Development of the turtle carapace: Implications for the evolution of a novel bauplan

The chelonian carapace is composed of the endochondral ribs and vertebrae associated with a specialized dermis. The ribs are found in an aberrant position compared to those of all other tetrapods; they are superficial and dorsal to the limb girdles. This morphological arrangement, which constitutes the unique chelonian Bauplan, is examined from a developmental perspective. Embryos of Chelydra serpentina were studied during stages of carapace development. Tissue morphology, autoradiography, and indirect immunofluorescent localization of adhesion molecules indicate that the outgrowth of the embryonic carapace occurs as the result of an epithelial–mesenchymal interaction in the body wall. A carapacial ridge composed of mesenchyme of the dermis and overlying ectoderm is formed dorsal to the ectodermal boundary between somitic and lateral plate mesoderm. It is the anlage of the carapace margin, in which the ribs will eventually terminate. The ectoderm of the carapacial ridge is thickened into a pseudostratified columnar epithelium, which overlies a condensation in the mesenchyme of the dermis. Patterns of cell proliferation and the distribution of N-CAM and fibronectin in the carapacial ridge are consistent with patterns seen in other structures initiated by epithelial–mesenchymal interactions such as feathers and limb buds. Based on an analogy to this developmental mechanism in the development of the limb skeleton, a further analogy with the evolution of the limbs from lateral fin folds is used to form a hypothesis on the evolution of the carapace from elements of the primitive reptilian integument.     

A Carapace-Like Bony ‘Body Tube’ in an Early Triassic Marine Reptile and the Onset of Marine Tetrapod Predation

Parahupehsuchus longus is a new species of marine reptile from the Lower Triassic of Yuan’an County, Hubei Province, China. It is unique among vertebrates for having a body wall that is completely surrounded by a bony tube, about 50 cm long and 6.5 cm deep, comprising overlapping ribs and gastralia. This tube and bony ossicles on the back are best interpreted as anti-predatory features, suggesting that there was predation pressure upon marine tetrapods in the Early Triassic. There is at least one sauropterygian that is sufficiently large to feed on Parahupehsuchus in the Nanzhang-Yuan’an fauna, together with six more species of potential prey marine reptiles with various degrees of body protection. Modern predators of marine tetrapods belong to the highest trophic levels in the marine ecosystem but such predators did not always exist through geologic time. The indication of marine-tetrapod feeding in the Nanzhang-Yuan’an fauna suggests that such a trophic level emerged for the first time in the Early Triassic. The recovery from the end-Permian extinction probably proceeded faster than traditionally thought for marine predators. Parahupehsuchus has superficially turtle-like features, namely expanded ribs without intercostal space, very short transverse processes, and a dorsal outgrowth from the neural spine. However, these features are structurally different from their turtle counterparts. Phylogeny suggests that they are convergent with the condition in turtles, which has a fundamentally different body plan that involves the folding of the body wall. Expanded ribs without intercostal space evolved at least twice and probably even more among reptiles.     

Evolutionary developmental perspective for the origin of turtles: the folding theory for the shell based on the developmental nature of the carapacial ridge

The body plan of the turtle represents an example of evolutionary novelty for acquisition of the shell. Unlike similar armors in other vertebrate groups, the turtle shell involves the developmental repatterning of the axial skeleton and exhibits an unusual topography of musculoskeletal elements. Thus, the turtle provides an ideal case study for understanding changes in the developmental program associated with the morphological evolution of vertebrates. In this article, the evolution of the turtle-specific body plan is reviewed and discussed. The key to understanding shell patterning lies in the modification of the ribs, for which the carapacial ridge (CR), a turtle-specific embryonic anlage, is assumed to be responsible. The growth of the ribs is arrested in the axial part of the body, allowing dorsal and lateral oriented growth to encapsulate the scapula. Although the CR does not appear to induce this axial arrest per se, it has been shown to support the fan-shaped patterning of the ribs, which occurs concomitant with marginal growth of the carapace along the line of the turtle-specific folding that takes place in the lateral body wall. During the process of the folding, some trunk muscles maintain their ancestral connectivities, whereas the limb muscles establish new attachments specific to the turtle. The turtle body plan can thus be explained with our knowledge of vertebrate anatomy and developmental biology, consistent with the evolutionary origin of the turtle suggested by the recently discovered fossil species, Odontochelys.     

