Campylobacter jejuni and Campylobacter coli isolates from commercial broiler farms was examined by multilocus sequence typing (MLST), with an assessment of the impact of the sample type and laboratory method on the genotypes of Campylobacter isolated. A total of 645 C. jejuni and 106 C. coli isolates were obtained from 32 flocks and 17 farms, with 47 sequence types (STs) identified. The Campylobacter jejuni isolates obtained by different sampling approaches and laboratory methods were very similar, with the same STs identified at similar frequencies, and had no major effect on the genetic profile of Campylobacter population in broiler flocks at the farm level. For C. coli, the results were more equivocal. While some STs were widely distributed within and among farms and flocks, analysis of molecular variance (AMOVA) revealed a high degree of genetic diversity among farms for C. jejuni, where farm effects accounted for 70.5% of variance, and among flocks from the same farm (9.9% of variance for C. jejuni and 64.1% for C. coli). These results show the complexity of the population structure of Campylobacter in broiler production and that commercial broiler farms provide an ecological niche for a wide diversity of genotypes. The genetic diversity of C. jejuni isolates among broiler farms should be taken into account when designing studies to understand Campylobacter populations in broiler production and the impact of interventions. We provide evidence that supports synthesis of studies on C. jejuni populations even when laboratory and sampling methods are not identical.The genetic diversity of  Campylobacter jejuni accounts for approximately 90% of human cases, followed by C. coli, which accounts for most of the remainder  (Broiler chickens are frequently colonized in the ceca by large numbers of  79.2%)  and in c (79.2%) . Coloniz9.2%  andCampylobacter isolates has resulted in major advances in the understanding of Campylobacter ecology and epidemiology , to classify and characterize emiology . MLST geemiology . These iemiology –15. Thermiology –. Howevermiology ––18.Campylobacter populations in poultry sources has been reported in different studies performed with a variety of genotyping methods  on the Campylobacter populations recovered from broiler flocks. The study also investigated the genetic diversity of C. jejuni and C. coli populations between and within commercial broiler farms before final depopulation.Molecular epidemiological studies and source attribution models are generally based on molecular comparisons of isolates from different points along the food chain, with data from different studies often combined to produce data sets describing populations of  species . Similarlobacter , 27, andpulation . In ordeCampylobacter detection and identification have been more fully described elsewhere (n = 3), Cary-Blair medium (CB) (n = 3), maximum recovery diluent (MRD) (n = 3), and Exeter broth (EX) (n = 3); a pooled cecal sample (n = 1); and pooled fecal dropping samples (n = 3). Bacteriological culture of boot swabs and cecal samples was performed by direct plating of all samples on modified charcoal cefoperazone desoxycholate agar  according to ISO 10272-1:2006 for the detection of Campylobacter spp. In addition, all samples were tested by culture on mCCDA after enrichment in Exeter broth. For logistical reasons, only one colony was selected from each of the suspected Campylobacter-positive samples and subjected to confirmation, species identification, and further characterization by MLST. We assumed that, on average, by systematically typing only one colony per sample from the end of the streak, we would detect the predominant isolates at the relative frequencies at which they occurred in all samples. Confirmation and species identification were carried out using a multiplex PCR based on the detection of partial sequences of two genes that allow the simultaneous identification of C. jejuni (mapA) and C. coli (ceuE)  were excluded from further characterization by MLST, as we would have had to select one of the two species, thus preventing us from selecting the predominant species in that isolate. In total, 751 Campylobacter isolates (645 C. jejuni and 106 C. coli) from 32 flocks and 17 farms, recovered from different sample types, were characterized by MLST. The number of C. jejuni and C. coli isolates characterized per flock and sample type is shown in Table S1 in the supplemental material.Isolates were obtained from a cross-sectional study of 40 conventional broiler flocks from 17 farms at the end of the rearing period. Details of sample collection and bacteriological culture methods for lsewhere . Brieflyi (ceuE) . The spr2–10% CO2–6% O2) generated in a gas-charged incubator . Chromosomal DNA was extracted by boiling a cell suspension in phosphate-buffered saline (PBS)  for 10 min on a heat block. The suspension was centrifuged at 13,200 rpm for 5 min, and the supernatant was retained frozen until MLST analysis.Isolates which had been stored at −80°C in a cryopreservative medium were revived on blood agar  (7% sheep blood and 100 mg of cycloheximide per liter) and incubated at 41.5 ± 1°C in a microaerobic atmosphere  were amplified by PCR, and the nucleotide sequence of the amplicons was determined using published oligonucleotide primers and reaction conditions  with sequences submitted for allele designation as appropriate.MLST was performed as described previously . Fragmennditions , 31. NucST). A value of 0 indicates that two populations are indistinguishable, while a value of 1 indicates maximum genetic differentiation between two populations. The resulting pairwise FST values were exported as genetic distances and visualized as neighbor-joining trees in MEGA5 .Campylobacter populations isolated from different samples and laboratory methods. This index takes into account the number of STs present (the “richness”) as well as the abundance (the “evenness”) for each ST. The Simpson's diversity index represents the probability that two individuals randomly selected from a sample belong to different species. The value ranges between 0 and 1; the greater the value, the greater the sample diversity (A modified version of Simpson's diversity index (1-D) and bootstrap 95% confidence intervals were calculated using StatsDirect  to compare the diversities of STs within the iversity , 36.Rarefaction analysis was performed by using the frequency of STs to investigate the relative levels of diversity or species richness among different sample types and laboratory methods. The rarefaction analysis was carried out using PAST  with a rC. jejuni and 7 C. coli) were identified among the 751 isolates and assigned to 15 CCs (14 C. jejuni and 1 C. coli). Six STs (five C. jejuni and one C. coli) accounting for 26.2% of the total isolates (18.4% of C. jejuni and 69.8% of C. coli isolates) did not belong to a known clonal complex at the time of the analysis and therefore do not have an assigned clonal complex. A total of 21 of the STs (17 C. jejuni and 4 C. coli) were represented by 5 or more isolates. The most common ST was ST-573 , followed by ST-2195  and ST-50 . Eight STs  accounted for more than 70% of the isolates , and the frequency of ST-2195 was significantly higher in feces (P = 0.036) than in all the other sample types. Interestingly, feces was the only sample type that did not yield ST-574, ST-1910, ST2197, ST-11, and ST-48  for boot swabs transported in MRD to 0.69  for boot swabs moistened in CB, with the confidence intervals of one method overlapping the point estimate of the other in all comparisons.Simpson's index of diversity for eces see . The genST, the genetic fixation index, was used to quantify the extent of genetic differentiation among populations. The index ranges from 0 to 1; a value of 1 indicates that the populations are completely different or separate . A slope of zero in the rarefaction curves indicated that the maximum genetic diversity had been reached and that it was unlikely that more genetic diversity would be identified if more samples were analyzed. The rarefaction analysis did not show any significant differences in diversity between sample types, although a trend toward greater diversity for P = 0.03, P = 0.017, and P = 0.009, respectively). C. jejuni ST-775 was the only type significantly more frequently identified after direct culture than after enrichment (P = 0.001) .Of the total of 47 STs isolated in this study, 8 were recovered by direct culture alone, 20 were recovered after enrichment only, and 19 were recovered by both methods. Multiple comparisons of the frequency distributions of STs between direct culture and enrichment revealed that only a few STs were statistically more frequently isolated using one method than using the other. ST-22, ST-48, and ST-830 were more frequently isolated from enrichment  than by direct culture (none), and these differences were statistically significant  for isolates recovered by direct culture and 0.92  for those obtained after enrichment  than by direct culture , although the confidence intervals of one overlapping the point estimate again indicated that these differences were not statistically significant . No significant difference was observed between direct culture and enrichment for C. jejuni isolates . Rarefaction analysis showed a greater diversity of C. jejuni and C. coli genotypes after enrichment than after direct culture . However, the confidence intervals of the rarefaction curves overlapped in both cases (data not shown), indicating that these differences should be interpreted with care.The Simpson's index of diversity for richment . For C. nificant . A smallThe number of CCs within a flock and within a farm ranged from 1 to 6 and from 1 to 7, with averages of 1.9 and 2.6, respectively. Some of the CC and STs were widely distributed within and between flocks and farms. CC573 was found in five farms and nine flocks, CC353 and CC354 were each found in five farms and six flocks, and CC21 was found in four farms and six flocks. The number of STs within a flock and within a farm ranged from 1 to 8, with averages of 4.4 STs per farm and 3.1 STs per flock. Although some STs were widely distributed within and between farms and flocks , only 10 of 47 (21.3%) STs were found on two or more farms. Other STs  were found in only one particular farm, where they predominated .C. jejuni isolates for the different levels of the population structure, with strong evidence of between-farm variability, which accounted for 70.5% of the total variance. There was also strong evidence of between-flock diversity within farms and of diversity within flocks, although this accounted for much smaller proportions of the total variance   and within flocks (25.1%)  . No effe (25.1%) . Figuresme farm 6.1% and wC. jejuni and C. coli at the farm and flock levels.This study assessed the effect of the sample type and laboratory method on the molecular types recovered from conventional broiler flocks and examined the genetic diversity of Campylobacter populations identified by a high number of different sampling approaches  were very similar, with far smaller differences among sample types than among different farms. Only three STs  were statistically more frequently identified in one sample type than in the others; however, due to the high number of multiple comparisons, these significant differences may have been due to chance. For C. jejuni isolates, similar and high degrees of genetic diversity were observed in isolates from broiler flocks by the use of different sampling strategies and no evidence of genetic differentiation among sample types was