Understanding evolutionary rescue and parallelism in response to environmental stress.

Evolutionary rescue, the process by which populations facing environmental stress avoid extinction through genetic adaptation, is a critical area of study in evolutionary biology. The order in which mutations arise and get established will be relevant to the population's rescue. This study investigates the degree of parallel evolution at the genotypic level between independent populations facing environmental stress and subject to different demographic regimes. Under density regulation, 2 regimes exist: In the first, the population can restore positive growth rates by adjusting its population size or through adaptive mutations, whereas in the second regime, the population is doomed to extinction unless a rescue mutation occurs. Analytical approximations for the likelihood of evolutionary rescue are obtained and contrasted with simulation results. We show that the initial level of maladaptation and the demographic regime significantly affect the level of parallelism. There is an evident transition between these 2 regimes. Whereas in the first regime, parallelism decreases with the level of maladaptation, it displays the opposite behavior in the rescue/extinction regime. These findings have important implications for understanding population persistence and the degree of parallelism in evolutionary responses as they integrate demographic effects and evolutionary processes.

The prevalence of copy number increase at multiallelic copy number variants associated with cave colonization.

Copy number variation is a common contributor to phenotypic diversity, yet its involvement in ecological adaptation is not easily discerned. Instances of parallelly evolving populations of the same species in a similar environment marked by strong selective pressures present opportunities to study the role of copy number variants (CNVs) in adaptation. By identifying CNVs that repeatedly occur in multiple populations of the derived ecotype and are not (or are rarely) present in the populations of the ancestral ecotype, the association of such CNVs with adaptation to the novel environment can be inferred. We used this paradigm to identify CNVs associated with recurrent adaptation of the Mexican tetra (Astyanax mexicanus) to cave environment. Using a read-depth approach, we detected CNVs from previously re-sequenced genomes of 44 individuals belonging to two ancestral surfaces and three derived cave populations. We identified 102 genes and 292 genomic regions that repeatedly diverge in copy number between the two ecotypes and occupy 0.8% of the reference genome. Functional analysis revealed their association with processes previously recognized to be relevant for adaptation, such as vision, immunity, oxygen consumption, metabolism, and neural function and we propose that these variants have been selected for in the cave or surface waters. The majority of the ecotype-divergent CNVs are multiallelic and display copy number increases in cavefish compared to surface fish. Our findings suggest that multiallelic CNVs - including gene duplications - and divergence in copy number provide a fast route to produce novel phenotypes associated with adaptation to subterranean life.

Phylogeographic relationships and morphological evolution between cave and surface Astyanax mexicanus populations (De Filippi 1853) (Actinopterygii, Characidae).

The Astyanax mexicanus complex includes two different morphs, a surface- and a cave-adapted ecotype, found at three mountain ranges in Northeastern Mexico: Sierra de El Abra, Sierra de Guatemala and Sierra de la Colmena (Micos). Since their discovery, multiple studies have attempted to characterize the timing and the number of events that gave rise to the evolution of these cave-adapted ecotypes. Here, using RADseq and genome-wide sequencing, we assessed the phylogenetic relationships, genetic structure and gene flow events between the cave and surface Astyanax mexicanus populations, to estimate the tempo and mode of evolution of the cave-adapted ecotypes. We also evaluated the body shape evolution across different cave lineages using geometric morphometrics to examine the role of phylogenetic signal versus environmental pressures. We found strong evidence of parallel evolution of cave-adapted ecotypes derived from two separate lineages of surface fish and hypothesize that there may be up to four independent invasions of caves from surface fish. Moreover, a strong congruence between the genetic structure and geographic distribution was observed across the cave populations, with the Sierra de Guatemala the region exhibiting most genetic drift among the cave populations analysed. Interestingly, we found no evidence of phylogenetic signal in body shape evolution, but we found support for parallel evolution in body shape across independent cave lineages, with cavefish from the Sierra de El Abra reflecting the most divergent morphology relative to surface and other cavefish populations.

The Difficulty of Predicting Evolutionary Change in Response to Novel Ecological Interactions: A Field Experiment with Anolis Lizards.

