Evol Ecol DOI 10.1007/s10682-010-9362-9 ORIGINAL PAPER
Clutch size and reproductive success in a female polymorphic insect Jessica Bots • Stefan Van Dongen • Luc De Bruyn Natalie Van Houtte • Hans Van Gossum
•
Received: 4 June 2009 / Accepted: 28 January 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Differences in reproductive success (RS) between different groups of individuals are of interest to researchers studying natural and sexual selection. Since it is often not feasible to quantify RS in the wild, researchers make use of proxies instead. One such proxy is clutch size. However, research on species providing parental care (mainly birds and mammals) has learned that a large clutch size does not guarantee a large number of offspring. In contrast, much less is known on the link between clutch size and RS for species lacking parental care, such as many reptiles and insects. Here, we ask whether clutch size provides a satisfactory estimate of RS for a polymorphic insect. Our study species is a damselfly showing two distinct female morphs for which RS (estimated by clutch size) has been studied to evaluate the evolutionary role of sexual conflict. However, in this system not only among family variation in offspring viability, but also differences between female morphs, may affect how clutch size relates to offspring number and quality. To evaluate the use of clutch size as estimate of RS, we examined how clutch size correlated with subsequent success measures of developing offspring by rearing damselfly from eggs to adults under two laboratory food treatments. In both treatments, we detected that clutch size correlated well with offspring number early in larval life, but that this relation is reduced by among family variation in survival in later developmental stages. Clutch size was moderately correlated with the number of offspring that successfully metamorphosed to winged adults. Patterns did not differ between female morphs and the nature of the correlation could not be explained from offspring quantity-quality trade-offs. Keywords Selection
Fecundity Female polymorphism Life history Odonates
J. Bots (&) S. Van Dongen L. De Bruyn N. Van Houtte H. Van Gossum Evolutionary Ecology Group, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium e-mail:
[email protected] L. De Bruyn Research Institute for Nature and Forest, Kliniekstraat (INBO) 25, 1070 Brussels, Belgium
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Introduction Studies on natural and sexual selection regularly examine whether different groups of individuals vary in reproductive success (RS) (Clutton-Brock 1988). Since it is often not feasible to quantify RS in the wild, researchers make use of simple proxies, such as clutch size. However, the use of clutch size as proxy of RS in evolutionary studies requires the assumption that it predicts the number offspring surviving to reproduction, while there is often little evidence for this in many animal taxa. Most theoretical and empirical work on the relationship between clutch size and RS has been conducted with species showing parental care (i.e. birds and mammals). Many of these studies indeed indicate that a large clutch size does not necessarily translate in many offspring (reviewed in Roff 1992). A reason for an imperfect linkage between clutch size and offspring number can be a trade-off between offspring quantity and quality, with offspring from large clutches suffering, for instance, reduced growth and survival probability (e.g. Dijkstra et al. 1990; Hardy et al. 1992; Koskela 1998). These negative effects are thought to be much caused by the inability of parents to gather sufficient food for their young (Roff 1992). In contrast, few studies have focussed on the link between clutch size and RS in species lacking parental care, such as many insects and reptiles (but see Madsen and Shine 1998; Fincke and Hadrys 2001), although factors affecting offspring viability can be expected to differ profoundly. Specifically in insects, gregarious parasitoids (i.e. multiple larvae develop together in a single host) have thus far received most attention when studying natural and experimental variation in clutch size and its effect on the number and size of surviving offspring (e.g. Godfray 1987). Results learn that larger clutches produce more offspring, but that size is reduced due to competition within the host. Producing smaller larvae may entail a fitness cost as larval size is positively correlated with fecundity and longevity (e.g. Hardy et al. 1992; Zaviezo and Mills 2000). In other insect groups there are indications that larger clutches may suffer reduced offspring success due to crowding and resource (maternal and environmental) limitations or, alternatively, have augmented success because of reduced predation (Allee effect) and protection against environmental extremes (reviewed in Godfray et al. 1991; Roff 1992). However, studies examining the relationship between clutch size and RS in detail are currently scarce, whereas the question is pertinent as clutch size has been