Combined Immunotherapy Improves Outcome for Replication-Repair-Deficient (RRD) High-Grade Glioma Failing Anti-PD-1 Monotherapy: A Report from the International RRD Consortium.

Immune checkpoint inhibition (ICI) is effective for replication-repair-deficient, high-grade gliomas (RRD-HGG). The clinical/biological impact of immune-directed approaches after failing ICI monotherapy is unknown. We performed an international study on 75 patients treated with anti-PD-1; 20 are progression free (median follow-up, 3.7 years). After second progression/recurrence (n = 55), continuing ICI-based salvage prolonged survival to 11.6 months (n = 38; P < 0.001), particularly for those with extreme mutation burden (P = 0.03). Delayed, sustained responses were observed, associated with changes in mutational spectra and the immune microenvironment. Response to reirradiation was explained by an absence of deleterious postradiation indel signatures (ID8). CTLA4 expression increased over time, and subsequent CTLA4 inhibition resulted in response/stable disease in 75%. RAS-MAPK-pathway inhibition led to the reinvigoration of peripheral immune and radiologic responses. Local (flare) and systemic immune adverse events were frequent (biallelic mismatch-repair deficiency > Lynch syndrome). We provide a mechanistic rationale for the sustained benefit in RRD-HGG from immune-directed/synergistic salvage therapies. Future approaches need to be tailored to patient and tumor biology. SIGNIFICANCE: Hypermutant RRD-HGG are susceptible to checkpoint inhibitors beyond initial progression, leading to improved survival when reirradiation and synergistic immune/targeted agents are added. This is driven by their unique biological and immune properties, which evolve over time. Future research should focus on combinatorial regimens that increase patient survival while limiting immune toxicity. This article is featured in Selected Articles from This Issue, p. 201.

Founder events and subsequent genetic bottlenecks underlie karyotype evolution in the Ibero - North African endemic Carex helodes.

BACKGROUND AND AIMS: Despite chromosomal evolution being one of the major drivers of diversification in plants, we do not yet have a clear view of how new chromosome rearrangements become fixed within populations, which is a crucial step forward for understanding chromosomal speciation. METHODS: In this study, we test the role of genetic drift in the establishment of new chromosomal variants in the context of hybrid dysfunction models of chromosomal speciation. We genotyped a total of 178 individuals from seven populations (plus 25 seeds from one population) across the geographic range of Carex helodes (Cyperaceae). We also characterized karyotype geographic patterns of the species across the distribution range. For one of the populations, we performed a detailed study of the fine scale, local spatial distribution of its individuals and their genotypes and karyotypes. KEY RESULTS: Synergistically, phylogeographic and karyotypic evidence show two main genetic groups: southwestern Iberian Peninsula vs. northwestern African populations, and within Europe our results suggest a west-to-east expansion with signals of genetic bottlenecks. Additionally, we have inferred a pattern of descending dysploidy, plausibly as a result of a west-to-east process of post-glacial colonization in Europe. CONCLUSIONS: Our results give experimental support to the role of geographic isolation, drift, and inbreeding in the establishment of new karyotypes which is key in the speciation models of hybrid dysfunction.

Unveiling underestimated species diversity within the Central American Coralsnake, a medically important complex of venomous taxa.

Coralsnakes of the genus Micrurus are a diverse group of venomous snakes ranging from the southern United States to southern South America. Much uncertainty remains over the genus diversity, and understanding Micrurus systematics is of medical importance. In particular, the widespread Micrurus nigrocinctus spans from Mexico throughout Central America and into Colombia, with a number of described subspecies. This study provides new insights into the phylogenetic relationships within M. nigrocinctus by examining sequence data from a broad sampling of specimens from Mexico, Guatemala, Honduras, Nicaragua, Costa Rica, and Panama. The recovered phylogenetic relationships suggest that M. nigrocinctus is a species complex originating in the Pliocene and composed of at least three distinct species-level lineages. In addition, recovery of highly divergent clades supports the elevation of some currently recognized subspecies to the full species rank while others may require synonymization.

