Coregonus sp.) into Austrian Alpine lakes have created ‘artificial hybrid zones’, threatening the genetic integrity of native lineages. We evaluate the genetic structure of Coregonus in Austrian lakes and characterize hybridization and introgression between native and introduced lineages. Fifteen populations (N= 747) were assessed for allelic variation at eight microsatellite loci and a reduced set (N= 253) for variation across two mtDNA genes (cyt b and NADH-3). Bayesian approaches were used to estimate individual admixture proportions  and classify genotypes as native, introduced or hybrids. q-value distributions varied among populations highlighting differential hybridization and introgression histories. Many lakes revealed a clear distinction between native and introduced genotypes despite hybridization, whereas some locations revealed hybrid swarms. Genetic structure among lakes was congruent with morphological divergence and novelty raising speculation of multiple taxa, including a population south of the Alps, outside the putative native range of Coregonus. Although statistically congruent with inferences based on nuclear markers, mitochondrial haplotype data was not diagnostic with respect to native and non-native lineages, supporting that the Alpine region was colonized post-glacially by an admixture of mtDNA lineages, which coalesce >1 Ma. Mechanisms promoting or eroding lineage isolation are discussed, as well as a high potential to conserve native Alpine lineages despite the extensive historical use of introduced Baltic stocks.Translocations of Baltic whitefish ( Coregonus) are a typical example among salmonid fishes subject to intensive management as well as conservation concerns due to threats associated with introduced or domesticated lineages.Increasing habitat degradation and translocation of plants and animals has been linked to increasing rates of hybridization. The consequences of such anthropogenic hybridization are of high interest to conservation when introgression with a non-native or domesticated lineage threatens the existence or genetic integrity of native populations in the wild . WhitefiC. lavaretus complex) are specifically noted to have undergone rapid  radiation in lacustrine systems of the Central European Alps. For example, in Swiss lakes, more than two species per lake are commonly reported  were exported into Bohemian aquaculture in the 19th century from Lake Miedwie (Poland)  . This Ba(Poland) . C. mara maraena  without Coregonus in Austria has never been carried out. Because the hatchery Maräne are extensively used in Austrian management regimes, it is hypothesized that the original genetic integrity of native whitefish populations has been lost and this view supports neglect concerning the conservation of native populations  promote varying degrees of reproductive isolation. We augment our genetic data with measurements of quantitative morphological traits both to raise the resolution in detecting introgression and to ask if there is a quantitative phenotypic affect associated with varying levels of introgression. We further speculate that historical ecological deterioration and concomitant stock collapse may have left lakes more vulnerable to introgression with Maräne than lakes that have remained ecologically stable. Additionally, we draw further inferences on the evolutionary history of the native stocks and discuss the implications of such results for conservation planning.We provide the first view of the population genetic structure of Austrian Coregonus, we use an ‘operational unit’ (OU) approach with no a-priori assumption of the number of biological units involved. Accordingly Coregonus stocks were classified as either OU-native or OU-introduced.Due to the inconsistent and controversial taxonomic nomenclature applied to European Adult fish were sampled from 12 Austrian lakes using monofilament gillnets. All Austrian populations belong to the Danube drainage, and to our knowledge only one of them (Achensee) has been integrated into previous studies . Three aCoregonus: ClaTet1, ClaTet5, ClaTet6, ClaTet9, ClaTet12, ClaTet13, ClaTet15 and ClaTet18 (AN) and the observed and expected heterozygosity  was calculated using genetix software 4.05 (AR) as well as deviations from Hardy–Weinberg equilibrium (FIS per population) was calculated using fstat version 2.9.3.2  we attempted to identify and classify OU-native, OU-introduced and hybrids at the global scale; second (b) we further evaluated the potential number of genetic units within the OU-native taxon at the global scale; third (c) we tested for potential multiple genetic units within each lake; and finally (d) we evaluated the pattern of introgression within each lake.structure 2.1  five independent Markov chain Monte Carlo (MCMC) simulations were run. We derived the most likely value of K using the second-order rate of change L′′ (K) following http://taylor0.biology.ucla.edu/struct_harvest). Following this approach the most likely population structure consisted of two clusters, reflecting our expectations of one divergent introduced  lineage and one or more native lineages. Thus, for subsequent assignment of individuals to pure and hybrid categories, K was set to 2. For comparison, we applied a second Bayesian clustering approach as implemented in newhybrids ver. 1.1 . However, considering the number of loci applied , which minimizes the number of misidentified pure-bred fish while maximizing the efficiency of assigning hybrids  of diploid genetic data can be carried out based on the principles outlined in genetix 4.05. We depicted the first two axis of an FCA on individual genotypes, whereby each data point was colour-coded according to the assignment categories produced in our structure analysis. This depiction was repeated for a more stringent (0.90) and less stringent (0.75) assignment threshold.For identification of OU-native, OU-introduced and hybrid individuals a model-based clustering method was used as implemented in ver. 1.1 , applyin applied  we used ficiency . For botq-values of >0.90 (according to structure) were used to evaluate patterns of genetic variation within the indigenous gene pool. Among population differentiation was quantified using FST and RST as calculated in fstat and Arlequin ver. 3.1.1 (fstat) and 10 100 (Arlequin) permutations. Neighbour-Joining trees of populations, based on the model-free shared allele distances (DAS), were constructed with the program populations 1.2.30  of the global structure analysis (12 lakes and the WAL reference). We additionally produced simulated hybrid populations and compared their q-value distributions to those in actual lakes to gain confidence in our lake-specific inferences. Synthetic hybrids were created using hybridlab 1.0 (N= 102 for each), and a second based on a reduced set of individuals (N= 79 for each) using the q-threshold of 0.90 (see results below). For both simulations, 100 synthetic genotypes were generated.To display the pattern of lake-specific introgression we used the mean N= 253) of our samples. MtDNA sequence variation was analysed for these individuals across segments of the cytochrome oxidase b (cyt b) and the NADH-3 subunit (ND3) as described in mega 4.1  for a reduced set . The software tracer  and the coefficient of variation (CV) for 10 morphological (i.e. ratio-variables) traits. The expectation is that the variance in these quantitative traits should be positively correlated to an increase in additive genetic variance due to introgression, reflected by higher admixture proportions of the OU-introduced lineage. As we were not interested in accounting for the obvious increased population phenotypic variance due to the inclusion of genetically recognized introduced or hybrid individuals, CVs for only OU-native individuals were used, whereby the analysis was repeated for two different thresholds as above.Because our eight-locus microsatellite characterization may not detect minor levels of introgression at the individual level, we further incorporated morphological information as a surrogate for quantitative traits, to evaluate the potential effects of introgression on the native stocks. Accordingly, we evaluated the correlation between the mean lake admixture proportion (AN) was high with 17  to 56 (ClaTet12) alleles per locus, with mean allelic diversities across lakes ranging from 6.3 (KLO) to 14.5 (MIL) . Analysis of linkage disequilibrium (LD) for the global data set revealed six highly significant comparisons out of 28. However, after removal of the OU-introduced (see identification criteria below) no evidence of LD for any locus was observed, and thus the signal of LD presumably reflects recent genetic admixture between two divergent gene pools. At the population level, signals of gametic disequilibrium were limited to one or two locus pairs within FUS, two within MIL and three within MON.Allelic variation . Overall 661 individuals from the 12 lakes and the WAL reference were assigned to one of these three groups. q-values from structure assigned 359 individuals to the OU-native, 123 to the OU-introduced and 179 as hybrids. newhybrids assigned a similar number of individuals to the OU-native, but assigned considerably more hybrids at the expense of OU-introduced . The genetic relationship of individuals with structure assignment  was depicted with a bi-variate FCA plot  implying that non-mutational mechanisms (i.e. genetic drift and gene flow) are responsible for this level of differentiation  supported four groups of genotypes corresponding to the geographical regions (provinces), Salzburg and Upper Austria, Carinthia, Tyrol and the Baltic region, whereas the KLO and ACH populations were by far the most divergent with regard to the OU-native .K= 1 through K= 3 of the OU-native in each lake (N= 12) did not show any sign of significant population structure. Thus it can be assumed that only one native species was present in our data set for each lake.Structure analysis simulations with q-value of 0.09, with 79 out of 102 individuals fulfilling the criteria of ‘pure’ OU-introduced. Simulated admixture analyses were run with both the entire WAL sample, as well as the 79 ‘pure’ individuals, though little difference was revealed  and MON (20.2%). Hybrid proportions varied from 10.3 (WOL) up to 74.3% (ZEL). Most lakes revealed an s-formed distribution of assignment , although with an abrupt slope clearly distinguishing the two OUs and their hybrids compared to the simulated hybrid populations (including parents). Three lakes  exhibited a more linear pattern indicating extensive introgression with a deficiency of pure OU-introduced individuals, suggesting the result of historical rather than contemporary processes. Three lakes ACH, KLO and KOP revealed no signal of hybridization reflecting the fact that no stocking with the OU-introduced has occurred  to 0.97 (KLO).Within population structure and levels of hybridization differed among lakes . The WALrevealed . Six of occurred . Mean q-b and ND3 combined) was aligned and used for analyses. We found 23 different haplotypes six of which  were previously described in A total of 509 bp of mtDNA (cyt N= 76) revealed a similarly weak clade structure as shown in Network analysis of all published and novel haplotypes (A second group (clade R) harboured haplotypes appearing in three different geographic regions: Denmark, the Baltic Sea area and the European Alps (The most common haplotypes were A1 (51%) found in each population, and R1 25%; . P1 haplP< 0.001). The OU-native lineage carried all clades, whereby the OU-introduced lacked haplotypes from sub-clade Z and carried a low proportion from clade R (7.7%). The highest disparity between lineages was seen for clade R, which was very frequent (43.4%) in the OU-native and carried by only three individuals of the OU-introduced. Much more than half of all hybrids carried a haplotype from clade A (63.1%), with the remaining from clade R (35.4%) and sub-clade Z (1.5%). There was no significant linkage between mtDNA clades and microsatellite allele diversity, whereby only a reduced set (N= 203) of the data could be tested.Using the microsatellite assignment-based categorization, the distribution of the main mtDNA clades  among OU-introduced, OU-native and their hybrids differed strongly  values ranged from just under 1 Myr to more than 2.3 Myr, in any case time periods that span over most if not all of the