Hylomyscus) and shrew (Sylvisorex) species complexes, with an aim to better determine how historical paleoenvironmental processes might have contributed to the EABH’s high diversity. We analyzed complete SNP matrices of > 50,000 RAD loci to delineate populations, reconstruct the history of isolation and admixture, and discover geographic patterns of genetic partitioning. These analyses demonstrate that persistently unsuitable habitat may have isolated multiple populations distributed across montane habitat islands in the Itombwe Massif and Albertine Rift to the west as well as Mt Elgon and Kenyan Highlands to the east. We detected low genetic diversity in Kenyan Highland populations of both genera, consistent with smaller historical population sizes in this region. We additionally tested predictions that Albertine Rift populations are older and more persistently isolated compared to the Kenyan Highlands. Phylogenetic analyses support greater historical isolation among Albertine Rift populations of both shrews and mice compared to the Kenyan Highlands and suggest that there are genetically isolated populations from both focal genera in the Itombwe Massif, Democratic Republic of Congo. The Albertine Rift ecoregion has the highest mammalian tropical forest species richness per unit area on earth. Our results clearly support accelerating efforts to conserve this diversity.The Eastern Afromontane biodiversity hotspot (EABH) has the highest concentration of biodiversity in tropical Africa, yet few studies have investigated recent historical diversification processes in EABH lineages. Herein, we analyze restriction-site associated DNA-sequences (RAD-Seq) to study recent historical processes in co-distributed mouse ( Understanding the evolutionary processes that generate and maintain biodiversity is essential for identifying regions of global conservation importance ,2. Over Sylvisorex shrew and Hylomyscus mouse lineages [Recently, the first mammalian multi-locus comparative phylogeographic study of the EABH supported the persistence of currently disjunct shrew and rodent populations over multiple Pleistocene climatic cycles. This same study also uncovered morphologically cryptic diversity in several lineages . Demos elineages  with 2–3Due to the previous study’s limited genetic sampling (3–6 loci), we regard these findings as provisional and in need of further testing ,12. PopuHylomyscus denniae complex (African montane wood mouse) and the Sylvisorex granti complex (Grant’s forest shrew). Within the H. denniae complex we obtained RAD-Seq genomic data from H. denniae, H. vulcanorum, H. endorobae and H. kerbispeterhansi individuals that were sampled from 8 mountain ranges (populations) across the Albertine Rift and Kenya Highlands  allow us to explore Pleistocene refugial population structure and pulses of admixture and characterize genetic variation across the entire range of two co-distributed African montane endemics. Although we base inference from 1–6 individuals per each of 10 localities, data from large numbers of unlinked loci allow highly resolved inference even with few individuals [Here we use high-throughput-sequencing to investigate genome-wide patterns of population structure and gene flow within and between two co-distributed EABH montane-endemic species complexes; the ighlands . In addiividuals ,21. Furtividuals –24.Several aspects of the geological and climatic history of the EABH lead to straightforward expectations for biodiversity patterns. First, both paleo-climatic and geological data from many studies indicate that the Albertine Rift is older, more topographically complex, and more climatically stable than the Kenya Highlands to the east ,26. It iHylomyscus denniae complex (African montane wood mouse) and the Sylvisorex granti complex (Grant’s forest shrew). The combination of broad geographic sampling in this study and deep population sampling made possible by genome-enabled phylogeographic data enable a more biogeographically informative and better supported evolutionary history of these species groups that offer significant departures from earlier studies.To test this hypothesis, we use high-throughput genome-wide sequencing to estimate historical demographic parameters underlying relative isolation times, historical effective population sizes, levels of population structure and post-divergence gene flow within and between two co-distributed EABH montane-endemic species complexes: the Hylomyscus and Sylvisorex from 8 and 9 montane blocks across the Eastern Afromontane region, respectively. Within the AR and KH, members of the H. denniae and S. granti complexes are co-distributed on every mountain. We collected 2–4 individuals of each genus on 7 mountain ranges where they co-occur. All specimens and tissue samples were deposited in the Field Museum of Natural History (FMNH) under the approval of the FMNH Institutional Animal Care and Use Committee  for library preparation and high-throughput sequencing following the methods outlined in Baird et al. , HohenloThe two RAD references came from a single individual per genus after preliminary analysis identified which had the greatest number of unique RAD tag clusters. Custom perl scripts were used to group identical sequences represented by 5 to 500x coverage. Reads for each individual were aligned to the RAD pseudo-reference genome using BOWTIE  using sei.e. q > 15, 4x coverage, and 65% population coverage) and applied our own custom filtering pipeline to minimize both false positive genotype detection of heterozygotes and the impact of paralogous regions. VCFtools was used to retain only those SNPs for which a minimum and maximum of 2 alleles were present. To ensure coverage adequate for correctly evaluating heterozygous sites and control for paralogous sequence variants [We used the relaxed filter applied by Floragenex to call genotypes  and the Sylvisorex granti species group . Two closely related outgroups per species group were also sampled. Within Kenya, a newly described species of Hylomyscus from the Hylomyscus anselli species group is co-distributed with Sylvisorex in western Kenya [H. denniae and S. granti complexes are co-distributed on the majority of mountain ranges sampled to date  were estimated using components of VCFtools v4.0 package. To visually explore how many populations the samples were derived from and how best to assign individual genotypes from these populations, we implemented a principal component analysis (PCA) using the software SmartPCA [Hylomyscus denniae complex and species therein n = 56,194; Sylvisorex granti complex and species therein, n = 52,580).Summary statistics (SmartPCA  in EIGENSmartPCA  we used K) of hypothetical ancestral populations and then infers the proportion of each individual’s genome derived from these ancestral populations. We estimated ancestry coefficients across a range of K values equal to the number of sampled mountain ranges plus one (1–11 for the Sylvisorex group and 1–8 for the Hylomyscus group). Separate analyses were carried out for individual species distributed on two or more montane blocks across a range of K values. Ten replicates were run for each value of K for both the inter- and intra-specific data sets. Cluster membership coefficients were identified using the cross-entropy criterion implemented in sNMF based on the prediction of masked genotypes (5% of the data set). This was done to evaluate the error of ancestry estimation and guide choice of the number of ancestral populations. Variance among runs of the cross-entropy statistic was evaluated by carrying out 10 replicates for every K value. sNMF results were visualized in R [K values. To further visualize population structure, we used the Neighbor-Net algorithm [We further examined population structure and assignment of genotypes to populations using the population clustering method implemented in sNMF , a new mzed in R  across algorithm  implemenlgorithm  to genern = 1–6 within Hylomyscus; n = 1–10 within Sylvisorex) including possible admixture with the closest known outgroup in both analyses. The best-supported number of migration events was inferred by dividing the residual covariance between each pair of populations, i and j, by the average standard error across all pairs. Because the TreeMix model is heavily parameterized, the program author’s caution against placing strict confidence on p-values generated from the Wald statistic. Instead, they recommend implementation of the three-population test using the f3 statistic [f4 statistic. These tests were carried out and compared with the Wald migration edge p-value.To find the population graph that best described the relationships among populations in the datasets, we used the software TreeMix  that indtatistic  and the http://tree.bio.ed.ac.uk/software/tracer/). All parameters had effective sample size (ESS) values > 100. Convergence of the posterior topology was assessed visually from trace plots. After removal of the first 20% of sampled trees as burn-in, maximum clade credibility trees were generated with TreeAnnotator using BEAST 2.0. No estimate of nuclear mutation rate is available for Hylomyscus or Sylvisorex, so we used the mutation rate 1.0 × 10−8 per year estimated from divergence in mammals [We estimated population relationships from SNPs in SNAPP (part of BEAST 2.0) which is software used to infer population trees and demography from independent bi-allelic markers . This pa mammals ,48 and aSylvisorex lunaris for Sylvisorex, and Hylomyscus kerbispeterhansi for Hylomyscus). Maximum likelihood analyses were carried out in Mathematica 8 using the supporting notebooks from Lohse & Franz [To test whether patterns of shared polymorphism between AR and KH populations are due to admixture, ancestral structure, or incomplete lineage sorting in ancestral panmictic populations, we implemented the maximum likelihood method described in Lohse et al.  and Lohs & Franz  and Hear & Franz . We asseHylomyscus and 238,291,000 single-end reads from 31 individuals within Sylvisorex. We extracted 56,194 SNPs from 20,021 RAD loci shared by all individuals in the Hylomyscus denniae complex and 52,580 SNPs from 18,658 RAD loci shared by all members of the Sylvisorex granti complex. With outgroups included, there were 47,427 and 18,385 SNPs in the Hylomyscus and Sylvisorex data sets, respectively.We obtained 143,726,000 single-end Illumina reads of 90 bp length from 28 individuals within Hylomyscus consistent with current taxonomy at the species level  received only slightly less support. Sylvisorex granti and S. mundus were supported as independent while S. vulcanorum was grouped into 3 clusters representing populations from Itombwe, Kahuzi, and three combined populations from eastern AR. Population clustering analyses at the intraspecific level within Sylvisorex support K = 3 for S. vulcanorum and K = 2 for S. mundus; they are congruent with the inferences made from the