Phylogenetic comparative methods (PCMs) are fundamental tools for understanding trait evolution across species. While linear models are widely used for continuous traits in ecology and evolution, their application to discrete traits, particularly ordinal and nominal traits, remains limited. Researchers sometimes recategorise such traits into binary traits (0 or 1 data) to make them more manageable. However, this risks distorting the original data structure and meaning, potentially reducing the information it initially contained. This paper promotes the use of phylogenetic generalised linear mixed-effects models (PGLMMs) as a flexible framework for analysing the evolution of discrete traits. We introduce the theoretical foundations of PGLMMs and demonstrate how univariate and multivariate versions of binary PGLMMs, which might be more familiar to evolutionary biologists, can be conceptually extended to model ordinal and nominal traits. Specifically, we describe ordered and unordered multinomial PGLMMs for ordinal and nominal traits, respectively. We then explain how to interpret regression coefficients and (co)variance components, including associated statistics (e.g., phylogenetic heritability and correlation) from PGLMMs for discrete traits. Using real-world examples from avian datasets, we illustrate the practical implementation of PGLMMs to reveal evolutionary patterns in discrete traits. We also provide online tutorials to guide researchers through the application of these models using Bayesian implementations in R. By making complex models more accessible, we aim to facilitate a more precise and insightful understanding of the evolution and function of discrete traits, which have received relatively limited attention in evolutionary biology so far.

Flight is a crucial activity for winged insects, involving diverse behaviours, and wing morphology has often been proposed as a key factor influencing flight capacity. Traits such as wing loading, wing:ratio, and wing aspect ratio have been suggested as targets of natural selection, exhibiting environmental and genetic variability. Here, we evaluate the relationship between morphological traits and two aspects of flight performance: flight duration (PTF) and its robustness (CVPTF) in Drosophila melanogaster. Additionally, we investigate the genetic basis of PTF and CVPTF. Our findings highlight two main insights. First, while genetic variation was detected in both morphological and flight traits, variability was considerably lower in the first case compared to the second one. This likely explains the absence of a strong relationship between these traits. Furthermore, among 107 candidate genes detected for flight traits, only a few were associated with wing morphology, reinforcing the weak link between morphology and performance. Second, we observed a negative relationship between PTF and CVPTF across sexes. However, CVPTF was associated with more polymorphisms and candidate genes than PTF, and only three genes (if, fru, and nAChRα5) were associated with both traits. These results suggest that both flight traits present distinct genetic bases.In conclusion, our results challenge the hypotheses that emphasize that wing morphology is determinant for flight performance. We also provide a first approximation to the genetic basis underlying two flight estimators, advancing our understanding of this behavioural trait in Drosophila.

Genomic differentiation usually accompanies speciation, but that differentiation is often highly heterogeneous across the genome. Understanding what parts of the genome are more prone to differentiation can inform us about genomic regions and evolutionary processes that may be central to the speciation process. Here, we study genomic variation among 3 hybridizing species of North American woodpecker: red-breasted, red-naped, and yellow-bellied sapsuckers (Sphyrapicus ruber, S. nuchalis, and S. varius). We use whole genome resequencing to measure genetic variation among these species and to quantify how the level of differentiation varies across the genome. We find that regions of high relative differentiation between species (FST) tend to have low absolute nucleotide distance between species (πB), indicating that regions of high relative differentiation often have more recent between-population coalescence times than regions of low relative differentiation do. Most of the high-FST genomic windows are found on the Z chromosome, pointing to this sex chromosome as being particularly important in sapsucker differentiation and potentially speciation. These results are consistent with a model of speciation in which selective sweeps of globally advantageous variants spread among partly differentiated populations, followed by differential local adaptation of those same genomic regions. We propose that sapsucker speciation may have occurred primarily via this process occurring on the Z chromosomes, resulting in genetic incompatibilities involving divergent Z chromosomes.

