Disanthus cercidifolius subsp. longipes is an endangered species in China. Genetic diversity and structure analysis of this species was investigated using amplified fragments length polymorphism (AFLP) fingerprinting. Nei's gene diversity ranged from 0.1290 to 0.1394. The AMOVA indicated that 75.06% of variation was distributed within populations, while the between-group component 5.04% was smaller than the between populations-within-group component 19.90%. Significant genetic differentiation was detected between populations. Genetic and geographical distances were not correlated. PCA and genetic structure analysis showed that populations from East China were together with those of the Nanling Range. These patterns of genetic diversity and levels of genetic variation may be the result of D. c. subsp. longipes restricted to several isolated habitats and “excess flowers production, but little fruit set”. It is necessary to protect all existing populations of D. c. subsp. longipes in order to preserve as much genetic variation as possible. Disanthus Maxim. (Hamamelidaceae) is a monotypic genus endemic to China and Japan Disanthus cercidifolius subsp. longipes is the only subspecies in Disanthus which is distributed in China , while its sister, D. c. subsp. cercidifolius, is endemic to Japan D. c. subsp. longipes was first reported by Cheng DisanthusDisanthus was a sister population of D. c. subsp. cercidifolius, which is distributed in warm and humid forests of the Cathayan Land, and also was a Tertiary relic. D. c. subsp. longipes was characterized by its morphology and preference for humid, acid soils and shady habitats. The inflorescences are paired, axillary; Capitula are 2-flowers, purple; Leaves are heart shaped, green then turning to purple, orange, and red. It is usually a small tree, 2–3 m high, occasionally reaching heights of 6–8 m in forests when growing along streams. Because of severe habitat fragmentation that caused population decline, Disanthus was listed in the 1992 Red List of Endangered Plant Species of China www.iucnredlist.org), the Key Wild Plants under State Protection Disanthus has attracted many investigators D. c. subsp. longipes based on nine allozyme loci and found a higher genetic variation within populations as well as significantly lower variation among populations. However, sampling of Xiao's study was limited to part of the distribution area of D. c. subsp. longipes only. So a comprehensive study of the populations' genetic structure at different geographic scales is still needed.Although study of Preserving the genetic diversity of endangered species is one of the primary goals in conservation planning. Because survival and evolution of species depended on the maintenance of sufficient genetic variability within and among populations to accommodate new selection pressures caused by environmental changes Field studies were approved by Hunan Provincial Bureau of Forestry for collection in Xinning County (1XN), the Dupanglin National Nature Reserve (2DP), the Mangshan National Nature Reserve (3MS), and approved by Guangdong Provincial Bureau of Forestry for collection in Nanling National Nature Reserve (4NL), and approved by Zhejiang Provincial Bureau of Forestry for collection in QianJiang Source National Forestry Park (5QJ) and Zhulong town, Longquan City (6ZL), and approved by Jiangxi Provincial Bureau of Forestry for collection in Guanshan National Nature Reserve (7GS), and Mount Sanqingshan National Park (8SQ).Cyclobalanopsis and Sorbus, etc.) at altitudes of 450–1200 m. The exception (3MS) was the one growing in bamboo forests in the Mangshan Mountains of Yizhang. For molecular analysis, 10–11 individuals per population were sampled. The locations and information of populations are provide in The specimens were collected from eight populations from April to June of 2008. All populations but one grew in evergreen and deciduous broad-leaved mixed forests (often lived with species such as in genus Genomic DNA was extracted from silica gel-dried leaves using the cetyl trimethylammonium bromide (CTAB) method 2O). A pre-selective polymerase chain reaction (PCR), using PTC-200 thermocycler  was done using primer pairs with a single selective nucleotide extension. The reaction mix  consisted of 4 µl template DNA from the restriction/ligation step, 1 µL primer (EcoRI/MseI), and 15 µL AFLP Core Mix , 1 U Taq DNA polymerase, and 10× H buffer). After an initial incubation at 94°C for 2 min, 20 cycles at 94°C for 20 s, 56°C for 30 s, and 72°C for 2 min, with a final extension at 60°C for 30 min, were performed. The PCR products of the amplification reaction were diluted 10-fold with dH2O and used as a template for the selective amplification using two AFLP primers, each containing three selected nucleotides.AFLP reactions were performed following the method reported by 2O), and mixed with 10 µL formamide and 0.2 µL Size standard-600 . The mix was used for sequence analysis.Nine primer combinations labeled with fluorescent 6-carboxy fluorescein (6-FAM) were probed for selective amplification, and only primer combinations with the greatest number of bands  were selected. Selective amplification was done using the following touchdown PCR conditions: 94°C for 2 min first, then 10 cycles at 94°C for 20 s, and 66°C for 30 s, with a 1°C decrease per cycle then extension at 72°C for 2 min; followed by 20 cycles at 94°C for 20 s, 56°C for 30 s, and 72°C for 2 min. After amplification, 3 µL of the samples were diluted 3-fold with sterile deionized water (dHRaw data were collected on a CEQ8000 Sequencer (Beckman Coulter). AFLP products were resolved using a Beckman Coulter CEQ8000 genetic analyzer. Semi-automated fragment analysis was performed using the fragment analysis software of the CEQ8000. The chromatograms of fragment peaks were scored as present (1) or absent (0), and a binary qualitative data matrix was constructed. A total of 82 individuals were run twice with all primer combinations.tot), the percentage of polymorphic fragments (Fragpoly), Nei's (1973) gene diversity (H), and Shannon's information index (I). In addition, the number of unique fragments (Fraguni) and DWs, frequency-down-weighted marker values www.r-project.org).As a measure of population diversity, the binary data matrix was input to POPGENE version 1.32 D. c. subsp. longipes populations were calculated using the software POPGENE version 1.32 Nei's genetic distance www.r-project.org), was used to convert the AFLP data matrix to the ARLEQUIN input format. The AMOVA was performed by partitioning genetic variation among and within populations regardless of their geographic distribution. In this study, the traditional F-statistics Φ-statistics F-statistics. The significance of Φ values was tested by 1000 permutations. Φ-statistics are computed from a matrix of Euclidean squared distances between every pair of individuals Φst as an overall measure of population divergence , and then we divided the populations into two groups, the East China region and the Nanling Range .ARLEQUIN version 3.0 Φst between every pair of populations was calculated in ARLEQUIN as a measure of interpopulation genetic differentiation, from which 1000 bootstrapped replicate matrices were then computed, so gene flow (Nm) based on ΦstΦst) between every pair of populations and applying the Mantel test, using NTSYSpc version 2.1e z-statistic value was tested non-parametrically by creating a null distribution of z using 1000 random permutations and comparing the observed z value A distance matrix of D. c. subsp. longipes genotypes was carried out using SPSS version 16.0 The AFLP data were also subjected to a principal components analysis (PCA), which may help reveal unexpected relationships among a large number of variables, reducing them to two or three new uncorrelated variables so they retain most of the original information D. c. subsp. longipes comes purely from one of the populations. It was applied using a “no admixture” model, 100 000 burn-in period, and 50 000 MCMC replicates after burn-in. The approach requires that the number of clusters K be predefined, and the analysis then assigns the individuals to clusters probabilistically STRUCTURE version 2.3.1 DIC) criteria, Dbar, Dhat, and pD, for assessing the importance of inbreeding were computed using the default values: Burn-in 5000; sample 100 000; thin 20 Population structure was also investigated by HICKORY version 1.1 tot) varied between 198 (7GS) and 227 (5QJ). The percentage of polymorphism (Fragpoly) across the eight populations ranged from 43.71% (7GS) to 50.11% (5QJ). The number of fragments that only occur in one population (Fraguni) varied between 6 (7GS) and 16 (8SQ). The DW ranged from 56.85 (5QJ) to 111.19 (1XN), mean 76.88, and SD  = 21.11. The diversity within a population (H) ranged from 0.1290 (2DP) to 0.1394 (5QJ)  .Φst value of 0.2328 (P<0.001), with of 23.28% variation among populations and 76.72% within populations  and gene flow (Nm) matrix were used to establish the level of genetic divergence among the populations (P<0.001) for the most closely related populations (4NL and 6ZL), to 0.3428 (P<0.001) in the most divergent populations (3MS and 8SQ). The gene flow (Nm) among populations ranged from 0.4792 to 1.5830. Except for one population in Nanling National Nature Reserve (4NL), most of populations' Φst were above 0.2, and the corresponding Nm were below 1. These results indicate that the genetic distance of D. c. subsp. longipes is not close, yet nor dependent on geographic distance.The pairwise genetic distance , the highest estimate of the likelihood of the data, given the number of clusters chosen, was k = 10 (ten clusters). We can't get a clear knee in Lnp(D) . Evanno'D. c. subsp. longipes  and Shannon's information index (I) are higher than those of endemic species based on allozyme Disanthus cercidifolius subsp. longipes is a Tertiary relic, which is distributed in warm and humid forests of the Cathayan Land in the Tertiary Disanthus cercidifolius subsp. longipes may be one of them and preserved mutation during the relatively long glacial period. So it shows high genetic diversity D. c. subsp. longipes compared to other endangered species Accurate estimates of genetic diversity are useful for optimizing sampling strategies aiming at the conservation and management of genetic resources Results of studies of genetic diversity within populations show that all populations were on the similar level, except for the Dupanglin National Nature Reserve (2DP) which is shown to be lower than the others. The lack of genetic diversity resulting from the total existing individual in population 2DP is limited . In practice, a larger population often has higher genetic variation stΦ value 0.23283) and only a small portion (23.28%) is due to differences among groups. Some other studies of endemic and endangered species show a similar pattern Nm) based on stΦ 0.23283 was 0.82374, which means that the number of migrants per generation is lower than one. The present distribution of D. c. subsp. longipes is restricted to several isolated habitats Disanthus was wide spread in warm and humid forests in the Tertiary Disanthus species was the result of population fragmentation and contraction after the Quaternary glacial cycles. Considering that D. c. subsp. longipes has poor success in pollination, “excess flowers production, but little fruit set” D. c. subsp. longipes.In this study AMOVA showed that the largest portion (76.72%) of genetic variance is contributed by genetic variation within populations  indicated significant genetic structuring. The populations of D. c. subsp. longipes spread from east to west , it would adopt inbreeding or mixed systems to reduce the risk of reproductive failure. This result is consistent with Xiao's conclusions D. c. subsp. longipes. If we use co-dominant markers, it most likely would provide an affirmative result and better understanding of the processes.HICKORY software analysis suggests that longipes , becauseD. c. subsp. longipes in the Junfeng Mountains, Jiangxi Province, according to Pan's data D. c. subsp. longipes in the Jinggang Mountains, Jiangxi Province, might have been transplanted from Guanshan National Nature Reserve based on the data D. c. subsp. longipes in the Jinggang Mountains in October 2009, evidence that the present distribution of D. c. subsp. longipes appears to be relic from a once extensive range and population.Hierarchical cluster analysis  revealedGenetic structure analysis of the individual samples using STRUCTURE shows that six gene pools are represented in the data, and each gene pool is relatively independent except a few mixtures from other clusters. 