Microsatellites reveal regional population differentiation and isolation in Lobaria pulmonaria, an epiphytic lichen

Many lichen species produce both sexual and asexual propagules, but, aside from being minute, these diaspores lack special adaptations for long-distance dispersal. So far, molecular studies have not directly addressed isolation and genetic differentiation of lichen populations, both being affected by gene flow, at a regional scale. We used six mycobiont-specific microsatellite loci to investigate the population genetic structure of the epiphytic lichen Lobaria pulmonaria in two regions that strongly differed with respect to anthropogenic impact. In British Columbia, L. pulmonaria grows in continuous old-growth forests, while its populations in the old cultural landscape of Switzerland are comparably small and fragmented. Populations from both British Columbia and Switzerland were genetically diverse at the loci. Geographically restricted alleles, low historical gene flow, and analyses of genetic distance (upgma tree) and of differentiation (amova) indicated that populations from Vancouver Island and from the Canadian mainland were separated from each other, except for one, geographically intermediate population. This differentiation was attributed to different glacial and postglacial histories of coastal and inland populations in British Columbia. In contrast to expectations, the three investigated Swiss populations were genetically neither isolated nor differentiated from each other despite the long-lasting negative human impact on the lichen's range size in Central Europe. We propose that detailed studies integrating local landscape and regional scales are now needed to understand the processes of dispersal and gene flow in lichens.     

Fungus-specific microsatellite primers of lichens: application for the assessment of genetic variation on different spatial scales in Lobaria pulmonaria

We isolated 12 microsatellite loci for the epiphytic lichen-forming ascomycete Lobaria pulmonaria and studied their patterns of variation within and among populations from Canada and Switzerland. Even though several microsatellites exhibited high levels of variability at different spatial scales, we did not find any evidence for intrathalline variation. Most of the genetic variation was attributed to differences among individuals within populations. High genetic variation was also detected among L. pulmonaria samples taken from individual trees, suggesting that either multiple colonization events had occurred or that local recombination is frequent. The geographically structured distribution of alleles from several microsatellites indicated that L. pulmonaria from Canada and Switzerland represent two distinct evolutionary lineages. The potential to identify multiple alleles, and their transferability to closely related species, make microsatellites an ideal tool to study dispersal, population differentiation, and microevolution in lichens.     

Exploring symbiont management in lichens

Lichens are unique among fungal symbioses in that their mycelial structures are compact and exposed to the light as thallus structures. The myriad intersections of unique fungal species with photosynthetic partner organisms (green algae in 90% of lichens) produce a wide variety of diverse shapes and colours of the fully synthesized lichen thallus when growing in nature. This characteristic complex morphology is, however, not achieved in the fungal axenic state. Even under ideal environmental conditions, the lichen life cycle faces considerable odds: first, meiotic spores are only produced on well-established thalli and often only after achieving considerable age in a stable environment, and second, even then in vivo resynthesis requires the presence of compatible algal strains where fungal spores germinate. Many lichen species have evolved a way around the resynthesis bottleneck by producing asexual propagules for joint propagation of symbionts. These different dispersal strategies ostensibly shape the population genetic structure of lichen symbioses, but the relative contributions of vertical (joint) and horizontal (independent) symbiont transmission have long eluded lichen evolutionary biologists. In this issue of Molecular Ecology, Dal Grande et al. (2012) close in on this question with the lung lichen, Lobaria pulmonaria, a flagship species in the conservation of old growth forests. By capitalizing on available microsatellite markers for both fungal and algal symbionts, they show that while vertical transmission is the predominant mode of reproduction, horizontal transmission is demonstrable and actively shapes population genetic structure. The resulting mixed propagation system is a highly successful balance of safe recruitment of symbiotic clones and endless possibilities for fungal recombination and symbiont shuffling.     

Genetic structure in a fragmented Northern Hemisphere rainforest: large effective sizes and high connectivity among populations of the epiphytic lichen Lobaria pulmonaria

An extraordinary diversity of epiphytic lichens is found in the boreal rainforest of central Norway, the highest-latitude rainforest in the world. These rainforest relicts are located in ravine systems, and clear cutting has increased the distance between remaining patches. We hypothesized that the relatively small lichen populations in the remaining forest stands have suffered a depletion of genetic diversity through bottlenecks and founder events. To test this hypothesis, we assessed genetic diversity and structure in the populations of the tripartite lichen Lobaria pulmonaria using eight SSR loci. We sampled thalli growing on Picea abies branches and propagules deposited in snow at three localities. Contrary to expectations, we found high genetic diversity in lichen and snow samples, and high effective sizes of the studied populations. Also, limited genetic differentiation between populations, high historical migration rates, and a high proportion of first generation immigrants were estimated, implying high connectivity across distances <30km. Almost all genetic variation was attributed to variation within sites; spatial genetic structures within populations were absent or appeared on small scales (5-10m). The high genetic diversity in the remaining old boreal rainforests shows that even relict forest patches might be suitable for conservation of genetic diversity.     

