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Ontogenetic dietary shifts in European common frog (Rana temporaria) revealed by stable isotopes ARTICLE in HYDROBIOLOGIA · APRIL 2011 Impact Factor: 2.21 · DOI: 10.1007/s10750-011-0804-3
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Hydrobiologia (2011) 675:87–95 DOI 10.1007/s10750-011-0804-3
PRIMARY RESEARCH PAPER
Ontogenetic dietary shifts in European common frog (Rana temporaria) revealed by stable isotopes Giedrius Trakimas • Timothy D. Jardine Ru¯ta Barisevicˇiu¯t_e • Andrius Garbaras • Raminta Skipityt_e • Vidmantas Remeikis
•
Received: 5 January 2011 / Revised: 21 May 2011 / Accepted: 18 June 2011 / Published online: 6 July 2011 Ó Springer Science+Business Media B.V. 2011
Abstract During migrations and ontogeny amphibians change their habitat and feeding, and thus are important in linking terrestrial and aquatic ecosystems. We measured d13C and d15N values of early stages (egg, embryo, tadpole) and toes of adult frogs Rana temporaria, collected from a small wetland in Lithuania. We compared the isotopic composition of these tissues with potential food sources, excrements of tadpoles, and filled intestinal tracts. We found that d13C values in R. temporaria tadpoles were markedly depleted in comparison to adults, eggs or embryos, demonstrating a terrestrial to aquatic shift in energy sources. After the onset of feeding, tadpoles approached isotopic equilibrium with available food (algae and litter). Tadpoles had higher d15N than both algae and litter, differing by 3.6 and 2.4%,
Handling editor: Lee B. Kats G. Trakimas (&) R. Skipityt_e Center for Ecology & Environmental Research, Vilnius University, M.K. Cˇiurlionio 21/27, LT-03101 Vilnius, Lithuania e-mail:
[email protected] T. D. Jardine Australian Rivers Institute, Griffith University, 170 Kessels Road, Nathan Brisbane, QLD 4111, Australia R. Barisevicˇiu¯t_e A. Garbaras V. Remeikis Nuclear & Environmental Research Laboratory, Center for Physical Sciences & Technology, Savanoriu ave. 231, LT-02300 Vilnius, Lithuania
respectively, and similar d13C to these sources. However, tadpole excrements and body tissue diverged, with mean d13C values of excrements (-30.3 ± 1.6% SD) more similar to litter (-31.7 ± 1.2% SD) and body tissue d13C (-34.8 ± 0.7% SD) more similar to algae (-34.2 ± 4.1% SD). This suggests that algal resources are critical in early life stages of this anuran, particularly at stages characterized by high growth and low development (stages: 25–35). Keywords Metamorphosis Tadpoles Algae Litter Excrements Wetlands
Introduction Amphibians are important consumers in both aquatic and terrestrial habitats. They play a significant role in linking terrestrial and aquatic ecosystems (Regester et al., 2006; Whiles et al., 2006), as they switch their habitat and feeding mode during migrations and ontogeny. Eggs and embryos potentially reflect energy and materials transferred from terrestrial ecosystems to aquatic predators, while late stage tadpoles may transfer aquatic energy to terrestrial systems following metamorphosis. An assessment of trophic position and sources of carbon sustaining growth of this critical group is therefore essential to our understanding of ecosystem ecology, fluxes of energy between habitats, and for the planning of conservation measures for this group that is currently
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under threat from a variety of natural and humaninduced stressors (Houlahan et al., 2000). Diet of multiple life stages of amphibians and turnover of tissues, including eggs, embryos, tadpoles and adults is useful to reveal the role of this group in temperate wetlands. Because mortality of amphibians is high in early life stages (Zahn, 1997), an understanding of diet shifts can help identify the critical resources needed to provide energy for growth and metamorphosis (Alvarez & Nicieza, 2002). Tadpoles are abundant grazers in many freshwater ecosystems and also are largely predated (Lardner & Loman, 1995); however, this important group of freshwater consumers is still poorly studied (Altig et al., 2007). The relative importance of terrestrial (litter) and aquatic (algae) organic matter in contributing to the growth of animals remains a controversial topic (Brett et al., 2009; Cole et al., 2011). Therefore, studies aimed at determining the sources of food for common and abundant taxa are useful in determining the role of these materials in sustaining food webs. Stable isotopes are a useful tool for quantifying energy and nutrient flow in ecosystems (Fry, 2006; Jardine et al., 2006). Using stable isotopes in ecological research, the most common elements are carbon (13C/12C) as its isotopic signatures reveal source material, and nitrogen (15N/14N), as its signatures reflect the trophic position of animals (Post, 2002). Stable isotope analysis is a powerful analytical tool that complements classical studies because it provides data about the partitioning of nutrients to body tissues, revealing the source of food that is assimilated rather than simply ingested. This is particularly important for species that consume mixed diets of living and dead organic matter such as anurans. Despite these advantages, isotopic data of amphibians are scarce (e.g., Verburg et al., 2007; Jefferson & Russell, 2008) though much needed (Dalerum & Angerbjorn, 2005). Rana temporaria is the most widespread brown frog in Europe and in many areas is the most common species (Arnold & Ovenden, 2002). This species therefore provides a good model system for examining nutrient and energy flow in freshwater wetlands as it occurs in high densities in tadpole or adult stages (Loman & Lardner, 2009) and has a wide geographic range. Patterns observed in this common species may also help in understanding diet and ontogeny in other rare or threatened amphibian species. We studied the
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isotopic composition of early stages of R. temporaria (egg, embryo, and tadpole), potential food sources, excrements of tadpoles, and isotopic fractionation between carcasses of tadpoles and filled intestine tracts. These measures were made to better understand sources and fate of organic carbon in the critical life stages of this common amphibian.
Materials and methods Samples of frog tissues and basal resources were collected from a small (\50 m2) shallow (\0.5 m) wetland at Vingis Park, Vilnius, Lithuania (54°400 5900 N; 25°150 5000 E). The study site was in a depression surrounded by suburban lawns with tall trees. The waterlogged area was partially covered by wood club rush (Scirpus sylvaticus) (&20%) and shaded by Salix sp. trees (&70%). Detritus from the emergent wood club rush makes up the majority of the deposited litter, particularly in areas where tadpoles frequent, while leaves from Salix sp. form a small fraction of the litter. Leaf litter from trees in adjacent park areas is also deposited at the edges of the study site but typically does not become submerged in the wetland and thus is not available to the aquatic food web. The area was sampled 15 times between 10 April 2009 and 10 August 2009. The majority of the wetland was dry by the end of the summer. Adult European common frogs (R. temporaria) were sampled during the breeding season (early April). To reduce our impact on the study population (Kelly et al., 2006), we used toe clippings to sample adult frogs (n = 9). The longest toe of the right hind foot was clipped for each adult frog captured in order to obtain tissue samples. After this procedure frogs were released at the same place where they were found. Eggs of brown frogs were collected from 15 different clutches (early April). We collected two eggs from each clutch, however, two of them were lost during the lab procedures. Both eggs and egg jelly that is produced on the outer layer of the eggs were collected, but only eggs had sufficient C and N to obtain isotopic data. Embryonic (n = 32) and larval (n = 20) stages ranging from stage 18–45 (Gosner, 1960) were collected by dipnet at different points in the season. Pre-feeding individuals (stages
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18–24) were collected from 15 April 2009 to 2 May 2009. Larvae of stages 25–45 were collected from 27 April 2009 to 4 July 2009. Algal and litter samples were collected during each sampling. Twenty-one samples of litter (S. sylvaticus) were collected from the waterlogged area to represent isotope values from terrestrial production. These litter samples were chosen because they contained little or no visible algae, and they were further cleaned prior to analysis to ensure a purely terrestrial isotopic signal. Thirteen algal samples, scraped from the surfaces of S. sylvaticus litter with a spatula, were taken. In these samples the most abundant taxa were Mougeotia sp., Ulothrix sp., Stigeochlonium sp., and Ophyocitium sp. To determine if there were differences between ingested and assimilated food, we also sampled excrements of tadpoles to compare with the isotope ratios of tadpoles. For a subset of samples, we used whole tadpoles to compare with recent studies (Jefferson & Russell, 2008). Tadpoles collected in the field (n = 20) were separated into glass vials with clean water, and we were able to obtain samples of excrements from a subset of 13 (stages 29–39). After 1 h tadpoles were removed and excrements were collected with a pipette. To further link the recent diet of tadpoles with isotope ratios of body tissues, full guts from 