Evolution Of Traits Research Articles

Genetic independence between traits separated by metamorphosis is widespread but varies with biological function.

Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life stages, similarly to how it enables some limbs and organs to develop at specific life stages? We tested this hypothesis by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 30% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 46% of the traits showed some level of genetic independence between stages. Gene ontology terms enrichment revealed that across stages correlated traits were often involved in proteins synthesis, insecticide resistance and innate immunity, while a vast number of genes expression traits associated with energy metabolism were independent between life stages. We compared our results to a similar case: genetic constraints between males and females in gonochoric species (i.e. sexual antagonism). We expected selection for the separation between males and females to be higher than between juvenile and adult functions, as gonochorism is a more common strategy in the animal kingdom than metamorphosis. Surprisingly, we found that inter-stage constraints were lower than inter-sexual genetic constraints. Overall, our results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life stages.

Diadromy Drives Elevated Rates of Trait Evolution and Ecomorphological Convergence in Clupeiformes (Herring, Shad, and Anchovies).

AbstractMigration can have a profound influence on rates and patterns of phenotypic evolution. Diadromy is the migration between marine and freshwater habitats for feeding and reproduction that can require individuals to travel tens to thousands of kilometers. The high energetic demands of diadromy are predicted to select for ecomorphological traits that maximize swimming and locomotor efficiency. Intraspecific studies have shown repeated instances of divergence among diadromous and nondiadromous populations in locomotor and foraging traits, which suggests that at a macroevolutionary scale diadromous lineages may experience convergent evolution onto one or multiple adaptive optima. We tested for differences in rates and patterns of phenotypic evolution among diadromous and nondiadromous lineages in Clupeiformes, a clade that has evolved diadromy more than 10 times. Our results show that diadromous clupeiforms show convergent evolution for some locomotor traits and faster rates of evolution, which we propose are adaptive responses to the locomotor demands of migration. We also find evidence that diadromous lineages show convergence into multiple regions of multivariate trait space and suggest that these respective trait spaces are associated with differences in migration and trophic ecology. However, not all locomotor traits and no trophic traits show evidence of convergence or elevated rates of evolution associated with diadromy. Our results show that long-distance migration influences the tempo and patterns of phenotypic evolution at macroevolutionary scales, but there is not a single diadromous syndrome.

Diverse O-methyltransferases catalyze the biosynthesis of floral benzenoids that repel aphids from the flowers of waterlily Nymphaea prolifera.

Nymphaea is a key genus of the ANA grade (Amborellales, Nymphaeales, and Austrobaileyales) of basal flowering plants, which serve as a key model to study the early evolution of floral traits. In this study, we comprehensively investigated the emission, biosynthesis, and biological function of the floral scent in a night-blossoming waterlily Nymphaea prolifera. The headspace volatile collection combined with GC-MS analysis showed that the floral scent of N. prolifera is predominately comprised by methylated benzenoids including anisole, veratrole, guaiacol, and methoxyanisole. Moreover, the emission of these floral benzenoids in N. prolifera exhibited temporal and spatial pattern with circadian rhythm and tissue specificity. By creating and mining transcriptomes of N. prolifera flowers, 12 oxygen methyltransferases (NpOMTs) were functionally identified. By in vitro enzymatic assay, NpOMT3, 6, and 7 could produce anisole and NpOMT5, 7, 9, produce guaiacol, whereas NpOMT3, 6, 9, 11 catalyzed the formation of veratrole. Methoxyanisole was identified as the universal product of all NpOMTs. Expression patterns of NpOMTs provided implication for their roles in the production of the respective benzenoids. Phylogenetic analysis of OMTs suggested a Nymphaea-specific expansion of the OMT family, indicating the evolution of lineage-specific functions. In bioassays, anisole, veratrole, and guaiacol in the floral benzenoids were revealed to play the critical role in repelling waterlily aphids. Overall, this study indicates that the basal flowering plant N. prolifera has evolved a diversity and complexity of OMT genes for the biosynthesis of methylated benzenoids that can repel insects from feeding the flowers. These findings provide new insights into the evolutional mechanism and ecological significance of the floral scent from early-diverged flowering plants.

