Spatiotemporal maintenance of flora in the Himalaya biodiversity hotspot: Current knowledge and future perspectives.

Mountain ecosystems support a significant one-third of all terrestrial biodiversity, but our understanding of the spatiotemporal maintenance of this high biodiversity remains poor, or at best controversial. The Himalaya hosts a complex mountain ecosystem with high topographic and climatic heterogeneity and harbors one of the world's richest floras. The high species endemism, together with increasing anthropogenic threats, has qualified the Himalaya as one of the most significant global biodiversity hotspots. The topographic and climatic complexity of the Himalaya makes it an ideal natural laboratory for studying the mechanisms of floral exchange, diversification, and spatiotemporal distributions. Here, we review literature pertaining to the Himalaya in order to generate a concise synthesis of the origin, distribution, and climate change responses of the Himalayan flora. We found that the Himalaya supports a rich biodiversity and that the Hengduan Mountains supplied the majority of the Himalayan floral elements, which subsequently diversified from the late Miocene onward, to create today's relatively high endemicity in the Himalaya. Further, we uncover links between this Miocene diversification and the joint effect of geological and climatic upheavals in the Himalaya. There is marked variance regarding species dispersal, elevational gradients, and impact of climate change among plant species in the Himalaya, and our review highlights some of the general trends and recent advances on these aspects. Finally, we provide some recommendations for conservation planning and future research. Our work could be useful in guiding future research in this important ecosystem and will also provide new insights into the maintenance mechanisms underpinning other mountain systems.

Hierarchical structuring of genetic variation at differing geographic scales in the cultivated sugar kelp Saccharina latissima.

The cultivation of macroalgae for biofuels, food and fertilisers has increased dramatically in recent years. The demand for such algal-derived products means that large scale cultivation in coastal waters will become necessary to provide sufficient algal biomass. As part of the process of establishing new macroalgal farms, the potential for gene flow between cultivated specimens and natural populations needs to be taken into consideration. Consequently, in the present study we have used a combined population genetic and hydrodynamic modelling approach to determine potential levels and patterns of gene flow in the kelp Saccharina latissima. Microsatellite analysis of 14 populations sampled across the northern part of the Irish Sea indicated four distinct genetic clusters. These were consistent with dispersal patterns indicated by the particle tracking model and show a combination of isolation by distance and genetic structuring due to local hydrodynamic conditions. At smaller scales (less than a few 10s of km), gene flow appears to be fairly extensive, with evidence of local population connectivity due to local currents. At larger scales, however, factors such as freshwater efflux and open water would appear to represent barriers to gene flow. Together, these patterns suggest that factors other than simple geographical distance and proximity need to be taken into account when planning the siting of kelp farms with the aim of minimizing gene flow to and from natural populations.

Testing Darwin's transoceanic dispersal hypothesis for the inland nettle family (Urticaceae).

Dispersal is a fundamental ecological process, yet demonstrating the occurrence and importance of long-distance dispersal (LDD) remains difficult, having rarely been examined for widespread, non-coastal plants. To address this issue, we integrated phylogenetic, molecular dating, biogeographical, ecological, seed biology and oceanographic data for the inland Urticaceae. We found that Urticaceae originated in Eurasia c. 69 Ma, followed by ≥ 92 LDD events between landmasses. Under experimental conditions, seeds of many Urticaceae floated for > 220 days, and remained viable after 10 months in seawater, long enough for most detected LDD events, according to oceanographic current modelling. Ecological traits analyses indicated that preferences for disturbed habitats might facilitate LDD. Nearly half of all LDD events involved dioecious taxa, so population establishment in dioecious Urticaceae requires multiple seeds, or occasional selfing. Our work shows that seawater LDD played an important role in shaping the geographical distributions of Urticaceae, providing empirical evidence for Darwin's transoceanic dispersal hypothesis.

Evidence for facultative protocarnivory in Capsella bursa-pastoris seeds.

