Long-term observation of amphibian populations inhabiting urban and forested areas in Yekaterinburg, Russia

This article presents data derived from a 36 year-long uninterrupted observational study of amphibian populations living in the city and vicinity of Yekaterinburg, Russia. This area is inhabited by six amphibian species. Based on a degree of anthropogenic transformation, the urban territory is divided into five highly mosaic zones characterized by vegetation, temperature, and a distinctive water pollution profile. Population data is presented year-by-year for the number of animals, sex ratio, and species-specific fecundity including the number and quality of spawns for the following amphibian species: Salamandrella keyserligii, Rana arvalis, R. temporaria, Lissotriton vulgaris, and Pelophylax ridibundus. These data provide an excellent opportunity to assess an urban environment from an animal population-wide perspective, as well as revealing the forces driving animal adaptation to the anthropogenic transformation of habitats.


Background and Summary
In the history of the biosphere, urbanization is a recent factor and therefore, its long-term effects on animal populations are largely unexplored. A city is a special type of environment where animals are exposed to a complex mixture of pollutants, often acting synergistically, and it is difficult to forecast the long-term consequences of such a synergism 1,2 . Previous work suggested that since amphibian development is environmentally sensitive, changes in urban amphibians at individual and/or population level could serve as an indication of environmental pollution 3 . Accordingly, many studies tried to analyze how urbanization affects various aspects of amphibian biology. However, published data usually lacks comprehensiveness 4 or spans only short periods. To evaluate the long-term effects of urbanization, we are conducting a multi-year comprehensive ecological and population-wide study of amphibians inhabiting Yekaterinburg, one of the largest Russian industrial centers located in Ural Mountains (Figure 1).
Yekaterinburg is a typical urban territory with a well-developed city infrastructure, dense human population, and a long history of industrial pollution. The city's landscape can be divided into 5 zones reflecting various levels of anthropogenic transformation 5 . Each zone is characterized by a particular vegetation community, water chemistry profile and temperature regime (see Vegetation_description_eng. csv and Temperature_April_May_eng.csv (Data Citation 1). Further details are presented below in Landscape typification section. Zone I is the most transformed area lacking open water sources and since amphibian development strictly depends on water, no amphibian species is found there. The distribution of amphibians in other urban areas is remarkably discontinuous because roads, buildings and other artificial barriers efficiently separate individual habitats ( Figure 2). The urban Zones II-IV and the forested area around the city (Zone C, native environment) are inhabited by the following five amphibian species (listed in order from the most to the least abundant): Rana arvalis, Lissotriton vulgaris, Pelophylax ridibundus, Salamandrella keyserlingii, and R. temporaria (see Populations_status_1977_2013_eng.csv  (Data Citation 1)). The sixth species, Bufo bufo only occasionally appears, in Zone C and therefore, no data is shown.
In the course of our study, we monitored the number of eggs (and, in some cases, their size) and the number of adult females through number of spawns (see corresponding files on fecundity, egg size, number of spawns, and populations' status). Importantly, extinction of amphibian populations resulted from habitat destruction has been recorded at~40% of sites. Thus, S. keyserlingii has disappeared from one site, L. vulgaris-from seven, R. arvalis-from eleven, R. temporaria and P. ridibundus-from five each. Of note, P. ridibundus has also appeared at five new urban and some natural sites thus demonstrating that this species is currently actively expanding in the Ural region 6 . See also the file on these populations' statuses.
Since amphibians spawn and develop in ponds, we regularly test the quality of water sources (see Water_1980_2013_basic_eng.csv (Data Citation 1)). There are environmental gradients between zones that are quite steep and remarkably stable over time including differences in pH, temperature, and the level of pollution. For example, ponds located in Zones II-IV have average pH 7.3, while ponds in Zone C have average pH 6.6. Higher chemical oxygen demand and mineral content is characteristic of ponds located in most urbanized Zones II and III compared to those in recreational Zone IV or forest areas (Zone C). In addition, average water temperatures are approximately 2-3°C higher in ponds in Zones II-IV than in Zone C, although temperatures in Zone IV have increased only during the last decade (Tables 1 and 2 and Temperature_April_May_eng.csv (Data Citation 1)).
During our period of observation, R. temporaria has significantly declined in Zone III and completely disappeared from Zone II. Contrary to that, P. ridibundus has emerged in Zone IV (see Populations_status_1977_2013_eng.csv (Data Citation 1)). Apparently, P. ridibundus is an ecologically malleable species and, thus, not only prevails in natural ecosystems but also can successfully reproduce in urban areas. Similarly, L. vulgaris usually reproduces successfully in Zone III, although it is absent in the most transformed sites in Zone II. Both species belong to an evolutionary younger taxon compared to B. bufo and S. keyserlingii, both of which belong to older taxonomic groups. Neither of the latter species perform well in the urban environment. Thus, B. bufo can never be found within the city and S. keyserlingii is absent from Zone II and is very rare in Zone III and in some of the most transformed places in Zone IV (see Populations_status_1977_2013_eng.csv (Data Citation 1)).
We suggest that the dynamics of amphibian populations in an urban environment is associated with ecological plasticity of the species, which in turn seems to relate to their evolutionary origin. Thus, polymorphic species belonging to evolutionary younger taxons have a survival advantage in an urban environment.

