Regina Berjano

Background and Aims: Floral mimicry is a form of deceptive pollination in which flowers or inflorescences imitate specific resources relevant to potential flower visitors, such as food, brood sites or mating partners (sexual mimicry). A plant genus containing many unstudied species with potentially highly diverse deceptive systems is the fly-pollinated Aristolochia (Aristolochiaceae), famous for its trap flowers. A few species with strongly male-biased pollinator assemblages have been suggested to mimic the female sex pheromones of flies. However, so far, neither the floral scents nor the female sex pheromones of the fly species involved are known. Methods: In this study on the Mediterranean Aristolochia lutea and A. paucinervis, we identified male-biased pollinator assemblages, analysed the floral scents and female-specific fly volatiles by gas chromatography/mass spectrometry (GC/MS) and (enantioselective) synthetic chemistry, and performed field bioassays with synthetic floral scents to characterize these pollination systems. Key Results: We found that the two Aristolochia species were pollinated by different assemblages of exclusively or primarily male phorid species (mostly Megaselia). The floral scents of both Aristolochia species were dominated by a small, largely overlapping set of structurally related short-chain, methyl-branched aliphatic alcohols and ketones, several of which we also identified as female-specific compounds in phorids [e.g. 4-methyl-2-pentanone, (E)-3-methyl-3-penten-2-one, 5-methyl-3-hexanone]. In field bioassays, synthetic floral scent resembling A. lutea attracted males of two pollinator species of A. lutea (Megaselia giraudii, M. pleuralis) and the main pollinator of A. paucinervis (M. pumila). A mixture resembling A. paucinervis attracted also a pollinator of A. lutea (M. giraudii). No female phorids were attracted in these assays. Conclusions: Both Aristolochia species mimic female-specific volatiles to deceive male phorid pollinators. Future studies must test whether the compounds involved function as female sex pheromones of the pollinating flies. If confirmed, this would be the first case of sex pheromone mimicry outside of orchids.

Functional traits express the ecological and evolutionary attributes of individuals that compose taxa, providing robust criteria to detect speciation when integrated with phylogenies. We tested this approach in Sonchus asper (L.) Hill, a putatively self-fertilizing annual weed that has recently been reported to exhibit extensive floral and life-form variation across the northern Mediterranean. This variability was already documented in the nineteenth century, but it is incongruent with current subspecific classifications based on leaf arrangement. We sampled 40 populations encompassing this variation, assigning each to (a) one of the four morphotypes defined by floral traits and life forms and (b) a leaf arrangement group. To assess their taxonomic relevance, we tested grouping characters as functional traits and analysed the consistency of these assignments with the phylogeny (ITS/ETS and chloroplast DNA markers) and genetic structure [amplified fragment length polymorphism (AFLP) markers]. Phylogenetic analysis using floral traits and life forms identified three well-supported clades that contradict leaf-arrangement taxonomy. The earliest-diverging clade comprises the common, annual, self-fertilizing S. asper. The remaining two clades consist of self-incompatible plants: one contains the rhizomatous morphotype, whereas the other groups annual and biennial plants. These life-history strategies exhibited distinct AFLP-based genetic profiles, alongside complex cpDNA phylogeographical patterns. The congruence between DNA analyses and functional value of floral and life-form traits corroborates the proposed morphotypes as distinct evolutionary lineages. This reveals unaccounted-for biodiversity and supports reinstating historical taxa. Our study shows that combining functional traits with genealogical relationships provides a powerful operational taxonomic criterion that integrates ecological and evolutionary species concepts.

Background and Aims: Delimitation of species remains a major challenge in understanding biodiversity, particularly in recently diverged lineages, where extensive morphological variability complicates taxonomic inference. In this study, we investigate patterns of morphological and ecological variation within Thymus sect. Mastichina, a Mediterranean lineage that includes the endangered species Thymus albicans. Previous genomic analyses revealed clear differentiation between two major groups corresponding to ploidy levels, in addition to additional genetic structure among diploid lineages. Methods: Here, we integrate this genomic framework with detailed analyses of floral morphology and ecological niches to assess the correspondence between phenotypic, ecological and genetic variation. Key Results: Our results reveal substantial overlap among genetic lineages at both morphological and ecological levels. Although some differentiation is associated with ploidy level, particularly in floral traits, extensive variability within several lineages obscures clear taxonomic boundaries based on discrete morphological characters. In contrast, some diploid lineages (notably the Cádiz and Algarve groups) show lower morphological variability but still both overlap in morphology and ecological niche. We morphologically characterized a recently discovered diploid lineage (Hercynian) that appears to be cryptic with the tetraploid T. mastichina. Patterns of admixture involving this Hercynian lineage, especially with the diploid Doñana group, contribute to pronounced phenotypic overlap, whereas no significant effect of tetraploid introgression on diploid morphology was detected. Ecological niche analyses indicate that niche differentiation is associated primarily with ploidy level rather than among diploid lineages, except for the Hercynian group. Conclusions: Overall, our results demonstrate a marked discordance between phenotypic variation and genetic structure, supporting the interpretation of Thymus sect. Mastichina as a complex of cryptic evolutionary lineages. These findings highlight the limitations of morphology-based taxonomy and emphasise the need for an integrative framework combining morphology, genome size and geographical distribution for taxa delimitation.

