Open data and digital morphology.

Over the past two decades, the development of methods for visualizing and analysing specimens digitally, in three and even four dimensions, has transformed the study of living and fossil organisms. However, the initial promise that the widespread application of such methods would facilitate access to the underlying digital data has not been fully achieved. The underlying datasets for many published studies are not readily or freely available, introducing a barrier to verification and reproducibility, and the reuse of data. There is no current agreement or policy on the amount and type of data that should be made available alongside studies that use, and in some cases are wholly reliant on, digital morphology. Here, we propose a set of recommendations for minimum standards and additional best practice for three-dimensional digital data publication, and review the issues around data storage, management and accessibility.

Comparative and Functional Anatomy of the Ectothermic Sauropsid Heart.

The heart development, form, and functional specializations of chelonians, squamates, crocodilians, and birds characterize how diverse structure and specializations arise from similar foundations. This review aims to summarize the morphologic diversity of sauropsid hearts and present it in an integrative functional and phylogenetic context. Besides the detailed morphologic descriptions, the integrative view of function, evolution, and development will aid understanding of the surprising diversity of sauropsid hearts. This integrated perspective is a foundation that strengthens appreciation that the sauropsid hearts are the outcome of biological evolution; disease often is linked to arising mismatch between adaptations and modern environments.

Malformations of the sacculus and the semicircular canals in spider morph pythons.

Spider morph ball pythons are a frequently-bred designer morph with striking alterations of the skin color pattern. We created high-resolution µCT-image series through the otic region of the skulls, used 3D-reconstruction software for rendering anatomical models, and compared the anatomy of the semicircular ducts, sacculus and ampullae of wildtype Python regius (ball python) with spider morph snakes. All spider morph snakes showed the wobble condition (i.e., twisting movements of the head, impaired locomotion, difficulty striking or constricting prey items). We describe the inner ear structures in wildtype and spider morph snakes and report a deviant morphology of semicircular canals, ampullae and sacculus in the latter. We also report about associated differences in the desmal skull bones of spider morph snakes, which were characterized by wider semicircular canals, ampullae widened and difficult to discern in µCT, a deformed crus communis, and a small sacculus with a highly deviant X-ray morphology as compared to wildtype individuals. We observed considerable intra- and interindividual variability of these features. This deviant morphology in spider morph snakes could easily be associated with an impairment of sense of equilibrium and the observed neurological wobble condition. Limitations in sample size prevent statistical analyses, but the anatomical evidence is strong enough to support an association between the wobble condition and a malformation of the inner ear structures. A link between artificially selected alterations in pattern and specific color design with neural-crest associated developmental malformations of the statoacoustic organ as known from other vertebrates is discussed.

Comparative Assessment of Computed Tomography and Magnetic Resonance Imaging of Spider Morph and Wild Type Ball Pythons (Python regius) for Evaluation of the Morphological Correlate of Wobble Syndrome.

There is general awareness of artificial selection and its potential implications on the health and welfare of animals. Despite growing popularity and increasing numbers of reptile breeds of atypical colour and pattern variants, only a few studies have investigated the appearance and causes of diseases associated with colour morphs. Ball pythons (Python regius) are among the most frequently bred reptiles and breeders have selected for a multitude of different colour and pattern morphs. Among those colour variants, the spider morph of the ball python is frequently associated with wobble syndrome. The aim of this study was to determine whether a morphological variant can be found and associated with the clinical occurrence of wobble syndrome in spider ball pythons, using computed tomography and magnetic resonance imaging as in-vivo diagnostic methods. Data from five spider and three wild type ball pythons was assessed and evaluated comparatively. We were able to identify distinctive structural differences in inner ear morphology in spider ball pythons, which were highly likely related to wobble syndrome. To our knowledge, this is the first report of these anomalies and provides a basis for further anatomical and genetic studies and discussion of the implications for animal welfare in reptile breeding.

Testing evolutionary hypotheses about the phylotypic period of zebrafish.

