Locomotor and postural diversity among reptiles viewed through the prism of femoral microanatomy: Palaeobiological implications for some Permian and Mesozoic taxa.

The water-to-land transition by the first tetrapod vertebrates represents a key stage in their evolution. Selection pressures exerted by this new environment on animals led to the emergence of new locomotor and postural strategies that favoured access to different ecological niches and contributed to their evolutionary success. Today, amniotes show great locomotor and postural diversity, particularly among Reptilia, whose extant representatives include parasagittally locomoting erect and crouched bipeds (birds), sub-parasagittal 'semi-erect' quadrupeds (crocodylians) and sprawling quadrupeds (squamates and turtles). But the different steps leading to such diversity remain enigmatic and the type of locomotion adopted by many extinct species raises questions. This is notably the case of certain Triassic taxa such as Euparkeria and Marasuchus. The exploration of the bone microanatomy in reptiles could help to overcome these uncertainties. Indeed, this locomotor and postural diversity is accompanied by great microanatomical disparity. On land, the bones of the appendicular skeleton support the weight of the body and are subject to multiple constraints that partly shape their external and internal morphology. Here we show how microanatomical parameters measured in cross-section, such as bone compactness or the position of the medullocortical transition, can be related to locomotion. We hypothesised that this could be due to variations in cortical thickness. Using statistical methods that take phylogeny into account (phylogenetic flexible discriminant analyses), we develop different models of locomotion from a sample of femur cross-sections from 51 reptile species. We use these models to infer locomotion and posture in 7 extinct reptile taxa for which they remain debated or not fully clear. Our models produced reliable inferences for taxa that preceded and followed the quadruped/biped and sprawling/erect transitions, notably within the Captorhinidae and Dinosauria. For taxa contemporary with these transitions, such as Terrestrisuchus and Marasuchus, the inferences are more questionable. We use linear models to investigate the effect of body mass and functional ecology on our inference models. We show that body mass seems to significantly impact our model predictions in most cases, unlike the functional ecology. Finally, we illustrate how taphonomic processes can impact certain microanatomical parameters, especially the eccentricity of the section, while addressing some other potential limitations of our methods. Our study provides insight into the evolution of enigmatic locomotion in various early reptiles. Our models and methods could be used by palaeontologists to infer the locomotion and posture in other extinct reptile taxa, especially when considered in combination with other lines of evidence.

Delimitation of the embryonic thermosensitive period for sex determination using an embryo growth model reveals a potential bias for sex ratio prediction in turtles.

The sexual phenotype of the gonad is dependent on incubation temperature in many turtles, all crocodilians, and some lepidosaurians. At hatching, identification of sexual phenotype is impossible without sacrificing the neonates. For this reason, a general method to infer sexual phenotype from incubation temperatures is needed. Temperature influences sex determination during a specific period of the embryonic development, starting when the gonad begins to form. At constant incubation temperatures, this thermosensitive period for sex determination (TSP) is located at the middle third of incubation duration (MTID). When temperature fluctuates, the position of the thermosensitive period for sex determination can be shifted from the MTID because embryo growth is affected by temperature. A method is proposed to locate the thermosensitive period for sex determination based on modelling the embryo growth, allowing its precise identification from a natural regime of temperatures. Results from natural nests and simulations show that the approximation of the thermosensitive period for sex determination to the middle third of incubation duration may create a quasi-systematic bias to lower temperatures when computing the average incubation temperature during this period and thus a male-bias for sex ratio estimate.

Nest temperatures in a loggerhead nesting beach in Turkey is more determined by sea surface than air temperature.

While climate change is now fully recognised as a reality, its impact on biodiversity is still not completely understood. To predict its impact, proxies coherent with the studied ecosystem or species are thus required. Marine turtles are threatened worldwide (though some populations are recovering) as they are particularly sensitive to temperature throughout their entire life cycle. This is especially true at the embryo stage when temperature affects both growth rates and sex determination. Nest temperature is thus of prime importance to understand the persistence of populations in the context of climate change. We analysed the nest temperature of 21 loggerheads (Caretta caretta) originating from Dalyan Beach in Turkey using day-lagged generalised mixed models with autocorrelation. Surprisingly, the selected model for nest temperature includes an effect for sea surface temperature 4-times higher than for air temperature. We also detected a very significant effect of metabolic heating during development compatible with what is already known about marine turtle nests. Our new methodology allows the prediction of marine turtle nest temperatures with good precision based on a combination of air temperature measured at beach level and sea surface temperature in front of the beach. These data are available in public databases for most of the beaches worldwide.

