Degeneration of the Olfactory System in a Murid Rodent that Evolved Diurnalism.

In mammalian research, it has been debated what can initiate an evolutionary tradeoff between different senses, and the phenomenon of sensory tradeoff in rodents, the most abundant mammalian clade, is not evident. The Nile rat (Arvicanthis niloticus), a murid rodent, recently adapted to a diurnal niche through an evolutionary acquisition of daylight vision with enhanced visual acuity. As such, this model provides an opportunity for a cross-species investigation where comparative morphological and multi-omic analyses of the Nile rat are made with its closely related nocturnal species, e.g. the mouse (Mus musculus) and the rat (Rattus norvegicus). Thus, morphological examinations were performed, and evolutionary reductions in relative sizes of turbinal bone surfaces, the cribriform plate, and the olfactory bulb were discovered in Nile rats. Subsequently, we compared multiple murid genomes, and profiled olfactory epithelium transcriptomes of mice and Nile rats at various ages with RNA sequencing. The results further demonstrate that, in comparison with mouse olfactory receptor (OR) genes, Nile rat OR genes have experienced less frequent gain, more frequent loss, and more frequent expression reduction during their evolution. Furthermore, functional degeneration of coding sequences in the Nile rat lineage was found in OR genes, yet not in other genes. Taken together, these results suggest that acquisition of improved vision in the Nile rat has been accompanied by degeneration of both olfaction-related anatomical structures and OR gene repertoires, consistent with the hypothesis of an olfaction-vision tradeoff initiated by the switch from a nocturnal to a diurnal lifestyle in mammals.

An anthocyanin activation gene underlies the purple central flower pigmentation in wild carrot.

Many organisms have complex pigmentation patterns. However, how these patterns are formed remains largely unknown. In wild carrot (Daucus carota subsp. carota), which is also known as Queen Anne's lace, one or several purple central flowers occur in white umbels. Here, we investigated the unique central flower pigmentation pattern in wild carrot umbels. Using wild and cultivated carrot (Daucus carota subsp. sativus L.) accessions, transcriptome analysis, protein interaction, stable transformation, and CRISPR/Cas9-mediated knockout, a anthocyanin-activating R2R3-myeloblastosis (MYB) gene, Purple Central Flower (DcPCF), was identified as the causal gene that triggers only central flowers to possess the purple phenotype. The expression of DcPCF was only detected in tiny central flowers. We propose that the transition from purple to nonpurple flowers in the center of the umbel occurred after three separate adverse events: insertion of transposons in the promoter region, premature termination of the coding sequence (caused by a C-T substitution in the open reading frame), and the emergence of unknown anthocyanin suppressors. These three events could have occurred either consecutively or independently. The intriguing purple central flower pattern and its underlying mechanism may provide evidence that it is a remnant of ancient conditions of the species, reflecting the original appearance of Umbelliferae (also called Apiaceae) when a single flower was present.

Exploring the Macroevolutionary Signature of Asymmetric Inheritance at Speciation.

Popular comparative phylogenetic models such as Brownian Motion, Ornstein-Ulhenbeck, and their extensions, assume that, at speciation, a trait value is inherited identically by two descendant species. This assumption contrasts with models of speciation at a micro-evolutionary scale where descendants' phenotypic distributions are sub-samples of the ancestral distribution. Different speciation mechanisms can lead to a displacement of the ancestral phenotypic mean among descendants and an asymmetric inheritance of the ancestral phenotypic variance. In contrast, even macro-evolutionary models that account for intraspecific variance assume symmetrically conserved inheritance of ancestral phenotypic distribution at speciation. Here we develop an Asymmetric Brownian Motion model (ABM) that relaxes the assumption of symmetric and conserved inheritance of the ancestral distribution at the time of speciation. The ABM jointly models the evolution of both intra- and inter-specific phenotypic variation. It also infers the mode of phenotypic inheritance at speciation, which can range from a symmetric and conserved inheritance, where descendants inherit the ancestral distribution, to an asymmetric and displaced inheritance, where descendants inherit divergent phenotypic means and variances. To demonstrate this model, we analyze the evolution of beak morphology in Darwin finches, finding evidence of displacement at speciation. The ABM model helps to bridge micro- and macro-evolutionary models of trait evolution by providing a more robust framework for testing the effects of ecological speciation, character displacement, and niche partitioning on trait evolution at the macro-evolutionary scale.

