Parasite escape mechanisms drive morphological diversification in avian lice.

Organisms that have repeatedly evolved similar morphologies owing to the same selective pressures provide excellent cases in which to examine specific morphological changes and their relevance to the ecology and evolution of taxa. Hosts of permanent parasites act as an independent evolutionary experiment, as parasites on these hosts are thought to be undergoing similar selective pressures. Parasitic feather lice have repeatedly diversified into convergent ecomorphs in different microhabitats on their avian hosts. We quantified specific morphological characters to determine (i) which traits are associated with each ecomorph, (ii) the quantitative differences between these ecomorphs, and (iii) if there is evidence of displacement among co-occurring lice as might be expected under louse-louse competition on the host. We used nano-computed tomography scan data of 89 specimens, belonging to four repeatedly evolved ecomorphs, to examine their mandibular muscle volume, limb length and three-dimensional head shape data. Here, we find evidence that lice repeatedly evolve similar morphologies as a mechanism to escape host defences, but also diverge into different ecomorphs related to the way they escape these defences. Lice that co-occur with other genera on a host exhibit greater morphological divergence, indicating a potential role of competition in evolutionary divergence.

Increasing the impact of vertebrate scientific collections through 3D imaging: The openVertebrate (oVert) Thematic Collections Network.

The impact of preserved museum specimens is transforming and increasing by three-dimensional (3D) imaging that creates high-fidelity online digital specimens. Through examples from the openVertebrate (oVert) Thematic Collections Network, we describe how we created a digitization community dedicated to the shared vision of making 3D data of specimens available and the impact of these data on a broad audience of scientists, students, teachers, artists, and more. High-fidelity digital 3D models allow people from multiple communities to simultaneously access and use scientific specimens. Based on our multiyear, multi-institution project, we identify significant technological and social hurdles that remain for fully realizing the potential impact of digital 3D specimens.

Evolution of hyperossification expands skull diversity in frogs.

Frogs (Anura) are one of the most diverse vertebrate orders, comprising more than 7,000 species with a worldwide distribution and extensive ecological diversity. In contrast to other tetrapods, frogs have a highly derived body plan and simplified skull. In many lineages of anurans, increased mineralization has led to hyperossified skulls, but the function of this trait and its relationship with other aspects of head morphology are largely unexplored. Using three-dimensional morphological data from 158 species representing all frog families, we assessed wide-scale patterns of shape variation across all major lineages, reconstructed the evolutionary history of cranial hyperossification across the anuran phylogeny, and tested for relationships between ecology, skull shape, and hyperossification. Although many frogs share a conserved skull shape, several extreme forms have repeatedly evolved that commonly are associated with hyperossification, which has evolved independently more than 25 times. Variation in cranial shape is not explained by phylogenetic relatedness but is correlated with shifts in body size and ecology. The species with highly divergent, hyperossified skulls often have a specialized diet or a unique predator defense mechanism. Thus, the evolution of hyperossification has repeatedly facilitated the expansion of the head into multiple new shapes and functions.

Embryonic development of a parthenogenetic vertebrate, the mourning gecko (Lepidodactylus lugubris).

BACKGROUND: One goal of evolutionary developmental biology is to understand the role of development in the origin of phenotypic novelty and convergent evolution. Geckos are an ideal system to study this topic, as they are species-rich and exhibit a suite of diverse morphologies-many of which have independently evolved multiple times within geckos. RESULTS: We characterized and discretized the embryonic development of Lepidodactylus lugubris-an all-female, parthenogenetic gecko species. We also used soft-tissue µCT to characterize the development of the brain and central nervous system, which is difficult to visualize using traditional microscopy techniques. Additionally, we sequenced and assembled a de novo transcriptome for a late-stage embryo as a resource for generating future developmental tools. Herein, we describe the derived and conserved patterns of L. lugubris development in the context of squamate evolution and development. CONCLUSIONS: This embryonic staging series, µCT data, and transcriptome together serve as critical enabling resources to study morphological evolution and development, the evolution and development of parthenogenesis, and other questions concerning vertebrate evolution and development in an emerging gecko model.

Descriptive osteology and patterns of limb loss of the European limbless skink Ophiomorus punctatissimus (Squamata, Scincidae).

