Method and approach

The project consists of six work-packages, numbered A-F, each with its own set of activities.

A. Desk study

A1. Update the geo-database with present distribution localities and range in SE Romania, Central and Eastern Europe.

The existing database on the distribution of Pelobates sp. in the Balkans (Džukić et al. 2005; Szekely et al. 2009 a, b), and data provided by the online databases (e.g. GBIF, HerpNet) will be updated with our own data obtained from activity B1, as well as geo-referenced distribution records covering the entire range of the two species of concern from publications and available museum collections.

A2. Create a geo-database with past distribution records (paleoherpetology).

Paleontological records of the genus and the two species of concern will be georeferenced and included in the database.

Milestone: Database available by Month 12.

Expected results: An updated database with present and past distribution records of the two species of Pelobates (to be used in activities E1 and E2).

B. Studies conducted in the field

The study will be conducted in at least three locations: two with only one species (allotopic), and one with both species (syntopic). Fieldwork will be started after obtaining the permits from the Danube Delta Biosphere Reserve Administration, and derogation for P. fuscus from the Ministry of the Environment and Forests. A pilot study conducted by us has received permits before (e.g. Research Permit nr. 21/9.04.2010 from the DDBRA; Ministerial Order 1173/3.08.2010). Considering the yearly variation in pond availability the number of locations considered will be higher to minimize the risks.

B1. Field surveys for mapping species distributions, and estimate habitat use and habitat requirements

The habitat survey and spadefoot toad monitoring in SE Romania will allow to: (i) Complete the geo-database with present distribution localities (activity A1); (ii) Select the locations for intensive studies; (iii) Characterize breeding aquatic habitats (local level) and their surrounding terrestrial habitat (landscape level) based on a number of variables that will allow identifying the best habitat predictors (see Hartel et al. 2010 for details).

Milestones: (i) Locations selected by Month 10 (to be used in B2, B3 and B4). (ii) Habitat predictors identified by Month 12 (to be used in E2).

B2. Estimate population structure

a. The life history of individuals from both species will be studied in the field by intensively monitoring populations during reproduction to estimate the breeding population size, the reproductive effort (from the loss in weight of both sexes during clutch release) and fertility (i.e. egg number and size per clutch). We will check for assortative mating in the two species that differ in sexual dimorphism in conjunction with Activity D1 (see also table 1). The methods used will be according to Dodd (2010).

b. The range of the selected allotopic and syntopic populations will be delimited based on the extent of occurrence (a minimum convex poligon based on presence data) during night torch surveys. Adult animals from each population will be marked using pit-tags (Gibbons and Andrews 2004). Subsequent recaptures will allow estimating population size and individual range size using the available software MARK (White and Burnham 1999) and CAPTURE (Rexstad and Burnham 1991).

c. The body condition is correlated with population and habitat quality parameters (Băncilă et al. 2010) and computing it will allow multiple comparisons among populations, sexes, size classes and moments in time.

Hypotheses to be tested: (i) The absence of male combat in P. syriacus suggests that females prefer to mate with larger males, while in P. fuscus females are not selective and males select for larger females. (ii) Predator density is well correlated with the frequency of malformation in tadpoles and juveniles.

Milestone: A body condition index valid for both species available by month 24.

B3. Prey availability and food consumption (trophic niche): selectivity in feeding and competition for resources

A previous study has shown that P. fuscus has a sit-and-wait foraging strategy, and has a wide trophic niche (Cogălniceanu et al. 1998), but very little is known about the feeding of P. syriacus. Our study will be conducted in autumn 2012, involving a comparative study in three locations: one with syntopic populations, and one with each of the two species. Invertebrate prey availability will be estimated for two weeks, before and during the sampling and will involve pit-fall traps and vegetation netting. Both adults and juveniles spadefoot toads will be sampled. The methods used will be according to Cogălniceanu et al. 2001).

Hypotheses to be tested: (i) P. syriacus is more selective than P. fuscus in choosing the prey, preferring prey with higher nutritional value. (ii) P. syriacus has a higher feeding intensity compared with P. fuscus. (iii) Prey selectivity is lower in juveniles compared to adults.