detected. Similarly, the rarefaction analysis showed marginal differences between the relative diversities of STs obtained by each sample type when equal numbers of samples were used. Lower levels of genetic diversity and higher levels of differentiation were observed between C. coli populations from different sample types, but these differences were not statistically significant. For C. coli, our results are therefore consistent with anything from a minimal to a substantial impact of sampling. Overall, these findings indicate that the use of boot swab, cecal, or fecal samples for isolation of C. jejuni at the farm level had no discernible effect on the genetic profile of the flocks.The Campylobacter population from direct culture and enrichment were very similar, with the two methods identifying the main types and at similar frequencies. It has been suggested that the isolation protocols used with enrichment may bias the recovery toward C. jejuni genotypes with increased laboratory fitness, thus limiting the understanding of population genetics and genetic diversity of Campylobacter strains circulating in environmental and animal reservoirs (The genotypic profiles of the servoirs . In the C. jejuni. However, a small but significant degree of differentiation between direct culture and enrichment was observed for C. coli isolates, probably due to the increased isolation rate of ST-830 after enrichment, as discussed above. An increase in the genetic diversity after enrichment was observed for C. jejuni and C. coli, although the 95% confidence intervals overlapped, indicating a lack of statistical significance in both cases. Overall, the findings suggest that enrichment may be more sensitive at picking up strains that are present at low concentration in the samples, but the present study did not demonstrate a substantial bias in the nature of the strains identified by these two methods, so the impact on the inferred population structure should be small, in particular, for C. jejuni.No significant differentiation of genotypes obtained by different laboratory methods was observed for C. jejuni and C. coli isolates. In contrast, a higher level of genotypic richness has been reported by direct plating of neck skin, cecum, and meat samples than by enrichment in Preston and Bolton broths (Williams et al.  describen broths . The avan broths , and optCampylobacter populations from different sampling sites and sample matrices across the broiler food chain, but, to our knowledge, there are no published studies comparing different sample types from broiler flocks at the farm level. A recent study comparing cecal, neck skin, and meat samples found a greater level of genetic diversity in isolates from neck skin and cecal samples than in those from meat samples (Campylobacter genotypes found at slaughter may not be captured by on-farm sampling, although cross-contamination during the slaughter process is also likely to be responsible for the increased diversity (A few studies have compared the genetic diversities of  samples . Others iversity . This coCampylobacter population in broiler flocks.Poultry samples have been found to be contaminated with more than one ST , 40; theCampylobacter strains. The predominance of a particular genotype within flocks and farms may also suggest the presence of a more stable, adapted, and successful flock colonizer.Despite the great genetic diversity observed here, the presence of a limited number of predominant types shared by multiple flocks was also observed. Common poultry management practices may favor the recirculation of these strains among and within farms. The use of common vehicles and personnel across different farms, particularly in high-density poultry areas and within integrated companies, may have contributed to the transmission of particular C. jejuni isolates resided mainly among farms, with less variation observed within flocks and between flocks within the same farm. Other studies have identified a spatial relationship among genotypes, with isolates being more similar within rather than between cattle and sheep farms (–C. coli isolates, the majority of genetic variation occurred among flocks within the same farm, with the within-flock diversity accounting for a much smaller proportion of the variation. These findings are in line with other work which suggested that C. coli may form a more stable population within a broiler flock than C. jejuni (The analysis of molecular variance showed that the genetic variation of ep farms –44. Howe. jejuni .The dynamics of strain diversity within flocks are complex and may reflect variation in phenotypic properties such as infectivity, virulence, and stress response, which could determine survival time, persistence, or different susceptibility properties. Several groups have also demonstrated that the genotype can affect colonization of the gastrointestinal tract of poultry –49 and t–50–Campylobacter MLST database revealed that most of the STs and CCs identified in this study have been previously described among isolates from poultry and humans, reinforcing the hypothesis of the importance of broiler flocks as a reservoir for human infection. Interestingly, other chicken-associated lineages that have been previously reported frequently were not identified in the farms in the fairly large current study. This shows the need for very wide on-farm sampling to fully index the Campylobacter population affecting the broiler industry at the farm level.Interrogation of the Campylobacter at the farm level has no statistically supported effect on the genetic profile of C. jejuni population in broiler flocks. The report also shows that commercial broiler farms provide an ecological niche for a wide diversity of genotypes and illustrates the complexity of the population structure of these organisms in the broiler production. The higher level of genetic diversity between broiler farms should be taken into account in designing sampling strategies to understand the population structure of Campylobacter in the broiler production. Further investigations will be needed to better understand the factors responsible for the genetic diversity of C. jejuni and C. coli between and within broiler farms and flocks and the impact of control interventions altering Campylobacter populations as well as the overall quantitative impact of any interventions.This report shows that the use of different sampling strategies and laboratory methods for isolation of


The Basque Diaspora in Western USA and Argentina represents two populations which have maintained strong Basque cultural and social roots in a completely different geographic context. Hence, they provide an exceptional opportunity to study the maternal genetic legacy from the ancestral Basque population and assess the degree of genetic introgression from the host populations in two of the largest Basque communities outside the Basque Country. For this purpose, we analyzed the complete mitochondrial DNA control region of Basque descendants living in Western USA (n = 175) and in Argentina (n = 194). The Diaspora populations studied here displayed a genetic diversity in their European maternal input which was similar to that of the Basque source populations, indicating that not important founder effects would have occurred. Actually, the genetic legacy of the Basque population still prevailed in their present-day maternal pools, by means of a haplogroup distribution similar to the source population characterized by the presence of autochthonous Basque lineages, such as U5b1f1a and J1c5c1. However, introgression of non-Basque lineages, mostly Native American, has been observed in the Diaspora populations, particularly in Argentina, where the quick assimilation of the newcomers would have favored a wider admixture with host populations. In contrast, a longer isolation of the Diaspora groups in USA, because of language and cultural differences, would have limited the introgression of local lineages. This study reveals important differences in the maternal evolutionary histories of these Basque Diaspora populations, which have to be taken into consideration in forensic and medical genetic studies. Euskera), geographical location, cultural tradition and genetic structure . Th. Th27]. The DDA group from Western USA, despite the dominant presence of Eurasian mtDNA lineages (91.49%), also presented minor Native American (6.38%) and African (2.13%) contributions. Among the Eurasian lineages, the main haplogroup was R0 (46.81%) (excluding cluster HV0), followed by T (9.57%) and U (8.51%). Concerning the Native American haplogroups, all four major founding haplogroups were detected  . The mosIn Argentina, the DDV group presented a predominance of Eurasian mtDNA lineages with a frequency of 72.22%. The diversity of haplotypes of Eurasian origin was high (0.9902±0.0080), being R0 the most common haplogroup in the population , followed by U (12.50%) and J (8.33%). This population presented a substantially contribution of Native American lineages, with a value of 26.39%, being the lineage B4b (11.11%) the most frequent one. A minimal African component was detected, more particularly one individual belonging to L3f1b1a.The ARG-DDA group also presented a trihybrid structure with a major Eurasian component (75.41%), followed by Native American (22.13%) and African (2.46%) components. Haplogroup R0 was the most frequent lineage, representing 33.61% of the DDA sample (excluding HV0) and 44.57% of all Eurasian haplogroups. The second most abundant haplogroup was U with a frequency of 14.75%. Among the Native American lineages, the two most common belonged to A2 and B4b haplogroups, with a 5.74 and 4.92%, respectively. On the other hand, African lineages corresponded to L3h (1.64%) and L1c (0.82%).The maternal Basque contribution to the Diaspora groups here studied is well-established by the presence of autochthonous lineages from the Basque Country, namely U5b1f1a and J1c5c1 . In thisTo determine the genetic affinity of the Basque Diaspora populations in a global context, a MDS analysis, based on haplogroup frequencies, was performed . All theAdditionally, the PCA allowed a further understanding of the European contribution to the Basque Diaspora populations. The result shown in The Basque Diaspora in the Western USA and Argentina represents populations that have maintained strong cultural and social Basque roots. They settled in a completely different geographic context, consequently providing an outstanding opportunity in order to study the dynamics of Diaspora populations.Furthermore, the prior differentiation of two different groups in each population, one composed by individuals whose maternal ancestor surnames were Basques (DDV), and the other one, by individuals who descend from Basques but have not maintained their maternal Basque surnames (DDA), has allowed a deeper understanding of the genetic results of the present study. In this regard, non-admixed surname groups constitute useful populations in order to assess the presence of founder effects by comparison to their European Basque source population, whereas admixed surname groups allow us to determine the degree of genetic introgression of American lineages.Interestingly, mtDNA analysis highlighted a similar degree of haplogroup diversity between DDV groups from Western USA and Argentina and the source Basque populations, regarding to their European maternal input. Therefore, non-important founder effects would have occurred in these Diaspora groups.The determination of the mtDNA haplogroups in the Diaspora populations has allowed us to discern the origin of their maternal gene