Exploring Phylogenetic Signal in Multivariate Phenotypes by Maximizing Blomberg's K.

Due to the hierarchical structure of the tree of life, closely related species often resemble each other more than distantly related species; a pattern termed phylogenetic signal. Numerous univariate statistics have been proposed as measures of phylogenetic signal for single phenotypic traits, but the study of phylogenetic signal for multivariate data, as is common in modern biology, remains challenging. Here we introduce a new method to explore phylogenetic signal in multivariate phenotypes. Our approach decomposes the data into linear combinations with maximal (or minimal) phylogenetic signal, as measured by Blomberg's K. The loading vectors of these phylogenetic components or K-components can be biologically interpreted, and scatterplots of the scores can be used as a low-dimensional ordination of the data that maximally (or minimally) preserves phylogenetic signal. We present algebraic and statistical properties, along with two new summary statistics, KA and KG, of phylogenetic signal in multivariate data. Simulation studies showed that KA and KG have higher statistical power than the previously suggested statistic Kmult, especially if phylogenetic signal is low or concentrated in a few trait dimensions. In two empirical applications to vertebrate cranial shape (crocodyliforms and papionins), we found statistically significant phylogenetic signal concentrated in a few trait dimensions. The finding that phylogenetic signal can be highly variable across the dimensions of multivariate phenotypes has important implications for current maximum likelihood approaches to phylogenetic signal in multivariate data.

Integrative systematic revision of the Montseny brook newt (Calotriton arnoldi), with the description of a new subspecies.

The Montseny brook newt (Calotriton arnoldi), a glacial relict endemic to a small, isolated massif in northeast Spain, is considered the only Critically Endangered urodele in Europe. Its restricted range is divided by a deep valley that acts as an impassable barrier to dispersal, separating two isolated metapopulations (Western and Eastern) that correspond to independent lineages with different evolutionary trajectories, based on genetic and genomic data. Here, we address the ecological differentiation between lineages and discuss its potential effect on the phenotypic distinctness of each lineage. Based on multiple lines of evidence, we formally describe the Western Montseny brook newt as a new subspecies: Calotriton arnoldi laietanus ssp. nov. Finally, our study underscores the importance of considering taxonomic progress in the conservation policies of endangered species, ensuring appropriate management and protection of the newly described taxa.

The effect of miniaturization on the evolution of sexual size dimorphism in geckos.

The evolution of miniaturization can result in dramatic alterations of morphology, physiology, and behavior; however, the effects of miniaturization on sexual dimorphism remain largely unknown. Here we investigate how miniaturization influences patterns of sexual size dimorphism (SSD) in geckos. Measuring 1,875 individuals from 131 species, we characterized patterns of SSD relative to body size across two families. We found that miniaturized species were more female biased than non-miniaturized species. Additionally, one family that contained many miniaturized species (Sphaerodactylidae) displayed allometric patterns in SSD with body size, where larger species were male biased and smaller species were more female biased. Smaller species in this lineage also produced proportionally larger eggs. By contrast, another family containing few miniaturized species (Phyllodactylidae) displayed a more isometric trend. Together, these observations are consistent with the hypothesis that selection for increased reproductive success in small species of Sphaerodactylidae results in female-biased SSD in these taxa, which in turn drives the positive SSD allometry observed in this lineage. Thus, selection for increased miniaturization in the clade may be offset by selection on maintaining a female size in smaller taxa that ensures reproductive success.

Genomic insights into the Montseny brook newt (Calotriton arnoldi), a Critically Endangered glacial relict.

The Montseny brook newt (Calotriton arnoldi), considered the most endangered amphibian in Europe, is a relict salamandrid species endemic to a small massif located in northeastern Spain. Although conservation efforts should always be guided by genomic studies, those are yet scarce among urodeles, hampered by the extreme sizes of their genomes. Here, we present the third available genome assembly for the order Caudata, and the first genomic study of the species and its sister taxon, the Pyrenean brook newt (Calotriton asper), combining whole-genome and ddRADseq data. Our results reveal significant demographic oscillations which accurately mirrored Europe's climatic history. Although severe bottlenecks have led to depauperate genomic diversity and long runs of homozygosity along a gigantic genome, inbreeding might have been avoided by assortative mating strategies. Other life history traits, however, seem to have been less advantageous, and the lack of land dispersal has driven to exceptional levels of population fragmentation.

