The genomes of Darwin's primroses reveal chromosome-scale adaptive introgression and differential permeability of species boundaries.

Introgression is an important source of genetic variation that can determine species adaptation to environmental conditions. Yet, definitive evidence of the genomic and adaptive implications of introgression in nature remains scarce. The widespread hybrid zones of Darwin's primroses (Primula elatior, Primula veris, and Primula vulgaris) provide a unique natural laboratory for studying introgression in flowering plants and the varying permeability of species boundaries. Through analysis of 650 genomes, we provide evidence of an introgressed genomic region likely to confer adaptive advantage in conditions of soil toxicity. We also document unequivocal evidence of chloroplast introgression, an important precursor to species-wide chloroplast capture. Finally, we provide the first evidence that the S-locus supergene, which controls heterostyly in primroses, does not introgress in this clade. Our results contribute novel insights into the adaptive role of introgression and demonstrate the importance of extensive genomic and geographical sampling for illuminating the complex nature of species boundaries.

Dark-centred umbels in Apiaceae: diversity, development and evolution.

The wild carrot (Daucus carota) is famous for its dark flowers in the umbel centre. Several studies have been conducted to figure out their functional significance, but the evolution of the dark centre remains an enigma. In the present paper, we consider all known apioid species with dark-centred umbels to get a deeper understanding of their biology and evolution. Based on herbaria studies, literature and field work, we reconstructed the distribution area of 10 species (7 genera, 6 clades) of Apiaceae-Apioideae. To recognize homology of the dark structures, developmental studies were conducted in Artedia squamata and Echiophora trichophylla Field studies included architecture, flower morph distribution (andromonoecy) and flowering sequence within the plants, abundancy and behaviour of umbel visitors and preliminary manipulation experiments (removal/adding of dark structures). The dark structures are not homologous to each other. In the Daucus alliance, central flowers or umbellets are conspicuous, whereas in other species dark brush-like (A. squamata) or club-shaped structures (Dicyclophora persica, Echinophora trichophylla, Tordylium aegyptiacum, T. cappadocicum) develop from a naked receptacle. Species are andromonoecious, have a modular architecture and flower in multicyclic protandrous sequence. Among the many umbel visitors, beetles were the most abundant group. Only visitors found on umbels in both flowering phases were recognized as possible pollinators. Manipulation experiments indicated that the dark structures influence the behaviour of some, but not all umbel visitors. In Echinophora trichophylla, a massive gall infection was observed. It is evident that the dark structures evolved several times in parallel. The brush- and club-shaped structures are interpreted as the results of mutations affecting umbel development. Dark umbel centres are most likely stabilized by selection due to their general adaptive function. Their appearance in an area known as a hotspot of beetle pollination gives rise to the assumption that they may act as beetle marks.

East Asian-North American disjunctions and phylogenetic relationships within subtribe Nepetinae (Lamiaceae).

Biogeographic disjunctions, including intercontinental disjunctions, are frequent across plant lineages and have been of considerable interest to biologists for centuries. Their study has been reinvigorated by molecular dating and associated comparative methods. One of the "classic" disjunction patterns is that between Eastern Asia and North America. It has been speculated that this pattern is the result of vicariance following the sundering of a widespread Acrto-Teritary flora. Subtribe Nepetinae in the mint family (Lamiaceae) is noteworthy because it contains three genera with this disjunction pattern: Agastache, Dracocephalum, and Meehania. These disjunctions are ostensibly the result of three separate events, allowing for concurrent testing of the tempo, origin, and type of each biogeographic event. Using four plastid and four nuclear markers, we estimated divergence times and analyzed the historical biogeography of Nepetinae, including comprehensive sampling of all major clades for the first time. We recover a well-supported and largely congruent phylogeny of Nepetinae between genomic compartments, although several cases of cyto-nuclear discordance are evident. We demonstrate that the three disjunctions are pseudo-congruent, with unidirectional movement from East Asia at slightly staggered times during the late Miocene and early Pliocene. With the possible exception of Meehania, we find that vicariance is likely the underlying driver of these disjunctions. The biogeographic history of Meehania in North America may be best explained by long-distance dispersal, but a more complete picture awaits deeper sampling of the nuclear genome and more advanced biogeographical models.

