Patterns of genetic and morphometric differentiation in Melitaea (Mellicta) athalia (Lepidoptera: Nymphalidae)

Patterns of genetic and morphometric differentiation were surveyed in Melitaea (Mellicta) athalia populations of the Carpathian Basin. This species has a wide distribution and exists under a wide variety of ecological conditions. It has two ecotypes in Hungary: with either one or two broods per year. It is of particular interest to reveal the main factors driving differentiation patterns in this species. Samples in our study were obtained from five Hungarian and one Transylvanian (Romanian) regions. Enzyme polymorphism, wing characters and male external genitalia were analysed using traditional morphometric methods. Statistical methods were optimized to compare morphological and genetic data. The results of genetic surveys revealed a clear regional pattern of differentiation in M. athalia. Moreover, the results of principal component analysis, Bayesian clustering and the dendrogram all suggested that the regions can be classified into two groups corresponding to the East or West zones of the Carpathian Basin. In contrast, differentiation between the two ecotypes was less expressed in the genetic variation of M. athalia. Results of the analyses conducted on phenotypic variation also suggested a regional pattern for both sets of morphometric characters (wings and external genitalia). At the same time, neither East–West regional division nor ecotype differentiation was detected in the morphometric studies. In sum, our analyses confirmed that both genetic and phenotypic variations of M. athalia exhibit a regional pattern rather than the differentiation between the two ecotypes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

N equals two (times five). Exploring the effects of horse rewilding on five congeneric adult butterflies

Rewilding incomplete ecosystems by using ungulate megaherbivores represents a significant potential for sustainable management of habitats of declining species. Two xeric grasslands patches in the Podyjí National Park, Czech Republic, were rewilded by a feral horse breed, the Exmoor pony, in 2018. Before this in 2017, demography, mobility, and adult habitat use of five congeneric Melitaea butterflies co-occurring at the grasslands were investigated (Vodickova et al., J. Nature Conserv. 2019). In 2021, four seasons after the rewilding, we replicated the survey to assess the effects of the horse on the butterflies. Here, we compare the results of the two surveys and investigate changes in spatial patterns of adult distribution using Ripley’s K-functions.Total numbers of captures, and estimated population sizes, were consistently lower in 2021, with the largest drop for spring-flying M. cinxia. We cannot discern whether this was due to the cold 2021 spring, or due to reduction of grasses by the horse, possibly contributing to desiccation of M. cinxia host plants. Demographic parameters such as residency/longevity and capture probability changed only little. Mobility ranking among species remained identical, but within species, some mobility characteristics changed among years. Among early summer species, M. britomartis, second most abundant in 2017, switched to the first position with M. aurelia, and these two species displayed the most notable shift in adult habitat use between the two seasons. Short thorny shrubs avoided by the horse protect M. britomartis host plants; this threatened butterfly thus did not suffer from horse presence. M. athalia, a species of woodland edges, profited from decay of conifers caused by a series of dry years; and M. didyma, forming multiple generations, from increase of its host plant. Contrary to expectations, spatial distribution of most butterflies became more aggregated within rewilded pastures, probably due to regularities in home ranges use by the horses, which restructured the vegetation in a zonal, rather than patchy, way. A considerably larger areas should be rewilded by the herbivores to fully achieve the desired beneficial effects.     

The possibility of using characters of the female genital armature for species diagnostics and classification of the genus <Emphasis Type="Italic">Mellicta Billberg</Emphasis>, 1820 (Lepidoptera,Nymphalidae): 1. <Emphasis Type="Italic">Mellicta athalia</Emphasis> (Rottemburg, 1775) species group

The morphology of female genital armature of seven species of the genus Mellicta belonging to the M. athalia species-group from different localities of their ranges is analyzed. The main distinctive features are described and their variability and use for species identification are assessed. It is shown that some characteristics of female genitalia can be used to clarify the systematic position of species within the genus. Keys to species of the athalia group based on female genitalia are given.     