Trait-Fitness Associations via Fecundity and Competition in a Two-Million-Year-Long Fossil Record.

AbstractThe evolution of phenotypic traits is usually studied on generational timescales or across species on million-year timescales. We bridge this conceptual gap by using high-density sampling of a species lineage, Microporella agonistes (Bryozoa, Cheilostomatida), over 2 million years of its evolutionary history, to ask whether trait-fitness associations are consistent with evolutionary trait models often applied to phenotypic time series. We use average fecundity and competitive outcome as two different fitness components, where competitive outcome is a proxy for partial survival. Examining three quantitative traits in multivariate analyses, we present evidence that some traits experienced substantial selective pressures, in part controlled by past environments. A complex interplay of resource competition with an altering set of competitors and past temperatures has contributed to the changing patterns of phenotypes within the focal species. A comparison with congeneric species living in the same regional community suggests that size traits are more temporally variable and less constrained than shape traits. Our analyses also show that while controls on phenotypes are complex and varied in time, ecological and evolutionary processes that unfold on shorter timescales are not inconsistent with macroevolutionary patterns observed on longer timescales.

Repeated Divergence in Opsin Gene Expression Mirrors Photic Habitat Changes in Rapidly Evolving Crater Lake Cichlid Fishes.

AbstractSelection pressures differ along environmental gradients, and traits tightly linked to fitness (e.g., the visual system) are expected to track such variation. Along gradients, adaptation to local conditions might be due to heritable and nonheritable environmentally induced variation. Disentangling these sources of phenotypic variation requires studying closely related populations in nature and in the laboratory. The Nicaraguan lakes represent an environmental gradient in photic conditions from clear crater lakes to very turbid great lakes. From two old, turbid great lakes, Midas cichlid fish (Amphilophus cf. citrinellus) independently colonized seven isolated crater lakes of varying light conditions, resulting in a small adaptive radiation. We estimated variation in visual sensitivities along this photic gradient by measuring cone opsin gene expression among lake populations. Visual sensitivities observed in all seven derived crater lake populations shifted predictably in direction and magnitude, repeatedly mirroring changes in photic conditions. Comparing wild-caught and laboratory-reared fish revealed that 48% of this phenotypic variation is genetically determined and evolved rapidly. Decreasing intrapopulation variation as environments become spectrally narrower suggests that different selective landscapes operate along the gradient. We conclude that the power to predict phenotypic evolution along gradients depends on both the magnitude of environmental change and the selective landscape shape.

Fitness effects of a demography-dispersal trade-off in expandingSaccharomyces cerevisiaemats.

Fungi expand in space and time to form complex multicellular communities. The mechanisms by which they do so can vary dramatically and determine the life-history and dispersal traits of expanding populations. These traits influence deterministic and stochastic components of evolution, resulting in complex eco-evolutionary dynamics during colony expansion. We perform experiments on budding yeast strains genetically engineered to display rough-surface and smooth-surface phenotypes in colony-like structures called 'mats'. Previously, it was shown that the rough-surface strain has a competitive advantage over the smooth-surface strain when grown on semi-solid media. We experimentally observe the emergence and expansion of segments with a distinct smooth-surface phenotype during rough-surface mat development. We propose a trade-off between dispersal and local carrying capacity to explain the relative fitness of these two phenotypes. Using a modified stepping-stone model, we demonstrate that this trade-off gives the high-dispersing, rough-surface phenotype a competitive advantage from standing variation, but that it inhibits this phenotype's ability to invade a resident smooth-surface population via mutation. However, the trade-off improves the ability of the smooth-surface phenotype to invade in rough-surface mats, replicating the frequent emergence of smooth-surface segments in experiments. Together, these computational and experimental findings advance our understanding of the complex eco-evolutionary dynamics of fungal mat expansion.

Evolution in fossil time series reconciles observations in micro- and macroevolution.

