Galerucella birmanica Jacoby) is a pest of the water chestnut (Trapa natans L.). To analyze the phylogeny and biogeography of the beetle and provide evidence for the origin of T. natans in China, we conducted this by using three mitochondrial genes  and nuclear ITS2 ribosomal DNA of G. birmanica. As for mtDNA genes, the beetle could be subdivided into three groups: northeastern China (NEC), central-northern-southern China (CC-NC-SC) and southwestern China (SWC) based on SAMOVA, phylogenetic analyses and haplotype networks. But for ITS2, no obvious lineages were obtained but individuals which were from NEC region clustered into one clade, which might be due to sequence conservation of ITS2. Significant genetic variation was observed among the three groups with infrequent gene flow between groups, which may have been restricted due to natural barriers and events in the Late Pleistocene. Based on our analyses of genetic variation in the CC-NC-SC geographical region, the star-like haplotype networks, approximate Bayesian computation, niche modelling and phylogeographic variation of the beetle, we concluded that the beetle population has been lasting in the lower, central reaches of the Yangtze River Basin with its host plant, water chestnut, which is consistent with archaeological records. Moreover, we speculate that the CC-NC-SC population of G. birmanica may have undergone a period of expansion coincident with domestication of the water chestnut approximately 113,900–126,500 years ago.Water chestnut beetle ( Galerucella birmanica Jacoby (Coleoptera: Chrysomelidae), is an important pest of a floating annual aquatic plant, Water chestnut (Trapa natans L.), and is relatively host-specific, although it has been known to feed on water shield plants (Brasenia schreberi Gmelin) [G. birmanica is mainly distributed in habitat of T. natans, such as Yangtze River Basin and Pearl River Basin. In recent decades, many studies have focused on the biology [G. birmanica showing promise as potential biological control agents to the invasive plant, T. natans [G. birmanica, such as genetic diversity, population genetic structure and phylogeography.Water chestnut beetle,  Gmelin) , 2. In C biology –5, ecolo biology –8, and c biology . In Amer. natans , 11. HowT. natans), was introduced into North America around 1870 and has become invasive in the Northeastern United States and Canada [T. natans; for example, some scholars consider Africa and Eurasia as the origin of water chestnut, even though it is considered an endangered species in Europe [T. natans originated from Europe and Asia [The beetle’s host, Water chestnut   formallyCOI, COII and Cytb) and nuclear ribosomal internal transcribed spacer 2 (ITS2) region. On the other hand, our study may provide indirect evidence for the geographical origin of water chestnut, which has long been debated, based on genetic structure of the water chestnut beetle.Because mtDNA has some inherent characteristics including haploid uniparental inheritance and the absence of genetic recombination ; in addiG. birmanica and G. grisescens (outgroup) on T. natans from 20 populations in ten provinces and one city. This included most of the areas where water chestnut occurs in China including Liaoning, Shandong, Jiangsu, Zhejiang, Anhui, Hubei, Hunan, Jiangxi, Guangdong, Yunnan and Shanghai  were amplified from individual beetles using primer pairs C1-J-2183 (5’-CAACATTTATTTTGATTTTTTGG) and TL2-N-3014 (5’-TCCAATGCACTAATCTGCCATATT) [2, 4 μL 25 mmol/L dNTPs, 1 μL 20 μmol/L primers, 0.4 μL rTaq DNA polymerase (5U/μL), and ultra-pure water. PCR was conducted using the following conditions: 94°C for 4 min; 35 cycles of 94°C for 50 s, Tm for 50 s, 72°C for 1 min, and a final extension step of 72°C for 10 min. About 680 bp fragment of 5.8S-ITS2-28S region was amplified with primers: ITS2-F (5’-GCATCGATGAAGAACGCAGC) and ITS2-R (5’-TCCTCCGCTTATTGATATGC) [Escherichia coli DH5α cells. Finally, a total of 156 positive clones (three repeats) were picked and sequenced by IGE Biotechnology Co., Ltd  and Sangon Biotechnology Co., Ltd .Three fragments of the mitochondrial genome  , COII-F CCATATT) , and CytCCATATT) , respectGATATGC) . All ampThe three mtDNA gene sequences were initially aligned using CLUSTAL X v. 1.83  and thenCT. Analyses of molecular variance (AMOVA) were performed using Arlequin v. 3.5 [ST) values were also calculated. Moreover, using the formula FST= 1/ (1 + 2Nm), which is specific for organelle genetic data [m). Phylogenetic trees were constructed using MrBayes v. 3.2.1 [G. grisescens served as the outgroup. The best fit model for nucleotide alignments was determined by Modeltest 3.7 [Spatial analysis of molecular variance was performed using SAMOVA v. 1.0  to identn v. 3.5  based ontic data , we deriv. 3.2.1  and a PHv. 3.2.1 –48; G. gtest 3.7 . Accorditest 3.7 . Median-test 3.7  and usedG. birmanica. Step 1, a total of six scenarios were considered regarding the variation in population size and the split and admixture events  [http://www.esri.com/software/arcgis).We used ecological niche modelling to predict the geographic distribution of climatically suitable habitats for g MaxEnt . We obtaent.htm) . The ranent.htm) . The finD and Fu’s FS tests were used to test for neutrality. Population expansion time (τ) and the sum of squared deviation (SSD) between observed and expected mismatch distributions were similarly calculated. All parameters were evaluated based on 1,000 bootstrap replicates.The demographic history of all populations (pooled) and individual population groups identified by SAMOVA analysis was studied using mismatch distributions in ARLEQUIN. Tajima’s COI, COII and Cytb genes was 837, 681 and 462 bp, respectively. Among individuals from 20 populations, 33 COI, 23 COII and 20 Cytb haplotypes were identified; these were submitted to GenBank as accession numbers EF512802-EF512833, EF512778-EF512800 and EF512757-EF512776, respectively. The average pairwise sequence divergence among haplotypes of COI, COII and Cytb was 0.965, 0.883 and 0.947, respectively . 34 unique sequences generated during this research were submitted to GenBank (accession numbers: KR401224-KR401257). However, ITS2 haplotype and nucleotide diversity were relatively low (H = 0.660 and π = 0.00234).G. birmanica retain high levels of genetic diversity.We also calculated the number of unique haplotypes (n), haplotype diversity (H), nucleotide diversity (π) and the average number of nucleotide differences (K) for each population . OverallCT values were analyzed by SAMOVA, three population groups were suggested as the optimal number for COII (FCT = 0.88007), Cytb (FCT = 0.76822) and ITS2 (FCT = 0.31787); however, five groups were suggested when COI was considered (FCT = 0.78653) (When F0.78653) . Populat0.78653) . The thr0.78653) . Interes0.78653) . However0.78653) .COI, 78.65%; COII, 88.01%; Cytb, 76.82%) of the variation was distributed between groups  and within individual populations  (CT (between groups), FSC (among populations within groups) and FST (within populations) were all highly significant (P<0.01) for the three mtDNA genes  revealed that most (n groups . Genetic 14.10%) . FixatioNA genes . The hieNA genes .Median-joining networks had star-like conformations with limited substructures . For thrST values for the three regions were 0.732–0.963 for COI, 0.826–0.957 for COII, 0.728–0.952 for Cytb, but -0.025–0.200 for ITS2  ; Table 4G. birmanica populations had a larger distribution during the LGM with a range contraction between the LGM and its current distribution  for the test data was 0.991–0.994, indicating a high fit of the modelled and observed distribution , 58. Theribution . What’s ribution . As a whl period . In addiD statistical test generally resulted in negative values with no significance; an exception was COII in the CC-NC-SC region  and Fu’s FS  indicated that the whole set of G. birmanica samples studied here did not fit a simple model of neutral evolution.For mtDNA genes, neutrality tests using Tajima’s ilibrium . As for FS test was utilized, significant, negative values were obtained for COI  and Cytb  as well as multimodal curves were obtained for all groups  and a unimodal curve, which demonstrates population expansion within the CC-NC-SC region  and FS = 0.5887 in NEC (COII) may indicate recent population bottlenecks in the two regions (P > 0.05).Besides, positive  regions . The sumCOI, Cytb and ITS2 ribosomal DNA, respectively, but only 0.902 for COII  was 10.814, whereas τ for COII was 0.000.The τ value (mean of population expansion time) of CC-NC-SC region that was estimated for the sudden expansion model was approximately 4.387, 2.689 and 1.480 mutation units for for COII . The τ pG. birmanica has limited dispersal ability; e.g., normally 15–20 m under favorable conditions [Anopheles funestus [As mentioned above, greater genetic differentiation was observed between the three groups versus between populations within groups and within populations based on mtDNA genes but relatively lower revealed by ITS2 ribosomal DNA . The gennditions . It is afunestus  and mitefunestus , which mfunestus , 62.G. birmanica has three genetically-diverse, geographically-localized clades in China; these are the NEC, CC-NC-SC and SWC clades. According to AMOVA, more than 75% of the variability was observed between these three regions with some differences arising between populations based on the three mtDNA genes; this indicates that the three groups have infrequent gene flow and are distinct entities. Thus it is tempting to hypothesize that the natural barriers present in the study area  may deter or limit gene flow; whereas gene flow in the CC-NC-SC region was frequent. Similar results have been obtained for the Chilo suppressalis [Based on analyses of three mtDNA sequences using AMOVA, phylogenetic tests, and haplotyping, we conclude that ressalis , where rressalis . HoweverCOI, COII, and Cytb in the CC-NC-SC region is about 113,900 (τ = 4.387 mutation units), 28,800 (τ = 0.902 mutation units) and 126,500 years (τ = 2.689 mutation units), respectively. Due to the lower number of sample sites in the NEC and SWC regions and reduced haplotype diversity, divergence times in these two regions could not be accurately predicted.Based on a proposed standard rate of 2.3% divergence per million years (My) for the insect mitochondrial genome   and ITS2 haplotype 6 and 11 are derived from this group. In addition, the scenario tests based on ABC method revealed that the CC-NC-SC area is the most likely origin of the beetle in China. Coupled with this area acted as refugia during glacial period, therefore, we speculate that the water chestnut beetle originated in the lower, central reaches of the Yangtze River Basin. What’s more, populations at the center of origin generally exhibit the greatest variation, which is consistent with the perspective that ancestral populations possess higher genetic diversity than derived populations [Analyses of the ulations . The larT. natans in Asia, including wild T. natans, T. japonicum, domesticated T. bicornis and T. bispinosa. In China, 15 species and variants of water chestnut have been identified, whereas only wild T. natans occurs in Europe and North America. Furthermore, water chestnut is now considered an endangered species in Europe and Russia [Potamogeton, Nymphaea, and Nuphar, which also have a floating leaf habit [T. natans on a global scale. However, based on our work with the beetle and archaeological findings, we believe that the lower, central region of the Yangtze River basin is one of origin centers for T. natans. We show a proposed expansion route for the water chestnut beetle (G. birmanica) in T. natans in the Late Pleistocene. Our study provides insight into the origin of water chestnut by examining population genetics of its pest, water chestnut beetle. Our results suggest that the lower, central region of the Yangtze River basin is one of the potential origin centers for the beetle, and we speculate that this dispersal was concordant with the host plant. Most importantly, we provide a plant-insect co-evolutionary system example on revealing the origin and dispersal history of a pest insect and its host.Water chestnut beetles are likely to have co-evolved with the host plant, water chestnut; thus, it has been suggested that the insect and pest share a common origin, which we speculate is located in the lower, central region of the Yangtze River Basin. Recent studies suggest that the Yangtze Valley is a potential center of origin for domesticated, aquatic vegetables , 23, 67,d Russia . These daf habit . Howeveraf habit . It is aaf habit . A similaf habit . Furtheraf habit , 19. In S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOC)Click here for additional data file.