Chromosome-level genomes of three key Allium crops and their trait evolution

Chromosome-level genomes of three key Allium crops and their trait evolution

Exposing the error hidden in plain sight: A critique of Calder's (1983) group selectionist seed-dispersal hypothesis for mistletoe "mimicry" of host plants.

Some mistletoe species (Loranthaceae) resemble their host plants to a striking degree. Various mechanisms have been proposed for the developmental origins of novel traits that cause mistletoes to appear similar to their hosts, as well as for the adaptive phenotypic evolution of such traits. Calder (1983) proposed a logically flawed group selectionist seed-dispersal hypothesis for mistletoes to resemble their hosts. Calder's (1983) hypothesis does not provide a viable potential explanation for mistletoe resemblance to hosts.

Single-nucleotide variants within heart enhancers increase binding affinity and disrupt heart development.

Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.

Hydrological niche regulation induced by different resistance strategies facilitates coexistence of P. longipes and L. communis under drought stress.

Under global warming, the availability of water resources is one of the most important factors affecting trait evolution and plant species distribution across terrestrial ecosystems, and the relationships between drought resistance strategies and the hydrological niche characteristics of plants are worth studying. We continuously monitored physiological drought response parameters such as gs , Tr , proline, soluble sugar, gene expression and activities of SOD, POD, and CAT to assess drought resistance strategies of Platycarya longipes and Lindera communis; determined plant soil hydrological niche separation by stable H and O isotope analysis; and analysed the effects of interspecific water competition by comparing the differences in morphological and physiological parameters between solo and mixed planting. Under drought stress, L. communis exhibited a drought avoidance strategy, and P. longipes exhibited a drought tolerance strategy. L. communis utilized the water within the shallow soil layer, while P. longipes mainly utilized the water in the deeper soil layer; there were fewer parameters with significant differences between the solo planting and the mixed planting of L. communis compared to P. longipes. Overall, P. longipes benefited from coexistence with L. communis under drought stress, which may be because L. communis employs a drought avoidance strategy, reducing soil water consumption in the drought environment. These results suggested that differences in functional traits or resistance strategies among species benefit species' coexistence in a community under drought stress.

Distribution and frequency of ace-1 and kdr mutations of Culex pipiens subgroup in the Republic of Korea

Mosquitoes in the Culex pipiens subgroup are the primary vectors of the West Nile virus. Two members, Culex pallens and Culex pipiens f. molestus, are present in the Republic of Korea (ROK). Because the Culex pipiens subgroup occurs in large amounts, often near human habitation, it is frequently exposed to various insecticides, which is probably responsible for the rapid evolution of insecticide resistance traits. Experiments related to insecticide resistance in the Culex pipiens subgroup conducted in the ROK have been performed without discrimination below the species level. This study categorized Culex pipiens mosquitoes subgroup from 13 regions in the ROK into Culex pallens and Culex pipiens f. molestus, and target site genotypes for acetylcholinesterase-1 (ace-1) and voltage-gated sodium channel (vgsc) genes were identified for each taxon. Screening for ace-1 did not identify a resistant allele (G119S) in Cx. pipiens f. molestus, and heterozygous resistance (AGC/GGC) was identified in one Cx. pallens collected in Mokpo. In vgsc, knockdown resistance (kdr) mutations [TTT(L1014F) and TCA(L1014S)] were present in both taxa, with Cx. pipiens f. molestus having homozygous resistance (TTT/TTT): 44%, heterozygous resistance (TTT/TTA): 28%, and homozygous susceptibility (TTA/TTA): 28%, whereas Cx. pallens showed homozygous resistance (TTT/TTT or TCA/TCA): 26%, heterozygous resistance (TTT/TTA, TTT/TCA, or TCA/TTA): 26%, and homozygous susceptibility (TTA/TTA): 48%. Furthermore, the unique vgsc allele was present in both Cx. pipiens f. molestus and Cx. pallens. This was the first experiment to analyze the Culex pipiens subgroup living in the ROK below the species level, and its results could be used in the future for more detailed mosquito control.

