Copepoda: Calanoida - Oxford Journals - Oxford University Press

JOURNAL OF PLANKTON RESEARCH

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Life cycle traits of two transatlantic populations of Eurytemora affinis (Copepoda: Calanoida): salinity effects DELPHINE BEYREND-DUR1,2, SAMI SOUISSI1*, DAVID DEVREKER1,3, GESCHE WINKLER4 AND JIANG-SHIOU HWANG2 1

UNIVERSITE´ DES SCIENCES ET TECHNOLOGIES DE LILLE- LILLE 1, LABORATOIRE D’OCEÁNOLOGIE ET DE GEÓSCIENCES, UMR CNRS 8187 LOG, 28 AVENUE 2 3 FOCH, F-62930 WIMEREUX, FRANCE, INSTITUTE OF MARINE BIOLOGY, NATIONAL TAIWAN OCEAN UNIVERSITY, KEELUNG, TAIWAN 202, ROC, HORN POINT LABORATORY, UNIVERSITY OF MARYLAND CENTER FOR ENVIRONMENTAL SCIENCE,

4

ISMER, UNIVERSITE´ DE QUE´BEC A` RIMOUSKI, QUEBEC, CANADA G5L

2020 HORNS POINT ROAD, PO BOX 775, CAMBRIDGE,

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21613, USA

AND

3A1

*CORRESPONDING AUTHOR: [email protected] Received December 17, 2008; accepted in principle February 11, 2009; accepted for publication February 24, 2009; published online 18 March, 2009 Corresponding editor: Mark J. Gibbons

While the populations of the copepod Eurytemora affinis are often morphologically (i.e. taxonomy) indistinguishable, the species complex is composed of genetically distinct clades, representing divergent evolutionary histories. The most distant clades, genetically and morphologically (i.e. phylogeny), are transatlantic clades: North American and European (Lee, 2000). The study of the life cycle strategies of two populations from St. Lawrence salt-marshes (Canada) and from the Seine estuary (France) at three salinities (5, 15 and 25) revealed differences in their salinity tolerance. Individuals from the Seine exhibited high mortality under the highest salinity suggesting that the St. Lawrence population tolerated a wider salinity range. At the lowest salinity, the development time of St. Lawrence individuals was longer than that of individuals from the Seine suggesting that the Seine population was more adapted to low salinity. The clutch size and the longevity of St. Lawrence adults were on average two times higher compared to Seine adults. Thus, the St. Lawrence population exhibited a higher fitness relative to the Seine population. Such differences could be due to genetic differences resulting from divergent evolutionary history, to phenotypic plasticity and/or to the acclimation to culture conditions. We confirmed that a gamma density function is an appropriate fitting function for copepod development time, based on a large data set on development time. It can therefore be integrated into individual-based models of copepod population dynamics.

I N T RO D U C T I O N Estuarine ecosystems are characterized by important fluctuations in physicochemical and biological parameters that may have a strong effect on zooplankton distribution. Estuarine copepods are often exposed to wildly fluctuating environmental conditions that affect the life cycle traits and subsequently their population dynamics. Estuarine copepods (i.e. true estuarine and estuarine-marine copepod species, Collins and Williams, 1981) are known to have wider thermal and salinity tolerances than oceanic copepods (Mauchline, 1998 and references therein).

The success of physiological adaptations to fluctuating salinity varies among life-history stages in aquatic invertebrates. Consequently, natural selection may act differently on each ontogenetic stage, so that the establishment of a species in a given habitat depends on the ability of each stage to adapt to the environment (Willmer et al., 2000; Charmantier and Wolcott, 2001). For example, one strategy to optimize recruitment when the young life-history stages are the most sensitive to fluctuating conditions (or less adapted), is that these stages have a narrow habitat range where conditions are stable, compared to a broader habitat range with

doi:10.1093/plankt/fbp020, available online at www.plankt.oxfordjournals.org # The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]


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fluctuating conditions for well-adapted adults (Willmer et al., 2000; Charmantier and Wolcott, 2001). To actually test the adaptation of ontogenetic stages of a species and compare differentiated adaptation between populations, eco-physiological studies with standard experiments (i.e. experiments using same protocol) are required (Drillet et al., 2008). Until now, adaptive strategies in estuarine species are not well understood, and few studies have investigated the evolution of physiological responses of estuarine and marine copepod species (Halsband-Lenk et al., 2002; Lee et al., 2003). The calanoid copepod Eurytemora affinis has been the focus of many ecological studies because of its dominance as a primary grazer and its importance as a food source for higher trophic levels throughout the Northern Hemisphere (Mauchline, 1998 and references therein; Gasparini et al., 1999; Koski et al., 1999; Appeltans et al., 2003; Winkler and Greve, 2004). Abiotic and biotic environmental factors such as temperature, hydrodynamics, salinity, oxygen, predation or competition influence the distribution of E. affinis in the field (Roddie et al., 1984; Viitasalo et al., 1994; Castel, 1995; Escaravage and Soetaert, 1995; Vuorinen et al., 1998; Appeltans et al., 2003; David et al., 2005). Most of these factors also affect the life cycle of this species in laboratory studies (Vijverberg, 1980; Roddie et al., 1984; Escaravage and Soetaert, 1993; Ban, 1994; Lee et al., 2003; Devreker et al., 2004). Eurytemora affinis is broadly distributed in estuaries, salt-marshes and brackish waters in the Northern Hemisphere from subtropical to subarctic regions of North America and temperate regions of Asia and Europe. Eurytemora affinis is recognized as a sibling species complex, marked by morphological stasis, high genetic divergence among clades and by reproductive isolation between several nearby populations, representing divergent evolutionary histories (Lee, 2000; Lee and Frost, 2002). Within this species complex, the North American clade and the European clade have probably been separated for millions of years (Lee, 2000). Within the past 60 years, some of these clades have independently invaded freshwaters habitats (Lee, 1999; Winkler et al., 2008). The broad habitat range of E. affinis and its recent invasions into freshwater habitats have often been attributed to its wide salinity tolerance (Wolff, 2000). To examine physiological adaptations of life cycle traits among clades of this copepod species complex, the use of standard experiments allows direct comparison, avoiding bias in comparing results from different studies conducted with different protocols. The purpose of this study was to examine whether the evolutionary history of two genetically divergent clades of the E. affinis species complex from two transatlantic locations, the St. Lawrence salt-marsh population

