Summary Significant variation in plant organic compound hydrogen stable isotope (δ2H) values among species from a single location suggests species biochemistry diversity as a key driver. However, the biochemical mechanisms and the biological relevance behind this species‐specific δ2H variation remain unclear. We analyzed δ2H values of cellulose and n‐alkanes across 179 eudicot species in a botanical garden sampled in 2019, and cellulose, n‐alkanes, fatty acids and phytol δ2H values from 56 eudicot species sampled in 2020. We utilized the observed species variation in δ2H values to determine phylogenetic structure and mechanistic constraints for biochemical 2H‐fractionation. A strong phylogenetic signal in lipid compound δ2H values implies that the drivers of species variation in lipid δ2H values are evolutionarily conserved. By contrast, species variation in cellulose δ2H values was not strongly linked to phylogeny. Generally low‐explanatory power of relationships between δ2H values of different compounds (R2 < 0.26) implies nonubiquitous drivers of species variation in plant organic compound δ2H values. Historically, variable biochemical 2H‐fractionation was often attributed to δ2H values of H incorporated from NADPH. Instead, the results from this study suggest that species variation in biochemical 2H‐fractionation largely occurs independently within biosynthetic pathways. For lipids, these mechanisms appear strongly linked to evolutionary history.

The impact of large-scale chromosomal rearrangements, such as fusions and fissions, on speciation is a long-standing conundrum. We assessed whether bursts of change in chromosome numbers resulting from chromosomal fusion or fission are related to increased speciation rates in Erebia , one of the most species-rich and karyotypically variable butterfly groups. We established a genome-based phylogeny and used state-dependent birth-death models to infer trajectories of karyotype evolution. We demonstrated that rates of anagenetic chromosomal changes (i.e., along phylogenetic branches) exceed cladogenetic changes (i.e., at speciation events), but, when cladogenetic changes occur, they are mostly associated with chromosomal fissions rather than fusions. We found that the relative importance of fusion and fission differs among Erebia clades of different ages and that especially in younger, more karyotypically diverse clades, speciation is more frequently associated with cladogenetic chromosomal changes. Overall, our results imply that chromosomal fusions and fissions have contrasting macroevolutionary roles and that large-scale chromosomal rearrangements are associated with bursts of species diversification.

Genetic diversity is heterogeneously distributed among populations of the same species, due to the joint effects of multiple demographic processes, including range contractions and expansions, and mating systems shifts. Here, we ask how both processes shape genomic diversity in space and time in the classical Primula vulgaris model. This perennial herb originated in the Caucasus region and was hypothesized to have expanded westward following glacial retreat in the Quaternary. Moreover, this species is a long-standing model for mating system transitions, exemplified by shifts from heterostyly to homostyly. Leveraging a high-quality reference genome of the closely related Primula veris and whole-genome resequencing data from both heterostylous and homostylous individuals from populations encompassing a wide distribution of P. vulgaris, we reconstructed the demographic history of P. vulgaris. Results are compatible with the previously proposed hypothesis of range expansion from the Caucasus region approximately 79,000 years ago and suggest later shifts to homostyly following rather than preceding postglacial colonization of England. Furthermore, in accordance with population genetic theoretical predictions, both processes are associated with reduced genetic diversity, increased linkage disequilibrium, and reduced efficacy of purifying selection. A novel result concerns the contrasting effects of range expansion versus shift to homostyly on transposable elements, for the former, process is associated with changes in transposable element genomic content, while the latter is not. Jointly, our results elucidate how the interactions among range expansion, transitions to selfing, and Quaternary climatic oscillations shape plant evolution.