population PCA data  ranged from 0.005 to 0.123 in Hylomyscus and from 0.008 to 0.090 in Sylvisorex  but were highly diverged from each other among three populations of Sylvisorex  while in S. mundus all KH populations were notably divergent from other populations in pairwise comparisons (i.e. Fst = 0.184–0.310).Populations from the western side of the AR (Kahuzi and Itombwe) and across the AR valley (Mgahinga and Kahuzi) grouped together for Hylomyscus and 11 trees (m = 0–10) inferred for populations within Sylvisorex. These population graphs are consistent with population relationships found in previous phylogenetic analyses [Hylomyscus, the H. denniae population was consistently recovered as sister to H. vulcanorum, whereas previous species tree analyses at this node were unresolved. Analysis of residuals for m = 0–6 showed that the graph that best fit the data inferred four interspecific migration events, the largest one from the ancestor of central Kenyan H. endorobae (Mt Kenya + Mau) to the ancestor of H. vulcanorum populations from Kahuzi + Mgahinga that had a weight  of 8%; three additional interspecific migration events had weights between 1–4%  are among S. vulcanorum populations from the AR .To further examine these migration events we next used three and four population tests ,51 to stH. endorobae and S. mundus) and AR species  to have the highest ML scores (S. mundus into S. vulcanorum and S. granti) was not significantly different from a strict divergence model that accounted for ancestral polymorphism . In Hylomyscus, the best-supported model involved admixture from H. endorobae into H. denniae and H. vulcanorum  yet the timing of admixture for that model was indistinguishable from the divergence time estimate between H. denniae and H. vulcanorum such that it collapses to a strict divergence model  were similar to previous estimates obtained in *BEAST using mtDNA and intron sequence data  with theulations . The SylHylomyscus and Sylvisorex populations at 3–5 loci [Hylomyscus and Sylvisorex specifically, the AR may retain greater population structuring and intra-specific genetic diversity either as a result of having larger and more stable populations, or because it is also conforms to a meta-population with more demes having lower local extinction rates and/or lower migration rates.This study used RAD-Seq to generate representational genomic data sampled from two co-distributed sets of populations of endemic Afromontane small mammals to test the hypothesis that AR populations are older and more persistently isolated than KH populations. To this end, we used the data and subsequent parameter estimates and model tests to compare with the predictions of greater genetic diversity, older intra-regional divergence times and greater population structuring in the AR regions for both species complexes. Our analyses are consistent with processes of long-term isolation within and among both species complexes, and we estimate parameters relevant to population relationships and demographic histories that were previously inferred from only one or a few independent loci ,6,7. AltThese patterns of phylogenomic structuring within the AR support the long-standing hypothesis that the eastern margins of the Congo Basin, with the AR, supported refugia as old as the Miocene. This is further supported by patterns of endemism, species richness and discontinuities in the geographic distributions of mammals ,53, birdHylomyscus, the topology of the ML tree inferred in TreeMix under the assumption of no admixture supports longer term persistence and isolation among AR lineages compared to those from the KH. Terminal population branch lengths are significantly longer in the SNAPP phylogenies for AR endemic H. vulcanorum and S. vulcanorum compared to KH endemic H. kerbispeterhansi and S. mundus. This pattern is replicated to a lesser degree for the same taxa in the TreeMix graphs with migration . This high level of partitioning between Mt Elgon and Mau populations was unexpected given the presence of lowland forest between these two montane forest blocks. This is particularly noteworthy when compared to the lower level of differentiation between Mau and Mt Kenya, which are separated by xeric non-forested Gregory Rift Valley habitat (S. mundus FST = 0.184). One possibility is that Mt Elgon and Mau populations are more stable than those in Mt Kenya. In this case, the Mt Kenya area could have been recolonized after late Pleistocene local extinction, whereas Mt Elgon and Mau populations persisted through multiple late-Pleistocene climate cycles thereby accumulating more inter-population genetic divergence with limited inter-population gene flow through the intervening lowland forest habitat.Within the KH, populations of H. kerbispeterhansi and S. mundus based on population clustering methods. However, the timing of the divergence of Mt Elgon populations from Mau in Hylomyscus, and Mt Elgon from Cherangani + Mau + Mt Kenya in Sylvisorex, are much more recent than divergence times within the AR based on estimates of split times in the SNAPP population tree  are consistent with Mt Elgon being a refugial center within the KH. Geologically, the extinct Mt Elgon volcano is much older than Mt Kenya in the East (Miocene vs. Late Pliocene-Early Pleistocene) although it does not possess more endemic mammal species than the Mt Kenya–Aberdare Mts ecosystem that is surrounded by a more arid matrix of dry woodland habitats [Intraspecific population structure is also apparent within the KH for ion tree ; these rhabitats ,57. FurtSylvisorex granti mundus from the Ruwenzori Mts endemic, S. granti granti. This is congruent with the recognition of S. mundus as a valid species. A recent revision of the Hylomyscus denniae complex similarly elevated H. denniae endorobae from Kenya to species. [Several lines of evidence support the pre-Pleistocene divergence and persistence of KH focal taxa across multiple glacial cycles with associated pulsed expansions and contractions of montane forest. Based on rough estimates of divergence times derived from population trees and reciprocal monophyly of the three endemic KH mouse and shrew species we sampled, there is little evidence for persistent connections between these two centers of Afromontane endemism. Data presented here also strongly supports the genetic isolation and deep divergence of Kenyan populations of species. .Hylomyscus denniae group  and the recognition of Sylvisorex mundus as a valid species sister to S. granti and S. vulcanorum. This is consistent with an earlier phylogeographic study using far fewer loci (14). Also congruent with these earlier analyses are phylogenetic analyses presented here that show longer branch lengths among lineages within the Albertine Rift. In contrast, the large genomic dataset analysis provides several new insights. This includes a strong signal of historic and cryptic isolation of populations within both Hylomyscus and Sylvisorex. This is apparent for montane populations from the Itombwe Massif and for two additional S. vulcanorum lineages from the AR. Multiple analyses support the AR as harboring a substantial number of cryptic lineages and this is consistent with a long regional history of isolation and environmental stability. These data also support a potential response of populations within the Sylvisorex granti group across the Albertine Rift region to climatic shifts during the Plio-Pleistocene that periodically reduced and fragmented suitable habitat. This climatic periodicity is thus congruent with earlier inferences based on multi-locus species trees and paleo-distribution models, but the strength of the response indicated by this study is much more evident. Finally, our results also strongly support the historical isolation and evolutionary independence of the Itombwe Massif populations from other Albertine Rift mountain block populations. This is consistent with previous studies that found this sub-region to house the highest species diversity within the Albertine Rift [All analyses strongly support the evolutionary independence of the three recognized species within the ine Rift ,59.S1 Appendix(XLSX)Click here for additional data file.


Glionnetia sericea is restricted to altitudes between 450 m and 900 m where the native forest vegetation has been largely lost and replaced with exotic invasives over the last 200 years. This study explores the genetic and ecological consequences of population fragmentation in this species by analysing patterns of genetic diversity in a sample of adults, juveniles and seeds, and by using controlled pollination experiments. Our results show no decrease in genetic diversity and no increase in genetic structuring from adult to juvenile cohorts. Despite significant inbreeding in some populations, there is no evidence of higher inbreeding in juvenile cohorts relative to adults. A Bayesian structure analysis and a tentative paternity analysis indicate extensive historical and contemporary gene flow among remnant populations. Pollination experiments and a paternity analysis show that Glionnetia sericea is self-compatible. Nevertheless, outcrossing is present with 7% of mating events resulting from pollen transfer between populations. Artificial pollination provided no evidence for pollen limitation in isolated populations. The highly mobile and specialized hawkmoth pollinators  appear to promote extensive gene flow, thus mitigating the potential negative ecological and genetic effects of habitat fragmentation in this species. We conclude that contemporary gene flow is sufficient to maintain genetic connectivity in this rare and restricted Seychelles endemic, in contrast to other island endemic tree species with limited contemporary gene flow.Long-distance gene flow is thought to be one prerequisite for the persistence of plant species in fragmented environments. Human influences have led to severe fragmentation of native habitats in the Seychelles islands, with many species surviving only in small and isolated populations. The endangered Seychelles endemic tree  In fragmented landscapes the degree of genetic exchange may be important for species to maintain their genetic diversity, and subsequently ensure their long-term survival. Negative genetic effects, such as inbreeding depression and loss of genetic diversity, have been observed to follow population isolation and restricted gene flow Glionnetia sericea, an endangered endemic tree of Mahé, the main island of the Seychelles archipelago. Populations of G. sericea have been reduced to small scattered patches surrounded by forests that are heavily dominated by alien invasive plants. Other studies on the Seychelles and elsewhere have demonstrated that under such circumstances native isolated plant populations can become vulnerable to loss of genetic diversity and inbreeding G. sericea is able to maintain extensive pollen flow due to its hawkmoth pollinators and