Orchid Orchis Research Articles

Narrow mycorrhizal specialization and its effect on early development in the Mediterranean orchid Orchis italica

Abstract The contribution of individual orchid mycorrhizal (OrM) fungi to orchid germination is poorly understood. We isolated Tulasnella OrM fungi from 11 populations representing nine Italian regions associated with the orchid Orchis italica. We performed in vitro germination experiments using both single and two OrM isolate combinations of Tulasnella and Ceratobasidium isolates found in O. italica and co-occurring orchids. We performed an in situ reciprocal translocation experiment of O. italica seeds in two populations and we experimentally planted seeds into two unoccupied sites to test whether the presence of suitable OrM may limit its distribution. In vitro, Tulasnella isolates initiated germination, but only two isolates resulted in seedling development, while seeds did not germinate in the presence of Ceratobasidium isolates. Combinations of two Tulasnella isolates had lower germination rates compared to single isolates. Reciprocal translocations revealed that seeds from the same population had higher germination rates. Protocorms developed in one unoccupied site, with the associated fungus identified as Tulasnella spp. These results suggest that Tulasnella vary in their efficacy in seed germination and O. italica has a narrow specialization on Tulasnella. Such information is important for understanding the range limits of orchids and their ecological dependency on OrM fungi.

Floral Trait and Mycorrhizal Similarity between an Endangered Orchid and Its Natural Hybrid

Hybridization can often lead to the formation of novel taxa which can have traits that resemble either or both parental species. Determining the similarity of hybrid traits to parental taxa is particularly important in plant conservation, as hybrids that form between rare and common taxa may more closely resemble a rare parental species, thereby putting the rare parental taxon at further risk of extinction via increased backcrossing and introgression. We investigated the floral (morphological and chemical) traits and orchid mycorrhizal (OrM) fungal associations of the endangered orchid Orchis patens, its more common sister species O. provincialis, and their natural hybrid O. × fallax in natural sympatric populations. We found that both morphological and chemical floral traits of O. × fallax are shared by the parents but are more similar to O. patens than O. provincialis. OrM fungi were shared among all three taxa, indicating that the availability of OrM fungi should not represent a barrier to establishment of individuals of any of these taxa. These results suggest that O. × fallax may be able to expand its distribution within a similar niche to O. patens. This highlights the importance of quantifying differences between hybrids and parental taxon in species conservation planning.

Sources of floral scent variation in the food-deceptive orchid Orchis mascula

Intraspecific variation of floral signals, and particularly floral scent, is widespread in orchid species. Pollinator-mediated selection is often hypothesized to govern such polymorphism. For example, circadian rhythms of floral emissions have been associated to the daily activity patterns of pollinators in several plant species. However, most of the studies on floral scent variation usually consider only one factor of variation (diel variation, pollination, flower age …). In this study, we investigated simultaneously seven sources of variation susceptible to induce changes in floral scent emissions in the food-deceptive orchid Orchis mascula: circadian rhythm, flower age, pollination, inflorescence morphology, herbivory, leaf spots and habitat. We found that all the factors studied, except plant morphology (spots on leaves, inflorescence morphology), were associated with significant changes in the volatile profile of O. mascula inflorescences. Electroantennography experiments with Bombus terrestris, the main pollinator of O. mascula, revealed that bumblebees were able to detect almost all the compounds involved in floral scent variation. Whether pollinator behaviour is influenced by changes in floral odours induced by these different factors, and whether it can affect plant reproductive success will have to be tested in further studies.

Lack of strong selection pressures maintains wide variation in floral traits in a food-deceptive orchid.

Angiosperms vary remarkably in traits such as colour, size and shape of flowers, yet such variation generally tends to be low within species. In deceptive orchids, however, large variation in floral traits has been described, not only between but also within populations. Nonetheless, the factors driving variation in floral traits in deceptive orchids remain largely unclear. To identify determinants of variation in floral traits, we investigated patterns of fruit set and selection gradients in the food-deceptive orchid Orchis purpurea, which typically presents large within-population variation in the colour and size of the flowers. Using long-term data, fruit set was quantified in two populations over 16 consecutive years (2004-2019). Artificial hand pollination was performed to test the hypothesis that fruit set was pollinator-limited and that selfing led to decreased seed set and viability. Annual variation (2016-2019) in selection gradients was calculated for three colour traits (brightness, contrast and the number of spots on the labellum), flower size (spur length, labellum length and width) and plant size (number of flowers, plant height). Fruit set was, on average, low (~12 %) and severely pollinator-limited. Opportunities for selection varied strongly across years, but we found only weak evidence for selection on floral traits. In contrast, there was strong and consistent positive selection on floral display. Selfing led to reduced production of viable seeds and hence severe inbreeding depression (δ = 0.38). Overall, these results demonstrate that the large variation in flower colour and size that is regularly observed in natural O. purpurea populations is maintained by the consistent lack of strong selection pressures on these traits through time.