Efficacy of Nivolumab in Pediatric Cancers with High Mutation Burden and Mismatch Repair Deficiency.

PURPOSE: Checkpoint inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory nonhematologic cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch repair deficiency (MMRD). PATIENTS AND METHODS: Twenty patients were screened, and 10 were ultimately included in the response cohort of whom nine had TMB >10 mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5 and 10 mut/Mb. RESULTS: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% [95% confidence interval (CI), 27-93]. Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37 months (range, 32.4-60), culminating in 2-year OS of 43% (95% CI, 18.2-100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with an MMR-proficient tumor and TMB 7.4 mut/Mb. CONCLUSIONS: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint inhibitors, including upfront management of cancer. See related commentary by Mardis, p. 4701.

Using comprehensive genomic and functional analyses for resolving genotype-phenotype mismatches in children with suspected CMMRD in Lebanon: an IRRDC study.

Constitutional mismatch repair deficiency (CMMRD) is an aggressive and highly penetrant cancer predisposition syndrome. Because of its variable clinical presentation and phenotypical overlap with neurofibromatosis, timely diagnosis remains challenging, especially in countries with limited resources. Since current tests are either difficult to implement or interpret or both we used a novel and relatively inexpensive functional genomic assay (LOGIC) which has been recently reported to have high sensitivity and specificity in diagnosing CMMRD. Here we report the clinical and molecular characteristics of nine patients diagnosed with cancer and suspected to have CMMRD and highlight the challenges with variant interpretation and immunohistochemical analysis that led to an uncertain interpretation of genetic findings in 6 of the 9 patients. Using LOGIC, we were able to confirm the diagnosis of CMMRD in 7 and likely exclude it in 2 patients, resolving ambiguous result interpretation. LOGIC also enabled predictive testing of asymptomatic siblings for early diagnosis and implementation of surveillance. This study highlights the varied manifestations and practical limitations of current diagnostic criteria for CMMRD, and the importance of international collaboration for implementing robust and low-cost functional assays for resolving diagnostic challenges.

Historical biogeography and diversification of ringless Amanita (section Vaginatae) support an African origin and suggest niche conservatism in the Americas.

Ectomycorrhizal fungi (ECM) sustain nutrient recycling in most terrestrial ecosystems, yet we know little about what major biogeographical events gave rise to present-day diversity and distribution patterns. Given the strict relationship between some ECM lineages and their hosts, geographically well-sampled phylogenies are central to understanding major evolutionary processes of fungal biodiversity patterns. Here, we focus on Amanita sect. Vaginatae to address global diversity and distribution patterns. Ancestral-state-reconstruction based on a 4-gene timetree with over 200 species supports an African origin between the late Paleocene and the early Eocene (ca. 56 Ma). Major biogeographic "out-of-Africa" events include multiple dispersal events to Southeast Asia (ca. 45-21 Ma), Madagascar (ca. 18 Ma), and the current Amazonian basin (ca. 45-36 Ma), the last two likely trans-oceanic. Later events originating in Southeast Asia involve Nearctic dispersal to North America (ca. 20-5 Ma), Oceania (Australia and New Zealand; ca. 15 Ma), and Europe (ca. 10-5 Ma). Subsequent dispersals were also inferred from Southeast Asia to East Asia (ca. 4 Ma); from North America to East Asia (ca. 11-8 Ma), Southeast Asia (ca. 19-2 Ma), Northern Andes (ca. 15 Ma), and Europe (ca. 15-2 Ma), respectively; and from the Amazon to the Caribbean region (ca. 25-20 Ma). Finally, we detected a significant increase in the net diversification rates in the branch leading to most northern temperate species in addition to higher state-dependent diversification rates in temperate lineages, consistent with previous findings. These results suggest that species of sect. Vaginatae likely have higher dispersal ability and higher adaptability to new environments, in particular compared to those of its sister clade, sect. Caesareae. Overall, the much wider distribution of A. sect. Vaginatae, from pan-tropical to pan-arctic, provides a unique window to understanding niche conservatism across a species-rich clade of ECM fungi.