Phylogeography,Population Structure,and Conservation of the Javan Gibbon (<Emphasis Type="Italic">Hylobates moloch</Emphasis>)

An understanding of phylogeography and population genetics is needed for a comprehensive long-term conservation management strategy. The Javan gibbon (Hylobates moloch), an Endangered species endemic to the island of Java, has been protected since 1924 but is threatened by ongoing habitat loss, habitat degradation, and the wildlife trade. We studied the phylogeography and population genetic structure of the Javan gibbon, to define the number of Evolutionary Significant Units (ESUs) in the species, and the population genetic structure in each ESU. We sampled 47 individuals, analyzing 35 for variation in mitochondrial DNA control region, 41 for variation in 8 nuclear DNA microsatellites, and 13 for variation in 45 nuclear DNA single nucleotide polymorphisms (SNPs). We found support for two ESUs across the species range: a western ESU, extending from Ujung Kulon to Gunung Gede–Pangrango, and a central ESU, extending from Gunung Masigit–Simpang–Tilu to Gunung Slamet. Analysis of molecular variance and population structure analysis indicate significant structuring in the western ESU between Ujung Kulon and Gunung Halimun–Salak–Gede–Pangrango, and little to moderate structure in the central ESU, underscoring the importance of conserving as many populations as possible to preserve the full array of genetic diversity in this species. Our results will inform future more comprehensive population genetic surveys and the conservation genetic management of the Javan gibbon. This study demonstrates the importance of genetics when designing conservation management strategies for endangered primates.     

Conservation genetics and population diversity of Erigeron breviscapus (Asteraceae), an important Chinese herb

Erigeron breviscapus (Vant.) Hand.-Mazz. is an important Chinese herb in treating cerebral infarction and heart diseases, which is increasingly endangered because of over-harvesting and habitat fragmentation. We applied AFLP method to investigate its genetic diversity and population structure in order to make efficient conservation strategies. The results indicated a relatively low intraspecific genetic diversity and feeble genetic differentiation among populations, and no Evolutionarily Significant Units (ESUs) was detected. The relationships between population genetic diversity and populations' distributions (latitude, longitude and altitude) were tested, and the genetic diversity of each population (H_POP) positively correlated with the populations' distribution altitudes. The suitable germinating and growing conditions and less human disturbance at the higher latitude regions might be attributable for this phenomenon. For conservation purpose, an ex situ conservation measure for the populations of E. breviscapus with large size in higher altitude regions should be conserved in priority.     

Phylogeographic pattern of range expansion provides evidence for cryptic species lineages in Silene nutans in Western Europe

As a result of recent or past evolutionary processes, a single species might consist of distinct Evolutionary Significant Units (ESUs), even corresponding to cryptic species. Determining the underlying mechanisms of range shifts and the processes at work in the build-up of divergent ESUs requires elucidating the factors that contribute to population genetic divergence across a species'' range. We investigated the large-scale patterns of genetic structure in the perennial herbaceous plant species Silene nutans (Caryophyllaceae) in Western Europe. We sampled and genotyped 111 populations using 13 nuclear microsatellite loci and 6 plastid single-nucleotide polymorphisms. Broad-scale spatial population genetic structure was examined using Bayesian clustering, spatial multivariate analyses and measures of hierarchical genetic differentiation. The genotypic structure of S. nutans was typical of a predominantly allogamous mating system. We also identified plastid lineages with no intra-population polymorphism, mirroring two genetically differentiated nuclear lineages. No evidence of admixture was found. Spatial trends in genetic diversity further suggested independent leading-edge expansion associated with founding events and subsequent genetic erosion. Overall, our findings suggested speciation processes in S. nutans and highlighted striking patterns of distinct stepwise recolonisation of Western Europe shaped by Quaternary climate oscillations. Two main potential ESUs can be defined in Western Europe, corresponding to Eastern and Western nuclear-plastid lineages. In situ preservation of populations and genetic rescue implying ex situ conservation techniques should take the lineage identity into account. This is particularly true in Great Britain, northern France and Belgium, where S. nutans is rare and where distinct lineages co-occur in close contact.     

