Light and scanning microscopic studies of integument differentiation in the grass snake Natrix natrix L. (Lepidosauria,Serpentes) during embryogenesis

We analysed the differentiation of body cover in the grass snake (Natrix natrix L.) over the full length of the embryo's body at each developmental stage. Based on investigations using both light and scanning electron microscopes, we divided the embryonic development of the grass snake integument into four phases. The shape of the epidermal cells changes first on the caudal and ventral parts of the embryo, then gradually towards the rostral and dorsal areas. In stage V on the ventral side of the embryo the gastrosteges are formed from single primordia, but on the dorsal side the epidermis forms the scale primordia in stage VII. This indicates that scalation begins on the ventral body surface, and spreads dorsally. The appearance of melanocytes between the cells of the stratum germinativum in stage VII coincides with changes in embryo colouration. The first dermal melanocytes were detected in stage XI so in this stage the definitive skin pattern is formed. In the same stage the epidermis forms the first embryonic shedding complex and the periderm layer begins to detach in small, individual flakes. This process coincides with rapid growth of the embryos.     

Development of the duct system during exocrine pancreas differentiation in the grass snake Natrix natrix (Lepidosauria,Serpentes)

We analyzed the development of the pancreatic ducts in grass snake Natrix natrix L. embryos with special focus on the three‐dimensional (3D)‐structure of the duct network, ultrastructural differentiation of ducts with attention to cell types and lumen formation. Our results indicated that the system of ducts in the embryonic pancreas of the grass snake can be divided into extralobular, intralobular, and intercalated ducts, similarly as in other vertebrate species. However, the pattern of branching was different from that in other vertebrates, which was related to the specific topography of the snake's internal organs. The process of duct remodeling in Natrix embryos began when the duct walls started to change from multilayered to single‐layered and ended together with tube formation. It began in the dorsal pancreatic bud and proceeded toward the caudal direction. The lumen of pancreatic ducts differentiated by cavitation because a population of centrally located cells was cleared through cell death resembling anoikis. During embryonic development in the pancreatic duct walls of the grass snake four types of cells were present, that is, principal, endocrine, goblet, and basal cells, which is different from other vertebrate species. The principal cells were electron‐dense, contained indented nuclei with abundant heterochromatin, microvilli and cilia, and were connected by interdigitations of lateral membranes and junctional complexes. The endocrine cells were electron‐translucent and some of them included endocrine granules. The goblet cells were filled with large granules with nonhomogeneous, moderately electron‐dense material. The basal cells were small, electron‐dense, and did not reach the duct lumen.     

Ultrastructural studies of epidermis keratinization in grass snake embryos Natrix natrix L. (Lepidosauria, Serpentes) during late embryogenesis

The changes and biochemical features of the epidermis that accompany the differentiation and embryonic shedding complex formation in grass snake Natrix natrix L. embryos were studied ultrastructurally and immunocytochemically with two panels of antibodies (AE1, AE3, AE1/AE3; anti-cytokeratin, pan mixture, Lu-5 and PCK-26). All observed changes in the ultrastructure of the cells forming the epidermal layers were associated with the physiological changes that occurred in the embryonic epidermis, such as changing of the manner of nutrition and keratinization leading to the embryonic shedding complex formation. The layers that originated first (basal, outer and inner periderm and clear layer) differentiated very early and rapidly. Rapid differentiation was also observed in the layers that are very important for the functioning of the epidermis in Natrix embryos (oberhäutchen and beta-layers). They started to differentiate at developmental stage IX, and then fused and formed the embryonic shedding complex at developmental stage XI. During the embryonic development of the grass snake the smallest changes appeared in the ultrastructure of the cells in the mesos and alpha-layers because they perform supplementary functions in the process of embryonic molting. They were undifferentiated until the end of embryonic development and started to differentiate just before the first adult molting. AE1/AE3, anti-cytokeratin, pan mixture, Lu-5 and PCK-26 antibodies immunolabeled clear layer, oberhäutchen and beta-layers at the latest phase of developmental stage XI. It should be noted that these antibodies did not immunolabel the alpha-layer until hatching. The presence of alpha-keratin immunolabeling in layers that were keratinized, particularly in the oberhäutchen and beta-layers in embryos, indicated that they were not as hard as in fully mature individuals.     