Pleistocene.Median values of the TMRCA under the strict and uncorrelated lognormal molecular clock were quite similar: 1.302 × 10C. maraena), showed a clear split of the south Alpine KLO population, whereby the WOE population grouped with KLO when a higher threshold (0.90) was applied. The next split in both trees involved the hatchery reference (WAL). The remaining populations formed a cluster, in which there was some bootstrap support in both trees for MIL and WOL, whereby MIL is a rehabilitated population that has received brood from WOL.There was little allometry in morphological measurements globally, whereby the first two PCA factors accounted for over 78% of the global variance, yet neither factor was correlated with size. Closer inspection of individual variables showed a clear allometric pattern to eye size, determined primarily by the small-sized and large-eyed fish in ACH and to some degree KLO and NIE, and large-sized and small-eyed fish of the Baltic outgroup (OST) population. Eye measurements, however, did not load heavily on any PC factor. Much of the variance in the data set was determined by gill raker and lateral line scale counts (first two PCA factors) and body depth (third PCA factor). Nonetheless, we used the first six factors accounting for 90% of the total variance for our morphological distance matrix, as we aimed to include minor components of the global variance that may account for differentiation of single populations. The UPGMA dendrograms  as well as RST values  when the 0.75 threshold for defining OU-native individuals was applied, whereby statistical significance was only substantiated for FST values when applying a 0.90 threshold .The phenotypic distance matrix was correlated to both q-values when applying a 0.90 threshold. However, when using a threshold of 0.75 a negative trend was revealed, whereby KLO was the outlier representing an OU-native population with very high morphological variation. Removal of KLO resulted in significant negative correlations between the population CVs of all traits and mean q-values .The coefficient of variance (CV) for all morphological traits of the OU-native was not correlated to mean population Coregonus in Austrian lakes reveals both ongoing and historical introgression with an introduced Baltic lineage. Introgression beyond the F1 generation is supported despite large credible intervals of assignment values, which do not allow the designation of individuals to backcross classes. The consequences of this introgression on the conservation potential of individual native stocks is highly variable and lake specific. Many lakes appear to harbour pure or nearly pure native genotypes despite the long history of regional stocking and only a few lakes appear to contain hybrid swarms. Thus, regionally, there are a number of lakes that should be given conservation priority, and shielded from introductions, a fact that was not considered prior to this study. For the lakes that do reveal non-native genotypes, a broad range of introgression intensity is assumed, most likely the result of the interaction between intrinsic isolating mechanisms and human-mediated ecological factors, long hypothesized to play a major role in promoting hybridization in freshwater fishes  reasons. Additionally, the native Alpine lineage exhibits substantial geographic structure, which needs to be recognized by resource managers. This structure is concordant with the possibility that the native range of Coregonus extends to the south slopes of the Alps.The genetic structure of r fishes . We posestructure analysis delineates two major lineages of Coregonus in Austrian lakes; the OU-introduced, commonly referred to as Maräne, and the OU-native. structure and newhybrids identified a similar number of OU-native individuals using a threshold of 0.90 whereby the number of OU-introduced detected by structure was much higher than that revealed by newhybrids. This differential result is not unusual  is graphically depicted in ual e.g. . Our resThe two lineages (OU-native and OU-introduced) were not distinguishable with mtDNA data because the major clades were randomly distributed within the OU-native lineage among lakes. The TMRCA estimate supports that the two major clades (A and R) have a much older origin than the colonization of Central Europe, which was post-glacial (approximately 20 000 years ago) as all sampled lakes lie in previously glaciated regions. Thus we assume that the mtDNA lineages were already admixed prior to post-glacial expansion and have subsequently sorted out randomly among lakes, similar to the scenario reported for Swiss lakes  are currently not stocked with OU-introduced although they were extensively managed with these stocks in the past. Our data reflect the lack of pure OU-introduced individuals and thus there can be no F1 hybrids as the parental OU-introduced is absent. The q-value distributions in these three lakes thus reflect introgressed or hybrid swarm populations  on Austrian ulations . Furtherq-values) for MON, FUS, NIE and OBE , whereas hybrids carry at least some of the diversity exhibited by OU-native. Further, the mtDNA haplotype distribution in hybrid individuals supports that at least some hybrids stem from female native fish that have mated with introduced males.The morphological correlation analysis provides an interesting assessment of the effect of a differing threshold (0.90 vs. 0.75) for defining hybrids. Clearly, individuals with some hybrid background, reflected by increased morphological CVs were assigned to the OU-native when using a threshold of 0.75. However, a threshold of 0.90 increased the efficiency of assigning hybrids, and thus there was no remaining correlation between morphological CVs of OU-native individuals and the level of introgression present in a lake.Coregonus , the Drau south of the Alps , and the Traun . Within the large Inn drainage, ZEL, NIE and OBE are all in a major sub-drainage  quite distant from ACH, and are furthermore all heavily introgressed . Thus, toregonus . Honsig-Coregonus is indeed native to some lakes south of the Alps, then it is logical to assume that they were also found in the WOE population, one of the largest lakes in the Drau drainage. WOE contained a number of novel haplotypes genealogically related to the common haplotype in KLO, as well as several common private microsatellite alleles (data not shown) providing at least some evidence of their possible common ancestry.Furthermore, KLO individuals exhibit an atypical number of gill rakers, with a mean of 43 (SD 3.9), while the means of all other populations ranged from 32 to 38  and MON at 480 m a.s.l., connected by a very narrow pass that reaches 620 m a.s.l. Early post-glacial evidence suggests that both WOL and MON where about 50 m higher than today, and nearby Attersee  was 132 m higher  implyingThe KOP sample stems from the main tributary of HAL, where the lake’s native stocks are known to spawn . In thisCoregonus sp. of Austrian lakes provided insights into the extent of hybridization and introgression between the native and an introduced Baltic lineage as well as the genetic structure of native populations. We were able to identify these two major lineages and their corresponding hybrids, albeit the pattern of population structure varied considerably among lakes. Indeed, we revealed different levels of hybridization and introgression and hypothesize that historical environmental degradation as well as other ecological factors like spawning segregation has played a role in the differential outcomes of introductions. Conservation planners need to be aware that generalizations concerning hybridization and introgression, even within a species, or in a specific region may be difficult to reach. Our identification of native populations and the extent of genetic effects by introductions of foreign stocks will be indispensable for effective conservation and management strategies in the region. We strongly urge managers to shield the remaining native gene pools from further invasion of non-native lineages, but also emphasize the notion that the maintenance of ecological integrity in ecosystems in general may be among the most important tools in shielding native fauna and flora from the potential negative effects of introduced species.Our study of eight microsatellite loci, two concatenated mtDNA segments and various morphological traits in


Hordeum chilense is one of the wild relatives with a high potential for cereal breeding, due to its high crossability (both interspecies and intergenera) and polymorphism for adaptation traits. In order to analyze the genetic structure and ecogeographical adaptation of this wild species, it is necessary to increase the number of polymorphic markers currently available for the species. In this work, the possibility of using syntenic wheat SSRs as a new source of markers for this purpose has been explored.Multi-allelic microsatellite markers have become the markers of choice for the determination of genetic structure in plants. Synteny across cereals has allowed the cross-species and cross-genera transferability of SSR markers, which constitute a valuable and cost-effective tool for the genetic analysis and marker-assisted introgression of wild related species. H. chilense germplasm. The H. chilense-Triticum aestivum addition lines were used to test the chromosomal location of the new polymorphic microsatellite markers. The genetic structure and diversity was investigated in a collection of 94 H. chilense accessions, using a set of 49 SSR markers distributed across the seven chromosomes. Microsatellite markers showed a total of 351 alleles over all loci. The number of alleles per locus ranged from two to 27, with a mean of 7.2 alleles per locus and a mean Polymorphic Information Content (PIC) of 0.5.From the 98 wheat EST-SSR markers tested for transferability and polymorphism in the wild barley genome, 53 primer pairs (54.0%) gave cross-species transferability and 20 primer pairs (20.4%) showed polymorphism. The latter were used for further analysis in the According to the results, the germplasm can be divided into two groups, with morphological and ecophysiological characteristics being key determinants of the population structure. Geographic and ecological structuring was also revealed in the analyzed germplasm. A significant correlation between geographical and genetic distance was detected in the Central Chilean region for the first time in the species. In addition, significant ecological influence in genetic distance has been detected for one of the population structure groups (group II) in the Central Chilean region. Finally, the association of the SSR markers with ecogeographical variables was investigated and one marker was found significantly associated with precipitation. These findings have a potential application in cereal breeding. Wild species usually exhibit large genetic variability, which serves as a resource for adaptability to changing environments. On the contrary, cultivated plants are usually more limited in number and display less genetic variability, as a result of the genetic bottlenecks occurring at domestication, translocation and transition from landraces to modern breeding . A conseHordeum chilense Roemer et Schultes, a native South American diploid wild barley (2n = 2x = 14), offers a high potential for cereal breeding among the species of the genus Hordeum, because of its high crossability with other members of the Triticeae tribe and its agronomically interesting characteristics. Crosses between wheat and H. chilense lead to fertile amphiploids named tritordeums. They represent the basic genetic material for introducing genetic variability from H. chilense into wheat breeding programs [H. chilense to wheat. The analysis of the germplasm genetic structure is the basis of management, research and utilization of such germplasm [H. chilense has been found in a wide range of environments, and shows high genetic as well as phenotypic diversity [H. chilense. To solve this problem, the transferability of wheat and barley microsatellite markers  to wild related species was evaluated [Aegilops longissima to 100% for wheat subspecies (Triticum compactum), but was also good for less related species such as rye (72.8%) or maize (40.4%). In barley, Varshney et al. [H. chilense genome [Thus, wild species related to cultivated crops represent interesting