Ecological interactions of the sexually deceptive orchid Orchis galilaea

ABSTRACTPlant species dependent on highly specific interactions with pollinators are vulnerable to environmental change. Conservation strategies therefore require a detailed understanding of pollination ecology. This two-year study examined the interactions between the sexually deceptive orchid, Orchis galilaea, and its pollinator Lasioglossum marginatum. Relationships were investigated across three different habitats known to support O. galilaea (garrigue, oak woodland, and mixed oak/pine woodland) in Lebanon. Visitation rates to flowers were extremely low and restricted to male bees. The reproductive success of O. galilaea under ambient conditions was 29.3% (±2.4), compared to 89.0% (±2.1) in plants receiving cross-pollination by hand. No difference in reproductive success was found between habitat types, but values of reproductive success were positively correlated to the abundance of male bees. Pollination limitation can have negative impacts on the population growth of orchids, and this study provides clear evidence for more holistic approaches to habitat conservation to support specific interactions.

A new locality of orchid Orchis purpurea Huds. in Cieszyn Foothills (Czech Republic)

Abstract Orchis purpurea Huds. is protected species both in the Czech Republic and Poland. In 2014 a new locality was found in Kojkovice (district Třinec) in the Cieszyn Foothils near border with Poland. The only one blooming individual was observed in 2014 and 2015 but it was accompanied by other 6 orchid species, that were much more abundant, as: Orchis pallens, Orchis mascula subsp. signifera, Listera ovata, Platanthera bifolia, Cephalanthera damasonium, Neottia nidus-avis. The Kojkovice forest deserves to be protected area due to abundant occurrence of many orchids.

Analysis of the TCP genes expressed in the inflorescence of the orchid Orchis italica

TCP proteins are plant-specific transcription factors involved in many different processes. Because of their involvement in a large number of developmental pathways, their roles have been investigated in various plant species. However, there are almost no studies of this transcription factor family in orchids. Based on the available transcriptome of the inflorescence of the orchid Orchis italica, in the present study we identified 12 transcripts encoding TCP proteins. The phylogenetic analysis showed that they belong to different TCP classes (I and II) and groups (PCF, CIN and CYC/TB1), and that they display a number of conserved motifs when compared with the TCPs of Arabidopsis and Oryza. The presence of a specific cleavage site for the microRNA miRNA319, an important post-transcriptional regulator of several TCP genes in other species, was demonstrated for one transcript of O. italica, and the analysis of the expression pattern of the TCP transcripts in different inflorescence organs and in leaf tissue suggests that some TCP transcripts of O. italica exert their role only in specific tissues, while others may play multiple roles in different tissues. In addition, the evolutionary analysis showed a general purifying selection acting on the coding region of these transcripts.

The analysis of the inflorescence miRNome of the orchid Orchis italica reveals a DEF-like MADS-box gene as a new miRNA target.

Plant microRNAs (miRNAs) are small, regulatory non-coding RNAs involved in a wide range of biological processes, from organ development to response to stimuli. In recent years, an increasing number of studies on model plant species have highlighted the evolutionary conservation of a high number of miRNA families and the existence of taxon-specific ones. However, few studies have examined miRNAs in non-model species such as orchids, which are characterized by highly diversified floral structures and pollination strategies. Therefore, we analysed a small RNA library of inflorescence tissue of the Mediterranean orchid Orchis italica to increase the knowledge on miRNAs in a non-model plant species. The high-throughput sequencing and analysis of a small RNA library of inflorescence of O. italica revealed 23 conserved and 161 putative novel miRNA families. Among the putative miRNA targets, experimental validation demonstrated that a DEF-like MADS-box transcript is cleaved by the homolog of miR5179 of O. italica. The presence of conserved miRNA families in the inflorescence of O. italica indicates that the basic developmental flower regulatory mechanisms mediated by miRNAs are maintained through evolution. Because, according to the “orchid code” theory, DEF-like genes exert a key function in the diversification of tepals and lip, the cleavage-mediated inhibitory activity of miR5179 on a OitaDEF-like transcript suggests that, in orchids, miRNAs play an important role in the diversification of the perianth organs.

