Phylogenetic Comparative Methods Research Articles

Evolutionary paths toward multi-level convergence of lactic acid bacteria in fructose-rich environments

Convergence provides clues to unveil the non-random nature of evolution. Intermediate paths toward convergence inform us of the stochasticity and the constraint of evolutionary processes. Although previous studies have suggested that substantial constraints exist in microevolutionary paths, it remains unclear whether macroevolutionary convergence follows stochastic or constrained paths. Here, we performed comparative genomics for hundreds of lactic acid bacteria (LAB) species, including clades showing a convergent gene repertoire and sharing fructose-rich habitats. By adopting phylogenetic comparative methods we showed that the genomic convergence of distinct fructophilic LAB (FLAB) lineages was caused by parallel losses of more than a hundred orthologs and the gene losses followed significantly similar orders. Our results further suggested that the loss of adhE, a key gene for phenotypic convergence to FLAB, follows a specific evolutionary path of domain architecture decay and amino acid substitutions in multiple LAB lineages sharing fructose-rich habitats. These findings unveiled the constrained evolutionary paths toward the convergence of free-living bacterial clades at the genomic and molecular levels.

Who cares? Elucidating parental care evolution in extant birds.

Extant birds stand out among vertebrates in the diversity of parental care types they present, spanning absence of care to uniparental care by either sex, biparental care, or even cooperative care. Despite years of research, key questions remain regarding parental care evolution in birds. Firstly, the parental care type in the most recent ancestor of extant birds is a matter of controversy, with proposed ancestral states including no care, uniparental male or female care, and biparental care. Another unsolved question is the direction, order, and frequency of transitions between parental care types. We address these key questions using a database of 5,438 bird species (~50% of extant diversity) and modern phylogenetic comparative methods controlling simultaneously for model and phylogenetic uncertainty as well as potential confounding effects of state-dependent diversification. Our results indicate that the most likely ancestral state for extant birds is male-only care, with a posterior probability of 0.8. Transition rates across parental care types were generally low and heterogeneous; loss of parental care virtually never occurs and transitions away from female-only or cooperative care most often lead to biparental care. Given the low transition rates, future research should analyze the factors favoring the maintenance of care types.

An evolutionary disruption of the buzz pollination syndrome in neotropical montane plants.

Under pollinator limitations, specialized pollination syndromes may evolve toward contrasting responses: a generalized syndrome with increased pollinator attraction, pollinator reward, and pollen transfer capacity; or the selfing syndrome with increased self-pollen deposition, but reduced pollinator attraction and pollen transfer capacity. The buzz-pollination syndrome is specialized to explore female vibrating bees as pollinators. However, vibrating bees become less-active pollinators at montane areas of the Atlantic Forest (AF) domain. This study investigated whether the specialized buzz-pollination syndrome would evolve toward an alternative floral syndrome in montane areas of the AF domain, considering a generalized and the selfing syndromes as alternative responses. We utilized a lineage within the buzz-pollinated Miconia as study system, contrasting floral traits between montane AF-endemic and non-endemic species. We measured and validated floral traits that were proxies for pollinator attraction, reward access, pollen transfer capacity, and self-pollen deposition. We inferred the evolution of floral trait via phylogenetic comparative methods. AF-endemic species have selectively evolved greater reward access and more frequently had generalist pollination. Nonetheless, AF-endemic species also have selectively evolved toward lower pollen transfer capacity and greater self pollination. These patterns indicated a complex evolutionary process that has jointly favored a generalized and the selfing syndromes. The buzz pollination syndrome can undergo an evolutionary disruption in montane areas of the AF domain. This floral syndrome is likely more labile than often assumed, allowing buzz-pollinated plants to reproduce in environments where vibrating bees are less-reliable pollinators.

Correlated evolution of conspicuous colouration and burrowing in crayfish.

Conspicuous colours have fascinated biologists for centuries, leading to research on the evolution and functional significance of colour traits. In many cases, research suggests that conspicuous colours are adaptive and serve a function in sexual or aposematic signalling. In other cases, a lack of evidence for the adaptive value of conspicuous colours garners interest from biologists, such as when organisms that live underground and are rarely exposed to the surface are nevertheless colourful. Here, we use phylogenetic comparative methods to investigate colour evolution throughout freshwater crayfishes that vary in burrowing ability. Within the taxa we analysed, conspicuous colours have evolved independently over 50 times, and these colours are more common in semi-terrestrial crayfishes that construct extensive burrows. The intuitive but not evolutionarily justified assumption when presented with these results is to assume that these colours are adaptive. But contrary to this intuition, we discuss the hypothesis that colouration in crayfish is neutral. Supporting these ideas, the small population sizes and reduced gene flow within semi-terrestrial burrowing crayfishes may lead to the fixation of colour-phenotype mutations. Overall, our work brings into question the traditional view of animal colouration as a perfectly adapted phenotype.

