain Monte Carlo (MCMC) replicates after a 100,000-step burn-in for each run to evaluate consistency. The best grouping number was evaluated by ΔK  was used to examine the similarity and divergence of genetic components among populations. The BCA was performed using STRUCTURE 2.3.4  was used for distinguishing bar and tpe under common environmental pressures with recent interspecific gene flow (secondary contact) after completed speciation  of the pseudodata and transform them by the pls. To validate the best scenario, the general linear model (GLM) post-sampling regression adjustments on the best 5,000 simulated parameters were retained for obtaining the marginal density of models by the ABCestimator. The best evolutionary scenario was selected according to the Bayes factor and the ratio of marginal densities.Based on the estimated genetic variation and genetic structure and current geographic distributions, two evolutionary scenarios were proposed: the first is the demographic bottleneck of both n Figure ; the secs Figure  or in a r Figure . Testingr Figure . We usedhttp://www.worldclim.org/bioclim). Principal component analysis (PCA) of independent climatic variables to reduce the dimensionality defining the niche space allowed comparison of the integrity of Grinnellian niches between bar and tpe. Firstly, the variables with a high variance inflation factor (VIF) were removed to reduce the multicollinearity. This procedure was repeated until the VIF values of all remaining variables were < 10 and finally six bioclimatic variables were retained for the further analyses. Species occurrence data were collected from the Global Biodiversity Information Facility , herbarium records, public records available on the web, and our collecting sites. Unclear records and wrong identifications were ruled out. Finally, 56 records of bar and five records of tpe were used for the PCA. We further used multinomial logistic regression (MLGR) to test the effect of bioclimatic variables on the prediction of species occurrence. Because the simpler model of no correlation among predictors (bioclimatic variables) could not be rejected by the model of correlated predictors , we used the model with independent predictors for MLGR analysis. The significance of each predictor was tested by the type-II analysis of variance (ANOVA) Wald χ2 test.Grinnellian niches are defined as the scenopoetic environmental variables a species requires to survive, such as the temperature and precipitation, etc.  was used as the genetic distance metric. Geographic distance was estimated using the Euclidean distance according to three dimensional factors . Environmental distance was also calculated using the Euclidean distance with six retained Grinnellian niches variables. A partial Mantel test between genetic and environmental distances controlled for the geographic distance was also done. A multiple matrix regression with randomization (MMRR) was further performed to test whether the genetic distance responds to the variation of geographic and/or environmental distances. We first examined the correlation between the genetic distance and geographic distance and between genetic distance and environmental distance. Autocorrelation between the geographic and environmental distances was further examined. Finally, the joint effect of both geographic and environmental distances on genetic distance was examined. Regression coefficients of Mantel test (ρ) and MMRR (β) and their significance were determined based on 9,999 random permutations.To test how the geographic distance and environmental differences affect the genetic composition, the Mantel test of Spearman correlation was performed between genetic, geographic, and environmental (climatic) distances. Pairwise on Nei's  approachon Nei's  estimatebar and tpe, partial distance-based redundancy analyses  were performed. The first five principal components (PC1–PC5) were used to represent the genetic components, and six retained climatic variables were used as predictors conditioning on the geographic distribution (latitude and longitude). Analysis of variance was further used to test the significance of each predictor. The partial dbRDA was conducted using the R package vegan. We further analyzed the distribution of climatic variables along ordination axes using the GLM. We also used the MLGR to test the effect of bioclimatic variables on populations. The type-II ANOVA Wald χ2 test was used to test the significance of each bioclimatic factor.To understand whether the climatic variables (Grinnellian niches) explain the genetic distribution of populations of bar and tpe are variable and can be affected by growth and nutrient conditions, we specifically checked the seed coat patterns, another distinguishing trait in the reproductive organs, to confirm the stability of this character for species delimitation. Based on our observations, there is slight variation in the seed size among individuals but no obvious difference between species  for the outlier analysis for the microsatellite loci. No deviation from zero in Tajima's D  and no positive or negative outliers of microsatellite loci  is 0.143, 0.154, and 0.051, respectively, and the index θW is 0.314, 0.322, and 0.237, respectively. In total, when considering the indels six haplotypes were obtained, and the haplotype frequencies, distributions, and relationships  are illustrated in Figure He) in bar and from 0.189 ± 0.080 to 0.230 ± 0.069 in He in tpe  with a relatively small proportion of genetic variation within populations  is 3 and the second-best K is 2 based on the ΔK , a broader range of genetic variation was detected in l Figure . A furthr Figure . The clu2 = 32.33, df = 1, P = 1.301e–08) revealed species divergence between bar and tpe but no species-level differences were found in PC1 (P = 0.872), LD1 (P = 0.810), or LD2 , much lower than the founder-speciation scenario . The estimated ancestral population size of bar is suggested to have decreased under the founder-speciation scenario , but the founder populations of tpe have a very small effective population size  of each locus per generation in secondary contact scenario has been estimated  of tpe  than the single origin , indicating a rejection of the monophyly of tpe. Similar to the estimation above, the effective population size of populations of tpe is smaller than that of bar  recent divergence and (2) sharing common ancestral polymorphisms that results in long coalescence times. This result also suggests that the divergence time is insufficient for complete speciation. Therefore, populations of both bar and tpe are combined into a single taxonomic group for the further analysis of local climatic adaptation. However, the very small migration rate on average , named the Mean Diurnal Range], bio8 (mean temperature of wettest quarter), bio9 (mean temperature of driest quarter), bio13 (precipitation of wettest month), bio18 (precipitation of warmest quarter), bio19 (precipitation of coldest quarter). Among these six bioclimatic variables, the first three  belong to the temperature dimension, while the last three  are the precipitation dimension.bar and tpe well  value for all models is 0.999, indicating that 99.9% of the records were correctly predicted. The climatic suitability of each species is proportional to similar climatic variables. Bio18 (precipitation of warmest quarter) and bio2  are the first two contributors to the species distribution model in bar  and in tpe .Ecological niche modeling (ENM) was further used to predict the suitable distributions of both tpe  are roughly consistent with the currently known distributions, except for a certain probability (< 50%) of suitable habitats predicted in the Hengchun Peninsula of southern Taiwan for the P = 0.0872), between genetic and geographic distances , or between genetic and environmental distances  and non-significant effects of geographic  and environmental distances  on the change of genetic distance were obtained by MMRR analyses  to be meaningful. If only the geographic clusters are considered  without genetic information, all the bioclimatic variables cannot predict the occurrence of populations by MLGR independently. Nevertheless, the joint effect of the four factors, bio8 × bio13 × bio18 × bio19, can predict the populations by MLGR significantly .Despite nonlinear correlation between the climatic differentiation and genetic distance, we still wondered whether the local climatic heterogeneity explains the genetic variation of populations. When conditioning on the geographic distribution, we found that 59.5% of the variation is explained by climatic variables  and the rounded concentric tuberculae type of nutlet coat that distinguished it from bar  in contrast to the long coalescent time of the Taiwanese populations of bar , the time that Taiwan Island was most recently connected to continental Asia . Multiple origins from a single progenitor usually are seen as an evolutionary mechanism for the recent and rapid radiation of island species rather than the mature radiation that sometimes leads to a high diversity of continental species  and tpe (24.66 ± 2.947°C) in means  and variance . This is probably because the overlapping distribution ranges of bar populations and tpe populations result in a similar climatic situation, thus causing a false positive for species prediction by the climate factor.The Grinnellian niche that is comprised of temperature and precipitation is important and related to plant speciation, in particular in those budding speciation cases with peripheral ranges or distributional overlaps . These two variables belong to the precipitation dimension, standing for the precipitation of the warmest quarter and the precipitation of the wettest month, respectively (Supplementary Table P = 1.75e–12). According to the common characteristics of bio13 and bio18 as well as their significant correlation, we suggest that the main determinant governing the genetic structure of populations is actually the precipitation from late summer to early autumn. This period is during the typhoon season (late summer and early autumn) in Taiwan and just after the flowering and fruiting season . Precipitation during seed germination and growth could affect the demographic size and could also influence the success of seed colonization. In addition, since the seed dispersal of Scutellaria species is thought to be spread by water  and the warmest quarter of all recorded populations is July to September  in Taiwan (MOST 102-2621-B-003-005-MY3) and also subsidized by the National Taiwan Normal University (NTNU), Taiwan.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.


