Phylogéographie des principales vipères européennes (Vipera ammodytes, V. aspis et V. berus), structuration génétique et multipaternité chez Vipera berus

Résumé: Les vipères du genre Vipera sont des serpents venimeux distribués dans la totalité du Paléarctique. Malgré cette répartition considérable, elles sont extrêmement menacées, leur déclin étant principalement dû à la destruction et à la fragmentation de leur habitat ainsi qu'à la persécution humaine. Afin d'apporter de nouveaux éléments dans le contexte de la protection de ce groupe de reptiles, nous avons utilisé durant ce travail de thèse différents marqueurs moléculaires pour étudier la structuration génétique à petite et à large échelle chez trois espèces appartenant au genre Vipera. La première étude, une phylogéographie moléculaire de la vipère ammodytes (Vipera ammodytes), a montré dans l'ensemble de l'aire de répartition une forte structuration génétique provenant d'isolements antérieures au Pléistocène. La présence d'un nombre important de clades dans le centre des Balkans suggère que cette région a fourni de nombreux refuges isolés durant les glaciations. Ces dernières ont également eu un impact considérable sur la diversité génétique au sein de la majorité des clades, suite à d'importants goulots d'étranglement durant le Pléistocène. L'étude de la phylogéographie de la vipère aspic (Vipera aspis) a montré une différenciation génétique entre les populations présentes de chaque côté des Alpes, mais également une forte structuration interne avec la mise en évidence d'un refuge en France. Cette étude est la première à établir clairement l'utilisation d'un refuge français pour un vertébré terrestre. La troisième partie de cette thèse a étudié la phylogéographie de la vipère péliade (Vipera berus), espèce cible de ce travail. En plus de la mise en évidence d'un groupe génétique inattendu (localisé dans le nord de l'Italie, le sud de l'Autriche, le nord de la Slovénie et l'extrême sud-est de la Suisse), la variabilité génétique au sein du groupe nordique (comprenant les animaux de l'entier de l'aire de répartition de l'espèce à l'exception des individus du groupe italien et les animaux provenant des Balkans) est suffisamment importante pour conclure à l'utilisation de refuges glaciaires nordiques durant les dernières glaciations, en complément des refuges habituellement décrits pour la majorité des espèces animales (soit les péninsules ibérique, italienne et balakanique). Ces résultats nous ont conduit à effectuer une étude morphologique (quatrième partie) comparant les vipères péliades du "clade italien" et du "clade nordique" décrits ci-dessus. Seules de petites différences morphologiques ont pu être mises en évidence, malgré une séparation de ces groupes estimée à plus d'un million d'années. Une étude à plus petite échelle, centrée sur le Massif jurassien et certaines populations alpines et françaises, a été entreprise afin d'estimer leur diversité génétique et d'évaluer la structuration génétique entre les populations à l'aide de marqueurs microsatellites (cinquième partie). Une importante structuration a été observée entre les populations distantes de plus de 3 kilomètres, la structuration entre les populations plus proches étant plus limitée. De plus, une diversité génétique plus faible dans les populations jurassiennes et alpines comparativement aux populations du massif central et de la côte atlantique a été constatée, probablement due à une perte de diversité génétique lors de la recolonisation post-glaciaire. La sixième étude s'est intéressée au succès reproducteur des mâles de vipères péliades en conditions naturelles. Une corrélation entre la taille des mâles et leur succès reproducteur a été relevée, les individus de plus grande taille ayant un succès reproducteur plus élevé. Le taux de multipaternité a aussi été investigué, démontrant que la proportion de pontes issues de plusieurs pères est élevée (69%) malgré la faible densité de vipères observée sur le site étudié. Finalement, aucun lien entre le nombre de pères au sein d'une ponte et la mortalité des jeunes à la naissance n'a pu être mis en évidence, contrastant avec des travaux précédents. En conclusion, l'observation de la structuration très marquée chez les vipères péliades devrait permettre d'affiner les méthodes de protection de l'espèce dans le massif jurassien. A plus large échelle, l'importante structuration génétique observée chez les vipères ammodytes, aspic et péliade résultant de l'utilisation de nombreux refuges glaciaires, complémentaires aux refuges habituellement utilisés par les espèces animales, démontre l'intérêt de l'analyse phylogéographique