Biodiversity is the diversity of life at all scales, from genes to ecosystems. Predicting its patterns of variation across the globe is a fundamental issue in ecology and evolution. Diversity within species, that is, genetic diversity, is of prime importance for understanding past and present evolutionary patterns, and highlighting areas where conservation might be a priority. Using published data on the genetic diversity of species whose populations occur in the Mediterranean basin, we calculated a coefficient of correlation between within-population genetic diversity indices and longitude. Using a meta-analysis framework, we estimated the role of biological, ecological, biogeographic, and marker type factors on the strength and magnitude of this correlation in six phylla. Overall, genetic diversity increases from west to east in the Mediterranean basin. This correlation is significant for both animals and plants, but is not uniformly expressed for all groups. It is stronger in the southern than in the northern Mediterranean, in true Mediterranean plants than in plants found at higher elevations, in trees than in other plants, and in bi-parentally and paternally than in maternally inherited DNA makers. Overall, this correlation between genetic diversity and longitude, and its patterns across biological and ecological traits, suggests the role of two non-mutually exclusive major processes that shaped the genetic diversity in the Mediterranean during and after the cold periods of the Pleistocene: east-west recolonization during the Holocene and population size contraction under local Last Glacial Maximum climate in resident western and low elevation Mediterranean populations. Whereas equitability measures are more sensitive to higher frequency alleles and how they are distributed within populations, richness measures respond more to the presence and quantity of rare alleles. Thus, the two measures are needed concurrently to document and test for demographic events, such as bottlenecks and expansions.GDpop . First, longitudinal trends could result from genetic drift due to long distance dispersal and founder effects during Holocene recolonization from refugia  clinal imprint left by the climate of the last glacial cycle on resident populations in refugia was stronger than the imprints left by the incongruent Holocene recolonization patterns of different species from different refugia ;their bioclimatic requirements ; andtheir location within distribution areas .We collected published population genetic studies of terrestrial plant and animal species whose distributions were at least partially found within the Mediterranean basin, from endemic to widespread . For thiGDpop estimates that were included within the Mediterranean basin. We used the delineation of the Mediterranean basin defined by Olson et al. . Also, not all organisms remained in the close vicinity of their glacial refugia after Holocene warming , where r is the partial correlation coefficient. Finally, effect-sizes  were weighted by the inverse of their asymptotic variance (see Aloe and Becker Vz = 1/(n−3), where n is the number of sampled populations in the source study. Finally, summary-effects (Zr) were computed as the weighted mean of the individual effect-sizes using a fixed-effect meta-analysis model. We chose a fixed-effect model , we tested the effect of non-independence on Type I error rates as well as on the precision of summary-effects , whereas the meta-analysis procedure was performed using MetaWin , then we may expect that the wider the range of population sampling across the Mediterranean basin in the primary studies was, the greater the probability of detecting a positive mean effect-size Zr would be. Also, Zr might be affected by the position of the range within the Mediterranean basin. We tested these relationships by regressing each Zr and their corresponding longitudinal span calculated as the absolute value of the difference in degrees of longitude between the easternmost and the westernmost populations and each Zr and their corresponding mean longitudinal coordinate.If we hypothesize a general and homogeneous link of GDpop and geography because they may reflect different processes acting at different spatial and time scales . They may also reflect different demographic histories via their different effective population size or sex-related transmission. Discrepancies have classically been found by phylogeographers between the nuclear and the mitochondrial DNA .The choice of the genetic marker may impact the sign of the correlation between We tested biogeographic effects by categorizing the effect-sizes as either northern or southern Mediterranean, and as either from continents or islands.GDpop in the Mediterranean.There may be a strong confounding effect between taxonomy and marker type in our general dataset. Specifically, cpDNA effects may be due to the type of DNA used or to traits specific to plants as this type of DNA is not present in animals. Thus, we used the part of our dataset restricted to plants to retest for marker-type effects on overall trends and also to test for the imprint of biological attributes and ecological requirements on GDpop differences. For example, generation time is a life-history trait that will moderate the imprint of past demographic events on the measured GDpop. As a first general and very coarse proxy of those traits, we used taxonomy and we characterized all species by their family, class, and kingdom names.Along with demographic processes, many life-history traits can be responsible more or less deterministically for spatial http://www.tela-botanica.org/ consulted in April 2009). We coded the altitudinal thermo-climatic belts  where each plant species was predominantly found. Bioclimatic requirements have been found to affect GDpop  leading to the tests of the categorical moderators reported below. The general longitudinal trend in GDpop was affected by sampling range and range type, by marker and metric types, by taxonomy (phylogeny), by biological traits, and by ecological requirements.Overall, there was a positive and significant correlation between erranean a. Withinerranean ; Data S4Zr . Widely distributed species and species from the eastern part of the Mediterranean tended to have more significantly positive Zr than others. The Zr was five times higher for continents than for islands and almost three times higher for the southern than for the northern Mediterranean  were not significantly different between the northern and southern Mediterranean , although they were between continents and islands .Studies in our dataset sampled a majority of northern Mediterranean populations (399 effect-sizes in the north compared with 53 for the south). Differences in Zr computed for the animal and plant kingdoms were both positive and significant. Zr for animals was more than three times higher than that of plants. The two Q-tests rejected homogeneity of Zr within plants and animals  and in the Lamiaceae, Nymphalidae, and Pottiaceae  confirmed the patterns observed when excluding phanerophytes except for the southern Mediterranean Zr, which became non-significant. In animals, two families showed a positive trend (Buthidae and Tephritidae), whereas two others showed the opposite trend (Plethodontidae and Torymidae)  .Zr of the same order of magnitude, although mitochondrial Zr was not significantly different from zero  was positive but non-significant (not shown), indicating that GDpop for maternally inherited markers does not significantly increase with longitude. Zr for both “equitability” and “richness” indices were significant, positive, and similar in magnitude. Excluding island populations from the dataset increased Zr values. Zr for “equitability” was three times higher when restricting the dataset to the southern Mediterranean envelope, whereas Zr for “richness” stayed constant, but became non-significant (not shown).Nuclear and organelle markers showed a positive rom zero a. When aThe summary-effects for bi-parentally and maternally inherited markers decreased and became  non-significant after excluding animals from the dataset (not shown). In plants, positive and significant effects-sizes were thus found for plastidial DNA and paternally inherited DNA .Zr and was the only significant group. The supra-Mediterranean group had a positive but non-significant Zr. The category with the highest sample size (mountain-Mediterranean) had a negative and non-significant Zr, and the gymnosperms contributed predominantly to this group (70 of the 82 effect-sizes). The group with the highest requirements in terms of temperature (thermo-Mediterranean) had an almost null Zr. Heterogeneity was significant in all groups.We were able to assign 201 effect-sizes of plant species to a bioclimatic belt without ambiguity b. The reZr (Zr (see above). Dicots among phanerophytes showed a contrasting pattern depending on the geographic envelope: at Mediterranean basin level, the effect was positive but non-significant; whereas it was strongly positive for the continental group  and strongly negative for islands . Contrasting with the other moderator analyses, the southern group yielded a negative trend while the northern group showed a positive one. Chamaephytes  were the only group showing a negative  Zr.Phanerophytes (trees and shrubs) represented approximately 2/3 of the plants in our dataset and showed a positive and significant Zr c. Among Zr values: Anemochorous  and barochorous (gravity-dispersed) plants showed a significant and positive Zr, whereas zoochorous  plants had a non-significant Zr  plants showed a homogeneous negative trend (Zr and anemogamous (wind-dispersed pollen) species had a positive and significant Zr.Seed dispersal mode affected icant Zr d. Pollenve trend e, whereaOrganization of genetic diversity in Europe mostly follows latitudinal routes of recolonization dating from the Holocene  that commonly affected glacial refugia. A meta-analysis, using a fixed-effect model, precisely makes this assumption  in mobile or high gene flow organisms, and more to local climatic effects in sessile or low gene flow organisms. With perhaps a difference in intensity between the two trends, that due to migration being less pronounced because of high gene flow and recurrent genetic exchange between populations blurring post recolonization foundation effects. Our data show that reality may be more complex because both highly mobile (insects) and more sessile (arachnids) animals showed trends similar in direction and magnitude. In arachnids, a group only comprising scorpions from the southern part of the Mediterranean basin in our dataset, the effect could be related to local past-climate effects. The pattern shown by insects could reveal a link between their contemporary genetic structure and the mirrored structure of the plants they exploit. Recent studies have indeed illustrated the link between levels of diversity in keystone organisms such as trees and in their phytophagous-associated organisms  displaying a negative trend in LGM climate on genetic diversity. Conversely, animal-dispersed species depend on their dispersers' behavior for survival  overlaps with that of wind-dispersed species and may reflect both local demographic effects under LGM climate and long distance Holocene recolonization. Seed dispersal by gravity, however, does not allow for rapid and long-range dispersal. Thus, the positive trend found in gravity-dispersed plant species matches the expectation of an effect of local GDpop from low- to high-elevation (from less to more cold tolerant) plant species. The rationale for this expectation was that the unfavorably cold LGM climate should affect more strongly population size in species with higher temperature requirements as they became trapped in reduced-size habitats compared with those of lower temperature requirement species. In contrast, species adapted to colder climates such as supra- and mountain-Mediterranean species for example, benefiting from larger habitats during the LGM, should not have suffered demographic bottlenecks as did the more truly Mediterranean group. Thus, a west to east oriented clinal climate at the LGM should result in a stronger west to east clinal GDpop structure in true Mediterranean species  by Guzmán and Vargas , both being valuable food crop.When assessing the impact of ecological (temperature) requirement on plants, we expected to find an increasing positive effect on  “Me” in b than fod Vargas . FinallyStudies using maternally inherited markers are most of the time designed to detect phylogeographic signals and capture differentiation effects, for example, those due to the imprints of contraction and recolonization to and from Pleistocene refugia. Studies using paternally inherited  or bi-parentally inherited markers are more often designed to detect local demographic signals  resulting from current environmental drivers, in addition to phylogeographic signals. This confirms that the overall trend detected in our study is a within-population demographic bottleneck effect, more weakly detected in maternally than paternally and bi-parentally inherited genomes. Phylogeographic studies have shown that the two different metric types we have used here, richness and equitability, can be negatively correlated along the distribution range of species , strong ecological importance  as well as endangered flagship species . The taxonomic groups that were the most heavily sampled  showed a congruently positive summary-effect, that is, an increasing genetic diversity from west to east. These abundant taxonomic groups are thus good models for detecting trends and patterns affecting biodiversity in general. The propensity of the genetic diversity of trees to be an excellent testimony of the imprint of ancient evolutionary and demographic processes (given their long generation time) has been mentioned for some time  and their congruence tested (Devictor et al.


