Arabidopsis arenosa. Here we conduct a population genomic study to test the hypothesis that adaptation to WGD has been mediated by adaptive gene flow between A. arenosa and A. lyrata. We find that A. lyrata underwent WGD more recently than A. arenosa, suggesting that pre-adapted alleles have rescued nascent A. lyrata, but we also detect gene flow in the opposite direction at functionally interacting loci under the most extreme levels of selection. These data indicate that bidirectional gene flow allowed for survival after WGD, and that the merger of these species is greater than the sum of their parts.Adaptive gene flow is a consequential phenomenon across all kingdoms. Although recognition is increasing, there is no study showing that bidirectional gene flow mediates adaptation at loci that manage core processes. We previously discovered concerted molecular changes among interacting members of the meiotic machinery controlling crossover number upon adaptation to whole-genome duplication (WGD) in  Arabidopsis arenosa could rescue the nascent A. lyrata from extinction following WGD.Whole genome duplication (WGD) presents new challenges to the establishment of optimal allelic combinations and to the meiotic machinery. Here, the authors show that adaptive gene flow from  Recent progress in evolutionary genomics underscores the high frequency of WGD at both ancient and recent time scales4, and population genomic approaches reveal widespread evidence of gene flow between the most diverse species6. Both processes have therefore been associated with adaptive benefits. However, WGD and hybridisation are large-effect mutations, often leading to a host of genomic instabilities, including epigenetic shock, perturbed gene expression patterns and meiotic instability, with direct negative impacts on fertility. Perhaps the most challenging issue is the most immediate: that of stable meiotic chromosome segregation following WGD. How nascent polyploids establish meiotic stabilisation remains an unresolved question.Whole-genome duplication (WGD) and hybridisation are key drivers of genomic novelty, promoting diversification in all kingdoms of lifeArabidopsis genus have recently emerged as fruitful models for the study of genome stabilisation following WGD7. Arabidopsis arenosa is a largely biennial outcrossing relative of the model Arabidopsis thaliana, which forms distinct lineages of diploids and autotetraploids throughout Central Europe11. Initial resequencing of a handful of autotetraploid A. arenosa individuals suggested selective sweep signatures at genes involved in genome maintenance, including DNA repair, recombination and meiosis12. Later, a targeted resequencing effort focused on patterns of differentiation between diploid and autotetraploid A. arenosa, revealing evidence of highly localised selective sweeps directly overlapping eight loci whose gene products interact during prophase I of meiosis13. These eight loci physically and functionally interact to control crossover designation and interference, strongly implying that a modulation of crossover distribution was essential for polyploid establishment in A. arenosa15. Cytological evidence of a reduction in crossover numbers in the autotetraploids indicated that the selected alleles had an effect13. Similar to its sister species A. arenosa (arenosa hereafter), Arabidopsis lyrata (lyrata hereafter) also naturally occurs as diploids and tetraploids across its distribution range18. Although there is little evidence for gene flow among diploids of each species, there have been reports of gene flow between tetraploid arenosa and lyrata and, less pronounced, gene flow between diploids and tetraploids20.The wild outcrossing members of the lyrata compared with arenosa and the possibility of adaptive gene flow between the two species. Specifically, we ask (1) whether the same or different loci may be involved in adaptation to WGD in lyrata as we observed in arenosa; and (2) whether these adaptations arose independently or via introgression from one species into the other. Using whole-genome sequence data from 92 individuals of lyrata, arenosa and outgroup species Arabidopsis croatica and Arabidopsis halleri, we first analyse population structure and demography, concentrating on assessing admixture and the degree and timing of population divergences. Then, to estimate the relative degree of adaptation to WGD across the ranges of lyrata and arenosa, we cytologically assess meiotic stability in key populations. Finally, after scanning the lyrata genomes for signatures of selective sweeps, we compare the most differentiated regions with those we previously found in arenosa13 and test whether these selective sweep signatures overlap with fine-scale conspicuous introgression signals. Overall, our results reveal the molecular basis by which WGD has been stabilised in both species and indicate that WGD-facilitated hybridisation allowed for stabilisation of meiosis in nascent autotetraploids by highly specific, bidirectional adaptive gene flow.Here we investigate the molecular basis of parallel adaptation to WGD in lyrata and arenosa throughout Central Europe along with outgroups, sequenced at a depth of ~15× per individual  and tetraploids from the Western Carpathians (TRE), and most of the Alpine tetraploids  harboured essentially pure arenosa genomes , as well as the lyrata eastern tetraploid (Let hereafter) populations  and the tetraploid KAG population  displayed only slight admixture with arenosa and populations at the Wachau margin  showed stronger admixture, probably due to the increased proximity to the Hercynian and Alpine arenosa lineages21. Compared with the Wachau, where lyrata occurs on the slopes and hilltops along the Danube river surrounded by arenosa populations outside of the valley, there is a classical hybrid zone in the eastern Austrian Forealps: the parental species are found at the two poles of the zone  generations ago using fastsimcoal223  and 226k generations ago for arenosa , which approximately mark periods of glacial maxima26. Noting this, we next asked if either species experienced substantial historical bottlenecks. Using pairwise sequentially Markovian coalescent (PSMC) model27 we could not reach ages as ancient as 130–300 kya  ~25 kya  and ~20 kya (PEQ), whereas diploid Dinaric arenosa (BEL) peaked earlier, at ~30 kya. Interestingly, diploid Western Carpathian arenosa SNO, the population that founded several widespread autotetraploid lineages9, gave a strong signal of continuous expansion. These results suggest that diploid lyrata and partly arenosa underwent a bottleneck after the last glacial maximum 30–19 kya. PSMC does not accommodate autotetraploid data, but using fastsimcoal2 we detected a strong bottleneck at WGD for lyrata and none for arenosa  and lyrata to arenosa gene flow at the same level  , which by STRUCTURE and PCA analyses exhibited the lowest levels of admixture , dXY, Fst, Rho and the number of fixed differences. Although the comparison of the most pure lyrata tetraploid populations, represented by the Let group, to lyrata diploids is the most stringent test of which loci are under selection in a purely lyrata genomic context, we extended our tetraploid lyrata sampling to populations from the Wachau, which frequently showed admixture with arenosa . As the Let and part of the Lwt populations grow in contrasting edaphic conditions , we used this approach to maximise our chances of capturing differentiation specifically related to ploidy and not local adaptation. In addition, we observed that differentiation between these two tetraploid lyrata groups is stronger than differentiation between the tetraploid arenosa lineages studied here  enrichment analysis of these 196 genes identified significant overrepresentations in categories related to meiotic and homologous chromosome segregation, but also diverse processes including epidermal cell differentiation, trichoblast maturation, root hair cell and epidermal differentiation, root hair cell development and elongation, and others such as indole-containing compound metabolic process and mRNA catabolic process , as well as the endopolyploidy genes CYCA2;3 and MEE22, and the global transcriptional regulator TFIIF, among others. We observed selective sweep signatures at the majority (6/11) of coding loci of known function that were found as the very top outliers in arenosa  having primary functions of mediating meiosis, endopolyploidy and transcription. In particular, outlier loci participating in meiotic crossover formation, including ASY1, ASY3, PDS5-like, PRD3, and SYN1 exhibited tight peaks of divergence directly over single gene-coding loci  were low genome-wide with only two peaks reaching above 0.5  in this system21, finding a very rapid reduction specifically in the autotetraploid cytotype (50% lower mean correlations at 1 kb distance), suggesting that tight introgression signals may be formed rapidly in this system.Similar to the divergence outlier windows, Twisst peaks, only one represents topology 6. The others represent topologies 11 and 14, in which the tetraploid alleles are nested within the diploids, implying that they arose after speciation and subsequently introgressed , but among those with the highest levels of selection in Lwt, Let, and arenosa, a majority (11/16 where direction can be inferred) harbour evidence of an arenosa origin , and the Wachau . We found an additional tetraploid lyrata population in Hungary (GYE) and included it in this study. Diploid lyrata populations were chosen from the Wienerwald , which are the geographically closest diploid populations to the Let and Lwt, and therefore likely serve as source populations.Central European tetraploid arenosa, representative populations of tetraploids from the Hercynian  and Alpine lineages  were selected, as well as additional arenosa populations from the Western Carpathians , which is the centre of arenosa genetic diversity and the region of origin of the tetraploid cytotype9. For breadth, we selected several more diploid arenosa populations from the Pannonian  and Dinaric (BEL) lineages, as well as the following populations from the hybrid zone in the eastern Austrian Forealps: HAL, ROK, FRE, OCH and KEH. To complement our sampling with diploid lyrata from across its entire distribution range, we selected samples from the Hercynian , arctic-Eurasian  and arctic-North American lineages . A. croatica (CRO) and A. halleri  were included as outgroups32. The majority of lyrata and hybrid samples were collected as seeds, cultivated and flash-frozen prior to DNA extraction, whereas samples for three populations  were collected and silica-dried. Silica-dried material from GYE was obtained from Marek Šlenker and Karol Marhold. Arenosa samples were collected and sequenced as part of a different study21. In addition, 16 accessions were downloaded from the NCBI Sequence Read Archive (SRA), bringing the total sample number to 92 . Genomic libraries for sequencing were prepared using the Illumina TRUSeq PCR-free library kit with 500 ng to 1 μg extracted DNA as input. We multiplexed libraries based on the Qubit concentrations, and those multiplexed mixes were run on an initial quantification lane. According to the yields for each sample, loading of the same multiplex mix on several lanes was increased to achieve a minimum of 15× coverage. Samples that had less than our target coverage were remixed and run on additional lanes. Libraries were sequenced as 125 bp paired-end reads on a HiSeq2000 by the Harvard University Bauer Core Facility .For 45 to identify and remove adapter sequences with a minimum read length of 25 bp and a maximum error rate of 0.15. We then quality trimmed reads using TRIMMOMATIC46 (LEADING:10 TRAILING:10 SLIDINGWINDOW:4:15 MINLEN:50). Samples sequenced on several lanes were then concatenated using custom scripts. Reads were deduplicated using MarkDuplicates in picard v.1.103. Broadinst and readgroup names were adjusted utilising AddorReplaceReadGroups within the picard package. Reads were then mapped to the North American lyrata reference genome (v.247) using bwa-mem in the default paired-end mode48. Indels were realigned using the Genome Analysis Toolkit (GATK) IndelRealigner. Prior to variant discovery, we excluded individuals that had fewer than 40% of bases <8× coverage . Our final dataset for analysis