des reptiles pour la compréhension des phénomènes de colonisation et d' extinction des populations durant la fin du Tertiaire et le Quaternaire. La mise en évidence chez les différentes espèces de vipères étudiées de nombreux groupes génétiques distincts (ESUs) devrait conduire à des modifications de la taxonomie ainsi qu'au statut de protection de ces espèces. Abstract: The vipers of the genus Vipera are venomous snakes widespread throughout the Palaearctic regions. Despite a large distribution area, several species are extremely threatened, especially due to the destruction and fragmentation of their habitats, as well as by human persecution. In order to increase the knowledge on these species and to improve their protection, several molecular markers have been used to investigate the genetic structure on small and large scales, within three species of the genus Vipera. The first study, a molecular phylogeography of the nose-horned viper (Vipera ammodytes), showed a considerable structuring throughout the distribution area, due to isolation into refugia before the Pleistocene. A high number of clades in the centre of the Balkans suggests that this region harboured numerous isolated glacial refugia during the last glaciation. Moreover, low genetic diversity within several clades implies that most populations of nose-horned vipers have suffered bottlenecks during the Pleistocene. The study of the phylogeography of the asp viper (Vipera aspis) showed genetic differentiation between populations on each side of the Alps, as well as considerable internal genetic structure, suggesting the use of a glacial refugium in France. This study is the first to establish firmly the occurrence of a French refugia for a terrestrial vertebrate. The third part of this work involved a phylogeographic study of the adder (Vipera berus), the target species of this thesis. Three clades were revealed: a Balkan clade (corresponding to the subspecies V. b. sachalinensis), an unexpected Italian clade (limited to northern Italy, southern Austria, northern Slovenia and southeasternmost corner of Switzerland) and a Northern clade clade (including adders of the whole distribution area excepted animals from the Balkan and the Italian clades). The genetic variability within the Northern clade is sufficiently high to conclude that a northern glacial refugia during the last glaciation, in addition to those refugia already described for the main species (Iberian, Italian and Balkan peninsula). These results motivated a morphological study (part four) comparing the adders from the Italian and the Northern clades describe above. Only small morphological differences have been found, despite the split between these two clades have taken place more than 1 million years ago. A study on a local scale, focused on the Jura Mountains, on a few populations in the Alps and France was, performed to estimate the genetic diversity and the genetic structure between populations using microsatellite markers (part five). Considerable structure was observed between populations separated by more than 3 kilometres, whereas the structure between closer populations is less marked. Moreover, lower genetic diversity in the populations from Jura Mountains and Alps was noticed compared to populations from Massif Central of Atlantic coast. Such loss of genetic variation probably followed post-glacial recolonisation. The sixth study focused on the reproductive success of male adders in the wild. A positive correlation between body length and reproductive success was observed. Multiple paternity was also observed in most of clutches (69%) despite the low density of adders in the study area. Finally, no relationship was found between the number of fathers in a clutch and the survival of offspring at birth, contradicting previous studies. To conclude, the observation of a significant genetic structure in Vipera berus will enable recommendations to be made to improve protection of this species in the Jura Mountain. On a larger scale, the considerable genetic structure found within Vipera ammdoytes, V. aspis and V. berus, resulting from isolation in additional glacial refugia to those already described for other species, demonstrates the relevance of phylogeographic studies of reptiles to better understand the colonisation and disappearance during the last Tertiary and the Quaternary. The observation of several groups of evolutionary significant units (ESUs) within the three studied species might lead to a revision of the taxonomy, as well as their conservation status.

Micronucleus test on Triturus carnifex as a tool for environmental biomonitoring.

