The matrilineal long-finned pilot whale presents an antitropical distribution and is divided into two subspecies, one in the temperate seas of the Southern Hemisphere and the other restricted to the North Atlantic and Mediterranean. Until now, population genetic and phylogeographic studies have included localities of most of its Northern Hemisphere distribution, while only the southwestern Pacific has been sampled in the Southern Hemisphere. We add new genetic data from the southeastern Pacific to the published sequences. Low mitochondrial and nuclear diversity was encountered in this new area, as previously reported for other localities. Four haplotypes were found with only one new for the species. Fifteen haplotypes were detected in the global dataset, underlining the species’ low diversity. As previously reported, the subspecies shared two haplotypes and presented a strong phylogeographic structure. The extant distribution of this species has been related to dispersal events during the Last Glacial Maximum. Using the genetic data and Approximate Bayesian Calculations, this study supports this historical biogeographic scenario. From a taxonomic perspective, even if genetic analyses do not support the subspecies category, this study endorses the incipient divergence process between hemispheres, thus maintaining their status and addressing them as Demographically Independent Populations is recommended. Physeter macrocephalus1 and the orca Orcinus orca2. Other species, especially small-sized odontocetes, generally present coastal and more restricted distributions, like the extreme case of the vaquita, Phocoena sinus3.Cetaceans have a diverse -and many times contrasting- array of geographic distributions. Some of the widest ranging species include the cosmopolitan sperm whale 4. Examples include the mysticete genus Eubalaena, with E. borealis inhabiting the North Pacific, E. japonica the North Atlantic and their austral equivalent in the Southern Hemisphere, E. australis5. The phocoenid species pair Phocoena phocoena and P. spinipinnis is another example, found in the Northern Hemisphere and the coasts of South America, respectively6. Among delphinids, the two Lissodelphis species also present this antitropical distribution pattern; L. peronii is present around the Southern Hemisphere, while L. borealis is found in the North Pacific7. A similar antitropical distribution is presented by the two subspecies of long-finned pilot whales Globicephala melas, where G. m. edwardii inhabits the temperate to subpolar waters of the Southern Hemisphere, while G. m. melas is restricted to the North Atlantic8. Extinct populations of long-finned pilot whales have been reported in the North Pacific, from Japan9 and Alaska10, dating back 8 000–12 000 years and 2 500–3 500 years, respectively10.Some cetaceans have a particular distribution pattern known as disjunct or antitropical, in which the taxon is present at high latitudes in both hemispheres while being absent from lower latitudes11 that can increase to hundreds through aggregations13. These groups are structurally based on strong matrilineal associations8 of closely related females and their descendants14. Pilot whales are among the most common cetaceans involved in mass strandings13. Southern Hemisphere long-finned pilot whales were originally described as a distinct species, G. leucosagmaphora15, but eventually ranked as one of the two subspecies of G. melas16, based on observations of the coloration pattern and morphology17. Sergeant (1962) stated that he found only minor differences in coloration and none in external morphology between specimens from the two hemispheres, albeit being in agreement with the subspecies classification proposed by Davies (1960)16. The author also expressed the need of including samples from additional localities, in order to assess the variation of the colour pattern in the subspecies, as also recommended by other authors18. A more recent study found differences in skull morphometry of North and South Atlantic specimens19, but no studies were found that account for geographic variation within each area. Little genetic evidence exists to support their classification status using mitochondrial DNA. Oremus et al., (2009) performed the first inter-hemisphere comparison of the taxa, between the southwestern Pacific and the North Atlantic20. The authors also stated that the two subspecies do not qualify as Evolutionary Significant Units (ESU) according to the reciprocal monophyly criterion of Moritz (1994)21, only reporting restrictions to gene flow among the areas of distribution mainly based on differences in the frequency of shared haplotypes. In the study of Oremus et al., (2009), preliminary genetic data support a biogeographic scenario previously proposed by Davies (1960)22. A colonization event from south to north would have taken place through a founder effect, followed by demographic population growth. Additional genetic research on pilot whales has been conducted mostly at the population level in the North Atlantic and Mediterranean Sea25, however, an integration of this available information is missing, together with the sampling of other areas such as the southeastern Pacific.Pilot whales are a highly social matrilineal odontocete, thought to be among the most gregarious cetaceans. The species is known to form groups with a mean size around tens of individualsIn this study, we include new genetic data and results from samples collected in two mass standings in southern Chile, in order to improve the global phylogeographic overview of long-finned pilot whales. We also evaluate the historical biogeographic processes that originated the extant antitropical distribution of this species and discuss its taxonomic status.In total, 90 mtDNA sequences were obtained, defining four haplotypes. One haplotype was previously unreported (hereon referred to as haplotype R2). Haplotype and nucleotide diversities were low: Hd = 0.62 and π = 0.23% Table .Table 1SHo = 0.655; He = 0.700 and nA = 7.5 (Table Microsatellites were successfully genotyped in n = 44 samples (32 from Isla Clemente and 12 from Isla Navarino), across fourteen polymorphic loci. DlrFCB6, and GT51 presented an excess of homozygotes, but were not used in the comparisons among hemispheres. Regional diversity values of usable loci were 26, a total of 15 haplotypes were obtained after the elimination of the site that was generating phylogeographic noise in the total 1012 sequences. This led to the combination of four previously described haplotypes in the following pairs: S with R, P with U, Q with Y, and E with G , originally of a consensus fragment size of 703 bp, were also merged into haplotype S + R.After adding previously published sequences from other oceanic basinsG. m. edwardii (SP) and G. m. melas (NA and MED), and (2) one haplotype was shared by all SP localities but was absent from NA and MED  two haplotypes were shared between  two haplG. melas subspecies  of haplotypes encountered in the NA and half (4) of those exclusive to the SP.Haplotypes P + U and S + R were the only ones detected within the distribution range of both es Table , Fig. 2aG. m. edwardii than in the range of G. m. melas, where it was detected in lower numbers in three localities . One of the haplotypes that originates from P + U, haplotype Q + Y, is exclusive to the South Pacific and is present in all three localities. Ten of the total fifteen haplotypes (67%) were exclusively present in one of the following five localities: TAS (3), NZ (3), CL (1), IB (2) and NWA (1). In the South Pacific, 70% of haplotypes were private to one of the three localities; it was the only basin with at least one private haplotype in each. Three private haplotypes were encountered in the North Atlantic: one in the NWA and two in the IB.In the haplotype network by locality, haplotype S + R is present in all ten studied areas and originates much of the local diversity within them Table , Fig. 2bi.e. South Pacific with North Atlantic and Mediterranean . The Snn test for genetic differentiation was statistically significant among the two disjoint distribution areas of the two subspecies . Subsequently, samples were organized in this same way for the AMOVA. Similar structure results were obtained grouping the samples by the 10 worldwide localities. Average φST values were highest within the SP (avg. φST = 0.283), lowest in the NA (avg. φST = 0.06), and intermediate among the two Mediterranean localities (avg. φST = 0.174)  among the subspecies G. m. edwardii and G. m. melas resulted in dA = 0.00158 (SD = 0.00019).In the Correspondence Analysis, the first axis separated the localities according to their respective hemisphere of origin. The three South Pacific localities  were clustered separately from those of the Northern Hemisphere. The second axis divided the Strait of Gibraltar and the Mediterranean Sea from the remaining localities in the Northern Hemisphere Fig. . The calGlobicephala melas was explored with DIYABC, using two historical biogeographic models associated to the Last Glacial Maximum (LGM). The first model, scenario 1, represented a colonization event from the Southern Hemisphere to the Northern Hemisphere, followed by isolation and population growth in the Northern Hemisphere. The second model, scenario 2, tested a possible event of vicariance among both distributions.The origin of the current disjunct distribution of −8.Based on the genetic data provided, both scenarios were realistic Fig. , even th27 and the North Atlantic28. The diversity by locus reported28 is shown in Table vs. 9), 415/416 (9 vs. 5), 464/465 (9 vs. 6), EV37 (10 vs. 6) and EV94 (7 vs. 7). Two of these loci were also present in our database of 44 analysed individuals from the southeastern Pacific (464/465 and EV37), with 9 and 15 alleles in total. The upper-tail exact test among the five shared loci of G. m. edwardii27 and G. m. melas28 showed that South Pacific samples had significantly more alleles per locus (p = 0.028). By contrast, the upper-tail exact test performed with the 10 loci shared between G. m. edwardii from the southwestern27 and southeastern Pacific (this study) did not detect a statistically significant difference (p = 0.166).Previous data on microsatellite diversity was available for the southwestern PacificPontoporia blainvillei29, bottlenose dolphin Tursiops truncatus30, Cuvier’s beaked whale Ziphius cavirostris31 and the short-beaked common dolphin Delphinus delphis32. The long-finned pilot whale is a species for which almost all genetic data has been obtained from sampling mass strandings. A large part of its distribution range has been already sampled in the northwestern and northeastern Atlantic26, but within their broader distribution in the Southern Hemisphere, only the southwestern Pacific has been covered20. This study presents new data from two recent mass standings in southern Chile, in order to produce the first genetic information for Globicephala melas in the southeastern Pacific and to integrate this new information into the global phylogeography of the species.Sampling cetacean species for population genetic studies has proven to be logistically challenging. A large number of individuals need to be sampled for this kind of studies, but they are generally widespread geographically and low in density. Mass strandings of cetaceans represent unique opportunities to collect large numbers of