Trialeurodes vaporariorum), a cosmopolitan invasive herbivore, and examined the genetic structure of this species in Northern and Southern Europe. In Finland, cold temperatures limit whiteflies to greenhouses and prevent them from overwintering in nature, and in Greece, milder temperatures allow whiteflies to inhabit both fields and greenhouses year round, providing a greater potential for connectivity among populations. Using nine microsatellite markers, we genotyped 1274 T. vaporariorum females collected from 18 greenhouses in Finland and eight greenhouses as well as eight fields in Greece.To predict further invasions of pests it is important to understand what factors contribute to the genetic structure of their populations. Cosmopolitan pest species are ideal for studying how different agroecosystems affect population genetic structure within a species at different climatic extremes. We undertook the first population genetic study of the greenhouse whitefly  together with longitude explained genetic structure in Greece, whereas in Finland, genetic structure was explained by host plant species. Furthermore, there was no temporal genetic structure among populations in Finland, suggesting that year-round populations are able to persist in greenhouses.Taken together our results show that greenhouse agroecosystems can limit gene flow among populations in both climate zones. Fragmented populations in greenhouses could allow for efficient pest management. However, pest persistence in both climate zones, coupled with increasing opportunities for naturalization in temperate latitudes due to climate change, highlight challenges for the management of cosmopolitan pests in Northern and Southern Europe. The dispersal of phytophagous insect pests can be enhanced by worldwide trade and human movement ,2]. In . In 2]. Tetranychus urticae Koch , al, alTetraTrialeurodes vaporariorum Westwood) is an invasive pest which was brought to Europe (UK) on Orchidaceae from Mexico in 1856  . P). PT. va country . Unlike al genes ,37]. Re. ReT. vabarriers . Howeverenhouses .T. vaporariorum populations in Finland and in Greece, representing boreal and Mediterranean climate zones, and evaluate the influence of host plants and agricultural practices on the spatial and temporal population genetic structure of this invasive species. We hypothesize that T. vaporariorum populations in Northern Europe are more likely to be genetically differentiated than populations in Southern Europe, because this species is expected to be restricted to greenhouses in the North.To understand how insect pests respond to different environmental conditions, such as climate, habitat, and crop management practices, and the role of agroecosystems in shaping population genetic structure, comparative studies of population genetic diversity of pests in different climate zones are necessary. In this study we present the first extensive genetic data on population structure of the greenhouse whitefly. We compare the genetic structure of In Finland we sampled commercial greenhouses that operate year-round and produce primarily tomato and cucumber crops of various cultivars Table . SamplesB. tabaci species complex; . Th. Th58]. ribution ,26]. In. InTrialtructure . Howevern Greece ,60]..TrialeurT. vaporariorum by limiting dispersal and gene flow among populations, and that populations in the North are more genetically differentiated than populations in the South. In Finland, our samples of T. vaporariorum were limited to greenhouses , whereas in Greece we sampled from both greenhouses and fields. The extent of the area inhabited by homogeneous populations was approximately 1–10 km straight line distance in Finland and approximately 100 km in Greece, indicated in our analyses as samples with non-significant differences in pairwise FST , and shared cluster assignment in Bayesian analysis . Whiteflies sampled at these spatial scales also showed significant isolation by distance. Although whiteflies are poor fliers, natural dispersal of up to 20 km has been observed previously for the related B. tabaci species ..T. vaporAlthough frequent high mortality events occur in agroecosystems in both climate zones , the high abundance of less enclosed/hermetic agroecosystems  and suitable climate in the South reduces effectiveness of pest management. On the other hand, isolation of populations in more enclosed agroecosystems, such as those in the North, can create an opportunity for the development of insecticide resistance through natural selection . In openT. vaporariorum populations indicated a larger gene pool and overall population size and possibly a lower frequency of bottlenecks in the South than in the North. This poses a threat to effective pest management in the Mediterranean region. However, we observed higher than expected homozygosity in samples from both countries , as well as increase in between-population genetic differentiation . Higher T. vaporariorum is able to persist over years in the same greenhouses in Finland. Samples collected from the same greenhouses in 2010 and 2011 were not genetically differentiated from each other in all but one case. Prevalence of the pest year-round might eventually allow further spread into natural ecosystems. Pest persistence in agroecosystems can create propagule pressure to natural habitats and favor utilization of wild host plants that surround greenhouses and fields . Adaptation to crop species could also lead to the development of a preference for particular wild host plants with similar chemistry, as was observed for Tetranychus urticae..T. vaporT. vaporariorum to specialize and adapt to particular host plant species or their cultivars. Although T. vaporariorum is a polyphagous insect, it is able to develop preference not only for certain plant species [Occupation of agroecosystems and differences in their individual management in the two climate zones could potentially allow  species , but als species . In FinlT. vaporariorum prefer cucumber hosts to tomato, and have higher fecundity and shorter development time on cucumber as well [F statistics than samples from cucumber. This might also be due to differences in individual management of agroecosystems. Cucumber crops are changed every three to four months, whereas tomato crops are maintained in greenhouses for nine to ten months, leading to more frequent reductions in pest population size in greenhouses growing cucumber than in those growing tomato. The result might also be a sampling effect: there were more greenhouses growing only tomato than those growing only cucumber   Table . HoweverT. vaporariorum populations might alternatively reflect different introduction sources, as well as varying frequencies of repeated introductions [T. vaporariorum to this cultivar, KR is also somewhat geographically isolated from other agroecosystems and is surrounded by forests, which potentially limits insect dispersal. Therefore, it is difficult to determine if its genetic differentiation is due to adaptation or isolation. Other samples from Finland collected from cucumber (except NL) clustered together  clustered with distant locations in the mainland  together with longitude explained population genetic structure. In contrast, host plant species (tomato vs. cucumber) explained population genetic structure in Finland. The differing influence of type of agroecosystem and potential host plant adaptation on population genetic structure of the pest in different climate zones highlights challenges for the management of a cosmopolitan invasive pest species.Greenhouse agroecosystems contribute to population genetic structure in The authors declare that they have no competing interests.IV, IO, AT were the authors of original idea. IO, LL and IV took part in sample collection in Finland, whereas DEK and AT collected samples from Greece. NG participated in microsatellite development. DEK extracted DNA of samples from Greece, whereas IO and KEK carried all further sample and data analysis steps of all samples. KEK, NG and AT advised on data analysis methods. IO drafted the manuscript. All authors participated in preparation of the manuscript, read and approved the final manuscript.


