DNA markers identify hybrids between butternut (Juglans cinerea L.) and Japanese walnut (Juglans ailantifolia Carr.)

Butternut (Juglans cinerea L.) is a temperate deciduous hardwood native to the eastern USA and southern Canada valued for its nuts and wood. Butternut’s survival is threatened by butternut canker, a disease caused by the exotic fungus Sirococcus clavigignenti-juglandacearum Nair, Kostichka & Kuntz. Field observations indicate that trees commonly called buartnut (a hybrid of butternut and its close congener Japanese walnut (Juglans ailantifolia × J. cinerea)) may be more resistant to butternut canker than is either parental species. Hybrids are difficult to distinguish morphologically from butternuts, and scientists have expressed concern over the possibility of range-wide genetic invasion by Japanese walnut via hybridization with butternut. We used pair-wise combinations of 40 random primers to screen bulked DNA pools of butternut, Japanese walnut, and buartnuts to identify genomic regions unique to Japanese walnut. We ultimately identified one ITS region marker, one chloroplast marker, one mitochondrial marker, and six nuclear markers. The utility of the markers for identifying hybrids was tested and verified using more than 190 genotypes. The markers will be used to identify buartnut hybrids based on the presence of introgressed genomic fragments inherited from Japanese walnut. We confirmed that hybrids have a complex genetic history and present features of the parental species in all possible combinations. These results will assist in the identification and testing of (non-hybrid) butternut for breeding and reintroduction of the species to its former habitats.     

Four cleaved amplified polymorphic sequence (CAPS) markers for the detection of the Juglans ailantifolia chloroplast in putatively native J. cinerea populations

Hybridization between butternut (Juglans cinerea), a forest tree native to eastern North America, and Japanese walnut (J. ailantifolia), a tree tolerant to the lethal fungal disease butternut canker, casts doubt on the genetic identity of the remaining butternuts. We report a diagnostic test to distinguish the J. cinerea chloroplast from the J. ailantifolia chloroplast using cleaved amplified polymorphic sequences resolvable in 1.5% agarose gels. J. ailantifolia maternal ancestry in naturally regenerated stands provides a site selection criterion for studies of introgression dynamics when the non-native parent and the hybrids tolerate a disease to which the native species is susceptible.     

Population genetic diversity of the rare hardwood butternut (<Emphasis Type="Italic">Juglans cinerea</Emphasis>) in the northeastern USA

Populations of butternut tree (Juglans cinerea) have undergone range-wide extirpation. A fungal pathogen, Ophiognomonia clavigignenti-juglandacearum, of unknown origin has been recognized as the causal factor. This population collapse has allowed for observations of a broadleaf hardwood in rapid decline. This study made use of six neutral microsatellite markers to describe the present genetic diversity of butternut in the northeastern USA. Our results indicated weak population differentiation (F _ST?=?0.084), further supported by an absence of regional genetic structure. Despite reports of high mortality rates, genetic analysis revealed no sign of a recent bottleneck. Population statistics and Bayesian analysis indicated significant historical gene flow among butternut populations of the northeast. Attention should be given to genetic differences between upland and riparian habitat as riparian populations appear to contain greater allele diversity.     

Geographically extensive hybridization between the forest trees American butternut and Japanese walnut

We investigate the question of naturally occurring interspecific hybrids between two forest trees: the native North American butternut (Juglans cinerea L.) and the introduced Japanese walnut (Juglans ailantifolia Carrière). Using nuclear and chloroplast DNA markers, we provide evidence for 29 F₁ and 22 advanced generation hybrids in seven locations across the eastern and southern range of the native species. Two locations show extensive admixture (95% J. ailantifolia and hybrids) while other locations show limited admixture. Hybridization appears to be asymmetrical with 90.9 per cent of hybrids having J. ailantifolia as the maternal parent. This is, to our knowledge, the first genetic data supporting natural hybridization between these species. The long-term outcome of introgression could include loss of native diversity, but could also include transfer of useful traits from the introduced species.     

