Cypripedium japonicum, is conducive to the development of conservation strategies. Here, we examined the levels and partitioning of inter-simple sequence repeat (ISSR) diversity (109 loci) in five populations of this orchid to gain insight into its genetic variation and population structure in Eastern and Central China. It harbored considerably lower levels of genetic diversity both at the population (percentage of polymorphic loci (PPL) = 11.19%, Nei’s gene diversity (H) = 0.0416 and Shannon’s information index (I) = 0.0613) and species level  and a significantly higher degree of differentiation among populations  = 0.698) than those typical of ISSR-based studies in other orchid species. Furthermore, the Nei’s genetic distances between populations were independent of the corresponding geographical distances. Two main clusters are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis and the STRUCTURE program. In addition, individuals within a population were more similar to each other than to those in other populations. Based on the genetic data and our field survey, the development of conservation management for this threatened orchid should include habitat protection, artificial gene flow and ex situ measures.Knowledge about the population genetic variation of the endangered orchid,  Orchidset al.  suggesteet al. . From a et al. . Swarts  [et al.  pointed Cypripedium japonicum Thunb. is a 35–55 cm-tall lady’s slipper orchid that grows in damp and humus-rich soil in forests or on shady slopes along ravines at an altitude of 1000–2000 m; it is mainly distributed in China, Japan and Korea [C. japonicum is at great risk of extinction due to habitat loss and anthropogenic threats, such as over-collection for its high horticultural potential and limited medicinal value. Consequently, this species is categorized as a conservation priority plant species [et al. [C. japonicum populations in South Korea, and there is only one report describing the use of DNA molecular markers to reveal the genetic diversity of wild C. japonicum populations in Zhejiang province [nd Korea . In Chin species . To date species  and repr species . However [et al.  found a province . The genAmitostigma hemipilioides [Calanthe tsoongiana [Cattleya elongate [Cymbidium goeringii [Piperia yadonii [The maintenance or restoration of genetic diversity is now regarded as a primary objective for conserving endangered and threatened species, which has resulted in the application of molecular methods and phylogenetic studies to design and implement conservation strategies . Becauseilioides , Calanthoongiana , Cattleyelongate , Cymbidioeringii , Piperia yadonii  and otheC. japonicum populations distributed in Anhui, Hubei, Zhejiang and Henan province in China with the following specific objectives: (1) to document the level of genetic diversity within C. japonicum populations and roughly estimate genetic diversity at the species level; (2) to identify the degree of genetic differentiation among populations to determine factors that have influenced genetic structure; and (3) to discuss the implications for effective conservation according to the basic genetic information.In the present study, we employed ISSR markers to investigate the genetic composition of five natural PPL) ranged from 7.34% (TMS) to 15.60% (DMS), with an average of 11.19%. The average effective number of alleles per locus (Ne) was 1.0728, while the observed number of alleles (Na) was 1.1119. Assuming Hardy–Weinberg equilibrium, Nei’s gene diversity (H) varied between 0.0297 and 0.0587, with an average of 0.0416, and Shannon’s information index (I) ranged from 0.0435 to 0.0863, with an average of 0.0613. However, the H and I values were 0.1273 and 0.1928, respectively, at the species level  summarized by Hamrick and Godt [The fourteen selected ISSR primers chosen for analysis produced a total of 109 bands among 128 individuals from five populations. Of these, 42 loci (38.53%) were polymorphic . At the es level . The speand Godt .C. japonicum populations was 67.12% (Gst) (Gst reviewed in Orchidaceae reviewed by Hamrick and Godt [p = 0.001) genetic diversification among the five C. japonicum populations (Φpt (0.698) from the AMOVA analysis and the Gst (0.6712) from the POPGENE analysis provide additional support for the statistics used in this study and robustness of the results [According to Nei’s analysis of gene diversity, the percentage of genetic variation among 2% (Gst) , which wand Godt , indicatulations . With re results .C. japonicum populations varied from 0.0828 to 0.1616   .C. japonicum formed the other group, which could be divided into two subgroups. One subgroup included the TMS population, and the other contained populations from SNJ to BTM. Furthermore, the dendrogram at the individual level based on Jaccard’s similarity matrix showed that plants from different population origins separated clearly, and individuals within a population were more similar to each other than to those in other populations (The arithmetic average (UPGMA) tree based on Nei’s genetic distance clustered all populations into two major groups . The DBSulations .In agreement with the UPGMA cluster analysis, principal coordinate analysis (PCoA) revealed that individuals from each population formed a separate plot and could be clearly distinguished from those of other populations . The indK value with the highest δ K value for the five populations was K = 2  was shown, as well, indicating that the six populations were isolated relatively independently. The relationships revealed by this program were strongly consistent with that implied by the UPGMA cluster and the PCoA analysis. In addition, we further confirmed the weak association between populations in relation to their geographic location.In the ISSR admixture analysis using STRUCTURE, the real as K = 2 , indicatividuals . The nexC. japonicum possessed considerably low genetic diversity both at the species  and population levels  compared to other orchid species  using the same molecular makers (ISSR)  and the UPGMA and PCoA results suggested limited gene flow, even between the less distant populations (TMS and DMS populations), as well as marked differentiation between populations, which was supported by the AMOVA results (Φpt = 0.698), indicating that the majority of genetic diversity was explained by variation among populations. The species’ ISSR genetic diversity of C. japonicum was also lower than some other self-compatible plants (such as Camellia japonica [Lilium tsingtauense [Viola pubescens [Carex moorcroftii [Mentha cervina [Psammochloa villosa [s (ISSR) . The higjaponica , Lilium gtauense  and Violubescens ) and asercroftii , Mentha Cypripedium orchids has come from allozyme markers. Chung et al. [Cypripedium species and concluded that genetic diversity within the Cypripedium genera can vary substantially, and those with traits, such as wide distribution and broad habitat preference, harbored much greater genetic diversity than rare species with narrower habitat amplitudes. Therefore, commonness and habitat diversity appear to be appropriate predictors for genetic variation in Cypripedium. From an ecological perspective, C. japonicum is a habitat specialist that is only found on rich humus soils under old, shady, wet deciduous forests on mountainous hillsides, partly due to the narrow mycorrhizal specificity for seed germination and seedling development [Much of our knowledge of genetic diversity and population structure in g et al.  summarizelopment  under loC. japonicum does not produce food rewards (nectar or edible pollen); rather, it has evolved a food-deceptive pollination system, like most other members of the Cypripedium genus [C. japonicum probably attracts pollinators visiting the flowers through false nectar guides; The pollinator enters the labellum via a front orifice. To escape, the insect should squeezes out of the rear exit hole, forcing it to pass under the receptive stigma and then pick up a new mass of pollen. Suetsugu et al. [Bombus ardens and Bombus diversusdiversus were the effective pollinators in the Japanese C. japonicum population, whereas two bumblebee species, Bombus remotus and Bombus imitator, have been shown to be the effective primary pollinators of C. japonicum in the SNJ population. However, the remarkable genetic differentiation among populations in studied C. japonicum populations does not match what is expected, indicating that some other more important factors shape their current genetic pattern. The same situation was observed in Changnienia amoena, another non-rewarding orchid [et al. [et al. [C. japonicum individuals under natural conditions. One reason for the low genetic diversity in C. japonicum populations can also be explained by the less successful fruit production. The mobility of bumblebees and the highly self-compatibility of C. japonicum [The biological properties of species, such as the types of reproduction and breeding systems, are stressed as one of the most important factors shaping the genetic diversity and extent of plant population differentiation . Rewardium genus . C. japou et al.  found thg orchid . We hold [et al.  suggeste [et al.  found a aponicum  did not et al. [C. japonicum were also observed in severely fragmented habitats, which makes this species very vulnerable to habitat disturbance and climate change. Population genetics theory predicts that rare orchid species occurring in small, spatially isolated populations will lead to the random fixation of alleles within populations and subsequent low levels of genetic variation; these populations will exhibit a high degree of population genetic divergence, primarily as a result of stochastic events, genetic drift and inbreeding [C. japonicum localities, may be exposed to reduced gene flow, which is also considered to be an important factor increasing genetic divergence between populations. The lack of correlation between geographic and genetic distances of the studied C. japonicum, revealed by the Mantel test, UPGMA clustering and PCoA, might be also caused by population fragmentation, genetic drift and restricted gene flow.Most orchid species have experienced acute declines in abundance due to a wide range of human activities that have resulted in habitat fragmentation, and the remaining populations tend to be small and isolated . Izawa eet al.  pointed breeding . Higher C. japonicum populations and the low fruit set likely reduced the probability of seeds being dispersed over long distances between populations. Limited seed dispersal distances have also been reported for other Cypripedium orchids. For example, Chung et al. [Cypripedium macranthos seeds fall close to the maternal plants on the basis of the significant fine-scale spatial genetic structure, and Brzosko et al. [Cypripedium calceolus seed dispersal occurred mostly at short distances within or around the fruiting ramets. Infrequent long-distance seed dispersal combined with a