used repeatedly for evaluating selection on females. Here, we evaluate whether clutch size provides a satisfactory predictor of RS for an insect that lacks parental care. Specifically, we selected a species that shows polymorphism in the female sex. Therefore, we not only add to knowledge on whether clutch size correlates with RS, but also provide insights on whether this relationship varies between different female morphs. Female polymorphism occurs in a variety of vertebrates and invertebrates, but is particularly common within damselflies (Insecta: Zygoptera) (Van Gossum et al. 2008; Svensson et al. 2009). Coexistence of multiple female morphs is considered consequence of sexual conflict, with males pursuing higher mating rates than females, resulting in excessive male sexual harassment towards unreceptive females (e.g. Arnqvist and Rowe 2005; Ha¨rdling and Bergsten 2006; Svensson et al. 2009). The phenomenon has attracted considerable attention during recent years, since it provides an attractive system for evaluating contemporary ideas on sexual selection, sexual conflict, and female trait differentiation (Gavrilets and Waxman 2002; Hayashi et al. 2007; Svensson et al. 2009). The approach typically taken to examine whether male harassment differentially affects female morphs fitness, is to determine fecundity (i.e. the number of eggs in a clutch) and use this as a proxy for RS (e.g. Sirot and Brockmann 2001; Svensson and Abbott 2005; Gosden and Svensson 2007). However, this approach inevitably brings
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the question whether clutch size is a satisfactory predictor of RS. For example, female morphs may adhere to an r or K strategy, laying many but low quality eggs versus few but high quality eggs (see Sinervo et al. 2000). The relationship between clutch size and RS was studied at multiple developmental stages for the female colour polymorphic damselfly Enallagma cyathigerum (Charpentier). Since the presence of offspring quality differences between families and/or female morphs may depend on the environment experienced, offspring of both female morphs were reared from egg to adult under two food treatments (‘‘high’’ or ‘‘low’’). This design is based on the observation that nutritional conditions are known to influence offspring development in odonates (Plaistow and Siva-Jothy 1999). Hence, we (1) evaluated among family variation in survival for several stages of development from egg hatching to final metamorphosis, (2) tested for possible trade-offs between offspring quantity and quality, by examining the effect of clutch size on offspring developmental success (i.e. egg hatching success, short- and long-term survival probability, metamorphosis success, developmental time and mass at emergence) and (3) considered whether different female morphs vary in these measures of offspring success.
Materials and methods Study species Enallagma cyathigerum (Odonata: Zygoptera) is a common damselfly distributed throughout Eurasia. The species can be found near all types of ponds, lakes and slow running rivers (Askew 2004; Dijkstra and Lewington 2006). In northwest Europe, where our study was conducted, the species is univoltine. Males and females reproduce alongside shoreline vegetation. After copulation, sperm is stored in specialised female storage organs, while fertilisation only occurs just prior to egg laying (Corbet 1999). Females lay eggs in different clutches in submerged vegetation (Doerksen 1980), with periods between successive clutches used to ripen new follicles. Last male sperm precedence occurs in Enallagma damselflies, i.e. the last male to mate a female fertilises the majority of eggs within a clutch (Fincke 1984). Eggs hatch shortly after oviposition and offspring pass through winter as aquatic larvae. Larvae metamorphose to flying adults during the next reproductive season. Enallagma cyathigerum exhibits a colour polymorphism only occurring in females. In males, body colouration is blue with a limited amount of black markings on the abdomen. Females are characterised as gynomorphs and andromorphs based on body colouration (e.g. Corbet 1999). In gynomorphs, body colour is brown to greenish-brown whereas andromorphs are blue and more closely resemble the conspecific male’s phenotype. Both female morphs differ from the conspecific male in showing more extensive black abdominal patterning (for illustrations see Dijkstra and Lewington 2006). Experimental design Female E. cyathigerum were collected near the edge of a fen in the North of Belgium (51°250 5500 N, 4°260 1000 E) on the 3rd of July 2006 using an insect net. Only copulating females were captured to increase the probability that females would have sperm in their storage organs. Females were transported to the university in small cages and housed in oviposition chambers during 24 h. Chambers were glass recipients covered with a mesh, provided with a stem to allow the damselfly to roost and with moist filter paper lined on the bottom in which eggs could be deposited (Van Gossum et al. 2003).
