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Department of Physics, Chemistry and Biology Final Thesis Does an evolutionary change in the water sowbug Asellus aquaticus L. alter its functional role? Md. Maidul Islam Choudhury LiTH-IFM- Ex--11/2419--SE Supervisor : Anders Hargeby, Linköping University Examiner : Bo Ebenman, Linköping University Department of Physics, Chemistry and Biology Linköpings universitet SE-581 83 Linköping, Sweden 1 Upphovsrätt Detta dokument hålls tillgängligt på Internet – eller dess framtida ersättare –från publiceringsdatum under förutsättning att inga extraordinära omständigheter uppstår. Tillgång till dokumentet innebär tillstånd för var och en att läsa, ladda ner, skriva ut enstaka kopior för enskilt bruk och att använda det oförändrat för ickekommersiell forskning och för undervisning. Överföring av upphovsrätten vid en senare tidpunkt kan inte upphäva detta tillstånd. All annan användning av dokumentet kräver upphovsmannens medgivande. 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Maidul Islam Choudhury 2 Presentation Date Department and Division IFM Biology 27.05.2011 Publishing Date (Electronic version) 07.02.2012 Language Type of Publication ISBN (Licentiate thesis) × English Other (specify below) Licentiate thesis × Degree thesis Thesis C-level Thesis D-level Report Other (specify below) ISRN: LiTH-IFM- Ex--11/2419--SE Number of Pages 23 Title of series (Licentiate thesis) Series number/ISSN (Licentiate thesis) URL, Electronic Version http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva- xxxxx Publication Title Does an evolutionary change in the water sowbug Asellus aquaticus L. alter its functional role? Author(s) Md. Maidul Islam Choudhury Abstract The ecology behind evolutionary diversification is a well studied area of research, whereas the effects of evolution on ecosystems get little attention. In line with ecological theory, evolutionary diversification of a species could influence different ecosystem aspects such as food web composition, energy flow, nutrient cycling etc. The main objective of this study was to investigate whether two diverging ecotypes (reed and chara) of Asellus aquaticus differ regarding their role in two aquatic ecosystem processes: decomposition of terrestrial leaves and grazing of periphyton. Their role in ecosystem process as well as treatment effects on fitness, measured as growth and survival, were investigated in a laboratory experiment with various levels of intra-specific competition and inter-specific interactions with the amphipod Gammarus pulex. The isopods were collected from two Swedish lakes: Lake Tåkern and Lake Fardume. These two lakes represent different history of ecotype divergence. The experimental design consisted of 2-L aquaria, each providing elm leaves (Ulmus glabra), oak leaves (Quercus roburleaves) and periphyton as food sources. Ten treatments with five replicates were applied for each lake and the experiment lasted for four weeks. The study showed that there was no significant difference between chara and reed ecotype in their functional role. However, the rate of ecosystem processes per individual decreased in competitive interactions. In high density, decomposition per dry weight consumer was low and total algae biomass was high at the end of four weeks due to intra-specific competition. Moreover, ecosystem processes were lowest in inter-specific competition between Gammarus pulex and each ecotype. Present study also shows that ecotypes from the different lakes, having different history, had different responses to mortality and growth. Keywords Asellus aquaticus, eco-evolutionary dynamic, ecosystem process, Gammarus pulex, intra-specific, inter-specific. 3 1 2 3 4 5 6 7 8 Abstract ……………………………………………………………………………….. Introduction …………………………………………………………………………… 2.1 Objective of the project ………………………………………………………. Material and methods ...……………………………………………………………….. 3.1 Study organisms …...…………………………………………………………. 3.2 Periphyton ……………………………………………………………………. 3.3 Leaves ………………………………………………………………………... 3.4 Experimental set-ups …………………………………………………………. 3.5 Measurements of periphytic algae biomass, decomposition and animal interactions …………………………………………………………………… 3.5.1 Algae biomass ………………………………………………………. 3.5.2 Decomposition ……………………………………………………… 3.5.3 Species interactions ………………………………………………… 3.6 Statistical analysis ……………………………………………………………. Results ………………………………………………………………………………… 4.1 Periphytic algae biomass ……………………………………………………... 4.2 Leaf decomposition …………………………………………………………... 4.3 Mortality ……………………………………………………………………… 4.4 Growth ……………………………………………………………………….. Discussion …………………………………………………………………………….. 5.1 Preiphytic algae biomass ……………………………………………………... 5.2 Leaf decomposition …………………………………………………………... 5.3 Mortality ……………………………………………………………………… 5.4 Growth ……………………………………………………………………….. Conclusion ……………………………………………………………………………. Acknowledgement ……………………………………………………………………. References …………………………………………………………………………….. 