Gasterosteus aculeatus L.) forms are numerous and common throughout the species distribution, offering an opportunity to examine these contrasting hypotheses in greater detail. This study reports on an interesting new contact zone located in a tidally influenced lake catchment in western Ireland, characterised by high polymorphism for lateral plate phenotypes. Using neutral and QTL-linked microsatellite markers, we tested whether the high diversity observed in this contact zone arose as a result of introgression or reproductive isolation between divergent forms: we found strong support for the latter hypothesis. Three phenotypic and genetic clusters were identified, consistent with two divergent resident forms and a distinct anadromous completely plated population that migrates in and out of the system. Given the strong neutral differentiation detected between all three morphotypes (mean STF = 0.12), we hypothesised that divergent selection between forms maintains reproductive isolation. We found a correlation between neutral genetic and adaptive genetic differentiation that support this. While strong associations between QTL linked markers and phenotypes were also observed in this wild population, our results support the suggestion that such associations may be more complex in some Atlantic populations compared to those in the Pacific. These findings provide an important foundation for future work investigating the dynamics of gene flow and adaptive divergence in this newly discovered stickleback contact zone.Contact zones between divergent forms of the same species are often characterised by high levels of phenotypic diversity over small geographic distances. What processes are involved in generating such high phenotypic diversity? One possibility is that introgression and recombination between divergent forms in contact zones results in greater phenotypic and genetic polymorphism. Alternatively, strong reproductive isolation between forms may maintain distinct phenotypes, preventing homogenisation by gene flow. Contact zones between divergent freshwater-resident and anadromous stickleback ( Divergent natural selection can lead to the evolution of adaptive differences between populations inhabiting contrasting environments ,2. DisruContact zones, i.e. regions of overlap between divergent populations or species, are often characterised by high levels of gene flow, which give rise to hybrid zones ,9. AlthoGasterosteus aculeatus L.) occur throughout the distribution of the species [Contact zones between adaptively divergent populations of three-spined stickleback  and Lough Furnace a smaller brackish lake  which lies ca. 200 m to the south of Feeagh [The Burrishoole catchment is a postglacial drainage system situated in the Nephin Beg mountain range in County Mayo, western Ireland see , compris2 ,32. Thref Feeagh . Lough Ff Feeagh ; anadromThree-spined sticklebacks (N = 414) were sampled at 17 sites across the Lower Burrishoole from March-June in both 2009 and 2010 . Individa priori grouping variables using principal components analysis (PCA).To analyse shape, the left side of each stickleback (N = 414) was photographed using a CANON EOS 1000D Digital SLR camera fitted with a macro lens. Seventeen landmarks based on configurations used by Albert et al  and whicLinear morphological measurements (±0.1 mm) were taken from digital photographs using ImageJ . These iEcotdysplasin (Eda) gene  wereFST and Jost’s [D—were calculated using the diveRsity R package [FST and DJost were calculated with all markers included, QTL markers excluded and for QTL markers only. All microsatellite data used in this study are available at the Dryad digital repository (Dryad DOI).Individual microsatellite genotypic data were grouped by location and plate morphology . Locus specific heterozygosity estimates, Hardy-Weinberg tests and two pairwise genetic differentiation statistics—Weir & Cockerham’s  FST andd Jost’s  D—were c package . For botK 2 to 6 using a burn-in of 100 000 and 250 000 MCMC steps with five iterations per K estimate. The most probable value of K was then determined using the Evanno et al. (2005) method implemented in Structure Harvester [In order to determine cryptic population structure in the Burrishoole, two Bayesian cluster assignment programs, STRUCTURE 2.3.3  and NewHarvester ,53.While STRUCTURE is suited for assigning individuals of unknown origin to clusters, the Bayesian algorithm implemented in NewHybrids specifically tests for the presence of hybrids between genetic differentiated clusters by assigning individuals to genotype categories . Although NewHybrids was originally developed to test for hybrids between species, it has also been successfully applied to examine zones of overlap between genetically divergent populations within species –56. Sincq-value for a given cluster was greater than or equal to 0.9, a value resulting in the highest efficiency in hybrid assignment [adegenet R package [While microsatellite markers can be useful for hybrid detection, it is important to carry out prior evaluations of markers and their performance using different analyses ,60. To tsignment . These ‘ package . For eacPST, a measure analogous to FST based on phenotypic measurements [PST values were calculated for mean lateral plate number, a heritable phenotypic trait, using the following formulae:2GB denotes the variance of a given phenotypic trait among all populations and σ2GW is the average variance of the same trait within populations [PST-FST comparisons can be informative for identifying the relative roles of drift versus selection between populations [PST compared to neutral FST estimates indicate that selection rather than drift contributes to population divergence [lme4 R package [PST estimates were then calculated and bootstrapped using 1000 replicates with resampling to determine 95% confidence intervals with custom R scripts (DRYAD). Pairwise PST estimates were compared to pairwise Weir & Cockerhams [FST estimates with bootstrapped 95% confidence intervals derived from all microsatellite markers, neutral markers only and QTL markers only using the diveRsity R package [In an attempt to examine the role of selection in constraining gene flow in the Burrishoole catchment, comparisons involving measures of phenotypic and genetic differentiation among morphotypes were carried out. Adaptive phenotypic divergence was quantified using urements . Pairwisulations . PST-Fulations ,64. Highvergence . Varianc package . PST esckerhams  FST est package .All linear phenotypic measurements were size-corrected to mean body-size using an ANCOVA based method with centroid size as a covariate ,39. Phen2 = 0.67, F3, 392 = 26.16, P < 0.0001), although the difference between lows and partials from Furnace was not significant at the 5% level (P = 0.06). Variation along PC2 largely represented differences amongst individuals and not plate morphs (P = 0.07); increasing values along this axis also reflected an increase in body depth, an elongated snout and a more compressed caudal peduncle.Principal components analysis of body shape described 50% of the total variance with the first two principal components . Shape variation along PC1 indicated a shift from a laterally compressed, deeper-body shape to an elongated, shallow bodied form see , support2 = 0.94, F3, 351 = 1062.53, P < 0.0001, Figs st dorsal spine, 2nd dorsal spine and pelvic spine length) resulted in a single axis explaining 97% of the total variance (PCAP hereafter). Loadings for the three defensive spines were highly positive ; Furnace completes had the largest defensive spines compared to all other types  but did not differ between Furnace lows and partials  was lowest in Feeagh lows (0.86±0.12) and greatest in Furnace completes .Following Bonferroni correction for multiple tests, 3–5 markers were not in HWE in each lateral plate location grouping . With thFST and DJost between phenotype classes were typically greater for estimates made using QTL markers (FST = 0.23 & DJost = 0.36) than those made using neutral markers only  and QTL microsatellite markers  occur in Burrishoole; one originating from Lough Feeagh and the other two in Lough Furnace.  and QTL markers . However, while FST estimates using all three marker sets were the lowest for Furnace lows and partials, PST between these forms remained relatively high  separated freshwater and anadromous forms clearly, with anadromous individuals completely fixed for a single homozygous genotype for both markers .Two  markers . Heteroz2 = 0.18, F2, 262 = 31.76, P < 0.0001). Most individuals homozygous for the EDAC (240 bp) allele were completely plated while those homozygous for the EDAL (172 bp) allele were low plated. Nonetheless, phenotype-genotype correlation at this locus was not perfect; 57% of all fish typed were heterozygous for the EDA marker, irrespective of their lateral plate phenotype  and the relatively low number of individuals genotyped.In contrast, to clear associations with the three Studies of secondary contact zones between anadromous and freshwater stickleback forms are important for understanding the nature of introgression between these divergent ecotypes. The Burrishoole catchment in western Ireland offers a particularly intriguing example of anadromous and freshwater resident populations existing in close contact. Based solely on morphological data—i.e. body shape, anti-predator traits and lateral plate morphology—the system appears to have a phenotypically intermediate hybrid zone between highly divergent freshwater ecotypes from upper Lough Feeagh and anadromous migrants from the Atlantic Ocean in the tidally influenced Lough Furnace . Howeverin situ divergence or secondary contact when examining anadromous freshwater-resident species pairs.Stable hybrid zones extending over ecological gradients provide evidence for the role of ecological adaptation in maintaining reproductive isolation . While tFST of 0.21 between parental populations is required to efficiently detect F1 hybrids. Here we used both neutral and QTL markers with a mean pairwise FST of 0.28 between freshwater and anadromous populations, suggesting our approach had considerable power to identify hybridisation. Our simulations further support this conclusion, indicating that it would be possible to detect hybridization accurately at least two generations after it has occurred using STRUCTURE . Since strong selection against all completely plated resident fish every generation is unlikely, plate number variation in the Furnace resident fish is likely to be caused by a locus other than Eda. The Eda complete allele has probably been lost in both the Feeagh and Furnace resident fish. This hypothesis is supported by the fact the two intronic microsatellite markers  show a clear distinction between anadromous and freshwater forms ’s Delta K method and log likelihood of the probability of K for A) full marker set, B) neutral markers only and C) QTL marker set.(EPS)Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 FigError bars represent 95% confidence intervals based on 1000 bootstraps with replacement.(EPS)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableHWE denotes exact P-values for Chi-squared goodness of fit tests, bold values indicate loci significant after Bonferonni correction.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table95% confidence intervals derived from bootstrapping with replacement.(DOCX)Click here for additional data file.


