Despite being located at the crossroads of Asia, genetics of the Afghanistan populations have been largely overlooked. It is currently inhabited by five major ethnic populations: Pashtun, Tajik, Hazara, Uzbek and Turkmen. Here we present autosomal from a subset of our samples, mitochondrial and Y- chromosome data from over 500 Afghan samples among these 5 ethnic groups. This Afghan data was supplemented with the same Y-chromosome analyses of samples from Iran, Kyrgyzstan, Mongolia and updated Pakistani samples (HGDP-CEPH). The data presented here was integrated into existing knowledge of pan-Eurasian genetic diversity. The pattern of genetic variation, revealed by structure-like and Principal Component analyses and Analysis of Molecular Variance indicates that the people of Afghanistan are made up of a mosaic of components representing various geographic regions of Eurasian ancestry. The absence of a major Central Asian-specific component indicates that the Hindu Kush, like the gene pool of Central Asian populations in general, is a confluence of gene flows rather than a source of distinctly autochthonous populations that have arisen in situ: a conclusion that is reinforced by the phylogeography of both haploid loci. The Hindu Kush covers the mountainous regions of Afghanistan and north Pakistan, including areas on the western borders of the Pamir Mountains; since ancient times it has been the crossroad of the more densely settled regions of South and Central Asia and of historical Persia. The Hindu Kush mountains have forests above 800–1000 meters and alpine meadows below; several old Iranian texts, such as the Avesta, refer to this territory as being rich in vegetal resources The earliest archaeological evidence of modern humans in the area dates back some 30,000 years; it was found in the northwest of Pakistan on the South Asian side of the Hindu Kush Later historical and linguistic evidence points to the Hindu Kush as being a region reached by the early expansion of the Indo-Iranian languages After circa 1000 BC this extensive Indo-Aryan layer was in turn overlapped by their close relatives, the Iranians. They practiced horseback nomadism across Asia, from the borders of Rumania to Xinjiang  with some of them also settling in the Hindu Kush (Bactrians), the Tien Shan area (Sogdians), and as far west as present-day Iran  Pastoral nomadism in western Central Asia, and in parts of eastern Central Asia, was characterized by Indo-European speakers first, followed by Indo-Iranian, then Iranian, until Turkic, Altaic-speaking people finally took over. The Kalash from Chitral in northwestern Pakistan provide an isolated illustration of such movements: there is no evidence of admixture between them and East Asians History shows commerce and conquests meandering through the Hindu Kush region. Alexander the Great's army subdued the area around 330 BC Around 600 AD, the western part of central Asia was invaded by nomad Turkic tribes who established the currently Turkic speaking areas The presence of nomad Turkic tribes was first reported around 200 BC with the creation of the first Central Asian nomad empire. The Turkic conquests went on for a thousand years when they were interrupted by the Mongol expansion after 1200 AD: Genghis Khan's vast empire stretched from the lower Danube to the Pacific, including much of Siberia, northern-central China and the Il-Khanate' that covered the Anatolian and Persian areas south of the Black and Caspian Seas and of the Hindu Kush Several late migrations took place simultaneously around 1000 AD: the western Iranian-speaking Baluchis moved eastward from eastern Turkey into Baluchistan, the Dravidian-speaking Brahui migrated north from Central India, and the Romani (Gypsies) migrated westward out of India Despite this rich and complex history, the few genetic studies devoted to Afghanistan have been restricted to Y-chromosome and/or to the Pashtun population Although the historic Mongol incursions have been strongly supported by genetic studies However, mitochrondrial DNA (mtDNA) haplogroup diversity in populations living in Turkey, Georgia, Iran, and Central Asia suggested that the predominant direction of gene flow was from west (the Fertile Crescent) to east (Pakistan) Whether Central Asia was a source or a convergent zone of sub-continent gene flows remains unresolved. To address this issue, we present mt-DNA and Y-chromosome data on more than 500 Afghan samples from 5 main ethnic groups inhabiting the mountainous region of the Hindu Kush: Tajik, Turkmen, Pashtun, Hazara and Uzbek. Mt-DNA analysis consisted in HV1 sequence and polymorphism analysis in the coding region. Y-chromosome analysis covered 102 binary SNPs, among which there were 6 new markers, and 39 Y-STRs. A representative subset of each ethnic group was also analyzed for autosomal markers by Illumina 650 K SNP. This Afghan data was completed with the same Y-chromosome analyses of 672 original male samples from Iran, Kyrgyzstan, Mongolia and updated Pakistani samples (HGDP-CEPH). Finally, the results were compared to databases built up from published literature for the purpose of the present study using autosomal results from 1183 individuals; 14,308 HV1 sequences concerning mt-DNA analysis and results from 34 harmonized Y haplogroups including 8,111 individuals.A total of 516 samples of blood obtained by venipuncture were collected from 5 ethnically distinct populations in the Hindu Kush region of Afghanistan: Hazara, Tajik, Uzbek, Turkmen, and Pashtun . 478 addMinistère de l'Enseignement Supérieur et de la Recherche in France .All samples were obtained from unrelated male volunteers after approval of the informed consent protocols. The study protocol was registered by the DNA was purified from blood using the Qiamp blood kit  and DNA concentrations were determined by spectrometry.http://evolutsioon.ebc.ee/MAIT/public_data/index.html.Autosomal genetic variation was analyzed in a subset of 5 Hazara, 5 Tajik, 5 Uzbek, 4 Turkmen and 5 Pashtun samples using Illumina 650 K SNP array. In addition 1485 samples were taken from published data Quality control2>0.4) in a window of 200 SNPs (sliding window of 25 SNPs) We used PLINK 1.07 To study the population's genetic structure in the dataset of over 232,000 genome-wide SNPs, we used a structure-like A color is assigned to each K. Each individual is partitioned into K colored components, which represent the individual's estimated fractions of shared genetic background, or ancestry component (AC). In order to depict the spatial distribution of the ACs detected in Eurasia, the proportions of ACs 3, 4, 6, 7, 8 and 9 as resolved at K = 9 were then displayed on a color-graded map.Correlation between spatial distribution of the ACs detected in Eurasia and each proportion of ACs 1 to 9 as resolved at K = 9 was tested with the Pearson test  using XLSTAT 7.5.2 software.ST index. PCA and FST calculations were performed using the SmartPCA program As the inclusion of African samples results in the first Principal Component (PC) sorting African samples versus non-African samples, the former were excluded from the analysis. The LD pruning procedure was repeated on the 1183 samples left. Pairwise genetic differentiation between populations with sample size >5 was estimated with the Fth) Mitochondrial DNA was analyzed from 90 Pashtun, 146 Tajik, 78 Hazara, 75 Turkmen and 127 Uzbek. Samples were sequenced between nucleotide positions 15900 and 16569. Further analyses were performed by RFLP and/or direct sequencing of polymorphisms of the coding region at 29 nucleotide positions: 1406, 1438, 3010, 3816, 3915, 3992, 4188, 4336, 4580, 4769, 4793, 7735, 7789, 8598, 8818, 10217, 10556, 11696, 12007, 13167, 14178, 14182, 14582, 14668, 14872, 15259, 15908, 15940, and 15968. The mutations were scored compared to RSRS Eighty seven Pashtun, 142 Tajik, 77 Hazara, 74 Turkmen and 127 Uzbek obtained successful Y-chromosome analysis. In order to analyze a representative geographical coverage at the same level of resolution, additional populations also had Y-chromosome analysis: 9 populations from Iran totalizing 186 individuals, 6 populations from Kyrgyzstan totalizing 150 men including Dungan and Uygur individuals, 4 populations from Mongolia totalizing 160 samples and the eight Pakistani populations from the HGDP-CEPH DNA collection Following PCR amplification, binary marker genotyping was accomplished by either Denaturing High Performance Liquid Chromatography (DHPLC), RFLP analysis, Taqman® (Applied Biosystems) assay or direct sequencing methodology. Nomenclature assignments were defined according to the International Society of Genetic Genealogy Haplotype 2012 Tree Additionally, a total of 39 Y-STRS n, DYS462, DYS463, GGAAT1B07, YCAIIa-b, YGATAA10, YGATAC4/Y_DYS635, YGATAH4) were genotyped using two multiplex reactions. Electrophoresis of the amplified fragments, mixed with formamide and 500 LIZ internal Size Standard was carried out in an ABIPRISM 31030XL Genetic Analyzer. Interpretation was performed by GeneScan ID 3.2 fragment analysis software.We gathered mtDNA and Y-chromosome haplogroup frequency data from published data focused on Central Asian populations.th) Concerning mtDNA, a total of 14,308 HV1 sequences from 214 populations were included . HaplogrConcerning the Y-chromosome, the data set was initially built from 442 Eurasian populations totalizing 23,800 men from 68 bibliographic references. Since the studies have not all used the same level of resolution for SNP genotype samples, we needed to determine the consensus level of phylogenetic depth in the Y-chromosome tree. We therefore agreed to 34 male lineages and summed all frequencies within each: C-M130(xPK2), C3a-PK2, D-M174, E-M96(xP147xM75), E1-P147, E2-M75, F-M89, G-M201, G1-M285, G2-P287, H-M69, H1a-M82, K-M9, I-M258, I1-M253, I2-M438, J-M304, J1-M267, J2-M172, J2a-M410, L-M11, L1a-M76, L1b-M317, M-P256, N-M231, O-M175, P-M74, Q-M242 (xM25), Q1a2-M25, R-M207 , R1a-M449, R1b-M343, R2-M479, and T-M70. Then sample sizes of less than 10 individuals were eliminated. The final Y-chromosome