Extrapolating microevolutionary models does not always provide satisfactory explanations for phenotypic diversification on million-year time scales. For example, short-term evolutionary change is often modeled assuming a fixed adaptive landscape, but macroevolutionary changes are likely to involve changes in the adaptive landscape itself. A better understanding of how the adaptive landscape changes across different time intervals and how these changes cause populations to evolve has the potential to narrow the gap between micro- and macroevolution. Here, we analyze two fossil diatom time series of exceptional quality and resolution covering time intervals of a few hundred thousand years using models that account for different behaviors of the adaptive landscape. We find that one of the lineages evolves on a randomly and continuously changing landscape, whereas the other lineage evolves on a landscape that shows a rapid shift in the position of the adaptive peak of a magnitude that is typically associated with species-level differentiation. This suggests phenotypic evolution beyond generational timescales may be a consequence of both gradual and sudden repositioning of adaptive peaks. Both lineages are showing rapid and erratic evolutionary change and are constantly readapting towards the optimal trait state, observations that align with evolutionary dynamics commonly observed in contemporary populations. The inferred trait evolution over a span of a few hundred thousand years in these two lineages is therefore chimeric in the sense that it combines components of trait evolution typically observed on both short and long timescales.

Eltrombopag modulates the phenotypic evolution and potential immunomodulatory roles of monocytes/macrophages in immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease. Loss of immune tolerance plays a crucial role in the pathogenesis of ITP. Monocytes and macrophages play an indispensable role in the pathophysiology of hematopoietic malignancies and have been implicated as key players in platelet destruction. Approximately 80% of adult patients with ITP exhibit corticosteroid treatment failure or become dependent, requiring novel therapy. Thrombopoietin (TPO) receptor agonists (TPO-RAs) have been used clinically to manage ITP effectively, however, little is known about the effect of TPO-RAs on monocyte and macrophage modulation in adult ITP. In this study, we investigated the phenotypic evolution and potential immunomodulatory roles of monocytes/macrophages in ITP patients receiving eltrombopag therapy. Results showed that the peripheral monocyte count positively correlated with IFN-γ/IL-4 ratio in ITP patients. Moreover, numerous phenotype-associated genes in ITP macrophages exhibited diverse responses, and ITP macrophages exhibited more M1-related characteristics. After eltrombopag therapy, the peripheral monocyte count and IFN-γ/IL-4 ratio significantly decreased in ITP patients. M1-related characteristics of ITP macrophages were partially reversed by eltrombopag. Therefore, this study revealed eltrombopag restored the monocyte dynamics and the associated Th1/Th2 imbalance, and partially reversed the M1-related characteristics of the ITP macrophages, which suggest the potential vital roles of TPO-RAs in regulating the monocyte/macrophage plasticity in ITP.

What is the context? Primary immune thrombocytopenia (ITP) is an acquired autoimmune hemorrhagic disease. Loss of immune tolerance plays a crucial role in the pathogenesis of ITP.Monocytes and macrophages play an indispensable role in the pathophysiology of hematopoietic malignancies and have been implicated as key players in platelet destruction.Approximately 80% of adult patients with ITP exhibit corticosteroid treatment failure or become dependent, requiring novel therapy. Thrombopoietin (TPO) receptor agonists (TPO-RAs) have been used clinically to manage ITP effectively, however, little is known about the effect of TPO-RAs on monocyte and macrophage modulation in ITP.What is new?In this study, we investigated the phenotypic evolution and potential immunomodula-tory roles of monocytes/macrophages in ITP patients receiving eltrombopag therapy.The expansion of peripheral monocytes positively correlated with IFN-γ/IL-4 ratio in ITP patients.ITP macrophages exhibited more M1-related characteristics.After eltrombopag therapy, the peripheral monocyte count and IFN-γ/IL-4 ratio significantly decreased in ITP patients.M1-related characteristics of ITP macrophages were partially reversed by eltrombopag.What is the impact?This study provides evidence that the potential vital roles of TPO-RAs in regulating the monocyte/macrophage plasticity in ITP.

The latitudinal gradient in rates of evolution for bird beaks, a species interaction trait.

