Pheidole megacephala is considered one of the world's most harmful invasive species.Biological invasions are recognized as a major cause of biodiversity decline and have considerable impact on the economy and human health. The African big-headed ant P. megacephala, one inhabiting urban areas and the other rainforests. Urban populations belong to the same phylogenetic group than those introduced in Australia and in other parts of the world. Behavioural analyses show that the eight populations sampled make up four mutually aggressive supercolonies. The maximum distance between nests from the same supercolony was 49 km and the closest distance between two nests belonging to two different supercolonies was 46 m. The genetic data and chemical analyses confirmed the behavioural tests as all of the nests were correctly assigned to their supercolony. Genetic diversity appears significantly greater in Africa than in introduced populations in Australia; by contrast, urban and Australian populations are characterized by a higher chemical diversity than rainforest ones.To better understand its ecological and demographic features, we combined behavioural (aggression tests), chemical  and genetic  data obtained for eight populations in Cameroon. Molecular data revealed two cryptic species of P. megacephala in Cameroon adopt a unicolonial social structure, like invasive populations in Australia. However, the size of the supercolonies appears several orders of magnitude smaller in Africa. This implies competition between African supercolonies and explains why they persist over evolutionary time scales.Overall, our study shows that populations of  The recent increase in human activity and commerce has, intentionally or unintentionally, greatly contributed to the dispersal of exotic species and their introduction into new habitats. In most cases, introduced species do not survive and, among those introduced species that do, many do not cause problems of any sort. However, some species' overwhelming invasive success upsets the balance in the invaded ecosystems and constitutes an important threat to biodiversity i.e. the Allee effect Biological invaders represent a paradox for evolutionary biologists http://antbase.org/ – December 2011) have successfully established populations outside their native range continuum, they are unicolonial and consist of a single, huge supercolony.Ants are among the most ecologically successful groups of organisms Linepithema humileWasmannia auropunctataThe evolutionary forces leading to unicoloniality may be diverse Pheidole megacephala forms a complex including at least ten subspecies, all described in Sub-Saharan Africa P. megacephala is dominant in the many areas it has invaded P. megacephala have been widely documented in the Pacific The African big-headed ant P. megacephala in Cameroon, west Central Africa  that evolved independently from a very long time. These two phylogenetic groups exactly match with the two ecological zones studied, P. megacephala var. 1 corresponding to the specimens collected in urban areas and P. megacephala var. 2 to those sampled in rainforest. The position of P. sexspinosa and P. xerophila on the phylogenetic tree, two sister species of P. megacephalaP. megacephala var. 1 (i.e. urban samples) and of Australian specimens belong to the same phylogenetic group. They are also very close to those isolated from samples collected in Mauritius (http://www.antweb.org/ and http://www.boldsystems.org/). Furthermore, the Basic Local Alignment Search Tool (BLAST) shows high homologies between sequences obtained from P. megacephala var. 1 and sequences published for samples collected in Seychelles , and BLAST for P. megacephala var. 2 gave the highest scores with samples collected in Gabon and Comoros (http://www.antweb.org/ and http://www.boldsystems.org/).The phylogenetic tree reconstructed on the basis of mtDNA sequences  reveals Minor were lower in P. megacephala var. 1 (urban) than in P. megacephala var. 2 (rainforest) populations . Morphological observations at electron microscope also show that minor of P. megacephala var. 1 have shorter spines on the propodeum than minor of P. megacephala var. 2 . Behavioural assays testing the responses of two major or two minor from different nests yielded consistent results . By contrast, encounters between individuals from different populations resulted in aggressive responses in 60.10% of the 386 trials   and 3.38% in YS ; in populations of P. megacephala var. 2, 9.09% of aggressive interactions was observed in AK , 12.94% in CA  and 7.14% in CB   and YN  of P. megacephala var. 1 or CC  of P. megacephala var. 2  . Agonist8±0.116) . Howevera var. 2 .P. megacephala. Two supercolonies, SC-1 and SC-2, cohabit in population YU where workers displayed aggressive behaviours in roughly one test out of two  and 52 in P. megacephala var. 2 populations (i.e. rainforest); 23 alleles were found in both species. Measurements of genetic diversity computed for P. megacephala var. 2  Ar[22] = 4.233±0.306 and genetic diversity H = 0.609±0.033) were higher than for P. megacephala var. 1 . The inbreeding coefficient computed for P. megacephala var. 2 (f = 0.097±0.053) was lower than for P. megacephala var. 1 (f = 0.120±0.067) (p = 0.011); however, they did not differ by their number of alleles, their genetic diversity or their inbreeding coefficient .One hundred and eight alleles over eight microsatellites loci were found across all of the eight populations sampled. Thirty-three alleles were detected exclusively in 0±0.067) . The allP. megacephala var. 1 and P. megacephala var. 2, respectively; P. megacephala var. 1 and 0.181±0.097  for P. megacephala var. 2). Accordingly, genetic differentiation estimated by the Fst index confirmed significant levels of population structure between supercolonies of a same phylogenetic group  and low gene flows between supercolonies belonging to different phylogenetic groups (Fst varied from 0.216 (Nm = 0.91) to 0.319 (Nm = 0.53)). In line with these results, Bayesian cluster analyses of the microsatellite data set detected genetic substructures even without including the sampling locations of the individuals. The resulting posterior probabilities were highly concordant between the replicated runs, with the highest average value indicating the most likely number of population groups, K. Our whole data yielded a best estimate of K = 2  was statistically lower than differentiation between nests collected from the Campo Forest Reserve  . The maximum distance between nests in the same supercolony varied from 1 to 49 km; on the other hand, the minimum distance between nests belonging to two different supercolonies was 46 m  where α is the frequency of sib-mating Estimates of genetic diversity, inbreeding and relatedness coefficients within supercolonies are given in P. megacephala var. 1) with those previously described for Australian populations P. megacephala var. 1: mean ± se = 0.933±0.009; Australia: 0.938±0.002; Mann-Whitney U-test, p = 0.260). Conversely, diversity of profiles isolated from individuals collected in rainforest  was significantly lower than that observed in Australian populations . Ionisation mass spectrometry revealed the presence of lipids including linear alkanes, methyl-branched alkanes and alkenes Cuticular lipids yielded 47 peaks ranging in size from C19 to C40. Electronic and chemical ionizations did not show qualitative nor quantitative differences of CHC profiles isolated from urban populations  allowed 71.7% of the Cameroonian nests to be accurately classified in their phylogenetic group  . The firI did not differ between nests, nor between supercolonies . The similarity in chemical profiles estimated by the Euclidean distances E was different between the two ecological zones. The chemical differentiation between nests was lower in P. megacephala var. 1 (urban) than in P. megacephala var. 2 (rainforest) populations  and spatial distances . In contrast, chemical distances had no influence on aggressiveness . In P. megacephala var. 2, aggressiveness was not associated with any of the three distances estimated . Genetic differentiation, CHC profile dissimilarity and spatial distances were all positively correlated , except in P. megacephala var. 2 where chemical and spatial distances were not associated . For both cryptic species, the diversity of cuticular compounds measured by the Nei index I was associated with neither the allelic richness , nor with the genetic diversity (p>0.366).In populations of STF) of which 0.352 (RTF) was due to the among-cryptic species component and 0.200 (SRF) was due to the among-population within-cryptic species component , the total genetic differentiation among populations was 0.482 . Second, mating takes place in the nests between related individuals (r = 0.694±0.038 between a queen and her mate), and results in a relatively high level of inbreeding. This reproductive pattern and the significant correlation between the genetic and geographical distances indicate that the main mode of colony reproduction occurs via dependent foundation, whereby a queen and a few workers leave the natal nests to start a new colony nearby. These breeding and dispersal strategies contribute maintaining genetic homogeneity in each supercolony, and simultaneously generate high levels of inbreeding. In Hymenoptera, a direct cost of inbreeding is the production of homozygous diploid and sterile males. However, in spite of the high inbreeding coefficient, no such diploid males were found in our sample . As suggested for other Hymenoptera Our results show that two cryptic species of P. megacephala is dominant in the human-disturbed zone and could negatively affect native ant fauna  and Odontomachus troglodytes and Myrmicaria opaciventris. In contrast to Australian populations, P. megacephala in Cameroon does not form a huge, single unicolonial population spreading over thousands of kilometres, but a mosaic of mutually aggressive supercolonies. Such a difference between populations has been documented for two other invasive ants between invaded and native ranges. Supercolonies of the Argentine ant Linepithema humile have been found spanning 6000 kilometres across Europe Wasmannia auropunctata has been reported in New Caledonia The two cryptic species show life history traits that are characteristic of introduced populations, both in Yaoundé where preliminary field observations indicate that P. megacephala var. 1 populations; present study), South Africa F>14.151, p<0.001; RTF = 0.340) is attributed to divergence among countries, 9% (SRF = 0.135) to populational differences within region, and 57% (STF = 0.429) to individual differences within populations  and Costus afer Ker-Gawler (Costaceae)) where they tended aphids. Chemical differentiation between urban and rainforest populations may therefore be induced by environmental conditions, but it may also stem from phylogenetic differences. Whether these chemical divergences lead to or result from reproductive isolation could remain crucial for understanding the evolutionary history of the two cryptic species.Cuticular lipids present on the surface of the cuticle of ants mediate nestmate