The limbless skink Ophiomorus punctatissimus is a cryptozoic species found in the Peloponnese region of Greece and on the Greek island Kythira. To provide the first thorough description of the cranial and postcranial osteology of this species, both disarticulated specimens and X-ray computed tomographies of wet-preserved specimens were examined in detail. Resulting from this, an anatomical atlas of this species is provided. Two separate considerations, an evolutionary and an ecomorphological one, are made based on the observed adaptations related to limb loss in this skink. The structure of the girdles shows a particular pattern of reduction: whereas the pelvic girdle is mostly vestigial, the pectoral girdle is instead well developed, with all the elements typical of limbed lizards except for the actual limbs. This led us to hypothesize an asynchronous pattern of limb reduction during the evolution of this species, in which the hindlimbs regressed earlier than the forelimbs. Furthermore, considerations based on overall body morphology, osteology and the structure of the inner ear led to the recognition of this species as a burrowing ecomorph. In contrast to the morphology normally displayed in this ecomorph, O. punctatissimus is characterized by the retention of autotomic vertebrae in its tail. This is consistent with the habitats in which it lives, where active burrowing would be difficult because of the hard, rocky terrain. Instead, this skink hides among rocks on the surface and is, therefore, subject to greater predation risk.

Structure of the Ambrosia Beetle (Coleoptera: Curculionidae) Mycangia Revealed Through Micro-Computed Tomography.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) rely on a symbiosis with fungi for their nutrition. Symbiotic fungi are preserved and transported in specialized storage structures called mycangia. Although pivotal in the symbiosis, mycangia have been notoriously difficult to study, given their minute size and membranous structure. We compared the application of novel visualization methods for the study of mycangia, namely micro-computed tomography (micro-CT) and laser ablation tomography (LATscan) with traditional paraffin sectioning. Micro-CT scanning has shown the greatest promise in new organ discovery, while sectioning remains the only method with sufficient resolution for cellular visualization. All three common types of mycangia (oral, mesonotal, and pronotal) were successfully visualized and presented for different species of ambrosia beetles: Ambrosiodmus minor (Stebbing) 1909, Euplatypus compositus (Say) 1823, Premnobius cavipennis Eichhoff 1878, Scolytoplatypus raja Blandford 1893, Xylosandrus crassiusculus (Motschulsky) 1866 and X. amputatus (Blandford) 1894. A reconstruction of the mycangium and the surrounding musculature in X. amputatus is also presented. The advantages of micro-CT compared to the previously commonly used microtome sectioning include the easy visualization and recording of three-dimensional structures, their position in reference to other internal structures, the ability to distinguish natural aberrations from technical artifacts, and the unprecedented visualizations of the anatomic context of mycangia enabled by the integrated software.

The first full body diffusible iodine-based contrast-enhanced computed tomography dataset and teaching materials for a member of the Testudines.

Diffusible iodine-based contrast-enhanced Computed Tomography (diceCT) is now a widely used technique for imaging metazoan soft anatomy. Turtles present a particular challenge for anatomists; gross dissection is inherently destructive and irreversible, whereas their near complete shell of bony plates, covered with keratinous scutes, presents a barrier for iodine diffusion and significantly increases contrast-enhanced CT preparation time. Consequently, a complete dataset visualizing the internal soft anatomy of turtles at high resolution and in three dimensions has not yet been successfully achieved. Here we outline a novel method that augments traditional diceCT preparation with an iodine injection technique to acquire the first full body contrast-enhanced dataset for the Testudines. We show this approach to be an effective method of staining the soft tissues inside the shell. The resulting datasets were processed to produce anatomical 3D models that can be used in teaching and research. As diceCT becomes a widely employed method for nondestructively documenting the internal soft anatomy of alcohol preserved museum specimens, we hope that methods applicable to the more challenging of these, such as turtles, will contribute toward the growing stock of digital anatomy in online repositories.

Anatomy of the female reproductive tract organs of the brown anole (Anolis sagrei).

Female reproduction in squamate reptiles (lizards and snakes) is highly diverse and mode of reproduction, clutch size, and reproductive tract morphology all vary widely across this group of ~11,000 species. Recently, CRISPR genome editing techniques that require manipulation of the female reproductive anatomy have been developed in this group, making a more complete understanding of this anatomy essential. We describe the adult female reproductive anatomy of the model reptile the brown anole (Anolis sagrei). We show that the brown anole female reproductive tract has three distinct anterior-to-posterior regions, the infundibulum, the glandular uterus, and the nonglandular uterus. The infundibulum has a highly ciliated epithelial lip, a region where the epithelium is inverted so that cilia are present on the inside and outside of the tube. The glandular uterus has epithelial ducts that are patent with a lumen as well as acinar structures with a lumen. The nonglandular uterus has a heterogeneous morphology from anterior to posterior, with a highly folded, ciliated epithelium transitioning to a stratified squamous epithelium. This transition is accompanied by a loss of keratin-8 expression and together, these changes are similar to the morphological and gene expression changes that occur in the mammalian cervix. We recommend that description of the nonglandular uterus include the regional sub-specification of a "cervix" and "vagina" as this terminology change more accurately describes the morphology. Our data extend histological studies of reproductive organ morphology in reptiles and expand our understanding of the variation in reproductive system anatomy across squamates and vertebrates.

