Ursus arctos) from the Eastern, Southern, and Western Carpathians. Carpathian Mountains (Europe). Nearly 400 noninvasive brown bear DNA samples from the Western (Poland) and Eastern Carpathians  were collected. Together with DNA isolates from Slovakia and Romania, they were analyzed using the set of eight microsatellite loci and two mtDNA regions (control region and cytochrome b). A set of 113 individuals with complete genotypes was used to investigate genetic differentiation across national boundaries, genetic structuring within and between populations, and movement between populations. Transboundary brown bear subpopulations (Slovakia and Poland) did not show significant internal genetic structure, and thus were treated as cohesive units. All brown bears from the Western Carpathians carried mitochondrial haplotypes from the Eastern lineage, while the Western lineage prevailed in the brown bears from the Bieszczady Mountains. Despite similar levels of microsatellite variability, we documented significant differentiation among the studied populations for nuclear markers and mtDNA. We also detected male‐biased and asymmetrical movement into the Bieszczady Mountains population from the Western Carpathians. Our findings suggest initial colonization of the Western Carpathians by brown bears possessing mtDNA from the Eastern lineage. Genetic structuring among populations at microsatellite loci could be a result of human‐mediated alterations. Detected asymmetric gene flow suggests ongoing expansion from more abundant populations into the Bieszczady Mountains and thus supports a metapopulation model. The knowledge concerning this complex pattern can be implemented in a joint Carpathian brown bear management plan that should allow population mixing by dispersing males.The abundance and distribution of large carnivores in Europe have been historically reduced. Their recovery requires multilevel coordination, especially regarding transboundary populations. Here, we apply nuclear and mitochondrial genetic markers to test for admixture level and its impact on population genetic structure of contemporary brown bears ( Its populations formerly occupied all of Europe  diversity for brown bear has been extensively researched to elucidate phylogeographic trends in Europe ; hairs found on marking trees and fences crossed by bears (n = 134); dried blood found on a vehicle destroyed by a bear; 14 buccal swabs or hairs collected from bears captured for telemetry studies; an individual rescued from poacher's snare; another one captured due to public safety; and from four dead individuals occasionally found in the field. Samples were collected in Polish (n = 376), Slovakian (n = 20), and Ukrainian (n = 3) Carpathians. Preliminary results of microsatellite DNA analyses of a subsample from Poland were reported by Śmietana, Rutkowski, Ratkiewicz, and Buś‐Kicman (n = 35) and Romania (n = 16) that were previously analyzed by Straka et al.  were performed in the Laboratory of Molecular Biology , and the second set of samples  were performed by Wildlife Genetics International . Within analyses of the first set of samples, we chose and organized 12 autosomal and unlinked microsatellite markers . Four microsatellite loci  produced unreliable results for stool samples  and therefore were discarded from multiplex sets after initial screening. PCR reactions were performed from two to four times depending on the consistency of obtained PCR products. During initial screening, samples with <4 genotyped loci were removed and not included in any further analysis. For samples which passed this step but did not produce reliable genotypes at one up to four loci, we attempted to fill in missing or weak data by performing additional PCRs (in multiplex sets or targeting single loci). All pairs of remaining unique genotypes were compared in search of similar genotypes showing one to maximum of three mismatches, then scrutinized for potential scoring errors. If necessary, samples were rerun in selected markers. Each group of matching multilocus genotypes was considered an individual animal. The frequency of null alleles was assessed in cervus 3.0.3 . Microsatellite DNA analyses of the first set of samples . All microsatellite loci analyzed by WGI were amplified separately. Genotype scores were obtained following protocols for low DNA quality and quantity samples, highly relying on: (a) contamination prevention, (b) strict sample quality control and scoring convention, (c) error‐checking and considering samples with missing data on a case‐by‐case basis, and (d) extensive comparisons of similar genotypes followed by reamplification and reanalysis if necessary , while remaining individuals were identified based on one sample.The second set of samples was analyzed by Wildlife Genetics International (WGI) with the use of the same set of eight selected microsatellite markers, and ZFX/ZFY marker for sex identification (instead of b (345 bp) gene was amplified together with flanking tRNA‐Thr (70 bp) and tRNA‐Pro (9 bp) using newly designed primers in fastpcr . In addition, the amplification of 385–388 bp of the mitochondrial control region together with 10 bp of tRNA‐Pro were performed using primers CR_F—AGGAAGAAGCAACAGCTCCACTA, CR_R—CCATCGAGATGTCCCATTTGAAG. The PCRs for both fragments were performed in 5 µl reaction volume containing 2 µl genomic DNA (~20 ng), 1.7 µl Qiagen Multiplex PCR Master Mix (1×), 0.3 µl mix of primers (0.2 µM of each primer), and 1 µl RNase‐free water. The reaction conditions followed the same protocol as used for the microsatellites, it ran for 40 cycles with annealing at 57°C. Sequencing reactions in both directions were performed using the BigDye™ Terminator Cycle Sequencing Kit (Applied Biosystems). The reaction conditions were as follows: 25 cycles with denaturation at 95°C for 20 s, annealing at 50°C for 15 s, extension at 76°C for 60 s. The detection of sequencing reaction products was carried out on ABI 3,130 Genetic Analyzer. Sequences were aligned manually in the bioedit sequence editing program  and MG254049–MG254058 (for mtDNA control region).MtDNA analyses were only performed in the Laboratory of Molecular Biology. The partial sequence of the cytochrome am Hall, . Sequenc2.3b and control region haplotypes (809–813 bp long), we constructed a neighbor‐joining tree with mega v.5.05  was used as an outgroup. Haplotype network reconstruction was performed in network v4.6.1.0  and average number of alleles per locus (NA), allelic richness (AR), allele size range, gene diversity (GD), departures from Hardy–Weinberg equilibrium (HWE), and inbreeding coefficient (FIS) in brown bear populations studied. Linkage disequilibria (LD) between pairs of loci were estimated and corresponding tests were based on permutations in fstat 2.9.3.We used  Goudet,  to estimstructure 2.2.3 , admixture level between them and to detect possible migrants. The test for migrants was additionally computed with geneclass2  using the Bayesian assignment algorithm implemented in BayesAss , haplotype diversity (h), nucleotide diversity (π), number of segregating sites (S), and mean number of pairwise differences (PD) for concatenated mtDNA control region and cyt b using software packages arlequin  that corresponded to 56 individuals. While 121 samples analyzed by Wildlife Genetics International yielded 94 reliable genotypes (78%) which corresponded to 41 individuals. Nineteen out of these 41 individuals were identified earlier by the Laboratory of Molecular Biology (they were present in the first set of samples). Thus, with an addition of DNA isolates from Slovakia and Romania, our microsatellite analyses were based on 121 individuals while mtDNA analyses augmented by the samples from Ukraine comprised of 119 individuals (for detailed information about sample usage see Supporting Information Table A) ranged from 6 (at 3 loci) to 13 alleles (Mu59); on average 8.63 alleles  among the studied brown bear populations.For the total sample, the number of alleles per locus  as well as between Polish and Slovakian parts of Bieszczady Mountains . Thus, the studied samples from Poland and Slovakia were pooled and regarded as two genetically distinct groups: Western Carpathians  and Bieszczady Mountains  for further analyses. Pairwise genetic differentiation values for microsatellite loci between brown bear populations from the Western Carpathians and Bieszczady Mountains were moderate for FST (0.106) or great (RST = 0.197) and significantly different from zero at p < 0.001. The genetic differentiation values between the population from Romania and the other two populations were low to moderate  while RST was great .We did not find significant genetic differentiation at microsatellite loci between the “transboundary subsamples” in Western Carpathians in Poland and Slovakia . When usepopinfo was used, with K = 3 and migrprior set at 0.05, six individuals in the Bieszczady Mountains were found to be immigrants or F1 backcrosses at p < 0.05 to p < 0.001: 4 from Western Carpathians  and two from Romania with H5 haplotype. These six individuals with the highest inferred ancestry were all males and they were present all over the Bieszczady Mountains population area. geneclass2 and bayesass also found the same four male immigrants from Western Carpathians (p < 0.01), however did not detect any immigrants from Romania into the Bieszczady Mountains. All but one pairwise comparison of relatedness between six males with the highest immigrant ancestry revealed they were not related. A single pair showed significant relatedness at the level of cousins. The statistical analysis of pairwise relatedness coefficient between all 17 admixed individuals found in Bieszczady Mountains without usepopinfo revealed four possible parent‐offspring pairs, two full siblings pairs, three half‐siblings pairs, and five cousins pairs, indicating recent effective dispersal and admixture of brown bears from Western Carpathians. Eight other brown bears that possessed H1 or H2 haplotype  that were present in Bieszczady Mountains did not show considerable admixture. bayesass found little recent gene flow (about 1%) from the BM and ROM to the WC populations . The same was from the ROM to the BM . The short‐term gene flow from the WC to the BM and from BM to the ROM population was about 2% . The largest level of recent gene flow was