1XN and 2DP are the first cluster separated from the other populations; 5QJ and 8SQ form the distinct cluster 2 from the others; 3MS and 4NL also cluster with each other and become distinct from rest populations. These three clusters show high correlation with geographic distribution. And the rest three clusters mixed in 6ZL and 7GS. There is a close relationship between 6ZL and 7GS. It shows that 6ZL is related to 7GS rather than to 5QJ or 8SQ, although 6ZL is close to 5QJ and 8SQ in geographic location. It seems likely that there were some populations between current populations 6ZL and 7GS, or gene flow could have been accomplished by pollen or seed dispersal. The results of PCA based on AFLP data indicate a different result, which the eight populations form a triangle that has 1XN and 2DP at one angle, 5QJ and 8SQ at a second angle, but 3MS and 4NL mixed with 6ZL and 7GS in the third angle. The reasons for divergence of the East China region and the Nanling Range are not clear.D. c. subsp. longipes, with its narrow archipelago-like distribution, habitat fragmentation, and reproductive-physiologic barriers, is typical species that can be easily affected. Although D. c. subsp. longipes is endangered in China, it has not yet been given high priority for protection. The original aim of this work is to provide some insight into the population biology of D. c. subsp. longipes in order to guide conservation measures. The guides which can be suggested by our study listed below. Firstly, Most of the genetic variation is located within populations, so we need to protect all the existing populations of D. c. subsp. longipes in order to preserve as much genetic variation as possible. Secondly, we should promote the further research and technology development to enable better reproduction of Disanthus. Finally, it is necessary to enforce the governmental law to forbid people stealing or purchasing wild Disanthus.In the face of the species extinction generated by human beings Disanthus cercidifolius subsp. longipes show a pattern of high within-population diversity and low among-population divergence. Although the distribution of D. c. subsp. longipes could be grouped as the East China region and the Nanling Range, the population divergence is clear. The estimates of genetic differentiation and gene flow suggest that the species primarily outcrosses, but can resort to mixed reproductive strategies. These patterns of genetic diversity and levels of genetic variation may be the result of D. c. subsp. longipes restricted to several isolated habitats and “excess flowers production, but little fruit set”. The status of relic and their low reproductive success results in D. c. subsp. longipes being endangered.The results of the AFLP on populations of


Pycnonotus plumosus) in Singapore, a highly urbanized island which retains <5% of its original forest cover in fragments. Combining our own collected and sequenced samples with those from the literature, we conducted phylogenetic and population genetic analyses. We revealed high genetic diversity, evidence for population expansion, and potential presence of pronounced gene flow across the population in Singapore. This suggests increased chances of long‐term persistence for the olive‐winged bulbul and the ecosystem services it provides within this landscape.With increasing urbanization, urban‐fragmented landscapes are becoming more and more prevalent worldwide. Such fragmentation may lead to small, isolated populations that face great threats from genetic factors that affect even avian species with high dispersal propensities. Yet few studies have investigated the population genetics of species living within urban‐fragmented landscapes in the Old World tropics, in spite of the high levels of deforestation and fragmentation within this region. We investigated the evolutionary history and population genetics of the olive‐winged bulbul ( Increasing urbanization has led to a sharp increase of fragmentation worldwide. Such fragmentation may result in small, isolated populations that face great threats from demographic and environmental events Lande  as well 2 per year , a South‐East Asian island city situated at the southern tip of the Malay Peninsula. Singapore has lost more than 95% of its original forest cover  surveyed, although reportedly at a declining overall population trend  between end of April 2013 and mid‐January 2014 in various locations across Singapore   and the Burke Museum of Natural History and Culture .b (cyt‐b). DNA extraction from field‐obtained samples was initially carried out using target blood samples. However, visualization of the PCR products in 0.8% agarose gel consistently gave smears or multiple bands. Following this, all DNA extractions were carried out using feather samples instead of blood, using the GeneAll® Exgene™ Clinic SV mini kit, following the manufacturer's protocol for hair. DNA extraction from the 10 museum‐sourced tissue samples was conducted using either the GeneAll® Exgene™ Clinic SV mini kit following the manufacturer's protocol for animal tissue, or via phenol/chloroform extraction , NADH dehydrogenase subunit 2 (ND2), and cytochrome‐™ Thermal Cyclers  for COI, ND2, and cyt‐b. Detailed laboratory procedures can be found in Appendix S1. Post‐PCR products were purified using ExoSAP‐IT® , and cycle‐sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit  and the same primers used for PCR. Cycle‐sequenced products were then purified using ALINE PureSEQ Solution  following the manufacturer's protocol and subsequently sequenced in an ABI 3130xl Genetics Analyzer (Applied Biosystems). Sequences were then blasted in GenBank to confirm species identity, and reconciled into gene sequence contigs using CODONCODE ALIGNER v4.2.3 . Contigs from the three genes were concatenated and were subsequently aligned using the ClustalW option in Mega 5.2  was then conducted on C1000P. plumosus sequences from South‐East Asia, generating a total of 80 target sequences from throughout the region (see Table S1 for GenBank sequence information). The second dataset comprised freshly obtained sequences spanning all three mitochondrial genes  (henceforth three‐gene dataset). Due to issues with cyt‐b amplification, this dataset had a reduced representation comprising 29 individuals all of Singaporean origin.Upon inclusion of preexisting GenBank sequences, two subsets of data were defined to maximize number of individuals and number of loci examined, respectively. The first dataset – based on ND2 alone – contained the largest number of sequences upon inclusion of 31 GenBank ND2 For all subsequent analyses except the phylogenetic analyses , all sites with gaps or ambiguity codes were removed across all samples. The final ND2 dataset comprised 77 individuals spanning 825 base pairs, while the concatenated three‐gene dataset, giving 29 individuals spanning 1812 base pairs. Details for each analysis are provided in Appendix S1.To investigate haplotypes in the dataset, GenAlEx 6.501  was used as the out‐group taxon, following recent phylogenetic studies showing babblers to be close relatives of bulbuls (Pycnonotidae) , maximum parsimony (MP), and maximum likelihood (ML) analyses were independently carried out for each data subset, using Mega 5.2  were carried out on both datasets in ARLEQUIN v3.5.12 (Excoffier and Lischer π) for each of the five geographical regions , and mean p‐divergence values between the five geographical regions. MEGA 5.2 , giving rise to a total of 28 haplotypes across the region. Of these, 15 belonged to the Singaporean population, five belonged to the Sabah population, five belonged to the Sarawak population, and one belonged to the Palawan population. The single remaining haplotype was shared between Sabah and Sarawak. The final concatenated three‐gene dataset used for the Singaporean haplotype analysis comprised 29 samples. This dataset spanned 1812 base pairs, of which 33 were SNPs. Analysis of this dataset revealed 16 haplotypes within the Singapore population, seven of which were shared across more than one locality.In the haplotype network Fig. , the SinSingapore‐specific phylogenetic analysis produced topologies congruent across all three analytical methods  Fig. . In thisπ) and p‐divergence among the five general collecting localities of Singapore (Table S2), and the results from AMOVA using Singaporean samples (Table S3). The π and p‐divergence values were generally similar across localities, although p‐divergence values between individuals in the northern part of Singapore and those of other Singaporean localities (0.00436–0.00543) were much higher than p‐divergence values between other localities (0.00268–0.00325) (Table S2). Similarly, AMOVA attributed almost all of the variation to within‐locality variation (91.52%), and very little to among‐locality variation (8.48%) (Table S3). The nonsignificant P‐value among localities (0.094) and the low Fst estimate (0.08) further indicate that differences among localities were insignificant.This result was further supported by the population values of nucleotide diversity  produced virtually the same topologies Fig. , with dest estimates were also high (0.90) among the three regions   Table , indicatD) Table . The maxAnalysis of the Singaporean olive‐winged bulbul population revealed a high level of genetic diversity Table ; Fig. 5,2 in size , is vast for an island only slightly more than 710 kmP‐values from mismatch distribution analysis and the roughly unimodal plots of pairwise differences suggest that the population in Singapore has undergone a recent population expansion. This finding is in agreement with the widespread deforestation that has occurred or is occurring in this region  . As the species is nonmigratory, this pattern of gene flow might be reflective of a baseline, year‐round pattern within the local population, unaffected by migratory individuals or seasonal movements. This finding also stands in contrast to other Singaporean species such as the short‐tailed babbler  and p‐divergence  among the five general collecting localities of Singapore .Table S3. Analysis of molecular variance (AMOVA) of Singaporean olive‐winged bulbul populations, grouped into five general collecting localities .Click here for additional data file.