Model selection, hummingbird natural history, and biological hypotheses: a response to Sazatornil et al.

We have previously suggested that a shift from bee to hummingbird pollination, in concert with floral architecture modifications, occurred at the crown of Salvia subgenus Calosphace in North America ca. 20 mya (Kriebel et al. 2020 and references therein). Sazatornil et al. (2022), using a hidden states model, challenged these assertions, arguing that bees were the ancestral pollinator of subg. Calosphace and claiming that hummingbirds could not have been the ancestral pollinator of subg. Calosphace because hummingbirds were not contemporaneous with crown subg. Calosphace in North America. Here, using a variety of models, we demonstrate that most analyses support hummingbirds as ancestral pollinators of subg. Calosphace and show that Sazatornil et al. (2022) erroneously concluded that hummingbirds were absent from North America ca. 20 mya. We contend that "biological realism" - based on timing and placement of hummingbirds in Mexico ca. 20 mya and the correlative evolution of hummingbird associated floral traits - must be considered when comparing models based on fit and complexity, including hidden states models.

Whole-genome analyses disentangle reticulate evolution of primroses in a biodiversity hotspot.

Biodiversity hotspots, such as the Caucasus mountains, provide unprecedented opportunities for understanding the evolutionary processes that shape species diversity and richness. Therefore, we investigated the evolution of Primula sect. Primula, a clade with a high degree of endemism in the Caucasus. We performed phylogenetic and network analyses of whole-genome resequencing data from the entire nuclear genome, the entire chloroplast genome, and the entire heterostyly supergene. The different characteristics of the genomic partitions and the resulting phylogenetic incongruences enabled us to disentangle evolutionary histories resulting from tokogenetic vs cladogenetic processes. We provide the first phylogeny inferred from the heterostyly supergene that includes all species of Primula sect. Primula. Our results identified recurrent admixture at deep nodes between lineages in the Caucasus as the cause of non-monophyly in Primula. Biogeographic analyses support the 'out-of-the-Caucasus' hypothesis, emphasizing the importance of this hotspot as a cradle for biodiversity. Our findings provide novel insights into causal processes of phylogenetic discordance, demonstrating that genome-wide analyses from partitions with contrasting genetic characteristics and broad geographic sampling are crucial for disentangling the diversification of species-rich clades in biodiversity hotspots.

Flower-like meristem conditions and spatial constraints shape architecture of floral pseudanthia in Apioideae.

BACKGROUND: Pseudanthia are multiflowered units that resemble single flowers, frequently by association with pseudocorollas formed by enlarged peripheral florets (ray flowers). Such resemblance is not only superficial, because numerous pseudanthia originate from peculiar reproductive meristems with flower-like characteristics, i.e. floral unit meristems (FUMs). Complex FUM-derived pseudanthia with ray flowers are especially common in Apiaceae, but our knowledge about their patterning is limited. In this paper, we aimed to investigate both the genetic and morphological basis of their development. RESULTS: We analysed umbel morphogenesis with SEM in six species representing four clades of Apiaceae subfamily Apioideae with independently acquired floral pseudanthia. Additionally, using in situ hybridization, we investigated expression patterns of LEAFY (LFY), UNUSUAL FLORAL ORGANS (UFO), and CYCLOIDEA (CYC) during umbel development in carrot (Daucus carota subsp. carota). Here, we show that initial differences in size and shape of umbel meristems influence the position of ray flower formation, whereas an interplay between peripheral promotion and spatial constraints in umbellet meristems take part in the establishment of specific patterns of zygomorphy in ray flowers of Apiaceae. This space-dependent patterning results from flower-like morphogenetic traits of the umbel which are also visible at the molecular level. Transcripts of DcLFY are uniformly distributed in the incipient umbel, umbellet and flower meristems, while DcCYC shows divergent expression in central and peripheral florets. CONCLUSIONS: Our results indicate that umbels develop from determinate reproductive meristems with flower-like characteristics, which supports their recognition as floral units. The great architectural diversity and complexity of pseudanthia in Apiaceae can be explained by the unique conditions of FUMs-an interplay between expression of regulatory genes, specific spatio-temporal ontogenetic constraints and morphogenetic gradients arising during expansion and repetitive fractionation. Alongside Asteraceae, umbellifers constitute an interesting model for investigation of patterning in complex pseudanthia.