pool. As expected, the European ancestry was predominant in the lineages observed in the Diaspora groups. More concretely, the maternal contribution from the Basque source population has been stated in these groups, as it is pointed out by the presence of autochthonous Basque haplogroups such as U5b1f1a and J1c5c1, but in different degree. In this regard, the DDV group in Western USA was the closest group to the European Basque populations, as it can be seen in the MDS plot. Accordingly, no genetic substructure was observed between these populations (p≥0.05). Contrarily, the preservation from the maternal European Basque input in the DDA group in Western USA as well as the two Argentinean Diaspora groups has been lower, as these groups displayed significant differentiation from the source Basque population (p<0.05). In fact, these groups presented a tryhibrid ancestry, with a major European component, followed by Native American and, in a minor proportion, African lineages. Consequently, the introgression of lineages from the local populations would have contributed to differentiate these groups, along with the presence of lineages coming from other immigrants from different regions of Spain, France or the rest of Europe, who likely mixed with Basque descendants. This result is in agreement with a previous study, based on X-STRs, reporting high genetic contribution of host or local populations in these admixed Basque Diaspora groups in Western USA and Argentina, which may have aroused the differentiation from the founder population .The geneAdditionally, the heterogeneity of haplogroups in the Diaspora groups in Argentina is in accordance with the diversity observed in the present-day multi-ethnic population . In thisThe data from the present study has also revealed that no genetic substructure existed among the two Basque Diaspora groups in Western USA, neither into the Argentinean Diaspora groups (p>0.05). However, the North-American Diaspora groups are clearly differentiated from the Argentinean Diaspora groups, as well as from their corresponding host populations. This genetic stratification may have important implications in Forensic Genetics and Genetic Epidemiology. On the one hand, forensic studies cannot ignore the population substructure, as the evaluation of the weight of the evidence can be biased . Hence, S1 TableFor data retrieved from GenBank database, GenBank accession numbers are given.(XLSX)Click here for additional data file.S2 TableRange included: 16024–576.(XLSX)Click here for additional data file.S3 TableThe relative frequency (%) of each haplotype is indicated.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Genetic relationships were established by two analysis approaches based on geographic origin (five populations) and genetic origin (based on a previous study). We identified six polymorphic sites, including five insertion/deletion (indels) types and one transversion. The overall nucleotide diversity was low for both approaches (geographic = 0.0032 and genetic = 0.0038). Conversely, we obtained moderate to high haplotype diversity (0.66 and 0.80) with 10 and 12 haplotypes, respectively. The common haplotype (H1) for both networks included cacao trees from all geographic locations (geographic approach) and four genetic groups (genetic approach). This common haplotype (ancient) derived a set of intermediate haplotypes and singletons interconnected by one or two mutational steps, which suggested directional selection and event purification from the expansion of narrow populations. Cacao trees from Soconusco region were grouped into one cluster without any evidence of subclustering based on AMOVA (FST = 0) and SAMOVA (FST = 0.04393) results. One population (Mazatán) showed a high haplotype frequency; thus, this population could be considered an important reservoir of genetic material. The indels located in the trnH-psbA intergenic spacer of cacao trees could be useful as markers for the development of DNA barcoding.Cacao trees have been cultivated in Mesoamerica for at least 4,000 years. In this study, we analyzed sequence variation in the chloroplast DNA  Theobroma cacao L.) is widely cultivated in Central and South America, Africa, Indonesia and Malaysia. It is considered an economically important crop because its seeds are used in the chocolate industry  and its markers have been increasingly used for studies of genetic population structure, evolution, gene flow, haplotype frequency and phylogenetic relationships. Due to its high conservation due to maternal uniparental inheritance, cpDNA is the main data source used for the construction of phylogenetic relationships in plants . In addiAlthough cpDNA has not been commonly used in cocoa studies, the technique was employed to analyze population genetic variability and to elucidate the complex origins of cocoa varieties. trnH-psbA, which has shown high variability and can be used to elucidate genetic relationships at the intraspecific level  using the variations in chloroplast DNA revealed by the trnH-psbA spacer sequence.Nonetheless, the use of indels for diversity and phylogenetic analysis has been questioned  because trnH-psbA spacer and analyzed, and seven sequence accessions were downloaded from GenBank as references. A total of 28 of the 38 sequenced trees were selected from plantations in Soconusco  based on a previous characterization  and stored at −20 °C prior to processing.A total of 45 cacao samples were included in this study. Thirty-eight trees were sequenced for the rization  using 10rization . Additiorization . Theobrow/v), 20 mM ethylenediaminetetraacetic acid (EDTA), 1.4 M NaCl, 100 mM Trizma® base, pH adjusted to 8 with HCl and 1% 2-mercaptoethanol (v∕v)). DNA extractions were performed with chloroform-isoamyl alcohol and precipitated with isopropanol. The extracted DNA was then purified with a mixture of phenol:chloroform:isoamyl alcohol (25:24:1). The DNA was dissolved in 60 µL of Milli-Q water and its integrity verified on 0.8% agarose gels. The purity was obtained from the 260/280 absorbance ratios while quantifications were estimated from absorbances at 260 nm. Absorbance readings were performed on Jenway, Genova Spectrophotometer .The total DNA extraction was performed by modifying the method described by trnH-psbA intergenic spacer was conducted using the forward primer 5′-CGCGCATGGTGGATTCACAATCC-3′ and reverse primer 5′-GTTATGCATGAACGTAATGCTC-3′ (2 (50 mM), 0.5 µL of dNTP Mix , 0.05 mM of each primer and 2.5 U of Taq DNA polymerase (Vivantis™). Following one cycle of 5 min at 94 °C, 35 PCR cycles of 30 s at 94 °C, 30 s at 53 °C and 1 min at 72 °C and a 10 min 72 °C final extension were performed in a TC3000 thermal cycler . To verify the presence of amplicons, the PCR products were separated on 6% polyacrylamide gels using 0.5X TBE buffer at 110 V for 210 min, stained with ethidium bromide (0.6 ng/µL) for 15 min, visualized under UV light and photographed with a Gel Doc™ EZ Imager gel documentation system . Fragment sizes were estimated using Image Lab  and integrating the GeneRuler™ 100 bp DNA Ladder Plus (Fermentas®) as a molecular weight marker.The cpDNA amplification of the TGCTC-3′ . The PCRThe PCR products were directly sequenced using the Dye Terminator Cycle Sequencing with Quick Start Kit (GenomeLab™) on a CEQ™ 8000 automatic DNA sequencer (Beckman Coulter™). To validate the results, the DNA was extracted twice and amplified independently. The sequences were verified by comparison with their forward and reverse sequences when applicable.S), the number of haplotypes, the haplotype diversity (Hd) and the nucleotide diversity (πd) were estimated following the methods of The sequence quality was checked and the electropherograms were edited using BioEdit© . SequencTo infer evolutionary relationships at the intraspecific level, we produced a network. The method used was median-joining (MD) based on parsimony criteria  and was Fst values and statistical analyses of molecular variance , FST (test performed by permuting genotypes among populations but within groups) and FSC (test performed by permuting genotypes among groups)) were estimated using Arlequin© version 3.0 , pairwise sion 3.0 . Signifision 3.0  using haD test , CRIOLLO-22 ( JQ228379.1) AMELONADO ( JQ228380.1) SCA 6 ( JQ228382.1), ICS 1 ( JQ228381.1), ICS 6 ( JQ228383.1) and ICS 39 ( JQ228387.1).Seven accessions from the NCBI database were included as references in the genetic origin approach analysis: MATINA 1/6  and nucleotide diversity (πd) values were 0.66 and 0.0032, respectively . The πd was low (0.0025–0.006) for all groups, which was similar to the results obtained with geographic approach. The Forastero-reference and Trinitario-reference groups did not present singletons  was found in the Admixture group . In contngletons . SequencFST = 0. In the spatial analysis of molecular variance (SAMOVA), the value K = 2 extended the FST to 0.0439 and generated two groups: the first contained only Population 4 (Mazatán) and the second grouped the other geographic populations  was not significant and had a value of ulations .D for all populations. Although, in Population 4 the Tajima’s D value was negative (D = − 0.93302) because this population including a transversion. All other populations showed values of D = 0; however, the overall value for this test was D = − 0.13329(P > 0.1).The neutrality tests showed non-significant values in the Tajima’s trnH-psbA region. In the data analysis, we included the indels as informative character states, and the high interspecific divergence of the spacer region allowed their use as a marker for DNA barcoding  and moderately high haplotype diversity (Hd = 0.7331) (our results for the geographic populations were πd = 0.0032 and Hd = 0.66). The results of those authors supported the use of one intergenic spacer to reveal nucleotide polymorphisms similar to our findings.In this study, high haplotype variation was found in the chloroplast DNA from cacao trees grown in the Soconusco region. No inversions or transitions were found, although they were reported to be common in other plants . Howeverarcoding . The molOur haplotype diversity results are contrary to those reported by The number of haplotypes was higher than the number of polymorphic sites . This feTheobroma cacao. To test those hypotheses, additional studies are needed using high-throughput sequencing of chloroplast genomes.Several explanations are possible for the presence of more than one Criollo haplotype. First, only the maternal line gave rise to the eight Criollo haplotypes by mutation. Second, the “Criollo” phenotype had multiple provenances, indicating that the ancient haplotypes persisted over time in the Soconusco cacao farms. Third, some samples were misclassified as “Criollo” . However, haplotype H1 located in this population is considered the common ancestor because it is shared by all populations (Population 7 (Selva Lacandona) exhibited no haplotype diversity  in Population 4 (Mazatán) could be related to a “bottleneck” event, which would indicate population expansion and not natural expansion because it was a cultivated population. The occurrence of unclear events in the past  may have caused the almost complete disappearance of populations established by the people in the Mesoamerican region (Codex Florentino). Rapid expansion due to recolonization of the populations and the probable introduction of other varieties of cacao trees not native to the region would have subjected the populations to a bottleneck events in very recent periods. However, these are presumptive weak inferences of the population history based on a single