Adansonia digitata, to the Indian subcontinent, we examined 10 microsatellite loci in individuals from Africa, India, the Mascarenes and Malaysia, and matched this with historical evidence of human interactions between source and destination regions. Genetic analysis showed broad congruence of African clusters with biogeographic regions except along the Zambezi (Mozambique) and Kilwa (Tanzania), where populations included a mixture of individuals assigned to at least two different clusters. Individuals from West Africa, the Mascarenes, southeast India and Malaysia shared a cluster. Baobabs from western and central India clustered separately from Africa. Genetic diversity was lower in populations from the Indian subcontinent than in African populations, but the former contained private alleles. Phylogenetic analysis showed Indian populations were closest to those from the Mombasa-Dar es Salaam coast. The genetic results provide evidence of multiple introductions of African baobabs to the Indian subcontinent over a longer time period than previously assumed. Individuals belonging to different genetic clusters in Zambezi and Kilwa may reflect the history of trafficking captives from inland areas to supply the slave trade between the fifteenth and nineteenth centuries. Baobabs in the Mascarenes, southeast India and Malaysia indicate introduction from West Africa through eighteenth and nineteenth century European colonial networks.To investigate the pathways of introduction of the African baobab,  Many of these have geographically widespread or disjunct distributions, with no historical records for when and how they may have been dispersed from their places of origin to new sites . SeveralAdansonia  occurs in Africa, Madagascar and Australia, with a different lineage present in each land mass [Adansonia digitata L., or the African baobab, is widely distributed across continental Africa. Adansonia digitata s.l. (i.e. in the broad sense) encompasses two described species, the tetraploid A. digitatas.s. and the diploid A. kilima [A. digitata s.s. extends across much of Sub-Saharan Africa, from the northern Sahel to South Africa's Limpopo province, but does not occur in the rainforest areas of central Africa, or higher altitude localities (above 800 m elevation) of eastern Africa [A. digitata s.l. in eastern and western Africa to be genetically distinct, with the eastern populations monophyletic within a paraphyletic grade of western populations [Adansonia kilima is recorded in higher altitude localities (650–1500 m elevation), partially overlapping with the range of A. digitata s.s., from the eastern slopes of Kilimanjaro to northern South Africa, and west to the Caprivi Strip, Namibia [A. digitata and A. kilima are genetically similar, suggesting that tetraploidy evolved relatively recently [A. digitata and hereafter use its common name, baobab.The charismatic tree genus and mass –13. Moleand mass ,14. Adan. kilima . The disulations . Adanson Namibia . Phylogerecently . In thisBiogeographic studies of baobabs in the African continent show that there are close associations between past human settlements and the presence of the species ,16. The Saccharum spp.) plantations [et al. [Adansonia in northwest Australia arrived from Africa via long-distance oceanic dispersal in the Miocene. The fruit of the African baobab has been demonstrated to remain viable after immersion in seawater for six months [The role of human agency in baobab dispersal is evident in its presence in the Caribbean and parts of tropical South America, where enslaved people were transported from West Africa between the sixteenth and nineteenth centuries to work on sugarcane , pearl millet (Pennisetum glaucum) and finger millet (Eleusine coracana), and legumes, such as cowpea (Vigna unguiculata), and hyacinth bean (Lablab purpureus) from Africa into the Indian subcontinent during the second and first millennia BCE [Bos indicus) in the reverse direction [Tamarindus indica) from the Sudano-Sahelian region to the Indian subcontinent [The few published surveys of baobabs in the Indian subcontinent attribute the introduction of baobabs to Arab traders ,27 or mennia BCE –42, and irection . There wontinent –46. We hIn this study, we investigate the history and pathways of introduction of baobabs from Africa to the Indian subcontinent and other parts of the Indian Ocean region. We combined genetic analysis of inferred ancestry and phylogeography with historical evidence of oceanic trade and migrations across the Indian Ocean to: (i) determine source populations of baobabs by examining genetic variation and relationships within and between African, Indian subcontinent and other Indian Ocean populations, (ii) associate these source populations to specific dispersal pathways based on historical trade routes and networks, and (iii) infer possible time periods of introductions based on genetic and historical evidence.2.2.1Our sampling in Africa focused on the eastern lineage, based on the assumption that this was the most likely source for dispersal of baobabs across the Indian Ocean. Thorough surveys of baobabs were conducted in Mozambique and Tanzania, with further sampling in Kenya and South Africa. A single sample was included from Senegal as a reference sample for baobabs in the West African genetic group . BaobabsA. digitata-specific primers would amplify them. To confirm that this had not occurred, we checked results from these samples for the presence of more than four bands per locus, to confirm that no extraneous DNA had amplified.Leaves were collected into plastic bags and dried with silica gel. If trees were not in leaf, then bark samples were used. Although bark samples are more likely than leaf samples to contain DNA from other organisms, such as endophytes and insects, it is highly unlikely that the Since baobab trees tend to occur as isolated individuals throughout their range rather than forming distinct populations, in many locations one tree was sampled. However, for some populations, samples were taken from up to 19 individuals. Voucher specimens were retained for a representative subset of individuals and deposited in the National Herbarium of Victoria (MEL).2.2et al. [et al. [Plant DNA was isolated from samples using DNeasy Plant Mini Kits (QIAGEN), with modifications to the manufacturer's protocol as described by Pettigrew et al. . The micet al.  were amp [et al. , modifie2.3in situ evolution, which may have occurred under the scenario of an ancient introduction. Specifically, we used GenoDive to test whether populations and loci were in Hardy–Weinberg equilibrium (HWE), and we used SPAGeDi v. 1.3a [Fis) and number of private alleles.Allele frequencies and measures of genetic diversity were examined for three sets of data: the entire range of the baobab, African populations only and Indian populations only. This allowed us to assess whether any changes could be detected in response to founder effects or admixture of lineages, which may be associated with recent introduction to India, and to test for  v. 1.3a  to calcuottleneck v. 1.2.02 [ottleneck software to compare observed versus expected heterozygosities. The ‘standardized differences test’ is unsuitable for less than 20 loci [To examine whether there was a recent reduction in effective population size of baobabs, as would be expected from a founder effect, deviations from mutation-drift equilibrium were tested using B. 1.2.02 . Expecte 20 loci . We ther2.4tructure v. 2.3.3 [a priori grouping of individuals into populations. In tetraploid species such as the baobab, the genotype of partial heterozygotes  is ambiguous. To avoid making assumptions about the genotypes of these individuals, we created a binary matrix of presence or absence of allele variants and treated these as dominant markers [K). Simulations consisted of a burn-in of 50 000 iterations followed by 100 000 Markov chain Monte Carlo iterations. Ten runs for each K=1–15 were carried out on an SGI Altix XE Cluster through the Victorian Life Sciences Computing Initiative. The value(s) of K that best explained the structure in the data was/were determined using Structure Harvester web v. 0.6.93 [K method of Evanno et al. [K were combined using Clumpp v. 1.1.2 [istruct v. 1.1 [hyloGeoViz [A Bayesian classification scheme was used to identify genetic clusters and evaluate their spatial distribution, in order to determine potential connections between source and introduced populations and to assess overall genetic structure. This analysis was implemented in Sv. 2.3.3 , without markers . An admi. 0.6.93 , followio et al. . To assiv. 1.1.2 , and plot v. 1.1 . ClusterloGeoViz .tructure analysis, for selected values of K, was tested using analysis of molecular variance (AMOVA) [rlecore v. 3.5.1.3 [tructure cluster to which the individuals of that population belonged. Samples from West Africa, the Mascarene Islands and Malaysia were excluded due to insufficient numbers. Analyses were based on the FST measure of genetic diversity, with 9999 permutations.The statistical significance of spatial genetic clustering identified in the S (AMOVA)  performe 3.5.1.3 . Populatenalex v. 6.41 [enalex. Since geographical structure in the form of genetic clustering and in the form of IBD can be confounded, further Mantel tests and partial Mantel tests were carried out to limit this effect using the vegan package [Isolation by distance (IBD) was examined among individuals through a Mantel test ; 999 permutations) conducted in G v. 6.41 . Genetic package  in R [62 package .enalex v. 6.41 [tructure at K=7. Clusters with disjunct distribution were subdivided into multiple populations and samples with missing data were excluded from the analysis. This left a total of seven groups under consideration. Principal coordinates were calculated based on the simple genetic distance  between each pair of samples, via a covariance matrix with data standardization. The first two principal coordinates were retained and plotted.A principal coordinates analysis (PCoA), conducted using G v. 6.41 , was alstructure at K=7 were used, with the inclusion of West Africa and the Mascarenes, and individuals were assigned to these regions. Loci were bootstrapped to generate 1000 pseudoreplicates and genetic distances between regions were calculated with the DC method [hylip v. 3.69 [hylip.Intraspecific phylogenetic analysis was used to investigate the relationships between regions and to provide further evidence for inferring