Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species.

BACKGROUND: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. RESULTS: Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. CONCLUSIONS: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection.

Shades of white: The Petunia long corolla tube clade evolutionary history.

Delimiting species is challenging in recently diverged species, and adaptive radiation is fundamental to understanding the evolutionary processes because it requires multiple ecological opportunities associated with adaptation to biotic and abiotic environments. The young Petunia genus (Solanaceae) is an excellent opportunity to study speciation because of its association with pollinators and unique microenvironments. This study evaluated the phylogenetic relationships among a Petunia clade species with different floral syndromes that inhabit several environments. We based our work on multiple individuals per lineage and employed nuclear and plastid phylogenetic markers and nuclear microsatellites. The phylogenetic tree revealed two main groups regarding the elevation of the distribution range, whereas microsatellites showed high polymorphism-sharing splitting lineages into three clusters. Isolation by distance, migration followed by new environment colonization, and shifts in floral syndrome were the motors for lineage differentiation, including infraspecific structuring, which suggests the need for taxonomic revision in the genus.

Disentangling the causes of high polymorphism sharing in sympatric Petunia species from subtropical highland grasslands: insights from nuclear diversity.

Genetic polymorphism sharing between closely related and sympatric plant species could result from common ancestry, ancient or recent hybridization. Here we analyzed four Petunia species from the subtropical highland grasslands in southern South America based on nuclear diversity to disentangle the causes of high polymorphism sharing between them. We genotyped microsatellite loci, employed population genetic methods to estimate variability, species limits, and ancient and recent gene flow, and assigned individuals to genetic and taxonomic groups. Finally, we modeled evolutionary processes to determine the impact of Quaternary climate changes on species phylogenetic relationships. Our results indicated that genetic diversity was strongly influenced by expansion and habitat fragmentation during the Quaternary cycles. The extensive polymorphism sharing is mainly due to species' common ancestry, and we did not discard ancient hybridization. Nowadays, niche differentiation is the primary driver for maintaining genetic and morphological limits between the four analysed Petunia species and there is no recent gene flow between them.

Neutral and outlier single nucleotide polymorphisms disentangle the evolutionary history of a coastal Solanaceae species.

Speciation begins with the isolation of some individuals or subpopulations due to drivers promoting a diverging genetic distribution. Such isolation may occur, followed by different processes and pressures. Isolation-by-distance (IBD), isolation-by-adaptation (IBA), and isolation-by-colonization (IBC) have been recognized as the main divergence patterns. Still, it is not easy to distinguish which one is the main pattern as each one may act at different points in time or even simultaneously. Using an extensive genome coverage from a Petunia species complex with coastal and inland distribution and multiple analytical approaches on population genomics and phylogeography, we showed a complex interplay between neutral and selective forces acting on the divergence process. We found 18,887 SNPs potentially neutral and 924 potentially under selection (outlier) loci. All analyses pointed that each subspecies displays its own genetic component and evolutionary history. We suggested plausible ecological drivers for such divergence in a southernmost South Atlantic coastal plain in Brazil and Uruguay and identified a connection between adaptation and environment heterogeneity.

Molecular evolution and structural analyses of proteins involved in metabolic pathways of volatile organic compounds in Petunia hybrida (Solanaceae).

The association between plants and their pollinators is essential for increasing the diversity in angiosperms. Morphological and physiological traits, mainly floral scent, can influence the pollination dynamics and select pollinators for each plant species. In this work, we studied two proteins involved in producing volatile organic compounds in plants, conyferyl alcohol acyltransferase (CFAT) and benzoyl-CoA:benzyl alcohol/phenyl ethanol benzoyl transferase (BPBT) genes. We aimed to understand these proteins with respect to evolutionary and structural aspects and functions in Solanaceae using phylogenetic methods and comparative molecular modeling. We used Bayesian inference to describe the proteins' evolutionary history using Petunia x hybrida as a query to search for homologs in the Solanaceae family. Theoretical 3D models were obtained for both proteins using Panicum virgatum as a template. The phylogenetic tree included several different enzymes with diverse biological roles in Solanaceae, displaying the transferase domain. We identified only one sequence of CFAT in the databases, which belongs to Petunia x hybrida, and found several BPBT sequences from the genera Nicotiana, Solanum, and Capsicum. The 3D structures of CFAT and BPBT have two different domains, and we have identified the amino acid residues essential for the enzymatic activity and interaction with substrates.

What could be the fate of secondary contact zones between closely related plant species?