locus. The bottleneck event could also be related to the loss of alleles , which is much greater than the loss of genetic variance per se. Although these rare alleles contribute little to the total genetic variability, they can provide unique responses against evolutionary challenges similar to the high number of unique haplotypes found in this study (3 singletons in Population 4). The presence of both common and rare haplotypes can be the result of a directional-purifying selection process or expansion events from small populations . This finding provides some explanations regarding the demographic history of T. cacao trees, indicating that the populations formed a priori and experienced gene flow, resulting in population homogenization. The spatial analysis revealed the highest differentiation between groups when K = 2 was tested; K = 3 (FST = 0.00088) grouped trees from the Yucatán, Selva and Cacahoatán in the same genetic population. This grouping is unusual because the geographic distance is longer among the three localities and may be associated with the distribution of trees in the past  grouped individuals from Selva; one mutational step resulted in the origination of the individuals from the Yucatán, which in turn originated the individuals at Cacahoatán by the same event . Followme event .trnH-psbA spacer region of cacao trees can distinguish individuals that are indistinguishable in other marker systems or separated by only few SSR markers, and further support use of these cpDNA markers.The molecular analysis showed low nucleotide diversity but high haplotype diversity possibly due to population bottleneck events. These results were confirmed by the negative Tajima’s D and the arrangement of the haplotype network. We identified 10 different haplotypes (from cultivated trees) of which H3–H8 resulted in singletons because they were not associated with other cacaos or with those reported in the molecular databases. The presence of these haplotypes accompanied by the low number of mutational steps might suggest a very short evolutionary history or events that led to disappearing-expanding populations in southern Mexico. These results suggest confirmation of selection of fruits from few mother trees (even as few as one) that were moved by human agents from South America into Mexico and that the Criollo complex may be homogenous based on maternal influence. Increasing sampling numbers would go a long way in establishing whether a true sub-structure of maternal origin exists. One geographic population  consisted of high frequency haplotypes, which makes this zone an important reservoir of genetic material at the chloroplast and possibly phenotypic levels because an abundance of pod morphology was also observed in this population. The genetic differentiation between populations was zero, suggesting that gene flow homogenized the populations.Indels and one transversion located in the chloroplast DNA 10.7717/peerj.1855/supp-1Supplemental Information 1trnH-psbA intergenic spacer of 39 Theobroma trees collected from Soconusco, Mexico.Passport descriptors and sequences of chloroplast DNA Click here for additional data file.10.7717/peerj.1855/supp-2Supplemental Information 2trnH-psbA intergenic spacer of 39 Theobroma trees collected from Soconusco, Mexico.Passport descriptors and sequences of chloroplast DNA Click here for additional data file.


Here, we study DNA sequences at the mitochondrial control region along with STR markers for over 680 specimens from all the main red deer populations in Spain and other west European areas. Our results from mitochondrial and genomic DNA show contrasting patterns, likely related to the nature of these types of DNA markers and their specific processes of change over time. The results, taken together, bring support to two distinct, cryptic maternal lineages for Iberian red deer that predated the last glacial maximum and that have maintained geographically well differentiated until present. Haplotype relationships show that only one of them contributed to the northern postglacial recolonization. However, allele frequencies of nuclear markers evidenced one main differentiation between Iberian and northern European subspecies although also supported the structure of both matrilines within Iberia. Thus, our findings reveal a paraphyletic nature for Iberian red deer but also its genetic identity and differentiation with respect to northern subspecies. Finally, we suggest that maintaining the singularity of Iberian red deer requires preventing not only restocking practices with red deer specimens belonging to other European populations but also translocations between both Iberian lineages.Red deer populations in the Iberian glacial refugium were the main source for postglacial recolonization and subspecific radiation in north‐western Europe. However, the phylogenetic history of Iberian red deer  During the Quaternary ice ages, most of northern Europe was covered by ice, while permafrost extended through Central Europe, shaping the distribution of many temperate species to ice‐free glacial refugia during the Late Pleistocene Hewitt . AccordiVarious phylogeographic analyses have pointed to the Iberian Peninsula as the main refugium for postglacial recolonization of north‐western Europe for different animal species  is the most widely distributed species of large wild mammal in the main the Palearctic  phylogeography was recently performed by Fernández‐García et al.  or antlers (antler‐bone shavings) from 611 individuals from wild populations of red deer in Spain (Iberian Peninsula) (Table N = 19), Cardeña , Hornachuelos , and Montes de Toledo . Additionally, we selected 20 samples from areas in which the western European lineages were described , and 50–100 ng of template DNA. The thermal cycling profile was 94°C (3 min) followed by 35 cycles at 94°C (1 min), 56°C (1 min), and 72°C (1 min) with a final extension step of 72°C (1 min).DNA extraction was performed by a standard salting‐out procedure  with an incubation of 37°C for 30 min followed by 85°C for 15 min. We sequenced the mt CR by using the PRO primer, as it has been shown that this captures most of the nucleotide variation along this region in red deer . Sequencing reactions were purified using the Montage SEQ96 Sequencing Reaction Clean‐up Kit  and analyzed in a DNA sequencer ABI PRISM 3130 (Applied Biosystems).Fragment was sequenced using BigDyeFor nuclear loci, samples were genotyped with 15 microsatellite markers optimized in three multiplex PCRs and two capillary electrophoresis runs [MP1: CelJP15, MM12, OarFCB304, OarFCB193, ETH225, BM1818; MP2: OarFCB5, TGLA53, CelJP38; MP3: CSSM19, RME25, CSSM22, CSSM43, OarCP26, CSPS115 (Buchanan and Crawford π).For mtDNA analyses, we used SeqScape v2.5 (Applied Biosystems) to visually inspect and correct sequences. Alignment was conducted using Bioedit Hall , and seqTo address the phylogeographic and evolutionary relationships among haplotypes, we carried out four types of phylogenetic analyses: (1) maximum likelihood (ML), (2) maximum parsimony (MP), (3) neighbor‐joining (NJ), and (4) Bayesian inference (BI). We used PAUP* 4.0b10 Swofford  with “trCervus elaphus barbarus) , Sardinian red deer (Cervus elaphus corsicanus) (AF291885), Italian red deer (Cervus elaphus hippelaphus) (AF291886 and AF291887), Spanish red deer (Cervus elaphus hispanicus) (AF291889), and Cervus elaphus (U12867) were obtained from the NCBI nucleotide data bank and were included in phylogenetic analysis.To determine the appropriate model of sequence evolution, we used the program MODELTEST 3.7  between groups was performed with DNAsp v5.10 , expected heterozygosity (He), and mean number of alleles per locus (A) with Genetix version 4.05 , 10 independent runs of K = 1–9 were carried out with 50,000 iterations, following a burn‐in period of 1,000,000 iterations. We assumed admixture and a correlation among genetic frequencies. We used an ad hoc statistic ΔK to identify the true number of genetic clusters (K)  for all populations with GENETIX.In order to check for structure in our data with different approaches, firstly pairwise Finally, we compared the geographical distance and genetic relatedness between all possible pairs of individuals in our populations with SPAGeDI. We considered as the geographical location of individuals the mean point of the area each population represents. For the analyses, we used the genetic relatedness coefficient described in Wang . To asseKT202236‐KT202280), defined by 61 polymorphic sites (8.1%) of which 44 (5.8%) were parsimony informative. Base composition was biased to a deficiency of guanine .The mtDNA CR sequence alignment (757 bp including sites with gaps) of 682 deer samples was collapsed in 45 haplotypes , C. e. hispanicus showed the highest nucleotide diversity value (π × 100 = 0.999), indicating a great amount of divergence between haplotypes within Iberian (Spanish) red deer. The lowest values of genetic diversity were found in the sampled population of C. e. elaphus (Hd = 0.255 and π × 100 = 0.108).Genetic diversity indices for the red deer populations analyzed in this study are summarized in Table C. e. scoticus and C. e. atlanticus distribution ranges and H6 that was very common in southern Spain and also corresponded to the haplotype identified in a trophy from Germany dated to 1872. In our sample, haplotype H6 was the most frequent (28%), although it was mostly restricted to Sierra Morena , followed by H16 and H9 .Most haplotypes were unique among subspecies. Among the 45 haplotypes found, only two were sampled in more than one subspecies: H19 was sampled in both C. e. hippelaphus sequences from GenBank and individuals sampled in Italy, where it is known that populations were restocked with C. e. hippelaphus , which has been more precise than the hierarchical likelihood ratio test  before present for the coalescence of the ancestors of both branches. Within the haplogroup that includes central‐south Iberian and north European populations, the main central‐south Iberian clade was inferred to diverge at approximately 88 Kyr (node 4), while the remaining central‐south Iberian and north European lineages diverged at approximately 76 Kyr. Thus, the phylogenetic pattern for Western European red deer indicates a deep division dated at 109 Kyr that differentiated both Iberian lineages, and subsequent differentiation events within these clusters that were dated at 88 and 76 Kyr, giving rise to the radiation within central‐south Iberian–north European main haplogroups and within the west Iberian lineage, respectively.Significant deviation from neutrality was detected only within the phylogroup Sw with Tajima's D 2,84**) but significance disappeared when more powerful statistic was applied  using DNAsp software  between the genetic clusters identified by STRUCTURE analyses. Figure Bayesian methodology implemented by STRUCTURE showed that ers Fig. A. Plottions Fig. A. In the= 3 Fig. B. Indiviany Fig. B. In theers Fig. C and twoers Fig. C. In thiers Fig. C: one maP < 0.001; Fig. Genetic relatedness between pairs of individuals declined with geographic distance  can be supported on the basis of nuclear DNA, while mtDNA uncovers for the west European clade a differentiated refugial lineage for western Spain populations.Phylogenetic differentiation of red deer in Europe based on mtDNA assumes three primary clades  may be a paraphyletic subspecies that encompasses two genetic lineages that predated the last glacial maximum with one of them leading to different subspecies during the Holocene when recolonized northern areas in Europe. The genetic differentiation of the western