found from WC to the ROM population , but possibly this result was affected by small sample sizes and low differentiation between WC and ROM.3.2K; H1–H4) or the Balkan branch of the Western lineage, for example, clade 1 (H5–H10). The haplotypes from these two distinct clades differed with 28–38 substitutions . Brown bears from Western Carpathians possessed only haplotypes from the Eastern mtDNA lineage (clade 3a1), while in the Eastern  and Romanian Carpathians, both mtDNA lineages (clades 1 and 3a1) were present  yielded nine haplotypes and the tenth haplotype (H10) was found in Greece . The same pattern was observed in the Bieszczady Mountains . On the other hand, genetic differentiation values for mtDNA between brown bear populations from the Western Carpathians and Bieszczady Mountains were very great  and significantly different from zero (p < 0.001). The genetic differentiation (FST) between population from Romania and other two populations was also great and significant  between Romanian Carpathians and Bieszczady Mountains (POL + SVK) populations did not differ significantly from zero.Similar to microsatellite loci, mtDNA pairwise genetic differentiation values between the “transboundary subsamples” from Western Carpathians in Poland and Slovakia were small and not significantly different from zero  in the Bieszczady Mountains may indicate recent admixture from the Western Carpathian population that resulted in significant LD among three pairs of microsatellite loci in this population. However, admixture from Western Carpathians soon after World War II may have occurred as other eight brown bears that possessed H1 or H2 haplotypes were present in Bieszczady Mountains, albeit were assigned local ancestry. In addition, the estimate of recent gene flow from the WC to the BM population obtained in bayesass (about 2%) is lower than genetic admixture in the latter population . This suggests that the observed admixture level cannot be explained by short‐term gene flow between these populations.Straka et al.  in theirHabitat fragmentation is assumed to be a general factor that may increase female philopatry  in the mountainous areas of the western Great Basin, USA  after World War II have been a minority (Jakubiec, Our results suggest that the studied Carpathian brown bear populations, despite being divergent at mtDNA and microsatellites, should not be considered as distinct conservation units (sensu Taberlet, Swenson, Sandegren, & Bjärvall, The authors declare no competing interests.WS, MM, LP, and MR conceived and developed the initial idea; SJ, WS, and MR acquired funding for data collection; WS, LP, JŠ, DK, and A‐TB collected and provided samples; MM and MC conducted laboratory work; MM, WS, and MR analyzed the data; MM, MR, WS, and LP led the writing with contributions from all authors. All authors approved the final version of the manuscript. Click here for additional data file.


Phyllonorycter platani is a widely distributed insect species on plane trees and has a well-documented colonisation history in Europe over the last century. However, phylogeographic data of the species are lacking.The plane leaf miner, We analysed 284 individuals from 38 populations across Europe, Asia, and North America. A 1242 bp fragment of the mitochondrial COI gene and an 893 bp fragment of the 28S rDNA has been Sanger sequenced. Twenty-four haplotypes were detected on the COI gene, and two alleles were identified on the 28S rDNA. We revealed two distinct clades for both markers reflecting the geographic origins, Asia and Europe. The genetic distance between the two main clades is 2.08% on the COI gene and 0.10% on the nuclear DNA.An overlapping zone of the two clades was found across Eastern Europe and the Anatolian Peninsula. We detected heterozygote individuals of the 28S rDNA gene in Moldavia, Ukraine and in the southern part of Turkey. These suggest that the two clades can hybridise. Furthermore, the presence of European type homozygote individuals has been confirmed in the southern part of Turkey as well.P. platani. The genetic patterns revealed at least two refugia during the last ice age: one in the Balkan Peninsula and the other in the Caucasus region. Recent expansion was detected in some European and Central Asian populations. The two main clades (Europe/Asia) show definite genetic differences; however, several hybrid individuals were found in the overlapping zone as well (stretching over Eastern Europe and the Anatolian Peninsula). Discrepancies in mitochondrial and nuclear data indicate introgressions in the southern part of the Anatolian Peninsula.We have shown that both post-glacial recolonization and recent expansion events influenced the present genetic structure of The online version of this article (10.1186/s12862-018-1240-z) contains supplementary material, which is available to authorized users. Phyllonorycter platani , is one of the most important invasive Gracillariidae species [Biological invasions have been considered a major economic and conservation problem recently , 2. The  species , 4. Its  species , 5. The Phyllonorycter platani  has a large distribution area across Europe, the Anatolian Peninsula, Near-East and Central Asia. Šefrová [Platanus orientalis) is native. Lopez-Vaamonde et al. [P. platani as unknown.According to the Global Taxonomic Database of Gracillariidae  Phyllono Šefrová , 5 suggee et al.  considerPhyllonorycter felinella HEINRICH, 1920 in California (US), but Deschka [Lithocolletis platani STAUDINGER, 1870. The actual situation of the taxon was, until recently, uncertain. [Heinrich  describe Deschka  synonymicertain. , [7, 12–certain. .Phyllonorycter [Platanus hybrida BROT., P. occidentalis L., P. orientalis L. and P. racemosa NUTTAL [P. orientalis  and P. hybrida  .Leaf mining is a special kind of phytophagy where monophagous species are dominant. Host shifts are rare in the genus norycter  and the norycter ): Platana NUTTAL . HoweverPlatanus orientalis is native to the eastern part of the Mediterranean, across the Anatolian Peninsula and the Caucasus to Central Asia, especially in the coastal areas and river valleys [P. platani is of high significance [ valleys –20. The  valleys , 20–23.  valleys . Therefoificance , 25, 26.Phyllonorycter species represent taxonomic works and use the mtDNA barcode fragment [Phyllonorycter [Most genetic analyses of fragment –29. Othefragment , 30. Hownorycter , 32 or onorycter .P. platani before this study. They include phylogenetic works of Phyllonorycter species [Cameraria microsatellite markers [Only a limited number of sequences were available in the various databases  for  species , 30, out markers , and par markers . In one  markers .P. platani and its possible refugial areas. (iii) To reveal factors influencing the recent genetic pattern, especially the isolation by distance and/or geographic isolation of certain populations.Our aims were (i) to assess the current phylogeographic pattern of this species. (ii) To determine the origin of P. platani, two population of P. issikii, and one of P. maestingella  GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich), b) E.Z.N.A.® Tissue DNA Kit and c) AquaGenomic Kit following the manufacturer’s protocol. Eluted DNA was stored at − 20 °C.A 1242 bp-long region of the COI gene was amplified for 284 individuals by using Pat, (5’-TCCAA TGCACTAATCTGCCATATTA-3′) and Lep2F (5′- ATTCAACAATCATAAAGATATTGG-3′) primers , 38, as We amplified an 893 bp fragment of the 28S rDNA for 103 individuals from five selected populations  at the Eurofin’s Laboratory . Sequences are available via GenBank with accession numbers KY952988–KY953017.P. issikii and P. maestingella were used as outgroups. Genetic distances were calculated with MEGA 5.02 [For nuclear DNA (28S) analyses, 103 individuals were used and 284 individuals were used for mitochondrial DNA (COI) analyses (Table EGA 5.02 .Maximum likelihood (ML) analysis was performed under GTR + I model with MEGA 5.02. The level of support for individual nodes was evaluated by bootstrapping with 5000 replicates. We used jModeltest 2.1.2 , 44 to sPatterns of molecular diversity based on the mtDNA sequences between and within populations were assessed by estimating: nucleotide diversity (π) , transitPopulation dynamics analyses were performed on different geographical scales . For the estimation of Tajima’s D statistics  and Fu’sCT reached a plateau [Spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA v1.0 . The pro plateau .In addition, alternative geographical groups were tested with Analysis of Molecular Variance (AMOVA) –58 with Isolation by distance was evaluated using Mantel test  with MANStatistical parsimony network (SP)  was creaTwenty-four haplotypes were detected on the 1242 bp long fragment of the COI gene (Table Phyllonorycter species (8.52–10.68%).There were 22 mutational steps between the Asian and European clades. Pairwise genetic distances between Asian (HT13–16) and European  or between Asian (HT13–16) and North American  haplotypes are lower (1.80–2.30%) than the interspecific distance among related Average sequence divergence between Asian and European clades 2.08%) was higher than the intrapopulation level . Divergence data shows that the population from the southern part of Anatolia (HT16) is closer to the Caucasian and Central Asian group (0.46%) than to the European group (1.88%). The genetic divergence between European and North American haplotypes was rather low (0.1–0.4%) in comparison to the outgroups (8.4–11.0%). ML tree support two clades with 100% probability: 1) the Asian and 2) the European (including the North American haplotypes)  Table . Haplotyr = 0.361, Ρ = 0.005), or a weak correlation: European clade ; and European population only .Genetic population structure correlated with geographic distances (Mantel test). The correlation coefficient (r) indicates either a moderate , but ca. 2.55% (Vb) of variance is still found among populations within groups (p < 0.001). We can detect only slight gene flow between the two main groups. In the second arrangement, when we used the European clade (without the East-European samples which constitute the hybrid zone), the first group contains Northern and North-Western European populations , and the second one contains Southern and Central European populations with the North American samples . In the third arrangement we used only the European samples (without Eastern European and North American samples) and received similar results .As the Fns Table . On the p = 0.00) in the populations from North America (R2 = 0.156), Caucasus (R2 = 0.162), and Eastern Europe (R2 = 0.132).Most of the Tajima’s D and Fu’s Fs indices are not significant Table ; we deteTwo alleles were identified on the 893 bp long fragment of the 103 specimens sequenced from 11 selected populations Fig. , Fig. 3.Phyllonorycter genus especially, an extremely high interspecific divergence was observed  [Phyllonorycter ulicicolella species group, Laštůvka et al. [Phyllonorycter species is 6.04% on the barcode fragment [The interspecific divergence on the COI gene is high in the Lepidopteran families –69. In tictella) . In the a et al.  noted a fragment .Macrosaccus species, 0.49% within Cameraria ohridella, 0.17–2.07% Choristoneura sp., 0.11–3.22% Hyles sp., 1.30–2.50% Sciadia tenebraria, 1.50–4.10% Epirrita autumnata, 1.93% within P. salictella [Phyllonorycter species [P. platani haplotypes are similarly high to another plane feeding Lepidopteran species of Acalyptris platani (1.80–2.30%) [P. issikii, the average distance (5.13 ± 0.003%) between the two clades is approximately two times higher [P. platani (2.08 ± 0.369%). The maximum intraspecific divergence is 2.96% for P. issikii and 1.38% for the putative new species [P. platani.A wide range of varying intraspecific divergence exists for the different Lepidoptera species: e.g. 0.00–0.50% within lictella , 68–70.  