Variability in the analysis of a single neuroimaging dataset by many teams.

Data analysis workflows in many scientific domains have become increasingly complex and flexible. Here we assess the effect of this flexibility on the results of functional magnetic resonance imaging by asking 70 independent teams to analyse the same dataset, testing the same 9 ex-ante hypotheses1. The flexibility of analytical approaches is exemplified by the fact that no two teams chose identical workflows to analyse the data. This flexibility resulted in sizeable variation in the results of hypothesis tests, even for teams whose statistical maps were highly correlated at intermediate stages of the analysis pipeline. Variation in reported results was related to several aspects of analysis methodology. Notably, a meta-analytical approach that aggregated information across teams yielded a significant consensus in activated regions. Furthermore, prediction markets of researchers in the field revealed an overestimation of the likelihood of significant findings, even by researchers with direct knowledge of the dataset2-5. Our findings show that analytical flexibility can have substantial effects on scientific conclusions, and identify factors that may be related to variability in the analysis of functional magnetic resonance imaging. The results emphasize the importance of validating and sharing complex analysis workflows, and demonstrate the need for performing and reporting multiple analyses of the same data. Potential approaches that could be used to mitigate issues related to analytical variability are discussed.

Circadian-Dependent Intermittent Fasting Influences Ischemic Tolerance and Dendritic Spine Remodeling.

BACKGROUND: Preconditioning by intermittent fasting is linked to improved cognition and motor function, and enhanced recovery after stroke. Although the duration of fasting was shown to elicit different levels of neuroprotection after ischemic stroke, the impact of time of fasting with respect to the circadian cycles remains unexplored. METHODS: Cohorts of mice were subjected to a daily 16-hour fast, either during the dark phase (active-phase intermittent fasting) or the light phase (inactive-phase intermittent fasting) or were fed ad libitum. Following a 6-week dietary regimen, mice were subjected to transient focal cerebral ischemia and underwent behavioral functional assessment. Brain samples were collected for RNA sequencing and histopathologic analyses. RESULTS: Active-phase intermittent fasting cohort exhibited better poststroke motor and cognitive recovery as well as reduced infarction, in contrast to inactive-phase intermittent fasting cohort, when compared with ad libitum cohort. In addition, protection of dendritic spine density/morphology and increased expression of postsynaptic density protein-95 were observed in the active-phase intermittent fasting. CONCLUSIONS: These findings indicate that the time of daily fasting is an important factor in inducing ischemic tolerance by intermittent fasting.

Incorporating plant phenological responses into species distribution models reduces estimates of future species loss and turnover.

Anthropogenetic climate change has caused range shifts among many species. Species distribution models (SDMs) are used to predict how species ranges may change in the future. However, most SDMs rarely consider how climate-sensitive traits, such as phenology, which affect individuals' demography and fitness, may influence species' ranges. Using > 120 000 herbarium specimens representing 360 plant species distributed across the eastern United States, we developed a novel 'phenology-informed' SDM that integrates phenological responses to changing climates. We compared the ranges of each species forecast by the phenology-informed SDM with those from conventional SDMs. We further validated the modeling approach using hindcasting. When examining the range changes of all species, our phenology-informed SDMs forecast less species loss and turnover under climate change than conventional SDMs. These results suggest that dynamic phenological responses of species may help them adjust their ecological niches and persist in their habitats as the climate changes. Plant phenology can modulate species' responses to climate change, mitigating its negative effects on species persistence. Further application of our framework will contribute to a generalized understanding of how traits affect species distributions along environmental gradients and facilitate the use of trait-based SDMs across spatial and taxonomic scales.

A new nuclear phylogeny of the tea family (Theaceae) unravels rapid radiations in genus Camellia.

Molecular analyses of rapidly radiating groups often reveal incongruence between gene trees. This mainly results from incomplete lineage sorting, introgression, and gene tree estimation error, which complicate the estimation of phylogenetic relationships. In this study, we reconstruct the phylogeny of Theaceae using 348 nuclear loci from 68 individuals and two outgroup taxa. Sequence data were obtained by target enrichment using the recently released Angiosperm 353 universal probe set applied to herbarium specimens. The robustness of the topologies to variation in data quality was established under a range of different filtering schemes, using both coalescent and concatenation approaches. Our results confirmed most of the previously hypothesized relationships among tribes and genera, while clarifying additional interspecific relationships within the rapidly radiating genus Camellia. We recovered a remarkably high degree of gene tree heterogeneity indicative of rapid radiation in the group and observed cytonuclear conflicts, especially within Camellia. This was especially pronounced around short branches, which we primarily associate with gene tree estimation error. Our analysis also indicates that incomplete lineage sorting (ILS) contributed to gene-tree conflicts and accounted for approximately 14 % of the explained variation, whereas inferred introgression levels were low. Our study advances the understanding of the evolution of this important plant family and provides guidance on the application of target capture methods and the evaluation of key processes that influence phylogenetic discordances.