Vertebrate embryos pass through a period of morphological similarity, the phylotypic period. Since Haeckel's biogenetic law of recapitulation, proximate and ultimate evolutionary causes of such similarity of embryos were discussed. We test predictions about changes in phenotypic and genetic variances that were derived from three hypotheses about the evolutionary origin of the phylotypic stage, i.e. random, epigenetic effects, and stabilizing selection. The random hypothesis predicts increasing values for phenotypic variances and stable or increasing values for genetic variances; the epigenetic effects hypothesis predicts declining values for phenotypic variances but stable or increasing values of genetic variances, and the stabilizing selection predicts stable phenotypic variances but decreasing genetic variances. We studied zebrafish as a model species, because it can be bred in large numbers as necessary for a quantitative genetics breeding design. A half-sib breeding scheme provided estimates of additive genetic variances from 11 embryonic characters from 12 through to 24 hr after fertilization, i.e. before, during (15-19 hr), and after the phylotypic period. Because additive genetic variances are size dependent, we calculated narrow-sense heritabilities as a size independent gauge of genetic contributions to the phenotype. The results show declining phenotypic variances and stable heritabilities. In conclusion, we reject the random and the stabilizing selection hypotheses and favor ideas about epigenetic effects that constrain the early embryonic development. Additive genetic variance during the phylotypic stage makes it accessible for evolution, thus explaining in a simple and straightforward way why the phylotypic period differs among vertebrates in timing, duration, and morphologies.

Morphology of the avian yolk sac.

The avian yolk sac is a multifunctional extraembryonic organ that serves not only as a site of nutrient (yolk) absorption, but also for early hemopoiesis, and formation of blood vessels. Although the yolk sac membrane being specialized to function as an extraembryonic absorptive organ, it is neither morphologically nor functionally part of the embryonic gut. Yolk absorption is by the phagocytic activity of the extraembryonic endoderm. I used cryohistology and resin embedding histology of complete developmental series of Japanese quail to document the development of the avian yolk sac and changes of the microscopic anatomy throughout development. This material is complemented by complete series of MRT-scans of live ostrich embryos from beginning of incubation through hatching. Considerable changes of size and shape of the yolk mass are documented and discussed as resulting from water flux from albumen to yolk associated with the biochemical activation of yolk sac proteins. During embryogenesis, the yolk sac endoderm forms villi that increase the absorptive surface and reach into the yolk ball. The histology of the absorptive epithelium is specialized for phagocytic absorption of yolk. During early developmental stages, the extraembryonic endoderm is single layered, but it eventually becomes several layers thick during later stages. The extraembryonic mesoderm forms an extensive layer of hematopoietic tissue; deep in this tissue lie the yolk sac vessels. During late stages of development, the erythropoietic tissue disappears, blood vessels are obliterated, and the yolk sac epithelium becomes apoptotic. Results are discussed in the light of the evolutionary history and phylogeny of the amniote egg.

Phylogeny and evolutionary history of the amniote egg.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates-the amniote egg presenting one "solution" that enabled the conquest of land for reproduction.

Developmental plasticity, modularity, and heterochrony during the phylotypic stage of the zebra fish, Danio rerio.

We studied early embryonic development of zebra fish and tested if changes in the external raising conditions could elicit phenotypic changes during the phylotypic stage which, classically, is considered as a conserved embryonic stage. In particular, we tested for internal constraints, plasticity, and heterochrony during the early embryonic development. Our tested hypotheses predict (i) no change associated with developmental stability/internal constraints, (ii) change of the rate of development associated with developmental flexibility, and (iii) heterochronic disruption of developmental pattern associated with a modular organization of the embryo. We measured 14 traits of embryos raised in different conditions (temperature, salinity, oxygen concentration). The results of our study show that zebra fish embryos respond flexibly to changes in external parameters even during the conserved "phylotypic stage." It also showed that internal constraints canalize early development when exposed to moderate external challenges. Hypoxic conditions, however, elicited a heterochronic delay of the onset of the development of the Anlagen of the eye and the otic vesicle from the remaining embryo. Therefore, we concluded that the eye and the otic vesicle are modules that may develop, to a certain degree, independently of the rest of the embryo. Because these modules become recognizable only under specific raising conditions, we suggest that the modularization acts as buffering mechanism against extreme developmental deviations. Our results provide support to the idea that modularity is present during the phylotypic stage, but it is not effective under normal conditions.

Fuel switching and energy partitioning during the postprandial metabolic response in the ball python (Python regius).

Digestion, absorption and assimilation of the meal are active processes that require start-up energy before the energy contained in a meal can be utilized. The energetic costs associated with feeding (specific dynamic action, SDA) are high in sit-and-wait foraging snakes that tolerate long fasting periods. We used (13)C-labelled prey to partition between endogenous energy sources (i.e. snakes' own resources) and exogenous energy sources (i.e. prey). A linear mixing model was then applied to determine the portion of (13)C originating from the different sources. The snakes showed a normal and typical postprandial response. By four hours after feeding, the delta(13)C-values indicated fuel switching from endogenous to exogenous. From then on, fuel mixing continuously increased until, at 20 h after feeding, 75% of fuel was exogenous. Resource partitioning showed that throughout SDA, the amount of exogenous energy increased to approximately 60% of SDA, which was equivalent to approximately 4.5% of the energy contained in a meal.

