American Journal of Botany

Little is known about environmental drivers of opportunities for hybridization, but its phylogenetic distribution across species and areas is heterogeneous, suggesting that ecological traits may play an important role in concert with postzygotic isolation. Because plant-pollinator interactions are responsible for gene flow in most plant species, differences in the mosaic landscape of plant-pollinator interactions could explain why some plants are particularly prone to hybridization. Prezygotic isolation is mediated by sometimes complex pollen presentation; conversely, conserved pollination strategies would lead to evolutionary constraints on pollinator assemblage divergence in the speciation process and therefore predict higher opportunities for gene flow, although this hypothesis has yet to be tested. The plant taxonomic tribe Heuchereae (Saxifragaceae) is a well-characterized system for pollinator interactions and particularly for floral scent, the primary pollinator attractant in the group. Floral volatile organic compounds (VOCs) in this clade are hypervariable at the population level and are thought to be responsible for pollination selectivity, leading to divergent pollinator assemblages. Observing a contrast of hybridizing and non-hybridizing species, the levels of attractant divergence may therefore predict levels of hybridization. We investigated pollination biology in the plant genus Heuchera, notable for frequent interspecific gene flow compared to tribal relatives, asking whether high rates of hybridization may be associated with low interspecific divergence of VOCs and the pollinator assemblages they shape, using as our system the hybrid zone between H. americana var. americana and H. richardsonii in the midwestern USA. We optimized a closed-space collection and GC-MS (gas chromatography-mass spectrometry) protocol to characterize VOCs in Heuchera flowers. To identify floral visitation and effective pollinators we conducted pollination observations at 40 Heuchera populations over the span of two field seasons. GC-MS data from 89 Heuchera specimens representing 69 populations suggests that classes of VOCs, and to a large extent individual compounds, are shared within the hybrid complex while other Heuchera that are not thought to hybridize with these species have distinct species-specific compounds. Pollination observations and metabarcoding of pollinator pollen loads confirm shared effective pollinators in the hybrid zone and between adjacent parental populations. Attractant and visitation data considered together suggest that conservatism of pollinator interactions may be a typical feature associated with frequent hybridizers, perhaps arising from developmental or biochemical constraints on prezygotic isolation, and more broadly that the macroevolution of isolation mechanisms may be predictive of natural hybridization rate.

Gene flow to marginal populations at a species range edge can facilitate rapid adaptation by increasing genetic diversity, reducing inbreeding depression, and introducing novel alleles. In highly inbred populations, hybrid vigor is often observed in the first generation (F1), but hybrid breakdown may diminish fitness in subsequent generations. Thus, benefits of gene flow may be overestimated when only F1 performance is assessed. We tested whether gene flow among populations of the annual plant Erythranthe laciniata (A. Gray) G.L. Nesom, from similar and contrasting environments, confers persistent fitness advantages across F1 and F2 generations at the high-elevation edge of its range in the California Sierra Nevada. Gene flow was experimentally introduced through pollen transfer between cold-edge populations, between cold edge and central populations, and within local cold edge populations, and compared to self-fertilized offspring, the predominant mating strategy of E. laciniata. For F1 progeny, we measured morphological, phenological, and fitness traits in a common garden located near the cold-climate range limit during 2008-2009, a relatively average year, and for F2 progeny in 2009-2010, a relatively wet year. Although F1 crosses showed no initial performance advantage measured in the previous year, F2 progeny from center-to-edge and edge-to-edge crosses significantly outperformed selfed and locally outcrossed lines in fruit mass, total pedicels, biomass, and height. Our findings demonstrate that gene flow can confer long-term fitness benefits, especially among populations adapted to similar selective pressures, and highlight the potential of assisted gene flow to bolster or rescue peripheral populations facing climate change. SIGNIFICANCE STATEMENTSpecies living at the edges of their geographic ranges often have small, isolated populations with limited genetic diversity, which can restrict their ability to adapt to environmental change. Gene flow from other populations may increase adaptive potential, but its long-term consequences remain uncertain because most studies evaluate only first-generation hybrids. Using experimental crosses in the mountain wildflower Erythranthe laciniata, we show that gene flow can produce stronger fitness benefits in second-generation hybrids than in the first generation at a high-elevation range edge. These results suggest that recombination among populations can generate advantageous genetic combinations that emerge over multiple generations. Our findings highlight the potential for assisted gene flow to enhance adaptation and persistence of range-edge populations under climate change.