Post-stroke depression: epigenetic and epitranscriptomic modifications and their interplay with gut microbiota.

Epigenetic and epitranscriptomic modifications that regulate physiological processes of an organism at the DNA and RNA levels, respectively, are novel therapeutic candidates for various neurological diseases. Gut microbiota and its metabolites are known to modulate DNA methylation and histone modifications (epigenetics), as well as RNA methylation especially N6-methyladenosine (epitranscriptomics). As gut microbiota as well as these modifications are highly dynamic across the lifespan of an organism, they are implicated in the pathogenesis of stroke and depression. The lack of specific therapeutic interventions for managing post-stroke depression emphasizes the need to identify novel molecular targets. This review highlights the interaction between the gut microbiota and epigenetic/epitranscriptomic pathways and their interplay in modulating candidate genes that are involved in post-stroke depression. This review further focuses on the three candidates, including brain-derived neurotrophic factor, ten-eleven translocation family proteins, and fat mass and obesity-associated protein based on their prevalence and pathoetiologic role in post-stroke depression.

Reducing the Likelihood of Opioid Overdose Fatalities on College and University Campuses: An Action Plan and Model.

For more than two decades there has been a continuous rise in opioid overdose related deaths. The majority of the deaths include the age range when, traditionally, individuals are likely to attend college or university. As a result, Vassar College has taken the important initiative and created and implemented a new opioid overdose intervention strategy and action plan called AED+. AED+ expands on the Model of Greater Awareness, Training and Increased Availability of and Accessibility to Life Saving Intervention Devices; a model that was created based on AED devices improving outcomes of out-of-hospital cardiac arrest emergencies. Similar to AED's improving out-of-hospital cardiac arrest outcomes, the + component of the AED+ initiative increases awareness and provides basic, targeted education about naloxone and its use. Furthermore, the education includes information about naloxone's greater availability and its more immediate access across the campus by students, staff, faculty, administrators, and visitors in the event of a suspected opioid overdose. Starting in May 2023, members of the school's Health Service and senior administrative leaders identified it necessary to be proactive and not reactive to managing an opioid overdose in the campus community. Although Vassar College has not recently experienced an opioid overdose, it is confidently projected that these targeted actions will proactively and positively reduce the likelihood of opioid-related fatalities on campus. Furthermore, it is the purpose of this article to share the AED+ model so other colleges and universities can modify it to best fit their unique setting in order to improve opioid overdose outcomes.

Pleistocene glaciation advances the cryptic speciation of Stellera chamaejasme L. in a major biodiversity hotspot.

The mountains of Southwest China comprise a significant large mountain range and biodiversity hotspot imperiled by global climate change. The high species diversity in this mountain system has long been attributed to a complex set of factors, and recent large-scale macroevolutionary investigations have placed a broad timeline on plant diversification that stretches from 10 million years ago (Mya) to the present. Despite our increasing understanding of the temporal mode of speciation, finer-scale population-level investigations are lacking to better refine these temporal trends and illuminate the abiotic and biotic influences of cryptic speciation. This is largely due to the dearth of organismal sampling among closely related species and populations, spanning the incredible size and topological heterogeneity of this region. Our study dives into these evolutionary dynamics of speciation using genomic and eco-morphological data of Stellera chamaejasme L. We identified four previously unrecognized cryptic species having indistinct morphological traits and large metapopulation of evolving lineages, suggesting a more recent diversification (~2.67-0.90 Mya), largely influenced by Pleistocene glaciation and biotic factors. These factors likely influenced allopatric speciation and advocated cyclical warming-cooling episodes along elevational gradients during the Pleistocene. The study refines the evolutionary timeline to be much younger than previously implicated and raises the concern that projected future warming may influence the alpine species diversity, necessitating increased conservation efforts.