Where is evolution fastest? The biotic interactions hypothesis proposes that greater species richness creates more ecological opportunity, driving faster evolution at low latitudes, whereas the 'empty niches' hypothesis proposes that ecological opportunity is greater where diversity is low, spurring faster evolution at high latitudes. We tested these contrasting predictions by analysing rates of beak evolution for a global dataset of 1141 avian sister species. Rates of beak size evolution are similar across latitudes, with some evidence that beak shape evolves faster in the temperate zone, consistent with the empty niches hypothesis. The empty niches hypothesis is further supported by a meta-analysis showing that rates of trait evolution and recent speciation are generally faster in the temperate zone, whereas rates of molecular evolution are slightly faster in the tropics. Our results suggest that drivers of evolutionary diversification are either similar across latitudes or more potent in the temperate zone, thus calling into question multiple hypotheses that invoke faster tropical evolution to explain the latitudinal diversity gradient.

Recapitulating Evolutionary Divergence in a Single Cis-Regulatory Element Is Sufficient to Cause Expression Changes of the Lens Gene Tdrd7.

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved noncoding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than 2-fold upregulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

Rampant False Detection of Adaptive Phenotypic Optimization by ParTI-Based Pareto Front Inference.

Organisms face tradeoffs in performing multiple tasks. Identifying the optimal phenotypes maximizing the organismal fitness (or Pareto front) and inferring the relevant tasks allow testing phenotypic adaptations and help delineate evolutionary constraints, tradeoffs, and critical fitness components, so are of broad interest. It has been proposed that Pareto fronts can be identified from high-dimensional phenotypic data, including molecular phenotypes such as gene expression levels, by fitting polytopes (lines, triangles, tetrahedrons, and so on), and a program named ParTI was recently introduced for this purpose. ParTI has identified Pareto fronts and inferred phenotypes best for individual tasks (or archetypes) from numerous data sets such as the beak morphologies of Darwin's finches and mRNA concentrations in human tumors, implying evolutionary optimizations of the involved traits. Nevertheless, the reliabilities of these findings are unknown. Using real and simulated data that lack evolutionary optimization, we here report extremely high false-positive rates of ParTI. The errors arise from phylogenetic relationships or population structures of the organisms analyzed and the flexibility of data analysis in ParTI that is equivalent to p-hacking. Because these problems are virtually universal, our findings cast doubt on almost all ParTI-based results and suggest that reliably identifying Pareto fronts and archetypes from high-dimensional phenotypic data are currently generally difficult.

A phylogeny of Antirrhinum reveals parallel evolution of alpine morphology.

Parallel evolution of similar morphologies in closely related lineages provides insight into the repeatability and predictability of evolution. In the genus Antirrhinum (snapdragons), as in other plants, a suite of morphological characters are associated with adaptation to alpine environments. We tested for parallel trait evolution in Antirrhinum by investigating phylogenetic relationships using restriction-site associated DNA (RAD) sequencing. We then associated phenotypic information to our phylogeny to reconstruct the patterns of morphological evolution and related this to evidence for hybridisation between emergent lineages. Phylogenetic analyses showed that the alpine character syndrome is present in multiple groups, suggesting that Antirrhinum has repeatedly colonised alpine habitats. Dispersal to novel environments happened in the presence of intraspecific and interspecific gene flow. We found support for a model of parallel evolution in Antirrhinum. Hybridisation in natural populations, and a complex genetic architecture underlying the alpine morphology syndrome, support an important role of natural selection in maintaining species divergence in the face of gene flow.

Novel genome sequence of Chinese cavefish (Triplophysa rosa) reveals pervasive relaxation of natural selection in cavefish genomes.

All cavefishes, living exclusively in caves across the globe, exhibit similar phenotypic traits, including the characteristic loss of eyes. To understand whether such phenotypic convergence shares similar genomic bases, here we investigated genome-wide evolutionary signatures of cavefish phenotypes by comparing whole-genome sequences of three pairs of cavefishes and their surface fish relatives. Notably, we newly sequenced and generated a whole-genome assembly of the Chinese cavefish Triplophysa rosa. Our comparative analyses revealed several shared features of cavefish genome evolution. Cavefishes had lower mutation rates than their surface fish relatives. In contrast, the ratio of nonsynonymous to synonymous substitutions (ω) was significantly elevated in cavefishes compared to in surface fishes, consistent with the relaxation of purifying selection. In addition, cavefish genomes had an increased mutational load, including mutations that alter protein hydrophobicity profiles, which were considered harmful. Interestingly, however, we found no overlap in positively selected genes among different cavefish lineages, indicating that the phenotypic convergence in cavefishes was not caused by positive selection of the same sets of genes. Analyses of previously identified candidate genes associated with cave phenotypes supported this conclusion. Genes belonging to the lipid metabolism functional ontology were under relaxed purifying selection in all cavefish genomes, which may be associated with the nutrient-poor habitat of cavefishes. Our work reveals previously uncharacterized patterns of cavefish genome evolution and provides comparative insights into the evolution of cave-associated phenotypic traits.

