Kevin M. Middleton

Evolve-and-Resequence studies combine experimental evolution with molecular sequencing, enabling associations between complex trait phenotypes and evolved genomic changes. For this, a targeted complex trait is subjected to artificial laboratory selection in genetically variable populations. Using populations of fruit flies Drosophila melanogaster from the DSPR background, we have conducted a short-term selection study on a highly complex trait – flight performance. We selected for improved flight performance using a custom-built flight tunnel system, with light as a motivator and airflow as a resistive factor. As a result, flight performance significantly improved in selected populations compared to controls. We also observed signatures of evolution in flight related traits, such as activity level and wing shape, and in life-history related traits such as adult dry mass and fecundity. We used whole genome pooled sequencing on the selected and control populations, revealing significantly differentiated genomic sites. Gene ontology and enrichment analyses revealed the evolution of pathways involved in response to stimuli, growth, and development. We identified several genes of interest (e.g., Sdc , dally ). The range of evolved phenotypes and associated genomic changes show that flight performance is an ideal complex trait for future Evolve-and-Resequence studies.

Objectives: Rudabánya II (RII) is a 10‐million‐year‐old fossil locality in Hungary that preserves a rich assemblage of vertebrate fossils, including specimens attributed to the pliopithecoid Anapithecus hernyaki . The RII dental remains of this stem catarrhine vary in mandibular molar morphology, particularly in the length and degree of tapering of the M ₃ . The goal of this study is to quantify metric variation in the RII fossil pliopithecoid mandibular dental sample and determine if it exceeds variation in extant anthropoid taxa. Materials and Methods: Using a bootstrapping approach, we compared variation in RII pliopithecoid mandibular molar shape ratios and relative size to a taxonomically broad sample of extant anthropoids. We also used 2D geometric morphometrics to compare the occlusal morphology of the RII pliopithecoid sample with other fossil and extant catarrhines and explored the relationship between mandibular molar shape in the more sexually dimorphic taxa. Results: In many cases, the RII pliopithecoid mandibular molars are more variable in shape than the comparative extant samples when comparing ratios of linear measures and in geometric morphometric analysis. In shape space, the RII pliopithecoid mandibular molars are distinct from other pliopithecoids. Mandibular molar size is not related to shape. Discussion: We address two potential sources of elevated variation in the RII pliopithecoid sample: (1) a high degree of sexual dimorphism, which exceeds that of extant anthropoid species, and (2) the presence of two pliopithecoid species in the RII fossil assemblage. Sexual dimorphism is not likely to be the cause of elevated variation in this sample.

Understanding craniofacial growth is foundational for research into intra‐ and interspecies variation, evolution, and clinical care. The Craniofacial Growth Consortium Study (CGCS), combines cephalographs from historical growth studies to create a dense longitudinal record of growth from 6 to 22 years of age. Originally comprised of six historical studies, two new collections (Burlington Growth study and the ADA Forsyth Study) are described and added to the CGCS increasing the sample to 2,100 individuals (1,056 males, 1,044 females; median of 9 cephalographs/individual). Coordinate data for 16 cephalometric landmarks, and 48 semilandmarks, were collected from each cephalograph. Cephalometric points were collected independently by three assessors. Previously established error envelopes for each landmark are used to compare the three assessments. If two or more coordinates for a landmark grossly differ, the assessors have the option to retrace; if acceptable, they are averaged for each point. A total of 32 measures were calculated from the coordinates. A multilevel double logistic equation was implemented to estimate growth trajectories fitting each trait separately by sex. The models successfully identified growth milestones (e.g., age at peak growth velocity) for all but a single trait (N‐ANS in females). Results demonstrate similarity among milestone estimates and the overall shape of the growth curve for most traits describing craniofacial. A subset of measures of small magnitude had early estimates of age at peak growth velocity and a small peak growth velocity. The six neurocranial traits were characterized as an asymptotic curve with difficulties in milestone estimation.

Skeletal morphology is determined by a combination of genetic background and phenotypic plasticity induced by mechanical loading across the lifespan (e.g., exercise), among other factors. Changes in limb morphology associated with locomotor activity experienced throughout individual ontogeny and in species over evolutionary time can provide insight into the adaptation of vertebrate locomotor systems. Here, a mouse line artificially selected for increased voluntary wheel‐running distance is used to explore the effects of selective breeding on the genetic “baseline” morphology of the hind limb skeleton and to describe its plastic response to chronic exercise, including potential genotype by environment interactions. This experimental design additionally allowed for testing two hypotheses: (1) that bone plasticity itself is a trait that can evolve in direction and magnitude, and (2) that bone plasticity can influence the trajectory of selection for locomotor phenotypes. Using a combination of 3D shape analyses, we find that 82 generations of selective breeding result in substantial changes in the morphology of the hind limb skeleton, as well as subtle changes in the skeletal response to exercise that support the evolvability of bone plasticity (i.e., skeletal plasticity differs based on genetic background). We additionally find that the evolved bone shape of selected mice does not resemble the plastic exercise response of control‐line animals. These results provide further evidence of genetic variations among populations in the plastic bone response to mechanical loading and inform our understanding of the plastic and evolutionary lability of the skeletal system in response to locomotor demands.

