Genomic incongruence accompanies the evolution of flower symmetry in Eudicots: a case study in the poppy family (Papaveraceae, Ranunculales).

Reconstructing evolutionary trajectories and transitions that have shaped floral diversity relies heavily on the phylogenetic framework on which traits are modelled. In this study, we focus on the angiosperm order Ranunculales, sister to all other eudicots, to unravel higher-level relationships, especially those tied to evolutionary transitions in flower symmetry within the family Papaveraceae. This family presents an astonishing array of floral diversity, with actinomorphic, disymmetric (two perpendicular symmetry axes), and zygomorphic flowers. We generated nuclear and plastid datasets using the Angiosperms353 universal probe set for target capture sequencing (of 353 single-copy nuclear ortholog genes), together with publicly available transcriptome and plastome data mined from open-access online repositories. We relied on the fossil record of the order Ranunculales to date our phylogenies and to establish a timeline of events. Our phylogenomic workflow shows that nuclear-plastid incongruence accompanies topological uncertainties in Ranunculales. A cocktail of incomplete lineage sorting, post-hybridization introgression, and extinction following rapid speciation most likely explain the observed knots in the topology. These knots coincide with major floral symmetry transitions and thus obscure the order of evolutionary events.

A 160 Gbp fork fern genome shatters size record for eukaryotes.

Vascular plants are exceptional among eukaryotes due to their outstanding genome size diversity which ranges ∼2,400-fold, including the largest genome so far recorded in the angiosperm Paris japonica (148.89 Gbp/1C). Despite available data showing that giant genomes are restricted across the Tree of Life, the biological limits to genome size expansion remain to be established. Here, we report the discovery of an even larger eukaryotic genome in Tmesipteris oblanceolata, a New Caledonian fork fern. At 160.45 Gbp/1C, this record-breaking genome challenges current understanding and opens new avenues to explore the evolutionary dynamics of genomic gigantism.

The Catalan initiative for the Earth BioGenome Project: contributing local data to global biodiversity genomics.

The Catalan Initiative for the Earth BioGenome Project (CBP) is an EBP-affiliated project network aimed at sequencing the genome of the >40 000 eukaryotic species estimated to live in the Catalan-speaking territories (Catalan Linguistic Area, CLA). These territories represent a biodiversity hotspot. While covering less than 1% of Europe, they are home to about one fourth of all known European eukaryotic species. These include a high proportion of endemisms, many of which are threatened. This trend is likely to get worse as the effects of global change are expected to be particularly severe across the Mediterranean Basin, particularly in freshwater ecosystems and mountain areas. Following the EBP model, the CBP is a networked organization that has been able to engage many scientific and non-scientific partners. In the pilot phase, the genomes of 52 species are being sequenced. As a case study in biodiversity conservation, we highlight the genome of the Balearic shearwater Puffinus mauretanicus, sequenced under the CBP umbrella.

Intra-leaf modeling of Cannabis leaflet shape produces leaf models that predict genetic and developmental identities.

The iconic, palmately compound leaves of Cannabis have attracted significant attention in the past. However, investigations into the genetic basis of leaf shape or its connections to phytochemical composition have yielded inconclusive results. This is partly due to prominent changes in leaflet number within a single plant during development, which has so far prevented the proper use of common morphometric techniques. Here, we present a new method that overcomes the challenge of nonhomologous landmarks in palmate, pinnate, and lobed leaves, using Cannabis as an example. We model corresponding pseudo-landmarks for each leaflet as angle-radius coordinates and model them as a function of leaflet to create continuous polynomial models, bypassing the problems associated with variable number of leaflets between leaves. We analyze 341 leaves from 24 individuals from nine Cannabis accessions. Using 3591 pseudo-landmarks in modeled leaves, we accurately predict accession identity, leaflet number, and relative node number. Intra-leaf modeling offers a rapid, cost-effective means of identifying Cannabis accessions, making it a valuable tool for future taxonomic studies, cultivar recognition, and possibly chemical content analysis and sex identification, in addition to permitting the morphometric analysis of leaves in any species with variable numbers of leaflets or lobes.

The Plant DNA C-Values Database: A One-Stop Shop for Plant Genome Size Data.

Genome size is a plant character with far-reaching implications, ranging from impacts on the financial and computing feasibility of sequencing and assembling genomes all the way to influencing the very ecology and evolution of species. The increasing recognition of the role of genome size in plant science has led to a rising demand for comprehensive and easily accessible sources of genome size data. The Plant DNA C-values database has established itself as a trusted and widely used central hub for users needing to access available plant genome size data, complemented with related cytogenetic (ploidy level) and karyological (chromosome number) information where available. Since its inception in 2001, the database has undergone six major updates to incorporate newly available genome size information, leading to the most recent release (Release 7.1), which comprises data for 12,273 species across all the major land plant and some algal lineages. Here we describe how to use the database efficiently, making use of its different query and filtering settings.