samples, as in the case of the franciscana 33, while the Isla Navarino individuals were sampled five days after stranding34, precluding tissue the deterioration encountered in the former event. This highlights the importance of swift responses to stranding events, in order to secure the best tissue quality possible.Contrasting tissue quality was found between both strandings, which we attribute to the time of sampling of each event. Our sampling of the Isla Clemente stranding took place an estimated two to three months after occurringG. melas was already detected20, but not taken into account for the genetic analyses. Homoplasic sites have been described to hinder the resolution of mtDNA gene trees35 and have also been pointed out as potential confounders of evolutionary analysis in the mtDNA control region of humpback whales36 and human mtDNA coding regions37, among others.Prior to conducting mtDNA analyses, we first decided to remove one polymorphic site that exhibited a strong homoplasy signal. This problem in mtDNA sequences of G. melas. Such low mtDNA diversity has been reported for other matrilineal odontocetes, including orcas (π = 0.52%)38, false killer whales (π = 0.01–0.30%)39 and sperm whales (π = 0.131–0.407%)40. In contrast, cetaceans with more labile social cohesion such as mysticetes and non-matrilineal odontocetes, generally present much higher nucleotide diversity, a feature that appears to be common among these species41. Cultural hitchhiking has been regarded as a driver of their low mitochondrial diversity42. An analogous effect of behaviour on mitochondrial diversity was described in a resident coastal bottlenose dolphin population in central Chile43. This particular population operates akin to pilot whales as it is also composed of adult females and their descendants, both male and female. That study found that the genetic diversity of this matrilineal population  was lower than that of the non-matrilineal transient-pelagic group adjacent to them , suggesting the importance of social structure in shaping the pattern of genetic diversity in cetaceans (at least in odontocetes).Despite the addition of 90 new sequences from the southeastern Pacific to the set of available sequences in the literature -over 9000 km from New Zealand, the nearest sampled locality- the global haplotype network was expanded by just one private low-frequency haplotype, further confirming the worldwide low mitochondrial diversity of 26, our results show that southeastern Pacific long-finned pilot whales (n = 90) have low genetic diversity, in particular for haplotype richness (h = 4) and nucleotide diversity  samples were similar to the values obtained for Tasmania. Nevertheless, genetic diversity in these two localities was very different from that of New Zealand. Despite accounting for 54% of the samples in this basin, the genetic and nucleotide diversity of pilot whales from New Zealand were the lowest  (Table φST = 0.086). The absence of obvious geographic or oceanographic barriers between New Zealand and the other localities in the South Pacific does not allow a simple interpretation of the pattern of genetic structure found here. This genetic differentiation could have been attained through ecological specialization, as previously pointed out20. Similarly, sea surface temperature and its influence on prey distribution has been regarded as a possible ecological factor underlying genetic differentiation in extant G. melas in North Atlantic waters28 and similar trends have been observed on the Scotian shelf44.As previously reported for other areasst Table , particuCephalorhynchus eutropia between two differing coastal habitats along the uninterrupted Chilean coastline, attributed to habitat adaptation and specific hunting strategies45. Also, the Eastern Pacific Barrier has been proposed as a driver behind the genetic differentiation of the short-finned pilot whale Globicephala macrorhynchus into two subspecies46.Differentiation over relatively short distances without any conspicuous geographical barriers has also been detected in other odontocetes. For example, genetic differentiation was detected in the Chilean dolphin 47. Such changes in environmental conditions, probably associated to a shift in the distribution of marine biota, may have provoked a typical population contraction-expansion in the long-finned pilot whale population in this area, as described for cold-temperate and polar marine species48, including cetaceans50.The haplotype network of the specimens from New Zealand presents a typical star-like shape Fig. , suggest25. In this case, geographic and oceanographic discontinuities between the Mediterranean Sea and the Atlantic Ocean provide a robust explanation for the observed genetic structure. The separation of Mediterranean populations from North Atlantic ones has been previously reported in various marine species, such as shallow water crustaceans51, sea stars52, white sharks53 and other odontocetes, like in sperm whales54, striped dolphins Stenella coeruleoalba55 as well as Cuvier’s beaked whales Ziphius cavirostris31 and Risso’s dolphins Grampus griseus56.A similar case of strong genetic differentiation among populations of long-finned pilot whales is observed between the North Atlantic and Mediterranean populations, where the latter exhibits high phylogeographic and genetic differences with the North Atlantic localitiesBalaenoptera physalus, with one fixed difference between South and North Atlantic samples, thus presenting no shared haplotypes57, the harbour porpoise Phocoena phocoena, with high divergence among ocean basins58 and the more closely related false killer whale Pseudorca crassidens39. In the latter species, although the study had a low sample size in the North Atlantic, no shared haplotypes were found and at least 10 substitutions separated the Atlantic populations from those in the Indo-Pacific. In the case of G. melas, despite its antitropical distribution and the large geographic discontinuity between northern and southern distribution areas, the subspecies shared their two most abundant haplotypes. This genetic pattern may reflect (1) contemporary gene flow between hemispheres, or alternatively (2) an ancestral polymorphism resulting from an incipient divergence process. The strong phylogeographic structure detected among the subspecies supports the second hypothesis.Widespread cetacean taxa occurring in both the Northern and Southern Hemispheres generally exhibit strong phylogeographic structure and genetic divergence between regions. Such genetic differentiation has been generally exemplified by fixed substitutions in mtDNA control region sequences. This is the case of the fin whale 22, with a split in distribution occurred after the Last Glacial Maximum, 10 000–15 000 years ago. This hypothesis was further expanded20, mentioning that a trans-equatorial dispersal event rather than vicariance might have taken place, on account of the lower diversity in the Northern Hemisphere subspecies. Thus, the hypothesis is supported by (1) the contrasting haplotype networks  following the criterion of Moritz (1994)21. It is likely that not enough time has passed to sort lineages, or some level of gene flow still occurs59, as they still share haplotypes, but in differing frequencies20. Additionally, net nucleotide divergence dA and Percent Diagnosability (PD) had been previously calculated for the long-finned pilot whale subspecies60, obtaining values of dA = 0.00128 and PD = 0.84286. Our calculation of net nucleotide divergence, which included new sequences from the southeastern Pacific and integrated other published sequences from the Northern Hemisphere, resulted in a slightly higher value of dA = 0.00158, which still is below the proposed subspecies interval61. Therefore, addressing them as Demographically Independent Populations (DIP), a transitional state between a panmictic population and separate ESUs59 might be more accurate.The South Pacific network holds much of the species’ genetic diversity and is therefore very similar to the global network in overall shape Fig. . In contrks Fig. , 2) the the22, wrks Fig.  the clearks Fig. . The valnce Fig. . PosteriFinally, from a conservation perspective, even if genetic analyses do not support the subspecies category, we recommend maintaining their current taxonomic status, since these DIP might be undergoing a recent divergence process which has yet to mature into fully sorted lineages.62. As previously stated, such molecular studies should include nuclear markers together with mitochondrial DNA, even more when delimitating species and subspecies18. However, to date, the mtDNA control region is the only molecular marker for which sequences are available with sufficient sample size from different ocean basins to perform a worldwide phylogeographic study in G. melas26. To incorporate new genetic markers to a global phylogeographic study would require tremendous sampling effort and expenses, and may also strongly depend on the occurrence of mass strandings in all these regions of the world.The results here presented should be considered as preliminary evidence, as the use of a single mtDNA marker for phylogeographic and demographic inferences has been deemed problematic beforeG. melas and to lay the groundwork for future research on these topics. Modern genetic tools, such as complete mitogenome and SNPs, have been already used to revise the taxonomic status of short-finned pilot whales46, which could be replicated in long-finned pilot whales.Finally, we believe that collaborative studies surveying uncharted areas, especially within the greater distribution range of long-finned pilot whales in the Southern Hemisphere, are fundamental to obtain complete data on the worldwide phylogeography and taxonomy of Globicephala melas were sampled from the mass stranding event that occurred in Isla Clemente 45°35′57.50″S; 74°34′30.32″ W. All samples were preserved in 90–95% ethanol.Tissue samples were collected from twelve individuals in a stranding event that occurred in Isla Navarino in 2006 , 2 µL 50 mM MgCl2 (Invitrogen), 2 µL 10pM dNTPs (Invitrogen), 1 µL 10pM of each primer , 0.5 µL Taq polymerase (Invitrogen) and 70–150 ng of DNA. A Thermo Hybaid PxE 0.5 thermal cycler was used for all amplifications, with the following profile: Preliminary denaturation of 2 minutes at 94 °C; followed by 30 cycles of: denaturation for 30 s at 94 °C, annealing for 40 s at 56 °C, polymerase extension for 40 s at 72 °C; and a final polymerase extension for 10 minutes at 72 °C and an infinite hold temperature of 4 °C. Each PCR run included positive and negative controls. Fragments were run in a 1% agarose gel, each well containing 3 µL of PCR product mixed with an equal volume of loading dye with 0.3% Gel Red and visualized in a gel documentation system (Maestrogen SMU-01).The mtDNA control region was amplified using the primers M13 Dlp1.5 5′-TGTAAAACGACAGCCAGTTCACCCAAAGCTG RARTTCTA-3′ (forward) and 8 G 5′-GGAGTACTATGTCCTGTAACCA-3′ (reverse)64 and polymorphic sites were visually checked. Prior to molecular analyses, the species for each sample was corroborated with two platforms of comparative analysis of sequences: BLAST  and DNA Surveillance65.PCR product purification and sequencing in both directions were done at Macrogen Inc., Seoul, South Korea with a 3730XL DNA Analyzer (Applied Biosystems). All sequences obtained were aligned manually