4 1 1 3 3 4 4 4 4 5 5 5 6 6 6 6 8 10 11 13 13 14 14 15 15 15 15 1 Abstract The ecology behind evolutionary diversification is a well studied area of research, whereas the effects of evolution on ecosystems get little attention. In line with ecological theory, evolutionary diversification of a species could influence different ecosystem aspects such as food web composition, energy flow, nutrient cycling etc. The main objective of this study was to investigate whether two diverging ecotypes (reed and chara) of Asellus aquaticus differ regarding their role in two aquatic ecosystem processes: decomposition of terrestrial leaves and grazing of periphyton. Their role in ecosystem process as well as treatment effects on fitness, measured as growth and survival, were investigated in a laboratory experiment with various levels of intra-specific competition and inter-specific interactions with the amphipod Gammarus pulex. The isopods were collected from two Swedish lakes: Lake Tåkern and Lake Fardume. These two lakes represent different history of ecotype divergence. The experimental design consisted of 2-L aquaria, each providing elm leaves (Ulmus glabra), oak leaves (Quercus roburleaves) and periphyton as food sources. Ten treatments with five replicates were applied for each lake and the experiment lasted for four weeks. The study showed that there was no significant difference between chara and reed ecotype in their functional role. However, the rate of ecosystem processes per individual decreased in competitive interactions. In high density, decomposition per dry weight consumer was low and total algae biomass was high at the end of four weeks due to intra-specific competition. Moreover, ecosystem processes were lowest in inter-specific competition between Gammarus pulex and each ecotype. Present study also shows that ecotypes from the different lakes, having different history, had different responses to mortality and growth. Key words: Asellus aquaticus, eco-evolutionary dynamic, ecosystem process, Gammarus pulex, intra-specific, inter-specific. 2 Introduction Evolutionary Biology mainly focuses on the ecological explanation of evolutionary diversification, whereas the effects of evolution on ecosystems get little attention (Thompson 1998, Fussmann et al. 2007, Yoshida et al. 2003). Although, some ecologists in 1950s and 1960s formally hypothesized the influence of evolution on ecology, it took time for biologists to test these influences specifically. Industrial melanism in peppered moths (Biston betularia) is perhaps the best well known example of ecological influence on evolution (Pelletier et al. 2009). Previous studies on various systems, such as sparrows in North America (Johnston & Selander 1964), mice in islands (Berry1964), Caribbean Anolis lizard (Losos 2009), Galapagos finches (Grant and Grant 2008) or African cichlids (Seehausen et al. 2008) concentrated on how environmental factors regulate the speciation and adaptation of species. Moreover, recent studies (Schluter 2000; Seehausen et al. 2008) revealed that adaptive radiation is influenced by ecological factors. However, Luke et al 2009 showed that adaptive radiation also significantly affects the ecosystem over a short period of time. In line with ecological theory, evolutionary diversification of a species can shape different ecosystem aspects such as predator–prey interactions, community composition, food web structure, energy flow, nutrient cycling, productivity etc. (Luke et al 2009; Pelletier et al. 2009; Loreau et al. 2001; Schluter 2000; Seehausen et al. 2008 ;Abrams 2000; Palkovacs and Post 2009). To regret, generally, little attention is paid to the influence of evolution on ecology at short-time scale. Pelletier et al. 2009 mentioned two general reasons behind it: the challenges of measuring feedbacks between evolution and ecology; scientists’ assumption that evolutionary process is too slow to exert changes in ecological processes. However, many empirical studies on contemporary evolution and eco-evolutionary dynamics have recently 5 presented, for example, on the Darwin finches (reviewed by Hairston et al. 2005), Trinidadian guppies (Palkovacs et al. 2009), Alewives study (Post & Palkovacs 2009), Populus tree (Bailey et al. 2009) and interaction between rotifer and algae (Yoshida et al. 2003; Jones et al. 2009), that evolution can occur in nature within short time-scale which has potential to change the ecosystem processes. Like most biological processes genes, the evolution of phenotypic traits and demographic rates are interacting as an integrated system (Pelletier et al. 2009). Changes in one part can influence many levels (Coulson et al. 2006a) of the system. This can be expressed in many ways (e.g. Coulson et al. 2006a; Fussmann et al. 2007; Bailey et al. 2009; Post & Palkovacs 2009), but the main theme is a relationship among fitness, phenotype and genes which could be illustrated as a simple eco-evolutionary dynamics (Fig. 1). Natural selection and population dynamics are intimately associated (Coulson et al. 2006b; Pelletier et al. 2009). For example, if natural selection translates into changes in phenotypic traits (e.g. body size, pigmentation), that will effect population through reproduction, survival or growth. Thus, in turn, natural selection will influence population dynamics. Finally, many ecosystems services e.g. decomposition, nutrient cycling and, primary productivity are modified due to changes in the numbers of individuals, their utilization of available resources and efficiency of resource utilization (Thompson 1998) and interaction with other species in the food web . Accordingly, laboratory experiments and field studies of natural population suggest habitat quality mediated genetic variance (reviewed in Hoffmann & Merilä 1999; Charmantier & Garant 2005). Evolutionary changes of phenotypic traits (e.g. body size, color) Ecosystem aspects (Decomposition, primary production, intra and interspecific interactions) Population dynamics (e.g. Reproduction, survival, growth) Figure 1: Conceptual framework of eco-evolutionary dynamics. In freshwater ecosystems there is a wide range of interactions between individuals and species for instance in macroinvertebrate communities (Wellborn 2002; Wellborn et al. 1996). Beside predation, competition itself has potential to affect species composition or community structure and species identity (Brown 