An antioxidant and anti-ER stress combo therapy decreases inflammation, secondary brain damage and promotes neurological recovery following traumatic brain injury in mice.

The complex pathophysiology of post-traumatic brain damage might need a polypharmacological strategy with a combination of drugs that target multiple, synergistic mechanisms. We currently tested a combination of apocynin (curtails formation of reactive oxygen species; ROS), tert-butylhydroquinone (promotes disposal of ROS), and salubrinal (prevents endoplasmic reticulum stress) following a moderate traumatic brain injury (TBI) induced by controlled cortical impact in adult mice. Adult mice of both sexes treated with the above tri-combo showed alleviated motor and cognitive deficits, attenuated secondary lesion volume, and decreased oxidative DNA damage. Concomitantly, tri-combo treatment regulated post-TBI inflammatory response by decreasing the infiltration of T cells and neutrophils and activation of microglia in both sexes. Interestingly, sexual dimorphism was seen in the case of TBI-induced microgliosis and infiltration of macrophages in the tri-combo treated mice. Moreover, the tri-combo treatment prevented TBI-induced white matter volume loss in both sexes. The beneficial effects of tri-combo treatment were long-lasting and were also seen in aged mice. Thus, the present study supports the tri-combo treatment to curtail oxidative stress and endoplasmic reticulum stress concomitantly as a therapeutic strategy to improve TBI outcomes.SIGNIFICANCE STATEMENTOf the several mechanisms that contribute to TBI pathophysiology, oxidative stress, endoplasmic reticulum (ER) stress, and inflammation play a major role. The present study shows the therapeutic potential of a combination of apocynin, tert-butylhydroquinone, and salubrinal to prevent oxidative stress and ER stress and the interrelated inflammatory response in mice subjected to TBI. The beneficial effects of the tri-combo include alleviation of TBI-induced motor and cognitive deficits and lesion volume. The neuroprotective effects of the tri-combo are also linked to its ability to prevent TBI-induced white matter damage. Importantly, neuroprotection by the tri-combo treatment was observed to be not dependent on sex or age. Our data demonstrate that a polypharmacological strategy is efficacious after TBI.

Cerebroprotective Role of N6-Methyladenosine Demethylase FTO (Fat Mass and Obesity-Associated Protein) After Experimental Stroke.

BACKGROUND: FTO (fat mass and obesity-associated protein) demethylates N6-methyladenosine (m6A), which is a critical epitranscriptomic regulator of neuronal function. We previously reported that ischemic stroke induces m6A hypermethylation with a simultaneous decrease in FTO expression in neurons. Currently, we evaluated the functional significance of restoring FTO with an adeno-associated virus 9, and thus reducing m6A methylation in poststroke brain damage. METHODS: Adult male and female C57BL/6J mice were injected with FTO adeno-associated virus 9 (intracerebral) at 21 days prior to inducing transient middle cerebral artery occlusion. Poststroke brain damage (infarction, atrophy, and white matter integrity) and neurobehavioral deficits (motor function, cognition, depression, and anxiety-like behaviors) were evaluated between days 1 and 28 of reperfusion. RESULTS: FTO overexpression significantly decreased the poststroke m6A hypermethylation. More importantly, exogenous FTO substantially decreased poststroke gray and white matter damage and improved motor function recovery, cognition, and depression-like behavior in both sexes. CONCLUSIONS: These results demonstrate that FTO-dependent m6A demethylation minimizes long-term sequelae of stroke independent of sex.

Complex climate-mediated effects of urbanization on plant reproductive phenology and frost risk.

Urbanization can affect the timing of plant reproduction (i.e. flowering and fruiting) and associated ecosystem processes. However, our knowledge of how plant phenology responds to urbanization and its associated environmental changes is limited. Herbaria represent an important, but underutilized source of data for investigating this question. We harnessed phenological data from herbarium specimens representing 200 plant species collected across 120 yr from the eastern US to investigate the spatiotemporal effects of urbanization on flowering and fruiting phenology and frost risk (i.e. time between the last frost date and flowering). Effects of urbanization on plant reproductive phenology varied significantly in direction and magnitude across species ranges. Increased urbanization led to earlier flowering in colder and wetter regions and delayed fruiting in regions with wetter spring conditions. Frost risk was elevated with increased urbanization in regions with colder and wetter spring conditions. Our study demonstrates that predictions of phenological change and its associated impacts must account for both climatic and human effects, which are context dependent and do not necessarily coincide. We must move beyond phenological models that only incorporate temperature variables and consider multiple environmental factors and their interactions when estimating plant phenology, especially at larger spatial and taxonomic scales.