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belonging to the Atlantic clade (referred here as North American clade) and the Seine Estuary population (European clade), induced differences in life cycle traits in response to different salinities. To achieve this objective, E. affinis from the two transatlantic populations were reared under identical laboratory conditions, and the post-embryonic development ( post-EDT), survival and reproduction at three different salinities were compared. Variability in individual performance is an important variable in population dynamics and persistence, regulating population size and the capacity of a population to adapt to long or short term fluctuating environmental conditions (Jimeńez-Melero et al., 2007). Thus, our experiments, conducted at satiating food levels, included individual observations to take into account the individual variability (Ban, 1994; Souissi and Ban, 2001; Devreker et al., 2007; Devreker et al., 2008) of each trait within each population. We then fitted our data on copepod stage development time to a gamma distribution (Klein Breteler et al., 1994; Souissi et al., 1997).

METHOD Cultures Eurytemora affinis from the North American clade (Lee, 2000) were collected in salt-marsh tidal ponds at Isle Verte in the St. Lawrence estuary (Canada) in May 2001. In these ponds, the salinity ranges from 5 to 40 depending on tidal flooding frequency, precipitation and evaporation pattern (Winkler, unpublished data). This population has been in culture at the University of Wisconsin, Department of Zoology, USA, by Dr C. E. Lee since 2001. Since September 2004, this population has also been cultured at the Marine Station of Wimereux, France. The copepods were cultivated in 20– 38 L aquariums at 15 salinity (mix of 0.45 mm filtered sea water and deionized water) and at 108C on a 12 L:12 D photoperiod. Such conditions were found to be optimal for the development and reproduction of the populations. Cultures were fed with a mixture of Rhodomonas marina (15 mm diameter) and Isochrysis galbana (5 mm diameter) at a 2:1 cell-ratio for several generations. The water in the culture was changed twice a week and food algae were supplied at the same time. Eurytemora affinis from the European clade were collected from the Seine estuary, France, under the Normandy Bridge in May 2004 using an oblique net haul (200 mm mesh). In this area of the Seine estuary, the salinity fluctuates daily from 0.5 to 25 (Devreker et al., 2008). This population was cultured at the

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Marine Station of Wimereux (France) under the same conditions as the St. Lawrence population, except at a temperature of 158C. One generation of this culture was acclimated at 108C before beginning the treatments. Throughout this paper, the population from St. Lawrence salt-marshes is referred to as “American St. Lawrence (ASL)” whereas the population from the Seine estuary is referred to as “European Seine (ES)”.

Individual post-EDT We chose the representative environmental conditions in our experiments to compare post-EDT rates between the populations. The 108C temperature represented the lowest temperature in the Seine estuary where the maximum abundance of E. affinis occurred (Mouny and Dauvin, 2002). This experimental temperature was also encountered in the salt-marsh tidal ponds of the St. Lawrence where the temperature range varied between 5 and 278C from May to August (Ward and Fitzgerald, 1983; Castonguay, 1988). Salinities of 5, 15 and 25 cover the salinity range of estuaries from polyto oligo-haline zones and are often encountered by E. affinis in both habitats. The effect of salinity on each individual life cycle was studied using three different salinities (5, 15 and 25) of autoclaved water, and we fed mixtures of the algal cultures in excess (20 000 cells/mL). Such a mixture has been shown to promote proper development and reproduction of E. affinis in culture (Ban, 1994; Lavens and Sorgeloos, 1996; Devreker, 2007). All experiments were performed under the same photoperiod as provided for the cultures and individual beakers were stored to avoid evaporation. Water was renewed every week and food was given every 2 days.

St. Lawrence Due to logistic problems, two sets of experiments were performed. First, we tested two salinity conditions of 5 and 15 (respectively, labelled ASL5 and ASL15a) wherein the copepods were fed with the same algalmixture as the cultures. Ten ovigerous females were selected individually from the culture and placed in water with salinities 5 and 15 in 30 mL beakers. The second set of experiments was conducted 10 months later at salinities 15 (ASL15b) and 25 (ASL25) and individuals here were fed only Rhodomonas baltica. The use of the same reference salinity 15 was to allow us to verify any possible differences between the two temporally separated experiments. Five ovigerous females from the culture were selected and individually placed into 30 mL beakers with salinities 15 and 25. Each beaker was observed every 6 to 12 h to obtain newly hatched

nauplii, and then nauplii were individually isolated in 15 mL beakers, from ASL5, ASL15a, ASL15b and ASL25, respectively, at the same salinity and food supply as those provided to their mothers.

Seine For each experiment, nine ovigerous females were selected from the culture and individually placed in the different salinity conditions in a 30 mL beaker. The experimental conditions for the Seine were called ES5, ES15 and ES25 with salinities: 5, 15 and 25, respectively. The experiments were carried out simultaneously. Each beaker was observed every 6 to 12 h for newly hatched nauplii. The nauplii were isolated individually in 15 mL beakers, each with the same conditions as the mother. They were fed with the same algal mixture as the cultures. To study the post-EDT, each individual from each experiment was observed frequently (every 6 to 12 h) with a stereo-microscope, and moulting times were noted until the individual died. Developmental stages were identified based on Katona (Katona, 1971). Duration of intermoult periods (i.e. stage duration) was calculated as age at moulting from development stage n to n + 1 for each individual. Initial conditions and number of copepods observed per developmental stage are summarized in the Table I.

Individual reproduction To estimate reproductive parameters of individual females from both clades, we used the same protocol as Devreker et al. (Devreker et al., 2009; see their Fig. 1). The time of maturation from C5 to adult female corresponded to day 0 of the reproductive life. The female – male pairs from treatments ASL5, ASL15a, ASL15b, ASL25, ES5, ES15 and ES25 were observed frequently (every 6 to 12 h) with a stereo-microscope and their reproductive states were noted until their death. We determined the reproductive parameters clutch size (CS), maturation time [MT; time between moulting from C5 (F) to female and spawning of the first clutch], hatching success (HS; (No. of nauplii/No. of eggs) 100), latency time (LT; time between hatching of clutch “x” or detachment of clutch “x” and spawning of clutch “x+1”), EDT (time between one clutch spawning and its hatching), inter-clutch time (ICT; time between spawning of clutch “x” and spawning of clutch “x+1” and ICT equals to LT plus EDT), and the egg production rate (EPR; the number of eggs produced by a female per day during its reproductive life).