Geographic location
Yekaterinburg is situated on the eastern side of the central part of the Ural Mountains. The Urals are a natural barrier between European and Asian plant and animal populations and are characterized by a considerable heterogeneity of landscapes including forested hills, lakes, and agricultural land. The area is characterized by a continental climate with four winter months (October-February) and three months of summer (June-August). Average winter temperature is −16°C (average high +6, average low −18) and average summer temperature is +20°C (average high +23, average low +10).

Collection sites
The material has been collected at 26 locations in the city and vicinity of Yekaterinburg (Table 3) See also Habitats_coordinates.csv (Data Citation 1). Every spring (1977-2013) 55 ponds were searched by the authors for an assessment of spawn numbers. In summer and autumn every habitat mentioned above was searched for adults and juveniles. Both procedures were carried out at least four times during the season.

Landscape typification
Urban landscape was typified based on land use and other results of human activity, such as height and concentration of buildings, human population density, level of pollution, types of vegetation, etc. 5 Closely placed multistory buildings and most of the ground covered by asphalt and concrete characterize Zone I (city center), which also has the highest human population density. Since there are no ponds or open springs, no amphibians live in Zone I. Although dense multistory development and compact human population are also characteristic to Zone II, it has some open soils and water streams as well as small ponds, which amphibians are able to use for spawning and development. Amphibian habitats in Zone II are isolated from each other by roads, long buildings, and other infrastructural elements. Zone III has lower human population density and a prevalence of low-rise building. There are many open grounds in gardens and city parks and a variety of small ponds, springs, and rivers, thus, providing amphibians with many opportunities to breed and grow. Zone IV is the least polluted urban territory as it is primarily an area of parks and recreation that are connected to forests around the city. Zone IV also has the lowest human population and a low industrial presence. Finally, Zone C (control) is comprised of the forests surrounding the city. Samples from Zone C were collected at a site located 23 km from the city line, which we consider a natural environment for amphibian populations.

Morphological measurements
Morphological measurements (snout-vent body length of specimens) have been made by manual caliper from 1977 to 2002. After that, a digital caliper from (Kraftool, Germany) has been used. Both calipers have the same scale interval −0.01 mm. Measurement of body, liver and heart mass was performed on a digital and torsion balance (Shimadzu) with a scale of 0.2 mg. Throughout the entire observation, we used the same volumetric laboratory glassware (Yugoslavia) and binocular microscope (LOMO, Russia). Methodology on the survey techniques are presented below (Fecundity and Eggs collection methods section).