Palynomorphological diversity among the Asteraceous honeybee flora: An aid to the correct taxonomic identification using multiple microscopic techniques.

This study aimed to characterize the palynological morphology of melliferous species of family Asteraceae belonged to seven tribes which were categorized into 15 genera and were studied under light and scanning electron microscopy. The pollen grains were acetolyzed, measured, and described qualitatively. Quantitative data were analyzed by descriptive and multivariate statistical analysis. The species were analyzed considering 11 quantitative pollen characteristics. The study revealed that the pollen grains are circular, triangular, angular, semi-angular, quadrangular, pentagonal and hexagonal amb, and 3-colporate or 3-colpate. The size of the pollen is variable among the species, ranged from 56.1 to 23.2 µm. Three pollen types: oblate spheroidal (eight species), prolate spheroidal (six species), and suboblate (two species) were observed. The surface pattern of the exine varies from echinoperforate, echinate, echinate microperforate, fenestrate, echinoperforate reticulate, microreticulate, echinate perforate, tectate, and scabrate. Exine thickness was calculated minimum in Cichorium intybus (2.47 µm) and maximum in Taraxacum campylodes (5.15 µm). A key to studied bee floral species, based on the morphological features of pollen grains, is also provided. The palynomorphological characters here analyzed can be potentially used to correctly identify the Asteraceous honeybee floral species most commonly plants visited by honeybees in the study area.

Sage Insights Into the Phylogeny of Salvia: Dealing With Sources of Discordance Within and Across Genomes.

Next-generation sequencing technologies have facilitated new phylogenomic approaches to help clarify previously intractable relationships while simultaneously highlighting the pervasive nature of incongruence within and among genomes that can complicate definitive taxonomic conclusions. Salvia L., with ∼1,000 species, makes up nearly 15% of the species diversity in the mint family and has attracted great interest from biologists across subdisciplines. Despite the great progress that has been achieved in discerning the placement of Salvia within Lamiaceae and in clarifying its infrageneric relationships through plastid, nuclear ribosomal, and nuclear single-copy genes, the incomplete resolution has left open major questions regarding the phylogenetic relationships among and within the subgenera, as well as to what extent the infrageneric relationships differ across genomes. We expanded a previously published anchored hybrid enrichment dataset of 35 exemplars of Salvia to 179 terminals. We also reconstructed nearly complete plastomes for these samples from off-target reads. We used these data to examine the concordance and discordance among the nuclear loci and between the nuclear and plastid genomes in detail, elucidating both broad-scale and species-level relationships within Salvia. We found that despite the widespread gene tree discordance, nuclear phylogenies reconstructed using concatenated, coalescent, and network-based approaches recover a common backbone topology. Moreover, all subgenera, except for Audibertia, are strongly supported as monophyletic in all analyses. The plastome genealogy is largely resolved and is congruent with the nuclear backbone. However, multiple analyses suggest that incomplete lineage sorting does not fully explain the gene tree discordance. Instead, horizontal gene flow has been important in both the deep and more recent history of Salvia. Our results provide a robust species tree of Salvia across phylogenetic scales and genomes. Future comparative analyses in the genus will need to account for the impacts of hybridization/introgression and incomplete lineage sorting in topology and divergence time estimation.

An updated tribal classification of Lamiaceae based on plastome phylogenomics.