The long-term performance of different selection strategies was evaluated via simulation using the example of a local cattle breed, German Angler cattle. Different optimum contribution selection (OCS) approaches to maximize genetic gain were compared to a reference scenario without selection and truncation selection. The kinships and migrant contribution (MC) were estimated from genomic data. Truncation selection achieved the highest genetic gain but decreased diversity considerably at native alleles. It also caused the highest increase in MCs. Traditional OCS, which only constrains kinship, achieved almost the same genetic gain but also caused a small increase of MC and remarkably reduced the diversity of native alleles. When MC was required not to increase and the increase of kinship at native alleles was restricted, the MC levels and the diversity at native alleles were well managed, and the genetic gain was only slightly reduced. However, genetic progress was substantially lower in the scenario that aimed to recover the original genetic background. Truncation selection and traditional OCS selection both reduce the genetic originality of breeds with historical introgression. The inclusion of MC and kinship at native alleles as additional constraints in OCS showed great potential for conservation. Recovery of the original genetic background is possible but requires many generations of selection and reduces the genetic progress in performance traits. Hence, constraining MCs at their current values can be recommended to avoid further reduction of genetic originality. The traditional approach of OCS  provides a solution to solve the first problem. It aims to maximize genetic gain while controlling the rate of inbreeding by optimizing the genetic contribution of each selection candidate to the next generation  (High-density marker panels of single-nucleotide polymorphisms (SNPs) allow us to obtain more accurate estimates of kinship than pedigrees, as it is common for pedigrees to contain errors (te (IBS) . The secte (IBS) . Moreovete (IBS) . The useoptiSel . The QTL effects were sampled from a γ distribution with a shape parameter of 0.4  of animal optiSel . The first and second principal components (PC1 and PC2) separated animals in the simulated base generation according to their breed . However, they were slightly larger because some segments were split by crossing-over into small pieces, so they did not contribute to the kinship estimated from offspring haplotypes. This indicates that the estimate obtained from offspring haplotypes is slightly biased. The rate of inbreeding estimated from segments remained 0.5% per generation in traditional OCS, in accordance with the constraint level setting.The predictions from Kinship In general, it must be distinguished whether MCs predominantly originate from closely related ancestors originating from a single high-yielding breed, or if different unrelated breeds have been used for upgrading. In the Angler breed, they predominantly originated from related Holstein ancestors, so reducing MC in OCS-III was meant to reduce the amount of genetic material contributed by Holstein cattle, which had a positive effect on the genetic diversity. Thus, the mean kinship It has been suggested that the marker density of the SNP chip used, the minimum length of the shared segment, the number of genotyping errors allowed, and the minimum number of SNPs allowed in a single shared segment are likely to remarkably influence kinship estimates based on shared segments (Estimated parameter values for MC, segments  until thi.e., the average observed heterozygosity Different parameters can be used to measure genetic diversity, such as the percentage of polymorphic loci, the number of alleles per locus, expected heterozygosity, etc. . The gen alleles .The additive genetic variance r Effect  and the Advanced OCS strategies enable the achievement of a balance between the different breeding goals of populations with historic introgression, which are to improve the genetic progress, recover the original genetic background, and conserve genetic diversity. Here, truncation selection and traditional OCS achieved the highest genetic gain, but both reduced the genetic originality of the breed by depleting diversity at native alleles and increasing MCs. However, maintaining genetic originality is crucial for conserving breeds with historical introgression. The inclusion of MC and kinship at native alleles as additional constraints in OCS showed great potential for conservation. Recovering the original genetic background is possible but requires many generations of selection and reduces the genetic progress. Thus, it is essential to set an appropriate constraint for MC in order to balance both breeding goals, which are to achieve genetic progress and to recover the original genetic background of local breeds.www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300272/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Millepora platyphylla colonies, a hydrocoral species known for its extensive morphological plasticity among reef habitats. We aimed to determine whether mosaicism and chimerism were related to specific reef habitats and/or colony morphologies. Our results show that intracolonial genotypic variability was common (31.4%) in M. platyphylla at Moorea, French Polynesia, with important variations in its frequency among habitats (0–60%), while no effect of morphology was observed. Mosaicism seemed responsible for most of the genetic heterogeneity (87.5%), while chimerism was rarer. Some mosaics were shared among fire coral clones indicating that mutations could be spread via colony fragmentation. Further, the genotypic variability among clones suggests that colonies produced asexually through fragmentation have the potential to accumulate their own mutations over time. Such mutation dynamics might have important implications for the adaptive potential of long-lived reef-builder populations that are predominantly sustained through asexual reproduction.Intracolonial genotypic variability is described in many colonial organisms and arises from mosaicism (somatic mutation) and/or chimerism . Both processes provide an additional source of genotypic variation in natural populations and raise questions on the biological significance of colonies having more than one genotype. Using fifteen microsatellite markers, we screened for potential genetic heterogeneity within  An individual is intrinsically defined as reproductive, physiologically autonomous, genetically unique and homogeneous3, but there are many studies that question this definition4. For instance, it has been notably recognised that for colonies of social insects, physiological unity is not respected because individuals cooperate with others to form a “superorganism”, acting as though the colony was one single individual5. Asexual reproduction is also common in natural populations of countless plants and animals, where individuals are not genetically unique6. At last, the occurrence of genetic heterogeneity within a single individual has been documented in populations of protists, fungi, plants and animals4 and is now considered a common phenomenon.Understanding evolutionary strategies in species largely relies on the concept of individuality, where each individual represents the unit on which selection pressures occur7. In contrast, chimerism originates from the fusion of at least two individuals of the same species  and requires specific environmental conditions and species’ life history traits8. Based on such definitions, chimerism is expected to generate a greater genetic variation within the successfully merged colony compared to mosaicism. Chimerism is generally rarer than mosaicism in natural populations due to restricting allorecognition systems4. This process mostly occurs in seaweeds and colonial marine organisms with a dispersive pelagic phase, such as sponges, hydroids, bryozoans, ascidians and corals11. Mosaicism, by contrast, is a widespread mechanism of many clonal plants and animals with long life-spans14.There are two main processes that can lead to intra-individual genotypic variability in natural systems: mosaicism and chimerism. Mosaicism is the outcome of intrinsic genetic changes within a single colony caused, among other processes, by somatic mutations16 and intra-individual competition18. However, the co-occurrence of many different genotypes within an individual also generates additional genotypic variation in natural populations. Such variability may result in more versatile phenotypic traits , which can further increase the potential for adaptation21 through intra-individual selection pressures 23. Even so, this additional genotypic diversity will only affect the fitness of an individual and not its population, unless it is possible for the genetic variation to be passed on to the gametes. Individuals having more than one genotype may thus facilitate the adaptive potential of long-lived organisms that reproduce primarily through asexual reproduction. As both chimerism and mosaicism generate intra-individual genotypic variation, evaluating the occurrence of these processes in threatened species such as reef-building corals may carry important implications for their conservation.Chimeras and mosaics may confer both benefits and disadvantages at the individual level. Theoretically, intra-individual genotypic variability may promote disruptive internal conflicts threatening an organism’s ability to function, such as developmental instability29. Due to the existence of multipotent stem cells (MPSCs) in colonial reef organisms30, the propagation of somatic mutations within a colony is likely to occur14 when mutations are not detrimental. These mutations are potentially passed on to the next generation of gametes due to the capacity of MPSCs to generate germline stem cells (GSCs)31. In partially clonal reef organisms, somatic mutations can also be spread by means of asexual reproduction pathways, which include fragmentation, budding, polyp bail-out, asexually produced planula and embryo breakage33.Many studies have shown the occurrence of intracolonial genotypic variability in scleractinian corals34, sponges35 and scleractinian corals36) has improved over the last decade. Until very recently, such information was unavailable for Millepora hydrocorals37  despite their major contribution to the reef framework in some reef ecosystems38. Millepora species inhabit a wide range of habitats40 and often grow into large colonies that pre-empt space and compete with scleractinian corals41. Fire corals alternatively reproduce by shifting from asexual pathway of fragmentation to sexual reproduction via gonochoric broadcasting of both medusoids and planula larvae42. The simultaneous use of sexual/asexual reproductive modes has been recorded in Millepora platyphylla at Moorea, French Polynesia, where habitat specific environmental conditions are thought to determine the levels of clonality and intraspecific variations in colony morphology43. Such variability in life history traits leads to marked differences in population structure40 and genotypic diversity among reef habitats43. However, the sets of the biological and environmental conditions influencing intraorganismal genotypic variability are still ambiguous.Our understanding of intracolonial genotypic variability in colonial reef species ; two on the fore reef: mid slope and upper slope, and three in the lagoon: back reef, fringing reef and patch reef  to test for intracolonial genetic variability in fire corals . To determine whether the colony morphology influences the prevalence of intracolonial genetic variability, we classified each colony in one of the following morphologies: 1) massive: solid colonies, roughly hemispherical in shape, 2) encrusting: thin colonies growing against the substratum or 3) sheet tree: encrusting bases with vertical bladelike outgrowths40. The size of each colony (standardised as the projected surface in cm²) was estimated from 2D photographs using ImageJ 1.4 f software44. Photographs were taken from above the colony and included a plate of known dimensions positioned next to each colony. Overall, a total of 255 small fragments (51 colonies × 5 samples) of tissue-covered skeleton (<2 cm3) were collected and preserved in 80% ethanol and stored at −20 °C until DNA extraction. Field experiments were approved by the Presidency of French Polynesia (#0085) and performed in accordance with relevant Polynesian regulations.Between April and December 2013 field surveys were conducted on the north shore of Moorea, French Polynesia, at three different locations  across five reef habitats; two in the fore reef: mid slope (13 m depth) and upper slope (6 m depth), and three in the lagoon (<1 m depth): back reef, fringing reef and patch reef . The PCR protocol included an initial denaturing step of 5 min at 95 °C, followed by 40 cycles of 30 sec at 95 °C, 90 sec at 57–63 °C, and 30 sec at 72 °C, and by a final 30 min elongation step at 60 °C. PCR products were sent to GenoScreen platform  for fragment analysis and were visualised using an Applied Biosystems 3730 Sequencer. An internal size ladder  was used for accurate sizing and alleles were scored and checked manually using GENEMAPPER v.4.0 . Samples that were ambiguous in their scoring were re-amplified and re-scored, as for missing alleles. Alleles were individually re-scored by a second and third person to ensure accurate genotyping. All peak profiles that were faint or ambiguous (i.e. multiple peaks) were considered as missing data and only samples with no more than two missing loci were retained for further genetic analyses.Samples were incubated at 55 °C for 1 hr in 450 µL of digest buffer with proteinase K . Genomic DNA was extracted using a QIAxtractor automated genomic DNA extraction instrument, according to manufacturer’s instructions. Samples were amplified and genotyped at fifteen microsatellite loci shown to be coral-specific and polymorphic in 47. Multilocus genotypes (MLGs) were produced for each sample and compared within each colony to detect the occurrence of intracolonial genotypic variability. The genotype probability (GP) was estimated for each locus and for a combination of all loci using GENALEX v.6.548. Repeated multilocus genotypes were also identified in GENALEX and were considered as clone mates at GP < 0.001  or chimerism . In previous studies, mosaic individuals were identified based on the number of divergent loci from the main genotype, i.e. only one or two loci (as in28) since mutations remain rare events49. In contrast, a greater number of loci and allelic differences were expected in chimeras, i.e. when two genetically distinct colonies merge. Based on the stepwise mutation model of microsatellite markers50, we estimated the number of repeat units that were added or subtracted during a mutation event. Divergent alleles from the main genotype caused by multiple-step mutations and large allele differences are most likely due to chimerism rather than somatic mutations. Stepwise mutations were identified over all loci for each deviating genotype and averaged per habitat and morphology (percentage of stepwise mutations).Controls for the presence of null alleles and large allele dropout were performed with MICRO-CHECKER v.3.729. Here, mosaic individuals and chimeras were identified based on a Bayesian clustering analysis using STRUCTURE51. Clustering analyses were performed to ensure non-biased detection of deviating genotypes following the protocol used in Schweinsberg et al.29. Initial STRUCTURE runs were used to determine the most likely number of clusters (K) in each population of M. platyphylla, i.e. within the five reef habitats: mid slope, upper slope, back reef, fringing reef and patch reef. Runs were performed with the default setting, a burn-in period of 50 000, 50 000 MCMC repeats and 10 iterations per K. The results were uploaded to STRUCTURE HARVESTER52 and the most likely K was retained for a second run in STRUCTURE with a burn-in period of 500 000, 500 000 MCMC repeats, 10 iterations and uniform prior setting. The results were once again uploaded to STRUCTURE HARVESTER and the resulting merged dataset was analysed to estimate cluster assignment. Based on our definitions of chimeras and mosaics using microsatellite data, we assumed that only fragments of the same colony having a genetic variation of at least 60%, whatever the number of divergent loci, were the result of chimerism29. All other deviating genotypes were considered as mosaic colonies.Bayesian clustering analyses have been used to identify chimeras based on their cluster assignment probability. Chimeras have to include genotypes that differ from the main genotype and belong to a different cluster53.Chi square tests with Monte Carlo simulation (1000 replicates) were used to assess for differences in the relative numbers of colonies that harbour single genotypes, somatic mutations and chimeras among the five reef habitats and three colony morphologies. Differences in stepwise mutations  among habitats, morphologies and between mosaic colonies and chimeras were also tested using Chi square tests with Monte Carlo simulation. Pearson’s correlation coefficient was used to determine whether the occurrence of intracolonial variability increased with the colony size. All statistical analyses were performed in the R programming environment v2.15.1M. platyphylla, 65 multilocus genotypes were identified (Table 2) compared to other habitats . The occurrence of intracolonial genotypic variability differed significantly among the five surveyed habitats  harboured more than one single genotype  and a second one (M5) had four divergent loci (mostly from one- and two-step mutations) and was shared with one of its clone mates  resulted in another deviating genotype in the third clone . In the mid slope, the two mosaic colonies differed by five to six loci from the main genotype mostly due to one- to three-step mutations.Based on our definitions of mosaicism and chimerism, 14 mosaic colonies and 2 chimeras were identified in total among the 51 colonies of M. platyphylla growth forms with most of the somatic mutations occurring in massive (7 out of 14) and sheet tree morphologies , while important variations in its frequency were found among habitats. Our results reveal that chimerism is restricted to the patch reef habitat and thus seemed rarer compared to mosaicism. Even so, the fusion between siblings may increase opportunities for the development of chimeras in fire corals. Furthermore, the genetic dissimilarity among clones revealed that asexual reproduction affects the prevalence of mosaic colonies in fire coral populations. Somatic mutations that arise within a colony can be spread in the population via its fragmentation and asexual fragments produced from the same mother colony have the ability to accumulate their own mutations over time. Such propagation and accumulation of somatic mutations may represent an efficient means to increase the genetic diversity in M. platyphylla populations at Moorea, which are predominantly sustained through asexual reproduction43.This study demonstrates that the occurrence of intracolonial genotypic variability in 18. However, recent investigations have demonstrated that genotypic heterogeneity is widespread in scleractinian corals29. Our results show that intracolonial genotypic diversity is also common in hydrozoans, such as the fire coral M. platyphylla, with important variations in its frequency among reef habitats. Our genotypic data from 51 colonies (with five replicates sampled per colony) revealed a high proportion of genetically heterogeneous colonies (31.4%). Such occurrence of intracolonial genotypic variability corroborates recent observations in branching Acropora corals , but is much higher when compared to another branching coral, Seriatopora hystrix . A recent study based on limited sampling (10 colonies) and less molecular markers (6 microsatellite loci) has demonstrated that intracolonial genotypic variability was a rare phenomenon in M. platyphylla at Moorea (10%)37. Here, our panel of fifteen microsatellite loci showed that populations of M. platyphylla can display up to 60% of genetically heterogeneous colonies, which is much higher than what was previously observed in both scleractinian and hydrozoan corals37. Increasing the number of loci and colonies screened most likely increases the discriminative power to detect intracolonial genotypic variability in natural populations, a phenomenon that may have been underestimated in previous studies. However, it has to be noted that our definitions of mosaicism and chimerism varies from some other studies. Some have compared allelic differences within a heterogeneous colony to the one observed at the population level, i.e. allelic differences expected from sexual reproduction27. This approach is less conservative compared to the one we used and yet our proportions of heterogeneous colonies were higher. When compared to other studies with the same approach that we used37, the occurrence of intracolonial genotypic is similar or higher to the ones recorded in scleractinians and hydrocorals.The occurrence of intracolonial genotypic diversity was traditionally assumed to be a phenomenon of rare exception54. Colonies were collected at different depths leading to varying exposure to UV-induced DNA damage55, which often derives in somatic mutations56. Variations in the proportions of heterogeneous colonies among the five surveyed habitats (0–60%) seemed unrelated to differences in their exposure to solar radiation. In Moorea, fire corals are exposed to high solar irradiance in the back and fringing reefs57. However, both of these shallow habitats (<1 m depth) were characterised by low proportions of heterogeneous colonies  and were similar to those of the mid slope population (20%) where irradiance is lower. Rather than UV radiation variability, the occurrence of intracolonial genotypic variability in M. platyphylla might be related to habitat specific life history strategies. Populations of fire corals can evolve differences in reproductive and morphological traits among contrasting reef habitats43, which may further influence the opportunity for chimerism and mosaicism to occur36.Fire corals were sampled in five different reef habitats where colonies were exposed to different environmental conditions. The variability of environmental settings can potentially influence the rate of somatic mutational divergence within a species29). This result further suggests that chimeric fusions between conspecific are rare events in dynamic environments such as coral reefs. Two chimeras were detected in the patch reef habitat, where M. platyphylla primarily reproduce through fragmentation rather than sexual reproduction43. In Moorea, fusion between siblings is likely to occur as fire corals have limited dispersal abilities and are often aggregated due to the co-settlement of their larvae . Puill-Stephan et al.58 demonstrated that high levels of relatedness between juvenile corals correlated with late maturation