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Eggs of andromorph and gynomorph females (15 families of each morph) were maintained in Petri dishes (14 cm in diameter) filled with dechlorinated tap water. After 2 days, fertilised eggs had changed in colour from transparent to brown (Cooper et al. 1996) and were counted. Clutch size was quite large and therefore only ±20% of randomly selected eggs of each female was retained for laboratory rearing (details on the number of offspring studied at each developmental stage are presented in Table 1). Retained eggs were kept in Petri dishes, randomised in a climate room over two shelves (21°C, 8D/16L photoperiod). Eggs started hatching about 2 weeks after they were laid. Hatching success of eggs was monitored daily, until no newly hatched larvae were observed in the Petri dishes for 3 days. Newly hatched larvae were transferred to plastic cups (9 cm in diameter, filled with dechlorinated tap water) in groups of ten individuals per cup and fed with nauplii of Artemia salina on a daily base. In accordance with previous research showing that larvae of E. cyathigerum do not exhibit cannibalism (Chowdhury and Corbet 1989), larvae were not observed to consume conspecifics. 10 days after hatching, short-term survival was determined for each family by counting the number of surviving larvae in each cup (Table 1). Then, each surviving larvae was assigned to an individual plastic cup (6.5 cm in diameter, filled with dechlorinated tap water). Cups were given a unique number and larvae were further reared until they Table 1 Details on the sample sizes of maternal females and their offspring at each stage of the experiment Experimental stage
Housing in climate room
Treatment
Total
Average per family (± SE)
Variance per family
Number of wild caught maternal females (i.e. families)
–
–
A: 15 G: 15
–
–
Total number of eggs oviposited by maternal females
Oviposition chamber
–
A: 9861 G: 8683
A: 657 ± 33 G: 579 ± 36
A: 435–840 G: 336–904
Number of eggs retained for rearing*
Filter paper in Petri dish
–
A: 1809 G: 1362
A: 120 ± 8 G: 91 ± 9
A: 76–182 G: 35–146
Number of larvae studied for short-term survival (10 days after hatching)
Cups with 10 larvae
–
A: 1195 G: 1039
A: 80 ± 9 G: 69 ± 7
A: 14–147 G: 35–127
Number of larvae studied for long-term survival and developmental time to emergence*
Individual cups
High food
A: 540 G: 466
A: 36 ± 2 G: 31 ± 3
A: 6–44 G: 17–40
Low food
A: 539 G: 563
A: 36 ± 2 G: 31 ± 3
A: 7–44 G: 16–40
Number of larvae studied for metamorphosis success
Individual cups
High food
A: 268 G: 226
A: 18 ± 2 G: 15 ± 2
A: 2–29 G: 5–28
Low food
A: 83 G: 74
A: 5.5 ± 0.9 G: 4.9 ± 0.9
A: 0–12 G: 0–11
High food
A: 221 G: 184
A: 15 ± 2 G: 12 ± 2
A: 1–28 G: 2–23
Low food
A: 63 G: 52
A: 4.2 ± 0.7 G: 3.5 ± 0.7
A: 0–8 G: 0–10
Number of winged offspring studied for weight at emergence
Individual cups
Numbers are presented separately for maternal females belonging to the andromorph (A) and gynomorph type (G). Stages at which the number of eggs or larvae had to be reduced due to space limitation in the climate rooms are indicated with an asterisk (*). Note that offspring were only exposed to a low or high food treatment when they survived the first 10 days after hatching and were moved to individual cups
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metamorphosed or died. Larvae of each family were randomly divided over two treatments: (1) a ‘high’ food group where larvae were fed daily with Artemia nauplii and (2) a ‘low’ food group which was only fed every other day. We adhered to the approach of De Block et al. (2008) in that Artemia nauplii were the only food item used throughout the rearing period. Cups with larvae were distributed randomly over two identical climate rooms (both 21°C and 8D/16L photoperiod). Maximally 80 larvae per family were kept (Table 1). Long-term survival of larvae in their individual cups was monitored on a monthly base. When first larvae reached the penultimate stage, cups were provided with a piece of mosquito netting allowing larvae to crawl from the water. Also, cups were covered with cotton mesh preventing winged damselflies from escaping. Once the first larvae started final metamorphosis, cups were checked daily for newly emerged individuals. Sex and metamorphosis success of all emerged individuals was determined (Table 1). Metamorphosis was considered successful when the damselfly was able to remove head, thorax, legs and abdomen from the larval skin and subsequently fully expand abdomen and all four wings. All successfully metamorphosed damselflies (Table 1) were weighted 24 h after emergence (Kern balance, accuracy 0.0001 g). Unfortunately, we were unable to determine female morph type (andromorph or gynomorph), because survival of winged individuals in the insectaries in our laboratory was short (\1 week) and no individuals with a mature body colour were observed (which is required to distinguish female morphs). No information is currently available in the literature on the maturation time of E. cyathigerum, but our observation suggests that development of mature body colouration in this damselfly may take longer ([1 week). The experiment was ended on the 7th of June 2007, when the majority (87%) of larvae had emerged or died. Larvae for which