Protective microbes are known for their service to hosts. While they allow hosts to survive infection, microbes, too, benefit from successful inhibition of incoming pathogens. Under constant pathogen exposure, protective symbionts should thus be selected for. Yet, it is less clear if, and how, microbe-mediated protection is maintained in symbionts in the absence of pathogen pressure. Addressing the stability of protective symbiosis during bacterial adaptation to healthy hosts, we studied microbe-mediated protection of Pseudomonas lurida MYb11 against pathogenic Bacillus thuringiensis Bt247 in its natural host Caenorhabditis elegans MY316. Specifically, we assessed host protection and in vitro inhibition of the pathogen for a collection of derived MYb11 isolates, which were previously evolved during serial passaging in healthy C. elegans hosts. We found that all evolved MYb11 isolates continued to inhibit the pathogen in vitro, while most, albeit not all, continued to protect hosts. We focused on two of these isolates, MT5 and MT11, one with and one without protection, and found that intact protection is associated with high symbiont colonization and resulting lower pathogen proportions. In sum, our study dissects the stability of a natural protective symbiosis and suggests that high colonization ability ensures microbe-mediated protection, even if microbes adapt to host association in the absence of pathogen selection.

New genes can emerge de novo from non-genic genomic regions. In budding yeast, computational predictions have shown that intergenic regions harbour a higher-than-expected propensity to encode transmembrane domains, if theoretically translated into proteins. This propensity seems to be linked to the high prevalence of predicted transmembrane domains in evolutionarily young genes. However, what accounts for this enriched propensity is not known. Here, we show that specific arrangements of polyA/T tracts, which are abundant and enriched in yeast intergenic regions, explain this observation. These tracts are known to function as nucleosome-depleted regions, which prevent or reduce nucleosome formation to enable transcription of surrounding genes. We provide evidence that these polyA/T tracts have been repeatedly coopted through de novo gene emergence for the evolution of novel small genes encoding proteins with predicted transmembrane domains. These findings support a previously proposed "transmembrane-first" model of de novo gene birth and help explain why evolutionarily young yeast genes are rich in transmembrane domains. They contribute to our understanding of the process of de novo gene evolution and show how seemingly distinct but potentially interacting levels of functionality can exist within the same genomic loci.

Spermatozoa exhibit striking morphological variation across the animal kingdom. In passerine birds, sperm exhibit considerable variation in size, yet the basic sperm phenotype is highly conserved; sperm are filiform, the head is corkscrew-shaped, and the midpiece is elongated and twisted around the flagellum. A significant departure from this typical sperm morphology has been reported in the sister species, the Eurasian bullfinch (Pyrrhula pyrrhula) and Azores bullfinch (P. murina). Here, we report a second evolutionary shift in passerine sperm phenotype in the nominate subspecies of the red-browed finch (Neochmia temporalis temporalis); sperm are nonfiliform, with an ellipsoid head and an extremely short midpiece restricted to the nuclear-axoneme junction. Additionally, we show that the sperm phenotype of the red-browed finch is similar to the putatively neotenous sperm described in the two bullfinch species. Using whole-genome data, we found no evidence that the unusual sperm phenotype of the red-browed finch is associated with reduced genetic variation or a population bottleneck. In contrast, using data on relative testes size, we find some support for the hypothesis that relaxed postcopulatory sexual selection, via a lack of sperm competition, may, at least in part, explain the unusual sperm of the red-browed finch. We also discuss the possible roles of mutation, genetic drift, and genetic hitchhiking in the evolutionary origins and maintenance of neotenous sperm phenotypes. Finally, we suggest that these dramatic evolutionary shifts in sperm phenotype warrant further investigation and highlight the need for a greater understanding of the developmental and genomic basis of sperm phenotype.

Complex eukaryotes generally express different traits as they grow and develop to facilitate their function in different ecological niches. Theory suggests the evolution of differentiation between life stages is facilitated by the expression of different genes at different points throughout ontogeny, which alleviates evolutionary constraints. Therefore, ascertaining what contributes to specialized patterns of gene expression across ontogeny is fundamental to understanding the evolution of ontogenetic complexity. Expression divergence between duplicate genes and gene organization have been identified as important features of relatively complex ontogenies. Therefore, one could predict that their link to transcriptional specialization may be weaker across relatively simpler ontogenies. Here, we investigated the links between gene duplication, gene organization, and stage-specific patterns of expression across the relatively simple Caenorhabditis elegans life cycle. We found that signatures of stage-biased chromosomal regions were weaker in C. elegans than what has been previously described in organisms with more complex ontogenies. Furthermore, we found the extent that duplicate genes varied in ontogenetic expression pattern was more constrained in C. elegans. Overall, these findings help generalize our understanding of the association between gene duplication, gene organization, and the evolution of complex ontogenies.