source populations and dispersal pathways. The biogeographic regions as defined by SC method  for each v. 3.69 . These d3.3.1Fis values were similar and a small number of private alleles were present in the Indian samples . Tests for deviations from mutation-drift equilibrium, based on the ‘sign test’ and ‘Wilcoxon test’ in Bottleneck, were non-significant under all statistical tests, and allele frequencies showed a normal L-shaped distribution. This implies that there have been no recent bottlenecks or reductions in effective population size [In general, genetic diversity parameters were found to be lower in baobabs from India compared to Africa. The number of alleles per locus and heterozygosity were both lower in India, although  samples . Measureion size . Howeverion size ,66, we c3.2tructure runs at different values of K increased rapidly with increasing K, up to K=4, after which the likelihood increased more slowly or decreased, and the standard deviations were greater (a). The highest value for ΔK, indicating the number of clusters that best explained the data [K=2 (b). A peak at K=2 in Structure analyses can be an artefact , so , so trucK-values –4. Most K=2, the samples were divided into an Inland and a Coastal cluster . The Inland cluster included individuals from inland East Africa, southeast India (Chennai), Malaysia and Senegal. The Coastal cluster included individuals from coastal and lowland East Africa and India (excluding Chennai). Individuals from Tete, in Mozambique's Zambezi River valley, and from Réunion and Mauritius included a mixture of both clusters.At  cluster a,b. The K=4, the clusters were: (1) southern Africa (SA) and Malaysia, (2) southern coast of East Africa between Mozambique and southern Tanzania (SCEA), (3) inland Tanzania (IT), and (4) coastal region between Mombasa–Dar es Salaam and India (MDES). Baobabs from Tete included individuals with similarity to the SA cluster and others similar to MDES, and those from Senegal, the Mascarenes and Chennai equal likelihood of belonging to all four clusters .At clusters a,b.FiguK=7, the clusters were: (1) SA, (2) SCEA, (3) inland Tanzania excluding Kilimanjaro (ITXK), (4) Kilimanjaro (KM), (5) Senegal, Chennai, Mascarenes and Malaysia (WA), (6) western India (WI), and (7) central India (CI). Individuals from the coastal region between Mombasa and Dar es Salaam were unable to be assigned to clusters, but showed similarities to clusters 4, 5, 6 and 7 .At  6 and 7 a,b.Figutructure analysis at K=2, 4 and 7 (K), genetic clustering was significantly higher than expected by chance. The partitioning of genetic variance among clusters, among populations within clusters, and within populations was similar across the different population structures that we tested. For subsequent analyses, we defined regions based on the structuring at K=7, as a more fine-scaled explanation of the population structure.The AMOVA supported the clustering patterns identified in the S 4 and 7 . For alltructure analysis. The Mantel test for IBD across the entire range was significant , demonstrating that individuals from distant populations were more genetically divergent than individuals from nearby populations. When permutations were carried out only within K=7 genetic clusters, the Mantel test was no longer significant (p=0.142). The partial Mantel test of the K=7 genetic clusters, with geographical distance as a covariate, was significant . The latter tests showed that the pattern identified in the first Mantel test could be explained better as genetically cohesive clusters of populations, rather than a more gradual pattern of isolation by distance. Statistical support from AMOVA and Mantel tests aligned well with the Structure analysis, justifying use of the K=7 clusters for further analyses.Mantel tests and partial Mantel tests provided additional support for the clustering by the Structure analysis, with substantial overlap between clusters in coordinate space , individuals from the MDES, SCEA, KM, CI and WI clusters tended to have positive values; those from the SA cluster tended to have negative values, and those from ITXK were closer to zero. The second principal coordinate , differentiated CI from WI individuals, and KM from SCEA, but with some overlap between adjacent clusters. Considering both coordinates, the WI individuals appear closer to those from SCEA and the CI individuals closer to MDES.The PCoA demonstrated weak differentiation between the clusters identified in the Ste space . For thetructure analysis  are closely related to (and likely derived from) the Mombasa-Dar es Salaam populations. However, they also differed from African clusters and contained private alleles.The low genetic diversity in the Indian baobab populations compared with those from Africa is consistent with introduction to the Indian subcontinent. The Sin situ, suggesting that these populations have been present for more than one generation. Hence the presence of private alleles could imply that the existing trees are not founding individuals. Around 20% of the trees sampled in India for the current study are above 1400 cm girth and, using Swart's estimate of baobab age based on girth measurement and radiocarbon dating [There could be two reasons for the presence of private alleles. First, mutations may have occurred n dating , could btructure analysis revealed individuals with ancestry from multiple clusters at K=4 for SCEA and ambiguously assigned individuals at K=7 for MDES, which either indicates dispersal from different regional clusters or the presence of unique individuals not matching other sampled populations. The phylogenetic analysis shows that MDES populations were the most likely African source for introductions to the Indian subcontinent, but the long branches leading to CI and WI may indicate relationships to other non-sampled sources, perhaps to those from further north near the Horn of Africa. However, the evidence of individuals assigned to both MDES and SCEA clusters suggests that both may have been source populations for the Indian introductions. The PCoA shows some clustering of populations, but with substantial overlap. This is consistent with substantial and repeated dispersals between African sources and Indian populations, preventing complete differentiation. The PCoA supports the close genetic similarity of WI and CI baobabs with the MDES cluster.Within the African clusters, the S4.2tructure analysis provides evidence for SCEA as an additional source for introductions to India. The history of trade interactions between East African coastal centres and those in the Indian subcontinent suggests that the MDES populations were possibly the main source for introductions to CI, and the SCEA populations a significant source for WI introductions [The long history of trade networks across the Indian Ocean can provide evidence for reconstructing the introduction of baobabs from Africa to the Indian subcontinent and other regions of the Indian Ocean –39,69,70ductions ,52,71,72et al. [et al. extends across most of northeast Africa including the Horn, most of Kenya and the eastern half of Ethiopia; their Ethiopian Region includes significant elements of White's Sudanian and Afromontane (e.g. KM) regions. Hence the private alleles found in the Indian clusters could indicate introductions from populations in present-day Sudan, Ethiopia and Somalia, all of which have had histories of interactions with the Indian subcontinent extending over more than four millennia [As noted earlier, the presence of private alleles in the Indian populations may indicate introductions from biogeographic regions of Africa not sampled in this study. These could be from the Sudanian Region or the Sahelian Transition Zone as per White's  chorologet al. . The Somillennia .K=4, 7). These source cluster areas match White's [et al.'s [The presence of individuals in Tete-Mocuba with ancestry in the SA and SCEA clusters suggests that baobab dispersal occurred from these neighbouring populations into this area ; K=4, 7. White's  Zambeziaet al.'s  biogeogret al.'s ,74. A stet al.'s . Captiveet al.'s .tructure analysis at K=7  plantations in Oman and around the Persian Gulf [The activities of the Portuguese in the Indian Ocean region between the sixteenth and nineteenth centuries provide insights for the short genetic distance between SCEA and WI clusters. During this period, Portuguese colonists and traders in eastern Africa obtained soldiers and slaves from present-day Malawi, northern Mozambique and southern Tanzania for their territorial enclaves in western India ,79. As tian Gulf .The grouping of baobab individuals from West Africa, Mascarene Islands, southeast India and Malaysia in the same genetic clusters is consistent with the history of Dutch, English and French colonial networks between Africa and Asia. During the eighteenth and nineteenth centuries, both the English and Dutch East India Companies recruited ship crews, soldiers and labourers from West Africa for their factories and forts in southeast India, Ceylon and the East Indies ,81. In a5.By combining genetic analysis of baobabs with historical evidence of oceanic trade, this study provides three important findings that improve current understanding of the history of their dispersal across the Indian Ocean.First, it shows that there have been multiple instances of dispersal from continental Africa to the Indian subcontinent, possibly even extending back into prehistoric times. Second, we infer that the dispersal of baobabs in the Indian subcontinent and around the Indian Ocean was due to the agency of African migrants. Their long-standing familiarity with the baobab fruit and the tree's ubiquitous presence along inland trade routes would have made it a free and important food for surviving long journeys on land and by sea. The evidence of multiple introductions, and sources from different regions of continental Africa show, without doubt, that the presence of baobabs in the Indian subcontinent, the Mascarenes and Penang signals the forgotten history of African migrants to these places .Finally, our study highlights the need for further genetic analyses of African baobab populations incorporating additional samples from West Africa, Somalia, Ethiopia, Sudan, coastal Yemen, Oman, Iran, Pakistan and Sri Lanka. These analyses can be combined with more detailed historical, cultural and linguistic evidence to establish new hypotheses of baobab dispersal across the Indian Ocean.