Quality of water
Water samples were collected twice a season: at the end of spawning and at the peak of metamorphosis. Sample volume was 1.5 liters. File Water_1980_2013_basic_eng.csv (Data Citation 1) contains data on quality of water in spawning ponds in 1980-2013, which was an important part of landscape typification. In the city, amphibians reproduce in small ponds, which are closed water bodies that accumulate pollutants washed in from the terrain and surrounding roads, brought in by atmospheric precipitation and dumped in by businesses and households. • Acidity (pH); • Mineralization-an integrated parameter of general level of inorganic substances in water; • Chlorides-an indirect measurement of inorganic substances brought in from the terrain; • Sulfates-an indirect measurement of inorganic substances originating from the air; • Oxidisability-a measurement of total level of organic matter (carbon) in water samples; • Biochemical Oxygen Demand (BOD, mg/dm 3 )-an indicator of organic water pollution (level of eutrophication), which is measured as the amount of dissolved oxygen necessary to break down organic material present in water by aerobic biological organisms; • Chemical Oxygen Demand (COD, mg/dm 3 )-an indicator of the total level of oxidizable matter in water including both organic and inorganic substances.

Temperature of water
Temperature_April_May_eng.csv contains data on water temperature (t°C) in spawning ponds taken within a month after amphibian clutches were laid. The temperature was taken manually between the end of April and the end of May using a mercury thermometer with a scale interval 0.5°C (standard thermometer:ТП-22, Thermopribor, Russia). The thermometer was calibrated in the factory and needed no further calibration. During measurements, the thermometer was positioned at approx. 20 cm below the surface and out of direct sunlight. All measurements were done in the morning (8-10 AM).

Fecundity
Files Spawns_number_Salamandrella_eng.csv, Spawns_number_Rana_arvalis_eng.csv, and Spawns_ number_Rana_temporaria_eng.csv (Data Citation 1) contain data on the number of spawns in habitats of S. keyserlingii, R. arvalis, and R. temporaria, respectively. The number of eggs within each clutch/spawn is presented in files Fecundity_Rana_temporaria_eng.csv, Fecundity_Rana_arvalis_eng.csv, and Fecundity_Salamandrella_eng.csv (Data Citation 1). Egg clutches in every pond under investigation were counted manually. Eggs of each clutch of Salamandrella keyserlingii were counted separately to determine the clutch size. Originally, Prof Vershinin counted eggs in the field (at the collection site). This was necessary to avoid the transportation of the eggs to the laboratory, which could destroy the material. Starting in 2009, however, eggs were counted in the laboratory using digital images taken at the site of the collection. Importantly, preparations for egg counting were identical, with or without digital photographs, eggs being manually spread as a single-egg layer over a light plastic surface (Figure 3). Prof Vershinin took all photographs and counted eggs. The number of eggs in a clutch of brown frogs (R. arvalis Nilss. and R. temporaria L.) was calculated by sparing technique 7 . In each case, firstly, the volume of a clutch was determined with a graduated cylinder (scale interval 5 ml); secondly, the volume of 100 separated eggs was measured; thirdly, the number of eggs in a whole clutch was calculated by dividing the first number by the second. Fecundity was determined only for those three species in which it can be done in vivo. To ensure reproducibility and consistency of the data collection, the same laboratory equipment (chemical glasses and cylinders) has been used throughout the whole period of the observation with one exception. The following example demonstrates our approach to reduce data collection bias.

Egg collection methods
To

Sample collection strategy
We used the following basic strategy in our sampling program. First of all, in spring we always collect biological material and perform on-site measurements on clear sunny days. This ensures the best visibility throughout the collection area and thus prevents losing or unintentionally destroying the material. Secondly, it is important to be able to access any spot within the collection area and, since the terrain is not always 'user-friendly', the collector always wears full hip boots. Thirdly, sufficient length of the collection period is very important, as spawnings in different ponds and in different seasons end at variable times in the season. The procedure begins after time when mating is finished to be sure that all of spawns from the ponds were counted. Every year (1977-2013) 55 ponds were searched by author for spawn's number evaluation, adults and juvenile's and water sampling. Juveniles were collected by hands near the ponds soon after metamorphose finishing. Mentioned procedures were made 4-5 times during the season (in spring, summer and autumn). So we spent about 623.1 person hours per pond. Therefore, to ensure the completeness of the data, we visit all collection sites regularly until no fresh spawns can be found. Finally, the same person, Prof Vershinin has performed the collection of all samples. Based on many years of field zoological work, this is the best approach to providing consistency of data.

Data Records
The following files are publicly available (Data Citation 1):