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Entomological Science (2015) 18, 479–488

doi:10.1111/ens.12139

ORIGINAL ARTICLE

Test for latitudinal variation of life history, behavior and mortality in the strictly univoltine damselfly Sympecma fusca (Zygoptera: Lestidae) Szymon SŃIEGULA and Maria J. GOŁA˛B Department of Ecosystem Conservation, Institute of Nature Conservation, Polish Academy of Sciences, Cracow, Poland

Abstract A wealth of evidence shows that combinations of ecological stressors interact in shaping life history traits, but little is known about how ecological stressors combine with different seasonal time constraints to shape life history, behavior and mortality across populations. We studied life history, behavior and mortality rate in two latitudinally distant populations of the strictly univoltine, adult-overwintering damselfly Sympecma fusca. Results from laboratory common-garden and outdoor experiments indicated countergradient variation of larval development time and growth rate: the more time-constrained larvae showed faster development and a higher growth rate. This finding led to larger size at emergence in the more time-constrained individuals. Under conditions of intraspecific interaction (outdoor experiment), northern individuals showed lower survival than southern ones, presumably due to cannibalism. In the absence of intraspecific interactions (laboratory experiment), northern and southern larvae did not differ in survival. Finally, laboratorygrown northern and southern larvae did not differ in activity level. This is the first time that compensation for seasonal time constraints has been shown in a temperate odonate species that overwinters in the adult stage. Key words: activity rate, countergradient variation, development time, growth rate, size at emergence, time constraint.

INTRODUCTION Life history traits such as time to and size at maturity are closely connected with fitness (Speight et al. 2008; Dmitriew 2011). In organisms with complex life cycles, time constraints imposed by seasonality are expected to cause a trade-off between development time and size at maturity (Blanckenhorn 2000). Faster growth in timestressed individuals may effectively decouple this tradeoff, however, by increasing the development and growth rates, so that it takes less time to reach a given size at emergence (Rowe & Ludwig 1991; Abrams et al. 1996). This decoupling mechanism is likely to occur in situa-

Correspondence: Szymon Sńiegula, Department of Ecosystem Conservation, Institute of Nature Conservation, Polish Academy of Sciences, Mickiewicza 33, 31-120 Cracow, Poland. Email: [email protected] Received 8 July 2014; accepted 15 November 2014.

© 2015 The Entomological Society of Japan

tions in which animals are not exposed to ecological stressors such as food shortage, low temperature, high competition, predation and cannibalism (Kause et al. 1999; Johansson et al. 2001; De Block & Stoks 2004a,b; Berger & Gotthard 2008; De Block et al. 2008; Lind et al. 2008; Nilsson-Örtman et al. 2012). In reality such ecological factors do commonly occur in nature (Gotthard & Nylin 1995; Speight et al. 2008). The presence of a compensating mechanism such as rapid growth may entail costs, however (Gotthard 2001). For instance, fast-growing individuals have lower resistance to suboptimal temperature, which may be maladaptive, especially at higher latitudes or altitudes (Karl et al. 2008; Stoks & De Block 2011). Seasonally delayed larvae are also more active, presumably to search for food, but at the same time they are under a higher risk of mortality as foraging activity often entails the risk of predation or cannibalism (Sih 1982; Johansson & Rowe 1999). There is limited empirical evidence that development and growth accelerate under