recognition between individuals P. megacephala forms unicolonial populations in Cameroon, both in natural habitats and urban areas. It has been suggested that unicoloniality is a key factor responsible for the ecological dominance of ants. Many invasive ant species  due notably to a high kinship among breeders. Our study emphasizes a fundamental distinction between African and introduced populations in Australia in terms of nest structure. In Australia, populations are organized into a single supercolony spreading over 3000 km P. megacephala forms a mosaic of mutually unrelated supercolonies within which individuals are related. In this context, competition (e.g. conquering new territories or food sources) between supercolonies favours reproductive altruism within supercolonies Our study shows that the African big-headed ant P. megacephala var. 1 share behavioural and evolutionary traits that are characteristic of introduced of P. megacephala populations in Australia. Moreover, field observations in Cameroon indicate that this species largely dominates other native ants in urban habitats. The ERH may act in synergy with other factors facilitating the introduction and naturalization of P. megacephala. First, propagules composed of a queen and a few workers may experience high survivorship and rapid growth. A study conducted in Australia showed that the invasion by P. megacephala is consistent with the introduction of only six haploid genomes P. megacephala combines a high aggressiveness towards other ant species with highly efficient predatory capacities. In invaded ranges, this species captures many different types of prey and is more efficient than native ants in preying on termites Wasmannia auropunctata, one of the most invasive and destructive pests in the world i) Its clonal reproductive system, whereby males and females are clonally produced ii) Its unicolonial population structure provides numerical and competitive advantages over multicolonial ants iii) Its proximity to humans allows it access to profoundly modified habitats that may, over time, increase its ability to become invasive W. auropunctata is the human activities Solenopsis invictaAnoplolepis gracilipesLinepithema humileParatrechina longicornisLasius neglectusOne commonly proposed explanation for invasive success is the enemy release hypothesis (ERH), which states that the lack of natural predators and competitors in an exotic environment favours the successful establishment and spread of species outside of their native range P. megacephala. These species are restricted to certain ecological zones: P. megacephala var. 1 is found in urban areas and P. megacephala var. 2 in rainforests. Urban populations are phylogenetically close to populations in Australia, Mauritius, South Africa and Madagascar. Rainforest populations have high homologies with populations in Gabon, Madagascar and Comoros. Populations of both cryptic species are organised into a mosaic of supercolonies and adopt a unicolonial structure. Further analyses should decipher the cryptic species complex of P. megacephala and to identify the sub-species  particularly suited to adapt to and invade new environments. The ecological and evolutionary strategies adopted in native areas should be considered as a first phase in the expansion of and invasion by this species. More attention should also be placed on how anthropogenic activities affect the biotic and abiotic conditions of the environment, and to what extent invasional meltdown processes P. megacephala will be to investigate the routes and means of introduction to prevent new invasions.In conclusion, our study shows that Cameroon hosts two cryptic, reproductively isolated species of the big-headed ant i.e. an aggregation of workers, brood, and/or queens in a single place) were sampled from eight populations in Cameroon in February 2009  and Costus afer Ker-Gawler (Costaceae), or, more rarely, in dead branches or termite mounds. Different species of the genus Pheidole and species belonging to other genera  were sympatric with P. megacephala. In contrast, the vegetation in the Yaoundé urban area belongs to the semi-deciduous forest type, but currently is a mixture of forest relics on hill summits and garden crops (urban agriculture) along river and inland valleys. In this zone, Pheidole megacephala is opportunist; it nests under stones or dead branches, and is found in gardens, kitchen-gardens and dwellings. Only a few other species lived in the proximity of the P. megacephala nests, namely Odontomachus troglodytes, Monomorium bicolor, Crematogaster clariventris, Lepisiota sp. and Myrmicaria opaciventris. Samples of workers and, when available, queens and males were collected from each nest and used for the behavioural assays. Pheidole megacephala has a pronounced worker caste polymorphism: major workers are considerably larger than minor and have disproportionately large heads. The minor are more active than the major, notably in exploring the ground and in alerting nestmates of the presence of intruders . A scanning electron microscope  was used to study the shape and surface details of two minor and one major of each ecological zone. To this purpose, samples were dehydrated in ethanol, critical point air-dried and coated with gold using an ion sputter .Morphological characteristics of specimens were assessed using a stereomicroscope and a scanning electron microscope. The maximum head width (across compound eyes) of 12 minors per population was determined at the nearest 0.005 mm by using a stereomicroscope  at a magnification of 40×. Head width is an accurate estimate of size, commonly used as a dependent variable in studies of allometry minor according to previously described protocols minor, were conducted for each pair of nests and the highest score was averaged across trials.Behavioural assays were conducted by confronting minor workers. Intraspecific aggressiveness was quantified between pairs of major from different nests to confirm that workers from both castes did not behave differently when confronted with non-nestmates.Interactions between individuals belonging to different nests, whether from the same or different sites, were considered as experimental assays. Interactions between individuals from the same nests were considered as control assays. In addition, we set up encounters between minor per nest and placed in 1 ml of cyclohexane during 5 min; the cyclohexane was evaporated and the extract was re-diluted in 5 µl of cyclohexane. We injected 1 µl of the extract into a Thermo Polaris Q™ electron impact ion trap mass spectrometer interfaced to a Thermo Trace GC Ultra™ gas chromatograph . Qualitative and quantitative data were obtained by running the Thermo Xcalibur™ data system . Cuticular profiles obtained for three nests of the population YM did not present clear patterns, and were therefore discarded from the analyses. We used the relative amount (i.e. area) of the peaks corresponding to cuticular lipids as a quantitative measurement for each profile. Compounds with a relative area of less than 2% in all profiles were excluded from the data. Cuticular lipids were identified by analysing their mass spectra produced by both electron impact and chemical ionization with methane.Protocols for the extraction of cuticular hydrocarbons and gas-chromatography, mass-spectrometry (GC-MS) analyses are described elsewhere I and Euclidian distances E, respectively We estimated the chemical diversity and similarity of cuticular hydrocarbon profiles using the Nei index DNA was isolated through phenol/chloroform extractions and ethanol precipitation by following standard protocols 5′-ATTCAACCAATCATAAAGATATTGG-3′; LepR1: 5′-TAAACTTCTGGATGTCCAAAAAATCA-3′). PCRs were carried out in 25 µl reaction volumes containing 14 µl of distilled water, 5 µl of genomic DNA, 2.5 µl of Qiagen 10× buffer , 2 µl 2.5 mM of dNTP, 0.625 µl 100 mM of forward and reverse primers and 0.250 µl of Qiagen Taq DNA polymerase. The PCR thermocycling profile consisted of one cycle of 2 min at 94°C, five cycles of 40 sec at 94°C, 40 sec at 45°C, and 1 min at 72°C, followed by 36 cycles of 40 sec at 94°C, 40 sec at 51°C, and 1 min at 72°C, with a final step of 5 min at 72°C. Products were visualized on a 2% agarose gel and samples containing clean single bands were sequenced using BigDye v3.1 on a 48-capillary 3730 DNA Analyzer (Applied Biosystems). The forward and reverse sequences for each sample were inspected and aligned using CodonCode Aligner 3.7.1.1 . Programs freely available from the phylogeny.fr web server uscle for multiple alignment blocks for alignment curation hyML for phylogeny reeDyn for tree drawing Pheidole sexspinosa and P. xerophila were used as sister species and those of Aphaenogaster senilis as outgroup The phylogenetic relationships between collected samples were inferred from a 650 base-pair (bp) region near the 5′ terminus of the CO1 gene. COI sequences were generated from a subset of the individuals sampled  using barcoding protocols eneMapper software (Applied Biosystems) and used to construct a multi-loci genotype for each individual.Genetic diversity and population genetic structure were investigated by genotyping eight statistically independent microsatellite loci i.e. the number of alleles, allelic richness, expected and observed heterozygosities) and fixation indices were estimated using Fstat 2.9.3 elatedness 5.08 minor was estimated by including all individuals as reference. The effect of inbreeding on relatedness was corrected by using the estimator r* minorGenetic diversity parameters (K) reflecting the population genetic sub-structure of our data set was characterised with the Bayesian model-based clustering method implemented in Structure v2.3.2 K from K = 2 to K = 64. The ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values was used to find the most likely value of KThe most likely number of genetic clusters .Correlations between the geographical origin, genetics, chemical composition and behaviour of i.e. collection sites) nesting within the two ecological zones (i.e. rainforest and human-modified habitats) were conducted to quantify the distribution of genetic and chemical variations between populations, zones and populations within zones. The significance of each variance component was tested using a random permutation test (999 permutations) using GenAlEx 6 Hierarchical analyses of molecular variance (AMOVA) of the eight populations  and P. megacephala var. 2 (B) in lateral views. Magnification and scale bar are indicated for each scanning electron microscopic image.(TIF)Click here for additional data file.Figure S2Lateral views of the propodeum showing shorter spines in P. megacephala var 1. than in P. megacephala var. 2 (B). Magnification and scale bar are indicated for each scanning electron microscopic image.(TIF)Click here for additional data file.Figure S3Details in lateral views of hairs present on the petiole. Magnification and scale bar are indicated for each scanning electron microscopic image. The terminal part of the hairs forms a point in P. megacephala var. 1 (A) and a brush in P. megacephala var. 2 (B).(TIF)Click here for additional data file.