in ProSeq 3.5et al., (2009)20 ; (2) Siemann (1994)26 ; (3) Monteiro (2013)24 ; (4) Verborgh (2015)25  and (5) Miralles et al., (2016)23   Oremus  Oremus eFST structure was detected among them in that study25  including the localities TAS, NZ and CL and G. m. melas from the North Atlantic (NA), including NWA, FI, UK, IB, NEA and the Mediterranean (GIB and MED).Samples were grouped in two ways: 1) first in ten groups, according to their respective sampling locality: Tasmania (TAS), New Zealand (NZ), Chile (CL), northwestern Atlantic (NWA), Faroe Islands (FI), United Kingdom (UK), Iberian Peninsula (IB), northeastern Atlantic (NEA), Strait of Gibraltar (GIB) and the Mediterranean Sea (MED). NEA included samples ranging from UK to IB, and was combined with the locality Euskadi from the original study, since no significant  first in66. With an exploratory examination of the global haplotype network, it was noted that site 156 of the alignment generated three loops in the network. This hypervariable site was considered to be interfering with the phylogeographic signal of the data and was consequently removed, in order to eliminate a potential homoplasy signal. Additionally, a repeated TA motif starting at position 90 was identified as a possible microsatellite. We modified the sequences by deleting one of the nucleotide positions within each repeat, so each motif was considered as a single mutational step, instead of each nucleotide separately. Thus the final fragment length was of 345 bp.After alignment and trimming, a haplotype network was constructed in Network 5.0π) and pairwise differences between sequences (Π) were estimated in Arlequin v3.5.267. Analyses of genetic structure (FST), phylogeographic structure (φST) and analysis of molecular variance (AMOVA) were conducted in Arlequin v3.5.2 with 1000 permutations and a significance level of 0.05. Phylogeographic structure was also explored with Snn tests of genetic differentiation68, performed in DnaSP 5.10.0169. For the AMOVA, the ten localities were grouped according to the distribution of each subspecies. A Correspondence Analysis (CA) was performed on all localities with the software Past 3.1970, using the matrix of Table The genetic diversity indices number of haplotypes (h), number of polymorphic sites (S), haplotype diversity (Hd), nucleotide diversity  net nucleotide divergence 71 was calculated among the two putative subspecies in DnaSP. According to these guidelines, the net nucleotide divergence among two subspecies should be within the range of dA = 0.004–0.04.Additionally, as suggested by the guidelines for the delimitation of cetacean subspecies using genetic data of Taylor 72. This software evaluates population histories using Approximate Bayesian Computation (ABC) with genetic data, by testing scenarios built through the combination of population divergence, admixture and population size changes. Two models were evaluated. The first model was defined based on a scenario previously proposed22, together with evidence from the genetic results provided in the present study. The model considers a trans-equatorial, Last Glacial Maximum (LGM)-associated dispersal event from the Southern Hemisphere to the Northern Hemisphere, followed by a split in distribution and instantaneous population growth  of ancient population = 1 000–100 000; Effective population size of founder effect (Nf) = 10–1 000; time of dispersal event t2 = 10 000–35 000; time of instantaneous population growth t1 = 2 000–15 000 (with t2 > t1) and mutation rate u = 1.5 e−7–1.5 e−8. In accordance with the recommendations of the authors of the software, we performed 6 000 000 simulations.The population history of the species was tested on the program DIYABC v2.1.0wth Fig. . The alt73, DlrFCB1, DlrFCB674, Ev1, Ev14, EV3775, GATA5376, GT6, GT5177, GT23, GT211, GT509, GT57578, MK5, MK979 and PPHO13158. PCR reactions were done with a Multiplex PCR kit (Qiagen), each reaction containing: 12.5 µL of water, 5 µL of MM2x Buffer and 1 µL of each primer at 10 pM. Between two and four loci with different fluorescent dyes were combined in each reaction. Allele scoring was done with the software GeneMarker v2.6.0 (www.softgenetics.com) with a 500 liz standard. The dataset was tested for scoring errors, allele dropout and null alleles in Micro-Checker v.2.2.380. Observed heterozygosity (Ho), expected heterozygosity (He), average number of alleles per locus (nA) and genetic structure (FST) were estimated in Genetix v4.05.281. Published microsatellite data on this species was available from Tasmania and New Zealand27, NWA, UK28, Faroe Islands28, Iberian Peninsula23, northeastern Atlantic, Strait of Gibraltar and the Mediterranean25. However, because of the differences in the loci used in these studies, only a partial comparison of genetic diversity could be performed between populations of G. m. melas and G. m. edwardii. Only comparisons of allelic richness could be done, which were carried out in Rundom Pro 1.182 with 10 000 randomizations. Comparisons were intra-subspecies among southwestern and southeastern Pacific samples, and inter-subspecies among southwestern Pacific and North Atlantic samples.A total of 19 loci were amplified: 409/470, 464/465We confirm that all methods were carried out in accordance with relevant guidelines and regulations. Samples were taken from stranded, deceased animals with permission from the National Fisheries Service . All experimental protocols were approved by the Postgraduate Evaluation Committee at the Faculty of Science of the Universidad de Chile.Haplotype R2 (GenBank accession number pending. Submission number #2305260).


Eutetranychus orientalis (Klein) is a major pest of citrus in many countries including Saudi Arabia (SA). The morphological variations among the different populations of E. orientalis were reported. In the present study, phenotypic variations based on 40 different morphological characteristics were evaluated in 10 E. orientalis populations collected from different hosts and regions of SA. Further, ITS2-rDNA sequences were used to confirm the identity of these phenotypically varying populations. Phenotypic variations in all populations were found in the shape and length of dorsal setae, striation pattern between setae d1 and e1, and leg chaetotaxy. The rDNA sequence analysis of these morphologically varying populations resulted in 10 different ITS2 Saudi haplotypes. The phenotypic and genetic variations were more related to the host plants rather than their geographic distribution. The E. orientalis population collected from Phoenix dactylifera was phenotypically distinct and genetically divergent. The populations collected from citrus species were also more phenotypically and genetically related to each other than to populations collected from non-citrus host plants. The haplotypes recovered from Ziziphus sp., Morus sp., and Azadirecta indica from different regions were grouped in the same sub-clade. Further, the ITS2 haplotypes of Saudi E. orientalis recovered from Citrus reticulata from Riyadh and Al Ula were 100% identical to the ITS2 haplotypes recovered from Citrus sp. from Israel. It is concluded that phenotypic variations exist among different populations of E. orientalis inhabiting different host plants. This species should be identified carefully by considering phenotypic intraspecific variations.The oriental red spider mite,  Eutetranychus orientalis (Klein), is a major pest of many economical shrubs and fruit trees including Citrus sp. and Prunus sp. They are widely distributed in many countries of Oriental, Afrotropical, and Palaearctic regions, including Saudi Arabia (SA) and have been found inhabiting more than 200 host plants [E. orientalis populations [E. orientalis resulted in misidentifications. Recently, the morphological characteristics of the Eutetranychus species were tabulated with descriptions and illustrations; in addition, seven Eutetranychus species as junior synonyms of E. orientalis were suggested based on phenotypic variations [E. orientalis populations from Israel [E. orientalis populations could be caused by either vast geographic distribution or polyphagous feeding behavior of the species [The oriental red spider mite, t plants –4. Diffeulations –9; thesem Israel . It was  species –14. Mole species , 15 and  species , 17. DNA species –21.E. orientalis collected from different localities and host plants in SA and (2) confirm the intraspecific nature of these phenotypic variations through ITS2-rDNA.The aims of the present study were to (1) assess the phenotypic variations in different populations of E. orientalis were collected from diverse host plants from different localities in six regions  of SA during 2009–2018  and ITS2-reverse (5'-GTARCCTCACCTRMTCTGAGATC-3') were used to amplify the ITS2 region of rDNA .Total genomic DNA was extracted from a single female, representing a population collected from 10 different locations (including seven different host plants), using a DNeasy mini kit . The data of the collection localities and hosts are provided in ® Green Master Mix , 0.75 μL of each 10 μM primer, 2 μL (approximately 20 ng) DNA template and 11.5 μL Nuclease free water (Promega). The PCR cycling conditions were as follows: denaturation step for 5 min at 94°C, followed by 35 cycles of 60 s at 94°C, annealing at 52°C for 90 s and 72°C for 60 s, and the final extension step at 72°C for 10 min. The PCR products were assayed in 1.2% agarose gel stained with Acridine Orange dye in 1× TAE buffer. These gels were later visualized under UV light using gel documentation system. Further, the PCR products were purified using a MoleQule-On PCR product purification kit .The PCR reaction was performed in a 30 μL reaction volume with 15 μL GoTaqE. orientalis obtained in the present study and those retrieved from GenBank were aligned using a Clustal W multiple alignment tool. The final sequences were submitted to the NCBI database; the accession numbers of each population are provided in The purified PCR products were directly sequenced using the same primers used in the PCR amplification; the sequences were then run on ABI 3500 . The obtained sequences were cleaned and analyzed using the BioEdit software . Thereafwww.minitab.com). To assess genetic variations among 10 Saudi E. orientalis ITS2 sequences, neighbor-joining (NJ) trees were constructed using the MEGA X tool. The ITS2 haplotypes were determined manually by aligning the DNA sequences using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Single nucleotide polymorphisms (SNPs) were detected and each ITS2 haplotype had its unique SNPs. Similarly, another NJ tree was constructed to assess the genetic variability among the Saudi haplotypes and 12 ITS2 E. orientalis sequences retrieved from the NCBI database. The evolutionary distances were computed using the Tamura-Nei method [The morphological variations between the collected populations were assessed based on different characteristics including length and shape of setae, length and chaetotaxy of legs, and dorsal striation patterns and shape of stylophore. Based on these phenotypic character, an UPGMA dendrogram was constructed using a Minitab 18 data analysis software (2010)  rather than their geographic distribution. In both the UPGMA and NJ phylogenetic trees clade 1 contained one E. orientalis phenotype/haplotype, found on Phoenix dactylifera (Arecaceae) (a monocot) from Riyadh, with