Analyses of S Protein Homology Using the Genomes of SARS-CoV-2 Specimens Unveil Missing Links in the Temporal Order of Mutations in Its Variants.

(1) Background: Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the evolutionary traits of its variants have been revealed. However, the temporal order of the majority of mutations harbored by variants after the closest ancestors (or precursors), as "missing links", remains unclear. In this study, we aimed to unveil such missing links based on analyses of S protein homology by focusing on specimens with incomplete sets of S protein mutations in a variant. (2) Methods: Prevariant and postvariant mutations were defined as those before and after the variant's development, respectively. A total of 6,758,926 and 14,519,521 genomes were obtained from the National Center for Biotechnology Information and the GISAID initiative, respectively, and S protein mutations were detected based on BLASTN analyses. (3) Results: The temporal order of prevariant mutations harbored by 12 variants was deduced. In particular, the D950N mutation in the Mu variant shows V-shaped mutation transitions, in which multiple routes of evolution were combined and resulted in the formation of a V-shaped transition, indicating recombination. (4) Conclusions: Many genome data for SARS-CoV-2 unveiled the candidate precursors of Mu variant based on a data-driven approach to its prevariant mutations in each nation.

Sexually concordant selection on floral traits despite greater opportunity for selection through male fitness.

Pollinators are important drivers of floral trait evolution, yet plant populations are not always perfectly adapted to their pollinators. Such apparent maladaptation may result from conflicting selection through male and female sexual functions in hermaphrodites. We studied sex-specific mating patterns and phenotypic selection on floral traits in Aconitum gymnandrum. After genotyping 1786 offspring, we partitioned individual fitness into sex-specific selfed and outcrossed components and estimated phenotypic selection acting through each. Relative fitness increased with increasing mate number, and more so for male function. This led to greater opportunity for selection through outcrossed male fitness, though patterns of phenotypic selection on floral traits tended to be similar, and with better support for selection through female rather than male fitness components. We detected directional selection through one or more fitness component for larger flower number, larger flowers, and more negative nectar gradients within inflorescences. Our results are consistent with Bateman's principles for sex-specific mating patterns and illustrate that, despite the expected difference in opportunity for selection, patterns of variation in selection across traits can be rather similar for the male and female sexual functions. These results shed new light on the effect of sexual selection on the evolution of floral traits.

Reference Genome Choice and Filtering Thresholds Jointly Influence Phylogenomic Analyses.

Molecular phylogenies are a cornerstone of modern comparative biology and are commonly employed to investigate a range of biological phenomena, such as diversification rates, patterns in trait evolution, biogeography, and community assembly. Recent work has demonstrated that significant biases may be introduced into downstream phylogenetic analyses from processing genomic data; however, it remains unclear whether there are interactions among bioinformatic parameters or biases introduced through the choice of reference genome for sequence alignment and variant calling. We address these knowledge gaps by employing a combination of simulated and empirical data sets to investigate the extent to which the choice of reference genome in upstream bioinformatic processing of genomic data influences phylogenetic inference, as well as the way that reference genome choice interacts with bioinformatic filtering choices and phylogenetic inference method. We demonstrate that more stringent minor allele filters bias inferred trees away from the true species tree topology, and that these biased trees tend to be more imbalanced and have a higher center of gravity than the true trees. We find the greatest topological accuracy when filtering sites for minor allele count (MAC) >3-4 in our 51-taxa data sets, while tree center of gravity was closest to the true value when filtering for sites with MAC >1-2. In contrast, filtering for missing data increased accuracy in the inferred topologies; however, this effect was small in comparison to the effect of minor allele filters and may be undesirable due to a subsequent mutation spectrum distortion. The bias introduced by these filters differs based on the reference genome used in short read alignment, providing further support that choosing a reference genome for alignment is an important bioinformatic decision with implications for downstream analyses. These results demonstrate that attributes of the study system and dataset (and their interaction) add important nuance for how best to assemble and filter short-read genomic data for phylogenetic inference.