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake,Natrix natrix L. (Lepidosauria,Serpentes)

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron‐dense crystalline‐like core that was polygonal in shape and an electron‐dense outer zone. The A granules had a spherical electron‐dense eccentrically located core and a moderately electron‐dense outer zone. The D granules were filled with a moderately electron‐dense non‐homogeneous content. The PP granules had a spherical electron‐dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.     

Development of endocrine pancreatic islets in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

Differentiation of the pancreatic islets in grass snake Natrix natrix embryos, was analyzed using light, transmission electron microscopy, and immuno-gold labeling. The study focuses on the origin of islets, mode of islet formation, and cell arrangement within islets. Two waves of pancreatic islet formation in grass snake embryos were described. The first wave begins just after egg laying when precursors of endocrine cells located within large cell agglomerates in the dorsal pancreatic bud differentiate. The large cell agglomerates were divided by mesenchymal cells thus forming the first islets. This mode of islet formation is described as fission. During the second wave of pancreatic islet formation which is related to the formation of the duct mantle, we observed four phases of islet formation: (a) differentiation of individual endocrine cells from the progenitor layer of duct walls (budding) and their incomplete delamination; (b) formation of two types of small groups of endocrine cells (A/D and B) in the wall of pancreatic ducts; (c) joining groups of cells emerging from neighboring ducts (fusion) and rearrangement of cells within islets; (d) differentiated pancreatic islets with characteristic arrangement of endocrine cells. Mature pancreatic islets of the grass snake contained mainly A endocrine cells. Single B and D or PP–cells were present at the periphery of the islets. This arrangement of endocrine cells within pancreatic islets of the grass snake differs from that reported from most others vertebrate species. Endocrine cells in the pancreas of grass snake embryos were also present in the walls of intralobular and intercalated ducts. At hatching, some endocrine cells were in contact with the lumen of the pancreatic ducts.     

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos.     

Seasonal changes of circulating blood parameters in the grass snake Natrix natrix natrix L.

• 1.1. Seasonal changes of circulating blood parameters of Natrix n. natrix were evident and involved both sexes to the same extent.
• 2.2. A significant decrease in red cell count, haematocrit and haemaglobin concentration in the mating period, and an increase in those parameters and mean cell volume in autumn were observed, and haemodilution during winter torpor.
• 3.3. The changes during the breeding season had probably a hormonal background; in winter, they resulted first of all from a decreased erythropoietic activity and, to a lesser extent, from an increased red blood cell breakdown rate. However, the possibility that some erythrocytes were withdrawn from the circulation cannot be excluded.
• 4.4. Winter lymphocytopenia, eosinocytopenia and neutrophilic granulocytosis in females during egg laying were expressions of changes of leucocyte formula.
• 5.5. Seasonal cyclicity was found only with respect to the white cell count in males and the eosinophile fraction in males and females.
• 6.6. Probable reasons for, and mechanisms of the changes in blood composition are discussed.

     

Novel microsatellite loci in the grass snake (Natrix natrix) and cross-amplification in the dice snake (Natrix tessellata)

Six novel polymorphic microsatellite loci are presented for the grass snake (Natrix natrix), a species with declining populations in many regions. The number of alleles per locus ranged from two to seven. Four dice snake (Natrix tessellata) microsatellites were polymorphic in the grass snake with three to four alleles. At two loci, the expected heterozygosity differed significantly from observed heterozygosity. Cross-amplification of the grass snake markers in the dice snake showed two polymorphic microsatellites with two and four alleles.     

Structure of muscle fibres and motor end plates in extraocular muscles of the grass snake, Natrix natrix L.

Six extraocular muscles of the grass snake, Natrix natrix L. together with their motor end plates were examined in the light and electron microscope, and the measurements of the diameter of muscle fibres and the area of their motor end plates were performed. Morphologically, two types of muscle fibres: tonic and red phase ones were distinguished. The former fibres, 2,3 to 14,5 mum in diameter possess single or multiple (up to five on a single fibre) "en grappe" motor end plates, without postsynaptic junctional folds. The latter fibres, 10...40 mum in diameter have single, "en plaque" motor end plates, with numerous postsynaptic junctional infoldings. The morphological features of muscle fibres and motor end plates as well as the correlation between the diameter of muscle fibres and the area of motor end plates are discussed.     