Anomalocardia brasiliana were tested for mutual genetic homogeneity, using data from 123 sequences of the mtDNA cytochrome oxidase c subunit I gene. A total of 36 haplotypes were identified, those shared being H3  and both H5 and H9 (Prainha and Acupe). Haplotype diversity values were high, except for the Camurupim population, whereas nucleotide values were low in all the populations, except for that of Acupe. Only the Prainha population showed a deviation from neutrality and the SSD test did not reject the demographic expansion hypothesis. Fst values showed that the Prainha and Acupe populations represent a single stock, whereas in both the Canela Island and Camurupim stocks, population structures are different and independent. The observed structure at Canela Island may be due to the geographic distance between this population and the remainder. The Camurupim population does not share any haplotype with the remaining populations in northeastern Brazil. The apparent isolation could be due to the rocky barrier located facing the mouth of the Mamanguape River. The results highlight the importance of wide-scale studies to identify and conserve local genetic diversity, especially where migration is restricted.Four Brazilian populations of  Anomalocardia brasiliana  is a venerid clam that occurs in the West Indies and South America from Surinam to Uruguay . Higher levels of dispersal are more common in species with this kind of larval phase than in those with direct development , indicating the occurrence of wide larval dispersion, in spite of the direction of ocean currents. Furthermore, five populations of Crassostrea gasar (= C. brasiliana) from Pará  to Piauí states  were found to belong to a single large stock (unpublished data). However, studies on Mytella guyanensis, also with the use of partial COI gene sequences, showed a clear structure level for the Camurupim population (Paraíba state) when compared with populations from the North and East of Brazil, thus implying that local environmental factors may be influencing larval dispersal (unpublished data). The Camurupim estuary is surrounded by reefs , from four populations of by reefs , these aAnomalocardia brasiliana, using COI partial sequences of mitochondrial DNA. The COI gene has already been used to study mollusk phylogeography , in Pará State, northern Brazil and from three other localities in northeastern Brazil: Prainha , in Ceará State; Camurupim , in Paraíba State; and Acupe , in Bahia State  and one of Lirophora mariae (GenBank DQ458483). The evaluation of statistical confidence was based on bootstrapping with 1,000 pseudo-replicates for ML  tree were performed with the PHYML version 2.4.4 , with gas for ML . The cris for ML  was usedDnaSP version 4.10 . This teh; Fs test , 14.8% (cytosine), 23.3% (adenine) and 20.9% (guanine). There were no indels and no termination codons. There was no evidence of saturation. Amongst the four populations studied, a total of 36 haplotypes were identified for the COI gene.The COI amplicons were approximately 650 bp long. One hundred and twenty three unambiguously aligned sequences from four populations were obtained for 580 bp of the COI coding sequence from A. brasiliana haplotypes and the Mercenaria mercenaria (GenBank EU360429) and Lirophora mariae (GenBank DQ458483) sequences used to generate the ML tree were: base frequencies , gamma distribution shape parameter (α = 1.3625) and substitution model rate matrix , the proportion of invariable sites being 0.6548. The rooted ML tree showed weak bootstrap values not supporting geographical haplotype groups , gamma distribution shape parameter (α = 0.4630) and substitution model rate matrix , the proportion of invariable sites being 0.8999. The distance matrix based on the General Time-Reversible model , from the same geographic region, revealed significant differences between the substitutions of the two species from Canela Island . However, most nucleotide mutations of A. brasiliana sequences from Canela Island were synonymous (N = 25), and only those found in haplotypes H32 and H33 were exclusive for that population  values showed that the Prainha and Acupe populations were not significantly different from one another . Fst values of the other populations ranged from 0.366 (Camurupim and Acupe) to 0.813 (Canela Island and Camurupim), all being highly significant , geographic distances and the direction of marine currents.The larval stage in le range , and theMytella guyanensis  have already been observed (unpublished data).On considering the latitude of forking in the South-Equatorial marine current  criteria, the low haplotype and nucleotide diversities found in this population suggest that it may have undergone a recent bottleneck or founder event.Moreover, in the The present isolation of the Camurupim population may be partially explained by the presence of a rocky barrier located at the mouth of the Mamanguape River see . BeachroA. brasiliana larvae to become homogenous throughout the northern Brazilian coast. On the other hand, it could be expected that the pronounced coastal environmental differences, due to the enormous discharge of fresh water and sediments from Amazonian rivers during the rainy season, could put pressure on the population, selecting for particular alleles. However, this does not seem to be the case in COI, in spite of the great differences observed between nucleotide sequences from Canela Island, where few differences were observed in the resulting amino acid sequences. Moreover, salinity on Canela Island ranged from 20 to 40 between August 2001 and December 2003 , being similar to that observed in other studies on this species  generates the North Brazilian current . These results indicate that this region should be studied in detail in order to verify whether this pattern of isolation exists for other aquatic species from the Mamanguape estuary. The COI molecular studies of Macoma balthica in Europe, that appears to have originated during the Pleistocene, some of the current populations appearing to be connected by gene flow whereas others are isolated. On the other hand, Scapharca roughtonii from Korea, also found differences between the population from Jinhae and six other Korean populations. The authors state that the presence of a narrow avenue for exchange with offshore waters affords limited dispersal. According to According to The observed partial isolation of the Camurupim population highlights the importance of wide-ranging conservation programs involving several different regions, in order to preserve local genetic diversity, especially if migration is partially or completely restricted.


Management of wild peppers in Mexico has occurred for a long time without clear phenotypic signs of domestication. However, pre-domestication management could have implications for the population's genetic richness. To test this hypothesis we analysed 27 wild (W), let standing (LS) and cultivated (C) populations, plus 7 samples from local markets (LM), with nine polymorphic microsatellite markers. Two hundred and fifty two alleles were identified, averaging 28 per locus. Allele number was higher in W, and 15 and 40% less in LS and C populations, respectively. Genetic variation had a significant population structure. In W populations, structure was associated with ecological and geographic areas according to isolation by distance. When LM and C populations where included in the analysis, differentiation was no longer apparent. Most LM were related to distant populations from Sierra Madre Oriental, which represents their probable origin. Historical demography shows a recent decline in all W populations. Thus, pre-domestication human management is associated with a significant reduction of genetic diversity and with a loss of differentiation suggesting movement among regions by man. Measures to conserve wild and managed populations should be implemented to maintain the source and the architecture of genetic variation in this important crop relative. Capsicum annuum var. glabriusculum  Heiser and Pickersgill syn. C. annuum L. var. aviculare (Dierbach) Biodiversity provides numerous services, one among them being food production C. annuum var. glabriusculum is considered the ancestor of the cultivated chili and bell pepper, C. annuum var. annuumCapsicum species. C. annuum var. glabriusculum is a perennial bush distributed from Colombia to the South Western United States Pepper huasteco yellow vein virusC. annuum var. glabriusculum population from Arizona (USA) and of ex situ accessions from Sonora, Chihuahua (Mexico) and Guatemala found genetic variation that was absent from other C. annuum accessions C. annuum have found a slightly higher variation in wild chiltepin population than in landraces or varieties of domestic C. Annuum var. annuumsensu Casas et al. The analysis of the effect of human management on the genetic variation and structure of chiltepin based on the study of both wild and managed populations at a large geographic scale has not been undertaken. This is the subject of this work. We sampled neighbouring wild, let standing and cultivated chiltepin populations  depending on the population size. Between 1 and 3 young branches with fresh leaves were collected per plant.Chiltepin plants were sampled during the summers of 2007–2009 at different sites over the species distribution in Mexico . A totalA second set of samples came from seeds contained in ripe chiltepin fruits purchased at local markets in the same regions where field surveys were conducted . In all C. annuum cultivars: loci with high and low number of alleles were included, and the ten loci belonged to different linkage groups Total nucleic acids were extracted by grinding 200 mg of fresh leaf tissues in three volumes of 200 mM Tris-HCl pH 9, 25 mM EDTA, 1% SDS, 400 mM LiCl, followed by phenol-chloroform extraction. A set of 10 nuclear microsatellites markers  were selected on the basis of their genetic variation in Na), unbiased expected heterozygosity (HS) Ho) and allelic richness (RS) FIS) was also estimated for each population. These parameters were estimated using FSTATv.2.9.3 FSTestD was estimated using SMOGD v1.2.5 K) in the whole data set and to confirm the putative geographic origin of the alleles of sampled individuals. This is especially relevant in the case of samples from anthropic habitats or those from fruits acquired in local markets, in which translocations may occur. To this end, microsatellite genotypes were analysed using the admixture model with correlated allele frequencies and without prior geographic information. The algorithm was run with 40000 Markov Chain Monte Carlo (MCMC) iterations of burn-in length and 40000 after-burning iterations for parameter estimation. The number of ancestral populations (K) was determined by doing 10 test runs with K values ranging from 10 to 36. The final K value was chosen based on the likelihood value that was significantly higher than the K-1 values using a Wilcoxon test. With the chosen K value, 20 more runs were performed with 60000 MCMC length and 60000 after-burning iterations. Individuals were organized in clusters as indicated by the run of the largest likelihood from the most probable value of K. The analysis of the most probable number of ancestral clusters within wild populations was performed using the same method but for K values from 2 to15, again with 10 runs of the algorithm.Genetic variation was measured as the mean number of alleles sampled (http://ibd.sdsu.edu/~ibdws/) AD genetic distance and Dest distance values between pairs of populations were log transformed, and to assess the