Presence, distribution and effect of white, pink and purple morphs on pollination in the orchid Orchis mascula

How floral polymorphism of flowering plants can be maintained in evolutionary time has long intrigued ecologists and is still debated. In particular, how floral colour polymorphism influences reproductive success is still poorly understood. Here, we investigated the case of Orchis mascula, a deceptive orchid species in which the presence of rare white-flowered individuals is known to increase the percentage pollination of co-occurring coloured morphs. In a brief review, we report all the orchid species for which rare colour morphs are recorded and show that colour polymorphism occurs in most orchid genera occurring in France. In this study, more than 20,000 individuals of O. mascula were surveyed and some rare clear pink morphs were recorded. The frequencies of white-flowered and clear pink-flowered individuals were 0.59% and 0.28%, respectively. These two rare-colour flowered individuals were not randomly distributed and restricted to a few populations. In addition, the presence of pink-flowered individuals and the use of experimental pink lures resulted in an increase in the percentage pollination of surrounding purple-flowered individuals, as previously shown for white-flowered individuals and white lures. These new observations favour kin selection as the means by which floral colour polymorphism is maintained in this species. We suggest conducting comparative studies of other species in order to evaluate the importance of this mechanism in orchid pollination and that of other plant families.

The AP2-Like Gene OitaAP2 Is Alternatively Spliced and Differentially Expressed in Inflorescence and Vegetative Tissues of the Orchid Orchis italica

The AP2/ERF proteins are plant-specific transcription factors involved in multiple regulatory pathways, from plant organ development to response to various environmental stresses. One of the mechanisms that regulates the AP2-like genes involves the microRNA miR172, which controls their activity at the post-transcriptional level. Extensive studies on AP2-like genes are available in many different species; however, in orchids, one of the largest plant families, studies are restricted to a few species, all belonging to the Epidendroideae subfamily. In the present study, we report the isolation of an AP2-like gene in the Mediterranean orchid Orchis italica (Orchidoideae). The OitaAP2 locus includes 10 exons and 9 introns, and its transcript is alternatively spliced, resulting in the long OitaAP2 and the short OitaAP2_ISO isoforms, with the latter skipping exon 9. Both isoforms contain the conserved target site for miR172, whose action is demonstrated by the presence of cleaved OitaAP2 mRNA. The OitaAP2 and OitaAP2_ISO mRNAs are present in the tepals and lip before and after anthesis at different expression levels. In addition, the OitaAP2_ISO isoform is expressed in the ovary before pollination and in the root and stem. The isoform-specific expression pattern suggests a functional differentiation of the OitaAP2 alternatively spliced transcripts. The expression profile of miR172 is complementary to that of the OitaAP2 isoforms in inflorescence tissues before anthesis, whereas after anthesis and in ovary tissue before and after pollination, this relationship disappears, suggesting the existence of OitaAP2 inhibitory mechanisms in these tissues that differ from that involving miR172.

The OitaAG and OitaSTK genes of the orchid Orchis italica: a comparative analysis with other C- and D-class MADS-box genes

According to the ABCDE model of flower development, the C- and D- class MADS box genes are involved in the formation of male and female reproductive organs (fused to form the column in orchids) and in ovule maturation (triggered by fertilization in orchids). In the present study, we report the isolation of the Orchis italica genes OitaAG and OitaSTK, homologs of the C-class AGAMOUS and the D-class SEEDSTICK genes of Arabidopsis, respectively. Analysis of their expression profiles reveals high levels of mRNA in columns and ovaries, particularly after pollination. However, weak expression is also detectable in the inner tepals (OitaAG) and the lip and root (OitaSTK). This expression profile is only partially overlapping with those reported in other orchid species and may be the consequence of a different evolutionary history of these functional gene classes in orchids. The genomic characterization of the OitaAG and OitaSTK genes shows that a high number of traces of mobile elements are present in introns and could have contributed to the size expansion of some of them (e.g., intron 2 and 3 of OitaAG and intron 3, 4 and 5 of OitaSTK). Nucleotide sequences of intron 1 of the OitaSTK gene and other STK-like genes do not share regulatory motifs, whereas sequence comparison of intron 2 of the OitaAG gene with that of intron 2 of other AG-like genes reveals, for the first time in an orchid species, the presence of conserved cis-regulatory boxes and binding sites for transcription factors that positively (e.g., LEAFY and WUSCHEL) or negatively (e.g., BELLRINGER) regulate the expression of the AG homologs in dicots and monocots.