Premise of the ResearchThe prevalence of sexual dimorphisms, which evolve due to contrasting strategies to maximize reproductive success in males and females, is variable among dioecious plant species. In the Salicaceae, many traits have been assessed across many studies, but direct or indirect associations between these traits and reproductive allocation are often neglected. Given the dynamic evolution of sex determination systems and the strong interest in wood production in the family, we wondered whether sexual dimorphisms related to reproduction may have gone unreported. Here, we assess sexual dimorphism in reproductive traits in two species of Salix. Recognition of reproductive sexually dimorphic traits will contribute to understanding the evolution of sex determination systems in the Salicaceae. MethodologyWe conducted observational studies in natural populations to assess the presence of sexual dimorphisms in early spring bud density, catkin number, and flower number per catkin across four sampling periods in Salix exigua. We also analyzed flower number and catkin number per flower in Salix nigra. Pivotal ResultsWe observed no sexual dimorphism in pre-season buds per branch in S. exigua but did find that males produced more flowers per catkin and more catkins per branch than females in both S. exigua and S. nigra. ConclusionsHigher flower numbers in males compared to females is consistent with expectations from intra-sexual selection among males. The presence of reproductive sexual dimorphisms in Salix suggests that sexual selection may affect the evolution of mating strategies in Salix species, and the evolution of the sex determination system within this genus.

The genus Solidago represents a taxonomically challenging group due to its sheer number of species, putative hybridization, polyploidy, and shallow genetic divergence among species. Here we use a dataset obtained exclusively from herbarium specimens to evaluate the status of Solidago ulmifolia var. palmeri, a morphologically subtle taxon potentially confined to Alabama, Arkansas, Mississippi, and Missouri. A multivariate analysis of both discrete and continuous morphological data revealed no clear distinction between S. ulmifolia var. palmeri and Solidago ulmifolia var. ulmifolia. Solidago ulmifolia var. palmeris status was also assessed with a phylogenomic and SNP clustering analysis of data generated with the "Angiosperms353" probe kit. Neither analysis supported Solidago ulmifolia var. palmeri as a distinct taxon, and we suggest that this name should be discarded. The status of Solidago delicatula (formerly known as Solidago ulmifolia var. microphylla) was also assessed. Both morphological and phylogenic analyses supported the species status of S. delicatula and we suggest maintaining this species at its current rank. These results highlight the utility of the Angiosperms353 probe kit, both with herbarium tissue and at lower taxonomic levels. Indeed, this is the first study to utilize this kit to identify genetic groups within a species.

Recent reviews of isolating barriers in plants conclude that prezygotic barriers play an outsized role in plant speciation; yet these conclusions derive overwhelmingly from studies of sympatric, perennial herbs in temperate zones, and at later stages of speciation. Trees possess several traits that are expected to influence barrier evolution, including prolonged generation times and reproduction, predominant outcrossing, and long-distance gene flow. We examined early-evolving post-pollination barriers between ecologically diverged, vegetatively distinct varieties of the tree species, Metrosideros polymorpha, that have a common floral morphology and highly overlapping flowering times. We performed controlled crosses between each of Hawaii Islands four varieties and maternal trees of the high-elevation variety and examined pollen-tube growth, fruit set, seed germination, and seedling phenotype. We then monitored survivorship, maturation rate, and fertility of F1 hybrids over [≥]8 years alongside parental controls and a fourth F1 genotype derived from companion studies. The four F1 crosses showed four contrasting patterns and strengths of predominantly postzygotic isolation, including high F1 mortality that manifested over several years. Results from this and other tree studies suggest that ecological speciation in trees follows the classical speciation model of early postzygotic barrier formation followed by reinforcement, whenever stable environments promote recurring hybridization.