Rusa unicolor) populations from the South, Central and North India.The mitochondrial DNA (mtDNA) control region is extensively used in the phylogeography of species. We examined sequence variations in the mtDNA control region of sambar (Most of the samples collected from the south India exhibited a 40 bp insertion in the mtDNA control region. This insertion was not observed in the North and Central Indian populations.This study provided a potential marker for molecular screening and identification of sambar populations in the form of a distinct 40 bp insertion. Some populations in South India did not exhibit this insertion. It indicates that there could be an ecological barrier that might be preventing the expansion of insertion-positive sambar population. Rusa unicolor) is the largest cervid species in Southeast Asia. In India, it is widely distributed from the Himalayan foothills to the southern limits. Seven subspecies of sambar are recognised, with R. u. unicolor occurring in India and Sri Lanka [The sambar (ri Lanka . DespitePanthera tigris [Sus scrofa [Cervus nippon [Capreolus capreolus [Odocoileus virginianus [Hydropotes inermis inermis [Muntiacus crinifrons [I (HVR-I) were compared among the sambar populations of the South, Central and North India for evaluation of genetic variation.The mitochondrial DNA (mtDNA) control region has been used extensively in studying the population genetics of wild species, for example the tiger, a tigris ; wild pis scrofa ; cervidss scrofa ; sika des nippon ; roe deeapreolus ; white-tginianus ; Chinese inermis  and blacinifrons . In the I region demonstrated high variability among sambar populations, and 26 distinct haplotypes were identified  region that is found mostly in the sambar population from the South India. This unique molecular feature differentiated it from the other populations of sambar from India suggesting further investigation. It is apparent from genetic data that INDEL has a significant role in population structuring in sambar.In several studies, insertion–deletion (INDEL) markers have been used in population genetics and forensics –12 as itThis study provided further insight into the genetic makeup of sambar. Analysis of samples collected from various parts of India revealed a high level of genetic variations among different populations. The percentage similarity and pairwise distance indicate that the insertion-positive haplotypes  were significantly related and had a higher genetic distance from the other haplotypes (Table b gene [http://www.cites.org) and Convention on Biological Diversity  were also complied with during the study. High standards were maintained during sample collection, and no animals were harmed during this study. Six tissue samples and one shed antler sample were collected from predator kills from Panna Tiger Reserve, Madhya Pradesh. Seven and four individual’s hair samples were collected from confined populations in deer parks in Andhra Pradesh and Goa, respectively. These hair samples were collected from the metal grills and concrete wall of the captive area. Ten faecal samples were collected from forests in Kerala and Tamil Nadu. Since no animal handling was involved, approval of animal ethical committees was not required. Whole genomic DNA was extracted using phenol–chloroform [A total of 64 biological samples  were used in this study. Of these, 26 were from South India, and 38 were from Central and North India  and 1 µl (~30 ng) of template DNA. The PCR conditions were initial denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 sec, annealing at 54 °C for 40 sec and extension at 72 °C for 75 sec. The final extension was at 72 °C for 10 min. The efficiency and reliability of the PCR reactions were monitored using control reactions. PCR amplification was confirmed by electrophoresis on 2.2 % agarose gel stained with ethidium bromide (0.5 mg/ml) and visualised under a UV transilluminator.The DNA extracted from above samples was used for PCR amplification of an approximately 600 bp long sequence of the mtDNA control region. A primer pair specific to the family Cervidae (deer) was used . The seqI and shrimp alkaline phosphatase for 15 min each at 37 and 80 °C. A BigDye terminator kit (version3.1) and an ABI 3130 Genetic Analyzer (Applied Biosystems) were used to generate DNA sequences from both the directions. The generated sequences were aligned by eye using Clustal W [http://www.fluxus-engineering.com) was used to generate median-joining network tree (Fig. The PCR products were treated with exonuclease-lustal W , availablustal W . Since tlustal W  (Table 1lustal W  and MEGAlustal W  (Table 2


Poecilia latipinna) genetic diversity among three geographically close mangrove salt marsh flats in the Florida Keys compared to populations in southern and northern Florida. The questions we are asking are whether there is sufficient genetic variation among isolated estuarine fish within populations and whether there are significant divergences among populations. Additionally, we want to know if GBS approaches agree with previous studies using more traditional molecular approaches. We are able to identify large genetic diversity within each saltmarsh community (π ≈ 36%). Additionally, among the Florida Key populations and the mainland or between southern and northern Florida regions, there are significant differences in allele frequencies seen in population structure and evolutionary relationships among individuals. Surprisingly, even though the cumulative FST value using all 955 SNPs within the three Florida Key populations is small, there are 29 loci with significant FST values, and 11 of these were outliers suggestive of adaptive divergence. These data suggest that among the salt marsh flats surveyed here, there is significant genetic diversity within each population and small but significant differences among populations. Much of the genetic variation within and among populations found here with GBS is very similar to previous studies using allozymes and microsatellites. However, the meaningful difference between GBS and these previous measures of genetic diversity is the number of loci examined, which allows more precise delineations of population structure as well as facilitates identifying loci with excessive FST values that could indicate adaptive divergence.Global climate change and increases in sea levels will affect coastal marine communities. The conservation of these ecologically important areas will be a challenge because of their wide geographic distribution, ecological diversity and species richness. To address this problem, we need to better understand how the genetic variation of the species in these communities is distributed within local populations, among populations and between distant regions. In this study we apply genotyping by sequencing (GBS) and examine 955 SNPs to determine Sailfin molly ( Global climate change (GCC) is a driving force behind many disturbances affecting global ecosystems , 2. Amoni.e. genetically diverse populations are more resilient in the face of disturbances than those that are less diverse [International Union for Conservation of Nature (IUCN) has declared the conservation of genetic diversity to be an necessity [Several studies have shown that ecosystems displaying broader ranges of biodiversity or species richness tend to be more resistant or resilient to disturbances –9. More  diverse , 10–12. ecessity . Geneticecessity –16. Thusvia the exchange of migrants, and c) whether there is evidence that populations occupying even slightly different habitats may show adaptive differentiation. Addressing these issues provide the necessary data to help inform conservation efforts.South Florida Keys coastal habitats may be particularly susceptible to GCC. These ecosystems have been under constant threats of habitat loss and reduction in species diversity due to rising sea levels , 18. In Poecilia latipinna) inhabiting mangrove saltwater flats. Sailfin mollies are a well-adapted species of topminnow with extraordinary resilience to salty, fresh and brackish waters that inhabit Florida saltwater flats. We apply Genotyping by Sequencing (GBS) to define the variation at 955 single nucleotide polymorphic sites (SNP). We use these data to address two broad questions: i) do GBS approaches agree with previous studies using more traditional molecular approaches and ii) what is the level of genetic variation within and among populations. Similar to previous studies using allozymes and microsatellites [In this study we focus on populations of the euryhaline fish, sailfin mollies  were collected from South Florida populations; sixteen individuals were collected from the North Florida population. Fish were collected near shore using minnow traps and dip nets. All fish were returned to the local environment after removing small (<10mm2) fin clips. Collected fish were measured for standard length, and fin clips were stored in 320 ul of Chaos buffer  and stored at 4°C prior to processing. Genomic DNA was isolated using a silica column [via gel electrophoresis, and concentrations were quantified using Biotium AccuBlue™ Broad Range dsDNA Quantitative Solution according to the manufacturer’s instructions.Sailfin mollies were collected from five populations in the spring of 2014 . These fa column . GenomicAse I. Adaptors (0.4 pmol/sample) were ligated to 50 ng of gDNA. The GBS library was sequenced on a single lane using Illumina HiSeq 2500 with a 100 bp single end read . The UNEAK GBS analysis pipeline, TASSEL [GBS was preformed as described  using th, TASSEL , was use, TASSEL .ST values, and outlier FST identification using the “ape” package in the R statistical computing environment [ST values calculated by FDist2 LOSITAN [ST [We examined pairwise differences within and among populations , heterozygosity, Hardy-Weinberg equilibrium, migration rates, Fironment , Arlequiironment  and LOSIironment . FST val LOSITAN  and Arle LOSITAN  were verITAN [ST . EstimatITAN [ST  and a BaITAN [ST  using > ITAN [ST , and RaXITAN [ST . IsolatiITAN [ST , 33. IBDversus southern populations. For the last FDist2 comparison (North vs. South), the four South Florida populations were treated as a single population. Comparisons were run using 500,000 simulations at a 99% confidence interval and a false discovery rate (FDR) of 0.01. Arlequin outlier analyses were conducted using a hierarchical model [Analyses for outlier SNPs were performed using LOSITAN (FDist2)  and Arleal model  with 10,To identify the genes with outlier SNPs, we searched GenBank at the National Center for Biotechnology Information (NCBI) using the 64 bp sequence tags containing these SNPs. Specifically, the nucleotide Basic Local Alignment Search Tool (nBLAST) at NCBI was searched among Actinopterygi sequences , using highly similar sequences search algorithm (megaBLAST) and accepting matches with E values less than 1oE-5. All other parameters were used as default.Poecilia latipinna is not endangered or protected status, and small marine minnows do not require collecting permits for non-commercial purposes. All fish were captured in minnow traps with little stress and released in less than 1 hour. Fish were returned to the site of capture with little obvious effect of sampling small fin clips (< 4mm x 4mm). IACUC  approved procedures were used for non-surgical tissue sampling with captures and release.Ethics Statement: Fieldwork was completed within publically available lands and no permission was required for access. There is a nested set of five Florida populations with a total of 120 individuals , Table 1Population sizes were estimated using a Bayesian approach . These dPoecilia latipinna genomic DNAs (gDNA) were used to create bar-coded, reduced representation, genomic libraries [2 > 0.2). This resulted in 955 SNPs with 6.48 millions reads. The average number of reads per SNP was 6,787 . Excluding individuals with no reads , the average number of reads per SNP per individual was 64 (range: 8–137). Individuals with no reads are excluded because they do not contribute to measures of allele frequencies, or population genetic measures.ibraries . Each SNversus 0%; Among populations, there were 30 fixed differences between the northern FL and southern FL populations  the