Setaria italica (L.) Beauv) is one of the earliest domesticated grains, which has been cultivated in northern China by 8,700 years before present (YBP) and across Eurasia by 4,000 YBP. Owing to a small genome and diploid nature, foxtail millet is a tractable model crop for studying functional genomics of millets and bioenergy grasses. In this study, we examined nucleotide sequence diversity, geographic structure, and levels of linkage disequilibrium at four nuclear loci  in representative samples of 311 landrace accessions across its cultivated range. Higher levels of nucleotide sequence and haplotype diversity were observed in samples from China relative to other sampled regions. Genetic assignment analysis classified the accessions into seven clusters based on nucleotide sequence polymorphisms. Intralocus LD decayed rapidly to half the initial value within ~1.2 kb or less.Foxtail millet ( Since the inception of the human agropastoral transition, many crops have been domesticated and served as important food sources. In East Asia, foxtail millet, known in Chinese as ‘xiao mi’ or ‘su’ is one of the most important and earliest domesticated grain crops. The earliest archeological evidence of domesticated foxtail millet was found at the Nanzhuangtou 11.5–11.0 YBP) and Donghulin (11.0–9.5 YBP) in the North China Plain , 3. Worl–11.0 YBPBrachypodium distachium has been sequenced as a means of providing more ready access to the large genomes of Triticeae species such as barley (Hordeum vulgare L.), rye , and wheat (Triticum aestivum L.) [Panicum virgatum L.), pearl millet (Pennisetum glaucum (L.) R.Br.), and napiergrass (Pennisetum purpureum Schumach.) [http://www.phytozome.net/Setariaitalica), short generation time makes foxtail millet a readily tractable system to rapidly explore the genetic basis of resistance to abiotic stress, agronomic traits, and phenotypic variation in Paniceae grasses that have applications as bioenergy crops [Setaria genome research is the release of the first assembled reference genome of cultivated and wild foxtail millet [There is also renewed interest in foxtail millet as a model system for bioenergy research –9. Just ivum L.) , foxtailhumach.) . With angy crops –12. A mal millet , 14, whil millet . Severall millet ; identifl millet ; identifl millet .Both association studies  and genoLevels of nucleotide sequence diversity and geographic structure are important in association studies , 23, subApplying a collection of 311 foxtail millet accessions across its historical range of cultivation, we used resequencing data from four loci to: (1) document the nucleotide diversity within and among geographic regions; (2) examine the genetic structure; (3) investigate the extent of intralocus linkage disequilibrium (LD). Based on this information, the application of association studies in foxtail millet was addressed.A total of 311 foxtail millet landrace accessions were sampled. China, Central Asia, and European accessions constitute a large portion of the total sample because these regions have been the major regions of cultivation and/or putative domesticated centers . Seeds fTPI1), glyceraldehyde 3-phosphate dehydrogenase (G3PDH), Alcohol dehydrogenase 1 (ADH1), the intergenic spacer 1 (IGS1) region of ribosomal DNA. Because the IGS1 region is a portion of nuclear ribosomal DNA that occurs as tandem array, which is subjected to concerted evolution [IGS1 among geographic regions but did not include it in estimates of average diversity. The PCR and sequencing primers for ADH1, G3PDH, IGS1 and TPI1 were designed from Zea mays. Primer information is detailed in Oryza sativa and Zea mays, thus these primers have potential utility across the Poaceae.Genomic DNA was extracted from young leaf tissue from a single individual of each accession using the modified CTAB method . We examvolution , we comp2+), 2.5 mM of dNTP, and 1 unit of Taq DNA polymerase. PCR reactions (except G3PDH) used the following cycling conditions: 94°C for 3 min; 35 cycles ; a final extension of 10 min at 72°C, the annealing temperature of G3PDH is 56°C. Amplified products were purified with EasyPure PCR Purification Kit , and were directly sequenced on an ABI  3730. The majority of PCR amplicons were directly sequenced. Amplicons from accessions that were determined to be heterozygous within a locus were cloned using pEASY kit . Not all samples were sequenced successfully, thus the sample size of final data matrix for ADH1, G3PDH, IGS1, TPI1 are 296, 285, 293, and 289, respectively.Polymerase chain reaction (PCR) was performed in a total volume of 25 μl containing 50 ng of template DNA, 10 μM of each primer, 2.5 μL 10 × PCR buffer , and edited sequences were aligned using Clustal X , and werθ = 4Neμ, based on the number of segregating sites (θw) [θπ) [T [T was tested by 10,000 replicate coalescent simulations.Descriptive statistics for nucleotide sequence diversity and tests of neutrality were estimated using tools from the libsequence C++ software library . Insert/es (θw)  and the w) [θπ) . We also[θπ) [T , a test KST* and Snn methods of Hudson [Snn (nearest-neighbor statistic) is a powerful statistic for detecting genetic differentiation using sequence-based analysis over a wide range of sample size and levels of variation [Snn is expected to be near one. KST* measures genetic differentiation using resequencing data and can be more powerful for detecting population structure than SNP frequency-based estimates of FST, especially in the case of high haplotype diversity [The sample was partitioned into nine geographic regions: China (CH), Central Asia (CA), South Asia (SA), Near East (NE), Korea and Japan (KJ), Southeast Asia (SEA), Europe (ER), North America (NA), and Africa (AF). We tested the extent of geographic structure in and among these partitions of the sample using f Hudson , 50 applf Hudson . Snn  in the model was systematically varied from 1 to 10, with the median likelihood of each K value estimated from the 20 runs. We used the △K method [Genetic differentiation was investigated using genetic assignment in STRUCTURE 2.3.4 , 53. BurK method  represenK method .ρ = 4Ner, where Ne is the effective population size and r is the recombination rate per generation [B [Rm) [Multiple methods were applied to estimate levels of LD in the data: a parametric estimate of the recombination rate: neration , 57; a cneration ; and intation [B , a summa[B [Rm)  were alsr2 [http://www r-project.org) was used to plot LD versus distance.LD between pairs of polymorphic sites was calculated based on squared correlation in allele frequency, r2 , betweenr2 . The staADH1, G3PDH, IGS1, and TPI1 are 875, 1390, 970, and 870 bp, respectively, and the combined total length of aligned sequence for four loci is 4105 bp , respectively. None of the Tajima’s T values show significant deviation from expectations for a panmictic population evolving under neutrality. Nucleotide diversity estimated by the number of pairwise differences among haplotypes (θπ) ranges from 2.86–10.05 × 10−3 for the four loci, with on average of 6.98 × 10−3  for all four loci. KST*statistics in locus of IGS1 (P < 0.05) also demonstrates significant geography structure  by physical distance between sites in foxtail millet are shown in r2 against distance in base pairs between SNPs  also suggests similar pattern of intralocus LD.Plots of squared allele frequency correlations  with average diversity θw = 4.04 × 10−3, θπ = 3.63 × 10−3. The level of genetic diversity is lower than wild green foxtail (Setaria viridis (L.) P. Beauv.), where θw = 5.9 × 10−3 [Oryza sativa) (θπ = 3.04 × 10−3) [−3) [Foxtail millet has relatively low diversity for the three single copy genes ( × 10−3) . There w−3) [−3) , Liu et −3) [−3) . Then th−3) [−3) . Many ge−3) [−3) , SSR mar−3) [−3) , 35, tra−3) [−3) , which cS. viridis [θπ) for loci G3PDH and TPI1, Europe for locus ADH1, and Central Asia for locus IGS1 showed. Europe has the highest haplotype diversity for loci ADH1 and IGS1, China for locus G3PDH, and Central Asia for locus IGS1. All the four regions except Near East contain high nucleotide diversity, haplotype diversity and private SNPs for certain locus, which should be major regions for germplasm conservation. Korea and Japan (KJ) have rarely been included in previous studies, high diversity  was revealed in this study. The Snn test indicates significant geographic structure for four loci studied (P = 0). The ADH1 locus also show significant population structure with KST* (P < 0.05). Given that the number of loci sampled is limited, the geographic structure of foxtail millet populations will require further exploration, but current data suggest a high degree of differentiation among geographic regions.Cytological and genetic studies indicated that the wild ancestor of this crop is  viridis , 67–69.  viridis , 70–72. Arabidopsis. thaliana (within 10 kb) [Boechera stricta (within ~ 10 kb or less) [O. sativa) (< 1 kb) [Generally speaking, LD decays more rapidly in outcrossing than selfing species  because n 10 kb)  and Boecor less) ; more ra(< 1 kb) , and ver(< 1 kb) –78. FoxtOur results show that low levels of LD in inbreeding organisms are not exceptional and could be explained by the following hypotheses. First, low LD may be resulted from a species-wide scale of sampling, which incorporates the entire history of polymorphism and recombination within a species over thousands of generations , 75, 77.There are also some previous studies about linkage disequilibrium of foxtail millet. Wang et al. reported similar level of LD as ours (extends to 1 kb) applied 9 gene loci . HoweverS1 Fig(TIF)Click here for additional data file.S1 TableGG = geographic group.(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Reticulitermes grassei (Isoptera: Rhinotermitidae), sampled from a population in southwestern France. Three haplotypes were detected, one of which was found exclusively in the southern part of the study area (near the Pyrenees). After genotyping 6 microsatellite loci for 512 individual termites, we detected a significant degree of isolation by distance among individuals over the entire range; however, the cline of genetic differentiation was not continuous, suggesting the existence of differentiated populations. A spatial principal component analysis based on allele frequency data revealed significant spatial autocorrelation among genotypes: the northern and southern groups were strongly differentiated. This finding was corroborated by clustering analyses; depending on the randomized data set, two or three clusters, exhibiting significant degrees of differentiation, were identified. An examination of colony breeding systems showed that colonies containing related neotenic reproductives were prevalent, suggesting that inbreeding may contribute to the high level of homozygosity observed and thus enhance genetic contrasts among colonies. We discuss the effect of evolutionary and environmental factors as well as reproductive and dispersal modes on population genetic structure.In population genetics studies, detecting and quantifying the distribution of genetic variation can help elucidate ecological and evolutionary processes. In social insects, the distribution of population-level genetic variability is generally linked to colony-level genetic structure. It is thus especially crucial to conduct complementary analyses on such organisms to examine how spatial and social constraints interact to shape patterns of intraspecific diversity. In this study, we sequenced the mitochondrial COII gene for 52 colonies of the subterranean termite  Subdivisions within populations can provide valuable insights when deciphering the ecological and evolutionary processes operating in natural populations. In most species, population