data set encompassed 8,111 individuals from 187 populations .For both haploid markers, we identified the most discriminative lineages. We estimated the chi-square values based on haplogroup frequencies and selected the haplogroups with significant (p<0.05) differences of frequencies between at least one couple of populations Y-Chromosome haplotype and haplogroup diversities were calculated for each population with the ARLEQUIN v3.5.1.2 package For both of the haploid markers, gene diversity indexes and AMOVA were performed with the ARLEQUIN v3.5.1.2 package Concerning the Y-chromosome, we used the 37 populations from Afghanistan, Iran, Kyrgyzstan, Mongolia and Pakistan screened for the high-resolution 102 Y-SNPs. In order to fairly compare the genetic structure of the female population with that of the male one, we selected a subset of 27 populations from Iran, Mongolia and Kyrgyzstan  from the mtDNA database described above  and compWe ran two levels of factorial correspondence analysis (FCA) using XLSTAT 7.5.2 software. Given the depth of resolution of the 102 Y-SNPs herein examined, we first focused on the genetic relationships between the 84 most-derived male lineages in 37 populations from Afghanistan, Iran, Pakistan, Kyrgyzstan and Mongolia. Afterwards, we extended the comparison between populations to a sub-continental scale using our databases described above .Networks were constructed by the median joining method using Network 4.5.0.2, where ε = 0 and microsatellite loci were weighted proportionally to the inverse of the repeat variance observed in each haplogroup To further explore the links between Y-chromosome distribution and geography, we first conducted a Mantel test using 37×37 matrices of Fst and geographic distance (in kilometers using version 1.2.3 of the Geographic Distance Matrix Generator). To elucidate the nature of this association, we investigated the role of latitude and longitude on the frequency distribution of the major Eurasian Y haplogroup, suggested as genetic markers of the most striking historical events : C3b2b1-M401, J2a1-Page55 and R1a1a-M198 Autosomal variation in Eurasian populations was analyzed via genetic structure in a dataset of over 232,000 genome-wide SNPs, depicted by a structure-like clustering approach implemented in ADMIXTURE. None of the genetic structure simulations  . The firUsing haplogroup frequencies , we focuFirst and second axes of the Factorial Correspondence Analysis are represented in The intergroup variance between the Hindu Kush populations and data from published literature ranges from 1.29% when sorted according to language  to 1.76% when sorted according to geography .We then tested numerous combinations of population clustering to deduce the best population structure based on our observations from the autosomal PCA  and haplPhylogenetic relationships, haplogroup frequencies and haplogroup and haplotype diversities are presented in Kyrgyz and Pashtun display the lowest Y-chromosome genetic diversity, whereas populations from Iran show the highest Y-chromosome genetic diversity -A. HaploWe ran a FCA on the populations for which the first two axes addressed 20% of total variance . Figure FC plots in Median Joining Networks were performed on specific lineages chosen for their correlation with geography or for the large size of the sample. The Mantel test showed significant rank correlation between genetic and geographic distances . Haplogroup C3-M401 correlated positively with latitude and longitude, whereas J2a1-Page55 correlated negatively. Haplogroup R1a1a-M198 showed no correlation with either latitude or longitude .AMOVAWe performed an AMOVA analysis of Y haplogroup frequencies in our 37 populations by comparing language families  and geography . Language grouping was not significant (Fct = −1.65%) whereas geography was significant .Central Asia, defined as the region containing Kazakhstan, Uzbekistan, Turkmenistan, Kyrgyzstan, Tajikistan, Afghanistan and the northern part of Pakistan, has gathered a growing and ongoing interest from archaeologists and anthropologists. Retracing the main historical events in the gene pool of the present Afghan populations has been strongly restricted, because of sampling work in this country being inadvised, with the exceptions of recent Y chromosome studies We confirmed that the Hazara showed a high degree of East Asian admixture for autosomal and both haploid loci; in accordance with previous reports using genome-wide genotyping data sets Several studies reported C3 Y-chromosome haplogroup in Mongols The enhancement of resolution within haplogroup C3 has important implications for future studies. First, it should allow tracking of the Mongol invasions by Genghis Khan and identification of affiliated descendants since the 13th century, as well as detection of possible dispersal of C3 lineages during prehistoric migrations Our haploid data support the scenario of a limited number of family members accompanying Mongol soldiers on foreign expeditions. Family accompaniment was probably subject to further restriction when permanent occupation with subsequent colonization was planned, since these operations required full-scale nomadic life with strict military discipline. Under these circumstances, mixing with the local population was probably extensive. This hypothesis is also supported by the fact that within one century after occupying Southeastern Europe, the Mongols were already speaking Kypchak Turkic. Similarly, the absence of East Asian ancestry components in the classical Persian heartland, clearly shows that political and military control by Genghis Khan and his sons had limited effects on the genetic structure of heavily populated areas like Iran, the Indus Basin or South Caucasus.Central Asia displays very high genetic diversity Our autosomal and haploid data suggested that the Afghan Hindu Kush populations exhibit a blend of components from Europe, the Caucasus, Middle East, East and South Asia. This juxtaposition of autosomal and haploid markers could reflect important male and female influences contributing to the Afghan populations' genetic make-up. Considering autosomal data, all ancestral components displayed a decreasing gradient of their frequencies when approaching Afghanistan. Finding the highest genetic frequencies in a region does not necessarily mean that this region was the original source: it has been shown that geographic distributions can result from various modalities besides natural selection such as geographic barriers, subsequent migrations, replacement, isolation, and the surfing effect Our population data gives continuous genetic cover across Asia independent of language. Whereas the Eurasian main subcontinent components  are consistent with the linguistic spectrum of Macro-Caucasian in the west  (AC3 & AC6), Indo-Iranian in the north (AC4), Dravidian Brahui in the south (AC7) and Turkic and Mongol in the east (AC8 & AC9); such a linguistic correlation is not to be found in our Afghan samples. In the Hindu Kush region, the autosomal and haploid genetic structure can be explained better by geography than by language or ethnicity; this is in accordance with two recent studies on autosomal STR and blood group from these Afghan samples and compared to published data from surrounding regions The Inner Asian Mountain Corridor (IAMC) proposed by Frachetti Although the modern Afghan population is made up of ethnically and linguistically diverse groups, the similarity of the underlying gene pool and its underlying gene flows from West and East Eurasia and from South Asia is consistent with prehistoric post-glacial expansions, such as an eastward migration of humans out of the Fertile Crescent in the early Neolithic period, and the arrival of northern steppe nomads speaking the Indo-Iranian variety of Indo-European languages. Taken together, these events led to the creation of a common genetic substratum that has been veneered with relatively recent cultural and linguistic differences.Figure S1Admixture analysis from K = 2 to 15. Each individual is represented by a vertically (100%) stacked column of ancestry fractions in the constructed population.(PDF)Click here for additional data file.Figure S2Admixture analysis at K = 7 and K = 9. Each individual is represented by a vertically (100%) stacked column of ancestry fractions in the constructed populations. The Hindu Kush populations are labeled in purple. On the zoomed out panel on the right, language families are color coded.(PDF)Click here for additional data file.Figure S3Correlation of latitude and longitude and AC frequencies defined at K = 9 in the admixture analysis. Triangles and squares respectively depict correlation with latitude and longitude. Black plots indicate significant correlation. Correlation was calculated using the Pearson test.(PDF)Click here for additional data file.Figure S4Pairwise FST distances between Central Asia and neighboring populations, ranging from red (low) to blue (high), based on autosomal data. The populations (data from this study and published data (PDF)Click here for additional data file.Figure S5Central Asia mt-DNA tree. Hierarchic phylogenetic relationships and frequencies (percentages) of the mitochondrial haplogroups observed in the 516 Afghan samples analyzed in the present study. The mutations are scored relative to the RSRS (2); ! denotes a back mutation to ancestral status. Some of the tips are color coded to reflect the most likely geographical origin , and their overall frequencies reported. WA: West Eurasia, SA: South Asia, EA: East Eurasia.