S. Sńiegula and M. J. Goła˛b

time constraints created by a short growth season at high latitudes (Dmitriew 2011). A few experimental designs have compared the responses of individuals from populations sourced from different latitudes (Laurila et al. 2008; Sńiegula & Johansson 2010; Sńiegula et al. 2012a,b; Stoks et al. 2012). These few experiments were run predominantly under laboratory conditions, which could bias the results (Birch 1953; Johansson 1992; De Block & Stoks 2004a; Speight et al. 2008). Exposing insects to time constraints in a natural field situation and then comparing those results with laboratory results should indicate how realistic the laboratory conditions were. Here we present the results of a comparative study of larvae of Sympecma fusca (Vander Linden, 1820) sampled from populations separated by 850 km in latitude. The results are based on both laboratory and seminatural outdoor experiments. We also estimated whether larval activity levels differ between more and less seasonally time-constrained populations. Following the above argument, we predicted that: (i) high-latitude (northern) larvae, which are more time constrained, would show shorter development time and a higher intrinsic growth rate than low-latitude (southern) larvae, under both laboratory and outdoor conditions; (ii) as a result, larvae from the two studied regions would not differ in size at emergence, since we predict decoupling between development time and size at emergence; that is, northern individuals would perfectly compensate shorter development time by growing faster to reach the same size at emergence as southern individuals; (iii) northern individuals would show an activity level higher than southern ones because the northern larvae should compensate seasonal time constraints by being more active in order to obtain food; and (iv) northern individuals would show a significantly higher mortality rate than southern individuals under the outdoor experimental conditions because of higher cannibalism among time-stressed individuals. Higher cannibalism would result from more frequent encounters during higher activity.

MATERIALS AND METHODS Study species Sympecma fusca is a damselfly common in large parts of Europe. It and the congeneric S. paedisca (Brauer) are the only two European damselflies that overwinter in the adult stage and reproduce the following spring. The next generation emerges in summer, making the species obligately univoltine (one generation per year, Dijkstra 2006). The short aquatic growing season before the start of winter makes the larvae seasonally time stressed. This

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stress is reflected in rapid larval development, which is completed in about 2 months (Askew 2004).

Field collection Damselflies from northeastern Bulgaria (the southern population) were collected from two wide eutrophic ditches along the Danube River, 2.3 km apart (44°01′N, 26°29′E and 44°02′N, 26°28′E). Those from a southern Polish locality (the northern population) were collected in a naturally formed eutrophic oxbow lake along the Vistula River (50°01′N, 19°31′E). All sites supported fish populations. The southern population was sampled on 12 April 2012, and the northern population on 26 April 2012. We sampled nine families from the southern and four families from the northern region. For adult size measurements we also took adult males and females from each locality and preserved them in 70% ethanol. Eggs from copulating females were sampled by a standard method (Sńiegula & Johansson 2010). Females oviposited within 3 days of capture. Filter paper with eggs was placed in 50 mL plastic test tubes filled with dechlorinated tap water. The eggs were transported to Cracow, Poland, by car; transportation took 1 h from the northern site and 18 h from the southern sites. During transport the eggs were exposed to a natural photoperiod and room temperature. On arrival, each egg clutch was divided in half and transferred to plastic containers (12 × 8 cm, 5 cm height) filled with 200 mL dechlorinated tap water. Half of the eggs from each clutch were placed in a climate chamber; these eggs were used for the laboratory experiment. The other half of each clutch was left in laboratory room conditions with a window; these larvae were later used for the outdoor experiment. Five southern and three northern egg clutches were used for the outdoor experiment, since the rest of the clutches held too few eggs to perform additional outdoor experiments and hence were used only for the laboratory experiment.

Laboratory common-garden experiment The eggs in the climate chamber were exposed to a photoperiod corresponding to natural day length at 44°N latitude, including civil twilight (cf. Lutz & Jenner 1964; Saunders 2002); that is, the photoperiod experienced by the population of damselflies sampled in the southern region. We set the climate chamber to that photoperiod because in the outdoor experiment (described below) the southern and northern individuals would both be exposed to the same natural day length as at the northern sampling site. This experimental setup exposed larvae to both latitude photoperiods but it has the disadvantage of using different photoperiods for the

Entomological Science (2015) 18, 479–488 © 2015 The Entomological Society of Japan

Latitudinal variation of insect traits

laboratory and outdoor experiments. To simulate the natural progress of light–dark conditions, the photoperiod was shifted every Saturday, following the natural light–dark conditions at the southern locality. The climate chamber temperature was set to constant 22°C. Previous studies of damselflies showed that larval mortality is lowest and larval growth is fastest at or near that temperature in the laboratory environment (Johansson & Rowe 1999; De Block & Stoks 2003; Sńiegula & Johansson 2010; Sńiegula et al. 2014). This kind of experimental setup has been used in other laboratory experiments on larval development and growth (Johansson et al. 2001; Stoks et al. 2005; Sńiegula & Johansson 2010; Sńiegula et al. 2012a,b, 2014). Hatching began on 30 April (southern larvae) and 19 May 2012 (northern larvae) and was synchronous. Immediately after hatching, similar numbers of individuals from each family and each region were individually placed in round plastic containers (7 cm diameter, 4 cm height). The experiment was started with 80 individuals total (2 regions × 40 individuals); from that time, larvae were fed daily with 290 ± 15.4 (SE, n = 10) laboratory-cultured brine shrimp Artemia sp., a high food ration (Johansson et al. 2001; De Block & Stoks 2004b).