Male reproductive investment, in particular pollen production, is a crucial and ecologically relevant component of a plant's phenotype and reproductive success. Its evolutionary trajectory, however, remains understudied, partly due to a lack of convenient methods to assess it. We developed a protocol for pollen quantification by flow cytometry and applied it to 107 flowers from 38 Papaveraceae species differing widely in floral traits (e.g., floral symmetry, stamen number), pollination syndromes (e.g., wind and insect pollination) and reproductive systems (e.g., degree of autogamy). We phylogenetically tested whether pollen number evolved in association with ovule, carpel, stamen and flower numbers per inflorescence, and if there were interacting effects between floral symmetry and/or self-compatibility with pollen and ovule production. Compared to manual counts, results using flow cytometry were similar, but much faster to obtain and more precise. Pollen and ovule numbers per flower varied > 39,000x and > 550x, respectively, among species. Pollen production correlated positively with ovule, carpel and stamen numbers. Lineage-specific trajectories to pollen-to-ovule ratio reduction (to values < 300) are observed. One involved increased female investment in ruderal species belonging to the subfamily Papaveroideae, while the other occurs through decreased male investment and is associated with the evolution of floral traits towards greater specialisation. The impact of reproductive systems on male and female investment is limited to ovule production in non-actinomorphic flowers. Taken together, these results revealed that the evolutionary associations between reproductive systems, floral traits, and pollen and ovule production are lineage-specific. Given the profound contrasts at the subfamily level of Papaveraceae, broader surveys across the diversity of flowering plants are clearly needed to better understand factors driving the evolution of reproductive investment. Such studies will certainly be facilitated by our new high-throughput pollen counting method outlined here. ### Competing Interest Statement The authors have declared no competing interest.

Mountains are among the most biodiverse places on Earth, and plant lineages that inhabit them have some of the highest speciation rates ever recorded. Plant diversity within the alpine zone - the elevation above which trees cannot grow—contributes significantly to overall diversity within mountain systems, but the origins of alpine plant diversity are poorly understood. Here, we quantify the processes that generate alpine plant diversity and their changing dynamics through time in Saxifraga (Saxifragaceae), an angiosperm genus that occurs predominantly in mountain systems. We present a time-calibrated molecular phylogenetic tree for the genus that is inferred from 329 low-copy nuclear loci and incorporates 73% (407) of known species. We show that upslope biome shifts into the alpine zone are considerably more prevalent than dispersal of alpine specialists between regions, and that the rate of upslope biome shifts increased markedly in the last 5 Myr, a timeframe concordant with a cooling and fluctuating climate that is likely to have increased the extent of the alpine zone. Furthermore, alpine zone specialists have lower speciation rates than generalists that occur inside and outside the alpine zone, and major speciation rate increases within Saxifraga significantly pre-date increased rates of upslope biome shifts. Specialisation to the alpine zone is not therefore associated with speciation rate increases. Taken together, this study presents a quantified and broad scale perspective of processes underpinning alpine plant diversity.

Connaraceae is a pantropical family of about 200 species containing lianas and small trees with remarkably diverse floral polymorphisms, including distyly, tristyly, homostyly, and dioecy. To date, relationships within the family have not been investigated using a targeted molecular phylogenetic treatment, severely limiting systematic understanding and reconstruction of trait evolution. Accordingly, their last infrafamilial classification was based only on morphological data. Here, we used phylogenomic data obtained using the Angiosperms353 nuclear target sequence capture probes, sampling all tribes and almost all genera, entirely from herbarium specimens, to revise infrafamilial classification and investigate the evolution of heterostyly. The backbone of the resulting molecular phylogenetic tree is almost entirely resolved. Connaraceae consists of two clades, one containing only the African genus Manotes (4 or 5 species), which we newly recognize at the subfamily level. Vegetative and reproductive synapomorphies are proposed for Manotoideae. Within Connaroideae, Connareae is expanded to include the former Jollydoreae. The backbone of Cnestideae, which contains more than half of the Connaraceae species, remains incompletely resolved. Reconstructions of reproductive system evolution are presented that tentatively support tristyly as the ancestral state for the family, with multiple parallel losses, in agreement with previous hypotheses, plus possible re-gains. However, the great diversity of stylar polymorphisms and their phylogenetic lability preclude a definitive answer. Overall, this study reinforces the usefulness of herbarium phylogenomics, and unlocks the reproductive diversity of Connaraceae as a model system for the evolution of complex biological phenomena.

Abstract Although species radiations on island archipelagos are broadly studied, the geographic and ecological modes of speciation that underlie diversification are often not fully understood. Both allopatry and sympatry play a role during radiations, particularly on islands with profound habitat diversity. Here, we use the most diverse Canary Island plant radiation, Aeonium (Crassulaceae), to phylogenetically test two hypotheses: (1) allopatric speciation, which predicts that closely related taxa are ecologically similar but do not co-occur, and (2) sympatric speciation, whereby closely related taxa co-occur geographically but are ecologically distinct. We fitted niche and spatial distribution models based on extensive field surveys to quantify geographic and ecological divergence among taxa integrated in a phylogenetic context. Contrary to our expectation, phylogenetically closely related species tend to occupy similar ecological niches, revealing that ecological niche divergence among species accumulates slowly, even in sympatry. This suggests that evolutionary young taxa may be partially reproductively isolated due to subtle phenotypic differences, such as reproductive morphology and phenology, rather than by ecological niche and may putatively exacerbate divergence among populations. Thus, allopatry and sympatry are complementary speciation mechanisms on oceanic islands, jointly spurring this enigmatic radiation.