pairwise differences (π) among individuals is substantial and similar to π among all individuals  among 3955 SNPs .ST values show a divergence pattern where WR, the most northern population, has the largest FST values (~0.45) when contrasted with any of the southern populations. Between southern Florida and the Florida Keys, the FST value is approximately 0.08, and within the Florida Keys the FST values are near 0.01. There are three additional sets of results that use the variation in allele frequencies to define divergences among populations  are shown in ST values exceeds this null expectation, dramatically so for the distinction between northern and southern Florida. Furthermore, Bonferroni’s corrected p-values (< 10−5) suggest that despite the small geographic distances among the Florida Keys populations, there are SNPs that have significant divergences. We can compare the number of FDist2 significant FST values  have shared significance.The overall Fulations . HoweverT values , Fig 4 whe AMOVA , nearly T values  were sigST values that lie outside the 99% distribution with 1% FDR . The majority of these (18) were for transcriptional factors or signaling factors . Among the three Florida Keys populations, which are less than 10 km apart with small overall pairwise genetic distances , there are eleven outlier SNPs with an average FST value of 0.104 (range: 0.020–0.230). These 11 SNPs have an average of 4,783 reads  among the 71 individuals in the three Florida Keys populations. Only one of these SNPs had meaningful annotation: FAM69A, a cysteine-rich type II transmembrane protein localized to the endoplasmic reticulum.Among the 411 outlier SNPs, the average number of reads for each SNP among the 120 individuals is 6,705, . Of these 481 outlier SNPs, 32 had significant nBLAST hits to all bony fish  uses anzygosity .ST values: 72 for the comparison between Northern versus Southern regions and 17 in southern Florida (between Crandon and the Keys) comparison.Results from the hierarchical outlier test are shown in ST values and outlier tests for FDist2 and hierarchical models. For comparison among all populations, 54 out of the 62 (87%) FDist2 outliers are outliers with the hierarchical model. For the comparison within the Southern Region (Crandon versus the Keys) 14 of the 23 (61%) FDist2 outlier are outliers with the hierarchical model. Many fewer outliers were shared when comparing the Northern and Southern region (In n region : only 20Poecilia latipinna) by comparing three geographically close Florida Keys populations to a mainland population in south Florida (Crandon Park) and a northern Florida, freshwater population.Among coastal habitats, mangrove estuarine forests are changing: they are extending further north with saltwater intrusion associated with sea level rise , 41, 42.P. latipinna populations have genetic divergence that increases with geographic distance [e.g., northern and southern Florida) with little substantial diversity among populations within a region . TheP. latipinna, [For population genetics it is common to examine polymorphic proteins, variable microsatellites or DNA sequences from one or a few genes. Proteins typically have two or few alleles that represent an unknown number of nucleotide changes. Microsatellites have many alleles , which is approximately 0.23 in the southern populations. In the northern population heterozygosity is much smaller: it is 0.14 or 60% of that found in the southern populations. These data are based on an average of 6,787 reads per SNP , with an average of 57 reads per SNP per individual (range: 6–137). Thus, it is unlikely that this variation is due to sequencing errors because all SNPs have sufficient replication. In comparison to other GBS studies, π values within these sailfin molly populations are similar to those measured in stickleback and natural populations of revisiae , 48.−4 at each amino acid. Although these are crude estimates, they do suggest that GBS provides much more information about polymorphism per locus than allozymes. This is not surprising because neutral divergence is a function of functional constraints [e.g., allozymes) there are fewer nucleotide substitutions that do not have a deleterious effect than in random genomic regions interrogated using GBS. Thus, one expects more variation in GBS studies than when using allozymes. The fact that GBS provides orders of magnitude more loci suggests that these data can reveal more detailed information about the conservation genetics of a species.These measures of genetic diversity using GBS data are substantial and seem to be much larger than other measures of genetic variation. For example, allozymes in populations from the same region have heterozyosity values of 0.05  with 13%straints , and in Ldh-B among populations from Georgia to Maine  and promoters for CYP1A among populations from New England , to DNA sequence variation. SNP's π per bp of 0.5% is larger than the π for DNA sequences captured from teleost fish promoters of  . The va  affects the analyses between the northern and southern regions. Thus, without a more explicit sampling design to ascertain geographic and environmental effects, the simplest explanation is that the divergence among the northern and southern regions represents neutral divergence.Without replicates of other freshwater samples or hyper-saline samples in northern Florida, it is difficult to discern what evolutionary factors are affecting the divergence between these two regions. However, the WR population in northern Florida has more monomorphic SNP and lower π and He values than any southern population, suggesting that this population has been historically smaller and more isolated. Both would contribute to greater drift. Additionally, the number of SNPs with significant Fe Tables  and 7. SST values comparing Crandon versus the Florida Keys are approximately 0.09 and within the Florida Keys FST values are 0.01 . These 44 SNPs have an average FST value of 0.22. Surprisingly 14 of these SNPs are significant outliers in both outlier tests (see below).Among the four southern populations (Crandon and the three Florida Keys), there is little over all genetic divergence: across all 955 SNPs, the Fare 0.01 . Yet, siare 0.01 . That is Crandon . This isST values are very small (0.01), and there is no support for differentiation based on phylogenetic or Structure analyses. However the discrimination analysis does suggest that there is sufficient allelic variation to distinguish NNK from the two other Florida Key sites (BPC and BPN). This can be seen in the 29 SNPs (9 with Bonferonni’s correction) that have significant FST values  and the fact that only 3% of SNPs have significant FST values (29/955), it is surprising that 11 of the SNPs are significant outliers.The genetic divergence among the three Florida Keys populations is small with little support for isolation among them: overall FT values . These FST values , an island model seems reasonable. For the Crandon versus the Keys, there are 23 FDist2 outliers . Most of these outliers (61%) were also found in the hierarchical analysis , natural selection has affected allele frequencies among these populations. The identification of potentially adaptive divergence among southern populations is where GBS SNP studies differ from previous studies. These results are possible because GBS examines many more loci and thus is more likely to find a locus with significant changes and statistically, one can distinguish loci with large significant FST values from neutral background by permutation of the data.For the FDist2 comparison among southern populations  suggest a near panmictic population. Using FDist2 (island model) there are 11 outlier SNPs . The average difference in allele frequencies for these 11 SNPs is 0.191 . These 11 SNPs have approximately 100-fold larger FST values and 20-fold greater changes in allele frequencies than the other SNPs. We could not apply a hierarchical outlier test within the Keys because migration rates were too high for these analyses. Thus, although the differences in allele frequency and extreme p-values . The large number of loci with small FST values strongly influences the larger estimates of Nm (effective population size times migration). Additionally, since nearly all of these loci appear to be neutral, we can more accurately detect loci with exceptionally high FST values. Thus, GBS SNP analyses appear to be similar to previously utilized techniques for determining population structure but provide a substantial improvement on those methods for detecting the potential signature of natural selection.Finally, we would like to remark on the similarity between our GBS SNP data and previous analyses of similar tellites , 21. Nea


Scrophularia ningpoensis Hemsl. We used chloroplast DNA and genomic AFLP markers to clarify not only the effects of domestication on genetic diversity, but also determine the geographic origins of cultivars and their genetic divergence from native populations. These results will allow both better management of cultivated populations, but also provide insights for crop improvement.Domestic cultivation of medicinal plants is an important strategy for protecting these species from over harvesting. Some species of medicinal plants have been brought into cultivation for more than hundreds years. Concerns about severe loss of genetic diversity and sustainable cultivation can potentially limit future use of these valuable plants. Genetic studies with comprehensive sampling of multiple medicinal species by molecular markers will allow for assessment and management of these species. Here we examine the population genetic consequences of cultivation and domestication in S. ningpoensis were identified. Wild populations contain all haplotypes, whereas only three haplotypes were found in cultivated populations with wild populations having twice the haplotype diversity of cultivated populations. Genetic differentiation between cultivated populations and wild populations was significant. Genomic AFLP markers revealed similar genetic diversity patterns. Furthermore, Structure analysis grouped all wild populations into two gene pools; two of which shared the same gene pool with cultivated S. ningpoensis. The result of Neighbor-Joining analysis was consistent with the structure analysis. In principal coordinate analysis, three cultivated populations from Zhejiang Province grouped together and were separated from other cultivated populations.Twenty-one cpDNA haplotypes of S. ningpoensis has experienced dramatic loss of genetic diversity under anthropogenic influence. We postulate that strong artificial selection for medicinal quality has resulted in genetic differentiation between cultivated and wild populations. Furthermore, it appears that wild populations in Jiangxi-Hunan area were involved in the origin of cultivated S. ningpoensis.These results suggest that cultivated  Plant domestication is one of the great milestones in human history. The process of domestication represents a continuum of increasing codependence between plants and people At present some medicinal plants are grown in home farms, and some are cultivated as field crops. But the number of medicinal plants species currently in high intensity cultivation for commercial production are varied world-wide. In India, 20 species are currently under cultivation, in Hungary 40 and in Germany 100 Artemisia judaicaScutellaria baicalensisCorydalis yanhusuoCoptis chinensisMagnolia officinalis subsp. bilobaFritillaria cirrhosaAs the demand for medicinal plant cultivation increased, many studies examined the impact of cultivation on genetic diversity, species such as Plant secondary metabolites are responsible for phytochemical diversity and therapeutic efficacy of medicinal species. The production of secondary compounds is influenced by both environmental and genetic factors Scrophularia ningpoensis Hemsl. is a perennial herb native to Southeastern China. Wild populations are distributed in forests along streams, thickets, and tall grasses below alt. 1700 m and reproduce from seed S. ningpoensis is listed as the Radix Scrophulariae S. ningpoensis populations is important and urgent for appropriate utilization, conservation and preservation of the