of allorecognition. The fusion of siblings could thus be related to a low conspecific acceptance threshold and/or a delay in allorecognition maturation for Millepora hydrocorals, as described in some hermatypic corals26. Such delay in the recognition system can increase opportunities for chimeric fusion between adjacent recruits (i.e. early life stages), which seems to offer advantages. Advantages include the establishment of a colony with an increased genetic repertoire and a reduced onset of reproduction, increased competitive capabilities and growth during early development and reduced colony mortality61. While enhancing intracolonial genetic heterogeneity, chimerism may result in different expressed phenotypes that might each withstand a different set of environmental pressures.A low proportion of chimeras was identified in the fire coral population , which is similar to earlier reports for scleractinian corals . This limitation in size will further lower the accumulation of mutations in adult corals. Nevertheless, mosaicism was found in colonies of various sizes , with some of the largest colonies being genetically homogeneous . In partially clonal organisms, such variability in the accumulation of mutations among size classes can be related to small recently fragmented clones that might have accumulated mutations before their fragmentation. For Millepora hydrocorals, clones are most likely produced by colony fragmentation as the production of asexual larvae has never been described so far for this genus. Furthermore, as suggested by our results, clones are potentially produced through different fragmentation events (multiple generations). Such a life history strategy highlights the importance of aging clones within genetic lineages (as described in62) rather than estimating colony size to better understand the mechanisms behind mosaicism in reef-building corals.Fourteen adult colonies exhibited genotypic variability due to mosaicism. This indicates that the accumulation of somatic mutations might well be a common phenomenon in M. platyphylla is also morphologically variable and can have massive, encrusting or sheet tree morphotypes. In this study, mosaicism was identified in all M. platyphylla growth forms, but mostly in the massive and sheet tree ones. In many colonial organisms, such as long-lived trees and corals, branching growth forms usually exhibit more deviating genotypes throughout the entire colony due to mutations that only occur within isolated branches63. On the contrary, the propagation of somatic mutations is less likely in massive growth forms because polyps are in close contact with one another. Such an interaction favours intracolonial competition, which often results in the elimination of alternative mutant cells18. In the upper slope, most of the colonies grew as isolated vertical blades on encrusting bases (i.e. sheet tree), which is similar to the branching morphology. Consequently, somatic mutations would be expected to be more abundant in sheet tree colonies. However, half of the mosaic colonies were observed within massive colonies and raise questions on whether colony morphology influences mosaicism processes. Schweinsberg et al.29 also demonstrated a conflicting pattern of mosaicism among coral growth forms, whereby the highest and lowest proportions of mosaic individuals were both detected in branching species. Whether more extensive studies could verify an increased accumulation of somatic mutations in branching corals (or tubular or sheet) compared to massive or encrusting growth forms remains to be determined.43. Furthermore, the morphological plasticity of fire corals has been reported to highly influence the level of clonality among reef habitats at Moorea due to differences in growth form’s sensitivity to fragmentation43. Regardless, there was no effect of the morphology on the occurrence of mosaicism within a colony. However, three clone mates were identified in the upper slope and were comprised of four different genotypes. Some genotypes were shared among asexual fragments (clones produced through fragmentation) and others were found only in one of the three fragments. This result indicates that when fire corals spread via colony fragmentation, the fragments inherit somatic mutations from their mother colony, in addition to acquiring their own over time. Hence, clonal reproduction can often result in mosaic colonies having deviating genotypes of more than four divergent loci, although mosaicism was commonly thought to induce divergent genotypes at one or two loci49. Furthermore, it is also possible that mosaic colonies result from multi-step mutations in long-lived organisms, such as fire corals. Consequently, there was no difference in the stepwise mutation pattern among heterogeneous colonies that were produced through mosaicism or chimerism, although this latter process was thought to induce more mutation steps within the merged colony. Considering the common occurrence of mosaicism in M. platyphylla, this process might have important implications for its population genetic variation.Despite our random sampling scheme, clone mates  were collected in almost all reef habitats with the exception of the mid slope. The absence of fire coral clones in this latter habitat must be related to the high investment in sexual reproduction reported at Moorea, where clones were less abundantM. platyphylla, a species that mostly relies on colony fragmentation for colonisation and population persistence43. Our microsatellite data showed that clonal genets can accumulate allelic mutations that can be spread in the population via asexual reproduction. However, further investigations are needed to ensure that there is no barrier to prevent somatic mutations of stem cells from being spread in the population via the next sexual generation in M. platyphylla. More studies on the occurrence of mutations occurring in coding and/or regulatory regions of the genome are also needed to examine whether such genetic variation become functionally variable under strong selection pressures. Such mutation dynamics can potentially result in more versatile phenotypic traits and facilitate adaptation processes of partially clonal organisms66. Nevertheless, the high level of standing genetic variation that results from sexual recombination in M. platyphylla43 might as well be far greater and thus more influential on adaptation of fire coral populations than somatic mutations. Even if chimerism is less common in the studied population, this process can confer various ecological advantages and can further benefit adaptation. Regardless of the intrinsic costs incurred with chimerism, such as cell competition and parasitism18, chimeras have the ability to present their best-fitted genetic combination in response to environmental changes24. It is thus imperative to expand studies of intracolonial genotypic variability based on neutral microsatellite markers and to further include functional genes that underpin coral physiology, which often correlates with adaptive advantages.Overall, our study reveals that mosaicism is a very promising process to increase genotypic variability in Supplementary Tables


Tympanuchus cupido pinnatus), where translocations (1992–1998) seemingly rescued genetically depauperate Illinois populations. We re-evaluated this situation after two decades by genotyping 21 microsatellite loci from 1831 shed feathers across six leks in two counties over 4 years (2010–2013). Low migration rates (less than 1%) established each county as demographically independent, but with declining-population estimates (4 year average N = 79). Leks were genetically similar and significantly bottlenecked, with low effective population sizes . Genetic structure was defined by 12 significantly different family groups, with relatedness r = 0.31 > half-sib r = 0.25. Average heterozygosity, indicating short-term survival, did not differ among contemporary, pre- and post-translocated populations, whereas allelic diversity did. Our results, the natural history of GRPC  and its controlled immigration suggest demographic expansion rather than genetic rescue. Legal protection under the endangered species act (ESA) may enhance recovery, but could exacerbate political–economic concerns on how best to manage ‘conservation-reliant’ species, for which GRPC is now an exemplar.A central question in conservation is how best to manage biodiversity, despite human domination of global processes (= Anthropocene). Common responses  forestall potential extirpations, yet have an uncertain duration. A textbook example is the greater prairie chicken (GRPC:  It has 1.1.Results are less controversial when impacts of the Anthropocene are measured on biodiversity. For example, many species are now listed as threatened and endangered (T&E) under the ESA (US Endangered Species Act), with recovery a prolonged process at best. Furthermore, 84% are now recognized as ‘conservation-reliant’ , meaning1.2.‘Genetic rescue’  is a mitYet despite these caveats, genetic rescue is viewed as a positive conservation tool, largely owing to its perceived success with iconic species: greater prairie chicken , Europea1.3.Yet translocations have drawbacks ,16, as dInterestingly, three of the four iconic species above revealed mixed results following re-examination. For European adder, the positive trajectory was reviewed  and subs1.4.Tympanuchus cupido pinnatus; Herein, we re-evaluated the fourth icon, the greater prairie chicken  estimates of genetic variability for GRPC differ significantly between pre- and post-translocation; and (ii) post-translocation estimates do not differ significantly from those more contemporary. We then explore our findings within a broader context that involves the ESA and the issue of conservation-reliant species, and propose several potential mitigation strategies that may separate GRPC from its status as an exemplar of conservation reliance.2.2.1.Approximately 4300 shed feathers were collected at six leks in Prairie Ridge State Natural Area  or linkage disequilibrium (LD), and rarefaction procedures for population genetic assays are provided in electronic supplementary material, part B.2.2.capture [Nc) of leks and counties. The mark/recapture of genotypes was then compared statistically with census estimates derived from visual counts of males at leks each year.Capture histories were imported into Rcapture , a packadNe: [Ne) for each county and lek. Breeding population size (Nb) was also estimated by county using a two-sex, no-sex change model in AgeNe [Nb/N and Nb/Ne ratios for management relevance.A linkage disequilibrium estimator (LdNe: ) was empin AgeNe , with thin AgeNe . Equal s2.3.Ho) empirically derived from our microsatellite data against expected heterozygosity (He) under Hardy–Weinberg mutation–drift equilibrium (HWE) [Ho was significantly greater than He for a population under HWE. A variety of statistical approaches and models of microsatellite evolution were employed in our bottleneck analyses, the most powerful being the Wilcoxon signed-rank test [Demographic parameters are important for long-term population persistence, yet are influenced by genetic attributes inherent to small populations . Here weum (HWE) . A bottlank test  and the ank test . A mode-2.4.tructure v. 2.3.3, [K = 1–16 (where K = numbers of aggregates). To test for consistency, the program was run for 1 000 000 generations, with the first 100 000 as burn-in, and with 20 iterations per K-value. The appropriate K-value was determined by deriving ΔK [tructure Harvester [K) so as to