Global phylogeny of the Shiitake mushroom and related Lentinula species uncovers novel diversity and suggests an origin in the Neotropics.

Lentinula (Basidiomycota, Agaricales) includes the most widely cultivated mushroom in the world, Lentinula edodes, also known as shiitake (Japanese) or xiang-gu (Chinese). At present, nine species are recognized in the genus, based on morphology, mating criteria, and geographic distribution. However, analyses of internal transcribed spacers (ITS) of ribosomal RNA genes have suggested that there are cryptic lineages. We analyzed a global-scale phylogenetic dataset from 325 Lentinula individuals from 24 countries in Asia-Australasia and the Americas plus Madagascar, with 325 sequences of ITS, 80 LSU sequences, and 111 sequences of translation elongation factor (tef1-α) genes. We recovered 15 independent lineages (Groups 1-15) that may correspond to species. Lineages in Asia-Australasia (Groups 1-5) and the Americas plus Madagascar (Groups 6-15) formed sister clades. Four lineages are represented only by sequences from single individuals and require further molecular sampling, including L. aff. raphanica (Group 7), L. ixodes (Group 8), L. boryana (Group 12), and L. aff. aciculospora (Group 14). Groups 1 and 5 are here referred to L. edodes and L. aff. edodes, respectively. However, these groups most likely represent the same species and are only recognized as (unsupported) monophyletic lineages by maximum likelihood analyses of ITS alone. Other putative species resolved here include L. lateritia (Group 2), L. novae-zelandieae (Group 3), L. aff. lateritia (Group 4), L. raphanica (Group 6), L. aff. detonsa (Group 9), L. detonsa (Group 10), L. guzmanii sp. nov. (Group 11), L. aciculospora (Group 13), and L. madagasikarensis (Group 15). Groups 9-12 represent the "L. boryana complex". Molecular clock and historical biogeographic analyses suggest that the most recent common ancestor (MRCA) of Lentinula can be placed in the middle Oligocene, ca. 30 million years ago (Ma), and had a likely presence in neotropical America. The MRCA of Lentinula in the Americas and Madagascar lived ca. 22 Ma in the Neotropics and the MRCA of Lentinula in Asia-Australasia lived ca. 6 Ma in Oceania. Given the current knowledge about plate tectonics and paleoclimatic models of the last 30 Myr, our phylogenetic hypothesis suggests that the extant distribution of Lentinula is likely to have arisen, in large part, due to long-distance dispersal. Lentinula collections include at least four dubious taxa that need further taxonomic studies: L. reticeps from the USA (Ohio); L. guarapiensis from Paraguay; Lentinus puiggarii from Brazil (São Paulo); and "L. platinedodes" from Vietnam. Approximately ten of the fifteen Groups are reported on Fagaceae, which appears to be the ancestral substrate of Lentinula.

Widespread misregulation of inter-species hybrid transcriptomes due to sex-specific and sex-chromosome regulatory evolution.

When gene regulatory networks diverge between species, their dysfunctional expression in inter-species hybrid individuals can create genetic incompatibilities that generate the developmental defects responsible for intrinsic post-zygotic reproductive isolation. Both cis- and trans-acting regulatory divergence can be hastened by directional selection through adaptation, sexual selection, and inter-sexual conflict, in addition to cryptic evolution under stabilizing selection. Dysfunctional sex-biased gene expression, in particular, may provide an important source of sexually-dimorphic genetic incompatibilities. Here, we characterize and compare male and female/hermaphrodite transcriptome profiles for sibling nematode species Caenorhabditis briggsae and C. nigoni, along with allele-specific expression in their F1 hybrids, to deconvolve features of expression divergence and regulatory dysfunction. Despite evidence of widespread stabilizing selection on gene expression, misexpression of sex-biased genes pervades F1 hybrids of both sexes. This finding implicates greater fragility of male genetic networks to produce dysfunctional organismal phenotypes. Spermatogenesis genes are especially prone to high divergence in both expression and coding sequences, consistent with a "faster male" model for Haldane's rule and elevated sterility of hybrid males. Moreover, underdominant expression pervades male-biased genes compared to female-biased and sex-neutral genes and an excess of cis-trans compensatory regulatory divergence for X-linked genes underscores a "large-X effect" for hybrid male expression dysfunction. Extensive regulatory divergence in sex determination pathway genes likely contributes to demasculinization of XX hybrids. The evolution of genetic incompatibilities due to regulatory versus coding sequence divergence, however, are expected to arise in an uncorrelated fashion. This study identifies important differences between the sexes in how regulatory networks diverge to contribute to sex-biases in how genetic incompatibilities manifest during the speciation process.