structure is affected by habitat characteristics, life history traits, and nonrandom mating among individuals  have been multiplying over the past fifteen years. Small-scale genetics studies have focused on characterizing colony breeding systems and delineating colony foraging areas, in both forests and urban areas; polymorphic microsatellite markers have been used to examine how genetic variation is partitioned within and among colonies . Indeed, colony breeding structure and modes of dispersal  act to shape complex termite populations that are also undergoing drift and selection. In particular, they can affect population inbreeding, which, in turn, can influence the degree of genetic differentiation among populations  gene for one individual from each of the 52 colonies using the primers TL2-J-3037 (5′– ATG GCA GAT TAG TGC AAT GG –3′) and TK-N-3785 (5′–GTT TAA GAG ACC AGT ACT TG–3′) . The final primer concentrations were 30 μmol/L for Rf21-1 and Rf24-2 and 100 μmol/L for Rf5-10 and Rf6-1. PCR products were separated by electrophoresis on 6% polyacrylamide gels run on a LI-COR 4000 L sequencer, and allele sizes were determined by comparison with a 50- to 350-bp IRDye 800™ standard . Alleles were scored using the program GeneProfiler, v. 4.03 .We genotyped 512 termite workers from 52 colonies at 6 microsatellite loci: -2 Vargo , and Rs7BioEdit 6.0.7 , which showed either a single consistent cloud of points (continuous cline) or several patches (distant populations) (Jombart We tested for the presence of an isolation-by-distance (IBD) pattern between colonies Slatkin . Using t Jombart .adegenet package  was applied. First, Moran’s I index was calculated to detect any spatial autocorrelation in the allele frequencies. Indeed, we expected that patches of similar allele frequencies could lead to a highly positive I because, within a population, the observed allele frequency of a colony should be positively correlated with the allele frequencies of its neighbors. The first sPCA eigenvalues that explained both variance and spatial autocorrelation were retained. The corresponding sPCA principal components (i.e. scores) were geographically mapped to reveal spatial patterns of interest. Second, global and local statistical tests (9999 permutations) were performed to detect global and/or local structuration forces, as per Jombart et al. (loadingplot in the adegenet package).To explore and detect spatial structure in populations, multivariate methods can be applied to genetic markers. Geography is explicitly integrated in the spatial principal component analysis  and to  assign colonies to clusters based on the multilocus genotype data. Both of the algorithms assume that clusters are panmictic units with distinct allele frequencies. In order to circumvent possible problems that might have arisen from the non-independence of genotypes within colonies, all the analyses were performed using data sets in which each colony was represented by a single, randomly chosen individual (see description above).To complement the latter frequency-based methods, we used two Bayesian clustering algorithms, Structure analyses, five runs, each using a different resampled data set, were conducted to look for consistency in the predicted K values. Each run consisted of a burn-in period of length 50,000 followed by 100,000 Markov chain Monte Carlo (MCMC) repetitions with 10 iterations using the admixture model. For the five runs, the optimal value of K was calculated using the ΔK methods . Given that the posterior distributions yielded two different modes, we then performed five additional runs to improve our estimation of K.We used Genepop . A colony was also considered to fall into the extended-family category if its genotype frequencies deviated significantly from those expected in simple-family colonies. Significance was determined by a G-test (P < 0.05) in which all the loci were combined. Colonies were categorized as mixed families when more than four alleles were found at one or more loci, a pattern that reveals that offspring were produced by more than two unrelated reproductives.Colonies were assigned to one of three family types by comparing their allele and genotype numbers and frequencies with those expected given standard criteria for the respective termite families Vargo . ColonieSequencing of 684 bp of the mtDNA COII locus revealed four polymorphic nucleotide sites that, taken together, resulted in three haplotypes: HT1, HT2, and HT3 . HT1 andGeneland, the mean values for overall expected unbiased heterozygosity were 0.505 and 0.483; the mean values for observed heterozygosity were lower, 0.244 and 0.256 , while the remaining variance (about 44%) could be attributed to variability within individuals . The observed correlation of r = 0.162  suggests that the matrices were positively associated. Although the scatterplot  compared to the others: It explained the high degree of variation in allele frequency. It differentiated all the termite colonies into two groups: a “northern” group and a “southern” group , which revealed that the observed allele frequencies were highly correlated among geographically close colonies. The second sPCA eigenvalue (λ2 = 0.03) revealed another spatial trend: There was some differentiation among “western” and “eastern” colonies, although the autocorrelation statistic was weaker , while the test for local population structure  was not significant (P = 0.289). We identified the alleles underlying this pattern. The alleles that contributed the most to sPC–1 were loc1.228 (locus Rf21-1) and loc3.134 (locus Rf5-10)  and loc1.228 (locus Rf21-1) alleles made the largest contributions to sPC-2  Fig.. The locPC-2 Fig..Structure results suggest that our data are best structured by grouping the colonies into two clusters (K = 2), according to two of the runs , according to three of the runs : 29 colonies were assigned to the first cluster and 23 colonies were assigned to the second cluster (numbers of colonies were averaged across the 6 runs). The maps of estimated cluster membership (based on the posterior modes) suggest that the first, main cluster has a more diffuse, northern distribution and that the second cluster has a more southern distribution (with the exception of colony #52). There is a distinct border between the clusters  and proposed a different version of this general pattern. On the map, it can be seen that the northern cluster (composed of 26 colonies) remains, but that there are two distinct clusters in the south  . For K = 3, the northern and southern clusters had significant FIS values , which were attributable to the following loci: Rf21-1, Rf6-1, and Rs76 for the northern cluster and Rf21-1 and Rf5-10 for the southern cluster . The pairwise FST values for the northern and southern clusters and for the northern and southwestern clusters were significant . In contrast, no significant difference was detected between the southern and southwestern clusters . There were no significant IBD patterns within any of the clusters .We conducted standard population genetic analyses on the results of two rs) Table. In bothGeneland clustering results, we estimated the percentages of each family type in the different clusters. For K = 2, in the northern cluster, 33% and 67% of the colonies were simple family and extended family, respectively; in the southern cluster, 40% and 60% of the colonies were simple family and extended family, respectively. Similarly, for K = 3, in the northern, southern, and southwestern clusters, 37.5%, 42%, and 22% of colonies were simple family, respectively, while 62.5%, 58%, and 78% were extended family, respectively.We identified 20 simple-family colonies (38.5%) and 32 extended-family colonies (61.5%)  using a Reticulitermes grassei colonies in our study area in southwestern France demonstrated limited mitochondrial DNA diversity: Only three haplotypes were observed. These haplotypes were previously described in a study that examined R. grassei and R. banyulensis phylogeography at other locations in southwestern France and on the Iberian Peninsula  may be attributable to a Wahlund effect. In social insects, homozygote excess can also be a consequence of inbreeding among related reproductives within colonies . First, it is clear from the pattern that the colonies were separated into a “northern” and a “southern” group, and the boundary between the two groups is demarcated by the near-zero sPCA scores for colonies #13, #40, #41, #42, and #43  uses allele frequency data to calculate spatial autocorrelation scores and thus tests for spatial patterns of genetic variability  assign colonies to clusters while accounting for individual genotypes and (2) detect genetic discontinuities. It is important to note that, while our data suggested the presence of an IBD pattern, the results of the clustering analyses identified two or three clusters, depending on the resampled data set. Although the e.g., the Adour River). In general, Reti culitermes winged primary reproductives naturally disperse only short distances  or inbreeding among related reproductives within colonies; the latter possibility will be discussed below. We did not detect a deficit in the southwestern cluster, possibly because its smaller sample size led to reduced statistical power.The next step was to look for landscape features that could account for the observed genetic patterns and discontinuities  play in determining population genetic structure in Reticulitermes. Our study has demonstrated that using complementary analyses is especially crucial when dealing with social species in which both spatial and social constraints interact to shape the distribution of intraspecific diversity. The next step is to conduct a study at the scale of the Landes’ forest, whose recent history (it was planted at the beginning of the 1800s) and its particularly large area (2.47 million acres covered mainly with the pine species Pinus pinaster) have probably helped the termite move northward and have influenced its population structure; human activities  may also have had an effect.Our findings have important implications for our understanding of the role different factors (