Interspecific hybridization has been fundamental in plant evolution. Nevertheless, the fate of hybrid zones throughout the generations remains poorly addressed. We analyzed a pair of recently diverged, interfertile, and sympatric Petunia species to ask what fate the interspecific hybrid population has met over time. We analyzed the genetic diversity in two generations from two contact sites and evaluated the effect of introgression. To do this, we collected all adult plants from the contact zones, including canonicals and intermediary colored individuals, and compared them with purebred representatives of both species based on seven highly informative microsatellite loci. We compared the genetic diversity observed in the contact zones with what is seen in isolated populations of each species, considering two generations of these annual species. Our results have confirmed the genetic differentiation between the species and the hybrid origin of the majority of the intermediary colored individuals. We also observed a differentiation related to genetic variability and inbreeding levels among the populations. Over time, there were no significant differences per site related to genetic diversity or phenotype composition. We found two stable populations kept by high inbreeding and backcross rates that influence the genetic diversity of their parental species through introgression.

Phylogenetic niche conservatism and plant diversification in South American subtropical grasslands along multiple climatic dimensions.

Phylogenetic niche conservatism can be investigated at multiple scales on an explicit geographical context. Haplotype-based comparative analyses of lineages occupying the same region, and thus subjected to similar environmental factors, allow decoupling shared evolutionary and ecological patterns, as well as multiple dimensions of adaptive diversification. Here we aimed to assess the role of environmental drivers on diversification of subtropical grassland, based on haplotypic diversity of two plant genera. We sampled two closely related and co-distributed grassland plant genera, Petunia and Calibrachoa, across their entire distribution area. Eigenvectors extracted from pairwise distances based on chloroplast DNA haplotypes were used to fit Phylogenetic Signal-Representation (PSR) curves to estimate evolutionary patterns in 19 bioclimatic variables and altitude. The PSR curves showed that altitude, precipitation, and temperature variables changed at different rates with haplotype differentiation. Altitude and temperature traits evolved under conditions closer to a neutral dynamics, whereas precipitation traits differentiated following more complex models. Our results indicated that the diversification in the two genera was more limited by precipitation conditions. Based on these novel findings, we suggest that future studies should test the possible impact of precipitation variables on the process of ecological differentiation in these genera.

Biogeographical, ecological, and phylogenetic analyses clarifying the evolutionary history of Calibrachoa in South American grasslands.

Calibrachoa is a charismatic South American genus of Solanaceae, closely related to Petunia, which encompasses approximately 30 species. Studies that were based solely on plastid molecular markers indicated the monophyly of the genus and distributed its species in two subgenera; to date no phylogeny has included a broad morphological variants and nuclear markers. Here, we present a phylogenetic analysis based on eight plastid and eight nuclear markers that cover the most extensive geographic distribution for the genus. We use this phylogeny to infer the biogeographic history of the genus and to understand the primary drivers for species diversification. Our results yield a fully supported tree where monophyly is confirmed to genus and subgenera. The species of Stimomphis subgenus that were previously considered uncertain, here emerge in four highly supported clades. The hypothesis of niche conservatism is confirmed, and adaptive radiation explains the species diversification. The lowlands are the most likely ancestral area of the genus, subgenera, and two clades of Stimomphis subgenus. Our results constitute an excellent starting point for further evolutionary and taxonomic studies and explain several uncertain evolutionary relationships in the group and the evolution of their distribution.

Phylogeny of the Neotropical tribe Jacarandeae (Bignoniaceae).

PREMISE: The tribe Jacarandeae includes Jacaranda (49 species) and Digomphia (3 species), two genera of trees and woody shrubs with Neotropical distribution. Jacarandeae is sister to the rest of the Bignoniaceae, but not much is known about interspecific and intergeneric relationships within this group. METHODS: We reconstructed the phylogeny of Jacarandeae using chloroplast (ndhF, rpl32-trnL, trnL-F) and nuclear (ETS, PPR62) markers. Evolutionary relationships within Jacarandeae were inferred using Bayesian, Maximum Likelihood, and species tree approaches. The resulting phylogenetic framework was used as the basis to interpret the evolution of key morphological character states (i.e., stamen and calyx traits) and revise the infra-generic classification of the group. RESULTS: Jacaranda and Digomphia belong to a well-supported clade, with Digomphia nested within Jacaranda. We propose the necessary taxonomic changes to recognize monophyletic taxa, including a broadly circumscribed Jacaranda divided into four sections: (1) Jacaranda sect. Nematopogon, species previously included in Digomphia and united by divided staminode apices and spathaceous calyces; (2) Jacaranda sect. Copaia, species with monothecal anthers and cupular calyces; (3) Jacaranda sect. Jacaranda, species with monothecal anthers and campanulate calyces; and (4) Jacaranda sect. Dilobos, species with dithecal anthers and cupular calyces, and including more than half of the species of the genus, all restricted to Brazil. CONCLUSIONS: As circumscribed here, Jacarandeae includes only a broadly defined Jacaranda divided into four sections. Each section is defined by a unique combination of anther and calyx morphologies.