whether apparently isolated G. sericea populations will maintain among-population pollen flow, and as a result will maintain high genetic diversity.Habitat fragmentation is often also accompanied by the spread of invasive plant species which have the potential to compete with native plants for pollinators G. sericea to explore whether the species has the potential to survive in relict populations, following habitat fragmentation. We assume that the current adult individuals pre-date recent fragmentation. We therefore address the following questions using adult , and juvenile and seed (contemporary gene flow/post-fragmentation) cohorts: (i) What is the extent of gene flow between discrete G. sericea adult populations? (ii) Does contemporary pollen or seed dispersal prevent genetic structuring between patches? (iii) Is G. sericea pollen limited in fragmented sites? We discuss the relevance of the results from the perspective of conservation of endangered island plant species.The aim of this study is to understand the variation in mating system in a species which is expected to have long-distance pollen-mediated gene flow and a naturally patchy distribution. We evaluate historical and contemporary gene flow among remnant populations of Glionnetia sericea (Rubiaceae) is endemic to the Seychelles archipelago. It is found on two islands, Mahé and Silhouette, and the total population size is estimated at fewer than 2500 individuals (Mahé and Silhouette Glionnetia sericea is representative of a plant community occurring in virgin forest remnants but is mostly found on inselbergs (granitic outcrops) at altitudes of about 450 m–900 m. These habitats were patchily distributed even before human colonisation. Nevertheless, current populations of G. sericea have been reduced in size by extensive deforestation during the 20th century Cinnamomum verum, Psidium cattleianum, Syzygium jambos, Falcataria moluccana, and Alstonia macrophylla have further isolated remaining populations.The rare tree G. sericea vary from extremely small  to relatively large and more continuous . The persistence of these small and fragmented communities is threatened by habitat degradation, increasing duration and frequency of drought due to global warming, and competition with invasive species G. sericea due to harsh environmental conditions (very thin acidic soils and xerophytic conditions), which invasives appear less easily to tolerate Known relict populations of Glionnetia sericea is a small to medium sized, slow growing tree (up to about 8 m), flowers are visited by two potential pollinator species, the hawkmoths Agrius convolvuli and Cenophodes tamsi which have long probosces adapted to access the nectar produced at the bottom of long narrowly tubular corollas G. sericea has non-fleshy fruits.We sampled leaf material for genetic analysis from adults, juveniles (seedlings and saplings with an estimated age range of 1 to 10 years) of six sites and seeds of five sites. Permission for these collections was received from the Seychelles Bureau of Standards on Mahé. The total number of sampled adults (181) represents 13% of the total estimated population size on Mahé. Sites included six from nine known sites on Mahé, including four inselberg habitats: Mt. Sebert: <20 trees (of which 11 sampled); Copolia: about 50 trees (of which 28 sampled); L’Exile: about 100 trees (of which 34 sampled); Morne Blanc: seven trees all of which were sampled and two mist forest sites at Mt. Jasmin and Congo Rouge  . It is pG. sericea adults, seedlings and seeds. To increase the power of our analyses we only included in our final data set individuals with a minimum of 8 typed loci. We thus analysed 181 adults and 178 juveniles (between 20 cm and 1 m height) at six sites and 595 seeds (from 24 mother trees) at five sites, see In 2009 we collected leaf material for DNA extraction from NA), observed and expected heterozygosities , and the number of private alleles (PA) were calculated using GenAlEx 6.5 RS) was calculated using FSTAT 2.9.3.2 FIS) and differentiation (FST), population specific FIS values (using 1023 permutations), and a hierarchical genetic variance analysis (AMOVA) to examine the organisation of overall genetic diversity, were calculated with Arlequin 3.5 FIS calculations we defined populations according to their contemporary geographic locations (6 populations) even though we cannot be sure that populations may be connected though gene flow. Nevertheless, calculations based on 6 populations should avoid a potential Wahlhund effect caused by ignoring sub-structuring in populations.Number of alleles  of 595 seeds we applied a maximum likelihood exclusion analysis in CERVUS 3.0, to assign the most likely candidate fathers, given known mothers Selfing rates were calculated as the number of cases where paternity analysis determined the father as the same tree as the known mother. Given that the seeds are collected directly from known mothers it is highly unlikely that the CERVUS analysis fails to detect all selfed individuals and unlikely that the unassigned seeds are selfed. Therefore we estimated the selfing rate as the proportion of selfed seeds to the total number of analysed seeds (595). Additionally, mating system was calculated using the software MLTR 3.2 The level of outcrossing and correlated paternity was estimated by a correlated-matings model implemented in MLTR 3.2 using the genotypes of the 595 seeds and their respective mothers. The software estimates the correlation of outcrossed paternity within progeny arrays (multilocus estimator).To test for the potential effects of pollinator limitation, and the relative benefits of pollen dispersal within and between populations, we conducted the following pollination experiments in 2009. For 16 trees at Copolia and 7 trees on Mt. Sebert, inflorescences, each with about 30 flowers at bud stage, were enclosed within Delnet pollination bags. In total 25 bags were used on Mt. Sebert, and 35 bags on Copolia. Within each bag the following treatments were applied, at Mt. Sebert: Selfing, using pollen sourced from flowers of the same tree; Within-population crosses using mixed pollen sourced from four or five other trees at Mt. Sebert; Between-population crosses using a random mix of pollen donors (four or five trees) from the Mt. Jasmin population; Between-population crosses using a random mix of pollen donors (four or five trees) from the Congo Rouge population; and unmanipulated (non-pollinated) flowers as complete pollen exclusion. On Copolia the same treatments were applied except for Between-population crosses which used pollen from the Congo Rouge population. As a control we determined seed set of unmanipulated flowers outside pollination bags, which were therefore openly accessible to pollinators. Pollen was transferred using a fine paint brush to viable stigmas of flowers. The period of stigma viability had been previously determined on other flowers using the Peroxtesmo Ko test Statistical analyses of the pollinator exclusion experiment were carried out in R, version 2.10.1 If possible, ripe and full fruits were collected for the controls. Often the ripe fruits were already opened and had released some or even most of their seeds, so that the number of seeds collected per tree was summed up independently from the number of collected fruits and taken as one sample for each tree. Thus, only 19 samples could be collected as a control group for Copolia and 9 for Mt. Sebert. We only sampled developed fruits for the control group and so we compared seed set values of the control group with only developed fruits of the other treatments. To test for significant differences the 95% confidence interval was calculated for the mean seed set values.FIS using INEST 2.0 HE, HO, FIS or allelic richness between groups, using a two-sided test of significance after 1000 permutations. Thus, there is no significant difference between historical and contemporary genetic diversity in G. sericea.At the species level the ten loci yielded between three and 15 alleles, with a total number of 82 alleles. To make sure that null alleles have no effect on our analyses we additional calculated FST value of 0.09, 0.07, 0.12  for adults, juveniles and seeds, respectively. FST was significantly different among all populations  as the most likely solutions indicating a delineation of L’Exile and Mt. Sebert populations from all other individuals  suggesting no genetic isolation by distance for G. sericea populations.The Mantel test showed no significant result  with 95% confidence, 55% (326 seeds) with 80% confidence. Results for the assignments with 95% and 80% confidence are presented in Realised pollen dispersal ranged between 0.34 m to 12.14 km, with nearly half of all seeds resulting from mating events of less than 20 metres . Within population pollen dispersal ranged from 0.34 m to 481.39 m and was mostly less than 10 m , between population pollen dispersal ranged from 160.71 m to 12.14 km and was mostly less than 6 km , see Fruit set was not significantly different when comparing between-population (0.31±0.06) and within-population crosses . Highest fruit set was obtained for the open controls (0.84±0.02) and this result was significantly higher than between-population  and within-population crosses , see The difference in seed set (as a proportion of developed seeds) when comparing, between-population (0.20±0.04) and within-population treatments  was not significantly different. There was no significant tree interaction effect with treatment (data not presented). The mean viable seed of the open control group was not significantly different than that from the other treatments . Viable seed set for selfed was high (0.28±0.04) and low for pollen exclusion (0.01±0.00), see Fruit set from between-population (Mt. Jasmin: 0.63±0.09 SE and Congo Rouge: 0.65±0.11 SE) and within-population crosses  was not significantly different. Highest fruit set was obtained for the open controls (0.76±0.03 SE), but this result was not significantly different to between-population  and within-population crosses , see Seed set from between-population (Mt. Jasmin: 0.36±0.04 SE and Congo Rouge: 0.35±0.06 SE) and within-population treatments  was not significantly different. There was no significant tree interaction effect with treatment (data not presented). The mean viable seed set of the control group was not significantly different from the other treatments : 0.66, 95% CI 0.61–0.70; between-population (Congo Rouge): 0.58, 95% CI 0.51–0.66; within-population: 0.62, 95% CI 0.56–0.67). Viable seed set for the selfed treatments was high (0.28±0.04) and low for the no-pollination treatments (0.01±0.00 SE), see Glionnetia sericea from the Seychelles illustrates the importance of long-distance gene flow for preventing loss of genetic diversity within populations in a highly fragmented habitat. Our results suggest extensive pollen flow across most