The only "lungless" frog has a glottis and lungs.

Blackburn et al. show using CT-scanning that the only previously reported "lungless" frog retains a glottis and lungs.

Rapid Targeted Assembly of the Proteome Reveals Evolutionary Variation of GC Content in Avian Lice.

Nucleotide base composition plays an influential role in the molecular mechanisms involved in gene function, phenotype, and amino acid composition. GC content (proportion of guanine and cytosine in DNA sequences) shows a high level of variation within and among species. Many studies measure GC content in a small number of genes, which may not be representative of genome-wide GC variation. One challenge when assembling extensive genomic data sets for these studies is the significant amount of resources (monetary and computational) associated with data processing, and many bioinformatic tools have not been optimized for resource efficiency. Using a high-performance computing (HPC) cluster, we manipulated resources provided to the targeted gene assembly program, automated target restricted assembly method (aTRAM), to determine an optimum way to run the program to maximize resource use. Using our optimum assembly approach, we assembled and measured GC content of all of the protein-coding genes of a diverse group of parasitic feather lice. Of the 499 426 genes assembled across 57 species, feather lice were GC-poor (mean GC = 42.96%) with a significant amount of variation within and between species (GC range = 19.57%-73.33%). We found a significant correlation between GC content and standard deviation per taxon for overall GC and GC3, which could indicate selection for G and C nucleotides in some species. Phylogenetic signal of GC content was detected in both GC and GC3. This research provides a large-scale investigation of GC content in parasitic lice laying the foundation for understanding the basis of variation in base composition across species.

Compound osteoderms preserved in amber reveal the oldest known skink.

Scincidae is one of the most species-rich and cosmopolitan clades of squamate reptiles. Abundant disarticulated fossil material has also been attributed to this group, however, no complete pre-Cenozoic crown-scincid specimens have been found. A specimen in Burmite (99 MYA) is the first fossil that can be unambiguously referred to this clade. Our analyses place it as nested within extant skinks, supported by the presence of compound osteoderms formed by articulated small ostedermites. The specimen has a combination of dorsal and ventral compound osteoderms and overlapping cycloid scales that is limited to skinks. We propose that this type of osteoderm evolved as a response to an increased overlap of scales, and to reduced stiffness of the dermal armour. Compound osteoderms could be a key innovation that facilitated diversification in this megadiverse family.

3D pollination biology using micro-computed tomography and geometric morphometrics in Theobroma cacao.

Premise: Imaging technologies that capture three-dimensional (3D) variation in floral morphology at micro- and nano-resolutions are increasingly accessible. In herkogamous flowers, such as those of Theobroma cacao, structural barriers between anthers and stigmas represent bottlenecks that restrict pollinator size and access to reproductive organs. To study the unresolved pollination biology of cacao, we present a novel application of micro-computed tomography (micro-CT) using floral dimensions to quantify pollinator functional size limits. Methods: We generated micro-CT data sets from field-collected flowers and museum specimens of potential pollinators. To compare floral variation, we used 3D Slicer to place landmarks on the surface models and performed a geometric morphometric (GMM) analysis using geomorph R. We identified the petal side door (an opening between the petal hoods and filament) as the main bottleneck for pollinator access. We compared its mean dimensions with proposed pollinators to identify viable candidates. Results: We identified three levels of likelihood for putative pollinators based on the number of morphological (body) dimensions that fit through the petal side door. We also found floral reward microstructures whose presence and location were previously unclear. Discussion: Using micro-CT and GMM to study the 3D pollination biology of cacao provides new evidence for predicting unknown pollinators. Incorporating geometry and floral rewards will strengthen plant-pollinator trait matching models for cacao and other species.

Semicircular canal size constrains vestibular function in miniaturized frogs.