sources of genetic variation, through the introgression of new and better performing alleles. The large genetic variability present in the wild cereals is an invaluable resource for cereal crop improvement. ermplasm , since iermplasm . H. chiliversity . The anaiversity -9. Moleciversity -21, allovaluated ,23. Compvaluated -27. The valuated . For exavaluated  tested Evaluated  reportedy et al.  reportedy et al.  reportede genome .H. spontaneum with a set of 42 EST-SSRs, revealing that wild barley populations can be divided into seven major genetically differentiated clusters, as well as the evidence of temperature and precipitation as environmental cues that have shaped the genetic makeup of wild barley. Pan et al. [T. dicoccum) populations using 25 EST-SSRs, detecting a considerable amount of genetic variation, partly related to ecological factors.In addition of their high degree of transferability across species, it was recently demonstrated that EST-SSRs are useful for genetic variability studies. For example, Gupta et al.  assessedn et al.  investigH. chilense; (ii) the genetic diversity of the H. chilense species, using a wide set of available microsatellite markers; (iii) the genetic structure of a natural collection of 94 H. chilense accessions; and (iv) the possible influence of spatial, morphological and environmental factors in the observed structure.The goals of the current study were to determine: (i) the transferability level of wheat EST-SSR markers and their usefulness for H. chilense. A SSR was considered as transferable when the PCR amplification of a band of the expected size and SSR pattern was observed. The primers that showed null alleles in some samples were tested at least twice, in order to avoid false negatives .The transferability of the 98 wheat EST-SSRs was evaluated on a set of eight accessions of H. chilense germplasm. Between two and 10 alleles per primer pair were observed and only 11.7% of the total alleles had the same size as the allele found in Triticum aestivum cv. 'Chinese Spring', confirming the good potential of H. chilense for wheat and barley genetic diversity improvement. These markers were first assigned to H. chilense chromosomes using the available set of wheat-H. chilense addition lines  showed cross-species transferability. The percentage of transferred markers was about 50% on each chromosome, thereby indicating a uniform distribution across the genome. Among the 53 transferable SSRs, 20 PCR primer pairs (20.4%) showed polymorphism in the accessions studied and were used for further analysis in the n lines . FifteeH. chilense  were wereH. cAdditionally, several rare or specific alleles were found among the analyzed germplasm. Out of the 351 alleles, 184 were found at a frequency lower than 5%, and were therefore considered as rare. Clustering showed that the germplasm can be separated into two groups (see below) with a total of 66 specific alleles found in group I (39 being rare) and 134 specific alleles found in group II (82 being rare). The gene diversity, polymorphic information content (PIC), and the number of specific alleles was lower in group I than in group II  Figure . We applTo find the key determinants to the inferred structure of these two groups, we investigated the geographical proximity, as well as the morphological, agronomical and ecological characteristics in the accessions belonging to each group. Figure r) between the geographic and the genetic distance matrices using the Mantel test [r value of 0.21  analysis. The first component , the accessions were grouped according to bioclimatic parameters. The results revealed admixed populations, except for some provinces that showed a more uniform structure , supporting the presence of a genetic structure.The unrooted Neighbor-Joining (NJ) tree Figure  distinguWe identified 12 outlier loci that detected high or low variability with respect to the expected neutrality. Among those, 11 loci  are candidates for balanced selection, while the locus CFE135 is a candidate for being subjected to positive selection. The 12 markers were assayed for their association with ecogeographical data. The marker GWM1302 exhibited four different alleles  among 10 alleles that were associated with low rainfall . This is likely due to the fact that wheat is evolutively more distant from H. chilense than barley. This result is relatively consistent with the findings of Zhang et al. [Brachypodium).The transferability of EST-SSRs across related species has been demonstrated in several species and genera -32,43-45chilense . In the g et al.  and Guptg et al. , who repH. chilense was high, indicating a good level of synteny between these two species. Only one disagreement was observed, that may be due to chromosomal rearrangements, which may be frequent during speciation [H. chilense have an added value as intergeneric syntenic markers, in addition to their direct application to analyze gene diversity. Since numerous additional wheat EST-SSRs are available in the public databases , this nH. chilense, about 40% exhibited polymorphism between at least two accessions, which represents 20% of the initial set of wheat EST-SSRs. This was lower compared to what was previously observed between wheat and barley . On the other hand, due to their wheat origin, they are not suitable for direct in-tube detection methods [H. chilense germplasm collections. In addition, they represent an invaluable tool for the introgression of H. chilense alleles to other cereal species.Among the 53 wheat EST-SSRs showing good transferability to ST-SSRs; ). This i methods . Neverth methods . TherefoThe microsatellite markers revealed a total of 351 alleles across all the 49 loci. The high level of genetic diversity detected could be an adaptive strategy in response to a heterogeneous environment. According to the marker's origin, the SSRs identified in EST databases detected a lower number of alleles and PIC than those obtained through general genomic libraries, including non-transcribed regions. Such is an expected result, due to the more conserved nature of EST-SSRs. The number of alleles detected and PIC found when using EST-SSRs from wheat was lower than with barley EST-SSR and non-transcribed genomic microsatellites, as expected, due to the evolutionary distance between species. Despite these facts, our results show a sufficient level of variation when using EST-SSRs (both from barley and wheat origins) to carry out genetic structure and future association mapping analysis. Therefore, this is one more case where the EST-SSR markers provide an opportunity to examine the functional diversity of germplasm collections, as shown by Eujayl et al. 2002 [The group I identified by Structure was fixed to one allele in eight loci. Seven of these loci could be assumed as being non-neutral, due to their origin from EST databases. The higher number of fixed alleles in non-neutral SSRs could be explained by temporal variation of external factors, generating selection pressures that maintain variation within populations . This maThe Spearman correlation showed that genetic diversity is influenced negatively by altitude and positively by temperature. Accessions in group II showed a higher number of alleles. Besides, they were found mainly in places with low altitude and higher temperature than accessions in group I. Therefore, accessions stressed by cold showed less genetic variation.H. chilense germplasm using wheat and barley gSSRs and EST-SSRs, we have defined two main germplasm groups (group I and group II). A previous analysis based on AFLP markers and a Principal-CoOrdinate analysis  and genetic distance approaches  of a set of 94 H. chilense germplasm. Thus, the two inferred groups are mainly in accordance with the agro-morphological clusters described by Vaz Patto et al. [The 'admixture model' implemented by Structure gave a better fit to the species ecophysiological clusters, as defined by Vaz Patto et al. , and it o et al. , as the H. chilense in South America, established initially in Central Chile from a long-distance continental dispersal from North America, followed by two independent dispersals to the North and to the South [H. chilense germplasm should be considered in future association mapping studies. The AMOVA detected higher differences among individuals within-population structure groups than among groups, which is consistent with findings from other studies, indicating that considerable genetic diversity is partitioned within rather than between wild barley populations [Several studies have been carried out to detect the population structure in barley. In most of them, the key factors affecting the genetic structure were growth habit or spike morphology and geographic origin ,20,55-57he South . On the ulations -63. The The loci that show unusually low or high levels of genetic differentiation are often assumed to be under natural selection . The accSignificant correlations between microsatellite markers and ecogeographical factors have been observed in several studies in wild wheat  and in wH. chilense germplasm. The second principal component explained by longitude and temperature significantly contributed to the separation of the two groups. Hübner et al. [Hordeum spontaneum and found a strong correlation of population structure with temperature and precipitation. In our study, the genetic structure of the analyzed germplasm showed a correlation with morphological and ecophysiological characteristics, influenced also to a minor extent by geographic and ecological factors.Genetic clustering in a principal component analysis revealed that the combination of geographic and ecological data, such as the latitude with rainfall, as the main contributor to the genetic structure of the r et al.  studied H. chilense, with a remarkably high level of polymorphism within this species, despite of the evolutionary distance between the wheat and barley genera. The current set of SSR markers available for Hordeum chilense, which includes wheat and barley gSSRs and EST-SSRs, is useful to analyze the genetic structure and ecogeographical adaptation of H. chilense wild barley populations. Both wheat and barley represent a good source of markers for genetic diversity and structure studies of H. chilense germplasm collections. In addition, they represent an invaluable tool for the introgression of H. chilense alleles into other cereal species, and are useful as anchors for the syntenic maps. The analyzed germplasm can be divided into two groups, with morphological and ecophysiological characteristics being key determinants of the population structure. Geographic and ecological structuring was also revealed in the analyzed germplasm. A significant correlation between geographical and genetic distance was detected in the Central Chilean region for the first time in the species. In addition, significant ecological influence in genetic distance has been detected for one of the population structure groups (group II) in the Central Chilean region. Finally, one marker was found significantly associated with precipitation. These findings have a potential application in cereal breeding.Our study shows the utility of barley EST-SSR for the genetic analysis of H. chilense collected during several expeditions to Chile [H. chilense DNA were used for selecting transferable and polymorphic microsatellite markers, using Triticum aestivum cv. 