Many plants derive nutrients by attracting, ensnaring and killing invertebrates, a process that is described as "protocarnivory". This has been observed in seeds of the weed Capsella bursa-pastoris, but it is unclear as to whether it confers any material benefit in terms of germination, establishment and development. In the present study, seeds were germinated in zero, low, medium and high nutrient soils in both the presence and absence of nematodes (Steinernema feltiae). Nematodes were attracted to the seeds, with many dying within three days. Germination rates and seedling fresh masses were higher at all nutrient levels, and seedling fresh lengths were higher in all but the zero nutrient treatment, in the presence of nematodes. After transplantation, young plant fresh root lengths and dried leaf and root masses were generally higher in plants that had been germinated in the presence of nematodes across all nutrient levels, with the majority of significant differences being observed in the low-nutrient treatment. Our findings suggest that protocarnivory may play a role in the germination, establishment and early development of C. bursa-pastoris, and that this process may be facultative, since differences between nematode and non-nematode treatments were generally more pronounced in soils with low nutrient levels.

Phylogenomic inference in extremis: A case study with mycoheterotroph plastomes.

PREMISE OF THE STUDY: Phylogenomic studies employing large numbers of genes, including those based on plastid genomes (plastomes), are becoming common. Nonphotosynthetic plants such as mycoheterotrophs (which rely on root-associated fungi for essential nutrients, including carbon) tend to have highly elevated rates of plastome evolution, substantial genome reduction, or both. Mycoheterotroph plastomes therefore provide excellent test cases for investigating how extreme conditions impact phylogenomic inference. METHODS: We used parsimony and likelihood analysis of protein-coding gene sets from published and newly completed plastomes to infer the phylogenetic placement of taxa from the 10 angiosperm families in which mycoheterotrophy evolved. KEY RESULTS: Despite multiple very long branches that reflect elevated substitution rates, and frequently patchy gene recovery due to genome reduction, inferred phylogenetic placements of most mycoheterotrophic lineages in DNA-based likelihood analyses are both well supported and congruent with other studies. Amino-acid-based likelihood placements are broadly consistent with DNA-based inferences, but extremely rate-elevated taxa can have unexpected placements-albeit with weak support. In contrast, parsimony analysis is strongly misled by long-branch attraction among many distantly related mycoheterotrophic monocots. CONCLUSIONS: Mycoheterotrophic plastomes provide challenging cases for phylogenomic inference, as substitutional rates can be elevated and genome reduction can lead to sparse gene recovery. Nonetheless, diverse likelihood frameworks provide generally well-supported and mutually concordant phylogenetic placements of mycoheterotrophs, consistent with recent phylogenetic studies and angiosperm-wide classifications. Previous predictions of parallel photosynthesis loss within families are supported for Burmanniaceae, Ericaceae, Gentianaceae, and Orchidaceae. Burmanniaceae and Thismiaceae should not be combined as a single family in Dioscoreales.

Cryptic species and parallel genetic structuring in Lethrinid fish: Implications for conservation and management in the southwest Indian Ocean.

Analysis of genetic variation can provide insights into ecological and evolutionary diversification which, for commercially harvested species, can also be relevant to the implementation of spatial management strategies and sustainability. In comparison with other marine biodiversity hot spots, there has been less genetic research on the fauna of the southwest Indian Ocean (SWIO). This is epitomized by the lack of information for lethrinid fish, which support socioeconomically important fisheries in the region. This study combines comparative phylogeographic and population genetic analyses with ecological niche modeling to investigate historical and contemporary population dynamics of two species of emperor fish (Lethrinus mahsena and Lethrinus harak) across the SWIO. Both species shared similarly shallow phylogeographic patterns and modeled historical (LGM) habitat occupancies. For both species, allele frequency and kinship analyses of microsatellite variation revealed highly significant structure with no clear geographical pattern and nonrandom genetic relatedness among individuals within samples. The genetic patterns for both species indicate recurrent processes within the region that prevent genetic mixing, at least on timescales of interest to fishery managers, and the potential roles of recruitment variability and population isolation are discussed in light of biological and environmental information. This consistency in both historical and recurrent population processes indicates that the use of model species may be valuable in management initiatives with finite resources to predict population structure, at least in cases wherein biogeographic and ecological differences between taxa are minimized. Paradoxically, mtDNA sequencing and microsatellite analysis of samples from the Seychelles revealed a potential cryptic species occurring in sympatry with, and seemingly morphologically identical to, L. mahsena. BLAST results point to the likely misidentification of species and incongruence between voucher specimens, DNA barcodes, and taxonomy within the group, which highlights the utility and necessity of genetic approaches to characterize baseline biodiversity in the region before such model-based methods are employed.

Low genetic diversity and potential inbreeding in an isolated population of alder buckthorn (Frangula alnus) following a founder effect.