Strategies of diaspore dispersal investment in Compositae: the case of the Andean highlands.

BACKGROUND AND AIMS: Understanding diaspore morphology and how much a species invests on dispersal appendages is key for improving our knowledge of dispersal in fragmented habitats. We investigate diaspore morphological traits in high-Andean Compositae and their main abiotic and biotic drivers and test whether they play a role in species distribution patterns across the naturally fragmented high-Andean grasslands. METHODS: We collected diaspore trait data for 125 Compositae species across 47 tropical high-Andean summits, focusing on achene length and pappus-to-achene length ratio, with the latter as a proxy of dispersal investment. We analysed the role of abiotic (temperature, elevation and latitude) and biotic factors (phylogenetic signal and differences between tribes) on diaspore traits and whether they are related to distribution patterns across the Andes, using phylogenomics, distribution modelling and community ecology analyses. KEY RESULTS: Seventy-five percent of the studied species show small achenes (length <3.3 mm) and 67% have high dispersal investment (pappus length at least two times the achene length). Dispersal investment increases with elevation, possibly to compensate for lower air density, and achene length increases towards the equator, where non-seasonal climate prevails. Diaspore traits show significant phylogenetic signal, and higher dispersal investment is observed in Gnaphalieae, Astereae and Senecioneae, which together represent 72% of our species. High-Andean-restricted species found across the tropical Andes have, on average, the pappus four times longer than the achene, a significantly higher dispersal investment than species present only in the northern Andes or only in the central Andes. CONCLUSIONS: Small achenes and high diaspore dispersal investment dominate among high-Andean Compositae, traits typical of mostly three tribes of African origin; but traits are also correlated with the environmental gradients within the high-Andean grasslands. Our results also suggest that diaspore dispersal investment is likely to shape species distribution patterns in naturally fragmented habitats.

A Bioinformatic Pipeline to Estimate Ploidy Level from Target Capture Sequence Data Obtained from Herbarium Specimens.

Whole genome duplications (WGD) are frequent in many plant lineages; however, ploidy level variation is unknown in most species. The most widely used methods to estimate ploidy levels in plants are chromosome counts, which require living specimens, and flow cytometry estimates, which necessitate living or relatively recently collected samples. Newly described bioinformatic methods have been developed to estimate ploidy levels using high-throughput sequencing data, and these have been optimized in plants by calculating allelic ratio values from target capture data. This method relies on the maintenance of allelic ratios from the genome to the sequence data. For example, diploid organisms will generate allelic data in a 1:1 proportion, with an increasing number of possible allelic ratio combinations occurring in individuals with higher ploidy levels. In this chapter, we explain step-by-step this bioinformatic approach for the estimation of ploidy level.

Giant Fern Genomes Show Complex Evolution Patterns: A Comparative Analysis in Two Species of Tmesipteris (Psilotaceae).

Giant genomes are rare across the plant kingdom and their study has focused almost exclusively on angiosperms and gymnosperms. The scarce genetic data that are available for ferns, however, indicate differences in their genome organization and a lower dynamism compared to other plant groups. Tmesipteris is a small genus of mainly epiphytic ferns that occur in Oceania and several Pacific Islands. So far, only two species with giant genomes have been reported in the genus, T. tannensis (1C = 73.19 Gbp) and T. obliqua (1C = 147.29 Gbp). Low-coverage genome skimming sequence data were generated in these two species and analyzed using the RepeatExplorer2 pipeline to identify and quantify the repetitive DNA fraction of these genomes. We found that both species share a similar genomic composition, with high repeat diversity compared to taxa with small (1C < 10 Gbp) genomes. We also found that, in general, characterized repetitive elements have relatively high heterogeneity scores, indicating ancient diverging evolutionary trajectories. Our results suggest that a whole genome multiplication event, accumulation of repetitive elements, and recent activation of those repeats have all played a role in shaping these genomes. It will be informative to compare these data in the future with data from the giant genome of the angiosperm Paris japonica, to determine if the structures observed here are an emergent property of massive genomic inflation or derived from lineage specific processes.

Novel Insights into the Nature of Intraspecific Genome Size Diversity in Cannabis sativa L.