Hybridization in palms (Arecaceae).

Hybridization has significant evolutionary consequences across the Tree of Life. The process of hybridization has played a major role in plant evolution and has contributed to species richness and trait variation. Since morphological traits are partially a product of their environment, there may be a link between hybridization and ecology. Plant hybrid species richness is noted to be higher in harsh environments, and we explore this hypothesis with a keystone tropical plant lineage, palms (Arecaceae). Leveraging a recent literature review of naturally occurring palm hybrids, we developed a method to calculate hybrid frequency, and then tested if there is phylogenetic signal of hybrids using a phylogeny of all palms. Further, we used phylogenetic comparative methods to examine the interaction between hybrid frequency and presence in dry environments, on islands, and the species richness of genera. Phylogenetic generalized least squares models had stronger support than models of random association, indicating phylogenetic signal for the presence of hybrids in dry and island environments. However, all p-values were >.05 and therefore the correlation was poor between hybridization and the trait frequencies examined. Presence in particular environments are not strongly correlated to hybrid frequency, but phylogenetic signal suggests a role in its distribution in different habitats. Hybridization in palms is not evenly distributed across subfamilies, tribes, subtribes yet plays an important role in palm diversity, nonetheless. Increasing our understanding hybridization in this economically and culturally important plant family is essential, particularly since rates are projected to increase with climate change, reconfiguring the dynamics and distribution of biodiversity.

The pronotum shape of scelimenine grasshoppers (Orthoptera: Tetrigidae) likely represents an exaptation for heterogeneous niche colonization

Abstract Insects are suitable model organisms for functional morphology research, especially in the context of exaptation, when the same morphological trait represents an advantage in disparate niches. Phylogenetically distant groups of pygmy grasshoppers (Orthoptera: Tetrigidae) have various pronotal projections defining their general appearance and body shape. However, body shape has never been related to niche occupation in these insects, thus the aim of this study is to investigate the relationship between pronotum shape and macrohabitat adaptation in Scelimeninae, a group of Asian and Papuan tetrigids encompassing amphibious and corticolous (bark-dwelling) representatives. With the use of geometric morphometrics and phylogenetic comparative methods, two morphological and functional groups were distinguished, with the body shape exhibiting a significant phylogenetic signal. The first group consists of elongated amphibious taxa (Scelimenini tribe) with highly uniform pronotum morphology, likely due to a strong selection for streamlined body shape. Stouter corticolous taxa (Discotettigini tribe) exhibit more conspicuous body shape variability, possibly increasing camouflage efficiency in tree bark habitats. Ecological divergence associated with macrohabitat adaptation may thus have been the primary driver of speciation in this insect group, but the evolutionary constraints leading to this divergence are still to be identified.

Small- to medium-sized mammals show greater morphological disparity in cervical than lumbar vertebrae across different terrestrial modes of locomotion.

During mammalian terrestrial locomotion, body flexibility facilitated by the vertebral column is expected to be correlated with observed modes of locomotion, known as gait (e.g., sprawl, trot, hop, bound, gallop). In small- to medium-sized mammals (average weight up to 5 kg), the relationship between locomotive mode and vertebral morphology is largely unexplored. Here we studied the vertebral column from 46 small- to medium-sized mammals. Nine vertebrae across cervical, thoracic, and lumbar regions were chosen to represent the whole vertebral column. Vertebra shape was analysed using three-dimensional geometric morphometrics with the phylogenetic comparative method. We also applied the multi-block method, which can consider all vertebrae as a single structure for analysis. We calculated morphological disparity, phylogenetic signal, and evaluated the effects of allometry and gait on vertebral shape. We also investigated the pattern of integration in the column. We found the cervical vertebrae show the highest degree of morphological disparity, and the first thoracic vertebra shows the highest phylogenetic signal. A significant effect of gait type on vertebrae shape was found, with the lumbar vertebrae having the strongest correlation; but this effect was not significant after taking phylogeny into account. On the other hand, allometry has a significant effect on all vertebrae regardless of the contribution from phylogeny. The regions showed differing degrees of integration, with cervical vertebrae most strongly correlated. With these results, we have revealed novel information that cannot be captured from study of a single vertebra alone: although the lumbar vertebrae are the most correlated with gait, the cervical vertebrae are more morphologically diverse and drive the diversity among species when considering whole column shape.

