Strong conservatism of floral morphology during the rapid diversification of the genus Helianthemum.

PREMISE: Divergence of floral morphology and breeding systems are often expected to be linked to angiosperm diversification and environmental niche divergence. However, available evidence for such relationships is not generalizable due to different taxonomic, geographical and time scales. The Palearctic genus Helianthemum shows the highest diversity of the family Cistaceae in terms of breeding systems, floral traits, and environmental conditions as a result of three recent evolutionary radiations since the Late Miocene. Here, we investigated the tempo and mode of evolution of floral morphology in the genus and its link with species diversification and environmental niche divergence. METHODS: We quantified 18 floral traits from 83 taxa and applied phylogenetic comparative methods using a robust phylogenetic framework based on genotyping-by-sequencing data. RESULTS: We found three different floral morphologies, putatively related to three different breeding systems: type I, characterized by small flowers without herkogamy and low pollen to ovule ratio; type II, represented by large flowers with approach herkogamy and intermediate pollen to ovule ratio; and type III, featured by small flowers with reverse herkogamy and the highest pollen to ovule ratio. Each morphology has been highly conserved across each radiation and has evolved independently of species diversification and ecological niche divergence. CONCLUSIONS: The combined results of trait, niche, and species diversification ultimately recovered a pattern of potentially non-adaptive radiations in Helianthemum and highlight the idea that evolutionary radiations can be decoupled from floral morphology evolution even in lineages that diversified in heterogeneous environments as the Mediterranean Basin.

Isolation of microsatellite markers for the endemic Phlomis lychnitis (Lamiaceae).

BACKGROUND: Phlomis lychnitis is a mostly endemic species of the Iberian Peninsula that frequently hybridizes with the narrow endemic P. crinita subsp. malacitana in southern Spain. Whenever they coexist they form homoploid hybrid zones. Unlike hybridization at the polyploid level, the process of hybridization at the homoploid level is much less well known. METHODS AND RESULTS: In this study we report the development of 22 microsatellite markers through next-generation sequencing technologies for P. lychnitis. We characterize the genetic diversity for two populations of this species for the 10 markers that resulted to be polymorphic. Further, we check the transferability of these polymorphic markers to one population of P. crinita subsp. malacitana to verify the potential use of these markers for hybridization studies. The values of expected heterozygosity for P. lychnitis were higher than in P. crinita subsp. malacitana, and the three analyzed populations displayed negative values for the inbreeding coefficient which is compatible with the frequent instances of hybridization and introgression between species. CONCLUSIONS: This set of polymorphic markers are useful for further studies aiming at a deeper understanding of the homoploid hybrid process between these species. Additionally, this is the first panel of microsatellite markers developed for the genus Phlomis, a genus very rich in endemic species and with medicinal properties that could benefit from the use of these new markers.

Screening for Selective Anticancer Activity of 65 Extracts of Plants Collected in Western Andalusia, Spain.

Finding cytotoxic drugs with a high selectivity towards cancer cells is crucial to improve the low survival rates of patients diagnosed with metastatic cancers. Since plants are an important source of anticancer drugs, we have screened 65 extracts from 45 plants collected in several areas of Western Andalusia (Spain) for cytotoxic activity on lung cancer cells versus lung normal cells. An extract from the leaves of Tetraclinis articulata (Vahl) Mast. (Cupressaceae) showed a marked cytotoxicity (IC50 = 0.37 ± 0.03 µg/mL) and selectivity (selectivity index = 378.3) against the lung cancer cells; cisplatin, 5-fluorouracil, and an extract from the leaves of Taxus baccata L. (Taxaceae) were less cytotoxic and selective. Extracts from Cascabela thevetia (L.) Lippold (Apocynaceae), Frangula alnus Mill. (Rhamnaceae), Iberis ciliata subsp. contracta (Pers.) Moreno (Brassicaceae), Juniperus macrocarpa Sm (Cupressaceae), and Pancratium maritimum L. (Amaryllidaceae) also showed selective cytotoxicity (selectivity index > 10). Active extracts were also tested against a panel of cancer cell lines from a variety tissues. The plants identified in this work are potential sources of natural compounds with selective toxicity towards cancer cells.

Biogeographic history and environmental niche evolution in the palearctic genus Helianthemum (Cistaceae).