PREMISE OF THE STUDYThe size and shape (physiognomy) of woody, dicotyledonous angiosperm leaves are correlated with climate and these relationships have been used to develop. proxies. These proxies assume that leaf morphology plastically responds to meteorological conditions and that leaf traits change isometrically through development.\n\nMETHODSWe used Digital Leaf Physiognomy (DiLP) to measure leaf characters of multiple Vitis species from the USDA Germplasm Repository in Geneva, NY from the 2012-2013 and 2014-2015 growing seasons. These growing seasons had different temperature and precipitation.\n\nKEY RESULTSWe found three primary results: (1) there were predictable significant differences in leaf characters in leaves of different developmental stages along the vine, (2) there were significant differences in leaf characters in leaves of the same developmental stage between the growing seasons, and (3) there were significant differences in leaf characters between growing seasons.\n\nCONCLUSIONSWe found that Vitis leaf shape had the strongest relationship with growing season meteorological conditions in taxa growing in their native range. In addition, leaves have variable phenotypic plasticity along the vine. We interpret that the meteorological signal was strongest in those leaves that have completed allometric expansion. This is significant for leaf physiognomic-paleoclimate proxies because these leaves are most likely to be preserved in leaf litter and reflect the type of leaves included in paleoclimate reconstructions. We found that leaf development does have the potential to be a confounding factor, but it is unlikely to exert a significant influence on analysis due to differential preservation potential.

SO_SCPLOWUMMARYC_SCPLOWO_LIThe tree of life is riddled with reticulate evolutionary histories, and some clades, such as the eastern standing Phlox, appear to be hotspots of hybridization. In this group there are two cases of reinforcement and nine hypothesized hybrid species. Given the historical importance of this group for our understanding of plant speciation, the relationships of these taxa and the role of hybridization and gene flow in their diversification require genomic validation. C_LIO_LIUsing phylogenomic analyses, we resolve the evolutionary relationships of the eastern standing Phlox and test hypotheses about if and how hybridization and gene flow were creative forces in their diversification. C_LIO_LIOur results provide novel resolution of the phylogenetic relationships in this group, including well-supported paraphyly across some species. We identify gene flow during one of two cases of reinforcement and find support for one of the five hypothesized homoploid hybrid speciation events. Additionally, we infer the ancestries of four allotetraploid hybrid species. C_LIO_LIHybridization has contributed to diverse evolutionary outcomes within this Phlox group; although, not as extensively as previously hypothesized. This study demonstrates the importance of phylogenomics in confirming hypothesized histories of non-model systems and adds to the growing evidence of interspecific genetic exchange in the generation of biodiversity. C_LI

PREMISE OF THE STUDYLife history is an important predictor of population genetic variation, although this link remains unexplored in numerous important plant lineages. One such lineage is the legume genus Strophostyles, which contains both annual and herbaceous perennial vines native to eastern North America. Specifically, it remains to be determined whether Strophostyles species with different life histories show different patterns of genetic differentiation and diversity, as well as if these species hybridize across their range. METHODSHere we sampled the perennial Strophostyles helvola and annual S. leiosperma in five sites across a latitudinal transect in the central United States, including three sites where the species occur in sympatry. Using genotyping-by-sequencing, we identified 5556 polymorphic SNPs across 166 individuals. KEY RESULTSThere is no evidence that Strophostyles helvola and S. leiosperma hybridize in the populations examined. Within species, Strophostyles helvola (perennial) displays admixture among populations, while S. leiosperma (annual) does not, although both species show more genetic diversity among rather than within populations. Patterns of genetic diversity are varied across populations of both species, with both heterozygote excess and deficiency. CONCLUSIONSThe complex patterns of genetic differentiation and diversity warrant further investigation of life history and population dynamics in Strophostyles, particularly mating system and modes of gene flow. This study exemplifies the diversity of population genetic patterns even within a small genus, and it reinforces the need to characterize such diversity in non-model systems to gain a more complete understanding of how life history contributes to population genetics.