species . The gen0–2.30%) . In the s higher  than bet species , while wPhyllonorycter [P. issikii revealed 7 diagnostic substitution differences on this fragment of the 28S rDNA [The interspecific genetic divergence on 28S rDNA varies 0.20–25.00%, while the average intraspecific divergence is lower than 0.50% in the genus norycter . We dete28S rDNA . The hig28S rDNA . In addiAglais urticae 0.96) is medium-high [Dioryctria species 0.03–0.35%, Hyles genus 0.03–2.71%, Papilio species 0.26–2.71%) [Cameraria ohridella  is lower [The haplotype diversity (h) of the plane leaf miner (0.68) compared to other Lepidopteran species (ium-high . The nuc6–2.71%) , 66, 72.is lower .P. 12platani populations [P. platani.Past and recent gene flow events determine the geographical pattern of populations . One of ulations , 5, 75; ulations . In our According to the coalescent theory, the most frequent haplotype is supposed to be the most ancient one . HoweverAll methods used for the statistical analyses  support the existence of two main clades (European and Asian) and the further differentiation of the Asian clade. The genetic divergence between the European and the Asian clade is high (2.08%), but this is typical for the members of the family Gracillariidae , 69. HapThe HT16 (which is represented in the southern part of Anatolia) is linked more closely to the Asian haplotypes (HT13–15) than to the European. This suggests that there may have been a connection among the refuges of plane leaf miner populations during the interglacial periods.r = 0.3605), and the variability value is high (Va = 94.32%) between the main two major clade.Mantel test results and SAMOVA also support the view that the species survived the ice ages in several refugia because the isolation by distance values are moderate . The population from Antalya has a homogenous haplotype pattern, which refers to a founder effect. Several little plant refugia are described from the Mediterranean Basin , but theThe star-like shape of the SP network Fig.  refers tP. platani in Europe and in the north of the Anatolian Peninsula may have gone through a rapid range expansion after bottleneck . Populations from the France-Germany borderline, the eastern Alps and the eastern border of Germany compose the edges of the “W-NW” population supported by SAMOVA (“W-NW” and “S-C”), which are common barriers within Europe [The populations of n Europe . The lown Europe . In the n Europe , 87. Pren Europe . The outHowever, while analysing our dataset we have to take into consideration that three factors  might have major influence on the quantification of population size changes . In ourThe results of the COI and 28S rDNA sequences show that the two main clades can hybridize. We found two possible hybrid zones. One of them is located in the eastern part of Europe; populations from Moldavia, Ukraine contain both of Asian and European haplotypes. In addition, we detected hybrid individuals from the Moldavian population with the nuclear marker. The other hybrid zone is located in the southern part of Anatolia. The detected unique COI haplotype (HT16) is more closely linked to the Asian haplotypes than to the European haplotypes. The revealed allelic pattern with the 28S rDNA marker shows the presence of heterozygotes and European-type homozygote individuals. This discrepancy with mitochondrial and nuclear data shows that there were introgressions in the southern part of Anatolia. In most cases, mito-nuclear discrepancies are the results of possible secondary contact zones after isolation , 90. TheLithocolletis felinella HEINRICH, 1920 to Phyllonorycter platani  is required, however a morphotaxonomic approval is desirable.Our results also confirmed that the synonymization of P. platani. The genetic patterns revealed at least two refugia during the last ice age: one in the Balkan Peninsula and the other in the Caucasus region. Recent expansion was detected in some European and Central Asian populations. The two main clades (Europe/Asia) show definite genetic differences; however, several hybrid individuals were found in the overlapping zone as well (stretching over Eastern Europe and the Anatolian Peninsula). Discrepancies in mitochondrial and nuclear data indicate introgressions in the southern part of the Anatolian Peninsula.We have shown that both post-glacial recolonization and recent expansion events influenced the present genetic structure of Additional file 1:Phyllonorycter platani. Number of COI haplotypes (HT1–24) in the countries/locations investigated. (DOCX 32 kb)COI haplotype distribution of


Many anthropological, linguistic, genetic and genomic analyses have been carried out to evaluate the potential impact that evolutionary forces had in shaping the present-day Sardinian gene pool, the main outlier in the genetic landscape of Europe. However, due to the homogenizing effect of internal movements, which have intensified over the past fifty years, only partial information has been obtained about the main demographic events. To overcome this limitation, we analyzed the male-specific region of the Y chromosome in three population samples obtained by reallocating a large number of Sardinian subjects to the place of origin of their monophyletic surnames, which are paternally transmitted through generations in most of the populations, much like the Y chromosome. Three Y-chromosome founding lineages, G2-L91, I2-M26 and R1b-V88, were identified as strongly contributing to the definition of the outlying position of Sardinians in the European genetic context and marking a significant differentiation within the island. The present distribution of these lineages does not always mirror that detected in ancient DNAs. Our results show that the analysis of the Y-chromosome gene pool coupled with a sampling method based on the origin of the family name, is an efficient approach to unravelling past heterogeneity, often hidden by recent movements, in the gene pool of modern populations. Furthermore, the reconstruction and comparison of past genetic isolates represent a starting point to better assess the genetic information deriving from the increasing number of available ancient DNA samples. Afterwards, the population size remained approximately constant until the last three centuries when it underwent a significant growth  = 164.96; p < 0.001) with only 21 out of the 62 defined lineages shared among the three main geographic regions  analysis was carried out on haplogroup frequencies, exploiting available literature data normalized to the highest possible level of phylogenetic resolution . The ploThe distribution, which is based on 27.84% of the total variance, shows an overall general agreement with geography: while the first component separates the populations according to longitude, the second component discriminates the populations approximately according to latitude. The continental populations from lands lying around the Mediterranean Sea are clearly separated at the periphery of the plot in five clusters . The populations of the Italian Peninsula and the Mediterranean islands follow a North-South cline with the northern-central Italians (together with Corsicans) closer to Spanish and French groups while the southern central Italians (and Sicilians) are closer to Caucasus-Anatolian and middle eastern populations. A similar pattern is also detected when looking at the entire genome, with Corsicans closer to the Central-North Italian groups , and SicThree haplogroups were previously identified as founding lineages: G2-L91, I2-M26 and R1b-V88.Haplogroup G2-L91  reaches The Network analysis of G2-L91 haplotypes  reveals Although the highest frequency of G2-L91 is observed in Corsica, the highest STR variation of the lineage is detected in the northern area of Sardinia , suggestHaplogroup I2-M26, likely of south-western European origin , accountThe dissection of this haplogroup into its main subclades  previously identified in Sardinia  highlighNetwork analyses reveal a massive expansion of sub-clade I2-L160delta in Sardinia  and a lo26df = 28.4, p-value = 0.3), higher frequencies of I2-L160delta D (PF4225) in North Sardinia and I2-L160delta I (Z26773) in the central-eastern area of the island are apparent. It is of note that with the only exceptions of a I2-L160delta L (PF4421) Y chromosome in Calabria and a I2-L160delta A (Y20194) Y chromosome in Andalusia (easily explained as a recent Sardinian contribution), the I2-L160 sub-lines are virtually only observed in the island, supporting a Sardinian origin of I2-L160delta clades. This interpretation seems to be confirmed by the results obtained by the YFull Tree results   revealed results .Haplogroup R1b-V88 is a scarcely represented early branch of haplogroup R1b, mainly observed in sub-Saharan Africa. Its highest frequency is reported