Size‐dependent flowering and costs of reproduction affect population dynamics in a tuberous perennial woodland orchid

Summary 1. In woodland herbs, the probability of flowering and costs associated with reproduction may strongly depend on environmental context (shade vs. light habitats) and on plant size. This may be particularly true for tuberous orchids that inhabit woodlands, as the amount of incoming radiation and total leaf area strongly determine photosynthetic capacity and hence the amount of carbohydrates that can be relocated to below‐ground storage organs that form next year’s rosette and flowering stalk. 2. To fully comprehend the impact of size‐dependent reproduction on population dynamics under varying light conditions, life cycle models should therefore include plant size in a continuous manner. In this study, annual changes in plant size and demographic behaviour of the tuberous perennial orchid Orchis purpurea were monitored during seven consecutive years (2003–2009) in open and shaded woodland. Integral projection models (IPMs) and life table response experiments (LTRE) were used to investigate the extent to which variation in plant size affected the overall population dynamics of this species and to decompose differences in population growth rates between populations of open and shaded woodland into contributions from growth, survival and reproduction. 3. Both plants in shaded and light environments needed to be a certain size to initiate flowering, but this threshold size was almost three times as large in shaded environments as in light environments. Plants in open woodlands flowered more frequently over the years, showed less size regression after flowering and produced significantly more fruits than plants in shaded environments, resulting in significantly larger population growth rates. 4. Our life cycle models revealed that costs of reproduction, measured at the population‐level, were small in the light environment, and more than buffered by the increase in survival of flowering plants compared to non‐flowering plants. In the shade environment, however, the costs of reproduction were significant and made the difference between a stable (current) and a growing (without reproduction costs) population. 5. Synthesis. Light penetration to the soil is a key variable determining population dynamics of woodland orchids. Our analyses show that differences in vital rates related to size‐dependent reproduction (flowering) and growth are essential drivers of changes in orchid population dynamics in different light environments. The combination of IPMs and LTREs thus proves to be very useful when studying the fitness consequences of size‐ and context‐dependent phenomena like flowering strategies and costs of reproduction.

Identification of two fungal endophytes associated with the endangered orchid Orchis militaris L.

A survey of the endangered orchid Orchis militaris populations was carried out in north-eastern Italy. The occurrence of fungal root endophytes was investigated by light and electron microscopies and molecular techniques. Two main sites of presence were individuated in the Euganean Hills, differing as to the percentage of flowering individuals and of capsules completing maturity. Fluorescence microscopy revealed an intracellular cortical colonization by hyphal pelotons. Two ITS PCR products co-amplified. Sequencing revealed for the former an identity and a high similarity (99%) with a Tulasnellaceae (Basidiomycota) fungus found within tissues of the same host in independent studies in Hungary and Estonia, suggesting an interesting case of tight specificity throughout the Eurosiberian home range. The second amplicon had 99% similarity with Tetracladium species (Ascomycota) recently demonstrated as potential endophytes. TEM revealed two different hyphal structures. Double fungal colonization appears to occur in Orchis militaris and the possible requirement of a specific fungal partner throws light on the causes of this plant's rarity and threatened status.

Seed limitation restricts population growth in shaded populations of a perennial woodland orchid

Seed production and seedling recruitment are thought to be of minor importance in determining population dynamics and long-term viability in long-lived perennial plants. Seed addition experiments, on the other hand, have amply shown that supplemental addition of seeds almost always, irrespective of longevity, results in increased seedling recruitment. Any change in the environment that affects fruit and seed production can thus be expected to affect seedling recruitment, but the extent to which increased fruit and seed production affect overall population dynamics remains relatively unknown. In this paper, we present demographic data of six populations of the long-lived woodland orchid Orchis purpurea that were monitored for seven consecutive years (2002-2008) occurring in two contrasting light environments. We use a nested life table response experiment (LTRE) at the vital rate level to disentangle the relative contributions of each of six annual transitions, six sites, and two light environments on the population dynamics of this species and to determine vital rate variations that contributed most to variation in population growth rate. Population growth rates (lamda) were significantly higher in the light environment than in the shaded environment (average lamda = 0.9930 and 1.0492 in the shaded and light environment, respectively). The LTRE analysis showed that variation in fecundity and, to a lesser extent, variation in growth made the largest total contributions to variation in lamda, whereas the contributions of variation in survival were almost negligible. Fruit production was two times larger and the net reproductive rate (R0) was approximately six times higher in the light environment than in shaded areas, suggesting that variables related to reproduction are the key drivers of population dynamics of this long-lived orchid species in different light environments. Our results indicate that light is an important factor affecting population dynamics of Orchis purpurea and illustrate that, even in long-lived species, flower and seed production can have important effects on the population dynamics.