Sexual dimorphism in plant growth and/or reproductive responses to the surrounding environment has been documented in some plant species. In gynodioecious plants, it is especially important to understand whether females and hermaphrodites differ in their response to environmental stressors, as the fitness of females relative to hermaphrodites determines the extent to which these separate sexes are maintained in natural populations. Soil nutrient availability is of particular importance given the different nutrient requirements of male and female sexual functions in plants. Here, we evaluated and compared the growth of females and hermaphrodites of Geranium maculatum in response to varying levels of nutrients. Using a greenhouse experiment, we manipulated the overall nutrient, nitrogen, and phosphorus levels in the soil and measured growth, allocation, and leaf quality responses in both females and hermaphrodites. We found that sexes responded similarly in their growth and allocation responses to nutrient availability, albeit evidence that female leaf chlorophyll content may have increased more than that of hermaphrodites across soil nitrogen levels. Our findings demonstrate that hermaphrodites differ from females in terms of their physiological response to varying nutrient levels, however these physiological differences did not translate into meaningful growth or reproduction differences.

PremiseIn plants, meristic traits, such as petal and sepal numbers, are usually considered invariant within taxa, yet certain species consistently exhibit great variability in these traits. The factors contributing to "atypical" counts are not well-known, published hypotheses include relaxation of pollinator selection, inbreeding, and hybridization, among others. The sand verbenas, Abronia (Nyctaginaceae), usually have five perianth lobes ( petals), yet certain taxa exhibit marked departures from this norm. MethodsHere we integrate an analysis of images from community science data (iNaturalist) and common garden experiments to evaluate a comprehensive set of adaptive and nonadaptive explanations for the production of these atypical flowers across an evolutionary transition from xenogamy (outcrossing) to autogamy (selfing) in the coastal sand verbena Abronia umbellata. Key resultsThe shift to autogamy in this lineage correlated with a higher frequency of atypical flowers from ~7% to ~20% and a significant reduction in mean petal number per inflorescence. Autogamous success did not change with petal number, and neither hybridization or up to three generations of inbreeding consistently increased production of atypical flowers or decreased mean petal number, all in contrast to previously-published hypotheses. In contrast, intra-inflorescence, inter-plant (intra-population), inter-population, and inter-variety comparisons demonstrated a correlation of reduced floral size with reduced petal number, suggesting correlated evolution due to a well-established relation between organ number and meristem size. ConclusionsThe reduction in petal number was probably a consequence of selection for smaller flowers associated with increased selfing. While we could not completely eliminate several alternative hypotheses, including a long-term history of inbreeding or relaxed selection on petal number constancy, those are less likely to explain the observed changes, though they may have contributed to the trend. In general, we develop a framework of hypotheses for evolutionary investigations of meristic variation in floral organs.

The genus Rumex is a unique member of the Polygonaceae (Buckwheat) family of plants. A source of intrigue for Rumex lies in the diversity of the reproductive systems associated with the subgenera, species, and subspecies within this genus. Four previously circumscribed subgenera, some 200 species, and a number of subspecies comprise the collective Rumex genus. These species exhibit monoecious, dioecious, synoecious (hermaphroditic), and polygamous reproductive systems. Moreover, some of the dioecious species contain sex chromosomes, a phenomenon that is very rare in angiosperms. Apart from the confirmed morphological and phytogeographical distinctions, two of the four described subgenera, Acetosa and Acetosella, are distinctive in their exhibited sex chromosome systems. For this study, we used three chloroplast markers, rbcL, trnH-psbA, trnL-F, and dense taxon sampling, to reconstruct a molecular phylogeny for Rumex.The reconstructed phylogeny for this work resolves six major clades and one large grade in Rumex. In addition, the species with known dioecious reproductive systems derived from unique sex chromosome systems are resolved in two different clades nested within "the dioecious clade". These results suggest that the species with divergent sexual systems are more closely related to each other than to other species comprising the rest of the Rumex genus. Furthermore, some species with known synoecious reproductive systems are resolved in a single clade which is also nested within "the dioecious clade". These results imply a possible reversal occurring over time which suggests the highly plastic nature of reproductive systems among Rumex species.