Body-axis organization in tetrapods: a model-system to disentangle the developmental origins of convergent evolution in deep time.

Convergent evolution is a central concept in evolutionary theory but the underlying mechanism has been largely debated since On the Origin of Species. Previous hypotheses predict that developmental constraints make some morphologies more likely to arise than others and natural selection discards those of the lowest fitness. However, the quantification of the role and strength of natural selection and developmental constraint in shaping convergent phenotypes on macroevolutionary timescales is challenging because the information regarding performance and development is not directly available. Accordingly, current knowledge of how embryonic development and natural selection drive phenotypic evolution in vertebrates has been extended from studies performed at short temporal scales. We propose here the organization of the tetrapod body-axis as a model system to investigate the developmental origins of convergent evolution over hundreds of millions of years. The quantification of the primary developmental mechanisms driving body-axis organization (i.e. somitogenesis, homeotic effects and differential growth) can be inferred from vertebral counts, and recent techniques of three-dimensional computational biomechanics have the necessary potential to reveal organismal performance even in fossil forms. The combination of both approaches offers a novel and robust methodological framework to test competing hypotheses on the functional and developmental drivers of phenotypic evolution and evolutionary convergence.

Evidence for general size-by-habitat rules in actinopterygian fishes across nine scales of observation.

Identifying environmental predictors of phenotype is fundamentally important to many ecological questions, from revealing broadscale ecological processes to predicting extinction risk. However, establishing robust environment-phenotype relationships is challenging, as powerful case studies require diverse clades which repeatedly undergo environmental transitions at multiple taxonomic scales. Actinopterygian fishes, with 32,000+ species, fulfil these criteria for the fundamental habitat divisions in water. With four datasets of body size (ranging 10,905-27,226 species), I reveal highly consistent size-by-habitat-use patterns across nine scales of observation. Taxa in marine, marine-brackish, euryhaline and freshwater-brackish habitats possess larger mean sizes than freshwater relatives, and the largest mean sizes consistently emerge within marine-brackish and euryhaline taxa. These findings align with the predictions of seven mechanisms thought to drive larger size by promoting additional trophic levels. However, mismatches between size and trophic-level patterns highlight a role for additional mechanisms, and support for viable candidates is examined in 3439 comparisons.

A New Diploid Parthenogenetic Whiptail Lizard from Sonora, Mexico, Is the "Missing Link" in the Evolutionary Transition to Polyploidy.

AbstractTransitions between sexual and unisexual reproductive modes have significant consequences for the evolutionary trajectories of species. These transitions have occurred numerous times in vertebrates and are frequently mediated by hybridization events. Triploid unisexual vertebrates are thought to arise through hybridization between individuals of a diploid unisexual lineage and a sexual species, although additional evidence that confirms this mechanism is needed in numerous groups. North American whiptail lizards (Aspidoscelis) are notable for being one of the largest radiations of unisexual vertebrates, and the most diverse group of Aspidoscelis includes numerous triploid lineages that have no known diploid unisexual ancestors. This pattern of "missing" ancestors may result from the short evolutionary life span of unisexual lineages or the selective advantages of polyploidy, or it could suggest that alternative mechanisms of triploid formation are operating in nature. We leverage genomic, morphological, and karyotypic data to describe a new diploid unisexual whiptail and show that it is likely the unisexual progenitor of an extant triploid lineage, A. opatae. We also resolve patterns of polyploidization within the A. sexlineatus species group and test predictions about the phenotypic outcomes of hybridization.

An Evolutionary and Ecological Community Model for Distribution of Phenotypes and Abundances among Competing Species.