A model to predict the thermal reaction norm for the embryo growth rate from field data.

The incubation of eggs is strongly influenced by temperature as observed in all species studied to date. For example, incubation duration, sexual phenotype, growth, and performances in many vertebrate hatchlings are affected by incubation temperature. Yet it is very difficult to predict temperature effect based on the temperature within a field nest, as temperature varies throughout incubation. Previous works used egg incubation at constant temperatures in the laboratory to evaluate the dependency of growtProd. Type: FTPh rate on temperature. However, generating such data is time consuming and not always feasible due to logistical and legislative constraints. This paper therefore presents a methodology to extract the thermal reaction norm for the embryo growth rate directly from a time series of incubation temperatures recorded within natural nests. This methodology was successfully applied to the nests of the marine turtle Caretta caretta incubated on Dalyan Beach in Turkey, although it can also be used for any egg-laying species, with some of its limitations being discussed in the paper. Knowledge about embryo growth patterns is also important when determining the thermosensitive period for species with temperature-dependent sex determination. Indeed, in this case, sexual phenotype is sensitive to temperature only during this window of embryonic development.

First Record of Kemp's Ridley Sea Turtle, Lepidochelys kempii (Garman, 1880), in the Waters of Martinique Island (Lesser Antilles).

An adult female Lepidochelys kempii was found trapped in drifting sargassum south of Martinique; this is the southernmost report of this taxon in the Lesser Antilles arc. Determining the limits of distribution and the existence of possible sympatry between L. kempii and L. olivacea in certain subregions of the Caribbean has been hindered by numerous misidentifications. We review the available data and propose a new distribution map in the Caribbean, which can serve as a basis for future studies.

Innovative monitoring scheme adapted to remote, scattered nesting aggregation reveals a major loggerhead turtle rookery in New Caledonia, South Pacific.

The loggerhead turtle Caretta caretta is a large marine turtle with a cosmopolitan repartition in warm and temperate waters of the planet. The South Pacific subpopulation is classified as 'Critically Endangered' on the IUCN Red List, based on the estimated demographic decline. This precarious situation engages an urgent need to monitor nesting populations in order to highlight conservation priorities and to ensure their efficiency over time. New Caledonia encompasses a large number of micro and distant nesting sites, localized on coral islets widely distributed across its large lagoon. Adequately surveying nesting activities on those hard-to-reach beaches can prove to be challenging. As a result, important knowledge gaps prevail in those high-potential nesting habitats. For the first time, an innovative monitoring scheme was conducted to assess the intensity of nesting activities, considered as a proxy of the population size, on an exhaustive set of islets located in the 'Grand Lagon Sud' area. These data were analyzed using a set of statistical methods specially designed to produce phenology and nesting activity estimates using Bayesian methods. This analysis revealed that this rookery hosts a large nesting colony, with a mean annual estimate of 437 nests (95% Credible Interval = 328-582). These numbers exceed that of the previous estimated annual number of loggerhead turtle nests in New Caledonia, highlighting the exceptional nature of this area. Considering the fact that similar high-potential aggregations have been identified in other parts of New Caledonia, but failed to be comprehensively assessed to this day, we recommend carrying out this replicable monitoring scheme to other locations. It could allow a significant re-evaluation of the New Caledonian nesting population importance and, ultimately, of its prevailing responsibility for the protection of this patrimonial yet endangered species.

The response of sea turtles to vocalizations opens new perspectives to reduce their bycatch.