the rearing was ended prematurely are treated as censored (33 individuals in the ‘high’ food and 252 in the ‘low’ food group, approximately equally distributed among maternal morphs). The probability of survival and developmental time to emergence of censored larvae was modelled using a Bayesian approach (see below). Statistical analysis Differences in the proportion of eggs that hatched were analysed with generalised linear model (GLM) with binomial distribution and logit error structure. Variation among families was tested by comparing the deviance and degrees of freedom of the final model with a chisquare test, since only one observation per family was available. In all other analyses, family and family 9 food treatment were included as random variable. Differences in the proportion of larvae that survived until day 10 after hatching (short-term survival) and the proportion of larvae that successfully completed final metamorphosis were analysed with a generalised linear mixed model (GLMM). In the analysis of short-term survival, it was taken into account that rearing cups contained groups of 10 larvae. The analysis of metamorphosis success was restricted to those individuals that initiated metamorphosis, thereby assuming that the censored larvae would not have differed in probability to metamorphose successfully. Variation in body mass at emergence was tested with a mixed model ANOVA. In all analyses, clutch size and maternal morph were included as independent variables. Also, shelf (egg developmental stage) or room (for larvae surviving first 10 days of development) were added to control for possible variation that could be caused by the position of the offspring in the climate rooms. In addition, for the analyses of metamorphosis success and body mass, the main effects food treatment and sex, and the interactions maternal morph 9 food treatment and clutch size 9 food treatment were added to the statistical models. Specifically for the analysis of body mass, also age was included as a
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covariable since it is known that age at emergence may affect body mass (e.g. De Block and Stoks 2005). Analyses were carried out in R (http://cran.r-project.org). Model selection started with the full model, including interactions, where after non-significant terms were removed until only significant terms remained, i.e. the most parsimonious model (Verbeke and Molenberghs 1997). Significance of independent factors was evaluated by comparing the deviance of the model with and without the factor using a likelihood ratio test. For the random variable family and the interaction family 9 treatment, significance was assessed using a permutation test with 1,000 iterations. Differences in the proportion of larvae that survived from 10 days after hatching to emergence (long-term survival) and the variation in developmental time to emergence were analysed in a single joint model using a Bayesian framework. This approach has the advantage that it allows to estimate a probability of surviving the larval stage for the 285 offspring that were censored (Van Dongen 2006). Simulation in Van Dongen (2006) showed that excluding these individuals from the analysis would lead to biased estimates of differences in time to emergence between treatments. In short, the probability of survival of each individual was modelled by a GLMM, with censored individuals included as missing values (i.e. survival status unknown). Individual survival probabilities were then generated in the Bayesian model conditional on the model parameters. As such, the GLMM generated prior distributions of individual survival probabilities for the model of time to emergence. This developmental time to emergence was analysed with a Cox Proportional Hazards (CPH) model, which was considered a count process with binary frailty (reflecting survival or not) incorporated as a zero-inflated Poisson model. Zeros or prior distribution from the GLMM for the censored individuals in this model represent the non-survivors. Detailed description and evaluation of robustness of the joint model can be found in Van Dongen (2006). Independent factors included in the analyses were: maternal morph, clutch size, sex, food treatment and the interactions maternal morph 9 food treatment and clutch size 9 food treatment. Family and also the interaction family 9 food treatment were treated as random variables. Analyses were performed in WINBUGS (http://www.mrc-bsu.cam.ac.uk/bugs). Posterior distributions were obtained using Monte Carlo Markov Chain (MCMC) simulations with 10,000 iterations. Using the previously described statistical models, we obtained the proportion of hatched, survived (both short- and long-term) and successfully metamorphosed larvae for each family. These proportions were used to estimate the family-specific number of offspring at each of these stages. The relation between clutch size and the number of remaining larvae at each stage was evaluated using Pearson’s correlation or Spearman Rank correlation when the assumption of normality was not fulfilled. In addition, the number of larvae remaining at a particular stage was also correlated against the number of larvae in the previous stages. Descriptive statistics are presented as means ± SE.