The amphibian micronucleus test has been widely used during the last 30 years to test the genotoxic properties of several chemicals and as a tool for ecogenotoxic monitoring. The vast majority of these studies were performed on peripheral blood of urodelan larvae and anuran tadpoles and to a lesser extent adults were also used. In this study, we developed protocols for measuring micronuclei in adult shed skin cells and larval gill cells of the Italian crested newt (Triturus carnifex). Amphibians were collected from ponds in two protected areas in Italy that differed in their radon content. Twenty-three adult newts and 31 larvae were captured from the radon-rich pond, while 20 adults and 27 larvae were taken from the radon-free site. The animals were brought to the laboratory and the micronucleus test was performed on peripheral blood and shed skins taken from the adults and on larval gills. Samples from the radon-rich site showed micronucleus frequencies higher than those from the radon-free site and the difference was statistically significant in gill cells (P < 0.00001). Moreover, the larval gills seem to be more sensitive than the adult tissues. This method represents an easy (and noninvasive in the case of the shed skin) application of the micronucleus assay that can be useful for environmental studies in situ. Environ. Mol. Mutagen. 56:412-417, 2015. © 2014 Wiley Periodicals, Inc.

Timeframe of speciation inferred from secondary contact zones in the European tree frog radiation (Hyla arborea group).

BACKGROUND: Hybridization between incipient species is expected to become progressively limited as their genetic divergence increases and reproductive isolation proceeds. Amphibian radiations and their secondary contact zones are useful models to infer the timeframes of speciation, but empirical data from natural systems remains extremely scarce. Here we follow this approach in the European radiation of tree frogs (Hyla arborea group). We investigated a natural hybrid zone between two lineages (Hyla arborea and Hyla orientalis) of Mio-Pliocene divergence (~5 My) for comparison with other hybrid systems from this group. RESULTS: We found concordant geographic distributions of nuclear and mitochondrial gene pools, and replicated narrow transitions (~30 km) across two independent transects, indicating an advanced state of reproductive isolation and potential local barriers to dispersal. This result parallels the situation between H. arborea and H. intermedia, which share the same amount of divergence with H. orientalis. In contrast, younger lineages show much stronger admixture at secondary contacts. CONCLUSIONS: Our findings corroborate the negative relationship between hybridizability and divergence time in European tree frogs, where 5 My are necessary to achieve almost complete reproductive isolation. Speciation seems to progress homogeneously in this radiation, and might thus be driven by gradual genome-wide changes rather than single speciation genes. However, the timescale differs greatly from that of other well-studied amphibians. General assumptions on the time necessary for speciation based on evidence from unrelated taxa may thus be unreliable. In contrast, comparative hybrid zone analyses within single radiations such as our case study are useful to appreciate the advance of speciation in space and time.

Icnología de vertebrados de las facies Buntsandstein de Mallorca

Se describen, por primera vez en Mallorca, hasta cinco tipos diferentes de icnitas y pistas de vertebrados continentales dentro de las facies cuarzoareniticas y limosas rojas de la facies Buntsandstein del Triásico inferior

Thermoregulation and microhabitat choice in the polymorphic asp viper (Vipera aspis).

In ectotherms, thermoregulation strongly depends on environmental conditions, as well as on intrinsic factors, such as skin colour. Indeed, due to its physical properties, melanin pigments allow melanistic morphs to benefit of a more efficient thermoregulation compared to non-melanistic ones. Despite thermal benefits of melanism have often been highlighted under experimental conditions, such field data remain scarce. In this study, we investigated the influence of colouration on body temperature and microhabitat choice in a montane population of colour polymorphic asp viper (Vipera aspis) characterized by a strong presence of melanism (64%). Results highlighted a difference in internal body temperature, but only within gravid females, with melanistic individuals having higher body temperatures compared to non-melanistic ones. No differences were found when considering both sexes. We also showed that melanistic and non-melanistic vipers were found in different microhabitat types, i.e. melanistic snakes used areas marked by a scarcer sun exposure and by higher vegetation cover compared to non-melanistic ones. This result has important implications. Indeed, besides providing a possible explanation for the lack of difference in body temperature (except for gravid females), it confirms that melanistic individuals can potentially use their efficient thermoregulation in order to inhabit less exposed and thermally unfavourable microhabitats.