Three genetically distinct ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).

Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of ferlaviruses into four genogroups, and criteria for definition of ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.

Morphological responses to feeding in ticks (Ixodes ricinus).

BACKGROUND: Ticks can survive long periods without feeding but, when feeding, ingest large quantities of blood, resulting in a more than 100-fold increase of body volume. We study morphological adaptations to changes in opisthosoma volume during feeding in the castor bean tick, Ixodes ricinus. We aim to understand the functional morphological features that accommodate enormous changes in volume changes. METHODS: Using light and electron microscopy, we compare the cuticle and epidermis of the alloscutum, the epithelium of the midgut diverticula, and the tracheae of adult female ticks when fasting, semi-engorged, and fully engorged. RESULTS: Our results add to an existing body of knowledge that the area of the epidermis increases by cellular differentiation, cellular hypertrophy, and changes in the shape of epithelial cells from pseudostratified to single layered prismatic in semi-engorged ticks, and to thin squamous epithelium in fully engorged ticks. We did not find evidence for cell proliferation. The midgut diverticula accommodate the volume increase by cellular hypertrophy and changes in cell shape. In fully engorged ticks, the epithelial cells of the midgut diverticula are stretched to an extremely thin, squamous epithelium. Changes in size and shape (and cell divisions) contribute to the accommodation of volume changes. Tracheae do not increase in size, but extend in length, thus following the volume changes of the opisthosoma in feeding ticks to secure oxygen supply to the internal organs. CONCLUSIONS: Changes of epithelial tissue configuration in the epidermis and the midgut diverticula are described as important components of the morphological response to feeding in ticks. We provide evidence for a previously unknown mechanism hosted in the endocuticle of the tracheae that allows the tracheae of castor bean ticks to expand when the body volume increases and the distance between the respiratory spiracle and the oxygen demanding tissue enlarges. This is the first report of expandable tracheae in arthropods.

Postprandial responses in the African rhombig egg eater (Dasypeltis scabra).

The African rhombic egg eater (Dasypeltis scabra) is a colubrid snake feeding exclusively on bird eggs. Frequency of feeding is governed by the seasonal availability of bird eggs; i.e., long fasting intervals change with relatively short periods when plenty of food is available. Intermittent feeding snakes show a remarkable postprandial increase of metabolic rate and digestive organ size. The postprandial increase in metabolic rate (specific dynamic action, SDA) in snakes is affected by meal size, temperature, and meal composition. A major portion of SDA in snakes is allocated to gastric function and the breakdown of the meal. We hypothesize that SDA in egg eaters is lower than in other snake species, because egg eaters feed on "liquid" food that does not require enzymatic breakdown in the stomach. We also hypothesized that other components of the postprandial response of egg eaters (e.g., size changes of the intestine and the liver) do not differ from other snakes. The standard metabolic rate and metabolic response to feeding were measured using closed-chamber respirometry. Size changes of small intestine and liver were measured using high-resolution transcutaneous ultrasonography. Standard metabolic rates of fasting egg eaters were in the same range of mass specific values as known from other snakes. Within 24h after feeding, oxygen consumption doubled and peaked at 2 days after feeding. At the same time, the size of the small intestine and the cross-sectional diameter of the liver increased. Within 2 days after feeding, the size of the mucosal epithelium doubled its thickness. Liver size increased significantly within 24h reaching maximum size 2-4 days after feeding. The size of both organs returned to fasting values within 7-10 days after feeding. The postprandial response of African rhombic egg eaters shows the same pattern and dynamics as known from other snake species. However, the factorial increase of metabolic rate during SDA is the lowest reported for any snake. A comparison with literature data supports the idea that SDA is mainly determined by gastric function and that it is low in egg eaters because they do not have to break down solid meals in the stomach as other snake species do.

Histological and ultrastructural analysis of the respiratory tracheae of Galeodes granti (Chelicerata: Solifugae).