in Central Sahel  where R1b-V88 sub-lineages underwent an expansion in Chadic-speaking groups ,55,56. OAncient genomes of a number of Sardinian specimens, 44 of which males, derived from various caves located in different areas of the island and belonging to different archaeological phases, have been recently analyzed ,63. On tG2-L91, G2-F872 characterized the oldest specimens (from Middle to Late Neolithic), R1b-V88 and I2-M223 were observed in samples from the Early Copper Age and especially from the Early Bronze Age and the Nuragic period. I2-M26 appeared starting from the Early Bronze Age, J2-L283 from the Nuragic period and J1-L862 and R1b-M269 from the Punic period. Finally, haplogroups E-L618 and I2-M423 appeared only in Punic and Medieval specimens .Haplogroup R1b-V88, observed in pre-Neolithic times in Balkan subjects , is the Genome-wide analyses of modern DNA place Sardinian samples in an outlier position in the European genomic landscape ,74,75,76Although archaeological data indicate that Sardinia has been inhabited since Paleolithic times, this early human presence might have been very limited, and the time of the first peopling is still a matter of debate. Modern and ancient mitogenome analyses  have shoAlthough the demographic and genetic features associated with insularity offer the opportunity to better evaluate the impact of evolutionary forces such as founder events, gene flow and genetic drift that acted on the present Sardinian population, the homogenizing effect caused by internal movements during the last 150 years is a major confounding element. Here, through the “monophyletic surname sampling” approach, we obtained, at least for the Y-chromosome variation, novel clues concerning the past genetic isolates of the island, thus bypassing the confounding effect of recent migrations. With this approach we obtained a picture of the island population at the time of the introduction of the use of surnames, an event that in Italy occurred in the Middle Age. The comparison of the Y-chromosome haplogroups of ancient samples dated from the Early Neolithic  with thoOn the whole, the Sardinian sample that we analyzed is characterized by a haplogroup heterogeneity of 0.8466 with high values in the northern (0.8814) and southern (0.8363) regions. The lowest heterogeneity in the central-eastern area (0.7469) reflects a reduction of the heterogeneity of all main haplogroups and can be explained by genetic drift due to long-term isolation in this region identified as the “archaic zone”. Indeed, it is reported that indigenous populations retreated to this mountainous region when Phoenicians and, later, Carthaginians colonized the southern part of the island . The extFor those haplogroups equally diversified in and outside Sardinia, it is difficult to discriminate between founder lineages and lineages that arrived later.In brief, this study shows that the analysis of the Y-chromosome gene pool coupled with a sampling method based on the origin of the family name provides a greatly improved picture of the genetic structure of past population isolates. The application of this approach to a sample of Sardinian subjects carrying territorial monophyletic surnames (whose origin can be assigned to a specific location) allowed us to confirm the peculiarity of the Sardinian population in the European context and to detect ancient heterogeneities among the three main geographical areas of the island. We observed differences in the distribution not only of founding lineages and of lineages acquired through subsequent migrations, but also of sub-lineages that are Sardinian-specific. In particular, the high homogeneity displayed by the central-eastern sample is in agreement with the long-lasting isolation and founder and genetic drift effects of the “archaic zone”. In addition, the comparison of the haplotype distribution in past isolates with that displayed by recent data on ancient DNA from different geographical areas and archaeological periods offered the unique opportunity to better understand the genetic history and demography of this peculiar insular population.The sample consisted of 603 Sardinian males carrying Sardinian territorial monophyletic surnames  whose pln > 1000) of apparently unrelated healthy males, whose three-generation Sardinian origin was ascertained by interview, gathered thanks to the collaboration of many laboratories on the Sardinian territory, during different campaigns over more than 20 years. The selected samples were then assigned according to the place of origin of their surnames to the three large macro-areas of the island .These subjects were selected from a larger collection  , followiAll samples were also analyzed at ten STRs loci  using two multiplex reactions and a 3730 Applied Biosystems sequencer, as previously described . NomenclConsidering the results previously obtained , three mHaplogroup distributions were compared through the Chi Square Test of independence using the Xlstat add-on for Excel. Haplogroup heterogeneity (H) was computed using Nei’s  standardST values [MDS was performed using the Xlstat add-on for Excel and RT values  calculatT values ) using A−4 per generation (25 years).Within specific haplogroups, median-joining (MJ) networks  were con


Environmental change can expose populations to unfamiliar stressors, and maladaptive responses to those stressors may result in population declines or extirpation. Although gene flow is classically viewed as a cause of maladaptation, small and isolated populations experiencing high levels of drift and little gene flow may be constrained in their evolutionary response to environmental change. We provide a case study using the model Trinidadian guppy system that illustrates the importance of considering gene flow and genetic drift when predicting adaptive response to acute stress. We compared population genomic patterns and acute stress responses of inbred guppy populations from headwater streams either with or without a recent history of gene flow from a more diverse mainstem population. Compared to “no‐gene flow” analogues, we found that populations with recent gene flow showed higher genomic variation and increased stress tolerance—but only when exposed to a stress familiar to the mainstem population (heat shock). All headwater populations showed similar responses to a familiar stress in headwater environments (starvation) regardless of gene flow history, whereas exposure to an entirely unfamiliar stress (copper sulfate) showed population‐level variation unrelated to environment or recent evolutionary history. Our results suggest that adaptive responses to acutely stressful environments are determined in part by recent evolutionary history and in part by previous exposure. In some cases, gene flow may provide the variation needed to persist, and eventually adapt, in the face of novel stress. What is “stressful” depends largely on the environmental conditions to which a population has adapted. For example, high temperatures and low water availability in desert conditions would most likely kill organisms restricted to cool and wet places. However, natural populations are increasingly exposed to novel stressors  or more severe environmental stress than what they have previously faced . Previous work documented increased genetic variation following gene flow from the upstream translocations using microsatellite data  and whether populations had previous exposure to the stress Figure . High teFST outliers) that could preserve adaptations to headwater‐specific stressors, potentially even in the face of gene flow.We also generated genomic data from all populations to characterize genetic factors that we would expect to influence responses to stress. We used this genomic data to characterize (a) population genomic diversity and differentiation, with the expectation that diversity would be lower and differentiation from the source population would be higher in headwater populations without a recent history of gene flow; (b) individual inbreeding and number and lengths of long runs of homozygosity, with the expectation that populations without a recent history of gene flow would show more evidence of genome‐wide inbreeding; and (c) the effects of local selective pressures in headwater populations, with the expectation that local selective pressures may maintain regions of genomic divergence (detectable as 22.1Poecilia reticulata Peters) using butterfly nets from each of six locations on the south slope of the Northern Range mountains in Trinidad. The comparison we were most interested in was between populations that experienced recent human‐mediated gene flow  and populations in similar environments that did not have a recent history of gene flow . These five populations were all from low‐order headwater streams with highly similar abiotic and biotic properties  bottles filled with local stream water and held in individual, aerated tanks. All individuals were anesthetized in dilute MS‐222, weighed, and photographed on the day of capture to estimate initial body condition (weight divided by standard length). Photographs were taken of fish spread laterally on a white background alongside a metric ruler using a Canon EOS Rebel XSi SLR digital camera .In future work, it will be important to evaluate acute stress response of both sexes, especially given that sex‐specific responses to stressors such as temperature are common  maximum temperature tolerance (30 guppies/population), (b) starvation (20 guppies/population), or (c) toxicity (15 guppies/population). All fish were held in constant laboratory environment conditions for two nights and one full day before experiments began.