The biogeographic history and the degree of environmental niche conservatism provide essential clues to decipher the underlying macroevolutionary processes of species diversification and to understand contemporary patterns of biodiversity. The genus Helianthemum constitutes an excellent case study to investigate the impact of the geo-climatic changes and the environmental niche shifts on the origins of plant species diversity in the Mediterranean hotspot. It is a palearctic species-rich lineage with c. 140 species and subspecies mostly belonging to three distinct evolutionary radiations, almost confined to the Mediterranean region and occurring across varied environmental conditions. In this work, we studied the ample and rapid diversification of the genus Helianthemum across its whole distribution range by performing phylogenetic reconstructions of ancestral ranges and environmental niche evolution. We observed a striking synchrony of biogeographic movements with niche shifts between the three major clades of the genus Helianthemum, likely related to the geo-climatic events occurred in the Mediterranean Basin since the Upper Miocene. In particular, Late Miocene and Early Pliocene were dominated by episodes of range expansions, the Late Pliocene by range contraction and vicariance events, and Pleistocene by most intense environmental niche shifts and in-situ diversification. Our study also provides evidence for four main environmental niches in Helianthemum (i.e., Mediterranean, subdesert, humid-montane and subtropical-insular) and a tendency toward environmental niche conservatism within different subclades, with few niche shifts mostly occurring from Mediterranean ancestors. The relative longer time spent in Mediterranean areas by the ancestors of Helianthemum suggests that the larger species diversity observed in the Mediterranean (i.e. Northern Africa and Southern Europe) may have been generated by a time-for-speciation effect reinforced by environmental niche conservatism. Overall, our work highlights the role of the Mediterranean Basin as a 'cradle of diversity' and an 'evolutionary hub', facilitating the environmental transitions and determining the building up of a global plant biodiversity hotspot.

Reconstruction of the spatio-temporal diversification and ecological niche evolution of Helianthemum (Cistaceae) in the Canary Islands using genotyping-by-sequencing data.

BACKGROUND AND AIMS: Several biogeographical models have been proposed to explain the colonization and diversification patterns of Macaronesian lineages. In this study, we calculated the diversification rates and explored what model best explains the current distribution of the 15 species endemic to the Canary Islands belonging to Helianthemum sect. Helianthemum (Cistaceae). METHODS: We performed robust phylogenetic reconstructions based on genotyping-by-sequencing data and analysed the timing, biogeographical history and ecological niche conservatism of this endemic Canarian clade. KEY RESULTS: Our phylogenetic analyses provided strong support for the monophyly of this clade, and retrieved five lineages not currently restricted to a single island. The pristine colonization event took place in the Pleistocene (~1.82 Ma) via dispersal to Tenerife by a Mediterranean ancestor. CONCLUSIONS: The rapid and abundant diversification (0.75-1.85 species per million years) undergone by this Canarian clade seems the result of complex inter-island dispersal events followed by allopatric speciation driven mostly by niche conservatism, i.e. inter-island dispersal towards niches featuring similar environmental conditions. Nevertheless, significant instances of ecological niche shifts have also been observed in some lineages, making an important contribution to the overall diversification history of this clade.

Maximize Resolution or Minimize Error? Using Genotyping-By-Sequencing to Investigate the Recent Diversification of Helianthemum (Cistaceae).

A robust phylogenetic framework, in terms of extensive geographical and taxonomic sampling, well-resolved species relationships and high certainty of tree topologies and branch length estimations, is critical in the study of macroevolutionary patterns. Whereas Sanger sequencing-based methods usually recover insufficient phylogenetic signal, especially in recently diversified lineages, reduced-representation sequencing methods tend to provide well-supported phylogenetic relationships, but usually entail remarkable bioinformatic challenges due to the inherent trade-off between the number of SNPs and the magnitude of associated error rates. The genus Helianthemum (Cistaceae) is a species-rich and taxonomically complex Palearctic group of plants that diversified mainly since the Upper Miocene. It is a challenging case study since previous attempts using Sanger sequencing were unable to resolve the intrageneric phylogenetic relationships. Aiming to obtain a robust phylogenetic reconstruction based on genotyping-by-sequencing (GBS), we established a rigorous methodological workflow in which we i) explored how variable settings during dataset assembly have an impact on error rates and on the degree of resolution under concatenation and coalescent approaches, ii) assessed the effect of two extreme parameter configurations (minimizing error rates vs. maximizing phylogenetic resolution) on tree topology and branch lengths, and iii) evaluated the effects of these two configurations on estimates of divergence times and diversification rates. Our analyses produced highly supported topologically congruent phylogenetic trees for both configurations. However, minimizing error rates did produce more reliable branch lengths, critically affecting the accuracy of downstream analyses (i.e. divergence times and diversification rates). In addition to recommending a revision of intrageneric systematics, our results enabled us to identify three highly diversified lineages in Helianthemum in contrasting geographical areas and ecological conditions, which started radiating in the Upper Miocene.