lowest phenotypic similarity percentage (6.26) and genetically highly distant from other nine haplotypes  and NJ  phylogenetic trees (Azadirachta indica (Meliaceae) in Qaseem) and P9 (from Citrus sp. (Rutaceae) grown within A. indica and Morus sp. (Moraceae) trees in Domat ul Jandal) showed highest affinity forming a cluster at 31.67% similarity level (Ziziphus sp. (Rhamnaceae) and Morus sp. from Wadi e Dawasir and Qaseem regions, respectively, joined a cluster at 22.42% phenotypic similarity level , whereas P7 and P3 clustered together (sharing 13 phenotypic characteristics) showing phenotypic similarity percentage of 29.05% in the UPGMA dendrogram  from both H1 recovered from Ficus sp. planted in citrus orchard from Al-Ula  and DQ656473 from Israel, were 100% identical to each other (P. dactylifera (Arecaceae), which was genetically divergent (0.016–0.025) from all other E. orientalis haplotypes, clustered with a Spanish haplotype (KP642048) from citrus agro-ecosystem in the NJ phylogenetic tree  [T. urticae, T. kanzawai, and Oligonychus gotohi adapted to different hosts were reproductively isolated and genetically different [T. urticae populations from the same host Viola sp. from Italy and France were more closely related when compared with those from different hosts from the same country. The species, Brevipalpus phoenicis, which is a host specific species, failed to adapt on alternate hosts and did not exhibit host based genetic variations [The present study revealed that different hosts associated with families . Host-asfamilies –33 and scae Koch –36, 13 acGregor) . Populatifferent ,38,39. Sriations . Howeverriations .T. urticae may form host races to combat toxic chemicals produced by different hosts influencing high selection pressure on herbivores to provide opportunity for prolonged existence on different hosts and strong association [The utilization of different host plants may cause sympatric speciation in arthropods . The phyociation .E. orientalis populations was associated with host plants they inhabited  [Amphitetranychus viennensis (Zacher) [E. orientalis populations from Israel have been reported as genetically distinct [E. orientalis haplotypes was in a similar range (0.002 to 0.017) as found in the Israeli E. orientalis haplotypes [In the present study, the genetic data of 10 Saudi nhabited . The phyociation . Similare (Koch)  and Amph(Zacher) . In a prFicus sp. in a citrus orchard was 100% identical to Israeli haplotype (DQ656474 from Citrus sp.) and shared the clade with other Saudi haplotypes collected from Citrus sp. Likewise, the genotype H9 from Domat ul Jandal on Citrus sp. under the A. indica trees was clustered in the phylogenetic tree and dendrogram with G4 from A. indica from Qaseem; this might be due to the colonization of E. orientalis on a new host. The herbivores adapt on new hosts with the passage of time after coping with the defense mechanisms of new hosts [E. orientalis indicate that this species has a strong ability to adopt to diversified hosts and cope with defenses exerted by the host species, thus making it possible to live successfully in different environmental conditions and topography. The mite species occurring on different host plants undergo marked changes in their behavior, morphology, and genetic structure. This commonly happens in polyphagous mite species where adaptation to different host plants results in genetic differentiation [The Saudi haplotype H1 collected from ew hosts , 47. Thentiation Lepidoglyphus destructor, which have active dispersal capabilities, have shown genetic differentiations among populations collected from localities that are far apart from each other [Panonychus citri, collected from different localities in China [In addition, the intraspecific genetic variations in mites could also be due to geographic distances. The geographic distances act as a barrier for gene flow and promote intraspecific differentiations. The mite species, ch other . In the ch other . The genin China .E. orientalis resulted in variations in phenotypic characteristics as well as genetic structures. The taxonomists should consider the phenotypic variations specially in the shape and length of dorsal setae and striation pattern between dorsal setae d1 and e1, while identifying different E. orientalis populations to avoid improper identifications. Further extensive studies are needed to investigate the occurrence of different races of E. orientalis associated with a specific host or locality. Also, its population structure needs consideration in different agro-ecosystems.The polyphagous behavior and wide distribution of S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Throughout the past few years, a lively debate emerged about the timing and magnitude of the human migrations between the Iberian Peninsula and the Maghreb. Several pieces of evidence, including archaeological, anthropological, historical, and genetic data, have pointed to a complex and intermingled evolutionary history in the western Mediterranean area. To study to what extent connections across the Strait of Gibraltar and surrounding areas have shaped the present-day genomic diversity of its populations, we have performed a screening of 2.5 million single-nucleotide polymorphisms in 142 samples from southern Spain, southern Portugal, and Morocco. We built comprehensive data sets of the studied area and we implemented multistep bioinformatic approaches to assess population structure, demographic histories, and admixture dynamics. Both local and global ancestry inference showed an internal substructure in the Iberian Peninsula, mainly linked to a differential African ancestry. Western Iberia, from southern Portugal to Galicia, constituted an independent cluster within Iberia characterized by an enriched African genomic input. Migration time modeling showed recent historic dates for the admixture events occurring both in Iberia and in the North of Africa. However, an integrative vision of both paleogenomic and modern DNA data allowed us to detect chronological transitions and population turnovers that could be the result of transcontinental migrations dating back from Neolithic times. The present contribution aimed to fill the gaps in the modern human genomic record of a key geographic area, where the Mediterranean and the Atlantic come together. Djezirat el Maghrib), but its isolation refers to the global territory because it is composed of a large number of disparate regions , which has crossing difficulties due to the strong winds and dangerous currents but also the Alboran Sea (∼380 km in length and a maximum width of 180 km) to the east. It contains the small island of Alboran, as an intermediate stopover, in the distance separating Spain and Morocco, which high mountains allow the sight of land during the crossing. All these characteristics have made the western Mediterranean area most suitable for maritime contacts involving the southern and northern shores. The conjunction of archeological, anthropological, historical, and genetic data confirms the rich scenarios of ancient human movements in the region.Migration events in the west Mediterranean were especially abundant over the Neolithic and Bronze Ages . Then, tHomo sapiens lineage in North Africa   has raised questions on population continuity or discontinuity  have reported the oldest remains of a ,000 ya) . The prentinuity . In the ntinuity . A harpontinuity . Evidenc14C dating procedures on different samples, placed the appearance of the “Neolithic package” in Iberia ∼5,500 cal. BC.Southern Iberia and the Maghreb share similar characteristics with respect to the Neolithization process, a finding that has been interpreted as the consequence of strategic relationships across the western Mediterranean area . The intvia de la Plata.” During the Roman Empire, the Emperor Othon (69 BC) aggregated the province of Mauritania Tingitana (around northern Morocco) into the Baetica province . In the AD 171, there was a prolonged Moorish invasion of Baetica and parts of Lusitania. In the late third century AD, Diocletian grouped Mauritania Tingitana together with other Iberian provinces to form the Diocesis Hispaniarum. Therefore, through the Roman Empire, there were recurrent human movements between the northern and southern coasts surrounding the Strait of Gibraltar, including the temporary passage of troops and tribal contingents. Muslim invasion was another period of great importance for the relationships between the Iberian Peninsula and North Africa. In the Andalusian region, the Islamic presence elapsed from the 711 to the middle of the 13th century, with the exception of its eastern part, where the Nazari Kingdom remained Islamic until 1492.The Tartessian culture (∼800–540 BC) located in southwestern Iberia was a key component of the Iberian protohistory. Tartessos possessed important mines, which attracted people of the eastern Mediterranean and were the origin of a way, connecting the southern and northern Iberian Peninsula in its western part, which accessed important deposits of gold and tin. This way was then improved as a Roman road known as “Over the past 5 years, a burst of interest in analyzing the Mediterranean basin both from Neolithization archeological processes  and geneAlthough the use of high-density SNP panels is highly efficient for understanding facets of the genetic and demographic histories in the Mediterranean area, the availability of genomic data with high coverage, particularly among their westernmost populations, is still scarce and more sampling and research should be performed. A particular and integrative human vision of this geographic region comprising samples from the southern Iberian Peninsula  and northwestern Africa has not been explored. Therefore, in the present study, we performed a GW screening of living western Mediterranean autochthonous people from southern Portugal, southern Spain , and Morocco . Fine details on population structure, demographic histories, and admixture dynamics of this metapopulation are provided here. Comparisons with dense SNP data based on modern and ancient DNA in other European, North African and Near Eastern populations shed new insights into the timing and extent of human migrations between continents and the gene flow connecting populations.P < 0.001) differential contribution of the sub-Saharan African ancestral panel among southern Iberian subpopulations  showed a significant , although differences among Berber populations were not significant. Moving forward into a European–Mediterranean landscape A, the prK levels (2–11)  were performed at different –11) see . The besulations C, and thK = 5 are presented graphically in The average ancestry proportions based on ADMIXTURE analysis K = 5 was further explored , whereas the sub-Saharan global sample showed a negative strong correlation . By contrast, the longitude yielded a positive significant correlation  when considering the Near East ancestral proportion. FST genetic distances estimated between populations with the heatmap depicting a clear division between sub-Saharan populations and the rest of the analyzed samples. The observed results are in close agreement with the genetic PCA findings analyzed above.The correlation between ancestry components and geography for explored  online. K ancestral sources leads to more complex patterns, suggesting the presence of microgeographic diversity in