Acropora tenuis, one of the dominant stony coral species in the Pacific, using 13 microsatellite markers to investigate 298 colonies from 15 locations across the Nansei Islands in southwestern Japan. Genetic diversity was not significant among sampling locations, even in possibly peripheral locations. In addition, our results showed that there are at least two populations of A. tenuis in the study area. The level of genetic differentiation between these populations was relatively low, but significant between many pairs of sampling locations. Directions of gene flow, which were estimated using a coalescence‐based approach, suggest that gene flow not only occurs from south to north, but also from north to south in various locations. Consequently, the Yaeyama Islands and the Amami Islands are potential northern and southern sources of corals. On the other hand, the Miyako Islands and west central Okinawa Island are potential sink populations. The Kerama Islands and the vicinity of Taketomi Island are potential contact points of genetic subdivision of coral populations in the Nansei Islands. We found that genetic population structure of A. tenuis in the Nansei Islands is more complex than previously thought. These cryptic populations are very important for preserving genetic diversity and should be maintained.To establish effective locations and sizes of potential protected areas for reef ecosystems, detailed information about source and sink relationships between populations is critical, especially in archipelagic regions. Therefore, we assessed population structure and genetic diversity of  Coral reefs are highly productive ecosystems that provide habitat for a great variety of marine organisms. Humans derive many benefits from them, including fisheries and tourism  are a 1200 km chain of approximately 200 islands that exhibit high levels of biodiversity and endemism , suggested that the Kerama Islands are a historical sink population, but not a source population for Okinawa Island, the mere 30‐km distance separating them notwithstanding  and quantified using a NanoDrop™ 1000 Spectrophotometer . Universal primer sets for 13 microsatellite loci, developed for the Genus We also conducted PCR using a multiplexed approach, based on the primers of Shinzato et al. . As forwxl Genetic Analyzers (Thermo Fisher Scientific). Fragment sizes were determined using GeneMapper software version 5.0 (Thermo Fisher Scientific) by comparison with a GeneScan™ 500 LIZ® (Thermo Fisher Scientific) internal lane size standard.Allelic variations of PCR products were analyzed with Applied Biosystems 3130 and Applied Biosystems 3730FIS), observed heterozygosity (Ho), expected heterozygosity (He), and Hardy–Weinberg equilibrium. Evidence and frequency of null alleles were tested using Micro‐Checker v 2.2.3 . CLUMPP . Then, data were averaged from waters shallower than 40 m, each May from 1990 to 2011, using a 0.5° grid. Current vector for velocity and direction were developed using SAGA software  version 2.2.0 . We also found that this species tends to be distributed along the northwestern coasts of islands, despite the fact that other Acropora species are normally distributed along southeastern coasts . These results suggest that the Nansei Islands have two mixed populations that co‐occur in different ratios in different locations , with most of the total variation (4.043) occurring within localities (AMOVA). Genetic variation between localities was generally low ; nonetheless, 56 of 105 pairs of locations differed significantly . We distinguished three clusters of A. tenuis in the Nansei. These are discernible in a heatmap based on pairwise FST values , the Kerama Islands shared between the first and second clusters  was found between pairwise FST values and geographic Euclidean distance, based on the isolation‐by‐distance model (Appendix S7). It infers that geographically restricted gene flow does not generate a genetic structure in the Nansei.Genetic variation among sampling locations is low , but significant . Therefore, migration rates were calculated separately for the northern, central, and southern parts of the Nansei using MIGRATE‐N 3.6.11  did not differ significantly, we calculated their migration rates (Appendix S8B). Most migration rates were symmetric between pairs of locations .Acropora species in Caribbean reefs  and a high frequency of null alleles were found at several loci and sites . In addition, ranges without significantly different FST values show that the dispersal range of most of A. tenuis larvae is up to several tens of kilometers  than A. tenuis (69 days). Reasons for different population structures among species include the following: population size, reproductive power, density of mature colonies relative to fecundity, and adaptability to environmental changes. Furthermore, Suzuki et al. . Moreover, MIGRATE‐N was unable to estimate gene flow between the Amami Islands and other localities in the southern Nansei (Appendix S8B). These results suggest that the microsatellites that we analyzed may not have enough resolution to detect differences between them. More high‐resolution markers, for example, whole‐genome SNPs, might be needed to reveal more detailed population relationships, probably reflecting repeated historical events, such as local extinction and recovery.The Amami Islands in the northern Nansei and some locations in the southern Nansei  are not significantly different Fig. . HoweverA. tenuis disperses from north to south. The southward return current along northern Okinawa Island during the middle of the spawning season of A. tenuis in May, from 1990 to 2014 also suggests the likelihood of southerly gene flow (Appendix S9). In addition, the geostrophic current from the Amami Islands to Okinawa Island, which flows into the Tokara Straits in opposition to the Kuroshio Current, has been reported , in the Kerama Islands, and west central Okinawa Island. The second is around Taketomi Island (11). The Kerama Islands are not the only source population for Okinawa Island, but they may have an interdependent relationship.On the whole, our results suggest that the Yaeyama Islands and the Amami Islands are potential source populations. On the other hand, the Miyako Islands, the Kerama Islands, and central western Okinawa Island are potential coral sink populations. In the southern Nansei, corals are recruited from south to north, as previously thought, due to the Kuroshio Current. Inside Sekisei Lagoon, migration data indicate a southwestward migration Fig. A and C. Acropora tenuis has formed the present distributional patterns in the Nansei based upon habitat preferences, complex ocean currents, and geographic and historical features. Genetic compositions of populations have continuously shifted in the process of distributional transition. Consequently, gradual but significantly different genetic populations have formed. These cryptic populations are very important for preserving genetic diversity and should be maintained. Black  and Shinzato et al.  and expected heterozygosity (He) for each sampling location. These did not differ significantly between locations .Appendix S4. Analysis with STRUCTURE  in the Nansei.Appendix S5. Estimated population structure using STRUCTURE  for multilocus pairwise FST estimates for 13 microsatellite loci from Acropora tenuis.Appendix S7. Genetic distance is unrelated to Euclidian distance among Nansei populations of Acropora tenuis.Appendix S8. Summary of estimated directions of gene flow for Acropora tenuis in the Nansei Islands.Appendix S9. Complex local ocean currents in May in the Nansei Islands, during the Acropora tenuis spawning season.Click here for additional data file.