Alder buckthorn (Frangula alnus) is one of Ireland's rarest tree species, and in Northern Ireland the species is now restricted to a single population in Peatlands Park, Co. Armagh numbering ca. 140 mature trees. Genotyping of 95% of the trees at nine nuclear microsatellite loci revealed that levels of genetic diversity within this population were generally lower than those reported from larger populations in Spain. Analysis of six chloroplast microsatellite loci revealed no variation. The level of F IS was significantly higher than that in the Spanish populations, as well as in other populations across Europe, potentially indicating inbreeding. Spatial autocorrelation analysis indicated some evidence of fine-scale genetic structuring, most likely due to limited seed dispersal, but the overall level of differentiation between subpopulations was low, indicating high levels of gene flow, probably due to cross-pollination by bees. Our results are consistent with a gradual population expansion from a limited number of individuals. We suggest that more immediate conservation efforts might be best focused on ensuring suitable habitat for the continued recovery of this isolated population.

Using MiddRAD-seq data to develop polymorphic microsatellite markers for an endangered yew species.

Microsatellites are highly polymorphic markers which have been used in a wide range of genetic studies. In recent years, various sources of next-generation sequencing data have been used to develop new microsatellite loci, but compared with the more common shotgun genomic sequencing or transcriptome data, the potential utility of RAD-seq data for microsatellite ascertainment is comparatively under-used. In this study, we employed MiddRAD-seq data to develop polymorphic microsatellite loci for the endangered yew species Taxus florinii. Of 8,823,053 clean reads generated for ten individuals of a population, 94,851 (∼1%) contained microsatellite motifs. These corresponded to 2993 unique loci, of which 526 (∼18%) exhibited polymorphism. Of which, 237 were suitable for designing microsatellite primer pairs, and 128 loci were randomly selected for PCR validation and microsatellite screening. Out of the 128 primer pairs, 16 loci gave clear, reproducible patterns, and were then screened and characterized in 24 individuals from two populations. The total number of alleles per locus ranged from two to ten (mean = 4.875), and within-population expected heterozygosity from zero to 0.789 (mean = 0.530), indicating that these microsatellite loci will be useful for population genetics and speciation studies of T. florinii. This study represents one of few examples to mine polymorphic microsatellite loci from ddRAD data.

Pure species in a continuum of genetic and morphological variation: sympatric oaks at the edge of their range.

BACKGROUND AND AIMS: Studies on oaks (Quercus spp.) have often been hampered by taxonomic confusion, a situation further compounded by the occurrence of extensive interspecific hybridization. In the present study, a combination of genetic and morphological analyses was used to examine sympatric populations of Q. petraea and Q. robur at the north-western edge of their ranges in Northern Ireland, since it had previously been suggested that hybridization could facilitate the apparent rapid, long-distance dispersal of oaks following the glaciations. METHODS: Samples were collected from 24 sites across Northern Ireland that had been previously designated as ancient or semi-natural woodland. Genotypes were obtained from a total of 950 trees using 12 nuclear microsatellite loci, and admixture coefficients were calculated based on a Bayesian clustering approach. Individuals were also classified as Q. petraea,Q. robur or hybrids based on two objective morphometric characters shown previously to delineate pure individuals effectively. Genetically 'pure' individuals of both species, as defined by the Bayesian clustering, were also genotyped for five chloroplast microsatellites. KEY RESULTS: Genetic and morphological analyses both indicated the presence of pure individuals of both species, as well as a continuum of intermediates. There was a good agreement between the molecular and morphological classification, with a generally clear separation between pure individuals. CONCLUSIONS: Despite millennia of hybridization and introgression, genetically and morphologically pure individuals of both Q. petraea and Q. robur can be found at the edge of their range, where both species occur sympatrically. The high proportion of individuals exhibiting introgression compared with previous studies may reflect the historical role of hybridization in facilitating dispersal following the glaciations. This is further supported by the significantly higher chloroplast diversity in Q. robur compared with Q. petraea.

Cryptic introgression into the kidney saxifrage (Saxifraga hirsuta) from its more abundant sympatric congener Saxifraga spathularis, and the potential risk of genetic assimilation.