Sexual Dimorphism through the Lens of Genome Manipulation, Forward Genetics, and Spatiotemporal Sequencing.

Sexual reproduction often leads to selection that favors the evolution of sex-limited traits or sex-specific variation for shared traits. These sexual dimorphisms manifest due to sex-specific genetic architectures and sex-biased gene expression across development, yet the molecular mechanisms underlying these patterns are largely unknown. The first step is to understand how sexual dimorphisms arise across the genotype-phenotype-fitness map. The emergence of "4D genome technologies" allows for efficient, high-throughput, and cost-effective manipulation and observations of this process. Studies of sexual dimorphism will benefit from combining these technological advances (e.g., precision genome editing, inducible transgenic systems, and single-cell RNA sequencing) with clever experiments inspired by classic designs (e.g., bulked segregant analysis, experimental evolution, and pedigree tracing). This perspective poses a synthetic view of how manipulative approaches coupled with cutting-edge observational methods and evolutionary theory are poised to uncover the molecular genetic basis of sexual dimorphism with unprecedented resolution. We outline hypothesis-driven experimental paradigms for identifying genetic mechanisms of sexual dimorphism among tissues, across development, and over evolutionary time.

Extreme mito-nuclear discordance in a peninsular lizard: the role of drift, selection, and climate.

Nuclear and mitochondrial genomes coexist within cells but are subject to different tempos and modes of evolution. Evolutionary forces such as drift, mutation, selection, and migration are expected to play fundamental roles in the origin and maintenance of diverged populations; however, divergence may lag between genomes subject to different modes of inheritance and functional specialization. Herein, we explore whole mitochondrial genome data and thousands of nuclear single nucleotide polymorphisms to evidence extreme mito-nuclear discordance in the small black-tailed brush lizard, Urosaurus nigricaudus, of the Peninsula of Baja California, Mexico and southern California, USA, and discuss potential drivers. Results show three deeply divergent mitochondrial lineages dating back to the later Miocene (ca. 5.5 Ma) and Pliocene (ca. 2.8 Ma) that likely followed geographic isolation due to trans-peninsular seaways. This contrasts with very low levels of genetic differentiation in nuclear loci (FST < 0.028) between mtDNA lineages. Analyses of protein-coding genes reveal substantial fixed variation between mitochondrial lineages, of which a significant portion comes from non-synonymous mutations. A mixture of drift and selection is likely responsible for the rise of these mtDNA groups, albeit with little evidence of marked differences in climatic niche space between them. Finally, future investigations can look further into the role that mito-nuclear incompatibilities and mating systems play in explaining contrasting nuclear gene flow.

Megaphylogeny resolves global patterns of mushroom evolution.

Mushroom-forming fungi (Agaricomycetes) have the greatest morphological diversity and complexity of any group of fungi. They have radiated into most niches and fulfil diverse roles in the ecosystem, including wood decomposers, pathogens or mycorrhizal mutualists. Despite the importance of mushroom-forming fungi, large-scale patterns of their evolutionary history are poorly known, in part due to the lack of a comprehensive and dated molecular phylogeny. Here, using multigene and genome-based data, we assemble a 5,284-species phylogenetic tree and infer ages and broad patterns of speciation/extinction and morphological innovation in mushroom-forming fungi. Agaricomycetes started a rapid class-wide radiation in the Jurassic, coinciding with the spread of (sub)tropical coniferous forests and a warming climate. A possible mass extinction, several clade-specific adaptive radiations and morphological diversification of fruiting bodies followed during the Cretaceous and the Paleogene, convergently giving rise to the classic toadstool morphology, with a cap, stalk and gills (pileate-stipitate morphology). This morphology is associated with increased rates of lineage diversification, suggesting it represents a key innovation in the evolution of mushroom-forming fungi. The increase in mushroom diversity started during the Mesozoic-Cenozoic radiation event, an era of humid climate when terrestrial communities dominated by gymnosperms and reptiles were also expanding.