contained 92 individuals.Newly generated sequencing data and SRA accessions were processed together from raw fastq reads. We first used CutadaptVariant calling was performed using the GATK HaplotypeCaller , followed by GenotypeGVCFs for genotyping. For each BAM file, HaplotypeCaller was run in parallel for each scaffold with ploidy specified accordingly and retaining all sites (variant and non-variant). We combined the single-sample GVCF output from HaplotypeCaller to multisample GVCFs and then ran GenotypeGVCFs to jointly genotype these GVCFs, which greatly aids in distinguishing rare variants from sequencing errors. Using GATK’s SelectVariants, we first excluded all indel and mixed sites and restricted the remaining variant sites to be biallelic. Additional quality filtering was performed using the GATK VariantFiltration tool . Then we masked sites that had excess read depth, which we defined as 1.6× the second mode (with the first mode being heterozygous deletions or mismapping) of the read depth distribution.arenosa from ref. 9. After quality filtering, these analyses were based on a genome-wide dataset consisting of 4,380,806 4dg SNPs, allowing for a maximum of 10% missing alleles per site (1.2% missing data) at a 5× coverage minimum for a given individual sample.All analyses dedicated to reveal population structure and demography were based on putatively neutral fourfold degenerate (4dg) single-nucleotide polymorphisms (SNPs) only. We used the 4dg filter generated for 49 to be superior in recognising admixture compared with STRUCTURE50, running fastSTRUCTURE on our dataset resulted in poor performance, in that the result did not coincide with the STRUCTURE results or other analysis. This misbehaviour was probably due to the inclusion of polyploid data, as fastSTRUCTURE does not accommodate polyploid genotypes. We had randomly subsampled two alleles per each tetraploid site, similar to ref. 32, using a custom script. However, evidently such a subsampling strategy dissolves the fine-scale differences in admixture between populations at this scale. Hence, STRUCTURE was preferred, and was run on all samples and both ploidies. As STRUCTURE accepts only uniform ploidy as input, with one row per each ploidy, we added two rows of missing data for our diploid samples, making them pseudo-tetraploid. In addition, input data were LD-pruned and singletons removed using custom scripts. Window size was set to 500 with a distance of 1000 between windows, allowing for 10% missing data, which resulted in a dataset of 32,256 SNPs genome wide. We performed ten pruning replicates using the admixture model with uncorrelated allele frequencies, and then ran each for K-values 2–10 with a burn-in period of 50,000 and 500,000 Markov Chain Monte Carlo (MCMC) replicates. We conducted PCA using the glPca function in the adegenet R package51.Although we expected fastSTRUCTUREfastsimcoal223 on 4dg sites. A minimum of two individuals per each population was required. Custom python scripts (FSC2input.py at https://github.com/pmonnahan/ScanTools/) were used to obtain the multi-dimensional allele frequency (DSFS) spectrum as well as bootstrap replicates of the DSFS for confidence interval estimation. For the bootstrap replicates, the genome was divided into 50 kb segments and segments were resampled with replacement until recreating a DSFS of equivalent size as the genome. Ultimately, we aimed to estimate demographic parameters and confidence intervals for a four-population tree corresponding to diploid and tetraploid lyrata and arenosa. For computational efficiency, three-population trees were initially used to establish the presence/absence of migration edges by comparing models with a single migration edge to a null model with no migration. Additional migration edges would then be added and compared with the initial simple model. For each model, 50 replicates were performed and values kept for the replicate with the highest likelihood. For each replicate, we allowed for 40 optimisation cycles and 100,000 simulations in each step of each cycle for estimation of the expected side frequency spectrum. Although the above process identified the key migration edges, it resulted in a four-population tree that was overly complex; the exercise suggested six migration edges in total  through time using information from whole-genome sequences of lyrata and arenosa diploids27. We generated plots of the most deeply sequenced representative of each of the diploid lyrata and arenosa populations, with the exception of distinct arenosa KZL and SZI. A consensus fastq sequence was created using samtools v.1.2 and bcftools v.1.2 using samtools mpileup -C50 -Q 30 -q 30 with the lyrata v.2 genome as the reference. The reference was masked at all sites at which read depth was more than twice the average read depth across the genome. Samtools mpileup was followed with bcftools call -c and vcfutils.pl vcf2fq -d 5 -D 34 -Q 30 to create a fastq reference file. Using PSMC, this was changed to a format that was required with PSMC by fq2psmcfa -q20, and psmc was run with parameters psmc -N25 -t15 -r5 -p “4 + 25*2 + 4 + 6” and psmc_plot.pl -R -g 2 -u 3.7e-8 to get a text file that could be plotted with R. We used the mutation rate estimate μ = 3.7 × 10−89 and a generation time of 2 years for both species, as arenosa is mainly biennial, and we estimate that lyrata generates the highest number of propagules in its second year after germination .PSMC model v.0.6.4 was used to infer changes in effective population size /16 h dark (4 °C) cycles. Plants were then grown in 16 h light (13 °C)/8 h dark (6 °C) cycles to encourage flowering. Buds were collected, fixed and anthers dissected for basic cytology as described in52 except that 50 mg (30 Gelatine Digestive Units) Zygest® Bromelain were added to the enzyme mixture, and incubation time was increased to 75 min. The prepared slides were stained and mounted with 7 µl 4′,6-diamidino-2-phenylindole (DAPI) in Vectashield (Vector Lab) and metaphase I chromosomes visualised using a Nikon 90i Eclipse fluorescent microscope with NIS Elements software. Chromosome spreads with all rod and/or ring bivalents were scored as stable meiosis  using primers 5SF 5′-AACCGAAATTGCGTGCATAG-3′ and 5SR 5′-AAACGGGAGGTGAGACGAG-3′ with Mimulus guttatus cloned genomic DNA that shares 96% nucleotide identity with A. lyrata in this region and the 45S pTa71 clone  by nick translation with digoxigenin (Jenna Biosciences). Streptavidin Dylight 594 and anti-digoxigenin Dylight 488 (Vector laboratories) were used as secondary fluorophores. Chromosomes were stained with DAPI in Vectashield (Vector Laboratories).Individual tetraploid sis Fig. , whereassis Fig. . FISH walyrata. We calculated the following metrics in adjacent nonoverlapping genomic windows: AFD, dXY, Fst53, Rho30 and the number of fixed differences between the lyrata diploids and the two lyrata tetraploid groups (Let and Lwt). We identified selective sweep candidates as the 1% outliers of the empirical distribution for each metric. To maximise our chances of capturing differentiation truly related to ploidy and not local adaptation, we selected the overlap between these two independent scans wherein the tetraploids contrast by edaphic (soil) preference and then focused on outliers that were identified in a highly stringent genome scanning approach in arenosa13.We grouped populations by ploidy level, species or hybrid affiliation, and affiliation to a biogeographic region in case of tetraploid dXY, Fst, Rho, and the number of fixed differences were calculated for additional populations. Arenosa populations were grouped by lineage, as identified in refs. 21, as arenosa Hercynian tetraploids (Aht) and arenosa Alpine tetraploids (Aat), which also corresponds to biogeographic groupings.To obtain insight into differentiation between population groups, AFD, A. thaliana gene identifiers. We retained levels 3-8 for biological process (Benjamini-Hochberg correction p ≤ 0.05).We performed gene function enrichment tests for each contrast using the CLUEGO app version 2.5.4 in CYTOSCAPE version 3.7.2 using GO information associated with orthologous 54 (10.4161%2Ff) with the manually added lyrata v.2 reference annotation55 (10.1371/journa). Variants annotated as missense (i.e. amino acid substitutions) were extracted using SnpSift54. Gene-coding loci were extracted from the whole-genome annotated VCF and per-population allele frequencies for each amino acid substitution calculated using GATK’s SelectVariants. Alternative allele frequencies (polarised against the lyrata reference) were visualised using the heatmap.2 function in the gplots package in R .We visualised allele frequencies of amino acid substitutions in form of a heatmap. Pre-processed VCF files were annotated using SnpEffTwisst)40 [www.github.com/simonhmartin/twisst/]. Twisst provides a quantitative measure of the relationships among a set of taxa when each taxon is represented by multiple individuals and the taxa are not necessarily reciprocally monophyletic. This provides a naive means to detect both introgression and ILS, and how these vary across the genome. We first inferred genealogies for 50 SNP windows across the whole genome using the BIONJ algorithm56 as implemented in PHYML57. As each individual carries two (for diploids) or four (for tetraploids) distinct haplotypes that represent different tips in the genealogy, it is necessary to first separate the haplotypes by phasing heterozygous genotypes. We used a heuristic approach to estimate phase that maximises the average extent of LD among all pairs of polymorphic sites in the window. This approach iteratively selects the best genotype configuration for each site, beginning with the site that has the most heterozygous genotypes. At each step, the optimal configuration is that which maximises the average LD between the target site and all previous target sites. This allows simultaneous phasing of diploids and tetraploids. We investigated the accuracy of this phasing approach using simulated sequences generated using the coalescent simulator msms58 and seq-gen59, following40, but here adding steps to randomise phase and then apply phase inference. As Twisst is robust to within-taxon phasing errors40, the relevant question here is the extent to which imperfect phasing would affect the estimated topology weightings. We therefore applied Twisst to the simulated data and compared the results with (i) perfect phase, (ii) randomised phase and (iii) randomised and then inferred phase. This confirmed that our heuristic phasing algorithm led to an improvement in the accuracy of the weightings, giving results that approached what is achieved with perfect phase information.To investigate how the relationships among diploid and tetraploid populations of the two species vary across the genome, we used topology weighting by iterative sampling of subtrees . In cases where this was not possible, approximate weightings were computed by randomly sampling combinations of haplotypes until the 95% binomial confidence interval for all fifteen topology weightings was below 0.05. Confidence intervals were computed using the Wilson method implemented in the package binom in R (R Core Team 2015).For running ies Fig. . AlthougFurther information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Supplementary Dataset 5Supplementary Dataset 6Source Data