12 tadpoles collected in the field (stages 24–39) were removed with tweezers under a binocular microscope. Tadpoles were cut lengthwise, the guts were removed through the incision and stored separately. The remainder of the body was processed whole for stable isotopes. All samples (n = 160) were placed in separate vials and labeled. Frozen amphibian tissues were homogenized, oven-dried at 60°C for 24–48 h, and then manually ground to a fine powder by mortar and pestle. Samples of algae, litter, and feces were ovendried at 60°C for 24 h then homogenized into a fine powder. Algae and feces were dried without prior homogenization. All samples were weighed in tin cups and combusted with an elemental analyzer (FlashEA 1112) connected to an isotope ratio mass spectrometer (Thermo Finnigan Delta Plus Advantage). Carbon and nitrogen isotope data are reported as dX values (where X represents the heavier isotope 13C or 15N) or differences from the given standards, expressed in parts per thousand (%) and are calculated according to the formula: dX = [Rsample/Rstandard - 1] 9 103,
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where Rsample = 13C/12C or 15N/14N of sample, Rstandard = 13C/12C or 15N/14N of standard. Analytical precision and calibration of reference gas CO2 (for d13C measurements) to PDB was evaluated by repeated analysis of certified reference material BCR 657, which gave an average d13C: mean ± SD = -10.76 ± 0.08% (certified value: mean ± SD = -10.76 ± 0.04%). For calibration of reference gases N2 (d15N measurements) to air the IAEA N-1 standard was used and had an average d15N: mean ± SD = 0.4 ± 0.2%. Because lipid content can confound interpretation of d13C data, in order to compare across tissue types that varied in their lipid content (maximum C/N in eggs = 6.2), we corrected animal tissue d13C using a proxy for lipid content (C/N), with formulas from Logan et al. (2008). Because C/N is a poor predictor of % lipid in plant material (Post et al., 2007), we did not lipid correct samples of algae, leaf litter, gut contents or excrements, all of which consisted wholly or mainly of plant material. To describe the d15N and d13C values for all samples, the arithmetic mean ± 1 SD was used. A Wilcoxon’s signed ranks test was used to compare isotope results of paired groups. A Kruskal–Wallis test, with a post-hoc multiple comparison test was applied for comparing multiple independent groups (Siegel & Castellan, 1988). Results were considered significant if P \ 0.05.
Results Carbon and nitrogen stable isotope ratios and C/N ratios of samples varied according to tissue type (Table 1). Overall d13C values were significantly different among toes of adults, eggs, embryos, and tadpoles (Kruskal–Wallis H = 44.81, d.f. = 3, P \ 0.0001). Toes of adults, eggs, and embryos were consistently enriched in d13C compared with tadpoles (Table 1, P \ 0.0001). Similarly, d15N values were significantly different among toes of adults, eggs, embryos, and tadpoles samples (Kruskal–Wallis H = 25.18, d.f. = 3, P \ 0.0001); the mean d15N value for tadpoles was significantly higher than for eggs (P \ 0.0001) and embryos (P \ 0.005). Early stage tadpoles had d13C that was similar to adults, eggs, and embryos. However, after the onset of feeding (stages 25–27), d13C rapidly approached
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90 Table 1 Stable isotopes of carbon and nitrogen (mean ± 1 SD) in the ontogenetic stages of R. temporaria, potential food sources, excrements and fractionation in tadpoles
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Type of sample
n
d13C (%)
d15N (%)
C/N
Terrestrial Toe of adult
9
-24.33 ± 0.6
3.9 ± 1.2
3.24 ± 0.4
Aquatic Egg
28
-25.16 ± 1.0
1.84 ± 1.9
5.53 ± 0.6
Embryo
32
-25.35 ± 0.8
2.5 ± 1.7
4.71 ± 0.5
Tadpole (all stages)
20
-31.64 ± 3.8
4.28 ± 1.4
4.31 ± 0.4
21 13
-31.74 ± 1.2 -34.23 ± 4.1
1.89 ± 2.0 0.7 ± 3.0
19.24 ± 6.9 9.03 ± 3.8
13
-30.25 ± 1.6
5.3 ± 1.5
9.78 ± 1.7
Aquatic/submerged food sources Litter Algae Excrements of tadpoles Tadpole early stages (G25–27) Body tissue
6
-26.35 ± 1.1
3.42 ± 1.5
3.93 ± 0.2
Gut with contents
6
-28.76 ± 0.8
2.23 ± 1.1
6.14 ± 0.6
Tadpole late stages (G31–39) Body tissue
6
-34.76 ± 0.7
5.41 ± 0.7
3.74 ± 0.1
Gut with contents
6
-34.72 ± 0.4
4.35 ± 0.5
6.37 ± 0.9
the isotopic value for available food in the wetland (Fig. 1). Overall, there was no difference in d13C values between tadpoles and potential food sources (algae, litter) (Kruskal–Wallis test, P [ 0.05) (Table 1). However, tadpoles had higher d15N than both algae and litter, differing by 3.6 and 2.4%, respectively, and these values were significantly different (Kruskal–Wallis H = 19.1, d.f. = 2, P = 0.0001; post-hoc multiple comparison test, P = 0.0004 and P \ 0.0001, respectively).