Adaptive and non-adaptive convergent evolution in feather reflectance of California Channel Islands songbirds.

Convergent evolution is widely regarded as a signature of adaptation. However, testing the adaptive consequences of convergent phenotypes is challenging, making it difficult to exclude non-adaptive explanations for convergence. Here, we combined feather reflectance spectra and phenotypic trajectory analyses with visual and thermoregulatory modelling to test the adaptive significance of dark plumage in songbirds of the California Channel Islands. By evolving dark dorsal plumage, island birds are generally less conspicuous to visual-hunting raptors in the island environment than mainland birds. Dark dorsal plumage also reduces the energetic demands associated with maintaining homeothermy in the cool island climate. We also found an unexpected pattern of convergence, wherein the most divergent island populations evolved greater reflectance of near-infrared radiation. However, our heat flux models indicate that elevated near-infrared reflectance is not adaptive. Analysis of feather microstructure suggests that mainland-island differences are related to coloration of feather barbs and barbules rather than their structure. Our results indicate that adaptive and non-adaptive mechanisms interact to drive plumage evolution in this system. This study sheds light on the mechanisms driving the association between dark colour and wet, cold environments across the tree of life, especially in island birds.

Evolution along allometric lines of least resistance: morphological differentiation in Pristurus geckos.

Species living in distinct habitats often experience unique ecological selective pressures, which can drive phenotypic divergence. However, how ecophenotypic patterns are affected by allometric trends and trait integration levels is less well understood. Here we evaluate the role of allometry in shaping body size and body form diversity in Pristurus geckos utilizing differing habitats. We found that patterns of allometry and integration in body form were distinct in species with different habitat preferences, with ground-dwelling Pristurus displaying the most divergent allometric trend and high levels of integration. There was also strong concordance between intraspecific allometry across individuals and evolutionary allometry among species, revealing that differences in body form among individuals were predictive of evolutionary changes across the phylogeny at macroevolutionary scales. This suggested that phenotypic evolution occurred along allometric lines of least resistance, with allometric trajectories imposing a strong influence on the magnitude and direction of size and shape changes across the phylogeny. When viewed in phylomorphospace, the largest rock-dwelling species were most similar to the smallest ground-dwelling species, and vice versa. Thus, in Pristurus, phenotypic evolution along the differing habitat-based allometric trajectories resulted in similar body forms at differing body sizes in distinct ecological habitats.

Rensch's rule: linking intraspecific to evolutionary allometry.

Sexual dimorphism describes phenotypic differences between the sexes; the most prominent of which is sexual size dimorphism (SSD). Rensch's rule (RR) is an allometric trend in which SSD increases in male-larger taxa and decreases in female-larger ones. Covariation between a trait and overall size within and across species can both be affected by sexual and natural selection. Thus, intraspecific allometric variation could influence the expression of RR. Here we used computer simulations to dissect how RR emerges under specific allometric patterns of intraspecific sexual differentiation in a trait. We found that sexual differentiation in static allometric slopes is the main determinant of RR. Based on our findings, RR and its converse can manifest in both body size and other traits. As a realistic showcase, we also examined RR and static allometry of different body parts in Mediterranean green lizards to establish whether intraspecific and evolutionary allometry are linked. Here, we identified RR and its converse for different traits, where the amount of sexual differentiation in static allometric slopes within species had a significant contribution to RR. Integrating the simulations and the empirical case we corroborate that sexual differentiation in static allometric slopes is a major parameter affecting evolutionary allometry.

Ecology, sexual dimorphism, and jumping evolution in anurans.