1982). In high density, individuals within a species compete with each other for available resources (e.g. food) whereas inter-specific competition occurs in presence of more than one species. An interference effect results from 6 heavy intra-specific competition among the individual within a species as well as interspecific competition between two or more species. In this case, increased density of interacting animals reduces the feeding or grazing rate. (Brown et al 1994, Soluk 1993). On the contrary, interaction hypothesis (Jonsson and Malmqvist 2000) states that intra-specific competition will be reduced in an addition of another species which in turn, will decrease the biomass of shared resources in great extent. Same scenario can be obtained from facilitation where one or more species is benefited from foraging together (Wilson et al 1999, Jonsson and Malmqvist 2003). So, intra- and inter-specific interactions have not only negative effect but also positive effect on growth, survival or fecundity. However, the result of competition is hard to predict because of its dependency on type of interaction and involved species (Hertonsson 2010). Recent studies show that the freshwater isopod Asellus aquaticus (Crustacea) differ locally in pigmentation within two Swedish lakes, Lake Tåkern and Lake Krankesjön (Hargeby et al. 2004; 2005; Eroukhmanoff et al. 2009). Both lakes have experienced periodical shift between submerged vegetation dominance and phytoplankton dominance (Blindow et al. 1993). In these lakes, isopods show lighter pigmentation in submerged vegetation dominated by stoneworts (Chara spp.) whereas, in adjacent stands of reed (Phragmites australis) and great fen sedge (Cladium mariscus L.) dark isopods are abundant. In addition, the bright A. aquaticus population is smaller in size than that of dark population (Eroukhmanoff et al. 2009). These different phenotypes resulted from evolutionary responses to local differences in natural selection. Hargeby et al. 2004 showed that this phenotypic difference has a genetic basis where diet and chromatophore adjustment to background color play a minor role. Moreover, they summarized that this genetic difference occurred during the colonization in a novel habitat, a situation that is known to promote adaptive evolution. In addition, rapid parallel divergence in the source habitat (reed stands) as well as phenotypic convergence in novel habitat (stonewotrs habitat) were also documented in these two lakes (Eroukhmanoff et al. 2009). In general, microevolutionary changes can occur in nature over short time-scales (Reznick and Ghalambor 2001). Lake Tåkern and Krankesjön shifted from phytoplankton dominant state to macrophyte state during the last 20 years and two ecotypes (reed ecotype and stoneworts ecotype) of Asellus emerged within this time-scale (Hargeby et al. 2004). However, similar differentiation has also been noted in other lake, Lake Fardume, with a different history (Hargeby et al. 2004). Lake Tåkern experienced alternative stable state during its lake-history. It became phytoplankton dominant (turbid stage) during 1995-1997 (Blindow et al. 1998) and again colonized by Chara spp. (clear water stage) in 1999 (Hargeby et al. 2004). However, Lake Fardume does not experience such periodic shift. It is always in clear water stage during its lake-history. Moreover, genetic distance between the populations of two ecotypes of Asellus is higher between Lake Tåkern and Fardume than within the lakes (Eroukhmanoff et al. 2009). So, in this study Lake Tåkern and Lake Fardume were selected for isopods collection because of their different lake history. This will also provide an opportunity to examine whether lake of origin has any signature in functional role as well as fitness of the both ecotypes of Asellus. Asellus aquaticus is a general freshwater isopod found in shallow lakes, ponds and slowflowing waterbodies in Europe and Asia (Whitehurst, 1991; Verovnik et al., 2005). Asellus are commonly detritivores (Smock and Harlowe 1983) and feed on decaying leaves (Zimmer and Bartholme´ 2003). This food source is likely to be important in reed habitat, where bottom substrate is mainly decaying parts of emergent vegetation (Hargeby et al. 7 2005). However, isopods colonized in stonewort habitat are more likely to feed on epiphytic algae e.g. periphyton (Arakelova 2001). If these expectations of local differences in feeding are true, the colonization in new habitat changed the functional role of the isopods, from detritivores to mesograzers. 2.1 Objective of the project The main objective of this project was to identify whether the evolutionary diversification of Asellus aquaticus affects its ecological function. My hypothesis was that these two ecotypes differ in their role as a grazer of epiphyton, or detritivore on coarse detrititus such as leaves. The role was tested both as the ecotype was living alone and in co-existence with other ecotype and with another macroinvertebrate, amphipod Gammarus pulex. 3 Materials and methods Lake Tåkern (58o 21´ N, 14o 50´ E) and Lake Fardume träsk(57o46´ N, 18o54´E) are located in Sweden. Both lakes are shallow and Lake Fardume is more oligotrophic in nature. Data on water chemistry is available on the web at http//www.ma.slu.se. Isopods were collected randomly from the following locations: in Lake Tåkern chara ecotype was collected from 58 o35´ 04´´ - 14 o 78´07´´ and reed ecotype from 58 o 33´21´´-14 o 81´88´´; in Lake Fardume chara ecotype was collected from 57 o 78´47´´ - 18 o 91´28´´ and reed ecotype from 57 o 78´03´´-18 o 91´07´´. 