Calotriton includes two species of newts highly adapted to live in cold and fast-flowing mountain springs. The Pyrenean brook newt , restricted to the Pyrenean region, and the Montseny brook newt , endemic to the Montseny massif and one of the most endangered amphibian species in Europe. In the present manuscript, we use an integrative approach including species distribution modeling (SDM), molecular analyses of mitochondrial and nuclear DNA sequence data and morphology to unravel the historical processes that have contributed to shaping the biogeography and genetic structure of the genus Calotriton, with special emphasis on the conservation of C. arnoldi. The results of the molecular analyses confirm that, despite having originated recently, being ecologically similar and geographically very close, there is no signal of hybridization between C. asper and C. arnoldi. SDM results suggest that tough environmental conditions on mountains tops during glacial periods, together with subsequent warmer periods could have prevented the contact between the two species. Within the critically endangered C. arnoldi, a high genetic structure is revealed despite its extremely small distribution range compared to C. asper. Haplotype networks, AMOVA and SAMOVA analyses suggest that two distinct groups of populations can be clearly differentiated with absence of gene flow. This is in concordance with morphological differentiation and correlates with its geographical distribution, as the two groups are situated on the eastern and western sides of a river valley that acts as a barrier. The genetic and morphological results are highly important for the ongoing conservation program of C. arnoldi and strongly justify the management of this species into at least two independent evolutionary significant units (eastern and western sectors) to guarantee the long-term population viability.The genus  The evolutionary history of species strongly depends on the variation of their distributions throughout time and space. For instance, it is well known that during the Pleistocene many species shifted their distribution ranges as a result of the climatic cycles  and the Montseny brook newt . According to Carranza & Amat 2 across the Pyrenean mountain chain (NE Iberian Peninsula) with some populations extending northwards and southwards, reaching the Prepyrenees 2 restricted to a few brooks in the Montseny massif C. arnoldi have been found, fragmented into two main population groups on both sides of the Tordera river valley separated by inhospitable habitat. The eastern and western sectors comprise three and four populations, respectively, with a total estimation of 1000–1500 mature individuals C. arnoldi is catalogued as Critically Endangered in the International Union for Conservation of Nature (UICN) Red List of Threatened Species. Currently, the conservation planning is in the process of development by the Catalan Government in order to ensure the survival of this species.The genus Calotriton provides an interesting opportunity to analyze in detail the effects of the Pleistocene climatic changes on the evolution of these two species. Additionally, understanding its population structure and evolutionary history is essential for formulating the appropriate conservation strategies and management proposals, such as identifying the management units . For the RAG-1 gene 33 and 75 specimens were analyzed, respectively. The number of individuals collected at each site and sampling locations are reported on C. arnoldi the three eastern populations are herein refereed as A1, A2, A3, and the four western populations as B1, B2, B3 and B4. Tissue samples consisted of tail tips or fingers preserved in absolute ethanol until further analysis. Genomic DNA was extracted using QiagenTM  DNeasy Blood and Tissue Kit following the manufacturer's protocol. A region of 374 bp of the cyt b gene was amplified using primers Cytb1EuprF and Cytb2EuprR et al. 5′-CGGTACGAGATCTGGCGGTCC-3′ and CaloRAG1-1362R 5′-TATCTCAGGCACGTGGGCTAGT-3′; the PCR program used for this region included an initial denaturing step at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and a final extension of 72°C for 5 min. Sequences were aligned using BioEdit 7.0.1 A total of 420 individuals were analyzed for the cytochrome Network approaches may be more effective than classical phylogenetic ones in order to represent intraspecific evolution F-statistics C. arnoldi populations from the two different sides of the Tordera river valley (western vs eastern populations) using ARLEQUIN ver 3.5.1.2 h) and variable sites (S), and haplotype (Hd) and nucleotide (Π) diversity indices, for each marker were calculated in DNASP v5 An analysis of molecular variance (AMOVA) calculating Calotriton arnoldi was excluded from the SDM as only seven localities are known to date separated each other by a maximum of 6 km. Unfortunately the number of populations is not enough to carry out a reliable prediction. Therefore, SDM was only performed for C. asper.C. asper were used. These localities included our own collection sites , and 16 additional species records derived from Milá et al.A total of 69 localities , coverinhttp://www.worldclim.org/) to form the present and past  climatic datasets http://www.ccsm.ucar.edu) and the Model Interdisciplinary Research on Climate .The study area comprised the northeast part of de Iberian Peninsula . Initially, a total of 19 BioClim variables were downloaded from the WorldClim database version 1.4 (C. asper were selected: Mean Temperature of Driest Quarter (BIO9), Annual Precipitation (BIO12) and Precipitation Seasonality (BIO15). Topogeographical variables and landcover were only available from the same database for current conditions. Altitude was downloaded from WorldClim database, slope was calculated using ARCGIS 10.0  and aspect was reclassified into four classes  using the same program. Landcover was downloaded from the Global Environment Monitoring database . To account for geological range constraints, we additionally used lithology  derived from the European Soil Database v. 2 C. asper dataset exceeded the 95th percentile of the null dataset AUCs. These randomly generated models can be used as a null-hypothesis against which to test the significance of species distribution models. If the AUC of the Pyrenean brook newt was significantly higher than the AUC of randomly generated models, it was considered as evidence that the species distribution model performs significantly better than expected by chance To avoid autocorrelation and over fitting of our data, colinearity among the initial 19 BioClim variables was tested using the Pearson's correlation coefficient in SPSS 17.0 Calotriton arnoldi were included in the morphological analyses, 57  and 106  from western and eastern sectors, respectively. Eight linear morphometric measurements were obtained using a digital caliper . All analyses were performed on log-transformed variables using Statistica v.5.5 software .One hundred and sixty-three adult specimens of  caliper : snout-vC. arnoldi (108 from the eastern sector and 81 from the western one) was examined focusing on the lack or presence of pale yellow spots on the dorsal side and patches on the head of the newts.The pattern of body coloration of 189 b gene (24 variable and 22 parsimony-informative positions). The nDNA data set included 530 bp . Of the 12 cyt b haplotypes identified, 8 were found in C. asper and 4 in C. arnoldi (C. asper and 3 to C. arnoldi (h) and variable sites (S), and estimates of nucleotide (Π) and haplotype (Hd) diversity for each marker in each sampled species are shown in C. asper than C. arnoldi. Instead, for the nDNA both values were, on average, higher for C. arnoldi. Within the Montseny brook newt, all 42 specimens from the western sector corresponded to the same unique haplotype for both mitochondrial and nuclear markers, thus Π and Hd were null.The final mtDNA data set included 374 bp of the cyt  arnoldi , whereas arnoldi . The numb and RAG-1 markers showing the relationship between both species are displayed in C. asper cyt b haplotype network comprised eight haplotypes, and adjacent haplotypes were separated by one single step. Individual haplotypes represented samples from between one and eight sampling sites. The C. arnoldi haplotype network for the same marker showed four haplotypes. The eastern sector included three haplotypes consecutively connected to each other by one step, and separated from the single western haplotype by two steps. The single RAG-1 parsimony network showed three steps between C. arnoldi and C. asper haplotypes, while one step connected haplotypes within each species.Geographical distribution of haplotypes and the haplotype networks for cyt Calotriton were divided into species groups , and for the nuclear marker, the overall FST value was 0.960 (P<0.001).Analyses of molecular variance were consistent with high levels of structure in both markers, when populations of the genus s groups . For theC. arnoldi, the genetic structure obtained by SAMOVA identified the same geographic structure found among the haplotype networks drawn in the phylogeographic analyses. The best partitioning of the genetic diversity by SAMOVA was obtained when samples were grouped into two groups, corresponding to the western and eastern sectors (data shown in K = 2). The genetic structure found in AMOVA analyses for the mitochondrial marker showed an overall FST  = 0.954 (P<0.0001). Among groups variation was statistically significant . Only 0.52% of variance was a result of differences among populations within these groups, and 4.59% within populations. The nuclear marker showed similar but slightly lower values . Most variation was explained by the among groups differences (83.16%), with variation values of 6.45% and 10.39% for among-populations within-groups and within-populations, respectively.Within C. asper under present and past conditions are shown in th percentiles of the null dataset were 0.618, 0.620, 0.629 and 0.619 for the present, CCSM, MIROC and LI, respectively). This indicates a good fit of the models.The predicted geographic distributions for C. asper during the last interglacial was restricted to the central areas of the Pyrenees, being almost absent southwards of the Montseny. During the last glacial maximum C. asper presented a range expansion. The predicted distribution spreads to lower altitudes, being absent from the highest parts of the mountain chain. A continuous predicted distribution from the Pyrenean chain to the