It takes two to tango – Phylogeography, taxonomy and hybridization in grass snakes and dice snakes (Serpentes: Natricidae: Natrix natrix, N. tessellata)

Using two mitochondrial DNA fragments and 13 microsatellite loci, we examined the phylogeographic structure and taxonomy of two codistributed snake species (Natrix natrix, N. tessellata) in their eastern distribution area, with a focus on Turkey. We found evidence for frequent interspecific hybridization, previously thought to be extremely rare, and for backcrosses. This underscores that closely related sympatric species should be studied together because otherwise the signal of hybridization will be missed. Furthermore, the phylogeographic patterns of the two species show many parallels, suggestive of a shared biogeographic history. In general, the phylogeographies follow the paradigm of southern richness to northern purity, but the dice snake has some additional lineages in the south and east in regions where grass snakes do not occur. For both species, the Balkan Peninsula and the Caucasus region served as glacial refugia, with several mitochondrial lineages occurring in close proximity. Our results show that the mitochondrial divergences in both species match nuclear genomic differentiation. Yet, in the former glacial refugia of grass snakes there are fewer nuclear clusters than mitochondrial lineages, suggesting that Holocene range expansions transformed the glacial hotspots in melting pots where only the mitochondrial lineages persisted, bearing witness of former diversity. On the other hand, the deep mitochondrial divergences in N. tessellata across its entire range indicate that more than one species could be involved, even though lacking microsatellite data outside of Turkey prevent firm conclusions. On the contrary, our microsatellite and mitochondrial data corroborate that N. megalocephala is invalid and not differentiated from sympatric populations of N. natrix. For Cypriot grass snakes, our analyses yielded conflicting results. A critical assessment of the available evidence suggests that N. natrix is a genetically impoverished recent invader on Cyprus and taxonomically not distinct from a subspecies also occurring in western Anatolia and the southern Balkans. Based on combined mitochondrial and nuclear genomic evidence we propose that for grass snakes the following subspecies should be recognized in our study region: (1) Natrix natrix vulgaris Laurenti, 1768, southeastern Central Europe and northern Balkans; (2) Natrix natrix moreoticus (Bedriaga, 1882), southern Balkans, western Anatolia, and Cyprus; and (3) Natrix natrix scutata (Pallas, 1771), eastern Anatolia, Caucasus region, Iran, northeastern distribution range (from eastern Poland and Finland to Kazakhstan and the Lake Baikal region). Thus, Natrix natrix cypriaca (Hecht, 1930) becomes a junior synonym of N. n. moreoticus and Natrix natrix persa (Pallas, 1814) becomes a junior synonym of N. n. scutata. Due to insufficient material, we could not resolve the status of Natrix natrix syriaca (Hecht, 1930) from the Gulf of İskenderun, southeastern Turkey.

Decline in body size and female fraction in the grass snake (Natrix natrix, Linnaeus 1758) population after 40 years (Southern Poland)

Depletion of free-living populations is often associated with changes in fitness-related traits, e.g., body size. Ongoing decrease in body size has been reported in most vertebrates, but reptiles remain understudied. Moreover, sexual size dimorphism, commonly observed in reptiles, indicates that environmental pressures on body size may appear sex-specific. This can also result in shifts in sex ratio, an aspect even less studied. We investigated body size and sex ratio in population of grass snake (Natrix natrix) surveyed over 40 years ago in comparison with the current state. We found that both sexes express similar magnitude in body size decline. The current sex ratio does not deviate from 1:1, while in the past, females outnumbered males. The observed changes are likely an outcome of several non-mutually exclusive factors. In the studied area, an increase in road traffic and human presence and a drop in prey availability have been documented. Both factors may exert higher pressure on larger individuals, particularly females, due to their high costs of reproduction. It is recorded here that increase in ambient temperatures and summer duration may additionally enhance the mortality risk and resource requirements. Shifts in body size and sex ratio can catalyze further declines in abundance and reproductive potential of the population.