Genetic diversity and differentiation in narrow versus widespread taxa of Helianthemum (Cistaceae) in a hotspot: The role of geographic range, habitat, and reproductive traits.

Unraveling the relationships between ecological, functional traits and genetic diversity of narrow endemic plants provide opportunities for understanding how evolutionary processes operate over local spatial scales and ultimately how diversity is created and maintained. To explore these aspects in Sierra Nevada, the core of the Mediterranean Betic-Rifean hotspot, we have analyzed nuclear DNA microsatellite diversity and a set of biological and environmental factors (physicochemical soil parameters, floral traits, and community composition) in two strictly endemic taxa from dolomite outcrops of Sierra Nevada (Helianthemum pannosum and H. apenninum subsp. estevei) and two congeneric widespread taxa (H. cinereum subsp. rotundifolium and H. apenninum subsp. apenninum) that further belong to two different lineages (subgenera) of Helianthemum. We obtained rather unexpected results contrasting with the theory: (a) The narrow endemic taxa showed higher values of genetic diversity as well as higher average values of pollen production per flower and pollen-to-ovule ratio than their widespread relatives; and (b) the two taxa of subg. Helianthemum, with larger corollas, approach herkogamy and higher pollen production than the two taxa of subg. Plectolobum, displayed lower genetic diversity and higher values of inbreeding. Altogether, these results disclose how genetic diversity may be affected simultaneously by a large number of intrinsic and extrinsic factors, especially in Pleistocene glacial refugia in mountains where the spatial context harbors a great ecological heterogeneity. On the other hand, differences in mating system and the significant effect of the substrate profile, both being highly diverse in the genus Helianthemum, in the genetic variability illustrate about the importance of these two factors in the diversification and species differentiation of this paradigmatic genus in the Mediterranean and open the field to formulate and test new hypotheses of local adaptation, trait evolution, and habitat diversification.

Screening for selective anticancer activity of plants from Grazalema Natural Park, Spain.

Since plants are an important source of anticancer drugs, we have carried out a random screening for selective anticancer activity of 57 extracts from 45 plants collected in Grazalema Natural Park, an area in the South of Spain of high plant diversity and endemism. Using lung cancer cells (A549) and lung non-malignant cells (MRC-5), we found that several extracts were more cytotoxic and selective against the cancer cells than the standard anticancer agent cisplatin. Five active extracts were further tested in cancer and normal cell lines from other tissues, including three skin cell lines with increasing degree of malignancy. An extract from the leaves of Daphne laureola L. (Thymelaeaceae) showed a striking potency and selectivity on lung cancer cells and leukemia cells; the IC50 values against these cancer cells were approximately 10,000-fold lower than against the normal cells. Daphnane-type diterpene orthoesters may be responsible for this highly selective anticancer activity.

Climatic and topographical correlates of plant palaeo- and neoendemism in a Mediterranean biodiversity hotspot.

BACKGROUND AND AIMS: Understanding the evolutionary and ecological forces contributing to the emergence of biodiversity hotspots is of outstanding importance to elucidate how they may withstand current climate changes. Here we explored patterns of phylogenetic and non-phylogenetic plant endemism in a Mediterranean biodiversity hotspot. We hypothesized that areas with wet and equable climatic conditions would be prone to long-term persistence of endemic lineages (palaeoendemism), whilst areas of recent local speciation (neoendemism) would be more related to harsher environmental conditions and to high topographical relief promoting speciation. METHODS: We focused on the Baetic-Rifan biodiversity hotspot (southern Iberian Peninsula and northern Morocco) in combination with molecular phylogenetic information and relative phylogenetic endemism (RPE), a recent phylogenetic measure of endemism, allowing us to discern centres of palaeo- from those of neoendemism. Using eco-geographical regions as study units, we explored correlations between both RPE and endemic species richness with precipitation- and temperature-related variables and with elevation range. KEY RESULTS: Centres of neoendemism were concentrated towards the easternmost part of the hotspot, while centres of palaeoendemism were clustered in the vicinity of the Strait of Gibraltar. The RPE index, indicating more palaeoendemism, was positively correlated with total annual precipitation, while endemic species richness showed a poor correlation. In contrast, elevation range and mean annual temperature were poor predictors of RPE, despite elevation range showing a strong correlation with endemic species richness. CONCLUSIONS: The Baetic-Rifan biodiversity hotspot shows clearly differentiated centres of neo- and palaeoendemism. Topographical relief may have driven evolutionary diversification of newly evolved species, while water availability seems more critical for the long-term persistence of ancient lineages in refuge areas of smoother topography. Given climatic trends towards increasing aridification, conservation planners should pay particular attention to preserve areas retaining older phylogenetic lineages, as these areas act as 'natural museums' of biodiversity within the Baetic-Rifan biodiversity hotspot.