AbstractHere, we propose a theory for the structure of communities of competing species. We include ecologically realistic assumptions, such as density dependence and stochastic fluctuations in the environment, and analyze how evolution caused by r- and K-selection will affect the packing of species in the phenotypic space as well as the species abundance distribution. Species-specific traits have the same matrix G of additive genetic variances and covariances, and evolution of mean traits is affected by fluctuations in population size of all species. In general, the model produces a shape of the distributions of log abundances that is skewed to the left, which is typical of most natural communities. Mean phenotypes of the species in the community are distributed approximately uniformly on the surface of a multidimensional sphere. However, environmental stochasticity generates selection that deviates species slightly from this surface; nonetheless, phenotypic distribution will be different from a random packing of species. This model of community evolution provides a theoretical framework that predicts a relationship between the structure of the phenotypic space and the form of species abundance distributions that can be compared against time series of variation in community structure.

Structure of the G-matrix in relation to phenotypic contributions to fitness.

Classical theory in population genetics includes derivation of the stationary distribution of allele frequencies under balance between selection, genetic drift, and mutation. Here we investigate the simplest generalization of these single locus models to quantitative genetics with many loci, assuming simple additive effects on a set of phenotypes and a linear approximation to the fitness function. Genetic effects and pleiotropy are simulated by a prescribed stochastic model. Our goal is to analyze the structure of the G-matrix at stasis when the model is not very close to being neutral. The smallest eigenvalue of the G-matrix is practically zero by Fisher's fundamental theorem for natural selection and the fitness function is approximately a linear function of the corresponding eigenvector. Evolution of genetic trade-offs is closely linked to the fitness function. If a single locus never codes for more than two traits, then additive genetic covariance between two phenotype components always has the opposite sign of the product of their coefficients in the fitness function under no mutation, a pattern that is likely to occur frequently also in more complex models. In our major examples only 1-2 percent of the loci are over-dominant for fitness, but they still account for practically all dominance variance in fitness as well as all contributions to the G-matrix. These analyses show that the structure of the G-matrix reveals important information about the contribution of different traits to fitness.

The self-healing of Bacillus subtilis biofilms.

Self-healing is an intrinsic ability that exists widely in every multicellular biological organism. Our recent experiments have shown that bacterial biofilms also have the ability to self-heal after man-make cuts, but the mechanism of biofilm self-healing have not been studied. We find that the healing process of cuts on the biofilm depends on cut geometries like its location or direction, the biofilm itself like the biofilm age, the growing substrate properties like its hardness, and also the environments such as the competitive growth of multiple biofilms. What is more, the healing rate along the cut is heterogeneous, and the maximum healing rate can reach 260 µm/h, which is three times the undestroyed biofilm expansion rate. The cut does not change the rounded shape growth of biofilms. Further study of phenotypic evolution shows that the cut delays bacterial differentiation; motile cells perceive the cut and move to the cut area, while the cut only heals when there are enough matrix-producing cells in the cut area. Our work suggests new ideas for developing self-healing materials.

How head shape and substrate particle size affect fossorial locomotion in lizards.

Granular substrates ranging from silt to gravel cover much of the Earth's land area, providing an important habitat for fossorial animals. Many of these animals use their heads to penetrate the substrate. Although there is considerable variation in head shape, how head shape affects fossorial locomotor performance in different granular substrates is poorly understood. Here, head shape variation for 152 species of fossorial lizards was quantified for head diameter, slope and pointiness of the snout. The force needed to penetrate different substrates was measured using 28 physical models spanning this evolved variation. Ten substrates were considered, ranging in particle size from 0.025 to 4 mm in diameter and consisting of spherical or angular particles. Head shape evolved in a weakly correlated manner, with snouts that were gently sloped being blunter. There were also significant clade differences in head shape among fossorial lizards. Experiments with physical models showed that as head diameter increased, absolute penetration force increased but force normalized by cross-sectional area decreased. Penetration force decreased for snouts that tapered more gradually and were pointier. Larger and angular particles required higher penetration forces, although intermediate size spherical particles, consistent with coarse sand, required the lowest force. Particle size and head diameter effect were largest, indicating that fossorial burrowers should evolve narrow heads and bodies, and select relatively fine particles. However, variation in evolved head shapes and recorded penetration forces suggests that kinematics of fossorial movement are likely an important factor in explaining evolved diversity.