southern Montseny massif is observed. The extent of range expansion is variable depending on the climatic model, being more apparent under the CCSM than under the MIROC. These models indicate that several areas of suitable habitat  existed in the glacial periods.SDM predictions based on current climate data showed a wide predicted distribution throughout the Pyrenean mountain chain (NE Iberian Peninsula), with some areas of suitable habitat southwards along the Montseny massif , althoug8 152 = 0.143; P<0.0001) and population  significant differences without interaction between these two factors . A posthoc test for unequal size showed no significant differences among sexes or populations in SVL and LI while HL and TH were the only sexually discriminant variables  and therefore is exclusive of the eastern populations. The coloration of the margin of snout is a new diagnostic character that is always present in males of the western sector, was found only in a female of the western sector, and it has never been found in male or female C. arnoldi of the eastern sector .Our analyses of coloration found striking differences between sectors defined by two traits: dorsal yellow spots and whitish margin of the snout . The firCalotriton species and within C. arnoldi, both with nuclear and mitochondrial genes. Despite being phylogenetically very closely related C. asper and all 150 alleles of C. arnoldi analyzed in the present study are unique for each species. Moreover, the analysis of all 315 mtDNA sequences of the cyt b gene of C. asper and all 105 sequences of C. arnoldi resulted in two independent networks with all sequences being unique for each species . Paleoclimatic studies have detected periglacial events during the last glaciations C. arnoldi is distributed), from the north face of the massif. It is possible that these harsh environmental conditions could have prevented the expansion of the Pyrenean species to the Montseny during the LGM period. Climatic conditions during LI were in many aspects similar to current ones in the Western Mediterranean basin  suggest that this widespread species presents a low level of genetic variability in the two genes analyzed here compared to its congener, the Montseny brook newt, which presents an extremely small distribution range (20 Km2). Morphological differences may appear under local selection pressure in response to population specific ecological conditions C. asper. The low genetic variability of C. asper may be explained by the much broader climatic suitability shown during the LGM that could allow the connection of populations and subsequent homogenization as a consequence of gene flow Rana pyrenaicaC. asper. Even though, owing to the discrepancies of high levels of variability found with AFLPs markers Regarding the SDM results and some fossil records from Cova Colomera  C. arnoldi.Despite having been described in 2005 and its relevance from a conservation and evolutionary point of view, this is the first attempt to assess the level of genetic and morphological differentiation of C. asper, the Montseny brook newt presents a similar level of genetic variability in the mtDNA and a higher level of genetic variability in the nDNA  have been found in land Euproctus platycephalus, a species that inhabits similar environments as C. arnoldi, presents a high genetic structure C. arnoldi not only presents a much smaller distribution range than E. platycephalus, but also its effective population size could be much smaller Here, the nucleotide and haplotype diversities have been analyzed to infer the genetic structure of the Montseny brook newt and the level of gene flow among populations on each side of the Tordera river valley. Our data show that despite having a distribution range 1000 times smaller than the nDNA ; Table 3C. arnoldi are concordant with the geographical structure within its distribution range. Our data show a strong genetic differentiation at both the mitochondrial and nuclear levels between eastern (A1–A3) and western (B1–B4) populations of C. arnoldi , population distribution is a significant factor of morphological differentiation in C. arnoldi is being reduced dramatically One of the major objectives of any conservation plan is the maintenance of genetic variability C. arnoldi shown here would justify the conservation of two distinct management units (ESU, Evolutionary Significant Units Population genetics is a useful tool for the planning and development of conservation programs Further genetic studies using microsatellites markers are in course to infer the current gene flow among populations and their possible isolation within sectors. This is of vital importance in order to establish whether further ESU's within sectors should be defined.


Rhyacophila pubescens a highly specialised freshwater insect that occurs in tufa springs, a very stable habitat.Dispersal rates, i.e. the effective number of dispersing individuals per unit time, are the product of dispersal capacity, i.e. a species physiological potential for dispersal, dispersal behaviour, i.e. the decision to leave a habitat patch in favour of another, and connectivity of occupied habitat. Thus, dispersal of species that are highly specialised to a certain habitat is limited by habitat availability. Species inhabiting very stable environments may also adopt a sedentary life-style. Both factors should lead to strong genetic differentiation in highly specialised species inhabiting stable environments. These two factors apply to our model species R. pubescens. We inferred the location of Pleistocene refugia and postglacial colonisation routes of R. pubescens, and examined ongoing local differentiation. Our results indicate intraregional differentiation with a high number of locally endemic haplotypes, that we attributed to habitat specificity and low dispersal rates of R. pubescens. We observed high levels of genetic diversity south of the Alps and genetic impoverishment north of the Alps. Estimates of migrants placed the refugium and the source of the colonisation in the Dauphiné Alps (SW Alps).We examined the genetic population structure and phylogeography using range-wide mtCOI sequence and AFLP data from 333 individuals of R. pubescens populations.This is the first example of an aquatic insect with a colonisation route along the western margin of the Alps to the Central European highlands. The study also shows that specialisation to a stable environment may have promoted a behavioural shift to decreased dispersal rates, leading to stronger local population differentiation than in less specialised aquatic insects. Alternatively, the occurrence of highly specialised tufa spring habitats may have been more widespread in the past, leading to range regression and fragmentation among present day  In recent years our knowledge of phylogeographic patterns of European animal and plant species has grown tremendously -4. From Compared to terrestrial niches, stream biotopes are erratically distributed, making their inhabitants particularly interesting for studying population genetics and phylogeography. Many highland aquatic insect species exhibit 'insular' distributions among mountain ranges, but also within mountain ranges where populations occur in isolated habitats with few or no interconnecting corridors of suitable habitat. This is due to the linear structure of stream habitats and the habitat specificity of many species , the resRhyacophila pubescens Pictet, 1834, is a highly specialised cold-stenotherm species that only occurs in permanent tufa spring brooks in limestone mountain ranges from the spring source to 5 km downstream [R. pubescens north of the Alps [R. pubescens postglacially recolonised its Central European range from a single refugial source. We also observed numerous private haplotypes and hypothesized recent or ongoing in-situ diversification. Since our sampling was limited to the northern ranges and excluded common European refugial areas, and we examined only mtDNA sequences, we could not test these hypotheses in our previous study.The caddisfly wnstream ,27. Usinthe Alps  and obseR. pubescens, which we predict to be associated with the south-western Alps based on calcareous Pleistocene refugia known from the region for plants [R. pubescens. Third, we ask if highly specialised inhabitants of stable environments - in this case R. pubescens - exhibit lower dispersal rates and higher levels of population differentiation than other species that are less specialised. We predict that R. pubescens exhibits high levels of population structure, even at a small geographic scale, due to its strong affiliation with isolated tufa spring environments. To address these objectives we use a range-wide sampling of both mtDNA sequence and nuclear Amplified Fragment Length Polymorphisms (AFLP) data. We employ both population genetic and phylogeographic methods to elucidate patterns of population differentiation, past migration rates and changes in demographic history.Our current study has three main objectives. First, we explicitly test the hypothesis of a postglacial colonisation of Central Europe from a single Pleistocene refuge. We predict that genetic data will show a connection between one, not several, southern refugia and the Central European populations. Second, we want to identify the location of the Pleistocene refugia of r plants  or with r plants -3,5. UpoR. pubescens from 51 sites across the entire distribution range  protocol followed Huck et al.  with minR. pubescens across the distribution range. Samples were grouped into 23 different geographic units, i.e., mountain regions, which we refer to as regions  [FST-values and geographical distance between all analysed populations. Analyses were performed with Arlequin 3.1 [D and Fu's FS. Significant negative D and FS values can arise under selective effects but can also indicate population expansion or bottlenecks [To examine genetic population structure we pooled the 51 sampled sites by mountain region and uniform geological units following Diercke Weltatlas  and Gons (AMOVA) : geograp (AMOVA)  for isolquin 3.1  using detlenecks ,42. We aFST-values and geographical distance between all analysed populations with 999 permutations in GenAlEx 6.1 [4), i.e. "the number of phenotypes expected at each AFLP locus when four individuals have been sampled from the population." Due to limited sample size we did not evaluate br4 in the Bilé Karpaty and Pieniny Mountains. The Shannon Index of phenotypic diversity S [A Mantel test  was condAlEx 6.1 . Using AAlEx 6.1  