'Chinese Spring' DNA as control, to corroborate the pattern of the microsatellites and the fragment sizes. In addition, a DNA set of T. aestivum-H. chilense addition lines developed by Miller [ch, 5Hch, 6Hch and 7Hch were disomic, whereas the addition lines for chromosomes 1HchS, 2Hch alpha arm, 5Hch, 6HchS arm and 7Hch alpha and beta arm were ditelosomic.The DNA collection, isolated by Castillo et al. , consistto Chile ,68. Thisy Miller  were useH. chilense genome, and 20 were selected for germplasm analysis. A set of 21 barley EST-SSRs and eight wheat and barley g-SSRs, previously identified as useful for the genetic analysis in H. chilense [H. chilense accessions. The polymerase chain reaction (PCR) amplification and fragment analysis were as previously described [A selection of 98 SSRs derived from wheat ESTs ,70 unifochilense ,68 was aescribed ,71,72, oescribed , with anhttp://www.diva-gis.org;. The geographic location of the study area was between 28°15' and 38°42' South latitude and between 70°18' and 73°24' West longitude. The altitude on the sites varied within a wide range, from sea level to high mountains (> 2000 m). Since only one accession is available in some provinces, the accessions were grouped in eight zones along Chile, from North to South, including in some cases various close provinces with similar ecological characteristics. The ecological data like rainfall of wettest and driest month and mean temperature of the warmest and coldest month were obtained for each site using DIVA-GIS. The ecological regions were described following the bioclimatic classification of DiCastri and Hajek [The geographic data  of 76 accessions were available [nd Hajek .The summary statistics including the number of alleles per locus, polymorphism information content (PIC) values and gene diversity were determined using the application PowerMarker version 3.25 . The unrA Bayesian model-based analysis for inference of population structure was performed using the program Structure (version 2.2)  to estimThe Distruct 1.1 software  was usedThe genetic structure of the population was also inferred by the Geneland package , implemeWe used the Lositan software  to identValues of environmental variables were first standardized and the Euclidean distance between the samples was computed using SPSS. The correlation between genetic distance and environmental distance in the collection was calculated by the Mantel test. Also, the Principal Component Analysis (PCA) was computed from environmental values, and the samples were plotted in genetic structure grouping. The Spearman rank correlation was used to assess differences in mean number of alleles and ecogeographic variables among the inferred groups.AC carried out the experiments and drafted the manuscript. CF, PS, GD and PH were involved in designing and planning the work, interpreting the results and critically editing the manuscript. PH, CF and PS conceived the study. PH coordinated the study and helped to draft the manuscript. All authors have read and approved the final manuscript.Estimated number of populations from Geneland analysis. (a) Posterior density distribution of the number of clusters estimated from analysis in five replicates; and (b) genetic assignment of H. chilense individuals.Click here for fileMantel test showing the relationship between genetic distance and geographic distance. (B1) Relationship between Nei's genetic distance and Napierian logarithm of geographic distances for group I accessions; and (B2) relationship between Nei's genetic distance and Napierian logarithm of geographic distances for group II-Center accessions.Click here for fileMantel test showing the relationship between genetic distance and environmental distance for group II accessions. Plot of genetic distance vs. Ln .Click here for file


Orthilia secunda (one-sided wintergreen) and Monotropa hypopitys (yellow bird's nest). Phylogeographic analysis was carried out to determine the distribution of genetic variation across the range of each species and to test whether both correspond to the "classic" model of high diversity in the south, with decreasing diversity at higher latitudes, or whether the cold-adapted O. secunda might retain more genetic variation in northern populations. In addition, projected species distributions based on a future climate scenario were modelled to assess how changes in the species ranges might impact on total intraspecific diversity in both cases.The aim of the present study was to use a combined phylogeographic and species distribution modelling approach to compare the glacial histories of two plant species with overlapping distributions, trnS-trnG region, nuclear ITS and microsatellites for O. secunda; chloroplast rps2, nuclear ITS and microsatellites for M. hypopitys) indicated that both species persisted throughout the Last Glacial Maximum in southern refugia. For both species, the majority of the genetic diversity was concentrated in these southerly populations, whereas those in recolonized areas generally exhibited lower levels of diversity, particularly in M. hypopitys. Species distribution modelling based on projected future climate indicated substantial changes in the ranges of both species, with a loss of southern and central populations, and a potential northward expansion for the temperate M. hypopitys.Palaeodistribution modelling and phylogeographic analysis using multiple genetic markers (chloroplast Orthilia secunda and Monotropa hypopitys appear to have persisted through the LGM in Europe in southern refugia. The boreal O. secunda, however, has retained a larger proportion of its genetic diversity in more northerly populations outside these refugial areas than the temperate M. hypopitys. Given that future species distribution modelling suggests northern range shifts and loss of suitable habitat in the southern parts of the species' current distributions, extinction of genetically diverse rear edge populations could have a significant effect in the rangewide intraspecific diversity of both species, but particularly in M. hypopitys.Both  The two most common haplotypes, Haplotypes 1 and 2 (depicted in blue and yellow), exhibited a largely east-west split. Only four populations exhibited any within-population variation  and of these, only the RORM population contained more than two haplotypes.The 320 bp chloroplast HQ864707-HQ865709). The distribution of these haplotypes was broadly congruent with that of the chloroplast rps2 haplotypes. Only the CHCH (Switzerland), SIDO (Slovenia), SKNT (Slovakia) and ROVG (Romania) populations exhibited any within-population variation, with all three haplotypes being found in the SIDO population.The 287 bp nuclear ITS alignment contained three distinct haplotypes (Table HE) calculated for populations with a sample size of N ≥ 4 ranged from 0.370 (IEST [Ireland]) to 0.750 (CHCH [Switzerland]), with a mean value of 0.629  and levels of expected heterozygosity  more likely represents a relatively recent mutation that has become fixed through genetic drift, rather than indicating an extreme northern refugium. For M. hypopitys, the modelling and genetic data both indicated a likely refugial area in southeastern Europe. The identification of two genetic clusters with a broadly northern/eastern vs. southern/western geographical distribution for both species based on microsatellite data could indicate isolation in separate refugia followed by differential recolonization after the retreat of the ice )igration ,53. Althe change -57, its e change . The site change , Beatty Orthilia secunda and Monotropa hypopitys appear to have persisted through the LGM in Europe in southern refugia. The boreal O. secunda, however, has retained a larger proportion of its genetic diversity in more northerly populations outside these refugial areas than the temperate M. hypopitys. Given that future species distribution modelling suggests northern range shifts and loss of suitable habitat in the southern parts of the species' current distributions, extinction of genetically diverse rear edge populations could have a significant effect in the rangewide intraspecific diversity of both species, but particularly in M. hypopitys.Both Both authors conceived and designed the study. GEB carried out the laboratory work. Both authors analysed the data and wrote the manuscript.


Epiphyas postvittana , is native to Australia but invaded England, New Zealand, and Hawaii more than 100 years ago. In temperate climates, LBAM can be a major agricultural pest. In 2006 LBAM was discovered in California, instigating eradication efforts and quarantine against Hawaiian agriculture, the assumption being that Hawaii was the source of the California infestation. Genetic relationships among populations in Hawaii, California, and New Zealand are crucial to understanding LBAM invasion dynamics across the Pacific.The light brown apple moth (LBAM), We sequenced mitochondrial DNA (mtDNA) from 1293 LBAM individuals from California (695), Hawaii (448), New Zealand (147), and Australia (3) to examine haplotype diversity and structure among introduced populations, and evaluate the null hypothesis that invasive populations are from a single panmictic source. However, invasive populations in California and New Zealand harbor deep genetic diversity, whereas Hawaii shows low level, shallow diversity.LBAM recently has established itself in California, but was in Hawaii and New Zealand for hundreds of generations, yet California and New Zealand show similar levels of genetic diversity relative to Hawaii. Thus, there is no clear relationship between duration of invasion and genetic structure. Demographic statistics suggest rapid expansion occurring in California and past expansions in New Zealand; multiple introductions of diverse, genetically fragmented lineages could contribute to these patterns. Hawaii and California share no haplotypes, therefore, Hawaii is not the source of the California introduction. Paradoxically, Hawaii and California share multiple haplotypes with New Zealand. New Zealand may be the source for the California and Hawaii infestations, but the introductions were independent, and Hawaii was invaded only once. This has significant implications for quarantine, and suggests that probability of invasion is not directly related to geographic distance. Surprisingly, Hawaiian LBAM populations have much lower genetic diversity than California, despite being older. In recent decades, globalization and the acceleration of anthropogenic trade and travel have increased both the frequency and scope of the introductions of alien species world-wide. Negative impacts of invasive species are evident in agriculture, native ecosystems, and human health Epiphyas postvittana, is native to Australia but has been established in England, Hawaii, and New Zealand for 100 years or more Invasive phytophagous insects are a major economic concern and constitute a primary target for agricultural quarantine inspections and eradication campaigns In Hawaii, LBAM is sparsely distributed, and appears to now be restricted largely to montane wet forest regions. It is unclear why LBAM never became a pest in Hawaii, and is now apparently retreating to a subset of its former range. Perhaps the climate in Hawaii is suboptimal, or parasitoid releases to control other pest moths over the past 100 years have had non-target impacts on LBAM, as they may have had on native Hawaiian moths In this investigation we specifically address the genetic relationships among invasive LBAM populations in New Zealand, Hawaii and California, what invasion pathways are indicated across these regions, and what patterns of genetic connectivity tell us regarding long-term invaded regions  compared with recent invasions (CA). Such information is essential in understanding whether there is a relationship between genetic diversity (mtDNA nucleotide and haplotype diversity) and invasion success. For example, in a recent molecular investigation of an explosive gall wasp invasion spanning the Pacific Basin, a single nuclear and mtDNA haplotype was found in multiple populations from Hawaii to China Because Australia represents the natal range of LBAM and likely the greatest diversity of haplotypes, thorough sampling across the continent was outside of the scope of this study and not among the objectives we wished to address regarding the invasive patterns in LBAM. This approach is justified because haplotypes present in invading populations represent subsets of those present in the source populations from the endemic range of the insect, and estimates of mtDNA mutation rates predict that novel haplotypes will not arise in less than two hundred years The head and thorax were dissected and used for genomic DNA extraction, while the remainder of each insect was stored at −80°C as a voucher. Genomic DNA was extracted from all specimens using the DNeasy™ Blood & Tissue kit (Qiagen) following standard protocols. Tissue was digested at 56°C for 24 hours with proteinase K, 200 µl of EB buffer was used to elute the DNA and extracts were stored at −20°C.COX1http://asgpb.mhpcc.hawaii.edu). For each sample, PCR products were sequenced in both sense and anti-sense strands. All COX1 sequences were aligned by eye using BioEdit 7.0.9 Polymerase chain reaction (PCR) was performed using a PTC-200™  We used primers LCO-1490, HCO-2198, Jerry and Pat2 to sequence 1318 base pairs across FS test statistic were used