Cannabis sativa has been used for millennia in traditional medicine for ritual purposes and for the production of food and fibres, thus, providing important and versatile services to humans. The species, which currently has a worldwide distribution, strikes out for displaying a huge morphological and chemical diversity. Differences in Cannabis genome size have also been found, suggesting it could be a useful character to differentiate between accessions. We used flow cytometry to investigate the extent of genome size diversity across 483 individuals belonging to 84 accessions, with a wide range of wild/feral, landrace, and cultivated accessions. We also carried out sex determination using the MADC2 marker and investigated the potential of flow cytometry as a method for early sex determination. All individuals were diploid, with genome sizes ranging from 1.810 up to 2.152 pg/2C (1.189-fold variation), apart from a triploid, with 2.884 pg/2C. Our results suggest that the geographical expansion of Cannabis and its domestication had little impact on its overall genome size. We found significant differences between the genome size of male and female individuals. Unfortunately, differences were, however, too small to be discriminated using flow cytometry through the direct processing of combined male and female individuals.

Hybridisation and chloroplast capture between distinct Themeda triandra lineages in Australia.

Ecotypes are distinct populations within a species that are adapted to specific environmental conditions. Understanding how these ecotypes become established, and how they interact when reunited, is fundamental to elucidating how ecological adaptations are maintained. This study focuses on Themeda triandra, a dominant grassland species across Asia, Africa and Australia. It is the most widespread plant in Australia, where it has distinct ecotypes that are usually restricted to either wetter and cooler coastal regions or the drier and hotter interior. We generate a reference genome for T. triandra and use whole genome sequencing for over 80 Themeda accessions to reconstruct the evolutionary history of T. triandra and related taxa. Organelle phylogenies confirm that Australia was colonized by T. triandra twice, with the division between ecotypes predating their arrival in Australia. The nuclear genome provides evidence of differences in the dominant ploidal level and gene-flow among the ecotypes. In northern Queensland there appears to be a hybrid zone between ecotypes with admixed nuclear genomes and shared chloroplast haplotypes. Conversely, in the cracking claypans of Western Australia, there is cytonuclear discordance with individuals possessing the coastal chloroplast and interior clade nuclear genome. This chloroplast capture is potentially a result of adaptive introgression, with selection detected in the rpoC2 gene which is associated with water use efficiency. The reason that T. triandra is the most widespread plant in Australia appears to be a result of distinct ecotypic genetic variation and genome duplication, with the importance of each depending on the geographic scale considered.

Genome Insights into Autopolyploid Evolution: A Case Study in Senecio doronicum (Asteraceae) from the Southern Alps.

Polyploidy is a widespread phenomenon across angiosperms, and one of the main drivers of diversification. Whilst it frequently involves hybridisation, autopolyploidy is also an important feature of plant evolution. Minority cytotypes are frequently overlooked due to their lower frequency in populations, but the development of techniques such as flow cytometry, which enable the rapid screening of cytotype diversity across large numbers of individuals, is now providing a more comprehensive understanding of cytotype diversity within species. Senecio doronicum is a relatively common daisy found throughout European mountain grasslands from subalpine to almost nival elevations. We have carried out a population-level cytotype screening of 500 individuals from Tête Grosse (Alpes-de-Haute-Provence, France), confirming the coexistence of tetraploid (28.2%) and octoploid cytotypes (71.2%), but also uncovering a small number of hexaploid individuals (0.6%). The analysis of repetitive elements from short-read genome-skimming data combined with nuclear (ITS) and whole plastid DNA sequences support an autopolyploid origin of the polyploid S. doronicum individuals and provide molecular evidence regarding the sole contribution of tetraploids in the formation of hexaploid individuals. The evolutionary impact and resilience of the new cytotype have yet to be determined, although the coexistence of different cytotypes may indicate nascent speciation.

The rise of grasslands is linked to atmospheric CO2 decline in the late Palaeogene.

Grasslands are predicted to experience a major biodiversity change by the year 2100. A better understanding of how grasslands have responded to past environmental changes will help predict the outcome of current and future environmental changes. Here, we explore the relationship between past atmospheric CO2 and temperature fluctuations and the shifts in diversification rate of Poaceae (grasses) and Asteraceae (daisies), two exceptionally species-rich grassland families (~11,000 and ~23,000 species, respectively). To this end, we develop a Bayesian approach that simultaneously estimates diversification rates through time from time-calibrated phylogenies and correlations between environmental variables and diversification rates. Additionally, we present a statistical approach that incorporates the information of the distribution of missing species in the phylogeny. We find strong evidence supporting a simultaneous increase in diversification rates for grasses and daisies after the most significant reduction of atmospheric CO2 in the Cenozoic (~34 Mya). The fluctuations of paleo-temperatures, however, appear not to have had a significant relationship with the diversification of these grassland families. Overall, our results shed new light on our understanding of the origin of grasslands in the context of past environmental changes.