Genetic diversity and population structure of naturally rare Calibrachoa species with small distribution in southern Brazil.

Calibrachoa is a South-American genus comprising 27 species, several considered endemic or rare; few were subjects in genetic studies. We attempted to generate new data about the phylogenetically related and rare species C. eglandulata, C. sendtneriana, C. serrulata, and C. spathulata concerning their genetic diversity and population structure, which, coupled with their known restricted distribution, could help access their conservation status and contribute to the study of the Brazilian biodiversity. We sequenced 88 individuals for plastid intergenic spacers and genotyped 186 individuals for five microsatellite loci. Compared among each other, C. sendtneriana and C. serrulata presented the highest values of genetic diversity [π% (sd) = 0.23 (0.14) and 0.43 (0.25), respectively], followed by C. spathulata [π% (sd) = 0.19 (0.12)] and C. eglandulata [π% (sd) = 0.02 (0.03)]. Population differentiation was evident for these latter species, whereas it was not significant for C. sendtneriana and C. serrulata. Factors such as habitat specificity and fragmentation, pollination syndrome, and life history could explain the observed patterns. Based on the new genetic data and the species' biology, a conservation status was assigned for C. sendtneriana and the status of the other three species was reviewed.

Molecular evolution and functional divergence of alcohol dehydrogenases in animals, fungi and plants.

Alcohol dehydrogenases belong to the large superfamily of medium-chain dehydrogenases/reductases, which occur throughout the biological world and are involved with many important metabolic routes. We considered the phylogeny of 190 ADH sequences of animals, fungi, and plants. Non-class III Caenorhabditis elegans ADHs were seen closely related to tetrameric fungal ADHs. ADH3 forms a sister group to amphibian, reptilian, avian and mammalian non-class III ADHs. In fishes, two main forms are identified: ADH1 and ADH3, whereas in amphibians there is a new ADH form (ADH8). ADH2 is found in Mammalia and Aves, and they formed a monophyletic group. Additionally, mammalian ADH4 seems to result from an ADH1 duplication, while in Fungi, ADH formed clusters based on types and genera. The plant ADH isoforms constitute a basal clade in relation to ADHs from animals. We identified amino acid residues responsible for functional divergence between ADH types in fungi, mammals, and fishes. In mammals, these differences occur mainly between ADH1/ADH4 and ADH3/ADH5, whereas functional divergence occurred in fungi between ADH1/ADH5, ADH5/ADH4, and ADH5/ADH3. In fishes, the forms also seem to be functionally divergent. The ADH family expansion exemplifies a neofunctionalization process where reiterative duplication events are related to new activities.

Ecological drivers of plant genetic diversity at the southern edge of geographical distributions: Forestal vines in a temperate region.

The Tropical Niche Conservatism hypothesis is one of the most relevant theories to explain why tropical diversity is high, although the mechanisms underlying this hypothesis require further clarification. A possible research avenue to address the underlying mechanisms includes determining population-level processes associated with such a hypothesis, in particular by trying to identify how adaptation may occur in extreme niche conditions at the edges of species ranges. However, the determinants of molecular diversity at the edges of geographical distributions of tropical taxa are still poorly known. Here we assessed which environmental variables determine diversity in nuclear and plastid genetic markers for populations of four Passiflora species in the southern limit of their geographical distributions. Climatic factors can drive genetic diversity, and their importance varies according to the marker. The primary predictors are variables representing higher temperatures during cold periods of the year and higher precipitation during dry periods. We concluded that, although these species are present in colder areas at the edge of their range, Tropical Niche Conservatism acts as a restraining force on genetic diversity in southern populations of Passiflora.

Multiple markers, niche modelling, and bioregions analyses to evaluate the genetic diversity of a plant species complex.