Multivariate patterns of geographic variation between the island and mainland populations of the Eastern grass snake (Natrix natrix natrix)

The substantial racial variation between the mainland and island populations of the Eastern grass snake ( Natrix natrix natrix ) is analysed by a range of multivariate methods, including principal component and canonical analysis. These techniques reveal a complex pattern of geographic variation which include sharp transition zones, gradual clines, a wide range of divergence of island populations and greater divergence per distance in the south than in the north. These patterns relate to the phylogenesis of this "incipient" species, and its post-Pleistocene range expansion as presented here and elsewhere. These racial patterns do not generally relate to physiographic features, conventional subspecies or CURRENT physical or biotic factors.     

Histochemical characterization of the mucins of the alimentary tract of the grass snake, Natrix natrix (Colubridae)

Characterization of mucins in the alimentary tract of the grass snake, Natrix natrix was performed by histochemical (PAS, Alcian Blue, pH 2.5 and pH 1.0, sialidase-Alcian Blue, pH 2.5, HID-AB pH 2.5) and lectin-histochemical (WGA, SWGA, PNA, sialidase-PNA, SBA, sialidase-SBA, DBA, sialidase-DBA, ConA, BSI-B4, AAA, UEA-1, LTA) techniques. Oesophageal lining epithelium consisted of ciliated and goblet cells, with no pluricellular glands. Mannosylated sialosulfomucins were observed. Fundic mucosa of stomach presented surface cells producing sialomucins with terminal sialic acid linked to galactose. In gastric glands neck and oxynticopeptic cells were found. Neck cells had sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose-α-(1,2)-linked residues. Cytoplasm of oxynticopeptic cells showed N-acetylgalactosamine and fucose residues. Secretion of surface cells in pyloric mucosa was similar to that of fundic ones, differing in having fucose. Goblet cells in the small intestine of N. natrix produced sulfo- and sialomucins, with sialic acid linked to galactose and N-acetylgalactosamine residues. Mucins also presented residues of mannose. Goblet cells in the large intestine presented sulfomucins only, with terminal N-acetylgalactosamine, galactose and N-acetylglucosamine. The glycosylation patterns found are probably related to protection against injuries, gastric juice and microorganisms, both pathogenic and decomposers, as well as to dietary adaptations.     

Morphology and taxonomic status of the grass snake, Natrix natrix (L.) (Reptilia, Squamata, Colubridae) on the island of Gotland, Sweden

The isolated population of grass snakes, Natrix natrix (L.), on the island of Gotland in die Baltic Sea is described and illustrated. Comparisons are made with the surrounding mainland grass snake. On the basis of morphological and ecological divergence, the conclusion is drawn that the Gotland population represents a new subspecies.     

Geographic variation in the Western grass snake (Natrix natrix helvetica) in relation to hypothesized phylogeny and conventional subspecies

Ordination analysis of the geographic variation in the Western grass snake reveals a V pattern with the northern populations at the apex and the south-eastern (Italian) populations differentiated clinally down one arm and the south western (Ibero-African) populations differentiated categorically along the other arm. There is a clear relationship between latitude and extent of differentiation; the populations in the south west being particularly highly differentiated. These phenetic patterns can be closely related to the numerically hypothesized phylogeny and can be explained by Pleistocene events. The relationship between the rejected conventional subspecies and these patterns is discussed and it is shown that some conventional subspecies are derived from arbitrarily sectioned clines and delimited by inappropriate physiographic features.     

Microevolution and taxonomy of European reptiles with particular reference to the grass snake Natrix natrix and the wall lizards Podarcis sicula and P. melisellensis