Vertical and horizontal photobiont transmission within populations of a lichen symbiosis

Lichens are widespread symbioses and play important roles in many terrestrial ecosystems. The genetic structure of lichens is the result of the association between fungal and algal populations constituting the lichen thallus. Using eight fungus- and seven alga-specific highly variable microsatellite markers on within-population spatial genetic data from 62 replicate populations across Europe, North America, Asia and Africa, we investigated the contributions of vertical and horizontal transmission of the photobiont to the genetic structure of the epiphytic lichen Lobaria pulmonaria. Based on pairwise comparisons of multilocus genotypes defined separately for the mycobiont and for the photobiont, we inferred the transmission mode of the photobiont and the relative contribution of somatic mutation and recombination. After constraining the analysis of one symbiont to pairs of individuals with genetically identical symbiotic partners, we found that 77% of fungal and 70% of algal pairs were represented by clones. Thus, the predominant dispersal mode was by means of symbiotic vegetative propagules (vertical transmission), which dispersed fungal and algal clones co-dependently over a short distance, thus shaping the spatial genetic structure up to distances of 20m. Evidence for somatic mutation generating genetic diversity was found in both symbionts, accounting for 30% of pairwise comparisons in the alga and 15% in the fungus. While the alga did not show statistically significant evidence of recombination, recombination accounted for 7.7% of fungal pairs with identical algae. This implies that, even in a mostly vegetatively reproducing species, horizontal transmission plays a role in shaping the symbiotic association, as shown in many coral and other symbioses in nature.     

Landscape-level gene flow in Lobaria pulmonaria, an epiphytic lichen

Epiphytes are strongly affected by the population dynamics of their host trees. Owing to the spatio-temporal dynamics of host tree populations, substantial dispersal rates--corresponding to high levels of gene flow--are needed for populations to persist in a landscape. However, several epiphytic lichens have been suggested to be dispersal-limited, which leads to the expectation of low gene flow at the landscape scale. Here, we study landscape-level genetic structure and gene flow of a putatively dispersal-limited epiphytic lichen, Lobaria pulmonaria. The genetic structure of L. pulmonaria was quantified at three hierarchical levels, based on 923 thalli collected from 41 plots situated within a pasture-woodland landscape and genotyped at six fungal microsatellite loci. We found significant isolation by distance, and significant genetic differentiation both among sampling plots and among trees. Landscape configuration, i.e. the effect of a large open area separating two forested regions, did not leave a traceable pattern in genetic structure, as assessed with partial Mantel tests and analysis of molecular variance. Gene pools were spatially intermingled in the pasture-woodland landscape, as determined by Bayesian analysis of population structure. Evidence for local gene flow was found in a disturbed area that was mainly colonized from nearby sources. Our analyses indicated high rates of gene flow of L. pulmonaria among forest patches, which may reflect the historical connectedness of the landscape through gene movement. These results support the conclusion that dispersal in L. pulmonaria is rather effective, but not spatially unrestricted.     

Molecular evidence for limited dispersal of vegetative propagules in the epiphytic lichen Lobaria pulmonaria

Propagation, whether sexual or asexual, is a fundamental step in the life cycle of every organism. In lichenized fungi, a great variety of vegetative propagules have evolved in order for the symbiotic partners to disperse simultaneously. For lichens with the ability of sexual and asexual reproduction, the relative contribution of vegetative dispersal is unknown but could, nonetheless, be inferred by studying genotype distribution. The genetic structure of three Lobaria pulmonaria (Lobariaceae) populations from Switzerland was investigated based on the observed variation at six microsatellite loci. All three populations had a clustered distribution of identical genotypes at small spatial scales. The maximum distance between identical genotypes was 230 m. At a distance of 350 m from a source tree, seemingly suitable habitat patches were too far apart to be colonized. Some multilocus genotypes were frequent within local populations but no genotypes were shared among populations. The restricted occurrences of common genotypes as well as the clustered distributions are evidence for a limited dispersal of vegetative propagules in L. pulmonaria. Gene flow among isolated populations will ultimately depend on the capacity of long-distance dispersal and thus probably depend on sexual reproduction.     