to evaluate whether demographic expansions or possible secondary contact had occurred for California, Hawaii and New Zealand populations. Mismatch distribution plots comparing pairwise frequency distributions of haplotype distances provide a graphic way to visualize the signature of population expansion. Mismatch plots and associated statistics were produced with Arlequin 3.11 TCS 1.21 was used to reconstruct statistical parsimony networks GSTh, and mean number of nucleotide substitutions per site between populations DxyFST) rely on a number of assumptions, including mutation-drift equilibrium and selective neutrality of the markers used. We tested for departures from equilibrium in each population with Tajima's D statistic F* and D* Maximum likelihood phylogenies were reconstructed using the GTR+Γ model in RAxML (Web-Server version) D contrasts estimates of the population mutation parameter θ, based on π, with those based on the number of segregating sites, for a given sample size. This test statistic is sensitive to demographic effects such as changes in population size. Fu and Li's statistics contrast estimates of θ based on mutations in internal vs. external branches of the gene tree. Designed to assess neutrality, these tests assume that more recent mutations occur near the tips of branches, while older substitutions are internal, and recent mutations confer a selective advantage, and therefore will increase in frequency rapidly under selection. Therefore positive selection results in an excess of identical haplotypes, or mutations in the external branches, and negative values of D* but population size expansions can also result in an excess of external substitutions and negative values of test statistics.Tajima's COX1 diversity within and among populations and among regions/groups, using Arlequin 3.11 F-statistic analogues (Φ-statistics), reflecting the correlation of haplotype diversity at different levels of hierarchical subdivision. Significance of Φ-statistics was tested by 1000 permutations of haplotypes among and within populations under the null hypothesis of panmixia. Significance of variance components was also tested using a permutational approach. Nei's average pairwise genetic distances were also computed with Arlequin.Analysis of molecular variance (AMOVA) was used for hierarchical analysis of the partitioning of Syndemis, (as310 and dr120) sequences were obtained from our work and Choristoneura (FSb53 and FSb216) was obtained from GenBank Altogether 1293 LBAM specimens were included in this population structure analysis, with 147 from across the introduced range on North and South Islands of New Zealand, 448 from Hawaii including all islands on which the species occurs , and 695 covering its recently invaded range in northern California. Three specimens from Australia, where the species is endemic, were included as phylogeographic outgroups. Four additional outgroup taxa were added for the maximum likelihood phylogenetic analysis. We used two genera of moth that, like LBAM, are archipine tortricids, COX1 resulted in a single clade with strong bootstrap support confirming the monophyly of all LBAM populations that we tested  generated for this study, via various approaches including analysis of molecular variance (AMOVA) (h) and nucleotide diversity (π) indices  , haploty indices , Jukes-Cmatrices , maximummatrices , haplotymatrices  and mismmatrices , 4, 5. TFST values among their subpopulations were quite high and significant, in the range of 0.29–0.57. Genetic distances and population statistics, including FST values, support isolation of Hawaii and California populations, while simultaneously suggesting that each region has an independent, genetic affinity with LBAM populations from New Zealand  as well as between New Zealand and Hawaii populations where two haplotypes are shared . Thus it is not possible to confirm that New Zealand, rather than Australia, is the source of both the Hawaiian infestation 100 years ago or the more recent California infestations, since Australia must be the ultimate source. While only one and two haplotypes are shared between California and New Zealand and Hawaii and New Zealand, respectively; all the California and Hawaii haplotypes are closely related to New Zealand haplotypes, even if they differ by a few base pairs. In the phylogenetic analysis New Zealand haplotypes fall amongst the California populations and the dominant Hawaiian haplotype. These results suggest that New Zealand may have served as the source for both invasions, and further sampling of New Zealand LBAM may confirm that all invasive haplotypes detected in the United States came from New Zealand. However, the genetic distinctiveness of the Hawaii and California populations is strongly supported by all analyses conducted , 3, 4, 5One puzzling finding during our sampling across the Hawaiian Islands was the apparent disappearance of LBAM from all lowland habitat and the near extinction of LBAM on the island of Oahu. About ninety years ago (1921), LBAM was recorded down to 600 m  elevation on Oahu (University of Hawaii Insect Museum specimen), on the outskirts of metropolitan Honolulu, and the moth occurred across both volcanoes constituting the Waianae and Koolau mountain ranges. Yet, despite extensive year-round pheromone and light trapping in these same areas and others, we never collected LBAM below 500 m on any island, except for one population on Hawaii Island at 100–450 m and 2 individuals caught on Maui at 136 m and 184 m. Furthermore, the moth has all but vanished from Oahu, no longer occurring in the Koolau Mountains where it was formerly found, and now restricted, ironically, to a single part of the pristine Mt. Kaala Natural Area Reserve for rare endemic species in the Waianae Mountains. This population is of relatively low density compared to both other Hawaiian Islands and California and New Zealand trapping efforts. The dynamics and reasons for the decline of LBAM across the Hawaiian Archipelago are unclear, but trapping revealed only a few localized populations which approached the density of the invasive populations in California and New Zealand, using trap catch as an indicator of population density. This decline bears further examination and may be important in understanding the potential spread and impact that LBAM may, or may not, ultimately have in California.LBAM haplotype diversity in California was relatively deep, suggesting the presence of at least 12 distinct mtDNA lineages, some of which are quite divergent. Hawaii, despite being an older invasion, has only 11 different haplotypes, and all of them are within one base pair of the dominant haplotype, shared by 96.7% of samples and only one of the other haplotypes is represented by more than one individual. This pattern suggests restricted, shallower genetic diversity than observed in California. Lower genetic diversity revealed in Hawaii could be the result of a founder effect following a population bottleneck associated with the release, in addition to a lack of continued introductions from genetically diverse populations in New Zealand and Australia.While our sampling from Australia was minimal, all haplotypes must be present in the native range of the moth; so extensive sampling would not conclusively demonstrate the source for California or Hawaii invasions, as long as those same haplotypes, or very similar ones, were also present in the New Zealand samples. This was largely the case, though only one of the haplotypes present in California was represented in our New Zealand samples, California's haplotypes were most similar to those from New Zealand. Based on the relatively high levels of genetic diversity in New Zealand and California, it is likely that there have been multiple invasions from Australia into New Zealand, and at least 12 unique female haplotypes have invaded California.The data support the idea that Hawaii was infested from Australia or New Zealand over 100 years ago, and a separate and recent introduction or series of introductions from New Zealand or Australia to California took place with no contribution from Hawaii. This result is surprising since Hawaii and California are not only geographically much more proximate but, as part of the same country, they share a brisk trade in goods and air traffic; California, in addition to being the closest continental landmass to Hawaii, is by far Hawaii's leading trading partner for agricultural goods and tourism. Thus, the data suggest that invasion pathways are not necessarily governed by levels of trade or geographic distance. Rather the level of the infestation in the source region appears to play the most significant role in determining invasion risk. For the past 100 years LBAM has been established in Hawaii and has still not made it to California. Yet, at least 12 LBAM haplotypes have successfully colonized California from a New Zealand/Australian source.The number of haplotypes present in California suggests that there have either been multiple invasions in a short period of time, or a single large infestation . The California invasion has maintained high genetic diversity, and this could be critical to the long-term persistence of LBAM in this novel habitat. Typically the homogenizing impacts of a strong founder event result in lower genetic diversity, LBAM's Californian diversity may help the moth adjust to adverse conditions. Regardless of whether the California infestation is eliminated, greater attention should be focused on inspections of imports, since other pests may arrive in a similar manner from New Zealand and Australia and be equally inconspicuous. Based on LBAM's apparently innocuous presence in Hawaii, there is a possibility that the moth may not cause severe damage in California, and after a period of population increase it may suffer the same decline that is typical of many invasive species over time From a scientific perspective, arguments for agricultural quarantine against Hawaii for LBAM appear unsubstantiated. Despite 100 years of intense trade, there is no evidence that LBAM from Hawaii has become established in California. Thus, California's quarantine of Hawaiian exports should be reconsidered. Despite the fact that Hawaii is geographically much closer to California, and is part of the same country, New Zealand or Australia is the source of the California invasion. This result demonstrates that trade trumps geographic and political boundaries in facilitating the spread of invasive species. Future efforts to control invasions through these pathways are essential, not just for LBAM but also for other pests from New Zealand and Australia that pose a risk to California's environmental and agricultural biosecurity. The data suggest that Hawaii was invaded only once, or possibly multiple times from a single geographic, genetically homogeneous population. Furthermore the data suggest that beyond distances that species can traverse naturally, whether actively or passively, geographic proximity does not enhance likelihood of invasion; New Zealand or Australia is the likely source of the California introduction, the distance from New Zealand is 11,073 km  and from Australia is 13,203 km  whereas Hawaii is 4,034 km  from California. Interestingly, survey data anecdotally support a recent population crash of LBAM in the Hawaiian Islands, and this may be a factor in the observed depauperate genetic patterns.Population genetic theory predicts that older established populations may have more pronounced genetic structure, due to opportunities for barriers to gene flow to arise, yet this pattern was not observed. LBAM sampling should be continued in California to establish a genetic baseline, and to try to detect whether introduction is ongoing. The molecular dataset presented could be used in a comparative framework for future sampling, from both additional invaded and native sources. In addition it would be worthwhile to obtain and compare samples from across the native range of LBAM in order to begin to understand the phylogeographic sources of invasive haplotypes characterized herein, in an effort that promises ultimately to shed light on the mechanisms of transport and release of this global pest species.Table S1Voucher identities, sample locations, collectors, and GenBank Accession numbers for LBAM (Epiphyas postvittana) specimens analyzed in this study.(DOCX)Click here for additional data file.