AbstractDetermining whether and how evolution is predictable is an important goal, particularly as anthropogenic disturbances lead to novel species interactions that could modify selective pressures. Here, we use a multigeneration field experiment with brown anole lizards (Anolis sagrei) to test hypotheses about the predictability of evolution. We manipulated the presence/absence of predators and competitors of A. sagrei across 16 islands in the Bahamas that had preexisting brown anole populations. Before the experiment and again after roughly five generations, we measured traits related to locomotor performance and habitat use by brown anoles and used double-digest restriction enzyme-associated DNA sequencing to estimate genome-wide changes in allele frequencies. Although previous work showed that predators and competitors had characteristic effects on brown anole behavior, diet, and population sizes, we found that evolutionary change at both phenotypic and genomic levels was difficult to forecast. Phenotypic changes were contingent on sex and habitat use, whereas genetic change was unpredictable and not measurably correlated with phenotypic changes, experimental treatments, or other environmental factors. Our work shows how differences in ecological context can alter evolutionary outcomes over short timescales and underscores the difficulty of forecasting evolutionary responses to multispecies interactions in natural conditions, even in a well-studied system with ample supporting ecological information.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Pervasive admixture and the spread of a large-lipped form in a cichlid fish radiation.

Adaptive radiations have proven important for understanding the mechanisms and processes underlying biological diversity. The convergence of form and function, as well as admixture and adaptive introgression, are common in adaptive radiations. However, distinguishing between these two scenarios remains a challenge for evolutionary research. The Midas cichlid species complex (Amphilophus spp.) is a prime example of adaptive radiation, with phenotypic diversification occurring at various stages of genetic differentiation. One species, A. labiatus, has large fleshy lips, is associated with rocky lake substrates, and occurs patchily within Lakes Nicaragua and Managua. By contrast, the similar, but thin-lipped, congener, A. citrinellus, is more common and widespread. We investigated the evolutionary history of the large-lipped form, specifically regarding whether the trait has evolved independently in both lakes from ancestral thin-lipped populations, or via dispersal and/or admixture events. We collected samples from distinct locations in both lakes, and assessed differences in morphology and ecology. Using RAD-seq, we genotyped thousands of SNPs to measure population structure and divergence, demographic history, and admixture. We found significant between-species differences in ecology and morphology, local intraspecific differences in body shape and trophic traits, but only limited intraspecific variation in lip shape. Despite clear ecological differences, our genomic approach uncovered pervasive admixture between the species and low genomic differentiation, with species within lakes being genetically more similar than species between lakes. Taken together, our results suggest a single origin of large-lips, followed by pervasive admixture and adaptive introgression, with morphology being driven by local ecological opportunities, despite ongoing gene-flow.

Las radiaciones adaptativas han demostrado ser clave para entender los mecanismos y procesos responsables de la diversidad biológica. La convergencia en forma y función, así como la mezcla genética y la introgresión adaptativa, son algo común en las radiaciones adaptativas. Sin embargo, distinguir entre estos dos escenarios sigue siendo un desafío para la biología evolutiva. El complejo de especies del cíclido de Midas (Amphilophus spp.) es un ejemplo paradigmático de radiación adaptativa, con diversidad fenotípica en varias etapas de diferenciación genética. Una de las especies, A. labiatus, que tiene labios grandes y carnosos, se asocia a zonas rocosas de los lagos, y tiene una distribución irregular en los lagos Nicaragua y Managua. En cambio, A. citrinellus, es una especie similar pero con labios finos, más común y con una distribución más amplia. Investigamos la historia evolutiva de la especie de labios grandes y, en concreto, si este rasgo ha evolucionado de forma independiente en los dos grandes lagos nicaragüenses a partir de poblaciones ancestrales de labios finos, o si por el contrario se ha dispersado mediante migración y/o mezcla genética. Colectamos muestras de distintas localidades en ambos lagos y evaluamos las diferencias en morfología y ecología. Genotipamos miles de SNPs utilizando RAD-seq para medir la estructura genética, la divergencia, la historia demográfica y la mezcla genética de las poblaciones. Encontramos diferencias significativas entre especies en ecología y morfología, diferencias intraespecíficas locales en la forma del cuerpo y rasgos tróficos, pero sólo una limitada variación intraespecífica en la forma de los labios. A pesar de las claras diferencias ecológicas, el análisis genómico desveló una intensa mezcla genética entre especies, y una limitada diferenciación genómica, encontrando mayor semejanza genética entre especies dentro de un mismo lago, que entre especies de distintos lagos. Nuestros resultados sugieren un origen único de la especie de labios gruesos seguido de mezcla genética e introgresión adaptativa, e indican que la morfología habría sido modelada por las oportunidades ecológicas locales, a pesar del flujo génico.