Dead-End Hybridization in Walnut Trees Revealed by Large-Scale Genomic Sequence Data

Although hybridization plays a large role in speciation, some unknown fraction of hybrid individuals never reproduces, instead remaining as genetic dead-ends. We investigated a morphologically distinct and culturally important Chinese walnut, Juglans hopeiensis, suspected to have arisen from hybridization of Persian walnut (J. regia) with Asian butternuts (J. cathayensis, J. mandshurica, and hybrids between J. cathayensis and J. mandshurica). Based on 151 whole-genome sequences of the relevant taxa, we discovered that all J. hopeiensis individuals are first-generation hybrids, with the time for the onset of gene flow estimated as 370,000 years, implying both strong postzygotic barriers and the presence of J. regia in China by that time. Six inversion regions enriched for genes associated with pollen germination and pollen tube growth may be involved in the postzygotic barriers that prevent sexual reproduction in the hybrids. Despite its long-recurrent origination and distinct traits, J. hopeiensis does not appear on the way to speciation.     

Population and Conservation Genetics in an Endangered Lemur, <Emphasis Type="Italic">Indri indri</Emphasis>, Across Three Forest Reserves in Madagascar

Population decline and fragmentation often lead to reduced genetic diversity and population differentiation. Habitat destruction throughout Madagascar has caused population decline and extinction of many endemic species. Lemur populations, including those of the largest extant lemur, Indri indri, have been fragmented into remaining forest patches. We assessed the level of genetic diversity in indri populations in three protected reserves by genotyping a total of 43 individuals at 17 microsatellite loci. Genetic diversity in terms of heterozygosity was high in all three reserves, with no differences between reserves. Population structure and F _ST analyses revealed Analamazaotra Forest Station and the Torotorofotsy Conservation Area, which are separated by ca. 18 km to be genetically differentiated from each other with some admixture. Betampona Strict Nature Reserve, which is separated from the other reserves by ca. 130 km, exhibited clear population genetic differentiation, with no signs of admixture with the other reserves. Our genetic diversity estimates are similar to those for other Indridae in similar habitats and may reflect past rather than current population processes, given that populations have declined recently. Our results suggest that Betampona may be genetically isolated and that it is important to maintain gene flow between remaining populations to prevent loss of genetic diversity for the future conservation of Indri indri.     

Regional patterns of declining butternut (Juglans cinerea L.) suggest site characteristics for restoration

Butternut trees dying from a canker disease were first reported in southwestern Wisconsin in 1967. Since then, the disease has caused extensive mortality of butternut throughout its North American range. The objectives of this study were to quantify changes in butternut populations and density across its range and identify habitat characteristics of sites where butternut is surviving in order to locate regions for potential butternut restoration. The natural range of butternut (Juglans cinerea L.) extends over a large region of eastern N. America encompassing New Brunswick south to North Carolina, north to Minnesota, and southwest to Missouri. Despite the species’ large range, it is typically not a common tree, comprising a relatively minor component of several different forest types. We evaluated change in butternut abundance and volume from current and historic data from 21 states in the eastern United States. We related abundance and volume at two time periods to a suite of ecological and site factors in order to characterize site conditions where butternut survived. We also assessed the current level of butternut mortality across its range. Since the 1980s, the number of butternut trees and butternut volume have decreased by 58% and 44%, respectively, across its US range. Substantial relative decreases in tree numbers and volume occurred in most ecoregion sections. Five environmental variables were found to be significant predictors of butternut presence. The potential impacts of butternut canker are particularly acute as the canker pathogen invasion pushes a rare tree species toward extinction, at least at a local scale. Based on the results presented here, large‐diameter maple/beech/birch stands in dry, upland sites in eastern Minnesota, western Wisconsin, and upstate New York appear to offer the most favorable conditions for butternut growth and survival and thus may be the best stands for planting resistant butternut trees.     

Conservation genetics of the annual hemiparasitic plant <Emphasis Type="Italic">Melampyrum sylvaticum</Emphasis> (Orobanchaceae) in the UK and Scandinavia

Melampyrum sylvaticum is an endangered annual hemiparasitic plant that is found in only 19 small and isolated populations in the United Kingdom (UK). To evaluate the genetic consequences of this patchy distribution we compared levels of diversity, inbreeding and differentiation from ten populations from the UK with eight relatively large populations from Sweden and Norway where the species is more continuously distributed. We demonstrate that in both the UK and Scandinavia, the species is highly inbreeding (global F _IS = 0.899). Levels of population differentiation were high (F’_ST = 0.892) and significantly higher amongst UK populations (F’_ST = 0.949) than Scandinavian populations (F’_ST = 0.762; P < 0.01). The isolated populations in the UK have, on average, lower genetic diversity (allelic richness, proportion of loci that are polymorphic, gene diversity) than Scandinavian populations, and this diversity difference is associated with the smaller census size and population area of UK populations. From a conservation perspective, the naturally inbreeding nature of the species may buffer the species against immediate effects of inbreeding depression, but the markedly lower levels of genetic diversity in UK populations may represent a genetic constraint to evolutionary change. In addition, the high levels of population differentiation suggest that gene flow among populations will not be effective at replenishing lost variation. We thus recommend supporting in situ conservation management with ex situ populations and human-mediated seed dispersal among selected populations in the UK.     