3.1 Study organisms Two ecotypes (Reed and Chara) of Asellus were collected from both lakes. Amphipod Gammarus pulex was collected from near by stream of Linköping University. No specific empirical study has done about the food of these two ecotypes of Asellus. Although it is assumed that reed ecotype is predominantly detritivore and periphyton is regarded as important food source for chara ecotype. So, my assumption was that chara ecotype acts as grazer whereas, reed ecotype is detritivore. However, the amphipod Gammarus pulex is both grazer and detritivore (Gladhill et al. 1993). In each lake, reed ecotypes were collected from reed stands while chara ecotypes from adjacent stonewort stand. All experimental animals were collected by means of hand net. Macroinvertebrates were kept in the laboratory in separate aerated aquaria filled with water from Lake Tåkern, supplied with periphyton and dead leaves as food. All experimental animals kept at least 3 week before the experiment start in order to adapt to the laboratory conditions. 3.2 Periphyton Tiles of (100x100) mm size were prepared to act as a refuge and substrate for periphyton. In order to offer shelter to the test animals the tiles were elevated approximately 10mm by fixing small silicon rubber tube with silicon glue. In each tube sufficient opening was made by means of cutting to avoid death of isopods due to trapping inside the tube. Four weeks prior to the experiment start one tile was placed in each experimental aquarium. Initially, periphyton was collected from plants from Lake Tåkern and the surface of rocks from the stream where Gammarus pulex was collected. Periphyton was then mixed into an algae inoculum that was added to each experimental aquarium along with fluid fertilizer (Blomstra, Cederroth, Sweden; P:N:1:5) to the concentration of 50μg P l-1 and 250 μg N l-1 (Hertonsson 2010). As the periphyton stopped growing before the start of the final experiment, the aquarium-water was replaced by filtered (400 µm mesh) lake water from Tåkern. The idea was to maintain more natural condition in term of water quality and nutrient. At the end of the experiment 8 periphyton on tiles was cleaned with a soft toothbrush and rinsed with dechlorinated tap water into separate containers then filtered onto a Whatman GF/C filter (pore size 1.2µm). Chlorophyll a was analysed according to the methodology of Jespersen and Christoffersen (1987) with extraction with ethanol and absorbance measured at 750 and 665 nm by means of a Hitachi spectrophotometer (model: U-2000). 3.3 Leaves Elm leaves (Ulmus glabra) and oak (Quercus roburleaves) leaves were collected recently fallen to the ground in September and November respectively, and dried in 60oC for 48 h. Then they were soaked so that they would not float and shad the periphyton covered tile. One week before to the experiment start, 0.99 (±0.03) g(DW) elm and 0.38 (±0.03) g (DW) oak of soaked leaves were added to each experimental aquarium. Oak leaves were added to provide secondary food source as they decay slowly compare to elm leaves. At the end of the experiment leaves were again dried in 60oC for 48 h and weighed. Leaf weight was adjusted in term of moisture absorption by using standard leave weight. 3.4 Experimental set-ups The experiment was conducted in the green house at Biology Department, Linköping University over four weeks between November and December, 2010. For each lake there were 10 different treatments. Protocols were: low density single species/ecotype (6 individuals); high density single ecotype/species (18 individuals); combination of both ecotypes (6 individuals from each); a combination of one ecotype and Gammarus pulex (6 individuals of each.) and three-form combination of both ecotypes and Gammarus pulex (6 individuals of each.) (Table 1). All the treatments were replicated five times for each lake. So, the experimental set-up was consisted of total 100 aerated plastic experimental aquaria (l x w x d: 200 x 170 x 100 mm) filled with 2 L of filtered (400 µm mesh) lake water from Tåkern. Each aquarium accommodated one periphyton covered tile and leaves. The aquariums were grouped into ten blocks and placed on two tables. Each block contained all the ten treatments. First table contained 3 blocks from Lake Tåkern and 2 blocks from Lake Fardume and second table contained 3 blocks from Lake Fardume and 2 blocks from Lake Tåkern. Table 1. Schematic illustration of the experimental set-up (C = chara ecotype of Asellus aquaticus (grazer), R = reed ecotype of Asellus aquaticus (detritivore), G = Gamarrus pulex (grazer- detritivore)) Treatment C R G C+R C+G R+G C+R+G 3xC 3xR 3xG No. of individual 6 6 6 6+6 6+6 6+6 6+6+6 6+6+6 6+6+6 6+6+6 In every second day 50ml of fish cue was added in each aquarium to maintain natural predation environment in the aquarium (Hertonsson 2010). Fish cue was collected from a water tank containing Perch (Perca fluviatilis) which is a benthivorous fish species noted to feed on Asellus in both chara and reed habitats of shallow Swedish lakes (Rask & Hiisivuori 1985). The fishes were fed with live Asellus and Gammarus. The day night regime was set to 9 12:12 h. Water temperature was 17 oC to 19 oC. The light intensity above the aquaria was measured by irradiameter (Kahl scientific instrument corporation, USA, model: 268 WA 310). In the first table, containing the three of the five blocks, the average light intensity was 9057μW whereas, in second table it was 9596μW. The difference in radiation among the aquaria was anticipated to be minor and randomly distributed among treatments. 3.5 Measurements of periphytic algal biomass, decomposition and animal interactions 3.5.1 Algal biomass The biomass of the periphytic algae on the tile from each aquarium was measured in term of amount of Chlorophyll a (chl a)(μg) at the end of the experiment. 