BACKGROUND: The classification of closely related plants is not straightforward. These morphologically similar taxa frequently maintain their inter-hybridization potential and share ancestral polymorphisms as a consequence of their recent divergence. Under the biological species concept, they may thus not be considered separate species. The Petunia integrifolia complex is especially interesting because, in addition to the features mentioned above, its taxa share a pollinator, and their geographical ranges show multiple overlaps. Here, we combined plastid genome sequences, nuclear microsatellites, AFLP markers, ecological niche modelling, and bioregions analysis to investigate the genetic variability between the different taxa of the P. integrifolia complex in a comprehensive sample covering the entire geographical range of the complex. RESULTS: Results from molecular markers did not fully align with the current taxonomic classification. Niche modelling and bioregions analyses revealed that taxa were associated with different ecological constraints, indicating that the habitat plays an important role in preserving species boundaries. For three taxa, our analyses showed a mostly conserved, non-overlapping geographical distribution over time. However, for two taxa, niche modelling found an overlapping distribution over time; these taxa were also associated with the same bioregions. CONCLUSIONS: cpDNA markers were better able to discriminate between Petunia taxa than SSRs and AFLPs. Overall, our results suggest that the P. integrifolia complex represents a continuum of individuals from distant and historically isolated populations, which share some morphological traits, but are established in four different evolutionary lineages.

Secondary structure of nrDNA Internal Transcribed Spacers as a useful tool to align highly divergent species in phylogenetic studies.

Recently, it has been suggested that internal transcribed spacer (ITS) sequences are under selective constraints to preserve their secondary structure. Here, we investigate the patterns of the ITS nucleotide and secondary structure conservation across the Passiflora L. genus to evaluate the potential use of secondary structure data as a helpful tool for the alignment in taxonomically complex genera. Considering the frequent use of ITS, this study also presents a perspective on future analyses in other plant groups. The ITS1 and ITS2 sequences presented significant differences for mean values of the lowest energy state (LES) and for number of hairpins in different Passiflora subgenera. Statistical analyses for the subgenera separately support significant differences between the LES values and the total number of secondary structures for ITS. In order to evaluate whether the LES values of ITS secondary structures were related to selective constraints, we compared these results among 120 ITS sequences from Passiflora species and 120 randomly generated sequences. These analyses indicated that Passiflora ITS sequences present characteristics of a region under selective constraint to maintain the secondary structure showing to be a promising tool to improve the alignments and identify sites with non-neutral substitutions or those correlated evolutionary steps.

Molecular evolution analysis of WUSCHEL-related homeobox transcription factor family reveals functional divergence among clades in the homeobox region.

Gene families have been shown to play important roles in plant evolution and are associated with diversification and speciation. Genes of WUSCHEL-related homeobox family of transcription factors have important functions in plant development and are correlated with the appearance of evolutionary novelties. There are several published studies related to this family, but little is known about the relationships among the main clades in the phylogeny and the molecular evolution of the family. In this study, we obtained a well-resolved Bayesian phylogenetic tree establishing the relationships among the main clades and determining the position of Selaginella moellendorffii WOX genes. Moreover, a correlation was identified between the number of genes in the genomes and the events of whole-genome duplications. The intron-exon structure is more consistent across the modern clade, which appeared more recently in the WOX evolutionary history, and coincides with the development of higher complexity in plant species. No positive selection was detected among sites through the branches in the tree. However, with regard to the main clades, functional divergence among certain amino acids in the homeodomain region was found. Relaxed purifying selection could be the main driving force in the evolution of these genes and in agreement with some genes have been demonstrated to be functionally redundant.

Could refuge theory and rivers acting as barriers explain the genetic variability distribution in the Atlantic Forest?

The Atlantic Forest is one of the most species-rich ecoregions in the world. The historical origins of this richness and the evolutionary processes that produced diversification and promoted speciation in this ecosystem remain poorly understood. In this context, focusing on Passiflora contracta, an endemic species from the Atlantic Forest distributed exclusively at sea level along forest edges, this study aimed to characterize the patterns of genetic variability and explore two hypotheses that attempt to explain the possible causes of the genetic diversity in this region: the refuge and riverine barrier theories. We employed Bayesian methods combined with niche modeling to identify genetically homogeneous groups, to determine the diversification age, and identify long-term climate stability areas to species survival. The analyses were performed using molecular markers from nuclear and plastid genomes, with samples collected throughout the entire geographic distribution of the species, and comparisons with congeners species. The results indicated that populations were genetically structured and provided evidence of demographic stability. The molecular markers indicated the existence of a clear structure and the presence of five homogeneous groups. Interestingly, the separation of the groups coincides with the geographical locations of local rivers, corroborating the hypothesis of rivers acting as barriers to gene flow in this species. The highest levels of genetic diversity and the areas identified as having long-term climate stability were found in the same region reported for other species as a possible refuge area during the climatic changes of the Quaternary.

Were sea level changes during the Pleistocene in the South Atlantic Coastal Plain a driver of speciation in Petunia (Solanaceae)?