Limacina helicina are widespread and can play an important role in the food webs and in biosedimentation in Arctic and Subarctic ecosystems. Previous publications have shown differences in the genetic structure of populations of L. helicina from populations found in the Pacific Ocean and Svalbard area. Currently, there are no data on the genetic structure of L. helicina populations in the seas of the Siberian Arctic. We assessed the genetic structure of L. helicina from the Kara Sea populations and compared them with samples from around Svalbard and the North Pacific.Pelagic pteropods L. helicina from three different locations in the Kara Sea via analysis of a fragment of the mitochondrial gene COI. We also compared a subset of samples with L. helicina from previous studies to find connections between populations from the Atlantic and Pacific Oceans.We examined genetic differences in L. helinica from the Kara Sea were sequenced to produce 19 different haplotypes. This is comparable with numbers of haplotypes found in Svalbard and Pacific samples . Haplotypes from different locations sampled around the Arctic and Subarctic were combined into two different groups: H1 and H2. The H2 includes sequences from the Kara Sea and Svalbard, was present only in the Atlantic sector of the Arctic. The other genetic group, H1, is widespread and found throughout all L. helicina populations. ϕ ST analyses also indicated significant genetic difference between the Atlantic and Pacific regions, but no differences between Svalbard and the Kara Sea.65 individual L. helicina populations from the Kara Sea and Svalbard: the majority of haplotypes belongs to the haplotype group H2, with the H1 group representing a minority of the haplotypes present. In contrast, in the Canadian Arctic and the Pacific Ocean only haplogroup H1 is found. The negative values of Fu’s Fs indicate directed selection or expansion of the population. The reason for this pattern could be an isolation of the Limacina helicina population during the Pleistocene glaciation and a subsequent rapid expansion of this species after the last glacial maximum.The obtained results support our hypothesis about genetic similarity of  Limacina helicina  are widespread in marine Arctic and Subarctic ecosystems, where their local abundance and biomass are comparable to or greater than that of copepods  or from the whole animal in case of small individuals (0.1–0.7 mm) using the ExtraGene™ DNA Prep 100 kit  per the manufacturer’s protocol.® 3500 Genetic Analyzer. Subsequently sequences were aligned and analyzed using MEGA 6.0  were amplified using Encyclo Plus PCR kit  using two standard primers: LCO-1490 (5′-GTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) . PCR wasMEGA 6.0 . The 503database . Low quadatabase  was thendatabase . Furtherdatabase  was useddatabase  softwareL. helicina from the Kara Sea. COI sequences were obtained from 65 of these samples, and eight sequences were discarded due to poor quality contigs, leaving 57 individual sequences for further analysis. The highest ϕST value found between the southern and northern parts was 0.023 (n. s.)  . Due to  GenBank . These s GenBank , and the GenBank . We also GenBank  for the  GenBank . Each haulations .H = 0.771). The diversity of haplotypes in the Kara Sea is similar (H = 0.672), despite a smaller number of analyzed individuals. The diversity of haplotypes in Pacific is significantly lower (H = 0.449) as well as nucleotide diversity ; however, the samples from the Kara Sea and Svalbard differed significantly from the Pacific .L. helicina populations from the Kara Sea would be genetically similar to those near Svalbard.The obtained results support our hypothesis that The haplotype network is very similar for populations from the Kara Sea and those from the North of the Atlantic near Svalbard , and theL. helicina were formerly widely distributed in the Arctic and the Pacific, but the populations were isolated in the Northern Atlantic during the glaciation. Haplotype group H1 may have persisted in a Pacific refuge, and H2—in an Atlantic refuge. Subsequently, during the retreat of the glacier about 131 ky BP, there was an increase in genetic diversity and distribution around Svalbard , but these differences are not significant. In the south (station AMK 5265 near the Kara Strait), the percentage H1 haplotype (14%) is lower than in the north at St. Anna or Voronin Troughs  (26%). This is in accordance with the penetration of water of different origins into the sea: in the south–west at station AMK 5265, the warm and salty water of the Barents Sea origin penetrates through the Kara Strait, while the northern part of the Kara Sea is strongly influenced by the Arctic saline and cold water . Since tL. helicina in the Kara Sea and makes an important contribution to zooplankton phylogeography by providing data on this large Arctic sea, which is not easily accessible. The comparison of our own data from the Kara Sea with the published data obtained in the Svalbard area, northwest Pacific, and Canadian Arctic, allowed us to conclude that the distribution of haplotypes in the Kara Sea is similar to that in Svalbard. Although no significant differences between habitats within the Kara Sea were found, the proportion of haplotypes H2 was higher near the Kara Strait than in the northern troughs. The analysis of the available data provides insight into the population structure of this pteropod species, indicating possible direction of post-glacial distribution of L. helicina in the Arctic. However, many questions regarding the genetics of this mollusk in the Arctic still remain unresolved, and in future studies we hope to better understand how far the western population of L. helicina penetrates and how the haplotypes are distributed over other Arctic seas.This study represents the first research on the genetic structure of


Centaurea × moncktonii) and its parental species (C. jacea and C. nigra). We sampled 20 populations from New York and Vermont, USA, and used genotyping by sequencing to identify single nucleotide polymorphisms in order to estimate genome-wide ancestry and classify individuals into hybrid genotype classes. We then tested for association between degree of genomic introgression and variation in a subset of traits diagnostic for the parental taxa, namely capitula morphology and monoploid genome size. Genomic clustering revealed two clearly defined lineages, as well as many admixed individuals forming a continuous gradation of introgression. Individual assignments to hybrid genotype classes revealed many advanced generation intercrosses and backcrosses, suggesting introgression has been extensive and unimpeded by strong reproductive barriers between taxa. Variation in capitula traits between the two unadmixed, presumed parental, lineages exhibited continuous, and in some cases transgressive, segregation among introgressed hybrids. Genome size was also divergent between lineages, although advanced generation hybrids had smaller genomes relative to additive expectations. Our study demonstrates deep introgression between the porous genomes of a hybrid invasive species complex. In addition to strong associations among genomic ancestry, genome size and morphology, hybrids expressed more extreme phenotypic values for capitula traits and genome size, indicating transgressive segregation, as well as a bias towards smaller genomes, possibly due to genomic downsizing. Future studies will apply these results to experimentally test how introgression, transgressive segregation and genome size reduction interact to confer invasiveness.Plant invasions are prime opportunities for studying hybridization and the nature of species boundaries, but hybrids also complicate the taxonomic treatment and management of introduced taxa. In this study, we use population genomics to estimate the extent of genomic admixture and test for its association with morphology and genome size in a hybrid complex of knapweeds invasive to North America: meadow knapweed ( Hybridization is thought to be a primary trigger underlying plant invasiveness, yet identifying hybrids and differentiating them from their parental species presents formidable challenges. Here, we present a multifaceted analysis of the invasive meadow knapweed complex that unravels the genetic make-up of hybrid populations in North America using DNA sequencing to infer the genomic ancestry of individuals, and associating ancestry with variation in floral traits and genome size. We find extensive genomic evidence of hybrids, with significant but incomplete correspondence with floral traits and genome size. Our results highlight the widespread yet often cryptic nature of hybridization in plant invasions. The invasion of numerous exotic plant species has been preceded or accompanied by interspecific hybridization. Consequently, hybridization has been hypothesized to be a key evolutionary mechanism stimulating invasiveness . AdmixtuOne potential constraint on hybridization and how it resolves is the difference in genome size between parental taxa. In plants, hybridization is often associated with genome size variation, either due to genome duplication (polyploidy) or smaller-scale gains and losses of chromosomes (aneuploidy) or chromosomal segments during homoploid hybridization. Population-level cytogenetic estimation of genome size has proven successful in resolving complex hybrid taxonomies, delimiting species boundaries and highlighting the origins of hybrids , and theAn additional consequence of the increased phenotypic variance that often accompanies hybridization is that it may be difficult or impossible to determine the ancestry status of hybrids and their parental types using morphological or cytological data alone. This can complicate the study and management of invasive species as a consequence of taxonomic confusion over invading taxa, which are often recognized based on visually diagnostic morphological traits. Recent developments in population genomics provide vast opportunity for investigations into the genomic consequences of hybridization, even in non-model organisms lacking a reference genome (Centaurea × moncktonii)—one of the invasive representatives of the extremely polymorphic Centaurea subg. jacea (Asteraceae), sometimes referred to as the C. jacea/nigra complex (e.g. C. jacea (brown knapweed) and C. nigra (black knapweed), with either brown or black knapweed as the female parent and the hybrid capable of freely backcrossing with either parental species . Our goal is to integrate multiple sources of evidence to deconvolute this apparent hybrid swarm into its constituent sources of ancestry and to determine the level of introgression that exists among parental species and their hybrids. Unravelling the extent of genomic introgression and mixed ancestry present within hybrid zones of introduced species is prerequisite for addressing the broader goal of how admixture and introgression affect fitness and contribute to invasion success  with morphometric analyses on reproductive traits and assessments of genome size for samples of the Centaurea jacea and C. nigra (Asteraceae) are short-lived, perennial knapweeds native throughout Europe and introduced to North America. The taxonomic classification of the C. jacea/nigra complex is highly controversial; treatments range from a single polymorphic species  compared to C. jacea (50–1300 m)  . The hybopenings . The preopenings .C. jacea and C. nigra (studied collectively as C. jacea s.l. in that paper) occur as two distinct cytotypes in Europe: a diploid (2n = 22) and a tetraploid (2n = 44) (n × 2n or 4n × 4n) and produces offspring with the same ploidy level as their parents. Segregation of allozyme loci in controlled crosses between tetraploid genotypes suggests inheritance patterns vary among loci from strict tetrasomic to intermediate between tetrasomic and disomic  . Therefodiploids .C. jacea/nigra complex were sampled from each of 10 locations in New York and Vermont. For each population, a whole young leaf (100 mg) and 1–2 mature capitula were collected from each of a minimum of 20 individuals. Leaf samples were transported back to the lab, and stored frozen at −80 °C until DNA extraction. Capitula were stored dried in envelopes in the lab until morphological analysis.In 2015, 20 populations see ; Table 1[see].Voucher specimens from each population were collected and have been submitted to the Liberty Hyde Bailey Hortorium at Cornell University (herbarium@cornell.edu) −1. Normalized DNA was used to prepare 96-plex genotype-by-sequencing (GBS) libraries following the Elshire protocol (ApeK1 (New England Bio Labs) for 16 h at 75 °C, followed by ligation with 1.2 ng barcoded adapter sequences  for 18 cycles, following conditions reported in We extracted total genomic DNA from leaf tissue using the DNeasy 96 Plant kit , quantified each sample fluorometrically using QUANT-IT (Invitrogen) and normalized concentrations to 10 ng μLhttp://hannonlab.cshl.edu/fastx_toolkit). We then used the genotyping pipeline GBS-SNP-CROP , and searched a range of k-mer lengths between 19 and 31 (command line: VelvetOptimiser.pl -s 19 -e 31 -f ‘-short -fastq CB_18.R1.fastq LH_3.R1.fastq MC_10.R1.fastq CH_15.R1.fastq LH_18.R1.fastq’). The optimal settings had a hash length of 29, which produced an assembly of 854 