Interestingly, although the European contact had a major negative demographic impact, we detect a previous decline in Mesoamerica that had begun a few hundred years before.The genetic characterization of Native American groups provides insights into their history and demographic events. We sequenced the mitochondrial D-loop region (control region) of 520 samples from eight Mexican indigenous groups. In addition to an analysis of the genetic diversity, structure and genetic relationship between 28 Native American populations, we applied Bayesian skyline methodology for a deeper insight into the history of Mesoamerica. AMOVA tests applying cultural, linguistic and geographic criteria were performed. MDS plots showed a central cluster of Oaxaca and Maya populations, whereas those from the North and West were located on the periphery. Demographic reconstruction indicates higher values of the effective number of breeding females (Nef) in Central Mesoamerica during the Preclassic period, whereas this pattern moves toward the Classic period for groups in the North and West. Conversely, Nef minimum values are distributed either in the Lithic period (i.e. founder effects) or in recent periods (i.e. population declines). The Mesomerican regions showed differences in population fluctuation as indicated by the maximum Inter-Generational Rate (IGRmax): i) Center-South from the lithic period until the Preclassic; ii) West from the beginning of the Preclassic period until early Classic; iii) North characterized by a wide range of temporal variation from the Lithic to the Preclassic. Our findings are consistent with the genetic variations observed between central, South and Southeast Mesoamerica and the North-West region that are related to differences in genetic drift, structure, and temporal survival strategies (agriculture  The peopling of the New World is still a controversial debate today. In fact, there is no consensus on whether the first settlers arrived in one ,2 or sevCurrent mtDNA studies in the new continent have focused on two new lines of research. On the one hand, the genetic characterization of skeletal remains using ancient DNA techniques provides information on the composition and genetic structure of pre-Columbian populations –13. On tAccording to Kirchhoff , MesoameConsidering cultural, geographical and historical criteria, Mesoamerica can be divided into different cultural areas –24. In tIn this paper these seven regions have been used to present a systematic classification of the results and as a starting point to explore the possible existence of genetic structure in the region.It is necessary to clarify that, although the work is focused on Mesoamerica, some northern populations come from Aridoamerica. This is an arid region north of Mesoamerica covering a territory that is distributed between northern Mexico and southern USA, traditionally inhabited by nomadic or seminomadic peoples .Another important aspect concerning Mesoamerica is its chronological classification according to archaeological data. In brief, it can be classified into five periods ,23,41. TSome of the most representative American cultures flourished in Mesoamerica, for example the Aztecs and Mayas, in addition to numerous indigenous groups, some of whom are the descendants of those ancient cultures. These populations emerged in a homogeneous pattern of rituals, politics and architecture, and shared a similar lifestyle based upon agriculture, as well as a similar social and commercial organization. This relative cultural homogeneity is also supported by the archaeological and anthropological data .Studies based on the genetic variations in native Mexican populations demonstrate that there is a high degree of differentiation between populations and a paternal heterogeneity correlated with geography ,44. HoweIn recent years, great efforts have been made to reconstruct the history and genetic relationships between human groups . Among tThe mtDNA control region of 520 individuals representing eight Mexican indigenous groups was sequenced. Five of the groups were of Maya filiation: Yucatan Maya (n = 40), Quintana Roo Maya (n = 74), Campeche Maya (n = 37), as well as Tojolabal (n = 74) and Tzotzil (n = 87) groups from Chiapas. The other three indigenous groups are Mazateco from Oaxaca (n = 41), Purepecha from Michoacán (n = 65), and Huichol from Nayarit (n = 102) , Fig 1.Throughout the text, different synonymous terms are used to refer to the populations studied. Native American refers to native populations but not necessarily from Mesoamerica. The terms native and indigenous are used interchangeably to refer to the Amerindian populations. Moreover, Native Mexican includes populations currently distributed in Mexican territory which may belong to either Mesoamerica or Aridoamerica. Whenever we refer to mestizo-and therefore non-indigenous- populations, this is specified in the text.DNA was extracted from fresh-blood samples by means of the salting-out method. Prior to their inclusion in our study, all volunteers signed an informed consent form according to the ethical guidelines of the Helsinki Declaration. This work was approved by the Research Ethics Committee of the CUCiénega, Universidad de Guadalajara, as part of the project “Comprehensive approach of genetic anthropology of the Mexican populations based on mtDNA, STRs and Y-chromosome” .40 using Big Dye version 1.1 of ThermoFisher . Unincorporated fluorescent ddNTPs were removed with the Performa V3 96-well short plate . Sequence data were generated on either the Applied Biosystems 3130xl using POP-6 polymer or the 3730 Genetic Analyzer using POP-7 polymer. Sequences were verified by two different analysts and an additional quality control check was performed by EMPOP [The control region for all 520 samples was amplified  using thhttp://www.mitotool.org/index.html); absolute frequencies were obtained by means of the gene counting method  applying different population clustering criteria were also performed, as indicated in the tables of the results section . For diagnostic purposes, hgs were identified based on polymorphic sites and using different online resources  using Bayesian Evolutionary Analysis Sampling Trees (BEAST 7.1) . Estimatg region . For a dg region .The scaled effective population size was converted to Nef, assuming a generation time of 25 years. Importantly, an assumption about the mutation rate and the generation time will only affect the scale of the Bayesian skyline plot, but not its shape . To asseThe use of the mtDNA control region for reconstructing demographic history by means of BSP is, in certain circumstances, very suitable. The BSP is fundamentally based on coalescence theory, which includes most of the properties that the BSP must meet. One of the most important is the selection and representativeness of the sample; the samples should ideally be obtained from individuals that have been randomly sampled from a panmictic population. In fact, the balanced sampling strategy whereby samples are distributed across several populations provides the best scheme for inferring demographic change over a typical time scale . Sample Another essential aspect which is not usually taken into account when applying BSP is the possible existence of population sub-structure –62, sincGenerally, however, increasing the amount of information in the alignment, either by increasing sequence length or by focusing on variable regions, will improve the precision of phylogenetic estimation of the genealogy.In fact, like the mitogenomes, methodologically the control region is treated as if it were a single locus , providiOne of the potential problems in using the control region is that certain drawbacks such as having a greater quantity of hotspots can alter the phylogenies and overestimate divergence times . This prIn addition, some other independent papers validate the use of the control region as a tool with which to reconstruct the demographic history of populations. One of these studies conducted on South American native populations provides results that largely coincide with our knowledge of the history of Amerindian populations . In anotA final point to note is that the control region has also been used to test correlations between demographic and paleoclimatic events ,76, examThe skyline results, represented by the median values, are shown in graphs for cultural regions: North, West, Central, Oaxaca and Maya region . Differen—Nef n-1) / Nef n-1)*100Both maximum and minimum periods for Mesoamerica were represented in order to simplify the demographic history comparison between different groups . Maximumn and Nefn-1 being the effective number of breeding females for a particular period and for the previous one, respectively. These calculations were performed for the 28 groups. The time evolution of the IGR for each of the 28 indigenous populations grouped by cultural area is shown in Figure C in NefThe time distribution of the maximum and minimum IGR values (IGRmax and IGRmin) by Mesoamerican periods is presented in www.empop.org). In addition, 20 Mesoamerican populations were included in the global analyses. The geographic, cultural and linguistic information of the total population sample is shown in We obtained the polymorphic sites in the mtDNA hypervariable regions I and II (HVRI and HVRII) detected in the 520 individuals from eight Native Mexican populations . Complete sequencing of the control region allows us to determine the most important sub-haplogroups (subhgs), for example D4h3a and B4b1. D4h3a was found in twelve individuals (2.3%), eight in Tzotziles (9.2%) and four in Mazatecos (9.8%). Conversely, the subhg B4b1 was detected only in Mayas from the Yucatan , followed by B2 , C1  and D1 , with significant intergroup variations . Tojolab Yucatan . Four AfThe highest genetic diversity rate (Ĥ) was detected in Mayan populations from the Yucatan Peninsula (Ĥ = 0.9974 ± 0.006), and the lowest in Tojolobal (Ĥ = 0.8911 ± 0.017) and Huichol_h (Ĥ = 0.7994 ± 0.026) . HoweverThe only populations with no significant differences, expressed by corrected average pairwise differences, are those from the Yucatan Peninsula . However, significant results were found when the same cluster (Culture II) was divided into two Mayan population groups: those from the Yucatan Peninsula and the rest . We also obtained significant results in the clusters 'Culture Area'  and 'Language' . A second set of analyses was then undertaken using these eight Mexican indigenous populations and a further 20 Native American groups. The latter came from the same geographical area and/or cultural background. The first step in this analysis was to calculate FST genetic distances (Table C in p <0.0001), which is an indication that the populations at the periphery of the distribution are those with less diversity. Thus, the MDS reflects not only inter-population genetic relationships, but also the genetic diversity of the populations. This latter parameter is related to demographic history and shows the effects of genetic drift. Moreover, the Mantel test, contrasting the FST distance matrix and the geographical distances between the 28 Native American groups, showed a significant correlation .We performed some AMOVA tests for the eight Mesoamerican populations according to different clustering criteria (Table B in S1). The results were not significant when Mayan populations were compared with the rest  were calculated  and Pima groups (Pima_k and Pima_a) from western and northern regions, respectively. Conversely, population samples closer to the Mesoamerican core  such as Nahua , Otomí (Otomi_v and Otomi_s) and Maya groups  show more homogeneous results; Tojolabales were the only exception.CT = 0.0858, p = 0.0000) and 'Cultural area' , while 'Linguistic groups' displayed no-significant p-value .Two of the new set of AMOVA tests performed on the 28 Native American populations indicated significant differentiation between groups   Figs  and 5. TCenter. In this region, the majority of the groups have high population sizes, with the exception of the Tepehua who show a maximum Nef of 31,302 individuals, compared with the 255,025 individuals for Nahuas_hu . In some cases the results were pooled by cultural region in order to facilitate a comparative analysis.The 520 samples analyzed herein belong to one of the six Pan American hgs, confirming the common origin and close relationship between Native Mexican