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Table I: Initial conditions in all seven experimental conditions for both populations of E. affinis at 108C St. Lawrence Salinity Food supply Date Labels Number of nauplii (N1)

5 a February 2005 ASL5 87

Seine 15 a February 2005 ASL15a 81

15 b November 2005 ASL15b 24

25 b November 2005 ASL25 41

5 a August 2004 ES5 31

15 a August 2004 ES15 25

25 a August 2004 ES25 30

The salinities, food supplies, dates, labels and numbers of nauplii used in the development time study are included in this table. a: fed with an algae blend of R. baltica and I. galbana. b: fed only with R. baltica.

Mathematical and statistical analysis Comparison between groups of treatments Differences between development times and reproductive parameters under each experimental condition were quantified using the non-parametric Wilcoxon test for independent data in MATLAB 6.5 software (The MathWorks Inc., 2002). To facilitate the comparisons, the paired tests of development times and of reproductive parameters were performed between treatments within a population and between treatments under the same salinity condition with a significance level of P , 0.001.

Probabilities of moulting Because of the short duration of naupliar stages and the difficulties identifying stages of live nauplii, we pooled the first naupliar stages (N1– N3) and the last naupliar stages (N4 – N6) to determine developmental time of the larval stages of E. affinis according to Souissi and Ban (Souissi and Ban, 2001). To simplify the graphic representation, we also pooled the first (C1 – C3) and the last copepodid (C4 – C5) stages. In order to predict the probability of moulting from one developmental stage to the next, the cumulative proportion of individuals moulting from one group of stages to the next was plotted against the ages of individuals. According to the shape of the cumulative moulting distributions, we fitted a gamma density function (GDF) (Souissi et al., 1997, see their formula) to the data, and determined the maximum likelihood estimates and confidence bounds of the GDF parameters a (the scale parameter) and b (the shape parameter), using the gamcdf [x/ a, b] function of the Curve Fitting Toolbox of MATLAB 6.5 software (The MathWorks Inc., 2002). The error between the adjustment and the real distribution corresponds to the sum of square residuals (SSE). The percentage of variance explained by the gamma adjustment corresponds to R-square (R 2). The difference between the values predicted by the

model and the values actually observed from the data modelled is the root mean squared error (RMSE). The closer the SSE and RMSE values are to 0 and closer the R 2 is to 1, the better the model fits. Once the parameters are known, the Median Development Time per stage (MDT) can be calculated using the command gaminv (included in the MATLAB software) which computes the inverse of the cumulative GDF at any probability. In this study, the probability is 0.5, which corresponds to 50% of individuals having moulted. To better understand the general patterns of the postembryonic development within both clades of E. affinis regardless of experimental condition, we used a multiple step approach. First, to standardize the data and to account for individual variability in development time, we used the Index of Development (ID). This allowed us to compare the individual development to the mean development of the population for each experimental condition: Di; j IDij ¼ Dj where the index of development is the ratio of the development time D of each individual i within the developmental stage (or the group of developmental stages) j(Di, j ) of the developmental to the mean development time D j ). Values stage (or the group of developmental stages) j (D for ID that are below (res. above) 1 for each developmental stage correspond to individuals developing rapidly (res. slowly) for that stage. Next, all IDs obtained for each individual from all experimental conditions (salinity, food and generation, population) were normalized (i.e. divided by the mean of the treatment) to eliminate their effects. Subsequently, to ensure a more accurate density function for a general pattern of E. affinis, the IDs were then fitted by three probability density functions: Normal, Lognormal and Gamma. The curve fitting toolbox of MATLAB software was used to obtain the best fitting parameters and the goodness of the fit.

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a: fed with an algae blend of R. baltica and I. galbana. b: fed only with R. baltica. M: male and F: female. Data on lines with the same letter superscript are not significantly different using Wilcoxon analysis with significant level at P , 0.001 between treatments within a population (ASL or ES) and between treatments at same salinity (5, 15 or 25). Data with the asterisk were not tested.

30 27 21 15 11 3 1 3 1 2 1 0.56 1.30 1.35 1.13 1.24 0.58 0 0.77 0 1.83 0 5.51 6.39c 3.30g,h 2.52c,e 3.04b,e 2.67b,e 3.75* 3.53b,e 6.00* 10.29* 28* 25 23 21 21 19 10 8 10 8 10 8 0.37 0.80 0.83 2.11 0.52 0.66 0.42 0.95 1.07 7.08 13.64 5.29 5.92b,c 2.36d,e,f,h 2.63c,d 2.15b,d 2.05b,d 3.31c,e 3.05b,d 5.81b 35.71b 40.64c,e 31 31 31 31 28 17 11 17 11 17 11 0.19 0.37 0.74 0.80 1.10 1.12 0.91 0.50 0.54 10.27 15.49 4.93 4.78 2.15d 2.00c 2.63b,c 2.68b,c 3.07c,d 3.07b,c 5.55b 34.54b 44.22c,d 41 38 32 29 29 16 9 16 9 14 8 0.86 1.53 1.23 0.84 1.29 0.49 0.74 0.54 0.78 26.57 18.59 5.57 7.74a 2.75b,c,g 2.12b,e 2.51a,e 1.88a,e 2.53a,b 2.80a,e 4.34a 46.86a 70.82a,b 24 24 24 24 24 11 13 11 13 9 13 0.68 1.78 0.43 0.38 0.64 0.27 0.68 0.62 0.50 18.03 17.10 5.99 6.21a,b 2.13c,f 1.97b,d 2.03a,d 1.78a,d 2.57a,b,e 2.13a,d 4.02a 76.83a 67.05a,b,e 80 79 77 75 74 40 34 40 33 35 28 1.01 1.54 0.83 1.09 0.77 0.61 0.99 0.95 1.11 24.39 32.42 5.28 5.50b 3.04a,b,e 2.48a,b,d 2.25a,d 2.24a,d 2.75b,e 2.50a,d 3.81a 65.67a 95.07b 87 87 82 81 81 34 46 34 46 34 45 0.58 2.70 0.87 0.73 0.73 0.64 1.35 0.83 1.23 24 27.75 4.78 8.29a 3.11a 2.64a 2.20a,c 2.41a,c 3.49a,d 2.75a,c 3.99a 65.18a 60.19a,d N1–N3 N4-N6 C1 C2 C3 C4 (M) C4 (F) C5 (M) C5 (F) Adult (M) Adult (F)