High Correlated Paternity Leads to Negative Effects on Progeny Performance in Two Mediterranean Shrub Species.

Anthropogenic habitat deterioration can promote changes in plant mating systems that subsequently may affect progeny performance, thereby conditioning plant recruitment for the next generation. However, very few studies yet tested mating system parameters other than outcrossing rates; and the direct effects of the genetic diversity of the pollen received by maternal plants (i.e. correlated paternity) has often been overlooked. In this study, we investigated the relation between correlated paternity and progeny performance in two common Mediterranean shrubs, Myrtus communis and Pistacia lentiscus. To do so, we collected open-pollinated progeny from selected maternal plants, calculated mating system parameters using microsatellite genotyping and conducted sowing experiments under greenhouse and field conditions. Our results showed that some progeny fitness components were negatively affected by the high correlated paternity of maternal plants. In Myrtus communis, high correlated paternity had a negative effect on the proportion and timing of seedling emergence in the natural field conditions and in the greenhouse sowing experiment, respectively. In Pistacia lentiscus, seedling emergence time under field conditions was also negatively influenced by high correlated paternity and a progeny survival analysis in the field experiment showed greater mortality of seedlings from maternal plants with high correlated paternity. Overall, we found effects of correlated paternity on the progeny performance of Myrtus communis, a self-compatible species. Further, we also detected effects of correlated paternity on the progeny emergence time and survival in Pistacia lentiscus, an obligate outcrossed species. This study represents one of the few existing empirical examples which highlight the influence that correlated paternity may exert on progeny performance in multiple stages during early seedling growth.

The building of a biodiversity hotspot across a land-bridge in the Mediterranean.

Many of the macroevolutionary processes that have shaped present-day phylogenetic patterns were caused by geological events such as plate tectonics and temporary land-bridges. The study of spatial patterns of phylogenetic diversity can provide insights into these past events. Here we focus on a western Mediterranean biodiversity hotspot located in the southern Iberian Peninsula and northwest Africa, two regions that are separated by the Strait of Gibraltar. We explore the spatial structure of the phylogenetic relationships within and across large-scale plant assemblages. Significant turnover in terminal lineages tends to occur between landmasses, whereas turnover in deep lineages tends to occur within landmasses. Plant assemblages in the western ecoregions of this hotspot tend to be phylogenetically overdispersed but are phylogenetically clustered on its eastern margins. We discuss our results in the light of potential scenarios of niche evolution (or conservatism) and lineage diversification. The significant turnover between landmasses suggests a common scenario of allopatric speciation that could have been facilitated by the intermittent joining of the two continents. This may have constituted an important stimulus for diversification and the emergence of this western Mediterranean biodiversity hotspot.

Disentangling environmental correlates of vascular plant biodiversity in a Mediterranean hotspot.

