Evolution & Development

Vertebrate appendages vary in length among species, despite a low divergence in bone configuration. In this study, we assessed whether the total sizes of anatomical features are constrained by the lengths of individual bones during development. We examined size control by expressing mutant genes related to allometric growth in zebrafish fins under cell type-specific promoters. Hyperactive potassium channel (kcnk5bW169L) or dominant-negative gap junction protein connexin (Cx43T154A) expression in epidermal cells increased or decreased fin size, respectively, but did not influence the lengths of fin bone segments. Osteoblast expression of these mutant genes altered fin bone segment length but not total fin size. The combination of kcnk5bW169L in epidermal cells and Cx43T154A in osteoblasts resulted in transgenic fish with large fins and short bone segments, and vice versa. These results clearly indicate that fin size and bone segment length are determined separately by independent regulatory systems, despite the use of the same genes. These findings shed new light on the evolution of allometric traits.

The repeated evolution of reduced skeletal armour in threespine stickleback provides an excellent model for understanding the genetic basis of morphological change. Here, we investigated the genetic and developmental mechanisms underlying the loss of the second dorsal spine in a freshwater stickleback population from North Uist, Scotland. Crosses between spineless freshwater and fully spined marine individuals confirmed a genetic basis for the trait, with inheritance patterns inconsistent with simple Mendelian expectations. A bulk segregant analysis of F3 hybrids revealed a strong genomic signal on chromosome VI, overlapping the hoxdb cluster, echoing previous findings in another spine-reduced stickleback population. Developmental series of the Scottish freshwater and marine populations using skeletal stainings and transcriptomic analyses of dorsal tissue showed delayed and incomplete cartilage formation and altered gene expression during critical stages of dorsal skeletal development in spineless fish, notably of several hoxdb genes. Our findings indicate that while the loss of dorsal spines has evolved repeatedly, it may involve distinct alleles across populations, with the same locus possibly playing a central role in this case.

The fins of fishes are remarkably diverse, and this variation is tied to the ecology and locomotor mode of a species. While numerous genetic factors are known to pattern fins in development, it is unclear how developmental plasticity shapes the fin skeleton. Here, we analyze the cichlid Satanoperca daemon, raised under three distinct feeding regimes, and show that plasticity is pervasive across the pectoral fin skeleton with foraging mode impacting patterning of both the endoskeleton and dermal skeleton. Radials and fin rays were {micro}CT scanned and analyzed using a combination of linear measures and geometric morphometrics. Anteroposterior patterning of both radials and fin rays are affected by feeding regime. Notably, S. daemon pectoral fin rays show distinct patterns of fin ray branching between treatments, suggesting altered fin stiffness. We argue that the observed changes in the fin likely reflect developmental plasticity resultant from altered swimming behaviors when fishes are challenged to forage in different ways. These data show how non-genetic mechanisms can shape both the endoskeleton and dermal skeleton of fins, and that foraging mode can induce plastic changes in skeletal elements that do not directly interface with food items.

Many animals have complex life cycles where larval and adult forms have distinct ecologies and habitats that impose different demands on their sensory systems. While the adaptive decoupling hypothesis predicts reduced genetic correlations between life stages, how sensory systems adapt across life stages at the molecular level is not well understood. Frogs are a compelling system to study this question in because most species rely on vision as both aquatic tadpoles and terrestrial adults, but these habitats present vastly different light environments. Here we used whole eye transcriptome sequencing to investigate differential expression between aquatic tadpoles and terrestrial juveniles of the southern leopard frog (Lithobates sphenocephalus). Because visual physiology changes with light levels, we also tested how constant light or dark exposure affected gene expression. We found 42% of genes were differentially expressed in the eyes of tadpoles versus juveniles, versus 5% for light/dark exposure. Analyses targeting a curated set of visual genes revealed significant differential expression between life stages in genes that control aspects of visual function and development, including spectral sensitivity and lens composition. Light/dark exposure had a significant effect on a smaller set of visual genes. Finally, microspectrophotometry of photoreceptors confirmed shifts in spectral sensitivity predicted by the expression results, consistent with adaptation to distinct light environments. Overall, we identified extensive expression-level differences in the eyes of tadpole and juvenile frogs related to observed morphological and physiological changes through metamorphosis, and corresponding adaptive shifts to optimize vision in aquatic versus terrestrial environments.