Comparative transcriptome analysis reveals lineage- and environment-specific adaptations in cacti from the Brazilian Atlantic Forest.

Natural selection influenced adaptive divergence between Cereus fernambucensis and Cereus insularis, revealing key genes governing abiotic stress responses and supporting neoteny in C. insularis. Uncovering the molecular mechanisms driving adaptive divergence in traits related to habitat adaptation remains a central challenge. In this study, we focused on the cactus clade, which includes Cereus sericifer F.Ritter, Cereus fernambucensis Lem., and Cereus insularis Hemsley. These allopatric species inhabit distinct relatively drier regions within the Brazilian Atlantic Forest, each facing unique abiotic conditions. We leveraged whole transcriptome data and abiotic variables datasets to explore lineage-specific and environment-specific adaptations in these species. Employing comparative phylogenetic methods, we identified genes under positive selection (PSG) and examined their association with non-synonymous genetic variants and abiotic attributes through a PhyloGWAS approach. Our analysis unveiled signatures of selection in all studied lineages, with C. fernambucensis northern populations and C. insularis showing the most PSGs. These PSGs predominantly govern abiotic stress regulation, encompassing heat tolerance, UV stress response, and soil salinity adaptation. Our exclusive observation of gene expression tied to early developmental stages in C. insularis supports the hypothesis of neoteny in this species. We also identified genes associated with abiotic variables in independent lineages, suggesting their role as environmental filters on genetic diversity. Overall, our findings suggest that natural selection played a pivotal role in the geographic range of these species in response to environmental and biogeographic transitions.

Wiring Between Close Nodes in Molecular Networks Evolves More Quickly Than Between Distant Nodes.

As species diverge, a wide range of evolutionary processes lead to changes in protein-protein interaction (PPI) networks and metabolic networks. The rate at which molecular networks evolve is an important question in evolutionary biology. Previous empirical work has focused on interactomes from model organisms to calculate rewiring rates, but this is limited by the relatively small number of species and sparse nature of network data across species. We present a proxy for variation in network topology: variation in drug-drug interactions (DDIs), obtained by studying drug combinations (DCs) across taxa. Here, we propose the rate at which DDIs change across species as an estimate of the rate at which the underlying molecular network changes as species diverge. We computed the evolutionary rates of DDIs using previously published data from a high-throughput study in gram-negative bacteria. Using phylogenetic comparative methods, we found that DDIs diverge rapidly over short evolutionary time periods, but that divergence saturates over longer time periods. In parallel, we mapped drugs with known targets in PPI and cofunctional networks. We found that the targets of synergistic DDIs are closer in these networks than other types of DCs and that synergistic interactions have a higher evolutionary rate, meaning that nodes that are closer evolve at a faster rate. Future studies of network evolution may use DC data to gain larger-scale perspectives on the details of network evolution within and between species.

Comparative Analysis of Maternal Gene Expression Patterns Unravels Evolutionary Signatures Across Reproductive Modes.

Maternal genes have a pivotal role in regulating metazoan early development. As such their functions have been extensively studied since the dawn of developmental biology. The temporal and spatial dynamics of their transcripts have been thoroughly described in model organisms and their functions have been undergoing heavy investigations. Yet, less is known about the evolutionary changes shaping their presence within diverse oocytes. Due to their unique maternal inheritance pattern, a high degree is predicted to be present when it comes to their expression. Insofar only limited and conflicting results have emerged around it. Here, we set out to elucidate which evolutionary changes could be detected in the maternal gene expression patterns using phylogenetic comparative methods on RNAseq data from 43 species. Using normalized gene expression values and fold change information throughout early development we set out to find the best-fitting evolutionary model. Through modeling, we find evidence supporting both the high degree of divergence and constraint on gene expression values, together with their temporal dynamics. Furthermore, we find that maternal gene expression alone can be used to explain the reproductive modes of different species. Together, these results suggest a highly dynamic evolutionary landscape of maternal gene expression. We also propose a possible functional dichotomy of maternal genes which is influenced by the reproductive strategy undertaken by examined species.