We determined the environmental correlates of vascular plant biodiversity in the Baetic-Rifan region, a plant biodiversity hotspot in the western Mediterranean. A catalog of the whole flora of Andalusia and northern Morocco, the region that includes most of the Baetic-Rifan complex, was compiled using recent comprehensive floristic catalogs. Hierarchical cluster analysis (HCA) and detrended correspondence analysis (DCA) of the different ecoregions of Andalusia and northern Morocco were conducted to determine their floristic affinities. Diversity patterns were studied further by focusing on regional endemic taxa. Endemic and nonendemic alpha diversities were regressed to several environmental variables. Finally, semi-partial regressions on distance matrices were conducted to extract the respective contributions of climatic, altitudinal, lithological, and geographical distance matrices to beta diversity in endemic and nonendemic taxa. We found that West Rifan plant assemblages had more similarities with Andalusian ecoregions than with other nearby northern Morocco ecoregions. The endemic alpha diversity was explained relatively well by the environmental variables related to summer drought and extreme temperature values. Of all the variables, geographical distance contributed by far the most to spatial turnover in species diversity in the Baetic-Rifan hotspot. In the Baetic range, elevation was the most significant driver of nonendemic species beta diversity, while lithology and elevation were the main drivers of endemic beta diversity. Despite the fact that Andalusia and northern Morocco are presently separated by the Atlantic Ocean and the Mediterranean Sea, the Baetic and Rifan mountain ranges have many floristic similarities - especially in their western ranges - due to past migration of species across the Strait of Gibraltar. Climatic variables could be shaping the spatial distribution of endemic species richness throughout the Baetic-Rifan hotspot. Determinants of spatial turnover in biodiversity in the Baetic-Rifan hotspot vary in importance between endemic and nonendemic species.

Extensive pollen flow but few pollen donors and high reproductive variance in an extremely fragmented landscape.

Analysing pollen movement is a key to understanding the reproductive system of plant species and how it is influenced by the spatial distribution of potential mating partners in fragmented populations. Here we infer parameters related to levels of pollen movement and diversity of the effective pollen cloud for the wind-pollinated shrub Pistacia lentiscus across a highly disturbed landscape using microsatellite loci. Paternity analysis and the indirect KinDist and Mixed Effect Mating models were used to assess mating patterns, the pollen dispersal kernel, the effective number of males (N(ep)) and their relative individual fertility, as well as the existence of fine-scale spatial genetic structure in adult plants. All methods showed extensive pollen movement, with high rates of pollen flow from outside the study site (up to 73-93%), fat-tailed dispersal kernels and large average pollination distances (δ = 229-412 m). However, they also agreed in detecting very few pollen donors (N(ep) = 4.3-10.2) and a large variance in their reproductive success: 70% of males did not sire any offspring among the studied female plants and 5.5% of males were responsible for 50% of pollinations. Although we did not find reduced levels of genetic diversity, the adult population showed high levels of biparental inbreeding (14%) and strong spatial genetic structure (S(p) = 0.012), probably due to restricted seed dispersal and scarce safe sites for recruitment. Overall, limited seed dispersal and the scarcity of successful pollen donors can be contributing to generate local pedigrees and to increase inbreeding, the prelude of genetic impoverishment.

Contrasting heterozygosity-fitness correlations between populations of a self-compatible shrub in a fragmented landscape.

The mechanisms underlying heterozygosity-fitness correlations (HFCs) are subject of intense debates, especially about how important population features such as size or degree of isolation influence HFCs. Here, we report variation in HFCs between Large and Small populations of a self-compatible shrub (Myrtus communis) occurring within an extremely fragmented landscape. In each of the five study populations, we obtained data on both heterozygosity and fitness for 9-12 maternal families (i.e. offspring from the same mother plant). Whereas heterozygosity explained most of the variance (60-86 %) in growth rate of seedling families within Large populations, this relationship was absent within Small populations. Our results suggest that inbreeding may explain the observed HFCs within Large populations, and that different genetic processes (such as genetic drift and/or selection) could have overridden HFCs within Small populations. While it is difficult to draw general conclusions from five populations, we think our results open new research perspectives on how different genetic processes underlie variation in HFCs under different population contexts. Our study also points to a need for further attention on the complex relationships between heterozygosity in self-compatible plants and their progeny in relation to mating system variation. Finally, our results provide interesting new insights into how population genetic diversity is maintained or lost in a highly fragmented landscape.

Isolation of microsatellite markers for the common Mediterranean shrub Myrtus communis (Myrtaceae).

PREMISE OF THE STUDY: The development of microsatellite markers was conducted in the Mediterranean common shrub Myrtus communis (myrtle) to assess levels of genetic diversity and patterns of gene flow across fragmented landscapes in southern Spain. METHODS AND RESULTS: Fourteen primer pairs were isolated showing clear and consistent patterns of amplification, three of which were apparently monomorphic. Levels of polymorphism in the other 11 markers were checked in 48 individuals from two populations. The number of alleles per locus ranged from 3 to 11 and the total number of alleles was 83. CONCLUSIONS: These highly polymorphic markers will allow us to improve our understanding of the genetic consequences of chronic fragmentation in Mediterranean landscapes.