Evolution creates and sustains biodiversity via adaptive changes in ecologically relevant traits. Ecologically mediated selection contributes to genetic divergence both in the presence or absence of geographic isolation between populations, and is considered an important driver of speciation. Indeed, the genetics of ecological speciation is becoming increasingly studied across a variety of taxa and environments. In this paper we review the literature of ecological speciation in the tropics. We report on low research productivity in tropical ecosystems and discuss reasons accounting for the rarity of studies. We argue for research programs that simultaneously address biogeographical and taxonomic questions in the tropics, while effectively assessing relationships between reproductive isolation and ecological divergence. To contribute toward this goal, we propose a new framework for ecological speciation that integrates information from phylogenetics, phylogeography, population genomics, and simulations in evolutionary landscape genetics (ELG). We introduce components of the framework, describe ELG simulations , and discuss design and experimental feasibility within the context of tropical research. We then use published genetic datasets from populations of five codistributed Amazonian fish species to assess the performance of the framework in studies of tropical speciation. We suggest that these approaches can assist in distinguishing the relative contribution of natural selection from biogeographic history in the origin of biodiversity, even in complex ecosystems such as Amazonia. We also discuss on how to assess ecological speciation using ELG simulations that include selection. These integrative frameworks have considerable potential to enhance conservation management in biodiversity rich ecosystems and to complement historical biogeographic and evolutionary studies of tropical biotas. Understanding how organisms adapt and diversify have been topics of fundamental importance in biology for over 150 years . It is nSpeciation – the continuous process that gives rise to biological diversity, is often intimately associated to changes in phenotypes and to adaptation to the ecological environment . NaturalThe increasing integration between molecular genetic approaches with theoretical and empirical ecological studies . For instance, 2013 has seen almost 200 publications and over 5,600 citations, compared to 60 articles and 1,600 citations in 2008. Interestingly, our evaluation shows that the majority of empirical studies (674 out of 862) used information from genetic datasets or genetic knowledge to improve pattern interpretation. Indeed, the genetics of ecological speciation is becoming increasingly studied across a variety of taxa and environments , genome scans, population genetics, phylogeography, and phylogenetics. We then use published population-level genetic datasets from five codistributed and taxonomically diverse Amazonian fish species  to illusWe suggest that this framework can assist in distinguishing the relative contribution of natural selection from biogeographic history in the origin and maintenance of biodiversity, even in a complex and understudied tropical ecosystem such as Amazonia. In addition, we show that it is feasible to frame ecological speciation in a spatially explicit ELG context that includes selection. We also discuss recent developments in environmental mapping, simulations and population genomics that allow clarification of population divergence across selection gradients.Figure 1) dealt with tropical species, and only 40 of those could be classified as empirical studies. Although our searches might have missed relevant studies that did not use the words “tropical” or “tropic(s)” in the abstracts, keywords, or titles, our inspection of the literature indicates that research effort in ecological speciation is indeed largely biased toward temperate regions. This conclusion is consistent with the empirical literature reviewed in a recently published book on ecological speciation  out of the 996 previously identified articles , a large proportion of studies used information from molecular phylogenetics (63%), phylogeography (50%) and morphology (42%). To our surprise, only few surveys of population genetic structure were conducted in the topics , with a handful of those also incorporating information from genome scans of selection (10%) or from landscape genetics (7.5%). As expected for any integrative field of research, ecological speciation was usually assessed by combining two or more approaches (80% of articles). Most combinations (83%) included phylogenetic or phylogeographic datasets, exemplifying the reliance of the field on molecular genealogical information.Research trends in tropical ecological speciation were collected by inspecting the 40 recovered empirical articles for key selected categories. In terms of biological realm, most publications focused on terrestrial (65%), rather than marine (20%), or freshwater (15%) organisms. Terrestrial plants and terrestrial invertebrates were the best represented taxonomic groups , followed by fishes (17%), aquatic invertebrates (15%), birds (12%), herpetofauna (10%), and mammals (3%). Comparisons involving two or more species (65%) were more common than intraspecific surveys (35%). Interestingly, 45% of studies included comparisons across habitat gradients, and 35% of the 40 articles assessed patterns of gene flow. In terms of the main approaches used to generate and analyze data  are the scarcest. The latter is consistent with the positive correlation found in another review  to clarify temporal and spatial patterns of diversification in tropical ecosystems. These surveys and meta-analyses have invariably compared lineages at or above the species level and framed pattern interpretation within historical biogeographic and macroevolutionary contexts. They have contributed substantially to our understanding of tropical diversity – such as improving the quality of the contentious debate about Tertiary versus Quaternary speciation timing . Although the primary impetus is to assess the role of ecology as a driver of biodiversity, this type of research also generates valuable information for several data-deficient areas in tropical research. These include improvements of biological inventory databases and taxonomy, the discovery and delineation of cryptic species, selection gradients, ecotones and regional hotspots for conservation management, and the clarification of speciation timing, patterns of connectivity and metapopulation structure.We now describe a framework to study ecological speciation in the tropics that integrates genomic (or genetic) data from populations with simulation modeling specific for ELG (summarized in Box II). Although this framework was conceived to assess ecological speciation in the tropics, it can be applied to other ecosystems and to most sexually reproducing species. This is particularly true for biotas in temperate regions for which a priori information about traits subject to divergent selection is more abundant and for which better logistics allow greater experimental tractability  distributed across heterogeneous environments or ecotones while controlling for spatial genetic autocorrelation and vicariant history. In this scenario, evidence for ecological speciation is obtained if a positive correlation is found between genetic differentiation and environmental (or phenotypic) divergence, after the effects of geographic distance  and histtability  comparedBelow we introduce the main components of the framework and discuss design and experimental feasibility within the context of tropical research. We also provide a more detailed description of simulations and ELG  and their expected contributions. This is followed by a performance assessment of the proposed research framework based on published studies of population diversification and ecological speciation of Amazonian fishes.Figure 3) is valuable for comparative surveys because the applicability of individual approaches for assessing ecological speciation scenarios is expected to vary among groups of organisms  or of traits related to reproductive isolation can be fundamentally important to generate hypotheses about divergence across environmental gradients and to inform sampling strategies. In cases where a priori information is available about the distribution of phenotypically divergent ecotypes within species or between closely related lineages  is required to ensure rigorous statistical analyses. Within lineages, sampling should be hierarchical to enable analyses of spatial dependence: the design should be spatially nested regarding dispersal potential to capture the degree of autocorrelated genetic data  and the environmental-species relationships  should contribute to improving and accelerating initiatives supporting bioinventory collections and population sampling in the tropics. Population genetic surveys should tag along to ensure that tissue samples for DNA analysis , are collected together with voucher specimens and other key associated data such as phenotypic information.The rigorous sampling regime proposed here contrasts remarkably with the sparse sampling strategies generally used in phylogeographic and phylogenetic studies of tropical biotas . AlthougLandscape (or riverscape or seascape) mapping is an important step that should be carried out in parallel with sampling design. A landscape is an area that is heterogeneous with regard to at least one variable of interest . A landsThe integration of molecular genealogical information from phylogenetic and phylogeographic analyses into the framework is a prerequisite for subsequent analyses of intraspecific  gene flow. Two main reasons account for this requirement. First, these analyses enable the discovery and spatial delineation of historically isolated lineages and hidden biodiversity, such as evolutionarily significant units (ESUs) and cryptic species. Levels of species richness and morphologically cryptic biodiversity have been grossly underdocumented in assessments of tropical biodiversity e.g.,  because The second reason for integrating molecular genealogical studies is that they add an essential component to the understanding of patterns of population structure and levels of reproductive isolation: time. Temporal changes in the physical and biotic environment of a population lead to demographic variations that are correlated with the structure of population genealogies . PhylogeIn this step, DNA markers are used to genotype individuals across heterogeneous environments with the aim of clarifying patterns of gene flow and identifying the spatial locations of genetic discontinuities . This is a key step in the framework that should be done at the metapopulation level after historical diversification has been accounted for (as above). The expectation is that during ecological speciation, ecological or environmental distance , reduces homogenizing gene flow and correlates to genetic population differentiation. These patterns are now considered common in nature and usually referred to as isolation-by-ecology , or isolAlthough isolation-by-ecology can be detected with relatively small neutral molecular datasets such as microsatellites and AFLPs . Using aBox II). Simulations have provided many important findings in various disciplines  on 1- or 2-dimensional regular lattices, or in irregular  coordinate space. Specific rules in the model then define how individuals move and interact across space, for example by defining the distances they can move away from their birth location, or the distance within which they can find a mating partner. Population genetic simulations are often also spatially explicit e.g., , but whiThe ELG simulation modeling implemented in our ecological speciation framework includes an additional component: it specifically integrates landscape genetics and evolutionary genetics, focusing on how space and selection impacts on the evolution of reproductive isolation (speciation). For example, selection can be controlled via fitness landscape surfaces  that detFigure 4). These included freshwater representatives of marine-derived lineages from two orders; a tetraodontiform (the puffer Colomesus asellus) and a perciform (the croaker Plagioscion squamosissimus) and ancient freshwater lineages representing three Gondwana-relict orders; a characiform , a siluriform (the catfish Centromochlus existimatus) and a gymnotiform (the electric fish Steatogenys elegans). Nuclear and mitochondrial DNA (mtDNA) sequences were used for genealogical analyses and AFLP datasets were used for analyses of gene flow and genome scans. Readers interested about species-specific datasets, analytical methods, and detailed biogeographic reconstructions should refer to our five publications . Molecular dating indicates that these lineages all diverged during middle to late Miocene . In contrast, there was no evidence for cryptic species within the marine-derived taxa or within T. albus . Despite these differences, however, several congruous patterns that may actually reflect general forces shaping freshwater biodiversity in Amazonia were detected. Below we introduce the study region and summarize key comparative findings about inferred spatial arrangements of neutral and putatively