Juncus bufonius L. s.l. is a species complex with several ploidy levels, for which species delimitation remains unclear due to a lack of reliable morphological characters and the paucity of molecular studies. To clarify taxonomic and geographic relationships in the complex, we combined genomic, cytometric and morphological data from a broad latitudinal range from England down to Spain. We collected morphometric and cytometric data from 31 populations, and genomic data were obtained through Hyb-Seq using the Angiosperm353 kit for a subset of individuals. These three datasets were combined to explore phylogenetic relationships, population structure, and the validity of four previously proposed morphospecies ( J. bufonius s.str. , a hexaploid; J. minutulus , a tetraploid; and J. ranarius and J. hybridus , both diploids). Sequencing supported the separation of diploids and polyploids as two distinct taxa, but morphometric characters used previously to describe morphospecies showed continuous variation with no diagnostic value, and were not congruent with genomic and cytometric data. Polyploids likely originated through allopolyploidisation from diploids and tetraploids. Phylogenetic lineages were extensively mixed geographically, both for diploid and polyploid taxa, which suggests repeated long-distance dispersal events for both diploids and polyploids, and no separation of taxa by geography. Splitting of diploids into J. ranarius and J. hybridus was not supported. We recommend J. ranarius be treated as a synonym of J. hybridus , and that tetraploids and hexaploids be grouped under J. bufonius . The observed geographical patterns are consistent with high rates of seed dispersal by migratory waterbirds.

Understanding gene flow between ploidy levels in polyploid complexes is essential for species delimitation and conservation. This study explores evolutionary dynamics in the polyploid complex Thymus sect. Mastichina (Lamiaceae), comprising three taxa: T. mastichina subsp. mastichina , T. mastichina subsp. donyanae , and the endangered T. albicans . Using Hyb‐Seq data, phylogenomics (nuclear orthologs), and population genomics (SNPs), we confirm the section consists of two sister groups with distinct ploidy levels: a diploid and a tetraploid one. The tetraploid group shows low genetic differentiation among its populations, probably indicating rapid expansion across diverse environments. In contrast, the diploid group exhibits more complex genetic structuring, potentially shaped by geomorphology, interploidy introgression, and incipient isolation. Four diploid subgroups (Algarve, Cádiz, Doñana, and Hercynian) are identified, with reticulate evolution. The dense reticulation observed is compatible with incomplete lineage sorting in diploid lineages due to recent and rapid divergence events. Phylogeographic analyses suggest isolation‐by‐distance, with two major riverbeds maybe playing a role in shaping genetic differentiation, while interploidy gene flow detected could have facilitated ancient and/or ongoing admixture between diploid and tetraploid lineages, despite geographic isolation. These findings highlight cryptic genetic diversity and emphasise the need for an integrative taxonomy that includes multiple lines of evidence: morphological, cytological, genomic, and ecological. Conservation efforts should prioritise protecting the four diploid subgroups, aided by flow cytometry, since they may harbour critical adaptive potential to both specific habitat types and/or environmental conditions. This work contributes to advancing our knowledge of evolution in polyploid complexes by combining genomic approaches and highlighting cryptic diversity in Thymus species. Future research should investigate morphometric and chemical data, hybridisation events, divergence times, diversification dynamics, and relationships with other Thymus species to further understand polyploid evolution and its impact on biodiversity.

