Canadian Journal of Zoology

Overwintering and cold tolerance in the moor ( arvalis, Anura) across its range

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2019-0179.R4

Manuscript Type: Article

Date Submitted by the 08-Aug-2020 Author:

Complete List of Authors: Berman, Daniil; Institute of Biological Problems of the North FEB RAS, FEB RAS Bulakhova, Nina; Institute of Biological Problems of the North FEB RAS, FEB RAS; Research Institute of Biology and Biophysics, Tomsk State University Draft Meshcheryakova, Ekaterina; Institute of Biological Problems of the North FEB RAS, FEB RAS Shekhovtsov, Sergey; Institute of Cytology and Genetics SB RAS; Institute of Biological Problems of the North FEB RAS, FEB RAS

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Rana arvalis, , geographic range, geographical variation of cold Keyword: tolerance, overwintering temperature conditions, lower lethal temperature, supercooling point

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Overwintering and cold tolerance in the moor frog (Rana arvalis, Anura)

across its range

D.I. Berman, N.A. Bulakhova, E.N. Meshcheryakova and S.V. Shekhovtsov

D.I. Berman. Institute of Biological Problems of the North FEB RAS, Portovaya St. 18,

685000 Magadan, (e-mail: [email protected]).

N.A. Bulakhova. Institute of Biological Problems of the North FEB RAS, Portovaya St. 18,

685000 Magadan, Russia; Research Institute of Biology and Biophysics, Tomsk State

University, Pr. Lenina 36, 634050 Tomsk, Russia (e-mail: [email protected]).

E.N. Meshcheryakova. Institute of Biological Problems of the North FEB RAS, Portovaya

St. 18, 685000 Magadan, Russia (e-mail:Draft [email protected]).

S.V. Shekhovtsov. Institute of Cytology and Genetics SB RAS, Pr. Lavrentieva 10, 630090

Novosibirsk, Russia; Institute of Biological Problems of the North FEB RAS, Portovaya St.

18, 685000 Magadan, Russia (e-mail: [email protected]).

Corresponding author: Nina A. Bulakhova (e-mail: [email protected]).

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D.I. Berman, N.A. Bulakhova, E.N. Meshcheryakova and S.V. Shekhovtsov

Overwintering and cold tolerance in the moor frog (Rana arvalis, Anura) across its range

Abstract: Only two species of boreal Holarctic (genus Rana) can survive freezing and overwinter on land; they are found in the subarctic and cold regions of (Rana sylvatica LeConte, 1825) and Eurasia (Rana arvalis Nilsson, 1842) and are an example of an unusual adaptive strategy of overwintering. Freeze tolerance (down to -16°C) of R. sylvatica has been thoroughly studied; however, little is known about cold resistance of R. arvalis in cold regions. We found that R. arvalis from European Russia and from West tolerate freezing down to -12 or -16°C, while frogs from Danish population survived freezing only to - 4°C (Voituron et al. 2009b). All of Draft these populations, according to mtDNA markers, are closely related. We suggest that the observed differences in cold tolerance (-4°C vs -12 or -

16°C) could be caused either by adaptations to climatic factors or by differences in experimental protocols. The northeastern boundary of the geographic range of R. arvalis in

Yakutia coincides with the transitional area between discontinuous and continuous permafrost; beyond this area, winter soil temperature sharply declines. The lower lethal temperature and overwintering of R. arvalis in Siberia are similar to those of the

North American R. sylvatica.

Key words: Rana arvalis, moor frog, geographic range, geographic variation of cold tolerance, overwintering temperature conditions, supercooling point, lower lethal temperature.

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Introduction

Only a few species inhabit cold northern and inner continental regions of

Eurasia and North America. They possess amazing adaptive abilities to survive under

extremely low winter temperatures. These adaptations have not been adequately studied yet,

although the available data on the Siberian salamander (Salamandrella keyserlingii

Dybowski, 1870), the (Rana sylvatica LeConte, 1825), and the Japanese tree frog

(Hyla japonica Günther, 1859) are of significant interest (Berman et al. 1984, 2016b, 2016c;

Storey and Storey 2004; Berman and Meshcheryakova 2012; Costanzo et al. 2013; Larson et

al. 2014).

In this context, cold tolerance of the moor frog (Rana arvalis Nilsson, 1842) deserves attention. This species is widespread inDraft the Palearctic, from France to southwestern Yakutia. It is found in the North, up to the zonal in Fennoscandia, in European Russia, and in

Siberia; to the south as far as northwestern China and Transbaikalia (Bannikov et al. 1977;

Borkin et al. 1984). Of all species of the genus Rana, only R. arvalis and R. sylvatica can

survive freezing. The Alaskan R. sylvatica tolerate exposure down to -16°C (Costanzo et al.

2013) and probably even to -18°C (Larson et al. 2014); this is the highest cold tolerance so far

observed within the genus Rana. In contrast to R. sylvatica, the studied populations of Rana

arvalis from Denmark could withstand only -4°C for 3-4 days (Voituron et al. 2009b).

Winter temperatures in northern are much lower than in Denmark. For example,

in the city of Olekminsk (southwestern Yakutia), the average January air temperature is below

-32°C with the absolute minimum of -59°C (Izyumenko 1989). Two hypotheses can be

suggested on how the moor frog manages to overwinter. The first is overwintering in water, a

highly effective way to avoid very low negative temperatures. Overwintering in water was

mentioned for moor frogs in Central Russia, northern West Siberia, and Yakutia (Krasavtsev

1939; Belimov and Sedalishchev 1979; Matkovskiy and Starikov 2011). However, Krasavtsev

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(1939) reported that overwintering in water is extremely rare for this species (only three findings with one or two individuals in each). Two other sources did not list any specific records. If R. arvalis overwinter in water, cold tolerance can be limited just to a few degrees below zero as known for frog species overwintering in water (Lotshaw 1977; Costanzo et al.

1993; Voituron et al. 2005, 2009a; Berman et al. 2017). The second option is overwintering on land, which requires much higher cold tolerance, as demonstrated for the wood frog (R. sylvatica) in . Indeed, the moor frog is known to overwinter on land almost everywhere across its range, in contrast to other Rana species of the northern Palearctic. It usually overwinters in pits with a thick layer of leaf litter, between tree and stump roots, etc.

(Krasavtsev 1939; Terentyev 1950). Therefore, in order to survive in Siberia, especially in its northeast, R. arvalis must have a muchDraft higher cold tolerance than that found in the Denmark population.

It is unclear how exactly the moor frog manages to survive in regions with harsh winter. It could be that cold tolerance of this species is geographically variable, similar to that observed for wood frogs (R. sylvatica). In contrast to northern populations (Fairbanks,

Alaska), wood frogs from southern populations have much lower cold tolerance; they survived at -5°C for 14 days in Pennsylvania, USA (Layne 1995) and at -6°C for 11 days in

Ottawa, (Storey and Storey 1984). In the same way, higher cold tolerance would be expected in moor frogs (R. arvalis) from Siberia than in those from Denmark.