DNA barcoding reveals twelve lineages with properties of phylogenetic and biological species within Melitaea didyma sensu lato (Lepidoptera,Nymphalidae)

The complex of butterfly taxa close to Melitaea didyma includes the traditionally recognized species Melitaea didyma, Melitaea didymoides and Melitaea sutschana, the taxa that were recognized as species only relatively recently (Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta) as well as numerous described subspecies and forms with unclear taxonomic status. Here analysis of mitochondrial DNA barcodes is used to demonstrate that this complex is monophyletic group consisting of at least 12 major haplogroups strongly differentiated with respect to the gene COI. Six of these haplogroups are shown to correspond to six of the above-mentioned species (Melitaea didymoides, Melitaea sutschana, Melitaea latonigena, Melitaea interrupta, Melitaea chitralensis and Melitaea mixta). It is hypothesized that each of the remaining six haplogroups also represents a distinct species (Melitaea mauretanica, Melitaea occidentalis, Melitaea didyma, Melitaea neera, Melitaea liliputana and Melitaea turkestanica), since merging these haplogroups would result in a polyphyletic assemblage and the genetic distances between them are comparable with those found between the other six previously recognized species.     

Habitat exploration in butterflies – an outdoor cage experiment

A large outdoor cage, measuring 7 × 30 m, was used to study the willingness of butterflies to move through unsuitable habitat in search of neighbouring patches. The area inside the cage was divided into two grassland parts by a 7 m long shady part of unsuitable habitat that the butterflies had to fly through to move between the grassland parts. In 1999 and 2000 we performed experiments on three Melitaeini species (Melitaea cinxia and Mellicta athalia were used both years and Euphydryas aurinia in 2000) and three additional species (Brenthis ino and Aphantopus hyperantus in 1999 and Clossiana euphrosyne in 2000). In both years the Melitaeini species moved at considerably lower rates through the shady part than the other species. Among the Melitaeini species Mell. athalia moved most frequently through the shady part while E. aurinia and M. cinxia moved at lower rates. The distribution of these butterflies differ from widespread to localized and the results are discussed in the context of their habitat preferences and distribution patterns.     

Genetic signatures of translocations and habitat fragmentation for two evolutionarily significant units of a protected fish species

Genetic population structure was evaluated for the White Sands pupfish (Cyprinodon tularosa), a protected fish species comprised of two Evolutionarily Significant Units (ESUs); the Malpais Spring ESU and the Salt Creek ESU. The Malpais Spring ESU is restricted to Malpais Spring, whereas the Salt Creek ESU includes the native Salt Creek population and two Salt Creek-derived populations at Mound Spring and Lost River; all three of these habitats are physically fragmented. We sampled the upper and lower reaches of the four populations, examining 13 DNA microsatellite loci from 40 individuals per population. As expected, significant genetic structure was observed between the two ESUs; Malpais Spring and Salt Creek. Substantial genetic drift was observed for the introduced Lost River population, with modest genetic drift for the introduced Mound Spring population. Taken together with ecological data, neither of the introduced populations successfully replicates the Salt Creek population. We also report significant reductions in genetic diversity for the upper reaches of both Salt Creek and Lost River, indicating that recent habitat changes have altered the genetic structure of these two populations. We consider these findings along with previously reported ecological data to develop guidelines for managing C. tularosa.     

Genetic structure of the alpine newt, Mesotriton alpestris (Salamandridae,Caudata), in the southern limit of its distribution: Implications for conservation

Tissue samples of Mesotriton alpestris veluchiensis were collected from 11 localities in Greece, and the sequences of two mitochondrial genes (cytochrome b and 16S rRNA), as well as frequencies of 18 allozyme loci, were used in order to describe levels and patterns of genetic variation, identify possible evolutionary units, and reveal aspects of their conservation status. Two major lineages, displaying considerable amount of genetic differences, were supported by both analyses. These lineages, which have been geographically separated since the middle Pleistocene, constitute separate Evolutionary Significant Units (ESUs) and correspond to populations from the Greek mainland and Peloponnisos, respectively. The particularly high inter-population differentiation within each region implies long-term isolation in fragmented habitats, while severe bottlenecking is proposed to have resulted in the observed lack of heterozygotes in the majority of populations. Conservational implications are also discussed, particularly in relation to environmental factors and human activities, which seem to have contributed to the genetic impoverishment of the most marginal populations studied.     