Miniaturization has evolved repeatedly in frogs in the moist leaf litter environments of rainforests worldwide. Miniaturized frogs are among the world's smallest vertebrates and exhibit an array of enigmatic features. One area where miniaturization has predictable consequences is the vestibular system, which acts as a gyroscope, providing sensory information about movement and orientation. We investigated the vestibular system of pumpkin toadlets, Brachycephalus (Anura: Brachycephalidae), a clade of miniaturized frogs from Brazil. The semicircular canals of miniaturized frogs are the smallest recorded for adult vertebrates, resulting in low sensitivity to angular acceleration due to insufficient displacement of endolymph. This translates into a lack of postural control during jumping in Brachycephalus and represents a physical constraint resulting from Poiseuille's law, which governs movement of fluids within tubes.

A new Early Cretaceous lizard in Myanmar amber with exceptionally preserved integument.

We here report on a well-preserved juvenile lizard specimen in Albian amber (ca. 110 mya) from the Hkamti site (Myanmar). This new taxon is represented by an articulated skull and the anterior portion of the trunk, including the pectoral girdle and forelimbs. The scleral ossicles and eyelid are also visible, and the specimen exhibits pristine detail of the integument (of both head and body). In a combined molecular and morphological analysis, it was consistently recovered as a scincoid lizard (Scinciformata), as sister to Tepexisaurus + Xantusiidae. However, the phylogenetic position of the new taxon should be interpreted with caution as the holotype is an immature individual. We explored the possibility of miscoding ontogenetically variable characters by running alternative analyses in which these characters were scored as missing data for our taxon. With the exception of one tree, in which it was sister to Amphisbaenia, the specimen was recovered as a Pan-xantusiid. Moreover, we cannot rule out the possibility that it represents a separate lineage of uncertain phylogenetic position, as it is the case for many Jurassic and Cretaceous taxa. Nonetheless, this fossil offers a rare opportunity to glimpse the external appearance of one group of lizards during the Early Cretaceous.

Between a rock and a hard polytomy: rapid radiation in the rupicolous girdled lizards (Squamata: Cordylidae).

Girdled lizards (Cordylidae) are sub-Saharan Africa's only endemic squamate family and contain 80 nominal taxa, traditionally divided into four genera: Cordylus, Pseudocordylus, Chamaesaura and Platysaurus. Previous phylogenetic analysis revealed Chamaesaura and Pseudocordylus to be nested within Cordylus, and the former genera were sunk into the later. This taxonomic revision has received limited support due to the study's poor taxon sampling, weakly supported results and possible temporary nomenclatural instability. Our study analyzes three nuclear and three mitochondrial genes from 111 specimens, representing 51 in-group taxa. Parsimony, likelihood and Bayesian analyses of concatenated and partitioned datasets consistently recovered a comb-like tree with 10, well-supported, monophyletic lineages. Our taxonomic reassessment divides the family into 10 genera, corresponding to these well-supported lineages. Short internodes and low support between the non-platysaur lineages are consistent with a rapid radiation event at the base of the viviparous cordylids.

Evolution of the locomotor skeleton in Anolis lizards reflects the interplay between ecological opportunity and phylogenetic inertia.

Anolis lizards originated in continental America but have colonized the Greater Antillean islands and recolonized the mainland, resulting in three major groups (Primary and Secondary Mainland and Greater Antillean). The adaptive radiation in the Greater Antilles has famously resulted in the repeated evolution of ecomorphs. Yet, it remains poorly understood to what extent this island radiation differs from diversification on the mainland. Here, we demonstrate that the evolutionary modularity between girdles and limbs is fundamentally different in the Greater Antillean and Primary Mainland Anolis. This is consistent with ecological opportunities on islands driving the adaptive radiation along distinct evolutionary trajectories. However, Greater Antillean Anolis share evolutionary modularity with the group that recolonized the mainland, demonstrating a persistent phylogenetic inertia. A comparison of these two groups support an increased morphological diversity and faster and more variable evolutionary rates on islands. These macroevolutionary trends of the locomotor skeleton in Anolis illustrate that ecological opportunities on islands can have lasting effects on morphological diversification.

Re-evaluating the morphological evidence for the re-evolution of lost mandibular teeth in frogs.