® Pro20 Multiparameter Meter . CTM was recorded as the temperature at which an individual showed initial loss of equilibrium. Usually, this point was preceded by several muscle spasms. As soon as a guppy reached CTM, it was removed with a plastic spoon and placed into a recovery tank. Three fish did not recover from the CTM experiment and were not included in analyses.Maximum temperature tolerance in guppy populations was determined by estimating critical thermal maximum (CTM) for 30 guppies per population. Guppies were starved for one day prior to the CTM experiments to avoid energy expenditure for digestion during the experiment. We used an insulated rectangular (34.5 × 21.1 × 56.6 cm) Coleman 25‐quart cooler filled with a constant volume (7.6 L) of water that was circulated with a 2.8 watt Rio Plus 90 Aqua pump and aerated with two air stones. We tested six guppies per CTM assay, randomly placing each individual into a mesh container that was secured to the bottom of the cooler. After a 5‐min adjustment period, water was heated at a constant rate of 0.4°C/min with two immersion coils connected to a PHC Enterprise voltage transformer set to 75 V. Temperature was recorded with a handheld YSI® tropical fish flakes  ad lib once a day at 18:00. We measured body condition of control fish before and after 120 hr. To standardize the effects of starvation across individuals and populations, we quantified a starvation index for each individual, defined as body condition at 0 hr minus body condition at 120 hr. A positive starvation index at the end of the experiment indicated a reduction in body condition, whereas negative values indicate the fish gained weight. Thus, we interpreted higher values of starvation index as corresponding to worsened ability to cope with a lack of resources. We used a Welch two sample t test to determine whether fish that were starved had overall higher starvation indices than control fish that were fed during the experiment. Although groups had unequal sample sizes, assumptions of equal variance and normality were met.To assess population differences in the ability to withstand low‐resource environments, we starved individually housed guppies for 120 hr. We based this interval on a previous study that used a similar duration for its guppy starvation treatment . Anthropogenic sources of copper pollution appear in aquatic systems from mine washing, agricultural leaching, and direct application as algaecide and molluscicide  and AmQuel PLUS® . Guppies in the toxicity experiment were fed ground fish flakes ad lib once a day at 18:00 p.m. Tanks were checked every 12 hr and dead fish were removed. We assessed the survival rate for each tank after 96 hr.Finally, we evaluated resistance to exposure to toxicity by subjecting group tanks to a toxic level of copper sulfate . A single RAD library was prepared with 96 individuals (Table ® AMPure XP beads (Beckman Coulter) were used to size select from the DNA fragments with an average of 600 bp. The final library was submitted to the RTSF Genomics Core at Michigan State University and sequenced in one lane with paired‐end 150‐bp reads on an Illumina HiSeq 4000.Caudal peduncle tissue from 30 individuals per population was collected from euthanized fish from the thermal tolerance experiment and preserved in 95% ethanol. Genomic DNA was purified from muscle tissue using Qiagenls Table  followinls Table . Briefly2.4We used linear mixed models to analyze the effects of population differences on CTM and starvation index. For modeling variation in CTM, we used stream as the fixed factor. Initial weight was included as a random effect as were hierarchically nested factors of CTM run and container position. For modeling variation in starvation index, we used stream as the fixed factor and included initial weight as a random effect. Both CTM and starvation index were modeled using maximum likelihood, and significance of the population effect was tested using likelihood ratio tests against the null model that included only random effects. Residual plots were used to determine whether model assumptions of normality and homoscedasticity were met. All models were carried out with package “lme4” in R  between the source population (GP) and the other populations (including those with and without a history of gene flow). We calculated both statistics over adjacent 50‐snp windows and plotted diversity and pairwise FST using local regression  using VCFtools for each individual. We characterized variation in inbreeding among individuals using g2, a metric of correlation of heterozygosity across markers  = −8.61, p < 0.0001. All headwater populations started with higher body condition compared to Guanapo (GP) guppies from the lowland mainstem site; however, Tumbassun (TU) guppies had significantly higher body condition than all other headwater populations  but not among the source, gene flow, and no gene flow groups .Finally, we documented substantial variation among populations with respect to survival rate when exposed to copper sulfate Figure . The two3.2Per‐SNP nucleotide diversity was highest for the Guanapo (GP) mainstem source site  and lowest for headwater populations Tumbassun  and Naranjo  with no recent history of gene flow. Headwater populations that experienced recent gene flow from the GP source exhibited diversity similar to that of the source , and headwater population QU also exhibited intermediate diversity  despite no known recent history of gene flow , intermediate between GP and the headwater population in the same drainage with no gene flow , and low between GP and the populations with a history of gene flow from GP  and highest for the two headwater populations that exhibited lowest nucleotide diversity . Variation in inbreeding was high across populations (g2 = 0.271) and generally near zero within populations  (>50) with high average LROH length .Individual inbreeding estimates were lowest for GP , endoplasmic reticulum function , and cyclic AMP production/signaling cascades .Both BayeScan analyses identified the same set of five loci as outliers with a false discovery rate of 0.05. All of these loci were variable in the mainstem GP population but fixed in headwater populations. Three loci were distributed throughout the genome , while two were in close association with one another (<100 kb apart) on linkage group 17. Three of the five loci were found within intronic regions of protein‐coding genes  inbreeding depression is often worse under stressful conditions , and populations experiencing different levels of heat stress in their natural environment exhibit differences in their ability to respond to heat stress through upregulation of HSPs  exhibited critical thermal maximums (CTMs) similar to a more outbred population (QU). As such, maladaptation to heat stress in this case seems to be tied more to local selective forces than random drift. However, headwater populations that had experienced recent gene flow exhibited CTMs similar to the source population, indicating that gene flow alleviated maladaptation to heat stress in this case, fitting the predicted scenario in Figure FST outliers provides support for the latter explanation, although increased plasticity may also contribute. Constant high gene flow between heterogeneous environments is expected to increase plasticity  in headwater environments is thought to be largely a result of density‐dependent selection driven by low‐resource availability and high population densities  may be predictable based on previous exposure of source and recipient populations. The ability to cope with increased and novel stresses will be extremely important in determining the fate of small populations in a changing world (Hoffmann & Sgro,  Click here for additional data file. Click here for additional data file. Click here for additional data file.


Anthus berthelotii), a passerine bird endemic to three archipelagos in the Atlantic. We first characterized genetic diversity and population structuring that supported previous inferences of a history of recent colonizations and bottlenecks. We then tested for regions of the genome associated with the ecologically important traits of bill length and malaria infection, both of which vary substantially across populations in this species. We identified a SNP associated with variation in bill length among individuals, islands, and archipelagos; patterns of variation at this SNP suggest that both phenotypic and genotypic variation in bill length is largely shaped by founder effects. Malaria was associated with SNPs near/within genes involved in the immune response, but this relationship was not consistent among archipelagos, supporting the view that disease resistance is complex and rapidly evolving. Although we found little evidence for divergent selection at candidate loci for bill length and malaria resistance, genome scan analyses pointed to several genes related to immunity and metabolism as having important roles in divergence and adaptation. Our findings highlight the utility and challenges involved with combining association mapping and population genetic analysis in nonequilibrium populations, to disentangle the effects of drift and selection on shaping genotypes and phenotypes.Island species provide excellent models for investigating how selection and drift operate in wild populations, and for determining how these processes act to influence local adaptation and speciation. Here, we examine the role of selection and drift in shaping genomic and phenotypic variation across recently separated populations of Berthelot's pipit ( Since the time of Darwin, evolutionary biologists have sought to understand how natural selection and random chance act to shape the extraordinary diversity we see in nature. Natural selection acts on individual differences that have a genetic basis, and these differences accumulate until new species eventually form. To fully grasp how natural selection operates in the wild, we need to understand the link between variation at the level of genes, individuals, populations, and species. In this study, we used genetic analyses to test how differences have evolved among island populations of Berthelot's pipit—a small passerine bird endemic to three North Atlantic archipelagos. Berthelot's pipits are separated into subspecies based on differences in bill length, and island populations differ markedly in their levels of disease. Until now, however, we did not know whether these differences were the product of natural selection, or random processes that are the feature of evolutionary histories of small island populations. We found a genetic variant associated with bill length in Berthelot's pipits, which is close to a gene involved in controlling bill shape in Darwin's finches. Importantly, we showed that variation in this genomic region—and therefore variation in bill length—is a product of the colonization history of the pipits. In addition, we identified two genes that may be associated with malaria infection—an ecologically important disease