Distinct in vitro and in vivo neutralization profiles of monoclonal antibodies elicited by the receptor binding domain of the ancestral SARS-CoV-2.

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.

Phylogenomics and taxon-rich phylogenies of new and historical specimens shed light on the systematics of Hypnea (Cystocloniaceae, Rhodophyta).

Cystocloniacae is a highly diverse family of Rhodophyta, including species of ecological and economic importance, whose phylogeny remains largely unresolved. Species delimitation is unclear, particularly in the most speciose genus, Hypnea, and cryptic diversity has been revealed by recent molecular assessments, especially in the tropics. Here, we carried out the first phylogenomic investigation of Cystocloniaceae, focused on the genus Hypnea, inferred from chloroplast and mitochondrial genomes including taxa sampled from new and historical collections. In this work, molecular synapomorphies (gene losses, InDels and gene inversions) were identified to better characterize clades in our congruent organellar phylogenies. We also present taxon-rich phylogenies based on plastid and mitochondrial markers. Molecular and morphological comparisons of historic collections with contemporary specimens revealed the need for taxonomic updates in Hypnea, the synonymization of H. marchantiae to a later heterotypic synonym of H. cervicornis and the description of three new species: H. davisiana sp. nov., H. djamilae sp. nov. and H. evaristoae sp. nov.

Species Tree Estimation and the Impact of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplication (WGD) occurs broadly and repeatedly across the history of eukaryotes and is recognized as a prominent evolutionary force, especially in plants. Immediately following WGD, most genes are present in two copies as paralogs. Due to this redundancy, one copy of a paralog pair commonly undergoes pseudogenization and is eventually lost. When speciation occurs shortly after WGD; however, differential loss of paralogs may lead to spurious phylogenetic inference resulting from the inclusion of pseudoorthologs-paralogous genes mistakenly identified as orthologs because they are present in single copies within each sampled species. The influence and impact of including pseudoorthologs versus true orthologs as a result of gene extinction (or incomplete laboratory sampling) are only recently gaining empirical attention in the phylogenomics community. Moreover, few studies have yet to investigate this phenomenon in an explicit coalescent framework. Here, using mathematical models, numerous simulated data sets, and two newly assembled empirical data sets, we assess the effect of pseudoorthologs on species tree estimation under varying degrees of incomplete lineage sorting (ILS) and differential gene loss scenarios following WGD. When gene loss occurs along the terminal branches of the species tree, alignment-based (BPP) and gene-tree-based (ASTRAL, MP-EST, and STAR) coalescent methods are adversely affected as the degree of ILS increases. This can be greatly improved by sampling a sufficiently large number of genes. Under the same circumstances, however, concatenation methods consistently estimate incorrect species trees as the number of genes increases. Additionally, pseudoorthologs can greatly mislead species tree inference when gene loss occurs along the internal branches of the species tree. Here, both coalescent and concatenation methods yield inconsistent results. These results underscore the importance of understanding the influence of pseudoorthologs in the phylogenomics era. [Coalescent method; concatenation method; incomplete lineage sorting; pseudoorthologs; single-copy gene; whole-genome duplication.].

Efficacy and safety of Oleogel-S10 (birch triterpenes) for epidermolysis bullosa: results from the phase III randomized double-blind phase of the EASE study.