Functional ecology and evolution of terrestrial and epiphytic species of Rhododendron section Schistanthe (Ericaceae)

Epiphytes are often characterized by small size, specifically adapted to a relatively dry and nutrient-poor habitat. However, this epiphyte syndrome is derived from studies on species-rich and habitat-dominating groups such as orchids and bromeliads. To better understand general trait syndromes of epiphytes, it is important to analyze plant groups that are more transitional in the evolution of epiphytism e.g., the Australasian vireyas (Rhododendron sect. Schistanthe; Ericaceae). Here, we studied the morphology, ecophysiology, and anatomy of 44 species of Rhododendron section Schistanthe from living collections and obtained data for 288 species related to their "life form" (holoepiphytes [= obligatory epiphytes], facultative epiphytes and terrestrial congeners) from the literature. For phylogeny and time-calibrated tree, we used the nuclear ITS (Internal Transcribed Spacers) region. Using different statistics and phylogenetic comparative methods, our results support the hypothesis that holoepiphytic vireyas are smaller in size than the terrestrial ones. Terrestrial vireyas are found at higher minimum and maximum elevation than either facultative or holoepiphytic congeners. We demonstrate that the diversification of tropical Rhododendron is not related to the evolution of the epiphytic life form. Regarding the evolution of traits that are important for water economy, holoepiphytic vireyas do not differ much from their terrestrial relatives indicating that evolutionary steps for specialization towards an holoepiphytic habit are in their initial stages in Rhododendron. Given the large species diversity of the group conclusions based on the subset used here must be considered preliminary. However, our results provide the basis for more detailed future studies.

Evolutionary trade-offs in dormancy phenology.

Seasonal animal dormancy is widely interpreted as a physiological response for surviving energetic challenges during the harshest times of the year (the physiological constraint hypothesis). However, there are other mutually non-exclusive hypotheses to explain the timing of animal dormancy, that is, entry into and emergence from hibernation (i.e. dormancy phenology). Survival advantages of dormancy that have been proposed are reduced risks of predation and competition (the 'life-history' hypothesis), but comparative tests across animal species are few. Using the phylogenetic comparative method applied to more than 20 hibernating mammalian species, we found support for both hypotheses as explanations for the phenology of dormancy. In accordance with the life-history hypotheses, sex differences in hibernation emergence and immergence were favored by the sex difference in reproductive effort. In addition, physiological constraint may influence the trade-off between survival and reproduction such that low temperatures and precipitation, as well as smaller body mass, influence sex differences in phenology. We also compiled initial evidence that ectotherm dormancy may be (1) less temperature dependent than previously thought and (2) associated with trade-offs consistent with the life-history hypothesis. Thus, dormancy during non-life-threatening periods that are unfavorable for reproduction may be more widespread than previously thought.

Evolutionary trade-offs in dormancy phenology

Seasonal animal dormancy is widely interpreted as a physiological response for surviving energetic challenges during the harshest times of the year (the physiological constraint hypothesis). However, there are other mutually non-exclusive hypotheses to explain the timing of animal dormancy, that is, entry into and emergence from hibernation (i.e. dormancy phenology). Survival advantages of dormancy that have been proposed are reduced risks of predation and competition (the ‘life-history’ hypothesis), but comparative tests across animal species are few. Using the phylogenetic comparative method applied to more than 20 hibernating mammalian species, we found support for both hypotheses as explanations for the phenology of dormancy. In accordance with the life-history hypotheses, sex differences in hibernation emergence and immergence were favored by the sex difference in reproductive effort. In addition, physiological constraint may influence the trade-off between survival and reproduction such that low temperatures and precipitation, as well as smaller body mass, influence sex differences in phenology. We also compiled initial evidence that ectotherm dormancy may be (1) less temperature dependent than previously thought and (2) associated with trade-offs consistent with the life-history hypothesis. Thus, dormancy during non-life-threatening periods that are unfavorable for reproduction may be more widespread than previously thought.

The Evolution of Body Size in Terrestrial Tetrapods

Body size is a fundamental trait in evolutionary and ecological research, given that it varies allometrically with several relevant features, such as life-history and physiological traits. Although previous studies uncovered many intriguing patterns, finding general principles of body size evolution in vertebrates has been elusive. In this study, we take advantage of recent advances in phylogenetic comparative methods and the availability of large-scale datasets to explore body size evolution in terrestrial vertebrates. Ancestral character estimation and disparity-through-time plots showed considerable variation in body size evolution, both across lineages and over time. In addition, regardless of the corresponding taxon, posterior predictive simulation demonstrated several consistent ways in which body size evolution in those groups departed from constant-rate models, namely: (1) there was considerable rate heterogeneity within each taxon, (2) there was a positive relationship between body size and its rate of evolution (i.e., large-bodied animals evolved faster than small-bodied ones), and (3) faster evolutionary rates near the present. Finally, geographical mapping of body mass and evolutionary rates revealed some similarities across taxa, but no clear latitudinal trends. Overall, these results indicate that there may be general patterns in the body size evolution on large scales in terrestrial vertebrates, with some intriguing taxon-specific differences.