paucity of suitable habitats suggests that the natural founding of new C. japonicum populations is a rare event. However, we cannot completely exclude great distance gene transfer via seeds through intensive tourism, forest management or some other random event. If long-distance seed dispersal occurs, founder effects are also likely to take place when populations are established by a small number of seed sources, resulting in a loss of genetic diversity during colonization [Generally, gene flow between populations occurs by one of only two methods: (1) pollen movement by pollinators; and (2) seed dispersal by the wind. Considering the former method, we expect that pollen transfer by insects is not likely when there are large geographical distances between different populations. Long-distance gene flow by pollen is not possible given the limited flight ranges of pollinators. Although orchids are commonly known to show wind seed dispersal over a wide range, our field observations showed that the dense forest structure surrounding the g et al.  stated to et al.  confirmenization . TherefoC. japonicum in this study belong to the “Sino-Japanese Floristic Region”, which was not affected by the major quaternary glaciations [Cypripedium parviflorum [Cypripedium reginae [et al. [C. japonicum in South Korea to their origination from the same genetically depauperate glacial refugium. However, our data are not sufficient to assess the influence of the Last Glacial Maximum on C. japonicum genetic diversity. Further studies involving a wider spectrum of populations from the geographic range of this orchid and the use of more genetic markers, including some codominant DNA markers, could help precisely determine how the glaciations shaped the genetic diversity in its distribution.Much literature documenting the influence of the Last Glacial Maximum on population genetic structure describe high levels of genetic diversity in previously unglaciated ranges and relatively low genetic variation in formerly glaciated areas. The regions of sampled ciations . Therefoviflorum  and Cypr reginae . Chung e [et al.  attributC. japonicum has been collected by plant sellers, herbalists and orchid hobbyists in recent decades, due to its horticultural and medicinal potential. This species has suffered from habitat loss and anthropogenic threats, including deforestation, building, agriculture and tourism. Many previously recorded populations have disappeared. Consequently, C. japonicum is at risk of extinction in China. Our genetic data and field observations suggest that the main explanatory factors for the low levels of genetic diversity and the shaping of the population genetic structure of C. japonicum are narrow habitat preference, genetic drift due to a small and isolated population size, restricted gene flow, historical events and human activity. Considering the current genetic diversity and structure of this orchid species, several conservation guidelines are suggested for the studied C. japonicum population.Knowledge about population genetic structure is considered an important element of conservation biology, so answers to these questions could help develop effective conservation management measures. The loss of genetic diversity will decrease adaptability to environmental changes, which reduces evolutionary potential. In addition, C. japonicum populations, the disappearance of each population would remove any unique biological characteristics that it may possess, and this could reduce the overall species biodiversity. Protection of habitats of standing populations in situ and prohibition of C. japonicum by law should be stressed to prevent habitat damage and harvest pressure on wild populations. Because the fruit set of C. japonicum in natural habitats is very low, nearby pollinator (bumblebees) populations and other plant species with floral rewards, if any, should also be safeguarded to ensure reproductive success. Furthermore, the method of artificial gene flow via seeds and hand-mediated artificial pollination suggested for Cypripediummacranthos var. rebunense [C. japonicum to rapidly increase the gene flow and relieve the effects of genetic drift. However, careful attention should be paid when implementing artificial gene flow. Concerning ex situ conservation measures, seed asymbiotic germination and tissue cultures would be helpful for the success of further reinforcement or reintroduction. Sampling for ex situ conservation should be taken from as many genets as possible in each population considering the current genetic structure of the studied C. japonicum.We recommend that all of the studied populations should be incorporated into conservation plans to maintain their total genetic diversity and, thus, prevent a further decline in the effective population size. Because each individual population comprises a unique genetic pool given the high genetic differentiation among the studied ebunense  may be aC. japonicum spreads by long, creeping rhizomes, we cannot rule out the possibility that vegetative reproduction could also have a pronounced effect on the genetic structure of populations. Therefore, further study of clonal diversity and structure is also necessary in this endangered orchid.To design and implement comprehensive and effective conservation actions for this threatened orchid species, more research is needed to investigate life-history traits, demographic dynamics, germination biology, seedling establishment and other population ecology parameters. In addition, as Cypripedium japonicum is a perennial herb with a 10–15-cm underground horizontal rhizome (diameter 3–4.5 mm). It has fibrous fleshy roots, with a diameter up to 2.5 mm. Each growing season, the underground rhizome produces a single new ramet or, more often, a cluster of shoots [f shoots . This hef shoots . The fruA total of 128 individuals corresponding to 5 populations with different geographic origins were included in the analysis , Table 5Total DNA was isolated with Dingguo Plant Genomic DNA Extraction Kits  following the manufacturer’s recommendations. The quality and yield of DNA samples were assessed on agarose gels and then precisely measured for DNA concentration using a Quawell Q5000 UV-Vis spectrophotometer . The DNA samples were diluted to 20 ng/µL in TE buffer (10 mM Tris-HCl (pH 8.0) with 0.1 mM EDTA) and stored at −20 °C until ready for use.All of the tested primers were synthesized by Sangon Biotech . A total of 100 primers  were initially screened, and 14 of them that yielded reproducible and discernible bands were used for the analysis of all 128 samples .2+, 0.5 mM each of dATP, dCTP, dGTP and dTTP, 0.3 µM primer, 30 ng DNA template and 1.5 U Taq DNA polymerase . A negative control with no DNA template was performed in each reaction to verify the absence of contamination. The amplification conditions were performed with the following program: Initial denaturation at 94 °C for 4 min, 40 cycles of 45 s at 94 °C to denature, 60 s for annealing at the primer-specific melting temperature and 90 s at 72 °C to extend; and the last cycle was followed by a final extension of 10 min at 72 °C. The amplified fragments stained with GelRed  were separated on 1.8% (w/v) agarose gels in 1× TBE buffer running at 100 V constant for 1.5 h, and then, the gels were photographed under UV light with a Furi imaging system . Bands with similar migration distances amplified from different individuals were considered homologous. The molecular sizes of the DNA fragments were estimated using a DL2000 ladder . To ensure the reproducibility of the banding patterns, each PCR amplification and gel running was repeated twice, and only the amplified ISSR products present in both runs were considered.PCR reactions were standardized and run in a Vapo-protect Gradient thermal cycler . Every 25-µL reaction contained 1.5 mM MgH, I, Na, Ne, PPL, Ht, Hs, S and D. Population differentiation was analyzed for polymorphisms between populations by Gst, which was estimated by Nei’s gene diversity statistics [Nm among these populations was quantified using the formula Nm = 0.5(1 − Gst)/Gst [Each band was assumed to represent the phenotype at a single diallelic locus, because ISSR markers are dominantly inherited . All cleatistics  and Shanatistics . Nm amonGst)/Gst .D) was also constructed implementing UPGMA to visualize the genetic relationships among the populations.The binary matrix was then employed to perform an individual-level cluster analysis using an UPGMA dendrogram with SAHN  in NTSYS-pc version 2.20 . AnotherTo test the correlation between Nei’s genetic distance between populations and geographic distances (in km) among populations, a Mantel test  was perfPCoA implemented with the GenAlEx version 6.5 program  was carrK (from 1 to 5), 10 independent runs were made, and for each run, a Markov chain Monte Carlo (MCMC) of 100,000 iterations was carried out after a burn-in period of 50,000 iterations. Taking the results from the STRUCTURE output file, the most likely value for assigning K clusters was determined using the Structure Harvester program [To identify population structure and genetic admixtures with a Bayesian approach, STRUCTURE v2.3.4  was used program , which i program .C. japonicum genetic diversity was remarkably low both at the species and population levels and that genetic differentiation between populations was considerably high. The Mantel matrix test revealed no positive correlation between genetic and geographic distances. The UPGMA and STRUCTURE analyses indicated that the five populations can be divided into two main groups. According to genetic data and field observations, narrow habitat preference, genetic drift as a result of a small and isolated population size, limited gene flow, historical factors and human disturbance have shaped the current population structure. Finally, we suggest a combination of conservation strategies, with in situ and ex situ conservation, to ensure the long-term survival of this endangered orchid species.Our results showed that