Range-wide distribution of genetic diversity in the North American tree Juglans cinerea: a product of range shifts, not ecological marginality or recent population decline

The spatial distribution of genetic diversity is a product of recent and historical ecological processes, as well as anthropogenic activities. A current challenge in population and conservation genetics is to disentangle the relative effects of these processes, as a first step in predicting population response to future environmental change. In this investigation, we compare the influence of contemporary population decline, contemporary ecological marginality and postglacial range shifts. Using classical model comparison procedures and Bayesian methods, we have identified postglacial range shift as the clear determinant of genetic diversity, differentiation and bottlenecks in 29 populations of butternut, Juglans cinerea L., a North American outcrossing forest tree. Although butternut has experienced dramatic 20th century decline because of an introduced fungal pathogen, our analysis indicates that recent population decline has had less genetic impact than postglacial recolonization history. Location within the range edge vs. the range core also failed to account for the observed patterns of diversity and differentiation. Our results suggest that the genetic impact of large-scale recent population losses in forest trees should be considered in the light of Pleistocene-era large-scale range shifts that may have had long-term genetic consequences. The data also suggest that the population dynamics and life history of wind-pollinated forest trees may provide a buffer against steep population declines of short duration, a result having important implications for habitat management efforts, ex situ conservation sampling and population viability analysis.     

Long-term human impacts on genetic structure of Italian walnut inferred by SSR markers

Life history traits, historic factors, and human activities can all shape the genetic diversity of a species. In Italy, walnut (Juglans regia L.) has a long history of cultivation both for wood and edible nuts. To better understand the genetic variability of current Italian walnut resources, we analyzed the relationships among the genetic structure of local walnut populations (inferred by SSR markers) and human migrations along ancient routes, using the territory of Royal Tratturo Candela-Pescasseroli (RT) as a case study. Sixteen J. regia provenances were collected along RT and compared with 13 Italian provenances and the landrace Sorrento. Although the level of SSR polymorphism we observed was moderately high, AMOVA revealed that most of the diversity was located within individuals (92.58%), and geographical differentiation was low (D _est = 0.076). Evidence for human-mediated domestication bottleneck events was detected in about 95% of walnut provenances. A Bayesian approach divided 456 walnut samples into three clusters: (1) Sorrento genotypes, (2) trees from the island of Sicily, and (3) the remaining germplasm. The UPGMA tree based on Nei's distances distinguished northeastern provenances and weakly grouped 12 of 16 provenances of RT. The observed genetic differences derived mainly from gradations in allele frequencies. Separation of the Sicilian provenance from the mainland may be explained in terms of founder effects and prolonged geographic isolation. Two contrasting forces, selection, and frequent inter-regional transfer of propagules, appear to drive the patterns of genetic variability for J. regia.     

Low genetic diversity and local adaptive divergence of Dracaena cambodiana (Liliaceae) populations associated with historical population bottlenecks and natural selection: an endangered long‐lived tree endemic to Hainan Island,China

Historical population bottlenecks and natural selection have important effects on the current genetic diversity and structure of long‐lived trees. Dracaena cambodiana is an endangered, long‐lived tree endemic to Hainan Island, China. Our field investigations showed that only 10 populations remain on Hainan Island and that almost all have been seriously isolated and grow in distinct habitats. A considerable amount of genetic variation at the species level, but little variation at the population level, and a high level of genetic differentiation among the populations with limited gene flow in D. cambodiana were detected using inter‐simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analyses. No significant correlation was found between genetic diversity and actual population size, as the genetic diversities were similar regardless of population size. The Mantel test revealed that there was no correlation between genetic and geographic distances among the 10 populations. The UPGMA, PCoA and Bayesian analyses showed that local adaptive divergence has occurred among the D. cambodiana populations, which was further supported by habitat‐private fragments. We suggest that the current genetic diversity and population differentiation of D. cambodiana resulted from historical population bottlenecks and natural selection followed by historical isolation. However, the lack of natural regeneration of D. cambodiana indicates that former local adaptations with low genetic diversity may have been genetically weak and are unable to adapt to the current ecological environments.     