Iberian lineage (Sw) was not strongly supported on the basis of the mtDNA phylogenetic tree, but the topology splitting both haplogroups within Spain was consistent in all consensus trees from all four methods used, while the central‐south Spanish populations showed very low genetic differentiation with respect to north European populations at mtDNA sequences. For nuclear DNA, however, the West European red deer was firstly split into two different groupings: Iberian and non‐Iberian populations. In the search for lower genetic discontinuities, the Iberian populations appeared then divided into two clusters corresponding to western and central‐south Iberian haplogroups.Therefore, our results indicate that Iberian red deer , mtDNA indicates an earlier differentiation during Würm glacial times in the Iberian Peninsula that maintains signatures in current populations at two main geographical areas in Spain, while postglacial recolonization of northern Europe has not produced noticeable differentiations at the level of mtDNA with respect to the Iberian refugial lineage to which they belong. Meanwhile, nuclear loci have revealed two additional facts. One of them (second subheading below) is that genomes of north‐expanding populations differentiated not only from their Iberian ancestors (haplogroup Sc) but also among themselves, especially affecting the northernmost Norwegian populations. Red deer might have disappeared at these northern ranges during the LGM or might have maintained some small stocks, but it seems most probable that all current native populations in northwestern Europe descend from the postglacial recolonization from the Iberian refugium  found five genetic clusters evidencing three glacial refugia within the Iberian refugium , it evidenced a genetic structure within Iberian red deer deeper than with distant populations in northern Europe. Haplotype diversity was lower for group Sw, but the phylogenetic relationships do not support a recent origin of Sw from the other Iberian lineage Sc. Rather, molecular divergence times indicate a differentiation that predated the LGM, which points to two refugial areas within Iberia. There are many examples of genetic differentiation at the level of mtDNA within Iberia during the Pleistocene for diverse taxa, from invertebrates to all vertebrate groups and many species of plants  of Iberian lineages in western (Sw) and central‐southern Spain (Sc) show a secondary contact zone located in NE‐SW direction at the mountain range occurring in eastern Extremadura (Villuercas and Sierra de Pela). Contact zones are common for other taxa in Iberia, although the location and direction are highly variable  differentiated from the other red deer subspecies, and in particular from the west European subspecies , on the basis of morphology  contributed to the extant populations in northern areas of western Europe. Along with morphology Geist , behaviogy Geist . Data frgy Geist .A debate exists in taxonomy on considering paraphyletic entities as real taxa  may be debatable Moritz , but it None declared.


Hypoderma bovis and H. sinense (Diptera: Oestridae) mainly parasitise cattle and yaks. The two parasites are pathogenic and cause economic losses that result from reduced amounts of livestock products, including milk, meat, and skin. Genetic diversity and population genetic structure of H. bovis and H. sinense have not been evaluated, but could be used to inform appropriate strategies to control these parasites.c  oxidase subunit I (COI) gene from 60 H. bovis isolates and 52 H. sinense isolates from five locations in Qinghai Province, China, to identify polymorphisms, and infer their phylogenetic relationships, historical population expansions, and divergence time.We cloned and sequenced part of the mitochondrial cytochrome H. bovis samples, and 23 COI haplotypes from the H. sinense samples. The haplotype and nucleotide diversities were 0.738 and 0.00202 for H. bovis, and 0.867 and 0.00300 for H. sinense, respectively, which indicates rich genetic diversity in H. bovis and H. sinense populations. Bayesian phylogenetic analysis revealed that the two species are monophyletic, and geographical structuring of haplotypes was significantly different in H. sinense (P < 0.05), but not H. bovis. Neutrality tests and mismatch distribution statistical analysis revealed that populations of the two species have undergone demographic expansions. The divergence three Hypoderma spp.  was estimated to have occurred approximately 4.5 million years ago (Mya), which indicates that the rapid uplift of the Qinghai-Tibetan Plateau during the late Miocene-Pliocene was associated with divergence of Hypoderma species.We identified 17 COI haplotypes from the H. bovis and H. sinense displayed high genetic diversity and widespread population genetic differentiation within and among populations; these data, along with the molecular phylogeny, demographic history, and divergence time estimation, provide new insight into evolutionary history of these species. These findings will help elucidate speciation in Hypoderma and provide theoretical basis for epidemiological surveillance and control of these species on the Qinghai-Tibetan Plateau.Results of the present study revealed that both  Hypoderma bovis and H. sinense (Diptera: Oestridae) are two species of flies in Oestridae and mainly parasitize cattle and yaks. The parasitizing flies are widely distributed in north and southwestern China  were calculated using DNASP 5.10 [COI sequences were aligned using MEGA 5.2 . IdenticASP 5.10  for eachH. bovis and H. sinense COI haplotypes were inferred using Bayesian inference (BI). We selected the best-fit model (GTR + I + G) for BI analyses for each data partition using Modeltest 3.7 [Phylogenetic relationships of test 3.7  in conjutest 3.7 . A Bayestest 3.7 , and MarH. bovis (AF497761) and H. sinense (AY350769) obtained from the GenBank database were used for phylogenetic analysis of the species in this study, and COI sequences from three other species of Hypoderma, H. lineatum (AF497762), H. tarandi (AF497764) and H. actaeon (AF497765), were used as in-group taxa for the phylogenetic analysis. Gasterophilus pecorum (AF497776) was selected as the out-group taxon to root the phylogenetic trees. Median-joining networks of all H. bovis and H. sinense haplotypes in this study were constructed using Network 4.6 [COI sequences of work 4.6  to visuaH. bovis and H. sinense population genetic structure in Arlequin 3.11 with 1000 permutations [ΦST). Phylogeographic structure of H. bovis and H. sinense populations was estimated using PERMUT (http://www.pierroton.inra.fr/genetics/labo/Software/Permut) with 1000 permutations. PERMUT tested phylogenetic structure by calculating GST (only haplotype frequencies) and NST (differences among haplotypes); phylogenetic structure is usually considered present when NST is higher than GST [Fs and Tajima’s D neutrality tests, and sum of squared deviation (SSD) and Harpending’s raggedness (rg) test statistics of mismatch distributions were calculated to detect evidence of past population expansions in Arlequin 3.11 [H. bovis and H. sinense populations were performed using DNASP 5.10 [Analysis of molecular variance (AMOVA) was used to evaluate utations . This stthan GST . Fu’s Fsuin 3.11 –28. In aASP 5.10  to test ASP 5.10 , whereasASP 5.10 .ut [t represents time in generations since expansion, and u = 2 μk, where μ is the mutation rate (COI was estimated to be approximately 2 % per million years for Hypoderma spp. ) [k is the length of the sequence [H. bovis and H. sinense generation time was estimated to be 1 y based on their life-cycle [Expansion time was estimated using the expectation τ = 2 ut , where τa spp. )  and k issequence . The H. fe-cycle .Hypoderma fossils, a secondary calibration approach was used. Based on previous research on divergence time in Hypoderma spp.  [H. tarandi and H. actaeon, and 4.2 Mya with 0.5 Mya for H. bovis and H. lineatum, respectively. The Markov chain Monte Carlo chain length was set to 10 million generations and sampled every 1000 generations. Chain convergence was assessed to determine effective sample sizes greater than 200 for all parameters using Tracer 1.5 [Divergence times were estimated using the Bayesian phylogenetic method implemented in BEAST 1.8.0 . The cloma spp.  , a mean acer 1.5 , and treacer 1.5 .H. bovis and H. sinense. A total of 19 nucleotide polymorphisms (12 singleton variable sites and seven parsimony-informative sites) for H. bovis and 31 nucleotide polymorphisms (24 singleton variable sites and seven parsimony-informative sites) for H. sinense were detected. In total, 17 haplotypes were detected in H. bovis populations and 23 haplotypes were detected in H. sinense populations  were connected in a star-like manner Fig. . The higH. bovis and H. sinense populations , which indicates remarkable genetic differentiation in H. bovis. For H. sinense, AMOVA showed that 7.83 % of the variation was among populations and 92.17 % was within populations. We also detected high genetic structure , which likewise indicates significant genetic differentiation, in the H. sinense isolates. Large pairwise FST values were found between H. bovis and H. sinense populations. For H. bovis, pairwise FST ranged from 0.009 to 0.190, and most pairwise values were statistically significant  occurred about 0.056 Mya. A permutation test showed that NST (0.141) was significantly higher than GST (0.048) for H. sinense (P < 0.05), whereas NST (0.116) was less than GST (0.122) for H. bovis, which indicates that significant phylogeographic structure is apparent in H. sinense, but not H. bovis.Demographic expansions were analysed for ve Table , and misve Table . From thHypoderma species with the 95 % highest posterior densities (95 % HPD) intervals are presented in Fig. Hypoderma species existed approximately 8.1 Mya (95 % HPD: 6.8–8.8 Mya). The split between two main clades of the three Hypoderma species  occurred about 4.5 Mya (95 % HPD: 3.1–5.3 Mya), and the divergence time between H. sinense and H. lineatum was approximately 3.7 Mya (95 % HPD: 2.2–4.9 Mya). Intraspecific divergence times all occurred within 3.1 Mya for H. bovis and H. sinense.The estimated evolutionary timescale of five h) and nucleotide (π) diversity are important indicators of genetic diversity in a population [h and π in H. bovis and H. sinense populations, which indicates rich genetic diversity of populations of the two species and might explain why the two species have a broad tolerance to environmental and habitat stresses; fast mutational processes inherent in individuals and populations may enable these two Hypoderma spp.  to successfully adapt to complex environments.The haplotype , which indicates that populations of the two Hypoderma species were closely related respectively, and speciation within Hypoderma might be relatively complex.The Bayesian phylogenetic analysis strongly supported the coalescence of COI haplotypes within ar study . This ispes Fig. , and theH. bovis and 92.17 % for H. sinense; among populations: 11.88 % for H. bovis and 7.83 % for H. sinense). Therefore, the majority of H. bovis and H. sinense genetic differentiation was intra-population. This result may be caused by gene flow. QTP uplift, which resulted in topography changes [Hypoderma species cannot fly long distances, and adults live for a very short time (only 5–6 days) [P < 0.001) for H. bovis and H. sinense based on AMOVA  than in H. sinense (−0.037 to 0.210), indicating greater genetic differentiation among H. sinense than in H. bovis populations. In addition, permutation test showed that H. sinense had greater geographic structure of h than H. bovis. Geographic structure of natural populations is determined by many factors, such as life history, population size, ecological traits, habitat, and historical events [H. bovis and H. sinense. However, geographic genetic structure was previously shown to negatively correlate with dispersal abilities [H. sinense exhibited greater geographic genetic structure than H. bovis, indicating that dispersal ability is higher in H. bovis than H. sinense. This finding is consistent with the life history of these species; H. bovis mainly parasitize cattle and yaks in plains, hills, and plateaus, whereas H. sinense mainly attack bovines in plateau settings [Hypoderma species with differences in habitat preference and dispersal ability.AMOVA indicated that genetic structure was substantially higher within than among populations  . Geneticulations . Our assl events , 42. Ourbilities . In the H. bovis and H. sinense populations, which indicates that population expansion events may have occurred in the demographic history of H. bovis and H. sinense. Additionally, the mismatch population test statistics rg and SSD for both H. bovis and H. sinense populations were small and not statistically significant; this indicates that the sudden expansion model, which corresponded with a unimodal curve of the mismatch distribution analysis, could not be rejected  diverged approximately 4.5 Mya, which indicates a late Miocene-Pliocene split among H. bovis, H. lineatum and H. sinense [Hypoderma spp.  