Sexual dimorphism (SD) is a common feature of animals, and selection for sexually dimorphic traits may affect both functional morphological traits and organismal performance. Trait evolution through natural selection can also vary across environments. However, whether the evolution of organismal performance is distinct between the sexes is rarely tested in a phylogenetic comparative context. Anurans commonly exhibit sexual size dimorphism, which may affect jumping performance given the effects of body size on locomotion. They also live in a wide variety of microhabitats. Yet the relationships among dimorphism, performance, and ecology remain underexamined in anurans. Here, we explore relationships between microhabitat use, body size, and jumping performance in males and females to determine the drivers of dimorphic patterns in jumping performance. Using methods for predicting jumping performance through anatomical measurements, we describe how fecundity selection and natural selection associated with body size and microhabitat have likely shaped female jumping performance. We found that the magnitude of sexual size dimorphism (where females are about 14% larger than males) was much lower than dimorphism in muscle volume, where females had 42% more muscle than males (after accounting for body size). Despite these sometimes-large averages, phylogenetic t-tests failed to show the statistical significance of SD for any variable, indicating sexually dimorphic species tend to be closely related. While SD of jumping performance did not vary among microhabitats, we found female jumping velocity and energy differed across microhabitats. Overall, our findings indicate that differences in sex-specific reproductive roles, size, jumping-related morphology, and performance are all important determinants in how selection has led to the incredible ecophenotypic diversity of anurans.

An effect size for comparing the strength of morphological integration across studies.

Understanding how and why phenotypic traits covary is a major interest in evolutionary biology. Biologists have long sought to characterize the extent of morphological integration in organisms, but comparing levels of integration for a set of traits across taxa has been hampered by the lack of a reliable summary measure and testing procedure. Here, we propose a standardized effect size for this purpose, calculated from the relative eigenvalue variance, V r e l $V_{rel}$ . First, we evaluate several eigenvalue dispersion indices under various conditions, and show that only V r e l $V_{rel}$ remains stable across samples size and the number of variables. We then demonstrate that V r e l $V_{rel}$ accurately characterizes input patterns of covariation, so long as redundant dimensions are excluded from the calculations. However, we also show that the variance of the sampling distribution of V r e l $V_{rel}$ depends on input levels of trait covariation, making V r e l $V_{rel}$ unsuitable for direct comparisons. As a solution, we propose transforming V r e l $V_{rel}$ to a standardized effect size (Z-score) for representing the magnitude of integration for a set of traits. We also propose a two-sample test for comparing the strength of integration between taxa, and show that this test displays appropriate statistical properties. We provide software for implementing the procedure, and an empirical example illustrates its use.

Variation in eye abundance among scallops reveals ontogenetic and evolutionary convergence associated with life habits.

Eyes are remarkable systems to investigate the complex interaction between ecological drivers and phenotypic outcomes. Some animals, such as scallops, have many eyes for visual perception, but to date, the evolution of multiple-eye systems remains obscure. For instance, it is unclear whether eye number changes over a lifetime or varies among species. Scallops are a suitable model group to investigate these questions considering the interspecific variation of adult size and ecological diversity. We tested whether eye abundance scales with body size among individuals and species and whether it varies with life habits. We performed comparative analyses, including a phylogenetic ANCOVA and evolutionary model comparisons, based on eye count and shell height (as a proxy of body size) across 31 scallop species. Our analyses reveal that patterns of increasing relationship with body size are not concordant among taxa and suggest ontogenetic convergence caused by similar ecologies. Accordingly, selective optima in eye numbers are associated with shifts in life habits. For instance, species with increased mobility have significantly more eyes than less mobile species. The convergent evolution of greater eye abundance in more mobile scallops likely indicates a visual improvement based on increased levels of oversampling of the surrounding environment.

Consilience of methods for phylogenetic analysis of variance.