BACKGROUND: Quaternary climatic changes led to variations in sea level and these variations played a significant role in the generation of marine terrace deposits in the South Atlantic Coastal Plain. The main consequence of the increase in sea level was local extinction or population displacement, such that coastal species would be found around the new coastline. Our main goal was to investigate the effects of sea level changes on the geographical structure and variability of genetic lineages from a Petunia species endemic to the South Atlantic Coastal Plain. We employed a phylogeographic approach based on plastid sequences obtained from individuals collected from the complete geographic distribution of Petunia integrifolia ssp. depauperata and its sister group. We used population genetics tests to evaluate the degree of genetic variation and structure among and within populations, and we used haplotype network analysis and Bayesian phylogenetic methods to estimate divergence times and population growth. RESULTS: We observed three major genetic lineages whose geographical distribution may be related to different transgression/regression events that occurred in this region during the Pleistocene. The divergence time between the monophyletic group P. integrifolia ssp. depauperata and its sister group (P. integrifolia ssp. integrifolia) was compatible with geological estimates of the availability of the coastal plain. Similarly, the origin of each genetic lineage is congruent with geological estimates of habitat availability. CONCLUSIONS: Diversification of P. integrifolia ssp. depauperata possibly occurred as a consequence of the marine transgression/regression cycles during the Pleistocene. In periods of high sea level, plants were most likely restricted to a refuge area corresponding to fossil dunes and granitic hills, from which they colonized the coast once the sea level came down. The modern pattern of lineage geographical distribution and population variation was established by a range expansion with serial founder effects conditioned on soil availability.

The phylogeny and evolutionary history of the Lesion Simulating Disease (LSD) gene family in Viridiplantae.

The Lesion Simulating Disease (LSD) genes encode a family of zinc finger proteins that play a role in programmed cell death (PCD) and other biological processes, such as plant growth and photosynthesis. In the present study, we report the reconstruction of the evolutionary history of the LSD gene family in Viridiplantae. Phylogenetic analysis revealed that the monocot and eudicot genes were distributed along the phylogeny, indicating that the expansion of the family occurred prior to the diversification between these clades. Sequences encoding proteins that present one, two, or three LSD domains formed separate groups. The secondary structure of these different LSD proteins presented a similar composition, with the ß-sheets being their main component. The evolution by gene duplication was identified only to the genes that contain three LSD domains, which generated proteins with equal structure. Moreover, genes encoding proteins with one or two LSD domains evolved as single-copy genes and did not result from loss or gain in LSD domains. These results were corroborated by synteny analysis among regions containing paralogous/orthologous genes in Glycine max and Populus trichocarpa. The Ka/Ks ratio between paralogous/orthologous genes revealed that a subfunctionalization process possibly could be occurring with the LSD genes, explaining the involvement of LSD members in different biological processes, in addition to the negative regulation of PCD. This study presents important novelty in the evolutionary history of the LSD family and provides a basis for future research on individual LSD genes and their involvement in important pathway networks in plants.

Pollen dispersal and breeding structure in a hawkmoth-pollinated Pampa grasslands species Petunia axillaris (Solanaceae).

BACKGROUND AND AIMS: The evolution of selfing is one of the most common transitions in flowering plants, and this change in mating pattern has important systematic and ecological consequences because it often initiates reproductive isolation and speciation. Petunia axillaris (Solanaceae) includes three allopatric subspecies widely distributed in temperate South America that present different degrees of self-compatibity and incompatibility. One of these subspecies is co-distributed with P. exserta in a restricted area and presents a complex, not well-understood mating system. Artificial crossing experiments suggest a complex system of mating in this sympatric area. The main aims of this study were to estimate the pollen dispersal distance and to evaluate the breeding structure of P. axillaris subsp. axillaris, a hawkmoth-pollinated taxon from this sympatric zone. METHODS: Pollen dispersal distance was compared with nearest-neighbours distance, and the differentiation in the pollen pool among mother plants was estimated. In addition, the correlation between genetic differentiation and spatial distance among plants was tested. All adult individuals (252) within a space of 2800 m(2) and 15 open-pollinated progeny (285 seedlings) were analysed. Genetic analyses were based on 12 polymorphic microsatellite loci. KEY RESULTS: A high proportion of self-pollination was found, indicating a mixed-mating system. The maximum pollen dispersal distance was 1013 m, but most pollination events (96 %) occurred at a distance of 0 m, predominantly in an inbreeding system. Both parents among sampled individuals could be identifed in 60-85 % of the progeny. CONCLUSIONS: The results show that most pollen dispersal in the hawkmoth-pollinated P. axillaris subsp. axillaris occurs within populations and there is a high proportion of inbreeding. This mating system appears to favour species integrity in a secondary contact zone with the congener species P. exserta.