SD DT (days) n SD

d,e c,d

DT (days) n SD DT (days) n SD

a b,e

DT (days) n SD DT (days) n SD n

b

ASL25 ASL15b

SD

b,c

DT (days)

a

ES25 ES15

DT (days)

The interpopulation comparison of the stage-specific development time at identical salinities showed some significant differences (Table II). At salinity 5, the development times from N4-N6 to C2 were significantly longer for the St. Lawrence population compared to

ES5

Interpopulation comparison: effect of salinity on the post-embryonic development

ASL15a

In the St. Lawrence population (Table II; Fig. 1), the development time of the first naupliar stages (N1 – N3) was significantly shorter at salinity 5 (ASL5) than at 15 and 25 (ASL15a, ASL15b and ASL25). In contrast, the development of the last naupliar stages (N4 – N6) was significantly longer at ASL5 than at ASL15a and was slightly longer than at ASL15b and at ASL25 (Table II). The development times of C3, C4 (M), C5 (M), C5 (F) and adult (M) were similar under all salinity conditions. In contrast, the development time of C4 (F) was significantly longer and the lifespan of adult (F) was significantly shorter at the salinity 5 (ASL5) compared to salinity 15 (ASL15a). Significant differences between stage development time were found within the first set of experiments (ASL5; ASL15a), but not in the second set of experiments (ASL15b; ASL25). This suggests that the St. Lawrence population was more sensitive to the lowest salinity than to the higher ones. Males developed faster than females, independent of experimental conditions (Fig.1); hence males spent less time within the last developmental stages, C4 and C5, compared to the females (Table II). The difference between food regimes in ASL15a and ASL15b seemed not to have affected the development time except in C1, which had differed significantly between the two treatments (AL15a versus ASL15b, Table II). For the Seine population (Table II; Fig. 1), the two groups of naupliar stages (N1-N3; N4-N6) showed a shorter development time at salinity 5 (ES5) than other experimental conditions (ES15, ES25). The development time of C1 (Table II) was shorter at ES5 than at ES25, but there was no difference in development time for all other stages (from C2 to adult) under any of the experimental conditions. As observed in the St. Lawrence population, Seine males developed faster than Seine females independent of the conditions (Fig.1), therefore males spent less time in the last developmental stages, C4 and C5, as compared to the females (Table II).

ASL5

Effect of salinity on the post-embryonic development within populations

Seine

R E S U LT S

n


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Table II: Mean (DT) and standard deviation (SD) of post-embryonic development time (days), and number of individuals (n) for seven different experimental conditions for both E. affinis populations



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Fig. 1. Cumulative proportion of individuals moulting from one group of stages to the next one as a function of age within developmental stage(s) for both populations of E. affinis studied at 108C. Groups of stages were aggregated for naupliar stages (i.e. N1– N3 and N4–N6) and copepodid stages (i.e. C1 –C3, C4– C5(M) and C4 –C5(F)). Observed data from ASL5 (A; C4 –C5F S), AS15a+b ( ;C4–C5F ), ASL25 (B; C4– C5F V) for the St. Lawrence on the left, and ES5 (W; C4 –C5F 4), ES15 ( ; C4 –C5F ) and ES25 (†) for the Seine on the right. versus expected data from the gamcdf [x/ a, b]fit (lines).

those observed from the Seine population. At salinity 5 and salinity 15, the C5 (F) of St. Lawrence population developed faster than those from the Seine (differences of 1.56, 2.00 and 1.79 days respectively between ASL5

and ES5, ASL15a and ES15, ASL15b and ES15) but C5 (M) was not significantly different. Additionally, the lifespans of the adult males from the St. Lawrence were longer than those observed from the Seine (differences

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of 30.64, 29.96 and 41.12 days respectively between ASL5 and ES5, ASL15a and ES15, ASL15b and ES15). In the second experimental set, developmental time of St. Lawrence N1-N3 was longer than Seine N1-N3 with a difference of 0.7 days; but in the first experimental set, they were not significantly different (P ¼ 0.016). In the first experimental set, St. Lawrence adult females lived significantly longer than Seine adult females by 54.43 days; but in the second experimental set, they were not significantly different (P = 0.005). At salinity 25, only the development time of N4-N6 was longer for the St. Lawrence population (1.35 days). A lack of data for the last copepodid stages due to high mortality rate (C4 to adults) for the Seine did not allow interpopulation comparison at this high salinity.

Table III: R2, SSE and RMSE estimations of the Gamma Density Function gamcdf [x/ a, b]fit to stage duration of E. affinis at 108C and different salinities, represented in Fig. 1 Salinity St. Lawrence

5

15

25

Individual variability Variability of the stage development time was evaluated by observing the distribution of moulting rates from one stage group to the next stage group. The distribution of moulting rates depended on salinity, sex, and population (Fig. 1). In the Seine population, the variability of development times was higher at salinity 25 compared to salinity 5 and 15. In the St. Lawrence population, the highest variability in the development time was observed at salinity 5, especially for N4-N6. The St. Lawrence population showed higher individual variability in naupliar development times (N1-N3 and N4-N6) compared to the Seine population. The values of the statistical parameters (R2, SSE and RMSE) of the cumulative GDF gamcdf [x/ a, b] are given in the Table III. The GDF produced good fits to the moulting distributions for both populations except for the groups N1-N3 at salinity 5 (R2 = 0.469; SSE = 1.378; RMSE = 0.218) and 15 (R2 = 0.724; SSE = 0.724; RMSE = 0.158), and the group N4-N6 at salinity 5 (R2 = 0.667; SSE = 0.863; RMSE = 0.173) of the Seine population. These exceptions were not used in Fig. 2, which shows the relationship between parameter b and the ratio (a / Median Development Time). The aggregated naupliar stages (N1-N6) of St. Lawrence population were widely dispersed as were the copepodid stages (C1-Adults) of the Seine population. The smallest dispersion was exhibited by the aggregated naupliar stages (N1-N6) of the Seine population.