groups. This contrasts with the hypothesis formulated in other studies supporting the idea of a dual origin for Mesoamerican populations .The small African admixture found in three Mexican indigenous groups (< 3%) is in agreement with previous results based on both mtDNA  and autoSome authors have described a pattern in the mtDNA frequency distribution in North America that consists of a north-south gradient for hgA2, a south-north gradient for hgC1 and hgD1, and a lack of distribution for hgB2 , 85. AccSix hgs have been described in this paper  and, as expected, haplogroup X2a has not been detected. This result is not surprising, however, since its distribution is typically restricted to northeastern North America . It is iD4h3 is a founder haplotype with an early presence in Native Americans , 12, andAlthough the B4b1 subhg is a clade initially defined exclusively for southeast Asian populations , it has ST shows the importance that geography had in population differentiation. In this context, indigenous populations started differentiating within and between cultural areas, which favored the important linguistic and cultural diversity nowadays found in the region. The fixation indices calculated for the eight populations support this approach, yielding significant values mainly between populations of different cultural areas. Although no studies analyze this phenomenon in earlier periods, the studies carried out for the Classic and Postclassic period support the idea that population movements took place primarily within these cultural areas and were influenced by geography, trade and politics [Our results show a colonization model characterized by a certain basal homogeneity common to all populations, but with differences in terms of cultural sub-regions . The propolitics ,40,94. TThe study of the influence of language also supports this idea; initially, at a micro-geographic level, language had an important role as a genetic barrier as suggested by the significant AMOVA results of the eight populations. However, the importance of this cultural component is diluted when the study region is expanded. These results are consistent with the hypothesis claiming that genetic differentiation in Native American groups occurred before the linguistic differentiation .Zea mays), and courgette (Cucurbita pepo), bean (Phaseolus vulgaris) and pepper (Capsicum annuum) crops led to a more sedentary lifestyle, an increase in social and urban complexity and the development of trade and migration routes. These trade-associated migrations were mainly performed within the same cultural areas and, on a second level, between areas [A second crucial event, which had important consequences in the population, was the adoption of agriculture. The development of maize domestication , the demographic decline began in Mesoamerica a few hundred years before. This result can be contrasted with paleo-climatic studies conducted in both Mesoamerica and the Mayan region. In Mesoamerica it has been suggested that during the middle Holocene weather conditions were relatively stable, but that a dry period began about 5,800 ybp . These dIn summary, our study–applying for the first time Bayesian skyline methodology to mitochondrial D-loop sequences–highlights the demographic changes that took place over time and in different geographical areas of Mesoamerica. These were the result of the complex interrelationship between geography, subsistence strategies, social structure and culture.S1 FileTable A. Genetic differentiation among the Mesoamerican populations studied herein. Corrected pairwise differences average  and p-values , among the eight Mexican Native populations based on the control region of mtDNA sequences.Table B. Population structure among the eight Mesoamerican populations studied herein. AMOVA based on historic, geographic, cultural and linguistic criteria for the eight indigenous populations studied herein.Table C. Genetic differentiation among 28 Native American populations. FST values between the 28 Native American populations based on the control region of mtDNA sequences.Table D. Population structure among Native American populations based on different criteria. AMOVA based on historic, geographic, cultural and linguistic and criteria among 28 Native American populations included in this study.Table E. Genetic relationships based on shared haplotypes between Native American populations. Number of shared haplotypes between the 28 Native American populations based on HVRI dataTable F. Estimates of Nef values for each of the Native American groups studied herein. Female effective population size (Nef) and corresponding maximum and minimum estimated for the studied Native American populations based on mitochondrial control region data.Table G. Demographic and temporary parameters estimated in the Native American groups studied herein. Temporary distribution of female effective population size (Nef), intergenerational growth rates (IGR) with maximum and minimum values, and period in which the IGR trend inversion occurred (major demographic changes) estimated in the Native American groups studied herein based on mitochondrial control region data.</SI_Caption>(PDF)Click here for additional data file.S2 FileFigure A. Bayesian skyline plot based on random samples from the 28 studied indigenous populations. A different number of samples were randomly selected to determine that sample size has no effect on the demographic profile.Figure B. The 28 studied indigenous populations grouped by cultural areas. The Nef median value is represented in the Bayesian skyline plot including confidence intervals.Figure C. IGR values temporary evolution for each indigenous group grouped by cultural areas. The Y-axis represents IGR percentage value and X-axis time in ybp.Figure D. Temporary distribution (bottom) according to Mesoamerican periods (top) in which the trend inversion occurred. The vertical red stripe indicates periods previous to 8,000 ybp.</SI_Caption>(PDF)Click here for additional data file.S3 FileAbsolute frequencies observed in each Mexican population studied herein are presented.(XLSX)Click here for additional data file.


Anableps anableps. This species is fully estuarine-resident, has internal fertilization, is viviparous and does not migrate across long distances. These features make the four-eyed fish an excellent model for the study of evolutionary processes related to genetic differentiation of species and populations in estuaries. The evolutionary history of A. anableps was inferred from phylogeographic and population analyses using sequences of the mitochondrial DNA Control Region of 13 populations distributed in the Amazon and Northeast Coast of Brazil from Calcoene (Amapa) to Parnaiba (Piaui). The 83 retrieved haplotypes show a pattern of four distinct mitochondrial lineages, with up to 3.4% nucleotide divergence among them. The evolutionary reconstruction suggests that these lineages diverged recently in the late Pleistocene/early Holocene after the Atlantic Ocean reaching current levels. Analysis of variability, neutrality and the genetic expansion pattern revealed that the lineages have distinct characteristics, which were shaped by the different geomorphological features of coastal regions combined with sea level oscillations over a very long period of time. Only few neighboring populations show a discreet gene flow. This study may also be helpful for designing new experiments to better understand the geomorphological evolutionary history of the estuaries of the Amazon and the Northeast Coast of Brazil using estuarine-resident species as a model.Historical events, such as changes in sea level during the Pleistocene glacial cycles, had a strong impact on coastal habitats, limiting connectivity and promoting the genetic divergence of various species. In this study, we evaluated the influence of climate oscillations and the possibility of estuary function as a barrier to gene flow among populations of the four-eyed fish,  Organisms that inhabit coastal waters such as estuaries or lagoons may show more genetic differentiation than strictly marine species due to the discontinuity of these natural ecosystems and the relative isolation of the great ocean currents Historical events, such as changes in the sea level during the Pleistocene glacial cycles, may have exerted a strong impact on coastal habitats by limiting connectivity and promoting genetic divergence between species The fluctuations in sea level in Brazil caused changes in coastal and estuarine regions, mainly in the Amazon coast. The Amazon River Delta is located in this region, which has the largest discharge of freshwater in the world In the Para Platform (estuaries 4 to 7), great cliffs are maintained by sediment from the Pirabas/Barreiras formation, which acts as barriers to the external action of waves and tidal currents. In this area, the estuaries are narrow, with a length of only two kilometers. The estuarine channels may reach up to 60 km on the mainland and have no interconnections between adjacent estuaries Anableps anableps Linnaeus, 1758. This species belongs to the family Anablepidae and is considered an estuarine-resident species Several studies indicate that marine environments can operate as barriers inhibiting gene flow between organisms that spend their entire life cycle in estuary ecosystems, as is the case for the four-eyed fish, A. anableps has attracted the attention of many researchers because its retina is split horizontally, allowing for an aerial view and an aquatic view simultaneously A. anableps extends from the Gulf of Paria in Venezuela to the Parnaiba Delta in the state of Piaui, Brazil. Moreover, the Anableps genus has two other species: A. microlepis Müller & Troschel, 1844, which is considered the sister species of A. anableps with whom it lives in sympatry in South America, and A. dowie Gill, 1861, which occurs in the Pacific  and is considered the most primitive species of the genus A. anableps shows that this fish has a low potential for dispersal, mainly because it does not have a period of planktonic larval dispersal (PLD) and is stenohaline and resides in estuarine regions throughout its life cycle; therefore, phylogeographic studies are of great importance in uncovering the role of historical events in the diversification of this species. Nevertheless, A. anableps is an excellent target for phylogeographic studies due to its previously described biological and ecological characteristics. In this context, the phylogeographic study of A. anableps will be of great importance for understanding the patterns of genetic variability, gene flow and levels of connectivity among populations in the estuaries of the Amazon and Northeast Brazil. Coalescence analyses may also shed light on how the evolutionary history of the estuaries correlates with the sea level alterations that occurred during the Pleistocene and the Holocene.The life history of Comparison among the 393 sequences obtained in the present work showed 70 variable sites (52 parsimony informative and 18 singleton variable sites) that defined 83 haplotypes. Forty-two haplotypes (43%) are represented by a single sequence in the whole sample, which are mostly concentrated in the Para Platform. The haplotype diversity varied from 0 in the Parnaiba estuary to 0.933 in the Amazonas river estuary.Nine haplotypes were detected for the two populations of the Amapa state coast: haplotype 1, which is the most frequent, is observed in the Calcoene and Amapa estuaries, which also share haplotype 3. Haplotype 2 was observed only in Calcoene estuary while haplotypes 4 to 9 occur only in specimens of the Amapa river estuary. The populations from the Amazonas river mouth to the Maracana estuary (3 to 6) do not share haplotypes as shown in Results from AMOVA show that the genetic variation among regions (54.07%) was higher than the variation among populations within regions (17.43%). However, the variation within populations (28.5%) was larger than the variation among populations within regions (17.43%) .Pairwise Fst values between populations were all significant except for the comparisons between the Calcoene (1) vs the Amapa (2), Urindeua (7) vs Japerica (8) vs, and Caete (10) vs Sao Marcos (12) . The higLineage 1 (the Amapa region), Lineage 2 (the Amazon river mouth estuary), Lineage 3 (the Para Platform) and Lineage 4 (from Japerica to Parnaiba). The haplotype network showed that the Amazon