Genetic diversity of present American populations results from very complex demographic events involving different types and degrees of admixture. Through the analysis of lineage markers such as mtDNA and Y chromosome it is possible to recover the original Native American haplotypes, which remained identical since the admixture events due to the absence of recombination. However, the decrease in the effective population sizes and the consequent genetic drift effects suffered by these populations during the European colonization resulted in the loss or under-representation of a substantial fraction of the Native American lineages. In this study, we aim to clarify how the diversity and distribution of uniparental lineages vary with the different demographic characteristics  and the different levels of admixture of extant Native groups in Colombia. We present new data resulting from the analyses of mtDNA whole control region, Y chromosome SNP haplogroups and STR haplotypes, and autosomal ancestry informative insertion-deletion polymorphisms in Colombian individuals from different ethnic and linguistic groups. The results demonstrate that populations presenting a high proportion of non-Native American ancestry have preserved nevertheless a substantial diversity of Native American lineages, for both mtDNA and Y chromosome. We suggest that, by maintaining the effective population sizes high, admixture allowed for a decrease in the effects of genetic drift due to Native population size reduction and thus resulting in an effective preservation of the Native American non-recombining lineages. Colombia was the major entrance point into South America during the peopling of the continent by the Paleoindians . From heThe demography of Native ethnic groups in Colombia endured several changes since pre-colonial times, through the Spanish domain and into present day. Several linguistic groups co-exist in Colombia since before the Spanish colonization, some of the most relevant being: the Chibchan, Carib and Arawakan, in the Atlantic coast, Chocoan in the Pacific coast and Paezan, Barbacoan and Quechua in the Southern Andean region  5–7]. T–7. T5–7]Colombia has been the target of several forensic and anthropological genetic studies –4,12–30.In the present work, we have focused on understanding how the different characteristics  of the remnant Native groups in Colombia have shaped the diversity and distribution of their uniparentally transmitted lineages, and whether the autosomal ancestry informative insertion-deletion (AIM-InDel) markers confirm these patterns.http://www.uma.net/e/policy/b3.htm).All samples involved in this study are long-lasting anonymized DNA extracts previously obtained from healthy individuals, from paternity cases, under informed consent for research purposes. The current study was approved by the institutional review board of IPATIMUP, and conducted in accordance with the ethical principles of the 2000 Helsinki Declaration of the World Medical Association  and H639 (5’-GGGTGATGTGAGCCCGTCTA-3’) ,38. The Samples for Y chromosome typing include the subset of male individuals from the total group that was typed for mtDNA. From all male samples, those that belong to Native American haplogroup Q were included in a previous study by Roewer et al. . The remMtDNA sequences were compared to the revised Cambridge Reference Sequence (rCRS)  using thwww.empop.org) [www.yhrd.org) [MtDNA sequences were submitted to EMPOP (pop.org) ,54 and Yhrd.org)  for qualhttp://www.fluxus-engineering.com). Phylogenetic networks were sequentially constructed applying reduced median and median-joining methods. In the Y-chromosome network the DYS385 marker was not considered and the number of repeats at DYS389II was calculated after subtracting the number of repeats at DYS389I. STR weighting was applied in accordance with Qamar et al.[For both mtDNA and Y chromosome data, haplogroup frequencies were calculated by direct counting, diversity indices considering individuals with Native American matrilineal or patrilineal ancestry were calculated using DNAsp v. 5  and Arlear et al. to obtaiAll Antioquia population samples and 55 Cauca samples were additionally genotyped for the 46 AIM-InDel polymorphisms described in Pereira et al. , accordiThe apportionment of genetic ancestral contributions was estimated using the software STRUCTURE v2.3.3 ,17. To eMtDNA control region haplotypes observed in the studied Colombian Native American population groups, together with their classification in haplogroups, are described in detail in The male haplotypes and haplogroups detected are described in All individuals except one (Cauca—haplogroup L) belong to the typical Native American mtDNA haplogroups A2, B2, C1 and D1 .Emberá-Chamí individuals (n = 38) belong to A2, B2 and D1 lineages (but not C), as previously reported for this ethnic group ,21. WithIn the Cauca population (n = 58) all four typical Native American haplogroups are present, with haplogroup C1 at a highest frequency (0.638), followed by A2 (0.190), D1 (0.086) and B2 (0.069). Because this population sample is heterogeneous in what concerns the origin of its individuals (both in location and in linguistic groups), we have analysed its major speaking groups separately.Guambiano-speaking group (n = 33) presents only Native American mtDNA haplogroups, with haplogroups A2 (0.091) and B2 (0.061) each one represented by one sub-lineage: A2ac and B2d. Within haplogroup C (0.758), the two most frequent sub-haplogroups observed were C1b (0.485) and C1d (0.273), both recognized as founder haplogroups in the Americas . The majChibcha speaking group (n = 14) presents one African haplogroup, L2a1c1 that descends from L2a1 which is a typical African lineage in Bantu populations . All fouThe Y chromosome haplogroup frequencies observed in our samples are indicated in Regarding Y chromosome haplogroups there is clearly a higher proportion of non-Native American lineages than for mtDNA, particularly in the populations from the Cauca department where various European lineages were found, in agreement with the asymmetric gender introgression pattern historically documented.The Emberá-Chamí individuals from Antioquia present only Native American haplogroups, with the M3 derived linage Q1a3a1a* at a higher frequency (0.833) than the M3 ancestral Q1a3* (0.167).In the Cauca populations, Native American (0.604), European (0.354), and sub-Saharan African (0.042) haplogroups were found.The Guambiano-speaking group shows both the Q1a3a1a*-M3 (0.483) and the Q1a3*-M346 (0.310) lineages at similar proportions, and the remaining 0.207 of the Y chromosomes belong to the European haplogroups R1b1-P25 and J2-M172.In the Chibcha-speaking group the Native American lineage Q1a3*-M346 is absent, and only four individuals present the M3 derived lineage Q1a3a1a* (0.333), while the non-Native American linages are present at a higher frequency (0.667) and represented by haplogroups R1b1-P25, G-M201, J2-M172, E1b1b1a1-M78 and E1b1b1c-M123 (0.500) that are typical in Europeans, and by two samples from the sub-Saharan African E1b1a1*-M2 haplogroup (0.167). Despite its small sample size, Sub-Saharan haplogroups were detected only in the Chibcha group, similarly to the results obtained for mtDNA.Maternal or paternal diversities were calculated only within lineages of Native American ancestry .MtDNA diversity indices showed generally higher values in the Cauca samples than in the Emberá-Chamí. While the Emberá-Chamí population presents only 7 different haplotypes in 38 samples (H = 0.751±0.051), the 57 Cauca individuals presented 38 different haplotypes (H = 0.958 ±0.018). Both major speaking-groups from Cauca present higher diversity values than the Emberá-Chamí population: the Guambiano-speaking group presents 19 different haplotypes in 33 individuals (H = 0.900±0.043), and the Chibcha-speaking group is highly diverse with 13 different haplotypes in 13 individuals (H = 1±0.030).Because the Chibcha sample presented few male individuals with Native American Y lineages (N = 4), the diversity indices for this population should be regarded with caution. For the other populations, however, the Y chromosome diversity indices follow the same trend than that described for mtDNA. The Emberá-Chamí population presents lower gene diversity (0.924±0.032) than the Guambiano (0.964±0.022) or the Cauca population considered as a whole (0.975±0.015), and the same is observed for the number of different haplotypes: 13 in 24 individuals for the Emberá-Chamí and 16 in 23 individuals for the Guambiano-speaking group. Despite this observation, the Emberá-Chamí population presents a slightly higher mean number of pairwise differences (MNPD) (9.214±4.390) than the Guambiano-speaking group (8.075±3.891) or the total Cauca population (8.325±3.971).The network analysis shows clearly the presence of the four typical mtDNA Native American lineages in the Colombian populations , with thIt is also noticeable that, for both mtDNA and Y chromosome, no haplotypes are shared between Antioquia and Cauca groups, although in each group there are various haplotype clusters distant from each other. This observation, together with the values of MNPD described above, is probably the result of bottleneck events acting on genetically differentiated and somewhat isolated Native American groups, as well as of the small sizes of the samples analysed.The genotyping results obtained for the 46 AIM-InDels are listed in The study of mtDNA, Y chromosome and autosomal AIM-InDel data in these groups contributes to a better knowledge of the genetic composition of Colombian populations.We show several important differences between the two samples, including different genetic composition and different degrees of admixture. Indeed, while the proportion of NAM matrilineal ancestry is well preserved in all samples, the proportion of paternal influx differed widely among samples, as reflected in the AIM-InDel data. The Emberá-Chamí sample from Antioquia presented exclusively Native American haplogroups both in mtDNA and Y chromosome, and only a residual proportion of non-Native ancestry in the autosomal AIM-InDels. The Cauca sample revealed a significant non-NAM male input (0.396), particularly within the Chibcha-speaking group, with its mtDNA haplogroups being almost exclusively NAM (only one AFR haplotype was detected), which reflects the traditional sex biased admixture between NAM and EUR men ,13,25. TIt is also noteworthy that the groups with a higher degree of Y chromosome non-NAM input show also a higher NAM mtDNA diversity. Although we can not exclude that larger Native populations may have attracted more non-Native admixture, we also suggest that populations exposed to a higher European admixture were less subjected to a decrease in effective population sizes and thus their Native non-recombining lineages were less prone to be erased by genetic drift. This protective effect of the admixture events on the genetic diversity of uniparental lineages is clear in the populations from Cauca, a less isolated and more cosmopolitan region than the Emberá-Chamí population from Antioquia.The study of the Native American genetic diversity has been complicated by the fact that, although the more isolated populations retain the Native American genetic identity, they paid the cost of losing most of its diversity by the decrease in effective population sizes after the European colonization. However this study demonstrates that, for lineage markers, it is possible to recover more of the Native American genetic diversity from the study of a comprehensive sampling of Native American ancestry individuals in admixed populations than from the analysis of isolated communities.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 FigThe haplogroups are named in accordance with Karafet et al..upgraded(DOCX)Click here for additional data file.