Incidental capture of non-target species poses a pervasive threat to many marine species, with sometimes devastating consequences for both fisheries and conservation efforts. Because of the well-known importance of vocalizations in cetaceans, acoustic deterrents have been extensively used for these species. In contrast, acoustic communication for sea turtles has been considered negligible, and this question has been largely unexplored. Addressing this challenge therefore requires a comprehensive understanding of sea turtles' responses to sensory signals. In this study, we scrutinized the avenue of auditory cues, specifically the natural sounds produced by green turtles (Chelonia mydas) in Martinique, as a potential tool to reduce bycatch. We recorded 10 sounds produced by green turtles and identified those that appear to correspond to alerts, flight or social contact between individuals. Subsequently, these turtle sounds-as well synthetic and natural (earthquake) sounds-were presented to turtles in known foraging areas to assess the behavioral response of green turtles to these sounds. Our data highlighted that the playback of sounds produced by sea turtles was associated with alert or increased the vigilance of individuals. This therefore suggests novel opportunities for using sea turtle sounds to deter them from fishing gear or other potentially harmful areas, and highlights the potential of our research to improve sea turtles populations' conservation.

New Method for Imputation of Unquantifiable Values Using Bayesian Statistics for a Mixture of Censored or Truncated Distributions: Application to Trace Elements Measured in Blood of Olive Ridley Sea Turtles from Mexico.

One recurring difficulty in ecotoxicological studies is that a substantial portion of concentrations are below the limits of detection established by analytical laboratories. This results in censored distributions in which concentrations of some samples are only known to be below a threshold. The currently available methods have several limitations because they cannot be used with complex situations (e.g., different lower and upper limits in the same dataset, mixture of distributions, truncation and censoring in a single dataset). We propose a versatile method to fit the most diverse situations using conditional likelihood and Bayesian statistics. We test the method with a fictive dataset to ensure its correct description of a known situation. Then we apply the method to a dataset comprising 25 element concentrations analyzed in the blood of nesting marine turtles. We confirm previous findings using this dataset, and we also detect an unexpected new relationship between mortality and strontium concentration.

Genome Size Variation of Chagas Disease Vectors of the Rhodniini Tribe.

The genome size of five Rhodnius species (R. milesi, R. nasutus, R. neivai, R. prolixus, and R. robustus) and two Psammolestes species (P. coroedes and P. tertius) were estimated using flow cytometry and/or k-mer distributions in genome sequences. Phylogenetic generalized linear mixed models highlighted significant genome size variations among species and between sexes, with R. prolixus showing the largest genome. In this study we provide the first data on female genome size in Triatominae. For five species, female genome size did not differ from males, except for R. robustus, where females had smaller genomes. Genome size estimations based on the k-mer distribution method were less than those estimated from flow cytometry, but both methods exhibited the same pattern of sexual differences. Further genomic studies are needed to infer whether genome size variation could be an adaptive trait in Rhodnius.

Fibropapillomatosis Prevalence and Distribution in Immature Green Turtles (Chelonia mydas) in Martinique Island (Lesser Antilles).

Fibropapillomatosis (FP) threatens the survival of green turtle (Chelonia mydas) populations at a global scale, and human activities are regularly pointed as causes of high FP prevalence. However, the association of ecological factors with the disease's severity in complex coastal systems has not been well established and requires further studies. Based on a set of 405 individuals caught over ten years, this preliminary study provides the first insight of FP in Martinique Island, which is a critical development area for immature green turtles. Our main results are: (i) 12.8% of the individuals were affected by FP, (ii) FP has different prevalence and temporal evolution between very close sites, (iii) green turtles are more frequently affected on the upper body part such as eyes (41.4%), fore flippers (21.9%), and the neck (9.4%), and (iv) high densities of individuals are observed on restricted areas. We hypothesise that turtle's aggregation enhances horizontal transmission of the disease. FP could represent a risk for immature green turtles' survival in the French West Indies, a critical development area, which replenishes the entire Atlantic population. Continuing scientific monitoring is required to identify which factors are implicated in this panzootic disease and ensure the conservation of the green turtle at an international scale.

Hatching Success Rather Than Temperature-Dependent Sex Determination as the Main Driver of Olive Ridley (Lepidochelys olivacea) Nesting Activity in the Pacific Coast of Central America.