estuary lineage (in blue) was slightly differentiated from the Para Platform population, but they are derived from a common ancestor. The populations from the Para Platform clearly show some degree of sub-structuring as a consequence of the relative isolation of populations from this region, a pattern demonstrated by a high number of unshared haplotypes. Conversely, the populations from Japerica to Parnaiba show a large number of shared haplotypes and a star-like pattern indicative of substantial gene flow. However, a surprising result was a close phylogenetic relationship of some haplotypes from Amapa and Calcoene  with some haplotypes from the Caete estuary . As these geographic areas are 500 km apart and no evidence of present day connectivity is observed between these two regions, this certainly suggests a historical relationship between these two populations.The haplotype network  clearly Maximum likelihood analysis produced the unrooted phylogenetic tree depicted in The results of Fu's Fs and R2 are presented in In perfect agreement with Fu's F and R2 results, the Bayesian Skyline Plot showed evidence of demographic expansion only in Lineage 4 (Japerica to Parnaiba), see . HoweverBayesian analysis of genetic structure identified four clusters : ClusterA. anableps from the North and Northeast coasts of Brazil.The genetic analyses using the mtDNA Control Region revealed a phylogeographic pattern with four distinct mitochondrial lineages in populations of Anableps along the coast. Therefore, it is possible that allopatric refuges due to variations in sea level during the Pleistocene-Holocene associated with the biology of the species, such as viviparity and low potential for dispersal may have been the main force that promoted the recent divergence observed between populations and for the phylogeographic pattern observed for this species today. The divergence among lineages of the four-eyed-fish are comparable to those found in other species such as Chelon haematocheilus, a fish species from Asia Many studies with estuarine fishes suggest Pleistocene glacial events as the main cause of the genetic structuring A. anableps can be explained by the geomorphological scenario of the study area, which was dramatically affected by sea level changes The Mantel test showed some evidence of isolation by distance when all populations where analyzed together , but when each region was analyzed separately, no evidence of IBD was found. The structure observed in A. anableps. Therefore, the genetic data obtained here clearly distinguish the Para Platform and the Caete Basin but do not show differences between populations of the Caete Basin and those of Sao Marcos and Parnaiba. Thus, the genetic pattern of Anableps mitochondrial lineages helps to distinguish four biogeographical regions in the whole area of study: Oceanic Amapa , the Amazonas river Mouth (near Macapa), the Para Platform  and Caete-Parnaiba .The geographic distribution of Lineages 2 (Amazonas), 3 (Para Platform) and 4 (Caete Basin to Parnaiba) matches the geomorphological landscape of the region. Therefore, it is highly probable that the wide distribution of tidal plains and the absence of interconnection between the estuaries have been essential for the isolation of Lineage 3 in the Para Platform. In contrast, there is an opposite pattern in the Caete Basin, which favored the demographic and spatial expansion of Lineage 4. Importantly, in contrast to the proposal of at least five geomorphological sectors The unexpectedly high genetic similarity between the four haplotypes from the Caete estuary and the haplotypes from Amapa (Lineage 1) may represent historical signatures of colonization of the Amapa estuaries by ancestral haplotypes from Caete that are widely distributed or are at least distributed from Caete to Amapa thousands of years ago, when the sea level was 130 m below the present level. Extinction of intermediate populations due to geomorphological changes, gradual increases in the sea level, formation of the Amazon plume, and oceanic currents may also have contributed. Lineage 1 is practically restricted to the Amapa State coast with no evidence of significant gene flow to neighboring populations, which is corroborated by the Bayesian Skyline Plot, suggesting that these populations did not experience a significant demographic expansion since colonization.A. anableps in northern Brazil, and both seem to be related to global events such as glaciations. The drop in the sea level during the Last Glacial period, which reached its lowest level (−130 meters) in Last Glacial Maximum .The present study revealed high genetic divergence among populations of A. anableps confirms that estuarine-resident organisms exhibit a degree of differentiation that is higher than strictly marine species Anableps.The discrepancy of the gene flow pattern observed along the coast of Para fits well with the geomorphological features that are known as the Para Platform and the Caete Basin. The former has no interconnections between adjacent estuaries, while in the latter, these connections are evident. This analysis identifies only four biogeographic regions  instead of the several geomorphological sectors described by geologists. The divergence observed in populations of Animal samples were collected and manipulated  under a permit (N° 12773-1) provided by the Environment Ministry (MMA) that was given to Iracilda Sampaio. All work was performed in compliance with and approved by the Ethics Committee of the University Federal do Para.For the present study, samples of muscle tissue were obtained from a total of 393 specimens collected in 13 estuaries from four Brazilian states: Amapa, Para, Maranhão and Piaui, practically covering the entire range of the geographic distribution of the four-eyed fish in Brazil ; Fig. 4.5′-CCTAACTCCCAAAGCTAGGTATTC-3′) and D-Loop H1(5′-TGTTTATCACTGCTGAGAGTTCCCT-3′) Total DNA was isolated from muscle tissue by digestion with proteinase K using a modified protocol with phenol chloroform and precipitation with sodium acetate and isopropanol F-statistics from haplotypes with 1000 permutations using Arlequin v. 3.5 The sequences obtained were aligned in the program CLUSTAL W The optimal model of sequence evolution was determined using the Akaike Information Criterion with Kakusan v.4 To test for past population expansion, two statistical tests were used: Fu's Fs, which uses the distribution of haplotypes , and the R2 Statistic, which is based on the relationships between singletons and the average number of nucleotide differences. Fu' Fs and R2 statistics −8The reconstruction of genealogy and demographic history was made in a Bayesian framework using the Bayesian Skyline Plot implemented in Beast v1.7.5 Ne) against time were drawn for each one of the following lineages: 1 , 3 , and 4 . Lineage 2 was not tested because it has only 10 individuals. The analyses were run in Beast v1.7.5 for 100 million iterations using default parameters and priors . The results of the analyses were visualized using Tracer v1.5 Effective population sizes (A. anableps correspond to a model of isolation by distance (IBD). Four options were used: 1) all 13 populations; 2) Caete Basin; (3) Para Platform; and (4) Amapa Basin. The matrices used were pairwise Fst and geographic distances among populations.The Mantel test was performed in Arlequin v. 3.5 To infer the number of subpopulations and to assign individual samples to these groups, we employed the BAPS6 software Figure S1Haplotype genealogy of the mitochondrial sequence based on a maximum-likelihood tree. Circles represent haplotypes; the size is proportional to the number of individuals, and small blue dots represent intermediate, unsampled haplotypes. The four lineages are identified by distinct colors.(TIFF)Click here for additional data file.Table S1(DOC)Click here for additional data file.


Sphenodon punctatus) from ten offshore islands of New Zealand. We observed a positive relationship that showed a proportional increase in the neutral diversity at synonymous sites (dS), with increasing geographical distance. In contrast we showed that diversity at evolutionarily constrained sites (dC) was elevated in the case of comparisons involving closely located populations. Conversely diversity was reduced in the case of comparisons between distantly located populations. These patterns were confirmed by a significant negative relationship between the ratio of dC/dS and geographic distance. The observed high dC/dS could be explained by the abundance of deleterious mutations in comparisons involving closely located populations, due to the recent population divergence times. Since distantly related populations were separated over long periods of time, deleterious mutations might have been removed by purifying selection.Population genetic models predict that populations that are geographically close to each other are expected to be genetically more similar to each other compared to those that are widely separate. However the patterns of relationships between geographic distance and molecular divergences at neutral and constrained regions of the genome are unclear. We attempted to clarify this relationship by sequencing complete mitochondrial genomes of the relic species Tuatara ( Tuatara are significant in terms of global biodiversity and the evolutionary history of reptiles35It is well known that genetic similarity decreases with increasing distances between the habitats of individuals or population8910http://www.doc.govt.nz/documents/science-and-technical/TSRP47.pdf). As a result of fragmented and small population sizes tuatara are a highly endangered reptile and therefore obtaining samples is difficult. We managed to obtain tuatara samples from ten different islands. We sequenced the complete mitochondrial genomes of a single tuatara sample from seven islands, two from Middle mercury, three from North Brother and 22 from Stephens Islands (which has the largest population). To examine the phylogenetic relationships among tuatara from ten distinct islands, a maximum likelihood tree was constructed using complete mitochondrial genome sequences  and reconstructed the ML tree using only the constrained sites. As shown in Since tuatara are the only surviving member of the order Sphenodontia there is no outgroup species available to root the tree. This is because the divergence time between tuatara and its closest relative, the Squamata reptiles is ~220 million years3ρ = 0.71) and dS (ρ = 0.92). These were highly significant using Mantel's test (P < 0.001). However a closer examination revealed that the relationship of dS with geographic distance is approximately linear and that of dC is non-linear. While dS estimates varied between 0.001 to 0.06 substitutions per site (s/s), dC range was much smaller between 0.001–0.01 s/s. However in ρ = −0.82) that was highly significant (P = 0.001). The declining trend of dC/dS could be seen clearly after rescaling the Y-axis to exclude the outlier in order to focus other data points (ρ = 0.56) as well as between geographic distance and net-dS (Spearman ρ = 0.91) were highly significant (P < 0.001) using Mantel's test. A highly significant negative relationship  was observed between geographic distance and the ratio of net-diversities at constrained and neutral sites (dC/dS).Our samples are from a wide geographic range, including locations on off-shore islands in the North Island and Cook Straight of New Zealand. While the shortest distance between two islands was 1.3 km (Green and Middle Mercury Islands) the longest was 631 km between Poor knight and Stephens Islands . We exama points    used in this study= 0.001) . This suFST) between tuatara populations in order to examine the relative contributions of drift and migration (gene flow) using the D-loop data. The mean inter-population D-loop diversity was estimated to be 0.03. In contrast, the average intra-population diversity was only 0.005. The resulting FST was 0.85, which suggests a much smaller proportion of within population diversity. This suggests that between-(island)population diversities estimated in this study were not significantly affected by intra-population diversities. To further examine this we correlated pairwise FST estimated for populations from different islands with the geographic distance between the islands. We observed a highly significant positive relationship  suggesting increase in population structure corresponds