Seine

5

15

25

Stage group

n

R2

SSE

RMSE

N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F) N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F) N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F) N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F) N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F) N1– N3 N4– N6 C1 –C3 C4 –C5(M) C4 –C5(F)

87 87 81 34 46 104 103 99 51 46 41 38 29 16 14 31 31 28 17 11 25 23 19 10 8 30 27 11 3 1

0.9180 0.9836 0.9608 0.9757 0.9680 0.9687 0.9867 0.9907 0.9726 0.9859 0.9780 0.9775 0.9407 0.9729 0.9491 0.4681 0.6667 0.9430 0.9455 0.9753 0.7242 0.9466 0.9653 0.9348 0.9335 0.8939 0.9658 0.9438 – –

0.5957 0.1192 0.2650 0.0685 0.1224 0.2701 0.1141 0.0774 0.1157 0.0542 0.0752 0.0712 0.1437 0.0360 0.0381 1.3777 0.8632 0.1325 0.0765 0.0226 0.7242 0.1025 0.0546 0.0538 0.0498 0.2652 0.0766 0.0516 – –

0.0837 0.0375 0.0579 0.0463 0.0527 0.0515 0.0336 0.0283 0.0486 0.0351 0.0439 0.0445 0.0730 0.0507 0.0737 0.2180 0.1725 0.0714 0.0714 0.0502 0.1579 0.0699 0.0567 0.0820 0.0843 0.0973 0.0554 0.0757 – –

n indicates the number of data points.

Fig. 2. Relationship between parameter b and the ratio a/MDT (Mean of Development Time) of the gamma density function, gamcdf [x/ a, b]. Parameter fits were obtained for the different groups of stages and for both populations of E. affinis.

General pattern of post-EDT in E. affinis The sample sizes throughout our data set varied among groups of stages within and between both populations due to mortality in the experimental treatments. To examine the general developmental pattern within the

two populations of E. affinis at 108C independently of salinity, we mixed the IDs of both populations and standardized them (i.e. neutralized the effects of

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experimental conditions and populations). This allowed us to increase the sample size for each stage group. An increased sample size was more representative of the distribution of the sampled population because the probability of finding scarce events increased (i.e. individuals with very slow or very rapid development compared to the mean of the population). Standardized IDs distributions for each stage group of E. affinis and their fitted probability density functions were plotted (Fig. 3). The maximum frequencies of each histogram correspond to the mode of the distribution (=1). The shape of all these distributions was asymmetrical. We fitted normal, lognormal and GDFs to these data. Confidence bounds of each fitted probability density functions were represented (Table IV).

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According to the values of SSE (close to 0), R2 (close to 1) and RMSE (close to 0), the GDF was the best fit model of the IDs distribution with the exception of the N1-N3 group (Table IV; Fig. 3A), where the best fit was obtained with a Lognormal Density Function.

Survival during post-EDT No mortality was observed for nauplii N1 to N4 in either population for any of the experimental conditions. However, survival during development differed among experimental conditions within and between the two populations (Fig. 4). Highest overall mortality was recorded in treatment ES25, where cumulative mortality resulted in 13% survival to copepodid stage

Fig. 3. Experimental and fitted probability density functions of the standardized index of development of the aggregated naupliar [N1–N3 (A); N4–N6 (B)] and aggregated copepodites [C1–C3 (C), C4– C5(M) (D) and C4– C5(F) (E)) stages. Histograms correspond to the experimental data and continuous lines represent the best-fitted distributions using three models: normal, lognormal and gamma.

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Table IV: Parameter values with 95% confidence intervals for the Normal Probability Density Function (m,s), the Log Normal Probability Density Function (m*,s*) and of the Gamma Probability Density Function (a, b) fit to development time for four groups of stages of E. affinis at 108C Function parameters (confidence bounds) Function

Stage group

m/m*/a

s/s*/b

R2

SSE

RMSE

N LogN Gamma N LogN Gamma N LogN Gamma N LogN Gamma N LogN Gamma

N1– N3 N1– N3 N1– N3 N4– N6 N4– N6 N4– N6 C1 –C3 C1 –C3 C1 –C3 C4 –C5(M) C4 –C5(M) C4 –C5(M) C4 –C5(F) C4 –C5(F) C4 –C5(F)

1.001 (0.9872, 1.015) 0.004423 (20.0074, 0.01625) 89.18 (68.48, 109.9) 0.9713 (0.9479, 0.9946) 3.213 1011 (**) 19.88 (16.27, 23.49) 0.9484 (0.934, 0.9628) 2.425 1010(**) 44.74 (36.95, 52.52) 0.9721 (0.9503, 0.9939) 1.784 1011 (**) 30.23 (23.46, 37) 0.9638 (0.9391, 0.9885) 1.957 1012 (**) 71.11 (42.18, 100)

0.1069 (0.09277, 0.121) 0.1058 (0.0941, 0.1176) 0.01129 (0.008655, 0.01393) 0.2176 (0.1942, 0.241) 0.2321 (0.2098, 0.2544) 0.05015 (0.04076, 0.05955) 0.1411 (0.1268, 0.1555) 0.1689 (0.1409, 0.1969) 0.02146 (0.01767, 0.02524) 0.1788 (0.1566, 0.201) 0.187 (0.1624, 0.2115) 0.0327 (0.02523, 0.04018) 0.1136 (0.08891, 0.1383) 0.1351 (0.1009, 0.1693) 0.01368 (0.008063, 0.1929)

0.9734 0.9811 0.9792 0.9770 0.9786 0.9825 0.9730 0.9239 0.9797 0.9749 0.9654 0.9779 0.9394 0.9040 0.9453

387.6 275.2 304.1 243.5 226.9 185.6 208 587 156.6 35.42 48.82 31.16 105.6 167.3 95.28

6.226 5.246 5.514 4.505 4.177 3.932 3.606 5.876 3.129 1.984 2.21 1.861 3.633 4.312 3.451

(**) indicates the confidence bounds could not been estimated.