B4. Breeding ponds survey for monitoring growth rate in tadpoles, duration to metamorphosis and reproductive success

The monitoring of breeding ponds will be run in conjunction with C1. Breeding ponds will be monitored weekly for shifts in water level, vegetation cover, predator presence and density and water chemistry (especially conductivity), and a random sample of 20 tadpoles will be measured and checked for injuries caused by predation (see B2). Water temperature will be monitored with automatic dataloggers. Incidence of malformation in tadpoles and juveniles will be used as a measure of predation impact (Szekely and Nemes 2003). The number of monitored ponds will vary between 4-10 as a risk insurance against drying before metamorphosis or not having water in subsequent years.

Hypotheses to be tested: (i) There is a threshold (minimum size and/or age) for metamorphosis, after which it is possible to accelerate it in response to certain environmental stimuli. Slower growers like Pelobates ssp. are predicted to transform closer to the threshold. (ii) Presence of predators will induce a faster metamorphosis.

Expected results: (i) Habitat and trophic resource use by the two species – focused on selective use and potential competition in the syntopic populations. (ii) Detailed population structure date: population size, breeding population size, size class structure, sex ratio. (iii) Reproductive effort and success. (iv) Fitness estimate of studied populations based on body condition.

C. Experimental studies

C1. Tadpole development to metamorphosis

Pond drying is a major cause of tadpole mortality impacting reproductive success (Newman 1992). We will evaluate for the two species the assumptions and test predictions of the Wilbur and Collins (1973) model for plasticity in age and size at metamorphosis. A second set of experiments will focus on the tolerance to salinity to identify both the impact of increased salinity on tadpole metamorphosis and the threshold that prevents tadpoles of the two species to metamorphose. The range of salinity tested will be established after aquatic habitat inventory and monitoring (see Activity B1). The third sets of experiments will involve aquatic predators (fish, dragonfly larvae, leeches): both as visual stimuli (i.e. separated by a glass wall from the tadpoles) or olfactive stimuli (i.e. water in the experimental tanks will flow from tanks stored with predators). Exact protocols (e.g. density of predators) will be decided based on the preliminary results of Activity B4.

Hypotheses to be tested: (i) Tolerance to salinity is higher in P. syriacus tadpoles. (ii) Increased salinity will trigger earlier metamorphosis of tadpoles (and smaller sizes). (iii) Predators will trigger earlier metamorphosis of tadpoles. (iv) Tadpoles manifest phenotypic plasticity in coping with environmental stress. (v) There is a correlation between larval period and limb length.

C2. Post metamorphic growth rate depending on food quality and quantity

The growth rate of freshly-metamorphosed juveniles will be estimated from individually-housed animals that are fed known quantities of live food (crickets and mealworms). Food quality and availability (2-4 feedings a week) will allow estimating the growth rate and the basal metabolism (e.g. Jørgensen 1989).

Hypotheses to be tested: (i) Juveniles metamorphosing at a smaller size will grow slower. (ii) P. syriacus has a lower basal metabolism. (iii) P. syriacus has a higher feeding rate. (iv) Growth rate differs between sexes.

Expected results: (i) Estimate plasticity and environmental thresholds that control metamorphosis.

(ii) Comparative post-metamorphic growth rates in the two species.

D. Laboratory analyses 

D1. Age structure assessment.

The age structure of a population is an integrative demographic parameter (Stearns and Koella 1986). Skeletochronology, i.e. the recording of periods of arrested growth in long bones like phalanges (Castanet and Smirina 1990), allows estimating the age structure of amphibian populations in natural conditions and provides detailed data on age and growth related parameters. Our approach will be to compare the age structure and the growth patterns and parameters among both syntopic and allotopic populations of the two species of spadefoot toads. Age will also be assessed for pairs in amplex, taking into consideration the assortative mating or sexual selection (in conjunction with Activity B2). The differences in age structure between the sexes can explain variation in size dimorphism (Monnet and Cherry 2002). The skeletochronology analysis will follow previous procedures (Castanet and Smirina 1990).