BACKGROUND: Epidermolysis bullosa (EB) is a heterogeneous group of rare, difficult-to-treat, inherited multisystem diseases affecting epithelial integrity. Patients with EB are affected by mechanical fragility of epithelial surfaces including the skin and, as a result, extensive recurrent blistering is a characteristic of the condition. Chronic wounds predispose patients with EB to the development of squamous cell carcinoma, which is a major cause of premature death. OBJECTIVES: EASE was a double-blind, randomized, vehicle-controlled, phase III study to determine the efficacy and safety of the topical gel Oleogel-S10 (birch triterpenes) in EB. EASE was funded by Amryt Research Limited. METHODS: Patients with dystrophic EB, junctional EB or Kindler EB and a target partial-thickness wound lasting ≥ 21 days and < 9 months that was 10-50 cm2, were enrolled and randomized via computer-generated allocation tables 1 : 1 to Oleogel-S10 or control gel - both with standard-of-care dressings. Study gel was applied to all wounds at least every 4 days. The primary endpoint was the proportion of patients with first complete closure of target wound within 45 days. RESULTS: A total of 223 patients were enrolled and treated (109 treated with Oleogel-S10, 114 with control gel). The primary endpoint was met; Oleogel-S10 resulted in 41·3% of patients with first complete target wound closure within 45 days, compared with 28·9% in the control gel arm (relative risk 1·44, 95% confidence interval (CI) 1·01-2·05; P = 0·013). Adverse events (AEs) occurred with similar frequency for Oleogel-S10 (81·7%) compared with control gel (80·7%). AEs were predominantly of mild-to-moderate intensity (4·6% were severe). CONCLUSIONS: Oleogel-S10 is the first therapy to demonstrate accelerated wound healing in EB. Oleogel-S10 was well -tolerated.

Herbarium specimens reveal herbivory patterns across the genus Cucurbita.

PREMISE: Quantifying how closely related plant species differ in susceptibility to insect herbivory is important for understanding the variation in evolutionary pressures on plant functional traits. However, empirically measuring in situ variation in herbivory spanning the geographic range of a plant-insect complex is logistically difficult. Recently, new methods have been developed using herbarium specimens to investigate patterns in plant-insect symbioses across large geographic scales. Such investigations provide insights into how accelerated anthropogenic changes may impact plant-insect interactions that are of ecological or agricultural importance. METHODS: Here, we analyze 274 pressed herbarium samples to investigate variation in herbivory damage in 13 different species of the economically important plant genus Cucurbita (Cucurbitaceae). This collection is composed of specimens of wild, undomesticated Cucurbita that were collected from across their native range, and Cucurbita cultivars collected from both within their native range and from locations where they have been introduced for agriculture in temperate North America. RESULTS: Herbivory is common on individuals of all Cucurbita species collected throughout their geographic ranges. However, estimates of herbivory varied considerably among individuals, with mesophytic species accruing more insect damage than xerophytic species, and wild specimens having more herbivory than specimens collected from human-managed habitats. CONCLUSIONS: Our study suggests that long-term evolutionary changes in habitat from xeric to mesic climates and wild to human-managed habitats may mediate the levels of herbivory pressure from coevolved herbivores. Future investigations into the potential factors that contribute to herbivory may inform the management of domesticated crop plants and their insect herbivores.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

Phenological displacement is uncommon among sympatric angiosperms.

Interactions between species can influence successful reproduction, resulting in reproductive character displacement, where the similarity of reproductive traits - such as flowering time - among close relatives growing together differ from when growing apart. Evidence for the overall prevalence and direction of this phenomenon, and its stability under environmental change, remains untested across large scales. Using the power of crowdsourcing, we gathered phenological information from over 40 000 herbarium specimens, and investigated displacement in flowering time across 110 animal-pollinated species in the eastern USA. Overall, flowering time displacement is not common across large scales. However, displacement is generally greater among species pairs that flower close in time, regardless of direction. Furthermore, with climate change, the flowering times of closely related species are predicted, on average, to shift further apart by the mid-21st century. We demonstrate that the degree and direction of phenological displacement among co-occurring closely related species pairs varies tremendously. However, future climate change may alter the differences in reproductive timing among many of these species pairs, which may have significant consequences for species interactions and gene flow. Our study provides one promising path towards understanding how the phenological landscape is structured and may respond to future environmental change.

Delimiting the cryptic diversity and host preferences of Sycophila parasitoid wasps associated with oak galls using phylogenomic data.