Simulation-based and permutation-based inferential methods are commonplace in phylogenetic comparative methods, especially as evolutionary data have become more complex and parametric methods more limited for their analysis. Both approaches simulate many random outcomes from a null model to empirically generate sampling distributions of statistics. Although simulation-based and permutation-based methods seem commensurate in purpose, results from analysis of variance (ANOVA) based on the distributions of random F-statistics produced by these methods can be quite different in practice. Differences could be from either the null-model process that generates variation across many simulations or random permutations of the data, or different estimation methods for linear model coefficients and statistics. Unfortunately, because the null-model process and coefficient estimation are intrinsically linked in phylogenetic ANOVA methods, the precise reason for methodological differences has not been fully considered. Here we show that the null-model processes of phylogenetic simulation and randomizing residuals in a permutation procedure are indeed commensurate, and that both also produce results consistent with parametric ANOVA, for cases where parametric ANOVA is possible. We also provide results that caution against using ordinary least-squares estimation along with phylogenetic simulation; a typical phylogenetic ANOVA implementation.

Is salamander arboreality limited by broad-scale climatic conditions?

Identifying the historical processes that drive microhabitat transitions across deep time is of great interest to evolutionary biologists. Morphological variation can often reveal such mechanisms, but in clades with high microhabitat diversity and no concomitant morphological specialization, the factors influencing animal transitions across microhabitats are more difficult to identify. Lungless salamanders (family: Plethodontidae) have transitioned into and out of the arboreal microhabitat many times throughout their evolutionary history without substantial morphological specialization. In this study, we explore the relationship between microhabitat use and broad-scale climatic patterns across species' ranges to test the role of climate in determining the availability of the arboreal microhabitat. Using phylogenetic comparative methods, we reveal that arboreal species live in warmer, lower elevation regions than terrestrial species. We also employ ecological niche modeling as a complementary approach, quantifying species-level pairwise comparisons of niche overlap. The results of this approach demonstrate that arboreal species on average display more niche overlap with other arboreal species than with terrestrial species after accounting for non-independence of niche model pairs caused by geographic and phylogenetic distances. Our results suggest that occupation of the arboreal microhabitat by salamanders may only be possible in sufficiently warm, low elevation conditions. More broadly, this study indicates that the impact of micro-environmental conditions on temporary microhabitat use, as demonstrated by small-scale ecological studies, may scale up dramatically to shape macroevolutionary patterns.

USING MULTIVARIATE ANALYSES TO EXPLORE DISEASE PROGRESSION OF FINCH MYCOPLASMOSIS.

Lesion severity scales have been developed for a number of wildlife diseases causing external pathology. Perhaps the best known and most widely used scoring system has been developed for finch mycoplasmosis in which observers measure conjunctival pathology along a four-point scale of increasing severity. We developed novel techniques to characterize variation in host phenotype based on occupancy of multidimensional trait space (disease space). First, we used shape analysis to track distortions of the inner and outer eye rims, defined by 16 anatomical landmarks. Then, we used community analysis to evaluate pathology based on the presence or absence of a unique set of binary descriptors. We applied these techniques to experimental infection data to relate differences in conjunctival pathology to stage of infection. Specifically, by comparing specimens that received the same severity score at different time points in infection, we asked if shape or community analyses could distinguish between individuals in early infection versus those in recovery. We found that individual eyes followed predictable loops through disease space, tracking further from their origin with more severe pathology. Also, certain pathological descriptors were more likely to appear earlier versus later in infection. Our results indicated that leveraging differences in pathology captured in complex trait space could complement severity scores by better resolving the time course of infection from limited data points.

Interspecific allometry for sexual shape dimorphism: Macroevolution of multivariate sexual phenotypes with application to Rensch's rule.