Rhagoletis zephyria (Snow) and the introduced quarantine pest R. pomonella  is occurring and may aid the spread of the latter into more arid commercial apple-growing regions of central Washington state, USA. Results for 19 microsatellites implied hybridization occurring at a rate of 1.44% per generation between the species. However, there was no evidence for increased hybridization in central Washington. Allele frequencies for seven microsatellites in R. pomonella were more ‘R. zephyria-like’ in central Washington, suggesting that genes conferring resistance to desiccation may be adaptively introgressing from R. zephyria. However, in only one case was the putatively introgressing allele from R. zephyria not found in R. pomonella in the eastern USA. Thus, many of the alleles changing in frequency may have been prestanding in the introduced R. pomonella population. The dynamics of hybridization are therefore complex and nuanced for R. pomonella, with various causes and factors, including introgression for a portion, but not all of the genome, potentially contributing to the pest insect's spread.Hybridization may be an important process interjecting variation into insect populations enabling host plant shifts and the origin of new economic pests. Here, we examine whether hybridization between the native snowberry-infesting fruit fly  Hybridization between closely related species can provide insights into the nature of species boundaries and the speciation process  state, a $2.25 billion annual industry accounting for 75% of apple production in the United States  from its native range in the eastern United States (AliNiazee and Penrose Crataegus spp.). In the eastern United States, the fly shifted to introduced, domesticated apple Malus domestica ∼160 ya, forming a new host race, the initial step in ecological speciation with gene flow  area . The two flies are parapatric in their distribution across the northern United States, overlapping extensively through the Midwest in Minnesota and Wisconsin  alleles from R. zephyria are introgressing into R. pomonella and potentially aiding its spread into the hotter and more arid central apple-growing region of the state. Comparisons of allele frequencies among these nine sites, as well as with potential source populations in the eastern United States, imply a complex pattern of low level hybridization and asymmetric introgression at some, but not all loci, implying a heterogeneous pattern of introgression throughout the genome.Here, using a set of 19 microsatellite loci, we genotyped flies infesting black hawthorn and snowberry across nine pairs of sites where the flies co-occur or are in geographic proximity from west of the Cascade Mountains into central WA to test the hypotheses that: (i) Infested fruit from snowberry bushes and black hawthorn trees were collected from nine pairs of ‘sympatric’ field sites in Washington state from July to September 2009 to 2012 where the flies co-occur less than 200 meters apart Fig. . The onlR. pomonella by Velez et al. . Consequently, the genetic survey was not limited to one region of the genome but screened a representative portion for evidence of hybridization and introgression.Flies were genotyped for 19 microsatellites originally developed for z et al. . The 19 n = 278 from black hawthorn and n = 327 from snowberry) were genotyped in the study. Genomic DNA was isolated and purified from pupae or adults using PUREGENE extraction kits . PCR amplification and genotyping of microsatellites were carried out as previously described for R. pomonella in Michel et al.  STRUCTURE analysis for all 18 populations. For this analysis, we used the admixture and correlated alleles model, investigating K = 1–18 as possible numbers of genetically distinct subpopulations existing across sites. Three replicate runs of 250 000 MCMC generations, following a burn in period of 250 000 generations, were assessed for each K value.To quantify genetic divergence between K values displaying the highest likelihood estimates in the initial STRUCTURE runs (K = 1–5). The follow-up analysis involved five replicate runs of 1 000 000 MCMC repetitions, following a burn in period of 500 000, for each K = 1–5 value. We used the ΔK method of Evanno et al.  separately for each paired site. Because the Evanno et al.  for each of three prior migration rates of 0.05 (the default), 0.01, and 0.1. The analysis produced posterior probabilities for each individual being derived from (i) cross of pure parental genotypes of natal host population origin ; (ii) a pure parental cross of non-natal host origin ; (iii) a hybrid cross between R. zephyria and R. pomonella (F1 hybrid); and (iv) a cross of a hybrid individual with one or the other parental types (backcross). Migration rates (gene flow levels) per generation between hosts could then be estimated as the number of migrant genotypes plus half the number of F1 hybrids divided by the total number of flies scored. Hybridization rates per generation similarly could be estimated as the number of F1 hybrids divided by the total number of flies scored.Fourth, potential migrants and hybrids were identified from STRUCTURE analyses performed at paired sympatric sites using the built-in function for population priors in cases when strong genetic structure exists between populations, as determined by step three above. The analyses for migrants and hybrids were conducted using a correlated allele frequency model and three replicate runs for vegan  were conducted using the package  Hijmans . Mantel R. pomonella and R. zephyria populations at the nine paired sites were also analyzed by linear regression against their geographic distance to the Tampico site near Yakima in central WA in R (R Development Core). To test the hypothesis that the genetic difference between species should decrease with proximity to Yakima given adaptive introgression from R. zephyria into R. pomonella, nonparametric, Monte Carlo simulations were used to determine significance levels for the regressions. In contrast to the Mantel tests which considered geographic distance between populations per se as the determinant of genetic differentiation regardless of ecology , the linear regression with distance to Yakima tested for microsatellite convergence of R. pomonella with R. zephyria along the primary axes of climatic change  varying from western to central WA hypothesized to underlie the adaptive introgression of alleles into R. pomonella. To test for statistical significance, microsatellite genotypes were randomly assigned within each species among the nine sites to determine how often a positive regression coefficient as large as that observed between allele frequency differences between the species at sites and the distance of sites to Yakima could be generated by chance. In addition, linear regressions were performed to test for an association between mean allele frequency differences and three environmental factors: (i) mean precipitation at sites from July to October, (ii) average high temperature at sites in July, and (iii) average low temperature at sites in January, as measured at recording stations near each site from 1981 to 2010, as listed on the Web sites for US Climate Data ( http://www.usclimatedata.com) and the Western Regional Climate Center ( http://www.wrcc.dri.edu).The overall mean and individual microsatellite locus allele frequency differences between R. pomonella and R. zephyria , while its highest frequency in any of the nine R. pomonella populations surveyed was 0.19. Conversely, allele 150 at locus p27 was present at a frequency of at least 0.18 in all R. pomonella populations, while its highest frequency in any R. zephyria population was 0.03. There were also a total of 196 private alleles for the 19 microsatellites found in one or more population of one of the species and not the other  than R. zephyria , consistent with the hypothesis of gene flow being primarily in the direction of snowberry into apple maggot fly populations. However, the difference could also be explained by larger effective population sizes and/or higher microsatellite mutation rates in R. pomonella, but given the history of recent introduction and generally lower densities of the apple maggot in the PNW, these two hypotheses seem less likely. Moreover, we genotyped fewer black hawthorn (n = 278) than snowberry-infesting (n = 327) flies, biasing the detection of private alleles in the direction of R. zephyria.None of the 19 microsatellites scored displayed a diagnostic difference between he other . These pR. zephyria and nine R. pomonella populations clustered distinctly and separately from one another with 100% bootstrap support in the neighbor-joining (NJ) network  across loci , had a consistently positive inbreeding coefficient such that the standard deviation of f values across loci did not overlap zero. Rhagoletis zephyria and R. pomonella did not differ in f , but R. zephyria populations did have lower observed heterozygosity than R. pomonella , consistent with the generally lower level of polymorphism found in the former species (see above). The results imply that deviations from random mating were not significant within local populations of either R. zephyria or R. pomonella and that the 19 microsatellites scored in the study did not possess high frequencies of null alleles.Microsatellite genotypes within populations of both species tended to be slightly heterozygote deficient, with low positive mean inbreeding coefficients , with the exception that more backcross individuals were identified for the m = 0.1 model. We therefore present the finding generated from the most conservative m = 0.01 model. One of the 46 flies reared from black hawthorn at the Beacon Rock . In contrast, no evidence for hybridization was detected between the two species on snowberries. The estimated rate of migration and gene flow from the snowberry into black hawthorn-infesting fly populations was 0.0072 per generation, while it was 0.0031 in the reverse direction.The finding of substantial population structure between black hawthorn versus snowberry-infesting fly populations at each of the nine paired sympatric sites allowed for posterior probabilities to be estimated for individuals to assess their genetic ancestry. Based on these probabilities, we found evidence for ongoing migration, gene flow, hybridization, and introgression between  WA Figs  and S2. ock Fig. B, two ofock Fig. C, and onock Fig. D possessock Fig. B. In addant Fig. A. None or = 0.032; P = 0.2785). Geographic distance was correlated with population genetic distance for R. zephyria populations analyzed separately , but not for R. pomonella populations .In a partial Mantel across all populations after species identity was accounted for, geographic distance was not significantly related to population genetic distance , consistent with the desiccation hypothesis. Mean allele frequency differences were also strongly correlated with summer high temperatures  and winter low temperatures . The seven significant loci displaying a relationship with distance to Yakima were distributed across all five of the chromosomes for which genetic markers were scored  being closest to snowberry flies in the genetic distance network . Consequently, while the microsatellites implied hybridization, gene flow, and a degree of genetic converge between R. zephyria and R. pomonella approaching central WA, there was also evidence for introgression being restricted between the species despite hybridization.Microsatellite allele frequency differences between WA Table , Fig. 4.WA Table , Fig. 4.ntral WA . MoreoveR. pomonella into the more arid apple-growing region of central Washington. We found no evidence for a hybrid swarm between R. pomonella and R. zephyria in central WA. However, the STRUCTURE analysis implied mainly unidirectional hybridization between R. zephyria into R. pomonella on black hawthorn occurring at a rate of 1.44% per generation , which is higher than that of 0.1% inferred from previous work , the effective introgression rate may be low, while potentially contributing to the adaptive process for R. pomonella. Such a scenario seems more in line with studies showing that larval survivorship is not high in reciprocal host transplant experiments  in black hawthorn populations in WA, implying introgression. However, most of these shared alleles between the two species in WA were present at low frequencies (<0.05%) and in only a subset of the nine R. pomonella populations surveyed. Indeed, in only eight of the 19 instances was the putatively introgressed R. zephyria allele found at modest to high frequencies in a majority of the R. pomonella populations sampled . Moreover, of these eight variants, only allele 301 at microsatellite p18 showed a clear pattern of increased frequency in central WA, although this was the locus displaying the most pronounced convergence with R. zephyria in the study. For the other significant microsatellites positively varying with proximity to Yakima, patterns were diverse. First, no shared and presumably introgressed unique R. zephyria allele was present at four loci ; second, for one locus (p16), a shared allele (292) was present at low frequency (0.0132) and only in one R. pomonella population (WSU); third, for the remaining locus (p80), there were several shared alleles distributed widely across black hawthorn fly populations, but not obviously increasing in frequency in central WA. Thus, for the majority of loci showing convergence with R. zephyria in central WA, the alleles can also be found in R. pomonella populations in the midwestern United States. As such, these genes may represent standing variation in R. pomonella that was present at the time of the fly's introduction prior to its spread into central WA. The origin of these alleles in R. pomonella could conceivably trace to past historical gene flow between R. zephyria and R. pomonella predating the western introduction. However, there is currently no evidence from the microsatellites that these genes recently introgressed into R. pomonella to facilitate the fly's movement into central WA. Proof or refutation of recent introgression will require much more detailed DNA sequence analysis. Consequently, certain variants at certain microsatellites appear to have introgressed and may be adaptive in R. pomonella , while other instances of observed geographic variation in WA could involve standing genetic variation.In total, we detected 19 of 50 ‘unique R. zephyria alleles into introduced R. pomonella populations. In central WA, larvae finish feeding, exit fruit, burrow into the soil, and form puparia during the hottest and driest period of the year. Studies suggest that R. zephyria pupae have a greater tolerance to desiccation than R. pomonella  that should lead to the eventual erosion of differentiation at equilibrium. The data presented here therefore add to the evidence that many ‘good’ species persist in the face of considerable hybridization (Mallet R. pomonella (Feder et al. Strong population genetic differentiation is maintained between n Mallet  and thatR. pomonella have suggested that past cycles of geographic isolation, genetic differentiation, secondary contact, and subsequent hybridization have contributed to the adaptive radiation of members of the R. pomonella sibling species complex onto a variety of novel host plants (Feder et al. R. zephyria and R. mendax may have given rise to a new honeysuckle-infesting population of Rhagoletis attacking Lonicera (Schwarz et al. R. zephyria and the recently introduced R. pomonella is contributing to the spread and adaptation of the apple maggot fly to harsher environmental conditions found in the commercial apple-growing region of central WA. Our results imply that standing variation may be important for R. pomonella's establishment in central WA. We found evidence that some of this variation could have its roots in R. zephyria and have recently introgressed into western R. pomonella populations, while a significant portion may have a deeper history in R. pomonella predating its introduction. Thus, a diversity of causes and factors, including hybridization, likely underlie the story of R. pomonella's invasion and spread in the western United States.In conclusion, there are a growing number of examples documenting differential adaptation of insect populations to commercially grown host plants (Kirk et al.