Results Food treatment Measures of larval developmental success differed between food treatments. In detail, larvae reared in the ‘low’ compared to the ‘high’ food treatment had a lower probability to survive until emergence (low food: 15 ± 1%; high food: 48 ± 2%), had longer developmental times (median obtained from CPH model: low food: 278.3 days, high food:
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Evol Ecol Table 2 Summary of statistical results for the analysis of hatching success (a), short-term survival (b), metamorphosis success (c) and mass at emergence (d) Effect
Test statistics
(a) Hatching success Family
v2 = 569.6, df = 28, P < 0.001 (v2 = 14.39, df = 26, P = 0.97)
Maternal morph
v2 = 0.41, df = 1, P = 0.52 (v2 = 0.62, df = 1, P = 0.43)
Shelve
v2 = 7.78, df = 1, P = 0.006 (v2 = 0.11, df = 1, P = 0.74)
Clutch size
v2 = 0.15, df = 1, P = 0.69 (v2 = 15.11, df = 1, P = 0.0001)
(b) Short-term survival Family
P < 0.001
Maternal morph
v2 = 0.01, df = 1, P = 0.91
Clutch size
v2 = 0.89, df = 1, P = 0.36
(c) Metamorphosis success Food treatment
v2 = 6.27, df = 1, P = 0.01
Family
P = 0.06
Family 9 food treatment
P = 0.52
Maternal morph
v2 = 0.61, df = 1, P = 0.44
Maternal morph 9 food treatment
v2 = 0.18, df = 1, P = 0.67
Clutch size
v2 = 0.86, df = 1, P = 0.35
Clutch size 9 food treatment
v2 = 2.10, df = 1, P = 0.15
Sex
v2 = 0.18, df = 1, P = 0.67
Sex 9 food treatment
v2 = 0.11, df = 1, P = 0.75
Room
v2 = 0.37, df = 1, P = 0.55
(d) Mass at emergence Food treatment
v2 = 210.13, df = 1, P \ 0.001
Family
P < 0.01
Family 9 food treatment
P = 0.50
Maternal morph
v2 = 0, df = 1, P = 1
Maternal morph 9 food treatment
v2 = 0.02, df = 1, P = 0.90
Clutch size
v2 = 0.15, df = 1, P = 0.69
Clutch size 9 food treatment
v2 = 0.13, df = 1, P = 0.72
Sex
v2 = 16.78, df = 1, P < 0.001
Sex 9 food treatment
v2 = 0.52, df = 1, P = 0.47
Age
v2 = 210.13, df = 1, P < 0.001
Age 9 food treatment
v2 = 3.90, df = 1, P = 0.05
Room
v2 = 4.72, df = 1, P = 0.03
The first three analyses were performed using generalised linear models, while for mass at emergence a mixed model ANOVA was applied. For the analysis of hatching success, the family effect was evaluated by comparing the deviance and degrees of freedom of the final model with a chi-square test since only one observation per family was available, whereas for all other analyses a permutation test was used. For hatching success, results of the analysis excluding families 3 and 19 are also provided between brackets. Note that larvae were only assigned to a high or low food treatment groups 10 days after egg hatching when housed in individual cups (i.e. after the short-term survival stage). Significant results are indicated in bold
213.0 days), were less likely to successfully metamorphose (low food: 77.2 ± 0.3%; high food: 86.7 ± 0.4%) and had a lower body mass at emergence (low food: 22.1 ± 0.3 mg; high food: 26.8 ± 0.2 mg) (Tables 2, 3; Fig. 1). These results confirm that we indeed
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Evol Ecol Table 3 Summary of statistical results of the joint Bayesian model evaluating variation in probability of long-term survival to emergence (a) and developmental time (b) Effect
Posterior mean
95% Credibility interval
(a) Long-term survival Family (variance) Food treatment (low vs. high)
0.34 21.80
0.15 to 0.67 22.10 to 21.53
Family 9 food treatment (variance)
0.12
0.01 to 0.46
Maternal morph (andromorph vs. gynomorph)
0.08
-0.44 to 0.59
Maternal morph 9 food treatment (variance)
-0.16
-0.69 to 0.38
Clutch size (variance)
-0.19
-0.44 to 0.06
Clutch size 9 food treatment (variance)
0.14
-0.12 to 0.40
-0.20
-0.58 to 0.19
Sex 9 food treatment (variance)
0.19
-0.73 to 1.11
Room (two vs. one)
0.86
0.60 to 1.13
Sex (males vs. females)
(b) Developmental time Family (variance) Food treatment (low vs. high)
0.16 21.84
0.60 to 0.33 22.13 to 21.57
Family 9 food treatment (variance)
0.05
0.01 to 0.18
Maternal morph (andromorph vs. gynomorph)
0.20
-0.17 to 0.57
Maternal morph 9 food treatment (variance)
-0.09
-0.57 to 0.39
Clutch size (variance)
-0.16
-0.34 to 0.01
0.11
-0.11 to 0.32
Clutch size 9 food treatment (variance) Sex (males vs. females)
0.23
0.03 to 0.43
Sex 9 food treatment (variance)
-0.15
-0.58 to 0.26
Room (two vs. one)
-0.18
-0.36 to 0.01
Significant results are indicated in bold