adaptive genetic diversity. These are discussed within the contexts of geomorphological history of Amazonia and divergent selection.The five species are largely codistributed, yet results indicate they have very different evolutionary histories. Phylogenetic analyses unexpectedly revealed five cryptic species within the catfish The Amazonian aquatic environment sustains dramatic hydrochemical and ecological gradients that impose physiological constraints upon its aquatic communities . These aFigure 4). The study area encompasses two putatively strong selection gradients or ecotones: where the black waters of the Negro River meets the white waters of the Amazon River, and where the clear waters of the Tapajós River meets the white waters of the Amazon River. Additionally, the transect allows testing for genetic structure geographically associated with river confluence. That is because two controls in which rivers of the same water color meet were included: the confluence of the black waters of the Branco and Negro Rivers and the confluence of the white waters of the Madeira and Amazon Rivers .The study system within the Amazon Basin consists of the five major rivers representing white, black and clear waters: the Amazon (white), Madeira (white), Branco , Negro (black), and Tapajós .Despite differences in ecology and phylogenetic history, strong evidence indicates that the biogeographic and ecological contexts of the Amazon Basin have promoted largely congruent fine-scale phylogeographic and population-level structuring. From a genealogical perspective, all species showed strong signals of demographic expansion and in every case the timing of these occurred well within the Quaternary period, with the exception of Figures 5). No significant barrier to gene flow was identified at the confluence of the Madeira and Amazon Rivers in any species. Indeed, based on both mitochondrial and nuclear datasets, population structure was strongly associated with ‘water color’ but not with river system. For instance, statistically significant differentiation was consistently found between populations from rivers of different colors, but not between those from rivers of the same color . For C. existimatus, the large number of inferred cryptic species resulted in insufficient data for intraspecific analysis. However, the C. existimatus cryptic species ‘D’ endemic to black waters of the Negro River, appeared recently derived and genetically distinct from white water lineages .From a population genetic perspective, all species and species complexes showed a predominant barrier to gene flow at the confluence of the Negro and the Amazon Rivers, and/or again at the confluence of the Tapajós and Amazon Rivers . These lineages likely exemplify “natural replicates of the ecological speciation in progress” , irrespective of river system and tributary arrangement.Using outlier loci approaches based on genome scans, divergent selection was quantified between populations of S. elegans , selection pressures were altered due to ‘water color’ between populations by considering three spatially explicit relative fitness surface scenarios. (i) No spatial selection gradient (‘uniform’): in this scenario, the three genotypes  were being selected against, but uniformly across the ‘water color’ riverscape scenario, thus having no spatial dependency. (ii) Gentle spatial selection gradient (‘gentle’): here, a ‘gentle’ spatial selection gradient corresponding to the three river color locations and three genotypes was used. (iii) Steep spatial selection gradient (‘steep’): for this scenario, stronger spatial selection gradients were assigned to each genotype for black, mixed, and white waters. For further details, see Simulations were conducted in the individual-based landscape genetics program CDPOP v1.2 . IndividS. elegans replicated system, population genetic analyses and FST-based genome scans showed that recent divergence appeared linked to a major hydrochemical gradient within each cryptic species . Results from ELG simulations further corroborate these findings. These ELG simulations show that neutral data can give a low population differentiation signal . They also show that selection-driven loci can respond with high population differentiation to the water color ecotone .In the The results link selection across an ecological gradient with reproductive isolation and it was speculated that assortative mating based on chemically different water types may be driving the divergence . The ratFigure 6). Moreover, dating of speciation events within Plagioscion, S. elegans and C. existimatus are consistent with the Miocene diversification of fishes observed in the fossil record, as well as in other molecular studies . These studies also suggest that the ancestral ecotype for each species was in Andean-derived white water .The palaeogeographic and paleoenvironmental changes in South America during the Miocene are known to have profoundly affected the evolution of the Amazonian fish fauna. These changes include the uplift of the Andes and associated neotectonic events, the incursion of marine waters into previously freshwater systems and the dramatic reorientation of major river drainages , ,c,d. Ind studies . Moving te water . These ete water . This asAlthough palaeogeographic events have clearly contributed to Amazonian fish diversity, the comparative analysis indicates that the force of divergent natural selection, in this case between water colors, deserves equal consideration. One of the primary tenets of ecological speciation theory is that high ecological opportunity, such as the colonization of new environments in the absence of predation and/or competition, will promote rapid population divergence resulting in speciation . Indeed,FST-based genome scans were employed to assess the role of selection in the origin and maintenance of population divergence between water colors. However, key to ecologically based divergent natural selection is that the process should be greater between selective environments than within (Table 4).Classic signatures of vicariant biogeographic history such as genetic drift, inbreeding and migration have genome wide effects, while selection usually leaves signatures only at those loci that are adaptive or tightly linked to adaptive loci via hitchhiking . For thin within . Indeed,C. asellus, divergent selection detected between the Tapajós and Amazon Rivers was a powerful yet recent phenomenon, such that genetic drift had not yet accumulated in selectively neutral genomic regions . For S. elegans, both the empirical landscape genetics and simulated ELG explicitly linked selection-driven population genetic structure to the water color ecotone, validating results of FST-based genome scans. By incorporating information about population history and by independently validating outlier results with simulations, we improved our ability to exclude type 1 errors normally associated with genome scans of selection .In the context of comparative biology, the use of genome scans revealed variable, yet valuable perspectives on the process of adaptive divergence across water colors, both within and between species. Although there is no certainty that ecologically diverged lineages will eventuate as reproductively isolated species , the obsThe evidence for the role of divergent natural selection in these studies occupies a relatively recent time frame and appears associated with the formation of the modern Amazon River and its aquatic ecotones. Considering that time erases the signature of divergent selection within the genome, resulting in a more diffuse pattern of genetic divergence , ecologiClarifying the role of ecology on population adaptation and divergence is crucial for understanding the origin of species and the evolutionary potential of biodiversity to respond to ongoing global change . AlthougAs illustrated in this review, studies of ecological speciation in the tropics should merge analyses of genealogical history and gene flow with recent developments in spatial modeling and ELG simulations. This integration provides valuable biogeographic and environmental contexts to disclose associations between landscape features and evolutionary processes, including divergent natural selection. Such approaches, when combined with NGS datasets and with information about divergent phenotypes can be used to identify adaptive loci across the landscape , and to Ecological adaptation and divergence in the face of homogenizing gene flow is still a controversial topic. Many studies of ecological speciation in nature appear as correlative, are based on results from lab experiments , and shoThe time is right for implementing studies that synergistically generate and explore information from adaptive phenotypes, phylogeography, population genomics and simulations in ELG to understand ecological divergence and speciation in the tropics. Such endeavors are expected to challenge results from current surveys that assess tropical diversity based on sparse population sampling and on geographic models that do not incorporate selection. In doing so, they will complement historical biogeographic and evolutionary studies of diversification of tropical biotas. Integrative frameworks such as the one illustrated here have considerable potential to enhance conservation management in biodiversity rich ecosystems and to contribute toward a better understanding of how ecology, space and time interact with the genome.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lolium perenne) is the most widely grown temperate grass species globally. Intensive plant breeding in ryegrass compared to many other crops species is a relatively recent exercise (last 100 years) and provides an interesting experimental system to trace the extent, impact and trajectory of undomesticated ecotypic variation represented in modern ryegrass cultivars. To explore germplasm dynamics in Lolium perenne, 2199 SNPs were genotyped in 716 ecotypes sampled from 90 European locations together with 249 cultivars representing 33 forage/amenity accessions. In addition three pseudo-cross mapping populations  were genotyped to create a consensus genetic linkage map. Multivariate analyses revealed strong differentiation between cultivars with a small proportion of the ecotypic variation captured in improved cultivars. Ryegrass cultivars generated as part of a recurrent selection programme (RSP) are strongly associated with a small number of geographically localised Italian ecotypes which were among the founders of the RSP. Changes in haplotype frequency revealed signatures of selection in genes putatively involved in water-soluble carbohydrate (WSC) accumulation (a trait selected in the RSP). Retrospective analysis of germplasm in breeding programmes (germplasm dynamics) provides an experimental framework for the identification of candidate genes for novel traits such as WSC accumulation in ryegrass.Perennial ryegrass ( Lolium perenne, has only recently (past 100 years) been subjected to selective breedingPlant species encounter a diverse set of selective pressures as they spread into new ecosystems, leading to changes in disease resistance, growth habit, production or partitioning of metabolites and gross morphology. Ecotypic material thus represents an important source of adaptive genetic variation which can be maintained by gene banks for use in conservation efforts or plant breeding. With the availability of high-density genotyping systems, this variation can be studied on a genome wide basis– both for understanding the genes which underpin adaptive traits, and also to facilitate their incorporation into plant breeding programmes for crop species1Lolium perenne  is the most widespread grass species in temperate