Larval activity To estimate larval activity, twenty 50-day-old larvae from each region were observed between 1100 and 1330 h. The larvae were removed from the climate chamber and individually transferred to rectangular plastic containers (24 × 14 cm, 4 cm height) with a bottom grid (1 × 1 cm) filled with 500 mL dechlorinated tap water. These larvae were in late pre-final stadia (F-1 and F-2 stadia, where F-0 stands for the final stadium, F-1 for the second-to-last stadium, and so on). Variation in larval and intra-ecdysis stages can influence larval activity (Corbet 1999) but previous studies of confamiliar species with similar instar differences (F-2 or F-3) showed no quantitative difference in activity (Brodin 2009). The activity data from individuals that stopped feeding and/or went through ecdysis were censored from later analyses. Activity observations were made before feeding. The larvae were left in the containers for 20 min before the observation trials. Each container was observed every 10 min for 140 min. During the trial, notes were taken on whether the larvae had moved. A move was recorded when a larva moved its head from one grid square to another grid square between observation events (0 to 13 moves). The number of moves was later used for statistical analysis. Similar methods of larval activity estima-


tion have been used in previous experiments on damselfly larvae (Johansson & Rowe 1999; Brodin 2009).

Outdoor experiment The larvae were grown in light-gray plastic buckets (30 cm diameter, 29 cm height) set in a research pond (2 × 4 m, 1 m depth) located in southern Poland, 95 km east of the northern sampling site (49°56′N, 20°50′E; Fig. 1). The pond did not support a fish population so it contained a large number of zooplankton. The buckets were submerged in water and extended 10 cm above the water surface. Each bucket was covered with a mosquito net preventing other insects from laying eggs, blocking entry by predators, and allowing collection of emerging adult damselflies. Several plastic-coated iron wires were attached vertically to the bottom inside each bucket, extending above the water surface so the larvae could crawl out and emerge. Pond water could flow into and out of the enclosures through two openings covered with fine mesh (7 × 7 cm, 139 μm mesh size). This size mesh excluded potential predators (Corbet 1999). Structure was provided by a thin layer of sediment, mainly dead leaves, and three stems of Potamogeton (Potamogetonaceae) with floating leaves in each bucket. Eight buckets in total were set out in the research pond (Fig. 1). Two-week-old southern larvae were transferred from the laboratory to outdoor conditions on 14 May 2012; two-week-old northern larvae were moved on 2 June 2012. Twenty larvae from each egg clutch (family) were placed in each bucket, giving a total of 160 larvae (5 buckets × 20 southern + 3 buckets × 20 northern). This setup corresponds to the intermediate larval density of a confamiliar damselfly reported in

Figure 1 In situ bucket experiment with southern (Bulgarian) and northern (Polish) larvae of Sympecma fusca in a research pond in southern Poland.

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S. Sńiegula and M. J. Goła˛b

nature (Duffy 1994). Larvae were provided with zooplankton prey sampled from the same pond and given at ration intervals of 20 days starting from the day the experiment was initiated. Approximately 500 Daphnia sp. were introduced to each bucket each time. Visual inspection before each addition of zooplankton indicated that daphnias were present in the buckets throughout the experiment and that there were no evident differences in the quantity of Daphnia between the buckets. From day 60 to the end of the experiment the buckets were checked daily for emerging damselflies. Temperature during the outdoor experiment was measured with a temperature data logger (Hobo UA-001– 08; Onset Computer Corporation, Bourne, MA, USA) placed in a control bucket containing no larvae, placed among the other experimental buckets. The logger was programmed to measure the temperature every day at 1500 and 0300 h.