However, we have not found any studies of the moor frog that would specifically address temperature regimes in the overwintering sites on land for this species. To search for such overwintering sites is extremely time-consuming and does not guarantee success. The only way to clarify this issue is to conduct experiments in order to determine a potential cold tolerance of the moor frogs from the regions with a harsh winter. Since the mechanisms of

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geographic variation of cold tolerance are yet unknown, one cannot exclude the possibility

that it is genetically determined (Larson et al. 2014).

The goal of this study was to determine cold tolerance of moor frogs from the regions

with strongly differing winter climates: European Russia and Siberia. We also conducted

phylogenetic (mtDNA marker) analysis of all studied moor frog populations in order to

determine their genetic similarity.

Materials and Methods

Animals

Live moor frogs were collected from two populations from the south of West Siberia

(near Karasuk town, Novosibirsk Оblast, 53°N, 78°E, n = 60; near Podgornoye village, Tomsk Оblast, 57°N, 82°E, n = 28), Draftas well as from one population from European Russia (near Chernogolovka town, Moscow Oblast, 56°N, 38°E, n = 47) (Fig. 1). The maximum cold

tolerance could be expected in the easternmost part of the species’ range (southwestern

Yakutia); however, collecting there was not possible since R. arvalis is listed as a rare species

in the local Red Book (2010).

Adult frogs from West Siberia (average mass 13.1±0.3 g) were collected in mid-

August – early September; in Chernogolovka (14±0.6 g), in September. In order to study their

cold tolerance, the were delivered to the Laboratory of Biocenology of the Institute of

Biological Problems of the North in Magadan in thermal containers.

Compliance with ethical standards

All procedures were carried out in accordance with the International Guiding

Principles for Biomedical Research Involving Animals (Council for International

Organizations of Medical Sciences, 1985). Rearing and experimental protocols (№ RAR-10)

were approved by the Bioethics Committee of the Institute of Biological Problems of the

North.

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Temperature measurements in the wintering sites

Temperature measurements in the wintering sites of moor frogs were performed from autumn 2009 to spring 2010 near Podgornoye (center of species' range, continental climate) and near Olekminsk (northeastern periphery of the range, highly continental climate).

Temperature loggers (iButton DS1922L, Maxim Integrated Products, San Jose, USA; temperature accuracy of ±0.5°C from -10 to +65°C) were placed 2 cm deep in soil near spawning water bodies (0.5–30 m from water edge). Cumulative negative soil temperatures were calculated as a sum of temperatures (°С×h) starting from the moment of soil freezing in the fall (0°С) to its thawing in the spring (0°С), according to the data from the loggers.

Information on air temperature, date of snow cover formation and its depth were obtained from the https://rp5.ru websiteDraft for the Podgornoye, Olekminsk, Karasuk, and Chernogolovka weather stations located 1.5–30 km away from the sampling sites or temperature measurement points.

Animal rearing and acclimation

Three to five frogs were placed in 1L transparent plastic containers filled with green moss (humidity about 90%). To check for potential temperature gradients, we placed in each container a calibrated temperature logger (iButton DS1922L, Scientific and technical laboratory "ElIn", Moscow, Russia; temperature accuracy within the 65 to -10°C range, ±

0.5°C; within -10 to -40°C – ±0.6°С).

The experiment used animals from three geographically remote localities with different climates. To equalize experimental conditions, an averaged regime of acclimation was selected reflecting cooling regime in the fall, observed in frogs’ natural . The acclimation of frogs within 5 to -1°C range was implemented on a 57-day scheme: 14 days at

5°С, 3 days at 3°С, 20 days at 1°C, and 20 days at -1°С. At 5°C, frogs were kept in cooling thermostats (TSO–1/80 SPU); at lower temperatures (3, 1, and -1°С), animals were kept in a

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WT 64/75 test chamber (Weiss Umwelttechnik GmbH, Reiskirchen-Lindenstruth, Germany).

Down to -1°С, frog condition was assessed visually.

Determining the supercooling point (SCP)

Supercooling point is the lowest temperature to which the organism can be cooled,

below its freezing/melting point, before ice spontaneously forms. Crystallization is

accompanied by a surge of heat output and a peak on the temperature diagram. Thus, an

is supercooled from its freezing/melting point to the supercooling point. An animal’s

survival after exposure to the temperatures below SCP means that it can withstand freezing.

The melting temperature for R. arvalis is unknown but it can be assumed to be close of that

for R. sylvatica, i.e. about -0.46°C (Costanzo et al., 2013). SCP was measured in a freezingDraft chamber, modified from an Okean-3 KSh-180 compression refrigerator. The evaporator was supplied with additional thermal isolation to

avoid temperature gradients inside the chamber. The chamber was also fitted with a regulator

that allowed to control the rate of temperature change (from 0.1°C per hour to several °C per

hour).

The temperature was measured with manganin-constantan thermocouples (wire

diameter, 0.12 mm). The thermocouple signal was converted using an analog-digital board

(ADC LA-TK5) via a DC voltage amplifier and recorded in a computer.

To determine SCP, the frogs (10 individuals from Karasuk and Chernogolovka

populations each) were acclimated from 5 to 1°C (the scheme is described in the previous

section) and placed into individual 10×8 cm Ziplock bags, perforated for ventilation. Through

a perforation hole, a thermocouple was attached between animal’s forelimbs using paper

adhesive tape. The contact of the thermocouple with frog’s skin was constantly monitored. A

bag containing the frog and the attached thermocouple was placed into a 5×8×8 cm copper

box with thick walls (2 mm) to provide uniform cooling. The box was transferred to the

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freezing chamber. As SCP was measured, the temperature was decreased by 0.1°C per hour, i.e., 2.4°С per day, which is the standard rate for such experiments (Costanzo et al. 1991).

Determining lower lethal temperatures

After acclimation, the survival rate was determined at several temperature points ranging from -2 to -16°C (Table 1). The frogs were cooled in the same 1L plastic containers filled with moss (3-5 frogs per container) as used for acclimation, placed in a WT 64/75 test chamber. Animal freezing was triggered by the ice crystals that form naturally on their wet skin as the wet moss is freezing; these ice crystals serve as multiple nuclei of contact inoculation. The chamber was cooled at a rate of 0.05°С per hour (1.2°С per day), following the experiments of Costanzo et al. (2013) for the wood frog. This rate was, in fact, far below than the maximal cooling rate recordedDraft in the upper centimeters of soil in the natural sites where frogs overwintered (Fig. 2). Frogs were incubated for two days at each of the indicated temperatures in Table 1, except -16°C where frogs from for different experimental series were left for a duration from 6 h to 16 days (exposure for 6 h at -16°C was chosen to directly compare our results to those for the wood frog (Costanzo et al. 2013)). After exposure to an experimental temperature, frogs were warmed to 5°С at the rate of 0.5°С per day, with 24 h pauses at -1, 0, and 1°C. When frogs were warmed to 1°C, they regained breathing movements and orientation behavior (i.e., turned over when placed on their back). As a rule, when these reactions did not return at 1°C, frogs failed to survive at higher temperatures; they were observed until obvious symptoms of death (sinking of eyes into the sockets, necrotic spots, etc.). Those frogs that completely regained typical behavioral reactions were considered as survived; they were kept in the laboratory for two weeks to check for delayed damage.