Conservation of genetic diversity in slippery elm (<Emphasis Type="Italic">Ulmus rubra</Emphasis>) in Wisconsin despite the devastating impact of Dutch elm disease

Forest pest epidemics are responsible for many population declines reported in forest trees. While forest tree populations tend to be genetically diverse, in principle mortality resulting from disease could diminish that genetic diversity and alter the genetic structure of the remnant populations with consequences for the ability of a species to adapt to changing environments. Slippery elm (Ulmus rubra Muhl.) is a long-lived, wind-pollinated forest tree with a native range covering essentially all of eastern North America. Dutch elm disease (DED) caused by an introduced fungal pathogen (Ophiostoma ulmi) devastated North American elm populations, including slippery elm, beginning in the 1930s. Estimates of the numbers of elms lost to DED are unknown but range into the hundreds of millions of trees given their former abundance. In this study, the genotypes of 77 herbarium specimens collected between 1890 and 2004 in Wisconsin, and of 100 slippery elm trees from five wild Wisconsin populations, were characterized using 13 microsatellite loci. Levels of genetic diversity were compared between the herbarium specimens collected pre- and post-DED spread in Wisconsin. In addition, the levels of genetic diversity and degree of genetic differentiation were quantified in the five wild populations. The allelic diversity and expected levels of heterozygosity were similar between the pre- and post-DED herbarium specimens. The five wild populations were only slightly differentiated and no genetic bottleneck was detected for any population. At least in Wisconsin, slippery elm apparently has maintained levels of genetic diversity that could facilitate adaptation to future climatic and environmental changes.     

Genetic insights into the historical distribution of <Emphasis Type="Italic">Polylepis pauta</Emphasis> (Rosaceae) in the northeastern Cordillera Oriental of Ecuador

Scattered patches of Polylepis forest that occur within the 3,000–4,500 m altitudinal belt of the Andean Cordillera from Venezuela to Argentina have been hypothesized to be remnants of once continuous forests whose range became fragmented through anthropogenic activities that probably preceded the Spanish conquest. Allozyme variation of Polylepis pauta from 12 forest populations in three adjacent watersheds in Northeastern Ecuador was investigated to assess whether observed patterns of gene diversity were consistent with a more continuous historical range of the species and to evaluate the populations’ potential for restoration. Genetic diversity and polymorphism in P. pauta populations were higher than mean values for most wind pollinated and dispersed temperate and tropical tree species with regional distributions. Genetic differentiation among watersheds was lower than among populations within each watershed. Isolation by distance was not evident and several populations from different watersheds were more genetically similar than populations from the same watershed. Larger forest patches with broader altitudinal ranges had more alleles. Forest patches on steeper slopes and at higher elevations supported populations with less genetic diversity; this might have resulted from the predominance of vegetative reproduction in these landslide prone areas. The amount of genetic diversity maintained by P. pauta, coupled with low genetic differentiation among populations within and among watersheds, is consistent with a more continuous historical range of the species in Northeastern Ecuador and point to the Oyacachi basin as having the highest levels of genetic diversity.     

Cryptic genetic subdivision in the San Benito evening primrose (Camissonia benitensis)

When rare plants are distributed across a range of habitats, ecotypic differentiation may arise requiring customized conservation measures. The rate of local adaptation may be accelerated in complex landscapes with numerous physical barriers to gene flow. In such cases, examining the distribution of genetic diversity is essential in determining conservation management units. We investigated the distribution of genetic diversity in the federally threatened Camissonia benitensis (Onagraceae), which grows in two distinct serpentine habitats across several watersheds in San Benito, Fresno, and Monterey Cos., CA, USA. We compared genetic diversity with that of its two widespread relatives, C. contorta and C. strigulosa, and examined the potential for hybridization with the latter species. Genotyping results using seven heterospecific microsatellite markers indicate that differentiation between habitat types was weak (F _ST = 0.0433) and in an AMOVA analysis, there was no significant partitioning of molecular variation between habitats. Watersheds accounted for 11.6 % of the molecular variation (pairwise F _ST = 0.1823–0.4275). Three cryptic genetic clusters were identified by InStruct and STRUCTURE that do not correlate with habitat or watershed. C. benitensis exhibits 5–11× higher inbreeding levels and 0.54× lower genetic diversity in comparison to its close relatives. We found no evidence of hybridization between C. benitensis and C. strigulosa. To maximize conservation of the limited amount of genetic diversity in C. benitensis, we recommend mixing seed representing the three cryptic genetic clusters across the species’ geographic range when establishing new populations.     