Dynamic evolution of locomotor performance independent of changes in extended phenotype use in spiders.

Many animals use self-built structures (extended phenotypes) to enhance body functions, such as thermoregulation, prey capture or defence. Yet, it is unclear whether the evolution of animal constructions supplements or substitutes body functions-with disparate feedbacks on trait evolution. Here, using brown spiders (Araneae: marronoid clade), we explored if the evolutionary loss and gain of silken webs as extended prey capture devices correlates with alterations in traits known to play an important role in predatory strikes-locomotor performance (sprint speed) and leg spination (expression of capture spines on front legs). We found that in this group high locomotor performance, with running speeds of over 100 body lengths per second, evolved repeatedly-both in web-building and cursorial spiders. There was no correlation with running speed, and leg spination only poorly correlated, relative to the use of extended phenotypes, indicating that web use does not reduce selective pressures on body functions involved in prey capture and defence per se. Consequently, extended prey capture devices serve as supplements rather than substitutions to body traits and may only be beneficial in conjunction with certain life-history traits, possibly explaining the rare evolution and repeated loss of trapping strategies in predatory animals.

Evolutionary stability of antigenically escaping viruses.

Antigenic variation is the main immune escape mechanism for RNA viruses like influenza or SARS-CoV-2. While high mutation rates promote antigenic escape, they also induce large mutational loads and reduced fitness. It remains unclear how this cost-benefit trade-off selects the mutation rate of viruses. Using a traveling wave model for the coevolution of viruses and host immune systems in a finite population, we investigate how immunity affects the evolution of the mutation rate and other nonantigenic traits, such as virulence. We first show that the nature of the wave depends on how cross-reactive immune systems are, reconciling previous approaches. The immune-virus system behaves like a Fisher wave at low cross-reactivities, and like a fitness wave at high cross-reactivities. These regimes predict different outcomes for the evolution of nonantigenic traits. At low cross-reactivities, the evolutionarily stable strategy is to maximize the speed of the wave, implying a higher mutation rate and increased virulence. At large cross-reactivities, where our estimates place H3N2 influenza, the stable strategy is to increase the basic reproductive number, keeping the mutation rate to a minimum and virulence low.

How seasonality influences the thermal biology of lizards with different thermoregulatory strategies: a meta-analysis.

Ectotherms that maintain thermal balance in the face of varying climates should be able to colonise a wide range of habitats. In lizards, thermoregulation usually appears as a variety of behaviours that buffer external influences over physiology. Basking species rely on solar radiation to raise body temperatures and usually show high thermoregulatory precision. By contrast, species that do not bask are often constrained by climatic conditions in their habitats, thus having lower thermoregulatory precision. While much focus has been given to the effects of mean habitat temperatures, relatively less is known about how seasonality affects the thermal biology of lizards on a macroecological scale. Considering the current climate crisis, assessing how lizards cope with temporal variations in environmental temperature is essential to understand better how these organisms will fare under climate change. Activity body temperatures (Tb ) represent the internal temperature of an animal measured in nature during its active period (i.e. realised thermal niche), and preferred body temperatures (Tpref ) are those selected by an animal in a laboratory thermal gradient that lacks thermoregulatory costs (i.e. fundamental thermal niche). Both traits form the bulk of thermal ecology research and are often studied in the context of seasonality. In this study, we used a meta-analysis to test how environmental temperature seasonality influences the seasonal variation in the Tb and Tpref of lizards that differ in thermoregulatory strategy (basking versus non-basking). Based on 333 effect sizes from 137 species, we found that Tb varied over a greater magnitude than Tpref across seasons. Variations in Tb were not influenced by environmental temperature seasonality; however, body size and thermoregulatory strategy mediated Tb responses. Specifically, larger species were subjected to greater seasonal variations in Tb , and basking species endured greater seasonal variations in Tb compared to non-basking species. On the other hand, the seasonal variation in Tpref increased with environmental temperature seasonality regardless of body size. Thermoregulatory strategy also influenced Tpref , suggesting that behaviour has an important role in mediating Tpref responses to seasonal variations in the thermal landscape. After controlling for phylogenetic effects, we showed that Tb and Tpref varied significantly across lizard families. Taken together, our results support the notion that the relationship between thermal biology responses and climatic parameters can be taxon and trait dependent. Our results also showcase the importance of considering ecological and behavioural aspects in macroecological studies. We further highlight current systematic, geographical, and knowledge gaps in thermal ecology research. Our work should benefit those who aim to understand more fully how seasonality shapes thermal biology in lizards, ultimately contributing to the goal of elucidating the evolution of temperature-sensitive traits in ectotherms.