The origin of the turtle body plan: evidence from fossils and embryos

The origin of the unique body plan of turtles has long been one of the most intriguing mysteries in evolutionary morphology. Discoveries of several new stem-turtles, together with insights from recent studies on the development of the shell in extant turtles, have provided crucial new information concerning this subject. It is now possible to develop a comprehensive scenario for the sequence of evolutionary changes leading to the formation of the turtle body plan within a phylogenetic framework and evaluate it in light of the ontogenetic development of the shell in extant turtles. The fossil record demonstrates that the evolution of the turtle shell took place over millions of years and involved a number of steps.     

Hepatocyte growth factor is crucial for development of the carapace in turtles

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan.     

The postembryonic transformation of the shell in emydine box turtles

A key trend in the 210‐million‐year‐old history of modern turtles was the evolution of shell kinesis, that is, shell movement during neck and limb retraction. Kinesis is hypothesized to enhance predator defense in small terrestrial and semiaquatic turtles and has evolved multiple times since the early Cretaceous. This complex phenotype is nonfunctional and far from fully differentiated following embryogenesis. Instead, kinesis develops slowly in juveniles, providing a unique opportunity to illustrate the postembryonic origins of an adaptive trait. To this end, we examined ventral shell (plastral) kinesis in emydine box turtles and found that hatchling plastron shape differs from that of akinetic‐shelled relatives, particularly where the hinge that enables kinesis differentiates. We also demonstrated shape changes relative to plastron size in juveniles, coinciding with a shift in the carapace‐plastron structural connection, rearrangement of ectodermal plates, and bone repatterning. Furthermore, because the shell grows larger relative to the head, complete concealment of the head and extremities is only achieved after relative shell proportions increase. Structural alterations that facilitate the box turtle's transformation are probably prepatterned in embryos but require function‐induced changes to differentiate in juveniles. This mode of delayed trait differentiation is essential to phenotypic diversification in turtles and perhaps other tetrapods.     

Developmental mechanisms of macroevolutionary change in the tetrapod axis: A case study of Sauropterygia

Understanding how developmental processes change on macroevolutionary timescales to generate body plan disparity is fundamental to the study of vertebrate evolution. Adult morphology of the vertebral column directly reflects the mechanisms that generate vertebral counts (somitogenesis) and their regionalisation (homeotic effects) during embryonic development. Sauropterygians were a group of Mesozoic marine reptiles that exhibited an extremely high disparity of presacral vertebral/somite counts. Using phylogenetic comparative methods, we demonstrate that somitogenesis and homeotic effects evolved in a co‐ordinated way among sauropterygians, contrasting with the wider pattern in tetrapods, in which somitogenetic and homeotic shifts are uncorrelated. Changes in sauropterygian body proportions were primarily enabled by homeotic shifts, with a lesser, but important, contribution from differences in postpatterning growth among somites. High body plan plasticity was present in Triassic sauropterygians and was maintained among their Jurassic and Cretaceous descendants. The extreme disparity in the body plan of plesiosaurian sauropterygians did not result from accelerated rates of evolutionary change in neck length, but instead reflect this ancestral versatility of sauropterygian axial development. Our results highlight variation in modes of axial development among tetrapods, and show that heterogeneous statistical models can uncover novel macroevolutionary patterns for animal body plans and the developmental mechanisms that control them.     

Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas)

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.     

Immune status of free-ranging green turtles with fibropapillomatosis from Hawaii

Cell-mediated and humoral immune status of free-ranging green turtles (Chelonia mydas) in Hawaii (USA) with and without fibropapillornatosis (FP) were assessed. Tumored and non-tumored turtles from Kaneohe Bay (KB) on the island of Oahu and from FP-free areas on the west (Kona/Kohala) coast of the island of Hawaii were sampled from April 1998 through February 1999. Turtles on Oahu were grouped (0-3) for severity of tumors with 0 for absence of tumors, 1 for light, 2 for moderate, and 3 for most severe. Turtles were weighed, straight carapace length measured and the regression slope of weight to straight carapace length compared between groups (KB0, KB1, KB2, KB3, Kona). Blood was assayed for differential white blood cell count, hematocrit, in vitro peripheral blood mononuclear cell (PBMC) proliferation in the presence of concanavalin A (ConA) and phytohaemagglutinin (PHA), and protein electrophoresis. On Oahu, heterophil/lymphocyte ratio increased while eosinophil/monocyte ratio decreased with increasing tumors score. Peripheral blood mononuclear cell proliferation indices for ConA and PHA were significantly lower for turtles with tumor scores 2 and 3. Tumor score 3 turtles (KB3) had significantly lower hematocrit, total protein, alpha 1, alpha 2, and gamma globulins than the other four groups. No significant differences in immune status were seen between non-tumored (or KB1) turtles from Oahu and Hawaii. There was no significant difference between groups in regression slopes of body condition to carapace length. We conclude that turtles with severe FP are imunosuppressed. Furthermore, the lack of significant difference in immune status between non-tumored (and KB1) turtles from Oahu and Kona/Kohala indicates that immunosuppression may not be a prerequisite for development of FP.     