populations on Mahé due to a strong flying and wide ranging hawkmoth pollinators, which prevents strong genetic differentiation between populations. Small and distant populations were not any more genetically isolated than larger or central populations. Artificial pollination experiments showed that despite human induced habitat fragmentation and high numbers of invasive plants, pollen quantity is not limited and pollen quality within population is high enough to prevent reduced reproductive outcome. We discuss our results in the context of the reproductive ecology of this species and their implications for persistence of moth pollinated plants in highly fragmented landscapes.G. sericea adults does not differ between small and large populations indicating historically stable populations with no apparent genetic depletion, genetic bottlenecks or genetic drift. The only population with comparatively low genetic diversity is Morne Blanc, which comprises only nine trees all of which are small in size  relative to the other populations. Morne Blanc might therefore be a relatively young population derived from a recent founder event. Across all populations, genetic diversity did not differ significantly between adults, juveniles and germinated seeds, indicating no genetic erosion from adult to juvenile cohorts. Contemporary gene flow therefore seems sufficient to maintain the existing genetic diversity across generations.The genetic diversity found in Vateriopsis seychellarum has highly inbred populations that currently show no obvious inbreeding depression Substantial inbreeding is not unusual for species that persist in small populations and have purged deleterious alleles Medusagyne oppositifolia where experimental pollinations resulted in greater viability of seeds and longer seedling survival of individuals derived from between-population crosses as compared to within-population crosses Medusagyne oppositifolia, substantial gene flow implies a greater degree of resistance to genetic erosion.For other populations, higher prevalence of inbreeding among seeds compared to juveniles or adults implies higher mortality of inbred individuals during the course of development. Selection against inbred offspring has already been observed for another endemic Seychelles tree FST and pairwise FST values indicate a certain degree of structure due to geographic isolation. We interpret this seemingly contradictory finding as a consequence of extensive pollen flow in combination with low seed dispersal. Moreover, most pollen is distributed among neighbouring trees , which could promote genetic differentiation between populations. Nevertheless, occasional long-distance pollen flow events appear sufficiently frequent to prevent strong genetic differentiation of populations. Low genetic differentiation between populations and even across islands in the Seychelles has also been shown in the coco-de-mer palm, Lodoicea maldivicaVateriopsis seychellarum showed strong genetic differentiation following habitat fragmentation The STRUCTURE analysis and the non-significant isolation by distance analysis suggest low genetic structuring, although significant overall G. sericea, then pollen could potentially be exchanged among most G. sericea populations on Mahé, the furthest distance between populations being about 12 km. Pollination by hawkmoths of the Costa Rican tree Pithecellobium elegans resulted in average pollen dispersal of 142 m and a maximum distance of 350 m G. sericea individuals and populations resulted in a reduced power to assign fathers. Nevertheless, this result shows  that long-distance pollen flow is relatively common for G. sericea and confirms the effectiveness of hawkmoth as long-distance pollinators. The low genetic structuring of the adult cohort across the whole island indicates extensive historic gene flow, possibly also due to small light-weight seeds that have the potential for wind dispersal. Genetic structuring does not increase in the juvenile cohort, supporting the view that contemporary gene flow is not restricted compared to past gene flow.Hawkmoth pollinators have been shown to move over long-distances G. sericea where even one of the smallest and most remote populations Mt Sebert (comprising only 11 known adults) receives pollen from the distal populations. Thus, population fragmentation might not necessarily have negative genetic effects subject to the dispersal capabilities of the pollinators. It may be that the highest conservation concern for G. sericea is the maintenance of its hawkmoth pollinators. Given the considerable habitat transformation in the Seychelles over the past 100 years, including widespread loss of the native forest and its replacement with exotic invasive trees and plants in both the canopy and understorey, it is possible that the larval host plant of these moths has also been much reduced. Agrius convolvuli larvae are, however, known to feed on plants from a wide range of families G. sericea-pollinator mutualism might be robust to such changes (though little is known of the host plants of the second pollinator Cenophodes tamsi). While many rare and highly fragmented plant populations in the Seychelles need urgent conservation action, our results suggest that priority should be afforded to those species that have poor pollen and seed dispersal, for which genetic rescue through artificial cross pollinations might be warranted.Compared to wind pollinated species, which can also exhibit long-distance gene flow G. sericea populations over the whole of Mahé, despite habitat fragmentation. Whether this is also the case for small  and isolated populations is not clear. Our artificial pollination experiments could not detect any pollen limitation in either of the small or large population suggesting that the hawkmoth pollinators are sufficiently abundant so far as pollination of G. sericea is concerned. Our results imply that non-manipulated, open pollinated flowers develop just as many if not more fruits and seeds compared to flowers that have been hand pollinated. Further, the small and isolated population, Mt. Sebert, had similar seed and fruit set values compared to the larger population Copolia. If pollen quantity can be associated with the amount of seed and fruit set in G. sericea this would imply similar numbers of pollination events in both populations.Historical and contemporary gene flow by pollen seems to have connected G. sericea may be vulnerable to a declining pollinator visitation frequency, for example due to a behavioural or numerical change in pollinators due to habitat transformation . Copolia has been shown  to be more isolated than the other populations and thus this lowered seed set for between-population crosses in combination with no reduced seed and fruit set for selfed flowers could hint toward potential outbreeding depression when flowers are being pollinated from distant populations. Non-pollinated flowers within bags (pollen exclusion) did produce fruits and seeds at both sites but at much lower frequencies showing that apogamy is possible but reproductive output and potentially seed viability is low. This also implies that ion e.g. . NeverthWe have to account for several caveats. Fruit set, yet not seed set, was higher for the open controls in both populations indicating that the pollination bags and/or hand pollinations might have had a negative effect on fruit development. To avoid any misinterpretation of our results we used both fruit and seed set to draw conclusions of the pollination study. The between-population crosses involved the transportation of pollen from one inselberg to another with pollen stored in open tubes overnight before being applied to flowers the following day. This may have resulted in reduced pollen viability for between-population compared to within-population treatments. Finally, in field pollination experiments, unintended pollen transfer may also result in some degree of contamination within pollination bags which could explain similar responses for the within-, between-population crosses and the controls.G. sericea is pollinated by hawkmoths that have the potential to disperse pollen over large distances and, crucially, among populations. This implies that conservation action should prioritise species with weakly dispersing pollination vectors.Despite these caveats, our study has shown that highly mobile pollinators (hawkmoths) can transport pollen effectively over relatively long-distances and that potentially wind dispersed seeds contribute to long-distance gene exchange between populations. We could also show that a possible decline in pollinator visitation frequency, which may follow habitat degradation or an increase in alien invasive plants, may decrease the species reproductive output and thus long-term population viability. So far, we could not detect strong evidence of pollen limitation even in distant populations, demonstrating a certain resistance to habitat fragmentation and/or the invasiveness of alien species of these hawkmoths species in the Seychelles. The results of this study stand in contrast to those of other recent studies on rare endemic tree species on the Seychelles that appear to be much more vulnerable to genetic differentiation and inbreeding


Castor fiber) throughout western and central Europe is considered a major conservation success. Traditionally, several subspecies are recognised by morphology and mitochondrial haplotype, each linked to a relict population. During various reintroduction programs in the 20th century, beavers from multiple source localities were released and now form viable populations. These programs differed in their reintroduction strategies, i.e., using pure subspecies vs. mixed source populations. This inhomogeneity in management actions generated ongoing debates regarding the origin of present beaver populations and appropriate management plans for the future. By sequencing of the mitochondrial control region and microsatellite genotyping of 235 beaver individuals from five selected regions in Germany, Switzerland, Luxembourg, and Belgium we show that beavers from at least four source origins currently form admixed, genetically diverse populations that spread across the study region. While regional occurrences of invasive North American beavers (n = 20) were found, all but one C. fiber bore the mitochondrial haplotype of the autochthonous western Evolutionary Significant Unit (ESU). Considering this, as well as the viability of admixed populations and the fact that the fusion of different lineages is already progressing in all studied regions, we argue that admixture between different beaver source populations should be generally accepted.The comeback of the Eurasian beaver ( Various subspecies were initially defined based on the geographic location of their refugia and subtle morphological differences sensu Moritz et al. et al. C. f. albicus Matschie 1907 (relict population