Prevalent bee venom genes evolved before the aculeate stinger and eusociality

BackgroundVenoms, which have evolved numerous times in animals, are ideal models of convergent trait evolution. However, detailed genomic studies of toxin-encoding genes exist for only a few animal groups. The hyper-diverse hymenopteran insects are the most speciose venomous clade, but investigation of the origin of their venom genes has been largely neglected.ResultsUtilizing a combination of genomic and proteo-transcriptomic data, we investigated the origin of 11 toxin genes in 29 published and 3 new hymenopteran genomes and compiled an up-to-date list of prevalent bee venom proteins. Observed patterns indicate that bee venom genes predominantly originate through single gene co-option with gene duplication contributing to subsequent diversification.ConclusionsMost Hymenoptera venom genes are shared by all members of the clade and only melittin and the new venom protein family anthophilin1 appear unique to the bee lineage. Most venom proteins thus predate the mega-radiation of hymenopterans and the evolution of the aculeate stinger.

Do male reproductive traits evolve at the intraspecific level in response to the amount of placentotrophy in a genus of viviparous fishes?

Placentotrophy is a particular type of maternal provisioning to developing embryos, in which mothers actively provide nutrients via complex placental structures. Placentotrophy implies less pre-fertilization investment, resulting in a shift from pre- to post-copulatory sexual selection. This change can potentially result in a conflict between females and males. This phenomenon has been demonstrated at the interspecific level in viviparous fishes of the family Poeciliidae, in which males of species that lack placentotrophy have evolved traits related to pre-copulatory sexual selection such as coloration, ornaments, and courtship behavior. Placentotrophic species, on the other hand, have evolved traits associated with post-copulatory sexual selection such as long intromittent organs (gonopodium) and increased sexual coercion behavior. Here we test, for the first time at the intraspecific level, whether there is a similar relationship between a higher degree of female placentotrophy and the evolution of male reproductive traits (larger testes and longer gonopodia) in three species of the genus Poeciliopsis (P. gracilis, P. infans, and P. prolifica). We observed a tendency towards longer gonopodia in males of P. gracilis as well as the largest testes of P. prolifica males in the populations with the highest degrees of placentotrophy. However, the statistical support for these findings was relatively weak. Therefore, we failed to support the hypothesis of a selective effect of female placentotrophy on male gonads and genitalia. We discuss other evolutionary forces that may have driven the observed intraspecific variation in male reproductive traits of Poeciliopsis fishes.

A derived weedy rice × ancestral cultivar cross identifies evolutionarily relevant weediness QTLs.