Evolution of the Nasal Structure in the Lower Tetrapods

The gross structure of the nasal cavities and the distributionof the various types of epithelium lining them are describedbriefly; each living order of amphibians and reptiles possessesa characteristic and distinctive pattern. In most groups thereare two sensory areas, one lined by olfactory epithelium withnerve libers leading to the main olfactory bulb and the otherby vomeronasal epithelium with fibers to the accessory bulb.All amniotes except turtles have the vomeronasal epitheliumin a ventromedial outpocketing of the nose, the Jacobson's organ,and have one or more conchae projecting into the nasal cavityfrom the lateral wall. Although urodeles and turtles possessthe simplest nasal structure, it is not possible to show thatthey are primitive or to define a basic pattern for either amphibiansor reptiles; all the living orders are specialized and the nasalanatomy of extinct orders is unknown. Thus it is impossible,at present, to give a convincing picture of the course of nasalevolution in the lower tetrapods.     

Dietary protein requirement for captive juvenile green turtles (Chelonia mydas)

Head-starting programs are extremely important for restoring the population of sea turtles in wild whereas husbandry conditions and feeding regimens of captive turtles are still limited. In the current study, the optimal dietary protein requirement for green turtle (Chelonia mydas) was investigated to support rearing in head-starting programs. Twenty-five-day-old turtles (44.5–46.2 g body weight, n = 45) were randomly distributed into 15 experimental plastic tanks, comprising three treatment replications of 3 turtles each. They were fed fishmeal-based feeds containing different levels of protein (30%, 35%, 40%, 45%, and 50%) for 8 weeks. At the end of feeding trial, growth performance (specific growth rate = 1.86% body weight/day) and feed utilization (protein efficiency ratio = 3.30 g gain/g protein) were highest in turtles fed with 40% protein in feed (p < .05). These nutritional responses were significantly supported by specific activities of fecal digestive enzymes, especially trypsin, chymotrypsin, amylase, and the amylase/trypsin ratio. Also, this dietary level improved the deposition of calcium and phosphorus in carapace, supporting a hard carapace and strong healthy bones. There were no negative effects in general health status of reared turtles, as indicated by hematological parameters. Based on a broken-line analysis between dietary protein levels and specific growth rate, the optimal protein level for green turtles was estimated as 40.6%. Findings from the current study support the use of artificial diets of specific protein levels to rear captive green turtle before release to natural habitats.     

Unintended backpackers: bio-fouling of the invasive gastropod Rapana venosa on the green turtle Chelonia mydas in the Río de la Plata Estuary, Uruguay

Here we report the first observations of the rapa whelk Rapana venosa massively bio-fouling immature green turtles Chelonia mydas. From November 2004 to July 2011, we examined 33 green turtles with rapa whelks attached to their carapaces in Uruguayan waters. The number of attached rapa whelks ranged from 1 to 49 individuals, representing up to 20 % of turtle weight. This previously unrecorded interaction may be of global importance to green turtles conservation because (a) immature green turtles from distant breeding populations utilize insular and coastal waters of the Río de la Plata estuary and Atlantic coast of Uruguay as developmental and foraging habitats and (b) attached whelks may reduce green turtle fitness by reducing buoyancy, increasing drag, and causing severe injuries to the carapace.     