correlations 1000 permutations tests were carried out. For the analyses, only data from wild populations were used as the input on the following sets: 1) all populations, 2) populations from the Western provinces CPS, CPA and SON, and 3) populations from the Eastern provinces YUC, SMO and AZP. In addition, for each W population, a spatial structure analysis was done using Structure. SJA-W was excluded from this analysis due to lack of data on the exact spatial position of sampled individuals in the field, and CER-W and HUJ-W because data were available for less than 20 individuals. The number of subgroups within each wild population was inferred in the same way as described above for K values ranging from 2 to 7.Mantel correlation tests to assess the relationship between geographic and genetic distance matrices were performed using the isolation by distance (IBD) web service  at, the number of years since the beginning of the expansion/decline and, (iii) µ, the mutation rate. This approach combines information from all loci for parameter estimation. The analyses were performed using the exponential growth model which is more suitable for modeling changes in population size on a shorter time scale 8 steps and was sampled every 10000 steps. Posterior density from individual runs was examined to check for overall consistency in shape, using Tracer 1.5 N0 = 1×10 3, N1 = 1×10 5, mutation rate (µ) = 1×10−4 and at = 1×103; for the second run were: N0 =  1×103, N1 =  1×104, µ = 1×10−4, at  =  1×10 3; for the third run were: N0 = 1×103, N1 =  1×105, µ =  1×10−4, at  =  1×104. Results of these runs are presented combined as obtained with LogCombiner 1.4.8 To explore historic demographic changes and possible bottlenecks, the coalescence-based MCMC method implemented by MSVAR 1.3 HS and Rs include a correction for uneven sample sizes, but the value of Na is expected to increase with the number of individuals analysed. Thus, a correction for sample size was included in the models for comparison of Na. To explore inbreeding levels with respect to levels of human management, comparisons of FIS were carried out. To test whether the different levels of human management affect the genetic variation, the fixed effect of level of human management  on the mean within population variation was analysed considering locus, population and biogeographical province as random effects. Na was transformed by the square root to meet the criteria of normality and homocedasticity. The remaining dependent variables were used without transformation. Analyses were performed with the JMP7 software .General linear mixed models were used to compare the within population genetic variation according to the level of human management. Plants derived from the seeds in fruits from local markets were not taken into account for these analyses. Estimates of RS and Na was found in populations from YUC, and the lowest in populations from SON > 4.10, P< 0.012).A total of 661 individuals were genotyped, 228 from wild habitats, 284 from anthropic habitats (LS and C), and 149 from seeds of fruits from seven LM. Marker CAMS-811 did not amplify in most individuals from SON and CPA populations. For the other populations, 33 alleles were found at this locus. The other nine microsatellite markers were amplified in all populations; the number of alleles per locus ranged from 7 (CAMS-020) to 102 (CAMS-885), averaging 28.6±27.2 alleles per locus. A total of 589 different genotypes were detected for 9 loci. Identical multilocus genotypes were frequent in some populations from anthropic habitats like LIB-CMC and POT-CHG, and extreme in HUA-CHG where all alleles were fixed for ten genotyped individuals. Mean number of alleles per population ranged from 1.00 to 11.11. Private alleles were observed in 17 populations, including 3 C populations, and plants from seeds from three LM. The number of private alleles varied from one allele in a single locus in SJA-W, MAU-W and TEM-CMC (with frequencies between 0.022 and 0.068) to 16 alleles distributed in six loci in DZI-W (frequencies between 0.022 and 0.196). The highest genetic variation, estimated by the sample size-corrected statistics Ho and Hs estimates. Conversely, FIS was highest in SON and lowest in YUC   =  3.63, P  =  0.02). The generally high FIS values most probably indicate high rates of selfing.The same trends were observed with −7) formed significant subgraphs (−9). Populations from YUC connect AZP populations with the rest. Interestingly, most LM populations grouped with populations from SMO, and not with populations from regions where fruits were purchased, exceptions being TOL-LM and BAT-LM  and significant (P < 0.05) values between most pairs of field populations, including all pairs of W populations . The lowest divergence value corresponded to populations PEL-W and HUJ-W, 112 km distant. Divergence estimates among some LM populations  indicated no genetic differentiation (Fst ranged between 0.005 and 0.032 and Dest between 0.005 and 0.024). Stepwise AMOVA showed significant latitude x longitude interaction when biogeographical province, level of human management and population were analysed, but no statistical significance was found for latitude or longitude. Population showed the greatest amount of differentiation (Φst|covariates  =  0.4722) followed by biogeographical province (Φst|covariates  =  0.2077) and level of human management (Φst|covariates  =  0.1244). Similar results were obtained when the analysis was restricted to the set of W populations.The structure of genetic variation was also examined by the fixation index, ulations . FST anAD) was positively correlated with geographic distance . The analysis was repeated for the Eastern and Western populations separately, showing that the positive correlation between geographic and genetic distance was due solely to the Western populations from CPS, CPA and SON , and not to the Eastern ones .To determine whether the distribution of genetic variation is structured geographically in the Mexican chiltepin population, isolation by distance (IBD) was analysed using data from W populations. Mantel test showed that genetic distance (P≥0.197), and a marginally significant correlation for BER-W (P  =  0.09), in which plants were sampled along two transects 1 km distant  to 4 (PEL-W) inferred groups for each population . The ave distant .Hs, Rs and Na was compared between W, LS and C populations. The mean value and standard error for these indices, estimated for the populations pooled according to the level of human management (W< LS < C), is shown in bold in F  =  4.41, P  =  0.027; F  =  5.39, P  =  0.014; F  =  4.83, P  =  0.020 for Hs, Rs and Na, respectively), and was lower in the C populations . The significant decrease in the genetic diversity of C populations relative to W ones is particularly clear when Rs and Na, are considered, with values being 63% and 68% lower, respectively, for C populations. These results did not vary when the population HUA-CHG was excluded from the analysis. No significant differences were found for any index between W and LSP+LSF populations, regardless of whether the average within population variation or the variation at each locus as repeated measures within populations were considered   =  0.00, P  =  0.949; F =  0.04, P  =  0.830; F  =  0.09, P  =  0.765 for Hs, Rs and Na, respectively). Similar analyses with inbreeding coefficient values did not show differences according to the level of human management   =  2.19, P  =  0.139). Comparable results were obtained when rarefaction analyses that correct for uneven sample sizes were done for the different levels of human management. W populations showed more haplotypes for the more diverse microsatellite (CAMS-885) than the LS+C populations, but LS populations in pastures supported more than twice the number of alleles than any of remaining managed populations  decrease of variation While tolerance or protection of plants in some anthropic habitats such as pastures did not result in a large loss of genetic diversity, in others, such as live fences, the levels of variation were lower . This obex situ preservation of crop varieties or landraces. The fraction of wild accessions in ex situ collections is still low, and large gaps in the species diversity most often remain to be covered in situ conservation, as it maintains the ability of the species to evolve in native conditions and, therefore, its potential genetic variation. In situ conservation has only been addressed rigorously over the last decade, when the growing rate of species extinction and its causes were identified. In situ conservation of chiltepin would require selecting areas including wild populations in the various geographical regions where individual clusters of genetic diversity have been identified, as well as areas of incipient domestication, which maintain the architecture of genetic variation. Due to the strong spatial genetic structure of chiltepin revealed here, to develop such a conservation programme would require a more detailed analysis of the genetic structure of wild chiltepin populations, with a denser coverage of its area of distribution than was attempted in this work. These analyses should not be delayed as population decline and fast alteration of native habitats may be a factor accelerating chiltepin domestication.Wild relatives of crops, such as chiltepin, represent a potential source of genetic diversity to cope with new needs in crop improvement, and their conservation has been emphasized as an important objective requiring international collaboration Figure S1Population graphs of A. wild, and B. let standing/cultivated populations of chiltepin. See ¤, CPA  =  Costa del Pacifico, #, SON  =  Sonora, £, SIN  =  Sinaloa.(PDF)Click here for additional data file.Figure S2Genetic structure of wild C. annuum var. glabriusculum populations. Two hundred and twenty eight genotyped individuals clustered into 7 groups. Each individual is represented by a vertical bar, which is divided into 7 coloured fractions representing the estimated portion of its genome that assigns the individual to each of 7 clusters. Black lines separate different clusters. Biogeographical provinces are indicated at the top of the chart.(PDF)Click here for additional data file.Figure S3Correlation between geographic and genetic distance in eleven wild populations of C. annuum var. glabriusculum. Log-transformed data are presented.(PDF)Click here for additional data file.Figure S4Local spatial structure in wild populations of C. annuum var. glabriusculum. (A) Within-population substructure was found in five out of eight wild populations tested. To facilitate comparisons, individuals are arranged according to the location within the sampled transects. Each individual is represented by a thin vertical bar which is divided into K coloured fractions representing the estimated portion of its genome that assigns the individual to each K cluster. Thin bars below grouped individuals according to their aggregation in the field and bold bars below represent the length of transects. (B) Correlation between geographic and genetic distances of individuals for populations BER-W and MOC-W. Log-transformed data are presented.(PDF)Click here for additional data file.Figure S5Rarefaction analyses for different habitats in C. annuum var. glabriusculum and the most variable microsatellite, CAMS-885.(PDF)Click here for additional data file.Table S1Values of the fixation index STF  and and Dest distance values  between pairs of Mexican populations of Capsicum annuum var. glabriusculum from Mexico.(DOCX)Click here for additional data file.