in animals. However, this association varied among islands, suggesting that resistance to malaria is not fixed, but constantly changing, as expected for host–pathogen coevolution. Finally, our analyses suggest that genes related to immune function and metabolism may under natural selection, which is consistent with studies on humans and other animals. Our study shows that combining genetic techniques with ecological study is a powerful way to understand how natural selection acts in island populations.The process of speciation is a continuum, beginning with small genetic differences between lineages that accumulate to produce reproductive isolation , a small passerine endemic to three volcanic island archipelagos in the Atlantic Ocean. The recent colonization of this species across a set of ecologically distinct islands makes it an interesting model for examining adaptive evolution in a natural setting. Within the last 2.5 million years Berthelot's pipits colonized the Canary Islands and diverged from their sister species, the tawny pipit , Mauritania (n = 4), and Morocco  for RAD‐seq, with efforts made to reduce the probability of sampling relatives by maximizing geographical coverage within each island, and to equalize the number of males and females where possible. In addition, 16 samples from tawny pipits were included; these were collected in Spain (SNP) at 1826 loci present in all samples, and the overall nucleotide diversity per locus (πRAD) as the sum of the nucleotide diversity at every site along a RAD locus, including invariable sites, divided by the length of the locus . Nucleotide diversity was calculated in VCFtools version 0.1.14015  analyses to identify loci associated with bill length and malaria infection. A central issue with GWAS is accounting for population structure .To check whether SNPs associated with bill length also covary with overall body size, we also ran a BSLMM with tarsus length as a candidate trait. Tarsus length is the strongest predictor of overall body size that can be accurately measured from a live bird , and viewed regions of interest using the University of Santa Cruz Genome Browser (http://genome-euro.ucsc.edu/cgi-bin/hgGateway).To identify genes located near outlier SNPs, we downloaded Ensembl gene predictions for zebra finch , we calculated allele frequencies for each island population and compared this against population‐level trait variation , using Pearson correlations. We also calculated pairwise https://github.com/gc5k/GEAR/wiki), to separate loci under divergent selection from the genome‐wide effects of drift between the archipelagos, using the “Berthelot's” dataset. This analysis uses eigenvector decomposition on the genotype data to characterize the underlying population genetic structuring, without the need to define discrete populations. This then enables the identification of SNPs under selection across gradients of population structure. EigenGWAS provides adjusted P values that use the genomic inflation factor λGC , 202,466,438 (second), and 184,660,891 (third) raw reads with GC contents of 52%, 51% and 50%, respectively. Following filtering, we produced six datasets containing between 1826 and 3330 polymorphic RAD loci per dataset, at a median read depth of between 176 and 198 reads per sample per locus . In line with expectations based on population sizes, tawny pipits had the highest levels of diversity , followed by Berthelot's pipit populations in the Canary Islands , Madeira , and Selvagens . There was moderate divergence between Deserta Grande and the rest of the Madeiran archipelago (see below), however, exclusion of Deserta Grande had little impact on measures of diversity (Table S2).We used two measures of nucleotide diversity, πThis trend was reflected throughout Berthelot's pipit archipelagos for linkage disequilibrium LD; Fig. B. BaseliK = 2) and then into the three archipelagos (K = 3). Further population structuring beyond the archipelago level was seen with Deserta Grande diverging from the other islands in the Madeiran archipelago (K = 4), and an east–west gradient in admixture across the Canary Islands , K = 4 (0.431), and K = 5 (0.434). ADMIXTURE plots for K = 7 to K = 13 are shown in Fig. S3. We found a moderate signal of isolation by distance within the Canary Island archipelago , although levels of structure within the archipelago were generally low (FST < 0.03).Population admixture analysis Fig.  first seP < 1.5 × 10−5; however, Bonferroni correction is often overly conservative, inflating the risk of type II errors  and bill length .Both LMMs and BSLMMs were used to find regions of the genome associated with malaria and bill length in the “Canary Islands” dataset, while accounting for population structure. The LMM analysis failed to find any SNPs significantly associated with either malaria or bill length when using a Bonferroni‐corrected significance threshold of P values in the LMM (Table IL‐16 gene. The next strongest association was with a SNP on chromosome 20 (7259s1) situated within an intronic region of RIMS4. Both of these SNPs had “A” and “T” alleles. The “AA” and “TT” homozygotes were associated with the highest and lowest levels of malaria, respectively, while heterozygotes had intermediate malaria prevalence ; however, this pattern was not observed when the populations from the bottlenecked northern archipelagos were included  or across all populations . Outlier FST values were correlated with pairwise geographic distance .In the BSLMM analysis for malaria, a median of 68.4% of phenotypic variation was explained by the genotype (95% CI 11.2–99.8%), of which 22.5% was explained by SNPs with nonzero effects, but the credible intervals on this estimate were very high (95% CI 0.0–91.3%). Five SNPs were found with a PIP > 0.1, after controlling for population structure , and across all Berthelot's pipit populations , and with geographic distance .The BSLMM for bill length found that genotype explained a median of 67.9% of phenotypic variation 95% CI 14.0–99.7%), with 14.3% explained by SNPs with nonzero effects . The strongest association with bill length was for a SNP on chromosome 5 .P values calculated by EigenGWAS that use genomic control to account for population structure.We used EigenGWAS to detect loci putatively under divergent selection between the archipelagos in the “Berthelot's” dataset. The first eigenvector (EV1) separated the Madeiran archipelago from the Canary Islands and Selvagens, and the second (EV2) separated Selvagens from Madeira and the Canary Islands . The first two eigenvectors captured a large proportion of the genetic variation, with eigenvalues of 31.1 and 14.6, respectively. Both EV1 and EV2 had considerable genomic inflation factors , suggesting high levels of population structuring .Using a Bonferroni‐corrected ome Fig. . A largeP = 0.1). A second SNP in the region also had an elevated (but not significant) PIP value in the BSLMM analysis and was much closer to the significant EigenGWAS candidate SNP . The regions identified in the BSLMM for association with malaria did not show signs of divergent selection between the archipelagos.None of the candidate SNPs identified in the BSLMM analyses was identified as outliers in the EigenGWAS analysis. An outlier SNP in the EigenGWAS analysis of EV1 was located close to the region on chromosome 5 that showed an association with bill length Fig. . HoweverUsing RAD sequencing, we examined fine‐scale population structure among Berthelot's pipit populations, finding within‐archipelago genetic structuring between island populations, as well as confirming the previously detected patterns of among‐archipelago variation. After controlling for demographic history, we identified candidate SNPs associated with the ecologically important traits of bill length and malaria resistance. Examining allele frequency variation at candidate SNPs revealed that: (i) population‐level candidate SNP variation was related to population‐level trait variation for bill length both within the founder Canary Islands archipelago and throughout all archipelagos, but only within the Canary Islands for malaria; and, (ii) candidate SNP variation was correlated with genome‐wide variation. Finally, although we found little evidence for divergent selection on bill length or malaria resistance, we found signatures of divergent selection between the archipelagos across the genome, including at genes involved in metabolism and immune function.Comparisons of genome‐wide diversity in Berthelot's and tawny pipits provided support for previous inferences of colonization history from microsatellites and mitochondrial DNA . Furthermore, we might not expect to be able to identify genotype–phenotype associations at SNPs under strongest divergent selection, as these are expected to have low variability within populations  and detecting rare genetic variants associated with phenotypes is very difficult . We also detected an EigenGWAS outlier SNP (8322s2) putatively under divergent selection within the wider IL‐16. This gene encodes a proinflammatory cytokine that has been implicated in susceptibility to various inflammatory disorders . This is in contrast to other studies that have shown almost perfect correlations between FST and EigenGWAS P values  error for K = 1 to K = 13 clusters, calculated by Admixture analysis of Berthelot's pipit populationsFigure S3. Admixture analysis at K = 7 to K = 13 clusters for Berthelot's pipitFigure S4. Pairwise genetic distance in relation to geographical distance across Berthelot's pipit populations in the Canary IslandsFigure S5. The relationship between BSLMM outlier SNP pairwise FST and A) pairwise genome‐wide FST; and B) pairwise geographic distance between all pairs of Berthelot's pipit populationsFigure S6. Genetic associations with tarsus length.Figure S7. Genetic associations with malaria and bill length in the "Berthelot's" datasetFigure S8. Selection across archipelagos.Figure S9. Region of interest for bill length on chromosome 5Figure S10. Individual observed heterozygosity averaged across SNPs taken from EigenGWAS outliers (candidate regions ‐ see main text) versus the rest of the genome (non‐candidate regions)Figure S11. Linkage disequilibrium between all pairs of outlier SNPs from EigenGWAS Eigenvector 1 and malaria BSLMM analysisClick here for additional data file.