446 contigs with an N50 of 100 bp, for a total assembled length of 94.2 Mb. While larger kmer values could have been searched and may have yielded longer contigs in some cases, larger kmer values are also computationally more demanding to search, and the highly fragmented nature of single-end GBS sequence ‘tags’ is not expected to assemble into large contiguous sequences  was very close but slightly smaller than obtained with Velvet, indicating that assembly of the GBS tags was not overly sensitive to assembly algorithm. We used the Velvet assembly as a reference sequence to map reads from all samples using the bwa-mem algorithm in BWA 0.7.12 with default parameters  to call heterozygotes. To eliminate SNPs that were not strongly bi-allelic, we required (vii) a minimum of 90 % of non-primary allele reads to be the secondary allele. This filter was specifically applied to reduce multi-allelic SNPs and enrich for loci exhibiting disomic inheritance. Lastly, we (viii) eliminated SNPs in contigs that likely represent microbial contamination by running nucleotide searches of the mock reference against the NCBI nr database with BLASTn  minimum and maximum average depth per genotype of 3 and 200 reads, respectively, (ii) a minimum frequency of 70 % of individuals with genotype data to accept a SNP and (iii) a minimum of three individuals possessing the putative secondary allele at a minimum depth of three reads. We applied the additional constraints of: (iv) minimum depth of three reads required to call homozygotes when the secondary allele depth was 0, (v) minimum primary read depth of 3 required to call homozygotes when the secondary allele depth was 1 and (vi) minimum read depth of 3 required for http://samtools.github.io/bcftools/) to convert genotypes with an imputed probability <0.9 to missing, followed by removing SNPs with >50 % missing values. Lastly, to eliminate loci departing from disomic, Mendelian segregation due to formation of tetravalents or gene paralogy, we filtered SNPs with significant heterozygote excess (P < 5 e−6 based on Bonferroni correction). Positions with heterozygote deficit were not removed since this is an expected phenomenon in a hybrid zone due to a Wahlund effect. The final data set contained SNP genotype data for 273 individuals at 10 348 polymorphic loci.After filtering, the genomic data set consisted of 25 999 polymorphic SNPs for 380 samples. To reduce the impact of missingness on downstream analyses, we imputed missing genotypes using the LD-kNNi algorithm as implemented in LinkImputeR   values > 0.9 in the K = 2 Admixture run . We emphasize the use of ‘cf.’ here, from the Latin ‘confer’ or ‘compare to’, as an indication of the resemblance of our sample to the above referenced taxa without asserting a formal taxonomic assignment. These data were further subdivided to use half the samples per parental taxon for marker selection and hybrid simulation, respectively.Assignments of genotypic ancestry can provide evidence for admixture in a sample, but cannot distinguish between different hybrid genotype classes . We used the program wHybrids  to exploture run . IndividNewHybrids assumes loci are independent (i.e. unlinked) and show strong allele frequency differences between parental taxa. To select an optimal subset of loci, we used the getTopLoc function to select diagnostic markers with low linkage disequilibrium within a parental taxon , probably reflecting unknown differences in genomic structure and recombination in each lineage. Rather than combine the two sets of markers into a single panel, we instead report hybrid assignments from each panel separately so that sensitivity and agreement in the hybrid class assignments can be assessed. We analysed simulated data sets of the two parental taxa as well as F1, F2 and F1 backcrosses using panels of 200, 300, 400 and 500 diagnostic markers. We then analysed each of nine replicates of the simulated data sets with the R package parallelnewhybrid , since these share the same global ancestry probabilities.C. jacea/nigra complex is unresolved, we assessed whether genetic clusters identified by Admixture were associated with differentiation in some of the morphological capitula traits previously used to characterize this species complex (Q > 0.9) from the K = 2 Admixture model  along with 86 individuals with mixed genomic ancestry randomly chosen from all sampled populations  as an internal reference standard loaded with each sample.As a further tool to help discriminate the ploidy level of our sample and to help decipher parental and hybrid taxon assignments, we measured genome size by flow cytometry. Genome size  reported in the literature for tetraploid post hoc tests for pair-wise comparisons of hybrid classes using the R package ‘multcomp’  as well as a large number of individuals with admixed genetic ancestry (C. cf. nigra and blue for C. cf. jacea). Geographic populations that were assigned by majority to one of the two unadmixed clusters usually also contained some individuals with mixed ancestry (i.e. maximum assignment probability < 0.9 in the K = 2 run).The composition of genetic ancestry in our sample estimated by K = 3, the model best supported by cross-validation in Admixture, most admixed individuals formed a separate cluster (orange), suggesting the presence of a distinct hybrid lineage with intermediate ancestry between C. cf. nigra and C. cf. jacea (C. cf. nigra (NY hybrids), with one additional individual in NY population MP  hybrids based on the C. cf. nigra panel, or as second (or higher) generation backcrosses towards C. cf. jacea based on the C. cf. jacea panel  hybrids in both NewHybrids analyses. No F1 hybrids and few first generation backcrosses were identified, suggesting most hybrids in our sample represent advanced generations of introgression.Inference of each individual’s hybrid genotype class by wHybrids  largely ea panel . By contAdmixture and NewHybrids results based on the C. cf. nigra marker panel  1, while individuals with hybrid ancestry were intermediate  and capitula morphology (see below), we hereafter focus on the hybrid class assignments made using the C. cf. nigra marker panel. The corresponding results based on the C. cf. jacea marker panel are presented in [see]. However, three traits differed significantly (and in accordance with expectations) between the two presumed parental taxa: the percentage of pectinate bract rows was significantly higher in C. cf. nigra than C. cf. jacea (χ 2(1) = 30.7, P < 0.001), whereas the ratio of bract appendage centre width to length (χ 2(1) = 6.2, P < 0.05) and the relative bract appendage centre width (χ 2(1) = 8.7, P < 0.01) were both significantly lower in C. cf. nigra than C. cf. jacea.Due to better correspondence of hybrid genotype classes with genomic ancestry  = 13.1, P < 0.05 for hybrid class main effect), whereas all classes of hybrids did not differ significantly from either of the presumed parental taxa in morphometric PC1 scores (C. cf. jacea than first generation backcrosses to C. cf. nigra (χ 2(1) = 14.8, P < 0.05 for hybrid class main effect), but this difference should be interpreted with caution since the sample size in the latter hybrid class was quite small  = 10.6, P < 0.01; PC2 χ 2(1) = 5.5, P < 0.05). These relationships were primarily driven by the clear morphological separation of genotypes classified as unadmixed parental taxa. By contrast, admixed hybrid individuals with intermediate values of genetic PC1 displayed highly variable capitula morphologies, but with a mean centred near the predicted value based on their ancestry , indicating we achieved good precision for estimating individual genome size. Despite the uniformity in DNA ploidy, we found considerable genome size differences that reflected genomic ancestry. Among NewHybrids genotype classes, C. cf. jacea as well as all types of backcrosses towards this parental taxon and F2 hybrids had significantly smaller genomes than C. cf. nigra (2(1) = 18.3, P < 0.01 for hybrid class main effect). The intermediate genome sizes of backcrosses to C. cf. nigra did not differ significantly from any other hybrid class. Interestingly, advanced hybrid generations (F2 and second generation backcrosses) consistently tended towards smaller genome sizes as compared to first generation backcrosses. Similar trends for hybrid class assignments were observed based on the C. cf. jacea marker panel [see].Holoploid (1C) genome size of our samples ranged between 1.83–2.28 pg, consistent with all sampled individuals being tetraploid (see2(1) = 5.03, P < 0.05). However, this effect was no longer significant when an extreme outlier  was excluded. Again, this relationship was mostly driven by genome size differences between the two parental taxa, whereas in the middle range of genetic PC1, genome size was highly variable. There were no significant relationships between genetic PC2 and genome size [see], or between morphometric PC scores and genome size [see].In accordance with these results, we found a significant relationship between genetic PC1 and genome size  both advanced generation hybrids as well as unadmixed parental types were found to occur in a geographically structured distribution across New York and Vermont; (ii) capitula morphology and genome size correspond significantly with genomic ancestry, reinforcing the phenotypic and genomic distinctiveness of the unadmixed lineages while also providing a detailed genetic view of the extent of their introgression; (iii) hybrid individuals showed great variation in floral traits and genome size, including transgressive segregation for capitula traits; (iv) many hybrids exhibited a C. cf. nigra-like capitula morphology although the majority of them had higher genomic ancestry from the C. cf. jacea parent; and (v) many advanced generation hybrids had relatively small genome sizes given their level of ancestry. We discuss these findings in detail below with an emphasis on how the combination of genomic, cytological and morphological data have provided our best view to date into the ancestry composition of this incredibly reticulate hybrid complex. The extensive introgression uncovered here offers opportunities for future research to shed light on how hybridization affects the ecological and evolutionary dynamics of invasive populations.Our results provide the first glimpse into the composition of genomic ancestry in the C. × moncktonii hybrids have reportedly largely replaced populations of the parental taxa  aspects of reproductive morphology to C. jacea and C. nigra. A third related taxon, C. nigrescens is also introduced to North America and is sometimes treated as part of the C. jacea s.l. complex, and C. × moncktonii is noted to sometimes combine features of C. nigrescens, notably the presence of involucres with distinct green phyllaries not fully covered by black appendages . Therefore, the presence of ray-like florets at sites assigned to C. cf. nigra indicates that the parent species as described in various treatments is not present (or deviates from the current understanding), given that the lack of ray-like florets is considered one diagnostic trait for C. nigra .Comparing individual assignments between the ure runs , we founure runs  compriseure runs . Most ofwHybrids , along wC. cf. jacea occurred exclusively in Vermont across seven populations, whereas populations with a predominantly unadmixed C. cf. nigra genetic background were more scarce and found in only one population in Vermont and three populations in New York  genomic and morphological features of its presumed parental lineages, and that these can be confidently separated using a combination of genomic, morphological and cytological evidence.Unadmixed New York  and 2. TC. × moncktonii hybrids in the Niagara region of New York . In contrast, hybrid class assignment based on the C. cf. jacea marker panel assigned most individuals as first or second generation backcrosses to C. cf. jacea (C. cf. nigra marker panel gave hybrid class assignments that more closely reflected the Admixture ancestry proportions (K = 2), and also gave results for the capitula morphometrics that fit with the degree of separation of the parental taxa, we suggest the analyses based on the C. cf. nigra marker panel are more robust. However, regardless of marker panel, both results point to advanced hybrid generations (either F2 or backcrosses) being commonplace.Whereas most of the Vermont hybrids are classified as advanced generation hybrids by f. jacea , upper, f. jacea , lower. C. cf. jacea populations, although the hybrid individuals had higher genomic ancestry from this parental taxon than from C. cf. nigra. It is possible that C. cf. jacea has been completely introgressed in this region. Alternatively, New York hybrids may be descendants of a hybrid lineage formed in the more distant past (possibly prior to introduction) and now represents a stabilized hybrid gene pool. This latter scenario would seem to fit the genetic differentiation of this hybrid group along PC2  persist. The potential for hybridization among co-invading reproductively compatible taxa is hypothesized to be a major mechanism for fuelling the establishment and spread of introduced plants (Centaurea could increase their propensity for invasion if hybrids have higher short-term fitness (e.g. heterosis) or increased quantitative genetic variance (evolvability) compared to the parental types. This study provides a foundation for further work aimed at addressing the impact of admixture, introgression and changes in genome size on the fitness of invasive hybrids.Invasive populations of Increasing attention is being paid to the role of genomic and cytogenetic features, including genome size  as potenhttps://github.com/SusanneLachmuth/Centaurea-moncktonii-AoB-Plants.All data and code necessary to reproduce these results are publically available from The following additional information is available in the online version of this article—Table S1.1. Specimen voucher information for Centaurea samples analysed in this study. Vouchers have been archived at the Liberty Hyde Bailey Hortorium at Cornell University.Table S1.2. Assignment criteria for Centaurea cf. jacea and C. cf. nigra and their hybrids using NEWHYBRIDS.Table S1.3. List of measured and derived morphometric capitula traits diagnostic for the Centaurea jacea/nigra complex.Figure S1.1. Boxplots of morphometric capitula traits  loci that showed highest global FST and no linkage disequilibrium among either 27 Centaurea cf. jacea individuals or among 27 C. cf. nigra individuals.Figure S1.3. Relationships of genetic ancestry and capitula morphology.Figure S1.4. Relationships of genetic ancestry and genome size.Figure S2.1. Genetic structure of 273 Centaurea jacea/nigra species complex individuals sampled in New York State and Vermont based on 10 348 single nucleotide polymorphisms (SNPs).Figure S2.2. Principal component analysis based on three morphometric capitula traits.Figure S2.3. Boxplots for variation in capitula morphology of the different hybrid classes.Figure S2.4. Relationships of genetic ancestry and capitula morphology.Figure S2.5. Relationships of genetic ancestry and genome size.Figure S2.6. Relationships of capitula morphology and genome size.plz055_suppl_Supplementary_Appendix_S1Click here for additional data file.plz055_suppl_Supplementary_Appendix_S2Click here for additional data file.This work was supported by a United States Department of Agriculture Co-Operative agreement to J.M., S.R.K. and L.M. (58-1907-4-032), and by the German Research Foundation (DFG) through a Research Fellowship grant to S.L. (grant LA 3434/3-1).J.M., L.M., and S.R.K. planned and designed the study. J.M. and L.M. conducted the field sampling. J.S. performed the flow cytometry for genome size determination. J.M. and S.L. performed measurements of morphological traits. S.R.K. oversaw the G.B.S. library construction and sequencing. S.L. performed the bioinformatics and statistical analyses under guidance from S.R.K. S.L. and S.R.K. interpreted the results, with contributions from J.M. and L.M. S.L. and S.R.K. drafted the manuscript, and S.L., S.R.K., J.M. and L.M. contributed to the submitted version and revisions.None declared.


The human demographic history of Mainland Southeast Asia (MSEA) has not been well studied; in particular, there have been very few sequence-based studies of variation in the male-specific portions of the Y chromosome (MSY). Here, we report new MSY sequences of ∼2.3 mB from 914 males and combine these with previous data for a total of 928 MSY sequences belonging to 59 populations from Thailand and Laos who speak languages belonging to three major Mainland Southeast Asia families: Austroasiatic, Tai-Kadai, and Sino-Tibetan. Among the 92 MSY haplogroups, two main MSY lineages (O1b1a1a* [O-M95*] and O2a* [O-M324*]) contribute substantially to the paternal genetic makeup of Thailand and Laos. We also analyze complete mitochondrial DNA genome sequences published previously from the same groups and find contrasting pattern of male and female genetic variation and demographic expansions, especially for the hill tribes, Mon, and some major Thai groups. In particular, we detect an effect of postmarital residence pattern on genetic diversity in patrilocal versus matrilocal groups. Additionally, both male and female demographic expansions were observed during the early Mesolithic (∼10 ka), with two later major male-specific expansions during the Neolithic period (∼4–5 ka) and the Bronze/Iron Age (∼2.0–2.5 ka). These two later expansions are characteristic of the modern Austroasiatic and Tai-Kadai groups, respectively, consistent with recent ancient DNA studies. We simulate MSY data based on three demographic models  of major Thai groups and find different results from mitochondrial DNA simulations, supporting contrasting male and female genetic histories. Thailand and Laos occupy a key location in the center of Mainland Southeast Asia MSEA; , which iHigham 2014Higham 2014The census size for Thailand was ∼68.41 million in 2017 and for Laos was ∼6.76 million in 2016 . There aIt is generally thought that AA languages were brought to the Thai/Lao region by Neolithic farmers from southern China, whereas TK languages were brought by a later, Bronze Age migration, also from southern China . The NeoSchliesinger 2000Previous studies have reported an influence of postmarital residence pattern on genetic variation in northern Thai hill tribes, with lower within-population genetic diversity coupled with greater genetic heterogeneity among populations for patrilocal groups than for matrilocal groups for the male-specific portions of the Y chromosome (MSY), whereas the opposite pattern is observed for mtDNA . HoweverThe MSY are paternally inherited and exhibit lineages specific to populations/geographic regions, making the MSY an informative tool for reconstructing paternal genetic history and demographic change . HoweverWe have previously carried out comprehensive studies of the maternal genetic history of the Thai/Lao region, based on 1,823 complete mtDNA genome sequences . In ordeWe generated 914 sequences of ∼2.3 mB of the MSY, which combined with 14 published sequences brings the total to 928 MSY sequences belonging to 59 populations from Thailand and Laos  online. U tests between AA and TK for MSY: h: Z = 3.37, P < 0.01; mean number of pairwise difference [MPD]: Z = 2.40, P < 0.05; haplogroup diversity: Z = 3.74, P < 0.01 and for mtDNA: h: Z = 4.33, P < 0.01; MPD: Z = 1.47, P > 0.05; haplogroup diversity: Z = 4.37, P < 0.01). After the Maniq (MN), who have no MSY variation, and the Mlabri (MA), who have no mtDNA variation, the Htin (TN1), Lawa (LW3), and Bru (BU) show very low diversity values of MSY, whereas the Htin (TN1–TN3), Khmer (KH2), and Seak (SK) show low mtDNA diversity . LW3 showed very low MSY haplogroup diversity  , who speak a Mon language, show the highest MPD value for the MSY  D, sugges the MSY C, which  the MSY A. MO3 an the MSY A.P < 0.01 and 18.49%, P < 0.01 without MN, the hunter–gatherer group from southern Thailand) than among the TK  and ST-speaking Karen groups . For the AA group with more than one population sampled, the greatest within-group variation by far was among the three Lawa populations , whereas the seven Mon populations showed very low  within-group variation  (P > 0.01), Khon Mueang groups from northern Thailand , and Lao Isan groups from northeastern Thailand , indicating overall genetic homogeneity among these major TK-speaking groups. In agreement with the MSY, larger mtDNA variation is observed in the AA groups  than the ST  and TK groups , but interestingly the largest within-group variation is not among the Lawa  but rather among the Htin populations . In contrast to the MSY, each of the TK groups with more than one population sampled showed significant within-group differences for mtDNA, especially the Khon Mueang  (P < 0.01) than for mtDNA  for AA groups, but about the same for TK groups , and the ST groups have larger among-population variation for mtDNA  than for the MSY   indicates that the variation among populations (within group) accounts for 11.12% of the total MSY genetic variance . There i < 0.01)  online.  < 0.01)  online.  < 0.01)  online. P < 0.01) than by language family  , and the total variation among populations within group reduces to 10.54%. Thus, neither geography nor language family is a good predictor of the MSY genetic structure of Thai/Lao populations, indicating that these two factors are not important in the broad view  , there ioad view .r = 0.3381, P < 0.01), resistance distance  and least-cost path distance . However, the correlations are no longer significant when the MN group is removed from the analysis: great circle distance , resistance distance  and least-cost path distance . In contrary, no significance was detected (P > 0.05) between matrices of mtDNA genetic distance and geographic distances with and without MN (great circle distance: r = 0.0776 and r = −0.0323), resistance distance (r = 0.1433 and r = −0.1105), and least-cost path distance (r = 0.0997 and r = −0.0253).There are significant correlations between matrices of MSY genetic and geographic distance, estimated by Mantel tests, for all three types of geographic distances, that is, great circle distance  was carried out. This analysis attempts to maximize among-groups genetic differentiation and minimize within-group genetic variation; the results showed considerable overlap among groups defined by either language family or geographic location in both MSY and mtDNA  online. In sum, all results indicate lower genetic diversity of the AA groups than the TK and ST groups, except the Mon and Nyahkur, who exhibit high genetic diversity. The AA groups also show greater genetic heterogeneity than the TK and ST groups.h and MPD values are higher for matrilocal groups, but not significantly ; however, haplogroup diversity is significantly higher for the matrilocal groups   (Φst = 0.3109 for MSY and 0.0774 for mtDNA) , whereas the matrilocal groups (Htin and Karen) also show higher average Φst for MSY (0.1859) than for mtDNA (0.1553), but these are not significantly different . Contrasting genetic differences for the MSY versus mtDNA of Lawa, Htin, and Karen are clearly seen in the multidimensional scaling (MDS) and DAPC plots  and the Palaung, another minority group in the mountainous area of northern Thailand but not officially recognized as a hill tribe. The Khmu (KA), Lawa , and Palaung (PL) groups practice patrilocality, whereas the Htin  are matrilocal, as are the ST-speaking Karen . If residence pattern is influencing genetic variation, then lower within-population genetic diversity coupled with greater genetic heterogeneity among populations is expected for patrilocal groups than for matrilocal groups for the MSY, whereas the opposite pattern is expected for mtDNA . The MSY < 0.05)  online.  > 0.05)  online.  > 0.05)  online. PC plots  online. PC plots  online.However, in general, the AA-speaking groups, whether identified as hill tribes or as other minorities, are patrilocal groups. The AMOVA result indicates that the variation among AA populations is higher in MSY (20.01%) than mtDNA (14.03%), in accordance with expectations if residence pattern is influencing genetic variation. Conversely, the TK populations, where neither patrilocal nor matrilocal residence is preferred, exhibit similar among-population variances for the MSY (4.48%) and mtDNA (4.33%)  online. Φst values . After excluding these MA and MN as outliers, the MDS for the MSY showed that almost all AA-speaking groups are located along the edges of the plot, whereas most of the TK groups cluster in the center of the plot  over time in each group reveal overall five different trends  or remains stable (Lao Isan and Laotian); the Lue and Phuan show two increases in Ne, at about ∼5 ka and ∼10 ka; the Lawa show a stable population size since ∼30 ka and then a decline during the last 2 ka with a sudden increase ∼1 ka; and the Karen differ only slightly from the common trend with a population increase ∼1 ka.The Bayesian Skyline Plots (BSPs) of effective population size change fig. 7). Ne increases, at ∼9–11 ka, ∼5 ka, ∼2.0–2.5 ka, and ∼1.0 ka .The BSP by each major MSY haplogroup show four pulses of paternal  ∼1.0 ka , in agrePreviously, we used mtDNA genome sequences and demographic modeling to test different hypotheses about the origins of TK groups. Specifically, we tested whether different TK groups were primarily related to local AA groups , to a TK group from southern China , or were related to both . We found that the Khon Mueang (from northern Thailand), Lao Isan (from northeastern Thailand), and Laotian most likely originated via demic diffusion from southern China without substantial gene flow from AA groups . HoweverΦst and corrected pairwise difference among groups of populations used in ABC tests to estimate their genetic relationships  people (Test 1), the highest posterior probability (0.80) and rather highly selected classification trees (0.58) were found for the demic diffusion model  online. ionships  online. In order to gain more insights into MSEA genetic history, we here investigate the paternal genetic variation and structure by sequencing ∼2.3 mB of the MSY from representative ethnolinguistic groups from Thailand and Laos. In sum, most of the studied populations exhibit two major MSY haplogroups, O-M324* and O-M95* in different proportions, indicating two major paternal sources. O-M324* was widely spread in the TK groups, whereas O-M95* is predominant in the AA groups. However, some TK populations (BT2 and IS3) and some AA populations  exhibited the opposite pattern  online. The hill tribes, who occupy the mountainous northern region of Thailand, are notable for their variation in patrilocal versus matrilocal residence pattern , as wellHere, we analyzed the sequences of mtDNA genome and ∼2.3 mB of the MSY of the Khmu, Palaung, and Lawa groups, who practice patrilocality, whereas the Htin are matrilocal, similar to the ST-speaking Karen. The within-population genetic diversity values are in agreement with expectations, that is, greater diversity of matrilocal than patrilocal groups for MSY and the opposite trend in mtDNA  online. One factor in particular that could influence the within-population genetic diversity and between-population differentiation is geographic isolation, which enhances genetic drift and inbreeding, thereby lowering within-population genetic diversity and increasing between-population differentiation. This could explain the very low internal diversity and high differentiation from other groups of some groups of Htin (TN1) and Lawa  online tMoreover, these results are in keeping with previous observations that although the expected difference between patrilocal and matrilocal groups holds in some regions , in otheThe Mon groups showed genetic differences from other AA populations but closer relatedness to the TK populations, especially the central Thai, in both MSY and mtDNA . In addiPrevious genetic studies of G6PD mutations reported a high prevalence of the Mahidol type G6PD deficiency in the Mon, Burmese, and Karen, different from Thai, Laotian, and Khmer groups exhibiting the Vientiane-type G6PD mutation . Thus, bA connection between Indian groups and the Mon is suggested by South/Central Asian MSY lineages in the Mon, for example, R*, H*, J*, L*, and Q* , consistNe increase during the Holocene is primarily driven by the O2a2a* and O2a1c* lineages  among the groups used in the simulations does support a real contrast in the maternal versus paternal histories for the major TK groups in each region, and also finds genetic heterogeneity among these major groups. The northern Thai people showed closer genetic relationship with the Dai than AA groups in both mtDNA and MSY, supporting the demic diffusion model, whereas the ethnic Lao are closer to Dai for mtDNA but for MSY they are related to both Dai and AA rather equally, suggesting demic diffusion for the maternal history and admixture for the paternal history. The central Thai MSY sequences could be of AA origin because they are genetically more similar to the AA groups than the Dai, supporting cultural diffusion, but for mtDNA they are related to both Dai and AA rather equally, supporting admixture in central Thailand as found previously , AMOVAs, and Mantel tests of the correlation between genetic and geographic distances.We combined the 914 newly generated sequences together with 14 published sequences  belong tadegenet 1.3-1 , the MDS heat plot with five dimensions, showing per-dimension standardized values between 0 and 1, and heat plots of the Φst distance matrix and the matrix of shared haplotypes.A correspondence analysis based on MSY haplogroup counts was performed using STATISTICA 13.0 . Haplogroup assignment was performed by yHaplo . The R phttps://sharehost.hms.harvard.edu/genetics/reich_lab/sgdp/Y-bams/Y.tar; last accessed June 25, 2018)  using Heffalump v0.2 . We subset the variant file to sites that were overlapping the regions present on our capture bait and to samples that had a major haplogroup that was also present in our data set. These samples were combined with our samples; we then removed variant sites for which <25% of the samples had genotype information, and samples that had >25% of all sites with missing genotype information. The resulting data set provides 16,684 variable sites, which was imputed using BEAGLE v4.1  , the 1005, 2018)  and the 5, 2018) . The resGLE v4.1 . Additio−10 substitutions/bp/year  and compared three competing scenarios: demic diffusion ; cultural diffusion  and continuous migration  that were developed based on known historical hypotheses  online. /bp/year , and a p/bp/year  online. We simulated the derived site frequency spectrum (unfolded-SFS) for 2,364,048 loci using the fastsimcoal simulator  with theAll reads that aligned to the region of the MSY that was targeted by the capture-enrichment array were deposited in the European Nucleotide Archive (ENA) (study ID: PRJEB31636).Molecular Biology and Evolution online.Supplement_Material_msz083Click here for additional data file.


The founding population grew significantly and reached its height during the 4th century BCE. We obtained nine complete mitochondrial genomes from skeletal remains from two Punic necropoli in Ibiza and a Bronze Age site from Formentara. We also obtained low coverage (0.47X average depth) of the genome of one individual, directly dated to 361–178 cal BCE, from the Cas Molí site on Ibiza. We analysed and compared ancient DNA results with 18 new mitochondrial genomes from modern Ibizans to determine the ancestry of the founders of Ibiza. The mitochondrial results indicate a predominantly recent European maternal ancestry for the current Ibizan population while the whole genome data suggest a significant Eastern Mediterranean component. Our mitochondrial results suggest a genetic discontinuity between the early Phoenician settlers and the island’s modern inhabitants. Our data, while limited, suggest that the Eastern or North African influence in the Punic population of Ibiza was primarily male dominated.Ibiza was permanently settled around the 7 Additional Phoenician settlements were established on the Balearic islands, Sardinia, Sicily, Malta, and Cyprus2. These Islands form a strategic arc across the northern Mediterranean allowing for island hopping from the Levantine homeland to the Iberian Peninsula and North African coast where they established their most dominant Western Mediterranean settlement in Carthage. The early Phoenician settlements in the Western Mediterranean are generally referred to as Western Phoenicia. From the middle of the sixth century BCE onwards, with the shift in Phoenician influence from the Levant to Carthage, they are typically referred to as Punic, and from the 6th century BCE onward this term has become synonymous to Phoenicians outside the Levant. Our previous research has shown that the Phoenician settlers who encountered indigenous communities already living on the islands that they settled were integrated to form the new Phoenician societies3.The Phoenician culture was one of the most prevailing and widespread in the history of the Mediterranean basin. From its rise in the northern Levant, the Phoenicians connected east and west for over a millennium through their established trade networks across the Mediterranean, reaching beyond the Straits of Gibraltar. One of their first western outposts was the city of Gadir, modern Cadiz, on the Atlantic coast of Spainth century BCE4. These Bronze Age settlements however, had relatively small populations compared to the larger Balearic Islands7. Ancient DNA analyses of human remains from early Ibizan sites can therefore provide evidence as to the origins of the Phoenician settlers of the island and the relationship between these early settlers and the modern population of the island. Further, when combined with other genetic data from Phoenician populations, analyses can provide key information about the process of Phoenician expansion and settlement of the Western Mediterranean.Archaeological evidence on the largest of the Balearic Islands, Mallorca and Menorca, indicates continuous settlement since the 3rd millennium BCE. Limited evidence favours some level of continuity of prehistoric settlement in Ibiza from the end of Bronze Age until the arrival of Phoenicians in the 78, arriving from west Phoenicia. They initially established a small settlement at the Bay of Ibiza and a second on Sa Caleta which was later abandoned. The first settlement gave rise to the Phoenician city of Ibusim or Ebusus9. The strategic importance of Ibiza did not escape the Phoenician navigators who realized that by controlling the Bay of Ibiza they could dominate maritime movement in all the North-western Mediterranean. Phoenician presence in these prime coastal locations asserted and ensured their maritime control over the entire Mediterranean and they controlled access to the Atlantic Ocean. For several centuries, the Phoenicians dominated metal trade from the Atlantic to the Eastern Mediterranean2.The archaeological evidence to date suggests that the Phoenicians first settled on the island of Ibiza around 654 BCE11. Burial data from this necropolis suggest three phases of the early demographic history of Ibiza11. The small founding population arrived from west Phoenicia, most probably Gadir, during the 7th century BCE, and they are identified by their funerary rituals, that primarily involved cremation. The second phase occurred between the 5th and 4th centuries BCE and is indicated by a significant and rapid expansion in population size, reaching nearly 4,000 inhabitants. This growth may have been driven by a population flow from Carthage and other Punic settlements in the Mediterranean and coincides with a period of prosperity and major development of the island. The third and last phase of this early settlement period was between the 3rd and 2nd centuries during which Ibiza witnessed a period of economic decline and the return of cremation funerary rituals. After the 2nd Punic War, Ibiza started a long process of integration in the Roman Empire, which culminated in 74 AD, when it became a Latin Municipium12. The Roman impact on the local inhabitants of Ibiza appears to have been minimal as is indicated by limited evidence of influence in the archaeological record. The Ibizan population remained somewhat isolated while under Roman political influence until the Islamic conquest of the island by Arabs and Berbers around 902 CE and they remained under Muslim rule for 333 years. Beginning around 1229 CE the Balearic Islands, including Ibiza in 1235, were impacted by immigrants from Catalonia, in mainland Spain, and the population of Ibiza slowly grew in number, but relative isolation of the autochthonous population continued and is still maintained today13. The extent of admixture between the indigenous Phoenician derived population of Ibiza and later