BACKGROUND: A robust molecular phylogeny is fundamental for developing a stable classification and providing a solid framework to understand patterns of diversification, historical biogeography, and character evolution. As the sixth largest angiosperm family, Lamiaceae, or the mint family, consitutes a major source of aromatic oil, wood, ornamentals, and culinary and medicinal herbs, making it an exceptionally important group ecologically, ethnobotanically, and floristically. The lack of a reliable phylogenetic framework for this family has thus far hindered broad-scale biogeographic studies and our comprehension of diversification. Although significant progress has been made towards clarifying Lamiaceae relationships during the past three decades, the resolution of a phylogenetic backbone at the tribal level has remained one of the greatest challenges due to limited availability of genetic data. RESULTS: We performed phylogenetic analyses of Lamiaceae to infer relationships at the tribal level using 79 protein-coding plastid genes from 175 accessions representing 170 taxa, 79 genera, and all 12 subfamilies. Both maximum likelihood and Bayesian analyses yielded a more robust phylogenetic hypothesis relative to previous studies and supported the monophyly of all 12 subfamilies, and a classification for 22 tribes, three of which are newly recognized in this study. As a consequence, we propose an updated phylogenetically informed tribal classification for Lamiaceae that is supplemented with a detailed summary of taxonomic history, generic and species diversity, morphology, synapomorphies, and distribution for each subfamily and tribe. CONCLUSIONS: Increased taxon sampling conjoined with phylogenetic analyses based on plastome sequences has provided robust support at both deep and shallow nodes and offers new insights into the phylogenetic relationships among tribes and subfamilies of Lamiaceae. This robust phylogenetic backbone of Lamiaceae will serve as a framework for future studies on mint classification, biogeography, character evolution, and diversification.

Pollinator shifts, contingent evolution, and evolutionary constraint drive floral disparity in Salvia (Lamiaceae): Evidence from morphometrics and phylogenetic comparative methods.

Switches in pollinators have been argued to be key drivers of floral evolution in angiosperms. However, few studies have tested the relationship between floral shape evolution and switches in pollination in large clades. In concert with a dated phylogeny, we present a morphometric analysis of corolla, anther connective, and style shape across 44% of nearly 1000 species of Salvia (Lamiaceae) and test four hypotheses of floral evolution. We demonstrate that floral morphospace of New World (NW) Salvia is largely distinct from that of Old World (OW) Salvia and that these differences are pollinator driven; shifts in floral morphology sometimes mirror shifts in pollinators; anther connectives (key constituents of the Salvia staminal lever) and styles co-evolved from curved to linear shapes following shifts from bee to bird pollination; and morphological differences between NW and OW bee flowers are partly the legacy of constraints imposed by an earlier shift to bird pollination in the NW. The distinctive staminal lever in Salvia is a morphologically diverse structure that has evolved in concert with both the corolla and style, under different pollinator pressures, and in contingent fashion.

Taxonomic significance of foliar epidermal morphology in Lamiaceae from Pakistan.

Foliar micromorphological features are useful to elucidate the taxonomy and systematics of the Lamiaceae species. Leaf epidermal morphology using scanning electron microscopy and light microscopy of 22 Lamiaceae species from 15 genera have been investigated with an aim to solve its taxonomic problem in the correct identification. Various foliar micromorphological features were observed to explain their importance in resolving the correct identification of Lamiaceae taxa. Two main types of trichomes were observed; glandular trichomes (GTs) and nonglandular trichomes (NGTs). GTs were further divided into seven subtypes including the capitate, subsessile capitate, sessile capitate, sunken, barrel, peltate, and clavate. Similarly, NGTs were also divided into simple unicellular and multicellular including conical, falcate, cylindrical, dendrite, papillose, and short hook shape. Quantitative measurement includes the length and width of the trichomes, stomatal complex, epidermal cells, stomata, and trichomes index. Based on the foliar micromorphological characters, a taxonomic key was developed to delimit and correctly identify studied taxa. Further molecular, other anatomical and phylogenetic studies are recommended to strengthen the systematics of Lamiaceae.