Genetic diversity and genetic structure of Persian walnut (<Emphasis Type="Italic">Juglans regia)</Emphasis> accessions from 14 European,African, and Asian countries using SSR markers

Persian walnut (Juglans regia L.) is the world’s most widely grown nut crop, but large-scale assessments and comparisons of the genetic diversity of the crop are notably lacking. To guide the conservation and utilization of Persian walnut genetic resources, genotypes (n = 189) from 25 different regions in 14 countries on three continents were sampled to investigate their genetic relationships and diversity using ten microsatellite (SSR) loci. The SSRs amplified from 3 to 25 alleles per locus, with a mean value of 11.5 alleles per locus. The mean values of observed and expected heterozygosity were 0.62 and 0.73, respectively. Based on Nei’s genetic identity, accessions from Bratislava (Slovakia) and Antalya (Turkey) showed the lowest similarity (0.36), while accessions from Algeria and Tunisia as well as accessions from Debrecen (Hungary) and Trnava (Slovakia) had the highest similarity (0.97). Two populations from Iran (Alborz and Ardabil) had the highest number of private alleles (7 and 5), but they were quite different as they also had the lowest genetic identity when compared to the remaining populations as well as to each other. Although overall differentiation among regions was relatively low (F _st  = 0.07), cluster analysis grouped accessions generally but not completely according to geography. STRUCTURE software confirmed these results and divided the accessions into two main groups, separating accessions collected from Europe and North Africa from those from Greece and the Near East. Results indicate the presence of a likely center of diversity for Persian walnut in Eastern and Southeastern Europe. They also provide information that can be used to devise conservation actions. Notably, the genetic diversity of threatened populations from two regions in Iran should be conserved.     

Genetic variation and plant performance in fragmented populations of globeflowers (Trolliuseuropaeus) within agricultural landscapes

The management of remnant populations in highly fragmented landscapes requires a thorough understanding of the processes shaping population persistence. We investigated relationships between population characteristics (i.e. size, density and pollinator abundance), offspring performance, genetic diversity and differentiation in Trollius europaeus, a plant with a nursery pollination system. In 19 populations of different sizes and located in north-east Switzerland, an area which has undergone widespread land use changes over the last decades, we assessed neutral genetic diversity (N _total = 383) using AFLPs and plant performance in a greenhouse experiment (N _total = 584) using competition and control treatments. Overall genetic differentiation was low (F _ST = 0.033) with a marginal significant isolation by distance effect (P = 0.06) indicating (historical) genetic connectivity among the populations. Mean expected heterozygosity was H _E of 0.309 (0.0257–0.393) while inbreeding coefficients (F _IS) were significant in only three populations. Genetic diversity was not related to population size, plant density or pollinator abundance. Plant performance was reduced under competition (P < 0.001) but the severity of competition was independent of genetic diversity and population size. In summary, remnant populations of T. europaeus retain genetic diversity and seem capable of persisting under the present conditions within an agricultural matrix. T. europaeus is a perennial herb, thus it may require several generations for the negative effects of fragmentation and isolation to manifest. Our findings indicate that small populations are as important as large populations for the conservation and management of genetic resources.     

Genetic Evidence of the Contribution of Ethnic Migrations to the Propagation and Persistence of the Rare and Declining Scrambling Shrub Caesalpinia bonduc L

This paper examines the contribution of human migrations to the propagation and maintenance of Caesalpina bonduc by means of an analysis of its population genetics and distribution patterns. One hundred and forty seven sites were surveyed in the three climatic zones of Benin and all individuals of the species were recorded. A set of individuals was randomly selected and sampled from seven populations and morphological variation and genetic diversity were assessed. The study confirmed the presence of the species in all climatic zones but its abundance varied greatly. Morphological variability between populations and zones was low in comparison with the high amount of variation within populations. AFLP and cpDNA fingerprinting revealed an extremely low genetic diversity within populations and a low genetic differentiation, suggesting parental links between populations. The results support the hypothesis of human involvement in Caesalpinia dispersal and persistence in Benin. However, the low genetic diversity may imply high risks for future extinction. We recommend that gene flow among the remaining populations be supported in order to conserve the species.     