in Germany), C. f. galliae Geoffroy 1803 (relict population in France), C. f. fiber L. 1758 (relict population in Norway); as well as beavers from the Voronezh breeding station in Russia (C. f. orientoeuropaeus Lavrov 1981), so far presumed to belong to the eastern ESU C. f. birulai Serebrennikov 1929 (relict population in China and Mongolia), C. f. tuvinicus Lavrov 1969 (relict population in West-Siberia), C. f. pohlei Serebrennikov 1929 (relict population in Middle Siberia), and C. f. belorussicus Lavrov 1981 (relict population in Belarus) are described Beavers were anthropogenically reduced to only few scattered relict populations in Eurasia C. fiber individuals of multiple origins, regardless of ESU assignment. In Western Europe, not only were all three schemes applied, but the underlying philosophies of each beaver reintroduction project also differed fundamentally. Several reintroductions were not monitored and the literature is scattered or inconsistent. Additionally, North American beavers (C. canadensis Kuhl 1820) that potentially escaped from captivity may have contributed to recolonisation While several reviews have attempted to reconstruct the complex reintroduction history of beavers in Eurasia e.g. In order to unravel the reintroduction and dispersal history of beavers in Germany and adjacent regions, we sampled tissue from animals found dead in the wild as well as noninvasively collected hair samples. Special emphasis was placed on known regions where beavers originate from different source populations. Mitochondrial CR haplotype analysis and nuclear microsatellite markers were applied in five study regions to reveal the degree of potential admixture between beavers from different origins. Beaver populations in these regions differ by origin of source animals as well as time since reintroduction, thus providing snapshots into different time windows of potential admixture within contact zones. Specifically, we addressed the following four questions.C. fiber, invasive C. canadensis) and relict populations (“subspecies”) contribute to the present distribution of beavers in the study region?Which species  in the study region?Is admixture ongoing and will it potentially lead to the disappearance of the classically recognised “subspecies” in the study region, including the relict population of Elbe beaver  were reintroduced to the Hessian Spessart Mountains in 1987–1988, while the southern part of this region is naturally colonised. Furthermore, we investigated a region in Eastern Germany  including the border to Poland (n = 53). Few individuals of the German relict beaver population C. f. albicus survived in region EG and the population was supported by several successful reintroductions of individuals from the Elbe river system since 1935. Since 1974 beavers from the Polish beaver farm in Popielno  were reintroduced to Poland and currently disperse to Germany where we investigated the admixture between the German and the Polish population. In the German federal states Bavaria and Baden-Württemberg  beavers of different origins  were reintroduced since 1966 (n = 64). Region IV (SW) comprises Switzerland including samples from the border to France and from Baden-Württemberg. Here, we analysed 32 samples from the contact zone where 141 individuals from the French relict population , from Norway (C. f. fiber) and from Voronezh, Russia, were reintroduced between 1956–1977. Additionally, we analysed 44 samples from Belgium, Luxembourg and the German federal states Rhineland-Palatinate and Northrhine-Westphalia, also known as The Greater Region , where beavers from the Elbe relict population, from Poland (wild catches and farmed beavers from Popielno) and Bavaria were reintroduced since 1981. Since 2006 the occurrence of C. canadensis is recorded for Luxembourg, Rhineland-Palatinate and Belgium. Detailed information for every region is provided in the In the German federal state Hesse  we investigated the potential hybrid zone between individuals from the Spessart Mountains and the population in southern Hesse and northern Bavaria (n = 42). Eighteen individuals from the German relict population . Culling was carried out by regional authorities during coordinated beaver management . Tissue samples of the invasive C. canadensis were obtained during a sterilisation programme by the federal state Rhineland-Palatinate with the aim to prevent the spreading of the North American beaver (Region GR). After sterilisation C. canadensis individuals were transferred to their original territory or zoos. Detailed information including sample location, sample material, and name of the collector providing the sample, as well as information about the means of sample acquisition for every sample and the approving authorities can be checked in No beavers were trapped or killed for this study. Noninvasive hair samples were collected using barbed wire traps without animal handling . Normally, one tuft of hair containing 20–30 wool hairs and 2–5 guard hairs was found at the traps. Beaver hair traps were set up in a height such that only adult beavers were sampled and barbed wire was sufficiently flexible as to avoid scratching animal skin (no signs of blood were ever observed during our hair trapping campaigns), and the wooden sticks to which the wire was attached was sufficiently loosely anchored to the ground that potential entanglement or suffocation of beavers  was impossible . Thus, no beavers or other wildlife were harmed following the placement of the hair traps and in the process of collecting the hair samples. Tissue samples of For DNA extraction from tissue we used the Qiagen Blood and Tissue Kit following the manufacturer’s instructions and diluted DNA to 6.5 ng/µl for further analyses. From areas where insufficient numbers of tissue samples were available we collected hair samples with barbed wire traps 5′-AATTACTTTGGTCTTGGTAAACC-3′) and 6R (5′-GCCCTGAAGTAAGAACCAGATG-3′) Horn Taq polymerase (New England Biolabs), 1.5 µL of 10× Taq polymerase buffer, 1.8 mM MgCl2, 0.2 mM each dNTP, 0.25 mg/ml BSA, and 0.3 µM each primer. We used the following thermal cycling parameters: 5 min at 94°C, 40 PCR cycles  plus 10 min at 72°C. PCR products were purified using Exo-Sap-it (Affymetrix). We sequenced the amplicons on a 3730 DNA Analyzer (Applied Biosystems) in both directions with Big-Dye Terminator v3.1 chemistry (ABI), aligned the sequences with ClustalW 1.83 We sequenced a part of the hyper-variable domain of the control region of mitochondrial DNA using the oligonucleotides 1F .A phylogenetic network was constructed in TCS 1.21 et al.2, 0.2 mM each dNTP, 0.2 µM each primer, 0.25mg/ml BSA, and 0.5 U/µl HotStar Taq-Polymerase (Qiagen). Fragments were amplified under the following cycle conditions: 15 min at 95°C, 45 cycles of 30 s at 94°C, 90 s annealing at 50°C, 60 s at 72°C, and final elongation of 30 min at 72°C. For all PCR reactions positive and negative controls were included. Hair samples were analysed in three replicates. We measured amplicon fragment length on an ABI 3730 DNA Analyzer (Applied Biosystems), using deionized formamide and Genescan size standard LIZ500 (Applied Biosystems), and analysed the raw data with Genemarker 1.6 (SoftGenetics). Six Loci showed inconsistent results with strong deviations from Hardy-Weinberg equilibrium (HWE) and were thus excluded from all further analyses. All following analyses are based on 13 microsatellites: Cca4, Cca8, Cca13, Cca18, CF05, CF06, CF07, CF19, CF31, CF32, CF33, CF41, CF44.We initially analysed 25 microsatellites see  originalimlet 1.3.3 rlequin 3.5 Genotyping errors  of hair samples were calculated using Gare 1.1) tructure with K = 2 and admixed individuals (q<0.8) were excluded. Here, rarefaction was applied according to the minimum sample size per sub-population within each region . Allelic richness for C. canadensis was not corrected for sample size because all individuals were sampled within the same region; no comparison to other regions is needed. For more details and calculations per marker see t.test, after we tested the data for normality using the Shapiro-Wilk Test, function shapiro.test, in R (R Development Core Team 2009). None of the data sets significantly deviated from normality when applying sequential Bonferroni correction (12 tests) We investigated two measures for allelic richness: the observed, uncorrected allelic richness  and corrected allelic richness (to prevent sample size bias) as determined by rarefaction . We tested a range of K from 1 to 20, with 10 replicates for each K for the complete data set as well as for the data set without C. canadensis. For analyses within the five regions we tested a range of K from 1–10 (10 replicates per K). The most likely K was inferred using Evanno et al.’s tructure HarvesterStructure we used Discriminant Analysis of Principal Components (DAPC) adegenetfind.clusters, we determined the most likely number of genetic clusters in each study group, using all principal components (PCs). To calculate the assignment probability of a beaver to each of these clusters we determined the optimal number of principal components (PCs) as advised in the manual. To avoid unstable assignments of individuals to clusters, we retained for every analysis a number of PCs equaling the respective sample size of the group to analyse divided by three, but used all discriminant functions in a preliminary DAPC run. We then used the optim.a.score function with 20 simulations to determine the optimal number of PCs, and a final DAPC was subsequently carried out with this optimal number of PCs.In addition to SewHybrids1 hybrids, F2 hybrids, backcrosses). We tested the four regions HE, EG, BB, and SW individually with a burn-in period of at least 200,000 repetitions followed by a run with minimally 800,000 steps for genetic admixture and excluded region GR because the two occurring species in this area, C. canadensis and C. fiber, do not hybridise NC. f. galliae DQ088703 (g), C. f. fiber DQ088702 (f) and C. f. albicus DQ088700 (a1) 1 JF264887 2 and r3), the only haplotype of the eastern lineage (e) and the haplotype of C. canadensis were not described so far  yielded eight CR haplotypes. Four of those were previously described: d so far . Geneticlineages , namely C. fiber . The nine microsatellites  were also polymorphic for C. canadensis . Together, in both species we found 75 alleles of which 12 were exclusively found in C. canadensis. Microsatellite genotype data is provided in an additional file  to a genetic cluster in DAPC than in Structure. For K = 6  DAPC assigned 228 individuals (97.02%) clearly to a group whereas Structure assigned 169 individuals to one of the populations (71.91%).The most likely number of genetic clusters for the whole data set was K = 6  and 4. Ter grey, . Within C. fiber we compared the runs K = 2–6 for DAPC . When analysing the data with K = 3 the third group represents individuals with a