Solifuges lack oxygen-carrying proteins like hemocyanins found in other chelicerates. For conduction of gases, therefore, we hypothesize that the tracheal system is divided into convective and respiratory parts, the latter having intimate association with respiring cells, tissues and organs. This hypothesis is supported by studies of tracheae in other arthropods. We used light, scanning, and transmission electron microscopy to examine the tracheae of Galeodes granti (Chelicerata, Solifugae). We studied tracheae in cheliceral and leg muscles, midgut and midgut diverticula, the heart and the supra- and subesophageal ganglia of the central nervous system. In all tissues examined, the tracheae penetrate into tissues and can be found between cells. Terminal branches with diameter less than one micron were found embedded into cell bodies of respiring tissue, thus reducing the diffusion distance. We suggest that the terminal branches are specialized for respiratory function due to their thin intima, with reduced endocuticle and exocuticle. Comparison with other tracheate chelicerates suggests parallel evolution of a common tracheal pattern based on homologous cellular building materials within the chelicerates and arthropods.

Morphology of the tracheal system of camel spiders (Chelicerata: Solifugae) based on micro-CT and 3D-reconstruction in exemplar species from three families.

We studied the tracheal system of exemplar species representing three families of Solifugae Sundevall, 1833, i.e., Galeodes granti Pocock, 1903, Ammotrechula wasbaueri Muma, 1962 and Eremobates sp., using µCT-imaging and 3D-reconstruction. This is the first comparative study of the tracheal system of Solifugae in 85 years and the first using high-resolution nondestructive methods. The tracheal system was found to be structurally similar in all three species, with broad major tracheae predominantly in the prosoma as well as anastomoses (i.e., connections between tracheal branches from different stigmata) in the prosoma and opisthosoma. Differences among the three species were observed in the presence or absence of cheliceral air sacs, the number of tracheae supplying the heart, and the ramification of major tracheae in the opisthosoma. The structure of the tracheal system with its extensive branches and some anastomoses is assumed to aid rapid and efficient gas exchange in the respiratory tissues of these active predators. The large diameter of cheliceral tracheae (air sacs) of taxa with disproportionally heavier chelicerae suggests a role in weight reduction, enabling solifuges to reach greater speeds during predation. The air sacs may also permit more rapid and efficient gaseous exchange, necessary to operate the musculature of these structures, thereby improving their use for predation in an environment where prey is scarce.

Morphological specializations for fetal maintenance in viviparous vertebrates: An introduction and historical retrospective.

In many viviparous vertebrates, pregnant females sustain their developing embryos and provide them with nutrients by means of placentas and a diversity of other types of specializations. With this article, we introduce a virtual (online) issue of the Journal of Morphology that presents 12 recent papers on fetal maintenance in viviparous vertebrates. We also outline the history of research in this area and document the central role of morphology in helping to explain the function and evolution of specializations for fetal nutrition. This virtual issue of the Journal of Morphology is an outgrowth of a symposium held under auspices of the International Congress of Vertebrate Morphology. The included papers reflect a diversity of taxa, research methods, and biological issues. To celebrate the publication of this virtual issue of the Journal of Morphology, the publisher is making freely available to readers a number of other relevant papers published in the journal over the past 128 years.

Alligators and Crocodiles Have High Paracellular Absorption of Nutrients, But Differ in Digestive Morphology and Physiology.

Much of what is known about crocodilian nutrition and growth has come from animals propagated in captivity, but captive animals from the families Crocodilidae and Alligatoridae respond differently to similar diets. Since there are few comparative studies of crocodilian digestive physiology to help explain these differences, we investigated young Alligator mississippiensis and Crocodylus porosus in terms of (1) gross and microscopic morphology of the intestine, (2) activity of the membrane-bound digestive enzymes aminopeptidase-N, maltase, and sucrase, and (3) nutrient absorption by carrier-mediated and paracellular pathways. We also measured gut morphology of animals over a larger range of body sizes. The two species showed different allometry of length and mass of the gut, with A. mississippiensis having a steeper increase in intestinal mass with body size, and C. porosus having a steeper increase in intestinal length with body size. Both species showed similar patterns of magnification of the intestinal surface area, with decreasing magnification from the proximal to distal ends of the intestine. Although A. mississippiensis had significantly greater surface-area magnification overall, a compensating significant difference in gut length between species meant that total surface area of the intestine was not significantly different from that of C. porosus. The species differed in enzyme activities, with A. mississippiensis having significantly greater ability to digest carbohydrates relative to protein than did C. porosus. These differences in enzyme activity may help explain the differences in performance between the crocodilian families when on artificial diets. Both A. mississippiensis and C. porosus showed high absorption of 3-O methyl d-glucose (absorbed via both carrier-mediated and paracellular transport), as expected. Both species also showed surprisingly high levels of l-glucose-uptake (absorbed paracellularly), with fractional absorptions as high as those previously seen only in small birds and bats. Analyses of absorption rates suggested a relatively high proportional contribution of paracellular (i.e., non-mediated) uptake to total uptake of nutrients in both species. Because we measured juveniles, and most paracellular studies to date have been on adults, it is unclear whether high paracellular absorption is generally high within crocodilians or whether these high values are specific to juveniles.