Several containers with frogs were cooled simultaneously but survival of the animals at the temperature points listed in Table 1, was checked only in one or two of them. Further cooling of the remaining containers started only when results from the previous step (i.e.,

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survival or death) were obtained, thus the time of exposure at some of the intermediate

temperature points increased (Table 2).

We also performed a second freezing of frogs from Siberian populations to compare

with the data from wood frogs (Larson et al. 2014). Six frogs that survived freezing to -8°C

were warmed to 1°C, and then frozen again to -8, -9 and -12°С; at each point frogs were kept

for 2 days. The rate of cooling was at 0.05° per hour (1.2°С per day), as in the first

experiment.

Cumulative negative temperatures were calculated as a sum of temperatures (°С×h)

accumulated by moor frogs Rana arvalis during each experiment (from 0°С to lower lethal

temperature and returning from lower lethal temperature to 0°С). Statistical analysis was performedDraft by standard methods using Statistica 10. All mean values are given with ± standard error. Correlation between survival of frogs and the value of

lower lethal temperatures was assessed by Spearman's rank correlation coefficient (rs).

Comparison of SCP values of frog from Karasuk and Chernogolovka populations was done

by Mann–Whitney U-test. Level of significance was set at 푃 < 0.05.

Genetic analysis

Genetic analysis was performed for nine individuals from Chernogolovka and seven

each from Podgornoye and Karasuk. DNA was extracted from amputated frog fingers using

kits and columns produced by Biosilica (Novosibirsk, Russia) according to manufacturer's

instructions. A fragment of the mitochondrial cytochrome b (cytb) gene was amplified using

specific PCR primers Rarv-cytb-F (5'-CATTGATCTCCCAACTCCTTC-3') and Rarv-cytb-R

(5'-AGGGTCTCCAAGCAGGTTAG-3') designed based on R. arvalis sequences from

GenBank. PCR reactions contained 1.5 mM MgCl2, 65 mM TrisHCl (pH 8.8), 16 mM

(NH4)2SO4, 0.05% Tween-20, 0.2 mM of each dNTP, 0.3 mM primers, and 1 unit of

recombinant Taq polymerase (SibEnzyme, Russia). We used the following PCR profile:

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initial denaturation for 2 min at 94°C; 37 cycles with denaturation for 20 s at 94°C, primer annealing for 20 s at 56°C, template synthesis for 1 min at 72°C; final synthesis for 10 min at

72°C. Sanger sequencing was performed in the SB RAS Genomics Core Facility

(Novosibirsk).

Sequences were processed using Chromas Lite v.2.0 (Technelysium Pty Ltd, South

Brisbane, Australia) and aligned using ClustalO (Goujon et al. 2010). The obtained sequences were compared to those available in GenBank using BLAST (www.ncbi.nlm.nih.gov/blast).

Haplotype network was constructed by Network v.5.0.0.3 (Fluxus Technology Ltd). Pairwise

Fst values were calculated using Arlequin v.3.0 (Excoffier et al. 2005).

For microsatellite analysis, we used primers taken or modified from Richter-Boix et al. (2011): WRA160-Fw (5'-TCAAGCCCTGCATTACGGTGTG-3';Draft labelled by 5'-FAM) and WRA160-Rv (5'-AGCTGCTGCAGGGAATGTTTCAG-3'); RTCA-Fw (5'-

GCCAGGGTATGTAAACTTATGAGC-3'; 5'-FAM) and RTCA-Rv (5'-

GTTTCAAATGTATATTATTGGTGCAATG-3'). PCR mixture contained the same components as described above; the following PCR profile was used: initial denaturation for 2 min at 94°C; 37 cycles with denaturation for 20 s at 94°C, primer annealing for 20 s at 58°C, template synthesis for 30 s at 72°C; final synthesis for 10 min at 72°C. Capillary electrophoresis was done on a Nanophore 05 analyzer (Sintol, Moscow, Russia), and microsatellite profiling was performed using Fragment Analysis v.73. Differences among populations were assessed using Arlequin v.3.0 (Excoffier et al. 2005).

Results

Temperature measurements in the wintering sites

Temperature measurements were performed at one of the sampled localities

(Podgornoye), as well as at Olekminsk, which is located at the easternmost boundary of the

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species’ range. In both localities, once the soil froze in the fall, it never thawed even at the

depth of 2 cm (Fig. 2).

While the minimum observed air temperatures were close in Podgornoye and

Olekminsk (-42.8 and -48.6°C), different snow depth (69 and 28 cm, respectively) caused

significant differences in minimum temperatures in the upper 2 cm of soil (-5.6 and -16.5°C)

(Figs. 2, 3a, b). The sums of negative soil temperatures at 2 cm depth for the period of late

October – late April ranged from -3189 to -12751°С×h in Podgornoye, and from -14036 to -

48076°С×h in Olekminsk.

We did not measure soil temperature using loggers in the two other sampling sites

(Karasuk and Chernogolovka); however, it can be assessed using extraction thermometer data from the relevant weather stations (TableDraft 3). Comparison of two adjacent West Siberian sampling sites, Podgornoye and Karasuk

(Figs. 3b, c), demonstrated that soil temperatures at 20 cm differ by 13°C (Table 3), with

harsher conditions observed for the southernmost studied population.

Frogs behavior during cooling

The behavior of frogs during cooling to low positive temperatures (1–0°С) was

stereotypic and uniform: they were hiding under the moss at the bottom of containers and

became passive but leapt away if disturbed. At slightly negative temperatures, frogs adopted

the typical overwintering posture in the frozen moss (pressed to the substrate with fore- and

hindlimbs clinging to the body); their skin color did not change. Ice crystals were present on

both moss and frog skin; however, no inoculation that would lead to freezing was observed. If

frogs were touched at these temperatures, their movement was limited to slight changes in

body and limb position.

Temperature of maximum supercooling

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Average SCP values for both Karasuk and Chernogolovka populations were -

2.7±0.2°C (varying between -1.9 and -3.3 or -3.6°C) with no statistically significant differences (U-test, p=0.9).

Strategy of cold tolerance and lower lethal temperatures

All frogs from Chernogolovka, which were cooled to -4°C (i.e, below the SCP), survived. This suggests that the moor frogs from the studied population, as well as those from the Danish population (Voituron et al. 2009b), could withstand freezing as their strategy of cold tolerance.