French ancestry of C. f. galliae  was K = 5 . The blue group is of French origin . The yellow cluster represents Swiss and EG beavers; in this eastern group we found an additional private allele. The higher K models did not reveal deeper, biologically meaningful structure and also no split between the geographically widely separated Rhine watershed in Switzerland and eastern Brandenburg in EG.To display all sub-structuring in the data set of for DAPC  Ib–f andtructure  IIb–f. As green;  in Regioae blue;  in DAPC  yellow; . For K =as K = 5 . Here, t1, representing the reintroduced C. f. albicus population and r1, likely stemming from reintroduced Russian individuals, as well as the individual HE29 carrying the French haplotype g. DAPC clearly separated individuals with haplotype a1 (except HE28) and a group with r1/g but Structure and NewHybrids identified a number of genetically admixed individuals (HE20–28). The individuals translocated to Hesse in 1987–1988 still reside in the region of original location in the Hessian Spessart Mountains. However, the population of southern Hesse spreads northwards to central and eastern Hesse (HE36), with a genetic impact already detectable in the core zone of the reintroduction area .We found two main CR haplotypes: a1, the indigenous German haplotype but also r1 (Russian haplotype). DAPC inferred two clusters roughly according to geography, namely a western (green) and an eastern part (yellow). Individuals EG37 and EG38 were not clearly assigned to either population. In Structure slightly more individuals had an intermediate genotype. NewHybrids, in contrast, suggested substantial admixture between Region EG and the immigrating Polish individuals.In north-eastern Germany we found two haplotypes: aC. f. fiber; n = 1, f), France  and Russia . Structure suggested admixed genetic patterns in the region in 56% (36/64) of all individuals, while DAPC assigned 77% with high posterior probability to one of the two clusters. According to the results of NewHybrids only eight individuals were assigned to parental group 1  and one was determined to be a pure P2 (assignment probability >0.8). All remaining individuals in the population BB were designated hybrids.In Bavaria individuals from multiple origins were reintroduced and also dispersed far into Baden-Württemberg. We detected individuals with haplotypes of three origins: Scandinavia  with seven individuals out of 32 showing an intermediate genotype in Structure. This admixture was confirmed by NewHybrids. This complies with a clear geographical distribution with Scandinavian haplotypes in the north-eastern and French haplotypes in western Switzerland.Mitochondrial haplotypes of the French (g) and the Scandinavian (f) relict populations were found in the Swiss region SW. DAPC and SC. canadensis). All 20 identified C. canadensis showed an identical haplotype. Across C. fiber, however, this region harboured the highest number of haplotypes. Maternal lineages from French beavers , Scandinavian beavers , the potential Russian lineage , and the German relict haplotype  were found. Additionally, we found one individual bearing a mitochondrial haplotype (e) of the eastern ESU. Microsatellite analysis of the C. fiber samples revealed no clear population structure, with single samples that clustered with an admixed genotype in the same group as the southern German admixed population, the German relict population  or the eastern European beavers.This region represented a special case in our study because of the co-occurrence of an additional beaver species .We found several significant deviations from HWE due to heterozygote deficits in all but the admixed BB population . HWE deptructure clustering revealed considerably lower values of genetic diversity, in particular in regions with pure occurrences of relict populations  or its descendants  were detectable whereas the allelic richness between the two subgroups within the regions HE and EG was significant different . Single populations sorted according to SBy investigating 235 samples and a combination of mitochondrial and nuclear DNA markers we provide insight into hidden recolonisation and admixture processes over different temporal scales of admixture, from recent population contact such as observed for the SW or HE regions to the well-admixed beaver population in Southern Germany, where beavers of various population origins might already have started to admix since the 1960s.The ESU is a fundamental concept in conservation biology. Moritz 1) is the most common among our German samples. Furthermore it is the most similar haplotype (3 bp differences) compared to the described haplotype of the Elbe beaver (a1). The haplotype r1 was detected in HE, BB, EG and GR and we assume two different origins in Central Europe. For region BB we expect a direct reintroduction from Voronezh to Bavaria from where it spread also to region HE. For EG the haplotype, r1, appears near to the Polish border, so these animals may derive from Poland. Beavers with that haplotype were potentially bred in the beaver farm in Popielno, Poland, and built the source for reintroduction projects in Poland . Given that many individuals from this farm came from Voronezh, Russia, 1 is from Russia. Because of the reintroduction of beavers from the Popielno beaver farm and wild catches from Poland 1 haplotype derive from that farm.Except for one sample from GR, all analysed beaver individuals in our study bear western ESU haplotypes. This is surprising as reintroduction of beavers from eastern regions (Russia) to Central Europe was commonplace. Beavers reintroduced from Russia to Bavaria originate mainly from a Voronezh stock and were assumed to be of pure eastern ESU origin 1 haplotypes in regions where descendants from Popielno beavers live, we presume that the e haplotype stems from the wild population in Poland, potentially harbouring this eastern haplotype. It is likely, therefore, that a contemporary contact zone between the two ESUs appears in Poland.The, so far, undescribed eastern lineage haplotype e was only found in GR. possibly implying that this lineage originated in Poland. Because we usually find only rC. fiber haplotypes within this study makes it likely that the general mitochondrial diversity is higher than expected. Further, ongoing natural dispersal and especially anthropogenic translocation increasingly leads to a breakdown of the historical, glaciation-induced, geographical separation of the major eastern and western lineages. The finding of western haplotypes in eastern regions  suggests that the differentiation between the two ESUs is today not linked to geography as was previously suggested et al.The phylogenetic separation between the two ESUs has been dated to 210,000 years ago et al.C. fiber should in future be updated with nuclear markers once more samples from eastern regions are available. Nevertheless, we note that the analysis of mitochondrial control region sequences is valuable because it enables tracing the origin of beavers due to unique haplotypes in the extant European relict populations.The conclusions of other studies based on Halley’s tructure and NewHybrids, DAPC seemed over-confident when assigning individual cluster membership scores. Only a few individuals were identified as genetically admixed in DAPC as compared to Structure. Also NewHybrids suggested relatively high levels of genetic admixture  in some regions. Methodologically, in DAPC there is a risk of over-fitting the discriminant functions when retaining too many principal components, which can lead to exaggerated and unstable posterior membership probabilities. The built-in DAPC function optim.a.score is supposed to balance discriminative power and over-fitting. However, while using this function as suggested, we suspected that for our data DAPC leads to results that under-estimate genetic admixture in the hybrid zones. Still, though to a lesser extent than Structure and NewHybrids, DAPC demonstrates the formation of hybrids zones where previously isolated beaver populations meet, and that these populations merge with on-going range expansion, most evident in BB. Additional evidence for higher extents of admixture as suggested by Structure and NewHybrids is the comparison with CR haplotype distribution. DAPC clustered one or several carriers of the CR haplotype of one population to the other population in HE, EG, and SW. NewHybrids identified all these unclear assignments as hybrids  and northern Switzerland  still show the a yellow; ; Fig. 8 C. fiber to be 1,880, a value well above the population census in all relict populations. Human-mediated reintroductions of few individuals from already bottlenecked populations to other areas resulted in even more severe bottlenecks and the low genetic diversity generally found in beaver populations today Only few beaver individuals survived human persecution in the known beaver relict populations throughout Eurasia C. f. albicus from the German Elbe river relict population were reintroduced C. f. albicus individuals still forms a population of less than 1,000 individuals. This population remained in the introduction region for ∼25 years, and have the lowest heterozygosity values in this study. On the other extreme, the Bavarian population, founded by around 43 individuals from different relict populations rose to a population of ∼14,000 individuals, which then actively recolonised adjacent areas over a 45 year period. Note, however, that the comparison of these populations is difficult because the population growth could be exponential, something we cannot see with our data. These observations provide evidence for the hypothesis that population growth rates and dispersal might be governed by the level of genetic diversity and inbreeding. Further research integrating ecological and demographic data will aid investigating this idea.A low population growth rate was documented for a Dutch population where 42 C. f. albicus population, which has been used for multiple reintroduction projects, has been observed in small beaver populations Click here for additional data file.Table S2Overview of successfully analysed samples for the five regions.(DOCX)Click here for additional data file.Table S3Error rates of the 57 analysed hair samples.(DOCX)Click here for additional data file.Table S4Microsatellites evaluated and used in this study.(DOCX)Click here for additional data file.Table S5Observed and expected heterozygosity and deviations from Hardy-Weinberg equilibrium per region and per locus.(DOCX)Click here for additional data file.Table S6Overview of haplotypes.(DOCX)Click here for additional data file.Table S7Allelic richness across regions.(DOCX)Click here for additional data file.Table S8Genotype data.(TXT)Click here for additional data file.Text S1Additional text describing details regarding the beaver population history in each region.(DOCX)Click here for additional data file.