Unravelling population processes over the Late Pleistocene driving contemporary genetic divergence in Palearctic buzzards.

Population range expansions and contractions as a response to climate and habitat change throughout the Quaternary are known to have contributed to complex phylogenetic and population genetic events. Speciation patterns and processes in Palearctic buzzards (genus Buteo) are a long-standing example of morphological and genetic data incongruence, attributed to panmixia, habitat range shifts, contact zones, and climate change. Here we assess the systematics, phylogeography and population genetic structure of three nominal species of Palearctic buzzards, Buteo buteo (including B. b. vulpinus), B. rufinus (including B. r. cirtensis) and B. hemilasius. Phylogenetic analyses inferred from mitochondrial data recover B. hemilasius as sister to the sister clades B. r. rufinus and B. buteo complex (B. b. buteo, B. b. vulpinus, but also including B. r. cirtensis). In contrast, we find an unresolved genetic delimitation inferred from four nuclear loci, suggesting an ancestral genetic pool for all species. Time-trees suggest population contractions and expansions throughout the Pleistocene, which likely reflect habitat change and contrasting ecological niche requirements between species. Microsatellite-based extended Bayesian skyline plots reveal relatively constant population sizes for B. hemilasius, B. r. rufinus, and B. b. vulpinus, in contrast to a dramatic population expansion in B. r. cirtensis within the last 3 kya. Overall, our study illustrates how complex population processes over the Late Pleistocene have shaped the patterns of genetic divergence in Palearctic buzzards, due to the joint effects of shared ancestral polymorphisms, population expansions and contractions, with hybridization at contact zones leading to admixture and introgression.

Biogeography of Korea's top predator, the yellow-throated Marten: evolutionary history and population dynamics.

BACKGROUND: Peninsulas often harvest high genetic diversity through repeated southward migrations of species during glacial maxima. Studies addressing within-species evolutionary responses to climate fluctuations in northeast Asia are limited compared to other regions of the world, and more so in the Korean Peninsula. In this study, we conducted the first population-level study of the yellow-throated marten, Martes flavigula, from the Korean Peninsula, Russian, Taiwanese and Chinese localities in a biogeographic framework using mitochondrial (cyt-b, nd2, cr) and nuclear gene sequencing (ghr). RESULTS: Bayesian analyses revealed a rather young population, with a split from the most recent common ancestor at around 125 kya. Martes flavigula likely colonized the Korean Peninsula from Mainland China through the Yellow Sea twice, ca. 60 kya and 20 kya. Korean martens diversified during the Late Pleistocene with at least two dispersal events out of Korea, towards the southwest to Taiwan (ca. 80 kya) and towards the North into Russia and eastern China; most likely after the Last Glacial Maxima (ca. 20 kya). We argue that the lack of population structure and mixed populations is possibly a consequence of the high dispersal capability of the species. The Bayesian skyline plot revealed a population decline within the last 5000 years, suggesting potential negative biotic and anthropogenic effects in the area. We find that local populations are not genetically differentiated, therefore no perceptible population structure within Korea was found. CONCLUSIONS: The topography and geography of the Korean Peninsula has played a pivotal role in its colonization. Connections between the Korean Peninsula and the Mainland through sea-level drops of the Yellow Sea at times of glacial maxima and the high dispersal capability of M. flavigula adds to the lack of geographical structure in this species and the paraphyly of Korean lineages.