visualize population assignments [To assess genetic structure, an admixture model  was appiving ΔK  using Starvester , and resignments . Microsaignments .FST values were derived to assess genetic divergence among leks and counties, whereas isolation by distance (IBD) was tested using a Mantel test . Average relatedness (r) within and among leks was estimated with 1000 bootstrap replicates ; second cousin (r = 0.063); first cousin (r = 0.125); half-sib (r = 0.25); parent/offspring or full-sib (r = 0.5). Average relatedness within and among gene pools was also derived and then tested for significance using a one-sample t-test [enalex v. 6.5; [Pairwise  v. 6.5; ). Averagncestry; ). A contncestry; , with rencestry;  gauged ae t-test . The cor v. 6.5; ).2.5.All temporal comparisons employed the six-locus dataset, in that pre- and post-translocation values were derived solely from these loci . Owing tHo) and allelic richness (AR) were compared among pre-translocation, post-translocation and contemporary Marion and Jasper counties populations (2010–2013), using Welch's t-test for unequal variances. This approach is more robust than Student's t-test and maintains type I error rates despite unequal variances and sample sizes. Values for contemporary populations (2010–2013) were derived from our data, whereas estimates for pre- and post-translocation were calculated using original summary statistics [http://stats.stackexchange.com/questions/30394/how-to-perform-two-sample-t-tests-in-r-by-inputting-sample-statistics-rather-tha) executed in R [http://www.ajdesigner.com/phpstatistics/standard_deviation_sample.php).Mean values for observed heterozygosity . These represented 88 unique males , with 963.2.N), under the assumption of a 1 : 1 gender ratio.Population estimates, as generated from the capture/recapture of male genotypes on each lek, did not differ significantly from those produced from visual count data . EstimatNe were uniformly low , with Jasper County Ne = 12.7 and Marion County Ne = 13.5 . Ne for leks averaged 15.9 and varied between 2.9 and 38.4 . The number of breeding adults (Nb) in Marion County was estimated as 24, whereas that for Jasper County was 17.Estimates for Ne/N effectively link demographic and evolutionary processes across a wide range of taxa, as reflected by the fact that half its variance is explained by only two life-history traits: age at maturity and adult lifespan [Nb/N, Ne/N and Nb/Ne, as averaged over both counties, were quite low. The 4 year average for GRPC (i.e. Ne/N = 0.189) is considerably lower than that recorded for sage grouse .3.3.FST estimates were significantly different between counties, but not among leks within counties  resident each year at the same lek. counties . Linear The mean migration rate averaged 0.9% between Marion and Jasper counties, establishing each as a demographically independent unit   rather than leks. Family groups were unrelated between counties, yet differed significantly within each across both 21- or six-locus datasets . Average relatedness within and between counties exceeded that of half-sib (i.e. r = 0.25), with Jasper r = 0.286 and Marion r = 0.337. The average for both counties was r = 0.311 (p < 0.006). Even though individuals reflected high lek fidelity, family groups did not, with most evenly distributed among leks within respective counties, a pattern consistent with natal dispersal.Each population was subdivided into six gene pools that equated to family groups  leks also reflected the signatures of a historic bottleneck .3.4.a). Heterozygosity is a reliable gauge for the loss of genetic variation and a good predictor of the potential for a population to evolve. However, our pairwise comparisons did not differ significantly among pre-, post- and contemporary evaluations (table 3b). Pre-translocation AR for Jasper County differed significantly from post-translocation and contemporary estimates . However, statistical significance can be questioned, given the undefined locations of the pre-translocation samples and the broad disparity in the timing of collection.The 21- and six-locus datasets  were used in the calculation of contemporary (2010–2013) genetic diversity by lek and county, and pre-/post-translocation diversity by county  and effective population (Ne) sizes allowed us to derive contemporary management parameters such as ratios of Nb/N, Ne/N and Nb/Ne. We also established the demographic independence of populations by quantifying immigration rates, and our estimates of genetic relatedness clearly underscored the management liabilities inherent to the mating system of GRPC, with only a few dominant males successfully reproducing.In our study, individual genotypes were verified using gender-specific markers. We then contrasted our genetically derived mark/recapture estimates against visual counts of individuals on leks, and by so doing detected male longevity extending over multiple years. Our estimates of the breeding population  and contemporary bottlenecks (i.e. less than five generations). Our results suggest that genetic rescue may have been an optimistic interpretation for GRPC, with genetic drift and a demographic expansion via translocation implicated as overriding factors [More importantly, we quantified those population genetic parameters for GRPC that have been missing to date, such as pairwise  factors . In hindBelow, we elaborate on our findings in more detail, particularly with regard to those extrinsic and intrinsic factors that integrate with the life history and ecology of GRPC and which promote its conservation reliance. We then offer several potential solutions that may contravene its status and promote recovery in Illinois.4.1.A key objective when managing conservation-reliant species such as GRPC is to restore its fitness environment to a state that existed before its decline. Here, two options prevail: ‘threat management’ (i.e. extrinsic to the species) and ‘population management’ (i.e. intrinsic to it)  figure . Threat Extrinsic factors are inherent to the declining-population paradigm, where numerical reductions are induced by habitat fragmentation, introduced predators, over-harvest or related impacts. Subsequent questions might focus on: How was the decline initiated? Is it perpetuated? Can it be rectified? In contrast, intrinsic factors are inherent to the small-population paradigm, with emphases on inbreeding, low effective population size and demographic isolation (among others). It is theory-rich  and rec4.2.Many of the management issues that underlie the conservation reliance of GRPC pertain to extrinsic factors (i.e. ‘threats’). Given this, a ‘threat management’ approach would address low abundances, with rehabilitation and/or extension of critical habitat as potential mitigation.GRPC is an ecological specialist with relatively narrow and inflexible habitat preferences  that undColonization would be facilitated and gene flow promoted if dispersal occurred among existing habitat fragments, but this process is severely constrained by the ecological characteristics of grouse. For example, more than 50% of yearling greater sage grouse will disperse to a non-natal lek (a positive characteristic), but with a downside that they remain faithful to it in subsequent years . Our molHabitat reductions and loss of connectivity are extrinsic factors reflected in the declining-population paradigm, yet their impacts can be modulated by intrinsic factors such as ecological specialization and dispersal behaviour. This, in turn, underscores the synergy among intrinsic and extrinsic factors , and how4.3.The intrinsic factors of conservation reliance are often reflected in the natural history of a species. They impede its capacity to persist within an anthropogenic-dominated landscape, and point to the small-population paradigm as a driver of conservation reliance. In this context, we interpret previous remediation strategies for GRPC as largely unsuccessful in that they worked against those limitations imposed by the life history of the species. This aspect has been overshadowed in GRPC by the benefits accrued from demographic expansion ,10.T. pallidicinctus) increases with age and thus reinforces consanguinity, but with declining survivorship [Ne) size, yet with few management solutions short of artificial propagation.As an example, lekking is a reproductive strategy that sustains the lifetime reproductive success of a few dominant males, as demonstrated by the fact that copulations are achieved by less than 20% of resident GRPC males ,55. In aivorship . InteresFemale fitness in lek-mating species is often at parity with that of males, and for several reasons: the existence of female dominance hierarchies ; a tendeOther gender-based variances in life-history promote subsidiary issues: females maintain larger home ranges, disperse farther and more frequently, and are thus more susceptible to the vagaries of climate change and a fragmented habitat ,60. They4.4.The relevance of extrinsic and intrinsic factors is mediated by the ecology and evolutionary history of a species . Here, gWas genetic rescue a success for GRPC in Illinois? Major caveats prevent a definitive answer. Pre-translocation genetic estimates were derived from 32 incidental mortalities without county of origin gathered over 19 years (i.e. approx. 0.6 sample per year or approx. one sample every 2 years). Unfortunately, these data were the only option available for the derivation of comparative metrics , and indAs a management approach, translocation seemingly addresses both extrinsic and intrinsic factors. It underpins genetic rescue, has roots in population genetic theory , invokes immigration as a mechanism for ecological remediation (i.e. ‘declining population’) and has been promoted as effective in recovering GRPC in Illinois . HoweverGRPC translocations in Illinois have a history of setbacks , with success either ambiguous or transitory, neither of which is sustainable. Translocations may avoid the costs of restoring connectivity but also invoke long-term intensive management (i.e. the perpetuation of conservation reliance). This will in turn, tax recovery such that it becomes unsustainable from an economic and political stance . In this4.5.Self-sustaining populations of GRPC in Illinois may be an elusive endpoint, particularly given the severe constraints imposed by ecological and evolutionary characteristics. A similar situation exists for the lesser prairie chicken . A reduchttp://www.dnr.state.il.us/orc/safe/). This, in turn, would promote critical habitat for GRPC, as well as long-term connectivity among prairie remnants. However, one particular downside for ESA protection is that its regulatory restrictions may be perceived negatively by private landowners, thus impeding appropriate management.Are potential solutions indeed plausible for GRPC? We consider four. Listing under the ESA affords legal protection for those populations that face extinction in parts of their range. In Illinois, GRPC is ‘state-endangered’ but federal listing would leverage increased ecosystem management, much as with the congeneric lesser prairie chicken. The ESA also offers protection for ‘distinct population segments’, and both populations qualify as they are demographically independent and significantly different from one another. An elevated listing would also reaffirm the ‘state acres for wildlife enhancement’ programme implemented by the Illinois Department of Natural Resources in an attempt to convert agricultural land back into prairie . Other unique endemics within this ecosystem should also be