First insight into the genomes of the Pulmonaria officinalis group (Boraginaceae) provided by repeatome analysis and comparative karyotyping.

BACKGROUND: The genus Pulmonaria (Boraginaceae) represents a taxonomically complex group of species in which morphological similarity contrasts with striking karyological variation. The presence of different numbers of chromosomes in the diploid state suggests multiple hybridization/polyploidization events followed by chromosome rearrangements (dysploidy). Unfortunately, the phylogenetic relationships and evolution of the genome, have not yet been elucidated. Our study focused on the P. officinalis group, the most widespread species complex, which includes two morphologically similar species that differ in chromosome number, i.e. P. obscura (2n = 14) and P. officinalis (2n = 16). Ornamental cultivars, morphologically similar to P. officinalis (garden escapes), whose origin is unclear, were also studied. Here, we present a pilot study on genome size and repeatome dynamics of these closely related species in order to gain new information on their genome and chromosome structure. RESULTS: Flow cytometry confirmed a significant difference in genome size between P. obscura and P. officinalis, corresponding to the number of chromosomes. Genome-wide repeatome analysis performed on genome skimming data showed that retrotransposons were the most abundant repeat type, with a higher proportion of Ty3/Gypsy elements, mainly represented by the Tekay lineage. Comparative analysis revealed no species-specific retrotransposons or striking differences in their copy number between the species. A new set of chromosome-specific cytogenetic markers, represented by satellite DNAs, showed that the chromosome structure in P. officinalis was more variable compared to that of P. obscura. Comparative karyotyping supported the hybrid origin of putative hybrids with 2n = 15 collected from a mixed population of both species and outlined the origin of ornamental garden escapes, presumably derived from the P. officinalis complex. CONCLUSIONS: Large-scale genome size analysis and repeatome characterization of the two morphologically similar species of the P. officinalis group improved our knowledge of the genome dynamics and differences in the karyotype structure. A new set of chromosome-specific cytogenetic landmarks was identified and used to reveal the origin of putative hybrids and ornamental cultivars morphologically similar to P. officinalis.

Evolutionary trends in the distylous genus Pulmonaria (Boraginaceae): Evidence of ancient hybridization and current interspecific gene flow.

The distylous genus Pulmonaria contains approximately 18 species that are widely distributed across Eurasia. Previous studies have shown that species delimitation in the genus is problematic, but have not yet explored the evolutionary history of the genus. Premating reproductive barriers between European species appear to be weak, as several species have strongly overlapping distribution areas, flower at the same time and share the same pollinators, suggesting that hybridization may have contributed to the evolutionary history of Pulmonaria. To test this hypothesis, phylogenetic analyses of nuclear ITS and plastid data (rps16, trnH-psbA, rpl16) from 48 allopatric and four sympatric populations were performed to (1) provide a molecular phylogeny for nine of the most common Pulmonaria species in Europe, (2) detect current and ancient hybridization events, and (3) assess the contribution of hybridization versus incomplete lineage sorting to the inferred phylogenetic patterns. Our results showed that gene trees displayed widespread, strongly supported incongruence associated with the conflicting position of hybrid samples rather than incomplete lineage sorting. Evidence was found of different degrees of hybridization, ranging from current interspecific gene flow at secondary contact zones to introgression at the population level and at least one event of hybrid speciation. Overall, these results suggest that hybridization and introgression were - and could still be - important processes affecting speciation in the genus Pulmonaria.

Differences in fine-scale spatial genetic structure across the distribution range of the distylous forest herb Pulmonaria officinalis (Boraginaceae).