Phylogeography and genetic effects of habitat fragmentation on endangered Taxus yunnanensis in southwest China as revealed by microsatellite data

It is not known how the profoundly complex topography and habitat heterogeneity generated by the uplift of the Qinghai‐Tibetan Plateau (QTP) during the late Tertiary affected population genetic structure of endangered Taxus yunnanensis. In addition, the effects of habitat fragmentation due to anthropogenic disturbance on genetic diversity and population differentiation of this species have not been studied. T. yunnanensis is an ancient tree/shrub mainly distributed in southwest China. Recently, the species has suffered a sharp decline due to excessive logging for its famous anticancer metabolite taxol, resulting in smaller and more isolated populations. To understand the phylogeography and genetic consequences of habitat fragmentation of this endangered species, using 11 polymorphic microsatellites, we genotyped 288 individuals from 14 populations from a range‐wide sampling in China. Our results suggest that two different population groups that were once isolated have persisted in situ during glacial periods in both areas, and have not merged since. Habitat fragmentation has led to significant genetic bottlenecks, high inbreeding and population divergence in this species. The two different population groups of T. yunnanensis could be attributed to restricted gene flow caused through isolation by geographical barriers and by habitat heterogeneity during uplift of the QTP, or the existence of two separate glacial refugia during the Pleistocene. In situ and ex situ conservation of the two Evolutionarily Significant Units (ESUs), artificial gene flow between populations and a comprehensive understanding of the pollination system in this endangered species are suggested from this study.     

Population genetic structure and conservation genetics of threatened Okaloosa darters (Etheostoma okaloosae)

Imperiled Okaloosa darters (Etheostoma okaloosae) are small, benthic fish limited to six streams that flow into three bayous of Choctawhatchee Bay in northwest Florida, USA. We analyzed the complete mitochondrial cytochrome b gene and 10 nuclear microsatellite loci for 255 and 273 Okaloosa darters, respectively. Bayesian clustering analyses and AMOVA reflect congruent population genetic structure in both mitochondrial and microsatellite DNA. This structure reveals historical isolation of Okaloosa darter streams nested within bayous. Most of the six streams appear to have exchanged migrants though they remain genetically distinct. The U.S. Fish and Wildlife Service recently reclassified Okaloosa darters from endangered to threatened status. Our genetic data support the reclassification of Okaloosa darter Evolutionary Significant Units (ESUs) in the larger Tom??s, Turkey, and Rocky creeks from endangered to threatened status. However, the three smaller drainages (Mill, Swift, and Turkey Bolton creeks) remain at risk due to their small population sizes and anthropogenic pressures on remaining habitat. Natural resource managers now have the evolutionary information to guide recovery actions within and among drainages throughout the range of the Okaloosa darter.     

Delimitation of evolutionary units in Cuvier’s dwarf caiman, <Emphasis Type="Italic">Paleosuchus palpebrosus</Emphasis> (Cuvier, 1807): insights from conservation of a broadly distributed species

An important goal of evolutionary and conservation biology is the identification of units below the species level, such as Evolutionarily Significant Units (ESUs), providing objectively delimited units for species conservation and management. In this study we tested the hypothesis that Cuvier’s dwarf caiman (Paleosuchus palpebrosus)—a species broadly distributed across several biomes and watersheds of South America—is comprised of different ESUs. We analyzed mitochondrial cytochrome b sequences of 206 individuals and 532 unlinked ddRAD loci of 20 individuals chosen from amongst the mitochondrial haplogroups. Analysis of the cytochrome b sequences revealed four mitochondrial clusters, while STRUCTURE analysis of ddRAD loci detected three genomic clusters with different levels of mixture between them. Using the Adaptive Evolutionary Conservation (AEC) framework we identified three ESUs: “Amazon”, “Madeira-Bolivia” and “Pantanal”; one of them composed of two different Management Units (MUs), “Madeira” and “Bolivia”. In general, based on the comparisons with other crocodilian species, genetic diversity of each lineage was moderate however, the Madeira MU showed fivefold lower genetic diversity than other geographic groups. Considering the particularities of each Paleosuchus palpebrosus conservation unit, we recommend that the conservation status of each is evaluated separately. Tropical biodiversity is largely underestimated and in this context the broadly distributed species are the most likely candidates to harbor distinct evolutionary lineages. Thus, we suggest that conservation research should not neglect species that are generally considered of Least Concern by IUCN due to the taxon’s broad geographic distribution.     