we calcuAlEx 6.1  in each AlEx 6.1 . High DWAlEx 6.1  we used AlEx 6.1  with rarersity S  was calcersity S . AMOVA [ersity S  was calcWe selected the regions where we sampled three or more streams  to assess ongoing or recent diversification among populations within regions. We calculated mean GST  among poFST-values for the first run. We conducted a second run using the estimates for theta and M of the first run as starting values. Both runs used 10 short chains and recorded 25,000 genealogies with a sampling increment of 20 , and two long chains that recorded 200,000 genealogies with as sampling increment of 50 . We used an adaptive heating scheme with four chains  and a swapping interval of one to ensure sufficient mixing. The analysis was based on the mtCOI dataset.The distribution of the ancestral haplotype H1 in the northern populations and the Western Alps (see Results), combined with the strongly diverged and differentiated haplogroups associated with the Ligurian Alps and the Apennines, indicates that the most probable location of the refuge and source for the northward colonisation is somewhere in the Western Alps. We therefore used Migrate 3.0.3  to invesEU885387-EU885414, GU186972-GU187002). The maximum difference was 70 bp (14.74%) between all haplotypes and 29 bp (6.11%) for the "Central European" haplotypes without the divergent haplotypes found in Liguria, the Apennines and Corsica. The unrooted median-joining haplotype network  than in the south (0.161948 +/- 0.080769). The percentage of polymorphic AFLP loci (95% confidence), band richness and Nei's gene diversities based on AFLPs were highest in the Ligurian Alps, followed by the Česky Kras, Cottic Alps, French Calcareous Alps and the Provence Alps  of all region pairs were significantly differentiated  and that variance among populations within regions was much lower . When only taking the mountain ranges north of the Alps into account, AMOVA of mtCOI data revealed more variation within regions  than among regions . In contrast, AMOVA of AFLPs from the northern populations showed 45.72% variation within regions and 54.28% variation among regions . In the southern regions there was less variation within regions  than among regions . The results illustrate that differentiation among mountain ranges north of the Alps was lower than among mountain ranges south of the Alps. In general, the differences were less pronounced in the AFLP data than in the mtCOI data. A weak isolation-by-distance effect was revealed by Mantel test . A Mantel test considering only the populations north of the Alps showed no correlation based on mtCOI data , but did show a weak correlation in the AFLP data . In both data sets a much stronger correlation was found in the southern populations , indicating that the southern populations were comparatively closer to equilibrium between genetic drift and gene flow than the northern ones.We examined ongoing or recent diversification using GST in the Northern Hessian Mountains, Franconian Alb, Northern Calcareous Alps, Swiss Jura and Northern Alpine slope. Mean GST among populations within each of the five regions was 0.54 in the Northern Calcareous Alps, 0.49 in the Swiss Jura, 0.45 in the Northern Alpine slope, 0.41 in the Northern Hessian Mountains and 0.14 in the Franconian Alb.D were found for the Swiss Jura, the French Calcareous Alps and for the dataset as a whole, indicating a high number of low frequency polymorphisms in the mtCOI dataset and potential population size expansion [FS test for mtCOI data were negative and significant for the Franconian Alb and the entire dataset, and highly significant for the Swiss Jura (results not shown). Negative, but non significant values for both tests were found in the Swabian Alb, Northern Calcareous Alps, Mittelland, Slovenské Rudohorie, Northern Hungarian Mountains and the Dauphiné Alps.In the regions studied north of the Alps, almost all mismatch distributions of mtCOI haplotypes were unimodal . Unimodaxpansion ,42 (resuWe used Migrate to test the hypothesis that the refuge and source for the northern populations was located in the south-western (SW) Alps. We calculated a stepping stone model with mtCOI data to estimate numbers of effective migrants and the direction of migration from the populations in the SW Alps to the northern populations. Both Migrate runs yielded similar results. Results of the second run are presented. Gene flow, measured as effective migrants, was detected from the Dauphiné Alps southward to the French Calcareous Alps, and from these to the Provence Alps Figure . There wRhyacophila pubescens is restricted to Central Europe and the Italian peninsula, the latter a region of many Pleistocene refuges [ refuges ,5. Circu refuges ,54,55. A refuges  - as posR. pubescens during the last glacial maximum was in the region of the French Calcareous or Dauphiné Alps, below the permafrost line and that northward expansion started from there. The results of gene flow analysis indicate that the Dauphiné Alps are the only region from which migration occurred northwards and southwards in the Western Alps , vast areas of Central Europe were covered by thick loess deposits . It is k records ,28,64 ilR. pubescens. Other examples of rapid northward recolonisation were shown to occur in the pond turtle Emys orbicularis [Chorthipppus parallelus [More frequent long distance dispersal or more widespread suitable habitat during this period of recolonisation would explain the presence of the common haplotype H1 over the entire northern part of the present range of icularis , and everallelus .Rhyacophila pubescens is the first example of a Central European aquatic insect that started postglacial recolonisation from a south-western alpine refugium along the western edge of the Alps to the former periglacial area north of the Alps. This pattern differs considerably from patterns of Pleistocene survival and postglacial recolonisation of Central Europe observed in other cold tolerant caddisflies and aquatic invertebrates, for example, multiple glacial refugia  or Centdiscolor ).R. pubescens provides another example that phylogeographic history appears to be largely species-specific in aquatic insects with no common patterns emerging to date. This is quite different to the situation in terrestrial species, where several common patterns are known [The case study in re known ,5. DiffeR. pubescens is adapted to a specialised habitat that also happens to be characterised by stable environmental conditions: small permanent headwater tufa streams and springs. While we sampled a variety of habitat types from all regions over several years, R. pubescens was only collected from tufa springs, underscoring its restriction to this habitat type. As is to be expected for any species that occurs in patchily distributed headwater stream environments with specific physico-chemical characteristics, R. pubescens exhibits regional differentiation. A similar pattern was also observed  in other highland caddisflies in Europe [R. pubescens, as evidenced by GST and private haplotypes. The presence of private haplotypes in almost all mountain ranges and in single streams across the entire distribution of the species indicates low dispersal rates between streams. This observation is supported by the absence of an isolation-by-distance effect in the northern populations. The lack of an isolation-by-distance pattern across much of the northern part of the range of R. pubescens indicates that higher genetic drift in marginal populations is not the main reason for the observed pattern [R. pubescens is rarely collected in large numbers [The caddisfly n Europe ,10,68. H pattern . R. pube numbers , and low numbers ,71.M. aurelia [Carabus variolosus. Molecular studies of European caddisflies have not yet examined differences among specialist and generalist species, but the available studies do allow for some comparisons. In Hydropsyche tenuis the genetic diversity and differentiation are lower than in R. pubescens and there is evidence for ongoing or recent long-distance dispersal surrounding the Alps in H. tenuis [Hydropsyche tenuis is less selective regarding its habitat, occurring in the very dynamic headwater and mid-range stream regions of calcareous and siliceous streams [Rhyacophila aquitanica, Drusus discolor, and Drusus romanicus are more strongly associated with cold habitats than H. tenuis, but occur in both siliceous and calcareous streams. They exhibit greater population differentiation than H. tenuis [R. pubescens. The comparison with the available studies does suggest that specialisation may lead to greater population differentiation in aquatic insects.Several studies suggest that highly specialised species are more isolated because of habitat availability than generalist species. A comparatively lower local dispersal rate may result in a high number of rare or locally restricted alleles as shown for the butterfly  aurelia , a calca aurelia  also inf. tenuis . Hydrops streams . Rhyacop. tenuis ,10,68, bR. pubescens apparently not dispersing among its specialised habitats? At first, this result seems at odds with the widespread occurrence of the dominant haplotype and the hypothesis of a rapid postglacial recolonisation of Central Europe. The observed patterns are consistent with a recent reduction of suitable habitat and resulting local allopatric fragmentation. The vast loess deposits over Central Europe in the early Holocene [R. pubescens. After the postglacial expansion and the decrease in loess deposits across Europe, the primarily sedentary species would have experienced allopatric fragmentation into more isolated outcrops of calcareous rocks where tufa streams form and persist to the present. Unfortunately with so little knowledge about tufa development in loess deposits and limited paleosol data, it is currently not possible to reconstruct the distribution of tufa habitats during the early Holocene to test this idea.Why is Holocene  may haveHolocene  suitableRhyacophila, both of which showed morphological attributes related to strong flight . However one species, R. fasciata was limited in the number of sites where it occurred (27%), while R. nubila occurred at all sites (100%). Thus, while both species have the physiological capacity for flight and dispersal, one species disperses less. Dispersal rates are not limited solely by flight capacity, i.e. the physiological ability to fly certain distances or for certain periods of time, but also by behaviour. If dispersal behaviour is linked to habitat stability [R. pubescens, which currently only inhabits environmentally stable tufa springs and headwater streams, has adopted a more or less sedentary life style with little lateral dispersal among streams. The widespread occurrence of a common haplotype suggests that long distance dispersal is possible in R. pubescens, but in this specialist caddisfly, dispersal rate may be responding to habitat availability and habitat persistence [R. pubescens could be promoting a predominantly sedentary behaviour.The contrast between widespread haplotypes and many private fragments and haplotypes is also consistent with a shift in dispersal rates either following a change in dispersal capacity of the species, or a change in dispersal behavior. We favour the explanation of a shift in dispersal behaviour over a relatively recently evolved morphological or physiological adaptation resulting in reduced dispersal capacity. Hoffsten  showed ttability -22, thensistence . CurrentR. pubescens may have adopted a more or less sedentary behavior with limited dispersal rates leading to rare exchange of genetic material among populations and thus the evolution of locally restricted haplotypes and AFLP fragments. Nevertheless diversity indices and shallow genetic population structure show that widespread postglacial dispersal from a southern refuge and occupancy of habitats north of the Alps was possible, highlighting the species' high physiological dispersal capacity. This apparent contradiction suggests a shift in dispersal behaviour or availability of habitat between the early postglacial and today. Our study species shows Pleistocene persistence and postglacial colonisation from a single refugial source in the southwestern Alps, a pattern hitherto unknown in aquatic insects. Both aspects highlight the specificity of aquatic species responses to past and potentially future climate change.Our study shows that changes in habitat availability through time or plasticity in ecological life history traits can shape a species' distribution pattern and genetic population structure. This is particularly true for high specialised species. In response to inhabiting a very stable but generally harsh environment (tufa springs), The authors declare that they have no competing interests.CHME, PH, and SUP conceived and designed the study. CHME carried out the molecular genetic studies. CHME and SUP analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.Regional differentiation based on mtCOI data.Click here for fileFST).Regional differentiation based on AFLP data (Click here for file


Dyckia distachya, an endangered bromeliad from southern Brazil, which will be useful to assess the population genetic structure and reproductive success in introduced and natural populations of this species. Twenty microsatellite loci were developed from an enriched genomic library, and nine of these were amplified. The loci were characterized in 43 individuals from introduced and wild D. distachya populations. All nine loci were polymorphic, with four to ten alleles per locus. In an introduced population the observed and expected heterozygosities ranged from 0.136–0.667 and 0.543–0.877, respectively, while in a wild population it ranged from 0.000 to 0.895 and from 0.050 to 0.811, respectively. The development of these microsatellite markers will contribute to investigations of the reproductive potential and viability of introduced populations of D. distachya as well as the single known wild population. Cross-amplification in other Bromeliaceae species was successful, with high rates in four loci, demonstrating the applicability of these microsatellite markers in other taxa.Microsatellite markers were isolated in  Dyckia distachya Hassler is a rare, endemic bromeliad (subfamily Pitcairnioideae) with a rupicolous, rheophytic habit that lives on riverbanks along the rapids of the Pelotas and Uruguay Rivers in southern Brazil [et al. [D. distachya is facing extinction due to increased hydroelectric construction along the rivers. Originally, eight wild populations of D. distachya were known along the Uruguay and Pelotas Rivers. Currently, only one wild population is known, located in Salto do Yucumã in Turvo State Park, Derrubadas, Rio Grande do Sul State, which is on the border between Brazil and Argentina. Therefore, D. distachya is on the Official List of Brazilian Endangered Species [D. distachya populations were rescued and conserved ex situ. However, the process occurred rapidly, without appropriate planning regarding the genetic structure of the populations. Initially, the plants were kept in nurseries before being introduced in environments similar to their origin. In addition to the long-term monitoring of introduced populations, knowledge of the genetic diversity of these populations is needed so that they can be managed appropriately and the success of the introduction is ensured.n Brazil . The spen Brazil ,3. Accorn Brazil  and Reis [et al. , D. dist Species . During D. distachya in one introduced population and in the only one known wild population. The cross-amplification of these loci was examined in 22 related species from three subfamilies of Bromeliaceae. The transferability of these microsatellites will be helpful in conservation genetics studies in many bromeliad taxa.Here, we report the isolation and characterization of a set of nine microsatellite loci for D. distachya loci amplified robustly and proved to be polymorphic. Markers isolation involved the development of a genomic enriched library. A total of 96 positive clones were captured and 48 colonies were sequenced, of which 24 clones showed simple sequence repeats (SSR) motifs. Twenty primer pairs were designed in which the product size ranged between 100 and 300 base pairs. Nine of these primer pairs generated consistent patterns of amplification and were used for further characterization of two D. distachya populations. The primer sequences and product sizes are shown in Nine from 20 p < 0.001; In the introduced population, the number of alleles per locus ranged from 4 to 10, with an average of 6.2, and the expected and observed heterozygosities ranged from 0.136 to 0.667 and from 0.543 to 0.877, respectively. In the wild population, the number of alleles per locus varied from 2 to 8, with an average of 3.8, and the expected and observed heterozygosities ranged from 0.000 to 0.895 and from 0.050 to 0.811, respectively . Four loD. maritima). Locus Dd10 and Dd20 amplified in all species tested, and locus Dd07 and Dd16 were successful in 86.3% of the species evaluated. The 12 genus tested correspond to three representative Bromeliaceae subfamilies from Atlantic Rainforest, indicating high potential for use of these four loci in phylogeography and population genetic studies of many Bromeliaceae species, mainly for Dyckia genus.Cross-species amplification was successful in different degrees . All locVriesea gigantea, another species of bromeliad [In our study, introduced population showed, on average, a higher number of alleles per locus, and higher observed and expected heterozygosities compared with the wild population . The resromeliad .D. distachya individual following the protocol of Doyle and Doyle [et al. [et al. [RsaI . The cleaved DNA was linked to two complementary oligo adapters. Polymerase chain reaction (PCR) was performed using one of the adapters as a primer. The PCR product was purified using a QIAquick PCR Purification Kit . Fragments that contained microsatellite repeats were selected using biotinylated probes (dCT)8 and (dGT)8 and isolated with Dynabeads M-280 Streptavidin magnetic particles . An additional PCR was performed using one of the adapters as a primer. The PCR products were inserted into the pGEM-T Easy vector  and used to transform XL1-Blue competent cells . Recombinant colonies were selected by blue/white screening. Ninety-six recombinant colonies were randomly selected, and 48 were double-sequenced using the BigDye Terminator version 3.1 Cycle Sequencing Kit  on an ABI PRISM 377 DNA Sequencer . Every the forward and reverse sequences obtained were aligned and edited in SeqMan , and primers were designed using the Primer3 program [To identify and characterize microsatellites, genomic DNA was extracted from fresh leaves of one nd Doyle . Microsa [et al.  with mod [et al. , using a program .D. distachya population introduced in Anita Garibaldi, Santa Catarina State, Brazil  and 21 individuals from the wild population were genotyped. The wild population was collected in Salto do Yucumã, in Turvo State Park, Derrubadas, Rio Grande do Sul State , southern edge of species original distribution and the introduced population came from the ex situ collection.To assess polymorphisms, 22 individuals from one TC-412 thermal cycler  in 10 μL reactions containing 10 ng of DNA template, 1× Taq buffer, 2 mM MgCl2, 200 μM of each dNTP, 1 pmol forward primer, 4 pmol reverse primer, 0.4 pmol universal M13 primer, and 0.5 U Taq DNA polymerase . The touchdown cycling program was used: 95 °C for 3 min, 10 cycles of 94 °C for 30 s, 58 °C decreasing to 48 °C at 1 °C per cycle for 30 s, 72 °C for 30 s, followed by 30 cycles of 94 °C for 30 s, 48 °C for 30 s, and 72 °C for 30 s, and concluding with a 10 min extension at 72 °C. The microsatellite alleles were resolved on a ABI 3100 DNA Analyzer Sequencer  and sized against the GS500 LIZ molecular size standard  using GeneMarker DEMO version 1.97 . The number of alleles, and expected and observed heterozygosities were analyzed with MSA 4.00 software [For each SSR, the forward primers were synthesized with a 19-bp M13 tail (5′-CACGACGTTGTAAAACGAC-3′) following the method of Schuelke , which isoftware  for eachsoftware .GelRed  and visualized under ultraviolet light. A locus was considered to have been amplified successfully when at least one band of the expected size was visualized. A 50-bp DNA ladder  was used as a molecular size marker. All voucher specimens of the species investigated are identified in The polymorphic markers were also tested for cross-transferability in 22 related species from three subfamilies of Bromeliaceae. The PCR conditions were the same as described above. The amplification products were separated on 2% agarose gels  stained with Dychia distachya was constructed and a total of nine polymorphic microsatellite DNA markers were characterized. The development of these markers will contribute to future population genetic studies in D. distachya and other bromeliad species. The nine loci will also be useful for identifying the effects of introduction on outcrossing and selfing rates, with a comparative analysis among introduced and wild populations of D. distachya using progeny arrays. The use of such markers will facilitate inferences about the reproductive potential and viability of the introduced D. distachya populations and will aid in the understanding of the role of genetics in plant species introduction.In the present study, microsatellite-enriched genomic library of