While the ultimate causes of most species extinctions are environmental, environmental constraints have various secondary consequences on evolutionary and ecological processes. The roles of demographic, genetic mechanisms and their interactions in limiting the viabilities of species or populations have stirred much debate and remain difficult to evaluate in the absence of demography-genetics conceptual and technical framework. Here, I computed projected times to metapopulation extinction using (1) a model focusing on the effects of species properties, habitat quality, quantity and temporal variability on the time to demographic extinction; (2) a genetic model focusing on the dynamics of the drift and inbreeding loads under the same species and habitat constraints; (3) a demo-genetic model accounting for demographic-genetic processes and feedbacks.Results indicate that a given population may have a high demographic, but low genetic viability or vice versa; and whether genetic or demographic aspects will be the most limiting to overall viability depends on the constraints faced by the species . As a consequence, depending on metapopulation or species characteristics, incorporating genetic considerations to demographically-based viability assessments may either moderately or severely reduce the persistence time. On the other hand, purely genetically-based estimates of species viability may either underestimate (by neglecting demo-genetic interactions) or overestimate (by neglecting the demographic resilience) true viability.Unbiased assessments of the viabilities of species may only be obtained by identifying and considering the most limiting processes , or, preferentially, by integrating them. The role of genetic deterioration in the extinction of endangered species or populations has long been controversial. For over 20 years, several authors have proposed that most populations go extinct for environmental or demographic reasons before genetic deterioration will affect them ,2. Yet, On the other hand, genetically based viability assessments generally neglect realistic ecological constraints . Contrary to demographic ones, insights on the "genetic viability" of species or populations use generic (not specific) data and come from two main categories of studies: (i) genetically determined minimum viable population sizes (MVPs), corresponding to the size necessary to compensate the loss of quantitative genetic variation (through genetic drift) by gains through mutations ; (ii) thCentaurea corymbosa, a narrow-endemic plant, provided guidelines for future reintroductions of the species regarding the number of seeds required and their initial distribution in space to limit the demographic effects of genetic self-incompatibility [Since the first formulation of the demographic-genetic  interactions , a few ttibility .Here, I compute projected times to metapopulation extinction using three different stochastic simulation models: (i) a demographic model assuming that population growth is finite and has a constant expectation; (ii) a genetic model accounting for variation in the drift and inbreeding loads. In this model, patch population sizes are assumed always equal to local carrying capacities. However, carrying capacities may vary through time in response to environmental variations, with subsequent effects on the dynamics of genetic loads. The metapopulation is assumed genetically extinct if the average genetic load reaches a certain threshold ; (iii) a demo-genetic model accounting for demographic (finite growth rate) and genetic processes (accumulation of loads) as well as demo-genetic feedbacks. Similar environmental and intrinsic constraints were applied to these systems and the demographic, genetic and demo-genetic extinction times that they provided were compared.extinct trajectories) are in excellent agreement; (ii) the effects of ecological variables on demographic (DT) and genetic (GT) viabilities are qualitatively similar. In all cases, D Tand G Twere positively related to dispersal rate, fecundity and total carrying capacity, while they decreased with fragmentation, as well as the frequency of perturbations (but see below for more details on the effects of fragmentation). The spatial correlation of environmental perturbations  always reduced demographic viability (as compared with the case where p C= 0) but had weaker and more complex effects on genetic viability.Comparisons between exponential and Weibull survival models suggested that all extinction rates increased with time  indicated that G Twas mainly related to metapopulation size and fragmentation  while D Tmostly depended on the basic growth rate and the perturbation regime  , but correlation increased when considering only scenarios with a constant environment (τ = 78%). In most situations, the demographic extinction time (TD) was longer than the genetic extinction time TG (median of TG/TD = 0.17) but the distribution of the TG/TD ratio was wide and skewed to the right . Further analysis indicated that this ratio strongly increases with total population size and the frequency of perturbations level and decreases with fragmentation  varied strongly, from 10-4 to 0.6 . Multiple regressions and HP indicated that this ratio was mostly influenced by the perturbation regime and the basic fecundity . The analysis indicated that this ratio was mostly influenced by population size and fragmentation  did not improve model fit (R2 = 64.6%), while using the minimum value increased the R2 to 78%, a value close to the R2 obtained by including both TG an TD in the model (without interaction). The interaction term between TG and TD was positive and highly significant but only marginally improved model fit (R2 = 80%).When considered as single predictors in D and TG and TDG, although the quantitative effects of F and P on TD may vary with the type of perturbations  on demographic, genetic and demo-genetic viabilities is illustrated in Figure The effect of the level of fragmentation (N) has less obvious effects (see below). However, despite these similarities, the processes leading to "demographic" and "genetic" extinctions are fundamentally different. Demographic viability is related to the mean and variance of the rate of increase [F and P). In contrast, genetic viability is associated with the inbreeding and drift loads, themselves primarily related to population size and fragmentation . TheWhile the genetic model presented here is a soft selection model , the integration of demographic processes  clearly adds a hard selection component (since the size and contribution of demes partly depend on their genetic loads). Theoretical work predicts that (1) With soft selection, population structure should reduce the efficiency of selection against mildly deleterious, nearly additive mutations  but willN and/or decreasing m has a negative effect on viability). This discrepancy is due to the fact that (i) the demo-genetic model is only a partial hard selection model, as local population size primarily depends on regulation processes (independent from genetic loads); (ii) potential genetic effects are masked by demographic effects  during the accumulation phase, stochastic variations in population size decrease the effective size and accelerate the rise of the genetic load; (2) during the extinction phase, the population decline increases the rate of mutation accumulation, which in turns accelerates population decline .extinction phase  is generally assumed much shorter than the accumulation phase in theoretical genetic models, and the extinction time is assumed equal to the duration of the accumulation phase . Here, in all scenarios, the initial decrease in fitness was more rapid with the demo-genetic model as compared with the genetic model , fragmentation has non obvious  qualitative effects on demographic and genetic viabilities. From a genetic view-point, most theoretical studies agree that subdivision has detrimental effects on fitness .  patches . The opt patches -44, see  patches ,46. In t patches ,47. In c patches ,45,46.Consequently, (i) demographically optimal levels of fragmentation may be much higher than genetically optimal levels; (ii) demo-genetic optimal levels are, in most cases, intermediary between demographic and genetic optimal levels Figure . This imUnderstanding the fine mechanisms of extinction is essential to the study of evolution and has crucial implications in the context of the current human induced mass extinction crisis . In the The present results indicate that (i) in most cases, the demo-genetic extinction time is lower than both the demographic and genetic extinction times, due to a demo-genetic interaction; (ii) while the demo-genetic extinction time is weakly correlated with demographic and genetic extinction times, it is substantially more correlated with the minimum between them, suggesting that considering the most limiting factors (demographic or genetic) to estimate population viability is a reasonable approach; (iii) a given population may have a high demographic, but low genetic viability or vice versa.At the evolutionary and ecological scales, most species extinctions are ultimately caused by changes in their environment. However, particular environmental constraints may primarily affect either demographic, genetic processes, or both of them. An illustration of this point is the contrast between captive and wild threatened populations. Small captive populations face important deleterious evolutionary changes  but are demographically safe (benign and buffered environment). On the other hand, wild populations with larger carrying capacities and sizes suffer less genetic problems but are ecologically vulnerable . While this antagonism is addressed in recent work in the particular context of the wild/captive population systems , there iN patches and a total carrying capacity tK.I used a monoecious, individual-based model with discrete generations to describe the dynamics of a metapopulation with L = 1000 different diploid loci that could carry either a wild-type or a deleterious allele. Following the approach of Jaquiéry et al. [s) and dominance (h) coefficients among loci and a negative correlation between them [L loci, with an average severity set to s = 0.05 [k, the dominance coefficient k hwas computed as k h= exp(-kcs)/2, where c was a constant computed so that the expected dominance of all mutations in the genome equaled h = 0.35 [U = 1.0, [Drosophila, nematodes or bacteria. These estimates are used in most theoretical studies, and should be viewed as conservative estimates . I assuspecies, ,54).k, the initial frequency of deleterious alleles was given by the mutation-selection balance [qk 0= U/(k hkL s). The genome of the initial population considered at t = 0 was implemented according to these frequencies (Bernoulli process). Thus, the individuals present at t = 0 derived from a large panmictic population .At each locus  balance : q0k = Ui was then given byI assumed that deleterious alleles acted on offspring viability. The proportional reduction in survival of the individual ki wwas equal to 1, 1-ksk hor 1-ks, for a locus k without mutation, with a heterogeneous deleterious mutation, or a homozygous mutation, respectively. w0 was the expected initial reduction in fitness due to deleterious alleles present at time zero, given byWhere t, the average reduction in survival of the metapopulation (W(t)) was computed.In