N. A. Bulakhova

Phylogeography and molecular adaptation of Siberian salamander Salamandrella keyserlingii based on mitochondrial DNA variation

We assessed the phylogeographic pattern of Siberian salamander (Salamandrella keyserlingii, Dybowski, 1870), which appear to be the most northern ectothermic, terrestrial vertebrate in Northern Eurasia, by sequence analysis of a 611-bp fragment of the mitochondrial cytochrome b gene in 159 specimens from different localities (Khabarovsk region, Sakhalin, Yakutia, Magadan region, Chukotka, Kamchatka and others). The data revealed that cytochrome b lineages of S. keyserlingii are divided into haplogroups A, B and C. Haplogroup A and B sequences are widespread in the Far East region, whereas haplogroup C consisting of several phylogenetic clusters (C1, C2, C3) is present in the all range of S. keyserlingii. Among them, cluster C3 appears to be specific for Sakhalin; most likely, it has arisen in situ in this island, with the entry time of the founder mtDNA estimated at about 0.4 MY. Analysis of cytochrome b gene variation by using different neutrality tests (including those based on K(A)/K(S)-ratio) has shown that differences between haplogroups were statistically insignificant, thus suggesting selective neutrality. However, analysis of amino acid changes allowed us to detect a signature of molecular adaptation, which might have led to appearance of adaptive cytochrome b variants in haplogroup C, originating most likely at the end of Eopleistocene (about 0.64 MY based on the haplogroup C divergence level). It seems probable that this adaptive mechanism could promote subsequent populating of new regions.

Geographic variation of life‐history traits in the sand lizard, Lacerta agilis: testing Darwin's fecundity‐advantage hypothesis

The fecundity‐advantage hypothesis (FAH) explains larger female size relative to male size as a correlated response to fecundity selection. We explored FAH by investigating geographic variation in female reproductive output and its relation to sexual size dimorphism (SSD) in Lacerta agilis, an oviparous lizard occupying a major part of temperate Eurasia. We analysed how sex‐specific body size and SSD are associated with two putative indicators of fecundity selection intensity (clutch size and the slope of the clutch size–female size relationship) and with two climatic variables throughout the species range and across two widespread evolutionary lineages. Variation within the lineages provides no support for FAH. In contrast, the divergence between the lineages is in line with FAH: the lineage with consistently female‐biased SSD (L. a. agilis) exhibits higher clutch size and steeper fecundity slope than the lineage with an inconsistent and variable SSD (L. a. exigua). L. a. agilis shows lower offspring size (egg mass, hatchling mass) and higher clutch mass relative to female mass than L. a. exigua, that is both possible ways to enhance offspring number are exerted. As the SSD difference is due to male size (smaller males in L. a. agilis), fecundity selection favouring larger females, together with viability selection for smaller size in both sexes, would explain the female‐biased SSD and reproductive characteristics of L. a. agilis. The pattern of intraspecific life‐history divergence in L. agilis is strikingly similar to that between oviparous and viviparous populations of a related species Zootoca vivipara. Evolutionary implications of this parallelism are discussed.

Variation of Reproductive Traits and Female Body Size in the Most Widely-Ranging Terrestrial Reptile: Testing the Effects of Reproductive Mode, Lineage, and Climate

The European common lizard, Zootoca vivipara, is the most widespread terrestrial reptile in the world. It occupies almost the entire Northern Eurasia and includes four viviparous and two oviparous lineages. We analysed how female snout-vent length (SVL), clutch size (CS), hatchling mass (HM), and relative clutch mass (RCM) is associated with the reproductive mode and climate throughout the species range and across the evolutionary lineages within Z. vivipara. The studied variables were scored for 1,280 females and over 3,000 hatchlings from 44 geographically distinct study samples. Across the species range, SVL of reproductive females tends to decrease in less continental climates, whereas CS corrected for female SVL and RCM tend to decrease in climates with cool summer. Both relationships are likely to indicate direct phenotypic responses to climate. For viviparous lineages, the pattern of co-variation between female SVL, CS and HM among populations is similar to that between individual females within populations. Consistent with the hypothesis that female reproductive output is constrained by her body volume, the oviparous clade with shortest retention of eggs in utero showed highest HM, the oviparous clade with longer egg retention showed lower HM, and clades with the longest egg retention (viviparous forms) had lowest HM. Viviparous populations exhibited distinctly lower HM than the other European lacertids of similar female SVL, many of them also displaying unusually high RCM. This pattern is consistent with Winkler and Wallin’s model predicting a negative evolutionary link between the total reproductive investment and allocation to individual offspring.