Phyllastrephuscabanisi), a cooperative breeder of Afrotropical rainforest, to quantify rates of gene flow and fine-grained genetic structuring within and among fragmented populations. We found genetic evidence for female-biased dispersal at small spatial scales, but not at the landscape level. Local autocorrelation analysis provided evidence of positive genetic structure within 300 m distance ranges, which is consistent with behavioral observations of short-distance natal dispersal. At a landscape scale, individual-based autocorrelation values decreased over time while levels of admixture increased, possibly indicating increased gene flow over the past decade.Dispersal affects both social behavior and population structure and is therefore a key determinant of long-term population persistence. However, dispersal strategies and responses to spatial habitat alteration may differ between sexes. Here we analyzed spatial and temporal variation in ten polymorphic microsatellite DNA loci of male and female Cabanis’s greenbuls ( Dispersal plays a major role in contemporary dynamics and the evolutionary potential of a population . HoweverIn many bird species, particularly those with a complex breeding system such as cooperative breeders, each sex faces different selective pressures and consequently shows dissimilar dispersal strategies. According to the resource hypothesis , female-a priori designation of population units may often lead to doubtful estimates of genetic structure [Genetic methods used to infer sex-biased dispersal can broadly be classified into three categories, i.e., models based on Wright’s F-statistics , assignmtructure –25. Alontructure . Despitetructure . Spatialtructure .Phyllastrephuscabanisi). Multivariate spatial autocorrelation previously revealed fine-grained genetic patterning in a variety of species [Here we apply all three methods to study both coarse-grained and fine-grained patterns of spatial genetic structure and sex-biased dispersal in a cooperatively breeding forest bird  represent the northernmost extreme of the Eastern Arc Mountains, a chain of mountains that run from south-eastern Kenya to southern Tanzania . They bo2 . Within 2 , mainly 2 ,43. At p2 ,44,45 , bandedWe genotyped two subsets of individuals that were first captured during one of two time periods of equal length: first period (1996-2000), second period (2006-2010). To avoid that temporal samples would differ in signature of dispersal because of unequal representation of young individuals, only birds aged >1 year upon first capture were included in the genetic analysis. Resulting sample sizes were as follows: NG=23F/42M, CH=28F/36M, FU=9F/5M, ND=10F/7M; second period (2006-2010): MB=18F/35M, NG=22F/32M, CH=35F/20M, FU=14F/10M, ND = 8F/8M. Individuals sampled during the first period and still alive during the second one, were included in the first period only. DNA was isolated by boiling in a 5% Chelex solution (Biorad) after an incubation period of 90 min at 55° C in the presence of 100 µg proteinase K . PCR proWe applied a Bayesian clustering algorithm as implemented in the software program STRUCTURE to assess the number of distinct genetic clusters and level of admixture in each period separately . We discWe assessed fine- and course-grained spatial patterns of genetic structure by quantifying the association between matrices of pairwise spatial and genetic distances through spatial autocorrelation analysis ,57,58. AWe used three different approaches to assess the extent of female-biased dispersal. First, we performed spatial autocorrelation analysis at a local (no GPS locations of captures available in fragment MB during the first period) and landscape scale (both sampling periods) and assessed similarities between male and female correlograms.IS, FST, mean (mAIc) and variance (vAIc) of the corrected assignment index AI [IS values. Allelic frequencies of the philopatric sex are more strongly driven by genetic drift than by gene flow and are hence predicted to show higher levels of genetic differentiation (FST) compared to those of the dispersive sex. Assignment indices reflect the probability that a multilocus genotype occurs in the sampled population, and the most dispersive sex is predicted to show lower mAIc values  and higher vAIc values (the dispersing sex is expected to contain an assembly of both residents and dispersers). Significance and null distributions of all four statistics were assessed using 10000 permutations.Second, we used four statistical descriptors as implemented in FSTAT 2.9.3.2: Findex AI ,61. WhenThird, we performed analyses of molecular variance (AMOVA) for each sex separately, i.e., to assess whether males and females showed similar patterns in genetic variation partitioning among and within fragments. Largest proportions of genetic variation attributed to the between-fragment component are expected in the most philopatric sex. Next we ran a hierarchical AMOVA on the combined male and female data set with sex as ‘region’ and fragment as ‘population’ to simultaneously estimate the differentiation among sexes and fragments. 999 random permutations were used to assess the level of significance and all AMOVA analyses were performed in GENALEX version 6.41 . Maximum Euclidian distance analysis showed that 100% of all male fledglings were recaptured within 300 m from their natal site during their first year, while 22% of all females showed dispersal distances larger than 300m. None of the birds however dispersed further than 600m in their first year . When inP > 0.05 after Bonferroni correction [There was no evidence for scoring errors due to large allele drop-out or stutter. Loci were highly polymorphic in all populations and consistent with Hardy-Weinberg equilibrium . There rrection , except We identified two genetic clusters (K=2) in period 1 (ΔK=183), whereby population CH was separated from all other populations . In peric values were significantly higher in females in period 2 (p=0.03). Under the assumption of equal dispersal rates among sexes, a permutation-based null distribution gave rise to smaller deviations between female and male vAIc values as compared to the observed difference in 97% of all cases (ST= 0.077) and males (FST= 0.092) were treated independently, and near-identical trends were observed in period 2 . Likewise, hierarchical AMOVA’s indicated similar levels of differentiation  scale, positive genetic structure was only observed in males, reflecting sex-biased dispersal at small spatial scale . AlthougBoth at a local (within-fragment) and regional (between-fragment) spatial scale, Cabanis’s greenbul populations of the Taita Hills forest archipelago showed evidence of genetic sub-structuring, the extent of which gradually decreased with increasing geographical distance. Within the large forest fragment (MB), dispersal was asymmetrical between sexes, with females appearing as the most dispersive sex. At a regional scale, however, such sex bias was no longer apparent. Genetic connectivity increased over time, but there was no unequivocal evidence that this was due to a disproportional increase in gene flow by the most dispersive sex.c is expected to outperform the other three methods [c estimation only.Patterns of dispersal are notoriously difficult to study, even when collecting large numbers of field observations or capture/recapture data over multiple years. Here we show that individual-based statistical analysis of indirect measures, such as those based on highly variable neutral genetic markers, allows inferences about sex-specific dispersal and population structure at high resolution and in a cost effective way. Results from our autocorrelation analysis support the central view that females are the most dispersive sex in birds, although this trend was not supported by subsequent assignment analysis. Because the sample size for within-fragment analysis was only moderate in our study, these results need to be interpreted cautiously. The lack of evidence for sex-biased dispersal at a regional scale, however, may be attributed to several, non-exclusive factors. First, local and regional spatial dynamics have been shown to be driven by different selection pressures. At small spatial scales, asymmetrical dispersal is generally assumed to result from selection against incestuous breeding or from differential resource acquisition ,18,63. LPcabanisi. , rather than dispersal limitation per se, is at the base of this fine-grained structuring. Pcabanisi.  is group-living, with adults occupying the same territory for up to 13 years (inferred from the long-term Taita ringing database). Within the study area, the species displays facultative cooperative breeding behavior with parents selecting identical nest sites during consecutive breeding cycles [Pcabanisi.  is likely generated by philopatry and short dispersal distances, a result that is consistent with studies on other cooperatively breeding species [Autocorrelation analysis yielded fine-grained  spatial genetic clustering in males only, indicating that males were genetically more strongly correlated than expected by chance when separated at distances smaller than 1000-1250m. As no obvious barriers were present in fragment MB and highly mobile species such as birds are expected to be able to cross such distances without much effort, we believe that the social (breeding) structure of g cycles . Albeit g cycles , . A decrease in genetic autocorrelation over time may reflect an increase in genetic drift and/or in long-distance gene flow . An incrt decade . Finallyt decade , which iIn conclusion, individual-based genetic analysis revealed female-biased dispersal at a local spatial scale but failed to demonstrate a similar pattern at a larger  scale. Such fine-grained genetic structuring remained undetected in earlier population-level studies on the same populations. While our analysis also suggested increased gene flow among isolated populations over time, the short time-frame involved  still hampers predictions on longer-term connectivity trends within bird populations of the Taita forest archipelago.