In marine turtles, sex is determined during a precise period during incubation: males are produced at lower temperatures and females at higher temperatures, a phenomenon called temperature-dependent sex determination. Nest temperature depends on many factors, including solar radiation. Albedo is the measure of the proportion of reflected solar radiation, and in terms of sand color, black sand absorbs the most energy, while white sand reflects more solar radiation. Based on this observation, darker sand beaches with higher temperatures should produce more females. As marine turtles show a high degree of philopatry, including natal homing, dark beaches should also produce more female hatchlings that return to nest when mature. When sand color is heterogeneous in a region, we hypothesize that darker beaches would have the most nests. Nevertheless, the high incubation temperature on beaches with a low albedo may result in low hatching success. Using Google Earth images and the SWOT database of nesting olive ridleys (Lepidochelys olivacea) in the Pacific coast of Mexico and Central America, we modeled sand color and nesting activity to test the hypothesis that darker beaches host larger concentrations of females because of feminization on darker beaches and female philopatry. We found the opposite result: the lower hatching success at beaches with a lower albedo could be the main driver of nesting activity heterogeneity for olive ridleys in Central America.

Inorganic elements in live vs dead nesting olive ridley marine turtles in the Mexican Pacific: Introducing a new statistical methodology in ecotoxicology.

This study reports the largest inorganic elements database in the blood of live marine turtles (Lepidochelys olivacea), with 241 live as well as 38 dead nesting turtles sampled and analyzed for 26 inorganic elements, including essential (Al, As, B, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Se, S, V, and Zn) and non-essential elements (Cd, Li, Pb, Sr, Ti, Tl, and Hg). We compared inorganic element concentrations in live and dead olive ridleys from the arribada beach "La Escobilla" located on the Pacific coast of southeastern Mexico. The most outstanding result of our study is the higher Cd concentration in dead (mean 4.27 µg g-1 ww: min 0.01-max 81.5) compared with live animals (mean 0.14 µg g-1 ww: min 0.02-max 0.52). This population has been previously reported to have the highest Cd concentration worldwide in kidney and liver samples from marine turtles (with 150.88 ±â€¯110.99 and 82.88 ±â€¯36.65 µg g-1 ww, respectively). Other important findings of this study include the low Hg concentration along with the decrease in Pb over the years in this population. The study also uses a new statistical method - the iconography of correlations - in which all available information is used without removing individuals or variables with missing information for the whole analysis, which is a common problem in ecotoxicology. A major advantage of this method compared to other multivariate methods is that the missing information can be easily handled, because the correlations (2 variables) and partial correlations (3 variables) are estimated only with the available data using a one-at-a-time strategy.

Recent advances on the estimation of the thermal reaction norm for sex ratios.

Temperature-dependent sex determination, or TSD, is a widespread phenomenon in reptiles. The shape of the relationship between constant incubation temperature and sex ratio defines the TSD pattern. The TSD pattern is considered a life-history parameter important for conservation because the wider the range of temperatures producing both sexes, the more resilient the species is to climate change impacts. We review the different published equations and methodologies that have been used to model TSD patterns. We describe a new flexible model that allows for an asymmetrical pattern around the pivotal temperature, which is the constant temperature producing both sexes in equal proportions. We show that Metropolis-Hastings with Markov chain produced by a Monte Carlo process has many advantages compared to maximum likelihood and is preferred. Finally, we apply the models to results from incubation experiments using eggs from the marine turtle Lepidochelys olivacea originating in Northeast Indian, East Pacific, and West Atlantic Regional Management Units (RMUs) and find large differences in pivotal temperatures but not in transitional ranges of temperatures.

Temperature-dependent sex determination and global change: are some species at greater risk?

In species with temperature-dependent sex determination (TSD), global climate change may result in a strong sex ratio bias that could lead to extinction. The relationship between sex ratio and egg incubation at constant temperature in TSD species is characterized by two parameters: the pivotal temperature (P) and the transitional range of temperature that produces both sexes (TRT). Here, we show that the proportion of nests producing both sexes is positively correlated to the width of the TRT by a correlative approach from sex ratio data collected in the literature and by simulations of TSD using a mechanistic model. From our analyses, we predict that species with a larger TRT should be more likely to evolve in response to new thermal conditions, thus putting them at lower risk to global change.

