Impact of water calcium on the phenotypic diversity of alpine populations of Gammarus fossarum
Knowledge of the contribution of the environment to intraspecific differentiation between neighboring populations is fundamental to understanding the ecological significance of geographic variation (for recent references, see Niewiarowski and Roosenburg 1993). A common approach is to identify differences between populations and to examine possible correlations between observed biological variation and environmental variation. In this respect, a synthetic approach that includes both comparative and experimental techniques plays an important role in identifying ecological sources of biological variation and their evolutionary significance (Niewiarowski and Roosenburg 1993). However, owing to the diversity of ambient parameters and their multiple interrelations, it is often difficult to identify a predominant factor responsible for phenotypic variation (Berven and Gill 1983).
Among freshwater crustaceans, Gammarus fossarum (Amphipoda, Gammaridae) has been considered a good model for studying intraspecific variation as it lives in clusters with less populated areas between (Siegismund 1988). Morphological and morphometrical characters were first used to differentiate neighboring populations of Gammarus fossarum (Dusaugey 1955, Roux 1970), as reported for other gammarid species (Bulnheim and Scholl 1981, Scheepmaker 1987, Kane et al. 1992). However, it soon became apparent that they were poor distinctive criteria because of their considerable amount of variability, not only between different populations, but also within the same population, going so far as to make the characterization of the species difficult (Pinkster 1983, Sheepmaker and Van Dalfsen 1989, Sheepmaker 1990). Moreover, identifying environmental sources of such morphological variation remained unsuccessful (Roux 1970). The diversity among geographic populations has been then considered at the ecophysiological level, leading to the concept of physiological races (Roux 1967). Of the ecophysiological traits investigated among different populations (Roux 1971), the calcium (Ca) requirement during molting is of key interest (Ormerod et al. 1988). Ca metabolism is a central parameter in the biology of gammarids (Graf 1974). As in other crustaceans, there is an important periodic Ca turnover through the cuticle during molt cycles throughout life (reviewed in Graf 1978, Greenaway 1985). Moreover, since Gammarus fossarum cannot store Ca before exuviation, as is the case for most gammarids (Vincent 1963, Graf 1965, Wright 1979, 1980), the Ca turnover during the molt cycle may depend upon the Ca availability in the water. Previous ecophysiological investigations at this level led to conflicting interpretations and a precise correlation between observed ecophysiological differences and clearly delimited environmental parameters was generally unclear (Roux 1971, Vincent 1971).
TABLE 1. Comparative analysis of ionic concentrations (Ca,
Mg, Na, K, HC[O.sub.3], Cl, S[O.sub.4], and N[O.sub.3] [mg/L])
and pH in water from the different sites [ILLUSTRATION FOR FIGURE 1 OMITTED].
and pH B3 B4 C2 V3 V1 C1
Ca 12.45 14.85 75.80 78.5 83.50 95.70
Mg 2.20 1.50 3.60 4.30 1.10 3.70
Na 1.40 0.83 2.10 3.00 4.90 3.50
K 0.15 0.13 0.50 0.80 0.80 0.90
HC[O.sub.3] 36.60 41.50 221.00 206.00 261.00 293.00
Cl 0.60 0.30 2.70 1.40 8.20 4.40
S[O.sub.4] 8.00 7.20 12.00 11.00 6.00 9.80
N[O.sub.3] 1.20 1.50 0 0.80 4.20 2.70
pH 7.76 7.73 8.23 8.24 8.25 8.17
In the field, the rearing boxes of control specimens were immersed in water from the collecting site (native water). Boxes of translocated specimens were transplanted to another site (alien water). To simplify the experimental design, not all combinations were performed (see Table 4). The average temperature of the water (11 [degrees] C) was determinated by periodic measurements in situ in all study sites during the period of experimentation.
At the laboratory, animals were reared in a climate-controlled room (constant temperature: 11 [degrees] C), with a daily cycle of 12 h of light. Control specimens were placed into water from their native site, whereas translocated specimens were reared in water from an alien site. The experimental design involved the same combinations as in the field experimentation (see Table 4). Water was changed weekly.
To further investigate the impact of the Ca concentration of the water on the duration of the molt cycle, some specimens from B3 and B4 populations were reared in their native water artificially enriched in Ca up to a concentration equivalent to that of C1 site by spectrophotometrically controlled addition of CaS[O.sub.4].
Both field and laboratory experiments were systematically performed on 600 animals divided into uniform samples each including 8 specimens of the same size, from all collecting sites. Experimentation occurred repeatedly and simultaneously in the field and in the laboratory from summer to autumn 1993 and from spring to autumn 1994.
Calcium balance analysis
At the end of each experiment, the total body Ca content of the dated specimens after 24 h of starvation was determined through atomic absorption spectro-photometry. [TABULAR DATA FOR TABLE 2 OMITTED] After measurement of their dry mass (Graf 1969), whole specimens were homogenized and then mineralized with [H.sub.2]S[O.sub.4] 6N (Meyran et al. 1987). The Ca concentration in homogenates was measured with a Varian apparatus at 422.7 nm after dilution in 0.2% lanthanum chloride. In order to avoid individual variation related to the animal’s size, results were standardized to a constant dry mass of 10 mg, following Graf (1964). In the same way, the Ca balance during the successive phases of the molt cycle was determined on 400 field-collected specimens at all stages of the molt cycle, directly originating from all geographic populations.
Data were analyzed using the SAS statistical package (SAS 1988).