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods(1,2). A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome(3,4). Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins(5-7). However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes(8). This 15-fold increase in genus-level sampling relative to comparable nuclear studies(9) provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade.

Aim: Many biogeographic analyses require some form of automated state assignment to tips of phylogenetic trees, reflecting a species presence or absence in a particular area, e.g., a biome. As datasets get exponentially larger, such procedures may increasingly induce errors (here called tip-state-error), but the specific algorithmic cause and consequence on downstream estimation of dispersal and extinction rates remains poorly known. We aim to improve automated tip-scoring methods in the context of the alpine biome by leveraging elevation information. We document the profound effect of tip-state-errors on Dispersal-Extirpation-Cladogenesis (DEC) models. Location: The European Alpine Arc. Taxon: Three thousand three hundred seventeen vascular plant species, emphasizing six focal genera: Campanula, Carex, Festuca, Ranunculus, Saxifraga, and Viola. Methods: We use GBIF data to classify whether species occur above the upper climatic treeline using a newly developed algorithm ElevDistr or a gridded landscape model of thermal belts, under various filtering thresholds. We compared classification performance using the Flora Alpina as validation data. To determine if tip-state-error biases the dispersal and extirpation rate estimation, we fit DEC models for selected clades using tip-states from different classification models. Results: ElevDistr is less error prone than other approaches. Filtering thresholds lower the false positive rate but increase the false negative rate. Inflated false positive rates bias the dispersal rate estimation upward, while inflated false negative rates lead to upward bias in extirpation rate estimation. Main Conclusions: Even moderate tip-state-error may lead to profound systematic bias in dispersal and extinction rate estimation if an unbalanced ratio between false positive and false negative rates occurs. Therefore, careful validation is imperative, though ElevDistr alleviates this problem in the context of the alpine environment. Overall, our results suggest contrasting rates of alpine biome shifts across the studied genera and have major implications for studies addressing the likelihood of niche evolution versus geographic dispersal.

The botanical heritage of multitalented naturalists Paul Benedict (1856–1929) and Karl Friedrich (Fritz) (1859–1942) Sarasin of Basel is poorly known. The second-degree cousins from Basel inherited great wealth, which funded their expeditions to the tropics in India, Sri Lanka, Sulawesi, and New Caledonia. Both were doctors in zoology with interdisciplinary interests from geography and anthropology to botany. They mixed race theories with traditional descriptions of biodiversity, blurring boundaries between anthropology and natural sciences. With the help of the local colonial governments where they travelled, they avidly collected many thousands of natural history objects and human artefacts, now kept in institutions across Europe.

BACKGROUND AND AIMS:The most species-rich and ecologically diverse plant radiation on the Canary Islands is the Aeonium alliance (Crassulaceae). In island radiations like this, speciation can take place either within islands or following dispersal between islands. Aiming at quantifying intra- and inter-island speciation events in the evolution of Aeonium, and exploring their consequences, we hypothesized that (1) intra-island diversification resulted in stronger ecological divergence of sister lineages, and that (2) taxa on islands with a longer history of habitation by Aeonium show stronger ecological differentiation and produce fewer natural hybrids. METHODS:We studied the biogeographical and ecological setting of diversification processes in Aeonium with a fully sampled and dated phylogeny inferred using a ddRADseq approach. Ancestral areas and biogeographical events were reconstructed in BioGeoBEARS. Eleven morphological characters and three habitat characteristics were taken into account to quantify the morphological and ecological divergence between sister lineages. A co-occurrence matrix of all Aeonium taxa is presented to assess the spatial separation of taxa on each island. KEY RESULTS:We found intra- and inter-island diversification events in almost equal numbers. In lineages that diversified within single islands, morphological and ecological divergence was more pronounced than in lineages derived from inter-island diversification, but only the difference in morphological divergence was significant. Those islands with the longest history of habitation by Aeonium had the lowest percentages of co-occurring and hybridizing taxon pairs compared with islands where Aeonium arrived later. CONCLUSIONS:Our findings illustrate the importance of both inter- and intra-island speciation, the latter of which is potentially sympatric speciation. Speciation on the same island entailed significantly higher levels of morphological divergence compared with inter-island speciation, but ecological divergence was not significantly different. Longer periods of shared island habitation resulted in the evolution of a higher degree of spatial separation and stronger reproductive barriers.