Fine scale geographic residence and annual primary production drive body condition of wild immature green turtles (Chelonia mydas) in Martinique Island (Lesser Antilles).

The change of animal biometrics (body mass and body size) can reveal important information about their living environment as well as determine the survival potential and reproductive success of individuals and thus the persistence of populations. However, weighing individuals like marine turtles in the field presents important logistical difficulties. In this context, estimating body mass (BM) based on body size is a crucial issue. Furthermore, the determinants of the variability of the parameters for this relationship can provide information about the quality of the environment and the manner in which individuals exploit the available resources. This is of particular importance in young individuals where growth quality might be a determinant of adult fitness. Our study aimed to validate the use of different body measurements to estimate BM, which can be difficult to obtain in the field, and explore the determinants of the relationship between BM and size in juvenile green turtles. Juvenile green turtles were caught, measured, and weighed over 6 years (2011-2012; 2015-2018) at six bays to the west of Martinique Island (Lesser Antilles). Using different datasets from this global database, we were able to show that the BM of individuals can be predicted from body measurements with an error of less than 2%. We built several datasets including different morphological and time-location information to test the accuracy of the mass prediction. We show a yearly and north-south pattern for the relationship between BM and body measurements. The year effect for the relationship of BM and size is strongly correlated with net primary production but not with sea surface temperature or cyclonic events. We also found that if the bay locations and year effects were removed from the analysis, the mass prediction degraded slightly but was still less than 3% on average. Further investigations of the feeding habitats in Martinique turtles are still needed to better understand these effects and to link them with geographic and oceanographic conditions.

Hen's teeth with enamel cap: from dream to impossibility.

BACKGROUND: The ability to form teeth was lost in an ancestor of all modern birds, approximately 100-80 million years ago. However, experiments in chicken have revealed that the oral epithelium can respond to inductive signals from mouse mesenchyme, leading to reactivation of the odontogenic pathway. Recently, tooth germs similar to crocodile rudimentary teeth were found in a chicken mutant. These "chicken teeth" did not develop further, but the question remains whether functional teeth with enamel cap would have been obtained if the experiments had been carried out over a longer time period or if the chicken mutants had survived. The next odontogenetic step would have been tooth differentiation, involving deposition of dental proteins. RESULTS: Using bioinformatics, we assessed the fate of the four dental proteins thought to be specific to enamel (amelogenin, AMEL; ameloblastin, AMBN; enamelin, ENAM) and to dentin (dentin sialophosphoprotein, DSPP) in the chicken genome. Conservation of gene synteny in amniotes allowed definition of target DNA regions in which we searched for sequence similarity. We found the full-length chicken AMEL and the only N-terminal region of DSPP, and both are invalidated genes. AMBN and ENAM disappeared after chromosomal rearrangements occurred in the candidate region in a bird ancestor. CONCLUSION: These findings not only imply that functional teeth with enamel covering, as present in ancestral Aves, will never be obtained in birds, but they also indicate that these four protein genes were dental specific, at least in the last toothed ancestor of modern birds, a specificity which has been questioned in recent years.

Maternal transfer of trace elements in leatherback turtles (Dermochelys coriacea) of French Guiana.

In sea turtles, parental investment is limited to the nutrients and energy invested in eggs that will support embryonic development. Leatherback females have the largest clutches with the biggest eggs of the sea turtles and the highest reproductive output in reptiles. The migration between foraging sites and nesting beaches also represents high energy expenditure. The toxicokinetic of pollutants in the tissues is thus expected to vary during those periods but there is a lack of information in reptiles. Concentrations of essential (Copper, Zinc, Selenium) and non-essentials elements (Cadmium, Lead, Mercury) were determined in blood (n=78) and eggs (n=76) of 46 free-ranging leatherback females collected in French Guiana. Maternal transfer to eggs and relationships between blood and eggs concentrations during the nesting season were investigated. All trace elements were detectable in both tissues. Levels of toxic metals were lower than essential elements likely due to the high pelagic nature of leatherbacks that seems to limit exposure to toxic elements. Significant relationships between blood and egg concentrations were observed for Se and Cd. Se could have an important role in embryonic development of leatherback turtles and Cd transfer could be linked to similar carrier proteins as Se. Finally, as multiple clutches were sampled from each female, trends in trace elements were investigated along the nesting season. No change was observed in eggs but changes were recorded in blood concentrations of Cu. Cu level decreased while blood Pb levels increased through the nesting season. The high demand on the body during the breeding season seems to affect blood Cu concentrations. Calcium requirement for egg production with concomitant Pb mobilization could explain the increase in blood Pb concentrations along the nesting season.