PremiseUnderstanding how arid-adapted plants have diversified in harsh environments is a central question in evolutionary biology. Linanthus (Polemoniaceae) occurs in biodiverse dry areas of Western North America and exhibits extensive floral trait variation, multiple color polymorphisms, differences in blooming time, and variation in life history strategies. Here, we reconstruct the evolutionary history of this group. MethodsWe generated restriction-site associated (ddRAD) sequences for 180 individuals and target capture (TC) sequences for 63 individuals, with complete species sampling. Using maximum likelihood and pseudo-coalescent approaches, we inferred phylogenies of Linanthus and used these phylogenies to model the evolution of phenotypic traits and investigate the geographic speciation history of this genus. Key resultsShallow relationships are consistent and well supported with both ddRAD and TC data. Most species are monophyletic despite rampant local sympatry and range overlap, suggesting strong isolating barriers. The non-monophyly of some species is possibly due to rapid speciation or issues with current species delimitation. Perenniality likely evolved from annuality, a rare shift in angiosperms. Night blooming evolved three times independently. Flower color polymorphism is an evolutionarily labile trait and is likely ancestral. No single geographic mode of speciation characterizes the radiation but most species overlap in range, suggesting they evolved in parapatry. ConclusionsOur results illustrate the complexity of phylogenetic inference for recent radiations, even with multiple sources of genomic data and extensive sampling. This analysis provides a foundation to understand aridity adaptations, such as evolution of flower color polymorphisms, night blooming, and perenniality, as well as speciation mechanisms.

Solanaceae are a globally distributed family of considerable economic importance. Recent phylogenetic work on various clades in the family has resulted in a plethora of names at various taxonomic levels. This results in a confusing set of names applied to clades at different ranks, and to the use of informal names that are often not accepted in online resources. To solve this problem, we present here a phylogenetic classification following the PhyloCode for the Solanaceae. Traditional taxon names previously recognized at the ranks of tribe, genus, and section were maintained when associated with clades. We base our clade definitions on a newly constructed phylogeny of the family using 10 plastid and nuclear markers with a broad sampling of 1,474 species, roughly half of the diversity of the family. We complement this marker-based analysis with a phylogenomic analysis of 110 taxa (including 97 of the 102 genera in the family) using the Angiosperms353 probe set. This pair of analyses produced highly concordant phylogenies and provides strong support for the clades included in the classification. In total, we propose and define rank-free names for 38 major clades, including 16 infrageneric taxa within the mega-diverse Solanum. This rank-free system intends to promote name stability as new data become available, avoiding name changes to well-known groups.