Allometric trends in the degree of sexual dimorphism with body size have long fascinated evolutionary biologists. Many male-biased clades display more prominent sexual dimorphism in larger taxa (Rensch's rule), with most examples documenting this pattern for body size dimorphism. Although sexual dimorphism in traits other than body size is equally functionally relevant, characterizing allometric patterns of sexual dimorphism in such traits is hampered by lack of an analytical framework that can accommodate multivariate phenotypes. In this article, we derive a multivariate equivalency for investigating trends in sexual dimorphism-relative to overall body size-across taxa and provide a generalized test to determine whether such allometric patterns correspond with Rensch's rule. For univariate linear traits such as body size, our approach yields equivalent results to those from standard procedures, but our test is also capable of detecting trends in multivariate datasets such as shape. Computer simulations reveal that the method displays appropriate statistical properties, and an empirical example in Mediterranean lizards provides the first demonstration of Rensch's rule in a multivariate phenotype (head shape). Our generalized procedure substantially extends the analytical toolkit for investigating macroevolutionary patterns of sexual dimorphism and seeking a better understanding of the processes that underlie them.

Macroevolution of desiccation-related morphology in plethodontid salamanders as inferred from a novel surface area to volume ratio estimation approach.

Evolutionary biologists have long been interested in the macroevolutionary consequences of various selection pressures, yet physiological responses to selection across deep time are not well understood. In this paper, we investigate how a physiologically relevant morphological trait, surface area to volume ratio (SA:V) of lungless salamanders, has evolved across broad regional and climatic variation. SA:V directly impacts an organisms' ability to retain water, leading to the expectation that smaller SA:Vs would be advantageous in arid, water-limited environments. To explore the macroevolutionary patterns of SA:V, we first develop an accurate method for estimating SA:V from linear measurements. Next, we investigate the macroevolutionary patterns of SA:V across 257 salamander species, revealing that higher SA:Vs phylogenetically correlate with warmer, wetter climates. We also observe higher SA:V disparity and rate of evolution in tropical species, mirrored by higher climatic disparity in available and occupied tropical habitats. Taken together, these results suggest that the tropics have provided a wider range of warmer, wetter climates for salamanders to exploit, thereby relaxing desiccation pressures on SA:V. Overall, this paper provides an accurate, efficient method for quantifying salamander SA:V, allowing us to demonstrate the power of physiological selection pressures in influencing the macroevolution of morphology.

Comparing the strength of modular signal, and evaluating alternative modular hypotheses, using covariance ratio effect sizes with morphometric data.

The study of modularity is paramount for understanding trends of phenotypic evolution, and for determining the extent to which covariation patterns are conserved across taxa and levels of biological organization. However, biologists currently lack quantitative methods for statistically comparing the strength of modular signal across datasets, and a robust approach for evaluating alternative modular hypotheses for the same dataset. As a solution to these challenges, we propose an effect size measure ( ZCR ) derived from the covariance ratio, and develop hypothesis-testing procedures for their comparison. Computer simulations demonstrate that ZCR displays appropriate statistical properties and low levels of mis-specification, implying that it correctly identifies modular signal, when present. By contrast, alternative methods based on likelihood (EMMLi) and goodness of fit (MINT) suffer from high false positive rates and high model mis-specification rates. An empirical example in sigmodontine rodent mandibles is provided to illustrate the utility of ZCR for comparing modular hypotheses. Overall, we find that covariance ratio effect sizes are useful for comparing patterns of modular signal across datasets or for evaluating alternative modular hypotheses for the same dataset. Finally, the statistical philosophy for pairwise model comparisons using effect sizes should accommodate any future analytical developments for characterizing modular signal.

Macroevolution of arboreality in salamanders.

Evolutionary theory predicts that selection in distinct microhabitats generates correlations between morphological and ecological traits, and may increase both phenotypic and taxonomic diversity. However, some microhabitats exert unique selective pressures that act as a restraining force on macroevolutionary patterns of diversification. In this study, we use phylogenetic comparative methods to investigate the evolutionary outcomes of inhabiting the arboreal microhabitat in salamanders. We find that arboreality has independently evolved at least five times in Caudata and has arisen primarily from terrestrial ancestors. However, the rate of transition from arboreality back to terrestriality is 24 times higher than the converse. This suggests that macroevolutionary trends in microhabitat use tend toward terrestriality over arboreality, which influences the extent to which use of the arboreal microhabitat proliferates. Morphologically, we find no evidence for an arboreal phenotype in overall body proportions or in foot shape, as variation in both traits overlaps broadly with species that utilize different microhabitats. However, both body shape and foot shape display reduced rates of phenotypic evolution in arboreal taxa, and evidence of morphological convergence among arboreal lineages is observed. Taken together, these patterns suggest that arboreality has played a unique role in the evolution of this family, providing neither an evolutionary opportunity, nor an evolutionary dead end.