Carapace asymmetry: A possible biomarker for metal accumulation in adult olive Ridleys marine turtles?

The Olive Ridley marine turtle (Lepidochelys olivacea) is characterized by individual morphological variability in the number and shape of scutes. The influence of pollutants on developmental instability and one of its consequences, the asymmetry of individuals, has been demonstrated in several species, especially invertebrates and some birds. However, the use of this asymmetry as a biomarker of contamination in adult individuals has never been explored. We developed an index to quantify developmental instability (DIx) based on the number and relative size of costal carapace scutes. The link between DIx and inorganic elements concentrations was explored in various tissues of stranded turtles from the Southern Mexican Pacific. The relationships between adult contamination and DIx could directly or indirectly reflect (i) the disruption of metal elimination in the adult stage dependent on embryonic perturbation and thus determining DIx, (ii) the difference in metal absorption dependent on DIx status, or (iii) DIx linked to other unknown factors.

Relationship between plasma biochemistry values and metal concentrations in nesting olive ridley sea turtles.

A hundred nesting olive ridley turtles were sampled to determine biochemical parameters (ALP, AST, ALT, creatinine, albumin, cholesterol, glucose, proteins, triglycerides, urea, and P-nitrophenyl acetate esterase activity). Esterase activity (EA) is a new biomarker very sensitive to metals. Most of the samples showed detectable levels. We also analyzed the concentration of 11 inorganic elements (As, Cd, Cr, Cu, Mn, Ni, Pb, Sr, Ti, Se, and Zn), some of them previously reported with very high concentrations in this population (especially cadmium with 82 and 150 µg g-1 ww in liver and kidney, respectively). Cadmium presented two negative relationships with creatinine and glucose. Some other understudied elements, Sr and Ti, for instance, presented five and four significant relationships with some biochemical parameters, respectively (most of them positive). EA was the parameter with most negative relationships (with Pb, Ti, As, Cr, and Se), reinforcing the results of other researchers in humans regarding the possible inhibition of EA by metals.

Molecular oxidative stress markers in olive ridley turtles (Lepidochelys olivacea) and their relation to metal concentrations in wild populations.

Due to their longevity and extensive migration areas, marine turtles are able to accumulate diverse contaminants over many years and as a consequence they represent an interesting bioindicator species for marine ecosystem pollution. Metals provoke toxicological effects in many aquatic animal species, but marine turtles have been under-investigated in this area. Thus, we have determined the presence of certain inorganic elements (As, Cd, Cu, Ni, Pb, Se and Zn) in olive ridley turtles (Lepidochelys olivacea) and related them to metallothionein (MT), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) transcription and/or enzymatic activities. Gene expression of sod, cat and gr was found to be higher in blood than liver or kidney but most of the significant relationships were found in liver, not only for gene expression but also for enzyme activities. This must be related to the role the liver has as the first filter organ. Several positive relationships of sod, cat and gr gene expression in the different tissues were found in this population, as well as very high Cd concentrations. This could mean that these turtles are adapting to the metals-production of ROS and damage through a high transcription of these antioxidants. Multiple positive relationships with GR seem to be part of its compensatory effect due to the decrease of SOD production against the high and chronic exposure to certain xenobiotics. CAT, on the other hand, seems not to be used much, and glutathione detoxification of H2O2 may be more important in this species. Finally, despite the very high Cd concentrations found in this population, no significant relationship was found in any tissue with metallothionein gene expression. These results, along with very high Cd concentrations and a negative relationship with Cu, lead us to consider some kind of disruption in mt gene expression in these turtles.