Biotic and abiotic factors and the phylogenetic structure of extinction in the evolution of Tethysuchia

AbstractCrocodylomorpha is a large and diverse clade with a long evolutionary history now restricted to modern crocodilians. Tethysuchia is a less-inclusive clade of semi-amphibious taxa that crossed two biological crises: the second Oceanic Anoxic Event (OAE 2) and the Cretaceous/Paleogene (K/Pg) crisis. Numerous studies have sought to find the driving factors explaining crocodylomorph evolution, producing contradictory conclusions. Studies of included groups may be useful. Here, we study factors driving tethysuchian evolution using phylogenetically informed statistical analyses. First, we tested the phylogenetic structure of tethysuchian extinction at the OAE 2 and K/Pg crises. We then used phylogenetic comparative methods to test the influence of intrinsic (body size, snout proportion) and extrinsic (temperature, paleolatitude) factors on the evolution of tethysuchian diversity at the OAE 2 and the K/Pg crises. Finally, we tested whether temperature influenced the evolution of body size. We conclude that (1) extinction was not random in regard to phylogeny for Tethysuchia at the OAE 2 and K/Pg crises; (2) while an important tethysuchian turnover follows OAE 2, the K/Pg crisis was followed by an explosion in diversity of tethysuchians, probably linked to the colonization of emptied ecological niches; (3) tethysuchians lived in warmer environments after the OAE 2 crisis, possibly because of both global warming and latitudinal distribution shifts; (4) there is a significant change of snout proportion after the OAE 2 and the K/Pg crises, likely caused by niche partitioning; and (5) there is a positive correlation between body size and temperature, possibly because of a longer growth season.

Shark genome size evolution and its relationship with cellular, life-history, ecological, and diversity traits.

Among vertebrates, sharks exhibit both large and heterogeneous genome sizes ranging from 2.86 to 17.05pg. Aiming for a better understanding of the patterns and causalities of shark genome size evolution, we applied phylogenetic comparative methods to published genome-size estimates for 71 species representing the main phylogenetic lineages, life-histories and ecological traits. The sixfold range of genome size variation was strongly traceable throughout the phylogeny, with a major expansion preceding shark diversification during the late Paleozoic and an ancestral state (6.33pg) close to the present-day average (6.72pg). Subsequent deviations from this average occurred at higher rates in squalomorph than in galeomorph sharks and were unconnected to evolutionary changes in the karyotype architecture, which were dominated by descending disploidy events. Genome size was positively correlated with cell and nucleus sizes and negatively with metabolic rate. The metabolic constraints on increasing genome size also manifested at higher phenotypic scales, with large genomes associated with slow lifestyles and purely marine waters. Moreover, large genome sizes were also linked to non-placental reproductive modes, which may entail metabolically less demanding embryological developments. Contrary to ray-finned fishes, large genome size was associated neither with the taxonomic diversity of affected clades nor with low genetic diversity.

Evolutionary dependence of host type and chasmothecial appendage morphology in obligate plant parasites belonging to Erysipheae (powdery mildew, Erysiphaceae)

ABSTRACT Evolutionary relationships between the morphological and ecological traits of fungi are poorly understood. The appendages of chasmothecia, which are sexual reproductive organs of Erysiphaceae, are considered to play a crucial role in the overwintering strategies of these fungi on host plants. Previous studies suggested that both the host type and appendage morphology evolved at the same nodes and transitioned from complex appendages on deciduous hosts to simple appendages on herb/evergreen hosts. However, the evolutionary dependence between host type and appendage morphology remains unproven owing to the limited species data used in analyses. To elucidate the evolutionary relationship between host type and appendage morphology, we used phylogenetic comparative methods (PCMs) to investigate the state transition, ancestral state, evolutionary dependence, and contingent evolution within Erysipheae, the largest and most diverse tribe in Erysiphaceae. Our PCMs, based on a comprehensive data set of Erysipheae, revealed that the most ancestral states were deciduous host types and complex appendages. From these ancestral states, convergent evolution toward the herb/evergreen host types and simple appendages occurred multiple times at the same nodes. For the first time in Erysiphaceae, we detected an evolutionary dependence between host type and appendage morphology. This is one of the few examples in which evolutionary dependence between host phenology and morphological traits in plant-parasitic fungi was demonstrated using PCMs. Appendage simplification on herb/evergreen hosts and complications on deciduous hosts can be reasonably explained by the functional advantages of each appendage type in different overwintering strategies. These expected appendage functions can explain approximately 90% of host type and appendage morphology combinations observed in the analyzed taxa. However, our results also highlighted the occurrence of evolutionary shifts that deviate from the expected advantages of each appendage morphology. These seemingly irrational shifts might be interpretable from the flexibility of overwintering strategies and quantification of appendage functions.