wild resources.S. ningpoensis is cultivated for its roots on a large scale in several regions of Central and Southeastern China. This species can trace its cultivation history back to Song dynasty (1000 years ago) beginning in backyard gardens S. ningpoensis is propagated vegetatively from rhizome. During the cultivation, large rhizomes are often selected for propagation; flowers are removed before opening so that resources are allocated to vegetative growth, in particular the rhizomes S. ningpoensis, accessions from Zhejiang Province produced higher concentrations of three main bioactive compounds, suggesting that the best quality of cultivated S. ningpoensis is from Zhejiang S. ningpoensis from Zhejiang has the best quality for medical use S. ningpoensis from Zhejiang is genetically differentiated for others is also important and indispensable to sustainable cultivation of S. ninpoensis.Today, S. ningpoensis with the aims of: (1) evaluating the genetic erosion of cultivated populations and the change in the pattern of genetic diversity under artificial selection; (2) unraveling the geographical origins and ancestral populations of cultivated S. ningpoensis; (3) clarifying the genetic divergence between cultivated populations from Zhejiang Province and the rest of cultivated S. ningpoensis; and (4) determining which wild populations are valuable and can be introduced for improvement of cultivation.Here we used two chloroplast DNA fragments and AFLP markers to study the population genetics and phylogeography of wild and cultivated Management Bureau of Mt. Jinggang National Nature Reserve issued the permit for Jinggang Mountain (JGW); Management Bureau of Mt. Tianmu National Nature Reserve issued the permit for Tianmu Mountain ; Management Bureau of Mt. Dapan National Nature Reserve issued the permit for Dapan Mountain (TWW). No specific permissions were required for other locations which are neither privately owned nor protected and the field study did not involve endangered or protected species.Scrophularia ningpoensis. Specific locations (GPS coordinates) of our field work are indicated in Field studies were conducted throughout distribution range of trnL–trnF psbA–trnH 2, 0.5 µm/L dNTP, 2.5 µl 10×buffer, 2.5 µm/L primer, 1 U Taq DNA polymerase (Takara Bio Inc.) and 20 ng DNA template. PCR products were identified on 1.5% agarose gels in a 0.5×TBE buffer, stained with ethidium bromide (0.5 µg/ml), visualized with ultraviolet light and photographed. DNA Marker DL2000 (Takara Bio Inc.) was used. The PCR products with a single band were cleaned by a GENECLEAN II Kit  for direct sequencing. Samples were sequenced in forward and reverse directions using the dideoxy chain termination method with fluorescent labeling with Big Dye Terminator (version 1.1). Sequences were determined with an ABI 377XL DNA sequencer and edited using Sequencher version 4.0 . Sequences of the two IGS regions were combined and aligned by Clustal W version 1.8 After preliminary screening for variation in the intergenic spacer (IGS) regions of chloroplast DNA, two regions: h) and nucleotide diversity (π) hS, πS) and overall  using DNASP 5.10 Scrophularia buergeriana that contains all linkages with >95% probability of being most parsimonious was drawn by TCS version 1.21 S. nignpoensis were assessed by Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods with S. buergeriana, S. spicata and S. dentate as the outgroup. For Bayesian inference, the substitution model was determined by MrModeltest 2.2 hT), within-population diversity (hS), U test (Nst/Gst) and population differentiation (GST) were calculated in HAPLONST D statistic D* and F* statistics Sequences from all individuals were characterized for their cpDNA haplotypes. Chloroplast DNA haplotype diversity (EcoRI-AAC (FAM)/MseI-CTC, EcoRI-ACA (FAM)/MseI-CTG, EcoRI-ACC (HEX)/MseI-CAC, EcoRI-ACC (HEX)/MseI-CTA, EcoRI-AGG (TAMAR)/MseI-CAG and EcoRI-AGG (TAMAR)/MseI-CTC. FAM, HEX and TAMAR are three differential fluorescence labels for multiplexed electrophoresis.AFLP method followed the protocol established by Vos 2O. The mixture was kept at 37°C for 3 hours, then 65°C for 10 minutes; (ii) the ligation reaction included 5.0 µl digested genomic DNA, 1.0 µl 10× T4 DNA ligase buffer, 1.0 µl Eco R I-adapters (5 µM), 1.0 µl Mse I adapters (50 µM), 0.1 µl T4 DNA ligase (3 U/µL) and 1.9 µl ddH2O. The ligation reaction was 16°C for 12 h and the product was diluted with 90 µl of ddH2O; (iii) pre-selective amplifications were carried out in 12.5 µl reactions: 2.5 µl diluted ligation product, 1.25 µl 10×PCR buffer, 1 µl MgCl2 (25 mmol/L), 2 µl dNTPs (2.5 mmol/L), 0.5 µl Eco R I - A primer (5 umol/L), 0.5 µl Mse I - C primer (5 umol/L), 0.05 µl Taq DNA Polymerase (5 U/µL), 4.7 µl ddH2O. PCR conditions for pre-selective amplification were 72°C for 2 min; 94°C for 20 sec, 56°C for 30 sec, 72°C for 2 min, for 20 cycles, 60°C for 30 sec. The product of the pre-selective PCR was diluted with 150 µl ddH2O; (iv) selective amplification reactions included 3 µl diluted pre-selective amplification products, 1.25 µl 10×PCR buffer, 1.25 µl MgCl2 (25 mmol/L), 2 µl dNTPs (2.5 mmol/L), 0.8 µl Eco R I primer (0.5 U/µL), 0.5 µl M primer (5 U/µL), 0.5 µl BSA (20 mg/mL), 0.1 µl Taq DNA Polymerase (5 U/µL), 3.6 µl ddH2O. PCR conductions were 94°C for 2 min; 13 cycles of 94°C for 30 sec, 65°C–56°C (−0.7°C/cycles) for 1 min, 72°C for 1.5 min; 23 cycles of 94°C for 30 sec, 56°C for 1 min, 72°C for 1.5 min; 72°C for 8 min. The product of the selective amplification was analyzed on ABI3730 (Applied Biosystems) according to the manufacture instructions with GS ROX-labelled 500 size standard (Applied Biosystems). For each primer combination, negative amplification controls without template DNA resulted in no amplification products.The protocols are as follows: (i) in the digestion step, 2.5 ul (0.25–0.3 ug) of genomic DNA was restricted in 2.5 ul of a reaction mixture that contained 0.2 µl Eco R I (15 U/µL), 0.15 ul Mse I (10 U/µL), 0.5 µl 10×Buffer and 1.65 µl ddHRaw AFLP data were scored and collected as presence/absence matrix firstly by Genemarker v1.7 (SoftGenetics LLC) and then each locus was manually inspected. Only unambiguously detectable fragments in the size range 75–450 bp were scored. To test the reproducibility of the AFLP data, the complete AFLP procedure  was repeated for 15 individuals (5% of the samples) and scored independently for each primer pair.Fp), the percentage of polymorphic fragments (PPF), Nei's h), Shannon's diversity index (H), coefficient of gene differentiation , and the level of gene flow: Nm = 0.5(1−GST)/GST. To explore the relationships among individuals Principal Coordinate Analysis (PCoA) was performed by software MVSP version 3.1  from pairwise Euclidian distances between individuals genotypes. A neighbor-joining (NJ) tree rooted by the populations of Scrophularia buergeriana was constructed by the PHYLIP 3.62 package Assuming a set of measures of wild and cultivated populations, population genetic statistics were generated by the program POPGENE version 1.31 S. ningpoensis. A total of 26 polymorphic sites were detected across the two chloroplast regions  was more variable than the trnL-trnF region . The combined cpDNA sequences of S. ningpoensis varied in length from 1255 to 1268 bp, with a consensus length of 1277 bp after alignment. When the two fragments were combined, 2.0% of all sites (26/1277) were polymorphic. Both Tajima's test  and Fu & Li's test  indicated no significant deviation from neutrality for the two regions. The combined sequences of the spacers conformed to the expectation of neutrality as well . Sequences of the eighteen psbA-trnH and six trnL-trnF haplotypes are available in GenBank database (GenBank accession numbers:KJ194143-KJ194166).Total 21 different cpDNA haplotypes (A-U) were identified among the 364 individuals from 27 populations of  regions ; 21 wereS. ningpoensis, eleven out of fourteen populations (78.6%) were polymorphic for haplotypes. In contrast, every cultivated population was fixed for a single haplotype. Wild populations had much higher estimates of haplotype diversity (hT = 0.919), the within-population component of diversity (hS = 0.444) and nucleotide diversity (πT = 0.00301) than do cultivated populations . In wild S. ningpoensis, haplotype diversity varied: populations LSW, TM2W had the highest haplotype diversity whereas populations TWW, PTW, THW from Zhejiang were each fixed for a single haplotype (ST = 0.71988) between cultivated and wild groups. And in cultivated S. ningpoensis all genetic variation was among populations, but in the wild populations, more variation occurred within populations (58.54%) than among wild populations (41.46%).Haplotype diversity and within-population diversity declined under anthropogenic influence during the course of cultivation . Wild poaplotype . An AMOVaplotype , which iS. buergeriana, S. spicata and S. dentata as the outgroup, resulted in 501 most parsimonious trees with a length of 87, a consistency index (CI) of 0.828, and a retention index (RI) of 0.583. Maximum likelihood optimization resulted in a final optimization likelihood of −2020.22, with the alpha parameter being 0.020000. The best-scoring ML tree had a length of 0.073087. The Maximum likelihood tree with statistical support indicated above branches was presented in S. ningpoensisis is shown in S. buergeriana is linked with the center of the whole network by two missing haplotypes. Two most common haplotypes A and B are on tip position linked with haplotype C and D to form one clade, while the majority of rare haplotypes forms a “star” shape dominated by haplotype E. The geographic distribution and haplotype frequencies within populations are shown in S. ningpoensis .Maximum parsimony analysis of these 21 haplotypes, with h = 0.0791–0.1614) than did cultivated populations (h = 0.0076–0.0875)  of which 261 fragments (90.31%) were polymorphic. The test for reproducibility yielded a mean error rate of 1.7% and confirmed the reproducibility of the AFLP fragments. Wild populations had much higher estimates of genetic diversity (–0.0875) . In wildtion NSC .The variation between cultivated and wild groups accounted for 19.62% of the overall genetic variation . In the S. ningpoensis. All populations were grouped into three main clusters  Culclusters .S. ningpoensis. The wild population HNW was sister to cultivated populations with a bootstrap value of 84%; LSW clustered with HNW and all cultivated populations supported by 100% bootstrap value  that had a close relationship with cultivated ap value . AlthougScrophularia ningpoensis is consistent with other domesticated crop species, even though the domestication history of S. ningpoensis is only one thousand years. Out of twenty-one cpDNA haplotypes in the species, only three haplotypes were detected in cultivated populations. Moreover, cultivated populations had little within-population genetic polymorphisms (πT) and haplotype diversity (hT) of cultivated populations were much less than that of the wild group  Metasequoia glyptostroboidesMalus pumilaZizania latifoliaFor many crop species the domestication process has left a signature in the pattern of genetic diversity as a result of founder effects, genetic bottlenecks and artificial selection orphisms . The cpDld group . AFLP dald group . These rS. ningpoensis was gathered from the wild. Most likely a limited number of individuals from the wild formed the founding populations of cultivated S. ningpoensis, the founders containing only a sample of the genetic diversity of wild populations. Although the domestication history of S. ningpoensis is short, under cultivation it is propagated vegetatively from rhizome and flowers are removed. These practices restrict gene flow between wild and cultivated S. ningpoensis populations and among individuals within cultivated populations. Moreover, farmers select large and healthy rhizomes to establish new populations. Such selection coupled with clonally propagation could quickly fix alleles carried by selected individuals. The increased FST values  relative to wild populations  reflect the different modes of reproduction in cultivated  and wild  populations.These large changes in genetic diversity are most likely the result