Managers of threatened species often face the dilemma of whether to keep populations separate to conserve local adaptations and minimize the risk of outbreeding, or whether to manage populations jointly to reduce loss of genetic diversity and minimise inbreeding. In this study we examine genetic relatedness and diversity in three of the five last remaining wild populations of dama gazelle and a number of captive populations, using mtDNA control region and cytochrome b data. Despite the sampled populations belonging to the three putative subspecies, which are delineated according to phenotypes and geographical location, we find limited evidence for phylogeographical structure within the data and no genetic support for the putative subspecies. In the light of these data we discuss the relevance of inbreeding depression, outbreeding depression, adaptive variation, genetic drift, and phenotypic variation to the conservation of the dama gazelle and make some recommendations for its future conservation management. The genetic data suggest that the best conservation approach is to view the dama gazelle as a single species without subspecific divisions. Nanger dama) Fragmented populations of endangered species present a conservation dilemma There are around 300 dama gazelles left in the wild The gazelle's range formerly spanned the entire Sahel and Atlantic Saharan region  from souNanger with Soemmering's gazelle (N. soemmerringii) and the Grant's gazelle complex (N. granti and related species) Gazella, molecular phylogenetic analysis revealed inclusion of these larger gazelles would make the genus Gazella paraphyletic and hence they have been separated into the genus NangerThe dama gazelle is the largest of the gazelle species N. dama ruficollis occurs east of c.15°E, N. dama dama occurs from c.7°E to c.15°E, and N. dama mhorr occurs west of c.7°E. This tri-part classification was also followed by Groves and Grubb dama and mhorr locating it further northwest, stating that mhorr did not occur further south than the Western Sahara , including Antilope nanguer, Antilope dama var. occidentalis, Gazella dama permista, Gazella mhorr reducta, Gazella dama damergouensis, Gazella dama weidholtziN. d. ruficollis , including Antilope addra, Antilope dama var. orientalisN. d. mhorr , including Gazella dama lozanoiN. d. mhorr is often referred to by the common names of mohor or mhorr gazelle and N.d. ruficollis is often called the addra gazelle. Here we use the term “dama gazelle” to cover all putative subspecies and populations of the species Nanger dama and for the time being we follow Cano's N. d. mhorr in Almeria (Spain) at the Parque de Rescate de Fauna Sahariana , with animals that had originated from the Dora-Hagunia and Tichla-Bir Ganduz of Western Sahara in 1958 N. d. mhorr were seen there in 1968 The early history of the captive stock is detailed by Cano N. d. ruficollis “addra” in favour of N. d. mhorrN. d. mhorr in favour of N. d. ruficollis. Therefore, the current situation is that N.d.ruficollis is subject to coordinated breeding efforts in the USA and breeding of N.d.mhorr is coordinated in Europe. A number of institutions in the Middle East hold both subspeciesThe decision was taken in 1995 by the European Association of Zoos and Aquaria (EAZA)  to phase out the captive management of ruficollis population is indeed ruficollis or in fact dama or a mixed subspecies population, but here we refer to it as ruficollis throughout, following the current consensus. Twenty-eight institutions across the USA hold a total of 168 animals ruficollis, because the original collecting locality is within this subspecies' range according to the Cano N. d. ruficollis in captivity in private collections, for which no additional founders are known.There has been historical debate over whether the Given the small number of animals left in the wild, the studbook-managed populations represent a considerable proportion of the species' global population . At least as many animals again exist in captivity outside the two studbooks, notably on private ranches in Texas The purpose of this study was to assess the relatedness and relative genetic diversity in extant populations and to use this information to assess the conservation value of different populations and the validity of the current subspecies classification system in order to inform future conservation management strategies for the dama gazelle.In total 124 samples were collected. This included 36 samples from the wild that were collected as a result of six collecting trips on public land between 2009 and 2013 with the authority and participation of the Termit & Tin Toumma National Nature Reserve, Niger and the Ouadi Rimé-Ouadi Achim Game Reserve and Government of Chad. Samples were collected opportunistically as pellets from distinctly separated fresh piles of faeces, in most cases following direct observation of wild dama gazelles. Samples from captive and reintroduced populations represent a mixture of blood and tissue samples taken from routine veterinary treatment or during autopsy  or via faecal sampling and were provided by the relevant institutions or from the collections of National Museums Scotland (see Supplementary Tissue and blood samples were extracted using DNeasy blood and tissue kits (Qiagen) and faecal samples with the QIAamp DNA Stool Mini Kit (Qiagen), according to standard protocols. In the case of faecal samples, a single pellet was used per extraction.cta tgt cct gtt acc att gac, nang-rev (5′ to 3′): gat tgt cca cat gca tat aag c. PCR amplification of the fragment was conducted with 1 µl of template DNA (10–50 ng ml−1)1 µl each of forward and reverse primer (10 µM) and 7 µl of Maxima Hot Start PCR Master Mix (Thermo-Fisher). Amplification was performed with an initial denaturation step of 5 mins. at 95°C, followed by 35 cycles of 1 min. (denaturation) at 95°C, 1 min.  at 58°C, 1 min. (elongation) at 72°C and ending with a 72°C extension for 10 mins. Negative controls were run as standard.Primers were designed from existing sequences on Genbank to amplify a 560 basepair fragment from the centre of the control region: nang-fwd (5′ to 3′): The fragments were examined by running them out on a 1% agarose gel and successfully amplified products were cleaned up by addition of 0.5 µl of the enzymes EXO1 and FastAP (Fisher) with an incubation step of 37°C×45 min and a denaturation step of 80°C×10 min. Fragments were sequenced in the forward direction using the BigDye Terminator Kit® (Applied Biosystems), using 3 µl of PCR product and conditions according to manufacturer's instructions. Sequences were run on a capillary ABI 3730 DNA Analyzer sequencer® (Applied Biosystems). In the case of faecal samples, sequencing was additionally conducted in the reverse direction and for a subset of samples with unique haplotypes the PCR was repeated and sequenced.Since cytochrome b evolves at a slower rate than the control region, a subset of 16 samples exhibiting unique control region DNA haplotypes were sequenced for a 421 base pair region of the mtDNA cytochrome b gene using the primers MCB 398 and MCB 869 Chromatograms were analysed with Geneious Pro v6.1.4 Tree building was performed separately for control region and cytochrome b fragments, using two different methods for each fragment. Firstly, a simple distance-matrix method was used to build the trees. This was done using neighbour joining with the Tamura-Nei model of genetic distance. The trees were built in Geneious Pro v6.1.4 and one-hundred bootstrap iterations were performed. Secondly, Bayesian inference was carried out with MrBayes v3.1.2 Nanger granti) as an outgroup and incorporating sequence JN632665 from a dama gazelle, originating from San Diego Zoo. Here it is referred to as control region haplotype S.Control region trees were built using the Genbank haplotype JN632666 . The network was examined at a number of different probabilities of parsimony .Haplotype networks of the control region data were created using TCS v1.21, treating gaps as a 5Nanger soemmerringii) and JN632666 (Nanger granti) as outgroups. Sequence JN632665 from the same dama gazelle, originating from San Diego Zoo, was also incorporated into the tree Cytochrome b trees were built, including Genbank sequences KC188777, JN632667, JF728776 . 16 haplotypes were discovered and these sequences have been named A to O & R and have been placed on Genbank under accession KJ848615–30. The majority of the haplotype diversity was spread between the wild populations (13 haplotypes). Captive and captive-derived populations  only contained three haplotypes, two within ulations . Haplotyulations . Nucleotulations .For the Bayesian inference of phylogeny the average standard deviation of split frequencies was 0.0069 after 2,000,000 Metropolis-coupled MCMC generations for both of the two replicates.Phylogenetic analysis of the mtDNA control region data revealed tree topologies that were roughly concordant with Neighbour-Joining and Bayesian methods. However, support was generally low apart from for clusters , ,  and  .Network analysis revealed a similar certainty surrounding resolution, with four clusters being resolved at a 95% connectivity limit , ,  and . Relaxation of the connectivity criteria to below 90% was required to draw a complete network, with 18 mutational steps required to link the entire tree . Pair-wiIn total seven haplotypes of cytochrome b were discovered, which were named MCB1–3, 5–8 and placed on Genbank under accession KJ848631–7. Individuals carrying control region haplotypes H and I corresponded to MCB Haplotype 1. MCB2 corresponded to control region B,O,A,D,K,J and R; MCB3 to C; MCB5 to L; MCB6 to N, MCB7 to G,M,F and MCB 8 to E. As previously mentioned, MCB4 corresponds to S . These cytochrome b haplotype groupings have been overlaid on the control region network .For the Bayesian inference of phylogeny the average standard deviation of split frequencies was 0.007 and 0.0042 after 2,000,000 Metropolis-coupled MCMC generations for the two replicates. Graphical output of the change in Log Likelihood during the analysis can be found in supplementary material 1.Resolution of the phylogeny was not much improved at the cytochrome b gene , with onPair-wise differences between cytochrome b haplotypes can be found in The purpose of this study was to assess the relatedness and relative