Variation in gastrointestinal morphology is associated with dietary specialization across the animal kingdom. Gut length generally correlates with trophic level, and increased gut length in herbivores is a classic example of adaptation to cope with diets with lower nutrient content and a higher proportion of refractory material. However, the genetic basis of gut length variation remains largely unstudied, partly due to the inaccessibility and plasticity of the gut tissue, as well as the lack of dietary diversity within traditional model organisms relative to that observed among species belonging to different trophic levels. Here, we confirm the genetic basis of gut length variation among recently evolved Lake Malawi cichlid fish species with different dietary adaptations. We then produce interspecific, inter-trophic-level hybrids to map evolved differences in intestinal length in an F2 mapping cross between Metriaclima mbenjii, an omnivore with a relatively long gut, and Aulonocara koningsi, a carnivore with a relatively short gut. We identify numerous candidate quantitative trait loci for evolved differences in intestinal length. These quantitative trait loci are predominantly sex-specific, supporting an evolutionary history of sexual conflicts for the gut. We also identify epistatic interactions potentially associated with canalization and the maintenance of cryptic variation in the cichlid adaptive radiation. Overall, our results suggest a complex, polygenic evolution of gut length variation associated with trophic level differences among cichlids, as well as conflicts and interactions that may be involved in evolutionary processes underlying other traits in cichlids. SummaryThis study examines the genetic basis of gut length variation in Lake Malawi cichlids, which exhibit different dietary adaptations. It highlights how cichlids recapitulate a broad taxonomic trend: gut length correlates with trophic level, with herbivores and omnivores having longer intestines than carnivores. By creating hybrids of Metriaclima mbenjii (omnivore) and Aulonocara koningsi (carnivore), we identify several quantitative trait loci and epistatic interactions underlying gut length differences. These genetic associations are predominantly sex-specific, suggesting historical sexual conflicts. The results indicate complex, polygenic evolution of gut morphology in these fish, and suggest evolutionary interactions and processes shaping dietary traits across species.

Osteoderms are bony plates which develop in the dermis of the skin of vertebrates, most commonly found in fishes and reptiles. They have evolved independently at least eight times in reptiles suggesting the presence of a gene regulatory network which is readily activated and inactivated. The absence of osteoderms in birds and mammals, except for the one example of armadillos, has prevented a comparative molecular approach to their evolution. However, following CT scanning, we have discovered that in two genera of Deomyinae, the spiny mouse Acomys and the brush-furred mouse, Lophuromys there are osteoderms present in the skin of their tails. We have studied osteoderm development within the dermis of the tail in Acomys cahirinus to show that they begin development before birth in the proximal part of the tail skin and they do not complete differentiation throughout the tail until 6 weeks after birth. This has allowed us to study the cellular differentiation of the osteoderms with histology and immunocytochemistry and perform RNA sequencing to identify the gene networks involved in their differentiation. There is a widespread down-regulation of keratin genes and an up-regulation of osteoblast genes and a finely balanced expression of signaling pathways as the osteoderms differentiate. Future comparisons with reptilian osteoderms may allow us to understand how these structures have evolved, why they are so rare in mammals and how they are position-specific.

Plumage pigmentation plays critical roles in survival and reproductive success in birds, from providing camouflage and thermoregulation to mediating elaborate mating displays. The genetic and developmental origins of diverse plumage pigmentation patterns remain incompletely understood in part due to limited intraspecific variation and high levels of genetic divergence between distantly related species. Domestic avian species are more tractable models for understanding the genetic architecture of plumage pigmentation, but the relevance of domestic phenotypes to plumage patterns observed in the wild is not clear. Here, we used comparative genomic approaches to examine coding variation in EDNRB2, a candidate gene associated with loss of plumage melanin in several species, in representative genomes from a diverse array of wild and domestic birds. We found widespread coding variation in EDNRB2 and in other pigmentation genes with limited pleiotropic roles in development. We also found that EDNRB2- mediated melanin loss may play a critical role in establishing bright non-melanin plumage colors. This work highlights EDNRB2 as a key candidate gene for mediating the development of both interspecific and intraspecific plumage variation and demonstrates the applicability of findings in domestic species to understanding avian plumage patterning more broadly.