3.5.2 Decomposition At the end of the experiment the remaining leaves from each experimental aquarium was collected, dried and, weighed. The reduction of leaf mass (mg) was used as a measure of decomposition. Leaf decomposition was measured for both elm and oak leaves separately. Decomposition = (Leaf start- Leaf end)…………………. (1) As macroinvertebrate species differ in size, decomposition was calculated per dry weight (DW)(mg) consumer.The following length-weight relationships were used to for Asellus aquaticus to calculate DW of the isopods (Hass et al. 2007). ln (Weight) = -4.212+ 2.268* ln (Length)……………. (2) For Gammarus pulex, thirty individuals were photographed and their bend length was analyzed with ImageJ v. 1.37v. The following regression equation was constructed by BoxCox transformation of the response with specified lambda = 0 in Minitab 16. ln (Weight) = -1,24998 + 0,239452 Length………… (3) 3.5.3 Species Interactions As a measure of species interactions mean growth and proportional mortality were used. All experimental animals were photographed at the start and end of the experiment. Animal size (body length) was analyzed with ImageJ v. 1.37v (http://rsbweb.nih.gov/ij) (Franken et al. 2005). Growth and proportional mortality were calculated according to: Growth= (Length end -Length start)………………..(4) Proportional mortality = (n start – n end)/ n start ………………..(5) Where n= number of individuals 3.6 Statistical analysis First, one-way ANOVA analysis was done to check the general differences among the ten treatments (listed on Table 1) in the four response variables: periphytic algae biomass, decomposition, mortality and growth respectively. Then more specific analysis was done by means of three-way ANOVA to find out the specific effects, and their interactions, of lakes, ecotypes and three types of competition on the above response variables. More specifically, the factors were ecotype (chara and reed ecotypes of Asellus), lake of origin (Tåkern and 10 Fardume) and three competition treatments such as, low competition (C, R); intra-specific competition (3*C, 3*R); inter-specific competition (C+G, R+G). The lower number of Gammarus (6 indv.) added in the inter-specific treatment compared with Asellus (12 indv.) in the intra-specific treatment was compensated by a higher individual biomass of Gammarus. Following issues were examined through three-way ANOVA: functional differences between two ecotypes from both lakes; whether the ecotypes from both lakes show any competition effect (or density dependence) on the response variables mentioned above and finally, their responses to functional role and fitness in presence of an additional competitor, Gammarus pulex. Before the analyses were performed, data were tested for normal distribution (Anderson-Darling goodness-of-fit statistic) and equality of variance (Levenes test). In ANOVA, data from the treatments C+(R) (for mortality); R+(C+G) and 3*G (for growth) were not normally distributed but had equal variances (Levenes test). So, data from those treatments were not log transformed to avoid negative mortality and growth values in the model. All the statistical analyses were done in Minitab 16. 4 Results 4.1 Periphytic algal biomass There was no general difference between chara and reed Asellus ecotypes regarding effects on periphytic algal biomass on the experimental tiles at the end of experiment (Fig. 2; Table 2). Periphyton biomass was not significantly different among the single species/ecotype treatments (One-way ANOVA; C vs R, C vs G and R vs G; Fig. 2). The amount of algae biomass was significantly higher (Fig. 2) in both of the inter-specific treatments than single ecotype treatments (One-way ANOVA, F1,18=12.52, P=0.002 and F1,18= 7.92, P= 0.011 respectively). So, the presence of Gammarus pulex tended to increase the algae biomass on the tiles. The same pattern could be seen in the C+R+G treatment in which periphyton biomass was significantly higher (Fig. 2) than in the intra-specific Asellus treatments (3*C, 3*R) (Oneway ANOVA, F1,18= 4.61, P= 0.046 and F1,18=5.04, P= 0.038 respectively). However, Gammarus pulex showed no significant difference between inter-specific and intra- specific treatments (F1,18=0,56 and P= 0,464). 11 A three factor ANOVA showed no significant difference between chara and reed ecotypes in their effects on periphytic algae. Similar result was observed for the ecotypes from both Lake Tåkern and Lake Fardume (Table 2, Fig. 3a). However, the amount of chl a was significantly different among the three selected competition treatments (Table 2). It was highest in the inter-specific treatment, relatively lower in intra-specific treatment. In contrast, it was inconsistent in low-competition treatment for two ecotypes (Fig. 3b). Table 2. Three factors ANOVA on the effect of Asellus ecotypes, lakes from which Asellus were collected and competition (single ecotype at two densities and along with Gammarus pulex) on periphytic algal biomass at the end of the experiment. Source Ecotype Lake Competition Ecotype*Lake Ecotype*Competition Lake*Competition Ecotype*Lake*Competition Error Total DF 1 1 2 1 2 2 2 48 59 Seq SS 0.0000026 0.0000103 0.0013929 0.0000193 0.0000510 0.0000398 0.0000038 0.0024980 0.0040177 MS 0.0000026 0.0000103 0.0006964 0.0000193 0.0000255 0.0000199 0.0000019 0.0000520 F 0.05 0.20 13.38 0.37 0.49 0.38 0.04 P 0.823 0.658 0.000 0.545 0.616 0.684 0.964 In line with treatment effect (Fig. 2), both chara and reed ecotypes showed a positive impact on algal biomass when an additional competitor, Gammarus pulex, was added in the interspecific treatment (Fig. 3b). Similar pattern was observed in both lakes (Fig. 3c). Finally, no interaction effect of selected factors was evident for algal biomass (Table 2). 