Numerous studies have applied genetic tools to the identification of source populations and transport pathways for invasive species. However, there are many gaps in the knowledge obtained from such studies because comprehensive and meaningful spatial sampling to meet these goals is difficult to achieve. Sampling populations as they arrive at the border should fill the gaps in source population identification, but such an advance has not yet been achieved with genetic data. Here we use previously acquired genetic data to assign new incursions as they invade populations within New Zealand ports and marinas. We also investigated allelelic frequency change in these recently established populations over a two-year period, and assessed the effect of temporal genetic sampling on our ability to assign new incursions to their population of source. We observed shifts in the allele frequencies among populations, as well as the complete loss of some alleles and the addition of alleles novel to New Zealand, within these recently established populations. There was no significant level of genetic differentiation observed in our samples between years, and the use of these temporal data did alter the assignment probability of new incursions. Our study further suggests that new incursions can add genetic variation to the population in a single introduction event as the founders themselves are often more genetically diverse than theory initially predicted. Unfortunately, many of these studies have been initiated long after the first incursion and this ‘snapshot’ of genetic diversity has been used as a starting point to decipher the many possible mechanisms that may be driving the observed patterns. For example, Perophora japonicaNew incursions, or recently established populations, may provide further insights into contemporary evolution. The initial period of invasion, which includes both founders and their progeny, is crucial for assessing the contribution of individuals to the persistence of the population and for predicting the evolutionary trajectory of the population. At this stage of the incursion, with relatively few colonisers, genetic changes are likely to occur as a result of stochastic processes in small populations Styela clava, was first recorded in New Zealand in 2005 in widely separated populations of the North (Hauraki Gulf) and South Islands . One recent and increasingly wide-spread ascidian invader, Styela clava individuals from hard surfaces within the top metre of the subtidal zone. Three levels of collections were made during 2007, and the data generated from this study were then compared with data collected from 2006 in two previous studies To address the aims of the study we collected Styela clava collected either from the hull of boats arriving to port (PAH), or from marinas where newly documented incursions had occurred , herein referred to as new incursions. These incursions were sampled during targeted surveillance and boat hull inspections in 2007, undertaken by the National Institute of Water and Atmospheric Research (NIWA) and MPI  and microsatellite genotyping  were done using the protocols of Goldstien We used mtDNA and genotypic data to investigate the equality of these molecular markers in accurately assigning new incursions. Data from previous studies on the global distribution of mtDNA haplotypes and microsatellite genotypes e) and allele frequency for loci and populations were estimated using Genalex6 e & FIS statistics between 2006 and 2007 samples we used the randomisation procedure All individuals sampled in 2007 were genotyped for comparison to locations sampled in 2006 and to investigate the role of temporal sampling in the assignment of the new incursions. Diversity indices such as expected heterozygosity (HST and pairwise distance statistics obs). Due to the occurrence of rare alleles, Chao's Jaccard estimator (Chao-Jacc-Est) of abundance-based similarity index FStyela clava sampled from four marinas and one boat hull in 2007. Considering the low sample size for these new incursions, the genotypic diversity  was high relative to populations previously sampled from New Zealand . The number of alleles per locus ranged from 8 (Sc2B12) to 19 (Sc1H1)  . Chi-squIS), allelic diversity (Ar), or heterozygosity (HE). Species  accumulation curves show that 85–92% of the diversity estimated by the Chao2 index was captured in each year for the Hauraki Gulf . In all cases the new incursions were assigned with high probability to populations sampled in North America . For all four of these data sets, the Hauraki Gulf had the highest number of individuals sampled, yet this does not appear to affect the results obtained. Temporal sampling increased the assignment to New Zealand, regardless of whether using only the shared populations or the whole data set, suggesting that this is not a function of increasing the sample size.Genotypic assignment of individuals from new incursions showed a low probability of assignment to New Zealand populations sampled in 2006 and 2007 , althougThe results obtained here highlight the importance of temporal genetic sampling and the application of baseline genetic data sets in determining the origin and spread of new incursions. To our knowledge this is the first study to use existing genetic data to assign individuals to sources as they arrive at new locations within a region. The key findings of this study are: 1) new incursions recorded in New Zealand exhibit high mitochondrial and nuclear genetic diversity and both markers were consistent in their assignment of individuals to populations; 2) genetic diversity has been maintained over a two-year period within approximately 10 years of the initial introduction; 3) significant spatial structure between the North and South Island populations is maintained over the two-year period, despite extensive regional admixture and population growth; 4) temporal sampling was important in the accurate assignment of new incursions.Styela clava is transported around the globe in biofouling on vessels and relies heavily on anthropogenic transport for international and domestic spread Carcinus maenas along the coast of Nova Scotia S. clava makes for a very “chaotic” pattern of genetic admixture and allelic shifts. Models of maritime transportation networks have highlighted how complexity in the transport pathways, coupled with stochastic demographic events  can drive an unpredictable sequence of invasion from primary and subsequent incursions, with potential for considerable re-assortment of populations of frequently transported species Styela clava is much more likely to be transported by recreational vessels than merchant vessels , and this is reflected in the admixture observed among populations and between years in our data. In contrast, the Port of Lyttelton receives a high number of merchant vessels but much less recreational traffic, which is reflected in the significant genetic differentiation between the Lyttelton and Hauraki Gulf populations, maintained over the two-year period, or about four generations for Styela clavaStyela clava were not resolved to specific source locations in this study, but they were proportionally assigned to northern and southern New Zealand populations, or to overseas populations for which data were available. The pooling of data from populations sampled in 2006 and 2007 significantly changed the proportion of assignments to northern and southern New Zealand from 20% and 25% to 35% and 37%. The change observed with the temporal approach could simply be a function of increasing the sample size and capturing more alleles. However, using only the shared populations actually decreased the sample size for the Hauraki Gulf. In addition, the data collected from 2006 and 2007 showed no significant difference and all alleles were captured in both datasets, albeit the distribution of these alleles did vary within years. It is more likely that there is a trade-off occurring here. For instance, the 2006 data for Hauraki Gulf includes the Port of Auckland, marinas and aquaculture farms; in 2007 Auckland Port was not sampled, but the marinas and aquaculture farms were sampled, as well as additional sites from natural habitats. By combining the 2006 and 2007 data, we have effectively sampled more individuals of the important populations around marinas and aquaculture farms, while the more minor sites have been eliminated. Analysis of this core data set, with a strong element of temporal resampling, indicates that temporal sampling may enhance the accuracy and likelihood of assignment to the true source region as unsampled (rare) alleles in the population may be important to assignments, particularly if these change in frequency from year-to-year.New incursions for The new incursions studied here highlight a key aspect of founding populations and the interplay of transport pathways and population dynamics in the genetic diversity of new incursions. Numerous genetic studies have shown that genetic diversity in populations of invasive species is higher than expected in founder populations under bottleneck conditions Rhagoletis completa using samples taken from the initial founding population and again 30 years after its initial introduction. Similarly, hybrid zones of Mytilus spp. along the coast of California were sampled in 1994–95 and again in 2005–07 showing a large range shift toward the equator in the subtropical species M. galloprovincialis following a decade of climatic oscillation, and Peres-Portela These data also provide a powerful model for further monitoring the change in genetic diversity with population dynamic shifts over time. Several other studies have successfully used historical genetic data to investigate genetic change over time r) from high or low fecundity species, and between marine and terrestrial invaders. For example, iteroparous species may have more temporal buffering of reproductive success because they are spreading the risk of failure over multiple reproductive events and years, as seen in the sea urchin Paracentrotus lividusPerhaps genetic equilibrium is rarely met in introduced species, particularly at small initial population sizes, where an individual's contribution to the next generation is amplified and is critical to successful establishment and population growth. However, the importance of temporal variation in allele frequencies may differ between species with short- and long-generation times, with different per capita rates of growth Click here for additional data file.