High-resolution analysis of salmonellae from turtles within a headwater spring ecosystem

Sediments and water from the pristine headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on the carapace and from the cloacae of 17 musk turtles (Sternotherus odoratus) and one snapping turtle (Chelydra serpentina serpentina) caught at the same site, were analysed for salmonellae by culture and molecular techniques. Whereas enrichment cultures from sediment and water samples were negative for salmonellae in PCR- and in situ hybridization-based analyses, both techniques detected salmonellae after enrichments from both carapace and cloacae of nine (i.e. of 53%) musk turtles. Further characterization of 10 isolates obtained from the enrichment cultures of four selected individuals and confirmed as salmonellae by PCR analysis was achieved by fingerprinting techniques (rep-PCR). The results show differences between individuals and, in one case, variation among isolates from a single individual. All isolates from two individuals displayed identical profiles. These profiles were different from those obtained from the isolates of the third individual, which were, themselves, also identical for all isolates. Salmonellae were much more diverse in samples from the carapace of the last individual with five different rep-PCR profiles retrieved. Serotyping of seven isolates representative for each rep-PCR profile identified all isolates as representing Salmonella enterica subspecies enterica serotype Rubislaw, which demonstrates the presence of different strains of potentially human pathogenic salmonellae naturally occurring on turtles even within pristine environments. The frequent detection of these organisms in biofilms on the carapace opens the door for speculations on the role of this habitat as a reservoir for salmonellae, and on potential implications for turtles acting as a dispersal vector.     

DEVELOPMENTAL BASIS OF TOOTHLESSNESS IN TURTLES: INSIGHT INTO CONVERGENT EVOLUTION OF VERTEBRATE MORPHOLOGY

The tooth is a major component of the vertebrate feeding apparatus and plays a crucial role in species survival, thus subjecting tooth developmental programs to strong selective constraints. However, irrespective of their functional importance, teeth have been lost in multiple lineages of tetrapod vertebrates independently. To understand both the generality and the diversity of developmental mechanisms that cause tooth agenesis in tetrapods, we investigated expression patterns of a series of tooth developmental genes in the lower jaw of toothless turtles and compared them to that of toothed crocodiles and the chicken as a representative of toothless modern birds. In turtle embryos, we found impairment of Shh signaling in the oral epithelium and early‐stage arrest of odontoblast development caused by termination of Msx2 expression in the dental mesenchyme. Our data indicate that such changes underlie tooth agenesis in turtles and suggest that the mechanism that leads to early‐stage odontogenic arrest differs between birds and turtles. Our results demonstrate that the cellular and molecular mechanisms that regulate early‐stage arrest of tooth development are diverse in tetrapod lineages, and odontogenic developmental programs may respond to changes in upstream molecules similarly thereby evolving convergently with feeding morphology.     

Demographic and tumour prevalence data for juvenile green turtles at the Coastal-Marine Protected Area of Cerro Verde,Uruguay

Marine Protected Areas are increasingly considered in coastal areas as an instrument to preserve threatened fauna and fragile habitats from the detrimental effects of human activities. For this reason baseline data are of utmost importance for the evaluation of the outcomes of ongoing conservation efforts. Along the Uruguayan coast, the area of Cerro Verde (declared protected since 2011) represents the most important foraging and development area for green turtles (Chelonia mydas). Between 2002 and 2009, a long-term capture-mark-recapture programme for green turtles was developed to gather data on demography, ecology and status of the species in the area. Turtles captured were juveniles ranging from 28.8 to 64.3?cm in length over the curve of the carapace (n?=?514), and results indicated a size-based habitat segregation. Tumour prevalence was 5.3% (n?=?27) and was positively correlated with carapace length. The mean body condition index was 1.25?±?0.14 (n?=?494). From the total number of tagged turtles 10.6% were recaptured during the study period. Green turtles showed high site fidelity; 81% of the turtles were recaptured within the same season and 76% were recaptured in different seasons but were found at the original capture spot. Mean annual growth rate was 1.6?±?0.9?cm year^?1. The catch per unit effort of 2008 differed from 2009, higher in 2009, but also significantly different between capture spots. The present study constitutes a baseline dataset for future monitoring of green turtles in the area and provides valuable information for wider analyses of population dynamics in the Southwestern Atlantic Ocean.     