Evolutionary drivers of sexual signal variation in Amazon Slender Anoles.

Phenotypic variation among populations, as seen in the signaling traits of many species, provides an opportunity to test whether similar factors generate repeated phenotypic patterns in different parts of a species' range. We investigated whether genetic divergence, abiotic gradients, and sympatry with closely related species explain variation in the dewlap colors of Amazon Slender Anoles, Anolis fuscoauratus. To this aim, we characterized dewlap diversity in the field with respect to population genetic structure and evolutionary relationships, assessed whether dewlap phenotypes are associated with climate or landscape variables, and tested for nonrandom associations in the distributions of A. fuscoauratus phenotypes and sympatric Anolis species. We found that dewlap colors vary among but not within sites in A. fuscoauratus. Regional genetic clusters included multiple phenotypes, while populations with similar dewlaps were often distantly related. Phenotypes did not segregate in environmental space, providing no support for optimized signal transmission at a local scale. Instead, we found a negative association between certain phenotypes and sympatric Anolis species with similar dewlap color attributes, suggesting that interactions with closely related species promoted dewlap divergence among A. fuscoauratus populations. Amazon Slender Anoles emerge as a promising system to address questions about parallel trait evolution and the contribution of signaling traits to speciation.

Parallel and non-parallel changes of the gut microbiota during trophic diversification in repeated young adaptive radiations of sympatric cichlid fish.

BACKGROUND: Recent increases in understanding the ecological and evolutionary roles of microbial communities have underscored the importance of their hosts' biology. Yet, little is known about gut microbiota dynamics during the early stages of ecological diversification and speciation. We sequenced the V4 region of the 16s rRNA gene to study the gut microbiota of Nicaraguan Midas cichlid fish (Amphilophus cf. citrinellus). Specifically, we tested the hypothesis that parallel divergence in trophic ecology in extremely young adaptive radiations from two crater lakes is associated with parallel changes of their gut microbiota. RESULTS: Bacterial communities of fish guts and lake water were highly distinct, indicating that the gut microbiota is shaped by host-specific factors. Among individuals of the same crater lake, differentiation in trophic ecology was weakly associated with gut microbiota differentiation, suggesting that diet, to some extent, affects the gut microbiota. However, differences in trophic ecology were much more pronounced across than within species whereas similar patterns were not observed for taxonomic and functional differences of the gut microbiota. Across the two crater lakes, we could not detect conclusive evidence for parallel changes of the gut microbiota associated with trophic ecology. CONCLUSIONS: A lack of clearly differentiated niches during the early stages of ecological diversification might result in non-parallel changes of gut microbial communities, as observed in our study system as well as in other recently diverged fish species. Video Abstract.

Genomic consequences of dietary diversification and parallel evolution due to nectarivory in leaf-nosed bats.

BACKGROUND: The New World leaf-nosed bats (Phyllostomids) exhibit a diverse spectrum of feeding habits and innovations in their nutrient acquisition and foraging mechanisms. However, the genomic signatures associated with their distinct diets are unknown. RESULTS: We conducted a genomic comparative analysis to study the evolutionary dynamics related to dietary diversification and specialization. We sequenced, assembled, and annotated the genomes of five Phyllostomid species: one insect feeder (Macrotus waterhousii), one fruit feeder (Artibeus jamaicensis), and three nectar feeders from the Glossophaginae subfamily (Leptonycteris yerbabuenae, Leptonycteris nivalis, and Musonycteris harrisoni), also including the previously sequenced vampire Desmodus rotundus. Our phylogenomic analysis based on 22,388 gene families displayed differences in expansion and contraction events across the Phyllostomid lineages. Independently of diet, genes relevant for feeding strategies and food intake experienced multiple expansions and signatures of positive selection. We also found adaptation signatures associated with specialized diets: the vampire exhibited traits associated with a blood diet (i.e., coagulation mechanisms), whereas the nectarivore clade shares a group of positively selected genes involved in sugar, lipid, and iron metabolism. Interestingly, in fruit-nectar-feeding Phyllostomid and Pteropodids bats, we detected positive selection in two genes: AACS and ALKBH7, which are crucial in sugar and fat metabolism. Moreover, in these two proteins we found parallel amino acid substitutions in conserved positions exclusive to the tribe Glossophagini and to Pteropodids. CONCLUSIONS: Our findings illuminate the genomic and molecular shifts associated with the evolution of nectarivory and shed light on how nectar-feeding bats can avoid the adverse effects of diets with high glucose content.