promoted for legal protection. This, in turn, would underscore the distinctiveness of the ecosystem, promote its public awareness and shape stakeholder perceptions regarding its conservation.Third, GRPC is particularly vulnerable to climate change, as typified by its steep decline during the Anthropocene. Species so recognized demonstrate seven life-history characteristics , five of2 levels and an extended growing season, but to the detriment of prairie landscapes [Midwestern North America now supports an elevated and energy-intensive economy whose greenhouse gas emissions exceed the national average by more than 20% . Furtherndscapes . Clearlyndscapes .ex situ propagation strategy may be a potential solution .5.Conservation threats in the Anthropocene are most often a result of extrinsic factors that are potentially manageable, but within a cost/benefit framework encompassing triage as an endpoint (where triage is viewed as a conservation strategy that provides the greatest benefit for multiple species and ecosystems). Additional complications occur when habitat has been subsumed by agriculture, and where protected areas are insubstantial in their number or dimensions to sustain a population expansion (as herein). However, restoring the vast North American prairie to a historic benchmark  is clearly unachievable in that the prairie has already been replaced by an agro-urban patchwork. In a similar fashion, those approaches that solely address intrinsic factors  also have a low probability of success and, as a consequence, promote rather than alleviate conservation reliance.However, it is counterproductive to question the success or failure of translocations and genetic rescue . Each is admirable with regard to effort , and insA major challenge for mitigation of GRPC, is the development of stakeholder initiatives that will drive its management at ecologically relevant scales. In this sense, we now have a clear understanding of GRPC, its population genetic status, and those extrinsic and intrinsic factors that tether it to conservation reliance. We must now assemble the economic coalition that will match the needs of management with the contemporary constraints of the private sector, and by so doing develop a conservation plan that will alleviate its conservation reliance.


Access to direct-to-consumer genetic testing services has increased in recent years. However, disparities in knowledge and awareness of these services are not well documented. We examined awareness of genetic testing services by rural/urban and racial/ethnic status.Analyses were conducted using pooled cross-sectional data from 4 waves (2011–2014) of the Health Information National Trends Survey (HINTS). Descriptive statistics compared sample characteristics and information sources by rural/urban residence. Logistic regression was used to examine the relationship between geography, racial/ethnic status, and awareness of genetic testing, controlling for sociodemographic characteristics.Of 13,749 respondents, 16.7% resided in rural areas, 13.8% were Hispanic, and 10.1% were non-Hispanic black. Rural residents were less likely than urban residents to report awareness of genetic testing . Compared with non-Hispanic whites, racial/ethnic minorities were less likely to be aware of genetic testing: Hispanic ; and non-Hispanic black .Rural-urban and racial-ethnic differences exist in awareness of direct-to-consumer genetic testing. These differences may translate into disparities in the uptake of genetic testing, health behavior change, and disease prevention through precision and personalized medicine. A major promise of genomic research is the transformation of healthcare practice and population health through earlier diagnosis, more effective prevention and treatment of disease, and avoidance of treatment side effects . PrecisiUnderserved groups have poorer access to genetic testing services within the general population . GeneticIn accordance with the evidence on access-related barriers to genetic testing services more broadly, findings from the Health Information National Trends Survey (HINTS) reveal that awareness of DTC genetic testing services, specifically, is lower among vulnerable populations, including racial and ethnic minorities and lower income individuals . HoweverData were pooled from 4 waves (2011–2014) of the HINTS, a publically available, population-based survey of non-institutionalized, US civilians who are ≥18 years . HINTS ionly as it was unavailable in other years. Participants reported their information source by answering “from which of the following sources did [they] read or hear anything about genetic tests?” with the option to choose one or more information sources .The main outcome of interest, awareness of DTC genetic testing services, was derived from the following question: “Genetic tests that analyze your DNA, diet and lifestyle for potential health risks are currently being marketed by companies directly to consumers. Have you heard or read about these genetic tests?” The source of genetic testing information was a secondary outcome observed in the 2013 wave The main predictor was rural-urban residence, defined using the US Department of Agriculture’s 2003 Rural-Urban Continuum (RUC) Code, with 10 categories based on county of residence in a metro or non-metro county and the population of the county. Codes 1–3 were designated as urban, representing commuting patterns to metro counties with populations of 250,000 or greater and codes 4–9 as rural, representing non-metro counties with populations ranging from 2500 to 20,000 [Data were analyzed with Stata 14 using jackknife replicate weights to estimate accurate variance estimates for statistical modelling. Descriptive statistics and bivariate analyses (using chi-square tests) were conducted to compare sample characteristics and information sources by rural/urban residence. A multivariate regression model of rural/urban residence as a predictor for awareness of genetic testing controlled for age, sex, race/ethnicity, education, income level, and survey year. We tested alternative models that controlled for family history of cancer and interaction terms between rural/urban residence and other respondent characteristics. Predicted marginals were derived for racial/ethnic minority group membership stratified by rural/urban residence.Of 13,749 total respondents across 4 years, 2078 (16.7% weighted) resided in rural areas, 1958 (13.8%) were Hispanic, 1942 (10.1%) were non-Hispanic black, and 903 (6.7%) were non-Hispanic of other races. Significant differences were observed across sample characteristics by geography  than urban residents to report awareness of DTC genetic testing Table . HoweverFurther, predicted marginals revealed rural-urban differences across all racial/ethnic groups Fig. . PredictTo our knowledge, this report is the first to examine rural-urban differences in awareness of DTC genetic testing using a nationally representative sample of US adults. After controlling for demographic characteristics and confounders, awareness of DTC genetic testing was lower among rural residents and racial/ethnic minorities compared with urban residents and non-Hispanic whites, respectively. Furthermore, the prevalence of awareness of genetic testing services among rural non-Hispanic whites was comparable to awareness among urban minorities.Among those who were aware of DTC genetic testing, we did not observe significant differences by rural vs. urban residence in the sources of information among participants who were aware of these tests. However, this measure was only available in one year of HINTS and the lack of significant differences may be due to insufficient power. Although not statistically significant, the proportion of rural residents reporting the Internet as an information source for genetic testing was almost ten percentage points lower than among urban residents. Given that the Internet is a major information source for genetic testing, differences in Internet use or access  maybe contributing to geographic disparities in access to health information .Meanwhile, health professionals were the least commonly reported information source, suggesting that there is an opportunity to improve delivery of or education regarding these services using interventions focused on health education and personalized medicine. However, it is worth noting that the question of interest specified DTC genetic testing, an approach to marketing genetic tests to consumers without the involvement of health professionals. DTC tests can have significant risks and limitations and health professionals may be well aware and rightfully do not discuss the tests with their patients. However, it is important for health professionals to be versed in these tests so that they properly advise patients and assist them with interpreting and acting upon the results.Accordingly, DTC genetic testing in itself is not a substitute for a precision medicine approach to risk assessment, prevention, diagnosis, or disease management. Whereas genetic testing within the context of precision medicine promotes individualized care, a population-based approach to its implementation will ensure that its reach is extended to underserved populations and reduce health disparities. More inclusive approaches for measuring disease and susceptibility could allow for better assessment of population health and development of policies and targeted programs for preventing disease . GeneticThis study has several strengths including the use of a large, multiyear, nationally representative sample of US adults to examine differences in awareness of genetic testing by geographic location. To our knowledge, this is the first study to report on awareness of genetic testing services across racial/ethnic groups stratified by rural-urban residence. As with any cross-sectional survey design, the limitations of this study include low response rates and inability to infer causation. In addition, we were unable to assess rural-urban differences in actual use of genetic testing because this follow-up measure was only available in 1 of the 4 waves of HINTS. Finally, because we used HINTS data dating back to 2011, we were unable to analyze all waves using the updated RUC codes from 2013, and therefore we applied the 2003 definitions for consistency. Finally, we were unable to control for health literacy, a potentially confounding variable that was unavailable in HINTS.In conclusion, we found significant differences in awareness of DTC genetic testing by rural-urban residence and these differences persisted across all racial and ethnic groups. Whereas differences in awareness of DTC tests may transcend to awareness of genetic testing more broadly, a distinction should be made between the level of awareness, the appropriate use of recommended genetic services and the currently limited evidence regarding health benefits following DTC testing. Accordingly, there are clear differences between the utility of information obtained from DTC genetic tests and the need for greater awareness of appropriate use of genetic services among high risk populations within the context of precision medicine. Nonetheless, it is likely that precision medicine programs or protocols that target rural or urban residence may suffer from limitations related to this knowledge gap. Future research should focus on interventions that reduce differences in awareness and examine whether improving awareness and subsequent utilization of indicated genetic testing translates to improvements in health outcomes.