The neoteny goldilocks zone: The evolution of neoteny in Ambystoma.

Neoteny is a developmental strategy wherein an organism reaches sexual maturity without associated adult characteristics. In salamanders, neoteny takes the form of individuals retaining aquatic larval characteristics such as external gills upon maturation. Mole salamanders (Ambystoma) occupy a wide range of habitats and areas across the North American continent, and display examples of non-neotenic, facultatively neotenic and obligate neotenic species, providing high variation for investigating the factors influencing the evolution of neoteny. Here, we use phylogenetic comparative methods to test existing hypotheses that neoteny is associated with elevational and latitudinal distribution, cave-associated isolation, and hybridisation-related polyploidy. We also test if neoteny influences the diversity of habitats a species can occupy, since the restriction to an aquatic life should constrain the availability of different niches. We find that neoteny tends to occur in a narrow latitudinal band between 20-30° North, with particularly narrow latitudinal ranges for obligate compared to facultative neotenic species (16-52° North). We also find that facultatively neotenic species occur at elevations more than twice as high as other species on average, and that species with a higher frequency of neoteny typically have lower habitat diversity. Our results suggest that evolutionary transitions between non-neotenic and facultative neoteny states occur at relatively high and approximately equal rates. Moreover, we estimate that obligate neoteny cannot evolve directly from non-neotenic species (and vice versa), such that facultative neoteny acts as an evolutionary 'stepping stone' to and from obligate neoteny. However, our transition rate estimates suggest that obligate neoteny is lost >4-times faster than it evolves, partly explaining the rarity of obligate species. These results support the hypothesis that low latitudes favour the evolution of neoteny, presumably linked to more stable (aquatic) environments due to reduced seasonality, but once evolved it may constrain the diversity of habitats.

Headhunting and warfare in Austronesia: A phylogenetic comparative analysis

Headhunting – the practice of acquiring human heads for ritual purposes – was historically widespread around the world. We hypothesize that headhunting represented a cultural response to frequent inter-tribal warfare and served as a mechanism to train warriors ready to defend their community. The practice was effective since, first, it allowed verification of warrior quality based on performance in headhunting raids and, second, it offered a system of rewards for men to develop and refine warfare skills. We use phylogenetic comparative methods and ethnographic data to empirically investigate this hypothesis in a sample of preindustrial Austronesian societies. Headhunting turns out to be substantially more prevalent in societies exposed to frequent warfare, accounting for shared cultural ancestry and a host of potentially confounding characteristics. Furthermore, Bayesian estimation of correlated evolution models suggests that, consistent with our hypothesis, the adoption of headhunting typically followed increases in warfare frequency and the decline of this practice was preceded by reduced intergroup conflict.

Blocking then stinging as a case of two-step evolution of defensive cage architectures in herbivore-driven ecosystems.

Dense branching and spines are common features of plant species in ecosystems with high mammalian herbivory pressure. While dense branching and spines can inhibit herbivory independently, when combined, they form a powerful defensive cage architecture. However, how cage architecture evolved under mammalian pressure has remained unexplored. Here we show how dense branching and spines emerged during the age of mammalian radiation in the Combretaceae family and diversified in herbivore-driven ecosystems in the tropics. Phylogenetic comparative methods revealed that modern plant architectural strategies defending against large mammals evolved via a stepwise process. First, dense branching emerged under intermediate herbivory pressure, followed by the acquisition of spines that supported higher speciation rates under high herbivory pressure. Our study highlights the adaptive value of dense branching as part of a herbivore defence strategy and identifies large mammal herbivory as a major selective force shaping the whole plant architecture of woody plants.