Pythons metabolize prey to fuel the response to feeding.

We investigated the energy source fuelling the post-feeding metabolic upregulation (specific dynamic action, SDA) in pythons (Python regius). Our goal was to distinguish between two alternatives: (i) snakes fuel SDA by metabolizing energy depots from their tissues; or (ii) snakes fuel SDA by metabolizing their prey. To characterize the postprandial response of pythons we used transcutaneous ultrasonography to measure organ-size changes and respirometry to record oxygen consumption. To discriminate unequivocally between the two hypotheses, we enriched mice (= prey) with the stable isotope of carbon (13C). For two weeks after feeding we quantified the CO2 exhaled by pythons and determined its isotopic 13C/12C signature. Ultrasonography and respirometry showed typical postprandial responses in pythons. After feeding, the isotope ratio of the exhaled breath changed rapidly to values that characterized enriched mouse tissue, followed by a very slow change towards less enriched values over a period of two weeks after feeding. We conclude that pythons metabolize their prey to fuel SDA. The slowly declining delta13C values indicate that less enriched tissues (bone, cartilage and collagen) from the mouse become available after several days of digestion.

Anatomy of the middle ear of the Japanese crane Grus japonensis (Gruidae: Aves).

Comparative anatomical studies of the avian middle ear provide structural data from which functional models can be derived. Subsequent phylogenetic analyses may reveal the phylogenetic history of taxon and the evolution of the functional model of the middle ear. We studied serial sections of a nestling of Grus japonensis. The microscopic anatomy of the middle ear (including skeletal parts, nerves, blood vessels, tendons, and tympanic recesses) was described with the help of a polystyrene plate reconstruction. These results were compared to those from skulls of adult cranes. Structural peculiarities of the crane middle ear are: 1) the ventral and anterior closure of the middle ear cavity by a dense plate of connective tissue, which ossifies during posthatching development; 2) the S-shaped infracolumellar process of the columella, of which the distal end forms a horizontal bar, and abuts against the ventral wall of the middle ear cavity; and 3) the absence of an intracolumellar hinge between the stapes and extracolumella. The crane middle ear shows the plesiomorphic pattern of middle ear structures of neognathous birds. We propose a one-lever-arm functional model for the middle ear of the crane, similar to that of paleognathous birds.

Bone microstructure and developmental plasticity in birds and other dinosaurs.

Patterns of bone microstructure have frequently been used to deduce dynamics and processes of growth in extant and fossil tetrapods. Often, the various types of primary bone tissue have been associated with different bone deposition rates and more recently such deductions have extended to patterns observed in dinosaur bone microstructure. These previous studies are challenged by the findings of the current research, which integrates an experimental neontological approach and a paleontological comparison. We use tetracycline labeling and morphometry to study the variability of bone deposition rates in Japanese quail (Coturnix japonica) growing under different experimental conditions. We compare resulting patterns in bone microstructure with those found in fossil birds and other dinosaurs. We found that a single type of primary bone varies significantly in rates of growth in response to environmental conditions. Ranging between 10-50 microm per day, rates of growth overlap with the full range of bone deposition rates that were previously associated with different patterns of bone histology. Bone formation rate was significantly affected by environmental/experimental conditions, skeletal element, and age. In the quail, the experimental conditions did not result in formation of lines of arrested growth (LAGs). Because of the observed variation of bone deposition rates in response to variation in environmental conditions, we conclude that bone deposition rates measured in extant birds cannot simply be extrapolated to their fossil relatives. Additionally, we observe the variable incidence of LAGs and annuli among several dinosaur species, including fossil birds, extant sauropsids, as well as nonmammalian synapsids, and some extant mammals. This suggests that the ancestral condition of the response of bone to environmental conditions was variable. We propose that such developmental plasticity in modern birds may be reduced in association with the shortened developmental time during the later evolution of the ornithurine birds.