(XLSX)Click here for additional data file.Figure S6S6-A. Highlight on the main linguistic phyla . S6-B. Altaic phylum dissection . S6-C. Indo-European phylum dissection . S6-D. Highlight on the main Eurasian regions . S6-E. Coordinates of the different variables.Mitochondrial DNA FCA. First and second axes of the Factorial Correspondence Analysis based on 50 lineages examined in five Afghan populations and 214 populations previously reported in published data. Population references are listed in (PDF)Click here for additional data file.Figure S7Central Asia Y-chromosome tree. Hierarchic phylogenetic relationships and frequencies (percentages), haplogroup and haplotype diversity of the 84 paternal haplogroups observed in the 87 Pachtuns, 142 Tajiks, 77 Hazaras, 74 Turkmens and 127 Uzbeks from Afghanistan. The following additional population samples were analyzed at comparable Y-chromosome resolution: 186 samples from Iran, 150 samples from Kyrgyzstan, 160 samples from Mongolia, plus 176 samples from Pakistan (HGDP-CEPH). M89, M429, M522, P326, M526  were not genotyped but were included for phylogenetic context. In addition, M356, M93, V68, V257, M293, V42, V92, M426, M253, M205, M340, M378, V88 and SRY1532.2 were typed in the present study, but no derived alleles were observed.(XLSX)Click here for additional data file.Figure S8S8-A. Y-chromosome haplotype and haplogroup diversities for each population under study. See S8-B Correlation of Y-chromosome haplotype and haplogroup diversities among populations under study .Y-chromosome haplotype and haplogroup diversities. (DOCX)Click here for additional data file.Figure S9S9-A. Highlight on the main linguistic phyla . S9-B. Altaic phylum dissection . S9-C. Indo-European phylum dissection . S9-D. Highlight on the main Eurasian regions . S9-E. Y-chromosome tree displaying the consensus lineages used for database construction. S9-F. Coordinates of the different variables.Y-chromosome FCA. First and second axes of the Factorial Correspondence Analysis based on 34 pooled lineages examined in 37 Central Asian populations and 187 additional ethnic groups previously reported in published data. Population references are listed in supplementary (PDF)Click here for additional data file.Figure S10Median-joining networks of Y STR with haplogroups C3b2b1-M401, J2a1-Page55 and R1a1a-M198.(PPT)Click here for additional data file.Table S1Description of Afghan, Mongolian, Kyrgyz and Iranian samples and HGDP-CEPH samples from Pakistan included in the study.(DOC)Click here for additional data file.Table S2List of the samples used for the autosomal analyses: Groups of population, Number of individuals (n), Country/Region of the population and Reference (source).(XLS)Click here for additional data file.Table S3Description of new Y-chromosome binary markers.(DOC)Click here for additional data file.Table S4References used for the mtDNA and the Y-chromosome database.(DOCX)Click here for additional data file.Table S5Y-Chromosome STR profile for each individual in populations from Afghanistan, Iran, Pakistan (CEPH), Mongolia, Kyrgyzstan.(XLS)Click here for additional data file.Table S6Spearman correlation between frequencies of C-M401, J-Page55, R-M17 and Latitude/Longitude of 37 populations.(DOCX)Click here for additional data file.


Aphis gossypii through host-plant choice and acceptance. We also assessed whether host-specialised genotypes corresponded exclusively to anholocyclic lineages regardless of the environmental conditions.Host plants exert considerable selective pressure on aphids because the plants constitute their feeding, mating and oviposition sites. Therefore, host specialisation in aphids evolves through selection of the behavioural and chemical mechanisms of host-plant location and recognition, and through metabolic adaptation to the phloem content of the host plant. How these adaptive traits evolve in an aphid species depends on the complexity of the annual life cycle of that species. The purpose of this field study was to determine how winged spring-migrant populations contribute to the evolution and maintenance of host specialisation in in natura host-plant-selection pressure by showing the great differences in genetic diversity between the spring-migrant populations  and the melon-infesting populations . Moreover, an analysis of the genetic composition of these alate and apterous populations in four geographic regions suggested differences in life-history strategies, such as host choice and reproductive mode, and questioned the common assertion that A. gossypii is an anholocyclic species throughout its distribution area, including Europe.The spring populations of cotton-melon aphids visiting newly planted melon crops exhibited an unexpectedly high level of genetic diversity that contrasted with the very low diversity characterising the host-specialised populations of this aphid species. This study illustrated in natura for host recognition by spring-migrant aphid populations that are not specialised on Cucurbitaceae. The agroecosystem structure and history of the four studied regions may have partially shaped the genetic structure of the spring-migrant populations of A. gossypii. Cucurbitaceae-specialised genotypes corresponded exclusively to anholocyclic lineages, regardless of the environmental conditions. However, some genotypes that were genetically close to the host-specialised genotypes and some genotypes that probably originated from wild plants had never been previously sampled; both were holocylic.Our results clearly demonstrate that the melon plant acts as a selective filter against the reproduction of non-specialised individuals. We showed that olfactory cues are unlikely to be decisive  The predictability and abundance of host resources is often assumed to favour ecological specialisation in populations of parasites that have a large spectrum of possible hosts through the disruptive selection of host preference, leading gradually to reproductive isolation and speciation if assortative mating occurs -6. Host-i.e., they remain on the same host species or closely related plant species throughout their life cycle [Like many parasites, aphids have evolved complex life cycles, with successive generations of individuals often specialised to cope with different ecological conditions. However, aphids are among the few organisms that show plasticity in their reproductive mode as an adaptive response to seasonal changes . The typfe cycle . In the fe cycle .Acyrthosiphon pisum [Sitobion avenae [i.e., on the primary host. In this case, specialisation could evolve through a reinforcement process whereby selection against hybrids - due to host-associated genetic trade-offs - can drive the increase of assortative mating [Myzus persicae [Aphis gossypii [M. persicae, host specialisation was evidenced for anholocyclic and holocyclic lineages, and assortative mating seemed to be promoted by differences in the pattern of sexual activity of both the males and mating females [A. gossypii, the specialised populations consisted of a few anholocyclic lineages [Only 5% of aphid species are polyphagous , comprison pisum -14 and tn avenae ,16, whicn avenae . Converse mating . Host-sppersicae -21 and tgossypii . These sgossypii . In M. p females . In A. glineages ,25,26, slineages .A. gossypii is distributed worldwide and is found in both tropical and subtropical areas and in temperate zones. This species has been detected on more than 900 plant species [Hibiscus syriacus, Catalpa bignoïdes and other ligneous plants . Sexualin Italy . Geneticractices ,34,35. Pvailable .A. gossypii through host-plant selection. We also assessed whether host-specialised genotypes corresponded exclusively to anholocyclic lineages in relation to the environmental conditions. The sampling was completed in areas where the studied agrosystems were dominated or not dominated by Cucurbitaceae (melon) crops. The host-plant selection and reproductive mode were evidenced from a comparison of the genetic structure of the alate spring-migrant populations that alighted on melon crops with the structure of the apterous populations that consisted of the offspring of the migrants.The aim of this field study was to investigate how winged spring-migrant populations may contribute to the evolution and maintenance of host specialisation in A. gossypii, we sampled alate and apterous aphids from 2004 to 2009 in four locations in the southeast (SE) of France, one location in the southwest (SW) and west (W) of France and one location in the Lesser Antilles (LA)  Figure . The aphAmong the 4792 aphids analysed, we discriminated 596 multilocus genotypes (MLGs). The majority of the MLGs were present as a single copy, and only four of the MLGs had a frequency higher than 5% of the total number of individual genotypes Table . We discAmong the 596 MLGs discriminated, 316 were observed within only the alate samples, 183 were observed within only the apterous samples and 97 were observed within both of the morphs Figure . The disR (number of clones relative to the sample size); the clonal diversity, D*; and the clonal evenness, V.Among each morph, the genotypic diversity of the samples was investigated at three levels: within an area, between years and between areas. Three diversity indexes were considered, reflecting the clonal richness, V) was 0.86 ± 0.05 in Saint-Andiol, 0.83 ± 0.03 in Montfavet and 0.81 ± 0.01 in Eyragues (with a confidence of 95%). No strong differences were observed in these three indexes over the different years in each location. The comparison between the alate and apterous populations showed a significant decrease in the clonal richness of the apterous populations, regardless of the location or the area  was significantly higher in the SW populations than in the SE populations, while the Simpson's evenness index (V) was significantly higher in the populations sampled from the SE area than in the populations sampled from the SW area , the year of sampling, the growing area and the location (Table P < 10-5), which explained 20% of the variance. The AMOVA analysis confirmed the absence of a significant difference of genetic structure within the populations from the SE region. Therefore, we pooled the samples from all of the SE locations. To search for clustering of the MLGs found in this study, we analysed the MLGs along with the 44 MLGs previously described and assigned to host races . Using a Bayesian structuring program, the likeliest numbers of the genetic clusters were K = 2 and K = 4  and Hibiscus spp. (Hib4) hosts. Therefore, cluster Z was called