The model zebrafish (Danio rerio) belongs to the Danioninae subfamily with a range of informative phenotypes. However, the craniofacial diversity across the subfamily is not fully described. To better understand craniofacial phenotypes across Danioninae we used microCT and 3D geometric morphometrics to capture skull shapes from nine species. The Danio species examined showed largely similar skull shapes, although D. aesculapii, the sister species to D. rerio showed a unique morphology. Two non-Danio species examined, Chela dadiburjori and Devario aequipinnatus showed distinct skull morphologies unique from those of other species examined. Thyroid hormone regulates skeletal development and remodeling, and we asked if changes in developmental thyroid hormone metabolism could underlie some of the craniofacial diversity across Danioninae. We reared two Danio species under altered thyroid profiles, finding that hypothyroid individuals from both species showed corresponding morphological shifts in skull shape. Hypothyroid Danios showed skull morphologies closer to that of Chela and unlike any of the examined wild-type Danio species. We provide an examination of the evolved craniofacial diversity across Danioninae, and demonstrate that alterations to thyroid hormone have the capacity to create unique skull phenotypes.

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespots central signaling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centers in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

Hox genes are transcriptional regulators that elicit cell positional identity along the anterior-posterior region of the body plan across different lineages of Metazoan. Comparison of Hox gene expression across distinct species reveals their evolutionary conservation, however their gains and losses in different lineages can correlate with body plan modifications and morphological novelty. We compare the expression of eleven Hox genes found within Streblospio benedicti, a marine annelid that produces two types of offspring with distinct developmental and morphological features. For these two distinct larval types, we compare Hox gene expression through ontogeny using HCR (hybridization chain reaction) probes for in-situ hybridization and RNA-seq data. We find that Hox gene expression patterning for both types is typically similar at equivalent developmental stages. However, some Hox genes have spatial or temporal differences between the larval types that are associated with morphological and life-history differences. This is the first comparison of developmental divergence in Hox genes expression within a single species and these changes reveal how body plan differences may arise in larval evolution.

Synovial joints, characterized by reciprocally congruent and lubricated articular surfaces separated by a cavity, are hypothesized to have evolved from continuous cartilaginous joints for increased mobility and improved load bearing. To test the evolutionary origins of synovial joints, we examine the morphology, genetic, and molecular mechanisms required for the development and function of the joints in elasmobranchs and cyclostomes. We find the presence of cavitated and articulated joints in elasmobranchs, such as the little skate (Leucoraja erinacea) and bamboo shark (Chiloscyllium plagiosum), and the expression of lubrication-related proteoglycans such as aggrecan and glycoproteins such as hyaluronic acid receptor (CD44) at the articular surfaces in little skates. Sea lampreys (Petromyozon marinus), a representative of cyclostomes, are devoid of articular cavities but express proteoglycan-linking proteins throughout their cartilaginous skeleton, suggesting that the expression of proteoglycans is primitively not limited to the articular cartilage. Analysis of the development of joints in the little skate reveals the expression of growth differentiation factor-5 (Gdf5) and {beta}-catenin at the joint interzone before the process of cavitation, indicating the involvement of BMP and Wnt-signaling pathway, and reliance on muscle contraction for the process of joint cavitation, similar to tetrapods. In conclusion, our results show that synovial joints are present in elasmobranchs but not cyclostomes, and therefore, synovial joints originated in the common ancestor of extant gnathostomes. A review of fossils from the extinct clades along the gnathostome stem further shows that synovial joints likely arose in the common ancestor of gnathostomes. Our results have implications for understanding how the evolution of synovial joints around 400 mya in our vertebrate ancestors unlocked motor behaviors such as feeding and locomotion. Author summaryWe owe our mobility and agility to synovial joints, characterized by a lubricated joint cavity between the bony elements. Due to the cavity, synovial joints function by bones sliding relative to each other, allowing an extensive range of motion and heightened stability compared to fused or cartilaginous joints that function by bending. Using histological and protein expression analysis, we show that reciprocally articulated, cavitated, and lubricated joints are present in elasmobranchs such as skates and sharks but not in cyclostomes such as the sea lamprey. Furthermore, the development of the little skate joints relies on genetic regulatory mechanisms such as BMP and Wnt-signalling, similar to tetrapods. Thus, our results show that synovial joints are present in elasmobranchs but not in cyclostomes. In conclusion, synovial joints originated in the common ancestor of jawed vertebrates. Furthermore, a review of fossil taxa along the gnathostome stem shows that cavitated joints that function by relative sliding of articulating surfaces originated at the common ancestor of all gnathostomes. Our results have consequences for understanding how the evolution of cavitated and lubricated joints in ancient vertebrates impacted behaviors like feeding and locomotion 400 million years ago.