The grass snake Natrix natrix and the wall lizards Podarcis sicula and P. melisellensis are used as examples to compare the procedure and achievements of the conventional approach to naming subspecies with the use of multivariate morphometries to investigate racial differentiation.
The conventional procedure, which has changed little over the last 50 years, fails to take into account the appropriate evolutionary facts or refer to any abstracted levels of divergence necessary for subspecific recognition. Consequently, the patterns of population differentiation are obscured by the recognition of a large number of rather meaningless subspecies. There is a tendency to section clines into artificial categories and arbitrarily delimit subspecies by physiographic features.
On the other hand, the use of multivariate morphometries reveals the patterns of population differentiation which can be related to geological events and patterns of differentiation in other species and species groups. The nature of 'hybrid' zones and population differentiation enables the relative importance of evolutionary forces such as gene flow, selection and genetic drift to be discussed and provides evidence concerning speciation mechanisms. These techniques also contribute to the discussion regarding the nature of species and provide abstracted and operational criteria for taxonomic decisions.
The difference between the results of multivariate analysis and the conventional approach cannot be explained solely on the basis of choice of characters. Some of the advantages and disadvantages of using multivariate morphometries, as opposed to other modern techniques, for investigating racial affinities are discussed.     

Spatial genetic analysis of the grass snake,Natrix natrix (Squamata: Colubridae), in an intensively used agricultural landscape

Both the conversion of natural habitats to farmland and efforts at increasing the yield of existing crops contribute to a decline in biodiversity. As a consequence of land conversion, specialised species are restricted to remnants of original habitat patches, which are frequently isolated. This may lead to a genetic differentiation of the subpopulations. We used seven microsatellite markers to examine the genetic population structure of the grass snake, Natrix natrix (Linnaeus, 1758), sampled in remnants of pristine habitat embedded in an intensively used agricultural landscape in north‐western Switzerland. The study area, a former wetland, has been drained and gradually converted into an agricultural plain in the last century, reducing the pristine habitat to approximately 1% of the entire area. The grass snake feeds almost entirely on amphibians, and is therefore associated with wetlands. In Central Europe, the species shows severe decline, most probably as a result of wetland drainage and decrease of amphibian populations. We found no genetically distinct grass snake populations in the study area covering 90 km². This implies that there is an exchange of individuals between small remnants of original habitat. Thus, gene flow may prevent any genetic differentiation of subpopulations distributed over a relatively large area. Our results show that a specialized snake species can persist in an intensively used agricultural landscape, provided that suitable habitat patches are interconnected. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 51–58.     

Mitochondrial phylogeography,contact zones and taxonomy of grass snakes (Natrix natrix,N. megalocephala)

Grass snakes (Natrix natrix) represent one of the most widely distributed snake species of the Palaearctic region, ranging from the North African Maghreb region and the Iberian Peninsula through most of Europe and western Asia eastward to the region of Lake Baikal in Central Asia. Within N. natrix, up to 14 distinct subspecies are regarded as valid. In addition, some authors recognize big‐headed grass snakes from western Transcaucasia as a distinct species, N. megalocephala. Based on phylogenetic analyses of a 1984‐bp‐long alignment of mtDNA sequences (ND4+tRNAs, cyt b) of 410 grass snakes, a nearly range‐wide phylogeography is presented for both species. Within N. natrix, 16 terminal mitochondrial clades were identified, most of which conflict with morphologically defined subspecies. These 16 clades correspond to three more inclusive clades from (i) the Iberian Peninsula plus North Africa, (ii) East Europe and Asia and (iii) West Europe including Corso‐Sardinia, the Apennine Peninsula and Sicily. Hypotheses regarding glacial refugia and postglacial range expansions are presented. Refugia were most likely located in each of the southern European peninsulas, Corso‐Sardinia, North Africa, Anatolia and the neighbouring Near and Middle East, where the greatest extant genetic diversity occurs. Multiple distinct microrefugia are inferred for continental Italy plus Sicily, the Balkan Peninsula, Anatolia and the Near and Middle East. Holocene range expansions led to the colonization of more northerly regions and the formation of secondary contact zones. Western Europe was invaded from a refuge within southern France, while Central Europe was reached by two distinct range expansions from the Balkan Peninsula. In Central Europe, there are two contact zones of three distinct mitochondrial clades, and one of these contact zones was theretofore completely unknown. Another contact zone is hypothesized for Eastern Europe, which was colonized, like north‐western Asia, from the Caucasus region. Further contact zones were identified for southern Italy, the Balkans and Transcaucasia. In agreement with previous studies using morphological characters and allozymes, there is no evidence for the distinctiveness of N. megalocephala. Therefore, N. megalocephala is synonymized with N. natrix.