Evolution of the therian face through complete loss of the premaxilla

The anatomical framework of the jawbones is highly conserved among most of the Osteichthyes, including the tetrapods. However, our recent study suggested that the premaxilla, the rostralmost upper jaw bone, was rearranged during the evolution of therian mammals, being replaced by the septomaxilla at least in the lateral part. In the present study, to understand more about the process of evolution from the ancestral upper jaw to the therian face, we re-examined the development of the therian premaxilla (incisive bone). By comparing mouse, bat, goat, and cattle fetuses, we confirmed that the therian premaxilla has dual developmental origins, the lateral body and the palatine process. This dual development is widely conserved among the therian mammals. Cell-lineage-tracing experiments using Dlx1-CreER^T2 mice revealed that the palatine process arises in the ventral part of the premandibular domain, where the nasopalatine nerve distributes, whereas the lateral body develops from the maxillary prominence in the domain of the maxillary nerve. Through comparative analysis using various tetrapods, we concluded that the palatine process should not be considered part of the ancestral premaxilla. It rather corresponds to the anterior region of the vomerine bone of nonmammalian tetrapods. Thus, the present findings indicate that the true premaxilla was completely lost during the evolution of the therian mammals, resulting in the establishment of the unique therian face as an evolutionary novelty. Reconsideration of the homological framework of the cranial skeleton based on the topographical relationships of the ossification center during embryonic development is warranted.     

A Standard System to Study Vertebrate Embryos

Staged embryonic series are important as reference for different kinds of biological studies. I summarise problems that occur when using ‘staging tables’ of ‘model organisms’. Investigations of developmental processes in a broad scope of taxa are becoming commonplace. Beginning in the 1990s, methods were developed to quantify and analyse developmental events in a phylogenetic framework. The algorithms associated with these methods are still under development, mainly due to difficulties of using non-independent characters. Nevertheless, the principle of comparing clearly defined newly occurring morphological features in development (events) in quantifying analyses was a key innovation for comparative embryonic research. Up to date no standard was set for how to define such events in a comparative approach. As a case study I compared the external development of 23 land vertebrate species with a focus on turtles, mainly based on reference staging tables. I excluded all the characters that are only identical for a particular species or general features that were only analysed in a few species. Based on these comparisons I defined 104 developmental characters that are common either for all vertebrates (61 characters), gnathostomes (26), tetrapods (3), amniotes (7), or only for sauropsids (7). Characters concern the neural tube, somite, ear, eye, limb, maxillary and mandibular process, pharyngeal arch, eyelid or carapace development. I present an illustrated guide listing all the defined events. This guide can be used for describing developmental series of any vertebrate species or for documenting specimen variability of a particular species. The guide incorporates drawings and photographs as well as consideration of species identifying developmental features such as colouration. The simple character-code of the guide is extendable to further characters pertaining to external and internal morphological, physiological, genetic or molecular development, and also for other vertebrate groups not examined here, such as Chondrichthyes or Actinopterygii. An online database to type in developmental events for different stages and species could be a basis for further studies in comparative embryology. By documenting developmental events with the standard code, sequence heterochrony studies (i.e. Parsimov) and studies on variability can use this broad comparative data set.     

不同生境间龟鳖类体型的差异

体型是动物重要的形态特征,影响动物的生境利用。为揭示龟鳖类体型与生境之间的关系,通过文献收集331种龟鳖(龟鳖目Tesudines总物种数的98.8%)的最大背甲长及其生境信息,将生境分为海洋、淡水、岛屿性陆地和大陆性陆地4种类型,再将淡水生境分为大静水、大流水、小静水、小流水和所有水域5种亚类型,大陆性陆地生境分为高地、平地和荒漠3种亚类型,从而比较不同生境类型或亚类型之间龟鳖类体型的差异。广义线性混合模型分析结果显示:1)海龟体型最大,岛屿性陆龟次之,淡水龟鳖和大陆性陆龟体型最小,且后两者差异无统计学意义。2)淡水龟鳖类的体型在5种亚类型生境间存在差异,大静水和大流水水域的体型均显著大于小静水和小流水水域,而体型在大静水与大流水水域、小静水与小流水水域之间的差异均无统计学意义,表明淡水龟鳖类体型与水域面积有关,而与水域是静水或流水无关。广布所有水域的淡水龟鳖类体型趋于中间型,且与其他4种亚类型生境中的体型之间的差异均无统计学意义。3)大陆性陆龟的体型从高地到平地再到荒漠有逐渐变大的趋势,但差异无统计学意义。本研究揭示龟鳖类的保护对策需要考虑其体型和生境面积的相关性。