Tibouchina papyrus (Melastomataceae), endemic to rocky savannas in Central Brazil, to test hypothesis of vicariance and dispersal in the origin of the disjunct geographical distribution. We sampled 474 individuals from the three localities where the species is reported: Serra dos Pirineus, Serra Dourada, and Serra de Natividade. Analyses were based on the polymorphisms at cpDNA and on nuclear microsatellite loci. To test for vicariance and dispersal we constructed a median-joining network and performed an analysis of molecular variance (AMOVA). We also tested population bottleneck and estimated demographic parameters and time to most recent common ancestor (TMRCA) using coalescent analyses. A remarkable differentiation among populations was found. No significant effect of population expansion was detected and coalescent analyses showed a negligible gene flow among populations and an ancient coalescence time for chloroplast genome. Our results support that the disjunct distribution of T. papyrus may represent a climatic relict. With an estimated TMRCA dated from ∼836.491 ± 107.515 kyr BP (before present), we hypothesized that the disjunct distribution may be the outcome of bidirectional expansion of the geographical distribution favored by the drier and colder conditions that prevailed in much of Brazil during the Pre-Illinoian glaciation, followed by the retraction as the climate became warmer and moister.Many endemic species present disjunct geographical distribution; therefore, they are suitable models to test hypotheses about the ecological and evolutionary mechanisms involved in the origin of disjunct distributions in these habitats. We studied the genetic structure and phylogeography of  The understanding of the ecological and evolutionary mechanisms involved in the origin of disjunct distributions in plant species has been improved by phylogeographic analyses e.g., . While f14C year BP (before present)], the climate was drier in most of the South American savannas and distribution of savanna-like vegetation in Central and Southeast Brazil was more extensive in early compared with the late Holocene  are particular vegetation communities associated to outcrops of sandstone and quartzite soils of the Brazilian Shield in the Cerrado Biome in Central and Southeast Brazil, with high number of endemic species and high influence of fire during the dry season . It is fTibouchina papyrus (POHL) Toledo (Melastomataceae) is an endemic tree species from outcrop quartzite and sandstone of the rocky savannas (cerrado rupestre) of the Cerrado biome. Flowers are purple and buzz-pollination is performed mainly by large bees, such as Xylocopa spp., Bombus spp., and Centris spp. (http://www.biodiversitas.org.br/florabr/lista_florabr.pdf) due to its natural endemism and habitat vulnerability. The species is reported only in three localities in the Brazilian Shield, Central Brazil  and Serra Dourada (SDO), Goiás (GO), and in Serra da Natividade (NAT), Tocantins (TO)  and SDO (66). In population NAT, we randomly sampled 192 individuals because this population was the largest, with ∼1500 individual trees. For each individual, expanded leaves were sampled and stored at –80°C. DNA extraction followed the standard CTAB procedure (We sampled ins (TO) . Notice rocedure . Distancrocedure : NAT–PIRpsbA and trnH (trnS and trnG (trnC and ycf6 (2), 250 µg of BSA, and 6.0 ng of template DNA. Amplifications were performed using a GeneAmp PCR System 9700  with the following conditions: 96°C for 2 min (1 cycle); 94°C for 1 min, annealing temperature for 1 min , 72°C for 1 min (30 cycles); and 72°C for 10 min (1 cycle). Polymerase chain reaction products were sequenced on an ABI 3100 automated DNA sequencer  using the DYEnamic™ ET terminator cycle sequencing kit , according to manufacturer's instructions. All fragments were sequenced in forward and reverse directions.For sequencing analyses, we randomly chose 32 adult individuals in each population. Three noncoding regions of the chloroplast genome (cpDNA) were sequenced: the region between the genes and trnH , trnS anand trnG , and trnand ycf6 . FragmenSequences were aligned using CLUSTALX , and chah) diversity (Populations were characterized for nucleotide (π) and haplotype ; (2) for each population, based on network relationships and high differentiation among populations (see results below) under the assumption that, after the species has attained the disjunction distribution, populations have undergone independent demographic histories, without gene flow among them. The mismatch distribution was obtained and the hypothesis of expansion was tested using the Harpending´s Raggeness Index R , g , where tθ=θnow exp(–gtµ) and t is time in mutational unit, and M= 2emN/θ (migration force or immigration rate) were estimated based on Bayesian estimation using Markov Chain Monte Carlo approach implemented in LAMARC 2.1.5 software , and ∼12.5 ng of template DNA. Amplifications were performed using a PE 9700 Thermal Controller  with the following conditions: 94°C for 5 min (1 cycle), 94°C for 1 min, 54°C–66°C for 1 min  were genotyped for 10 microsatellite loci, previously developed for  papyrus . Microsaf). Analyses were performed with FSTAT 2.9.3.2  than loss of observed heterozygosity (Ho). Hence, populations that have experienced recent reduction in effective population size may present higher Ho than Heq for a given number of alleles in the population , using LAMARC software. Time to most recent common ancestor (TMRCA) was estimated from overall θ. We used the lowest mutation rate reported for microsatellite markers in plants, 2.4 × 10–4 mutation per allele per generation   and used a generation time of eight years .Demographic parameters were estimated for microsatellite data based on the coalescent model as described above for sequencing data, correcting for diploid genome (θ= 4× 10–4]) , often qon e.g., . We chospsbA-trnH, trnS-trnG, and trnC-ycf6 generated fragments of 268 bp, 580 bp, and 294 bp, respectively. All sequences were submitted to GenBank database .Amplification of the noncoding regions psbA-trnH noncoding region presented eight different haplotypes, with five polymorphic sites. For the region trnC-ycf6, we found three haplotypes and two polymorphic loci. For trnS-trnG, we found five haplotypes and four polymorphic sites (psbA-trnH (h= 0.771) was very similar to the value found for trnS-trnG (h= 0.775) and nucleotide diversity  was higher than for trnS-trnG . The region trnC-ycf6 presented the lowest level of gene diversity (h= 0.341) and nucleotide diversity . The combined data from the three sequence regions generated a fragment of 1142 bp, with 17 haplotypes, and 11 polymorphic sites , and pairwise FST were significant and high for all pairs of populations .Analysis of molecular variance showed a high differentiation among populations . Coalescent analysis performed with LAMARC software also showed that populations are not expanding but have constant size with very low mutation parameter θ . Genetic diversity was higher at population NAT, but allelic richness was higher in SDO . RST also showed a high level of genetic differentiation , but FST and RST were not significantly different (P= 0.427). A significant amount of inbreeding was found , and nonrandom mating among populations . Pairwise FST were also significant and high for all pairs of populations .Populations were highly differentiated . Coalescent analysis also showed population expansion but not for population SDO , a species from rocky savannas that co-occurs with T. papyrus in SDO and PIR , that last from 455,000 to 300,000/380,000 year BP . In the us e.g., , Aracaurfer taxa . Based o and PIR , despiteulations .T. papyrus may represent a climatic relict and that long distance gene flow is unlikely. Tibouchina papyrus has undergone a recent bottleneck that may have caused loss in genetic diversity. Since migration among populations is negligible, the probability of local extinction due to demographic and genetic stocasticity may be very high. Tibouchina papyrus is a rare and endemic species of sandstone outcrop habitat, which is highly unstable and suffers high levels of disturbance caused by fire during the dry season and sandstone and quartzite disruption, mainly during the rainy season. These disturbances may be highly variable among local habitats and may cause sudden modifications in population size (T. papyrus populations are three different ESUs (Evolutionary Significant Units, In conclusion, our results strongly support that the disjunct distribution of ion size . An impo


Apis mellifera in the topographically heterogeneous East African region. The currently accepted “mountain refugia hypothesis” states that populations living in disjunct montane forests belong to a different lineage than those in savanna habitats surrounding these forests. We obtained microsatellite data, mitochondrial sequences, and morphometric data from worker honey bees collected from feral colonies in three montane forests and corresponding neighboring savanna regions in Kenya. Honey bee colonies from montane forests showed distinct worker morphology compared with colonies in savanna areas. Mitochondrial sequence data did not support the existence of the two currently accepted subspecies. Furthermore, analyses of the microsatellite data with a Bayesian clustering method did not support the existence of two source populations as it would be expected under the mountain refugia scenario. Our findings suggest that phenotypic plasticity rather than distinct ancestry is the leading cause behind the phenotypic divergence observed between montane forest and savanna honey bees. Our study thus corroborates the idea that high gene flow may select for increased plasticity.Identifying the forces shaping intraspecific phenotypic and genotypic divergence are of key importance in evolutionary biology. Phenotypic divergence may result from local adaptation or, especially in species with strong gene flow, from pronounced phenotypic plasticity. Here, we examine morphological and genetic divergence among populations of the western honey bee  Understanding the mechanisms and evolutionary processes leading to the distribution and phenotypes of species and populations is a central goal in biogeography. Divergent selection along environmental gradients may lead to phenotypic and genotypic differentiation, potentially resulting in reproductive isolation and speciation , and an alpine zone. Savanna vegetation usually occurs in areas below 1000 m a.s.l. Today the forests on the various mountains are isolated from each other, cover very little land area and are endangered by economically attractive alternative uses such as agricultural conversion and timber exploitation A. m. monA. m. monticola is a distinct lineage which evolved at an unspecified time in a high altitude forest area and had a large historical distribution during the last glacial maximum, between 21000 and 15000 years BP when forest vegetation shifted about 1000 m downslope . All six sampled populations are considered feral, as there are no reports of breeding efforts or successful introductions of foreign honey bees in these areas  were examined morphometrically .SL: length of the left antennal scape.HTL: length of the left hind tibia.Pigmentation – number of segments that are not completely dark among the following six segments: scutellum, tergit 1- tergit 5. In entirely dark specimens this value is zero . These statistical analyses were carried out in STATISTICA 8 (StatSoft (Europe) GmbH, Hamburg, Germany). Voucher specimens from the six populations have been deposited in the National Museum (Invertebrate Zoology Section) in Nairobi.For each colony, we calculated the mean values and used these data for principal component and discriminant function analyses. To test for linear relationships between these characters and altitude we calculated Pearson's st and Jost's D) analyses described below. We also obtained a larger dataset . For our smaller dataset . Microsatellite allele