Genetic diversity loss and homogenization in urban trees: the case of <Emphasis Type="Italic">Tilia </Emphasis>× <Emphasis Type="Italic">europaea</Emphasis> in Belgium and the Netherlands

Urban trees form a vital component of sustainable cities but the use of a restricted range of species and genotypes may pose a risk to global biodiversity. Despite several studies investigating tree species diversity, intraspecific genetic diversity of urban trees remains largely unexplored. Here, we characterized the genetic diversity of Tilia?×?europaea, one of the most widely planted urban tree species in Northwest Europe. We compared the genotypic diversity of historical plantings of Tilia spp. from the 17th century with the genotypic diversity of currently available planting stock in Belgium and the Netherlands. In total, 129 trees were sampled and genotyped with 14 microsatellite loci and 150 polymorphic Amplified Fragment Length Polymorphism markers. In Northwest Europe, homogenization of urban T.?×?europaea plantings already started at the 17th century. Genetic diversity within contemporary commercial planting stocks was extremely narrow and consisted mainly of two clones, sold under the name ‘Pallida’ and ‘Zwarte linde’. The genetic diversity found within the historical plantings was about four times higher than in the current commercial planting stocks. We recommend that tree nurseries should enlarge the genetic diversity of T.?×?europaea commercial planting stocks. The old clones have shown long-term disease resistance and could provide tree breeders with the valuable new genetic material. The range of available Tilia species and genotypes needs to be explored in future urban tree planning to optimize desired ecosystem services.     

Genetic diversity and population genetic structure of wild banana Musa ornata (Musaceae) in Mexico

The wild banana Musa ornata is an inhabitant of the tropical regions of Mexico characterized by patches of tropical rainforest. The overexploitation of its habitat has caused the extinction of several populations affecting diversity and population genetic structure of remaining ones. We used microsatellite markers to determine the genetic diversity and the population’s genetic structure of all extant populations. The thirty-two microsatellite loci previously characterized for M. acuminata and M. balbisiana were tested in M. ornata. Only twelve amplified. From these seven were polymorphic and were used for genetic analyses. The Nei’s diversity estimator shows low levels of genetic diversity (H _e = 0.263) with a mean of 4.40 alleles per locus. Excess homozygosity was evident in all populations indicating high levels of inbreeding. F _ST pairwise analyses and AMOVA indicated low genetic differentiation. However, 28 % of private alleles were registered, suggesting limited gene flow. Genetic distances, Jaccard’s coefficient and principal component analysis showed a good correspondence to geographical locations. The Mantel test performed was not significant. The results support the hypothesis of recent fragmentation events; therefore, not enough time has passed to detect differences between populations. However, it is also likely that results are caused by factors such as bottleneck, decline in pollinator populations, self-pollination and/or a tendency towards clonal reproduction. It is proposed that the preservation strategy focuses on maintaining all the remaining populations and ensuring their connectivity, so as to maintain gene flow and increase the genetic diversity of this species.     

Latitude drives diversification in Madagascar's endemic dry forest rodent Eliurus myoxinus (subfamily Nesomyinae)

Numerous hypotheses have been proposed for the historical processes governing the rich endemism of Madagascar's biodiversity. The ‘watershed model’ suggests that drier climates in the recent geological past have resulted in the contraction of forests around major watersheds, thereby defining areas of endemism. We test whether this hypothesis explains phylogeographical patterns in a dry forest‐dependent rodent, Eliurus myoxinus, an endemic species widely distributed through western Madagascar. We sequenced the mitochondrial cytochrome b locus and nuclear introns of the β‐fibrinogen and the growth hormone receptor genes for E. myoxinus. Using a parametric bootstrapping approach, we tested whether the mitochondrial gene tree data fit expectations of local differentiation given the watershed model. We additionally estimated population differentiation and historical demographic parameters, and reconstructed the spatial history of E. myoxinus to highlight spatial and temporal patterns of differentiation. The data do not support the watershed model as a clear explanation for the genetic patterns of diversity within extant E. myoxinus populations. We find striking patterns of latitudinal genetic structure within western Madagascar, and indicate possible roles for environmental and ecological gradients along this axis in generating phylogeographical diversity. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 500–517.