BACKGROUND AND AIMS: Although hybridization can play a positive role in plant evolution, it has been shown that excessive unidirectional hybridization can result in replacement of a species' gene pool, and even the extinction of rare species via genetic assimilation. This study examines levels of introgression between the common Saxifraga spathularis and its rarer congener S. hirsuta, which have been observed to hybridize in the wild where they occur sympatrically. METHODS: Seven species-specific single nucleotide polymorphisms (SNPs) were analysed in 1025 plants representing both species and their hybrid, S. × polita, from 29 sites across their ranges in Ireland. In addition, species distribution modelling was carried out to determine whether the relative abundance of the two parental species is likely to change under future climate scenarios. KEY RESULTS: Saxifraga spathularis individuals tended to be genetically pure, exhibiting little or no introgression from S. hirsuta, but significant levels of introgression of S. spathularis alleles into S. hirsuta were observed, indicating that populations exhibiting S. hirsuta morphology are more like a hybrid swarm, consisting of backcrosses and F2s. Populations of the hybrid, S. × polita, were generally comprised of F1s or F2s, with some evidence of backcrossing. Species distribution modelling under projected future climate scenarios indicated an increase in suitable habitats for both parental species. CONCLUSIONS: Levels of introgression observed in this study in both S. spathularis and S. hirsuta would appear to be correlated with the relative abundance of the species. Significant introgression of S. spathularis alleles was detected in the majority of the S. hirsuta populations analysed and, consequently, ongoing introgression would appear to represent a threat to the genetic integrity of S. hirsuta, particularly in areas where the species exists sympatrically with its congener and where it is greatly outnumbered.

Coexistence of congeneric native and invasive species: the case of the green algae Codium spp. in northwestern Spain.

We examined the patterns of distribution and abundance, and reproductive traits (presence of gametophytes and size at time of reproduction) in the invasive Codium fragile ssp. fragile and the native C. tomentosum and C. vermilara on intertidal habitats of NW Spain at two dates. All three species coexist in the locations and habitats studied, although abundances were low. We found a greater proportion of C. fragile ssp. fragile towards the east of the Cantabrian coast and on upper levels on the shore, where conditions are more stressful. The proportion of thalli bearing gametangia in C. fragile ssp. fragile was greater than in the native species in all habitats. The presence of gametangia was size-dependent for all species, with the invasive species maturing at a smaller size, which combined with the previous features, might confer competitive advantages to this species over the native species. We also demonstrated that molecular analyses are necessary for the correct identification of C. fragile subspecies.

Understanding macroalgal dispersal in a complex hydrodynamic environment: a combined population genetic and physical modelling approach.

Gene flow in macroalgal populations can be strongly influenced by spore or gamete dispersal. This, in turn, is influenced by a convolution of the effects of current flow and specific plant reproductive strategies. Although several studies have demonstrated genetic variability in macroalgal populations over a wide range of spatial scales, the associated current data have generally been poorly resolved spatially and temporally. In this study, we used a combination of population genetic analyses and high-resolution hydrodynamic modelling to investigate potential connectivity between populations of the kelp Laminaria digitata in the Strangford Narrows, a narrow channel characterized by strong currents linking the large semi-enclosed sea lough, Strangford Lough, to the Irish Sea. Levels of genetic structuring based on six microsatellite markers were very low, indicating high levels of gene flow and a pattern of isolation-by-distance, where populations are more likely to exchange migrants with geographically proximal populations, but with occasional long-distance dispersal. This was confirmed by the particle tracking model, which showed that, while the majority of spores settle near the release site, there is potential for dispersal over several kilometres. This combined population genetic and modelling approach suggests that the complex hydrodynamic environment at the entrance to Strangford Lough can facilitate dispersal on a scale exceeding that proposed for L. digitata in particular, and the majority of macroalgae in general. The study demonstrates the potential of integrated physical-biological approaches for the prediction of ecological changes resulting from factors such as anthropogenically induced coastal zone changes.

Genetic evidence for predominantly hydrochoric gene flow in the invasive riparian plant Impatiens glandulifera (Himalayan balsam).

BACKGROUND AND AIMS: Riparian systems are prone to invasion by alien plant species. The spread of invasive riparian plants may be facilitated by hydrochory, the transport of seeds by water, but while ecological studies have highlighted the possible role of upstream source populations in the establishment and persistence of stands of invasive riparian plant species, population genetic studies have as yet not fully addressed the potential role of hydrochoric dispersal in such systems. METHODS: A population genetics approach based on a replicated bifurcate sampling design is used to test hypotheses consistent with patterns of unidirectional, linear gene flow expected under hydrochoric dispersal of the invasive riparian plant Impatiens glandulifera in two contrasting river systems. KEY RESULTS: A significant increase in levels of genetic diversity downstream was observed, consistent with the accumulation of propagules from upstream source populations, and strong evidence was found for organization of this diversity between different tributaries, reflecting the dendritic organization of the river systems studied. CONCLUSIONS: These findings indicate that hydrochory, rather than anthropogenic dispersal, is primarily responsible for the spread of I. glandulifera in these river systems, and this is relevant to potential approaches to the control of invasive riparian plant species.