each generation t, in each patch j, all individuals paired randomly with possibility of self-fertilization at the random rate. The fecundity of each individual was determined by a Poisson trial of parameter F. Punctual negative environmental perturbations resulted in a reduction  of local offspring survival. The survival of each offspring i present in patch j at generation t was drawn from a Bernoulli function of expectation si 0= (1-j(t)p)iw, that depended on the genetic characteristics i wof the individual (see above) and on the occurrence of a local perturbation. The occurrence of a perturbation was determined by a Bernoulli trial of parameter P . If no perturbation occurred in patch j at time t, pj(t) was set to zero. If a perturbation occurred, the value of pj(t) was drawn from an empirical severity distribution [p Cdenoted the degree of spatial correlation of perturbations. I considered two extreme situations where perturbations were either independent (p C= 0) or fully correlated in time (p C= 1) across patches. An alternative method to model perturbations  is presented in results and Additional file In each generation ribution . The part/NK = K), and were initially at their carrying capacity. All parents died after reproduction. Local regulation consisted in a truncation of population size of the offspring to K in each generation for each patch. Truncation was made independently of the genetic qualities of individuals.I assumed that all patches had the same maximum local carrying capacity (m (Bernoulli process) of the individuals present in each patch after the selection and regulation steps (but before the reproduction step) emigrated (island model) in each generation. Extinction occurred when there were no more individuals in the metapopulation.Dispersal occurred by assuming that a proportion t, in each patch j, two parents were randomly selected to reproduce. Fertilization and new mutations occurred as described above to create the new individual i, that survived or not, according to its relative fitness i w(Bernoulli process). This operation (random selection of parents with replacement) was repeated until there was Kj(t) surviving offspring. Then all parents died. jKt)), where pt) j population dynamics were independent from Wt)  and p C and acted by temporally reducing local carrying capacities (same distribution of severity as in the demo-genetic model); (ii) "demographic extinction" could not occur . Environmentally-driven variations of population size  tended to reduce the long term effective size and increase the inbreeding level, with subsequent effects on selection. The metapopulation was assumed "genetically extinct" when the average offspring survival Wt) . Dispersal occurred as for the demo-genetic model.In each generation i wwas set to one for all individuals). The similarities and differences between the demographic, genetic and demo-genetic models are summarized in Table The demographic model worked similarly to the demo-genetic model, but the effect of selected genetic variation on population dynamics was removed . I was interested in investigating the viability of small to medium metapopulations (t Kranged from 50 to 2000) with a wide range of persistence times. With the demo-genetic model , typical persistence times were a few tens or hundreds of generations ; however, in some scenarios assuming large populations with high and stable growth, times to extinction could be very long, especially with the demographic or genetic sub-models. Therefore, for technical reasons, all trajectories were stopped after 1,000,000 generations. In scenarios for which one or several trajectories were non extinct after 1,000,000 generations, extinction times could not be computed. These extinction times were treated either as right-censored data . I used Monte Carlo simulations for 1476 combinations of the ecological input parameters  or as lI used survival models, generalized linear models (GLM) and hierarchical partitioning (HP) to examine the relationships between ecological input parameters and extinction times.D, TG Tand DGT) and ecological input parameters, considering all 1476 scenarios. The Weibull and exponential models were compared to describe whether extinction rates were constant over time or not [As a first step, exponential and Weibull survival models assuming right censored extinction events were fitted to examine the relationship between extinction times (e or not .DT/GT) and their relationships with ecological variables. HP uses all models in a regression hierarchy to distinguish those variables that have high independent correlations with the dependent variable. At all steps, results from the survival models, GLM and HP were compared to help interpretation. Although most extinction events occurred within the range 10-10,000 generations (> 80% of scenarios), the variance was high and the distribution of extinction times was non-normal. Thus, graphical results are presented using logarithmic scales and extinction times were log-transformed (Neperian logarithm) in all statistical analysis. Other dependent and independent variables were transformed using usual functions to achieve the best linearity (transformations provided in results). The aim of these analyses was not to test the significativity of regressions but rather to examine the direction and strength of relationships between ecological variables and viabilities [As a second step, GLM and HP were applied to those scenarios for which extinction times were available  to examine variations in the ratios of extinction times .Click here for fileEffect of fragmentation on extinction times: regime of spatially correlated perturbation.Click here for fileEffect of fragmentation on extinction times: complementary results .Click here for fileFitness and population size reductions: an illustration.Click here for file


Anopheles nili group of mosquitoes includes important vectors of human malaria in equatorial forest and humid savannah regions of sub-Saharan Africa. However, it remains largely understudied, and data on its populations’ bionomics and genetic structure are crucially lacking. Here, we used a combination of nuclear  and mitochondrial DNA markers to explore and compare the level of genetic polymorphism and divergence among populations and species of the group in the savannah and forested areas of Cameroon, Central Africa.The An. nili group. However, they revealed high genetic heterogeneity within An. nili s.s. in deep equatorial forest environment. Nuclear markers showed the species to be composed of five highly divergent genetic lineages that differed by 1.8 to 12.9% of their Internal Transcribed Spacer 2 (ITS2) sequences, implying approximate divergence time of 0.82 to 5.86 million years. However, mitochondrial data only detected three major subdivisions, suggesting different evolutionary histories of the markers.All the markers provided support for the current classification within the An. nili s.s. in the deep equatorial forest environment of South Cameroon, reflecting a complex demographic history for this major vector of malaria in this environment. These preliminary results should be complemented by further studies which will shed light on the distribution, epidemiological importance and evolutionary history of this species group in the African rainforest, providing opportunities for in-depth comparative studies of local adaptation and speciation in major African malaria vectors.This study enlightened additional cryptic genetic diversity within  Anopheles mosquitoes can and do transmit Plasmodium, the malignant agent of the disease, simultaneously or replace each other seasonally insuring year-round transmission Anopheles nili group of malaria vectors.In the equatorial forest regions of Africa, malaria remains endemic despite significant efforts in treatment of the disease and vector control An. nili group includes four closely related species namely An. nili sensu stricto , An. somalicus, An. carnevalei and An. ovengensis which can be identified through slight morphological diagnostic characters present at the larval and/or adult stages Anopheles nili s.s. is widespread and is found throughout sub-Saharan Africa. It is highly anthropophilic, biting man and resting indoors as well as outdoors Anopheles somalicus is not involved in malaria transmission because of its preference for non-human blood and exophilic habits An. nili s.s. is the major malaria vector of the group and emphasized the exophagic behavior of An. ovengensis and An. carnevaleiAn. ovengensis can reach 4.1% (n = 74), which is higher than that of An. gambiae in the same area  when compared to other populations from humid savannah of Central and West Africa in situ hybridization (FISH) mapping of the microsatellite loci on a recently developed cytogenetic map for An. niliAn. nili natural populations in equatorial Africa.A recent study using 11 microsatellite markers demonstrated a significant genetic differentiation of the forest An. nili group in South Cameroon, where all four members of the group are known to occur An. nili s.s., reflecting a complex demographic history. We advocate for further evaluation of the impact of this genetic heterogeneity and its relevance for malaria epidemiology and control.Here, we used nuclear (i.e. microsatellites and rDNA) and mitochondrial DNA markers to further explore the genetic structure of members of the This study received the approval of the National Ethics Committee of Cameroon (Ref: FWA IRB0001954). No other specific permits were required. No location was privately-owned or protected and field studies did not involve endangered or protected species. Consent was obtained from all volunteers participating in the study, and they were monitored for signs of fever and treated for all diagnosed malaria cases.An. nili s.l. mosquitoes were first sorted using morphological diagnostic characters Mosquitoes were collected from 2007 to 2009 in eight riverside villages situated in different ecological settings in Cameroon including Afan-Essokyé, Ako, Ekelemba, Kentzou, Moloundou, Mbébé, Nkolbisson, and Nyabessan , Table 1An. nili s.s.An. carnevalei, An. ovengensis and An. somalicus DNA. Genotypes were determined for individual mosquitoes as previously described Eleven microsatellite loci developed in Heterozygosity tests were implemented in BOTTLENECK V1.0.02 et al.Fst, monomorphic loci were excluded from the analysis and significance tests were conducted using a randomization approach as implemented in FSTAT.Bayesian clustering analysis was implemented in STRUCTURE V2.2 Nm) per generation between populations was estimated from pairwise FstFst/(1-Fst) on geographic distance, assuming migration in one direction  The number of putative migrants  and NADH deshydrogenase subunit IV (ND4) of mitochondrial DNA.Sequencing was performed using the primers set and protocol described in Ndo Drosophila melanogaster and D. virilis groups Basic sequence polymorphism statistics and various population parameters were computed using DnaSP V5.10.01 An. moucheti sequences (The Neighbour-Joining (NJ) method An. nili s.s., although some specimens from Moloundou and Ekelemba failed to amplify even after repeated attempts, possibly due to poor DNA quality. All individuals from Nyabessan, Afan-Essokyé and Mbébé were An. ovengensis, An. carnevalei and An. somalicus respectively, but An. nili s.s is also known to be present in the two later