regions globally due to its rapid establishment, persistence and nutritional value to ruminants. It is outbreeding and exhibits extensive genetic variation for morphological growth characteristics57911L. perenne are favoured due to their quick establishment, long growing season and high dry matter yield that is highly digestible to ruminants1314161718Due to the range of phenotypic traits exhibited by perennial ryegrass, it is commercially important as both a forage and amenity  species. Forage varieties of Lolium perenne with over 2,000 validated markers provided the opportunity to interrogate germplasm in new wayset al.The creation of a publically available genotyping array for In this study we exploit the Infinium array described previouslyTo evaluate levels of diversity and divergence, summary statistics were calculated within different groups of germplasm. Observed heterozygosity for each cultivar group was greater than the expected heterozygosity based on HW expectations, which was similar to the ecotype accessions . ConsequAn analysis of molecular variance (AMOVA) was conducted to examine the patterns of diversity between and within germplasm groups . As expeLolium perenne germplasm reflects their history of breeding for specific end user needs. Outliers from the central clusters were Aberystwyth’s amenity varieties, AberImp and AberSprite, and in the opposite direction on PC2 Aberystwyth’s Aurora and recurrent selection programme (ARSP) varieties . The ARSP shows progressive movement away from other forages towards 3 Italian ecotypes, IT7, 8 & 10. These ecotypes were, in fact, used in the original polycross with S23 (AF1 & 2) in the development of the ARSP (AF4-7). The breeding programme has focussed on recurrent selection for increased nutritional composition, in particular high WSC. Cultivar Aurora (AF3) is also descended from ecotypic material, CH6. A total of 21 tillers from accession CH6 were polycrossed and the progeny subjected to multiple rounds of crossing/selection for persistence, winter hardiness, early heading, yield and increased WSC, likely explaining the shift of AF3 away from its CH6 founder on In order to understand the distribution of genetic diversity contained in cultivars in comparison to the variation across European ecotypes, an unbiased principal component analysis (PCA) was performed on the allele frequency for each of the 2199 SNPs within each of the 90 sample locations (accession) in addition to the 33 cultivars . As prevL. perenne contigs that have top BLAST hits to succinate dehydrogenase. In addition to which, markers associated to NADH dehydrogenase and hexokinase are also listed. PC2 significantly separates the Aberystwyth amenity varieties and the other commercial amenity varieties; it also discriminates between clusters of the forages and the amenities. The top 20 markers contributing the variation seen on PC2 are shown in the lower half of To focus further on the differences between the cultivars, PCA was performed on only the cultivar accessions along with the Italian ARSP founder ecotypes . This anIn order to identify regions of the genome that have been under directional selection, a consensus linkage map was constructed from three distinct mapping populations that were genotyped with the Infinium array. Each mapping population generated its own linkage map with 1255, 1475 and 745 markers for the amenity x forage, AberMagic x Aurora and a F2 populations, respectively. Upon combining these three maps, 1386 markers of the 2199 validated markers were included in an integrated consensus map ; Table 4Lolium perenne populations across Europe (2 = 0.2). The genetic distance was slightly greater in commercial varieties  in natural s Europe . LD acroarieties . To focuarieties . ARSP inarieties  and ecotarieties . HoweverIn order to genetically locate regions under selection for functional use (amenity or forage), the top 20 markers with the highest loading in the PCA plot  are idenOf particular interest is the cluster of 3 SNPs at 86cM on LG4 from 2 different contigs that both BLAST to succinate dehydrogenase. These 2 contigs have a total of 5 markers on the array, with 2 currently unmapped. These 5 markers are all in the top 20 markers contributing to the loading on PC1, and therefore differentiating ARSP from OF and OA. SNP- based haplotype analysis was performed on these 5 markers for the ecotypes, Italian founders of the ARSP and other groups of interest . This idL. perenne is the largest, to date, in terms of individuals genotyped with a comprehensive, genomewide array of 2199 SNPs. It therefore provides insight into the extent and distribution of genetic diversity available in European ecotypes of Lolium perenne and how this variation has been incorporated and exploited in breeding programmes. As the most economically and agriculturally important temperate forage crop, understanding patterns and distribution of genetic variation will help breeders respond to the new challenges of sustainable intensificationThis study of natural variation in Lolium perenne. The effect at individual loci is non-significant and it is only due to the large number of markers and populations studied that the general effect is visible. We propose this effect arises from the action of SI and warrants further investigation.The European ecotype sample previously described222324Although the commercial varieties together capture a reasonable range of the genetic diversity observed across Europe, there was a surprising “geographic” divide seen between amenity and forage varieties genotypes. The amenities captured a greater expanse of the European ecotype diversity, with the forages forming a tight cluster on the PCA plot . This fiArabidopsis thaliana and Oryza sativa2931Lolium perenneet al.The ARSP genepool is differentiated from the other commercial forages, demonstrating the power of directional selection with a focus on increasing WSC. An individual from each of the ecotype accessions IT7, IT8 and IT10 were originally polycrossed with S23 and Ba9633 between 1978–1980. Recurrent selection of this population has led to a number of award-winning varieties, renowned for their increased WSC14Arabidopsis37Phytophthora infestans resistance in NicotianaAmenity varieties are selected for their suitability for lawns, sports pitches and recreation, requiring a tough sward that thrives on maintenance at a short sward height. This contrasts to forages that need to be highly digestible to animals and have rapid growth yields for grazing or cutting. The contrasting phenotypic traits and the genetic control of these can be dissected by the use of genetic markers. The differentiation between forage and amenities in L. perenne and the identification of key regions of the genome that are strongly associated with the differentiation of forage and amenity forms. In addition candidate genes implicated in the control of WSC accumulation have been identified based on a combination of multidimensional and haplotype analysis of genetic variation in ecotypes and commercial breeding germplasm, illustrating the power of crop diversity to both improve nutritional composition and the underlying genetic basis of WSC in ryegrass.In summary, this study has allowed quantification of genetic variation in ecotypes and cultivars of Lolium perenne was collected. These represented 9 accessions from commercial forage varieties (OF) and 7 accessions from Aberystwyth University’s forage breeding programme (AF), in addition to 15 accessions of commercial amenity varieties (OA) and 2 accessions of Aberystwyth amenity varieties (AA)  . DNA wasFor a more accurate representation of the effect of a recurrent selection breeding programme on the genetic diversity contained within a cultivar, the AF group was subsetted to Aberystwyth Recurrent Selection Programme (ARSP). The ARSP group contains the 4 most recent forage varieties generated by the programme AF4-7;  genotypeLolium perenne ecotypes as previously describedIn addition to the cultivars, 716 individual In order to generate a robust consensus linkage map, three mapping populations were genotyped. The first population was a back-cross mapping family of 162 individuals was derived from an initial cross between contrasting amenity-type (ex cv AberImp) and forage-type  ecotype collection (grid ref 52.1167 -4.6833) genotypes, followed by backcrossing of a single F1 genotype to the amenity type parentet al.All cultivars (n = 249), ecotypes (n = 716) and 3 mapping populations (n = 450), were genotyped using our custom Illumina Infinium iSelect array across 3425 SNPs. The cluster file trained using the diverse European ecotype panel  was calculated for both the ecotypes and the varieties using PopGen (1.0–3) package in R project and plotted against the genetic map distances between markers as generated from the consensus linkage map. LD was calculated for each linkage group, and then combined to produce genome wide LD plots. A LOWESS curve was included in each plot. This was repeated for the ecotypes, varieties  and ARSP.How to cite this article: Blackmore, T. et al. Germplasm dynamics: the role of ecotypic diversity in shaping the patterns of genetic variation in Lolium perenne. Sci. Rep.6, 22603; doi: 10.1038/srep22603 (2016).


Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory–secretory (ES) antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1%  = 17.1–21.1%). The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1%) of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of  Trichinella. Infection occurs after consuming larvae in the muscle of infected animals. Several outbreaks of trichinellosis have occurred in Laos since 2004, resulting in a substantial public health problem. The principal risk factor for trichinellosis is consumption of uncooked or partially cooked meat from domestic pigs and game. We visited communities in four ethnically diverse provinces of northern Laos to determine the seroprevalence of trichinellosis in the human population and explore the population and individual level risk factors of exposure. In addition, we also examined muscle samples collected from pigs post-slaughter to determine the prevalence of Trichinella infection and identify the species of Trichinella circulating in the domestic pig population. Our findings indicate that Trichinella spp. are endemic in the domestic environment of northern Laos and consumption of uncooked pork is common for all ethnic groups. Targeted preventative health measures, taking a one health approach by bringing together medical, veterinary and health sociology professionals, should be initiated to prevent the transmission of Trichinella and other foodborne pathogens in Laos.Trichinellosis is one of the most widely distributed parasitic zoonoses worldwide and is caused by infection with nematodes of the genus  TrichinellaTrichinella species involved and individual susceptibility Trichinellosis is one of the most widely distributed zoonoses worldwide and is caused by infection with nematodes of the genus Trichinella spp. are endemic throughout Southeast Asia (SE Asia), from southern China to the Indonesian archipelago Trichinella have been detected in the SE Asian region, the encapsulated T. spiralis and the non-encapsulated T. pseudospiralis and T. papuaeTrichinella spiralis has a regional distribution T. pseudospiralis and T. papuae have occurred in Thailand after consuming wild pig meat T. papuae have been detected in Papua New Guinea Trichinella infection in pigs and the species circulating in the domestic pig population. We report here the results of a cross-sectional serological survey of the human population and a concurrent survey in domestic pigs using muscle digestion in four provinces of northern Laos.Several