Cryptic species diversity is a major challenge regarding the species-rich community of parasitoids attacking oak gall wasps due to a high degree of sexual dimorphism, morphological plasticity, small size and poorly known biology. As such, we know very little about the number of species present, nor the evolutionary forces responsible for generating this diversity. One hypothesis is that trait diversity in the gall wasps, including the morphology of the galls they induce, has evolved in response to selection imposed by the parasitoid community, with reciprocal selection driving diversification of the parasitoids. Using a rare, continental-scale data set of Sycophila parasitoid wasps reared from 44 species of cynipid galls from 18 species of oak across the USA, we combined mitochondrial DNA barcodes, ultraconserved elements (UCEs), morphological and natural history data to delimit putative species. Using these results, we generate the first large-scale assessment of ecological specialization and host association in this species-rich group, with implications for evolutionary ecology and biocontrol. We find most Sycophila target specific subsets of available cynipid host galls with similar morphologies, and generally attack larger galls. Our results suggest that parasitoid wasps such as Sycophila have adaptations allowing them to exploit particular host trait combinations, while hosts with contrasting traits are resistant to attack. These findings support the tritrophic niche concept for the structuring of plant-herbivore-parasitoid communities.

Phytogeographic History of the Tea Family Inferred Through High-Resolution Phylogeny and Fossils.

The tea family (Theaceae) has a highly unusual amphi-Pacific disjunct distribution: most extant species in the family are restricted to subtropical evergreen broadleaf forests in East Asia, while a handful of species occur exclusively in the subtropical and tropical Americas. Here, we used an approach that integrates the rich fossil evidence of this group with phylogenies in biogeographic analysis to study the processes behind this distribution pattern. We first combined genome-skimming sequencing with existing molecular data to build a robust species-level phylogeny for c.130 Theaceae species, resolving most important unclarified relationships. We then developed an empirical Bayesian method to incorporate distribution evidence from fossil specimens into historical biogeographic analyses and used this method to account for the spatiotemporal history of Theaceae fossils. We compared our method with an alternative Bayesian approach and show that it provides consistent results while significantly reduces computational demands which allows analyses of much larger data sets. Our analyses revealed a circumboreal distribution of the family from the early Cenozoic to the Miocene and inferred repeated expansions and retractions of the modeled distribution in the Northern Hemisphere, suggesting that the current Theaceae distribution could be the remnant of a larger continuous distribution associated with the boreotropical forest that has been hypothesized to occupy most of the northern latitudes in the early Cenozoic. These results contradict with studies that only considered current species distributions and showcase the necessity of integrating fossil and molecular data in phylogeny-based parametric biogeographic models to improve the reliability of inferred biogeographical events. [Biogeography; genome skimming; phylogenomics; plastid genome; Theaceae.].

The Perfect Storm: Gene Tree Estimation Error, Incomplete Lineage Sorting, and Ancient Gene Flow Explain the Most Recalcitrant Ancient Angiosperm Clade, Malpighiales.

The genomic revolution offers renewed hope of resolving rapid radiations in the Tree of Life. The development of the multispecies coalescent model and improved gene tree estimation methods can better accommodate gene tree heterogeneity caused by incomplete lineage sorting (ILS) and gene tree estimation error stemming from the short internal branches. However, the relative influence of these factors in species tree inference is not well understood. Using anchored hybrid enrichment, we generated a data set including 423 single-copy loci from 64 taxa representing 39 families to infer the species tree of the flowering plant order Malpighiales. This order includes 9 of the top 10 most unstable nodes in angiosperms, which have been hypothesized to arise from the rapid radiation during the Cretaceous. Here, we show that coalescent-based methods do not resolve the backbone of Malpighiales and concatenation methods yield inconsistent estimations, providing evidence that gene tree heterogeneity is high in this clade. Despite high levels of ILS and gene tree estimation error, our simulations demonstrate that these two factors alone are insufficient to explain the lack of resolution in this order. To explore this further, we examined triplet frequencies among empirical gene trees and discovered some of them deviated significantly from those attributed to ILS and estimation error, suggesting gene flow as an additional and previously unappreciated phenomenon promoting gene tree variation in Malpighiales. Finally, we applied a novel method to quantify the relative contribution of these three primary sources of gene tree heterogeneity and demonstrated that ILS, gene tree estimation error, and gene flow contributed to 10.0$\%$, 34.8$\%$, and 21.4$\%$ of the variation, respectively. Together, our results suggest that a perfect storm of factors likely influence this lack of resolution, and further indicate that recalcitrant phylogenetic relationships like the backbone of Malpighiales may be better represented as phylogenetic networks. Thus, reducing such groups solely to existing models that adhere strictly to bifurcating trees greatly oversimplifies reality, and obscures our ability to more clearly discern the process of evolution. [Coalescent; concatenation; flanking region; hybrid enrichment, introgression; phylogenomics; rapid radiation, triplet frequency.].