Fig. 1 Ontogenetic changes in stable carbon isotope ratios of Rana temporaria. Toes clipped from adults were similar to eggs, embryos, and tadpoles of early stages (closed circles, stages 25–27). Older tadpoles (from 29 stage) approached isotopic equilibrium with a diet of algae (closed triangle). Average values are shown with 1 SE bars
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Excrements had higher mean d13C values than tadpoles; this difference was significant in matched pairs of tadpoles of later stages (stages 31–39) and their excrements (Wilcoxon test, W = 0, P = 0.002, Fig. 1). d15N values and C/N ratios were also significantly different between tadpoles and excrements (Wilcoxon tests, W = 0, P = 0.002, Fig. 2). Excrements were more enriched in d13C and d15N compared to potential food sources (Table 1). Overall
Fig. 2 Ontogenetic changes in stable nitrogen isotope ratios of Rana temporaria. Toes clipped from adults were similar to eggs, embryos and tadpoles of early stages (closed circles, stages 25–27). Older tadpoles (from 29 stage) approached isotopic equilibrium with a diet of algae (closed triangle), including a 3.6% diet-tissue fractionation. Average values are shown with 1 SE bars
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d13C and d15N values were significantly different among excrements, litter and algae (Kruskal–Wallis tests: H = 9.86, d.f. = 2, P = 0.0072 and H = 19.16, d.f. = 2, P = 0.0001, respectively). Although post-hoc multiple comparison tests revealed differences between specific samples (P \ 0.05), the mean d13C value for excrements was more similar to litter than it was to algae (Table 1; Fig. 1). The mean d15N value for excrements was significantly higher than for litter (P \ 0.001) and algae (P \ 0.0005) (Fig. 2). In early stages of tadpoles d13C values in body tissue were consistently enriched relative to guts. d13C values were significantly different among body tissue of tadpoles and gut contents in early stages (25–27) (Wilcoxon test, W = 0, P \ 0.05), however, there were no difference in late stages (31–39). In both early and late stages of tadpoles d15N values were significantly higher in body tissue compared to guts (Wilcoxon tests, W = 0, P \ 0.05). C/N ratios were significantly lower in body tissue compared to guts (Wilcoxon tests, W = 0, P \ 0.05) in both early and late stages of tadpoles.