the NM1 cluster.When K = 2, 203 MLGs of the 596 that were detected in our samples were assigned to cluster A and represented only 5% of the aphids, and 393 MLGs were assigned to cluster B, accounting for 95% of the aphids Figure . The Fsts Figure . ClusterWe analysed the MLGs that were assigned to cluster B and those that were assigned to cluster A separately using the same parameters of the Structure program. The number of genetic sub-clusters that best fitted the data was K = 3 for cluster B . Moreover, in all of the areas, the number of individuals with an MLG assigned to the cucurbit cluster or to the NM1 cluster (known for its ability to colonise Cucurbitaceae) was significantly higher in the apterous populations than in the alate populations . Notably, although the percentage of apterous individuals belonging to the Cucurbitaceae host race was equivalent in the SE and SW areas, the diversity among the cucurbit MLGs was considerably higher  in the SE region (n = 93) than in the SW region (n = 28)  Figure .is Findex. To reduce the Wahlund effect, we split the populations a) according to their cluster membership  and their area  , the linkage disequilibrium (LD) and the ) Tables  or accor) Tables .is Fvalue. These characteristics were observed in the LA region subpopulations in cluster X, in the SE area subpopulation in cluster Z (NM1) and in all of the subpopulations of cluster Y (cucurbit) except for the W subpopulation. The fingerprints of panmictic sexual reproduction were frequent HWE, infrequent LD and a positive is Fvalue close to 0. The only subpopulation with these characteristics was the W subpopulation in cluster Y. The fingerprints of sexual reproduction by inbreeding were a departure from HWE due to heterozygote deficiency, frequent LD and a positive is Fvalue close to 1. These fingerprints were observed in the SE area subpopulation in cluster X, the SW and W area subpopulations in cluster Z and the W area subpopulation in cluster A. We observed a fourth situation with a frequent HWE, rare LD and is Fvalues ranging from 0.23 to 0.61 for the SW and W area subpopulations assigned to cluster X and the SE and SW area subpopulations assigned to cluster A. When cluster A was split into six sub-clusters, HWE was observed for several loci (p > 0.05), and the number of significant LD was low  and the melon-infesting populations . Moreover, an analysis of the genetic composition of these alate and apterous populations in four geographic regions suggested differences in their life-history strategies and questioned the common assertion that A. gossypii is an anholocyclic species in most of its distribution area, including Europe [The spring populations of the cotton-melon aphids visiting newly planted melon crops exhibited an unexpectedly high level of genetic diversity that contrasted with the very low diversity characterising the host-specialised populations of this aphid species. The host races identified in l clones , as a rel clones ,34,35. Bg Europe .A. gossypii individuals discriminated only 44 MLGs. Each of these individuals corresponded to an apterous aphid taken from a different colony on vegetable crops of the Cucurbitaceae, Solanaceae, Malvaceae, Rutaceae and Rosaceae families in different countries [Using the same set of microsatellite markers, previous genetic analyses of more than 3600 ountries ,26. AlmoCucurbitaceae (NM1) and some that specialised on Malvaceae; these MLGs were found in 18% of all of the individuals. Cluster X contained MLGs from the Solanaceae and cotton host races; these MLGs were found in less than 5% of the aphids. In contrast, cluster A contained 203 MLGs that had never been observed in previous studies. However, these new MLGs represented less than 5% of the sampled aphids. The hypothesis that the aphids bearing these MLGs may have originated from a broad array of wild host plants is supported by the huge host-plant spectrum of A. gossypii, which consists of more than 900 plant species over 119 botanical families [The analysis of the 596 MLGs revealed four genetic clusters Figure . Three ofamilies .A. gossypii. These hyper-specialised clones are found all over the world on the same crops. Because these aphids specialise on annual crops, they are submitted to strong demographic bottlenecks, and they have to produce alate morphs at least twice during the annual cycle, first at the end of the crop season to disperse on refuge plants and again at the beginning of the next season to migrate back to the host crops. Therefore, the settlement of a new colony challenges alate aphids because they may have to make long flights to find a suitable host plant [in natura. In the present study, we observed alate individuals that were obviously not specialised on Cucurbitaceae on melon crops, i.e., individuals belonging to the clusters A and X. This finding suggests that olfactory cues are unlikely to be decisive in natura for host recognition, which probably occurs only after landing and plant probing. To test this hypothesis, we compared the diversity and the genetic structure of alate and apterous populations on melon crops at the very beginning of the crop season. As expected, the clonal richness was significantly lower in the apterous samples than in the alate samples in all of the studied fields  and pairwise st Fanalysis , and 4,000 ha in the Lesser Antilles (Guadeloupe and Martinique).The genetic diversity of the populations on the melon crops was geographically structured, as revealed by the AMOVA  , the evost Fanalysis, See additional file Cucurbitaceae crops, both across seasons and years in these three production areas. From an historical point of view, the melon crop has been of significance for many decades in the SE and SW regions, while in the W region, melons have been grown on a large scale for only ten years. Therefore, cucurbit-adapted MLGs have been favoured and selected for longer in the south than in the western region. In addition, in the SE region, Cucurbitaceae crops are grown from January to the end of the fall, either under cover or in open fields, while in the SW region, the plants are restricted to open fields in the spring and summer. The availability of the Cucurbitaceae resource throughout the aphid annual cycle varies considerably among regions, and that variation could explain the differences in the level of genetic diversity of the melon-adapted populations. In the SE area, the time window after which the Cucurbitaceae resource is no longer available is very small. Therefore, the Cucurbitaceae-specialised populations do not experience a harsh bottleneck during the winter, allowing for the maintenance of slightly divergent MLGs produced by mutation. The high genetic diversity observed in the Cucurbitaceae-specialised populations in the SE region probably results from both long-term selection and weak demographic bottlenecks during the winters. Another significant difference lies in the proportion of the alate aphids belonging to cluster A, which was ten times higher in the samples from the SW region (10%) than in the SE region (1%). This difference might be due to a difference in the uniformity of the agricultural environment between the two regions, resulting in a lower abundance of the overwintering colonies of specialised Cucurbitaceae clones in the SW region, and therefore, reducing the alate spring population size.When considering the populations from France, a geographical structure of the genetic diversity was detected (isF) strongly suggest that these individuals correspond to obligate parthenogenetic lineages, in accordance with previous genetic studies [Cucurbitaceae-specialised population collected in the W region  or because of a mix of asexual and sexual lineages.Another n France ,47. The ex Table . The freex Table . The posSolanum spp., pepper, citrus, strawberry and Hibiscus spp. . The origin of the new MLGs remains unknown.Cluster X was composed of 133 MLGs, of which 115 were unique, and the 18 MLGs left existed in two or three copies . Populations of R. padi were not genetically differentiated according to host-plant origin [S. avanae, a much higher proportion of sexual lineages were found on uncultivated host plants [nicotianae of M. persicae [A. gossypii populations depended on both the reproductive strategy and host plant. The Bayesian clustering analysis was used to first separate the populations into two groups. One group consisted of cluster A, with individuals assumed to reproduce sexually and to develop on wild plants, and the other group consisted of cluster B, in which asexual lineages co-existed with a few sexual lineages. The sub-clustering of B led to the identification of the cluster Y, containing mainly parthenogenetic lineages specialised on Cucurbitaceae, and the clusters X and Z, which likely displayed a mixed reproductive strategy. The high level of diversity detected in the spring alate aphids was generated by the reproduction of the sexual lineages. However, this huge diversity was counter-selected by the melon plants, as evidenced by the much lower diversity found in the apterous offspring. Moreover, none of the non-specialised offspring was able to develop a colony. We cannot exclude that some of the parthenogenetic lineages from the Cucurbitaceae host race are androcyclic, producing males and parthenogenetic females or are intermediate, investing into both sexual and parthenogenetic reproduction. Their sexual morphs could migrate to the primary host to mate either with an unrelated MLG or with a cucurbit MLG (assortative mating). Indeed, the cucurbit-specialised clone C9, which was found in high frequency in Cucurbitaceae crops all over the world [A. gossypii as a pest of melon crops would depend on the balance between the gain of genetic diversity and the loss of host-adapted gene combinations resulting from sexual reproduction. The evolution of parthenogenetic lineages in the Cucurbitaceae host race also relies on the selection of new mutants exhibiting a higher fitness in response to pest-management practices and to the clonal amplification of these