Permissivity of insect cells to Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Recent large scale studies questioning the presence of intracellular bacteria of the Chlamydiales order in ticks and fleas revealed that arthropods, similarly to mammals, reptiles, birds or fishes, can be colonized by Chlamydia-related bacteria with a predominant representation of the Rhabdochlamydiaceae and Parachlamydiaceae families. We thus investigated the permissivity of two insect cell lines towards Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae, three bacteria representative of three distinct families within the Chlamydiales order, all documented in ticks and/or in other arthropods. We demonstrated that W. chondrophila and E. lausannensis are able to very efficiently multiply in these insect cell lines. E. lausannensis however induced a rapid cytopathic effect, which somehow restricted its replication. P. acanthamoebae was not able to grow in these cell lines even if inclusions containing a few replicating bacteria could occasionally be observed.

Systematization, description and territory of the caudal cerebral artery of the brain in broad-snouted Caiman (Caiman latirostris)

Thirty heads with the neck segment of Caiman latirostris were used. The animals were provided from a creation center called Mister Caiman, under the authorization of the Brazilian Institute of Environment and Renewable Natural Resources (Ibama). Animals were sacrificed according to the slaughtering routine of the abattoir, and the heads were sectioned at the level of the third cervical vertebra. The arterial system was washed with cold saline solution, with drainage through jugular veins. Subsequently, the system was filled with red colored latex injection. Pieces were than fixed in 20% formaldehyde, for seven days. The brains were removed, with a spinal cord segment, the duramater removed and the arteries dissected. At the level of the hypophysis, the internal carotid artery gave off a rostral branch, and a short caudal branch, continuing, naturally, as the caudal cerebral artery. This artery projected laterodorsalwards and, as it overpassed the optic tract, gave off its I (the first) central branch. Penetrated in the cerebral transverse fissure, emitting the diencephalic artery and next its II (second) central branch. Still inside the fissure, originated occipital hemispheric branches and a pineal branch. Emerged from the cerebral transverse fissure, over the occipital pole of the cerebral hemisphere. Projected rostralwards, sagital to the cerebral longitudinal fissure, as interhemispheric artery. This artery gave off medial and convex hemispheric branches to the respective surfaces of the cerebral hemispheres, anastomosed with its contralateral homologous, forming the common ethmoidal artery. This artery entered the fissure between the olfactory peduncles, emerging ventrally and dividing into ethmoidal arteries, right and left, which progressed towards the nasal cavities, vascularizing them. The territory of the caudal cerebral artery included the most caudal area of the base of the cerebral hemisphere, its convex surface, the olfactory peduncles and bulbs, the choroid plexuses and the diencephalus with its parietal organs.

Effects of fasting and refeeding on the metabolic functions of the turtle Kinosternon scorpioides (Linnaeus, 1766) raised in captivity

The metabolic responses of adult and young freshwater Kinosternon scorpioides turtles raised in captivity were evaluated. Two experiments were performed: a) blood metabolite changes caused by food deprivation, and b) liver and muscle glycogen and total lipid differences after fasting and refeeding. Blood glucose concentration of young animals was susceptible to food deprivation. In both groups this metabolite decreased after 30 days of fasting. Feeding for 15 days did not recover blood glucose. Total seric proteins were not affected by food deprivation. Fasting decreased blood urea nitrogen and the highest difference was found around 30 days. Uric acid increased in young animals after 60 days of fasting. Triacylglicerol decreased after 15 days of fasting and refeeding for 15 days recovered the pre-fasting levels. Free fatty acid plasma tended to increase around 15 days of fasting. Liver glycogen decreased at day 15 of fasting, being stable thereafter while muscle glycogen decreased at a slower rate. Total liver lipid stabilized after 30 days and then decreased 70% after 60 days of fasting. Muscle lipids remained stable throughout fasting. It could be concluded that fasting of Kinosternon scorpioides led to metabolic adaptations similar to the one reported from reptiles and fish.