Tracking temporal shifts in area, biomes, and pollinators in the radiation of Salvia (sages) across continents: leveraging anchored hybrid enrichment and targeted sequence data.

PREMISE OF THE STUDY: A key question in evolutionary biology is why some clades are more successful by being widespread geographically, biome diverse, or species-rich. To extend understanding of how shifts in area, biomes, and pollinators impact diversification in plants, we examined the relationships of these shifts to diversification across the mega-genus Salvia. METHODS: A chronogram was developed from a supermatrix of anchored hybrid enrichment genomic data and targeted sequence data for over 500 of the nearly 1000 Salvia species. Ancestral areas and biomes were reconstructed using BioGeoBEARS. Pollinator guilds were scored, ancestral pollinators determined, shifts in pollinator guilds identified, and rates of pollinator switches compared. KEY RESULTS: A well-resolved phylogenetic backbone of Salvia and updated subgeneric designations are presented. Salvia originated in Southwest Asia in the Oligocene and subsequently dispersed worldwide. Biome shifts are frequent from a likely ancestral lineage utilizing broadleaf and/or coniferous forests and/or arid shrublands. None of the four species diversification shifts are correlated to shifts in biomes. Shifts in pollination system are not correlated to species diversification shifts, except for one hummingbird shift that precedes a major shift in diversification near the crown of New World subgen. Calosphace. Multiple reversals back to bee pollination occurred within this hummingbird clade. CONCLUSIONS: Salvia diversified extensively in different continents, biomes, and with both bee and bird pollinators. The lack of tight correlation of area, biome, and most pollinator shifts to the four documented species diversification shifts points to other important drivers of speciation in Salvia.

Morphological, genetic and epigenetic aspects of homoploid hybridization between Salvia officinalis L. and Salvia fruticosa Mill.

The inheritance of phenotypic, genetic and epigenetic traits in hybridization events is difficult to predict, as numerous evolutionary, ecological, and genetic factors can play a crucial role in the process of hybridization. In the middle Adriatic island of Vis, we investigated hybridization between Salvia officinalis and S. fruticosa at morphological, genetic and epigenetic levels. SSR results revealed that hybrid individuals were characterized by diploid set of chromosomes suggesting homoploid hybridization. A well-defined group that mostly comprised of F1 generation individuals was detected. For the majority of analysed morphological characteristics, hybrids were placed in-between parental taxa, while at the same time, values of different genetic parameters were mostly higher in hybrids than in parental species. The results revealed a high contrast in the levels of phenotypic variability and epigenetic excitation between parental taxa. Environmental niche modelling confirmed that in the studied location S. officinalis experiences optimal climatological conditions, while S. fruticosa struggles with unsuitable conditions. Very low levels of gene flow between the parental species were detected. In addition, contrasting levels of epigenetic excitation in the studied groups clearly demonstrated the importance of an epigenetic response to an altered environment and confirmed the trans-generational nature of the epigenetic changes.

The diversity and evolution of pollination systems in large plant clades: Apocynaceae as a case study.

Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades.

An expanded molecular phylogeny of Plumbaginaceae, with emphasis on Limonium (sea lavenders): Taxonomic implications and biogeographic considerations.