genetic diversity in extant populations of dama gazelle and to use this information to begin an assessment of the conservation value of different populations.mhorr populations  and the captive ruficollis populations samples  . However, we have not been able to survey genetic diversity extensively within the captive ruficollis population, having only sampled from derived European (MAR_R) and Arabian (AIN_R) captive populations, and not the original population brought from Chad to the USA. We can expect USA populations to retain some additional genetic diversity not found in the derived populations sampled as part of this study. Understanding how the captive ruficollis population in the USA, both within zoos and private collections, relates to dama gazelle populations globally, represents an important future task. It is interesting to note that the animals sampled from the ruficollis captive population exhibit haplotypes that are closely related to those found at OROA . Particularly striking is the much higher haplotype diversity in the wild populations as a whole  compared with the captive and captive-derived , MAR_R) . Low gen, MAR_R) . This su at OROA , as inde at OROA , althougCaptive populations of threatened species should be managed to maximise genetic diversity and to minimise inbreeding for two reasons: to reduce the likelihood of inbreeding depression, and to retain the greatest adaptive potential of the population.mhorr at La Hoya  that in highly inbred captive populations, pedigree estimates of inbreeding are often not reliable indicators of accumulated inbreeding, because base levels of inbreeding may already be high and assumptions of non-relatedness of founders may not be met. It seems likely that captive mhorr populations are suffering from other effects of inbreeding, but it is hard to measure these effects due to lack of statistical power. Similar studies have not been conducted for ruficollis.Inbreeding has been shown on numerous occasions to have a detrimental effect on fitness in naturally outbreeding species Inbreeding depression is not the only reason to be concerned about inbreeding in captive dama gazelles. Concurrent loss of genetic diversity may result in loss of adaptive potential, limiting the ability of the population to evolve Although genetic diversity in the wild appears to be considerably higher than in captivity, diversity is undoubtedly being lost in wild populations and this is of concern for the same reasons as mentioned above. Populations are small and isolated, and in the long term may become vulnerable to inbreeding, if the situation remains the same. Interestingly no haplotypes were shared between different populations despite lack of genetic structure (see below). This may indicate a lack of recent gene flow between sites and genetic drift in small fragmented populations, or may simply be an artefact of the small sample sizes available to this study. Additionally, the comparatively small number of haplotypes found in Termit versus the other wild populations  may indimhorr population  and differ by 12 base pair mutations , F (mhorr) and G (ruficollis/Manga) are associated with an identical cytochrome b haplotype.Critically, the clustering of these groups is also supported by the pattern of relatedness at the more slowly evolving cytochrome b gene, because closely related control region haplotypes are associated with identical or closely related cytochrome b haplotypes , 5. Notaruficollis subspecies grouping is likely to be polyphyletic with respect to both dama and mhorr  and rock pocket mouse (Chaetodipus intermedius) have been linked to variation in substrate colour exerting differential selection pressure on specific genes Jaculau jaculus) Agouti gene, a gene that has also been implicated in coat colour variation in a large number of mammals, including Soay sheep, Ovis ariesCanis familiaris, The lack of phylogeographical structure across the dama gazelle range does present a contradiction because the cline in pelage coloration  points tThere are distinct climatic differences between the Sahel and the Atlantic Sahara If the phenotype is not under selection, one possible explanation for the observed variation in pelage coloration is that genetic drift occurred in separate refugia, perhaps either side of palaeolake Mega-Chad, which was larger than the Caspian Sea 5,000–6,000 years ago and has undergone changes in size associated with climatic cycles for at least the last 23,000 years Addax nasomaculatus) and Arabian oryx (Oryx leucoryx), are thought to have evolved a predominantly white pelage to reflect solar radiation for thermoregulation and advertisement when in the open and to aid with camouflage when in the shade It seems unlikely however that pelage coloration is neutral to selection given the  presence of predators per se. Genetic management of dama gazelles should not seek to deliberately breed for phenotype as this will certainly result in further inbreeding of captive populations and may lead to selection for unforeseen traits, because genes controlling coat colour are known to have pleiotropic effects on other (often fitness-negative) traits Until evidence to the contrary is produced, phenotype should be assumed to have some selective relevance as this is the most conservative scenario for conservation. The key here is that it is the preservation of the diversity of genes controlling phenotype that is important as this is the raw material on which selection can act (in different environments), not the phenotype The issues behind the apparent cline in dama gazelle phenotype come to the fore when dealing with the conservation of its populations, creating a classic dilemma for modern-day conservation: are the now fragmented (wild and captive) populations distinct enough to justify being managed separately?et al.et al.By mixing populations that are distinct, the risk is that the population will suffer from hybrid sterility or outbreeding depression. The risk of maintaining them separately is inbreeding depression and loss of genetic diversity (see above). Frankham mhorr) and San Diego (ruficollis) in the form of a single chromosomal rearrangement  between the short acrocentric Y1 male-specific sex chromosome and an autosome of pair 14 in the San Diego animals. However a karyotypic polymorphism is also present within animals from Almeria (mhorr); in this case a Robertsonian polymorphism of chromosome 1 mhorr in a private collection at Taif in Saudi Arabia, although the origin of this animal is not known Gazella subgutturosa, G. gazella (from northern Israel), Eudorcas thomsonii and Antilope cervicapraIn the case of the dama gazelle it seems likely the range has been contiguous within the last 500 years and dama gazelles are known to migrate seasonally in search of water It must also be noted that the captive animals studied here are descended from few individuals see  and thusn subspecies” even if subspecies were not classified on a rigorous scientific basis. A large number of putative dama gazelle subspecies were recognised historically, based on rather limited evidence, which the paper by Cano a priori assumption of one. The levels of genetic divergence at cytochrome b (0.5% between captive mhorr and ruficollis haplotypes and 2.1% as a whole) are at within-species levels both for the best studied Saharan gazelle species, the dorcas gazelle The problem with the historical preference for splitting into subspecies is that the burden of proof rests with the contemporary researcher. The null hypothesis becomes: “there are In the light of this study and the evidence presented here, we conclude with the following statements relevant to the conservation of the dama gazelle:a priori reason to divide the cline into three discrete units and lack of coincident mtDNA genetic structure and the possible polyphyly of the nominate ruficollis subspecies supports this view.To the best available knowledge, phenotypic variation was originally more or less clinal across the species range. Today, we see phenotypic variation between populations, but also within captive and wild populations. Phenotypic differences between subspecies in captivity may be exaggerated by (in)breeding of the current populations from small numbers of founder individuals. Uneven and incomplete sampling of historical wild populations, and absence and fragmentation of contemporary populations, has in addition possibly presented a distorted view of the original phenotypic variation throughout the species' range. There is no a priori barrier to exchange between any populations provided the risks of exchange have been evaluated properly.The conservation of the dama gazelle will be greatly advanced if it is considered a single species without subspecies division, even though it exhibits phenotypic variation. Under the “three subspecies view” artificial impermeable boundaries are erected and only individuals which conform to a particular phenotype from within the same subspecies should be bred together, and used for reintroduction and population augmentation. Under the “monotypic species view” there is a continuum of suitability of donors to a population, where all else being equal, the geographically most proximate population is the most suitable, but there is no Inbreeding depression and loss of genetic diversity must be taken seriously during ongoing management efforts. Evidence for inbreeding depression in a captive population has already been found Unless there is evidence to the contrary, phenotype should be assumed to be under some degree of selection in the wild as this is the most conservative scenario to conservation. However, no attempt should be made to breed or select for “a true phenotype” in captivity as this will result in further loss of genetic diversity and possibly unintended selection for traits linked to phenotype.mhorr and ruficollis individuals should be conducted as part of a risk-benefit analysis for future actions The possible risks associated with interbreeding of animals from phenotypically distinct and geographically distant populations should be taken seriously, however the potential conservation benefits of such interbreeding should be evaluated. Small-scale scientifically monitored experimental crosses of captive Table S1Details of the individual samples used in this study.(XLSX)Click here for additional data file.