Although species radiations on island archipelagos are broadly studied, the geographic and ecological modes of speciation that underlie diversification are often not fully understood. Both allopatry and sympatry play a role during radiations, particularly on islands with profound habitat diversity. Here, we use the most diverse Canary Island plant radiation, Aeonium (Crassulaceae), to phylogenetically test two hypotheses: (1) allopatric speciation, which predicts that closely related taxa are ecologically similar but do not co-occur, and (2) sympatric speciation, whereby closely related taxa co-occur geographically but are ecologically distinct. We fitted niche and spatial distribution models based on extensive field surveys to quantify geographic and ecological divergence among taxa integrated in a phylogenetic context. While allopatry seems to be the main driver in speciation among islands, within-island speciation occurs in sympatry. Contrary to our expectation, phylogenetically closely related species tend to occupy similar ecological niches, suggesting that ecological niche divergence among species accumulates slowly, even in sympatry. This suggests that evolutionary young taxa, may be partially reproductively isolated due to subtle phenotypic differences, such as reproductive morphology and phenology rather than by ecology and may putatively exacerbate divergence among populations. Thus, allopatry and sympatry are complementary speciation mechanisms on oceanic islands, jointly spurring this enigmatic radiation.

Sapindales is an angiosperm order of high economic and ecological value comprising nine families, c. 479 genera, and c. 6570 species. However, family and subfamily relationships in Sapindales remain unclear, making reconstruction of the order's spatio-temporal and morphological evolution difficult. In this study, we used Angiosperms353 target capture data to generate the most densely sampled phylogenetic trees of Sapindales to date, with 448 samples and c. 85% of genera represented. The percentage of paralogous loci and allele divergence was characterized across the phylogeny, which was time-calibrated using 29 rigorously assessed fossil calibrations. All families were supported as monophyletic. Two core family clades subdivide the order, the first comprising Kirkiaceae, Burseraceae, and Anacardiaceae, the second comprising Simaroubaceae, Meliaceae, and Rutaceae. Kirkiaceae is sister to Burseraceae and Anacardiaceae, and, contrary to current understanding, Simaroubaceae is sister to Meliaceae and Rutaceae. Sapindaceae is placed with Nitrariaceae and Biebersteiniaceae as sister to the core Sapindales families, but the relationships between these families remain unclear, likely due to their rapid and ancient diversification. Sapindales families emerged in rapid succession, coincident with the climatic change of the Mid-Cretaceous Hothouse event. Subfamily and tribal relationships within the major families need revision, particularly in Sapindaceae, Rutaceae and Meliaceae. Much of the difficulty in reconstructing relationships at this level may be caused by the prevalence of paralogous loci, particularly in Meliaceae and Rutaceae, that are likely indicative of ancient gene duplication events such as hybridization and polyploidization playing a role in the evolutionary history of these families. This study provides key insights into factors that may affect phylogenetic reconstructions in Sapindales across multiple scales, and provides a state-of-the-art phylogenetic framework for further research.

Mountains represent ‘experiments by nature’ that permit testing ecological theory. Using herbarium samples of 92 Rhododendron species collected between 800 and 4500 m a.sl. in the Himalaya-Hengduan Mountains region, SW China, we explored congeneric elevational trends in key plant traits at the among- and at the within-species level. We aimed at identifying the contributing factors to phenotypic trait expression across a climatic gradient, distinguishing effects in response to elevation from those due to phylogenetic relatedness, non-phylogenetic species effect, and within-species trait variability. We found that a substantial fraction of the trait variation (up to 88

Investigating the impacts of global environmental change on plants remains a challenge. Experiments that scale from leaf to ecosystem have provided important insights into the ways plants and ecosystems respond to changing environmental drivers. Yet, experiments are often short-lived and can not realistically simulate long-term environmental changes. Monitoring programs and historical observations are thus valuable alternatives for studying the impact of environmental change on plants. These are, however, unfortunately rare and often poorly suited to identify the mechanistic and physiological basis by which changes in the environment impact plant life. Analyses of biological collections and in particular herbarium materials could be a valuable complementary approach that allows assessing functional responses of plants to global environmental change.