Convergent evolution in floral morphology in a plant ring species, the Caribbean Euphorbia tithymaloides.

PREMISE: Ring species have long fascinated evolutionary biologists for their potential insights into lineage divergence and speciation across space. Few studies have investigated the potential for convergent or parallel evolution along the diverging fronts of ring species. We investigated a potential case of parallel floral variation in the Caribbean spurge Euphorbia tithymaloides, the only plant system with molecular support as a ring species. The terminal populations of each front, despite being the most divergent, exhibit such similar floral traits that they were originally considered each other's closest relative. METHODS: We evaluated convergence in floral and leaf traits in relation to geography across 95 populations spanning the distribution of E. tithymaloides. We also reanalyzed available genetic data (from previous phylogenetic analyses) in an explicitly spatial framework. RESULTS: Floral morphology appears to have shifted in a convergent fashion along both geographic fronts of E. tithymaloides, resulting in shorter and more compact inflorescences in Antillean populations compared to the typical elongate "slipper-like" cyathia characteristic of the area of origin. Patterns of spatial genetic variation were more consistent with a two-fronted invasion of the Caribbean than with a simpler model of isolation-by-distance. CONCLUSIONS: Floral divergence in E. tithymaloides is consistent with convergent evolution along the two fronts of a ring species. We outline several (not mutually exclusive) mechanisms that could be driving patterns in morphology, including shifts toward generalized pollination with reduced reliance on hummingbirds, shifts in floral structure closely matching available hummingbird bill traits, and shifts toward increased selfing.

Island- and lake-like parallel adaptive radiations replicated in rivers.

Parallel adaptive radiations have arisen following the colonization of islands by lizards and lakes by fishes. In these classic examples, parallel adaptive radiation is a response to the ecological opportunities afforded by the colonization of novel ecosystems and similar adaptive landscapes that favour the evolution of similar suites of ecomorphs, despite independent evolutionary histories. Here, we demonstrate that parallel adaptive radiations of cichlid fishes arose in South American rivers. Speciation-assembled communities of pike cichlids (Crenicichla) have independently diversified into similar suites of novel ecomorphs in the Uruguay and Paraná Rivers, including crevice feeders, periphyton grazers and molluscivores. There were bursts in phenotypic evolution associated with the colonization of each river and the subsequent expansion of morphospace following the evolution of the ecomorphs. These riverine clades demonstrate that characteristics emblematic of textbook parallel adaptive radiations of island- and lake-dwelling assemblages are feasible evolutionary outcomes even in labile ecosystems such as rivers.

Predation drives morphological convergence in the Gambusia panuco species group among lotic and lentic habitats.

Fish morphology is often constrained by a trade-off between optimizing steady vs. unsteady swimming performance due to opposing effects of caudal peduncle size. Lotic environments tend to select for steady swimming performance, leading to smaller caudal peduncles, whereas predators tend to select for unsteady swimming performance, leading to larger caudal peduncles. However, it is unclear which aspect of performance should be optimized across heterogeneous flow and predation environments and how this heterogeneity may affect parallel phenotypic evolution. We investigated this question among four Gambusia species in north-eastern Mexico, specifically the riverine G. panuco, the spring endemics G. alvarezi and G. hurtadoi, and a fourth species, G. marshi, found in a variety of habitats with varying predation pressure in the Cuatro Ciénegas Basin and Río Salado de Nadadores. We employed a geometric morphometric analysis to examine how body shapes of both male and female fish differ among species and habitats and with piscivore presence. We found that high-predation and low-predation species diverged morphologically, with G. marshi exhibiting a variable, intermediate body shape. Within G. marshi, body morphology converged in high-predation environments regardless of flow velocity, and fish from high-predation sites had larger relative caudal peduncle areas. However, we found that G. marshi from low-predation environments diverged in morphology between sub-basins of Cuatro Ciénegas, indicating other differences among these basins that merit further study. Our results suggest that a morphological trade-off promotes parallel evolution of body shape in fishes colonizing high-predation environments and that changing predation pressure can strongly impact morphological evolution in these species.

Predictability and Parallelism of Multitrait Adaptation.