State-of the art methodologies dictate new standards for phylogenetic analysis.

The intention of this editorial is to steer researchers through methodological choices in molecular evolution, drawing on the combined expertise of the authors. Our aim is not to review the most advanced methods for a specific task. Rather, we define several general guidelines to help with methodology choices at different stages of a typical phylogenetic 'pipeline'. We are not able to provide exhaustive citation of a literature that is vast and plentiful, but we point the reader to a set of classical textbooks that reflect the state-of-the-art. We do not wish to appear overly critical of outdated methodology but rather provide some practical guidance on the sort of issues which should be considered. We stress that a reported study should be well-motivated and evaluate a specific hypothesis or scientific question. However, a publishable study should not be merely a compilation of available sequences for a protein family of interest followed by some standard analyses, unless it specifically addresses a scientific hypothesis or question. The rapid pace at which sequence data accumulate quickly outdates such publications. Although clearly, discoveries stemming from data mining, reports of new tools and databases and review papers are also desirable.

Geological and ecological factors drive cryptic speciation of yews in a biodiversity hotspot.

The interplay of orographic uplift and climatic changes in the Himalaya-Hengduan Mountains region (HHM) have had a key role in speciation and population demography. To gain further insight into these processes, we investigated their effects on Taxus wallichiana by combining molecular phylogeography and species distribution modeling. Molecular data were obtained from 43 populations of T. wallichiana. Nineteen climatic variables were analyzed alongside genetic discontinuities. Species distribution modeling was carried out to predict potential past distribution ranges. Two distinct lineages were identified, which diverged c. 4.2 (2.0-6.5) million years ago (Ma), a timescale that corresponds well with the recent uplift of the Qinghai-Tibet Plateau and subsequent climatic changes of the region. Correlations with climatic variables also suggest that ecological factors may have further reinforced the separation of the two lineages. Both lineages experienced population expansion during the last glaciation. The high genetic divergence, long-term isolation and ecological differentiation suggest a scenario of cryptic speciation in T. wallichiana associated with geological and climatic changes in the HHM. Our findings also challenge the notion of general population 'contraction' during the last glaciation in the HHM.

Sampling strategy and potential utility of indels for DNA barcoding of closely related plant species: a case study in taxus.

Although DNA barcoding has become a useful tool for species identification and biodiversity surveys in plant sciences, there remains little consensus concerning appropriate sampling strategies and the treatment of indels. To address these two issues, we sampled 39 populations for nine Taxus species across their entire ranges, with two to three individuals per population randomly sampled. We sequenced one core DNA barcode (matK) and three supplementary regions (trnH-psbA, trnL-trnF and ITS) for all samples to test the effects of sampling design and the utility of indels. Our results suggested that increasing sampling within-population did not change the clustering of individuals, and that meant within-population P-distances were zero for most populations in all regions. Based on the markers tested here, comparison of methods either including or excluding indels indicated that discrimination and nodal support of monophyletic groups were significantly increased when indels were included. Thus we concluded that one individual per population was adequate to represent the within-population variation in these species for DNA barcoding, and that intra-specific sampling was best focused on representing the entire ranges of certain taxa. We also found that indels occurring in the chloroplast trnL-trnF and trnH-psbA regions were informative to differentiate among for closely related taxa barcoding, and we proposed that indel-coding methods should be considered for use in future for closed related plant species DNA barcoding projects on or below generic level.

Unique genetic variation at a species' rear edge is under threat from global climate change.