occupiers has not been investigated at the genetic level, though genetic studies of the modern population of Ibiza indicate that they are distinct from the other Balearic Island and from mainland populations16. Pacelli and Márquez-Grant17 suggest that this difference may be due to the arrival of North African Punic settlers, a hypothesis that remains to be tested.The Phoenicians, and later the Punics, remained the main inhabitants of Ibiza for about seven centuries as demonstrated by the archaeological evidence from Puig des Molins, one of the largest necropoli in the Western Mediterranean, now a World Heritage site, with around 3000 tombsHere we report ancient DNA (aDNA) analyses of archaeological remains from two Punic necropoli on Ibiza, obtained through high-throughput sequencing. We determine the maternal ancestry of the founders of Ibiza through analyses of complete mitochondrial genomes (mitogenomes) of 8 archaeological samples from Ibiza. These ancient mitogenomes are compared to the first complete mitogenome data generated for 18 modern Ibizans to determine if there is genetic continuity between the original Phoenician settlers and the modern indigenous population of the island today and to elucidate the settlement history of Ibiza. The complete mitogenome from a Chalcolithic/Early Bronze-Age sample from the Island of Formentera was also obtained, providing evidence of the ancestry of the pre-Phoenician occupants of this island. Finally, we present the first genome-wide sequence data obtained from an ancient Phoenician and assess the likely ancestry of this individual from Ibiza. The accumulating aDNA evidence from Phoenicians from Ibiza, Sardinia, Tunisia and Lebanon is providing valuable information regarding the Phoenician expansion practices and the development of Phoenician communities in the Western Mediterranean.19; one sample from the Cas Molí site, in Sant Antoni de Portmany20, also from the island of Ibiza; and one sample from the megalithic chamber tomb from the site of Ca na Costa, on Formentera21. The archaeological context for these samples is provided in the Supplemental Data A total of 13 ancient tooth samples were obtained for aDNA analyses, from the Museu Arqueologic d’Elvissa i Formentera, in Ibiza, Spain: eleven samples from the urban Puig des Molins Necropolis, in the town of EivissaA total of 18 modern DNA samples, obtained from volunteers from Ibiza, initially sampled by researchers from the Institut de Biologia Evolutiva (IBE), Barcelona, as part of a study on Y chromosomes and surnames, were sent to the University of Otago for full mitogenome sequencing. Individuals were only included in the sampling if all four grandparents were born on Ibiza and carried Ibizan surnames.22. Double stranded Illumina libraries were constructed with dual barcodes for the ancient samples as described previously23, and the mitochondrial DNA was captured using hybridization with modern human mitochondrial bait, generated in our lab24.All ancient DNA processing was conducted in a dedicated aDNA facility at the University of Otago. DNA was extracted from archaeological teeth using standard protocols23.Modern DNA samples were sent by CSIC-UPF to the University of Otago where they were processed in a separate lab from the aDNA facility, and mitogenomes were amplified, prepared and sequenced as described previously25 with Phylotree build 1726. All aDNA reads generated have been submitted to the NCBI sequence read archive identified by lab sample number. All mtDNA haplogroup determinations for samples processed in this study and associated archaeological site names, dates, GenBank accession numbers, coverage and contamination estimates are shown in Table All ancient sequences obtained were assessed for potential contamination and DNA damage patterns. Deamination and fragment lengths were all as expected for ancient samples  and thus the individual was determined to be female, which is inconsistent with the sex determination based on skeletal characteristics. After mapping the shotgun reads to the UCSC reference genome hg19, the resulting bam file was aligned with the Human Origins SNP dataset to call a pseudo-haploid genotype28. This genotype was merged with previously published ancient30 and modern SNP data sets33, resulting in a total of 119,316 SNPs used in subsequent analyses (See Supplementary Table One ancient sample (MS10614) was selected for shotgun sequencing, based on the high genome coverage observed in the mitochondrial sequencing of this sample. Adequate preservation was confirmed with an estimated 9% endogenous DNA content. The sex of this sample was determined based on computing the number of alignments to the Y Chromosome (nY) as a fraction of the total alignments to both sex chromosomes (nX + nY)rd century BCE.A subsample of the tooth from which the DNA was extracted was sent to Beta Analytic Inc. (Miami) for AMS dating. It returned a conventional radiocarbon age of 2190 ± 30, which provided a calibrated date range of 361–178 cal BC , at 95.4% probability. This date is consistent with the archaeological context proposed by the archaeologists who excavated the site, suggesting 336 and modern North African37 populations is shown in Fig. Nine complete mitogenomes were successfully obtained from 13 ancient samples and their Hg were determined. In addition, mitogenomes and their Hg were successfully determined from 18 modern Ibizans  networks shown in Fig. To investigate the similarities between our Ibizans and other ancient samples from the Iberian mainland and the other Balearic islands we reduced our data to the comparable, publicly available HVR regions of 383 bp for Neolithic and Bronze age Iberian samples40, shown in Fig. To test for continuity between ancient and modern Ibizan haplotypes, we performed a temporal network analysisST) was calculated between modern Ibizans and multiple candidate source populations47. Pairwise FST between all populations were visualized in a MDS plot , Europe (CEU) and Africa (YRI) in which MS10614 was located closest to the European samples (when including both tranversions only and transitions and transversions)  and Levantine populations from Jordan, Syria, Lebanon, and Israel32. The Ibizan sample plotted within the Levantine cluster, outside of the modern Iberians  were first assessed by constructing a PCA with the 1000 genomeses Fig.  and see ses Fig. . The ancses Fig. .Figure 649  that impacts all of the Eastern Mediterranean populations and the modern Iberians. Sample MS10614 has an intermediate amount of this component, with more than modern Spanish, but less than modern Lebanese and ancient Levantines.The ancestry of MS10614 was also estimated using model-based clustering in ADMIXTURE3-statistics were used to measure shared genetic drift between MS10614 and other ancient and modern populations. These results indicated that this Phoenician individual is most closely related to Neolithic aged samples from Iberia and Anatolia, plotting away from North African samples, providing further evidence for this individual having ancestry from both mainland Europe and the Near East  of Neolithic and Early Bronze Age Iberian samples. Investigating coding region SNPs, they showed that among haplogroup H, 65% belonged to hg H1 and 14% to hg H3. Particularly high frequencies of H1 and H3 are also seen in ancient and modern Sardinians34. The majority of the remaining haplogroups present in the Phoenician population of Ibiza, U4a, U5b, and T2b have all been identified in Mesolithic or Neolithic populations in Western Europe including Iberia53. Haplogroup U5b3 frequencies, though generally low across Europe, are the highest today in Sardinia and Spanish Andalusia54 and are thus also consistent with an Iberian origin or possible contacts with Phoenician Sardinia. Haplogroup J1c is thought to have an eastern Mediterranean origin and has been identified in several Neolithic samples in Eastern Europe and in a Chalcolithic sample (2880–2630 BCE) from the El Mirador Cave site, Atapuerca, Spain55. Haplogroup J1c has not been found previously in any ancient samples in the Levant and appears to have been distributed across Europe with the Neolithic expansion, suggesting that it may have been picked up in Northern Anatolia30. Olivieri et al.34 found a J1c3 haplotype in one of their ancient samples from Sardinia (MA74), dated to 6190–6000 calBP and thus its presence in our Puig des Molins samples is not unexpected, as this lineage was clearly present in the Western Mediterranean region at the time that Ibiza was settled. The primary Phoenician settlers of Ibiza are likely to have be males who sailed from Gadir and the presence of these haplogroups provides additional support to our previous work suggesting that local female integration was a hallmark of Phoenician settlement history across the Mediterranean basin3.Our analyses of the mitogenomes of the ancient samples from Ibiza indicate a predominantly European maternal ancestry for the population Fig. . Haplogr3. We suggest a similar pattern is indicated for the Phoenician settlement of Ibiza. When we investigated the relationships of mitogenomes of Phoenician populations from Lebanon, Sardinia and Ibiza .Unfortunately, to really dissect population mtDNA histories generally, and those of southwest Europe or any region with high rates of haplogroup H in particular, complete mitogenome sequencing is necessary. Despite the large sample sizes for studies of HVR sequences for the Iberian Peninsulaet al.55 identified K1a1b1 in a Middle Neolithic Iberian sample (Mina3) with an archaeological context dated to 3900–3600 BCE. In their analyses of mtDNA hypervariable region sequences obtained from ancient samples from Neolithic and Iron-Age necropoli from Mallorca and Menorca, Simón et al.39 found that 4.34% belonged to Haplogroup K, with the highest frequency (10%) in the oldest (Neolithic) necropolis, Cova des Pas, on Minorca. Our K1a1b result may indicate that the initial settlement of Formentera, prior to the arrival of the Phoenicians, also likely came from the mainland or via Mallorca, an observation presented by Bellard4. Olivieri et al.34 found K1a and K1b1b1 in pre-Phoenician Sardinians, but we have yet to find haplogroup K in any of our Phoenician samples.In addition to the ancient Ibiza samples, we sequenced a complete mitogenome of a Late Chalcolithic/Early-Bronze Age sample (MS10589) from the Ca na Costa site on the island of Formentera which was determined to belong to the K1a1b haplogroup. Mathieson 13, based on HLA markers and blood groups in Ibizans, and earlier mtDNA14 and Y-chromosome15 studies which show affinities to North African and Near Eastern populations.Ibiza has had a complex history of resettlement periods since the time of the Phoenicians. In addition to a Phoenician founding population, modern Ibizans would have had potential genetic influences from later Punic settlements, like Carthage and our ancient DNA data are consistent with this scenario. Historic records would suggest that the Ibizan genetic signature was also likely influenced by the arrival of Arab and Berber populations during the first millennium CE and finally, from 1200 CE, it was said to be overwhelmed by mainland colonists. This might explain the discontinuity between the ancient and modern DNA results we observed, shown in Fig. 56. The modern Ibizan population is singularly unusual in the entire Western Mediterranean region for their apparent lack of these two common haplogroups. It is possible that the mtDNA frequencies are so different from the ancient samples due to random genetic drift, which is common in small island populations. The small sample size (n = 18) could also be a factor, though our result indicating that the modern Ibizans are genetically quite different to mainland populations is consistent with previous studies39.Our TempNet results showed little continuity between the Phoenician inhabitants of the island and the modern Ibizan population. No shared haplotypes were found and only one haplogroup, T2b, was found in both groups. Perhaps most surprising was that neither haplogroups H1 or H3 were found in the modern Ibizans. These two haplogroups were not only the most common in our ancient population but are found at high frequencies in both modern North African and Western European populations generally55. These lineages are