Acrocephalus sechellensis) – an ecological model species. By the 1960s, this species was on the brink of extinction, but its previous history is unknown. We used DNA samples from contemporary and museum specimens spanning 140 years to reconstruct bottleneck history. We found a 25% reduction in genetic diversity between museum and contemporary populations, and strong genetic structure. Simulations indicate that the Seychelles warbler was bottlenecked from a large population, with an ancestral Ne of several thousands falling to <50 within the last century. Such a rapid decline, due to anthropogenic factors, has important implications for extinction risk in the Seychelles warbler, and our results will inform conservation practices. Reconstructing the population history of this species also allows us to better understand patterns of genetic diversity, inbreeding and promiscuity in the contemporary populations. Our approaches can be applied across species to test ecological hypotheses and inform conservation.The importance of evolutionary conservation – how understanding evolutionary forces can help guide conservation decisions – is widely recognized. However, the historical demography of many endangered species is unknown, despite the fact that this can have important implications for contemporary ecological processes and for extinction risk. Here, we reconstruct the population history of the Seychelles warbler ( Evolutionary processes are often overlooked by biologists and policy makers interested in conserving endangered species. This is a problem, as understanding the demographic history of populations, and thus the evolutionary pressures that they may have faced, is of conservation importance. High levels of inbreeding in small populations can increase homozygosity, and thus the expression of deleterious recessive alleles, with negative fitness consequences , a small passerine endemic to the Seychelles archipelago in the Indian Ocean  and cats (Felis catus)  to allow the succession of natural pisona (Pisonia grandis) woodland, was successful, and the Cousin warbler population quickly recovered; since the 1980s, it has been at a carrying capacity of approximately 320 adults  and Marianne (n = 7) in 1876–1940 (Table S1). Although the temporal range of sampling of the museum specimens was wide, structure analyses suggested that they grouped into two populations (see Results), enabling us to group them for population genetic analyses. A small (approximately 1.5 × 1.5 × 3.0 mm) piece of skin was excised from the ventral surface of the foot and stored at room temperature in a sterile microfuge tube. Contemporary samples were collected as part of an intensive, long-term study of Seychelles warblers on Cousin Island  was added at incubation; and (iii) 1 μL carrier RNA  was added during the precipitation phase  and 1 μL DNA. The PCR cycling conditions were 15 min at 95°C, followed by 45 cycles of 30 s at 90°C, 1 min 30 s at 56°C and 1 min 30 s at 72°C. All pre-PCR work was carried out in the ‘clean room’. Each contemporary sample was molecularly sexed and genotyped at 30 polymorphic microsatellite loci combined into four multiplexes (Tables S2 and S3). PCR amplification of the contemporary samples was performed in 10 μL volumes containing 20–50 ng of template DNA, using a Qiagen Multiplex PCR Kit and the manufacturer's protocol. The PCR program used was as follows: 95°C for 15 min, followed by 30 or 35 (for the museum specimens) cycles of 94°C for 30 s, annealing temperature  for 90 s and 72°C for 60 s, followed by 60°C for 30 min. All PCR products were separated on a 48-well capillary ABI 3730 DNA analyser, and allele sizes assigned using GeneMapper 4.0 software . All samples were genotyped at least twice to assess repeatability, and new alleles  were only confirmed when observed in both reactions. Twelve of the 30 markers were successfully genotyped using the partially fragmented DNA extracted from the museum samples (Table S4), so only the corresponding loci from the contemporary samples were included in the data set used for comparison.The potential for amplification of each museum DNA sample was tested by molecular sexing using the Z-002D marker set Dawson . Each PChttp://www.r-project.org/). For each locus and population , we calculated observed and expected heterozygosity and tested for deviations from Hardy–Weinberg equilibrium (HWE) using GENEPOP version 4.0.10 , by dividing the number of alleles in a population (k) by the range in allele size (r).We used two approaches based on summary statistics to detect whether the warbler populations had undergone genetic bottlenecks. We first tested for heterozygosity excess, which occurs owing to the loss of rare alleles shortly after bottlenecks . For each scenario, we simulated 1 million data sets. As summary statistics, we used the mean number of alleles per locus, mean gene diversity and mean Garza–Williamson M index within each population, as well as pairwise FST across each pair of populations. The posterior probability of scenarios was estimated by (i) taking the 500 simulated data sets closest to the observed data set and calculating the proportion that belong to each scenario (direct estimate) and (ii) performing a logistic regression on the closest 1% of data sets to the observed data  and contemporary populations using the Bayesian approach implemented in ONeSAMP  so was excluded from further analysis. Two were genotyped at six loci ; population-level analyses carried out both with and without these two samples yielded the same results (data not shown), so they were retained, leaving sample sizes of 19 and 6 for Cousin and Marianne, respectively, and a total sample size of 126 individuals when the 1997 and 2011 populations were included. The genotyping error rate in the museum specimens was 2.5%. Observed and expected heterozygosity for each locus and population, along with results of tests for deviations from HWE and null allele frequency estimates, are given in Table S4.FST estimates between museum and contemporary samples were moderately large and highly significant . The only difference between the PCA and STRUCTURE analyses was that STRUCTURE, when K = 2, grouped the Marianne samples with the Cousin museum samples  – however, this is most likely the result of the low sample size on Marianne  and in both the 1997 and 2011 populations . There was no evidence for heterozygosity excess in the Marianne museum populations (P = 0.09–0.27), but the number of individuals (n = 6) is lower than the recommended minimum sample size for this analysis (n = 10), so this result must therefore be treated with caution. Mean (±SE) Garza and Williamson's M ratio was 0.72 ± 0.06 for the Marianne museum population, 0.81 ± 0.05 for the Cousin museum population and 0.66 ± 0.05 for both the 1997 and 2011 Cousin populations . We evaluated confidence in this scenario choice by simulating 500 pseudo-observed data sets under the constant Ne scenario, performing ABC analyses and calculating the number of times the bottleneck scenario was incorrectly chosen, and found that in no case did this occur (suggesting a Type 1 error rate of <0.002). That 14 of the 15 observed summary statistics were well within the range of the simulated data for the bottleneck scenario (Table S5) gives us further confidence in our scenario choice. Posterior estimates suggested that the Cousin population was reduced from an Ne of several thousand to <50 between 120 and 250 years ago, with relatively low bias and high precision estimates . We note that sample sizes lower than 20 individuals can cause problems with ONeSAMP  and therefore interpret absolute values from this program with caution.Simulations carried out in DIY-ABC strongly supported a bottleneck over the scenario of a constant The Seychelles warbler, like many bottlenecked species, is characterized by low levels of genetic diversity at neutral and functional genes  – two of the most geographically separated islands within the Praslin group , paradise-flycatcher (Terpsiphone corvina) and white-eye (Zosterops modestus; Collar and Stuart The loss of genetic diversity between museum and contemporary samples, the results from bottleneck tests, and the estimate of the bottleneck time from DIY-ABC all indicate a recent dramatic decline in population size in the Seychelles warbler. Most islands in the Seychelles were planted with coconuts in the late 19th and early 20th centuries, creating unsuitable habitat for Seychelles warblers Komdeur . This, aNe documented here, is that inbreeding avoidance mechanisms might not evolve in larger populations where inbreeding is infrequent (Jamieson Long-term study systems such as the Seychelles warbler provide unique insight into key processes in ecology and evolutionary biology (Clutton-Brock and Sheldon Jamieson . LikewisJamieson , the SeyJamieson ; howeverThe approaches used here, combining museum DNA with population simulations, have been and will be of use to other studies wishing to generate clear, testable hypotheses about ecological and evolutionary phenomena. These may be questions similar to those that we address using the Seychelles warbler system concerning inbreeding depression and the evolution of promiscuity. Alternatively, they may be questions related to older ecological and evolutionary phenomena, such as the role of climate fluctuations and prehistoric hunting pressures in historical population change (Fontaine and Snirc Ne was already dramatically reduced when the translocations were carried out, and the further bottlenecks associated with moving a subset of individuals (Wright et al. Genetic diversity is crucial for the long-term persistence of populations and species and is one of the IUCN's three global conservation priorities along with species and ecosystem diversity (McNeely et al.