localities An. somalicus on human baits owing to its zoophilic behavior. Similarly, attempts to collect An. nili s.s. on human baits in Kentzou were unsuccessful suggesting possible zoophilic behavior of the species in this locality.All mosquitoes from Ako, Ekelemba, Kentzou, Moloundou and Nkolbisson were morphologically and molecularly identified as An. nili s.s. populations  successfully amplified in Kentzou (9 polymorphic) and Moloundou (9 polymorphic), falling down to 8 (73%) in Ekelemba (with only 5 loci being polymorphic). Similarly, only 9 (82%) loci successfully amplified An. carnevalei and An. ovengensis DNA, among which 7 and 6 respectively were polymorphic. In An. somalicus, 8 (73%) loci were scored successfully, with only 5 showing more than one allele , whereas polymorphism was much lower in the other samples .A total of 363 individual mosquitoes were genotyped and there were marked differences in amplification profiles and polymorphism of loci among species, as well as between ulations . All 11 e allele . These dAn. nili s.s., the sample from Moloundou had the highest number of deviations. In fact, all 9 polymorphic loci were out of HWE, associated with positive Fis values suggesting population substructure, although null alleles could also account for some deviation. By contrast, no more than 2 significant deviations were observed in all other An. nili s.s. samples. The number of significant deviations from HWE detected in the other members of the group varied from 1 (out of 5 polymorphic loci) in An. somalicus to 3 (out of 7 polymorphic loci) in An. carnevalei  tests out of 63 were found out of HWE (P<0.001) after Bonferroni correction for multiple testing . Within rnevalei . The frernevalei , suggestAn. nili s.s. population resulted in 5 (2.6%) significant values (P<0.05) out of 192 comparisons after correction by the Bonferroni procedure. All these deviations were detected in Moloundou where all polymorphic loci were found out of HWE, and involved loci 1D80 and F41, 2Ateta and F41, B115 and F41, 2Ateta and 1A27, and B115 and F56. Because all these loci are located on different chromosomal arms An. carnevalei, An. ovengensis and An. somalicus populations.Exact tests for LD within each An. nili group. The optimal number of clusters detected based on the method of Evano et al.Anopheles carnevalei, An. somalicus and An. ovengensis samples each formed a distinct and homogeneous genetic cluster, while five clusters were defined within An. nili s.s. Specimens sampled in savannah (Ako) and degraded forest (Nkolbisson) areas were merged together into a single genetic cluster, whereas four clusters were detected among populations sampled within the evergreen forest block: one in Kentzou, one in Ekelemba and two in Moloundou, namely ‘Moloundou A’ and ‘Moloundou B’ in subsequent analyses.The Bayesian clustering analysis using the software STRUCTURE was implemented in the expectation that the genotypes would segregate into four main clusters corresponding to the four previously recognized species within the Fst estimate across all loci was 0.373 and was highly significant (P<0.0001). Mean pairwise Fst over all loci varied from 0.201 to 0.659 (P<0.0001) with corresponding Nm values in the range of 0.14 to 1.16, suggesting very restricted gene flow between species  and correspondingly low migration index (Nm: 0.28–1.07) were estimated between populations, except between Ako and Nkolbisson which belong to the same genetic cluster  and there was no indication of recent population contraction in any of the populations tested (data not shown).No significant correlation was detected between genetic and geographic distances (Rπ = 0) was detected within each geographical population, except in Moloundou where two highly divergent sequence variants were observed (π = 0.034), corresponding to the Moloundou A and Moloundou B microsatellites clusters. Haplotypes detected in An. carnevalei, An. ovengensis and An. somalicus populations, and in An. nili s.s. from Ako and Nkolbisson were identical to those published by Kengne et al.An. nili s.s., AJ429052 for An. ovengensis, AJ438690 for An. somalicus and AJ429051 for An. carnevalei). The four remaining An. nili s.s. haplotypes were new and found exclusively in deep forest populations (GenBank accession numbers: KC189970– KC189973). They differed from one another, and from the deposited An. nili s.s. haplotype by 13 to 16 fixed nucleotide substitutions and many indels. Genetic distances between species’ haplotypes ranged from 0.3 to 5%. Similar genetic distances were also estimated between An. nili s.s. haplotypes, ranging from 0.5 to 5.9%  and the alignment length, including insertions/deletions (indels), was 382 bp . No sequ to 5.9% .An. carnevalei, 503 bp for An. ovengensis, 513 bp for An. somalicus, 451 bp for An. nili s.s. from Ako and Nkolbisson, 445 bp for An. nili s.s. from Kentzou and Moloundou A and 480 bp for sequences from Ekelemba and Moloundou B  An. nili s.s. populations (GenBank accession numbers: KC189966– KC189969). They differed from one another, and from the deposited An. nili s.s. haplotype by 30 to 95 nucleotide substitutions and many indels, suggesting high genetic divergence  An. nili s.s. from savannah/degraded forest (Ako and Nkolbisson), 2) An. nili s.s. from deep forest group 1 (Kentzou and Moloundou A) and 3) An. nili s.s. from deep forest group 2 (Ekelemba and Moloundou B)   and 3. ICOII and ND4, respectively. Variations among sequences only included base substitutions. Absence of indels and heterozygous sites therefore suggests that there was no nuclear transposed copy.Mitochondrial DNA sequences were determined for a minimum of 81 individual mosquitoes and summary statistics for genetic polymorphism are given in An. nili s.s. populations was confirmed. All the phylogenetic trees were congruent, showing An. nili group as monophyletic, with six clades: 1-An. carnevalei, 2-An. ovengensis, 3-An. somalicus, 4-An. nili s.s. from Ako and Nkolbssion 5-An. nili s.s. from Kentzou and Mouloundou A (deep forest group 1), and 6-An. nili s.s. from Ekelemba and Moloundou B (deep forest group 2)   and 5. HCOII and ND4 genes, respectively (COII divergence time was estimated and found to vary approximately between 0.2 and 3 myr. All new mtDNA sequences have been deposited to GenBank (Accession numbers: KC189974 – KC190023).Sequence divergence between consensus haplotypes from each population ranged from 0.4 to 6.2% and from 0.5 to 11.2% for ectively . BecauseCOII, ND4) were used to assess the level of genetic variability and differentiation among species of the An. nili group, and between An. nili s.s. populations from different ecological settings in South Cameroon. All the markers provided support for the current classification in the group and revealed further cryptic genetic diversity within the major vector An. nili s.s., despite morphological similarities.Genotypes at 11 microsatellite loci and sequence variation in two rDNA regions  and two mtDNA genes  and could be used for population genetic studies in these species.In the present study, 11 An. nili s.s., by detecting highly divergent lineages in the deep equatorial forest, despite morphological similarities of all specimens analyzed. This genetic substructure was detected by a range of molecular markers with different evolutionary dynamics, providing strong support for phylogenetic inferences. Indeed, rDNA haplotypes found in these populations differed from one another, and from the An. nili s.s. haplotypes from savannah/degraded forest areas by a large number of fixed mutations and indels leading to genetic distance estimates 4 to 8 fold higher than those commonly reported among cryptic Anopheles species An. nili group suggests that these taxa might actually belong to distinct species groups. However, these results generated using a small sample size and on a limited sampling range should be considered as preliminary. Extending the study area throughout the equatorial forest block of Central Africa will allow improving knowledge on the taxonomic status and the geographic distribution of forest An. nili s.l. populations, their ecology and behaviour, and their epidemiological role in malaria parasite transmission.Previously unrecognized cryptic genetic diversity not correlated to geographical distance between sampling sites was evidenced within the nominal species of the group An. nili s.s. which is replaced in this area by the other members of the group, namely An. carnevalei and An. ovengensisAnopheles nili s.s. is rather more frequent at the savannah/degraded forest ecotone, where it is characterized by shallow population structure with a weak effect of geographical distance on genetic differentiation An. nili s.s. in this environment probably restricts opportunities for genetic exchange between geographically isolated populations, prompting for genetic divergence under the action of increased genetic drift An. nili provided further support for a major role of genetic drift in shaping the population genetic structure of An. nili in Central Africa, reflected in genome-wide differentiation patterns An. nili in search for signatures of selection in ecologically-relevant genes or genomic regions. Indeed, original information gained on the genetic structure, gene flow and species diversity within this major group of malaria vectors can further be used to explore global processes such as the genetics of ecological adaptation, speciation and susceptibility to Plasmodium, within a comparative framework that will use and complement information available for other major human malaria vectors such as mosquitoes from the An. gambiae complex and the An. funestus group.The evergreen forest block of Central Africa is known to be of low overall quality for the development of P. falciparum along with other Plasmodium species in great apes in Central African forest region P. falciparum acquired the parasite from humans (or from other primates), nor the pathogenicity of these infections in gorillas, the continuously increasing contact between humans and primate populations, mostly due to logging and deforestation, increases opportunities for transmission of new pathogens from primates to humans and vice versa An. nili group might contribute to the emergence of zoonosis in humans needs to be assessed.Moreover, the recent findings of circulation of Figure S1An. nili s.l. haplotypes. AN: An. nili s.s.; AO: An. ovengensis; AC: An. carnevalei; AS: An. somalicus. AK: Ako; NK: Nkolbisson; KT: Kentzou; MOA: Moloundou A; MOB: Moloundou B; EK: Ekelemba; AE: Afan-Essokyé; NY: Nyabessan; MB: Mbébé.Alignment of D3 sequences of (PDF)Click here for additional data file.Figure S2Alignment of ITS2 sequences of An. nili s.l. haplotypes. AN: An. nili s.s.; AO: An. ovengensis; AC: An. carnevalei; AS: An. somalicus. AK: Ako; NK: Nkolbisson; KT: Kentzou; MOA: Moloundou A; MOB: Moloundou B; EK: Ekelemba; AE: Afan-Essokyé; NY: Nyabessan; MB: Mbébé.(PDF)Click here for additional data file.Table S1Estimates of null alleles frequencies per locus and per geographical population of An. nili s.l. from Cameroon.(DOCX)Click here for additional data file.Table S2Pairwise genetic distance estimates between An. nili s.l. D3  and ITS2  haplotypes from Cameroon.(DOCX)Click here for additional data file.Table S3Pairwise genetic distance estimates between An. nili s.l. ND4  and COII  haplotypes from Cameroon.(DOCX)Click here for additional data file.