After freezing, the frogs’ skin became darker, limb muscles became rigid, eyes became dull, and ice was detectable by touch under the skin of the animals. Similar changes were observed in the wood frogs upon Draftfreezing (Costanzo et al. 2013). High survival rate for the frogs from Siberian populations was observed down to -8°C, with no mortality. However, already at -10°C mortality for both Siberian populations

(Podgornoye and Karasuk) reached 13–25%; at -12°C, it was 43–50%. At -14 to -16°C, only frogs from Karasuk survived. Same survival rate was found for Chernogolovka, where frogs survived down to -12°C (Fig. 4).

At temperatures from -10 to -16°C freezing experiments were done in multiple batches, each containing 3–5 frogs. The experiments were highly reproducible since the similar survival rate was observed in the different series tested at the same temperature.

Out of six frogs that underwent the second freezing experiment, only three survived

(one that was cooled down to -8°C, and two, to -9°C); three other frogs died (one that was cooled down to -8°C, and two, to -12°C).

Each frog continuously spent 21 days in supercooled condition (i.e., before reaching the SCP); and from several days to almost three months in frozen condition (Table 4).

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The sum of cumulative negative temperatures differed among samples depending on

the amount of time the animals spent at intermediate temperatures (Table 5). It is clear even

from our limited sample that the cumulative exposure to negative temperatures could hardly

be a defining factor in frog survival. Among the frogs from the Karasuk population, survival

was observed only at -12896°C×h; however, no frogs survived at -6320°C×h. Among the

frogs from the Chernogolovka population, 50% survived at -6936°C×h (-12°C) but none

survived at -6548°C×h (-14°C). At the same time, a moderate association was found between

survival and the lower lethal temperature (rs = 0.6, p=0.02).

Genetic analysis

We obtained sequences of the mitochondrial cytb gene fragment for 23 individual frogs, seven each for Karasuk and Podgornoye,Draft and nine for Chernogolovka. A total of nine cytb haplotypes were found among the 23 sampled animals. Of these, the majority (15) of

individuals belonged to the R. arvalis haplotype A2 (Babik et al. 2004); among them were

representatives of all three studied populations. Six haplotypes differed from A2 by one

nucleotide substitution, and two, by two substitutions. Pairwise Fst values among the studied

populations were low (0–0.004) and not statistically significant. On the microsatellite level,

all three studied populations had high levels of haplotype diversity (0.71–0.75), and the most

frequent haplotypes were shared among all populations. Pairwise Fst values were also low (0–

0.079) and not statistically significant.

Discussion

Cold tolerance strategy of the moor frog

The cold tolerance strategy of the moor frog in all sampled populations is based on the

ability to survive freezing after supercooling at slightly negative temperatures (down to -

3.6°C). Frogs can persist in supercooled condition for a long time; in our experiment, at least

for 21 days.

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Although the frog’s skin was covered by numerous ice crystals, there was no rapid inoculation leading to freezing. This is noteworthy since frog skin is thin and wet, and inoculation would be expected, triggered by ice crystals (Lee and Costanzo 1998). Such cases were observed for juveniles of the turtle Malaclemys terrapin Schoepf, 1793, the tree frog

Acris crepitans Baird, 1854, and the wood frog Rana sylvatica (Layne et al. 1990; Irwin et al.

1999; Baker et. 2006). In the absence of moisture and ice, M. terrapin was supercooled to -

15°C, and A. crepitans, to -4.3 to -6.8°C, but under contact with ice crystals freezing started at

-0.8 to -1.6°C and -0.5 to -0.8°C, respectively. R. sylvatica remained supercooled for 3 h at -

1.5 to -2.0°C in the absence of ice, but in its presence, freezing started in 30 s.

However, we have observed that certain and reptiles are not immediately inoculated by ice forming in their environmentDraft and even on their skin. This was confirmed for the Siberian salamander Salamandrella keyserlingii, the Japanese tree frog Hyla japonica, several species of toads (Bufo bufo L., 1758, Strauchbufo raddei (Strauch, 1876), Bufotes viridis (Laurenti, 1768)) and frogs ( Boulenger, 1886, R. dybowskii Gunther,

1876, R. temporaria L., 1758, Pelophylax nigromaculatus Hallowell, 1861), as well as the viviparous lizard Zootoca vivipara Jacquin, 1787 and the common European viper Vipera berus L., 1758 (Berman et al. 2016a, 2016b, 2016c, 2017; Bulakhova et al. 2017a, 2017b; our unpublished data). However, our experimental animals did eventually freeze (after several days) and we only can suggest that their freezing depended on the time of contact of their skin with ice crystals, or on the abundance of those crystals. This issue requires a special study.

SCP in moor frog populations

SCP in two moor frog populations located more than 2 800 km apart in different climates (moderately continental and continental) was found to be identical. Unfortunately,

Voituron et al. (2009b) did not determine SCP for the Danish population. In the wood frog,

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this characteristic varied from -3°C in southern populations vs -1.6°C in the north (Layne

1995; Larson et al. 2014).

Below the SCP point

As demonstrated above, moor frogs survive the temperature below the SCP point in

frozen condition, as do wood frogs. Long-term (about three months) survival in this state in

the laboratory suggests that existing with ice in their tissues is normal for this species. On the

other hand, in Siberia and in the north of European Russia, the overwintering sites of R

arvalis such as leaf litter, a few upper centimeters of soil, burrows of small rodents, etc., could

have negative temperatures for almost half a year (November to March). The sums of

negative temperatures in the surface soil horizons near Podgornoye were similar to those in the experiments with animals from theDraft same population. However, in certain habitats near Olekminsk, low temperatures last much longer and their sums were twice as high. Therefore,

the death of animals in our experiments should not be explained by the length of exposure.

The maximum cold tolerance of moor frogs from Podgornoye and Chernogolovka was

-12°C, and from Karasuk, -16°C. The latter value could be close to the maximum possible for

R. arvalis; however, in natural overwintering conditions it might be even lower, as observed

in R. sylvatica (-16°C in the laboratory and -18°C in nature) (Costanzo et al. 2013; Larson et

al. 2014). The value obtained for R. arvalis in our laboratory experiments (-16°C) coincides

with that known for R. sylvatica.

The observed high cold tolerance suggests that the moor frog can overwinter on land

even in very cold regions. Available reports on overwintering in water for R. arvalis probably

refer to exceptional, although noteworthy, situations. Note that we could not find any

published references to, or discussion of, overwintering in water for R. sylvatica, which is

widespread in the northern part of North America.

Geographic variation of cold tolerance

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Our experimental results obtained on Siberian and Chernogolovka populations, compared with the data on Denmark (Voituron et al. 2009b) suggest that cold tolerance increases from West to West Siberia. In the Danish population, only two of the total

14 individuals survived freezing at -4°C for 81 h, while moor frogs from Russia could tolerate

-12°C for 16 days, and -16°C, for two days.