Inter-observer and intra-observer differences in measuring body length: a test in the common lizard, Zootoca vivipara

The snout-vent length (SVL), a conventional measure of overall body size in lizards and snakes, is used in a wide variety of ecological, evolutionary, and taxonomical studies. Trends in SVL variation are often analysed using data from several researchers (observers), but possible confounding effects due to inter-observer differences in measurement protocols have never been appropriately examined. This study reports inter-observer biases between eleven herpetologists who measured the same specimens of the Eurasian common lizards (21 adult specimens were examined by eight observers and additional 192 specimens by two observers). Intra-observer bias over time (1.5-15 months between measuring sessions) was also estimated. In the vast majority of comparisons, mean difference between the first author and another observer varied from −1.0 to +0.8 mm, or from −1.9 to +1.6% if expressed as a percent of the specimens SVL value. Some non-regular effects of sex and study sample on the studied bias were revealed, and their possible reasons are discussed. We are advising the researchers who intensively collect SVL and other morphometric data to consider testing intra-observer and inter-observer biases and to establish etalon samples available for re-examinations.

Genetic structure of Schrenck newt Salamandrella schrenckii populations by mitochondrial cytochrome b variation

The nucleotide sequence variation of the mitochondrial cytochrome b gene was studied in Schrenck newt Salamandrella schrenckii (Strauch, 1870) from populations of Primorye and the Khabarovsk region. Phylogenetic analysis revealed two haplotype clusters, southern cluster 1 and northern cluster 2, with a divergence of 3%. Analysis of the mtDNA and cytochrome b amino acid sequence variations made it possible to assume that the modern range of Schrenck newt was colonized from south Primorye northwards. In contrast to the southern cluster, the northern one demonstrated all the signs of demographic expansion (a unimodal distribution of pairwise nucleotide differences, specific results of tests for selective neutrality of mtDNA variation, and a good correspondence of genetic parameters to those expected from demographic expansion models).

Polymorphism of the Mitochondrial Cytochrome b Gene, Phylogeography, and Molecular Adaptation of the Siberian Salamander (Salamandrella keyserlingii, Amphibia, Caudata)

We performed an analysis of nucleotide variation of the mitochondrial DNA (mtDNA) cytochrome b gene in populations of the Siberian salamander (Salamandrella keyserlingii Dybowski, 1870) from Khabarovsk krai; the Jewish autonomous oblast; Amur, Sakhalin, and Magadan oblasts; Sakha; and other regions of northern Eurasia. Two major phylogenetic haplotype groups, AB and C, were found using phylogenetic analysis. The degree of intergroup divergence is 1.87%, which indicates that these groups diverged about 1.5 Ma ago. Siberian salamanders with the AB haplogroup and with four of the five subgroups of the C haplogroups are found only in the Far East, including Sakhalin; the rest of the geographical range is occupied by one of the haplogroups, C1. Analysis of the distribution of nonsynonymous and synonymous substitutions demonstrated that the cytochrome b gene variation of the Siberian salamander is neutral. However, nucleotide sequences of the AB and C groups differ significantly in the region corresponding to the cd-loop of the Q0 redox center of cytochrome b protein. In addition, the radical serine-to-leucine substitution in position 160 of the cd2-loop changes the physicochemical properties of the cd region of the C group sequences by increasing its hydrophobicity. The obtained results suggest that the changes in the sequence of the cytochrome b gene of the Siberian salamander group C are adaptive.