specific territories. The ADMIXTURE model for K = 11 yielded a CV value (0.5139) similar to that found for K = 5 (0.5115). The former plot permits subdividing continental groups into finer details. The European block makes clear that a specific Iberian cluster arose (in light blue color) with overrepresentation in French and Spanish Basques and a differential weight in the other Iberian populations. This cluster probably represents a signature of the descendants of people living there during the Last Glacial Maximum (LGM). Finns are fairly distinguished by a deep blue color that is almost absent in Iberians, while an intermediate blue denotes the rest of western Europeans. Non-Basque Iberians present relatively similar admixture proportions of North Africa, eastern Mediterranean’s, and Near East ancestry. The Near East appears subdivided into three groups: Druzes (in light green), Bedouins , and Palestinians (in dark green). The genetic impact of Palestinians on the Iberian people is remarkable when compared with the other Levantine groups. According to the geographic position of Italy in the central Mediterranean, the sample analyzed here harbors a greater ancestry fraction from the Near East. In the case of North Africans, characterized by varying shades of ochre, the model permits us to distinguish Mozabite Berbers (light ochre), Tunisian Berbers (Chenini) (intermediate ochre), and Moroccan Berbers (dark ochre). The latter is the northern African component more predominant in Iberia. Sub-Saharan from western Africa and the two emerging groups  used in the present work are weakly represented in Iberia.The increase in N = 41 clusters), western Mediterranean individuals are embedded in five North African and four Iberian clusters . British and CEU samples show a relative affinity with the Basque group. Near Eastern samples are completely differentiated from those from North Africans with the exceptions of most Egyptians and a few Libyans and Bedouins. Egyptians and Eastern Libyans are populations closer to the Near East than to the Maghreb. This could be mainly due in part to the barrier of the Libyan Desert. Most Berbers are placed in different clusters: C9TUN, C15MOZ, and C10MOR, probably reflecting a clear Berber population structure associated with the old kingdoms and contemporary nations in the region.A and B, Clusters built with fineSTRUCTURE were subsequently used for reconstructing past admixture events using GLOBETROTTER and the results are presented in supplementary table S4Admixture events inferred were defined by two population sources (1 and 2) with different contributions, the best proxy of each source and admixture dates. Most of the estimated admixture episodes for both the Iberian and North African recipient clusters were unique (one-date admixture events). GLOBETROTTER detected two additional admixture pulses for the most diverse clusters on the data set as a reflection of their complex history.Point estimates of dates spanned from ∼54 to 4 generations ago, that is, from the 7th to 19th centuries CE see  online. A, supplementary table S4B, With respect to Iberian recipients, virtually all cases in the present study included an admixture event between a major European-like source (82.3 ± 6.5% as an average) and an African source. The latter source is best represented by a North African population and not by a sub-Saharan population. Since we set two different scenarios, C10MOR or C14MAGH2, as North African donors, it should be interesting to search for potential differences along the five Iberian recipient clusters considered . When first using C10MOR as the proxy for North African ancestry, this component is displaced as best-source 1 in two cases  and Tunis (Tunisia) are 4,500 km apart, and within Libya, the broad desert territory between Tripoli and Benghazi, has been scarcely populated from antiquity to recent times. Likewise, Berbers in the Maghreb are genetically differentiated according to ethnic origins  indicating a prolonged isolation among them. The European component present in Berbers, at least those settled in northwestern Africa (present study), is generally higher than that observed from the Near East, signifying more intense contacts with the west than eastern Mediterranean. A portion of that European component comes from the Basque genome, indicating old relationships with Iberia. Unfortunately, with data emerging from the present study, it is not possible to discern whether most of the Near East ancestry component in Berber genomes is a result of Islamic expansion or due to more ancient times.Some genetic diversity studies have observed closer relationships between North Africans and Europeans than the former with sub-Saharans . Howevervia de la Plata” Roman road. Our genomic data reflect that this road, that enabled military and commercial activities, could have acted as a vehicle for the spread of (African) genes, and that have significantly defined the contemporary internal structure within the Iberian Peninsula  at least for the basal situation of three ancestries tested see C. The imsula see .Although current available methods for dating admixture events from GW data are likely biased toward more recent episodes , global In conclusion, the great complexity of the analyzed area regarding geographic and climatic conditions, demographic history, and archeological record has shaped the genomic diversity of their contemporary populations. Western Iberia—its southernmost part in particular—is the European hotspot regarding African genomic contribution. The Maghreb harbors several ancestry sources enriched by an Iberian influx dating back from Neolithic times. Broadly, the geographic link-up of Iberia and the Maghreb should be considered a transition area between two continents, acting as a bridge for genetic exchanges. Although GW studies have the potential to provide an accurate overview of the human past, including population origins, migration and admixture events, and demographic history of populations, haploid marker analysis should still be explored for tracing deep structure and relationships between populations. The present contribution aimed to fill the gaps in the modern human genomic record of a key geographic area, where the Mediterranean and the Atlantic come together.In the present work, we performed a GW screening of six western Mediterranean populations. The southern Iberian sample comprises three autochthonous populations, one from Portugal, a national territory separated from Spain by a border from medieval times, and two others from the western and eastern sides of the Spanish Andalusian region, from Huelva and Granada provinces, respectively, and Moroccan Berber native people from Asni, Bouhria, and Figuig representing the three North African samples. Individuals analyzed here were unrelated, and samples are the result of a random selection from their respective source population sample sets. Each participant provided written informed consent as well as information on familiar geographic origins.Biological samples from both southern Iberia and Morocco analyzed in the present study have been previously used in other genetic diversity studies based on uniparental and autosomal markers . DetailsCentro Nacional de Investigaciones Oncológicas) in the Human Genotyping laboratory (CeGen). The raw data genotyped consisted of 2,372,784 SNP markers with a mean genotyping rate of 99.5%. A duplicated sample was used as an internal control (HapMap code Na12775) with a reproducibility of >99.9%. The genotypes from the individuals analyzed in this study can be accessed from the EGA repository . Mitochondrial DNA (mtDNA) haplogroups were inferred from chip data by using Haplogrep  from Hardy–Weinberg equilibrium, and a minor allele frequency threshold of 1%. Recent genetic relatedness was excluded by applying the PC-Relate approach . Data from the Illumina and Affymetrix platforms were merged on physical position. The final merged data set (Database 2A) resulted in 84,586 SNPs for 1,523 individuals across 36 present-day populations. Details on this database are shown in r2> 0.2) to obtain 465,598 (Database 1B) and 63,189 (Database 2B) SNP sets, respectively. Each database was used for different methodological procedures  phase 3  to obtait-test with the Welch correction when the variances between the pairs of groups differed significantly.Database 1A was first phased with SHAPEIT2 . Then, wsmartpca program in the EIGENSOFT package (The LD-pruned high-resolution database (Database 1B) was used to check for possible outliers or errors in data merging by means of an exploratory PCA using the  package  (data noK = 2 to K = 13 clusters). A balanced sample size was used across populations. A cross-validation (CV) test was performed to validate the best-fitting estimation . The RFMix approach, as indicated above, was complemented with the development of a model-based estimation of global ancestry using ADMIXTURE . Models approach . In geneapproach  online. FST (a measure of genetic differentiation) between pairs of populations.Database 2B was also used to evaluate population structure by means of PCA  and the chromosome painting multistep approach for inferring ancestry blocks (“chunks”), and it allowed us to classify individuals into genetically homogeneous clusters and to sequentially date evolutionary events. First, we developed an “all vs. all” analysis for all samples included in our extended data set (Database 2A) considering the 1,523 individuals both as donors and recipients in ChromoPainter v2 (Mut (mutation rate) and Ne (switch rate) parameters were estimated by an initial analysis with ten EM steps in four chromosomes , and the values were then incorporated into the program to perform analysis with all chromosomes. The resulting matrices showed the amount (counts) and size (lengths) of DNA copied from each donor individual by each recipient individual per chromosome. Data were combined into single files by using CHROMOCOMBINE, and the normalization parameter “c” (effective number of chunks or independent genetic elements) was estimated as well.Migration timing modeling was performed by using a haplotype-based methodology. Specifically, we used a inter v2 . The MutThe combined coancestry matrix was used for fineSTRUCTURE analysis  by perfoIndividuals were grouped into 119 clusters. We performed a manual exploration of the tree to define genetically homogeneous groups by considering a minimum size of ten individuals per cluster. The 41 clusters finally established showed a fair correlation with the geographic affiliation of populations included  were first explored to analyze the proportion of the genome copied from each surrogate cluster by each target cluster. Both the total length of genomic material copied from donors and the total number of haplotype segments were standardized to sum up 100% in each recipient cluster.For the new ChromoPainter runs, we again estimated the outputs were used in GLOBETROTTER to identify and date recent admixture events in the Iberian and North African clusters using the painting profiles and surrogates defined for each scenario , making it difficult at these K-tests to envision genomic relationships between ancient and modern samples.We implemented two strategies for merging aDNA individuals with our modern sample sets following IGENSOFT  with themsz288-Supplementary_DataClick here for additional data file.