The observed increase in moor frog cold tolerance from West Europe to Siberia along the gradient of continentality highly resembles the northward gradient detected in North

America for R. sylvatica: from -5 to -6°C in Pennsylvania and Ottawa to -16°C in Alaska

(Storey and Storey 1984; Layne 1995; Costanzo et al. 2013). Although these two frog species are not closely related phylogenetically (Yuan et al. 2016), both overwinter on land at low negative temperatures in a frozen state;Draft this common ecological characteristic could result from either common origin or from convergent evolution.

The dramatic variation in cold resistance within each species’ range may have several explanations. Most probable of those are genetic and climatic hypotheses (Costanzo et al.

2013; Larson et al. 2014).

Genetic variation among the studied populations

Analysis of the cytb gene and nuclear microsatellites showed that the three studied populations of R. arvalis (Karasuk, Podgornoye and Chernogolovka) were genetically similar.

This is in agreement with the findings of Babik et al. (2004) and Knopp and Merilä (2009), who suggest that current populations of R. arvalis in Russia originated recently from a West

European refugium.

The phylogenetic study of R. arvalis by Knopp and Merilä (2009) included the same

Roskilde population (Denmark) that was assessed for cold tolerance by Voituron et al.

(2009b). Knopp and Merilä (2009) suggested that their entire sample could be divided into two groups, western and eastern; the former included Roskilde, and the latter, all Russian

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populations. However, the differences among these groups seem to be minimal: the clades on

the microsatellite tree were not supported by bootstrap, and the mitochondrial haplotypes of

Roskilde and most of our samples differ by only one nucleotide substitution (Fig. 5).

Unfortunately, we cannot compare microsatellite data of the Roskilde population to

that of our sample directly. However, in addition to Roskilde, the study of Knopp and Merilä

(2009) included several populations from Russia; among them were populations from the

Moscow and Tomsk Oblasts, which shared cytb haplotypes with our sample. Therefore, there

are only minor genetic differences between R. arvalis from Denmark and Russia.

Therefore, since all the moor frog populations are genetically very similar, we can

suggest that the geographic differences in cold tolerance are unlikely to be due to genetic variation as it was suggested for the woodDraft frog (Larson et al. 2014). The influence of climate on cold tolerance

All moor frog sample sites in this study, as well as the Danish population studied by

Voituron et al. (2009b), lie at approximately the same latitude but in highly different climates.

Denmark has a humid maritime climate with mild winters; in Moscow Oblast

(Chernogolovka) and more especially in Siberia, there is a higher degree of continentality and

colder winters (Fig. 3). One can postulate that the higher cold tolerance of the eastern

populations results from an adaptation to survive colder winters.

However, soil temperature data that we obtained for the moor frog sampling sites in

West Siberia and southwestern Yakutia as well as weather station data from other localities

show that minimum winter temperatures are expectedly moderated by snow cover. Due to the

difference in the thickness of snow cover, the soil temperatures in Podgornoye, Karasuk, and

Olekminsk differ dramatically even at the depth of 20 cm (from -2 to -15°C) while minimum

air temperatures in all three localities similar (-39 and -40°C). On the other hand, minimum

soil temperatures at the depth of 5 cm happen to be unexpectedly close in Denmark and

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Siberia (-3 and -5.6°C, respectively), despite the fact that the minimum winter air temperatures differed dramaticaly (-10°C in Denmark vs. -40°C in Siberia). It is unlikely that the differences in cold tolerance can be explained by distinct overwintering temperatures.

Other climatic factors that differ between Denmark and Siberia could contribute to this variation, among them: the dynamics of freezing in the fall season (rate of cooling, number of freeze/thaw cycles), the length of winter, early spring frost spells, and duration of the time with negative temperatures.

In discussing this issue, one should remember that the behavioral choice of overwintering sites is statistical (i.e., animals do not always survive). Study of this behavior is a complex task, and thus microclimatic conditions for overwintering determined by experiments (such as in this study) mayDraft not reflect those experienced by frogs in nature. At the same time, the differences in cold tolerance found by us and Voituron et al.

(2009b) could also be an artifact due to different experimental procedures. The most important of those differences are discussed below.

(1) Acclimation and cooling at slightly negative temperatures. In the study by

Voituron et al. (2009b), frogs from Denmark were acclimated in nature with multiple transitions through 0°C (from 2 to -2°С). It is generally assumed that multiple freeze-thaw cycles facilitate higher levels of glucose, which confers a higher cold tolerance of northern populations compared to southern ones (Larson et al. 2014). Therefore, we could expect that the Danish population would demonstrate higher cold tolerance than Siberian populations; however, we observed just the opposite. Siberian climate differs from European climates, especially those of maritime regions, by continuous cooling of soil in the fall without thaws and once frozen then only thaw several months later (Fig. 2). This cooling regime was simulated in our experiments on the frogs within programmed thermostats.

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(2) Immobilization for freezing. Frogs from the Danish population were fixed with

adhesive tape inside a wet tube made of rubber foam. We found that this procedure is

uncomfortable for animals, as indicated by some frogs secreting mucous on immobilization.

In our experiments, frogs were placed in containers with wet moss, and temperature loggers

were placed next to the animals in the moss; loggers attached directly to animals can impede

adoption of wintering poses (see Results).

(3) Inoculation mode. Inoculation of Danish frogs was initiated by using a water

sprayer at slightly negative temperatures, with immediate freezing. This method was also

applied to other amphibians (Voituron et al. 2009а). In contrast, we used gradual cooling in

containers with wet moss (see Materials and Methods), which is much closer to processes occurring naturally. A similar methodDraft was employed for determining cold tolerance of the wood frog by Costanzo et al. (2013).

(4) Frog size and age. Voituron et al. (2009b) used juvenile animals (3.2±0.7 g) while

hypothesizing that adults could be more cold tolerant. We, on the contrary, used only adult

frogs. It is known that juveniles of some amphibians are more cold tolerant than adults (as we

found for the Siberian salamander; see Berman et al. 2016c).

Summarizing the factors that could possibly influence the results of determination of

cold tolerance, we should emphasize that it is difficult to address all these factors in a single

experiment. The experiments of Costanzo et al. (2013) on the wood frog were exemplary in

this respect, since they were conducted in the same laboratory using identical procedures.

Their study demonstrated that wood frogs from the Inner Alaska have higher cold tolerance

compared to those from temperate regions of North America, and that these differences most

probably are genetic.