of both founder effects and selection for medicinal quality, and importantly, the clonal mode of reproduction. The extent of the loss of genetic diversity depends in part on population size during domestication and time over which domestication occurred T values , Table 6S. ningpoensis, while other six wild populations  located in geographically separated places were only involved in origin of three cultivated populations. Moreover, the wild population JGW harboring both haplotype B and E is also in Jiangxi Province as the wild population LSW. Thus, the Jiangxi region is a critical site for the origin of cultivated S. ningpoensis.Locating the geographical site of domestication and assessing the demographic consequences of wild-domesticated species evolution can help to design strategies of use and management of genetic resources S. ningpoensis. Although three cultivated populations, PAC, XJC and GZC, grouped to another cluster in PCoA analysis  found in cultivated populations. But the wild population HNW didn't share any cpDNA haplotype with cultivated populations; only the native population LSW harbored haplotype A. These data suggest that this entire region of China may have served as the source of medicinal S. ningpoensis rather than a specific population. The ancestors of cultivated populations YLC, SCC and NSC were probably introduced from wild population similar to the extant HNW; harvesting and cultivation may, by chance, collected all individuals which had the same cpDNA haplotypes with cultivated populations. In addition, the nearest wild population to HNW is JGW, which shared two cpDNA haplotypes, B&E, with cultivated populations. But the very high value of Dw in HNW (21.92) and JGW (24.79) suggests a long-term history of isolation S. ningpoensis lead to a complicated domestication history for this species.Considering the discordance between chloroplast and nuclear markers,the origin of cultivated S. ningpoensis from Zhejiang Province is recognized to have the best medicinal quality S. ningpoensis has also revealed that materials cultivated in Zhejiang Province produce the highest content of bioactive compounds compared to other cultivated populations S. ningpoensis from Zhejiang  of most wild S. ningpoensis were higher than 0.1 (S. ningpoensis to ensure that it provides an adequate supply of the medicinal plant and the quality of the plant is enhanced. Furthermore, the utilization of wild populations with appropriate guidelines for collection and for regulating management could be an efficient way to find, conserve and deploy desirable agronomic traits which would be beneficial for Radix Scrophulariae cultivation.The results of this study can be used to select the most diverse natural populations for such a breeding program. Using the AFLP data, the values of Nei's than 0.1 , except than 0.1 . Thus, T


Fundulus heteroclitus). Surprisingly, the magnitude and phenotype of DLC tolerance is similar among these killifish populations that have adapted to varied, but highly aromatic hydrocarbon-contaminated urban/industrialized estuaries of the US Atlantic coast. Multiple tolerant and neighboring sensitive killifish populations were compared with the expectation that genetic loci associated with DLC tolerance would be revealed.The most toxic aromatic hydrocarbon pollutants are categorized as dioxin-like compounds (DLCs) to which extreme tolerance has evolved independently and contemporaneously in (at least) four populations of Atlantic killifish (Since the aryl hydrocarbon receptor (AHR) pathway partly or fully mediates DLC toxicity in vertebrates, single nucleotide polymorphisms (SNPs) from 42 genes associated with the AHR pathway were identified to serve as targeted markers. Wild fish (N = 36/37) from four highly tolerant killifish populations and four nearby sensitive populations were genotyped using 59 SNP markers. Similar to other killifish population genetic analyses, strong genetic differentiation among populations was detected, consistent with isolation by distance models. When DLC-sensitive populations were pooled and compared to pooled DLC-tolerant populations, multi-locus analyses did not distinguish the two groups. However, pairwise comparisons of nearby tolerant and sensitive populations revealed high differentiation among sensitive and tolerant populations at these specific loci: AHR 1 and 2, cathepsin Z, the cytochrome P450s (CYP1A and 3A30), and the NADH dehydrogenase subunits. In addition, significant shifts in minor allele frequency were observed at AHR2 and CYP1A loci across most sensitive/tolerant pairs, but only AHR2 exhibited shifts in the same direction across all pairs.The observed differences in allelic composition at the AHR2 and CYP1A SNP loci were identified as significant among paired sensitive/tolerant populations of Atlantic killifish with multiple statistical tests. The genetic patterns reported here lend support to the argument that AHR2 and CYP1A play a role in the adaptive response to extreme DLC contamination. Additional functional assays are required to isolate the exact mechanism of DLC tolerance. Fundulus heteroclitus, is one of the more sensitive fish species  supST were considered. Although these two SNPs are synonymous and do not result in amino acid changes, they are both located in the transactivation domain of their respective AHR genes and in close proximity to non-synonymous SNPs found to be under selection in Reitzel et al. [ST modeling approach identified the AHR1_1530 SNP as a significant outlier in only one of the comparisons (BI/NBH), when genotype data from all DLC-sensitive populations were pooled and compared to that of all DLC-tolerant populations AHR2_1929 was the only locus found to deviate from neutral expectations. Similar patterns of genetic variation in AHR1 and AHR2 loci is not surprising given that these two genes are arranged in tandem (and therefore linked) within the killifish genome [Since functional variation in AHR-ligand binding initiates the AHR pathway cascade  of the h genome . To deteh genome ,9. Studih genome ,48; whilh genome . Taken tST values and large differences in minor allele frequencies were observed at the CYP1A locus in all four population comparisons and the FST modeling approach identified CYP1A as a significant outlier in three out of the four comparisons (BI/NBH excluded). However, the shift in allele frequency, although significant, was not in the same direction across all comparisons. A full genome scan analysis of three of the same tolerant killifish populations (excluding BP) and their sensitive counterparts also identified a SNP marker in the CYP1A promoter region as the only locus (out of 354 screened) under selection in all three DLC-tolerant populations surveyed, but again, the direction of the allele frequency shift between DLC-sensitive and tolerant populations was not uniform [in vitro, contradicting the well-documented refractory response of CYP1A to DLC exposure among tolerant populations in vivo. These previously reported results, coupled with the knowledge that the CYP1A SNP included in this analysis is located in the 3’ untranslated region of the gene [Consistent with the refractory induction patterns for CYP1A (an early and sensitive marker of AHR pathway activation) observed among DLC-tolerant killifish populations in laboratory studies ,29,49, t uniform . In a fo uniform  found ththe gene  make theThe interpretation of CYP1A as a candidate for selection must also take into account that tolerance has evolved in response to different AHR ligands. Specifically, the role of CYP1A in AHR-mediated toxicity varies by ligand class. Classically described, AHR agonists induce the production of enzymes (predominantly CYP1A) that metabolize PAHs, but not DLCs. Unlike DLC toxicity, PAH toxicity is self-limiting, due to AHR-enhanced PAH elimination, and includes components that are not AHR-mediated. For example, CYP1A knockdown studies in zebrafish embryos have demonstrated the protective value of CYP1A during developmental PAH exposures . That toST; many additional loci were identified as candidates for selection . In addition to the AHRs and CYP1A, these populations appear to be highly differentiated at cathepsin F, cathepsin Z, CYP3A30, and the NADH ubiquinone oxidoreductase MLRQ subunit loci. Cathepsins are a large group of proteolytic enzymes that have been implicated in cardiomyopathies and cardiovascular disease, ultimately resulting in impaired pump function [Among nearby sensitive/tolerant killifish population comparisons, the BI/NBH pairing was the most genetically differentiated based on multi-locus Ffunction ,56. Givefunction , it is rfunction  and has function . In killfunction ; howeverfunction ,37. Mutafunction . Again, function . Moreovefunction . The repIt is not known whether the identification of additional candidate loci for strong selection in the BI/NBH pair only suggests unique tolerance-related or biologically-relevant differences  or technical artifacts . While similar phenotypes among the four tolerant populations examined suggest a conserved biochemical basis for intra-specific DLC tolerance in killifish, whether that similarity is constrained to identical nucleotide changes remains to be seen. The genetic mechanisms of adaptation could vary among DLC-tolerant populations. Alternatively, the differences detected may be a reflection of additional unique stressors encountered by each population pair.F. heteroclitus unigene database resulted in an enriched but incomplete set of candidate genetic markers for DLC toxicity and (presumably) tolerance. Genetic resources being made available through the Fundulus genome consortium as well as a parallel, unbiased, Quantitative Trait Locus (QTL) approach to the discovery of the genetic mechanism of DLC tolerance in killifish will greatly increase our understanding of this dramatic example of anthropogenically induced, rapid adaptation in the wild.The purpose of this study was to identify genetic polymorphisms associated with DLC sensitivity in Atlantic killifish from a suite of candidate loci involved in the AHR and interacting pathways; whether the polymorphisms in and of themselves are responsible for the drastic differences in DLC sensitivity among populations was beyond the scope of this work. Two loci, AHR2 and CYP1A, displayed patterns of variation consistent with selection in each of four pairwise comparisons. Additional loci with specific alleles significantly overrepresented in some, but not all, DLC tolerant populations underscore the possibility that the genetic variation in each population may have been shaped by similar yet unique selection pressures. Although the intention was to include all components of the AHR and interacting pathways in this population screen, the underrepresentation or absence of key genes ) in the Data from: Targeted approach to identify genetic loci associated with evolved dioxin tolerance in Atlantic Killifish (Fundulus heteroclitus), doi:10.5061/dryad.2355t Data files: Proestou_etal_Killifish_SNP_sequences.The sequences for the SNPs analyzed in this article are available through the Dryad Digital Depository and can be accessed by searching for the data package title DLC: Dioxin-like compound; AHR: Aryl hydrocarbon receptor; PCB: Polychlorinated biphenyl; PAH: Polycyclic aromatic hydrocarbon; BI: Block Island; NBH: New bedford harbor; BP: Bridgeport; FLAX: Flax pond; SH: Sandy hook; NWK: Newark; ER: Elizabeth river; KC: Kings creek.The authors declare they have no competing interests.DAP, PF, DC, and DN contributed to study design, sample collection and processing. DP performed data analysis. DP and DN wrote the manuscript. All authors read and approved the final manuscript.List of all genes considered for SNP analysis and their fate during the marker development process. Sequences provided by Sibel Karchner were derived from either 26  or 10 individuals (not cDNA libraries) collected from Scorton Creek, MA. In some cases, Qualitiy SNP identified multiple available contigs from a single unigene entry. The number of SNPs genotyped does not equal the number of SNPs in the analysis because 8 of the markers failed to provide quality genotype information. N = No, Y = Yes, N/A = Not available, U = Unknown.Click here for file