Measurements To estimate adult size at emergence in the laboratory and the outdoor experiments, head width was measured with a digital caliper to the nearest 0.1 mm. Fieldcollected adults were measured the same way. Head width is positively correlated with other body size estimates and hence is suitable for estimates of larval growth rate and adult size (Corbet 1999). Larval development time was calculated as the number of days from hatching to emergence. We made additional analyses based on day-degree because in the outdoor experiment the larvae were started on different dates and hence different water temperatures. Growth rate was calculated as adult head width divided by number of days from hatching to emergence.

Statistical analysis All statistics were calculated with R v2.10.1 (R Core Team 2009). To test for larval survival during the laboratory and field experiments we ran a two-sample test for equality of proportions. Separate two-way analyses of variance (ANOVA) were run to assess the effect of region and sex on larval development time, size at emergence and growth rate in the laboratory experiment. For the outdoor experiment we used separate one-way ANOVA to determine the effect of region on development time (based on both raw and day-degree data), size at emergence and growth rate. For the outdoor experiment we pooled the sexes because only the males of the northern group survived to emergence. The sexs did not differ in any of the measured traits among the southern individuals in the outdoor experiment (for all variables F1,26 < 1.53, P > 0.23); from this we inferred that pooling the sexes would not bias the results. Two-way

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ANOVA with region and sex as explanatory variables and adult size as a response variable was used to estimate differences among the field-sampled individuals. No data transformation was needed since in all cases the models nicely fitted the data (visual inspection). Full models with all interaction terms were used initially, but non-significant interactions were removed from the final models. A Student’s t-test was run to check for regional differences in larval activity based on movement counts.

RESULTS Laboratory common-garden experiment Northern larvae took less time for development than southern ones (Fig. 2a; Table 1). Overall, females took a longer time for development (mean 91.2 days for southern and 83.2 days for northern larvae) than males (mean 84.3 days for southern and 75.3 days for northern larvae). There was no interaction between region and sex (Table 1). Size did not differ between regions, and sex and the interaction between region and sex were not significant (Fig. 2b; Table 1). Regions and sexes did not differ in growth rate, though the sex effect bordered on significance (Fig. 2c; Table 1); females showed slower growth than males (mean 0.057 mm/day for males and 0.052 mm/day for females). The effect of interaction between region and sex on growth rate was not significant (Table 1). Larval survival did not differ between southern and northern regions (χ2 = 0.05, P = 0.82; Fig. 3). Most larvae died during early developmental stages. Sex cannot be recognized in early larval stages (Corbet 1999) so we could not determine sex-specific survival rates. Regions did not differ in activity level (12.05 ± 0.39 moves for southern and 12.35 ± 0.23 moves for northern larvae; t29.73 = 0.66, P = 0.51; not shown graphically).

Outdoor experiment Northern larvae required less development time than southern larvae (results based on untransformed raw data are shown in Fig. 4a and Table 1). Analyses based on day-degree data gave the same results qualitatively (F1,29 = 40.01, P > 0.001; mean day-degree 1521.9 for southern and 1039.7 for northern larvae; not shown graphically). Size at emergence was larger for northern than for southern individuals, and northern larvae grew significantly faster than southern ones (Fig. 4b,c; Table 1). Southern larvae showed significantly higher survival than northern larvae (χ2 = 6.11, P = 0.01; Fig. 5). We could not determine sex-specific survival rates since most larvae died or were cannibalized by other larvae during early larval stages when, as mentioned, sex cannot be recognized.


Latitudinal variation of insect traits

Table 1 Results of ANOVA comparing life history traits between southern (Bulgarian) and northern (Polish) populations of Sympecma fusca larvae under laboratory and outdoor conditions df (num, den) Laboratory experiment Response: development time Region Sex Region × sex Response: size at emergence Region Sex Region × sex Response: growth rate Region Sex Region × sex Outdoor experiment Response: development time Region Response: size at emergence Region Response: growth rate Region

F

P

1, 44 1, 44 1, 44

5.74 4.33 0.02

0.02* 0.04* 0.89

1, 12 1, 12 1, 12

0.08 0.19 0.11

0.78 0.67 0.75

1, 12 1, 12 1, 12

0.33 3.74 0.33

0.57 0.07 0.58

1, 29

42.89