outbreaks and sporadic cases of trichinellosis have occurred in Laos over the past five years Written informed consent was obtained from all participants 15 years and older and from the parents or legal guardians of children <15 years of age. The age of consent for this study was 15 years old. The study protocol was reviewed and approved by the Murdoch University Human Ethics Committee (Project no. 2008/266) and the Lao Ministry of Health National Ethics Committee for Health Research (no. 239/NECHR) before commencing this study. For the pig study, the protocol was reviewed and approved by the Murdoch University Animal Ethics Committee (Project no. R2108/07), which adheres to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.Laos is an ethnically diverse country with 49 distinct ethnic groups classified into four ethno-linguistic families , comprising 67%, 24%, 8%, and 1% of the population, respectively The human survey was conducted in the dry season during January–March 2009 to maximize study participation and minimize negative impacts on seasonal labour demands. The survey design, sample size calculations and methodology have been described in detail elsewhere Trichinella excretory secretory antigens (ESA) prepared from T. spiralis larvae T. spiralis muscle larvae were harvested from 3 month old CD1 female mice weighing 25 g which had been infected with 500 muscle larvae each 40 days before, by HCl–pepsin digestion and then maintained in culture for 18 h. Five hundred thousand larvae were washed three times for 20 min each time by sedimentation in a sterile 50 ml conical tube with 45 ml of warm sterile phosphate buffered saline (PBS), pH 7.3, supplemented with Penicillin and Streptomycin . At each change of the washing solution, larvae were gently shaken to dislodge adherent bacteria. The washing solution was removed after the final sedimentation of larvae and washed an additional five times by sedimentation in a sterile 50 ml conical tube with 45 ml of warm RPMI 1640 media supplemented with Penicillin and Streptomycin, as above. The larvae were resuspended in warm RPMI 1640 media supplemented with 5,000 mg/mL of Penicillin, 5,000 U/mL of Streptomycin, 200 mM Glutamine and 100 mM sodium pyruvate at a concentration of 5,000 larvae/ml and placed in 25 ml tissue culture flasks. The flasks were incubated in 5% CO2 at 37°C for 16–18 h. The larvae were separated from the medium by sedimentation in 50 ml conical tubes. The medium was filtered through a 0.2 µm filter and the larvae were discarded. The filtered medium was then concentrated (100x) in a pressure concentrating chamber using a YM-3 filter at 4°C and clarified by washing with PBS in the same chamber or by dialysing in PBS for at least 4 h. The protein concentration was checked by spectrophotometer at 260 nm and 280 nm, and each batch was aliquoted and lyophilized at 500 µg of total proteins per vial. The ESA and control sera were reconstituted in analytical grade water, aliquoted and stored at −20°C according to the manufacturer's instructions immediately prior to use. Reconstituted ES antigens were further diluted in carbonate buffered saline (pH 9.6) to a working concentration of 5 mg/ml and the reconstituted positive control sera were further diluted 1/200 in blocking solution  for use in the assay. A panel of eight negative control serum samples were sourced from Lao people with no reported history of trichinellosis or of consuming raw pork. Negative control serum and test serum were diluted 1/200 in blocking solution for use in the assay.The ES ELISA was performed in Laos at the National Centre for Laboratory and Epidemiology (NCLE) using a validated protocol Sixty-eight samples that had an ES ELISA test ratio ≥1 were randomly selected from the pool of positive samples and sent to the EURLP for confirmatory testing. Samples were tested by the ES ELISA and western blot according to methods described elsewhere The abattoir survey design has been described elsewhere Tongue and diaphragm muscle samples were artificially digested by the magnetic stirrer method in 1% pepsin  and 1% hydrochloric acid (HCl) after removal of fat and fascia Trichinella ES antigens was calculated for three diagnostic cut-offs in the ES ELISA, test ratios ≥1, ≥1.2 and ≥1.4. The level of agreement between the ES ELISA results from Laos and EURLP, and the level of agreement between the ES ELISA results from Laos and the western blot test were calculated for the three diagnostic cut-offs using the Kappa statistic. Sensitivity and specificity could not be calculated since no ES ELISA negative samples from Laos were subjected to further testing at the EURLP.The prevalence of human serum reactivity with The questionnaire and laboratory test data were entered into a spreadsheet  and subsequent analysis was carried out in STATA/IC version 10 . The socioeconomic status of each household was calculated by use of principal component analysis of household assets P≤0.20) in the univariate analyses were included in a multivariate random effects logistic regression model adjusting for the effect of household clustering. The results are reported as adjusted odds ratios and 95% confidence intervals (CIs). The final analysis only considered persons with serologic and questionnaire data.Univariate logistic regression without adjustment was used to test associations between ES ELISA reactivity and gender, location, ethnicity, age, wealth status and uncooked meat consumption habits. Risk factors significant or borderline significant  and age, breed, sex and production system at last point of sale.very poor or most poor (68.5%) and this was reflected in the finding that Mon-Khmer people were the most impoverished ethnic group and Lao-Tai people were the least poor overall.A total of 1,582 persons in 332 households were eligible to participate in this survey. Of these persons, 1,419 (89.7%) individuals from 324 households aged 6–91 years provided a blood sample, a completed questionnaire, and had valid laboratory test results. The final survey population consisted of 583 Lao-Tai (93.6% compliance), 564 Mon-Khmer (95.4% compliance), and 272 Hmong-Mien (73.4% compliance). No Sino-Tibetan persons were recruited into this study. Survey population structures stratified by province are shown in Sixty-eight samples with a test ratio ≥1 in the ES ELISA in Laos were tested at the EURLP by the ES ELISA method and all but one were confirmed positive, corresponding to 98.5% agreement. In comparison with the western blot test, only 35.3% (24/68) of the Lao samples had three diagnostic bands detected, a banding profile consistent with clinically confirmed trichinellosis  [23], [2Trichinella antibodies detected by ES ELISA were 19.1%, 12.7% and 7.5%, respectively  of which 13 were identified as T. spiralis by molecular typing. Two isolates were not identified to the species level due to damaged DNA that may have occurred during tissue storage and muscle digestion. Prevalence of Trichinella spp. infection in pigs varied significantly (P<0.05) by province whereby the highest prevalence was recorded in Xiengkhuang (4.8%) and Oudomxay (2.8%) provinces  of tongue tissue was detected in 10 pigs, 1–10 lpg was detected in three pigs and greater than 10 lpg was detected in two pigs. The highest recorded intensity of infection was 69 lpg in a pig slaughtered in Xiengkhuang province.Of the 15 pigs infected with T. spiralis in the pig population of Laos together with a spatial difference in prevalence of T. spiralis infection in pigs with worm burdens sufficient to cause severe human disease. One of the principle aims of the present study was to determine population and individual level risk factors associated with human exposure to Trichinella spp. larvae. Therefore we conducted a randomised cross-sectional survey of the human population in four northern provinces of Laos and used the ES ELISA as a serological measure of exposure. A high prevalence of Trichinella antibodies was detected by ES ELISA, with significant increased risk being associated with increasing age, Lao-Tai ethnicity, residing in Oudomxay province, being male and regular consumption of fermented pork sausage.Trichinellosis is endemic in Southeast Asia with a concentration of outbreaks occurring in the ethnically diverse regions of northern Thailand, northern Vietnam and Laos An important limitation of this study was the low participation rate of people from the Hmong-Mien ethnic group. The reasons for this low participation rate have been discussed elsewhere Trichinella ES ELISA lacks specificity owing to the relatively large population of antigens resulting in the detection of non-specific cross-reacting antibodies No diagnostic test for trichinellosis, in any host species, has been validated for cross-sectional studies in Southeast Asia. The Trichinella larvae in the human population. Increasing the diagnostic cut-off in the ES ELISA resulted in improved agreement with the western blot test, likely as a consequence of improved specificity at the expense of sensitivity. For this reason, prevalence was calculated for a range of diagnostic cut-offs in the ES ELISA to test the effect on the subsequent risk factor analysis. The pattern of risk for age, province of residence, gender, ethnicity and wealth status remained essentially the same as the diagnostic cut-off increased. For raw meat consumption, only the self-reported consumption of fermented pork sausage on a weekly basis was significantly associated with antibody detection in the ES ELISA at a cut-off test ratio ≥1.2. The consumption of fermented pork has previously been linked with an outbreak of trichinellosis in Bolikhamxay province in central Laos Despite these limitations, we were able to demonstrate widespread serological evidence of exposure to The lack of association with raw meat consumption, particularly raw pork, was somewhat unexpected since previously reported outbreaks of trichinellosis in Oudomxay and Bolikhamxay provinces have been linked with consumption of raw pork at festivals Trichinella antibodies detected by ES ELISA was highest in Oudomxay province compared to all other provinces. This finding may be an artefact of the large and widespread outbreak of trichinellosis that occurred in this province in 2005 In all risk models, the risk of having T. spiralis larvae were detected had a worm burden of less than 1 lpg and the infecting dose for clinically apparent trichinellosis has been estimated to range from ∼70-150 larvae The majority of pigs in which Trichinella antibody detection in the ES ELISA increased significantly with increasing age and Lao-Tai people were at significantly greater risk. Regionally, trichinellosis has been associated with ethnically diverse mountainous areas of northern Vietnam and Thailand Trichinella and other foodborne pathogens in Laos.The risk of Checklist S1STROBE checklist.(DOCX)Click here for additional data file.Figure S1Western blot (Wb) patterns of reactivity of excretory/secretory antigens (ESA) with human sera that tested positive by ELISA with ESA. M, molecular weight marker in kD; line 1, serum from a person with confirmed trichinellosis, positive control serum; line 2, Trichinella-specific proteins detected by Wb on an ELISA-positive serum from a Lao person; lines 3-5, non-diagnostic proteins recognized by Wb on ELISA-positive sera from three Lao people; line 6, ELISA negative control serum. The three-band pattern considered to be diagnostic is boxed in red.(JPG)Click here for additional data file.Table S1Adjusted odds ratio (AOR) of population characteristics associated with Trichinella ES-ELSA reactivity, as determined by random effects multiple logistic regression modelling controlling for household clustering.(DOC)Click here for additional data file.