BACKGROUND: Geographical ranges of plants and their pollinators do not always entirely overlap and it has been suggested that the absence of specialized pollinators at range margins may induce changes in mating systems. Because a species' mating system is known to have a considerable effect on within-population pollen movement, the extent of fine-scale spatial genetic structure (SGS) can be expected to differ between populations located at different parts of their geographical range. To test this prediction, we compared the fine-scale SGS between two core and two disjunct populations of the distylous forest herb Pulmonaria officinalis. Because in disjunct populations of this species the heteromorphic self-incompatibility system showed relaxation in the long-styled morph, but not in the short-styled morph, we also hypothesized that the extent of fine-scale SGS and clustering differed between morphs. RESULTS: Spatial autocorrelation analyses showed a significant decrease in genetic relatedness with spatial distance for both core and disjunct populations with the weakest SGS found in one of the core populations (Sp = 0.0014). No evidence of stronger SGS in the long-styled morph was found in the center of the range whereas one disjunct population showed a significantly (P = 0.029) higher SGS in the long-styled morph (SpL = 0.0070) than in the short-styled morph (SpS = 0.0044). CONCLUSIONS: Consistent with previous analyses on distylous plant species, we found weak, but significant spatial genetic structure. However, the extent of SGS varied substantially between populations within regions, suggesting that population characteristics other than mating system (e.g. local pollinator assemblages, population history) may be as important in determining variation in SGS.

Strong differences in genetic structure across disjunct, edge, and core populations of the distylous forest herb Pulmonaria officinalis (Boraginaceae).

PREMISE OF THE STUDY: Populations at the edge of a species' distribution area are often small and have low levels of gene flow resulting in lower genetic variation and higher differentiation compared to core populations. This study examined genetic variation among populations of the distylous temperate forest herb Pulmonaria officinalis located in the core, the edge, and outside the species' main distribution range. METHODS: We compared patterns of genetic variation for eight microsatellite loci between disjunct (Belgium), edge (western Germany), and core (eastern Germany) populations of P. officinalis. KEY RESULTS: Disjunct populations contained only a subset of alleles found in edge and core populations and had significantly lower within-population genetic variation. No significant differences, however, in within-population genetic variation were found between edge and core populations, except for allelic and genotypic richness. Genetic differentiation was highest among disjunct (F(ST) = 0.11) and lowest among core populations (F(ST) = 0.03). Significant (P < 0.01) isolation by distance was found for disjunct and edge populations (r(M) = 0.29 and 0.50, respectively), but not for core populations (r(M) = 0.18). CONCLUSIONS: The results are best interpreted from a "dynamic range" point of view in which the observed low levels of genetic diversity and high genetic differentiation in disjunct populations are best explained through historical processes, most likely the introduction of the species in medieval times. Lower levels of gene flow caused by the pronounced fragmentation of forests in Belgium may further have contributed to the genetic structure of P. officinalis in these disjunct populations.

Biased morph ratios and skewed mating success contribute to loss of genetic diversity in the distylous Pulmonaria officinalis.

BACKGROUND AND AIMS: In heterostylous plant species, skewed morph ratios are not uncommon and may arise from a range of factors. Despite the recognized importance of skewed morph ratios on overall reproductive success within populations, little is known about the impact of skewed morph ratios on population genetic diversity and differentiation in heterostylous species. This study specifically aimed to clarify the effect of population size and morph bias on population genetic diversity and differentiation in the temperate forest herb Pulmonaria officinalis. This species is characterized by a distylous breeding system and shows morph-specific differences in reproductive success. METHODS: Genetic diversity was determined for 27 P. officinalis populations in northern Belgium by using eight recently developed microsatellite markers. Multiple regressions were used to assess the relationship between genetic diversity, morph bias and population size, and F(ST)-values were calculated for short- and long-styled morphs separately to study genetic differentiation as a function of morph type. KEY RESULTS: For all genetic measures used, morph bias was more important in explaining patterns of genetic diversity than population size, and in all cases patterns of population genetic diversity followed a quadratic function, which showed a symmetrical decrease in genetic diversity with increasing morph bias. However, probably due to the reproductive advantage of L-morphs relative to S-morphs, maximum genetic diversity was found in populations showing an excess of L-morphs (60·7 % L-morph). On the other hand, no significant difference in pairwise genetic distances between populations was observed between L- (0·107) and S-morphs (0·106). CONCLUSIONS: Our results indicate that significant deviations from equal morph ratios not only affect plant reproductive success but also population genetic diversity of heterostylous plant species. Hence, when defining conservation measures for populations of heterostylous plant species, morph ratios should be considered as an important trait affecting their long-term population viability.

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.