Lichen endozoochory by snails

Endozoochory plays a prominent role for the dispersal of seed plants. However, for most other plant taxa it is not known whether this mode of dispersal occurs at all. Among those other taxa, lichens as symbiotic associations of algae and fungi are peculiar as their successful dispersal requires movement of propagules that leaves the symbiosis functional. However, the potential for endozoochorous dispersal of lichen fragments has been completely overlooked. We fed sterile thalli of two foliose lichen species (Lobaria pulmonaria and Physcia adscendens) differing in habitat and air-quality requirements to nine snail species common in temperate Europe. We demonstrated morphologically that L. pulmonaria regenerated from 29.0% of all 379 fecal pellets, whereas P. adscendens regenerated from 40.9% of all 433 fecal pellets, showing that lichen fragments survived gut passage of all snail species. Moreover, molecular analysis of regenerated lichens confirmed the species identity for a subset of samples. Regeneration rates were higher for the generalist lichen species P. adscendens than for the specialist lichen species L. pulmonaria. Furthermore, lichen regeneration rates varied among snail species with higher rates after gut passage of heavier snail species. We suggest that gastropods generally grazing on lichen communities are important, but so far completely overlooked, as vectors for lichen dispersal. This opens new ecological perspectives and questions the traditional view of an entirely antagonistic relationship between gastropods and lichens.     

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.     

Local genetic structure in a North American epiphytic lichen, Ramalina menziesii (Ramalinaceae)

Epiphytic lichens possess unique life history traits that can have conflicting effects on genetic structure: symbiotic mutualism between a fungus with its algal or cyanobacterial photobiont, association with a host plant, and ability to reproduce sexually and asexually. Our study species, Ramalina menziesii, has small ascospores that can facilitate long-distance gene movement, and it is capable of clonal reproduction. The goals of this study are to test whether different haplotypes were differentially distributed across host plant species, to look for evidence of asexual vs. sexual reproduction, and to assess the local genetic structure of the population. We sampled individuals from multiple trees of three oak species in four lichen subpopulations within a savanna ecosystem. Using DNA sequence data from four fungal nuclear loci, we found no tendency for host specialization. Alleles were randomly distributed across subpopulations. The frequency of multilocus genotypes was consistent with a randomly mating population. Sexual reproduction involving relichenization appeared to be the predominant mode of reproduction of R. menziesii at this study site. We found no significant local genetic structure suggesting widespread gene flow at the local scale. The genetic structure of this lichen is comparable to that of widely distributed epiphytic plants.     

Variogram analysis of the spatial genetic structure of continuous populations using multilocus microsatellite data

A geostatistical perspective on spatial genetic structure may explain methodological issues of quantifying spatial genetic structure and suggest new approaches to addressing them. We use a variogram approach to (i) derive a spatial partitioning of molecular variance, gene diversity, and genotypic diversity for microsatellite data under the infinite allele model (IAM) and the stepwise mutation model (SMM), (ii) develop a weighting of sampling units to reflect ploidy levels or multiple sampling of genets, and (iii) show how variograms summarize the spatial genetic structure within a population under isolation-by-distance. The methods are illustrated with data from a population of the epiphytic lichen Lobaria pulmonaria, using six microsatellite markers. Variogram-based analysis not only avoids bias due to the underestimation of population variance in the presence of spatial autocorrelation, but also provides estimates of population genetic diversity and the degree and extent of spatial genetic structure accounting for autocorrelation.     

SEXUAL PARTNERS FOR THE STRESSED: FACULTATIVE OUTCROSSING IN THE SELF-FERTILIZING NEMATODE CAENORHABDITIS ELEGANS

Sexual reproduction shuffles genetic variation, potentially enhancing the evolutionary response to environmental change. Many asexual organisms respond to stress by generating facultative sexual reproduction, presumably as a means of escaping the trap of low genetic diversity. Self-fertilizing organisms are subject to similar genetic limitations: the consistent loss of genetic diversity within lineages restricts the production of variation through recombination. Selfing organisms may therefore benefit from a similar shift in mating strategy during periods of stress. We determined the effects of environmental stress via starvation and passage through the stress-resistant dauer stage on mating system dynamics of Caenorhabditis elegans , which reproduces predominantly through self-fertilization but is capable of outcrossing in the presence of males. Starvation elevated male frequencies in a strain-specific manner through differential male survival during dauer exposure and increased outcrossing rates after dauer exposure. In the most responsive strain, the mating system changed from predominantly selfing to almost exclusively outcrossing. Like facultative sex in asexual organisms, facultative outcrossing in C. elegans may periodically facilitate adaptation under stress. Such a shift in reproductive strategy should have a major impact on evolutionary change within these populations and may be a previously unrecognized feature of other highly selfing organisms.     