succeeded in studying the fate of larvae under two contrasting food conditions. Within a food treatment, longer developmental time resulted in higher mass at emergence (Table 2d). Furthermore, male offspring developed faster (Table 3b), but weighted less at emergence (Table 2d), than female offspring (male: 24.9 ± 0.3 mg; female: 26.7 ± 0.3 mg). No differences between male and female offspring were observed for survival to emergence (Table 3a) or metamorphosis success (Table 2c). Family effects For most stages of development, we observed significant among family variation in survival and development, but there was little evidence that offspring developmental success of different families varied among food treatments (family 9 food treatment interaction) (Tables 2, 3). Specifically for egg hatching success, the family effect was driven by particularly low numbers of eggs that hatched for female 3 and female 19 (see Fig. 3a). Excluding these two families from the analysis resulted in the among family variation to disappear (Table 2a). Also, the significant effect of shelf appeared to be driven by low hatching success of offspring of these two families (Table 2a). In contrast, including or excluding family 3 and 19 in all other analyses, did not alter any results for the other
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Fig. 1 Larvae reared in the ‘low’ food treatment were less likely to survive to emergence (a), had a longer developmental time (b), had a lower probability to successfully metamorphose (c), and weighted less at emergence (d), than larvae in the ‘high’ food treatment Fig. 2 Clutch size had a positive effect on hatching success, but only when data of two families (3 and 19) with very low hatching probability of offspring were excluded from the analysis
measured traits. Therefore, for all developmental stages except hatching success, only the results of the analyses including all families are presented in Tables 2 and 3. Trade-off between quantity and quality of offspring Clutch size did not explain the developmental success of offspring for any of the studied traits for either treatment (‘high’ and ‘low’ food), indicating that quality differences between larvae from small and large clutches were not present (Table 2, 3). However, specifically for hatching success, reanalysing data excluding families 3 and 19 [which had very low proportions of hatched larvae (52 and 10%, respectively) in comparison to other
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Fig. 3 The relation between clutch size and the number of remaining offspring for each family at different developmental stages for both the ‘low’ and ‘high’ food treatment. Also, larval success between successive developmental stages was compared. All analyses were repeated excluding data of families 3 and 19, which had very low proportions of hatched larvae. Correlation coefficients of these additional analyses are indicated between brackets (all P \ 0.01)
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families (87 ± 1%)], showed different results. Clutch size was found to have a strong positive effect on the proportion of larvae that hatched (Fig. 2; Table 2a), indicating a superior quality for individuals of large clutches. Differences between female morphs Offspring of andromorphs and gynomorphs were equally successful at all stages of development in both the ‘high’ and the ‘low’ food treatment (Tables 2, 3). Patterns of offspring survival and clutch size as a predictor of reproductive success Finally, we evaluated whether the observed among family variation affected the relation between clutch size and the number of larvae surviving at each studied developmental stage (see Fig. 3). Clutch size correlated significantly with the number of hatched larvae (rs = 0.77, df = 28, P \ 0.001), (Fig. 3a). The relation between clutch size and the number of surviving larvae after 10 days remained somewhat similar (rs = 0.76, df = 28, P \ 0.001) (Fig. 3b), despite significant among family variation in short-term survival (Table 2b). This result is further supported by the strong correlation between number of hatched and surviving larvae after 10 days (rs = 0.99, df = 28, P \ 0.001) (Fig. 3c). However, much variation in the relationship with clutch size arose during the long period that larvae needed to survive until emergence (‘high’ food: rs = 0.48, df = 28, P = 0.01; ‘low’ food: rs = 0.34, df = 28, P = 