The origin of birds represents a pivotal transition in vertebrate evolution, marked by significant changes in both brain size and feeding biomechanics. The evolution of the avian skull involved dramatic modifications, such as a segmented palate and the development of powered cranial kinesis in neognath birds. Powered kinesis, the ability to move parts of the skull independently, is considered a key innovation behind the dietary diversity and evolutionary success of birds. However, the processes driving the emergence of avian kinesis have remained unclear until recently. By analyzing data from Mesozoic birds, including reinterpretations of palate homology, 3D jaw muscle biomechanics, and linkage analysis, researchers have quantified changes in muscle forces and their effects on palate mechanics during the transition from theropods to birds. As the neurocranium expanded in non-avian theropods, temporal muscles shifted to more rostrocaudal positions in birds, aiding in the segmentation of the pterygoid. This musculoskeletal transformation increased fore-aft muscle force in neognaths, enabling powered cranial kinesis. A critical change was the separation of the epipterygoid ossification from the braincase, leading to the breakdown of primitive kinematic linkages and the development of a new basicranial joint, which allowed for greater cranial flexibility. These findings shed light on how the neurosensory and feeding systems coevolved during bird origins and offer new methods for identifying cranial kinesis in extinct vertebrates.

Thermal tolerance is a fundamental physiological complex trait for survival in many species. For example, everyday tasks such as foraging, finding a mate, and avoiding predation are highly dependent on how well an organism can tolerate extreme temperatures. Understanding the general architecture of the natural variants within the genes that control this trait is of high importance if we want to better comprehend thermal physiology. Here, we take a multipronged approach to further dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource as a model system. First, we used quantitative genetics and Quantitative Trait Loci mapping to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, we used the RNAi system to (1) alter tissue-specific gene expression and (2) functionally validate our findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.

High-resolution imaging, 3D modeling, and quantitative analyses are equipping evolutionary biologists with new approaches to understanding the variation and evolution of the musculoskeletal system. However, challenges with interpreting DiceCT data and higher order use of modeled muscles have not yet been fully explored, and the error in and accuracy of some digital methods remain unclear. West Indian Anolis lizards are a model clade for exploring patterns in functional adaptation, ecomorphology, and sexual size dimorphism in vertebrates. These lizards possess numerous jaw muscles with potentially different anatomies that sculpt the adductor chamber of the skull. Here we test approaches to quantifying the musculoskeletal shape of the heads of two species of Anolis: A. pulchellus and A. sagrei. We employ comparative approaches such as DiceCT segmentation of jaw muscles, 3D surface attachment mapping, and 3D landmarking with the aim of exploring muscle volumes, 3D muscle fiber architecture, and sexual dimorphism of the skull. We then compare sources of measurement error in these 3D analyses while also presenting new 3D musculoskeletal data from the Anolis feeding apparatus. These findings demonstrate the accessibility and repeatability of these emerging techniques as well as provide details regarding the musculoskeletal anatomy of the heads of A. pulchellus and A. sagrei which show potential for further research of comparative biomechanics and evolution in the clade.

Dense, longitudinal sampling represents the ideal for studying biological growth. However, longitudinal samples are not typically possible, due to limits of time, prohibitive cost, or health concerns of repeat radiologic imaging. In contrast, cross-sectional samples have few such drawbacks, but it is not known how well estimates of growth milestones can be obtained from cross-sectional samples. The Craniofacial Growth Consortium Study (CGCS) contains longitudinal growth data for approximately 2000 individuals. Single samples from the CGCS for individuals representing cross-sectional data were used to test the ability to predict growth parameters in linear trait measurements separately by sex. Testing across a range of cross-sectional sample sizes from 5 to the full sample, we found that means from repeated samples were able to approximate growth rates determined from the full longitudinal CGCS sample, with mean absolute differences below 1 mm at cross-sectional sample sizes greater than ~ 200 individuals. Our results show that growth parameters and milestones can be accurately estimated from cross-sectional data compared to population-level estimates from complete longitudinal data, underscoring the utility of such datasets in growth modeling. This method can be applied to other forms of growth (e.g., stature) and to cases in which repeated radiographs are not feasible (e.g., cone-beam CT).

Thermal tolerance is a fundamental physiological complex trait for survival in many species. For example, everyday tasks such as foraging, finding a mate, and avoiding predation, are highly dependent on how well an organism can tolerate extreme temperatures. Understanding the general architecture of the natural variants of the genes that control this trait is of high importance if we want to better comprehend how this trait evolves in natural populations. Here, we take a multipronged approach to further dissect the genetic architecture that controls thermal tolerance in natural populations using the Drosophila Synthetic Population Resource (DSPR) as a model system. First, we used quantitative genetics and Quantitative Trait Loci (QTL) mapping to identify major effect regions within the genome that influences thermal tolerance, then integrated RNA-sequencing to identify differences in gene expression, and lastly, we used the RNAi system to 1) alter tissue-specific gene expression and 2) functionally validate our findings. This powerful integration of approaches not only allows for the identification of the genetic basis of thermal tolerance but also the physiology of thermal tolerance in a natural population, which ultimately elucidates thermal tolerance through a fitness-associated lens.