Furthermore, the divergence time between H. sinense and H. lineatum was estimated to be 3.7 Mya, which mainly corresponded to the rapid uplift of the QTP approximately 3.6 Mya [Hypoderma spp. More importantly, the rapid uplift of the QTP changed topography, strengthened the East Asia monsoon, and modified global climate [Hypoderma populations, and eventually caused speciation of H. bovis, H. lineatum and H. sinense.The COI gene is a global molecular clock gene . In this sinense , 48. Cli 3.6 Mya  and ther climate –52, whic climate –55. TherHypoderma spp. play a critical role in production losses and susceptibility of cattle and yaks to disease [Hypoderma evolutionary history and speciation, and provides important information for the future study of epidemiological surveillance and hypodermosis control on the QTP. disease , 56, 57.H. bovis and H. sinense populations in Qinghai Province, China. The results support the distinction of the two species of Hypoderma based on genetic diversity and divergence. Most genetic differentiation of H. bovis and H. sinense was found within populations, which may have been caused by QTP uplift and life history of the species. Further research including more molecular markers, increased sampling, and different phylogenetic analysis methods are necessary to elucidate genetic differentiation of Hypoderma spp. in more detail. In addition, the current findings provide fundamental evolutionary information regarding H. bovis and H. sinense. These findings also provide a molecular baseline for the control and elimination of Hypoderma spp. on the QTP.This is the first characterization of the genetic diversity and population structure using mitochondrial COI sequences of


Boechera fecunda, found in only several populations in each of two geographic regions (WEST and EAST) in southwestern Montana. We analyzed variation in quantitative traits, neutral molecular markers, and environmental factors and provided evidence that despite the restricted geographical distribution of this species, it exhibits a high level of genetic variation and regional adaptation. Conservation efforts therefore should be directed to the preservation of populations in each of these two regions without attempting transplantation between regions. Heritabilities and genetic coefficients of variation estimated from nested ANOVAs were generally high for leaf and rosette traits, although lower (and not significantly different from 0) for water-use efficiency. Measures of quantitative genetic differentiation, QST, were calculated for each trait from each pair of populations. For three of the five traits, these values were significantly higher between regions compared with those within regions . This suggested that natural selection has played an important role in producing regional divergence in this species. Our analysis also revealed that the B. fecunda populations appear to be locally adapted due, at least in part, to differences in environmental conditions in the EAST and WEST regions.Many biological species are threatened with extinction because of a number of factors such as climate change and habitat loss, and their preservation depends on an accurate understanding of the extent of their genetic variability within and among populations. In this study, we assessed the genetic divergence of five quantitative traits in 10 populations of an endangered cruciferous species,  Because of increasing habitat loss and/or fragmentation, climate change, and various other factors, many species currently are in decline and face potential extinction  between two major genetic groups of Boechera stricta. As in this example, most of these sorts of studies have used environmental factors and either neutral molecular or quantitative genetic traits. Here, we make use of both neutral molecular and quantitative genetic traits as well as environmental variables to understand population differentiation and conservation in Boechera fecunda (Brassicaceae), an endangered wild relative of Arabidopsis.A number of studies have shown that environmental variables can also contribute to population differentiation . Boechera fecunda is restricted to only 21 populations in two geographical regions (WEST and EAST) in western Montana (Rollins http://fieldguide.mt.gov/detail_PDBRA06290.aspx). Rollins  separateFST = 0.57; Song and Mitchell-Olds FST–QST regional comparisons and tests for divergent selection. We also analyzed a number of environmental variables to assess their contribution in producing differentiation among the populations. In particular, we were interested in testing for local adaptation in B. fecunda in the two genetically and geographically different regions, and if found, in determining whether differences in specific environmental variables might account for this divergence.Previous analyses of neutral genetic (microsatellite and nucleotide sequence) data in these populations showed that they possess levels of genetic variability comparable to a nonendangered congener and that they exhibit considerable differentiation (B. fecunda genotypes per population were collected from ten populations (generation S0), three in the WEST geographical region and seven in the EAST geographical region of their range in Montana  and height (RosH) were measured in cm. Rosette volume (RosV) was calculated as an additional trait by πr2h/3, where r = the radius and h = the height of the rosette (Lee and Mitchell-Olds N = 472 for RosV).During the 8th week of growth of each of the seedlings from the N = 230) was put in a separate cuvette from which three measurements were taken with a 10-s interval once the concentration of CO2 had stabilized. All measurements were made between 9 am to 5 pm with roughly 400 μmol−1/mol−1 CO2 and 26% relative humidity in the surrounding environment. The mean of the three measurements was used in further analyses.During the 10th week of growth, instantaneous water-use efficiency (WUE) was measured in each plant. WUE was calculated by dividing the carbon fixation rate (A) by the water transpiration rate (E). A and E were recorded on whole plants using a modified system and protocol  on the x-axis and the second principal component (PC2) on the y-axis for each of the 10 populations to discover patterns of dispersion among these populations.Prior to the quantitative genetic analysis, we first examined the distributions of each of the five  traits after adjustment for differences among populations . All were found to be normally distributed except RosV, which showed some skewness that was corrected with a logarithmic transformation. We calculated basic statistics (means and standard deviations) for each of the traits for populations in the separate EAST and WEST regions and conducted an ANOVA to test for differences in their means between these regions. We used a nested ANOVA model with regions as a fixed factor, populations as a random factor nested within regions, and families as a random factor nested within populations. Probabilities associated with the FST values  in R. To test for isolation by distance (IBD) among the studied populations, we used a Mantel's test of association of the FST values in each of the 45 population pairs with the matrix of geographical distances.Molecular data for 13 polymorphic microsatellite loci were retrieved from data collected by Song and Mitchell-Olds . A matri Slatkin  also werVR), populations nested within regions (VP), families nested within populations (VF), and the error (VE) from replicates within families. Regions was treated as a random factor in these analyses so that its percent contribution to the total variance could be assessed. Broad-sense heritabilities for each trait were calculated by VF/(VF + VE) within populations, and sampling with replacement (bootstrapping) among the families was performed with 1000 iterations to estimate 95% confidence intervals for the heritabilities. Beyond heritabilities, we also calculated the coefficient of genetic variability, CVG, for each trait by G is a measure of how much phenotypic change may occur given a unit of selection (“evolvability”).We used the MIXED procedure in SAS to estimate variance components for four random factors in a nested ANOVA model. The variance components for the factors affecting each of the five traits were regions  ideally should be compared to the distribution of FST values. QST values exceeding an appropriate threshold  FST value then are taken as evidence of divergent selection. Our total of 13 molecular markers genotyped for the B. fecunda populations, however, is far fewer than the minimum of 50 recommended by Whitlock  in magnitude, however, and Whitlock , where VP and VF were estimated from nested ANOVAs . VF estimates the genetic variance among families within populations and is equivalent to the VA term in the formula for QST shown by Whitlock and Gilbert  with a matrix of QST values, a 0/1 matrix defining population pairs within and between regions, and an FST matrix. Probabilities for the Mantel's correlations were estimated from matrix permutations  and evaluated using the FDR procedure. Positive and statistically significant correlations between pairwise QST values and the within/between region population relationship were taken to indicate that quantitative genetic differences between regions were greater than those within regions after adjustment for neutral genetic effects, and therefore that regional variation was ascribable to divergent selection. Saether et al.  than those in the EAST region. This is especially true for water-use efficiency (WUE) for which the mean in the WEST region is approximately 50% larger than that in the EAST region. Trait variability is comparable between the two regions, although as judged by coefficients of variation (not shown), all traits, especially WUE, show high levels of variation.Basic statistics for the five traits over the pooled populations in each of the two geographical regions (WEST and EAST) are shown in Table P < 0.01 in Student's t-tests), although none of these four traits are significantly correlated with WUE  for all five traits