Morphological and Genome-Wide Evidence of Homoploid Hybridisation in Urospermum (Asteraceae).

The genus Urospermum is distributed in the Mediterranean region and Macaronesia, and has been introduced to other extra-Mediterranean regions. Although the two species constituting the genus, U. dalechampii and U. picroides, are frequently found together, hybrids have so far only been reported once, from Morocco. However, we found certain individuals in Catalonia, whose intermediate morphology suggested a potential hybrid origin. In this study, we applied morphological and molecular methods to investigate the origin of those individuals. Intermediate features at phenotype, karyological, cytogenetic, and genomic levels were identified in morphologically intermediate individuals, supporting their homoploid hybrid origin. Chloroplast sequence data suggest that U. dalechampii is the maternal progenitor of the hybrid. Together with the intermediate traits displayed, the lack of fertile seeds suggests that hybrids are probably F1. Future monitoring studies will be, nonetheless, needed to evaluate the extent of hybridisation and its potential impact on the biology of the genus.

Lineage-specific vs. universal: A comparison of the Compositae1061 and Angiosperms353 enrichment panels in the sunflower family.

PREMISE: Phylogenetic studies in the Compositae are challenging due to the sheer size of the family and the challenges they pose for molecular tools, ranging from the genomic impact of polyploid events to their very conserved plastid genomes. The search for better molecular tools for phylogenetic studies led to the development of the family-specific Compositae1061 probe set, as well as the universal Angiosperms353 probe set designed for all flowering plants. In this study, we evaluate the extent to which data generated using the family-specific kit and those obtained with the universal kit can be merged for downstream analyses. METHODS: We used comparative methods to verify the presence of shared loci between probe sets. Using two sets of eight samples sequenced with Compositae1061 and Angiosperms353, we ran phylogenetic analyses with and without loci flagged as paralogs, a gene tree discordance analysis, and a complementary phylogenetic analysis mixing samples from both sample sets. RESULTS: Our results show that the Compositae1061 kit provides an average of 721 loci, with 9-46% of them presenting paralogs, while the Angiosperms353 set yields an average of 287 loci, which are less affected by paralogy. Analyses mixing samples from both sets showed that the presence of 30 shared loci in the probe sets allows the combination of data generated in different ways. DISCUSSION: Combining data generated using different probe sets opens up the possibility of collaborative efforts and shared data within the synantherological community.

Detecting Introgressed Populations in the Iberian Endemic Centaurea podospermifolia through Genome Size.

Based on results from previous studies, populations of the Iberian endemic Centaurea podospermifolia north of the Ebro River are considered genetically pure, while those southward are introgressed, with genetic input from C. cephalariifolia. This phenomenon is particularly relevant, especially given both the endangered and protected status for the species, which can have consequences in how to best apply conservation strategies to maintain genetic resources in the species. The main goal of this study was to evaluate whether genome size assessments using flow cytometry can help distinguishing between pure, hybrid and introgressed populations, and hence become a powerful and cost-effective tool to complement comprehensive population genetic surveys. The results indicate that there are significant genome size differences between populations of C. podospermifolia, which are coincident with previous considerations of pure and introgressed populations. Given the simplicity and reproducibility of this technique, flow cytometry could become an effective tool for monitoring pure populations of this species and, indeed, become an integral part of the management plans that are mandatory for listed taxa.

Low dispersal and ploidy differences in a grass maintain photosynthetic diversity despite gene flow and habitat overlap.

Geographical isolation facilitates the emergence of distinct phenotypes within a single species, but reproductive barriers or selection are needed to maintain the polymorphism after secondary contact. Here, we explore the processes that maintain intraspecific variation of C4 photosynthesis, a complex trait that results from the combined action of multiple genes. The grass Alloteropsis semialata includes C4 and non-C4 populations, which have coexisted as a polyploid series for more than 1 million years in the miombo woodlands of Africa. Using population genomics, we show that there is genome-wide divergence for the photosynthetic types, but the current geographical distribution does not reflect a simple habitat displacement scenario as the genetic clusters overlap, being occasionally mixed within a given habitat. Despite evidence of recurrent introgression between non-C4 and C4 groups, in both diploids and polyploids, the distinct genetic lineages retain their identity, potentially because of selection against hybrids. Coupled with strong isolation by distance within each genetic group, this selection created a geographical mosaic of photosynthetic types. Diploid C4 and non-C4 types never grew together, and the C4 type from mixed populations constantly belonged to the hexaploid lineage. By limiting reproductive interactions between photosynthetic types, the ploidy difference probably allows their co-occurrence, reinforcing the functional diversity within this species. Together, these factors enabled the persistence of divergent physiological traits of ecological importance within a single species despite gene flow and habitat overlap.