H. euphorbiae and Afro-Macaronesian H. tithymali. It has been suggested that these species merged into several hybrid swarm populations, although a mitochondrial phylogeography revealed substructure with local differentiation. We analysed a three-gene mt-dataset  and 12 microsatellite loci . Microsatellite analyses revealed an overall weak differentiation and corroborated the superordinate division into two clusters. The data indicate that the populations studied belong to only one species according to the biological species concept, refuting the opening hypothesis. A future taxonomic revision appears necessary to reflect the division into two subgroups. Ancestral mitochondrial polymorphisms are retained in H. euphorbiae, indicating gene flow within a broad ‘glacial refuge belt’ and ongoing postglacial gene flow. Diverse patterns of extensive mito-nuclear discordance in the Mediterranean and the Middle East presumably evolved by more recent processes. This discordance indicates introgression of H. tithymali-related mitochondrial haplogroups, accompanied (to a lesser degree) by nuclear alleles, into Italian and Aegean H. euphorbiae populations as recently as the late Holocene. The complex mosaic of divergence and reintegration is assumed to have been influenced by locally differing environmental barriers to gene flow.We test the morphology based hypothesis that the Western Palaearctic spurge hawkmoths represent two species, the Eurasian  The discipline of phylogeography has substantially advanced our understanding of the formation of geographically structured diversity within and among closely related species and thus enabled intriguing insights into the origin of species1242273628101112Hyles euphorbiae complex: HEC) represent a widespread, polymorphic and taxonomically controversial species complex that features enigmatic biogeographic patterns in an incipient stage of speciation in the Western Palaearctic141516H. dahlii and non Euphorbia-feeding H. zygophyllii from this study, although they are formally included in the HEC17et al.141516et al.14151618H. euphorbiae and the Arab-Afro-Macaronesian H. tithymali hybridise in the Mediterranean. They readily hybridise in captivity without any evidence for reduced hybrid fitness, i.e. postzygotic isolation ; on Malta, H. sammuti (*MAL); in the coastal and montane areas of the Maghreb, H. tithymali mauretanica ; and on the coast of north-western Spain, H. t. gallaeci . A morphological transition zone of putative hybrid origin is also reported for the relict populations on the Arabian PeninsulaH. robertsi, has been described for the Middle East and Central AsiaH. cretica, H. euphorbiae, H. robertsi, H. sammuti and H. tithymali). However, the two underlying assumptions of this paper are that (i) H. cretica and H. sammuti are hybrids of H. euphorbiae and H. tithymali, and (ii) H. robertsi is a local form of H. euphorbiae. We thus only use the two species names H. euphorbiae and H. tithymali.The spurge hawkmoths  and southern Aegean Islands (*CRE) suggesting these areas represent glacial refugia of distinct taxa rather than hybrid zones.Due to the complexity of mitochondrial haplotype distribution found in a first phylogeographic study on the HECH. euphorbiae (‘euphorbiae’ and ‘enigmatica’) were once mixed among the endemic ‘tithymali’-related haplogroups, ‘italica’ and ‘cretica’. It was only during the Twentieth Century that ‘italica’ increased in frequency in Italy from below 60 percent of the total population to near fixation. This was ascribed to genetic drift resulting from habitat destruction and/or to a spread of this haplogroup through the population facilitated by climate warmingH. euphorbiae and H. tithymali hybridisation in these areas12By analysing museum specimens from Italy  and southern Aegean Islands (*CRE), Mende & Hundsdoerfer12et al.1) to be tested is that the HEC populations marked with a * in H. euphorbiae and H. tithymali. We formulate the null hypothesis (H0) as follows: the HEC consists of a single gene pool without species boundaries within the Western Palaearctic.The drawbacks of phylogeographic inference from mtDNA alone are known26Larval tissue or legs (if specimens were reared to or caught in the adult stage) of 898 specimens were collected from various localities covering virtually the entire Western Palaearctic distribution range of the HEC  and storet al.14BioEdit 7.0.9.0In addition to DNA from 339 specimens that had already been isolated for mtDNA sequence analysisH. euphorbiae , ‘enigmatica’  and ‘italica’ (#5649) mtDNA haplogroups with the ‘Perfect Pure RNA Cell & Tissue Kit’ (5Prime) in a dedicated RNA laboratory. RNA concentration was measured with a NanoDrop ND-1000 (Thermo Scientific). The ‘RevertAid First Strand cDNA Synthesis Kit’ (Thermo Scientific) was used to synthesise cDNA from 5 μg RNA according to the manufacturer’s manual; however, with an extended DNA digestion at 37 °C for 1.5 h. A short fragment of COI (fragment ‘B’) and of COII (‘L’) was amplified for haplogroup assignment , PCR reaction buffer with a final concentration of 2.5 mM MgCl2 (Bioron), 0.2 mM of each dNTP (Fermentas), approximately 10–50 ng DNA template and infrared dye labelled forward and plain reverse primers. In addition, the reactions for loci developed by Hundsdoerfer et al.2. Primers were partly multiplexed and concentrated as follows: 0.18 μM Hti62 + 0.25 μM Hti63 + 0.38 μM Heu76; 0.33 μM Hti65 + 0.13 μM Hti66 + 0.13 μM Heu72; 0.13 μM Hyti13 + 0.2 μM Hyti41 + 0.2 μM Hyti49; 0.01 μM Hyti14; 0.1 μM Hyti22; 0.03 μM Hyti50. The PCR of loci from Mende et al.et al.We analysed the six microsatellite loci described by Mende Previous characterisation of the loci showed that highly variable flanking regions cause allele lengths that were inconsistent with the microsatellite repeat modulus29Network 4.5.1.0 (Fluxus Technology). Since the analysis is prone to errors in the presence of ambiguities, base positions with >10 ambiguities in the total alignment were deleted, leaving 2070 bp. The few remaining ambiguities at non-informative sites were overwritten to the respective base at their particular locus and those at parsimony-informative sites to the most common base of the corresponding haplogroup. Average p-distances between haplogroups were calculated with Mega 4.0.2ArcGIS version 10 (ESRI). DnaSP 5.0Structure analysis of microsatellite data (see below).Evolutionary relationships among mitochondrial sequences were evaluated by a median-joining network using Arlequin 3.1Nef) for each haplogroup by means of a Bayesian coalescent approach with 3 × 108 Markov chain Monte Carlo (MCMC) iterations  using Beast 1.8.034−8 per year based on a standard COI divergence rate of 3.54% per million years for univoltine insects−8 per generation; see Schenekar & Weiss17Tracer 1.6We tested for signals of population size changes for each haplogroup with Genepop ‘on the web’ 4.2Arlequin with 100,000 MC and 10,000 dememorisation steps. Significance of the results was evaluated after Bonferroni correction. Micro-Checker 2.2.3We tested for linkage equilibrium (LE) in each population containing ≥15 individuals using (FST) between HEC populations was calculated with Arlequin . We tested for correlation between genetic and geographic distance by performing a Mantel test implemented in IBDWS 3.23ST matrix computed by Arlequin, a geographic distance matrix calculated from the average population coordinates (Pairwise differentiation rdinates  by meansArlequin to test the data support (based on an average over 12 loci) of a priori population structure hypotheses. According to the traditional species distribution ranges and H1, “H. euphorbiae” was defined to contain the populations IRA, KAZ, CAU, TUR, BUL, GRE, SGR, *CRE, HUN, SLK, GER, BEL, FRC, ESP, CSP, SSP, *GAL, NIT, *EIT, *CIT, *SIT, *SIC and *PAN and “H. tithymali” STU, *MOR, SMO, cLZ, cFV, cGC, cTF, cLG, cEH, cLP, MAD, SAA, FOG and YEM (14H. euphorbiae-look” of moths) and mitochondrial assignment . The hypothesis for the a priori group definitions is thus based on other sources of characters than the ones tested, to avoid circular argument.A hierarchical AMOVA was performed using  and YEM . The popa priori groups. We performed Bayesian cluster analyses using Structure 2.3.2Geneland 4.0.345StructureGenelandStructureGeneland so as not to overestimate KGeneland was further provided with information of coordinates in decimal degrees (K = 1–20 with 106 MCMC iterations of which the first 2.5 × 105 were discarded as burn-in (2.5 × 103 of the 104 sampled iterations in Geneland). We reran the Geneland computation with the same parameters except for K being fixed for the best two values inferred from the first computation (see below) to obtain more reliable estimates of individual cluster assignmentΔK method of Evanno et al.K = 1 using mean lnP(D) values] for the Structure analysis and from the posterior probability distribution given by Geneland. Individual probabilities of cluster membership of the replicate run with the highest posterior probability were displayed as barplots with Distruct 1.1K = 2 and the second most probable partition K = 3 (see Results) to assess consistency of cluster boundaries and to reveal potential substructure  as implemented in the R package Adegenet 1.3-6Analysis of the data without defining  degrees  with an To provide a robust assessment of population structure in the HEC, we adopted two strategies:Structure and Geneland clusters was inferred using a locus-by-locus AMOVA (as a weighted average over loci) using Arlequin. We assessed genetic diversity within the entire HEC and the Structure clusters by counting the numbers of alleles and computing allelic richness  with Fstat 2.9.3.2Convert 1.3.1Arlequin. For these analyses, all samples of a geographically defined population were collectively assigned to a cluster according to the predominant assignment of its individuals.Differentiation of http://www.ebi.ac.uk/ena/data/view/LT595043-LT595594. MtDNA sequence alignment: Figshare doi: 10.6084/m9.figshare.3438317. Microsatellite genotype table: see MtDNA sequences: GenBank accession number AJ749460-469/472-486/488-494/499-500/503-521/524-528/532/535/537-563/565-567/570-575/577, FN386562-563/600-604, FR839374-532/534-577/579-580/582-583/585-615, European Nucleotide Archive (ENA) accession numbers for new sequences: LT595043-594, available at H. dahlii (H. dahlii GB Acc. No. AJ749458).The network analysis of mitochondrial sequences corroborated the previously found division of the HEC into seven haplogroupseuphorbiae’, the less frequent ‘enigmatica’ and the very distinct ‘melitensis’, in the entire Eurasian range of ‘pure’ H. euphorbiae , whereas none of the three occurs in the range of the Afro-Macaronesian H. tithymali , where it is mixed with ‘euphorbiae’ and ‘enigmatica’ . In contrast, the range of H. tithymali (see above) and continental African putative hybrid populations  is predominantly occupied by a single haplogroup, ‘tithymali’. This is absent from the range of morphologically ‘pure’ H. euphorbiae; its sole occurrence on the European mainland is in the putative hybrid populations of Central Italy .We detected three haplogroups, ‘ network . The ‘rogmatica’  in the seuphorbiae’ haplotypes, in contrast to its taxonomic status as H. t. gallaeci. Malta (*MAL) was corroborated as consisting of a mixture of haplotypes belonging to ‘tithymali’ and two haplogroups, ‘euphorbiae’ and ‘melitensis’, that also occur in mainland H. euphorbiae. In contrast, the other Mediterranean putative hybrid populations are dominated by haplogroups that are equally very closely related to ‘tithymali’ and each other  by the endemic ‘cretica’ . In turn, the H. euphorbiae haplogroups ‘euphorbiae’, ‘enigmatica’ and ‘melitensis’, show occasional occurrences among the dominant ‘italica’ in Southern Italy .Populations considered being of hybrid origin based on their intermediate larval morphology show remarkably different compositions of mitochondrial haplogroups . The increase becomes significant, i.e. when the 95% highest posterior density (HPD) limits loose overlap with older estimates, at about 6,500 years BP  of the genetic divergence is explained by geographic distance. The Mantel correlogram reveals a continuous and approximately linear decrease of Mantel correlation, i.e. a decrease in genetic similarity, with increasing geographic distance (Nuclear genetic and geographic distances between populations are significantly correlated across the HEC’s entire range (rdistance .p < 0.00001) of the total variability in the dataset is found between the two groups of populations defined a priori, supporting the division into H. euphorbiae and H. tithymali (sensu H1). Between populations within these groups we found 6.2% (p < 0.00001) of the total variability, i.e. less than between the two a priori defined groups. However, the greatest part of the variability in the dataset (83.8%) was found between individuals .The result of the hierarchical AMOVA (average over 12 loci) is that 9.9%  and largely congruent between both analyses except for the populations *TUN and *MAL , whereas it is substantially lower for the majority of loci and some values are even insignificant  and H. tithymali (red)  represent an unexpected exception to the congruence between microsatellite clusters and ‘pure’ taxa ranges, since they are predominantly assigned to the H. euphorbiae microsatellite cluster by both cluster analyses, in conflict with their morphological and mitochondrial affiliations (H. robertsi (IRA)  to the H. euphorbiae cluster contrasts with their partly ‘tithymali’-related ‘robertsi’ haplogroup mitochondria. Remarkably, the diversity of both nuclear and mitochondrial markers is distinctly higher in the H. euphorbiae than in the H. tithymali cluster  . However cluster  and 7a.  cluster . Yemeni liations  and 7a.  cluster .Structure, the African mainland  and Maltese (*MAL) populations are substantially admixed but predominantly assigned to H. tithymali. In contrast, Geneland assigns *TUN and *MAL to H. euphorbiae. The putative hybrid populations of Italy , and the southern Aegean Islands (*CRE) also include an increased number of admixed individuals according to Structure  also shows a considerable number of admixed individuals according to the Structure result. In contrast, all individuals from the north-western Spanish H. t. gallaeci population (*GAL) are assigned to the H. euphorbiae cluster in both analyses, in congruence with mtDNA.The geographic position of the main divide between the two microsatellite clusters falls within the Mediterranean putative hybrid populations, but is inconsistent between the two cluster analyses . Accorditructure . Howevercretica’  and 7a pK = 3, also deserve attention, since they reveal interesting substructure , whereas they are more closely connected to the main haplogroup of H. tithymali in the network of mitochondrial sequences . These discordances demonstrate an incomplete separation of H. euphorbiae and H. tithymali. The underlying causes are probably different for each of the haplogroups. The patchy admixture of ‘tithymali’-related ‘enigmatica’ with ‘euphorbiae’ indicates incomplete mitochondrial lineage sorting with a retained ancestral polymorphism in H. euphorbiae. This retained mitochondrial diversity in H. euphorbiae is accompanied by a higher nuclear diversity compared to the southern H. tithymali  exclusive prevalence of ‘italica’  and ‘cretica’ in their respective geographically confined areas  and their close relation to the ‘tithymali’ haplogroup suggest more recent introgressions of these mitochondrial haplogroups into H. euphorbiae  and confirming H0. The populations studied should thus be classified as belonging to a single species according to the biological species concept. Although a division into two clusters corresponding to H. euphorbiae and H. tithymali is well supported by the microsatellite data, H. euphorbiae is not a maternal lineage monophylum (mtDNA), in the sense that the taxon does not contain all descendants of its most recent common ancestor (MRCA).Our data further indicate ongoing nuclear gene flow far beyond the Mediterranean populations of putative hybrid origin. Individuals assessed as admixed by EC range  and the EC range . Congrueet al.tithymali’-related haplogroups in parts of Southern Europe.Accepting the Western Palaearctic HEC as one biological species is not inconceivable, given that HEC moths are considered strong fliers15H. dahlii, the spurge hawkmoth species endemic to Sardinia, Corsica and the Balearic Islands, which challenges the alleged effective reproductive isolation of this morphologically and genetically more distinct taxon141718Hyles dahlii individuals have been reported sporadically from the surrounding mainland coasts and interpreted as rare dispersal events14H. dahlii haplotype in the established HEC populations *PAN and *TUN suggests that dispersal across the Mediterranean Sea and subsequent hybridisation may be more common than presumed. Detecting genetic traces of such hybridisation should be rather unlikely, given the small chance that rare migrants’ genes can establish themselves in an established population due to the principle of ‘high density blocking’Moreover, we found evidence for mitochondrial introgression from H. euphorbiae has three  mitochondrial haplogroups . Massive interglacial gene flow could explain why the known postglacial re-colonization paradigms4H. euphorbiae. The data denote a scenario by which the organisms retreated into a ‘glacial refuge belt’ in the Circum-Mediterranean area with local differentiation and simultaneous ongoing gene exchange. In this scenario, moths would fly between refugia of favourable climatic conditions, maintaining gene flow and thus the geographic distribution of the mitochondrial diversity within this region. The recolonisation of Central Europe would have occurred by the northward expansion of moths carrying this mitochondrial mixture.The phylogeography of European biota, including mobile lepidopteran species, generally reveals well preserved spatial genetic patterns resulting from differentiation in isolated glacial refugia on the Mediterranean peninsulas and subsequent postglacial re-colonisation24logroups  occurrineuphorbiae’ haplogroup, roughly dated to the early Holocene  and Central European (SLK) populations, but has no core area. Our augmented dataset further reveals that ‘melitensis’, highly divergent from ‘euphorbiae’, is not endemic to Malta as previously assumedeuphorbiae’, ‘enigmatica’ and ‘melitensis’ must all have occurred in founder individuals coming from the ‘glacial refuge belt’ (see above), and simultaneously occupied the new habitats in Central Europe. The larval food plants of the genus Euphorbia need open habitats, which would have increased with the spread of Neolithic culture from 6,500 years B.P. onwardseuphorbiae’  mitochondrial haplogroupThe currently observed genetic admixture between  samples  and the amples 17 suggest italica’ haplogroup considerably later than the expansion of ‘euphorbiae’  in Italy by ‘italica’. This is corroborated by an analysis of museum specimens, which showed that this replacement was still in progress during the last centuryH. euphorbiae haplogroups throughout Italy. Accordingly, ‘euphorbiae’ on Malta is not as disjunct from the mainland as previously thoughtitalica’ into the European H. euphorbiae or the haplogroup’s geographic origin. Its present-day main occurrence would suggest that it initially separated from the ‘tithymali’ haplogroup only after it became isolated in Italy. However, the more frequent and widespread occurrence of ‘italica’ in the Maghreb  than previously founditalica’ was at a frequency below 60 percent of the total Italian population only a hundred years agoExtensive mito-nuclear discordance as observed in the Italian HEC populations  is a common result of introgressive hybridisationhorbiae’ . Furthertithymali’ is observed. Unfortunately, the small sample size of the ‘cretica’ haplogroup, which exclusively dominates the Aegean Islands today, does not allow for a conclusive assessment of its past demographic evolution. However, an analysis of museum specimens revealed that ‘cretica’ had once also been admixed with ‘euphorbiae’ as far south as Cretecretica’ may not be endemic to the Aegean Islands but possibly also have originated and still be present in Africa, like ‘italica’. However, more samples, e.g. from the relict HEC population in western EgyptInterestingly, the evolutionary history of the Aegean Islands population (*CRE) is suggested to parallel the Italian one. A similar mito-nuclear discordance with a mitochondrial haplogroup closely related to ‘H. tithymali–related haplogroups into the Italian  and Aegean Islands (*CRE) populations was evidently accompanied by a parallel nuclear introgression. This is indicated by the geographically congruent prevalence of intermediate morphotypesH. tithymali microsatellite cluster according to Structure  than to the abovementioned populations in the third cluster.Mitochondrial introgression from tructure  and 7a. gression . The assK = 2) could reflect an artefact caused by allele frequency gradients between the genetically more distinct H. tithymali and H. euphorbiae populations in Macaronesia and Eurasia, respectively. A linear decrease of the Mantel correlation with geographic distance  had also been considered a hybrid population based on morphology. Especially the frequent occurrence of horizontally connected upper eyespots in the larvae, a character frequently found on the western Canary Islandsitalica’ skyline plot  almost exclusively occurs from the local to the invading taxon due to demographic disparityine plot  and an iHolocene  coincidetithymali’ and related mitochondrial haplogroups across the wide Strait of Sicily and Libyan Sea into Southern Europe, this was not observed across the comparatively narrow Strait of Gibraltar, although southern Spain has a potentially suitable climate for these haplogroupsH. tithymali in SSP . The considerably different mitogenetic composition can be ascribed to the island’s geographic isolationIn contrast to dispersal of ‘i in SSP  and 7a. i in SSP , would ii in SSP , as repoA locally varying interplay of geographic barriers and climate variables, as well as incomplete lineage sorting and introgression, have shaped a truly complex phylogeography of this dispersive hawkmoths species complex. Our study highlights the importance of comprehensive sampling and analyses of multiple markers for a conclusive evaluation of a species’ evolutionary history. The data imply that the populations studied constitute a single gene pool, and thus belong to one biological species. Nevertheless, a division into two subgroups is predominant. Our data put forward a new pattern of a ‘glacial refuge belt’ with both local differentiation and ongoing gene exchange. A subsequent oscillating ‘admixture belt’ is postulated to follow climate fluctuations. The dominance of a distinct mitochondrial haplogroup in the Italian HEC would generally be considered to have evolved from long-term processes of vicariance during glacial periods and to have been static since the early Holocene2How to cite this article: Mende, M. B. et al. A comprehensive phylogeography of the Hyles euphorbiae complex (Lepidoptera: Sphingidae) indicates a ‘glacial refuge belt’. Sci. Rep.6, 29527; doi: 10.1038/srep29527 (2016).