Leaf shape and size track habitat transitions across forest-grassland boundaries in the grass family (Poaceae).

Grass leaf shape is a strong indicator of their habitat with linear leaves predominating in open areas and ovate leaves distinguishing forest-associated grasses. This pattern among extant species suggests that ancestral shifts between forest and open habitats may have coincided with changes in leaf shape or size. We tested relationships between habitat, climate, photosynthetic pathway, and leaf shape and size in a phylogenetic framework to evaluate drivers of leaf shape and size variation over the evolutionary history of the family. We also estimated the ancestral habitat of Poaceae and tested whether forest margins served as transitional zones for shifts between forests and grasslands. We found that grass leaf shape is converging toward different shape optima in the forest understory, forest margins, and open habitats. Leaf size also varies with habitat. Grasses have smaller leaves in open and drier areas, and in areas with high solar irradiance. Direct transitions between linear and ovate leaves are rare as are direct shifts between forest and open habitats. The most likely ancestral habitat of the family was the forest understory and forest margins along with an intermediate leaf shape served as important transitional habitat and morphology, respectively, for subsequent shifts across forest-grassland biome boundaries.

Phylogenetic ANOVA: Group-clade aggregation, biological challenges, and a refined permutation procedure.

Phylogenetic regression is frequently used in macroevolutionary studies, and its statistical properties have been thoroughly investigated. By contrast, phylogenetic ANOVA has received relatively less attention, and the conditions leading to incorrect statistical and biological inferences when comparing multivariate phenotypes among groups remain underexplored. Here, we propose a refined method of randomizing residuals in a permutation procedure (RRPP) for evaluating phenotypic differences among groups while conditioning the data on the phylogeny. We show that RRPP displays appropriate statistical properties for both phylogenetic ANOVA and regression models, and for univariate and multivariate datasets. For ANOVA, we find that RRPP exhibits higher statistical power than methods utilizing phylogenetic simulation. Additionally, we investigate how group dispersion across the phylogeny affects inferences, and reveal that highly aggregated groups generate strong and significant correlations with the phylogeny, which reduce statistical power and subsequently affect biological interpretations. We discuss the broader implications of this phylogenetic group aggregation, and its relation to challenges encountered with other comparative methods where one or a few transitions in discrete traits are observed on the phylogeny. Finally, we recommend that phylogenetic comparative studies of continuous trait data use RRPP for assessing the significance of indicator variables as sources of trait variation.

A phylogenetic comparative method for evaluating trait coevolution across two phylogenies for sets of interacting species.

Evaluating trait correlations across species within a lineage via phylogenetic regression is fundamental to comparative evolutionary biology, but when traits of interest are derived from two sets of lineages that coevolve with one another, methods for evaluating such patterns in a dual-phylogenetic context remain underdeveloped. Here, we extend multivariate permutation-based phylogenetic regression to evaluate trait correlations in two sets of interacting species while accounting for their respective phylogenies. This extension is appropriate for both univariate and multivariate response data, and may use one or more independent variables, including environmental covariates. Imperfect correspondence between species in the interacting lineages can also be accommodated, such as when species in one lineage associate with multiple species in the other, or when there are unmatched taxa in one or both lineages. For both univariate and multivariate data, the method displays appropriate type I error, and statistical power increases with the strength of the trait covariation and the number of species in the phylogeny. These properties are retained even when there is not a 1:1 correspondence between lineages. Finally, we demonstrate the approach by evaluating the evolutionary correlation between traits in fig species and traits in their agaonid wasp pollinators. R computer code is provided.