Biogeography and genome size evolution of the oldest extant vascular plant genus, Equisetum (Equisetaceae).

BACKGROUND AND AIMS: Extant plant groups with a long fossil history are key elements in understanding vascular plant evolution. Horsetails (Equisetum, Equisetaceae) have a nearly continuous fossil record dating back to the Carboniferous, but their phylogenetic and biogeographic patterns are still poorly understood. We use here the most extensive phylogenetic analysis to date as a framework to evaluate their age, biogeography and genome size evolution. METHODS: DNA sequences of four plastid loci were used to estimate divergence times and investigate the biogeographic history of all extant species of Equisetum. Flow cytometry was used to study genome size evolution against the framework of phylogenetic relationships in Equisetum. KEY RESULTS: On a well-supported phylogenetic tree including all extant Equisetum species, a molecular clock calibrated with multiple fossils places the node at which the outgroup and Equisetum diverged at 343 Mya (Early Carboniferous), with the first major split among extant species occurring 170 Mya (Middle Jurassic). These dates are older than those reported in some other recent molecular clock studies but are largely in agreement with a timeline established by fossil appearance in the geological record. Representatives of evergreen subgenus Hippochaete have much larger genome sizes than those of deciduous subgenus Equisetum, despite their shared conserved chromosome number. Subgenus Paramochaete has an intermediate genome size and maintains the same number of chromosomes. CONCLUSIONS: The first divergences among extant members of the genus coincided with the break-up of Pangaea and the resulting more humid, warmer climate. Subsequent tectonic activity most likely involved vicariance events that led to species divergences combined with some more recent, long-distance dispersal events. We hypothesize that differences in genome size between subgenera may be related to the number of sperm flagellae.

The Role of Botanical Families in Medicinal Ethnobotany: A Phylogenetic Perspective.

Studies suggesting that medicinal plants are not chosen at random are becoming more common. The goal of this work is to shed light on the role of botanical families in ethnobotany, depicting in a molecular phylogenetic frame the relationships between families and medicinal uses of vascular plants in several Catalan-speaking territories. The simple quantitative analyses for ailments categories and the construction of families and disorders matrix were carried out in this study. A Bayesian approach was used to estimate the over- and underused families in the medicinal flora. Phylogenetically informed analyses were carried out to identify lineages in which there is an overrepresentation of families in a given category of use, i.e., hot nodes. The ethnobotanicity index, at a specific level, was calculated and also adapted to the family level. Two diversity indices to measure the richness of reported taxa within each family were calculated. A total of 47,630 use reports were analysed. These uses are grouped in 120 botanical families. The ethnobotanicity index for this area is 14.44% and the ethnobotanicity index at the family level is 68.21%. The most-reported families are Lamiaceae and Asteraceae and the most reported troubles are disorders of the digestive and nutritional system. Based on the meta-analytic results, indicating hot nodes of useful plants at the phylogenetic level, specific ethnopharmacological research may be suggested, including a phytochemical approach of particularly interesting taxa.

Genome Size Evolution and Dynamics in Iris, with Special Focus on the Section Oncocyclus.

Insights into genome size dynamics and its evolutionary impact remain limited by the lack of data for many plant groups. One of these is the genus Iris, of which only 53 out of c. 260 species have available genome sizes. In this study, we estimated the C-values for 41 species and subspecies of Iris mainly from the Eastern Mediterranean region. We constructed a phylogenetic framework to shed light on the distribution of genome sizes across subgenera and sections of Iris. Finally, we tested evolutionary models to explore the mode and tempo of genome size evolution during the radiation of section Oncocyclus. Iris as a whole displayed a great variety of C-values; however, they were unequally distributed across the subgenera and sections, suggesting that lineage-specific patterns of genome size diversification have taken place within the genus. The evolutionary model that best fitted our data was the speciational model, as changes in genome size appeared to be mainly associated with speciation events. These results suggest that genome size dynamics may have contributed to the radiation of Oncocyclus irises. In addition, our phylogenetic analysis provided evidence that supports the segregation of the Lebanese population currently attributed to Iris persica as a distinct species.

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.