Population Genetic Patterns of Threatened European Mudminnow (Umbra krameri Walbaum, 1792) in a Fragmented Landscape: Implications for Conservation Management

The European mudminnow (Umbra krameri) is a Middle Danubian endemic fish species, which is characterised by isolated populations living mainly in artificial habitats in the centre of its range, in the Carpathian Basin. For their long term preservation, reliable information is needed about the structure of stocks and the level of isolation. The recent distribution pattern, and the population genetic structure within and among regions were investigated to designate the Evolutionary Significant, Conservation and Management Units (ESUs, CUs, MUs) and to explore the conservation biological value of the shrinking populations. In total, eight microsatellite loci were studied in 404 specimens originating from eight regions. The results revealed a pronounced population structure, where strictly limited gene flow was detected among regions, as well as various strengths of connections within regions. Following the results of hierarchical structure analyses, two ESUs were supposed in the Carpathian Basin, corresponding to the Danube and Tisza catchments. Our results recommend designating the borders of CUs in an 80–90km range and 16 clusters should be set up as MUs for the 33 investigated populations. How these genetic findings can be used to better allocate conservation resources for the long term maintenance of the metapopulation structure of this threathened endemic fish is discussed.     

Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.     

Genetic Diversity and Population Structure of the Pelagic Thresher Shark (Alopias pelagicus) in the Pacific Ocean: Evidence for Two Evolutionarily Significant Units

There has been an increasing concern about shark overexploitation in the last decade, especially for open ocean shark species, where there is a paucity of data about their life histories and population dynamics. Little is known regarding the population structure of the pelagic thresher shark, Alopias pelagicus. Though an earlier study using mtDNA control region data, showed evidence for differences between eastern and western Pacific populations, the study was hampered by low sample size and sparse geographic coverage, particularly a lack of samples from the central Pacific. Here, we present the population structure of Alopias pelagicus analyzing 351 samples from six different locations across the Pacific Ocean. Using data from mitochondrial DNA COI sequences and seven microsatellite loci we found evidence of strong population differentiation between western and eastern Pacific populations and evidence for reciprocally monophyly for organelle haplotypes and significant divergence of allele frequencies at nuclear loci, suggesting the existence of two Evolutionarily Significant Units (ESU) in the Pacific Ocean. Interestingly, the population in Hawaii appears to be composed of both ESUs in what seems to be clear sympatry with reproductive isolation. These results may indicate the existence of a new cryptic species in the Pacific Ocean. The presence of these distinct ESUs highlights the need for revised management plans for this highly exploited shark throughout its range.     

Phylogeographic-based conservation implications for the New Zealand long-tailed bat, (<Emphasis Type="Italic">Chalinolobus tuberculatus</Emphasis>): identification of a single ESU and a candidate population for genetic rescue

The New Zealand long-tailed bat (Chalinolobus tuberculatus) is an endemic species threatened with extinction. Since the arrival of humans, massive deforestation has occurred and invasive mammalian predators were introduced. As a result, C. tuberculatus’ distribution shrank dramatically and became fragmented. To aid the management of the remaining populations, two Evolutionary Significant Units (ESUs) were designated: one on each of New Zealand’s main islands. We utilised mitochondrial sequence data (cytb, 703 bp) and 10 nuclear DNA microsatellite loci to reconstruct the demographic history of this species, to characterise the level of genetic diversity in remaining populations, and to assess the current connectivity between them. Our results indicate that the North Island, with the highest genetic diversity, served as a glacial refuge, with a loss of diversity following the path recolonization to the south of the South Island. However, our data are also consistent with continued, or at least very recent, genetic exchange between colonies across the species distribution. The only exception is the Hanging Rock colony on the east coast of the South Island, which appears to be isolated. Thus, there was no support for the previously designated ESUs. Signatures of past population declines were found in three colonies, the most extreme of which was found in Hanging Rock. Consequently, we recommend that it be genetically rescued via translocation from a donor population. In general, future management priorities should treat Chalinolobus tuberculatus as a single unit, focusing on maintaining connectivity between remaining populations, together with continued roost protection and pest control.     