Fig. 4. Survival rate (%) during post embryonic-development and before maturation in response to different salinity (5, 15 and 25) for E. affinis from the St. Lawrence salt-marshes (ASL) and Seine estuary (ES). Curves represent percentage of survival per developmental stage from N1 to C5.

C5. In general, highest mortality was observed in stages just before and/or after metamorphosis (N6 and copepodid stages) in both populations. In treatments ES15 and ASLS25, copepods showed similar patterns of survival during development, resulting in 70% survival at stage C5. Treatments ASL5, ASL15a, ASL15b and ES5 also showed similar patterns of mortality despite differences in the experimental conditions (survival .90%).

Survival during adulthood In the St. Lawrence population, we found a salinity effect on adult survival in both males and females. Both sexes died earlier at salinity 25 compared to salinities 5

and 15, and the age of the oldest males and females in this treatment was approximately the same (Fig. 5). The median survival of males was between 93 and 102 days at salinities 5 and 15, respectively, and the longest living male was 146 days old. In contrast, the female median survival in treatment ASL15a was 120 days, and the oldest female died at an age of 173 days. In the Seine population, the differences in survival between experimental conditions were less pronounced (Fig. 5). At salinities 5 and 15, males reached the median and quartiles of survival at the same age. The last set of males all died at the age of 50 days. At salinity 15, female median survival was reached at 37 days; this was before the female median survival at salinity 5 which was 45 days. However, median survival of females at both salinities reached the first quartile at the same age, even though the last females died at approximately the same age: 63 days old at ES15 and 64 days old at ES5. At salinity 5, the median of survival for males was 35 days while the median survival of females was 45 days. Both median survival of males and females were similar at salinity 15. Survival rate during the adult stage differed dramatically between both populations. The St. Lawrence population showed a greater survival in all experimental conditions compared to the Seine population (Fig. 5). In fact, the median survival for St. Lawrence individuals was more than twice that of Seine individuals under the same experimental conditions (ASL5 versus ES5; ASL15a versus ES15). The maximum age was observed for St. Lawrence females, which lived three times

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extrude the next egg sac. No significant differences were found between treatments ASL15a and ASL15b for the reproductive parameters. Independent of experimental conditions, the CSs of both populations of E. affinis were very different. While St. Lawrence females produced a maximum 19 clutches with an average of 52 eggs per clutch during adulthood, Seine females produced a maximum 11 clutches with an average of 24 eggs per clutch. At salinity 5, the LT and the ICT were shorter for St. Lawrence than the Seine. The EDT at salinity 15 was lower for the St. Lawrence (ASL15a and ASL15b) than for the Seine (ES15). No differences in the reproductive parameters were observed among salinity treatments of the Seine population. Unlike the Seine population, the reproduction of the St. Lawrence population was enhanced at the lowest salinity, increasing the CS and decreasing the LT and the EDT which affected the ICT. Fig. 5. Survival rate (%) of males (A) and females (B) in response to salinity (5, 15 and 25), date and food (a and b) of two populations of E. affinis from St. Lawrence salt-marshes (ASL) and the Seine estuary (ES) at 108C. Only individuals within adult stage are represented and the absolute age zero corresponds to the hatching day. The first and third quartiles (– –) and the median of survival (---) are represented. The median of survival correspond to the ages of 50% dead adults. b: fed with R. baltica. Each point represents one individual. ES25 is not represented because a lack of data due to high mortality rate during post embryonic-development.

longer than Seine females. A higher survival rate of females compared to males was clearly observed in the Seine population under both experimental conditions. In contrast, in the St. Lawrence population, the last male and the last female died at approximately the same time (ASL5, ASL15b and ASL25) except under ASL15a.

DISCUSSION A broad salinity tolerance allows copepod species to expand their habitat over a wide geographic range and/or to live in environments with wide salinity variations such as estuaries and tidal ponds. This study compared the physiological performance of two genetically and morphologically (i.e. phylogeny) divergent populations of E. affinis in relation to salinity. From copepod cultures, a common individual-based protocol was used to compare development, survival, reproduction and longevity of both populations. The data resulting from this experiment can be useful to calibrate an individual-based model and gain insights into the genotypic variability within the species complex.

Methodology Effects of salinity on reproduction For the St. Lawrence population (Fig. 6), the CS was significantly higher under treatment ASL15b (75 eggs clutch21) than treatment ASL5 (52 eggs clutch21), although the highest number of clutches produced by a female (19 clutches) was found at salinity 5 (ASL5). Furthermore, the highest HS (98%) was observed at that low salinity, while HS was 91%, 92% and 86% at treatments ASL15a, ASL15b and ASL25, respectively. The ICT was 4.3 days at salinity 5 (ASL5) and 6.3 at salinity 25 (ASL25). The LT, EDT and ICT were significantly longer at salinity 25 than at salinity 5. Under all treatments, several zero LT were observed because the female lost or detached the egg sac before hatching to

Previous studies have shown that the GDF is an effective tool for estimating stage durations and development times, and describing the individual moulting rate from one stage to the next one (Souissi et al., 1997; Souissi and Ban, 2001; Devreker et al., 2007). Because of its asymmetrical shape, the GDF is useful to fit the asymmetrical distribution of IDs. The distributions of moulting rates were well fit by the GDF (see R 2 and SSE values in Tables II and III), confirming the usefulness of this function. This asymmetry is a result of the patterns of individual variability (see Fig. 3) inherent in copepod development (Souissi et al., 1997; Souissi and Ban, 2001). Other advantages of the GDF compared to other fitting functions were discussed by Klein Breteler et al.

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Fig. 6. Mean values of reproductive parameters of females observed in seven experimental conditions and for both E. affinis populations studied at 108C: Clutch size (eggs clutch21) (A), egg production rate (eggs female21 day21) (B), embryonic development time (days) (C), inter-clutch time (days) (D), maturation time (days) (E), latency time (days) (F). For ASL5, ASL15a, ASL15b, ASL25, ES5 and ES15, we observed 15, 15, 5, 9, 7 and 8 individuals, respectively. ES25 is not included due to low survival rate during development at that treatment. The standard deviation is represented by the vertical bars. Histograms with the same letter are not significantly different using Wilcoxon analysis with significant level at P , 0.001 between treatments within a population (ASL or ES) and between treatments at same salinity.