DNA barcoding: How many earthworm species are there in the south of West Siberia?

Earthworms are a widespread and ecologically important group of animals, which has the highest total biomass in some ecosystems and often defines the composition of soil fauna. Earthworms are known to have high cryptic genetic diversity. In this study we attempted to estimate earthworm species diversity in the south of West Siberia by DNA barcoding. This method employs short fragments of the genome to identify species, and allows one to work with specimens that cannot be identified by conventional techniques, as well as to search for new species and predict their phylogenetic affinities. As the target sequence we took a fragment of the mitochondrial cytochrome oxidase 1 (cox1) gene. The studied territory (Novosibirsk and Tomsk oblasts, Altai krai, and the Altai Republic) is known to contain 16 species and subspecies of earthworms. We analyzed 259 individuals from twelve locations and detected 27 genetic clusters. Ten of them correspond to known species (A. caliginosa, E. fetida, O. tyrtaeum, D. rubidus tenuis, D. octaedra, E. balatonica, E. sibirica, as well as three genetic lineages ofE. nordenskioldi nordenskioldi). Seventeen of the 27 clusters do not have close sequence similarity to any known earthworm species. Representatives of some of these novel clusters are morphologically similar to the Eisenia n. nordenskioldi/E. n. pallida species complex and may belong to new genetic lineages of this complex. The rest of the novel clusters probably represent new earthworm species. Therefore, we can conclude that a large portion of earthworm biodiversity in the south of West Siberia is still unexplored.

Extreme negative temperatures and body mass loss in the Siberian salamander (Salamandrella keyserlingii, amphibia, hynobiidae)

Frozen Siberian salamander safely tolerates long (45 days) stay at–35°C. Short-term (3 days) cooling down to–50°C was tolerable for 40% of adult individuals; down to–55°C, for 80% of the underyearlings. Generally, the salamanders lose about 28% of the body mass during the pre-hibernating period (before winter, at temperatures as low as 0°C) and during the process of freezing (as low as–5°C). The body weight remained constant upon further cooling (to–35°C). The frozen salamanders have no physiological mechanisms protecting from sublimation.

The Japanese tree frog ( Hyla japonica ), one of the most cold-resistant species of amphibians

The Japanese tree frog, a representative of the Manchurian fauna, is characterized by an outstanding cold resistance among the anuran amphibian species studied so far. Almost 70% of the specimens from the population inhabiting the middle Amur River withstand the cooling down to–30°C; some animals, down to–35°C. This exceeds more than twofold the cold hardiness of the wood frog (Lithobates sylvaticus LeConte, 1825), which has been considered earlier to be the most cold-resistant species. The ability of H. japonica to survive for four months in the frozen state at low temperatures makes this species independent of the temperature overwintering conditions.

The Siberian wood frog survives for months underwater without oxygen

Few of the amphibian species that occur in the Subarctic and in mountains are adapted to low sub-zero temperatures; most of these species overwinter underwater. It is believed that the distribution of the species that overwinter underwater can be limited by the low oxygen levels in waterbodies covered with ice. We show that the colonisation of the coldest areas of Northern Asia (to 71°N) by the Siberian wood frog (Rana amurensis) was facilitated by a unique adaptation, the ability to survive extreme hypoxia — and probably anoxia — in waterbodies during overwintering. The oxygen content in the overwintering waterbodies that we have studied in different parts of the range of this species fell to 0.2–0.7 mg/L without causing any large-scale mortality among the frogs. In laboratory experiments the R. amurensis survived for up to 97 days in hermetically sealed containers with water that contained less than 0.2 mg/L oxygen at temperatures of 2–3 °C, retaining the ability to respond to external stimuli. An earlier study of a broad range of frog species has shown that very few of them can survive even brief (up to 5–7 days) exposure to oxygen-free water. The revealed adaptation to prolonged extreme hypoxia is the first known case of this kind among amphibians overwintering in water.