The microscopic fungi of orchid species in the Őrség National Park

The wild orchids growing in Hungary are some of the most decorative and interesting members of the country's flora. The majority of species are rarely spotted, and some are only found in very few habitats, though others are quite common .All the species known in Hungary are protected, and 1 1 species are strictly protected. Itis thus important to monitor the health status of these plants, to determine what diseases affect them, what pathogens are found on them and how severely they are infected, and to take the necessary precautionary measures. Eleven of the 14 orchid species occurring in the 6rseg National Park were included in the study and the presence of microscopic fungi was detected on eight of these. Pathogenic species were found on lesser butterfly orchid (Platanthera bifolia), green-winged orchid (Orchis morio), burnt orchid (Orchis ustulara), sword-leaved helleborine (Cephalanthera longifolia), common twayblade (Listera ovata), autumn lady's-tresses (Spiranthes spiralis), western marsh orchid (Dacrylorhiza majalis) and broad-leaved helleborine (Epipactis helleborine). A detailed account is given of the symptoms of major diseases and of the microscopic traits of the pathogens. An attempt was made to determine to what extent the pathogenic fungal species found on protected orchid species influence the lives of these plants. Current knowledge on this subject is extremely deficient, as practically no data are available from Hungary.

Effects of population size and forest management on genetic diversity and structure of the tuberous orchid Orchis mascula

Due to societal changes and altered demands for firewood, the traditional forest management of coppicing has been largely abandoned. As a result, many forest herbs that are specifically adapted to regular opening of the canopy, have suffered significant declines in abundance, and the remaining populations of these species often tend to be small and isolated. Reduced population sizes and pronounced spatial isolation may cause loss of within-population genetic diversity and increased between-population differentiation through random genetic drift and inbreeding. In this study, we investigated genetic diversity and genetic structure of 15 populations of the food-deceptive orchid Orchis mascula using AFLP markers. Within-population genetic diversity significantly increased with increasing population size, indicating genetic impoverishment in small populations. Genetic differentiation, on the other hand, was rather low (ΦST = 0.083) and there was no significant relationship between genetic and geographic distances, suggesting substantial gene flow within the study area. However, strong differences in levels of within-population diversity and among-population differentiation were found for populations located in forests that have been regularly coppiced and populations found in forests that were neglected for more than 50 years and that were totally overgrown by shrubs. Our data thus indicate that a lack of coppicing leads to decreased genetic diversity and increased differentiation in this orchid species, most likely as a result of genetic drift following demographic bottlenecks. From a conservation point of view, this study combined with previous results on the demography of O. mascula in relation to forest management illustrates the importance of coppicing in maintaining viable populations of forest herbs in the long-term.

Fitness variation and genetic diversity in small, remnant populations of the food deceptive orchid Orchis purpurea

In much of Western Europe, orchid species have suffered dramatic declines in abundance, whereas the remaining populations often tend to be small and isolated, occur in ecologically marginal habitats and may show decreased reproductive output due to altered pollinator interactions or the absence of specialized pollinators. Furthermore, small and isolated populations are expected to suffer from genetic erosion and increasing genetic divergence among populations, through the effects of random genetic drift, increased levels of inbreeding and reduced gene flow, potentially leading to reduced possibilities of population recovery in the future. In this study, genetic diversity and fitness variation were studied in nine fragmented populations of the food deceptive orchid Orchis purpurea. Within-population genetic diversity and among-population differentiation were investigated using dominant AFLP markers. All studied populations were relatively small (range 12–302; mean: 90 individuals/population). Despite their small size, genetic diversity within populations was rather high (mean gene diversity H j: 0.21, range: 0.15–0.27) and genetic differentiation among populations was not higher than that typically observed in deceptive orchids ( F ST = 0.09). Nevertheless, Mantel tests showed a positive correlation between geographical and genetic distances indicating limited gene flow among populations. Fruit set, on the other hand, was very low (average fruit set: 5.5%), suggesting strong pollinator limitation, which, in turn, resulted in very low recruitment rates (average seedling recruitment per flowering individual: 0.12). Both measures significantly increased with increasing population size, indicating that a certain threshold value (>50 flowering individuals) has to be reached to produce a sizeable number of fruits and seedlings. These results suggest that although deceptive pollination clearly results in a fitness cost, it may serve as an effective means to maintain high gene diversity within and to counter high genetic differentiation among small orchid populations. From a genetic point of view, our results further suggest that small populations of orchid species should not be neglected as they may harbour as much genetic diversity as large populations.