The impacts and interactions between hatchery-bred fish and wild fish populations has been a topic of active investigation in recent decades. In some instances, the benefits of stocking can be overshadowed by negative effects such as genetic introgression with natural populations, loss of genetic diversity, and dilution of local adaptations. Methods that facilitate the identification of stocked fish enable us to estimate not only the effectiveness of stocking but also the level of natural reproduction and the degree of hybridization. The longest Baltic river, the Vistula, also has the second highest discharge. Historically, it hosted numerous populations of the anadromous form of brown trout (sea trout); however, dam construction has since interfered with and reduced spawning migration to a rate that is much lower than before. Reduced spawning has resulted in a population collapse and a negative flow-on effect on commercial catches. In response, Poland  initiated an intensive stocking program which continues today and which sees the average annual release of 700,000 smolts. As a consequence, today’s main-river and inshore catches come from stock-enhanced populations. High-throughput single-nucleotide polymorphism (SNP) genotyping was performed on samples of sea trout from southern Baltic populations; results suggest that a significant portion of the sea trout catches in the Vistula mouth region have direct hatchery origin and indicate the presence of Pomeranian specimens. SNP loci identified as outliers indicate a potential selection pressure that may be related with effects of hatchery breeding and mixing with natural populations. The brown trout SNP array applied in this study showed high effectiveness not only for population differentiation, but more importantly, it emerged as a sensitive tool to provide evidence of detection selection. Salmo salar L. and brown trout Salmo trutta L. [Mixed-stock fisheries may target multiple anadromous fish populations; one strategy for documenting stock composition is genetic stock analysis . This aprutta L. ,6 caughtrutta L. .Sea trout, the anadromous form of brown trout, resides as a native population in about 500 mainly small- and medium-sized rivers in the Baltic Sea region. In Poland, evidence suggest that 30 rivers have sea trout populations, and all of them have mixed status according to the Baltic Marine Environment Protection Commission (HELCOM) categorization . The VisArtificial stocking of the Vistula River began in the mid-nineteenth century. At that time, the majority of stockings used sea trout originating from spawners collected in the Dunajec River, about 800 km from the Vistula mouth and few other upper Vistula tributaries ; stockinSince 1985, it has been assumed that the released material should represent progeny from individuals directly caught in the river during spawning migration. However, in practice, due to difficulties with spawner collection caused by seal predation, high water levels, etc., much of the roe used for production has originated from hatchery spawners belonging to a handful of broodstock from hatcheries in northern Poland. The biggest farm, Aquamar, maintains a hatchery stock established in the 1990s as a reserve stock for the Vistula River and is comprised of a putative winter sea trout stock. In subsequent years, this broodstock was supplemented by single winter sea trout specimens, and in the early 2000s, these fish began to deliver stocking material for release into the Vistula River. This hatchery population comprised 12–63% (average of 25%) of the material released annually into the Vistula River between 2004 and 2013 . From thTagging studies exploring sea trout costal migrations have shown that individuals from smolt stocking which were released close to the mouths of the Pomeranian rivers had very high levels of straying individuals, which can explain a significant share of Pomeranian genotypes among sea trout collected in the Vistula firth area ,16. ResuRelative to wild stocks, salmonid hatchery fish often display a reduced performance and survival rate  and dispS. trutta populations in the Vistula River that were related to differential stocking strategies [Several studies sought to develop tools to genetically differentiate between southern Baltic Sea populations, mainly from the Pomeranian and Vistula rivers and local hatchery stocks. However, none using allozymes , mitochorategies . Lack ofrategies . Until nrategies  and may rategies .The main goal of the present study was to differentiate hatchery origin and Pomeranian genotypes in sea trout collected in the south of the Gulf of Gdańsk, a region representing the Vistula population. In addition, the genotyping of a significant number of SNP loci provided an opportunity to identify loci under selection and to highlight potential effects of breeding operations . Additionally, temporal genetic stability in a population from the Słupia River was tested in order to demonstrate the effect of genetic drift caused by artificial spawning using a small number of parental combinations.Sea trout samples from the Słupia River were collected in autumn 2008  and 2009  from adult fish caught in trap about 30 km from the river mouth in Słupsk City, 54.460538°, 17.039682°, during spawning migration. Sampled fish represented both sexes and were at sea age from 1 to 3+ years. Adult specimens collected in the Vistula mouth area  26 area) were harvested using gillnets in October 2009 by fishermen during several trials over a few fishing grounds located on fishing square T4. Sea trout were mainly female and at sea age from 1 to 3+ years. The other two samples originated from hatcheries. The first  was collected from smolts in 2009 taken from a brood stock created and maintained in the Department of Salmonid Fish Breeding at the Inland Fisheries Institute in Rutki. The second investigated hatchery line  was sampled in 2009 from adults in the hatchery farm Aquamar in Miastko. Fin clips were stored in 95% EtOH solution at 4 °C. Genomic DNA was extracted using Qiagen DNeasy 96 blood and tissue kit, as recommended by the manufacturer. DNA samples were analyzed in 1.0% agarose gels and quantified on a NanoDrop device.Salmo trutta) llumina iSelect SNP microarray at the Centre for Integrative Genetics (CIGENE), Norway, as in Linløkken et al. [Samples were genotyped using a custom-developed brown trout  for all loci and for each population were estimated in Arlequin 3.5.2.2 software  [FIS, pairwise weighted FST, and overall F-statistic were estimated by analysis of molecular variance (AMOVA). A sequential Bonferroni type method was employed to correct for multiple significance tests. Clustering of sea trout populations was calculated using the Structure 2.3.4 software  [The number of polymorphic loci, the mean number of alleles, and the observed and expected heterozygosity (zerland) . DepartuCA, USA)  assumingCA, USA) ; second CA, USA)  in StrucCA, USA) . The CLUCA, USA) .A distances [FST values outside the 99% quantile that were based on coalescent simulations were considered as candidates for diversifying selection [q-value) threshold of 0.05. For new SNP panels based on calculated outlier loci, the overall FST and pairwise comparisons were recalculated similar to Bayesian clustering in STRUCTURE. The identification of selection signatures can help to understand the genetic factors and patterns engaged in important biological traits because these regions might have adaptive and functional relevance underlying their selection [Genetic structure was also examined by Principal Coordinates Analysis (PCoA) done in GenAlex 6.502  . The POPistances , with 10istances . To deteistances  implemenistances . The hieelection . Outlierelection . Settingelection ,44. Putaelection ,46.p-values for the observed allelic association under the null hypothesis of random allelic assortment were done by Markov Chain Monte Carlo with 10,000 steps and 1000 dememorizations. Additionally, LD as r2 or D’ values was calculated in Multiallelic Interallelic Disequilibrium Analysis Software (MIDAS) [Blast2go   was used (MIDAS)  and TASS (MIDAS) . To buil (MIDAS)  and was The mean number of alleles in investigated stocks of individuals varied between 1.906 and 1.961 . The lowIS were insignificant (p < 0.05) in all stocks. Analysis of molecular variance was performed for three scenarios: “All samples” (TS9 + TS8 + TVS + TVR + TVA); “Sampled in wild” (TS8 + TS9 + TVS); and “Hatcheries” (TVR + TVA). In all scenarios, the largest amount of variance had its source in within-population diversity  was equal to 0.033, which indicates a low level of differentiation. In the tests performed for sea trout sampled in the wild, overall FST was even lower and reached 0.019. The highest general FST was observed for hatchery fish and reached 0.062. The dissimilarity of the hatchery lines, especially from the TVR samples, is well presented by pairwise comparisons. All tests were significant (p < 0.05)  . The higBayesian estimation of genetic structure and individual membership performed in STRUCTURE 2.3.4 indicated that the maximum value of Δκ was for K = 4 (ΔK = 140.86). Ln Pr(X|K) slowly increased from K = 1 to K = 4, and ΔK presented a sharp peak for K = 4 . In thisOther scenarios have lower ΔK values. The scenario with K=2 (ΔK = 37.80) grouped TS population with TVS and Aquamar hatchery TVA. For K = 3 ΔK = 3.04, and first clad has TS samples, the second clad comprised TVS and TVA, but TVR has own distinct cluster. The last scenario with K = 5 shows separate clusters for every investigated stock.Results from Principal Coordinates Analysis (PCoA) are in agreement with Bayesian clustering. The PCoA plot shows large differences between TVR and other samples. Additionally, the significant share of Pomeranian genotypes in the sample from the Vistula mouth was confirmed .For better presentation of patterns of the genetic compositions inside studied stocks, the individual self-assignment test was performed . ResultsGenetic relationships between investigated sea trout samples obtained from the phylogenetic analysis showed three clads with high bootstrap support. A neighbor-joining (NJ) tree  grouped FST from AMOVA performed only for 83 candidate loci detected in Arlequin reached 0.172 and was evidently higher than the global FST calculated for the 3843 polymorphic loci (0.033) for p = 0.001. Pairwise FST also showed much higher values, even between samples from consequent years in the Słupia River , which has been the subject of extensive sequencing and annotation. During scoring, many orthologs among annotated genes were generally observed between close evolutionary species from genus Oncorhynchus and from the Esocidae family, e.g., LOC106573934 in the Salmo salar, LOC109889095 for Oncorhynchus kisutch, and 105011366 (sgta) for Esox lucius, all coding glutamine-rich tetratricopeptide repeat-containing protein alpha-like.Atlantic salmon chromosome 18 (Ssa18) includes the greatest number of markers showing evidence of selection (n = 12); followed by Ssa15 (n = 9) and Ssa09 (n = 7); and then Ssa05, Ssa10, and Ssa13  . No outlSalmo salar chromosomes, we also tested 3404 (full matrix) possible linkage combinations in TASSEL. Results show 305 links with p < 0.01 with D’ values ranging between 0.44 and 1 (mean 0.80) and r2 values ranging between 0.12 and 1 (mean 0.34). Similar to the Arlequin method, the highest number of significantly linked loci were located on chromosome 18 and on loci Gdist:S541107 1153 and Gdist:S99221 1923 , Gdist:S2685113092 (42 links), and cDNA:S363339747 (41 links) . Four an221 1923 .S. trutta) was used to analyze the genetic relationship between sea trout specimens from the southern Baltic Sea. The experiment explored the proportion of Pomeranian and hatchery origin genotypes among sea trout harvested in the southern Gulf of Gdańsk in the vicinity of the Vistula River mouth. In rivers flowing to the investigated area, regular stocking with sea trout smolts, fry, and alevins has been conducted from the 1960s [In this study, a 5.5 K Illumina SNP iSelect genotyping array developed for brown trout  and NJ tree show that, among adult specimens harvested near the Vistula mouth (TVS sample), the Aquamar TVA hatchery line represents a significant share; roughly 30%. The comparison of share of the Pomeranian genotypes between TVS and TVA samples suggest that the real proportion of Pomeranian genotypes is lower because it is represented by a significant proportion in the TVA hatchery stock (37%). However, results from PCA analysis do not confirm this. Further, they show that some specimens from Aquamar are only close to Pomeranian genotypes where TVS partially overlap them. Bayesian analysis of genetic structure also shows the presence of another clade; it can be called the “Vistulian” clade. The highest share of these genotypes occurred within the TVS sample. This is important information indicating that some level of natural spawning in the Vistula Basin occurs. Despite the percent of Vistula clade in the Aquamar sample being lower than that in the TVS sample, it still exists in the hatchery line. The dissimilarity of samples from the TVR hatchery shows that sea trout imported from Dalälven are relatively distant  from contemporary Polish populations. The reason for this could be genetic drift during years of hatchery breeding. The theory that this line is close to the original Vistulian population from before the Włocławek Dam is unlikely, especially if we consider high genetic distance between present Polish populations and Dalälven. Furthermore, recent results from microsatellite analysis of the historical and contemporary Vistulian populations show lower genetic distance between them .Analysis of the samples from the Słupia River was performed not only as a comparison with the Vistula line but also for temporal stability test. There was an assumption that artificial spawning performed annually on this river has a strong influence on genetic structures in sea trout populations . The estOutlier analysis showed differences in the total number of detected loci between methods. The difference in the number of candidate markers could be either due to the inability of the Bayescan methods to detect signatures of selection at these loci or because a large number of the outlier loci detected by Arlequin 3.5 are false positives (type I error) . HoweverSalmo salar investigated using a custom 6.5K SNP array). In the study designed for discrimination of different classes of hybrid between wild Atlantic salmon and aquaculture escapees, where a 220K SNP array was used, the level of outliers was also reported at about 1%  [Salmo salar assembly [S. salar  of the analyzed loci were classified as outliers, a portion that has been reflected in other studies  . Howeverpproach) . A similpproach) . The avapproach)  (loci Gdpproach)  and collpproach) . Howeverassembly ; despite = 54–58 ) and S. (2n = 80 ) indicat(2n = 80 , mainly (2n = 80 ,65. The n et al.  as 1.87%Obtained results have revealed several facts that may prove important from a management perspective. The share of the hatchery origin specimens inhabiting the Vistula mouth area is high, which can be indicative of relatively good stocking effectiveness. The breeding line from Aquamar is genetically similar to the Pomeranian fish. The discovered “Vistulian” clad indicates some level of natural reproduction among the Vistula population. The analysis also showed disorder in genetic temporal stability within the population from the Słupia River, which is related to stocking practices and significant population fluctuations. The brown trout SNP array applied in this study showed high effectiveness not only for population differentiation but also, more importantly, as a sensitive tool for evidence of detection selection. Obtained outliers exposed that, in investigated sea trout populations, significant evidence for signatures of selection occurs and many candidate markers are linked with molecular functions related to the selection process of specific traits.