Glial cells of the central nervous system of Bothrops jararaca (Reptilia, Ofidae): an ultrastructural study

Abstract Although ultrastructural characteristics of mature neuroglia in the central nervous system (CNS) are very well described in mammals, much less is known in reptiles, especially serpents. In this context, two specimens of Bothrops jararaca were euthanized for morphological analysis of CNS glial cells. Samples from telencephalon, mesencephalon and spinal cord were collected and processed for light and transmission electron microscopy investigation. Astrocytes, oligodendrocytes, microglial cells and ependymal cells, as well as myelin sheaths, presented similar ultrastructural features to those already observed in mammals and tended to maintain their general aspect all over the distinct CNS regions observed. Morphological similarities between reptilian and mammalian glia are probably linked to their evolutionary conservation throughout vertebrate phylogeny.

Participation of nitric oxide in the nucleus isthmi in CO2-drive to breathing in toads

The nucleus isthmi (NI) is a mesencephalic structure of the amphibian brain. It has been reported that NI plays an important role in integration of CO2 chemoreceptor information and glutamate is probably involved in this function. However, very little is known about the mechanisms involved. Recently, it has been shown that nitric oxide synthase (NOS) is expressed in the brain of the frog. Thus the gas nitric oxide (NO) may be involved in different functions in the brain of amphibians and may act as a neurotransmitter or neuromodulator. We tested the hypothesis that NO plays a role in CO2-drive to breathing, specifically in the NI comparing pulmonary ventilation, breathing frequency and tidal volume, after microinjecting 100 nmol/0.5 µl of L-NAME (a nonselective NO synthase inhibitor) into the NI of toads (Bufo paracnemis) exposed to normocapnia and hypercapnia. Control animals received microinjections of vehicle of the same volume. Under normocapnia no significant changes were observed between control and L-NAME-treated toads. Hypercapnia caused a significant (P<0.01) increase in ventilation only after intracerebral microinjection of L-NAME. Exposure to hypercapnia caused a significant increase in breathing frequency both in control and L-NAME-treated toads (P<0.01 for the control group and P<0.001 for the L-NAME group). The tidal volume of the L-NAME group tended to be higher than in the control group under hypercapnia, but the increase was not statistically significant. The data indicate that NO in the NI has an inhibitory effect only when the respiratory drive is high (hypercapnia), probably acting on tidal volume. The observations reported in the present investigation, together with other studies on the presence of NOS in amphibians, indicate a considerable degree of phylogenetic conservation of the NO pathway amongst vertebrates.

Critical steps in the early evolution of the isocortex: Insights from developmental biology

This article proposes a comprehensive view of the origin of the mammalian brain. We discuss i) from which region in the brain of a reptilian-like ancestor did the isocortex originate, and ii) the origin of the multilayered structure of the isocortex from a simple-layered structure like that observed in the cortex of present-day reptiles. Regarding question i there have been two alternative hypotheses, one suggesting that most or all the isocortex originated from the dorsal pallium, and the other suggesting that part of the isocortex originated from a ventral pallial component. The latter implies that a massive tangential migration of cells from the ventral pallium to the dorsal pallium takes place in isocortical development, something that has not been shown. Question ii refers to the origin of the six-layered isocortex from a primitive three-layered cortex. It is argued that the superficial isocortical layers can be considered to be an evolutionary acquisition of the mammalian brain, since no equivalent structures can be found in the reptilian brain. Furthermore, a characteristic of the isocortex is that it develops according to an inside-out neurogenetic gradient, in which late-produced cells migrate past layers of early-produced cells. It is proposed that the inside-out neurogenetic gradient was partly achieved by the activation of a signaling pathway associated with the Cdk5 kinase and its activator p35, while an extracellular protein called reelin (secreted in the marginal zone during development) may have prevented migrating cells from penetrating into the developing marginal zone (future layer I).