Environments shape the traits of organisms. Environmental variation may rarely alter selection on only a few traits, but instead precipitate wholesale changes of the multidimensional selective regime-many traits might experience divergent selection across divergent environments. Such changes in selection can elicit multifarious evolution. How predictable (from theory) and how parallel (consistent occurrences) is multitrait divergence across replicated environments? Here, I address this question using the post-Pleistocene radiation of Bahamas mosquitofish (Gambusia hubbsi) inhabiting blue holes on Andros Island. These fish independently colonized numerous blue holes, some that harbor a major fish predator (bigmouth sleeper, Gobiomorus dormitor) and some that lack any major predators. I used 5 approaches to quantitatively explore the predictability and parallelism of multitrait divergence between predation regimes in Bahamas mosquitofish. Synthesizing data for 90 traits from 13 different types of character suites (e.g., body morphology, life history, genital morphology, coloration, mating preference, habitat use), I found widespread evidence for strong, predictable, and parallel divergence between predation regimes. Yet despite the great majority of traits showing predictable trajectories of change, and the majority of traits showing significant parallelism and strong magnitudes of predictable divergence, I uncovered that over half of the overall phenotypic variation among populations was not driven by variation in predation regime. Results suggest that focusing on few traits, or focusing on parallel aspects of divergence, can provide a misleading picture of adaptation, and nonparallel divergence appears widespread and warrants greater attention. Taking a multitrait perspective, and quantifying predictability and parallelism, can yield important insights.

Origins and recent radiation of Brazilian Eupatorieae (Asteraceae) in the eastern Cerrado and Atlantic Forest.

The remarkable diversity of Eupatorieae in the Brazilian flora has received little study, despite the tribe's very high levels of endemism and importance in the threatened Cerrado and the Atlantic Forest biodiversity hotspots. Eupatorieae are one of the largest tribes in Asteraceae with 14 of 19 recognized subtribes occurring in Brazil. We constructed the largest phylogeny of Brazilian Eupatorieae to date that sampled the nrITS and ETS, chloroplast ndhI and ndhF genes, and the ndhI-ndhG intergenic spacer for 183 species representing 77 of the 85 Brazilian genera of the tribe. Maximum likelihood and Bayesian phylogenetic analyses showed that these species are not collectively monophyletic, so their distribution reflects multiple introductions into Brazil. A novel clade was found that includes 75% of the genera endemic to Brazil (Cerrado-Atlantic Forest Eupatorieae, "CAFE" clade). This radiation of at least 247 species concentrated in the Cerrado and Atlantic Forest biomes of central eastern Brazil is <7 my old and exhibits several ecologically diverse life forms. Eight subtribes of Brazilian Eupatorieae (Ageratinae, Alomiinae, Ayapaninae, Critoniinae, Disynaphiinae, Eupatoriinae, Gyptidinae and Hebecliniinae) and 16 genera (Ageratum, Agrianthus, Austroeupatorium, Bejaranoa, Chromolaena, Critonia, Disynaphia, Grazielia, Hatschbachiella, Heterocondylus, Koanophyllon, Lasiolaena, Neocabreria, Praxelis, Stylotrichium, and Symphyopappus) were found to be polyphyletic. We attribute incongruities between the molecular phylogenetic results and the current classification of the tribe mostly to convergent evolution of morphological characters traditionally used in the classification of the tribe. We used these phylogenetic results to suggest changes to the classification of some subtribes and genera of Eupatorieae that occur in Brazil.

Unique evolutionary trajectories in repeated adaptation to hydrogen sulphide-toxic habitats of a neotropical fish (Poecilia mexicana).

Replicated ecological gradients are prime systems to study processes of molecular evolution underlying ecological divergence. Here, we investigated the repeated adaptation of the neotropical fish Poecilia mexicana to habitats containing toxic hydrogen sulphide (H2 S) and compared two population pairs of sulphide-adapted and ancestral fish by sequencing population pools of >200 individuals (Pool-Seq). We inferred the evolutionary processes shaping divergence and tested the hypothesis of increase of parallelism from SNPs to molecular pathways. Coalescence analyses showed that the divergence occurred in the face of substantial bidirectional gene flow. Population divergence involved many short, widely dispersed regions across the genome. Analyses of allele frequency spectra suggest that differentiation at most loci was driven by divergent selection, followed by a selection-mediated reduction of gene flow. Reconstructing allelic state changes suggested that selection acted mainly upon de novo mutations in the sulphide-adapted populations. Using a corrected Jaccard index to quantify parallel evolution, we found a negligible proportion of statistically significant parallel evolution of Jcorr  = 0.0032 at the level of SNPs, divergent genome regions (Jcorr  = 0.0061) and genes therein (Jcorr  = 0.0091). At the level of metabolic pathways, the overlap was Jcorr  = 0.2545, indicating increasing parallelism with increasing level of biological integration. The majority of pathways contained positively selected genes in both sulphide populations. Hence, adaptation to sulphidic habitats necessitated adjustments throughout the genome. The largely unique evolutionary trajectories may be explained by a high proportion of de novo mutations driving the divergence. Our findings favour Gould's view that evolution is often the unrepeatable result of stochastic events with highly contingent effects.