Environmental parameters: differences in water Ca concentrations
Successive measurements indicated no obvious difference in ionic concentration among water samples of the same study site from one period of experimentation to another. Data from Table 1, summarizing the first analysis, revealed that the most prominent differences among water samples from the different areas were Ca concentrations, which may be related to the geological differences of the leached substrata. High Ca titers were characteristic of waters from the limestone areas of Chartreuse and Vercors (from 75.8 mg/L in C2 to 95.7 mg/L in C1), whereas waters with low Ca concentration were localized within the crystalline area of the Belledonne (from 12.4 mg/L in B3 to 14.8 mg/L in B4). Differences in [Mathematical Expression Omitted] concentrations were also noticed, which may be responsible for the differences in pH between waters from the two different geological areas.
TABLE 3. Analysis of variance for the dry mass of animals and
the mean duration of the molt cycle in reared control
populations (NS = nonsignificant; * P [less than] 0.05; **
P [less than] 0.01; *** P [less than] 0.001) (localization
of experiments: field or laboratory).
Source df MS F
Animal’s dry mass
Site of origin (SO) 6 63.73 23.98(***)
Error 256 2.65
Mean duration of the molt cycle
Site of origin (SO) 6 55.74 11.25(***)
Localization of experiments (LOC) 1 13.7 12.77 NS
SO x LOC 4 5.1 81.05 NS
Error 109 4.95
The present study clearly relates phenotypic differences among alpine populations of Gammarus fossarum to the levels of environmental sources of Ca. The populations from limestone areas of Chartreuse and Vercors include specimens with a larger maximum size and a longer molt cycle than those from the crystalline site of Belledonne. The Ca balance of animals from limestone areas was different during both pre- and postmolt from that of specimens from crystalline sites. Experimental increasing of Ca in the environment can reduce the duration of the molt cycle and vice versa.
However, our experimental results appear to contradict the descriptive data. The comparison is biased at the descriptive level as the measurements were made only on adults of maximum size, which were largest in limestone populations. As previously stated, the duration of the molt cycle in smaller specimens from limestone populations was shorter than that observed for animals of the same size living in crystalline areas. Smaller animals have not been retained in our study as they are not representative for a rigorous comparison among different populations.
TABLE 6. Effects of translocation of B3 and B4 animals in their
native water, artificially Ca-enriched up to a concentration
equivalent to that of C1 (equivalent C1) on the mean duration
(mean no. days, with standard errors in parentheses) of the molt
cycle in laboratory experiments (N = number of observations) vs. the
mean duration of the molt cycle in B3, B4, and C1 control populations.
Average duration of the molt cycle
Originating Control specimens located in
site in native water Ca-enriched water
B3 39.08 (1.84) 34.92 (1.93)
N = 23 N = 9
B4 38.78 (1.47) 34.14 (1.41)
N = 25 N = 11
C1 42.10 (1.02)
N = 5
The ecopbysiological plasticity of Ca metabolism currently observed among alpine populations of Gammarus fossarum may be a determining factor in the dynamics of colonization of this species. Migration constitutes the central parameter of colonization processes in gammarids (review in Goedmakers and Pinkster 1981) but its relation with the environmental Ca availability is still controversial. Previous authors have considered Gammarus fossarum to be a preglacial inhabitant of the Alps (review in Scheepmaker 1990), largely represented in this area (Dusaugey 1955, Siegismund and Muller 1991), and rather independent from the Ca content of the water (Roux 1971). Such an independence may be limited, as suggested by our ecophysiological results and our field observations, which showed that the most flourishing populations were located in high Ca concentration areas. Moreover, current experimental data reveal that mortality rates increased in animals translocated to waters lower in Ca concentration. This relatively greater difficulty in adapting to lower environmental Ca levels, earlier observed in other gammarid species by Vincent (1971), allows us to suggest that the primitive populations of Gammarus fossarum probably lived in limestone areas and that colonization of the crystalline sites may have been secondary. This hypothesis agrees with the migration theory earlier proposed by Pacaud (1945a, b), which was based only upon biogeographic considerations.
The phenotypic variation among geographic populations of Gammarus fossarum observed in this study may not only reflect environmental diversity, as presently suggested, but may also have a possible genetic basis. In this respect, the concept of microgeographic races was proposed (Goedmakers 1980a, b, 1981a, b, Goedmakers and Pinkster 1981). Using enzymatic polymorphism analysis, Siegismund and Muller (1991) found that local populations of Gammarus fossarum show strong genetic differentiation compared to other gammarid populations. Such genetic differentiation is now being further considered at the molecular level through the comparison of mitochondrial DNA (Meyran and Taberlet 1996), as this genetic marker appears to evolve rapidly at the sequence level in crustaceans (Palumbi and Benzi 1991).
Finally, the present study clearly attests to the value of a synthetic approach including both comparative and experimental techniques in identifying environmental parameters responsible for biological variation among neighboring populations. Moreover, investigation of Ca metabolism during the molt cycle may provide a new powerful ecophysiological tool to supplement the criteria generally used to test the phenotypic variability among geographic populations of gammarids.
The author thanks A. Zganic and the Laboratoire Regional d’Analyse des Eaux, Universite J. Fourier, Grenoble, for the water chemistry analysis and the atomic absorption spectrophotometric dosage of calcium; Dr. I. Till-Bottraud for help in statistics, Dr. B. Serra-Tosio for helpful advice in the choice of the study sites, and L. Waits for help with the English. This work was funded by the Centre National de la Recherche Scientifique and the University J. Fourier (Grenoble, France).
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