How multilocus genotypic pattern helps to understand the history of selfing populations: a case study in Medicago truncatula

The occurrence of populations exhibiting high genetic diversity in predominantly selfing species remains a puzzling question, since under regular selfing genetic diversity is expected to be depleted at a faster rate than under outcrossing. Fine-scale population genetics approaches may help to answer this question. Here we study a natural population of the legume Medicago truncatula in which both the fine-scale spatial structure and the selfing rate are characterized using three different methods. Selfing rate estimates were very high ( approximately 99%) irrespective of the method used. A clear pattern of isolation by distance reflecting small seed dispersal distances was detected. Combining genotypic data over loci, we could define 34 multilocus genotypes. Among those, six highly inbred genotypes (lines) represented more than 75% of the individuals studied and harboured all the allelic variation present in the population. We also detected a large set of multilocus genotypes resembling recombinant inbred lines between the most frequent lines occurring in the population. This finding illustrates the importance of rare recombination in redistributing available allelic diversity into new genotypic combinations. This study shows how multilocus and fine-scale spatial analyses may help to understand the population history of self-fertilizing species, especially to make inferences about the relative role of foundation/migration and recombination events in such populations.     

Genetic variation within and among populations of the threatened lichen Lobaria pulmonaria in Switzerland and implications for its conservation

The foliose epiphytic lichen Lobaria pulmonaria has suffered a significant decline in European lowlands during the last decades and therefore is considered as endangered throughout Europe. An assessment of the genetic variability is necessary to formulate biologically sound conservation recommendations for this species. We investigated the genetic diversity of the fungal symbiont of L. pulmonaria using 143 specimens sampled from six populations (two small, one medium, three large) in the lowland, the Jura Mountains, the pre-Alps and the Alps of Switzerland. Among all nuclear and mitochondrial regions sequenced for this study, variability was found only in the internal transcribed spacer (ITS I), with three polymorphic sites, and in the nuclear ribosomal large subunit (nrLSU), with four polymorphic sites. The variable sites in the nrLSU are all located within a putative spliceosomal intron. We sequenced these two regions for 81 specimens and detected six genotypes. Two genotypes were common, two were found only in the more diverse populations and two were found only in one population each. There was no correlation between population size and genetic diversity. The highest genetic diversity was found in populations where the fungal symbiont is reproducing sexually. Populations with low genetic diversity included only the two same common genotypes. Our study provides evidence suggesting that L. pulmonaria is self-incompatible and heterothallic. Based on our results we give populations with sexually reproducing individuals a higher rank in terms of conservation priority than strictly asexual populations. The remaining lowland populations are so small, that one single catastrophic event such as a windthrow might destroy the entire population. Hence we suggest augmenting such populations in size and genetic diversity using small thallus fragments or vegetative diaspores collected in other populations. As we did not detect any locally adapted genotypes, these transplants can be taken from any other genetically diverse population in Switzerland.     

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.     

Growth in relation to microclimatic conditions and physiological characteristics of four Lobaria pulmonaria populations in two contrasting habitats

The aim of the present study was to compare the physiological characteristics of various populations of the lichen Lobaria pulmonaria in Portugal and Sweden. For this, indirect markers of algal (photobiont) and fungal (mycobiont) activity were measured, as well as their CO₂ gasexchange characteristics. Microclimatic conditions and the lichens growth performance in the two countries were compared using reciprocal transplantation. Two populations of L. pulmonaria represented each country: one collected from forest interior conditions and one from forest edge habitats. A non-transplanted "wild" population was also studied in each country, in order to evaluate any transplantation effects per se. The main hypothesis were that; 1) growth should be faster in Portugal due to higher light availability; 2) the energy use efficiency of lichen biomass gain should be similar for the native populations in their respective native habitat; 3) if the lichens were able to adapt to the environmental conditions in the foreign habitat this should be revealed as similar growth rates among all thalli transplanted at the same site, regardless of their origin. Physiologically, the Portuguese and Swedish populations were very similar, both concerning their CO₂ gas exchange characteristics and distribution of resources between photo- and mycobiont tissue. Environmental conditions were more advantageous for L. pulmonaria growth in Portugal, i.e. higher photon flux densities and ambient temperatures when the lichens were wet and active, and a lower fraction of the active time occurring in darkness. However, despite similar physiological characteristics of all the studied populations, the Swedish lichens were not able to grow as well in Portugal as the native, while all populations had similarly low growth rates in Sweden.     