(Klein Breteler et al., 1994). In our study, the efficiency of the GDF was improved by increasing the number of individuals. Mortality reduces the number of individuals during the life cycle experiments, and a low initial number of individuals can affect the accuracy of the parameter estimations of fitting GDF (Souissi and Ban, 2001). Thus, the initial number of individuals in each experiment needs to be large enough to compensate for these losses. When 87 individuals were used for the St. Lawrence population, only half of the individuals reached the adult stage. Moreover, the observation interval needs to be small enough to get a good resolution of moulting probabilities in each developmental stage. In our study, the early naupliar stages (N1 – N3) of

the Seine population developed rapidly, resulting in many individuals moulting within the same time interval. As a result, the accuracy of the fitted GDF was weak (R 2 , 0.9). Thus, we confirmed that the accuracy of GDF to fit individual-based experiment data is impaired by an inappropriate number of individuals and inappropriate observation intervals, as previously described by Hu et al. (Hu et al., 2007). If the experimental conditions are well chosen (experiments reared under controlled conditions at food satiation, initial number of individuals high enough and frequent observations), the GDF provides a robust fit to the data set. Thus, this GDF can be used in an individualbased model to represent the individual variability of the population being modelled (Dur et al., 2009).

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Laboratory individual-based experiments The use of a common protocol for the individual-based experiments allowed us to describe the reaction norms of each population (i.e. pattern of phenotypic plasticity regarding salinity in a life cycle trait), and their life history strategies under different experimental conditions. Differences in individual responses in life history traits of a group of individuals which were exposed to (or cultured under) similar controlled environmental conditions are due to intrinsic properties of each individual, related to its particular genotype. Hence, the physiological differences observed between the populations in our study might be partially due to genetic divergence and to acclimation to experimental conditions. Here, the acclimation to experimental parameters such as salinity and photoperiod was long (more than 10 generations) for both population but the acclimation of the Seine population to 108C was short (one generation) that probably decreased its life cycle performance (Devreker et al., 2009). Life cycle studies using individual-based experiments highlight individual variability. The Seine population exhibited higher variability in development time at the highest salinity (i.e. 25). According to Souissi et al. (Souissi et al., 1997), the variability in copepod development increases when they are reared under limiting conditions. Jimeńez-Melero et al. (Jimeńez-Melero et al., 2007) observed in another egg-bearing copepod, Arctodiaptomus salinus that the variability in development time (i.e. EDT and ICT) increased at high temperature where its survival was reduced. Comparing both populations, the development time of naupliar stages of the St. Lawrence population showed higher variability than that of the Seine population. As the heritability of the development time has been established at the individual scale by Lee et al. (Lee et al., 2003), the variability in development time, assuming it is a reaction norm, might be an inheritable trait at the population scale. Hence, a small proportion of the population should always be able to find optimal conditions within a fluctuating habitat and be able to invade new habitats such as freshwater reservoirs (Lee, 1999), because the phenotypic variability in development increases at stressful salinities. We observed only minor differences of postembryonic development between both populations in our study (Table II), in particular at salinity 15 (culture salinity), that might be due to the uniform selection pressure of constant laboratory conditions. Lee et al. (Lee et al., 2007) showed that salinity tolerance of two populations of E. affinis can be shifted under constant rearing conditions. For instance, they found that the freshwater tolerance (tolerance to salinity 0) of the

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freshwater population from Lake Michigan decreased following two generations of selection at salinity 5 (salinity of culture), but still persisted only for a small part of the population during the subsequent six generations. Selection processes within our cultures which were reared for more than 10 generations under constant salinity probably led to a slight shift in the physiological reaction norms. On the other hand, the highest variation was still found in the St. Lawrence population which was held for the longest time under laboratory conditions, indicating that it still maintained a broad response capacity to the conditions experienced in the experiments.

Salinity effects on population performance of E. affinis When copepods are not at their optimal salinity, they experience osmotic stress (Roddie et al., 1984; Hall and Burns, 2002). This stress is characterized by the allocation of a greater proportion of energy to the metabolic process of osmoregulation, by the synthesis or the degradation of proteins to maintain the concentration of solute in intracellular medium (Kimmel and Bradley, 2001). Thus, there is less energy available for development, probably resulting in slow development and/or high mortality. In this study at 108C, the highest mortality rate in post-EDT development was recorded for the Seine population at salinity 25, suggesting that conditions were stressful for this population. However, the salinity had only a slight effect on survival of early naupliar stages of both populations (see Fig. 4) which might be due to a greater osmotic tolerance of these premetamorphic stages compared to others. The slower development time of females relative to that of males is probably due to larger body size (Katona, 1971), and partly to the long MT of the oocytes (Smith and Lane, 1985). In the present study, we confirmed differences in development pattern between females and males (Table II) already observed in this species by Ban (Ban, 1994) and Devreker et al. (Devreker et al., 2007). The sexual size dimorphism could also explain their distinct physiological abilities. Females of E. affinis are bigger than males (Katona, 1970, 1971); thus, females have a lower surface to volume ratio than males (Wolvekamp and Waterman, 1960). Hence, their osmoregulation abilities might be different. During adulthood, we found differences in salinity tolerance (Fig. 5) between genders, also females showed a higher survival rate than males. Females of another estuarine calanoid copepod, Acartia tonsa also showed higher salinity tolerance than the males of the same species (Cervetto et al., 1999).