Dollo's law of irreversibility states that once a complex structure is lost, it cannot be regained in the same form. Several putative exceptions to Dollo's law have been identified using phylogenetic comparative methods, but the anatomy and development of these traits are often poorly understood. Gastrotheca guentheri is renowned as the only frog with teeth on the lower jaw. Mandibular teeth were lost in the ancestor of frogs more than 200 million years ago and subsequently regained in G. guentheri. Little is known about the teeth in this species despite being a frequent example of trait "re-evolution," leaving open the possibility that it may have mandibular pseudoteeth. We assessed the dental anatomy of G. guentheri using micro-computed tomography and histology and confirmed the longstanding assumption that true mandibular teeth are present. Remarkably, the mandibular teeth of G. guentheri are nearly identical in gross morphology and development to upper jaw teeth in closely related species. The developmental genetics of tooth formation are unknown in this possibly extinct species. Our results suggest that an ancestral odontogenic pathway has been conserved but suppressed in the lower jaw since the origin of frogs, providing a possible mechanism underlying the re-evolution of lost mandibular teeth.

Underground Down Under: Skull anatomy of the southern blind snake Anilios australis Gray, 1845 (Typhlopidae: Serpentes: Squamata).

The cranial anatomy of blindsnakes has been markedly understudied, with the small size and relative rarity of encountering these subterranean reptiles being significant limiting factors. In this article, we re-visit the skull anatomy of the Australian southern blind snake Anilios australis Gray, 1845 using microCT data, and produce the first complete atlas for the cranial anatomy of a representative of this speciose typhlopid genus. The skull is formed by 18 paired and four unpaired elements. We here produce a bone-by-bone description of each element as well as an inner ear endocast for each of two specimens differing in skull size. This approach has revealed the presence of a highly perforated dorsal plate on the septomaxilla-a structure convergent with the cribriform plate of the mammalian ethmoid bone-and a feature previously unknown for typhlopid snakes. This detailed anatomical study will facilitate ongoing taxonomic and systematic studies in the genus Anilios as well as provide comparative data for future studies on blindsnake anatomy more broadly.

Unveiling the elusive: X-rays bring scolecophidian snakes out of the dark.

Scolecophidian snakes have long posed challenges for scholars interested in elucidating their anatomy. The importance, and relative paucity, of high-quality anatomical data pertaining to scolecophidians was brought into sharp focus in the late 20th century as part of a controversy over the phylogeny and ecological origin of snakes. The basal position of scolecophidians in the phylogeny of snakes makes their anatomy, behavior, ecology, and evolution especially important for such considerations. The depauperate fossil record for the group meant that advances in understanding their evolutionary history were necessarily tied to biogeographic distributions and anatomical interpretations of extant taxa. Osteological data, especially data pertaining to the skull and mandible, assumed a dominant role in shaping historical and modern perspectives of the evolution of scolecophidians. Traditional approaches to the exploration of the anatomy of these snakes relied heavily upon serial-sectioned specimens and cleared-and-stained specimens. The application of X-ray computed tomography (CT) to the study of scolecophidians revolutionized our understanding of the osteology of the group, and now, via diffusible iodine-based contrast-enhanced computed tomography (diceCT), is yielding data sets on internal soft anatomical features as well. CT data sets replicate many aspects of traditional anatomical preparations, are readily shared with a global community of scholars, and now are available for unique holotype and other rare specimens. The increasing prevalence and relevance of CT data sets is a strong incentive for the establishment and maintenance of permanent repositories for digital data.

Size, microhabitat, and loss of larval feeding drive cranial diversification in frogs.

Habitat is one of the most important factors shaping organismal morphology, but it may vary across life history stages. Ontogenetic shifts in ecology may introduce antagonistic selection that constrains adult phenotype, particularly with ecologically distinct developmental phases such as the free-living, feeding larval stage of many frogs (Lissamphibia: Anura). We test the relative influences of developmental and ecological factors on the diversification of adult skull morphology with a detailed analysis of 15 individual cranial regions across 173 anuran species, representing every extant family. Skull size, adult microhabitat, larval feeding, and ossification timing are all significant factors shaping aspects of cranial evolution in frogs, with late-ossifying elements showing the greatest disparity and fastest evolutionary rates. Size and microhabitat show the strongest effects on cranial shape, and we identify a "large size-wide skull" pattern of anuran, and possibly amphibian, evolutionary allometry. Fossorial and aquatic microhabitats occupy distinct regions of morphospace and display fast evolution and high disparity. Taxa with and without feeding larvae do not notably differ in cranial morphology. However, loss of an actively feeding larval stage is associated with higher evolutionary rates and disparity, suggesting that functional pressures experienced earlier in ontogeny significantly impact adult morphological evolution.