Premise of researchWhat maintains mixed selfing and outcrossing is an enduring mystery in evolutionary biology. Cleistogamy, where individuals produce both potentially outcrossing chasmogamous and obligately selfing cleistogamous flowers, provides an ideal framework in which to study the evolutionary forces maintaining mixed-mating. Despite their promise for providing insight into mating system evolution, few studies of cleistogamous species have comprehensively investigated the relative costs and benefits of cleistogamous selfing. MethodologyWe quantified the necessary components to calculate the relative energetic cost of reproduction of the different flower types in three natural populations of the perennial Ruellia humilis Nutt (Acanthaceae). These components include flower dry mass, fertility (fruit set), and seed mass and number per fruit. We also measured pollen-ovule ratios for both flower types as another measure of relative energetic investment. We additionally tracked phenology of the two flower types and used the proportion of chasmogamous flowers to estimate maximum potential outcrossing rates. Pivotal ResultsWe found that the energetic cost of reproduction via cleistogamous flowers was about 4-10 times less than that of reproduction via chasmogamous flowers, and this energetic economy was from both reduced mass and increased fertility of cleistogamous flowers. Pollen ovule ratios in cleistogamous flowers were one third to one half those of chasmogamous flowers, providing additional support for their energetic economy. Maximum potential outcrossing rates in these populations based on chasmogamous flower production were between 43-61%, but chasmogamous flowers can autogamously self at rates of 33-75%. ConclusionsThe relative energetic economy of cleistogamous flowers suggest that progeny from chasmogamous flowers would have to have 4-10 times greater relative fitness to explain their evolutionary maintenance. These values are likely even greater considering the reduced investment in pollen production in cleistogamous flowers. Ongoing work will quantify potential advantages of chasmogamous flowers due to inbreeding depression and heterosis.

O_LIFloral nectar is a crucial energy source for many insects, yet its availability is rarely quantified at broad taxonomic and geographic scales. Mosquitoes are opportunistic nectar feeders whose survival and vectorial capacity depend on access to sugar resources, but the extent to which nectar sugar availability can be predicted from floral morphology across floras remains poorly understood. C_LIO_LIWe analysed relationships between corolla morphology and nectar traits in 156 flowering plant species from the UK and Zambia. Corolla length, base width and floral shape were measured alongside nectar volume and sugar concentration. Linear and linear mixed-effects models were used to assess predictive relationships in tubular and non-tubular flowers, and phylogenetic statistics (Blombergs K and Pagels {lambda}) were applied to test for evolutionary signal. C_LIO_LINectar volume and sugar content increased with tube length and width in tubular flowers, while corolla width and shape were strong predictors in non-tubular flowers. Phylogenetic analyses across 154 species (42 families) indicated weak phylogenetic signal in nectar volume. C_LIO_LISimple floral traits provide proxies for nectar sugar availability across lineages. Shape-specific scaling relationships suggest convergent responses to ecological and morphological constraints, offering a practical framework for predicting nectar resources and linking floral diversity to mosquito and pollinator ecology. C_LI

Using conventional statistical approaches there exist powerful methods to classify shapes. Embedded in morphospaces is information that allows us to visualize theoretical leaves. These unmeasured leaves are never considered nor how the negative morphospace can inform us about the forces responsible for shaping leaf morphology. Here, we model leaf shape using an allometric indicator of leaf size, the ratio of vein to blade areas. The borders of the observable morphospace are restricted by constraints and define an orthogonal grid of developmental and evolutionary effects which can predict the shapes of possible grapevine leaves. Leaves in the genus Vitis are found to fully occupy morphospace available to them. From this morphospace we predict the developmental and evolutionary shapes of grapevine leaves that are not only possible, but exist, and argue that rather than explaining leaf shape in terms of discrete nodes or species, that a continuous model is more appropriate.

Anthropogenic climate change and land-use changes threaten the health and survival of plants, particularly by altering the microclimatic conditions of habitats. Plant productivity is highly sensitive to these abiotic conditions that influence their morphological and physiological traits. I studied this relationship in plants of the Arctostaphylos genus, commonly known as manzanita, shrubs in chaparral ecosystems across California. I assessed microclimate and examined its effects on leaf morphological traits and plant productivity of A. crustacea ssp. crustacea across two sites in Alameda County, California. At the site of higher sunlight and less soil moisture (VWC), leaves had a greater mass per area (LMA) and were more steeply angled, and photosynthesis was significantly higher (11.14 {micro}molCO2/m2s) than leaves at the site of less sunlight and more VWC (7.94 {micro}molCO2/m2s). Results from linear mixed models showed that light level was the overall strongest predictor variable for plant traits, with vapor pressure deficit and VWC also contributing to LMA, and leaf temperature and leaf angle distribution also influencing photosynthesis. Overall, no individual microclimatic variable was the sole contributing predictor of a leaf morphological trait or photosynthesis. Rather a combination and interaction of microclimatic conditions influenced plant functional traits, though some conditions had greater influence than others. The functional traits of A. crustacea ssp. crustacea adjusted in response to microclimatic factors, showing intraspecific trait variation (ITV) of this species. ITV is an essential defense for plant resilience that allows for adaptations in the face of rapidly changing climatic conditions.