Discussion During their life history anurans undergo ontogenetic habitat (aquatic–terrestrial) and also dietary (herbivory–carnivory) shifts. These shifts during amphibian life stage transitions may be accompanied by marked changes in their isotopic signatures, as primary aquatic and terrestrial production tends to differ in d13C (e.g., Rau, 1980); while stepwise changes in trophic position are revealed by d15N (Minagawa & Wada, 1984). We found that d13C values in late stage R. temporaria tadpoles were markedly depleted in comparison with adults. Because the sampled wetland was largely dry by the end of summer, the diet of adult frogs in summer and autumn is composed of terrestrial insects (adult Tipulidae, Lepidopteran larvae) and gastropods (slugs) (Houston, 1973). While the d13C of toe samples from R. temporaria was significantly enriched in 13C compared to our litter samples, both adult toes and litter samples had values that were within the range of globally observed values for terrestrial d13C (-28 ± 0.9% SD, Finlay, 2001). Furthermore, adult toes were similar isotopically to invertebrates from temperate forests (Ponsard & Arditi, 2000); their enriched
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values are likely because they contain some inorganic C in bone fragments that is enriched in 13C relative to muscle (Kelly et al., 2006). Recently, a similar distinction between terrestrial and aquatic sources was also recorded for R. clamitans with adults having d13C = -28 to -26% and tadpoles having d13C = -36 to -30% (Jefferson & Russell, 2008), indicating a transition from terrestrially to aquatically derived carbon. Also, R. temporaria eggs and embryos had d13C that was similar to adults, demonstrating maternal inheritance of egg lipid and protein (Jardine et al., 2008) and suggesting a possibility for eggs and embryos to be used in ecological studies as signatures of terrestrially derived material and tracers of energy transfer through aquatic food webs. Body tissues in early stages of tadpoles were higher in d13C relative to their guts with contents because of the inherited carbon from terrestrially feeding carnivorous adults (Houston, 1973). Similarity of d13C values between body tissue and guts in late stages of tadpoles implies that, just before the start of toe development (at stage 31), body tissues have reached isotopic equilibrium with the diet. Both early and late stages of feeding tadpoles had d15N values that were higher in body tissue compared to guts with contents, likely due to fractionation between the nitrogen in the food (litter, algae) and body tissues. However, fractionation of nitrogen in body tissue compared with gut content samples was less (average of 1.2 and 1.1% for early and late stages, respectively) than typically expected (3.4 ± 1%, Post, 2002) for the next trophic level. Disparity in both d13C and d15N between guts and diet sources likely occurs because gut samples contained some tadpole gut tissue along with gut contents, thus causing the guts to more closely resemble body tissue (Jardine et al., 2005). The development of anuran larvae is characterized by two periods where growth slows and significant development occurs: embryogenesis through hatchling (1–24 stages) and premetamorphic stages ([35 stages); however, from the feeding stage (25th) until the premetamorphic stages tadpoles show elevated growth and very little development (Alford & Jackson, 1993; McDiarmid & Altig, 1999). This latter period is concordant with the observed step depletion of d13C in common frog tadpoles between 27 and 30 development stages, indicating a shift to exogenous feeding. This alteration in isotope ratios
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during rapid growth can be attributed to a ‘‘dilution’’ of the previous ratio (maternally derived terrestrial carbon in the egg) by added tissue of differing isotopic composition (the tadpole diet) (Jardine et al., 2004, 2008). The similarity in d13C between early stage tadpoles (25–27) and eggs, embryos, and adult toes suggests that while feeding may have begun, turnover has not yet occurred in tadpoles at these body sizes (typically 25–50 mg of wet mass). Alternatively, consumption of egg jelly with a similar isotopic composition as inherited carbon may occur and not be detected in the tissues at these early stages. However, egg jelly contains very little C and N, and thus consumption of egg jelly may be done to target a thin layer of algae that grows on the surface. Algaecovered floating remains of egg jelly is typically used for food by newly hatched common frog tadpoles (Loman, 2009), while older tadpoles may consume a considerable amount of litter (our observation). A difference in the mean d15N values between the guts with contents of early and late tadpole stages (Table 1) also suggests a shift in diet during early ontogeny of R. temporaria tadpoles. While nitrogen signatures can provide key insights into the trophic position of consumers (Peterson & Fry, 1987; Post, 2002), they may also be influenced by eutrophication of aquatic habitats (Karr et al., 2001; Jefferson & Russell, 2008) that tends to elevate d15N, or they may simply represent different food types that differ in their d15N despite being at the same trophic level (Bunn et al., 2003). Together, this makes interpretation of nitrogen isotopic signatures difficult in amphibians, whose biphasic life history is marked by habitat and dietary shifts during ontogeny. Enrichment of d15N by C3% (one trophic level) would be expected if adult common frogs are at higher trophic level than tadpoles. However, our adult data showed depletion in 15N in comparison with tadpoles that possibly reflects differences in baseline d15N between aquatic and terrestrial habitats, or changes in diet-tissue fractionation upon transition to adulthood. Jefferson & Russell (2008) found that green frog tadpoles in wetlands with high nitrate concentrations had greater d15N values than adults, while tadpoles from wetlands without N enrichment had lower d15N values. The authors suggested that enriched d15N signatures of green frog tadpoles compared with adults can be used as a relative measure of eutrophication in ponds. Because there