Landscape limits gene flow and drives population structure in Agassiz's desert tortoise (Gopherus agassizii).

Distance, environmental heterogeneity and local adaptation can strongly influence population structure and connectivity. Understanding how these factors shape the genomic landscape of threatened species is a major goal in conservation genomics and wildlife management. Herein, we use thousands (6,859) of single nucleotide polymorphism markers and spatial data from hundreds of individuals (n = 646) to re-evaluate the population structure of Agassiz's desert tortoise (Gopherus agassizii). Analyses resolve from 4 to 8 spatially well-defined clusters across the range. Western, central, and southern populations within the Western Mojave recovery unit are consistent throughout, while analyses sometimes merge other recovery units depending on the level of clustering. Causal modeling consistently associates genetic connectivity with least-cost distance, based on multiple landscape features associated with tortoise habitat, better than geographic distance. Some features include elevation, soil depth, rock volume, precipitation, and vegetation coverage, suggesting that physical, climatic, and biotic landscape features have played a strong evolutionary role restricting gene flow between populations. Further, 12 highly differentiated outlier loci have associated functions that may be involved with neurogenesis, wound healing, lipid metabolism, and possibly vitellogenesis. Together, these findings have important implications for recovery programs, such as translocations, population augmentation, reproduction in captivity and the identification of ecologically important genes, opening new venues for conservation genomics in desert tortoises.

Phylogeographic and population insights of the Asian common toad (Bufo gargarizans) in Korea and China: population isolation and expansions as response to the ice ages.

The effects of ice ages on speciation have been well documented for many European and North American taxa. In contrast, very few studies have addressed the consequences of such environmental and topographical changes in North East Asian species. More precisely, the Korean Peninsula offers a unique model to assess patterns and processes of speciation as it hosts the northern- and eastern-most distribution limit of some widespread Asian taxa. Despite this, studies addressing phylogeographic patterns and population genetics in the peninsula and surrounding countries are few and studies for most families are lacking. Here we inferred the phylogenetic relationships of the common toad (Bufo gargarizans) from South Korea and their North East Asian counterpart populations, based on mitochondrial data. Korean B. gargarizans GenBank BLASTs matched few individuals from nearby China, but the presence of a Korean clade suggests isolation on the Korean Peninsula, previous to the last glacial maximum, linked to sea level resurgence. Molecular clock calibrations within this group were used to date the divergence between clades and their relationship to paleo-climatic events in the area. Lack of genetic structure among South Korean populations and strong homogeneity between the Korean and some Chinese localities suggest weak isolation and recent expansion. Geographical projection of continuous coalescent maximum-clade-credibility trees shows an original Chinese expansion towards the Korean Peninsula through the Yellow Sea circa two million years ago with colonisation events dating circa 800 thousand years ago (K. y. a.). Following this colonisation, the data point to outgoing Korean Peninsula dispersal events throughout different periods, towards the North through land, and West through land bridge formations over the Yellow Sea during sea level falls. In accordance, demographic analyses revealed a population expansion in the Koran Peninsula circa 300 K. y. a., likely attributed to glacial cycle fluctuations.

The Gondwanan connection - Southern temperate Amanita lineages and the description of the first sequestrate species from the Americas.

Amanita is a diverse and cosmopolitan genus of ectomycorrhizal fungi. We describe Amanita nouhrae sp. nov., a new hypogeous ('truffle-like') species associated with Nothofagus antarctica in northern Patagonia. This constitutes the first report of a sequestrate Amanita from the Americas. Thick-walled basidiospores ornamented on the interior spore wall ('crassospores') were observed consistently in A. nouhrae and its sister epigeous taxon Amanita morenoi, a rarely collected but apparently common species from northern Patagonia that has sometimes been misidentified as the Australian taxon Amanita umbrinella. Nuclear 18S and 28S ribosomal DNA and mitochondrial 16S and 26S DNA placed these two species in a southern temperate clade within subgenus Amanita, together with other South American and Australian species. Based on a dated genus-level phylogeny, we estimate that the southern temperate clade may have originated near the Eocene/Oligocene boundary (ca. 35 Ma ± 10 Ma). This date suggests a broadly distributed ancestor in the Southern Hemisphere, which probably diversified as a result of continental drift, as well as the initiation of the Antarctic glaciation. By comparison, we show that this clade follows an exceptional biogeographic pattern within a genus otherwise seemingly dominated by Northern Hemisphere dispersal.