The potential for variation and the capacity to evolve in response to ecological opportunity are important aspects of an adaptive radiation. Identifying the origin of phenotypic variation, in which natural selection might act upon, is a major goal of evolutionary developmental biology. The New World leaf-nosed bats (phyllostomids) are a textbook example of an adaptive radiation. Their cranial morphology is diverse along relative facial length, which is related to their diets. We previously used geometric morphometrics to reveal peramorphosis, a type of heterochrony, in the cranial evolution among phyllostomid bats. We then demonstrated that the mechanism of peramorphic diversity in phyllostomid rostrum length resulted from altered cellular proliferation. Here, we investigate the progenitors of the face, the cranial neural crest, and a key signaling pathway related to their proliferation and differentiation into mature tissues: the bone morphogenetic protein (BMP). With geometric morphometrics, immunofluorescence, and confocal imaging--in three phyllostomid species and one outgroup bat species--we show the molecular patterns that underlie the adaptive and innovative traits seen in phyllostomid bats. Then, with mouse genetics, we mimic the BMP molecular pattern observed in nectar feeding bats and recapitulate the elongated morphological variation in mice. Surprisingly, we also observe an expansion in the nose-tip of mice, akin to the expanding leaf-nose tissue in phyllostomid bats. These data, combined with the mouse genetics literature on BMP signaling, suggest the BMP developmental pathway plays a central role in shaping the craniofacial variation necessary for adaptation in bats. Further, we speculate that the BMP signaling pathway could underlie other bizarre facial phenotypes in mammals that are derived from frontonasal mesenchyme, such as the proboscis. Overall, this study combines a comparative framework to developmental data, with a genetic approach, to directly investigate the role of development on complex morphology.

Elasmobranchs (sharks and rays) exhibit a wide range of body forms adapted to various ecological niches. Body form differs not only between species, but between life stages of individual species as a result of ontogenetic allometry. In sharks, it has been proposed that these ontogenetic shifts in body form result from shifts in trophic and/or spatial ecology (the allometric niche shift hypothesis). Alternatively, it has been suggested that ontogenetic allometry may result from intrinsic morphological constraints associated with increasing body size - e.g. to counteract shifts in form-function relationships that occur as a function of size and could compromise locomotory performance. One major limitation affecting our understanding of ontogenetic scaling in sharks is that existing studies focus on postpartum ontogeny - ignoring the period of growth that occurs prior to birth/hatching. In this study, we report ontogenetic growth trajectories from 39 near-term brown smooth hound (Mustelus henlei) embryos taken from manually collected measurements. We found that unlike most other species and later ontogenetic stages of M. henlei, these embryos predominantly grow isometrically, and appear to display relatively high levels of morphological disparity. These results provide rudimentary support for the allometric niche shift hypothesis (as in the absence of ontogenetic niche shifts isometry dominates body-form scaling) and provide important insight into early shark ontogeny and morphological/developmental evolution.