with the increase in the geographic distance between the island populations  and hence any significant effects of population size on our results is unlikely. The higher dC/dS ratios (1.0) observed might also suggest the possibility of positive selection. However it is well known that the effect of genetic drift is much higher in small populations, which reduces the efficacy of natural selection13The positive relationship between geographic distance and neutral diversity suggests a direct relationship between geographic distance and time of divergence between the island populations. Further evidence for this prediction comes from our dC analysis. It is well known that at short timescales deleterious mutations are expected to segregate in populations due to the power of genetic drift. However they are selected against over time and are eventually eliminated from a population1315Studies of population genetics, evolution and ecology routinely examine the relationship between genetic and geographic distances in order to infer the demographic history of populations. However many studies use genetic data to estimate population genetic parameters without distinguishing whether the sequences are under selective constraints or evolve neutrally. Here we have shown that these two types of genomic regions behave quite differently. This suggests that future studies need to test their sequence data for neutrality to obtain unbiased relationship between genetic and geographic distances.The temporal pattern of deleterious non-synonymous polymorphisms has been demonstrated by a number of previous studies151718Blood samples were collected from 34 tuatara individuals from ten islands around New Zealand (number of samples in parenthesis): Poor Knights (1), Cuvier (1), Stanley (1), Green Mercury (1), Plate (1), Hen (1), Red Mercury (1), Middle Mercury (2), North Brother (3) and Stephens Islands (22) . DNA washttp://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) was used. For limiting the DNA amplification to the long fragments and avoiding the amplification of shorter sequences, particular PCR conditions were used. This resulted in competition between the templates in each cycle in favor of the long template from the preceding cycle. After purification each fragment was sequenced in both directions using forward and reverse primers using an ABI 3730 sequence analyzer. This was important to verify (any) nucleotide variants. A third PCR was performed in the case of any inconsistencies. All methods were carried out in accordance with the approved guidelines of Griffith University, Nathan, Australia. All experimental protocols were approved by Griffith University, Nathan, Australia.The list of primers used are given in the SeaViewThe complete mitochondrial genomes belonging to 34 tuatara individuals along with the reference (NC 004815) genome were aligned using the program Ad = XYd − (Xd + Yd)/2, where XYd is inter-population diversity and Xdand Yd are intra-population diversitiesTo estimate pair-wise diversities at synonymous sites (dS) we used third codon positions (3182 bp) or four-fold degenerate sites (1161 bp). Although both produced almost identical results we used the former for all analysis due to its size. For constrained sites (dC) we concatenated nonsynonymous (first two codon positions) sites of protein-coding genes, all tRNAs and two rRNAs . To estimate genetic diversities within and between populations we used HKY + Gamma model for constrained and synonymous sites using the alpha values of 0.05 and 0.11 respectively. This model was chosen using the program MODELTEST and it corrects for multiple substitutions, accounts for base compositional bias as well as corrects for rate variation among sites. To estimate the net diversity between populations we used Nei and Li's method DistanceFromTo (http://www.distancefromto.net/). To test the significance of any correlation between geographical distance and genetic divergence we used the program Isolation by distanceGenepopP < 0.007). To estimate pairwise FST between tuatara populations we used the software DNASPGeographic distances between individuals from pairs of islands were obtained from the online resource S.S. and D.L. conceived the project. E.M. performed sequencing and all other laboratory analyses. C.D.M. contributed to sample collection and laboratory analyses. S.S. conducted data analysis and hypothesis testing. S.S. wrote the paper with inputs from all authors. D.L. and C.D.M. edited the paper.Supplementary information


Panthera leo) in West/Central Africa is largely based on mitochondrial markers. Previous studies using mtDNA only have shown this region to hold a distinct evolutionary lineage. In addition, anthropogenic factors have led to a strong decline in West/Central African lion numbers, thus, the conservation value of these populations is particularly high. Here, we investigate whether autosomal markers are concordant with previously described phylogeographic patterns, and confirm the unique position of the West/Central African lion. Analysis of 20 microsatellites and 1,454 bp of the mitochondrial DNA in 16 lion populations representing the entire geographic range of the species found congruence in both types of markers, identifying four clusters: 1) West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India. This is not in line with the current taxonomy, as defined by the IUCN, which only recognizes an African and an Asiatic subspecies. There are no indications that genetic diversity in West/Central Africa lions is lower than in either East or Southern Africa, however, given this genetic distinction and the recent declines of lion numbers in this region, we strongly recommend prioritization of conservation projects in West/Central Africa. As the current taxonomic nomenclature does not reflect the evolutionary history of the lion, we suggest that a taxonomic revision of the lion is warranted.The evolutionary history of a species is key for understanding the taxonomy and for the design of effective management strategies for species conservation. The knowledge about the phylogenetic position of the lion ( Panthera leo) that occupy large geographic ranges within which disjunct populations may not allow for natural dispersal and gene flow. Increasing habitat fragmentation and variable anthropogenic factors have created a growing need to manage lions at the population level [Identifying and describing patterns of mitochondrial (mtDNA) and nuclear genetic variation is a crucial component to fully understanding the evolutionary history of a species. High quality phylogeographic data that represent the underlying genetic complexity are important for taxonomy and contribute to designing effective conservation strategies. This is of particular importance for species such as the lion (on level . In addion level –4.Panthera leo leo), ranging throughout sub-Saharan Africa with the exception of dense rain forest, and the Asiatic lion (Panthera leo persica), which exists as a single population in the Gir forest, India. Although all African lion populations are considered as belonging to the African subspecies (P. l. leo), distinct subgroups have been recognized based on morphology [Syncerus caffer) [Hippotragus equinus) [Alcelaphus buselaphus) [Giraffa camelopardalis) [Acinonyx jubatus) [Two subspecies of lion are officially recognized by the IUCN, based on genetic data ,6: the Arphology ,8 and gerphology –5,9–12. rphology . Lions frphology ,8. This rphology   ,16, roanequinus) , hartebeelaphus) ,19, giraardalis) ,21 and cjubatus) ,23.et al. (2008) to explain incongruent patterns in their lion data based on mtDNA and autosomal markers. Taxonomic revisions have potentially far-reaching ramifications with regard to management  and, therefore, should be approached cautiously. Ideally, proposed revisions should be supported by a combination of biogeographic, mtDNA and autosomal DNA, and morphological data.Due to the genetic differentiation within the African lion and the nested position of the Asiatic lion subspecies within the West/Central Africa clade based on mtDNA, the current taxonomic division is challenged –4. HowevIn this study, we analyzed 20 microsatellite loci for lions from thirteen wild populations, one of which is located in West Africa (Benin) and four in Central Africa . Furthermore, we included microsatellite data from another West African population in Senegal and from two distinct zoo populations of Ethiopian lions representing the region where the two major genetic lineages  may connect. To compare the phylogenetic clusters derived from the microsatellite data and to check for congruence with previously published patterns, we included data from 1,454 base pairs (bp) of the mitochondrial DNA for each sampling location. Using this approach, we are aiming to contribute to the ongoing discussion about lion taxonomy by answering four questions: 1) Do autosomal data support previously described phylogenetic groupings in the lion in general and the distinct position of the West/Central African lion in particular?, 2) Can an effect of sex-biased gene flow be detected?, 3) How genetically distinct are the sampled populations, at both the continental and regional scales, and do levels of genetic diversity vary amongst regional subdivisions, with a special focus on West/Central Africa? and 4) Are there signs for reduced genetic diversity in particular lion populations with an emphasis on West/Central Africa? Our study is the first to include multiple lion populations from West/Central Africa, using both autosomal and mtDNA markers in a phylogenetic context covering the entire current geographic range of the lion.We processed a total of 48 samples from eight populations, including one population from West Africa (Benin), four populations from Central Africa , two populations from East Africa (Ethiopia2 (captive) and Kenya) and one population from Southern Africa (Zambia). Except for Ethiopia2, all samples originated from free-ranging lions, with no known history of anthropogenic introductions of lions from other populations. Samples were collected in full compliance with specific permits . Details on permits, sample storage, DNA extraction, polymerase chain reaction (PCR) amplification, fragment analysis and quality control are given in Generated microsatellite data were supplemented by published data for the same 20 loci from another six populations , togetheA distance in POPTREE2 using 1,000 bootstraps [STRUCTURE 2.3.3  was usedotstraps .et al. (2011) (see www.fluxus-engineering.com). A repeat region of cytosines of variable length was excluded due to unknown homology (positions 1382–1393) and all remaining characters were included with equal weighting.For each sampling location, a mitochondrial region of 1,454 bp that encompassed cytochrome B (cytB), tRNAThr, tRNAPro and part of the control region was included for a number of individuals . DetailsST and Nei’s genetic distances were computed with GenAlEx 6.501 [For AMOVA of Dataset 1, individuals for which all 20 loci were analyzed were included as either 1) without an indicated substructure , 2) following IUCN classification , 3) following a North/South division as was indicated from the haplotype network, or 4) using the four groups identified by STRUCTURE . Isolation By Distance (IBD) was assessed by correlating geographic to genetic distances and using a Mantel’s permutation test with 999 permutations, as implemented in GenAlEx 6.501 . In addiEx 6.501  for micrEx 6.501 .p) was calculated with HP-Rare 1.1 [IS per population was calculated in FSTAT [The average number of alleles per locus (Na) was calculated using ARLEQUIN 3.5 . PrivateRare 1.1  includinRare 1.1  was usedRare 1.1 . To obtain FSTAT  and the in FSTAT ,44. The Based on the STRUCTURE results of Dataset 1, Structure Harvester identified that the observed genetic structure is best described by four clusters representing the following geographic areas: 1) West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India . There iA total of 87 sequences of 1,454 bp were analyzed. Nucleotide diversity (π) was 0.102. Based on 43 polymorphic sites, 15 different haplotypes were distinguished. The haplotype network  and the Results from AMOVA of the microsatellite data show that using the clusters identified by STRUCTURE to assign populations to four groups resulted in a relatively high percentage of the molecular variance being attributed to