Ixodes. Understanding the origin, maintenance, and spread of these ticks contributes much to our understanding of the spread of LB and other disease agents borne by these ticks. We collected 1232 Ixodes scapularis ticks from 17 east coast sites ranging from New Hampshire to Florida and used mtDNA, three nuclear genetic loci, and incorporated Bayesian analyses to resolve geographically distinct tick populations and compare their demographic histories. A sparse, stable, and genetically diverse population of ticks in the Southeastern US, that is rarely infected with the agent of LB is genetically distinct from an abundant, expanding, and comparatively uniform population in the Northeast, where epidemic LB now constitutes the most important vector borne disease in the United States. The contrasting geography and demography of tick populations, interpreted in the context of the geological history of the region, suggests that during the last glacial period such ticks occupied distinct refugia, with only the northern-most site of refuge giving rise to those ticks and pathogens now fueling the epidemic.Lyme borreliosis (LB) is the archetypal emerging zoonosis and is dependent on transmission by ticks in the genus  Ixodes scapularis is the main vector tick of Borrelia burgdorferi, the bacterium that causes Lyme disease. In the United States, most human Lyme disease cases are reported in the Northeast and upper Midwest, while cases in western and southern regions of the country are low2. This contrast is also evident in the entomological record where the frequency of B. burgdorferi is 2–8% in the main vector I. scapularis adult ticks in the South4, compared to infection rates averaging 50% in New England5. Environmental factors may affect survival rates of immature I. scapularis and the geographical distribution of Lyme disease6. In addition to the persistent difference in Lyme infection rates, there are major differences of questing behavior, human attacking rate, host preferences, and survival rate between northern and southern populations of I. scapularis ticks11. There are also vast differences in abundance, with I. scapularis tick densities in the South being much lower than in the North12.Lyme disease is the most commonly reported vector borne disease in North America though its risk varies widely by geographic regionI. scapularis populations. In 1985, the original description of the north population as a new species I. dammini was driven in part by the report of an abundant I. ricinus-like tick with northern distribution several hundred miles disjunct from that of the southern I. scapularis13. However, the morphological differences between northern and southern populations were cryptic, tending to manifest only during particular life stages. In 1993, Oliver and colleagues suggested that I. dammini should be reduced to a junior synonym of I. scapularis14. Population genetic studies showed no differences in chromosome morphology or allozyme frequencies among populations15. The nuclear ribosomal DNA sequences of the ITS-l and ITS-2 regions were also continuously distributed among populations17. However, the distribution of mitochondrial haplotypes is distinct and forms two clades: (1) the American clade, found in the northeastern US down to the Carolinas, and (2) the Southern clade, which overlaps in the Carolinas and extends into the southeastern states. More genetic variation and diversity have been observed in ticks collected from southern states21 and extremely abundant single nucleotide polymorphisms (SNPs) have been found in I. scapularis22. SNPs analysis for ten ticks showed that samples from New Jersey and Virginia formed a homogeneous group with low genetic diversity, whereas southern ticks from Georgia and Mississippi consisted of two separate groups, each with high genetic diversity. SNPs analysis also revealed a predominantly North to South gene flow23.Due to these phenotypic variations, it is not surprising that large genetic differences occur between north and south 24. Although ticks from northern and southern populations appear to be equally competent B. burgdorferi vectors in laboratory conditions26, data on the prevalence of the Lyme spirochete has not been linked to mitochondrial Southern clade ticks. The present study is based on mtDNA and three nuclear genetic loci and incorporated Bayesian analyses to detect evidence of population structure and geographically distinct tick populations. We have also used ecological niche modeling to determine the historical distribution and spread of these ticks. We found that northeastern (northern) and southeastern (southern) I. scapularis tick populations are genetically distinct with different histories and Lyme spirochete infection rates.While the population genetic structure of blacklegged ticks is a fundamental basis for understanding and predicting Lyme disease epidemiology, relevant studies have mainly utilized phylogenetic methods based on mitochondrial markers from a small number of individuals at limited collection sites. As a result, we still do not know the border between the northern and southern populations. Studies of nuclear, segregating genetic markers that could elucidate the evidence for genetic exchange across the broad geographic range of the east coast of the United States are limitedI. affinis N = 3; and I. brunneus N = 1) based on the mitochondrial 16 S sequences. Fragments of mitochondrial 16 S ribosomal RNA (mt DNA 16 S), nuclear (nuc) Calreticulin (CRT), Glycerol-3-phosphate dehydrogenase (G3PD) and Isocitrate dehydrogenase (IDH) genes were amplified for 1232 adult I. scapularis ticks.A total of 1236 adult ticks were flagged from vegetation from 17 east coast sites in November to December 2007  on the two Last Glacial Maximum climate reconstructions (CCSM and MIROC) suggested that the northern tick population was derived from a small refuge located just south of the Pleistocene ice sheet on the Atlantic continental shelf off the current Delmarva Peninsula. The southern Borrelia infection rates and post-glacial histories.Based on haplotype distribution, population comparison, phylogenetic analysis, demographic history and Bayesian analysis of population structure, we conclude that there are two distinct blacklegged tick populations (northern and southern) along the US eastern coast, with distinct 20, this study not only suggested that blacklegged ticks are geographically structured along the east US coast, but also suggested that two distinct clades meet at North Carolina and Virginia. The southern I. scapularis population is characterized by greater heterogeneity and absence of widely distributed haplotypes. In contrast to the results from the ITS-2 gene17, analysis of three nuclear genes confirms the existence of two major structured populations of I. scapularis ticks. Both mitochondrial and nuclear genes are comparatively homogeneous in northern I. scapularis populations, but more diverse in southern I. scapularis populations. We found nuclear genes suggested a boundary between the northern and southern population in Virginia, whereas mtDNA suggested this boundary to be positioned in North Carolina.In agreement with previous mtDNA studiesI. scapularis populations are suggested to be derived from a small refugia located to the south of the Pleistocene ice sheet and on the Atlantic continental shelf off the current Delmarva Peninsula. Although this region has been submerged as sea level raised from melted glacial ice, it was a broad coastal plain and well covered with land vegetation during the Last Glacial Maximum27. The southern I. scapularis population may have originated from a suitable refuge that is more to the South, located in the Southern coastal plain. The later expansion of the southern population seemed to occur through much of the Southeastern quadrant of the United States. However, since I. scapularis is expanding its range and has recently shown tolerance for colder conditions in northern US and southern Canada, the current distribution of I. scapularis may not represent the full spectrum of its habitable environment, and the inferred historical range might be underestimated. The New York-Connecticut region20 and the areas further north of Delmarva have been suggested as a likely location of refugia populations of I. scapularis28.The demographic model strongly supports a recent and rapid increase in population size for all Northeastern sites. By contrast, only several southern sites  showed no evidence of changes in historical population size. The ecological niche model suggests that there were two possible distinct refugia in eastern North America during the Last Glacial Maximum. The northern 19, we found that the two major mitochondrial haplogroups co-occur throughout the south. All northern ticks carry only Haplogroup A, while Haplogroups A and S occur mixed throughout the South. This has previously been explained as evidence of continuous gene flow among populations and a northward movement of the southern population3. We, however, found that the relative frequency of A and S haplogroups is a latitudinal cline with S increasing towards the South. The introgression of the northern population mitochondrial DNA might suggest that gene flow between two tick populations has migrated southwards. Positive selection or migrating birds may play an important role for the directional gene flow because they would carry ticks southward more effectively than northward28. However, the expansion of I. scapularis includes southward as well as northward expansion. For example, the recent northward invasion of I. scapularis and B. burgdorferi in Canada has occurred via the introduction by migratory birds29. The environmental conditions and selective pressure from warmer temperatures may change the host-seeking behavior of southern tick population6. Alternatively, the northern mtDNA in the Southeastern population may represent a genetic footprint left after the southern population displaced an expanding northern population31. Bayesian Analysis of Population Structure revealed two populations with a possible hybridization zone located in Virginia.We detected a homogenous