O_LIStudies into the evolution and development of leaf shape have connected variation in plant form, function, and fitness. For species with consistent leaf margin features, patterns in leaf architecture are related to both biotic and abiotic factors. However, for species with inconsistent leaf margin features, quantifying leaf shape variation and the effects of environmental factors on leaf shape has proven challenging. C_LIO_LITo investigate leaf shape variation in species with inconsistent shapes, we analyzed approxi-mately 500 digitized Capsella bursa-pastoris specimens collected throughout the continental U.S. over a 100-year period with geometric morphometric modeling and deterministic techniques. We generated a morphospace of C. bursa-pastoris leaf shapes and modeled leaf shape as a function of environment and time. C_LIO_LIOur results suggest C. bursa-pastoris leaf shape variation is strongly associated with temperature over the C. bursa-pastoris growing season, with lobing decreasing as temperature increases. While we expected to see changes in variation over time, our results show that level of leaf shape variation is consistent over the 100-year period. C_LIO_LIOur findings showed that species with inconsistent leaf shape variation can be quantified using geometric morphometric modeling techniques and that temperature is the main environmental factor influencing leaf shape variation. C_LI

As global commitments to restoration are underway, science is needed to support capacity to achieve meaningful gains for ecosystems and human communities. In Chile, identification and generation of appropriate plant material is a barrier to achieving major restoration goals under the Paris Climate Agreement. Understanding genetic differentiation among plant populations is needed to maximize restoration success. For Araucaria araucana, a highly threatened iconic South American tree, this information is greatly needed to guide restoration and conservation efforts because this species occurs across a strong climate gradient. We grew seedlings from 12 populations of A. araucana across its range in Chile in a common garden to assess regional (coastal versus Andes mountain ranges) and population variation in key plant traits and relate this variation to environmental variables. We demonstrate that A. araucana is differentiated within regions and populations across its range in Chile by a suite of traits, particularly branch number and length (showing plant architectural differences) and needle width (showing leaf investment differences). We show that this variation is at least partly explained by climate and soil variables, with the most variation explained by differences between regions in temperature annual range. Thus, we recommend that restoration efforts focus on conserving genetic variation among and within regions and their populations and preventing the translocations of genotypes between coastal and Andes populations.

Mycorrhizal fungi form essential mutualisms with the majority of land plants, yet their role on plant community composition and diversification remains poorly understood. Orchids provide a model system for studying these interactions because the orchid life cycle obligatorily depends on mycorrhizae. Historically, studies have emphasized the role of niche partitioning and competition avoidance resulting in distinct mycobiome compositions among coexisting orchids. Since closely related orchid species have been found to associate with similar groups of fungi, it has been speculated that different fungal species are needed for coexistence but not for speciation. However, fungi have often been examined at lower resolution levels (i.e., class, order, family, or genus). Using third-generation long-read sequencing (PacBio) of the full ITS region, we characterized fungal communities at fine scale with high accuracy. We evaluated alpha- and beta-diversity of fungal communities in four closely related, narrowly endemic epiphytic orchid species from the rapidly diversifying genus Lepanthes in one of the worlds richest biodiversity hotspots. Our analyses reveal that orchid species have distinct mycobiont assemblages, with differences in composition unevenly distributed across species. These results suggest that shifts in fungal partners may contribute to speciation and rapid diversification in Lepanthes. This study highlights the potential evolutionary role of mycorrhizal fungi in orchid diversification and demonstrates the value of high-resolution sequencing in uncovering cryptic fungal diversity.