among-groups for microsatellite data (17.4%) and mtDNA data (52.3%) . While iST values ranged from 0.064 to 0.736 and were significant for all pairwise comparisons   . Within to 2.018 .IS values illustrated a significant heterozygosity excess in Benin (P<0.01) and Cameroon1 (P<0.01) and a significant heterozygosity deficiency in Zambia (P<0.01), RSA1 (P<0.05) and Ethiopia1 (P<0.05). Results of the bottleneck analysis showed that there was a significant excess of heterozygotes found in Cameroon1 (P<0.01), Kenya (P<0.05) and Ethiopia1 (P<0.05), possibly indicating a recent reduction in population size.Diversity indices  show thaHere we describe the distinct position of lions in West/Central Africa, compared to other populations across the lion’s current geographic range, based on phylogenetic analyses of microsatellite and mtDNA datasets. Moreover, we assessed levels of genetic diversity across different geographic scales to detect signs of low genetic diversity.et al. (2012), remained after inclusion of a population from Zambia which is geographically intermediate between Tanzania and RSA [et al. (2012) concluded that the Ethiopia1 population forms a unique clade, as it showed to be distinct from India, East Africa and Southern Africa [et al. (2012) [Analysis of microsatellite data (STRUCTURE) identified three clusters in the African lion: 1) West/Central Africa, 2) East Africa, and 3) Southern Africa, in addition to a cluster comprising the Asiatic subspecies. Although the high level of fixation of alleles in the Asiatic lion is likely to contribute to the identification of this population as a distinct cluster, genetic structure is found within the African subspecies. This supports the genetically distinct position of lions from West/Central Africa reported previously and found again here based on mtDNA data . In addiet al. 20, remaineet al. 20, remaineet al. 20, remaine. (2012)  west of . (2012) ,9,47,48.A as a measure of genetic distance [A is commonly accepted as the most suitable measure for inferring phylogenetic relationships [et al. (2008) and attributed to sex-biased gene flow. To further assess congruence between mtDNA and autosomal markers, a Mantel test was performed based on corrected Nei’s genetic distances for both datasets  West/Central Africa, 2) East Africa, 3) Southern Africa and 4) India. The deepest split in the haplotype network distinguishes a North group consisting of the West/Central African lion together with the Asiatic subspecies, and a South group consisting of lions from East and Southern Africa. Within a single country, only one or two closely related haplotypes are found, with two exceptions where more divergent haplotypes are present: 1) Ethiopia, which could be explained by the geographic location of the country as previously noted, and 2) RSA2, likely due to past translocations to and amongst small reserves in RSA . The neidistance . Despiteionships ,52 and, datasets . This ilP. l. leo) very likely to be extinct and the Cape lion (P. l. melanochaita) a possible con(sub)specific with P. l. krugeri [P.l.bleyenberghi (South West Africa) and P.l.azandica (North East DRC) as fully differentiated lineages. The remaining four subspecies, P. l. persica and P. l. senegalensis in the northern part of the range, and P. l. nubica and P. l. krugeri in the southern part of the range correspond to India, West/Central Africa, East Africa, and Southern Africa respectively, and reflect the deepest split in the haplotype network. Although sample size was limited, the close genetic relationship of West/Central African lions to the Asiatic subspecies, was later reconfirmed by craniometric data [The four lineages we describe based on autosomal and mtDNA data are further corroborated by the distinction of four groups based on morphological data . Up to e krugeri . Of the ric data .ST and Nei’s genetic distance), we conclude that all sampled populations are significantly differentiated from each other.IBD explains the genetic distances on a continental scale and on a regional scale. The strong slope of the trend line in IBD analysis for West/Central Africa, compared to Southern and East Africa, is suggestive of near complete isolation between populations in the West/Central region. This is also supported by the high among-groups variance in the AMOVA. Based on the genetic distances  may be explained by the mating system as was also shown for prides in Selous GR [IS value found in RSA1 is congruent with previous findings [IS value in the Ethiopia1 lions can be explained by the breeding history of the population, which was founded by five males and two females in 1948 [et al. (2014), following the deepest ancestral split found in the haplotype network, recognizing a North group and a South group. Primarily, as mtDNA, autosomal markers and morphological data show a congruent pattern, we believe that it is enough to support a taxonomic split within the African subspecies of the lion.It was hypothesized that lion populations in West Africa and parts of Central Africa were especially vulnerable to declining levels of genetic diversity since fragmentation of the habitat is particularly severe in this region. However, we did not find significant heterozygotic deficiencies, reduced number of alleles or fixed loci in any of the six sampled populations in this region. The significantly negative Felous GR , howeverelous GR . The strfindings  and a hi in 1948 . In addi in 1948 , althougS1 File(DOCX)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(FAS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations.This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot.stR values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations.Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other. Human Y chromosome short tandem repeats (Y-STRs) are repeating regions with 2-7 bp long repetitive units found in the non-recombining region of Y chromosome. Y-STRs are characterized by male inheritance pattern. They are the most widely used Y chromosome markers due to simple typing and a high level of diversity. Typing is performed using polymerase chain reaction (PCR), which is a reliable procedure tolerant to degraded DNA. Thus, Y-STRs can be used in forensics for the investigation of sexual assault cases, for deficient paternity testing when the alleged father is not available for testing, in gang rape situations , for the investigation of genetic reasons of male infertility, in genealogical research, particularly for surname testing, in population genetic studies, for the verification of amelogenin Y-deficient men, and in genetic epidemiology -9. In thMore than 400 years of shared history of Bosnia and Herzegovina and Ottoman Empire shaped cultural features of both populations, with consequences visible even in the modern era. However, according to a previously published study , there wstR) and associated probability values  with 10 000 permutations were calculated between the studied population and the neighboring populations. Multi-dimensional scaling (MDS) plots were generated using genetic distances for the comparison of different populations’ haplotype data found in the Y Chromosome Haplotype Reference Database .This study included Turkish students currently studying in Sarajevo as a representative sample of the Turkish population that has recently settled in Sarajevo. The aim of the study was to provide the haplotype polymorphisms and distributions in the Turkish population and estimate their forensic parameters. In addition, population pairwise genetic distances  healthy male students from Turkish population living in Sarajevo and originating from different geographical regions of Turkey were sampled for the Y-STR analysis. DNA samples were collected at the International Burch University in the period between March, 2013 and January, 2014. The informed consent form was obtained from all participants. Ethical approval was received from the International Burch University, Sarajevo.Buccal swabs were collected and used as a DNA source (n = 100). Air-dried buccal swabs were placed in paper envelopes and stored at +4°C for DNA extraction. Genomic DNA was isolated from buccal swabs using the salting out method  and extrPCR amplification of 23 STR loci  on Y chromosome was performed using PowerPlex Y 23 System (Promega Corporation) according to the manufacturer’s recommendations . PCR wasi2p)*n/(n-1), where n is the sample size and pi is the ith’s haplotype frequency. Gene diversity (GD) was calculated as 1- ∑ i2p, where ip is the allele frequency. Arlequin software version 3.5 was used  (stR) and associated probability values  between the studied population and the neighboring populations with 10 000 permutations. MDS plots were generated using genetic distances for the comparison of different populations’ haplotype data present in YHRD. For the population comparison, recently published haplotype data present in YHRD were used and the analyses were performed on 23 Y-chromosome STR loci. Compared population samples with the number of haplotypes were as follows: Macedonian (n = 101), Italian (n = 335), Lebanese (n = 555), Iraqi (n = 124), Greek (n = 109), Slovenian (n = 104), Hungarian (n = 143), German (n = 131), Croatian (n = 125), Bosnian (n = 100), and Turkish (n = 100).Allele and haplotype frequencies were calculated by counting method. Haplotype diversity was estimated by Nei’s formula : HD = (1was used . The matpulation . In ordehrd.org) . AMOVA ohrd.org)  from YHRWe calculated haplotype frequencies of the sample population and detected 165 alleles at the 23 Y-STR loci . All 100Average GD for the study population was 0.703, ranging from 0.470 to 0.942. The initial analysis of GD values indicated that the highest GD was detected at DYS385a/b loci with a value of 0.942 and the lowest GD at DYS391 locus with a value of 0.470, which is consistent with the polymorphism findings presented above in this study.Two microvariant alleles were detected at DYS458, namely 17.2 and 18.2. The total frequency of the variant alleles at DYS458 was 6% of all study samples. We detected duplicated allele  in the two samples at DYS19.st = 0.001, P = 0.289) and Iraqi  populations than to Italian  and Greek  populations. Also, it was closer to Hungarian , German , and Slovenian  populations than to Macedonian  and Croatian  populations. The greatest genetic distance was detected between Turkish and Bosnian  population , which is not usually observed in the literature and confirms that the tested individuals are indeed paternally unrelated. Such results are probably a consequence of using PowerPlex Y 23 kit, which types results on 23 Y-STR loci, thus enabling increased haplotype diversity to be observed. A large number of identified alleles also confirm high haplotype diversity due to the use of 23 Y-STR loci, while previous studies of Turkish population showed a much smaller number of alleles due to the use of fewer Y-STR loci ,21-24.Six loci that were included and analyzed for the first time in PowerPlex Y 23 kit  were found to be highly informative and to contribute to the uniqueness of the haplotypes in the present population. DYS385a/b loci showed a high degree of polymorphism, which was expected as these loci were analyzed as a duplicated locus in the generated haplotypes. It was also found that DYS458 was one of the most informative markers with 12 different alleles. This marker was shown to be highly polymorphic in a study of Turkish population from Anatolia as well .The least informative locus was DYS391, with GD value of 0.470 and with only 3 alleles, which is in concordance with the previously published data for Turkish population ,23,24 thTwo of 12 alleles on locus DYS458 were microvariant alleles (17.2 and 18.2). The appearance of variant alleles was expected as it is a specific feature of the locus DYS458 . These aDuplication was found in two individuals at DYS19 locus, which was also expected according to the theoretic and experimental data from the literature . Both inTo conclude, 23 Y-STR loci had high haplotype diversity and the kits using this number of Y-STR loci represent great progression in the field of Y chromosome-related testing of individuals for the purposes of forensic investigation, paternity testing, population studies, and genealogical research. Also, selected 23 Y-STR markers within PowerPlex Y 23 kit are very useful in the investigation of genetic relationships between examined populations.