Species delimitation remains a major challenge in understanding biodiversity, particularly in recently diverged lineages, where extensive morphological variability complicates taxonomic inference. In this study, we investigate patterns of morphological and ecological variation within Thymus sect. Mastichina , a Mediterranean lineage that includes the endangered species T. albicans . Previous genomic analyses revealed clear differentiation between two major groups corresponding to ploidy levels, as well as additional genetic structure among diploid lineages. Here, we integrate this genomic framework with detailed analyses of floral morphology and ecological niches to assess the correspondence between phenotypic, ecological, and genetic variation. Our results reveal substantial overlap among genetic lineages at both morphological and ecological levels. Although some differentiation is associated with ploidy level, particularly in floral traits, extensive variability within several lineages obscures clear taxonomic boundaries based on discrete morphological characters. By contrast, some diploid lineages (notably the Cádiz and Algarve groups) show lower morphological variability but still both overlap in morphology and ecological niche. We morphologically characterized a recently discovered diploid lineage (Hercynian) that appears to be cryptic with the tetraploid T. mastichina . Patterns of admixture involving this Hercynian lineage, especially with the diploid Doñana group, contribute to pronounced phenotypic overlap, whereas no significant effect of tetraploid introgression on diploid morphology was detected. Ecological niche analyses indicate that niche differentiation is primarily associated with ploidy level rather than among diploid lineages, except for the Hercynian group. Overall, our results demonstrate a marked discordance between phenotypic variation and genetic structure, supporting the interpretation of Thymus sect. Mastichina as a complex of cryptic evolutionary lineages. These findings highlight the limitations of morphology-based taxonomy and emphasize the need for an integrative framework incorporating genome size, genomic information, and geographic distribution for species delimitation.

Understanding gene flow between ploidy levels in polyploid complexes is essential for species delimitation and conservation. This study explores evolutionary dynamics in the polyploid complex Thymus sect. Mastichina (Lamiaceae), comprising three taxa: T. mastichina subsp. mastichina , T. mastichina subsp. donyanae , and the endangered T. albicans . Using Hyb-Seq data, phylogenomics (nuclear orthologs), and population genomics (SNPs), we confirm the section consists of two sister groups with distinct ploidy levels: a diploid and a tetraploid one. The tetraploid group shows low genetic differentiation among its populations, probably indicating rapid expansion across diverse environments. In contrast, the diploid group exhibits more complex genetic structuring, potentially shaped by geomorphology, interploidy introgression, and incipient isolation. Four diploid subgroups (Algarve, Cádiz, Doñana, and Hercynian) are identified, with reticulate evolution. The dense reticulation observed is compatible with incomplete lineage sorting in diploid lineages, due to recent and rapid divergence events. Phylogeographic analyses suggest isolation-by-distance, with two major riverbeds maybe playing a role in shaping genetic differentiation, while interploidy gene flow detected could have facilitated ancient and/or ongoing admixture between diploid and tetraploid lineages, despite geographic isolation. These findings highlight cryptic genetic diversity and emphasise the need for an integrative taxonomy that includes multiple lines of evidence: morphological, cytological, genomic, and ecological. Conservation efforts should prioritise protecting the four diploid subgroups, aided by flow cytometry, since they may harbour critical adaptive potential to both specific habitat types and/or environmental conditions. This work contributes to advancing our knowledge of evolution in polyploid complexes, by combining genomic approaches and highlighting cryptic diversity in Thymus species. Future research should investigate morphometric and chemical data, hybridisation events, divergence times, diversification dynamics, and relationships with other Thymus species to further understand polyploid evolution and its impact on biodiversity.

Premise of the Study: Although an evolutionary link between breeding system and dispersibility has been proposed, to date empirical data and theoretical models of plants show contrasting trends.Methods: We tested two competing hypotheses for the association between breeding systems and dispersibility in the heterocarpic Hypochaeris salzmanniana (Asteraceae) by using both an experimental approach and surveys over 2 years of five natural populations along an environmental cline with a gradient of pollinator availability.Key Results: Hypochaeris salzmanniana produced two types of fruits, beaked (BF) and nonbeaked (NBF), which differ in their dispersal ability. The BF were lighter and had a lower dropping velocity and higher dispersal distance than the NBF. Potential for long‐distance dispersal, measured as BF ratio per head, had high narrow‐sense heritability. Greater dispersibility and selfing ability were linked at all the scales studied. Both selfed BF and NBF fruits had longer plumes and lower plume loading than outcrossed fruits, characteristics that promote farther dispersal. Natural populations with a higher percentage of self‐compatible plants showed a higher BF ratio. Moreover, selfing led to a higher BF ratio than outcrossing.Conclusions: The avoidance of inbreeding depression seems to be the most plausible selective pressure for the greater dispersibility traits of selfed seeds. Furthermore, the ability to modulate the BF ratio and thus the potential for long‐distance dispersal of offspring based on its selfed or outcrossed origin could be advantageous, and therefore selected, under unpredictable pollination environments that favor higher dispersive selfers, which overcome both pollen limitation and inbreeding avoidance.