The distinct cold tolerance found in moor frog populations may be the result of sample

size, the tested temperature, and how close it is to the lower lethal temperature, etc. As seen in

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Fig. 4, temperatures above -8°C may be considered a comfort zone (all animals from all three sampled populations, survived); at -10°C, low mortality was recorded. At -12°C animals from the three populations demonstrated different responses: both Siberian populations had near

50–57% survival rate, while the Moscow Oblast (Chernogolovka) population had only 40% survival. At -14°C (close to the minimal lethal temperature) the Karasuk population exhibited the 45% survival rate. At the same time, the sample size from Podgornoye was likely insufficient to estimate cold tolerance at -14°C. We believe that testing of larger samples from the Podgornoye population at -14 and -16°C could show that they have cold tolerance similar to that of Karasuk. The 40% survival at -12°C and 0% at -14°C in a large sample of frogs from Chernogolovka may indicate a slightly lower cold resistance compared to the Siberian (Podgornoye and Karasuk) frogs. Draft Cold tolerance and northeastern range boundary of Rana arvalis

High cold tolerance of the moor frog, and the resulting ability to overwinter on land at low negative temperatures, explain its enormous geographic range in Eurasia. This range includes the entire West Siberia and the southern part of East Siberia, where the species is found as far north as 58°N (Fig. 1). Rana arvalis is also found in Yakutia, the coldest region of Northern Asia, but only in a small portion of the Lena Valley down to the mouth of

Olekma, the right tributary of Lena.

This complex shape of the geographic distribution of R. arvalis is clearly limited by the boundaries of continuous permafrost (Fig. 1). We should emphasize that this frog species can be found only within discontinuous («island») permafrost areas with a significant proportion of seasonally freezing soils.

A portion of R. arvalis range lies in the Lena valley. This territory is an extension of island permafrost into a zone of solid permafrost (Ershov 1989). According to our data, near

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Olekminsk the soil temperatures at the depth of 2 cm were above -10°C (Fig. 2), and even

higher at the depth of 10 cm, well above lower lethal temperatures for the moor frog.

The distribution map (Fig. 1) shows that R. arvalis in absent from the area of solid

permafrost, implying that the harsher winter soil temperatures present a limiting factor for this

species. The barrier for further northeastern dispersal of the moor frog along the Lena Valley

is likely the well-known area of extremely low temperatures, from the interfluve of Lena,

Amga, and Aldan Rivers, and further downstream the Lena River. It is yet unclear why this

species is absent from the Lena Plateau that has relatively high soil temperatures.

Both R. arvalis and R. sylvatica are the most cold tolerant (with a similar degree of

tolerance) species among all currently studied frog species of the Rana group. At the temperatures down to -1.9 or to -3.6°DraftС moor frogs are supercooled; at lower temperatures they freeze and can survive as much as -16°C, albeit with high mortality rate; at -14°C,

mortality rate in the coldest region we studied was 55%. The frogs can withstand both these

states in laboratory for a long time: supercooling, for a 21 days; and freezing, for almost three

months.

Cold tolerance of moor frogs from the geographically remote populations (West

Siberia and European Russia) was high, but that of Danish frogs was found to be much less,

only down to -4°C. Although the genetic divergence between these populations seems to be

low (1–2 nucleotides as estimated by mtDNA cytb marker), it cannot be ignored, and further

studies of genetic differences are required. However, we believe that the observed differences

between our samples and the Danish population are more likely to be caused by differences in

experimental protocols, especially those for acclimation and cooling.

While the issue of geographic variation in cold tolerance in moor frog from Denmark

to West Siberia remains to be resolved, we can state that, in its cold tolerance and

overwintering mode the moor frog is similar, and maybe even identical, to the Alaskan

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populations of the wood frog. It would be very interesting to find out if this similarity is due to common phylogenetic origin or convergent adaptations, and on which biochemical mechanisms it is based.

The correlation of R. arvalis cold tolerance with winter soil temperatures strongly suggests that the northeastern range boundary of this species is defined by its insufficient cold resistance. This boundary coincides with the transition zone between the island permafrost and continuous one.

Acknowledgements

We are grateful to Yu. S. Korobeynikov and Yu. S. Ravkin for their assistance with frog collection, as well as to an anonymous reviewer for their help in improving the manuscript. Special thanks to our colleagueDraft V. Fet for language editing the text. The reported study was funded by RFBR (projects number 16–04–00082–а and 19-04-00312–а) and by

Budget project 0324-2019-0040-C-01.

Conflict of interest. The authors declare that they have no conflict of interest.

Data availability. Cytochrome b sequences were deposited in GenBank under the accession numbers MH401051-MH401055, MH401072-MH401073 (Karasuk), MH401056-

MH401060, MH401068-MH401071 (Chernogolovka), and MH401061-MH401067

(Podgornoye).

Author contributions. D.I.B. originally formulated the idea of the research and designed and controlled experiments. E.N.M. performed the laboratory experiments. S.V.Sh. performed the genetic analysis. D.I.B., N.A.B. and S.V.Sh. performed the field work and wrote the manuscript.

References

https://mc06.manuscriptcentral.com/cjz-pubs Page 23 of 38 Canadian Journal of Zoology

23

Babik, W., Branicki, W., Sandera, M., Litvinchuk, S., Borkin, L.J., Irwin, J.T., and Rafiński,

J. 2004. Mitochondrial phylogeography of the moor frog, Rana arvalis. Mol. Ecol. 13:

1469–1480. doi:10.1111/j.1365-294X.2004.02157.x.

Baker, P.J., Costanzo, J.P., Herlands, R., Wood, R.C., and Lee, R.E.Jr. 2006. Inoculative

freezing promotes winter survival in hatchling diamondback terrapin, Malaclemys

terrapin. Can. J. Zool. 84 (1): 116–124. doi:10.1139/z05-183.

Bannikov, A.G., Darevsky, I.S., Ishchenko, V.G., Rustamov, A.K., and Shcherbak, N.N.

1977. The determinant of amphibians and reptiles of the USSR. Obrazovanie

(Education), Moscow. (In Russian).

Belimov, G.T., and Sedalishchev, V.T. 1979. To the biology of a moor frog that inhabits Yakutia. Ecology, 5: 92–95. (InDraft Russian). Berman, D.I., and Meshcheryakova, E.N. 2012. Is the western boundary of the Siberian

salamander (Salamandrella keyserlingii, Amphibia, Caudata, Hynobiidae) range

determined by the specific features of its wintering? Dokl. Biol. Sci. 443: 97–100. doi:

10.1134/S0012496612020068.

Berman, D.I., Leirikh, A.N. and Mikhailova, E.I. 1984. Winter hibernation of the Siberian

salamander Hynobius keyserlingi on the Upper Kolyma. J. Evol. Bioch. Physiol. 20(3):

323–327. (In Russian).

Berman, D.I., Bulakhova, N.A., Alfimov, A.V., and Meshcheryakova, E.N. 2016a. How the

most northern lizard, Zootoca vivipara, overwinters in Siberia. Polar Biol. 39(12):

2411–2425. doi:10.1007/s00300-016-1916-z.

Berman, D.I., Meshcheryakova, E.N., and Bulakhova, N.A., 2016b. The Japanese tree frog

(Hyla japonica), one of the most cold-resistant species of amphibians. Dokl. Biol. Sci.

471: 276–279. doi:10.1134/S0012496616060065

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 24 of 38

24

Berman, D.I., Meshcheryakova, E.N., and Bulakhova, N.A. 2016c. Extreme negative

temperatures and body mass loss in the Siberian salamander (Salamandrella

keyserlingii, Amphibia, Hynobiidae). Dokl. Biol. Sci. 468: 137–141.

doi:10.1134/S001249661603011X.