Global climate change is having a significant effect on the distributions of a wide variety of species, causing both range shifts and population extinctions. To date, however, no consensus has emerged on how these processes will affect the range-wide genetic diversity of impacted species. It has been suggested that species that recolonized from low-latitude refugia might harbour high levels of genetic variation in rear-edge populations, and that loss of these populations could cause a disproportionately large reduction in overall genetic diversity in such taxa. In the present study, we have examined the distribution of genetic diversity across the range of the seaweed Chondrus crispus, a species that has exhibited a northward shift in its southern limit in Europe over the last 40 years. Analysis of 19 populations from both sides of the North Atlantic using mitochondrial single nucleotide polymorphisms (SNPs), sequence data from two single-copy nuclear regions and allelic variation at eight microsatellite loci revealed unique genetic variation for all marker classes in the rear-edge populations in Iberia, but not in the rear-edge populations in North America. Palaeodistribution modelling and statistical testing of alternative phylogeographic scenarios indicate that the unique genetic diversity in Iberian populations is a result not only of persistence in the region during the last glacial maximum, but also because this refugium did not contribute substantially to the recolonization of Europe after the retreat of the ice. Consequently, loss of these rear-edge populations as a result of ongoing climate change will have a major effect on the overall genetic diversity of the species, particularly in Europe, and this could compromise the adaptive potential of the species as a whole in the face of future global warming.

Comparative phylogeography of two related plant species with overlapping ranges in Europe, and the potential effects of climate change on their intraspecific genetic diversity.

BACKGROUND: The aim of the present study was to use a combined phylogeographic and species distribution modelling approach to compare the glacial histories of two plant species with overlapping distributions, Orthilia secunda (one-sided wintergreen) and Monotropa hypopitys (yellow bird's nest). Phylogeographic analysis was carried out to determine the distribution of genetic variation across the range of each species and to test whether both correspond to the "classic" model of high diversity in the south, with decreasing diversity at higher latitudes, or whether the cold-adapted O. secunda might retain more genetic variation in northern populations. In addition, projected species distributions based on a future climate scenario were modelled to assess how changes in the species ranges might impact on total intraspecific diversity in both cases. RESULTS: Palaeodistribution modelling and phylogeographic analysis using multiple genetic markers (chloroplast trnS-trnG region, nuclear ITS and microsatellites for O. secunda; chloroplast rps2, nuclear ITS and microsatellites for M. hypopitys) indicated that both species persisted throughout the Last Glacial Maximum in southern refugia. For both species, the majority of the genetic diversity was concentrated in these southerly populations, whereas those in recolonized areas generally exhibited lower levels of diversity, particularly in M. hypopitys. Species distribution modelling based on projected future climate indicated substantial changes in the ranges of both species, with a loss of southern and central populations, and a potential northward expansion for the temperate M. hypopitys. CONCLUSIONS: Both Orthilia secunda and Monotropa hypopitys appear to have persisted through the LGM in Europe in southern refugia. The boreal O. secunda, however, has retained a larger proportion of its genetic diversity in more northerly populations outside these refugial areas than the temperate M. hypopitys. Given that future species distribution modelling suggests northern range shifts and loss of suitable habitat in the southern parts of the species' current distributions, extinction of genetically diverse rear edge populations could have a significant effect in the rangewide intraspecific diversity of both species, but particularly in M. hypopitys.

High clonal diversity in threatened peripheral populations of the yellow bird's nest (Hypopitys monotropa; syn. Monotropa hypopitys).

BACKGROUND AND AIMS: Peripheral populations of plant species are often characterized by low levels of genetic diversity as a result of genetic drift, restricted gene flow, inbreeding and asexual reproduction. These effects can be exacerbated where range-edge populations are fragmented. The main aim of the present study was to assess the levels of genetic diversity in remnant populations of Hypopitys monotropa (syn. Monotropa hypopitys; yellow bird's nest) at the edge of the species' European range in Northern Ireland, since these remnant populations are small and highly fragmented. METHODS: Every plant found through surveys of 21 extant populations was genotyped for eight microsatellite loci to estimate levels and patterns of genetic diversity and clonality. KEY RESULTS: Levels of genetic diversity were relatively high in the populations studied, and the incidence of clonal reproduction was generally low, with a mean of only 14·45 % of clonal individuals. Clones were small and highly spatially structured. Levels of inbreeding, however, were high. CONCLUSIONS: The observed low levels of clonality suggest that the majority of genets in the populations of H. monotropa studied are fertile and that reproduction is predominantly sexual. As the species is highly self-compatible, it is likely that the high levels of inbreeding observed in the populations in the present study are the result of self-pollination, particularly given the small numbers of individuals in most of the patches. Given this extent of inbreeding, further genetic monitoring would be advisable to ensure that genetic diversity is maintained.