12 25 20 15 10 5 3* G 3* R 3* C C +R +G +G R +G C +R C G R 0 C Decomposition/DW(mg) 4.2 Leaf decomposition The experimental animals only fed on elm leaves during the four week of incubation and no decomposition of oak leaves was found at the end of the experiment. So, leaf decomposition/ DW consumer was measured for elm leaves only. I found no difference between chara and reed ecotypes in decomposition per DW (dry weight) consumer (Fig. 4 and Table 3). However, combined effect of chara and reed ecotypes on decomposition was significantly lower (One-way ANOVA, F1,18= 28.59; P<0.001 and F1,18=17.26; P<0.001) than their individual effect. Decomposition did not differ significantly when the two ecotypes co-existed with Gammarus pulex (Fig. 4; One-way ANOVA, F1,18= 0,25 and P= 0,623). In the high density treatments, both chara and reed ecotypes had significantly higher (One-way ANOVA, F1,18= Treatments Figure 4. Elm (Ulmus glabra) leaf decomposition/DW consumer at the end of experiment, after four weeks incubation. Means+sd, n=10 (abbreviations according to Fig. 2). 114.28; P<0.001 and F1,18= 113.59; P< 0.001) leaf decomposition in intra-specific treatment than inter-specific treatment. Gammarus pulex also had significantly higher leaf decomposition in intra-specific competition than inter-specific competition (Fig. 4). However, the individual effect of isopod and amphipod on leaf decomposition in inter-specific treatment was unknown and decomposition by microbes was not examined in the current study. Three factors ANOVA analysis also showed no significant difference in leaf consumption/DW consumer between chara and reed ecotypes irrespective of competition and lake history (Table 3). Lake of origin also showed no significant effect on leaf decomposition (Table 3 and Fig. 5a). However, the difference in leaf decomposition among the three selected competition treatments is highly significant (Fig. 5b and Table 3). In the low-competition treatment, chara as well as reed ecotype resulted in highest leaf consumption. However, the leaf decomposition/DW consumer in low-competition was marginally significantly higher for chara ecotype than reed ecotype (Table 3). But the three factor interaction among lake, ecotype and competition indicate that actually this tendancy may only relevant for one of the lakes. The presence of Gammarus pulex tended to decrease the decomposition process for both ecotypes. Finally, three-way interaction among the factors showed that both chara and reed ecotypes from Lake Fardume had high leaf consumption than those from Lake Tåkern when the ecotypes existed in low density. 13 Table 3. Three factors ANOVA on the effect of Asellus ecotypes, lakes from which Asellus were collected and competition (single ecotype at two densities and along with Gammarus pulex) on leaf decomposition/DW consumer. Source Ecotype Lake Competition Ecotype*Lake Ecotype*Competition Lake*Competition Ecotype*Lake*Competition Error Total DF 1 1 2 1 2 2 2 48 59 Seq SS 10,73 18,51 1214,86 0,01 42,28 2,39 50,98 360,97 1700,74 MS 10,73 18,51 607,43 0,01 21,14 1,20 25,49 7,52 F 1,43 2,46 80,77 0,00 2,81 0,16 3,39 P 0,238 0,123 0,000 0,971 0,070 0,853 0,042 4.3 Mortality In general, chara and reed ecotypes did not differ in mortality (Fig. 6; Table 4). One-way ANOVA results showed that chara and reed ecotypes did not have any impact on the mortality of each other. Similarly, Gammarus pulex had no significant influence on the mortality of any of the two ecotypes (Fig. 6) of Asellus. Three factors ANOVA showed that lake of origin had significant effect (Table 4) on the mortality of the isopods, but this effect differed between the ecotypes, shown by significant interaction between ecotype and lake. Mortality tended to be higher in the chara ecotype than reed ecotype among the isopods from Lake Tåkern, whereas, among the isopods from Lake Fardume the chara ecotype had lower mortality than reed ecotype (Fig. 7a; Table 4). So, ecotypes from different lakes had different responses to mortality. 14 1 0,8 0,6 0,4 0,2 3* G 3* R G C+ (R ) (C )+ R C+ (G ) (C )+ G R+ (G ) (R )+ C+ G (R +G R+ ) (C +G ) G+ (R +C ) 3* C R 0 C Proportional mortality The three selected competition treatment also had significant effect on the mortality of isopods (Table 4). This effect tended to differ in lake of origin which is indicated by significant interaction between lake and competition treatment but not in ecotype level (Table Treatments Figure 6. Proportional mortality of animals at the end of experiment, after four weeks incubation. Means+sd, n=10. Alphabet without parenthesis indicates the mortality of that species/ecotype in the combination (abbreviations according to Fig. 2). 4; Fig. 7b & 7c). Mortality was lowest in low-competition (or low density) treatment and higher in intra-specific competition (or high density) followed by inter-specific competition (or mixed species) treatment (Fig. 7b). Both ecotypes showed density dependence on mortality. However, the addition of Gammarus pulex tended to increase the difference on mortality between two ecotypes. Chara ecotype had higher mortality than reed ecotype when co-existed with Gammarus pulex. In general, lake of origin tended to influence the mortality of the isopods. 