Berman, D.I., Bulakhova, N.A., and Meshcheryakova, E.N. 2017. Adaptive strategies of

brown frogs (Amphibia, Anura, Rana) in relation to winter temperatures in the

northern Palaearctic. Zool. Zh. 96(11): 1392–1403. doi:10.7868/S004451341711003.

(In Russian).

Borkin, L.A., Belimov, G.T., and Sedalishchev, V.T. 1984. New data on the distribution of

amphibians and reptiles in Yakutia. In Ecology and faunistics of amphibians and reptiles of the USSR and neighboringDraft countries. Edited by L.A. Borkin. Zoological Institute of the USSR Academy of Sciences, Leningrad. pp. 89–101. (In Russian).

Bulakhova, N.A., Meshcheryakova, E.N., and Berman, D.I. 2017a. Cryoresistance of three

toad species from . In Proceedings of the 19th SEH European Congress of

Herpetology, University of Salzburg, Salzburg, Austria, 18–23 September. Facultas

Verlags- und Buchhandels AG Wien, Salzburg. p. 186.

Bulakhova, N.A., Meshcheryakova, E.N., Korosov, A.V., Ganjushina, N.D., and Berman, D.I.

2017b. Cold tolerance of the common adder, Vipera berus in Karelia. In Proceedings

of the 5th Biology of the Vipers Conference, 10–20 May. Chefchaouen, Morocco. p.

46.

Costanzo, J.P., Lee, R.E.Jr., and Wright, M.F. 1991. Effect of cooling rate on the survival of

frozen wood frogs, Rana sylvatica. J. Comp. Physiol. B. 161: 225–229.

Costanzo, J.P., Lee, R.E., and Lortz, P.H. 1993. Physiological responses of freeze-tolerant and

-intolerant frogs: clues to evolution of anuran freeze tolerance. Am. J. Physiol.

https://mc06.manuscriptcentral.com/cjz-pubs Page 25 of 38 Canadian Journal of Zoology

25

Regulatory Integrative Comp. Physiol. 265: 721–725. doi:

10.1152/ajpregu.1993.265.4.R721.

Costanzo, J.P., do Amaral, M.C.F., Rosendale, A.J., and Lee, R.E.Jr. 2013. Hibernation

physiology, freezing adaptation and extreme freeze tolerance in a northern population

of the wood frog. J. Exp. Biol. 216: 3461–3473. doi:10.1242/jeb.089342.

Ershov, E.D. (ed) 1989. Geocryology of the USSR: Eastern Siberia and the Far East. Nedra,

Moscow. (In Russian).

Excoffier, L., Laval, G., and Schneider, S. 2005. Arlequin ver. 3.0: an integrated software

package for population genetics data analysis [online]. Evol. Bioinform. Online, 1:

47–50. PMID: 19325852. Goujon, M., McWilliam, H., Li, W.,Draft Valentin, F., Squizzato, S., Paern, J., and Lopez, R. 2010. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids

Res. 38: 695–699. doi:10.1093/nar/gkq313.

Irwin, J.T., Costanzo, J.P., and Lee, R.E.Jr. 1999. Terrestrial hibernation in the northern

cricket frog, Acris crepitans. Can. J. Zool. 77(8): 1240–1246. doi.org/10.1139/z99-

087.

Izyumenko, S.A. (Editor) 1989. Climate of the USSR: an applied reference book. Series 3.

24(1). Gidrometeoizdat, Leningrad (in Russian).

Knopp, T., and Merilä, J. 2009. The postglacial recolonization of Northern Europe by Rana

arvalis as revealed by microsatellite and mitochondrial DNA analyses. Heredity, 102:

174–181. doi:10.1038/hdy.2008.91.

Krasavtsev, B.A. 1939. Materials for ecology of the moor frog. Iss. Ecol. Biocen. 4: 253–268.

(In Russian).

https://mc06.manuscriptcentral.com/cjz-pubs Canadian Journal of Zoology Page 26 of 38

26

Larson, D.J., Middle, L., Vu, H., Zhang, W., Serianni, A.S., Duman, J., and Barnes, B.M.

2014. Wood frog adaptations to overwintering in Alaska: new limits to freezing

tolerance. J. Exp. Biol. 217: 2193–2200. doi:10.1242/jeb.101931.

Layne, J.R.Jr. 1995. Seasonal variation in the of Rana sylvatica from

Pennsylvania. J. Therm. Biol. 20(4): 349–353.

Layne, J.R.Jr., Lee, R.E.Jr., and Huang, J.L. 1990. Inoculation triggers freezing at high

subzero temperatures in a freeze-tolerant frog (Rana sylvatica) and insect (Eurosta

solidaginis). Can. J. Zool. 68(3): 506–510. doi.org/10.1139/z90-074.

Lee, R.E.Jr., and Costanzo, J.P. 1998. Biological ice nucleation and ice distribution in cold-

hardy ectothermic animals. Annu. Rev. Physiol. 60: 55–72. doi: 10.1146/annurev.physiol.60.1.55.Draft PMID: 9558454. Lotshaw, D.P. 1977. Temperature adaptation and effects of thermal acclimation in Rana

sylvatica and Rana catesbeiana. Comp. Biochem. Physiol. A. 56: 287–294.

Matkovskiy, A.V., and Starikov, V.P. 2011. The ecological aspects of amphibians

reproduction in the north of Western Siberia. Izvestiya Samarskogo nauchnogo centra

Rossijskoj Akademii Nauk (Proceedings of the Samara scientific center of RAS), 13

(1-5): 1130–1132. (In Russian).

Red Data Book of the Republic of Sakha (Yakutia) 2: Rare and Endangered Species of

Animals (Insects, Fish, Amphibians, Reptiles, Birds, Mammals). 2010.

Sakhapoligrafizdat, Yakutsk. (In Russian).

Richter-Boix, A., Quintela, M., Segelbacher, G., and Laurila, A. 2011. Genetic analysis of

differentiation among breeding ponds reveals a candidate gene for local adaptation in

Rana arvalis. Mol. Ecol. 20 (8): 1582–1600. doi:10.1111/j.1365-294X.2011.05025.x.

Storey, K.B., and Storey, J.M. 1984. Biochemical adaption for freezing tolerance in the wood

frog, Rana sylvatica. J. Comp. Physiol. B. 155: 29–36.

https://mc06.manuscriptcentral.com/cjz-pubs Page 27 of 38 Canadian Journal of Zoology

27

Storey, K.B., and Storey, J.M. 2004. Physiology, biochemistry and molecular biology of

vertebrate freeze tolerance: the wood frog. In Life in the Frozen State. Edited by B.J.

Fuller, N. Lane and E.E. Benson. CRC Press, Boca Raton. pp. 243–274.