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Interpopulation comparison: life history evolution The two geographically distinct populations exhibited different ranges of salinity tolerance at 108C. Eurytemora affinis from the St. Lawrence salt-marshes seemed to be tolerant of a wide range of salinity (5 – 25) whereas E. affinis from the Seine tolerated salinities from 5 to 15 and exhibited low tolerance at 25. The North American clade (St. Lawrence population) and the European clade (Seine population) have probably been separated for millions of years (Lee, 2000), so that each clade experienced different environmental conditions. Thus, each environment has influenced individual physiological performance and ultimately the ecological performance of each population. The St. Lawrence salt-marsh population encounters a wide salinity range in its habitat, from 5 to 40 (Winkler, unpublished data), which exceeds the highest salinity tested in our experiments (25) and a wide temperature range from May to August between 5 and 278C (Ward and Fitzgerald, 1983; Castonguay, 1988). We assume that this population is able to tolerate salinities greater than 25. In the Seine estuary, the temperature ranges seasonally from 6 to 218C (Mouny and Dauvin, 2002) while salinity changes on a tidal cycle, 2 tides per day (Devreker et al., 2008). In this habitat, the Seine population of E. affinis dominates the zooplankton community in the oligo- to meso-haline zones (salinities 2.5 to 18) where it reaches concentrations of 149 000 ind. m23 (including only late copepodid stages and adults) during spring (Mouny and Dauvin, 2002) and 700 000 ind. m23 (all developmental stages included), during ebb tides (Devreker et al., 2008). In the area of Seine estuary where the population was sampled, the salinity varies daily from 0.5 to 25, with the majority of the population between salinities 0.5 and 15 and few individuals found at salinities over 20 (Devreker et al., 2008). Consequently, this population encounters a narrower range of salinity than the St. Lawrence population. Although in our study the F1 generation of the Seine population did not tolerate salinity 25 at 108C, it was able to develop and reproduce at this salinity at 158C in another study using the same protocol (Devreker et al., 2007). However, copepods of the Seine population were cultivated for 10 generations at 158C and then were acclimatized for only 1 generation at 108C prior to the experiments. The short acclimation time from 15 to 108C has been suggested to be insufficient to provide good adaptation to colder temperature, resulting in a decrease of female performance (Devreker at al., 2009). Furthermore, salinity 25 corresponds to the limit of the distribution of E. affinis adults and late

copepodites in the Seine estuary (Devreker et al., 2008). We postulate that this population might be well adapted to the range of salinity (salinities 2.5 to 18) where it dominates the zooplankton community in the Seine estuary. It might survive at higher salinities (salinities 18 to 25) for a short period of time (tidal scale), and females can reproduce after a period of acclimation to temperature variations that is greater than one generation, which corresponds to the seasonal succession of temperature. Interpopulation differences in salinity tolerance have already been shown within the E. affinis species complex. Lee et al. (Lee et al., 2003) studied the physiological tolerance of two populations of E. affinis, one population from Lake Michigan (freshwater, salinity ,0.5) and the other one from St. Lawrence saltmarshes (the same population used in our study). Four salinities were tested, 0, 5, 15 and 25, and they showed that salinities 5 and below were stressful for the St. Lawrence population (87% mortality). This might explain the increase in variability of development time of the last naupliar stages at salinity 5 in our study. In comparison, salinities 15 and above induced a high mortality (70%) for the Lake Michigan population. Thus, the salinity 5 seems to represent an intermediate salinity tolerated by all populations of E. affinis that have been tested to date. Lee et al. (Lee et al., 2003) also observed that the St. Lawrence population had an optimal salinity range from 5 to 25 (see their Fig. 4). Our experiments on the Seine population at salinity 25 showed that salinity affects the development rate and survival, and consequently the reproduction rate of individuals. We suggest that the optimal salinity of the Seine population corresponds to an intermediate salinity between the optima of the Lake Michigan and the St. Lawrence populations. Our study revealed that fitness, in terms of egg production rate and reproductive lifespan, was much higher in the St. Lawrence population than in the Seine population. The egg production rate of the Seine population was 12.6 eggs female21 day21 at 158C (Devreker et al., 2009) using the same protocol as ours, and the experimental temperature used the present study (or the acclimation period) was not optimal for its reproduction. In fact, this experimental temperature is relatively close to the average minimum temperature of 68C found in the Seine estuary. The maximum longevity of individuals in the Seine population is similar to those found in most of the subtemperate copepod species, 2 to 3 months (Ianora, 1998). In contrast, the maximum longevity of the St. Lawrence population is more than 5 months. The high reproductive output coupled with the long reproductive lifespan of the St. Lawrence

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population of E. affinis might be an evolutionary adaptive response to its habitat. In the Isle Verte’s saltmarshes, this population is found in two kinds of ponds: permanent and ephemeral tide ponds. In both types of ponds, the temperature changes quickly between day and night, up to 158C on a daily basis (McQuinn et al., 1983) while salinity changes due to precipitations on an irregular basis and/or flooding of the ponds by tides. Adaptive responses (i.e. individual variability in development, high reproductive output and long reproductive lifespan) might have been selected for maintenance of the population in permanent ponds and rapid colonization of temporary ponds despite the rapid fluctuations of abiotic factors. The responses found enhance the hypothesis that such adaptive responses may have helped this population to invade new environments, such as freshwater reservoirs as suggested by Lee et al. (Lee et al., 2003). In summary, the results of these experiments confirm that the GDF is well adapted to represent copepod development and indicates that large data sets and adapted frequency observations are required to improve its accuracy. We have shown that the life cycle strategies of both E. affinis populations in relation to salinity are different at 108C. The inter-population differences observed might be due to genetic and phenotypic differences resulting from divergent evolutionary history. Moreover, our study shows that more than one generation is necessary for acclimating individuals at low temperature before measuring life cycle traits of this copepod. All life cycle traits measured here suggested that the Seine population developed under stressed conditions resulting in its performance being lower than those observed in the field for the same temperature (Devreker, 2007). This paper confirms the importance of designing adequate and accurate experimental protocols to obtain valuable empirical data to develop individual-based models of these copepods (Souissi et al., 2005; Dur et al., 2009). Eurytemora affinis offers a good biological model to study the consequences of life history trait differences between divergent clades (or populations) using individual-based models. In the case of E. affinis, we suggest developing a specific model for a given clade (or population) to avoid erroneous representation of population dynamics of the cryptic species complex of E. affinis.

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affinis. We thank Dr Carol Eunmi Lee for permitting us to work on St. Lawrence population of E. affinis. We thank Pr. T. Ramakrishna Rao for his comments on this manuscript. This paper is a contribution to bilateral collaboration programs between France and Taiwan and between France and Canada. We are grateful to all S. Souissi’s students that contributed to the maintenance of copepod cultures in the Marine Station of Wimereux. We are grateful to James J. Pierson for his comments and improvements of the English. This manuscript was improved by insightful comments of anonymous reviewers.

FUNDING This work is a contribution to ZOOSEINE project funded by Seine-Aval IV program.

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This study is a contribution to Seine-Aval III program within the framework of the project aiming at building bioindicators based on the estuarine copepod Eurytemora

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