Host plant use by the Heath fritillary butterfly, Melitaea athalia: plant habitat, species and chemistry

We present a study of habitat use, oviposition plant choice, and food plant suitability for the checkerspot butterfly Melitaea athalia Rottemburg (Lepidoptera: Nymphalidae) in Åland, Finland. We found that in Åland, unlike in the mainland of Finland and many parts of its range, M. athalia flies mainly in open meadows. When offered an array of plants in a large (32 × 26 m) field cage, they predominately oviposited upon Veronica chamaedrys L., V. spicata L. and Plantago lanceolata L. (Plantaginaceae), which grow in open meadows. The relative abundance of the butterfly in Åland, and its habitat and host plant use there, may reflect local adaptation to land use practices and geology that maintain clusters of small open meadows with little successional change. At the scale of a plant patch, preferred species were used as frequently in mixed species patches as in mono-specific patches, and more oviposition occurred in open than in grassy patches. All of the host plants used by M. athalia are defended by iridoid glycosides (IGs). However, oviposition choice among species and among individual plants within species was largely independent of IG concentration. This contrast with the more discerning congener, M. cinxia, supports the idea that host discrimination decreases with increasing host range. Finally, although the adult butterflies chose specific plant species for oviposition, as larvae they performed well on twelve out of thirteen species of plants, including both known hosts and related novel plants that occur in Åland, indicating a much wider range of larval food plant species than adult oviposition species.     

Patterns of genetic variation in the Chinese endemic Psilopeganum sinense (Rutaceae) as revealed by nuclear microsatellites and chloroplast microsatellites

Psilopeganum sinense is a perennial herb endemic to Three-Gorges Reservoir Area (TGRA) in China. Genetic diversity of this endangered species was assessed by using 11 nuclear microsatellites and three chloroplast microsatellite (cpSSR) markers. A total of 8 haplotypes were identified in a survey of 212 individuals sampled from nine populations encompassing most of the natural range of the species. A low level of genetic diversity was detected: H_E = 0.301 for SSR and H_E = 0.28 for cpSSR. Populations were highly differentiated from one another: an AMOVA analysis that showed that 56.3% and 68.2% genetic variation resided between populations based on SSR and cpSSR analysis, respectively, and F_ST and F_STc (0.467 for SSR and 0.644 for cpSSR, respectively) were high. Significant differences were found between estimates of haplotypic differentiation calculated by using unordered alleles (G_STc = 0.857) and ordered alleles (N_STc = 0.728), which indicated the existence of phylogeographical structure in P. sinense. The indirect ratio of pollen flow/seed flow derived from estimates of haplotypic and nuclear DNA differentiation indicated that gene flow via pollen is less efficient than via seed. Two distinct evolutionary lineages (evolutionary significant units, ESUs) were recognized for P. sinense on the basis of both the PCoA and NCA analysis. Sampling strategies for conserving this endangered plant were discussed.     

Selection and localised genetic structure in the threatened Manauense Harlequin Frog (Bufonidae: Atelopus manauensis)

Knowledge of genome-wide variation and the processes influencing gene flow are critical to managing threatened species. Here, we characterise genetic diversity and the environmental features associated with connectivity for a narrowly distributed and threatened Amazonian frog, Atelopus manauensis. We sampled 94 individuals throughout the upper, middle and lower courses from each of the five major rivers covering the species’ known geographic range and genotyped each individual at 3859 single nucleotide polymorphisms (SNPs). Genetic variation was significantly subdivided into six groups, each mostly containing individuals sampled within the same major river system. The genetic distances among these groups increased with geographic distance, and open forests and the extremities of the altitude gradient were associated with less genetic connectivity. Using F_ST outlier approaches and environmental association analyses, we identified SNPs indicative of localised adaptation, with 28 SNPs significantly associated with forest biomass and altitude. Evidence of divergent selection among the six genetic clusters suggests the presence of six Evolutionary Significant Units (ESUs). Overall, the ESUs were characterized by low contemporary effective population sizes (N_E?<?100) suggesting that genetic variation will be lost by random genetic drift. We demonstrate surprisingly high levels of divergence across the limited distribution of A. manauensis and suggest that each of the six adaptively divergent lineages be considered in conservation planning.