Many factors such as divergence time, shared standing genetic variation, frequency of introgression, and mutation rates can influence the likelihood of whether populations adapt to similar environments via parallel or non-parallel genetic changes. However, the frequency of parallel vs non-parallel genetic changes resulting in parallel phenotypic evolution is still unknown. In this study, we used a QTL mapping approach to investigate the genetic basis of highly divergent craniofacial traits between scale- and snail-eating trophic specialist species across similar hypersaline lake environments in an adaptive radiation of pupfishes endemic to San Salvador Island, Bahamas. We raised F2 intercrosses of scale- and snail-eaters from two different lake populations of sympatric specialists, estimated linkage maps, scanned for significant QTL for 30 skeletal and craniofacial traits, and compared the location of QTL between lakes to quantify parallel and non-parallel genetic changes. We found strong support for parallel genetic changes in both lakes for five traits in which we detected a significant QTL in at least one lake. However, many of these shared QTL affected different, but highly correlated craniofacial traits in each lake, suggesting that pleiotropy and trait integration should not be neglected when estimating rates of parallel evolution. We further observed a 23-52% increase in adaptive introgression within shared QTL, suggesting that introgression may be important for parallel evolution. Overall, our results suggest that the same genomic regions contribute to parallel integrated craniofacial phenotypes across lakes. We also highlight the need for more expansive searches for shared QTL when testing for parallel evolution.

Reconstructing ancestral reproductive systems is essential for understanding the evolution of bilaterian body plans, yet the origin and development of reproductive organs remain poorly characterized. Here, we investigate postembryonic sexual development in the xenacoelomorph Hofstenia atroviridis, a basal acoel species, to gain insight into early bilaterian reproductive evolution. Individuals were reared from eggs to adulthood, and the ontogeny of reproductive organs was examined using histology and immunohistochemistry with muscle and neural markers. H. atroviridis is a protandrous simultaneous hermaphrodite, with sexual maturity correlated with body size rather than age. The male copulatory system comprises a seminal vesicle, granular vesicle, penis with a penile bulb, and a single copulatory stylet, accompanied by regionally specialized musculature and innervation. In contrast, the female reproductive system consists of paired, asaccate ovaries containing large, follicle cell-bound oocytes and lacks a discrete gonopore or copulatory organ. Fertilization occurs via traumatic insemination, and eggs are likely released through the mouth. Despite the organisms overall morphological simplicity, the male reproductive system exhibits pronounced structural differentiation. These findings suggest that sexual selection acting on a hermaphroditic ancestor may have contributed to the early diversification of bilaterian copulatory organs and establish H. atroviridis as a useful model for studying the evolutionary origins of animal reproductive systems.

A widely accepted model for the evolution of cave animals posits colonization by surface ancestors followed by the acquisition of adaptations over many generations. However, the speed of cave adaptation in some species suggests mechanisms operating over shorter timescales. To address these mechanisms, we used Astyanax mexicanus, a teleost with ancestral surface morphs (surface fish, SF) and derived cave morphs (cavefish, CF). We exposed SF to completely dark conditions and identified numerous altered traits at both the gene expression and phenotypic levels. Remarkably, most of these alterations mimicked CF phenotypes. Our results indicate that cave-related traits can appear within a single generation by phenotypic plasticity. In the next generation, plasticity can be further refined. The initial plastic responses are random in adaptive outcome but may determine the subsequent course of evolution. Our study suggests that phenotypic plasticity contributes to the rapid evolution of cave-related traits in A. mexicanus.

Comparing gene regulatory patterns between seemingly similar phenotypic traits can provide important insights on the molecular mechanisms underlying the evolution of those traits. In this study, we investigate the molecular basis of the formation of a spade-shaped caudal fin, which is a rare phenotype among teleost fish characterized by an elongated medial region of the fin. We examined the expression patterns of candidate fin-shape genes in the spade-shaped caudal fin of the related species Lamprologus tigripictilis, an East African cichlid in the tribe Lamprologini. The candidate gene set consisted of a previously identified gene regulatory network (GRN) associated with the elongation of fin regions in another Lamprologini cichlid species and further genes selected on the basis of co-expression data and transcription factor prediction. Unexpectedly, the anatomical features of elongated fin rays differed and gene expression patterns associated with fin elongation were only weakly conserved between the two related species. We report 20 genes and transcription factors (including angptl5, cd63, csrp1a, cx43, esco2, gbf1 and rbpj), whose expression levels differed between the elongated and the short caudal fin regions of L. tigripictilis, and which are therefore candidates for the regulation of the spade-like fin shape.