Lampetra fluviatilis (mostly from anadromous populations) and freshwater European brook lamprey Lampetra planeri from across 18 sites, primarily in the British Isles, were investigated for 13 polymorphic microsatellite DNA loci, and 108 samples from six of these sites were sequenced for 829 bp of mitochondrial DNA (mtDNA). We found contrasting patterns of population structure for mtDNA and microsatellite DNA markers, such that low diversity and little structure were seen for all populations for mtDNA (consistent with a recent founder expansion event), while fine-scale structuring was evident for nuclear markers. Strong differentiation for microsatellite DNA loci was seen among freshwater-resident L. planeri populations and between L. fluviatilis and L. planeri in most cases, but little structure was evident among anadromous L. fluviatilis populations. We conclude that postglacial colonization founded multiple freshwater-resident populations with strong habitat fidelity and limited dispersal tendencies that became highly differentiated, a pattern that was likely intensified by anthropogenic barriers.The tendency of many species to abandon migration remains a poorly understood aspect of evolutionary biology that may play an important role in promoting species radiation by both allopatric and sympatric mechanisms. Anadromy inherently offers an opportunity for the colonization of freshwater environments, and the shift from an anadromous to a wholly freshwater life history has occurred in many families of fishes. Freshwater-resident forms have arisen repeatedly among lampreys (within the Petromyzontidae and Mordaciidae), and there has been much debate as to whether anadromous lampreys, and their derived freshwater-resident analogues, constitute distinct species or are divergent ecotypes of polymorphic species. Samples of 543 European river lamprey  Although the abandonment of migration remains a poorly understood aspect of evolutionary biology, there is evidence to suggest that this phenomenon might act as an initiator for adaptive radiation et al. et al. et al. Salmo salar) undergo extended oceanic migrations, yet exhibit significant local adaptation and substantial reproductive isolation between populations owing to precise philopatry and a high homing fidelity to their natal river or tributary  generally show very low interpopulation differentiation across geographically distant river systems  and its nonparasitic freshwater-resident derivative the European brook lamprey (Lampetra planeri Bloch 1784), together with several L. fluviatilis populations that comprise potamodromous individuals that migrate within freshwater only . In Loch Lomond  are truly sympatric; however, in all other paired sites, L. planeri samples were obtained upstream (within the same river) of anadromous L. fluviatilis populations, which were usually separated by migration barriers  MgCl2, 2 μL dNTPs (2.0 mm), 0.2 μL of each primer (10 mm), 4 μL of Colorless GoTaq® Reaction Buffer (Promega), 0.1 μL GoTaq DNA polymerase (Promega) and 1 μL of template DNA. Cycle conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 30 cycles of; denaturation at 94 °C for 1 min, annealing temperature 57.1 °C for 1 min and extension at 72 °C for 2 min; and followed by a final extension at 72 °C for 2 min. The resulting PCR products were purified using the Qiagen PCR Purification kit and sequenced using an ABI PRISM 3730 DNA Analyser (DBS genomics Durham University).The PCR primers ATPfor and ATPrev  were included , and no loci with evidence for selection were found.Microsatellite loci were multiplex amplified using a Qiagen Multiplex kit. Thermal cycler conditions were as follows: initial denaturation at 95 °C for 15 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing temperature 60 °C for 90 s and extension at 72 °C for 60 s; and followed by a final extension at 60 °C for 30 min. PCR products were genotyped on a 3730 ABI DNA Analyser  and visualized with Geneious VR6 (Biomatters). Microsatellite loci were tested for null alleles, large allele dropout and scoring errors due to stutter peaks using geneious vR6 (Biomatters). The program dnasp 10.4.9  were calculated using the program fstat 2.9.3  repetitions and 500 000 burn-in using no population prior information and assuming correlated allele frequencies and admixture. structure was also used with a location prior (LOCPRIOR) to clarify population structure within the Loch Lomond system ] and geographic distances [Isolation by distance (IBD)] based on microsatellite DNA loci was carried out using a Mantel test (10 000 permutations) in genepop v4.2. Geographic distances were calculated between sample sites using linear referencing tools in Quantum GIS (Lisboa). A Mantel test was also carried out to test for association between genetic distances and number of physical barriers (defined as any anthropogenic feature larger than 0.5 m height at base river level which reaches the full width of the river) between sample sites. The 0.5 m value was subjective, based on the fact that many structures of this height or greater generate discrete water level differences (upstream–downstream) at base flows, on published and unpublished data on the impact of different height potential barriers on lamprey movement , and on our ability to identify potential barriers in field surveys and databases. Only river systems for which information on barriers was available were utilized in the Mantel tests .Patterns of microsatellite differentiation were subsequently examined using a factorial correspondence analysis (FCA) implemented in migrate-n (v 3.2.6) was used to estimate levels of historical gene flow between populations (Beerli & Felsenstein L. fluviatilis and L. planeri) at six locations , of which the latter three are all tributaries in the same river catchment, where samples from both species were available. To implement Bayesian inference in MIGRATE-N, the Brownian motion approximation was selected with an MCMC search of 100 000 burn-in steps followed by 5 000 000 steps with parameters recorded every 100 steps; exponential prior on theta ; and an exponential prior on migration . migrate-n was run with parameter values starting from FST-based estimates, and the distribution of parameter values was compared across runs to ensure overlap of 95% CI. bayesass 1.3  from six sampling sites  being statistically significant  were statistically significant ; however, only 45.4% of FST values for L. fluviatilis populations were significant, with a range from −0.00524 to 0.11945. When the freshwater-resident L. fluviatilis populations were not included, the FST values ranged from −0.00524 to 0.02537. FST values between L. fluviatilis and L. planeri populations ranged from 0.011 to 0.18554. Average allelic richness per locus ranged from 2.43 (Lp_003) to 14.9 (Lamper_4). Average FIS per site ranged from −0.095 [Wear (L. planeri)] to 0.028 [Lomond (anadromous L. fluviatilis)].A total of 543 lampreys were genotyped at thirteen loci. All loci were in Hardy–Weinberg equilibrium and not impacted by null alleles for most populations, and there were no consistent issues for any given population  in populations of either species showed this to be rare, 0% in some cases for both species, and no higher than 0.74%. One randomly chosen individual of each full-sibling pair was excluded, and analysis was repeated. There were no differences that affected inference in the results with full-siblings included or excluded, so all individuals were included in the analysis.structure analyses consistently identified L. planeri populations as being separate from L. fluviatilis populations (anadromous and freshwater-resident) and from each other   outcome]. There were several peaks for ΔK at K = 2, 5 and 8 (LnP(D) result (K = 9) was most informative, distinguishing all L. planeri and L. fluviatilis freshwater-resident populations .ther Fig.. The onlL. planeri populations were compared, the highest likelihood result identified all populations as distinct . The Loch Lomond system  was compared to an anadromous L. fluviatilis population (Nidd) and another freshwater-resident L. fluviatilis population (Bann). structure identified three populations with highest likelihood, while ΔK was 2 . However, when freshwater-resident L. fluviatilis populations were excluded (Bann and Loch Lomond), the positive relationship was much stronger  showed a highly significant positive correlation  ranged from 3.73 to 10.43 migrants/generation from L. fluviatilis to L. planeri, and from 4.18 to 16.28 from L. planeri to L. fluviatilis , but 95% confidence intervals were large and overlapping. The bayesass analysis indicated low-level contemporary gene flow between the putative species, and some comparisons also suggest the downstream direction from L. planeri to L. fluviatilis , with suitable riverine habitat available only during interglacial periods  years ago using tau and a mutation rate of 5% per million years, which roughly coincides with the last glacial maximum  that identified strong genetic differentiation between sympatric L. fluviatilis and L. planeri in the Iberian Peninsula with numerous fixed and diagnostic single nucleotide polymorphisms (SNPs) between the two putative species, some associated with genes related to osmoregulation  geographic populations also found evidence consistent with local adaptation  in Japan . Gene flow between the putative species may be possible owing to a combination of interspecific nest association  and coalescent (migrate-n) methods suggested directionality in genetic migration, favouring the direction of L. planeri to L. fluviatilis, although the confidence limits were broad. Asymmetric gene flow occurring in these types of freshwater systems can significantly influence the distribution of genetic variation, with downstream populations typically exhibiting higher genetic diversity than headwater populations , and consistent with expectations , the correlation with L. planeri was weak and showed a broad range of values for a given distance  populations in a Danish river system. Similar decreases of genetic diversity from downstream towards upstream populations have been observed in other fish species in relation to anthropogenic barriers  exhibited higher genetic differentiation  than most other population comparisons.The number of anthropogenic barriers between populations was found to be significantly positively correlated with genetic distance, and such barriers have been shown to limit the upstream migration of d et al.  also det. et al.  found fr , acceleration in genetic divergence may result in the subsequent establishment of allopatric speciation  and northern brook (I. fossor) lampreys occurring sympatrically , but did find differentiation among parapatric populations. Yamazaki et al. (Lethenteron camtschaticum) and its nonparasitic derivatives in the Ohno River, Japan.The system in Loch Lomond offers evidence of the potential for gene flow between morphologically differentiated ecotypes, indicating that where they are found sympatrically, gene flow between  (et al.  found noi et al.  also foubayesass analysis suggests that contemporary gene flow is occurring between all three populations in Loch Lomond, consistent with a tendency for interbreeding when there are no environmental barriers to limit connectivity. The divergence of the freshwater-resident L. fluviatilis population would then suggest a period of differentiation in isolation. Therefore, in Loch Lomond, the anadromous strategy is also paralleled by a population component with potamodromous behaviour, with some fish apparently showing migration mostly between the loch and spawning streams. While all three Loch Lomond populations were significantly differentiated from each other (Fig.L. fluviatilis and sympatric L. planeri populations both showed evidence of connectivity with the wider L. fluviatilis populations.The et al. et al. Lampetra fluviatilis from southern refugia and the subsequent establishment of multiple freshwater-resident Lampetra planeri populations. These may have been relatively small founder groups that retained some degree of reproductive isolation that was likely intensified, although perhaps not entirely determined, by the anthropogenic introduction of barriers. Moreover, it was ascertained that there is gene flow between L. fluviatilis and L. planeri in both long-term and contemporary timescales and the pattern of gene flow is apparently asymmetric. This has significant implications for the management of L. planeri populations and the extent to which this is underpinned by natural processes will have important evolutionary implications with respect to the mechanisms that generate diversity. Our data emphasizes the importance of founder events in the evolution of diversity among populations and as a frequent component of the speciation process (Templeton Lampetra divergence present in the Iberian Peninsula, the northern European populations appear to have been established relatively recently, and the process of differentiation is still ongoing. There may be a natural tendency towards speciation in freshwater-resident populations that remain environmentally stable over time, but a dynamic process instead at higher latitudes experiencing a cycle of habitat loss and release.Alternative life history strategies are common among fishes inhabiting postglacial lakes, often resulting from adaptation to different foraging strategies or environments (Robinson & Parsons