are high (average of 22.0), with WUE having the lowest value. CVG values for the leaf/rosette traits in the EAST region (mean = 23.3) exceed those for the WEST region (mean = 19.9), whereas WUE exhibits the reverse trend.Within populations, heritabilities estimated from the proportion of family variance over that for family plus error variance average a moderately high 0.53 for the five traits  vary from 0.087 to 0.849. The median values for these estimates within (0.405) and between regions (0.369) are quite similar. QST estimates for the five traits also varied considerably among the 45 pairs of populations. Median values for these QST values for population pairs between regions are greater than those within regions for all traits, including for RosD, RosV, and WUE where the median within regions is 0.QST values differed for population pairs within and between the WEST and EAST regions, after adjustment for neutral genetic variation , with the use of partial Mantel's tests as described earlier. Because of the non-normal distributions of the QST values, we used nonparametric Spearman rather than Pearson's correlations in the ECODIST package. These correlations between QST values and the between/within region index values were significantly different (P < 0.05) for Leaf, RosD, RosV, and WUE. Figure QST versus FST values for each trait and clearly illustrates that the QST values between regions predominantly are greater than those within regions.We tested whether the Results of the principal components analysis of the 26 environmental variables are shown in Supplemental Table S5. The first principal component accounts for 38.6% of the total variance, and primarily represents a contrast of the temperature variables  with the precipitation variables . The second principal component contributes 28.1% of the total variation and loads most heavily on the precipitation variables (mean loadings = 0.26) more so than the temperature variables (mean loadings = 0.14), but also with some contributions from other variables, especially altitude and flow direction. Principal component three contributes considerably less variation (10.6%) than either of the first two components, but represents an interesting contrast between BIO2, SLOPE and STREAM with BIO12 and especially ASPECT. PC4 contributes slightly <8% of the total variation and loads most heavily on CPI, with some contrast from SLOPE.Figure QST values for the five traits, none were significantly associated with the environmental distances, but QST values for three  of the five traits were significantly correlated with the environmental PC2 distances.Mantel's tests showed that environmental distances between pairs of populations were significantly associated with geographic distances, although not with regional differences Table . EnvironB. fecunda previously uncovered in a study using neutral genetic markers . Further, we conducted an environmental analysis to quantify environmental differences and to discover what environmental variables, if any, might have shaped differences in B. fecunda between the two regions.Optimal preservation strategies for endangered and threatened species are based on information about the extent of genetic variability within and between populations, and the forces  driving population differentiation. In general, the goal is to preserve adaptive genetic variability across the range of the species  compared with plants in the EAST populations and may well use a resource acquisitive strategy. The higher WUE level in the WEST populations is not consistent with the accelerated development hypothesis, but is consistent with the general trend that WUE tends to increase along gradients of declining moisture.In our species, we found both accelerated development and higher water-use efficiency in plants in the drier (WEST) region compared with the wet region (EAST), as did McKay et al. . PerhapsBoechera stricta living in two separate regions with divergent levels of water availability. Donovan and Ehleringer (QST < FST) was found in Helianthus anomalus  variances in these four morphological traits were comparable between the two regions . For WUE, the CVG of 20.4 for the WEST populations was much higher than that of 9.4 for the EAST populations. More traits will be measured in the near future to fully understand patterns of quantitative genetic variation for Boechera fecunda in each population and region.Song and Mitchell-Olds  previousBeyond family differences within populations, our analysis also provided evidence of population and regional divergence in the quantitative traits. Regional divergence was apparent from the significant differences in the means of four of the five traits in the WEST versus EAST regions, from the clear separation of the populations in these two regions in the principal components analysis of the phenotypic correlations of the traits and from the nested ANOVAs where regional differences for each trait contributed on average about 23% of the total variation. Divergence among populations within regions was much less (mean contribution = 9%). If we had analyzed divergence only among the 10 populations, the mean contribution would have increased to 20%, but we would have missed the heavy contribution of regional differences to this divergence. Clearly, it is important to understand the spatial scales on which divergence occurs . This implies that genetic drift alone cannot explain the regional genetic differentiation, and that is most likely ascribable to the action of divergent selection.In the B. fecunda populations. These variables are not exhaustive, and other environmental measures may be important, but they do represent a good sampling especially of temperature and precipitation measures. The principal components analysis of these environmental variables showed that the first two components were effective in separating the 10 populations. PC2 was particularly interesting, because it separated populations in the two regions. This component was a complex contrast, with high loadings especially on most of the precipitation variables, but with contributions from other variables. It was not surprising that a Mantel's test showed a significant association of environmental and geographic distances, because many of the environmental variables vary considerably among the populations, especially as the distance increases. More interesting was the finding that the QST values showed no association with the environmental distances for the population pairs, but three of the five QST values exhibited significant correlations with the environmental PC2 distances. These results suggest that the variables described by the second environmental principal component may be contributors to local adaptation of B. fecunda populations in the EAST and WEST regions.We used a suite of 26 environmental variables to probe what conditions in the EAST versus WEST regions may have played a role in shaping local adaptations in the B. fecunda. For this nonmodel species, next generation sequencing technology that is now available will allow genome-wide genotyping at reduced costs and thus provide a greater range of molecular markers from which to calculate FST distributions. Combined with additional phenotypic traits, this should give us a more detailed understanding of population divergence and ecological adaptation in this species and of the genetic basis of ecologically important complex trait variation as well.In summary, we used quantitative genetic, neutral genetic and environmental data to analyze diversity and ecological adaptation in


Our study documents a combination of demographic and genetic rescue over multiple generations under fully natural conditions. Within both recipient populations, we found substantial and long‐term increases in population size that could be attributed to high survival and recruitment caused by immigration and gene flow from the introduction sites. Our results suggest that low levels of gene flow, even from a divergent ecotype, can provide a substantial demographic boost to small populations, which may allow them to withstand environmental stochasticity.Genetic rescue, an increase in population growth owing to the infusion of new alleles, can aid the persistence of small populations. Its use as a management tool is limited by a lack of empirical data geared toward predicting effects of gene flow on local adaptation and demography. Experimental translocations provide an ideal opportunity to monitor the demographic consequences of gene flow. In this study we take advantage of two experimental introductions of Trinidadian guppies to test the effects of gene flow on downstream native populations. We individually marked guppies from the native populations to monitor population dynamics for 3 months before and 26 months after gene flow. We genotyped all individuals caught during the first 17 months at microsatellite loci to classify individuals by their genetic ancestry: native, immigrant, F Specifically, we tested the initial and sustained effects of gene flow between populations of guppies locally adapted to streams with different predator regimes. Guppies adapted to predators were introduced upstream of naturally occurring populations in headwater streams lacking most predators. These translocation experiments were designed by D. Reznick and colleagues to study eco‐evolutionary feedbacks in rapidly adapting populations  over multiple generations. This allowed us to assess whether gene flow from a divergent source results in an overall reduction or increase in fitness at the population level. We could also determine whether changes in population size were caused only by demographic factors, genetic factors, or both. If population growth were caused only by high survival and recruitment of immigrants and pure immigrant offspring, demographic but not genetic rescue would be invoked (Brown and Kodric‐Brown P. reticulata) are a model system in evolutionary ecology that has provided some of the best evidence for rapid adaptation in response to divergent selection  and the Caigual (80 m long) were chosen because they included the upstream extent of native guppies prior to introductions and were bound on either end by waterfalls, thereby preventing upstream movement. Due to the location of waterfall barriers, overall distance between our study sites and the introduction sites differed between streams .