Development of the vertebrate antral stomach and pyloric sphincter (antropyloric region) - involved in enzymatic breakdown and thoroughfare of food - is underpinned by a highly conserved developmental pathway involving the hedgehog, bone morphogenetic protein (BMP) and Wingless/Int-1 (Wnt) protein families. Monotremes are a unique lineage where acid-based digestion has been lost, and this correlates with a lack of genes for gastric acid and enzymes in the genomes of the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus). Furthermore, these species feature unique gastric phenotypes, both with truncated and aglandular antral stomachs and the platypus with no pylorus. Here, we explore the genetic underpinning of monotreme gastric phenotypes, investigating genes important in antropyloric development using the newest monotreme genome sequences (mOrnAna1.pri.v4 and mTacAcu1) together with RNA-seq data. We found that the pathway is generally conserved but, NK3 homeobox 2 (Nkx3.2) was pseudogenised in both platypus and echidna. We speculate that pyloric-like restriction in the echidna may correlate with independent evolution of Grem1 and Bmp4 sequences, and that the convergent loss of gastric acid and stomach size genotypes and phenotypes in teleost and monotreme lineages may be a result of eco-evolutionary dynamics. These findings reflect the effects of gene loss on phenotypic evolution and further elucidate the genetic control of monotreme stomach anatomy and physiology.

Heterochronic shifts are regarded one of the major evolutionary changes acting on developmental modules and underlying the origin of morphological disparity. Conserved characters, rarely subject to heterochronic shifts during the curse of evolution, in contrast could indicate underlying developmental or functional constraints. Here we use the development of the cranial musculature Siberian sturgeon (Acipenser baerii) as a model to investigate the role of heterochrony during the evolution of the craniofacial system of Actinopterygii. Using histology, fluorescent antibody staining and fast propagation-based phase contrast imaging in combination with 3D-reconstruction we describe the development of the branchial and hypobranchial musculature. We show that the development of the first branchial arch is accelerated compared to other basal-branching actinopterygians leading to a more synchronous development with the hyoid arch. A pattern that could relate to the derived migratory behaviour of the neural crest cells in sturgeons. In contrast, the developmental timing of the more posterior branchial musculature, including the cucullaris muscle in the Siberian sturgeon, appears to be highly conserved compared to other Actinopterygii and even Osteognathostomata. This could indicate the presence of functional or developmental constraints underlying the evolution of the muscles at the head/trunk interface.

We evaluated subtle-to-incipient pathology traits in coxofemoral joints from dry bone museum specimens of: Vulpes lagopus; Vulpes; Nyctereutes procyonoides; Urocyon cinereoargenteus; Canis lupus familiaris; and Canis latrans. Multiple intra-articular structures were evaluated on acetabula and proximal femora. Primary observations included multifocal, variable osteophytelike formations; osteophyte-like rimming of articular margins and femoral head (ligamentum teres attachment); and rough or worn bone. Within limitations on valid statistical applications, we observed little difference among the high trait frequencies across taxa, aligning with previous morphological observations. Additionally, for this study, we evaluated the known history of the taxa, from deep time to the present, to consider our data in a phylogenetic context. Potential introgression over the evolution of Canidae, along with early history of the canid genome, likely supported broad and deep conservation of pathophysiological processes associated with observable pathology at the same intra-articular foci, across taxa. We also evaluated the "modern" natural histories of the taxa, noting that coxofemoral joint impacts of their respective life habits did not appear to influence pathology trait outcomes differentially. We conclude that conservation of the physiology underlying subtle and incipient coxofemoral joint pathology that did not segregate among taxa. We hypothesize that the intersecting basic biology of growth-development and insult response, over long geological time, may owe in part to the evidently long histories of hybridization and generally high historical gene flow, with high levels of heterogeneity. These data argue for new research to advance an interdisciplinary, integrated understanding of relationships among canid growth-development, incipient-to-subtle joint pathology, influences of natural histories across related taxa, and implications for genomic interrelationships.