A spatially explicit analysis of seedling recruitment in the terrestrial orchid Orchis purpurea

Seed dispersal and the subsequent recruitment of new individuals into a population are important processes affecting the population dynamics, genetic diversity and spatial genetic structure of plant populations. Spatial patterns of seedling recruitment were investigated in two populations of the terrestrial orchid Orchis purpurea using both univariate and bivariate point pattern analysis, parentage analysis and seed germination experiments. Both adults and recruits showed a clustered spatial distribution with cluster radii of c. 4-5 m. The parentage analysis resulted in offspring-dispersal distances that were slightly larger than distances obtained from the point pattern analyses. The suitability of microsites for germination differed among sites, with strong constraints in one site and almost no constraints in the other. These results provide a clear and coherent picture of recruitment patterns in a tuberous, perennial orchid. Seed dispersal is limited to a few metres from the mother plant, whereas the availability of suitable germination conditions may vary strongly from one site to the next. Because of a time lag of 3-4 yr between seed dispersal and actual recruitment, and irregular flowering and fruiting patterns of adult plants, interpretation of recruitment patterns using point patterns analyses ideally should take into account the demographic properties of orchid populations.

Effects of coppicing on demographic structure, fruit and seed set in Orchis mascula

Summary Opening of the forest canopy may result in higher fitness of understory plants due to increased light intensity on the forest soil and increased nutrient availability. Especially, tuberous orchids may profit from increased light as photosynthetic assimilation products and nutrients are accumulated in their belowground storage organs. We investigated the effects of coppicing on demographic structure and fruit and seed set in 15 populations of the tuberous, perennial orchid Orchis mascula , seven of which were located in undisturbed, shaded woodland and eight in recently coppiced woodland. Coppicing resulted in a massive increase in flowering and in increased fruit set. On average, 42.9% of all individuals consisted of flowering plants in coppiced woodland, whereas in undisturbed woodland only 20.8% of all individuals flowered. The percentage fruit set varied between 20% and 55% in coppiced woodland and was strongly related to population size (measured as the number of flowering individuals). Mean fruit weight and fruit size were also significantly higher in populations located in coppiced woodland than in undisturbed woodland, whereas the proportion of viable seeds was not related to woodland type. Given that O. mascula is dependent on seed set to fill space with new individuals, these results suggest that the long-term survival of this species strongly depends on frequent opening of the canopy.

Fine‐scale genetic structure of life history stages in the food‐deceptive orchid Orchis purpurea

In natural plant populations, fine-scale spatial genetic structure can result from limited gene flow, selection pressures or historical events, but the role of each factor is in general hard to discern. One way to investigate the origination of spatial genetic structure within a plant population consists of comparing spatial genetic structure among different life history stages. In this study, spatial genetic structure of the food-deceptive orchid Orchis purpurea was determined across life history stages in two populations that were regenerating after many years of population decline. Based on demographic analyses (2001-2004), we distinguished between recruits and adult plants. For both sites, there was no difference in the proportion of polymorphic loci and expected heterozygosity between life history stages. However, spatial autocorrelation analyses showed that spatial genetic structure increased in magnitude with life history stage. Weak or no spatial genetic structure was observed for recruits, whereas adult plants showed a pattern that is consistent with that found in other species with a predominantly outcrossing mating system. The observed differences between seedlings and adults are probably a consequence of changes in management of the two study sites and associated demographic changes in both populations. Our results illustrate that recurrent population crashes and recovery may strongly affect genetic diversity and fine-scale spatial genetic structure of plant populations.