®  bottles filled with stream water and held in aerated tanks separated by location and sex. Before processing, fish were anesthetized with a dilute solution of MS‐222 to allow individuals to be marked and photographed. Guppies were marked under a dissecting microscope with visible implant elastomer tags  injected subcutaneously. Each fish was given a unique combination of marks using two or three out of eight discrete marking sites, and 12 possible colors. Concurrently, an identical capture‐mark‐recapture protocol was conducted in upstream introduction sites , we recorded every individual captured that had a standard length over 14 mm. We therefore sampled a total of 29 occasions over 30 months, three of which were before upstream introductions in March 2009. Unmarked individuals were given a unique mark for future identification. Guppies were caught using a combination of butterfly nets, hand nets, and minnow traps. We recorded the location of all pools and riffles within the streams in order for fish to be returned to their precise site of capture. Fish were transported to the lab in NalgeneIn total we uniquely marked and monitored 9590 individual guppies throughout 29 capture events (months) between 2009 and 2011. Of these, 4710 were captured in Taylor and 4880 were captured in Caigual. We recaptured 88 individuals in Taylor and seven in Caigual that had originally been marked as part of the upstream introduction experiment, and thus were confirmed immigrants.We conducted genetic analyses on all individuals from both streams captured during the first 17 (of 29) months of our study. Although we were limited to 17 months of genetic monitoring due to time and resources, this timeframe captured two consecutive wet and dry seasons and 3–4 guppy generations. We extracted genomic DNA from scale samples using Gentra Puregene Tissue Kits . Individuals were genotyped at 12 microsatellite markers developed for this study (Table S1). Microsatellite development and checks for neutrality are described in Appendix S1. We amplified loci using Qiagen Type‐It Microsatellite Multiplex PCR kits with reactions carried out following the manufacturer's recommended conditions. PCR products combined with HiDi formamide and LIZ size standard (500 GeneScan) were read on an ABI 3730xl automated sequencer (Life Sciences Core Laboratories at Cornell University). Microsatellites were visually scored using the microsatellite plug‐in with GENEIOUS 7.1.7  and the relative contributions of vital rates that contribute to population growth, apparent survival (ϕ) and recruitment (f), while accounting for detection probability (Pradel λ) was estimated using the Pradel model, which is a reverse‐time approach that simultaneously incorporates the contribution of survival (ϕ) and recruitment (f) on the rate of increase in population size between two periods (Pradel λ), we compared the most complex model, which included an interaction between sex, stream, and month, to all possible model simplifications including all two‐way interactions, single factors, and the constant model. We used a maximum likelihood approach to fit the models and compared among them using Akaike's Information Criterion adjusted for sample size AICc and AICc weights (Burnham and Anderson P) was modeled with stream by month interactions (described in Appendix S1).Individual capture‐mark‐recapture data allowed us to estimate population growth rate (y Pradel . We firss Pradel . RecruitN = 125 of 3298 fish) from these analyses. We used the survival and recruitment parameterization of the Pradel model to test the effect of genetic ancestry on vital rates. We implemented a sequential modeling approach where we first fit models using the most general structure for recruitment (f) and modeled variation in survival (ϕ) with a three‐way interaction of stream, sex, and genetic ancestry, and all simplifications. We then modeled variation in recruitment (f) in the same way while using the most general structure (and highest supported) for survival (ϕ). We were not able to include interactions between monthly variation and other factors in these models due to small sample sizes for some genetic classes per month. However, our primary goal here was to directly test overall impacts of genetic ancestry on population vital rates and we did not have a priori reasons to think that different genetic groups would experience more or less favorable environmental conditions in a given month. Models from each of the Pradel model sets were compared using AICc.A second set of models was used to test the role of gene flow on population vital rates  and to distinguish between demographic and genetic rescue. If demographic rescue were solely responsible for population growth, we would expect immigrants to have the highest vital rates and the rates of native and hybrid groups to be more or less equivalent. However, if genetic rescue contributed to population growth, we would expect hybrids to show higher relative fitness than native fish. Using capture histories from individuals genotyped and monitored during the first 17 months of the study, we grouped individuals by stream, sex, and genetic classification as determined by the NEWHYBRIDS analysis. We excluded individuals with unknown genetic ancestry  was extremely low within native focal sites of both streams . However, it is important to interpret these data with some caution because differences in detection through time and between streams are not reflected in the overall counts displayed in Fig. λ tended to be lowest during rainy season months (June–December) when resources are low (Reznick λ < 1) and increasing during the dry season . Strong support for this model provides evidence for sex and stream‐specific temporal changes in population growth rate Fig. . Seasona Reznick . For exaϕ) and recruitment (f) in our second set of analyses that only included capture histories from genotyped individuals, but the highest supported models differed between the two vital rates. The full structure  was the best supported structure for survival (ϕ) with 67% of the weight of evidence (Table S4). However, survival estimates did not differ dramatically across genetic groups , with 99% of the weight of evidence, consisted of the genetic group by stream interaction suggesting that sex differences did not explain much of the variation in recruitment, but differences between streams and genetic ancestry did (Table S4). Parameter estimates of recruitment revealed substantial differences in recruitment across genetic classification groups, with native individuals showing lowest recruitment in both streams. Additionally, F2 hybrids and both types of backcross hybrids had highest recruitment in both streams, although confidence intervals overlap with immigrants and F1 hybrids in Caigual and just immigrants in Taylor  in two natural populations. Immigration and subsequent hybridization with genetically and phenotypically divergent individuals led to an overall increase in within‐population genetic variation, abundance, and population vital rates, though dynamic differences were observed between streams and over time. Our results provide a detailed picture of how genetic and demographic rescue can operate in the wild and add to increasing evidence that intraspecific gene flow can be beneficial, even when immigrants are adaptively divergent.Prior to the onset of gene flow, the two native populations in our study were small and genetically depauperate. By the end of our genetic monitoring, spanning 17 months and 3–4 guppy generations, within‐population genetic diversity had more than doubled Fig. . By the 1 hybrids were especially successful toward the end of 2009 in Taylor and throughout the study in Caigual suggesting the possible role of heterosis. Additionally, in both streams F1 × immigrant backcrosses contributed to population size more than F1 × native backcrosses suggesting possible favorable selection for these genotypes.Genotyping each individual allowed us to distinguish between demographic and genetic rescue. If increases in population size were caused only by demographic rescue, immigrants and their offspring should be the only substantial contributors to the increase in fitness  showed highest recruitment in both streams, which suggests that the genetic benefits of crossing persisted beyond first generation hybrids. Genome‐wide SNP data collected from both Caigual and Taylor populations at the end of the study showed that native alleles persisted in both streams, providing additional evidence that hybrid individuals continued to do well and that native alleles were not fully lost from either population . To summarize, the occurrence of genetic rescue is evidenced by the sustained increase in population size and vital rates that can be attributed, at least in part, to the success of the hybrids.Estimates of vital rates based on genetic groups revealed minor differences in survival based on genetic ancestry Fig.  but dramThe variation in vital rates that we observed between sexes, streams, and over time is consistent with patterns previously observed in guppies. First, female guppies tend to have higher survival than males  would be sufficient to induce genetic rescue, as confirmed by the results from Caigual.Our study also illustrated how different rates of migration and gene flow can lead to drastic differences in genetic composition of the population. Unlike most management scenarios, we could not control the rate of immigration and gene flow into recipient populations. Differences in the location of introduced and recipient populations led to substantial differences between streams in the rate of gene flow. Over the first 17 months, we estimate that Taylor received an average of 182 migrants per generation, while Caigual received an estimated average of four migrants per generation. Overall, both streams experienced substantial and sustained increases in population size, regardless of these differences in migration rate. However, from a conservation standpoint, the lower migration rate in Caigual led to a more ideal outcome where the increase in population size was mostly due to success of the hybrids and pure native genotypes were maintained in the population. In contrast, high migration into Taylor led to a dramatic decline of the pure native genotype, which may have caused the loss of potentially important local alleles. Determining the appropriate level of gene flow to prevent inbreeding without swamping local adaptation is a high priority goal for conservation biologists. The classic rule of thumb is one‐migrant‐per‐generation Spieth , yet comUnderstanding the genetic factors that underlie demographic responses will improve our ability to manage connectivity and maintain healthy populations in the wild. The scenario we studied, where immigrants are adaptively divergent and the resident population has low genetic diversity, mimics a common situation faced by managers deciding whether to augment endangered populations. Although many questions remain, our results suggest that adaptive divergence should not, in itself, preclude the use of assisted gene flow for inducing fitness benefits, and also that low levels of migration can result in genetic rescue without replacing the native genetic signature. Future studies that employ genome‐wide markers  will aid in understanding the genomic basis of genetic rescue and hopefully provide additional insight on the optimal number and type of migrants, and how neutral versus selected loci respond to selection and gene flow. Ultimately, sufficient habitat is necessary for long‐term persistence, but genetic rescue may provide a demographic buffer that allows populations to persist through environmental disturbances, as well as the genetic variation needed to adapt to a changing world.http://dx.doi.org/10.5061/dryad.rn262Data available from the Dryad Digital Repository: Appendix S1. Development and characterization of 12 microsatellite loci for the Trinidadian guppy (Poecilia reticulata).Appendix S2. Modeling detection probability with capture‐mark‐recapture data.Table S1. Characteristics of 12 microsatellite loci in Poecilia reticulata.Table S2. Model selection results for detection probability (P) using the Pradel model.Table S3. Model selection results for population growth rate (λ) using Pradel models with the full capture‐mark‐recapture dataset with 29 capture occasions.Table S4. Model selection results for survival (ϕ) and recruitment (f) using Pradel models with the genotyped subset of capture‐mark‐recapture data with 17 capture occasions.Table S5. Parameter estimates and 95% confidence intervals from the highest supported models for survival (ϕ) and recruitment (f).Figure S1. Distribution of average overall performance scores  from the most parsimonious Pradel model (P(stream × month), Table S2).Click here for additional data file.