15 Table 4. Three factors ANOVA on the effect of Asellus ecotypes, lakes from which Asellus were collected and competition (single ecotype at two densities and along with Gammarus pulex) on the mortality of the ecotypes. Source Ecotype Lake Competition Ecotype*Lake Ecotype*Competition Lake*Competition Ecotype*Lake*Competition Error Total DF 1 1 2 1 2 2 2 48 59 Seq SS 0.02882 1.18363 0.17283 0.29454 0.00899 0.19888 0.03084 1.12018 3.03873 SS 0.02882 1.18363 0.17283 0.29454 0.00899 0.19888 0.03084 1.12018 MS 0.02882 1.18363 0.08642 0.29454 0.00449 0.09944 0.01542 0.02334 F 1.23 50.72 3.70 12.62 0.19 4.26 0.66 P 0.272 0.000 0.032 0.001 0.825 0.020 0.521 4.4 Growth Chara and reed ecotypes had no significant different in growth (Fig. 8 and Table 5). One-way ANOVA results showed no difference between chara and reed ecotypes both in low density (C vs R) and high density (3*C vs 3*R) (Fig. 8). Moreover, Gammarus pulex did not influence the growth of chara ecotype as well as reed ecotype. Similarly, chara and reed ecotypes did not influence the growth of one another. Additionally, in high density, intraspecific and inter-specific treatments had no significant impact on the growth of two ecotypes (Fig. 8). Growth(mm) 2,5 2 1,5 1 0,5 3G 3R 3C ) ) +C R G +( C+ G ) R +( R+ G )+ G C +( G ) (R +( R )+ G G ) (C +( )+ R C (C R) G C +( R C 0 Treatments Figure 8 Growth of animals at the end of experiment, after four weeks incubation. Means+sd, n=10. Alphabet without parenthesis indicates the growth of that species/ecotype in the combination (abbreviations according to Fig. 2). Three factors ANOVA showed significant lake effect (Table 5) on the growth of isopods. Isopods from Lake Tåkern showed higher growth than those from Lake Fardume (Fig. 9a). However, mean growth of chara ecotype was higher than reed ecotype among the isopods from Lake Tåkern whereas, mean growth of reed ecotype was higher than chara ecotype among the isopods from Lake Fardume (Fig. 9a). There was no significant effect of different competition treatments on the growth of both ecotypes (Table 5). However, the presence of Gammarus pulex in inter-specific competition influenced the growth of two ecotypes in opposite direction. 16 The effects of three selected competition treatments on isopods’ growth tended to differ between two lakes (Table 5). The growth of isopods was higher in Lake Tåkern for both intraspecific and low-competition treatments compare to Lake Fardume. However, in presence of Gammarus pulex, isopods from Lake Fardume had higher mean growth than those from Lake Tåkern. Three form interaction among ecotype-lake-competition showed that both chara and reed ecotypes from lake Tåkern had significant (Table 5) higher growth than those from lake Fardume when they existed in low density and high density. So, lakes had different effects on the growth of isopods. Table 5. Three factors ANOVA on the effect of Asellus ecotypes, lakes from which Asellus were collected and competition (single ecotype at two densities and along with Gammarus pulex) on the growth of the two ecotypes. Source Ecotype Lake Competition Ecotype*Lake Ecotype*Competition Lake*Competition Ecotype*Lake*Competition Error Total DF 1 1 2 1 2 2 2 48 59 Seq SS 0.0232 1.5806 0.1375 0.4493 0.5016 1.7292 2.4577 7.4919 14.3710 MS 0.0232 1.5806 0.0687 0.4493 0.2508 0.8646 1.2288 0.1561 F 0.15 10.13 0.44 2.88 1.61 5.54 7.87 P 0.702 0.003 0.646 0.096 0.211 0.007 0.001 5 Discussion 5.1 Preiphytic algal biomass The main objective of this study was to examine the functional role of two ecotypes of Asellus aquaticus. In term of feeding on periphytic algae, I found no significant difference between the two ecotypes in general. In addition, lake history generally had no effect on the feeding of algae by the two ecotypes. Both ecotypes showed almost similar impact on algal biomass in 17 response to different competition treatments. However, in the low density (low-competition) treatment, mean algal biomass on the tile was less (although not significant) for chara ecotype than reed ecotype (Fig 3b). This might have resulted from the observed higher mortality of chara ecotype than reed ecotype in low density treatment (Fig. 7b) as invertebrates add nutrient in water through excretion which also stimulate algae growth (Post & Palkovacs 2009). The presence of Gammarus pulex significantly increased the algal biomass in interspecific treatment than intra-specific or low-competition treatment for both ecotypes. This could be resulted from strong inter-specific interference effect between G. pulex and the two ecotypes of Asellus that inhibited the invertebrates to graze on the epiphytic algae. In contrast to my result, Hertonsson et al. 2008 in their experiment found that co-existed snails and mayflies significantly reduced the periphyton biomass compare to their single species treatment irrespective of density. However, G. pulex is both efficient detritivore and grazer and larger than Asellus aquaticus in body size. It may consume more algae and dead leaves and excrete more, which in turn would influence higher algae growth through nutrient input in the water. This idea also helps to explain higher algae biomass in inter-specific treatment for both ecotypes. 5.2 Leaf decomposition I found no significant difference between chara and reed ecotypes in the second functional role, in leaf consumption. The two ecotypes did differ neither between the lakes nor among the selected competition treatments. However, the three competition treatments tended to differ in impact on leaf decomposition. Leaf consumption per DW consumer was higher in low-competition treatment, lower in intra-specific competition and lowest in inter-specific competition when each ecotype co-existed with Gammarus pulex. So, the lower leaf consumption in high density treatment than low-competition treatment could be resulted from strong intra-specific interference effect within the individuals of chara and reed ecotypes respectively (Jonsson and Malmqvist 2000, 2003). The other reason could be higher grazing on periphytic algae than feeding on leaf in high individual treatment. Moreover, periphyton is regarded as more nutritious than leaf litter (Hertonsson 2010). So, species can gain more from grazing on periphyton than leaf consumption. Lowest leaf decomposition for both ecotypes in inter-specific treatment suggests strong inter-specific interference effect between G. pulex and A. aquaticus where both species shared the available food resource instead of decreasing the resource biomass (Hertonsson 2010, Brown et al. 1994) in large extent. 