Weedy rice (Oryza spp.) is a weedy relative of the cultivated rice that competes with the crop and causes significant production loss. The BHA (blackhull awned) US weedy rice group has evolved from aus cultivated rice and differs from its ancestors in several important weediness traits, including flowering time, plant height and seed shattering. Prior attempts to determine the genetic basis of weediness traits in plants using linkage mapping approaches have not often considered weed origins. However, the timing of divergence between crossed parents can affect the detection of quantitative trait loci (QTL) relevant to the evolution of weediness. Here, we used a QTL-seq approach that combines bulked segregant analysis and high-throughput whole genome resequencing to map the three important weediness traits in an F2 population derived from a cross between BHA weedy rice with an ancestral aus cultivar. We compared these QTLs with those previously detected in a cross of BHA with a more distantly related crop, indica. We identified multiple QTLs that overlapped with regions under selection during the evolution of weedy BHA rice and some candidate genes possibly underlying the evolution weediness traits in BHA. We showed that QTLs detected with ancestor-descendant crosses are more likely to be involved in the evolution of weediness traits than those detected from crosses of more diverged taxa.

How Does the Time-Varying Network Structure Evolve between the EU Carbon Futures Prices and Industrial and Energy-Related Indices? A Study Based on a Time-Varying T-Copula

Industrial and energy-related industries are major sources of carbon dioxide emissions, and their interdependence, as reflected in the financial field, has attracted the attention of scholars. For the purpose of exploring the evolutionary characteristics of the short-term dynamic correlation coefficient between the EU carbon futures price and the industrial and energy-related indices, this paper selected the settlement price of EU carbon emission quota futures, the MSCI energy I index on three dimensions, and the Dow Jones industrial index and West Texas crude oil futures price, as sample data. Using the time-varying t-copula model to measure the dynamic correlation coefficient between variables, the time-sliding window idea and coarse-grained method were combined to establish the correlation fluctuation mode, and a complex network theory and analysis methods were used to study the evolutionary traits of the time-varying network structure between the EU carbon price and the industrial and energy-related index. The results show that the transmission objects of the key correlation fluctuation modes in the network are stable and maintain their own state with a high probability. Second, the clustering effect exists in the transmission process. Some nodes with high mediating abilities are also the key correlation wave modes in the dynamic correlation evolution network. This study provides ideas for the study of the correlations between multiple variables and is also a useful reference for international investors.

Evolutionary dynamics of whole-body regeneration across planarian flatworms

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.

Accounting for Within-Species Variation in Continuous Trait Evolution on a Phylogenetic Network

Within-species trait variation may be the result of genetic variation, environmental variation, or measurement error, for example. In phylogenetic comparative studies, failing to account for within-species variation has many adverse effects, such as increased error in testing hypotheses about evolutionary correlations, biased estimates of evolutionary rates, and inaccurate inference of the mode of evolution. These adverse effects were demonstrated in studies that considered a tree-like underlying phylogeny. Comparative methods on phylogenetic networks are still in their infancy. The impact of within-species variation on network-based methods has not been studied. Here, we introduce a phylogenetic linear model in which the phylogeny can be a network to account for within-species variation in the continuous response trait assuming equal within-species variances across species. We show how inference based on the individual values can be reduced to a problem using species-level summaries, even when the within-species variance is estimated. Our method performs well under various simulation settings and is robust when within-species variances are unequal across species. When phenotypic (within-species) correlations differ from evolutionary (between-species) correlations, estimates of evolutionary coefficients are pulled towards the phenotypic coefficients for all methods we tested. Also, evolutionary rates are either underestimated or overestimated, depending on the mismatch between phenotypic and evolutionary relationships. We applied our method to morphological and geographical data from Polemonium. We find a strong negative correlation of leaflet size with elevation, despite a positive correlation within species. Our method can explore the role of gene flow in trait evolution by comparing the fit of a network to that of a tree. We find marginal evidence for leaflet size being affected by gene flow and support for previous observations on the challenges of using individual continuous traits to infer inheritance weights at reticulations. Our method is freely available in the Julia package PhyloNetworks.