Cajanus cajan) is an annual or short-lived perennial food legume of acute regional importance, providing significant protein to the human diet in less developed regions of Asia and Africa. Due to its narrow genetic base, pigeonpea improvement is increasingly reliant on introgression of valuable traits from wild forms, a practice that would benefit from knowledge of its domestication history and relationships to wild species. Here we use 752 single nucleotide polymorphisms (SNPs) derived from 670 low copy orthologous genes to clarify the evolutionary history of pigeonpea (79 accessions) and its wild relatives (31 accessions). We identified three well-supported lineages that are geographically clustered and congruent with previous nuclear and plastid sequence-based phylogenies. Among all species analyzed Cajanus cajanifolius is the most probable progenitor of cultivated pigeonpea. Multiple lines of evidence suggest recent gene flow between cultivated and non-cultivated forms, as well as historical gene flow between diverged but sympatric species. Evidence supports that primary domestication occurred in India, with a second and more recent nested population bottleneck focused in tropical regions that is the likely consequence of pigeonpea breeding. We find abundant allelic variation and genetic diversity among the wild relatives, with the exception of wild species from Australia for which we report a third bottleneck unrelated to domestication within India. Domesticated C. cajan possess 75% less allelic diversity than the progenitor clade of wild Indian species, indicating a severe “domestication bottleneck” during pigeonpea domestication.Pigeonpea ( One common feature of domesticated organisms is reduced genetic diversity compared to their wild relatives. Two major forces that cause the reduction in genetic diversity are small population sizes (“founder effect”) that occur during the initial formation of a domesticated lineage, and selective sweeps and/or directional selection for genes associated with domestication traits As much as domestication is a human-driven process, it can also be influenced by random gene flow from wild relatives. Many crops, particularly minor crops of regional importance, are still grown alongside their wild relatives, increasing the opportunity for gene flow between cultivated and non-cultivated populations. Although such gene flow reduces our ability to characterize domestication-related processes, its occurrence over protracted periods can allow for the contribution of novel traits from locally-adapted wild populations of related species into domesticated forms Cajanus cajan (L.) Millsp.) is a widely adapted, drought tolerant food legume crop cultivated throughout the semi arid tropics and subtropics. Though considered a minor crop, pigeonpea is of considerable importance in areas of South Asia (mainly on the Indian-subcontinent), Africa, the Caribbean and Latin America, where it is a prominent source of protein nitrogen in the human diet, as well as wood for fuel and light duty structural applications such as thatch for roofing. Grown on 4.63 million hectares, pigeonpea ranks 6th among grain legumes in production Cajanus is composed of 34 species C. cajan, a feature that has enabled the use of inter-specific crosses in breeding programs Cajanus species are endemic and confined either to Southern/South-Eastern Asia or Australia Cajanus species are parts of species complexes that arose through current or recent natural gene flow.Pigeonpea  in Africa C. cajanifolius), as well as archaeological remains, linguistic evidence, and a variety of uses in the daily cuisine within India Morphological evidence suggests that C. cajan is to introgress genetic diversity from the wild gene pool Pigeonpea germplasm represents a diverse set of landraces and heterogeneous feral forms that are adapted to various agro-ecological settings Cajanus species and inferring patterns of domestication, we examined allelic variation in domesticated pigeonpea and its wild relatives using a set of gene-based single nucleotide polymorphisms. The genetic signatures of domestication that we identify suggest a primary bottleneck within subtropical India, the likely center of domestication, and a nested bottleneck associated with pigeonpea that is cultivated in disperse tropical regions, which we speculate is the consequence of breeding for adaptation to a new environment. Moreover, we provide evidence of both modern and archaic gene flow between pigeonpea and wild relatives, including a third genetic bottleneck in Australian Cajanus species that is unrelated to the India-centric domestication of modern pigeonpea.With the objective of understanding genetic diversity among C. cajan (79 accessions) and its wild relative relatives (31 accessions)  were assayed in a total of 110 accessions representing cultivated essions) , all of C. cajan accession ICP 28 and C. scarabaeoides accession ICPW 94 in a set of low-copy orthologous genes. These two species span an evolutionary distance that is wider than the proposed domestication gradient from cultivated C. cajan to its presumed progenitor C. cajanifolius. Excluding failed sequencing reactions and monomorphic loci, single nucleotide polymorphisms were identified in a total of 670 unique genes from which 752 SNPs were used to design a GoldenGate genotyping assay  and thus C. scarabaeoides was used to root the Neighbor Joining tree. Similarly, a parsimony phylogenetic analysis was performed using all wild accessions and two representative samples of the domesticated (C. cajan) accessions. Both parsimony and Neighbor Joining trees resolved congruent topology with overall similarity on major clades  are sister to the large group of domesticated genotypes in maximum parsimony analysis with strong bootstrap support (91%), validating C. cajanifolius as the most recent progenitor for pigeonpea. These C. cajanifolius accessions, which we speculate are true wild representatives, are closely related to the wild non-scarabaeoides species of Indian origin with the expected affinities to coherent sets of C. platycarpus and C. sericeus genotypes, as well as to individual representatives of C. albicans and C. crassus.The tree topology in C. cajan C6364 and ICP 11975 were the most basal genotypes among the domesticated accessions. Interestingly, C. cajan C6364 is annotated as a naturally occurring, semi-domesticated and rarely found Australian woody herbaceous pigeonpea, while ICP 11975 is a genotype from the Philippines. The distant relationship of C. cajan C6364 to other Cajanus spp. of Australian origin, and its close affinity with domesticated C. cajan, is consistent with the origin of C. cajan C6364 as a feral genotype, and in fact both C. cajan C6364 and ICP 11975 show evidence of genetic admixture (see below). The data also suggest that at least three accessions are misclassified, as they are annotated as wild non-cajan species  but were well integrated into the domesticated group.Phylogenetic tools are not suited to analysis of individual genotypes with strongly reticulate histories, as is the case with genotypes from a single species or breeding pool. Nevertheless, Neighbor Joining analysis does identify similarity among sets of genotypes see  and manyC. scarabaeoides group (group I) from the remaining wild accessions of both Australian and Indian origin (group II). Within group II the Australian set forms a homogenous subgroup and the Indian genotypes form a more diverse assemblage, consistent with the previous phylogenetic analysis. Group III contained the domesticated C. cajan cluster. The low level of variation of the domesticated cluster is reflected in the tight clustering of most genotypes. Interestingly, ICP 11975 is an outlier from the main domestication group in C. cajan from the Philippines (ICP 12765), as well as C. cajanifolius accessions ICPW 29 and ICP 15629, and C. lineatus ICPW 46, were also outliers in the PCoA analysis  using GenAlEx v.6.3 analysis . AnalysiC. cajan, sampling of multiple accessions within a species was limited. Thus, the population genetic processes that STRUCTURE is sensitive to occur within the cultivated group of accessions, but not the more basal groups of diverged species. Nevertheless, in the combined analysis STRUCTURE served to delimit the primary subdivisions circumscribed by phylogenetic analysis and provided the basis to investigate the possibility of gene flow between groups.To investigate genetic relationships among accessions and to search for evidence of genetic admixture between cultivated and wild genotypes, we utilized the Bayesian algorithm STRUCTURE C. scarabaeoides accessions, congruent with previous phylogenetic and Principal Coordinate Analysis (PCoA)  of wild India accessions. By contrast, 12% of these 209 SNPs were shared between domesticated accessions and only one of the wild India species; for example, half of this set  was associated only with C. crassus, the most basal of wild-India species. This observation is consistent with admixture between domesticated accessions and C. crassus and