Widespread parallel population adaptation to climate variation across a radiation: implications for adaptation to climate change.

Global warming will impact species in a number of ways, and it is important to know the extent to which natural populations can adapt to anthropogenic climate change by natural selection. Parallel microevolution within separate species can demonstrate natural selection, but several studies of homoplasy have not yet revealed examples of widespread parallel evolution in a generic radiation. Taking into account primary phylogeographic divisions, we investigate numerous quantitative traits (size, shape, scalation, colour pattern and hue) in anole radiations from the mountainous Lesser Antillean islands. Adaptation to climatic differences can lead to very pronounced differences between spatially close populations with all studied traits showing some evidence of parallel evolution. Traits from shape, scalation, pattern and hue (particularly the latter) show widespread evolutionary parallels within these species in response to altitudinal climate variation greater than extreme anthropogenic climate change predicted for 2080. This gives strong evidence of the ability to adapt to climate variation by natural selection throughout this radiation. As anoles can evolve very rapidly, it suggests anthropogenic climate change is likely to be less of a conservation threat than other factors, such as habitat loss and invasive species, in this, Lesser Antillean, biodiversity hot spot.

Several origins of floral oil in the Angelonieae, a southern hemisphere disjunct clade of Plantaginaceae.

UNLABELLED: • PREMISE OF THE STUDY: Over the past 75 Myr, successive groups of plants have entered the "oil bee pollination niche," meaning that they depend on oil-collecting bees for their pollination. The highly dissimilar numbers of plant species and bee species involved in these mutualisms imply evolutionary host switching, asymmetric mutual dependencies, and uncoupled diversification. Among the clades with the best field data on oil bee behavior is the Angelonieae, which we here investigate to better understand the evolutionary time frame of this pollination syndrome.• METHODS: We generated nuclear and plastid data matrices for 56% of the Angelonieae species (plus outgroups) and used Bayesian methods of molecular clock dating, ancestral state reconstruction, and biogeographic inference.• KEY RESULTS: We found that Angelonieae have two major clades, Angelonia (including Monopera) and Basistemon, and Monttea, Melosperma, and Ourisia.• CONCLUSIONS: Angelonieae date back to the Uppermost Eocene, ca. 35 (26-47) Myr ago (Ma) and diversified in dry areas of southern South America; they switched from nectar to oil as a reward four or five times over the past 25 Ma. As predicted in a previous non-clock-dated study, dispersal to Australasia dates to the Miocene/Pliocene.

Repeated elevational transitions in hemoglobin function during the evolution of Andean hummingbirds.

Animals that sustain high levels of aerobic activity under hypoxic conditions (e.g., birds that fly at high altitude) face the physiological challenge of jointly optimizing blood-O2 affinity for O2 loading in the pulmonary circulation and O2 unloading in the systemic circulation. At high altitude, this challenge is especially acute for small endotherms like hummingbirds that have exceedingly high mass-specific metabolic rates. Here we report an experimental analysis of hemoglobin (Hb) function in South American hummingbirds that revealed a positive correlation between Hb-O2 affinity and native elevation. Protein engineering experiments and ancestral-state reconstructions revealed that this correlation is attributable to derived increases in Hb-O2 affinity in highland lineages, as well as derived reductions in Hb-O2 affinity in lowland lineages. Site-directed mutagenesis experiments demonstrated that repeated evolutionary transitions in biochemical phenotype are mainly attributable to repeated amino acid replacements at two epistatically interacting sites that alter the allosteric regulation of Hb-O2 affinity. These results demonstrate that repeated changes in biochemical phenotype involve parallelism at the molecular level, and that mutations with indirect, second-order effects on Hb allostery play key roles in biochemical adaptation.