Plumbaginaceae is characterized by a history of multiple taxonomic rearrangements and lacks a broad molecular phylogenetic framework. Limonium is the most species-rich genus of the family with ca. 600 species and cosmopolitan distribution. Its center of diversity is the Mediterranean region, where ca. 70% of all Limonium species are endemic. In this study, we sample 201 Limonium species covering all described infrageneric entities and spanning its wide geographic range, along with 64 species of other Plumbaginaceae genera, representing 23 out of 29 genera of the family. Additionally, 20 species of the sister family Polygonaceae were used as outgroup. Sequences of three chloroplast (trnL-F, matK, and rbcL) and one nuclear (ITS) loci were used to infer the molecular phylogeny employing maximum likelihood and Bayesian analyses. According to our results, within Plumbaginoideae, Plumbago forms a non-monophyletic assemblage, with Plumbago europaea sister to Plumbagella, while the other Plumbago species form a clade sister to Dyerophytum. Within Limonioideae, Ikonnikovia is nested in Goniolimon, rejecting its former segregation as genus distinct from Goniolimon. Limonium is divided into two major clades: Limonium subg. Pteroclados s.l., including L. sect. Pteroclados and L. anthericoides, and L. subg. Limonium. The latter is divided into three well-supported subclades: the monospecific L. sect. Limoniodendron sister to a clade comprising a mostly non-Mediterranean subclade and a Mediterranean subclade. Our results set the foundation for taxonomic proposals on sections and subsections of Limonium, namely: (a) the newly described L. sect. Tenuiramosum, created to assign L. anthericoides at the sectional rank; (b) the more restricted circumscriptions of L. sect. Limonium (= L. sect. Limonium subsect. Genuinae) and L. sect. Sarcophyllum (for the Sudano-Zambezian/Saharo-Arabian clade); (c) the more expanded circumscription of L. sect. Nephrophyllum (including species of the L. bellidifolium complex); and (d) the new combinations for L. sect. Pruinosum and L. sect. Pteroclados subsect. Odontolepideae and subsect. Nobiles.

Flies as pollinators of melittophilous Salvia species (Lamiaceae).

UNLABELLED: • PREMISE OF THE STUDY: Floral adaptation to a functional pollinator group does not necessarily mean close specialization to a few pollinator species. For the more than 950 species of Salvia, only bee and bird pollinations are known. Restriction to these pollinators is mainly due to the specific flower construction (lever mechanism). Nevertheless, it has been repeatedly suggested that Salvia flowers might also be pollinated by flies. Are flies able to handle the lever mechanism? Are they functionally equivalent pollinators? In this study, we compared and quantified pollen transfer by bees and flies to test whether flies are true pollinators in Salvia.• METHODS: We identified pollinators using field observations and photos. Video documentation of the visitation rate and the site of pollen placement on the pollinator body, morphometric measurements, quantification of pollen placement, pollen load, handling time, and stigma contact ratio were analyzed.• KEY RESULTS: Field investigations revealed that 19 insect species pollinated S. virgata and four pollinated S. verticillata, including 16 bee species from seven genera of the Apidae and three fly species from three genera of the Nemestrinidae and Tabanidae.• CONCLUSIONS: Flies have been found to be pollinators in primarily bee-pollinated Salvia species. This result demonstrates the potential of a given "melittoid" flower construction to broaden the range of pollinators to guarantee successful pollination and seed production. Though bees, particularly Bombus terrestris, were more efficient than flies, the study shows that flies significantly contribute to pollen transfer in Salvia.

Evaluation of cholinesterase inhibitory and antioxidant activities of wild and cultivated samples of sage (Salvia fruticosa) by activity-guided fractionation.

In European folk medicine, Salvia species have traditionally been used to enhance memory. In our previous study of 55 Salvia taxa, we explored significant anticholinesterase activity of cultivated S. fruticosa. In this study, we compared the inhibitory activity of dichloromethane, ethyl acetate, and ethanol extracts of 3 wild-grown samples and 1 cultivated sample of S. fruticosa against acetylcholinesterase and butyrylcholinesterase enzymes (which are associated with pathogenesis of Alzheimer's disease) by using the spectrophotometric Ellman method. Antioxidant activities were assessed by determining 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, iron-chelating capacity, and ferric-reducing antioxidant power. The dichloromethane extract of the cultivated sample was then subjected to fractionation by using open column chromatography and medium-pressure liquid chromatography to obtain the most active fraction by activity-guided fractionation. All fractions and subfractions were tested in the same manner, and inactive subfractions were discarded. The essential oil of the cultivated sample was analyzed by gas chromatography-mass spectrometry.