northern population and a heterogeneous southern population. The heterogeneity of the southern population is due to introgression from the northern population. In agreement with previous studiesI. scapularis populations appear to be at a demographic equilibrium, while northern populations have gone through a recent demographic expansion, especially after glacial retreat. However, human activities have accelerated the expanding of I. scapularis ticks in the eastern United States and southern Canada during the 20th century32. I. scapularis was first collected on Naushon Island, Massachusetts in 1920. With the return of white-tailed deer to other areas of the Northeast and Midwest, the range of I. scapularis began expanding28. During the 1960s, focal I. scapularis infestations were recognized on Long Island and Nantucket Island in the Northeast and in northwestern Wisconsin. By the mid-1970s, the known range of I. scapularis had expanded, particularly extending in a southerly direction along the middle-Atlantic coast28. Within 100 years, this tick has largely spread throughout most of the eastern United States and into southern Canada32. The expansion of I. scapularis and B. burgdorferi is determined by distinct environmental factors: climate, habitat and host range and abundance34. In this study, only climatic factors were included in the ecological niche models.We found that the southern Borrelia (43–74%) is mostly linked to the northern I. scapularis population and where Borrelia does occur in the southern I. scapularis population (as defined by nuclear DNA), it is always found linked to the northern mtDNA type. The infection rate drops substantially to 0–6% with a change of the nuclear DNA defined population boundary in Virginia. Whether the differences in the infection rate is environmentally determined, is a heritable trait for two populations or is a combination of genetic traits and hosts needs further study. For example, warmer temperatures and a lower density of host population may change the host-seeking behavior of southern tick population and eventually lower the incidence of Lyme disease in areas at low altitudes6. However, environmental factors alone cannot explain the virtual absence of infection rate in I. scapularis. Although different hosts were believed to be the reason for low infection rates in the South, a comparative study between two sites in eastern Virginia35 showed the infection of white-footed mice is 66.5% in the coastal site and 15.5% in the inland sites. Borrelia infection is 35.7% and 0% in the nymphal ticks removed from these mice in the coastal site and in the inland sites, respectively. Recently, the absence of detectable B. burgdorferi infection was found among blacklegged ticks in North Carolina36 and Tennessee37. These may suggest a genetic basis of tick behaviors that influence the epidemiology of vector-borne pathogens. It is very likely that the Northeastern tick population is the principal tick population responsible for transmitting Lyme disease. This same population is solely carrying two zoonotic agents in the Eastern US: Anaplasma phagocytophilum and Babesia microti13.The most interesting finding is that a high infection rate of I. scapularis populations continue to spread and grow38. It is worth noting that tick population ecology and spirochete transmission patterns are different between the northeast and northern Midwest. The two foci of B. burgdorferi in the northeastern and midwestern have a shared past and once belonged to an admixed population but are now isolated40. Transport of B. burgdorferi between northeastern and midwestern I. scapularis populations appears to be relatively infrequent.In this paper, we only sampled adult ticks from eastern coastal sites. Tick-borne disease in the Midwest of the US and Canada is increasing as Borrelia by Northeastern populations of I. scapularis is of significance for public health. We believe that, currently, the risk of acquiring Lyme disease through I. scapularis population is low in the South. First because they are low in density and they rarely bite humans9; more importantly, this population is not likely to be carrying the Lyme spirochete. However, this does not mean that there are no enzootic cycles of spirochetes in the South. Enzootic transmission of Lyme disease spirochetes among rodents and ticks has been documented in Southern and South-central states. Although they rarely bite humans, the sympatric tick species I. affinis, I. minor and I. dentatus may maintain a different enzootic cycle in the South41. In this study, two of three collected I. affinis ticks are Borrelia positive. In North Carolina, Borrelia DNA was detected in 63.2% of 155 I. affinis, but in 0% of 298 I. scapularis36; so-called “Ixodes scapularis” with a 35% Borrelia positive rate was confirmed as a non-accurate identification of I. affinis42. The current and potentially enhanced future role of the southern blacklegged tick population I. scapularis in Lyme epidemiology requires additional studies. Monitoring dynamics of the two populations will help us to predict future changes of tick and tick-borne pathogens.The suggestion of a genetic basis to the transmission of 2 white flannel flag through vegetation in November - December 2007  throughout the eastern United States by dragging a 1-mWe examined multiple genetic loci, including partial sequences of one mitochondrial gene and three nuclear genes. Both mitochondrial and nuclear gene primers were developed from the available sequences in GenBank. Primers TK16S_105F (5′-CGGTCTGAACTCAGATCATGTA-3′) and TK16S_546RC (5′-AATTGCTGTGGTATTTTGACTATAC-3′) were used to amplify a mitochondrial 16 S (441 bp) region. PCR was performed at 95 C 15 sec, 45 C 15 sec and 72 C 60 sec for 40 cycles. Primers CRT_849F (5′-TGGACGAGCCGATGGGTA-3′) and CRT_1657RC (5′-GGGCTGTACTCTGGGTTATCGA-3′), G3PD_3574F (5′-GCGGTGGCACCTTGGTAG-3′) and G3PD_4307RC (5′-CCGTCCTTGTCCCGTTTCT-3′) and IDH_707F (5′-GGTACTTCGTGACCCTCCTGC-3′) and IDH_1436RC (5′-CGACCATAGTTGCGACCAGAC-3′) were designed for genes encoding Calreticulin , Glycerol-3-phosphate dehydrogenase  and Isocitrate dehydrogenase , respectively. These three nuclear genes were amplified at 95 C 15 sec, 57 C 15 sec and 72 C 60 sec for 40 cycles. The PCR products were treated using Exosap-IT then sequenced with an ABI 3130 sequencer following the manufacturer protocols.B. burgdorferi) with a reaction volume of 20 μl, by using the Brilliant II QPCR Master Mix in a Stratagene MX3000P QPCR System . The detection of tick DNA served as an internal control with 300 nM of primers Tick16S_F (5′-AATACTCTAGGGATAACAGCGTAATAATTTT -3′) and Tick16S_R (5′-CGGTCTGAACTCAGATCAAGTAGGA-3′), and 125 nM of Tick16S_Probe (FAM-5′- AAATAGTTTGCGACCTCGATGTTGGATTAGGAT 3'- BHQ1). The detection of B. burgdorferi was modified from  with 700 nM primers Borrelia23S_F (5′-CGAGTCTTAAAAGGGCGATTTAGT-3′) and Borrelia23S_R (5′-GCTTCAGCCTGGCCATAAATAG-3′), and 300 nM of Borrelia_Probe (HEX-5′- AGATGTGGTAGACCCGAAGCCGAGTG-3′-BHQ1). Cycling conditions included an initial activation of the Taq DNA polymerase at 95 °C for 10 min, followed by 40 cycles of a 15 s denaturation at 95 °C and a 1 min annealing-extension step at 60 °C.Taqman real-time PCR assays were performed in a duplex format  method in PAUP 4.0b1044.DNA sequences were aligned using the ClustalW software. Individual mitochondrial haplotypes were identified and converted to Arlequin format using FaBox45 with default parameters. Genetic structure was examined by an analysis of molecular variance (AMOVA), implemented in Arlequin version 3.146. Structure analysis was performed using Structure47 and BAPS version 5.348. Structure analysis was carried out with 10 independent runs per each K value (K = 1–17), with a 100,000 burn-in period and 900,000 Markov chain Monte Carlo iterations. These analyses were performed using a model with admixture, correlated allele frequencies and with no a priori information on the sample location of individuals. The ΔK approach was used to estimate the most likely K value. For BAPS, a spatial clustering algorithm and a mixture analysis of individuals without geographic information was chosen, with 10 replicates from K = 2 to K = 17 ran. When we run clustering of 17 sample populations, K = 4 was calculated based on nuclear genes and K = 2 was justified based on mtDNA. We, therefore, set the maximum number of clusters to 2, 3 and 4 for BAPS.Nuclear haplotypes for heterozygotes were reconstructed using PHASE 2.150. These occurrences were separated into two partitions based on genetic results: northern I. scapularis population (n = 825) and southern I. scapularis population (n = 598). For climate layers we used bioclimatic variables for the present, the Mid-Holocene (~6Ka) and the Last Glacial Maximum (~21Ka) available from the WorldClim database 1.4 (http://www.worldclim.org) and resampled these to a 25 arcminute resolution (c. 50 × 50 km). We selected a subset of bioclimatic variables that show a Pearson’s correlation of r < 0.7, and are deemed biologically meaningful based on life history knowledge of the model system, in this case, seasonality: bio2 = mean diurnal temperature range (mean of the monthly difference between the minimum and maximum temperature), bio8 = mean temperature of the wettest quarter, bio9 = mean temperature of the driest quarter, bio16 = precipitation of the wettest quarter, and bio17 = precipitation of the driest quarter52 (Supplementary S2 Table We composed a data set of 1423 county-level reported occurrences of blacklegged ticks (See Supplementary S3)I. scapularis populations with Maxent 3.3.3k53. We restricted the feature type to hinge features as this facilitates extrapolation54. The area between 20 and 55 degrees latitude and -60 and -130 degrees longitude was chosen as environmental background. Ecological niche models were projected on the current and past climate layers. Statistical significance of ecological niche models was confirmed55 by testing their AUC values against a null model based on random localities created with ENMTools 1.356.We created ecological niche models for northern and southern Supplementary Table S3.Supplementary Information.