new variants [In conclusion, the genetic survey of the alate aphids revealed that sexual reproduction occurs in French populations of  regions . Approxienotypes . Populatn avenae  and Rhophum padi , revealet origin , whereast plants . Distincpersicae . Our stuhe world , could bhe world . New asehe world . Therefovariants .Four melon-growing areas were considered in this study - in the southeast, southwest, and west of France and in Guadeloupe in the Lesser Antilles Figure . In the ® was applied once a few days before transplanting). The fields contained plants without genetic resistance to aphids. One population was defined as a sample collected in a field a given year and with a given morph, alate or apterous. Melons were transplanted in fields at the end of March in the LA and in May in the SE, SW and W regions of France. We collected 15 to 228 alate morphs per field during the first three weeks after planting; the aphids were collected from the melon plants and constituted a sample of the spring migrants that alighted on the melon crops. Sampling of apterous morphs, which consisted of the spring migrants offspring, started three weeks after planting and was conducted over four to five weeks. We collected 80 to 491 apterous morphs per field. Each apterous aphid was collected either as an isolated individual or as one individual representing the colony that was produced by a single asexual founding mother. Each colony that was sampled was tagged to avoid any re-sampling.The aphids were sampled over five years  in seven melon fields . The DNA amplifications at eight microsatellite loci specific to the A. gossypii genome [The DNA of 4,792 individual aphids was extracted using a 5% (w/v) Chelex resin solution, as described by Fuller i genome  were peri genome . The allR) was computed as G is the number of distinct genotypes in the population and N is the total number of sampled individuals from the population. The unbiased Simpson's complement (D*) was calculated as i nis the number of occurrences of genotype i and N is the total number of sampled individuals from the population. This index can be interpreted as the probability that two individuals chosen at random have different genotypes and can thus be considered as an exact measure of the clonal heterogeneity. The Simpson evenness index (V) was computed as V is an equitability index that describes the distribution of the components and the relative amount of clones. R and D* range from 0, if all of the individuals in the population are clones (G = 1), to 1, when each sampled individual in the population bears a distinct genotype (G = N). The Simpson evenness index (V) varies between 0 and 1, which represent extreme skew and evenness, respectively. We used a jack-knifing procedure to estimate the standard error of R, D* and V: for each sample set, we recomputed the 3 indices, leaving out one observation at a time from the sample set. From this new set of observations, an estimate for the variance of the indices was calculated, and thereafter, their confidence interval at 5% was calculated [Three indices were computed for each population as proposed by , considelculated .st Ftests [We used hierarchical analyses of molecular variance, AMOVA, and pairwise st tests  in the Ast tests  to test Cucurbitaceae, cotton, Solanum and pepper) from numerous samples of A. gossypii collected over several years on a large geographical scale from annual crops of different plant families [K (varying from 1 to 30) were compared to check the consistency of the estimates and to determine the likeliest number of genetic clusters. We paid particular attention to those MLGs that clustered with the 12 cucurbit reference MLGs that were defined by Carletto et al. (2009) [In our assignment procedure, we will refer to the four host races that have been unambiguously identified (families . We assifamilies  using thfamilies . We used. (2009) .i.e., the alate samples, collected in the early season in the melon fields. We computed three parameters: departures from the Hardy-Weinberg equilibrium (HWE) for each locus, the linkage disequilibrium (LD) for each pair of loci and the is Fvalue using the exact tests (p = 0.05) available in the Arlequin version 3.1 program, considering repeated genotypes [is Findex can be thought of as a measure of the identity of alleles within individuals relative to the identity of alleles randomly drawn from two different individuals issued from the same subpopulation. The is Fvalue is interpreted in terms of the deviation from random mating caused by the breeding system of the organism under study: for a strictly clonal population, is F= -1; for a strictly sexual population, is F= 0; and for a strictly inbreeding population, is F= +1. To reduce the Wahlund effect, the samples collected in each of the growing areas were divided according to the genetic clusters evidenced using the Structure program. The calculations of the HWE, LD and is Fwere conducted on these sub-samples.The genetic fingerprints of sexual reproduction were studied in the spring-migrant populations, enotypes . The MLGThe authors declare that they have no competing interests.ST carried out the acquisition, analysis and interpretation of the data and drafted the manuscript. NB conceived and designed the study and was involved in the acquisition and interpretation of the data and in drafting the manuscript. FV was involved in the acquisition and interpretation of the data and in revising the manuscript. All author read and approved the final manuscript.Figure S1. Clonal diversity of the alate and apterous A. gossypii samples collected from 2004 to 2009 in the four locations in southeastern France. R is the index of clonal richness, D* is the unbiased Simpson's complement and is the probability that two individuals chosen at random have different genotypes and can thus be considered as an exact measure of the clonal heterogeneity and V is the Simpson evenness index and is an equitability index that describes the distribution of the components and the relative amount of clones. The confidence intervals derive from jack-knifing procedures (p = 0.05).Click here for fileFigure S2. Mean values of the ln likelihood and delta K obtained for ten simulations of each K after Structure analysis completed with a. all of the MLGs, b. the MLGs assigned to cluster B and c. the MLGs assigned to cluster A.Click here for fileTable S1. Genetic differentiation between the A. gossypii populations sampled in three areas of France and in the Lesser Antilles according to the pairwise stF.Click here for fileTable S2. Number of individuals (N), number of multilocus genotypes (G), and the number of unique MLGs (g) in the alate (Al) and apterous (Ap) A. gossypii populations sampled in France and the Lesser Antilles, grouped according to the clusters.Click here for file


Using DNA to determine an individual's ancestry from among human populations is generally interesting and useful for many purposes, including admixture mapping, controlling for population structure in disease or trait association studies and forensic ancestry inference. However, to estimate ancestry, including possible admixture within an individual, as well as heterogeneity within a group of individuals, allele frequencies are necessary for what are believed to be the contributing populations. For this purpose, panels of ancestry informative markers (AIMs) have been developed.We are presenting our work on one such panel, composed of 128 ancestry informative single-nucleotide polymorphisms (AISNPs) already proposed in the literature. Compared to previous studies of these AISNPs, we have studied three times the number of individuals  in three times as many population samples (119). We have validated this panel for many ancestry assignment and admixture studies, especially those that were the rationale for the original selection of the 128 SNPs: African Americans and Mexican Americans. At the same time, the limitations of the panel for distinguishing ancestry and quantifying admixture among Eurasian populations are noted.structure program to determine which groups cluster together and consequently influence the ability of a marker panel to infer ancestry. We demonstrate the strengths and weaknesses of this particular panel of AISNPs in a global context.We demonstrate the simultaneous importance of the specific set of population samples and their relative sample sizes in the use of the  In recent years, there have been many proposed ancestry informative markers (AIMs) and published sets of AIMs useful for particular purposes. Some sets have focused on estimating the admixture between specific ancestral populations such as the African and European genetic contributions to African Americans or European, Native American and African contributions to Latino populations e.g., -7. Other. Other7.Whatever the strategy for identifying them, AIMs are necessarily selected because they distinguish the specific population samples used. Therefore, replication with other samples of individuals from the same and/or closely related populations is necessary to verify the robustness of any set of AIMs. Such replication is onerous, costly and rarely undertaken.Given the broad interest in AIMs in genetics, medicine, anthropology and forensics, the development of an optimal set of AIMs for a broad range of uses needs to be based on multiple markers studied on moderate to large samples of multiple relevant populations; appropriate resources will probably not be available in any single lab. As we advocated in the case of single-nucleotide polymorphisms (SNPs) for individual identification ,26, multSeldin's group ,27 identet al. [et al. [et al. [Understanding that a set of AIMs  will only be broadly useful for population relationships and for identifying admixture if that set can be shown on a very large data set to be valid, we have tripled the size of the Nassir et al.  populati [et al. . We find [et al.  results et al. [et al. [We assembled a data set of samples of 4,871 individuals: those from the HapMap 3 , the Humet al. . Some of [et al.  are also [et al.  associatet al. [® . The HGDP marker data were downloaded from http://hagsc.org/hgdp/files.html[http://hapmap.ncbi.nlm.nih.gov/index.html.yo[The polymorphic sites were those reported by Kosoy et al. . The 3,0iles.html. The Hapx.html.yo. Of the Fst was calculated across all populations for each marker using the simple formula of Wright : σ2pq¯. http://www.xlstat.com/en/company/)) as one method to evaluate effectiveness of these SNPs for distinguishing among populations and to determine the major factors accounting for the population frequencies.Principal component analysis (PCA) analyses of population sample allele frequencies were performed using XLSTAT  [http://rosenberglab.bioinformatics.med.umich.edu/distruct.html) [ture.html-35) was pp.html) . Specifict.html) . The matThe allele frequencies for the 128 AISNPs for all 119 population samples are given in Additional file et al. [n) of Rosenberg et al. [SLC24A5, OCA2 (two SNPs), HERC2 and EDAR), and each of these genes is well known to have SNPs with marked global variation in allele frequency; but the best "known" SNPs are not part of this 128 AISNP set  and RTTN . There are 10 outliers at the low end of the reference Fst distribution with Fst <0.04. Only one of the AISNPs falls below the mode of the reference distribution: TWGS1 .The Fst distribution of the 128 AISNPs was compared to the distribution of 2,327 non-AISNPs typed in our lab  for each of the patterns  samples from two or more different populations, (2) the descendants of people from two or more populations who have "recently" intermarried, (3) descendants of people from two or more populations who have intermarried in the ancient past and (4) people discretely sampled from a single region along a geographic allele frequency cline established predominantly by random genetic drift.structure  willet al. [et al. [et al. [et al. [It is important to realize that the outcome of any analysis of admixture or other population structure depends heavily on both the population samples and the markers used. Though we have included some of the same HGDP populations as Kosoy et al.  did in t [et al.  as well  [et al.  or Nassi [et al.  reports.The results shown in Figure Structure attempts to find the set of K population allele frequencies that will give the best fit to all individual samples assuming Hardy-Weinberg ratios for each of the K populations. Structure does not consider or produce analyses of population relationships. Fortunately, this is not an issue of interest to forensic science. Rather, structure assigns individuals to clusters of genetically similar individuals. Obviously, if numbers of individuals differ greatly among different populations, a population sample with a large number of individuals will influence the allele frequencies of the particular cluster into which it falls more than a population with a small number of individuals. Thus, a small population from the middle of a cline with larger numbers in populations from the more extreme parts of the cline will appear "admixed." Such is seen at K = 3 for the South, Central and East Asian populations. However, a large population from that middle region will, at the same K value, cause the allele frequency estimates of the flanking clusters to move toward the center even if cluster assignments do not change. The consequences at higher K values may be that the "middle" population is a distinct group or, by shifting the estimates for the flanking clusters, cause a population at the extreme of the cline to "fall off" and become a separate cluster. The cluster assignments at K = 4 and K = 5 illustrate this  indicate no new strongly supported subgroups of populations and suggest, for example, that differentiating ancestry from among populations within East Asia will not be easy using the allele frequencies for this set of SNPs.The data for these SNPs can be used to "assign" regional ancestry to a single individual based on the genotypes at all or a significant fraction of these 128 SNPs. This would be done by calculating the likelihood of the multisite genotype based on the allele frequencies of each of the 119 population samples (frequencies are in ALFRED ). It is K = 8 structure analysis. Compared to the variation among individuals shown in Figure It is important to distinguish population averages from the variation among individuals  occurred in three of the panels and 26 other SNPs (about 4%) occurred in two panels. None of the 128 SNPs in the panel we have analyzed occurred in any of these other panels. The small number of overlapping SNPs across panels likely results from the different methods of selecting SNPs, the different data sets from which SNPs are selected and the different purposes of the panels: some are global, some are regional and some are for the four continental extremes. However, though the specifics of these panels are not relevant, it is clear that there is no single set of AISNPs that will be of value for all questions.K = 6-8). Distinguishing among East Asian populations is also not optimal with this set of AISNPs. Neither of these conclusions is surprising, since populations in those regions were not part of the selection of this set of AISNPs. Selection to identify SNPs with markedly different allele frequencies across East Asia will be necessary [structure analyses of Figures With our additional data and the analyses presented here, this panel of 128 AISNPs is the best documented and validated for broad global application to infer ancestry. However, it is not necessarily the optimal panel depending on the question being asked, and it is definitely not optimal at identifying ancestry within Europe and Southwest Asia (cf. Figures ecessary . Many usThe authors declare that they have no competing interests. FMDLV is employed by Life Technologies.All authors have read and approved the final manuscript. KKK and FMDLV were involved in the conception and design of the study, and KKK assisted in writing the manuscript. JRK supervised the genotyping assays and data analysis and wrote the manuscript. FRF and AJP assisted in the data analysis. WCS assembled and integrated the data set from the literature and laboratory.List of missing values and how they were handled in the PCA.Click here for filePopulation allele frequencies.Click here for fileList of Fst values for all 128 AISNPs.Click here for fileList of upper and lower outliers for Fst in Reference and AISNP distributions.Click here for fileLikelihood plot K = 2-12.Click here for fileStructure analysisIndividual Assignments in .Click here for fileStructure analysisPopulation Assignments in .Click here for file


Differences in ploidy levels among different fern species have a vast influence on their mating system, their colonization ability and on the gene flow among populations. Differences in the colonization abilities of species with different ploidy levels are well known: tetraploids, in contrast to diploids, are able to undergo intra-gametophytic selfing. Because fertilization is a post-dispersal process in ferns, selfing results in better colonization abilities in tetraploids because of single spore colonization. Considerably less is known about the gene flow among populations of different ploidy levels. The present study examines two rare fern species that differ in ploidy. While it has already been confirmed that tetraploid species are better at colonizing, the present study focuses on the gene flow among existing populations. We analyzed the genetic structure of a set of populations in a 10×10 km study region using isoenzymes. Genetic variation in tetraploid species is distributed mainly among populations; the genetic distance between populations is correlated with the geographical distance, and larger populations host more genetic diversity than smaller populations. In the diploid species, most variability is partitioned within populations; the genetic distance is not related to geographic distance, and the genetic diversity of populations is not related to the population size. This suggests that in tetraploid species, which undergo selfing, gene flow is limited. In contrast, in the diploid species, which experience outcrossing, gene flow is extensive and the whole system behaves as one large population. Our results suggest that in ferns, the ability to colonize new habitats and the gene flow among existing populations are affected by the mating system. Gene flow is the successful movement of genes among populations by mating or by the migration of diaspores, and it is one of the key factors determining the spatial genetic structure of populations The intensity of gene flow is, to a vast degree, influenced by the mating system of the species The fact that fertilization in ferns occurs on the gametophyte, which originates from spores, is of crucial importance for dispersal. In seed plants, fertilization takes place prior to dispersal, before the seed is formed. Theoretically, one seed is enough to colonize a new habitat. In ferns, fertilization is a post dispersal process on the haploid gametophyte. For ferns unable to use intra-gametophytic selfing, this means that the two spores must fall in close proximity and under favorable conditions on a new habitat. Fertilization can only occur after a gametophyte with archegonia/anteridia develops from both. Colonization of a new habitat is problematic in this case The differences in colonization abilities naturally result in very different patterns of genetic variation. Thus, the structure of genetic diversity was used as a source of information about the mating system of ferns in many studies Studies on the distribution of genetic diversity within and among populations are limited because it is difficult to distinguish between the gene flow among already established populations and historical processes during the colonization of new habitats . DespiteAsplenium adulterinum and A. cuneifolium, which differ in both ploidy level and their breeding system. In our model system, the two species occupy very similar habitats – serpentine rocks, which