Erica species from these Mediterranean heathlands that differ in their edaphic specificity, E. australis, sandstone specialist, and E. arborea, generalist. Average levels of within-population genetic diversity and gene flow between populations were significantly lower in Tangier (high sandstone patchiness) than in Algeciras (low patchiness) for the sandstone specialist, whereas no differences between both sides of the Strait were detected in the edaphic generalist. Since most endemic species in Mediterranean heathlands of the Strait of Gibraltar are sandstone specialists, these results highlight an increased vulnerability to loss of genetic diversity and local extinction of the heathland endemic flora in the Tangier side of the Strait of Gibraltar.Many habitat specialist species are originally composed of small, discontinuous populations because their habitats are naturally fragmented or patchy. They may have suffered the long-term effects of natural patchiness. Mediterranean heathlands, a representative habitat in the Strait of Gibraltar region, are associated with nutrient-poor, acidic sandstone soils. Sandstone soil patches in the African side of the Strait (Tangier) are, in general, smaller and more scattered than in the European side (Algeciras). In this study, we analyze the effect of this sandstone patchiness on the population genetic diversity and structure of two  Plant populations in small habitat fragments have frequently smaller population sizes and experience a higher degree of isolation among populations than those from continuous or less fragmented habitats Genetic diversity in a plant population is lost mostly by the effect of genetic drift and by reduced genetic communication (i.e. low gene flow and migration) among populations Habitat degradation and fragmentation have indeed become major research topics in both population genetics and conservation biology Tangier) are lower than those from the European side (hereafter Algeciras) Many endemic, and/or endangered plant species in the Mediterranean Basin, one of the world’s biodiversity hotspots, are habitat specialists − edaphic and/or orographic − restricted to uncommon, patchy habitats. Mediterranean heathlands of the Strait of Gibraltar region, at the western end of the Mediterranean, harbour an endemic-rich flora composed mainly of edaphic specialist species, restricted to nutrient-poor, highly acidic, sandstone soils  specificity: while E. australis is a sandstone specialist, virtually restricted to sandstone-derived soils on both sides of the Strait, E. arborea has a broader edaphic spectrum E. australis, but not for E. arborea.Here we explore whether an overall high level of sandstone patchiness is associated to low levels of within-population genetic diversity and of gene flow between populations of sandstone specialist species. To do so, we studied the genetic diversity and population structure of two heath species, This study provides valuable insights into the differential effects of natural habitat (sandstone) patchiness on the genetic diversity and structure of populations of habitat specialist and generalist species. Considering that most endemic species from these Mediterranean heathlands are sandstone specialists Junta de Andalucía (Spain). None of the two species used in this study is endangered or red-listed.Necessary permits for fieldwork and sampling were obtained through the University Abdelmalek-Essaadi (Morocco) and the Erica australis and E. arborea (Ericaceae) are two relatively abundant heath species in Mediterranean heathlands on acid, nutrient-poor, Oligo-Miocene sandstone soils at both sides of the Strait of Gibraltar n = 2x = 24; E. australis is a sandstone specialist, restricted to highly acidic, sandstone soils, whereas E. arborea has a broader edaphic spectrum. Within the regional context of this study , E. australis is virtually restricted to highly acidic, aluminium-rich sandstone soil patches whereas E. arborea occurs both within sandstone patches and out in the surrounding matrix on different, non-acid soils E. australis is endemic to the western end of the Mediterranean basin (western third of the Iberian Peninsula and NW tip of Africa), whereas E. arborea has a much wider, circum-Mediterranean and eastern African distribution E. australis and whitish in E. arborea. They are insect pollinated, with pollen shed in tetrads Erica australis sets flowers in late-winter to early-spring (December to April), whereas E. arborea is a spring-blooming species  than those of E. arborea  and 631 individuals of E. arborea (308 from Algeciras and 323 from Tangier). Samples were dried in silica gel and stored at room temperature until DNA extraction. Dry leaf material, approximately 100 mg per sample, was reduced to fine powder using stainless steel beads on a Mixer Mill MM400 cell disrupter . DNA was extracted using SpeedTools plant DNA extraction kit , and eluted in 50 µl in Tris-EDTA 0.1×buffer.Twenty-two localities (11 from Algeciras and 11 from Tangier) where  sampled , Fig. 1. reached . Thus, tet al. E. australis, and eight  in E. arborea. These microsatellite loci were unlinked Amplification of microsatellite loci followed Segarra-Moragues http://datadryad.org/) under accession number doi:10.5061/dryad.bj70k.PCR products were electrophoresed in an ABI3730 automated sequencer  using LIZ500 as internal lane size standard. Assignment of fragments to allele classes was carried out with Genemarker version 1.97 software . Genotypic matrices were deposited at DRYAD (A), and observed (HO) and unbiased expected (HE) heterozygosities F-statistics were estimated according to Weir & Cockerham et al. GENETIX v. 4.05 A*) estimated according to the rarefaction method of Hurlbert HO), average genetic diversity within populations (HS), and inbreeding coefficient (FIS), were compared using FSTAT version 2.9.3.2 FST values) for each species between Algeciras and Tangier.To evaluate, for each species, whether population diversity indices differed between Algeciras and Tangier groups of populations, average allelic richness per locus (K) in both heath species. STRUCTURE assigns individuals to the K different genetic clusters based on allele frequencies at each locus. Estimated number of K clusters ranged from 1 to 21, and analyses were based on an admixture ancestral model with correlated allele frequencies. In each run of the program Monte Carlo Markov Chain (MCMC) and burn-in period length consisted of 1.2×106 and 8×105 iterations, respectively. The amount of variation of the likelihood was evaluated by carrying out ten runs for each K. The most likely number of genetic clusters (K) was estimated following Evanno et al. ad hoc parameter (ΔK) to estimate the rate of change of likelihood values between successive K values. POPULATIONS version 1.2.3. beta DA genetic distances DA genetic distance matrices were used to compute eigenvalues and eigenvectors to perform Principal Coordinates Analyses (PCoA) for each species. Minimum Spanning Trees (MST) using DA distance matrices were constructed with NTSYSpc version 2.1 E. arborea and E. australis at each side of the Strait by matrix correlation analyses using a matrix of log-transformed pairwise geographical distances between populations and a matrix of a pairwise linearized FST values ; Nm), obtained directly from FST values by using Wright’s Nm values between populations at each of the two sides of the Strait may still be useful to explore for each species the existence of a decrease in gene flow associated with higher patchiness. For the sake of this study, we focused on geographical distances separating pairs of populations smaller than 30 km in order to factor out or minimize possible geographical effects other than sandstone patchiness.Bayesian analyses in STRUCTURE v. 2.1 Erica australis (HO) ranged from 0.505 (population T16) to 0.704 (population T06), and unbiased expected heterozygosities (HE) ranged from 0.554 (population T16) to 0.786  to 0.650 (population A17), and unbiased expected heterozygosities ranged from 0.534(population T06) to 0.623  was found in 22 out of the 23 populations studied of the sandstone specialist E. australis  and null alleles. Estimation of null allele frequencies gave significant results  in 24 out of 161 and 39 out of 176 locus × population combinations in E. australis and E. arborea, respectively. Most of these significant combinations were concentrated in one locus  in E. australis and two loci  in E. arborea. Although the actual presence of null alleles in the dataset may only be confirmed by progeny analyses, our large sampling sizes provided indirect evidence that the frequencies of null alleles could have been overestimated by Dempster et al.’s (1977) method used in GENEPOP. As an example, in locus Ecoc431 we should have found ca. 20 null homozygotes out of 446 individuals genotyped in 15 populations that showed significant estimated frequencies of null alleles, but none was found. Therefore, null alleles, if at all present, should have little impact in our datatset. Besides, repeated estimation of genetic diversity indices and population relationships did not change significantly after removing the two loci (data not shown). Accordingly, high FIS in these heath species may be related to pollen dispersal system favouring inbreeding and/or to population substructure, as it has been reported in other Erica species Significant deviation towards heterozygote deficiency  in both species were slightly lower in Tangier, where sandstone patchiness is more prominent  than Tangier ones, whereas no significant differences were detected in the edaphic generalist E. arborea  to 0.164 (T05–T16), whereas for E. arborea they ranged from zero (pair T01–T05) to 0.106 (pair T06–T16). Average FST values were significantly different from zero in the two species, but higher in the sandstone specialist E. australis (average FST = 0.074) than in the edaphic generalist E. arborea (average FST = 0.039). The comparison of average FST values between the two sides of the Strait revealed for the two species a slightly stronger genetic differentiation in Tangier populations, although these differences were not significant in either species  revealed a maximum modal value of ΔK for K = 2 for both E. australis and E. arborea  in E. australis did not correspond to the geographical membership of the populations, with some populations of each side showing a higher proportion of membership to cluster 1 and others to cluster 2  showed higher proportion of membership to more different genetic clusters than populations from the low patchiness one (Algeciras). On the other hand, in the generalist E. arborea, populations and individuals showed an almost symmetrical membership to both genetic clusters revealed at K = 2 in either Tangier or Algeciras  throughout the Strait of Gibraltar and within Tangier and Algeciras The method of Evanno orea see . These vectively . A seconustralis . The proluster 2 . For K = 2 and 4 . Intereslgeciras . The metE. australis the PCoA separated two population groups along the first axis, both of them including Algeciras and Tangier populations +MST revealed similar results to those from STRUCTURE, and also showed direct links between populations . In E. aulations . In E. ambership . The larmbership .E. australis showed significant isolation by distance (IBD) within each side of the Strait of Gibraltar (FST) and geographical distance was obtained for Algeciras populations, as denoted by their significantly steeper correlation line , both E. australis and E. arborea showed conspicuous differences between the two sides of the Strait. In E. australis, a significant decrease in gene flow levels was found in Tangier between pairs of populations separated less than 30 km from each other  compared to Algeciras , as evidenced by the non-overlapping confidence intervals. This difference vanished at distances longer than 30 km . In contrast, E. arborea showed similar levels of gene flow between populations at each side of the Strait, both for population pairs at distances shorter than 30 km  and longer than 30 km .Regarding estimated levels of gene flow between populations  and between-population gene flow levels (Nm) in the sandstone specialist E. australis were significantly lower in Tangier  than in Algeciras , whereas no differences concerning sandstone patchiness were detected in the edaphic generalist E. arborea  in Tangier populations of E. australis than in Algeciras ones  of the sandstone specialist E. australis in Tangier despite their significantly lower genetic diversity (HS) values in this side of the Strait  values between pairs of geographically close populations that fall in different, isolated sandstone patches  between populations of the sandstone specialist E. australis distant less than 30 km in this region compared to Algeciras, which was not found for the edaphic generalist E. arborea.While significant correlations between geographical and genetic distances  were detected for the sandstone specialist e Strait , the IBD arborea . The widtructure ; Fig. 3bficients . This weE. australis) than in those of an edaphic generalist (E. arborea). Since most endemic species in Mediterranean heathlands of the Strait of Gibraltar are sandstone specialist High patchiness leads to genetic impoverishment and isolation of populations, which makes them more prone to  extinction Figure S1E. australis (a) and of the eight microsatellite loci data for 22 populations of E. arborea (b) given K clusters, obtained through 10 runs of the STRUCTURE analysis for each K. Corresponding ΔK estimation  according to Evanno et al. (2005) showing maximum peaks of ΔK values at K = 2 and K = 4 for E. australis (c), and at K = 2 for E. arborea (d), indicating that those are the optimal solutions for K given the data. Log-likelihood of the seven microsatellite loci data for 23 populations of (DOC)Click here for additional data file.Table S1Allele frequencies for 7 microsatellite loci in 23 Erica australis populations. Numbers in brackets indicate sample sizes. Number of sampled individuals (N) in each population is indicated in paretheses.(DOC)Click here for additional data file.Table S2Allele frequencies for 8 microsatellite loci in 22 Erica arborea populations. Numbers in brackets indicate sample sizes. Number of sampled individuals (N) in each population is indicated in paretheses.(DOC)Click here for additional data file.