raises the possibility of significant rates of gene flow between domesticated lineages and other species within the wild-India clade.When one has knowledge of the biology and history of a set of accessions, analyzing the partitioning of accessions into “K” subgroups can be informative . At optis (PCoA) , 2 and 3s (PCoA) , and onlC. cajan accessions of Indian origin . Fifty-eight SNPs (29 loci) among 15 cultivated accessions were implicated in the simple admixture. We analyzed the cultivated accessions by themselves to determine if genetic structure could be detected without the confounding effect of far greater differences among the wild taxa. As shown in C. cajan ICP 11975 was there evidence of genetic contribution from the C. scarabaeoides gene pool to a domesticated genetic background. This is interesting given the status of ICP 11975 as a Philippines “C. cajan” accession. ICP 11975 was also unusual as an outlier in the PCoA analysis  was admixed in nearly equal proportions from both wild and domesticated backgrounds, and had been previously proposed as an admixed genotype based on morphology by van der Maesen .Only in the case of analysis , reflectscarabaeoides; Group II, wild-Australia and wild-India; Group III, domesticated-India, domesticated-tropical, and domesticated Philippines. As shown in To find groups with reduced genetic variation, we used Analysis of Molecular Variance (AMOVA) to partition variance among hierarchical sets of individual genotypes (three groups and six populations) that were circumscribed by a combination of phylogenetic analysis, geographical origin, breeding history, and the outputs of PCoA and STRUCTURE: Group I, wild-scarabaeoides), with rates ∼3-fold higher than within the wild-Australian group (∼37% polymorphism compared to ∼12%) (Genetic polymorphism was highest within wild groups of Indian origin (both wild-India and wild-to ∼12%) . In contscarabaeoides . Moreover, the results permit assignment of C. cajanifolius as the most probable progenitor species, and they allow us to infer the origin of modern cultivated pigeonpea from nested population bottlenecks, with an initial domestication in India and subsequent spread of cultivation to tropical regions beyond India.Here we have investigated the genetic diversity and population structure of domesticated pigeonpea and its wild relatives in the genus Crop domestication is accompanied by genome-wide reduction in genetic diversity C. cajan group (excluding the Philippines accessions) in comparison to 283 SNP markers that were polymorphic among the progenitor wild-India accessions. It is noteworthy that landraces (primitive cultivars) and improved (elite) cultivars that comprise the domesticated portion of our genotype panel  CajanusAlthough limited within-taxon sampling reduces our ability to assess genetic diversity in the wild species, we can still make preliminary assessments of diversity in the non-C. scarabaeoides wild-India species, including the sister relationship of these two lineages in Parsimony analysis  notes that multiplication of C. lineatus in experimental gardens is common practice in India and therefore the occurrence of spontaneous hybrids involving C. lineatus and nearby cultivated C. cajan should be expected and has been observed. Here we identified one highly admixed genome described as C. lineatus.Gene flow subsequent to initial domestication is also likely to have contributed to the character of cultivated pigeonpea, and the haplotypes of several accessions provided evidence of recent genetic admixture between wild and cultivated gene pools. The potential for gene flow is significant, as insect-aided natural out-crossing for pigeonpea may range up to 70% C. lineatus accession, the highest rates of admixture from the wild-India population were observed for two Philippines accessions (ICP 12765 and ICP 10880), ICRISAT reference set accession (ICP 11975), and two accessions of C. cajanifolius (C7847 and ICPW 32) collected from the field. Of particular interest are two accessions of C. cajan (ICP 15629 and ICPW 29) that possess genomes that are predominantly domesticated, but with 5–10% membership of the wild-Indian group. C. cajan ICP 15629 and ICPW 29 served as parental lines to develop a stable cytoplasmic male sterility (CMS) system in pigeonpea. The CMS accession, ICP 2039A, was derived from an inter-specific hybrid of ICPW 29 and cultivar ICP 11501 In addition to the above referenced C. cajanifolius is the most recent progenitor of cultivated pigeonpea and supports India as the most likely center of pigeonpea domestication. However, crop domestication is a progressive process that may involve both independent derivations within the range of the ancestral species . Thus pigeonpea breeding and improvement programs would benefit from the continued and expanded use of this bounty of genetic diversity prevailing in the wild gene pool.This molecular diversity study corroborates the long held alpha-taxonomic hypothesis that C. cajan and allied species used in this study derive from diverse environments in Asia, Australia, Africa and the Caribbean. With the exception of 12 wild accessions, which were acquired from the Western Australia Herbarium and were originally collected from the field in Australia, all other genotypes were obtained from the gene bank of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) or from germplasm resources at Panjabrao Deshmukh Agricultural University (PDAU), University of Agricultural Sciences (UAS)-Bangalore, or UAS-Dharwad. Within the cultivated genotypes several accessions derive from core and mini-core accessions circumscribed by ICRISAT based on various morphological descriptors, and agro-morphology traits and SSR diversity data As listed in C. cajan accession ICP 28 and C. scarabaeoides accession ICPW 94 (www.comparative-legumes.org/). Polymorphisms were identified by amplicon re-sequencing and sequence alignment involving C. cajan (ICP 28) and C. scarabaeoides (ICPW 94). The target sequences were a set of primarily single copy orthologous genes, whose orthology was inferred initially from legume EST data  and subsequently based on conserved genome location in a multi-species comparative genetic analysis . Individual SNPs meeting assay design criteria, determined by Illumina Inc. using their proprietary Assay Design Tool, were converted to a 768 Illumina GoldenGate genotyping assay. Together these SNP assays survey biallelic states at 670 distinct genes. For purposes of genotyping, DNAs were extracted from the 110 Cajanus accessions using the Qiagen DNeAsy protocol using a Retch mixer mill, according to manufacturer’s instruction, and delivered to the UC Davis Genome Center DNA Technologies Core for analysis (http://dnatech.genomecenter.ucdavis.edu/). Allele calls were curated using the Illumina Beadstudio software package . To minimize the confounding effects of technical error, all SNP calls with more than 10% missing data were excluded from the analysis. Details on sequence context of SNPs, and the curated genotyping data (locus x genotype) are provided in Genotyping was based on a set of 752 SNPs discovered in a comparison of 1440 sequenced amplicons between As our genotypes included both diverged species and samples from the breeding pool where interbreeding and/or admixture occurs or has occurred often in the recent past, we took both phylogenetic and population genetic approaches to the data. We first used phylogenetic methods to distinguish the major groups, having a greater number of informative markers than previous studies. We then took the least well-resolved group of domesticated germplasm and most closely related wild species and analyzed it with population genetic approaches. Phylogenetic analyses were conducted using both the character based phylogenetic analysis of maximum parsimony and the distance-based analysis of Neighbor Joining. The maximum parsimony analysis was based on the complete data set of the concatenated SNPs at 670 loci and the analysis was performed using PAUP* 4.0b10 ad hoc static ΔK based on the rate of change in the natural log probability of data between successive K values as described by Evanno and colleagues Genetic structure was analyzed using the program STRUCTURE 2.1 Based on the output of STRUCTURE and principal coordinate analysis (PCoA) (see below), combined with phylogenetic inferences and common knowledge of geography and breeding history, we circumscribed 6 subgroups for further analysis. Analysis of Molecular Variance (AMOVA) was conducted, based on the hierarchical model and permutational procedures of Excoffier and colleagues Table S1Details of individual Canajus spp accessions.(DOCX)Click here for additional data file.Table S2SNP assay design and genoytpe data.(XLSX)Click here for additional data file.Table S3Ln of the probability and its variance for K from 1 to 10, provided as supporting information for .(DOCX)Click here for additional data file.Table S4Ln of the probability and its variance for K from 1 to 5, provided as supporting information for .(DOCX)Click here for additional data file.