are scattered in an area of 10×10 km. From our previous study, we have additional information about the colonization rates of the species in the system In the present study, we analyzed the genetic structure of the populations to investigate gene flow among the populations of two rare fern species, Asplenium adulterinum and A. cuneifolium?, 2) What is the intensity of gene flow among the populations? and 3) Do the patterns differ between species, and do they correspond to the expected difference in mating systems?Our study addressed the following questions: 1) What is the distribution of genetic diversity within and among the populations of Furthermore, we compare the results from the current study about the genetic structure of the populations of the two species with information about the colonization rates in the system Asplenium adulterinum Milde. and a diploid, Asplenium cuneifolium Viv. (Aspleniaceae), both of which are restricted to the serpentine substrate in Europe. Distribution of both species is highly scattered, following serpentine rocks in Europe from the Mediterranean to Norway and from Greece to Spain  A. cuneifolium is generally more widespread than A. adulterinum.The study involves two fern species, a tetraploid, A. adulterinum is classified as a species of interest to the European ecological network Natura 2000 A. adulterinum is allotetraploid , A. cuneifolium is diploid Both species are rare and of interest to nature conservation groups throughout Europe A. adulterinum and A. cuneifolium are quite common in the area. A. adulterinum is more common and occupies rocks in both unforested habitat and the forest (dominated by Pinus sylvestris and Picea abies). In total, there are 66 populations located throughout the area, ranging from a few individuals to nearly 2000 individuals A cuneifolium prefers rocks under the forest canopy. The unforested rocks are rarely inhabited and the populations are very small. In total, there are 48 populations of A. cuneifolium in the study region, mostly in the central area, with several more distant localities, and the populations range from several individuals up to several hundred individuals This study was carried out in the region of Slavkovský les in western Bohemia, Czech Republic. In this region of ca. 10 × 10 km, 98 serpentine rocks are scattered across the landscape A. adulterinum occupies 81% of the suitable habitats and A. cuneifolium occupies 73%, indicating metapopulation dynamics in the study system For both species, unoccupied suitable habitats exist in the area; A. adulterinum and 12 localities in A. cuneifolium. Sampling design followed the distribution of the species in the study area (A. adulterinum has more populations). If available, 20 plants per population were sampled for each species. In total, we sampled 268 individuals of A. adulterinum and 227 individuals of A. cuneifolium.Samples for isoenzyme analysis were collected from 14 localities in Samples were evenly distributed over each locality to represent the entire range of variability within the population . We sampled 1–2 young leaves without spores per plant, being careful to not seriously damage the plant. The position of each plant was recorded using GPS  or marked on a map in the field, followed by digitalization of the map.Samples collected in the field were kept on ice for 24–48 hours until the isoenzymes were extracted in the laboratory. Electrophoresis was performed on the crude protein extracts of the leaf material. All enzymes were resolved on polyacrylamide gels using an 8.16% separating gel and a 4% stacking gel.Nine enzymatic systems were studied, 7 of which provided an interpretable pattern and were variable at least for one of the study species: LAP, DIA, 6-PGDH, SHDH, PGM, ADH and AAT. For a detailed methodology of the isoenzyme extraction, electrophoresis and staining procedures, see A. adulterinum is an allotetraploid, and it was rarely possible to assess the exact ratio of alleles from the intensity of the bands. Moreover, allotetraploids often have fixed pairs of alleles that always segregate together A. cuneifolium, the same approach of treating the data as a dominant marker was used to compare the species.Bands were interpreted in two ways. First, only the presence or the absence of alleles was recorded, and the data were further treated as a dominant marker  data were prepared in the program FAMD st values between populations and pairwise geographic distance (in meters) between centroids of localities hosting the populations  × the total number of samples.The 7 enzymatic systems provided a total of 9 interpretable loci: AAT, ADH-1, ADH-2, DIA, LAP-1, LAP-2, 6-PGHD, PGM and SHDH. In A. adulterinum, only one locus (LAP-1) could be reliably evaluated as allelic data. This locus either showed fixed heterozygosity  or fixed homozygosity (pattern AAAA), indicating diploid inheritance . Rarely, a clear pattern of AAAB was observed  in diploid A. cuneifolium. In A. adulterinum, only 14 haplotypes were present in the entire dataset, with separate populations containing 2–8 haplotypes. In A. cuneifolium, 96 haplotypes were present in the entire dataset, with separate populations containing 4–19 haplotypes.The mean gene diversity across the populations was 0.47 (ranging from 0.029 to 0.8) in tetraploid A. adulterinum, 40.6% of genetic variation was within populations and 59.4% was among populations . It contrast, in A. cuneifolium, 81.0% of genetic variation was within populations and only 19.0% was among populations , In A. adulterinum. In contrast, in A. cuneifolium, the relationship was not significant , A. adulterinum host more genetic diversity. This relationship was, however, only marginally significant . In A. cuneifolium, no relationship between the size of the population and the genetic diversity was observed (p = 0.290), The correlation between geographic and genetic distance between populations was highly significant  in A. adulterinum. As expected, the observed heterozygosity in A. cuneifolium was much more balanced in all enzymatic systems (According to the one enzymatic system (LAP-1) that made allowance for reliable co-dominant scoring, there was very high deficiency of heterozygotes in  systems .Asplenium adulterinum, have a high genetic differentiation, and the difference increases with geographical distance. However, the individuals are rather uniform within populations. In contrast, populations of the diploid, A. cuneifolium, are very similar to each other, but individuals within populations are genetically variable. Based on these results, we can deduce the probable mating system of the species and the intensity of gene flow between populations. Together with the results from our previous study The present study revealed considerable differences in the genetic structure of two populations of rare fern species differing in ploidy level. Populations of the allotetraploid, A. adulterinum (4n) and A. cuneifolium (2n) was based on their ploidy level and on the common expectation that diploids undergo selfing and polyploids undergo outcrossing Our original assumption about the differences in the mating system between Asplenium adulterinum, which is expected to undergo selfing, has 59.4% of the genetic variability partitioned among its populations, while A. cuneifolium, which is an expected outcrossing species, has 19% of the genetic variability partitioned among populations (based on the dominant marker).The mating system of a species is commonly estimated from the genetic structure of its populations, where the genetic variation is partitioned mainly within populations of outcrossing species and among populations of selfing species Asplenium adulterinum. This suggests a high level of inbreeding. In contrast, the observed heterozygosity in A. cuneifolium was rather balanced, as expected. Therefore, the genetic population structure suggests that selfing is the prevailing mating system for A. adulterinum, while outcrossing is the prevailing mating system for A. cuneifolium.Additionally, the enzymatic system, in which interpretation based on alelles was possible, shows a striking lack of heterozygotes – we found only 2.6% of heterozygotes in A. adulterinum is capable of outcrossing because we found heterozygotes, which are clearly a direct output from crossing two different genotypes. However, the frequency of individuals resulting from outcrossing is quite low. This finding corresponds to the conclusions of previous studies that gametophytic selfing is the main mating system in polyploid ferns Our data further showed that A. cuneifolium, the species with prevailing outcrossing, is capable of intragametophytic selfing, and thus, single spore colonization. However, single spore colonization has been confirmed in other diploid species, such as Asplenium scolopendriumUnfortunately, our data cannot provide information regarding whether A. adulterinum show strong genetic differentiation similar to other predominantly selfing fern species, e.g., A. cuneifolium, there is no relationship between genetic and geographic distance, and most of the genetic diversity can be found within populations. This result suggests that the whole system of this species functions as one large population with frequent dispersal throughout the area and a high level of gene flow, as is often found in various outcrossing fern species . Populations of es e.g., .When we combine the results of the present study about the genetic structure of the populations with information on the colonization ability of the species A. adulterinum is predominantly a good colonist of empty patches A. adulterinum, A. cuneifolium has a more difficult time colonizing empty patches. However, outcrossing facilitates effective gene flow between already established populations because the new genotype must be crossed with the local gametophytes to be incorporated into the population’s gene pool Click here for additional data file.Appendix S2Unbalanced isoenzyme banding pattern AAAB in locus LAP-1 in tetraploid A. adulterinum.(DOC)Click here for additional data file.


Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the existence of eastern refugia.The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns.  Castanopsis (Fagaceae)-type broadleaved evergreen forest community in Japan, which characterizes the biodiversity and endemism of the East Asia. We aimed to elucidate the effects of past climatic changes more clearly on the current genetic diversity of the species that inhabit warm-temperate zone, because the effects of climate change are particularly severe for these members of the community.The Quaternary climate cycles played an important role in shaping the distribution of biodiversity among current populations, even in warm-temperate zones, where the land was not covered by ice sheets Palynological evidence has indicated that the broadleaved evergreen forests in Japan experienced cold periods at least four times during the Quaternary Castanopsis, which is the dominant tree species in East-Asian broadleaved evergreen forests. In Japan, the plant genus Castanopsis holds two species, C. sieboldii and C. cuspidataC. sieboldii var. sieboldii and C. cuspidata var. cuspidata are independent species and whether the intermediate morphologies are a natural hybrid. However, the extremely low levels of intraspecific variation in the chloroplast DNA, have made it difficult to elucidate their phylogenetic and phylogeographic relationships.It is extremely important to reveal the genetic diversity and phylogeographical structure of the keystone species in the forests. We aimed to investigate C. sieboldii and C. cuspidata can be clearly distinguished on the basis of genetic information as well as morphologically observed data. Second, we attempt to elucidate the genetic structure within each of the two species. The study presented in this work was performed to analyze Expressed Sequence Tag (EST)-associated microsatellite genetic variation in Castanopsis. Finally, these analyses of the dominant trees in the broadleaved evergreen forests of Japan are then combined with previous results on the phylogeographic patterns of the plants growing in the same climatic zone to reconstruct the history of the forests.In the present study, we first aim to determine whether All necessary permits were obtained for the described sampling sites in verbal or written form. For sampling sites belonging to Japanese national forests, we obtained permits from regional forestry offices, and for the private sampling sites, permits was obtained from the owners. The University of Tokyo Chiba Forest and Kumamoto Prefectural Forestry Research Guidance Place also issued the permit. The plant materials did not involve endangered or protected species.Castanopsis (Fagaceae) is represented by two species, C. sieboldii and C. cuspidataCastanopsis sieboldii is found in the main islands and the Ryukyu Islands in Japan, and is divided into two varieties, sieboldii and lutchuensis  at the LGM existed in southwestern Kyushu  and northern Kyushu  as well as at Ryukyu Islands (10%) C. sieboldii var. sieboldii, C. cuspidata var. cuspidata, and hybrids We examined the epidermis of the leaves from each sampled individual, since this is the most effective way of discriminating between et al. et al. nd, QmC00288, was developed in the course of this work   method gtacaag) . The DNACastanopsis trees collected from 63 sites for the 32 EST-SSR loci. To evaluate the genetic diversity over all populations, we calculated the total number of alleles (NA), the average gene diversity within populations (HS) HO). We also calculated the fixation indices, FISFIS from zero and the linkage disequilibrium for all locus pairs was evaluated by permutation tests with sequential Bonferroni correction. We calculated coefficients of gene differentiation, GSTFST in the case of multiple alleles, to determine relative genetic differentiation among populations.We determined the genotypes of 1,502 NA), unbiased heterozygosity (HE) RS) Castanopsis species, we employed GIS program GRASS We used the following parameters calculated from the allele frequencies at all loci analyzed to evaluate the genetic diversity within each population: the average number of alleles (C. sieboldii populations (Nos. 1–40), C. cuspidata populations (No. 47–63) and mixed populations (No. 41–46) from seven districts  and the current environmental conditions in their habitats to examine the impact of local conditions on genetic diversity. This was done using a generalized linear model (GLM) created in R RS has continuous values. The environmental factors included in the model were latitude, longitude, altitude, precipitation in the coldest three months, precipitation in the warmest three months, minimum temperature of the coldest month, and mean temperature of the warmest three months, as shown in We also analyzed the relationships between the genetic diversity within each population  To assess the proportion of variance in FST values over all loci to their expected distributions under an island model with the assumption of neutrality using the LOSITAN program P values for each locus, 10,000 independent loci were generated and the simulated FST distribution was compared to the observed FST values. This made it possible to identify outliers in a one-step process by defining them as observed FST values falling outside the 99% confidence interval for the simulated group.Because STRUCTURE analyses are not suitable for studying loci under selection, we carefully checked the neutrality of each locus. We compared the distribution of the DA distances We estimated the genetic structure among populations by constructing a neighbor-joining (NJ) tree Castanopsis and within C. sieboldii and C. cuspidata, and to infer the most appropriate number of subpopulations (K) using STRUCTURE version 2.2 K (1–10) with 300,000 Markov chain Monte Carlo sampling runs after a burn-in period of 500,000 iterations, using the admixture model under the assumption of correlated allele frequencies. The most appropriate cluster number (K) was selected using the criterion of Evanno et al. K. These analyses were performed using two data sets, one covering all 32 loci and the other containing only loci without outliers. We used multiple regression analyses to investigate the relationship between the membership values calculated for each individual using STRUCTURE and the number of epidermal cell layers in its leaves.In addition, we used the Bayesian clustering method to elucidate the genetic structure among populations of C. sieboldii and C. cuspidata by comparing genetic distances to geographic distances (DA) between pairs of populations. The significance of the associations between the two types of distance was determined by the Mantel test We assessed the presence of isolation-by-distance patterns in C. cuspidata var. cuspidata and C. sieboldii var. sieboldii  were primarily found in the six populations (Nos. 41–46) in which single- and double- epidermal cell layer individuals were sympatrically distributed, although one or two individuals with intermediate morphology were also found in five other populations .Populations consisting mainly of individuals with single epidermal cell layers were distributed in inland areas, while populations in which individuals had double epidermal cell layers were distributed along the coastal areas . These dieboldii . IndividHS and HO over all loci were 0.644 and 0.591, respectively. Across all populations, the FIS values deviated significantly and positively from zero at 12 loci, and over all loci. No evidence of significant linkage disequilibrium was detected in any of a total of 9,920 permutation tests for linkage disequilibrium between loci. High levels of genetic diversity within populations were also observed in each population  for all Castanopsis populations. The GST value varied among loci, ranging from 0.053 at locus CcC02535 to 0.361 at locus CcC01513   .Castanopsis populations: eight of these had FST values exceeding the upper 99% CI  and the other consisting of individuals having a double epidermal cell layer (Nos. 1–40)  . Six popK = 2 provided a satisfactory explanation of the observed data . The results obtained when using data from only 11 loci and excluding 21 outliers .Multiple regression analysis showed that the Q values (which measure group membership) calculated for each individual using STRUCTURE correlated significantly with the number of cell layers in the epidermis of the leaves  and in C. sieboldii populations (P = 0.02), while no significant effect was observed in C. cuspidata populations (P = 0.35). Within C. sieboldii populations, those in western Japan (Kyushu) tended to have above average NA, RS, and rare allele values  tended to have above average NA and RS values , 15 populations of C. cuspidata , and all of the mixed populations deviated significantly from mutation-drift equilibrium under the IAM after sequential Bonferroni correction  of C. sieboldii populations , and that precipitation in the warmest three months significantly correlated with the genetic diversity of C. cuspidata var. cuspidata populations .An analysis of variance for the selected GLM model using the AIC revealed that latitude significantly correlated with the genetic diversity . AMOVA indicated that a small fraction of the observed gene diversity was attributable to differences between the various geographic regions in which C. sieboldii populations occur (1.3–2.0%) and differences among populations within groups (6.2–6.5%)  and differences among populations within groups (5.2%).The overall genetic differentiation among populations at the 32 loci was low (.2–6.5%) . In C. cC. sieboldii contained two major clusters corresponding to populations from the western (Cluster W) and eastern (Cluster E) regions (C. sieboldii var. lutchuensis) clustered strongly together to form a sub-cluster.The NJ tree of  regions . Within C. sieboldii. Bayesian clustering demonstrated that the highest ΔK value was achieved when K = 3, therefore the fraction of ancestry derived from each of three clusters was estimated. We analyzed the STRUCTURE analysis both with and without the two outlier loci and the results were in general congruent between these analysis. The frequency of cluster 1 was particularly high among populations from the Ryukyu Islands .In contrast,  NJ tree . BayesiaC. sieboldii .A Mantel test revealed a weak but significant correlation between the pair-wise genetic distance and geographical distance between populations for GST = 0.069 in C. sieboldii populations, and GST = 0.057 in C. cuspidata populations). Wind-pollinated and wind-dispersed tree species have previously been reported to exhibit low values of GST , as reviewed by Tsumura GST values: 0.144 in Camellia japonicaZanthoxylum ailanthoidesGST values of the Castanopsis species examined in this study were relatively low even though they are insect-pollinated and animal-dispersed. Yumoto Castanopsis attracts many taxonomic groups of insects, such as small bees, wasps, flies, beetles and butterflies. Moreover, flower-visiting insects have been observed to visit Castanopsis more often than other canopy-flowering tree species. This would be expected to result in relatively low levels of genetic differentiation among Castanopsis populations due to frequent large-scale gene flow.We found low levels of genetic differentiation among the populations that we examined . The two clusters identified in our genetic clustering analyses were largely consistent in terms of their leaf epidermal structure, indicating that this morphological trait is a useful characteristic for discriminating between the two genetic groups of Castanopsis. These results suggest that the two clusters should be treated as independent species, i.e. C. sieboldii and C. cuspidata. A far larger proportion of the total variance  (C. sieboldii (1.3 – 2.0%) and within C. cuspidata in Japan (−0.2%), further demonstrating their clear genetic differentiation.The Bayesian clustering analyses and the phylogenetic analysis revealed the presence of two well-separated groups of populations, one consisting of individuals with a single-layer epidermis and the other consisting of individuals with a dual-layer epidermis . MoreoveP<0.001)  was explC. sieboldii and C. cuspidata, and in consequence, the genetic diversity values of mixed populations (No. 41–46) were higher than those of the other populations  , clusters within C. sieboldii were not clearly differentiated. This was probably due to the extremely small number of mother trees (5–7) sampled from each population and to the limited geographical dataset . Our collecting strategy covered the distributional range of the species, and most populations, in areas where C. cuspidata does not coexist, exhibited clear genetic differentiation of three groups in C. sieboldii.The Bayesian and NJ tree clustering analysis of  islands , 8. The P<0.001) . These rC. sieboldii are observed in the populations of the Ryukyu Islands Click here for additional data file.Table S2Polymorphisms for each of the 32 EST-SSR loci investigated in this study based on 63 Castanopsis populations.(DOC)Click here for additional data file.Table S3Outlier loci (P<0.01) among Castanopsis populations and within C. sieboldii and C. cuspidata populations.(DOC)Click here for additional data file.