Terentyev, P.V. 1950. Frog. Sovetskaya nauka (Soviet science), Moscow. (In Russian).

Vertebrate animals of Russia (Available information system and integrated database on the

diversity of vertebrate animals of Russia). 2012. [online]. Available from

http://www.sevin.ru/vertebrates.

Voituron, Y., Joly, P., Eugène, M., and Barré, H. 2005. Freezing tolerance of the European

water frogs: the good, the bad, and the ugly. Am. J. Physiol. Regul. Integr. Comp.

Physiol. 288: 1563–1570. doi:10.1152/ajpregu.00711.2004. Voituron, Y., Barré, H., Ramlov, H.,Draft and Douady, C.J. 2009a. Freeze tolerance evolution among anurans: Frequency and timing of appearance. Cryobiology, 58(3): 241–247.

doi:10.1016/j.cryobiol.2009.01.001.

Voituron, Y., Paaschburg, L., Holmstrup, M.,·Barré, H., and Ramlov, H. 2009b. Survival and

metabolism of Rana arvalis during freezing. J. Comp. Physiol. B. 179: 223–230.

doi:10.1007/s00360-008-0307-3.

Yuan, Z.Y., Zhou, W.W., Chen, X., Poyarkov, N.A.Jr., Chen, H.M., Jang-Liaw, N.H., et al.

2016. Spatiotemporal diversification of the true frogs (genus Rana): a historical

framework for a widely studied group of model organisms. Syst. Biol. 65(5): 824–842.

doi:10.1093/sysbio/syw055.

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Fig. 1. Geographic distribution of the moor frog Rana arvalis. Line, boundary of the range; stars, sampling sites; circles, points of temperature measurement; dark grey, continuous permafrost; light grey, discontinuous permafrost. Figure was created using CorelDRAE version 12 and assembled from the following data sources (shapefiles): base map with permafrost (Berman et al., 2016a), boundary of the range (http://www.sevin.ru/vertebrates/), sampling sites and points of temperature measurement (this study). Adapted by permission from [Springer Nature and Copyright Clearance Center]: [Springer Nature] [Polar biology] [How the most northern lizard, Zootoca vivipara, overwinters in Siberia, Daniil I. Berman, Nina A. Bulakhova, Arcady V. Alfimov et al. [COPYRIGHT] (2016a).

Fig. 2. Soil temperatures at the depth of 2 cm for moor frog Rana arvalis habitats: (a) Olekminsk and (b) Podgornoye (determined from the data loggers).

Fig. 3. Minimal and maximal air temperatures and depth of snow cover in winter 2009–2010. Data from weather stations:Draft (a) Olekminsk; (b) Podgornoye; (c) Karasuk, and (d) Chernogolovka (https://rp5.ru).

Fig. 4. Survival of the moor frog Rana arvalis at various temperatures. Dotted line, Chernogolovka; full line, Podgornoye; dashed line, Karasuk.

Fig. 5. Network of haplotypes for the studied populations of moor frogs Rana arvalis. Black circles, Karasuk; grey, Podgornoye; white, Chernogolovka. A1 and A2, haplotype designations from Babik et al. (2004); dot, missing haplotype.

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Table 1. The general number of moor frogs Rana arvalis, survival of which was determined

at different temperatures*.

Population Temperature, °C -2 -4 -7 -8 -10 -12 -14 -16 Chernogolovka 2 4 5 – 4 10 12 – Karasuk – – – 4 8 7 11 20 Podgornoye – – – 3 4 4 4 13 * animals spent for 2 days at each temperature, except -16°C (from 6 h to 16 days for

different experimental series).

Draft

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Table 2. Cooling regimes (number of days at each temperatures) tested on moor frog Rana

arvalis.

Temperature, °С Duration of Population -2 -3 -4 -6 -7 -8 -10 -12 -14 -16 cooling* Chernogolovka 10 2 35 2 2 – 2 14 2 – 8 Karasuk 2 2 34 5 – 30 2 2 2 2-16 10 Podgornoye 2 2 34 5 – 2 30 2 2 0.25 10 * duration of temperature decrease (at 1.2°С day-1).

Draft

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Table 3. Minimal monthly soil temperatures at the depth of 20 cm (extraction thermometer

data from the weather stations, 2009–2010).

Month Olekminsk Podgornoye Chernogolovka Karasuk October -2.2 1.4 4 1.6 November -3.7 -1.7 2 -3.6 December -5.1 -0.8 -5.7 -7.5 January -7.6 -1.3 -6.4 -13.4 February -7.9 -2.1 -3.6 -15.4 March -8.3 -1.5 -1.3 -12 April -5 -0.2 0.2 -2.7

Draft

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Table 4. Total duration (days) spent in the supercooled state and maximal duration (days)

spent in the frozen state by surviving moor frog Rana arvalis.

Total duration* Population Supercooling† Freezing‡ Chernogolovka 21 (37) # 47 (2) Karasuk 21 (50) 83 (1) Podgornoye 21 (28) 77 (2) * the sum of cooling time and both scheduled and unplanned pauses;

† supercooling was calculated up to the highest values of SCP (-1.9°C);

‡ freezing was calculated starting from -4°C, assuming that all animals passed the SCP by that

point;

# in parentheses, the sample size for each group. Draft

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Table 5. Sum of cumulative negative temperatures accumulated by moor frogs Rana arvalis

during the experiment, % of surviving animals and time spent at the limiting temperature.

Testing Time spent at Cumulative, Population temperature, Survival, % the limiting °C×h °С temperature, h -17504 0 216 -17120 0 316 -16 -14816 0 48 -12896 25 48 Karasuk -6320 0 48 -11876 74 48 -14 -8588 0 48 -4316 67 48 -8623 0 6 Podgornoye -16 -7663 0 6 -5359 0 48 -8564 0 48 -14 -6548 0 48 Chernogolovka Draft -6936 50 48 -12 -3000 33 48

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Fig. 1. Geographic distribution of the moor frog Rana arvalis (from: http://www.sevin.ru/vertebrates/). Line, boundary of the range; stars, sampling sites; circles, points of temperature measurement; dark grey, continuous permafrost; light grey, discontinuousDraft permafrost. Adapted by permission from [Springer Nature and Copyright Clearance Center]: [Springer Nature] [Polar biology] [How the most northern lizard, Zootoca vivipara, overwinters in Siberia, Daniil I. Berman, Nina A. Bulakhova, Arcady V. Alfimov et al. [COPYRIGHT] (2016a).

182x100mm (600 x 600 DPI)

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Fig. 2. Soil temperatures at the depth of 2 cm for moor frog Rana arvalis habitats: (a) Olekminsk and (b) Podgornoye (determinedDraft from the data loggers). 85x47mm (300 x 300 DPI)

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Draft

Figure 3

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Figure 4. Survival of the moor frog Rana arvalis at various temperatures. Dotted line, Chernogolovka; full line, Podgornoye; dashed line, Karasuk.

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Draft

Figure 5.

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