also found in Berbers which could explain their presence in the modern Ibizans. Haplogroup L2c however, which was identified in two unrelated, modern Ibizans, has not been found in Berbers60. It is typically a West African, sub-Saharan lineage and it may have been introduced during the Islamic expansion which had significant exchange with the entire African continent. Alternatively, it could have been a more recent arrival, perhaps the result of the transatlantic slave trade59. Botigué et al.61, showed in their whole genome analyses that sub-Saharan ancestry was less than 1% in Europe, except for the Canary Islands, where Maca-Meyer et al.62 found L2c in both 17th and 18th century historic and modern individuals. Another sub-Saharan lineage, L1b has also been identified in a Late Chalcolithic population in central Iberia38 and in one 7th century CE sample from Mallorca39.Haplogroups T1 and V, found in the modern Ibizans, are lineages found at reasonably high frequencies in the Near East. Haplogroup V is also found in both ancient and modern populations from the Iberian Peninsula and in ancient samples from Mallorca and Minorca, and T1 is also found at low frequency in mainland Spain today63) or are widely distributed across Europe and the Near East and identifying the specific origin of the population influx that causes the discontinuity between ancient and modern Ibizans is difficult. The FST results for the complete mitogenomes shown in Fig. 64. Further whole genome analyses of the modern indigenous Ibizans would provide the necessary data to fully assess the origins of this population, but from a mitochondrial perspective, it appears that they are not directly related to the Phoenician and Punic founding populations and thus this is not the explanation for the unusual genetic signature.The remaining lineages identified in modern Ibizans are all either exclusively European markers . Despite this lack of continuity observed in the mitochondrial genomes, previous Y chromosome analyses suggest that there is still some Phoenician signature in the modern Ibizan population. This is consistent with historical evidence suggesting that Phoenician influence in the West was male dominated and indicates that there was not a total replacement of the Ibizan founding population. Comparisons of the ancient Phoenician whole genome data with whole genome data from modern Ibizans will help clarify this point further. Finally, the whole genome data obtained from the ancient Ibizan sample belonged to an individual with a significant Eastern Mediterranean component, suggesting an admixed Phoenician community in Ibiza during the 3rd century BCE. While this result is consistent with the archaeological evidence and further indicates that diversity was a hallmark of Phoenician societies, it also highlights the complexity of island population settlements and underscores the importance of the inclusion of ancient DNA analysis in population genetics.In conclusion, our results show a complex pattern of settlement of the island of Ibiza. We demonstrate a clear genetic discontinuity between the early Phoenician settlers and the modern inhabitants of the island based on the mtDNA results. Thus, the unusual genetic signature of modern Ibizans is not likely to be the result of their Phoenician ancestry, at least from a mitochondrial perspective. It appears that multiple population arrivals through invasions or other movements combined with periods of population instability since the early Phoenician settlement may have led to a total reshuffling of the genetic makeup of this island. Over the last several centuries Ibiza witnessed population growth supported by the arrival of mainland populations to the island, followed by significant population reduction resulting from the bubonic plague and malariaPermits and approval for this study were obtained from the office of the Director General of Antiquities in Lebanon  for all Lebanese samples and from the Museu Arqueològic d’Eivissa i Formentera for the Spanish samples and all methods were performed in accordance with the relevant guidelines and regulations.th century BCE, it spans an area around 50,000 m2, with close to 3000 tombs. At least three burial rituals can be distinguished in Puig Des Molins. The first between the 7th and 6th centuries BCE used cremation. The second coincided with significant population growth on the island during the 5th and 4th centuries BCE and corresponded to inhumation using the spectacular hypogea cut in the rock. The third, between the 3rd and 2nd centuries BCE witnessed the brief return of cremation. This necropolis has yielded many of the most important clues and indices that led to the understanding of the funerary rituals of the early Phoenician settlers of Ibiza.Puig des Molins (windmill Mountain) is one of the largest necropoli discovered to date in west Phoenicia. Used since the 7rd–2nd centuries B.C.Cas Molí is a Punic hypogeum situated near the Bay of Saint Antoni the Portmany, in the Western coast of the Island. Inside were recovered the bone remains of at least 15 individuals. The pottery found allow to situate the chronology of the site in 3Ca na Costa is a megalithic funerary site on the island of Formentera and dating from the Late Chalcolithic to early Bronze age (2000–1600 BCE). The site was excavated between 1974 and 1977 and the remains of 8 individuals along with associated funerary  items were recovered in the circular stone tomb.66. Saliva samples were collected with full written informed consent after revision and approval by the Institutional Review Board of the Comitè Ètic d’Investigació Clínica-Institut Municipal d’Assistència Sanitària (CEIC-IMAS) in Barcelona (2016/6723/I) and all methods were performed in accordance with the relevant guidelines and regulations. Modern DNA samples were amplified in two long range PCR products, produced using primers HUM-LR1 and HUM-LR2 described previously24. The PCR products were pooled and 1 µg of the products, in equal molar concentrations, were mechanically sheared using sonication to produce fragments of approximately 500 bp in length. Blunt end repair, ligation of sequencing adaptors, sample barcoding and pooling were carried out following Meyer and Kircher67 and Kircher and Kelso68 with modifications for Illumina sequencing adaptors. Pooled samples were sequenced on one lane of the Illumina MiSeq in a 2 × 250 base paired-end run with version 2 chemistry at the Otago Genomics and Bioinformatics Facility (OGBF).A total of 18 DNA samples were obtained from Ibizan individuals first sampled by researchers from the Institut de Biologia Evolutiva (CSIC-UPF), Barcelona, as part of a study on Y chromosomes and surnames. Previously, data on Y-chromosome SNPs and STRs in four of those samples had been part of a wider study on surnames and the Y chromosome69. DNA was extracted from 13 teeth recovered from two archaeological sites on Ibiza and one site on Formentera. All teeth were rinsed in 5% bleach, followed by washing with distilled water for multiple washes to remove any bleach residues and drying overnight. DNA was extracted from ~150 mg of ground tooth, following a silica-based extraction protocol70. One extraction negative was prepared alongside every five extracts and processed alongside extracts for the remainder of the lab work.All aDNA extraction and library preparation (until PCR) was carried out in a purpose built aDNA facility at the University of Otagoet al.23. Mitochondrial genome libraries were pooled and sequenced using a 2 × 75 bp paired-end sequencing kit on an Illumina MiSeq platform at the Otago Genomics and Bioinformatics Facility (OGBF).For the mitochondrial genome sequencing, libraries were prepared and hybridisation capture used to target the human mitochondrial genome according to the protocol described by Matisoo-Smith 3. Complete mitogenome sequences were subsequently aligned with one another and previously published mitochondrial sequences using mafft71.Resulting sequencing data from the ancient mitochondrial libraries were processed according to methods described previously72 was used during processing of sequencing data to check nucleotide mis-incorporation patterns and fragment length distribution of sequencing libraries, as an indication that the resulting data looked degraded . When the VCF was loaded into HaploGrep we obtained the haplogroup call of K1a1b  and confirmed 7 informative SNPs, ranging from between 2 and 4X coverage, thus we included this sample in further mitogenome analyses.Due to the relatively high contamination rate (14%) of sample MS10589, we used PMDtools with a damage threshold of 3 to select only those reads that were damaged75. A full list of populations included in each network is available in the Supplementary Tables Network Analyses – mitogenome and HVR: In order to identify relationships between the modern and Phoenician Ibizans and potential source populations, aligned mitochondrial sequences  were used to construct median joining networks in PopArt40 to assess whether the modern Ibizan population were directly descended from the Phoenician Ibizan population.Temp net: The modern and ancient mitogenome sequences generated in this study were aligned and used to create a TempNet temporal haplotype network76 and FST values were visualised in a two-dimensional MDS plot, using the pcoa function in the ‘ape’ package for R77.MDS: The genetic distance between modern Ibizans and candidate source populations, based on complete mitogenomes, were compared using a multi-dimensional scaling analysis. A MDS plot was also generated to compare mitogenome sequences from modern and Phoenician Ibizan individuals to modern, Phoenician and pre-Phoenician (depending on data availability) populations from the Spain, Sardinia, Lebanon and Egypt using a multi-dimensional scaling analysis. The genetic distances between populations were calculated using DnaSP v6https://github.com/stschiff/sequenceTools/tree/master/src-pileupCaller). The resulting genotypes were merged with ancient DNA datasets from Lazaridis et al.30 and Haber et al.29 and modern DNA datasets of Iberians (IBS) from the 1000 Genomes Project Consortium33, Tunisians from Lazaridis et al.32 and Lebanese from Haber et al.31.One individual, MS10614, was selected for shotgun sequencing based on the high coverage of the mitochondrial genome sequencing results. The amplified sequencing library prepared for hybridisation capture was submitted for shotgun sequencing using a 2 × 75 bp paired-end sequencing kit on one lane of a HiSeq at the Otago Genomics and Bioinformatics Facility. Sequencing reads were mapped to a human reference sequence (UCSC hg19), and genotypes were called by sampling a random read per SNP in the Human Origins SNP panel, using the ‘pileupCaller’ tool  to the total number of X and Y chromosome alignments  as described by Skoglund and colleaguesPCA: When we projected MS10614 into the context of the three worldwide modern populations .In all the considered scenarios involving modern populations and MS10614, the Principal Component Analyses were produced calculating the eigenvectors with smartpca; the ancient sample was projected setting the lsqproject parameter to yes in the smartpca parameter file. In every case we only took into account those SNPs that were common to the merged datasets and the ancient sample. No minor allele frequency filter, or LD pruning were performed. In modern samples, only variants and samples with many missing calls were filtered out . Also, variants failing a Hardy-Weinberg equilibrium exact test were discarded , ancient individuals from Lazaridis 49 was used on a subset of the above merged dataset, comprised of the Phoenician Ibizan, Neolithic Anatolians, Late Neolithic-Bronze Age Europeans, Bronze Age Iberians, Bronze Age Levantines, Neolithic Levantines, modern Lebanese, modern Spanish, Bronze Age Sidon samples, and modern Tunisian populations. The ancestry proportions of these populations were assessed by testing clusters of K = 1–10  where ‘X’ tests all populations in the merged dataset. Based on the results of the outgroup f3 statistics, the ‘qpDstat’ tool from the Admixtools package50 in the form of D, where ‘X’ and ‘Y’ tests populations identified as being ancestral to MS10614 in the f3 and PCA analyses (Supplemental Data FSupplementary TablesSupplementary Information