Lactuca sativa L., 2 n = 18). Molecular markers were first used to genotype 71 putative parental lines and to plan 89 controlled crosses designed to maximise recombination potentials. The resulting 871 progeny plants were then molecularly screened, and their marker allele profiles were compared with the profiles expected based on the parental lines. The average cross-pollination success rate was 68 ± 33%, so 602 F1 hybrids were completely identified. Unexpected genotypes were detected in 5% of cases, consistent with this species’ spontaneous out-pollination rate. Finally, in a later step of the breeding programme, 47 different F3 progenies, selected by phenotyping for a number of morphological descriptors, were characterised in terms of their observed homozygosity and within-population genetic uniformity and stability. Ten of these populations had a median homozygosity above 90% and a median genetic similarity above 95% and are, therefore, particularly suitable for pre-commercial trials. In conclusion, this study shows the synergistic effects and advantages of conventional and molecular methods of selection applied in different steps of a breeding programme aimed at developing new varieties of lettuce.The development of new varieties of horticultural crops benefits from the integration of conventional and molecular marker-assisted breeding schemes in order to combine phenotyping and genotyping information. In this study, a selected panel of 16 microsatellite markers were used in different steps of a breeding programme of lettuce ( Lactuca sativa L.) is a self-pollinating leafy vegetable species (2 n = 2 x = 18) of the Asteraceae family. It is cultivated on a large scale throughout the world for consumption as a fresh vegetable on its own or in combination with other ready-to-eat vegetable products [Lettuce  has started to be adopted in plant breeding programmes for various purposes, including identification of resistance genes ,14 or QuIn this work, SSR markers were used in three different steps of a conventional breeding scheme aimed at developing new varieties characterised by distinctiveness, uniformity, and stability .We first examined the genetic background of a number of superior pure lines in order to plan experimental matings to produce F1 hybrids and then derived F2 progenies manifesting morphological variability as a result of genetic segregation and recombination . Each ofPlant materials were developed and provided by Blumen Group SpA, Italy and belonged to five different lettuce cultivar types . SeventyFinally, to assess the uniformity of the 47 experimental lines, previously chosen for morpho-phenological traits and pathogen resistances, 940 samples belonging to the 47 F3 populations (labelled 1 to 47) were collected in the spring of 2018. Each experimental line comprised 20 individuals.® 96 Plant Kit (Qiagen), according to the manufacturer’s protocols. After extraction, the integrity of the gDNA was assessed by electrophoresis on 1% (w/v) agarose gel stained with SYBR Safe® 1 × DNA Gel Stain  in Tris-Acetate-EDTA (TAE) running buffer. Both the yield and purity of the extracted gDNA samples were evaluated using a NanoDrop 2000c UV-Vis Spectrophotometer . Following DNA quantification, all DNA samples were diluted to a final concentration of 20 ng/μL. A total of 100 mg of fresh leaves was collected from each of the 1882 lettuce samples  and ground to a fine powder using Tissue Lyser II . Genomic DNA (gDNA) was extracted with the DneasySixteen SSR marker loci were selected from those available in the scientific literature ,21, accoThe 16 primer pairs were first tested individually (singleplex reactions) using three randomly chosen lettuce gDNA to evaluate primer efficiency and to check the correspondence between expected and actual size of the bands; they were then evaluated in multiplex PCRs to assess possible primer interactions.® Multiplex PCR Master Mix (Thermo Scientific), 10% GC Enhancer (Thermo Scientific), 0.25 µM of non-tailed primer, 0.75 µM of tailed primer, 0.50 µM of fluorophore-labelled primer  and 20 ng of genomic DNA. Thermal cycling conditions were as follows for multiplex 1 and 2:94 °C for 5 minutes followed by 6 cycles of 94 °C for 30 seconds, 61 °C for 30 seconds, 72 °C for 45 seconds, with a 1 °C annealing temperature stepdown per cycle (from 61 °C to 56 °C). The annealing temperature for the following 35 cycles was set at 56 °C, with denaturation and extension phases as above and a final extension at 60 °C for 30 minutes. The multiplex 3 thermal cycling conditions were instead: 94 °C for 5 minutes followed by 6 cycles of 94 °C for 30 seconds, 56 °C for 30 seconds, 72 °C for 45 seconds, with a 1 °C annealing temperature stepdown per cycle (from 56 °C to 51 °C). The annealing temperature for the following 35 cycles was set at 51 °C with denaturation and extension phases as above and a final extension at 60 °C for 30 minutes. All amplifications were performed in a GeneAmp® PCR 9700 thermal cycler . PCR products were first checked on gel electrophoresis  then run on capillary electrophoresis with ABI 3730 DNA Analyzer (Applied Biosystem), using LIZ500 as the molecular weight standard. The size of each peak was determined with the Peak Scanner 1.0 software (Applied Biosystems).All amplification reactions (both singleplex and multiplex) were performed in a 10 µL reaction volume containing 1× Platinuma priori knowledge of the origin of the populations under study was available, the admixture model and then the correlated allele frequencies model were used. Ten replicate simulations were conducted for each value of K, with the number of founding groups ranging from 1 to 8, using a burn-in of 200,000 and 1,000,000 iterations. The most likely K Estimates were determined using the method described by Evanno et al. [The 71 potential parents were genotyped at 16 SSR loci and statistical analyses were performed using NTSYS  version 2.2 (Exeter Software) . Rohlf’setitions . The genetitions . Since no et al. . Estimato et al. . The 89 subsequent crosses were planned according to the following criteria: (i) high genetic dissimilarity values among parents within the same lettuce cultivar type and between them, (ii) availability of informative loci able to distinguish between the resulting offspring and individuals resulting from accidental self-pollitated. Only homozygous loci for different alleles were considered informative, whereas heterozygous loci were taken into account only if the origin of the parental alleles could be clearly discerned in the progenies. The resulting offspring (871 samples) were then screened, with the analysis restricted to those SSR loci which had previously proven to be informative for hybrid detection. This made it possible to determine whether individuals belonging to a given F1 population originated from cross-pollination or self-pollination. The successful crosses (S.C.) rate of 89 was calculated as follows: Finally, 940 samples from the 47 F3 populations were genotyped using the previously-described panel of SSR markers. The POPGENE software  was usedK = 2  had observed homozygosity values greater than 90%, while 30 of the 71 (42%) had a medium-high observed homozygosity (Ho) between 81% and 90%. Fourteen of the 71 parental lines (20%) had observed homozygosity ranging from 71% to 80%, and only 8 individuals had values lower than 70% .Using a combination of genotypic and phenotypic data, 89 cross combinations were planned . Before We were able to take advantage of these informative loci to calculate the success rate of each cross. In 30% of manual pollinations (27 out of 89), a success rate of 100% was achieved , whereas in 18% of crosses (16 out of 89), the S.C. ranged from 71% to 90%. A hybridity rate fluctuating between 51% and 70% was reported in 15% of cases (13 out of 89), while 26% of the crosses produced fewer than 50% hybrids each. Finally, in only 7% of crosses (6 out of 89) were all the offspring the result of self-pollination . OverallThe 47 F3 experimental lines were genotyped using the same set of 16 SSR loci as for the previous analyses. The homozygosity estimates of all samples (940) ranged from 67% to 93% a. Ten exThe median genetic similarity observed within each line was always greater than 90% b, and 37After assembling the data, we found 10 breeding populations, belonging to butterhead type , to haveL. sativa  facilitated selection of the best parents to cross in order to maximise the variability of the progeny both within the same cultivar type and among them, (ii) allowed accurate evaluation of the resulting offspring, and (iii) sped up the screening of experimental F3 lines for their stability and uniformity.The first part of our work focused on pre-screening 71 parental lines for crossing with the aim of maximising the gains obtained from each out-pollination within cultivar type and, in some cases, among them. As expected, the similarity matrix and the unweighted pair group method with an arithmetic mean (UPGMA) dendrogram showed varying levels of similarity among the different parental genotypes. Parental germplasm appeared to divide into two different groups, as revealed by the Principal Coordinates Analysis (PCoA) results and particularly by the genetic structure analysis. However, samples did not separate in UPGMA tree and PCoA according to the cultivar type, but we may assume that increasing the number of markers it could be possible to clarify this clustering. Although 53 parental lines were found to be fully distinguishable, with similarity values ranging from 53% to 98% and characterised by a unique genotypic profile, it was impossible to identify unequivocally the remaining 18. This is not surprising if we consider that some of the parental lines were closely related. We may speculate that increasing the number of SSRs would allow us to address these remaining issues. Given the aim of this study, these data were useful to avoid crosses between parents with 100% similarity. To introduce variability according to the phenotype and the lowest similarity values, we carried out 89 crossing combinations. Another aspect that needs to be considered when planning crosses is the stability of the parental line in terms of homozygosity. In our study, the median observed homozygosity of the parental lines was lower than expected (82%), especially in light of the strictly autogamous reproductive system of lettuce . OverallBefore proceeding with hand pollination, in order to distinguish between F1 individuals resulting from cross-pollination and those resulting from self-pollination, we first examined the informative loci among the parental lines used in the crosses. Only homozygous loci for different alleles in parental lines were considered informative. Our analysis showed at least 2 informative loci in 84% of the programmed crosses. It is worth pointing out that restricting the analysis to the informative loci brought us considerable savings in terms of time and costs.Overall, the molecular determination of hybridity was successful: F1 individuals represented at least 51% of the offspring in 67% of the manual crosses, and 100% of the offspring in 30% of the crosses (100% success rate), in agreement with the estimates originally reported by the developer of the pollination technique . UnexpecFinally, at an advanced step of the breeding programme, we genetically assessed 47 different experimental F3 populations (940 samples), previously selected for their morpho-phenotypic traits and resistances of interest . InteresFor the remaining experimental populations (white box blot, The results of this study demonstrate the advantages of mutual integration of traditional and biotechnological methods and show the added value that molecular markers can give to breeding programmes. We used microsatellite markers in three different steps of a conventional lettuce breeding program see  and demo