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Contient : I吳氏重訂本草綱目Wu shi chong ding ben cao gang mu.Traité des simples, réédition de Wu Yu chang ; préfaces, tables, avertissement, etc., livres 1 et 2 ; correspondance des médicaments et des maladies, livres 3 et 4 ; eaux, livre 5 ; feux, livre 6 ; terres, livre 7 ; métaux, livre 8 ; pierres, livres 8 à 11 ; herbes, livres 12 à 21 ; grains comestibles (gu), livres 22 à 25 ; légumes, livres 26 à 28 ; fruits, livres 29 à 33 ; arbres, livres 34 à 37 ; vêtements et ustensiles (effet et emploi), livre 38 ; reptiles, insectes, mollusques (chong), livres 39 à 42 ; animaux écailleux (lin), livres 43 et 44 ; animaux à carapace (jie), livres 45 et 46 ; volatiles, livres 47 à 49 ; quadrupèdes, livres 50 et 51 ; hommes, livre 52 ; II奇經八脉考Qi jing ba mo kao.Examen des huit vaisseaux dits qi jing (impairs) ; III瀕湖脉學Bin hu mo xue.Traité des vaisseaux, de Bin hu ; IV脉訣考證Mo jue kao zheng.Examen du Mo jue

Level and precision of behavioural thermoregulation in the bearded dragon, Pogona vitticeps : effects of hypoxia and environmental thermal quality /

Most metabolic functions are optimized within a narrow range of body temperatures, which is why thermoregulation is of great importance for the survival and overall fitness of an animal. It has been proposed that lizards will thermoregulate less precisely in low thermal quality environments, where the costs associated with thermoregulation are high; in the case of lizards, whose thermoregulation is mainly behavioural, the primary costs ofthermoregulation are those derived from locomotion. Decreasing thermoregulatory precision in costly situations is a strategy that enhances fitness by allowing lizards to be more flexible to changing environmental conditions. It allows animals to maximize the benefits of maintaining a relatively high body temperature while minimizing energy expenditure. In situations where oxygen concentration is low, the costs of thermoregulation are relatively high (i.e. in relation to the amount of oxygen available for metabolic functions). As a result, it is likely that exposures to hypoxic conditions induce a decrease in the precision of thermoregulation. This study evaluated the effects of hypoxia and low environmental thermal quality, two energetically costly conditions, on the precision and level of thermoregulation in the bearded dragon, Pogona vitticeps, in an electronic temperature-choice shuttle box. Four levels of hypoxia (1O, 7, 5 and 4% 02) were tested. Environmental thermal quality was manipulated by varying the rate of temperature change (oTa) in an electronic temperature-choice shuttle box. Higher oT a's translate into more thermally challenging environments, since under these conditions the animals are forced to move a greater number of times (and hence invest more energy in locomotion) to maintain similar temperatures than at lower oTa's. In addition, lizards were tested in an "extreme temperatures" treatment during which air temperatures of the hot and cold compartments of the shuttle box were maintained at a constant 50 and 15°C respectively. This was considered the most thermally challenging environment. The selected ambient (T a) and internal body temperatures (Tb) of bearded dragons, as well as the thermoregulatory precision (measured by the central 68% ofthe Ta and T b distribution) were evaluated. The thermoregulatory response was similar to both conditions. A significant increase in the size of the Tb range, reflecting a decrease in thermoregulatory precision, and a drop in preferred body temperature of ~2 °C, were observed at both 4% oxygen and at the environment of lowest thermal quality. The present study suggests that in energetically costly situations, such as the ones tested in this study, the bearded dragon reduces energy expenditure by decreasing preferred body temperature and minimizing locomotion, at the expense of precise behavioural thermoregulation. The close similarity of the behavioural thermoregulatory response to two very different stimuli suggests a possible common mechanism and neuronal pathway to the thermoregulatory response.