0.06) (Fig. 3d). In agreement, the number of larvae that survived to emergence was less strongly related to the number of larvae that hatched (‘high’ food rs = 0.68, df = 28, P \ 0.001; ‘low’ food: rs = 0.54, df = 28, P \ 0.001) (Fig. 3e) and survived after 10 days (‘high’ food rs = 0.69, df = 28, P \ 0.001; ‘low’ food: rs = 0.56, df = 28, P \ 0.001) (Fig. 3f). Among family variance in metamorphosis success was limited (Table 2c) such that the correlation between clutch size and the number offspring surviving to the next generation was not altered much further at the last developmental stage (‘high’ food: rs = 0.47, df = 28, P = 0.01; ‘low’ food: rs = 0.42, df = 26, P = 0.03) (Fig. 3g–j). Excluding families 3 and 19 from these analyses resulted in a limited increase of the correlation coefficient in most cases (all P \ 0.01, see Fig. 3).
Discussion In contrast to the ample literature on birds and mammals (reviewed in Roff 1992), much less is known on the relation between reproductive output (e.g. clutch size) and reproductive success (RS) in species lacking parental care, such as many reptiles and nonparasitoid insects. Here, we evaluated the correlation between clutch size and offspring number at subsequent developmental stages for the polymorphic insect Enallagma cyathigerum. Early in larval life, clutch size correlated very well with offspring number. Indeed, clutch size explained 58% (92% excluding families 3 and 19 with exceptionally low egg hatching success) of the variation in egg hatching and short-term survival success (Fig. 3, see r2 of the relation between clutch size and offspring number). Later in development, however, high among family variation in long-term survival was observed, leading to much lower associations between offspring survival and clutch size (maximally 23 and 37% excluding families 3 and 19). Little among family variation was detected in metamorphosis success, and correlations between clutch size and offspring number were not much further weakened. As such, clutch size maximally explained 22% (48%
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excluding families 3 and 19) of the variation in offspring success at this last stage of development. At all stages, the correlation between clutch size and offspring number was somewhat reduced in the ‘low’ compared to the ‘high’ food treatment. Furthermore, we did not observe a trade-off between offspring quantity (i.e. clutch size) and quality (i.e. survival probability, developmental time or mass) at any developmental stage as seen in other insect species (Hardy et al. 1992; Fox et al. 1996; Desouhant et al. 2000). Although such quantity-quality trade-off may only be present under situations when resources were limited (Gillespie et al. 2008), we did not detect the predicted trade-off in any food treatment. In contrast, we found evidence for a positive relation between clutch size and hatching probability, suggesting a quality advantage of large clutches. Possibly, females differ in energy reserves, with high energy quantities leading to both higher numbers and quality of eggs (Van Noordwijk and de Jong 1986). Anyway, an offspring quantity-quality trade-off does not seem to weaken the correlation between clutch size and offspring number in our study. Together, these results indicate that clutch size moderately predicts the number of surviving offspring such that it could be considered a useful proxy of RS in this study system. On the other hand, it is noteworthy that since our study was carried out under laboratory conditions, it provided a best case scenario for offspring survival and growth. Indeed, in our experiment, predation was excluded and larvae were reared in individual cups which ruled out food competition. In odonates, it has been shown that besides lowering survival probabilities, predation risk also affects fitness of developing larvae by decreasing growth rate and storage compounds (proteins and triglycerides), and increasing oxygen consumption and stress protein production (e.g. Stoks 2001; Stoks et al. 2005; Slos and Stoks 2008). In addition, competition may increase larval mortality through food shortage (as shown here in the low food treatment) and cannibalism (although E. cyathigerum has not been observed to eat conspecifics; Chowdhury and Corbet 1989) (De Block and Stoks 2004). Thus, because competition and predation can be