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were clear indications of eutrophication in the studied wetland (e.g., odor, abundant sediments, floating organic material, macrophyte cover), we suggest that elevated d15N values in tadpoles were likely due to N enrichment in the wetland. Alternatively, relatively high d15N in tadpoles may signal the presence of animal tissue in the diet. Evidences of omnivory in R. temporaria tadpoles have been documented by several authors. Banks & Beebee (1987) reported R. temporaria tadpoles feed on toad eggs, and insects and cladocerans have been found in gut contents of tadpoles (Savage, 1961), possibly due to scavenging. In our study, however, common frog tadpoles had diet-tissue fractionation relative to algae and litter of 3.6 and 2.4%, that is typical of trophic fractionation (average of 3.4%, Post, 2002), suggesting that algae and litter were the likely contributors to tadpole tissue. In the study system, there were few animal materials available for consumption by tadpoles. R. temporaria eggs hatch synchronously, so tadpoles would not obtain sufficient size to consume conspecifics, and eggs or tadpoles of other frog species are not present in the wetland. Also, tadpole gut contents had C/N [6 (Table 1), higher than would be expected if animal tissue formed a considerable fraction of the diet. Most temperate Rana tadpoles are generalized algal feeders (Seale, 1980), however, important food categories also include detritus, e.g., decomposed higher plants (Jenssen, 1967). That R. temporaria tadpoles were consuming litter was evident during this study, based on visual observations in the field and laboratory. Isotopic data showed that tadpole excrements and body tissue diverged, with mean d13C values of excrements consistent with litter and body tissue d13C similar to algae (Fig. 1). This implies that tadpoles were ingesting both terrestrial detritus and algae, but they were mostly excreting the detritus and assimilating the algae. This is likely due to differences in food quality: C/N ratios of algae were lower than litter (9.0 ± 3.8 vs. 19.2 ± 6.9, respectively) suggesting better food quality of algae (Elser et al., 2000). Nutrient concentrations in leaf litter are generally low (Bowen, 1987) and survival, growth, and development of some Rana tadpoles are worse when they were fed on a leaf litter diet compared with algae (Iwai & Kagaya, 2005). Greater availability of high quality algae can therefore reduce time spent foraging and increase size at metamorphosis, reducing predation
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risk (Anholt & Werner, 1998; Alvarez & Nicieza, 2002). While algal resources are critical in the early life stages of anurans, particularly during those developmental stages when growth rates are high (25–35), anurans may also play a role in the processing of leaf detritus in temporary aquatic habitats. In shaded, forested wetlands (e.g., our study site) leaf litter is often the main energy source for other consumers in food webs (Pieczynska, 1986; Oertli, 1993), thus consumption and processing of large amounts of litter by tadpoles, despite its non-assimilation, may have a direct or indirect effect on detritivore food webs in such habitats (Moore et al., 2004). Uncertainty remains about the diet of adults, given that their d15N is lower than those of tadpoles despite presumably feeding at a higher trophic level (Kam et al., 1995). Data for terrestrial insects and captive rearing experiments with adults on a diet of known isotopic composition would be useful in determining the mechanism for such low d15N in a known carnivore. Other studies of riparian insects have reported d15N (-3 to 2%) and d13C (-28 to -26%) values (Collier et al., 2002; Walters et al., 2008) consistent with the expected diet that we observed here for adult frogs. While some uncertainty remains about adult feeding, this study has demonstrated clearly that a shift in energy occurs from egg-derived protein that was likely derived from terrestrial sources to exogenous algal feeding, and the relative importance of litter and algae in fueling growth during these stages.
Conclusion Our study has shown that (1) carbon, likely of terrestrial origin, is present in eggs and embryos, representing an energy subsidy to aquatic systems, and (2) algal carbon (not litter) is more likely to be responsible for tadpole growth prior to metamorphosis and outmigration as adults. These findings highlight the dynamic nature of resource availability at the aquatic–terrestrial interface and the importance of fluxes across ecosystem boundaries as mediated by anurans. We encourage more lab- and field-based experiments to determine elemental turnover rates in different anuran tissues and to measure the allocation of nutrients to growth of somatic and reproductive
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tissues, further refining the ecological role and limits to production of these important species. Acknowledgments We thank Rasa Gvozdait_e for assistance in the lab, Jolanta Kostkevicˇien_e for identifying algae, and Laura Jardine for providing helpful comments on the manuscript. This research was partially supported through a Vilnius University research program (Ecosystems’ and Climate Changes, Preservation of Environment and Use of Natural Resources) to Giedrius Trakimas.
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