Leukocyte blood composition of Naturix natrix (Serpentes: Colubridae) in the Mordovian State Nature Reserve (Russia)

The state of the grass snake (Natrix natrix) population in the Mordovian State Nature Reserve was assessed using the hematological approach. The leukocyte blood count was estimated together with calculation of the associated leukocyte indices (neutrophil:lymphocyte (N:L), eosinophil:lymphocyte (E:L), heterophil:eosinophil (H/E) and heterophil:lymphocyte (H:L) ratios). The surface area of leukocytes (heterophils, basophils, eosinophils, azurophils, monocytes, and lymphocytes) was measured. The leukocyte blood composition of Natrix natrix was characterized by the predominance of agranulocytes, and the granulocyte fraction was 31– 37% (u = 1.99, p = 0.04). The population pattern of the leukocyte blood composition of Natrix natrix was as follows: heterophils – 8.07±0.6%, basophils – 12.33±0.95%, eosinophils – 8.33±0.65%, azurophils – 5.25±0.53%, monocytes – 9.77±0.42%, and lymphocytes – 56.22±1.7%. According to the averaged area, the leukocytes were arranged in the following order: eosinophils – monocytes – azurophils, basophils – heterophils – lymphocytes. The diameter of the largest cells (eosinophils) was (17.5±2.42) μm. The smallest lymphocytes had diameters within (7.22±1.21) μm. The absence of any differences in the quantitative content of all types of granulocytes and agranulocytes in the blood of the grass snake from different parts of the reserve indicated a comparable level of impact and the identity of the physiological mechanisms of adaptation that occur in the body of animals in protected habitat conditions. Most of the leukocyte integral indices revealed the same type of variability in the parameters of the white blood cell system of the Natrix natrix in three areas of the reserve, with the exception of an increased value of the heterophiles/lymphocytes ratio in the sample from the village Pushta. The increase in this indicator made it possible to draw a conclusion about the stress effect on the individuals of this sample. The principal component method was used to differentiate the samples of the grass snake with a more complete quantitative description of the leukocyte composition of the objects of study; the results obtained are presented in a visual, integrated and generalized form. The use of the principal component method made it possible to combine samples of Natrix natrix with similar indicators of leukocyte composition, as well as to identify a population of Natrix natrix that differs in its indicators in the urbanized territory of the Samara region. The blood pattern and the dynamics of blood leukocyte indices reflected the active response of the Natrix natrix body to the complex of environmental factors of the habitat on the territory of the Mordovian State Nature Reserve.

Decline in Body Size and Female Fraction in the Grass Snake (Natrix Natrix, Linnaeus 1758) Population After 40 Years (Southern Poland)

Depletion of free-living populations is often associated with changes in fitness-related traits, e.g. body size. Ongoing decrease in body size has been reported in most vertebrates, but reptiles remain understudied. Moreover, sexual size dimorphism, commonly observed in reptiles, indicates that environmental pressures on body size may appear sex-specific. This can also result in shifts in sex ratio, an aspect even less studied. We investigated body size and sex ratio in population of grass snake (Natrix natrix) surveyed over 40 years ago in comparison with the current state. We found that both sexes express similar magnitude in body size decline. The current sex ratio does not deviate from 1:1, while in the past females outnumbered males. The observed changes are likely an outcome of several non-mutually exclusive factors. In the studied area an increase in road traffic, human presence and a drop in prey availability has been documented. Both factors may exert higher pressure on larger individuals, particularly females, due to their high costs of reproduction. Recorded here increase in ambient temperatures and summer duration, may additionally enhance the mortality risk and resource requirements. Shifts in body size and sex ratio can catalyze further declines in abundance and reproductive potential of the population.

Different shades of snake: Peculiar coloration in an urban population of the Grass Snake, Natrix natrix (Linnaeus, 1758)

Although color variation in Natrix species is relatively well documented, information on a local scale can still be considered lacking. Here we report on melanistic Natrix natrix individuals in an urban population from Belgrade, along with the occurrence of other interesting color morphs simultaneously. The reasons behind the occurrence of such peculiar coloration are also discussed. At the same time, this is the first documented occurrence of melanism in N. natrix in Serbia.

Obtaining Oviparous Grass Snake, Natrix natrix (Serpentes, Colubridae), embryos at Early Developmental Stages by Caesarean Section

There is a specific feature in the developmental biology of oviparous snakes: embryos in the eggs, which were just laid, have already undergone significant development. This fact makes it significantly complicated to obtain data on organs’ development at early stages of embryogenesis. In addition, the fertilization time and the duration of snake pregnancy in the wild are unknown. In order to obtain the embryos of an oviparous grass snake Natrix natrix (Linnaeus, 1758) at successive developmental stages with minimal harm to gravid females we used caesarean section. The past known experience of performing caesarean section in snakes and anesthesia in reptiles were used. All the embryos were taken from the upper oviduct of a female simultaneously; in this way we eliminated the influence of medications on embryos’ development. The described method is valuable when it is necessary to obtain snake embryos and to preserve the life of the female and, possibly,its reproductive ability.