Fusarium strains collected from wheat (Triticum aestivum L.) throughout the Jiangsu province during 1976, 1983, 1998, 2006, and 2014. The results showed that 3-acetyldeoxynivalenol (3ADON) was consistently the predominant type in this region over 40 years, and the nivalenol (NIV) type has emerged since 1998. Long-term rotation of wheat and rice (Oryza sativa L.), rather than fungicide application, crop fitness, or weather conditions, might be the main cause of this phenomenon. The genetic diversity results from two toxin synthetic genes, Pks4 and Tri10, and variable number of tandem repeat (VNTR) markers revealed the largest variance within the population in 1998, which was also the year with the highest production of mycotoxins. Population differentiation analysis indicated that major temporal population comparisons from the same area were not significantly differentiated. Our results showed that dominant species could maintain genetic stability for a long time, and Pks4 would be of utility in genetic and population studies.Trichothecene genotype composition, mycotoxin production, genetic diversity, and population structure were analyzed, using 185  Triticum aestivum L.), barley (Hordeum vulgare L.), and other cereals, globally. This disease reduces crop yields, causing extensive economic losses. More importantly, harvested grain quality is often compromised, due to mycotoxins produced by pathogens. Consumption of contaminated grains and products might have pernicious effects on human and animal healthFusarium head blight (FHB) is one of the most destructive and widespread diseases of wheat (Fusarium graminearum species complex (FGSC) consists of at least 16 phylogenetically distinct species, and is the main causal agent of FHB23456Fusarium asiaticum is the predominant species in Asia891011F. graminearum sensu stricto (s. str.) is present in most FHB-occurring areas around the world, especially in America and Europe3413Tri5Tri6 and Tri10 are not located in that gene cluster, they were proven to be key regulators of synthetic genes202223242526Tri clusters have been used successfully in phylogenetic studiesPKS4 and PKS13, from the PKS gene cluster, are essential for ZEN production29PKS genes are useful in phylogenetic studies as their intraspecific divergenceWith the development of molecular biology, sequences and functions of members in the trichothecene biosynthetic gene cluster have been gradually elucidated. The first gene discovered in the trichothecene pathway was Genetic diversity and population structure, resulting from evolutionary forces over time and space, could be used to predict a pathogen’s response to disease management . High genetic variation in a small region might suggest high adaptive capacity of the pathogen to different environments3537st century. Scab was more severe throughout the Jiangsu province, due to the warm and humid climate during the flowering stage of wheat and other grains, which were heavily contaminated with high levels of mycotoxins. As a result, the Fusarium population dynamic genetic structure over the years, which reflects toxin-producing ability, merits further study.In China, the first recorded FHB severe outbreak was in 1936In this study, a large number of samples, from five representative years, were used to investigate species composition and analyze the factors affecting FHB population dynamics. Genetic diversity and population structure were measured with VNTR markers and toxin biosynthetic genes, the effect of new molecular markers was assessed. We also attempted to explain the relationship between genetic diversity and mycotoxin accumulation.F. asiaticum strains remained consistently high, always above 80%. F. asiaticum strains with the NIV genotype were not detected in the previous period. The NIV frequency was 8.33, 11.11, and 4.91% in 1998, 2006, and 2014, respectively. In F. graminearum s. str. strains, only 15ADON type was identified. The 15ADON ratio decreased between 1976 and 2014  and the greatest haplotype diversity  results showed that five populations had minimal genetic structure at Tri10, while F1976 was separated from the other populations for Pks4  higher compared to that of the remaining groups. Pairwise comparisons of this indices among the other four populations showed no obvious difference.Colony diameter and conidia production are listed in The yearly annual rainfall has fluctuated in the past 40 years, ranging from 560 to 1,510 mm. The mean temperature ranged between 14 to 16.5 °C in the same period. Large fluctuations in these two factors were observed, especially in adjacent years . Both faA similar trend was observed at the anthesis stage, especially in April, and two meteorological parameters were at their highest in 1998. In May, rainfall was significantly higher in 1998. This region was particularly cold in 1998 and the highest temperature was recorded in 2014 .et al.4142The FHB pathogen composition and population dynamics have been studied widely and intensively in North America and China. Ward F. asiaticum strains in the middle valley of the Yangtze River increased from 53% in 1999 to 87% in 200612In China, the 3ADON ratio of the F. asiaticum strains was 74.12% in 1999 and 85.71% in 2006, similar to the results obtained in the previous study. We previously analyzed a large number of FHB isolates in the Jiangsu and Anhui provinces in this region in 2011 and 2012, and discovered a slight, but non-significant, decrease in 3ADON productionFusarium population follows a unique evolutionary trajectory in a diverse region. The bottle-neck effect, or certain other factors, might cause this phenomenon and should be considered. Continuous monitoring of the FHB pathogen in multiple areas, and collection of related data, might shed more light on these results.Concerning the Jiangsu and Anhui provinces specifically, the frequency of the F. asiaticum, while F. graminearum isolates accumulate 15ADON891011There is no clear evidence to explain the chemotype dynamics. A number of previous studies indicated that the composition of chemotypes appeared to be phylogenetic species-dependent. In Asia, NIV and 3ADON are produced by Zea mays L.), cotton (Gossypium spp. L.), and soybean (Glycine max L.). Extensive wheat-rice rotation is critical for F. asiaticum overwintering, and perithecium production typically favors rice straw under warmer conditions. As a result, a 3ADON-producing F. asiaticum population could be present within this region for a long time, and we suggest this might have been the main factor underlying the absence of variation in trichothecene genotype frequencies from 1976 to 2014. Recently, more F. asiaticum isolates with the 3ADON genotype and DON content were detected in wheat samples when rice was the preceding crop (unpublished data). Many publications have also reported a profound influence of host prevalence on F. graminearum species and trichothecene chemotype composition9et al.The FHB pathogen population might be adapted to different plantsF. asiaticum occurs in warm (mean temperature >22 °C) and wet  areas, while F. graminearum is not limited by climatic conditions. The average annual temperature and rainfall data of our region showed remarkable variation during the past 40 years, particularly in consecutive years. The average temperatures were between 13.5 and 16 °C, which seemed to be lower than the results from modeling. In a previous study, 15 °C was recommended as the boundary between F. asiaticum and F. graminearumF. asiaticum.Environmental factors were closely correlated with FHB outbreak and the distributions of FHB pathogens, especially at anthesiset al.Fusarium species differed in their responses to air temperature and humidity, as mentioned in several previous reports. Low rainfall and relative humidity during the anthesis stage caused a lower number of 15ADON-producing F. graminearum strains, which were replaced in F. avenaceum with the 3ADON chemotype, and in F. poae with the NIV chemotype, in LuxembourgFusarium species.Yang Fusarium species. This resistance continued to increase over the period, suggesting minor effects of long-term changes in cultivar composition and species shifts. Low-level genetic differentiation, and the distances observed between the resistant and susceptible wheat populations in the US, proved that FHB cultivars have little effect on F. graminearum population divisionFusarium species and favorable effects on FHB. Long-term single fungicide application could only induce decreased sensitivity, but no changes in pathogen structure.Winter wheat cultivars, grown throughout the Jiangsu province between 1976 and 2014, varied over time. The planting areas for those varieties showed more resistance toward F. asiaticum strains were found to be more receptive to carbendazim under selection pressure, which could partially explain the predominance of 3ADON-producing F. asiaticum populations. Similarly, carbendazim resistance was only detected in F. asiaticum groups with 3ADON or NIV genotypes, from 2010 to 2012Population genetics analysis in a previous study revealed that two populations were genetically similar, based on the sensitivity to carbendazimet al.Fusarium composition41et al.F. graminearum strains of 3ADON and 15ADON genotypes, and found no differences. This further suggested that it was the result of recombination and natural selection; through which genes conferring phenotypic advantages might be present in any trichothecene genotypes.Ward There were no clear distinctions in mycelial growth or spore production among the five populations