Hypotheses to be tested: (i) Growth rate is higher in allotopic populations. (ii) Maximal size is higher in allotopic populations. (iii) Longevity is higher in males of P.  syriacus. (iv) Age at maturity is higher in P.  syriacus compared to P. fuscus. (v) Growth rate is higher in P. syriacus. (vi) Older males will mate with larger females.

D2. Measuring morphology and fluctuating asymmetry of the skull and of the body.

In P. fuscus different cranial elements respond differently to prolongation of larval development (Smirnov, 1992). Skull size and shape in metamorphs with different larval development periods will be studied from radiographs of dorsal and palatal sides taken with an X-ray system. About 20+ landmarks will be used on the photos obtained. Developmental instability was proposed as a useful tool for quantifying the degree of environmental and genetic stress. A common mean of assessing developmental stability is by analyzing fluctuating asymmetry (FA) in bilateral traits (Van Valen, 1962). We will use the landmark superimposition method (Procrustes superimposition method), which is a powerful morphometrics approach to study integrative morphological variation (Debat et al. 2000). Ten biometric symmetrical characters will be measured on both sides of the body from high quality pictures. On each picture landmarks will be digitized using TpsDIG software (Rohlf 2005). Data analysis will follow Băncilă et al. (2010).

Hypotheses to be tested: (i) Skull size and shape differences will increase with prolonged duration of the larval period. (ii) Skull size and shape are correlated with size at metamorphosis. (iii) Individuals from populations inhabiting extreme environments (arid and/or saline) will exhibit higher FA. (iv) FA is not influenced by sex or size class. (v) Longer legs indicate good dispersers and allow exploiting larger ranges thus increasing individual fitness.

Expected results: (i) The age structure and growth parameters for the studied populations. (ii) Quantification of the morphological changes induced by environmental constrains.

E. Data analysis and modeling

E1. Species Distribution Modeling at different spatial scales: regional (Central and SE Europe) and local (Dobrogea)

We shall compute a number of past, present and future species distribution models based on actual climate and future predicted climate for contrasting General Circulation Models using algorithms and their associated software: Maximum Entropy run with Maxent  (Philips et al. 2006), Bioclim run from within OpenModeller (Muñoz et al. 2009). The data used will be species occurrence data of spadefoot toads (provided by activity A1), our own expert knowledge about habitat requirements (from activity B1), a selection of environmental layers such as WorldClim climate variables (Hijmans et al. 2005) or different topographic or vegetation variables.

E2. Improving the models with field surveys for ground truth (presence/absence and density) and habitat availability and use.

Based on our own data from Activity B1 and data collected in Activity A1, we shall be able to apply thresholds, weights and re-run the models to improve them.

E3. Simulating viability of the studied populations of Pelobates

Modeling the extinction risk for vulnerable populations is an important tool in conservation (Akçakaya and Sjögren-Gulve 2000). The demographic and environmental data resulting from activities B1, B2, B4 and D1 will allow for a tentative Population Viability Analysis (PVA) using the software Vortex (Miller and Lacy, 1999) and Ramas Metapopulation (Akçakaya, 1998).

E4. Data analysis and integration of the results.

The data and results provided by the different activities will be jointly analyzed and integrated into a single framework.

Expected results: (i) Detailed predictive species distribution maps of the two species. (ii) Predictive shifts in range under different climate change scenarios. (iii) Environmental factors that control distribution of the two species. (iv) Estimates of extinction risk in the populations studied.

F. Action plan for the conservation of the two species of Pelobates

The results of the project will allow the preparation of an Action Plan (AP) for the two species of Pelobates. It will be publicly launched in Tulcea where the administrations of two large protected areas are located: Danube Delta Biosphere Reserve and Măcin Mountains National Park. Based on the objectives identified, the AP will provide a set of actions and targets, identifying the leading organizations and milestones within a clear timeframe. It will be within the frame of the National Biodiversity Action Plan and the EU Biodiversity Action Plan. A dedicated web page on will provide detailed information for the two species, including the results of the project.

 Milestones:  (i) Web site online by month 6. (ii) Action Plan ready by month 36.

Expected results: (i) A web page dedicated to the two species of Pelobates. (ii) An Action Plan prepared and disseminated.

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