5.3 Mortality The use of available food resource through grazing or decomposition affects the fitness components e.g. survival and growth of a species. I checked the mortality as well as growth of both ecotypes separately and in an interaction with Gammarus pulex. Irrespective of lake and competition effect, I found no significant difference in mortality between chara and reed ecotypes. However, different ecotypes from different lakes experienced variable mortality. For both ecotypes mortality was higher in Lake Tåkern compared to Lake Fardume. The chara ecotype from Lake Fardume had a lower mortality than reed ecotype from same lake, where as the chara ecotype from Lake Tåkern had higher mortality than the reed ecotype (Fig. 7a). So, lake origin tended to have a signature not only on the absolute, but also on the relative mortality of the ecotypes. A previous study (Eroukhmanoff and Svensson 2009) stated that the emergence of chara ecotype in two Swedish lakes (Tåkern and Krankesjön) is an example of parallel evolution where chara ecotypes have independent evolutionary origins. So, chara ecotypes from different lakes can exhibit different traits of fitness components that lead to differentiation on 18 mortality between two lakes in my experiment. Competition effect can also be translated into the survival and growth of the interacting species (Hertonsson et al. 2008). Chara and reed ecotypes showed almost similar pattern of mortality in low density (or low-competition) and high density (or intra-specific) competition (Fig. 7b). In inter-specific competition the chara ecotype had higher mortality than the reed ecotype. This can be driven by two interaction scenarios: observed higher mortality of chara ecotype from Lake Tåkern (Fig. 7a) and higher mortality for in inter-specific competition for Lake Tåken (Fig. 7c). However, it can’t be certain that which ecotype from Lake Tåkern had higher mortality in inter-specific competition. This observed mortality can also be explained by the strength of inter-specific interactions between chara and Gammarus pulex or reed and Gammarus pulex. Reed ecotype might strongly compete with the amphipod for shared resources where as chara was out competed by amphipod as chara ecotype has less endurance than reed ecotype (Eroukhmanoff and Svensson 2009). Moreover, high genetic variation is observed among the isopods from reed habitat than those from chara habitat in both Tåkern and Fardume (Eroukhmanoff et al. 2009). So, due to less genetic variation chara ecotype could have less resistance to environmental or competition stress which might result in higher mortality of chara ecotype in the laboratory study. In addition, exploratory behaviour study done by Eroukhmanoff and Svensson 2009 showed that in presence of both food substrates, dead leaves and periphyton, reed ecotype had higher tendency to forage on alternative substrate than chara ecotype. The weak exploratory behaviour could also increase the mortality of chara ecotype. The effects of competition were significantly different between the two lakes. Among Asellus from Lake Fardume, competition had no influence on mortality; where as, both intra- and inter-specific competition had a significant influence on mortality among Asellus from Lake Tåkern. So, lake history has potential to influence the fitness (e.g. mortality, growth) of species. Lake Tåkern experienced both turbid (dominated by phytoplankton) and clear (dominated by submerged vegetation) water states in past few decades. This shift also affected both biotic (e.g trophic web structure, benthic invertebrate, fish and waterfowl assemblage etc.) as well as abiotic ( e.g total phosphorus, organic nitrogen concentration) characters of the lake (Hargeby et al 2007, Hargeby et al 1994). This could also affect the fitness of ecotypes of Asellus aquaticus from Lake Tåkern. However, Lake Fardume did not experience such alternative shift. Moreover, genetic variation of ecotypes of Asellus is higher between the two lakes compared to within lakes (Eroukhmanoff et al. 2009). So, different lake can have different impacts on the fitness of isopods. However, a further result from several lake studies is needed to generalise this idea. 5.4 Growth In line with mortality, chara and reed ecotypes of Asellus showed no difference in growth at ecotype level. Similarly, ecotypes from Lake Tåkern had higher growth than those from Lake Fardume (Fig. 9a). This could be resulted from higher mortality of isopods in Lake Tåkern. As there were fewer individuals, the remaining isopods got more food and showed better growth. Same might happen for Lake Fardume where high survival resulted in reduced growth of isopods. Similar to mortality, isopods from different lakes have variable growth in different competition treatments (Fig 9c). So, lake history has potential to influence the growth of a species . 6 Conclusion To sum up, present study suggests that there is no general difference between chara and reed ecotypes in their potential role of leaf decomposition and grazing on periphytic algae. 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