Remnants fragments preserve genetic diversity of the old forest lichen <Emphasis Type="Italic">Lobaria pulmonaria</Emphasis> in a fragmented Mediterranean mountain forest

Fragmentation represents a serious threat to biodiversity worldwide, however its effects on epiphytic organisms is still poorly understood. We study the effect of habitat fragmentation on the genetic population structure and diversity of the red-listed epiphytic lichen, Lobaria pulmonaria, in a Mediterranean forest landscape. We tested the relative importance of forest patch quality, matrix surrounding fragments and connectivity on the genetic variation within populations and the differentiation among them. A total of 855 thalli were sampled in 44 plots (400 m²) of 31 suitable forest fragments (beeches and oaks), in the Sierra de Ayllón in central Spain. Variables related to landscape attributes of the remnant forest patches such as size and connectivity and also the nature of the matrix or tree species had no significant effects on the genetic diversity of L. pulmonaria. Values of genetic diversity (Nei’s) were only affected by habitat quality estimated as the age patches. Most of the variation (76%) in all populations was observed at the smallest sampled unit (plots). Using multiple regression analysis, we found that habitat quality is more important in explaining the genetic structure of the L. pulmonaria populations than spatial distance. The relatively high level of genetic diversity of the species in old forest patches regardless of patch size indicates that habitat quality in a highly structured forest stand determines the population size and distribution pattern of this species and its associated lichen community. Thus, conservation programmes of Mediterranean mountain forests have to prioritize area and habitat quality of old forest patches.     

Hidden genetic variance contributes to increase the short‐term adaptive potential of selfing populations

Standing genetic variation is considered a major contributor to the adaptive potential of species. The low heritable genetic variation observed in self‐fertilizing populations has led to the hypothesis that species with this mating system would be less likely to adapt. However, a non‐negligible amount of cryptic genetic variation for polygenic traits, accumulated through negative linkage disequilibrium, could prove to be an important source of standing variation in self‐fertilizing species. To test this hypothesis, we simulated populations under stabilizing selection subjected to an environmental change. We demonstrate that, when the mutation rate is high (but realistic), selfing populations are better able to store genetic variance than outcrossing populations through genetic associations, notably due to the reduced effective recombination rate associated with predominant selfing. Following an environmental shift, this diversity can be partially remobilized, which increases the additive variance and adaptive potential of predominantly (but not completely) selfing populations. In such conditions, despite initially lower observed genetic variance, selfing populations adapt as readily as outcrossing ones within a few generations. For low mutation rates, purifying selection impedes the storage of diversity through genetic associations, in which case, as previously predicted, the lower genetic variance of selfing populations results in lower adaptability compared to their outcrossing counterparts. The population size and the mutation rate are the main parameters to consider, as they are the best predictors of the amount of stored diversity in selfing populations. Our results and their impact on our knowledge of adaptation under high selfing rates are discussed.     

Genetic diversity and geographic differentiation in Tacca chantrieri (Taccaceae): an autonomous selfing plant with showy floral display

BACKGROUND AND AIMS: Despite considerable investment in elaborate floral displays, Tacca chantrieri populations are predominantly selfing. It is hypothesized that this species might possess considerable spatial or temporal variation in outcrossing rates among populations. To test this hypothesis, genetic variability and genetic differentiation within and among T. chantrieri populations were investigated to find out if they are in agreement with expectations based on a predominantly inbred mating system. METHODS: Genetic diversity was quantified using inter-simple sequence repeats (ISSR) in 303 individuals from 13 populations taken from known locations of T. chantrieri in China, and from one population in Thailand. KEY RESULTS: Of the 113 primers screened, 24 produced highly reproducible ISSR bands. Using these primers, 160 discernible DNA fragments were generated, of which 145 (90.62 %) were polymorphic. This indicated considerable genetic variation at the species level. However, there were relatively low levels of polymorphism at population levels, with percentages of polymorphic bands (PPB) ranging from 8.75 % to 55 %. A high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: G(ST) = 0.5835; AMOVA analysis: F(ST) = 0.6989). Furthermore, based on levels of genetic differentiation, the 14 populations clustered into two distinct groups separated by the Tanaka Line. CONCLUSIONS: High levels of differentiation among populations and low levels of diversity within populations at large spatial scales are consistent with earlier small-scale studies of mating patterns detected by allozymes which showed that T. chantrieri populations are predominantly selfing. However, it appears that T. chantrieri has a mixed-mating system in which self-fertilization predominates, but there is occasional outcrossing. Significant genetic differences between the two distinct regions might be attributed to vicariance along the Tanaka Line. Finally, possible mechanisms of geographic patterns based on genetic differentiation of T. chantrieri are discussed.