Adaptive radiation is a significant driver of biodiversity. Primarily studied in animal systems, mechanisms that trigger adaptive radiations remain poorly understood in plants. A frequently claimed indicator of adaptive radiation in plants is growth form diversity when tied to the occupation of different habitats. However, it remains obscure whether morphological adaptations manifest as growth form diversity per se or as its constituent traits. We use the classic Aeonium radiation from the Canary Islands to ask whether adaptation across climatic space is structured by growth form evolution. Using morphological sampling with site-associated climate in a phylogenetic context, we find that growth forms dictate adaptations to the local environment. Furthermore, we demonstrate that the response of specific traits to analogous environments is antagonistic when growth forms are different. This finding suggests for the first time that growth forms represent particular ecological functions, allowing the co-occurrence of closely related species, being a product of divergent selection during evolution in sympatry.

Phylogenetic analyses are increasingly being performed with data sets that incorporate hundreds of loci. Due to incomplete lineage sorting, hybridization, and horizontal gene transfer, the gene trees for these loci may often have topologies that differ from each other and from the species tree. The effect of these topological incongruences on divergence time estimation has not been fully investigated. Using a series of simulation experiments and empirical analyses, we demonstrate that when topological incongruence between gene trees and the species tree is not accounted for, the temporal duration of branches in regions of the species tree that are affected by incongruence is underestimated, whilst the duration of other branches is considerably overestimated. This effect becomes more pronounced with higher levels of topological incongruence. We show that this pattern results from the erroneous estimation of the number of substitutions along branches in the species tree, although the effect is modulated by the assumptions inherent to divergence time estimation, such as those relating to the fossil record or among-branch-substitution-rate variation. By only analyzing loci with gene trees that are topologically congruent with the species tree, or only taking into account the branches from each gene tree that are topologically congruent with the species tree, we demonstrate that the effects of topological incongruence can be ameliorated. Nonetheless, even when topologically congruent gene trees or topologically congruent branches are selected, error in divergence time estimates remains. This stems from temporal incongruences between divergence times in species trees and divergence times in gene trees, and more importantly, the difficulty of incorporating necessary assumptions for divergence time estimation. [Divergence time estimation; gene trees; species tree; topological incongruence.]

Background Wolbachia is an endosymbiont common to most invertebrates, which can have significant evolutionary implications for its host species by acting as a barrier to gene flow. Despite the importance of Wolbachia , still little is known about its prevalence and diversification pattern among closely related host species. Wolbachia strains may phylogenetically coevolve with their hosts, unless horizontal host-switches are particularly common. We address these issues in the genus Erebia, one of the most diverse Palearctic butterfly genera. Results We sequenced the Wolbachia genome from a strain infecting Erebia cassioides and showed that it belongs to the Wolbachia supergroup B, capable of infecting arthropods from different taxonomic orders. The prevalence of Wolbachia across 13 closely related Erebia host species based on extensive population-level genetic data revealed that multiple Wolbachia strains jointly infect all investigated taxa, but with varying prevalence. Finally, the phylogenetic relationships of Wolbachia strains are in some cases significantly associated to that of their hosts, especially among the most closely related Erebia species, demonstrating mixed evidence for phylogenetic coevolution. Conclusions Closely related host species can be infected by closely related Wolbachia strains, evidencing some phylogenetic coevolution, but the actual pattern of infection more often reflects historical or contemporary geographic proximity among host species. Multiple processes, including survival in distinct glacial refugia, recent host shifts in sympatry, and a loss of Wolbachia during postglacial range expansion seem to have jointly shaped the complex interactions between Wolbachia evolution and the diversification of its host among our studied Erebia species.

Field photographs of plant species are crucial for research and conservation, but the lack of a centralized database makes them difficult to locate. We surveyed 25 online databases of field photographs and found that they harboured only about 53% of the approximately 125,000 vascular plant species of the Americas. These results reflect the urgent need for a centralized database that can both integrate and complete the photographic record of the world's flora.