AbstractPontodriluslitoralis  occurs in a very wide range of subtropical and tropical coastal areas. Morphometrics on size variation  and genetic analysis using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequence were conducted on 14 populations of P.litoralis from Southeast Asia and Japan. Statistical inference on morphometric data revealed significantly different size variations in the body length and diameter among these 14 populations of P.litoralis. However, discordance between the morphometric and mitochondrial COI gene-based phylogenetic analyses was evident, where the size variations in P.litoralis showed a different pattern from the COI genetic differences. The update on the current distribution of P.litoralis is reported and revealed different aspects of the littoral habitat characteristics between Southeast Asia and Japan.The luminous earthworm  Annelida, Clitellata), except for a few semi-aquatic taxa, such as earthworms in the family Almidae and Eiseniellatetraedra  in freshwater habitats, and Pontodriluslitoralis , P.primoris Blakemore, 2000, and P.longissimus Seesamut and Panha, 2018 in marine littoral habitats  and Fukuoka (Kyushu island). Subsequently, the distribution of this species in Japan was been further studied  that were otherwise similar in morphological characters, and this was supported by DNA sequence analysis of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene  gene .P.litoralis , which led us to test this hypothesis based on their size variation coupled with a genetic analysis. The objective of this study, therefore, was to investigate the pattern between the size variations  and genetic  variations in the littoral earthworm P.litoralis from 14 populations across Southeast Asia  and Japan. Moreover, we report new data on the distribution and habitat types used by this species.Many distribution records have reported size variation within itoralis . HoweverP.litoralis were collected throughout the coastal areas of both the east and west sides of the Thai-Malay Peninsula  and Japan . Moreover, samples from Myanmar, Vietnam, Malaysia, Singapore, and Indonesia were collected  and principal component analysis (PCA) were performed to assess the significant variation among the three morphometric characters. The mean length and diameter were calculated separately both within each locality and a country scale, and those mean differences were analyzed by one-way ANOVA. The clustering analysis (CA) of the sampling sites was performed to construct a dendrogram depicting the morphological relationship based on the three morphometric measurements, CA were tested based on complete linkage and Euclidean distances. All statistical analyses were performed using the MINITab software v. 18.1 .Fourteen populations of ns Table . At leasCOI gene fragment was amplified using the Tks GflexTM DNA Polymerase (Takara) and the universal primers , 1 μL each of 10 µM LCO1490 (forward) and HCO2198 (reverse) universal primer, 19.5 μL of sterilized distilled water and 2.5 μL of crude lysate (ca 500 ng/μL DNA) with Lysis buffer. Thermal cycling was performed at 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 48 °C for 1 min and 72 °C for 2 min and then a final 72 °C for 5 min. The concentration and quality of the amplicons were determined visually after coresolution through a 1% (w/v) agarose gel against a DNA standard marker in 1x TAE buffer and detected under UV transillumination.Three specimens were chosen from each of the same 14 populations as in the morphometric analysis resulting in the total of 42 samples used for the molecular analysis , obtained using the BLASTn similarity search tool (http://www.ncbi.nlm.nih.gov). Corrected genetic distances were calculated using the Kimura two-parameter (K2P) model (ML) analysis in RAxML v. 8.1.20 (Pontodriluslongissimus was used as the outgroup (Accession number MK642683 and MK642684).For sequencing, the PCR products were directly sent to Macrogen Inc. (Japan) without purification. All . 7.0.18  and manuP) model  as imple. 8.1.20 , and 1,0. 8.1.20 . PontodrP.litoralis earthworms from all 14 sampling sites revealed a length range between 31.1–125.4 mm , followed by that between 50.0–59.6 mm (n = 44) and 70.5–79.6 mm (n = 38). Two specimens from JP2 were recorded as having a length >120 mm. The relationship between the total number of segments and the body length of P.litoralis . The longest length of P.litoralis was 125.4 mm, found in Japan (JP2), while the shortest was 31.1 mm, found in Vietnam (VT2). The mean ± S.D. and median length of P.litoralis were 62.6 ± 14.2 mm and 63.1 mm, respectively. The highest number of segments in P.litoralis was 119, found in Thailand (TA1) and Myanmar (MY1), while the lowest was 81, found in Indonesia (IN1). The mean ± S.D. and median of the total number of segments of mature P.litoralis were 101.7 ± 8.9 and 102, respectively. In addition, the largest body diameter of P.litoralis was 4.08 mm, found in Japan (JP2), while the smallest was 1.21 mm, found in Vietnam (VT2). The mean ± S.D. and median P.litoralis diameter were 2.12 ± 0.52 mm and 2.02 mm, respectively.The measurement of 212 individuals of ANOVA analysis revealed a significant difference (p < 0.05) in the mean length and diameter of P.litoralis among the 14 locations  and diameter (3.39 ± 0.6 mm), while the VT2 population from Vietnam showed the lowest mean body length (39.5 ± 5.4 mm) and diameter (1.55 ± 0.18 mm). Moreover, the highest mean body length (73.6 ± 16.4 mm) and diameter (2.86 ± 0.47 mm) were found in all the Japanese populations , while the lowest mean body length (52.4 ± 14.2 mm) and diameter (1.66 ± 0.25 mm) were found in all the Vietnamese populations .The e p < 0.0 in the mJP2 population from Japan  explained more than 60.6% of the variation in the dataset and had a variance  of 1.8174. The second and third PCs (PC2 and PC3) had a variance  of 0.8882 and 0.2944, respectively, which accounted for 29.6% and 9.8% of the data variability, respectively. The PC1 revealed that all the loadings were positive, whereas the PC2 showed both positive (number of segment) and negative loadings (body length and diameter). The loadings from the PC2 were less similar among themselves compared to the PC1. The PC1 had a large positive association with the body length and diameter as determined by loadings >0.5, so this PC1 primarily measured the size of the earthworms  from 42 individuals, three specimens from each of the 14 populations used in the morphometric analysis, were analyzed. The analysis yielded 158 variable (polymorphic) sites and 139 parsimony informative sites. No insertions, deletions, or stop codons were observed in any of the sequences. The K2P genetic distances among the 14 geographical locations within P.litoralis ranged from 0.3–12.8 %  and the longest population (JP2) samples clearly showed a significant difference in their body length , and body diameter  (p < 0.05), while the cluster analysis confirmed that the two clusters were separated, one contained 13 populations (included VT2) and the other contained only JP2. However, the genetic distance analysis showed a low genetic distance between VT2 and JP2 population  and JP2 population  , whereas the southernmost site was in Bantan, Indonesia . In this study, we reported the first record of P.litoralis in Singapore despite only juveniles being collected from the beach in West Coast Park . Among the localities in the sub-tropical areas, P.litoralis specimens were collected from various beaches in Japan, and the northernmost site was Matsushima Kaihin Koen in the Miyagi prefecture, where the synonym of P.litoralis (P.matsushimensis) was originally described from. In total, 29 localities were recorded in the distribution range of P.litoralis within Japan, including in Honshu, Kyushu, and the Ryukyu islands.In Southeast Asia,  Figures  and 2. TP.litoralis was found to occupy several types of habitats  and the shortest samples (VT2) formed separate groups with statistical differences in their size, whereas a low genetic distance between the two samples from each respective population was detected, revealing that the size variation of P.litoralis was independent of the genetic (COI gene) differences.According to the results of the one-way Metaphirepeguana  from Penang and neighboring states of Malaysia revealed significant differences in their morphometric variations that were not matched by their genetic difference but rather were affected by the type of habitat  earthworms from Germany and Canada and their mitochondrial cytochrome c oxidase II (COII) sequences, showing that small and large individuals were genetically distinct.Differences in the body length, diameter, and number of segments have also reported in other earthworms. The terrestrial earthworm  habitat . HoweverP.litoralis, in the coastal areas of Thailand, Japan , and some parts of Southeast Asia  based on field collections. This survey supported the assumption that P.litoralis is widely distributed in subtropical and tropical coastal ecosystems  differences. Thus, we propose that the food resource is the key factor underlying size variation in P.litoralis. Future analyses on the type of habitats, sand texture, and components of the food resources are necessary. Moreover, studies on salinity tolerance are needed to confirm the habitat preference of this littoral earthworm species.In conclusion, although morphometric examinations of size variation could make reliable distinctions among different populations of