Diversity-dependent cladogenesis throughout western Mexico: Evolutionary biogeography of rattlesnakes (Viperidae: Crotalinae: Crotalus and Sistrurus).

Rattlesnakes (Crotalus and Sistrurus) represent a radiation of approximately 42 species distributed throughout the New World from southern Canada to Argentina. Interest in this enigmatic group of snakes continues to accrue due, in part, to their ecomorphological diversity, contributions to global envenomations, and potential medicinal importance. Although the group has garnered substantial attention from systematists and evolutionary biologists for decades, little is still known regarding patterns of lineage diversification. In addition, few studies have statistically quantified broad-scale biogeographic patterns in rattlesnakes to ascertain how dispersal occurred throughout the New World, particularly among the different major biomes of the Americas. To examine diversification and biogeographic patterns in this group of snakes we assemble a multilocus data set consisting of over 6700bp encompassing three nuclear loci (NT-3, RAG-1, C-mos) and seven mitochondrial genes (12S, 16S, ATPase6, ATPase8, ND4, ND5, cytb). Fossil-calibrated phylogenetic and subsequent diversification rate analyses are implemented using maximum likelihood and Bayesian inference, to examine their evolutionary history and temporal dynamics of diversity. Based on ancestral area reconstructions we explore dispersal patterns throughout the New World. Cladogenesis occurred predominantly during the Miocene and Pliocene with only two divergences during the Pleistocene. Two different diversification rate models, advocating diversity-dependence, are strongly supported. These models indicate an early rapid radiation followed by a recent speciation rate decline. Biogeographic analyses suggest that the high elevation pine-oak forests of western Mexico served as a major speciation pump for the majority of lineages, with the desert biome of western North America colonized independently at least twice. All together, these results provide evidence for rapid diversification of rattlesnakes throughout the Mexican highlands during the Neogene, likely in response to continual orogenesis of Mexico's major mountain systems, followed by more recent dispersal into desert and tropical biomes.

In and out of refugia: historical patterns of diversity and demography in the North American Caesar's mushroom species complex.

Some of the effects of past climate dynamics on plant and animal diversity make-up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent-based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein-coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species.

High speciation rate at temperate latitudes explains unusual diversity gradients in a clade of ectomycorrhizal fungi.

Understanding the patterns of biodiversity through time and space is a challenging task. However, phylogeny-based macroevolutionary models allow us to account and measure many of the processes responsible for diversity buildup, namely speciation and extinction. The general latitudinal diversity gradient (LDG) is a well-recognized pattern describing a decline in species richness from the equator polewards. Recent macroecological studies in ectomycorrhizal (EM) fungi have shown that their LDG is shifted, peaking at temperate rather than tropical latitudes. Here we investigate this phenomenon from a macroevolutionary perspective, focusing on a well-sampled group of edible EM mushrooms from the genus Amanita-the Caesar's mushrooms, which follow similar diversity patterns. Our approach consisted in applying a suite of models including (1) nontrait-dependent time-varying diversification (Bayesian analysis of macroevolutionary mixtures [BAMM]), (2) continuous trait-dependent diversification (quantitative-state speciation and extinction [QuaSSE]), and (3) diversity-dependent diversification. In short, results give strong support for high speciation rates at temperate latitudes (BAMM and QuaSSE). We also find some evidence for different diversity-dependence thresholds in "temperate" and "tropical" subclades, and little differences in diversity due to extinction. We conclude that our analyses on the Caesar's mushrooms give further evidence of a temperate-peaking LDG in EM fungi, highlighting the importance and the implications of macroevolutionary processes in explaining diversity gradients in microorganisms.