sizes were further scrutinized manually to detect null alleles and errors in allele scoring. Null alleles refer to the failure to amplify, via PCR, a specific microsatellite, due to mutations in the primer binding site, for example. We excluded all individuals where at least one null allele was detected. Within each colony, alleles differences of 1 bp were assumed to be a genotype scoring error and were rounded up to the next higher value   Jost . Fst estCrawford . DepartuCrawford . SignifiA. m. scutellata savanna population, which represents a population with an extensive range with no obvious barriers to gene flow.Population structure estimates such as those produced by Bayesian clustering approaches  can be k) was used to estimate population structure using the software STRUCTURE v2.3  and to assign individuals to any of K populations, using the admixture model with correlated allele frequencies and 1,000,0000 iterations, with a burn-in of 2,00,000. We tested a range of k values of 1–12 populations, and for each k we ran this STRUCTURE routine three times to evaluate consistency. The method of ΔK ; first, not using the LOCPRIOR option and then using as prior each of the six sampling localities. The LOCPRIOR setting was used to detect hard-to-find structure, as this model has been shown to detect structure with lower levels of divergence, compared to other models and it is not supposed to be biased toward detecting false structure . Interestingly, all four characters showed a linear correlation with altitude , raising the question whether the observed pattern is due to the existence of two distinct morphological forms or rather represents continuous variation along an altitudinal gradient.The morphometric data suggest that the colonies from mountain forest and savanna areas belong to two distinct groups Fig. A. AlthouIn order to further test levels of differentiation between forest and savanna populations we used microsatellite data and mitochondrial DNA to evaluate genetic differentiation between populations. Overall our molecular analyses suggest there are no tangible differentiations between the two putative subspecies.Our tests for departure of HWE and LD suggest that there are only very small levels of both LD between loci and deviations from HWE for all populations in our small dataset, where we used one individual per colony. This dataset was used directly for subsequent genetic analyses.n = 56) and obtained mean number of alleles (Na) that ranges from 8.11 to 9.78 and average gene diversity  ranging from 0.80 to 0.85 (Table a and He not significantly different to each other (Table e = 0.83 ± 0.02 versus savanna He = 0.83 ± 0.02). Summary statistic for each of the nine microsatellites separately are given in Table We first measured basic population levels parameters including unbiased estimates of gene diversity for each of the six populations on the small data set  are not more similar to each other than to those in the other habitat type. As an interesting exception, the Fst and Dest values for MKF, NHF, and NHS are highest although closely located (Fig. st values obtained in this study are strikingly different from those obtained by Soland-Reckeweg et al. (A. mellifera in Switzerland, which are on average an order of magnitude larger than the Fst values we found. However, a study of the genetic differentiation among A. mellifera populations in Italy using microsatellites (Dall'Olio et al. st values between populations of A. m. ligustica similar to those found in our study. It should be noted, however, that the anthropogenic effect on honeybee population structure in Europe is much more pronounced than in Kenya. Consequently, naturally occurring gene flow among honeybee populations in Kenya most likely contributes to the low genetic differentiation we found.Our genetic analyses show extensive sharing of mitochondrial alleles between the montane forest and savanna populations studied, as well as low levels of population differentiation between the two groups, based on microsatellite data. These results are in remarkable contrast with previous reports on these two putative subspecies in Kenya, which found corresponding morphological and genetic differentiation between them, supporting their taxonomic status (Meixner et al. rs Table . Fst andted Fig. , which mg et al.  for compBayesian clustering analyses Fig.  show thaCombining the morphological data, which show clear differences between the two groups of populations and were significantly correlated with altitude Fig. A and B, A. m. monticola, the mountain refugia hypothesis was favored by Meixner et al. (A. m. monticola, found in different montane localities, should exhibit low genetic differentiation among themselves, as a reflection of their shared genetic past. We would also expect to find lower similarity between montane forest and close-by savanna populations, compared to levels between montane forest populations, as expected if they indeed belong to different subspecies. However, several honey bee life-history traits such as their high degree of panmixia (Franck et al. Although East Africa has been described as “mosaic of spatial and temporal refugia” by Lorenzen et al. , clear er et al. . The maiA. m. monticola. In order to address the question whether A. m. monticola needs to be synonymized, it will be necessary to examine samples of A. m. monticola from their type locality, Mount Kilimanjaro. Furthermore, it would be worthwhile to focus on the closely related subspecies A. m. litorea which occurs at lower altitudes at the East African coast as it cannot be excluded that all these three “subspecies” represent morphological variants of a single, highly plastic group within the East African region.Phenotypic plasticity is defined as the ability of a genotype to produce different phenotypes, depending on environmental conditions such as food, ambient light, temperature, and other ecological characteristics. From an evolutionary perspective, phenotypic plasticity can be adaptive because of past or ongoing selection regimens, allowing organisms to quickly respond to changing environmental conditions or to different conditions encountered after dispersal Agrawal , which i


A. assamensis is a phytophagous Lepidoptera from Northeast India reared on host trees of Lauraceae family for its characteristic cocoon silk. Source of these cocoons are domesticated farm stocks that crash frequently and/or wild insect populations that provide new cultures. The need to reduce dependence on wild populations for cocoons necessitates assessment of genetic diversity in cultivated and wild populations. Molecular markers based on PCR of Inter-simple sequence repeats (ISSR) and simple sequence repeats (SSR) were used with four populations of wild insects and eleven populations of cultivated insects. Wild populations had high genetic diversity estimates  and at least one population contained private alleles. Both marker systems indicated that genetic variability within populations examined was significantly high. Among cultivated populations, insects of the Upper Assam region  were genetically distinct (FST = 0.38 with both marker systems) from insects of Lower Assam . Sequencing of polymorphic amplicons suggested transposition as a mechanism for maintaining genomic diversity. Implications for conservation of native populations in the wild and preserving in-farm diversity are discussed. Antheraea assamensis is a silk-producing Lepidoptera indigenous to Northeast India and Indo-Burma Antheraea yamamai, the cocoon silk of A. assamensis is touted as the second most expensive silk in the world and its cultivation contributes significantly to livelihoods of indigent tribal peoples. A. assamensis larvae are reared outdoors, on trees of Persea bombycina and Litsea monopetala (family Lauraceae) for commercial silk production A. assamensis moths originating from cultivated populations or wild moths from forests. Cultivated populations were collected from two regions of Northeast India, Upper Assam and Lower Assam Antheraea species in NCBI database A. assamensis. Results obtained are discussed for their relevance to conservation strategies.This paper investigates genetic variability in fifteen populations of i = 0.37±0.02) estimated for all populations using all ISSR primers was similar to Nei’s expected heterozygosity value (HE = 0.37±0.25) obtained from SSR data. Both ISSR and SSR primers yielded variable numbers and sizes of amplicons. Bayesian heterozygosity estimates (HS) obtained with all ISSR primers was more circumspect than Hi. However, trends observed with the frequentist (Hi) and Bayesian estimates (HS) were consistent. For instance, AhISSR5 showed lowest percentage polymorphism, lowest Hi and HS values among all the ISSR primers tested (i and HS). Nei’s heterozygosity estimates could not be estimated for insect populations from this region, as a single allele (monomorphic locus) was amplified with each SSR primer tested. A wild population from the Shillong plateau  showed the highest percent polymorphism, highest number of haplotypes, Hi and HT estimates using both marker systems  and Mangaldoi (a cultivated population from Lower Assam).High genetic diversity estimates were obtained for the full data set comprising of fifteen populations  using bos tested . An exces tested . Table 4ST = 0.36 and 82% with SSR markers, FST = 0.18 at P-value = 0, CT = 0.18. Results with SSR marker data , approximately 20% of the total genetic variation (FCT = 0.2) was observed between the three groups with both markers , was applied. For ISSR data with 50 loci, and K = 3, discrete groups of populations comprising wild insects from Shillong Plateau region, cultivated insects from Upper Assam and Lower Assam regions were retrieved with both admixture and no-admixture models . Results893M) for Upper Assam (UA), Lower Assam (LA) and Shillong plateau regions (SP); (ii) polymorphic for LA and SP but absent from UA (NB5437P); (iii) polymorphic  for UA, LA and SP; (iv) polymorphic (GP131249P) and monomorphic (GP14607M) for moths of the same population; and (v) polymorphic (NB91316P) and monomorphic  for moths from different populations. Amplicons representing loci that were (i) monomorphic from the same moth of a population  and (ii) monomorphic  were also analyzed to determine if such monomorphic loci contain the same sequence. About 10 kbp of the A. assamensis genome was sequenced. Sequence analyses of these cloned loci revealed that without exception, primers based on the same repeat sequence but containing different mononucleotide anchor amplified unique, non-redundant loci across the A. assamensis genome.Several ISSR amplicons were cloned into pDrive vector and their sequences submitted to GenBank . These s1456P locus contained the largest copy number (103) of such a repeat. Amplicons obtained with tri-nucleotide repeat primers had 40%GC compared with primers based on tetra-nucleotide repeat sequence (34%GC). Most sequences were AT-rich and contained stop codons in all three frames especially at the 5′ end. Sequence of the TR41456P amplicon contained twenty copies of a TATAAAAT sequence while MN11064M locus contained a 40 bp AT-rich region. No tandem, perfect simple sequence repeats were observed in any amplicon using either SRF or mREP softwares. Analyses with RepeatMasker and Consensus softwares n was identified in TR41456P and TR41452P close to the Jockey-like stretch. About 50% of the ISSR amplicons sequenced in this study were associated with remnants/footprints of transposable elements. Furthermore, the (GATA)n primer binding regions along with an additional 12 to 28 bp were found to resemble hAT and the Harbinger elements from zebra fish in the sequences of GP16825M and GP131249P loci. During searches of the GenBank databases, the (GATA)n primer binding sequences along with 6–12 bases were found to resemble micro-satellite regions from a wide variety of organisms. BLAST hits of the EST databases often indicated that the 3′ ends of the amplicons contained regions of coding