expected to affect offspring survival, the correlation between clutch size and offspring number may be weaker under natural conditions. Nevertheless, results of this study are in strong contrast with results in other taxa lacking parental care, where clutch size has not been found to be predictive of RS (Madsen and Shine 1998; Fincke and Hadrys 2001). Fincke and Hadrys (2001) evaluated the RS of different families of a tree hole-breeding (monomorphic) damselfly using micro-satellite markers under natural conditions and showed that it did not correlate with clutch size. Instead, the time span between hatching of the first and the last egg within a clutch was detected to be the most appropriate estimate of RS. This was because, a larger hatching span increased the likelihood that some larvae encountered a window of opportunity during which the risks of being eaten by larger conspecifics were lower (Fincke and Hadrys 2001). Similarly, also in the water python Liasis fuscus, time of hatching, and not clutch size, was most predictive of reproductive success (Madsen and Shine 1998). Clutch size has also been used as a proxy of RS in several studies aiming at understanding the role of sexual conflict in the evolution and maintenance of female polymorphism (e.g. Svensson and Abbot 2005; Svensson et al. 2005). We found that offspring of female morphs did not differ in any of the studied developmental characteristics. It thus appears that, at least for E. cyathigerum, offspring quality differences between maternal morphs do not interfere with how clutch size predicts RS, but it is difficult to generalise across taxa. Indeed, in contrast to our study, Abbott and Svensson (2005) showed for the female polymorphic damselfly Ischnura elegans that offspring of different maternal morphs differ in developmental time. This result could, however, be due to the fact that for
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one of the female morphs, mothers from different populations were used in the rearing experiment, whereas development times differed across populations (Abbott and Svensson 2005). In conclusion, our results show that associations between clutch size and the number of offspring that successfully metamorphosed were moderate and that larval survival probabilities varied among families at several developmental stages. Still, females that laid the highest number of eggs produced the most offspring, with clutch size maximally explaining 22–48% of the observed variation in RS. However, it is important to consider that damselflies typically lay multiple clutches throughout their life-time (Corbet 1999) and that lifetime RS has been shown to be also shaped by adult survival probability, since a longer live span allows for more clutches to be laid (Banks and Thompson 1987). Similarly, in other insect taxa it has also been shown that longevity positively affects lifetime RS as longer lived females can invest more time and resources in reproduction (e.g. LopezVaamonde et al. 2009). Therefore, studies focussing on the fecundity of only one clutch may only poorly reflect lifetime RS when fecundity per clutch does not reflect total clutch size (for example due to changes in clutch size with age). Together, we suggest that clutch size can be a useful proxy of RS to evaluate selection on females, but researchers should be aware of the assumptions it requires (e.g. non-random offspring survival, link between fecundity per clutch and total clutch size, or specifically for this study that the observed among family variation does not result from negative genetic correlations). Future studies may want to evaluate whether the observed correlations between clutch size and RS remain significant when developing larvae are reared under more natural conditions (e.g. including the effects of predation or competition). Acknowledgments We are grateful to Tom Snijkers, Bert Van Den Branden, Tine Van Duffel, Ben Huysmans and Bie Van Linden for their help with rearing damselfly larvae. Robby Stoks and Marjan De Block kindly advised on rearing methods. The ‘‘Institute for the promotion of Innovation through Science and Technology in Flanders’’ (IWT-Flanders) supported this work (research grant to J.B.). H.V.G. is a postdoctoral fellow with the Fund for Scientific Research-Flanders (FWO-Flanders).
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