Spatiotemporal mating pattern variation in a wind-pollinated Mediterranean shrub.

Spatiotemporal variation in mating patterns is poorly known in wind-pollinated plant species. Here, we analysed mating patterns of the wind-pollinated dioecious shrub Pistacia lentiscus by genotyping 904 seeds from 30 mother plants with eight microsatellite markers in a high-density population in two consecutive flowering seasons. We found significant differences in some mating system estimates between years, particularly in the levels of correlated paternity. Overall, within-mothers correlated paternity was higher in 2007 than in 2006 (r(pWM) = 0.085 and 0.030), which translated into an effective number of fathers (N(ep)) of 11.8 and 33.6 respectively. Using a smoothing interpolation technique, we show that the effective pollen cloud was spatially structured in patches of high- and low-genetic diversity, which do not remain constant from year to year. In 2006, the among-mothers correlated paternity (r(pAM)) showed no trend with distance, suggesting no restriction of pollen dispersal. However, in 2007, r(pAM) was greater than zero at short distances, revealing the existence of small-scale patterns of pollen dispersal. The fact that the studied seasons were climatically homogeneous during the flowering time suggested that the observed differences might be ascribed to between-year phenological variation of individuals in the studied population or other (unknown) factors. Numerical simulations, based on the real data set, indicated that the clumping of males and decreasing plant density, which is related to different types of pollen limitation, greatly increase correlated mating in this wind-pollinated species, which is of relevance under the frame of the continuous anthropogenic habitat disturbance suffered by Mediterranean ecosystems.

Networks of spatial genetic variation across species.

Spatial patterns of genetic variation provide information central to many ecological, evolutionary, and conservation questions. This spatial variability has traditionally been analyzed through summary statistics between pairs of populations, therefore missing the simultaneous influence of all populations. More recently, a network approach has been advocated to overcome these limitations. This network approach has been applied to a few cases limited to a single species at a time. The question remains whether similar patterns of spatial genetic variation and similar functional roles for specific patches are obtained for different species. Here we study the networks of genetic variation of four Mediterranean woody plant species inhabiting the same habitat patches in a highly fragmented forest mosaic in Southern Spain. Three of the four species show a similar pattern of genetic variation with well-defined modules or groups of patches holding genetically similar populations. These modules can be thought of as the long-sought-after, evolutionarily significant units or management units. The importance of each patch for the cohesion of the entire network, though, is quite different across species. This variation creates a tremendous challenge for the prioritization of patches to conserve the genetic variation of multispecies assemblages.

Population genetic structure and hybridization patterns in the Mediterranean endemics Phlomis lychnitis and P. crinita (Lamiaceae).

BACKGROUND AND AIMS: The historical influence of gene flow and genetic drift after the last glacial phase of the Quaternary Period is reflected in current levels of genetic diversity and population structure of plant species. Moreover, hybridization after secondary contact might also affect population genetic diversity and structure. An assessment was made of the genetic variation and hybrid zone structure in Iberian populations of the Mediterranean Phlomis lychnitis and P. crinita, for which phylogenetic relationships are controversial, and hybridization and introgression are common. METHODS: Allozyme variation at 13 loci was analysed in 1723 individual plants sampled from 35 natural locations of P. lychnitis, P. crinita subsp. malacitana and P. crinita subsp. crinita in southern and eastern Spain. Standard genetic diversity parameters were calculated and patterns of genetic structure in each taxon were tested to fit the equilibrium between gene flow and genetic drift. Individual multilocus genotypes were subjected to Bayesian clustering analysis to estimate hybridization and introgression rates for both geographic regions. KEY RESULTS: Contrasting patterns in the distribution of genetic variation among the three taxa were found. Phlomis lychnitis showed no significant inbreeding, low genetic differentiation among populations and no evidence of isolation by distance. Phlomis crinita subsp. malacitana and P. crinita subsp. crinita showed high levels of genetic structure consistent with a pattern of gene flow-drift equilibrium. Higher instances of hybridization and introgression were detected in locations from southern Spain compared with locations from eastern Spain, matching unimodal and bimodal hybrid zones, respectively. CONCLUSIONS: High instances of historical gene flow, range expansion and altitudinal movement during the Quaternary Period, and lineage sorting can explain the diversity of patterns observed. The results suggest that P. lychnitis is the most differentiated lineage in the group; however, the relationship between the three taxa remains unclear.