sequences suggesting that intron-exon junctions may be spanned during amplification with these ISSR primers. This may explain an observation made earlier that the stop codons were generally clustered in the 5′ends of the sequences. In the case of GP131249P and SK7643P sequences obtained with primer AhISSR13 and AhISSR5, a 130 bp sequence was common in both amplicons immediately proximal to the forward primer sequence. In fact, several sequenced amplicons obtained with the same primers contained perfectly conserved sequences immediately proximal to the forward primers, suggesting the occurrence of multiple copies of repetitive regions in the A. assamensis genomes. Finally, we observed identical sequences within a monomorphic locus of 547 bp amplified from several individuals belonging to the three population clusters. This result supported a basic premise of ISSR-PCR that shared bands (scored as 1) may represent homologous loci.Results obtained after analyzing the data with Spectral Repeat Finder (SRF) indicated the presence of interspersed, imperfect repeats ranging from 2 to 10-mers with a low spectral frequency of.0005 to.001. The TR4oftwares  revealed1456P locus  contained the 1456 bp amplicon. Low stringency salt washes showed hybridization of the probe with heterologous sequences from A. proylei, A. mylitta but not B. mori and S. cynthia indicating that the probe was specific for Antheraea species (B. mori and S. cynthia (not shown). High stringency washes revealed that the probe  hybridized to multiple but distinct loci in the genomes of individual A. assamensis that came from different populations (Antheraea genomes and distributed differently in moths of different populations. Nei’s genetic distances between Boko (a cultivated population from Lower Assam region) and Kohargaon (a cultivated population from Upper Assam region) was 0.1644, Boko and Turah (a wild population from the Shillong plateau region) was 0.1839; Turah and Kohargaon was 0.3011  using frequentist methods and 0.24±0.01 (HS) while using Bayesian principles (i) and Bayesian approach (HS) were similar. Similar discrepancies in numerical values have been observed earlier and attributed to the different approaches for these measures E) estimated from SSR data was also high at 0.37±0.23 (A. assamensis using molecular markers O) observed in Lepidoptera using allozyme analysis is 0.127 E. mana to a high of 0.46 for O. brumata [30/19]. Results from this study  indicated the need to preserve forests in North-east India as repositories of diverse germplasm of A. assamensis. The highest number of haplotypes (12), and two private SSR alleles was detected in the Agropetal population which contained native insects. Similar reports have been reported earlier for wild populations from this region using single markers systems i = 0.24±0.17; HS = 0.21±0.01 for ISSR markers and HE = 0.21±0.11 with SSR markers. While several observations for cultivated populations held between the two marker systems, some did not. For example, in contrast to the ISSR data, cultivated populations of Upper Assam were monomorphic with all eleven SSR markers . Both Nei’s Hi and Bayesian HS values were the lowest for Titabor (a cultivated population from Upper Assam) which also had the lowest percentage polymorphism and least number of polymorphic bands . Reasons for these observations are unclear, but could be recent introduction of wild parents to invigorate cultivated populations of Lower Assam. The values from Upper Assam populations could be a snapshot of genetic diversity values in populations containing more inbred insects, prior to a stock invigoration. More work is needed to understand and monitor the genetic consequences of introducing wild insects into cultivated populations.High gene diversity estimates for wild populations from the Shillong plateau with both marker systems  when three groups of populations from different regions  were examined  on population genetics of cultivated insect populations.Both molecular marker systems indicated that genetic variation was partitioned within individuals of the fifteen populations rather than among groups of populations based upon topography or origin . The imp markers  and a lo markers . The SSRexamined 1, 5D1. Texamined 2, 5D2 coexamined . ReasonsA. assamensis, some mixing of genotypes especially between wild populations and the geographically proximal cultivated populations of Lower Assam was also indicated. Moderate genetic distance estimates  observed earlier  was evident . Positiv1456P amplicon, sequence motifs of more than one mobile genetic element were detected. The association of transposable elements with population genetic variation is well known for animals and plants Bilbo have been shown to maintain genetic variability within populations and influence population structure of Drosophila species D. melanogaster, transposable elements (some of which occur in introns) which show population heterogeneity have been attributed a major role in adaptation to local temperate climates Bombyx mori genome Lymantria dispar. The probe hybridized with DNA from A. assamensis, A. mylitta and A. proylei but not B. mori or S. cynthia, suggesting that it was specific for Antheraea species. In A. assamensis, the probe bound to multiple but identical loci within Hind III digested DNAs of four moths from different populations (not shown). In the case of Hae III digested DNAs of the same four A. assamensis moths, the probe hybridized to multiple loci, some of which were distinct for moths from different populations (A. assamensis. The probe was a fragment of an amplicon obtained with AhISSR 110 primer whose sequence was based on a (GATA)n repeat. Our Southern hybridization result is reminiscent of the autosomal, non-allelic hypervariable Bkm DNA loci in the moth Ephestia kuehniella known to be associated with mobile genetic elements Ostrinia nubilalis, Pectinophora gossypiella and B. moriAmplicons representing several polymorphic and monomorphic loci from moths within the same and different populations were sequenced . About 5ulations  suggestiA. assamensis. Further research is necessary to understand the role of transposable elements and other genomic mechanism(s) that can influence genetic variability within populations of A. assamensis. Taken together, the importance of monitoring the history and genetic consequences of introduction of wild moths (mixing) during invigoration of cultivated stocks is paramount. Molecular markers can be useful for such efforts. Both wild and cultivated populations of A. assamensis are endemic and restricted to Northeast India In conclusion, the use of two molecular markers as described in this study strongly indicates that wild populations need to be preserved in their natural habitats as invaluable germplasm for invigorating cultivated populations. Our results coincide with other reports based on single marker systems A. assamensis were randomly collected from forests, silk farmers, state, and central government insect rearing depots in Northeast India. To the best of our knowledge, no collection was made from a restricted or protected area. A total of 256 cocoons were collected representing fifteen collection sites (henceforth referred to as populations). The location, altitude and latitude of these fifteen sites were ascertained from governmental maps and Google earth , wing length Cocoons of le earth . CocoonsAntheraea species available in the public domain NCBI sequence database were collated and searched for sequence repeats using the software mREPS m of the primer used, Taq DNA polymerase in buffer containing 1.5 mM magnesium chloride provided by the manufacturer , 100 µM of each dNTPs, 5 pmol of each primer, and approximately 10 ng of genomic DNA from each moth as template. All reagents were of molecular biology grade and purchased from Sigma Aldrich Inc., USA, except for the primers, which were synthesized by IDT Inc., USA.High molecular weight, nuclear genomic DNA was isolated from an abdominal half of each moth using a proteinase K, SDS-based nuclei extraction method i) were obtained using two free-wares: POPGENE version 1.32 and TFPGA version 1.3 IS = 0), moderate (FIS = 0.5) and high disequilibrium (FIS>0.5). A Bayesian approach for estimating total average panmictic heterozygosity (HS) was determined reiteratively using Hickory version 1.03 O, HE and HT) and pair-wise tests for linkage disequilibrium Amplification products obtained consistently with each ISSR primer for each insect representing the fifteen populations were scored for the presence (1) or absence (0) of bands. Only repeatable bands in the range of 0.25 to 2.5 kbp were scored. Rare alleles were pruned from the dataset Ф-statistics) was estimated within populations and among populations using by AMOVA 5; no. of MCMC reps after burn-in = 105; Ancestry model with and without admixture; and correlated allele frequency between populations was assumed. Most likely value for assigning K clusters was determined from an ad-hoc statistic (ΔK) derived from second order rate of change in likelihood function was used derived from second order rate of change with respect to K of the likelihood function Data obtained was also converted into haplotype frequencies by AMOVA PREP Selected amplicons obtained from individual moths of various populations  were gelA. assamensis , B. mori, S. cynthia A. proylei and A. mylitta was digested completely with the restriction enzyme Hind III and Hae III . The probe used was an 800 bp amplicon obtained by nested PCR (using primers Fp2: 5′CTATGTCATCCACTGTACTGTAATT3’ and Ah16: 5′TTTCACGAACCCATACA GGC3’) from a plasmid clone DQ872512 containing a polymorphic locus TR41456P obtained with primer AhISSR110. This locus occurred in the dataset with a frequency of 37% and showed high sequence similarity to lepidopteran transposable elements. The 800 bp amplicon contained an internal restriction site for Hae III but lacked sites for Hind III. Low stringency washes (4X SSC +0.1%SDS) were done twice for 30 minutes at 65°C before overnight autoradiography at −80°C. Subsequent high stringency washes  for 30 minutes each at 65°C was followed by 2 days autoradiography at −80°C. Primers Fp2 and Ah16 used for nested PCR and amplification of the probe were also used to amplify DNA from the heterologous insects, B. mori, S. cynthia, A. proylei and A. mylitta. These PCRs were performed as described earlier except that a primer annealing temperature of 60°C was used.Southern hybridizations were performed after Mazumdar-Leighton and Broadway Figure S1A tree depicting clusters among 15 populations of A. assamensis based on Nei’s genetic distances. The number of loci supporting each node and proportion of similar replicates (bootstrap) calculated using TFPGA is indicated in parentheses. Population color codes are the same as (TIF)Click here for additional data file.Figure S2Mean (±SD) weight of moth weight (MW), cocoon weight (CW) and shell weight (SW) of male (M) □ and female (F) ▪ A. assamensis.(TIF)Click here for additional data file.Figure S3Relation between moth weight and cocoon weight in male (Δ) and female  A. assamensis. The r2 values are also indicated.(TIF)Click here for additional data file.Table S1Partitioning of genetic variation for the full set of A. assamensis moths using (A) ISSR marker data and (B) SSR marker data. *Significance tests were performed at 1023 permutations. **Bayesian analogue  was 0.32+0.02.(DOC)Click here for additional data file.Table S2Matrix showing pair-wise estimates of Nei’s genetic distance estimates based on 50 loci from ISSR data. Wild populations from Shillong plateau are shown in bold, cultivated populations from Lower Assam region are italicized. Populations showing the highest and lowest genetic distance estimates are italicized in bold.(DOC)Click here for additional data file.Table S3Details of thirty ISSR primers designed from intronic repeats within Antheraea genes and their ability to amplify lepidopteran genomic DNAs.(DOC)Click here for additional data file.