in our study. Although DON and ZEN levels varied, there were no significant effects on pathogen dynamics, as species composition remained stable. As 15ADON isolates were rare in all populations, we could not compare these attributes between two groups with different genotypes. Individual fitness might vary, and those isolates with advantages expand faster and dominate during evolution. It is possible that 3ADON populations replaced the others much earlier, as single 3ADON groups were predominately present in this region from the 1970s.The development of FHB, and accumulation of mycotoxins in infected wheat grains, are influenced by many factors from pre-sowing to harvest. We attempted to determine the causes of the relatively consistent presence of the FHB pathogen and found significant differences in rainfall for certain years. The heaviest rainfall occurred in 1998, and the highest levels of mycotoxins were detected in the population F1998. Based on these results, we speculate that rainfall at anthesis or throughout the year promoted mycotoxin productivity of most isolates in 1998. Unfortunately, we have no data on the mycotoxin contents in grains from 1976, 1983, and 1998. In a previous article, we found a clear relationship between higher precipitation and high levels of DON and ZEN5057F. graminearum was detected in 1992Fusarium strains to carbendazim gradually decreases with frequent use. This could partially explain the increased toxin production levels of Fusarium isolates from 1973 to 1998. The fact that the largest amount of toxins was in F1998 might be due to resistance to carbendazim, which could increase trichothecene production in vivo or in vitro. Similar phenomena have been reported, attracting attention since 2000, when more efficient fungicides, including tebuconazole and phenamacril, were introducedAnother factor worthy of attention is the application of carbendazim, which is effective for controlling FHB and toxin accumulationThe population F1998, which has the maximum genetic diversity, produced the largest amounts of mycotoxins as fitness-related traits. Extensive studies focusing on the relationship between genetic variability and population fitness, or fitness-related characteristics, have been conducted by evolutionary and conservation biologists62In the present study, some fitness-related traits were compared between populations and no differences were found in terms of mycelium growth or spore yields. These basic abilities ensure survival and reproduction of individual isolates. However, those populations with high genetic diversity or a high number of haplotypes showed increased capability in terms of toxin synthesis. Many published papers have indicated that low genetic diversity could induce inbreeding depression effects, while high genetic diversity increased population fitness, as mediated by heterosis63F. asiaticum populations in China were described previously, with groups from southwestern locations harboring the greatest diversity1249et al.F. asiaticum strains have been present within this region for at least 40 years. Yang et al.F. asiaticum isolates in the mountain-rich provinces to represent the older dominant populations in China, which have been replaced by DON producers. Zhang et al.F. asiaticum. Regional studies should be conducted to understand the spread of the 3ADON population.Varying levels of genetic diversity of Mycotoxin production of individual strains varied widely, even in the same population, and the process was under gene regulation. Some nucleotides of mutations were detected in high toxin-producing strains in two genes. Further study is required to determine if these mutations could influence a single individual’s ability to regulate the synthesis of secondary metabolism.F. asiaticum or F. graminearum populations from North America and East Asia reported a similar genetic structure, suggesting a pathogen population from a small, isolated disease nursery might remain stable for a certain period of time8356661215The VNTR data revealed significant population subdivision between F1976 and the subsequent population. High gene flow and low population differentiation in the latter groups indicated that individual temporal populations were part of a single metapopulation, at least from 1983 to 2014. Several studies focusing on Pks4 and Tri10. Genetic diversity based on those two genes was coincidental, although haplotype diversity values were higher for Pks4 than for Tri10 in the same population. There were inconsistent results regarding population differentiation by these two genes. At Tri10, population differentiation values were low and non-significant between each pair. At Pks4, F1976 was demonstrated to be significantly separated from the other populations, and this finding was very similar to that for the VNTR data. According to our results, some specific genes with rich nucleotide polymorphisms could be used in genetic and population characterization studies. We also tried to introduce Tri6 and Pks13, but failed to acquire long and single fragments in preliminary amplification; this work is continuing.The VNTR markers developed for FGSC have been successfully applied to population analyses in recent years. In this study, we used amplified fragments to characterize population differentiation and acquired divergence between Fusarium populations with two methods are rare. Ouellet and Seifert (1993)F. graminearum populations, and detected low population differentiation. In an earlier study on the same German isolates, these authors found higher Fst values with VNTR markersInconsonant genetic results pertaining to In total, 185 isolates were used for this study, including 26 collected in 1976, 28 collected in 1983, 35 collected in 1998, 46 collected in 2006, and 50 collected in 2014, from different counties in the Jiangsu province of eastern China. Those isolates were grouped according to the sampling time . The first three populations were stored in 20% glycerol at −80 °C for long-term storage. The other isolates were obtained from diseased wheat grain, following methods developed previouslyFor DNA extraction, mycelia, harvested from single-spore cultures on potato dextrose agar medium (PDA), was ground by drill and then pulverized with lysis buffer . The mixture was centrifuged at 12000rpm for 10 min and the supernatant was mixed with an equal volume of absolute ethanol at −20 °C for about 2 h. The DNA was precipitated after another centrifugation. After a wash with 70% ethanol, pure DNA was air dried and dissolved with sterilized water.F. graminearum species complex were used to identify each isolate by species. The 410-bp DNA fragment was amplified from the isolates belonging to F. graminearum, while the 497-bp fragment was generated from the isolates of F. asiaticum48et al.Tri3 geneF. graminearum complex, as 3ADON or 15ADON.Fg16F⁄R primers that produce polymorphic products (400–500 bp) with DNA from members of the et al.F. asiaticum strains. The forward primer of each marker was labeled with either fluorescent 6-FAM  or PEX  and PCR was performed as described previouslyNm), population differentiation or fixation index (Fst), allele frequencies, unbiased gene diversity (H), Nei’s unbiased genetic distance, and Nei’s unbiased genetic identity.Eight VNTR markers, developed by Suga Pks4 gene (1944 bp), and the complete Tri10 gene (1194 bp) of all F. asiaticum isolates, were amplified by PCR. The primer pairs for Pks4  and Tri10  were designed based upon the genome sequence of F. graminearum PH-1 (http://www.broad.mit.edu). PCR was performed in 40 μL reaction volumes with 10 ng of template DNA, 0.3 μL of Taq polymerase, 2.5 μL of Taq Buffer, 0.5 μL of dNTP, and 1 μL of each primer (10 mM). The amplification profile consisted of an initial denaturation of 94 °C for 3 min, followed by 35 cycles of denaturing  and an additional extension at 72 °C for 10 min. PCR-amplified DNA fragments were excised from the gel and purified using an AxyPrep DNA Gel Extraction Kit  according to the manufacturer’s instructions and sequenced by Shanghai Shenggong Biotechnological Ltd.Portions of the Nh), nucleotide diversity (π), and haplotype diversity (Hd) were calculated with DnaSP software (ver. 5). The degree of population differentiation was quantified using pairwise FST values in Arlequin (ver. 3.5). Genotypic differentiation between pairwise populations was detected by an exact test. To visualize these genetic relationships between populations, the pairwise FST matrix was then used to perform PCA in GenAlEx software (ver. 6.5)DNA sequences were aligned and edited using the MEGA software package, and the truncated sequences of the two genes were then concatenated into a data matrix for subsequent analysis. To assess the genetic variation within populations, the number of haplotypes  from the edge of a 3-day-old colony of each strain were placed on PDA plates at 25 °C. Radial growth was measured after 3 d. Ten plugs, as described above, were added to a conical flask containing 100 mL liquid CMC, and the flask was placed on a shaker for 7 d . Spore production was determined with a hemacytometer. Three replicates were used for each strain.http://data.cma.gov.cn). Climatic data during wheat anthesis (April and May) were also obtained at 24 observation stations, as listed in Yearly rainfall and average temperature records from available meteorological stations throughout the Jiangsu province, from 1976 to 2014, were downloaded from a Chinese meteorological data service system .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.