Defensive symbionts-organisms that confer protection to their hosts against natural enemies such as parasites, predators, or herbivores-are found throughout the natural world. Theoretical and empirical studies have shown that defensive symbionts can both interfere with ecological interactions between hosts and exploiters, as well as drive exploiter evolution. Defensive symbionts are also potential candidates for biocontrol agents to help manage infectious diseases or agricultural pests. The impact of defensive symbionts on parasite ecology and evolution has therefore recently received increased empirical and theoretical attention. In this theoretical study, we investigate the impact that a defensive symbiont which protects hosts from infection (conferred resistance) has on the evolution of parasite virulence. We also explore how the extent of protection conferred by the defensive symbiont coevolves with parasite virulence, and how symbiont and parasite evolution affect the ecology of the host population in both the short- and long-term. We show that, while costly resistance-conferring defensive symbionts always select for increased parasite virulence, the overall long-term ecological effect on the host population may still be positive due to reductions in disease prevalence. This contrasts with tolerance-conferring symbionts (which protect against virulence), where the long-term ecological effects on the host population are always negative. We also show when the defensive symbiont can successfully eliminate the parasite. Resistance-conferring defensive symbionts therefore offer more promise as evolutionarily robust biocontrols than those that only confer tolerance.

Dioecy is a breeding system with separate females and males, where sex can be determined by sex chromosomes (for instance, XY males and XX females). Dioecy is generally associated with the evolution of sexual dimorphism. In this study, we tested whether small RNAs (sRNAs) play a role in sexual dimorphism and sex chromosome evolution in Silene latifolia. We generated new data for female and male sRNAs and DNA methylation. We showed that sRNAs are most of the time female-biased in S. latifolia, suggesting that the presence of the Y chromosome globally impacts the epigenome by diluting sRNAs genome-wide in males. We found limited evidence for the regulation of sex-biased genes by sRNAs, but we nonetheless identified a male-biased transcription factor that may potentially be regulated by sex-biased RNA-directed DNA methylation. This transcription factor might contribute to male traits through the regulation of key factors in sex determination and phenotypic sexual dimorphism. Finally, we compared female and male sRNA mapping along the S. latifolia sex chromosomes. We found that X and Y genes are targeted by significantly more sRNAs in males compared to females and pseudoautosomal region genes. Our results suggest that Y genes silencing following Y degeneration leads to the formation of sRNAs that can interact with both X and Y genes in males due to X-Y sequence homology. Our work calls for future investigation of the impact of these sRNAs generated from the Y chromosome on X gene expression in males.

The role of ecological interactions and abiotic factors in shaping macroevolutionary patterns has been extensively debated. Yet, the effect of predator-prey interactions on diversification dynamics is unclear. Here, we test the influence of predator-prey interactions in the diversification of sabre-toothed cats, a subfamily of felids (Felidae: Machairodontinae) that developed a highly specialized morphology, the notoriously long upper canines. This morphology has been associated with a high degree of trophic dependence on large prey, which is assumed as one of the main reasons for the group's demise. To test the hypothesis that prey availability affected the diversification of sabretooths we examined whether shifts in speciation and extinction rates were linked with changes in the species richness of their potential prey clades. The only association we detected was between the increase in extinction rates of North American sabretooths and the decline in Equidae richness. However, when accounting for the prey richness per species, regardless of taxonomic affiliation of the prey, we found that the endpoint of most sabretooth taxa occurred when prey richness for that taxon was at its lowest. Our analyses then suggested that reduced prey richness was associated with increased extinction risk. These results support the hypothesis that the extinction of sabretooths was associated with the decline in megafauna, but extend it to more remote times, millions of years before their final demise, suggesting that prey availability contributed to drive the diversification dynamics of the group. More broadly, our findings highlight the importance of ecological interactions in shaping macroevolutionary patterns.