IMPACT OF WATER CALCIUM ON THE PHENOTYPIC DIVERSITY OF ALPINE POPULATIONS OF GAMMARUS FOSSARUM

1579

Ecology, 78(5), 1997, pp. 1579–1587q 1997 by the Ecological Society of America

IMPACT OF WATER CALCIUM ON THE PHENOTYPIC DIVERSITY OFALPINE POPULATIONS OF GAMMARUS FOSSARUM

JEAN-CLAUDE MEYRAN

Laboratoire de Biologie des Populations d’Altitude, CNRS UMR 5553, Universite Joseph Fourier,BP 53 F-38041, Grenoble Cedex 9, France

Abstract. As an example of the relative impact of a specific ambient parameter onintraspecific phenotypic differentiation, the contribution of environmental sources of cal-cium on the geographic variation between alpine populations of the freshwater crustaceanGammarus fossarum was investigated through a comparative analysis of six different pop-ulations in the torrents of the Grenoble region of France.

Major differences in water calcium levels were observed in correlation with lithologicaldifferences (average 13.6 mg/L in the crystalline massif of the Belledonne and 83.4 mg/Lin the limestone areas of the Chartreuse and Vercors). The comparative analysis of thepopulations was performed through field and laboratory experimentation at morphometricaland ecophysiological levels. Uniform samples of male adult specimens from six differentsites (two sites in the Belledonne, two sites in the Chartreuse, and two sites in the Vercors)were compared using three easily measurable parameters: maximum dry mass, duration ofthe molt cycle, and calcium balance during the molt cycle.

Animals from waters that flow over limestone show a larger maximum size and a longermolt cycle than those from waters that flow over crystalline rocks. Spectrophotometricalanalysis of their calcium balance during the molt cycle reveals that, whereas all animalsare equally calcified during the intermolt, the decalcification during the premolt and therecalcification during the postmolt are more rapid in animals from limestone areas than inthose from crystalline sites. Translocation experiments in both natural and artificially cal-cium-enriched waters confirm these differences. A significant increase in the duration ofthe molt cycle was observed in animals translocated to water of lower calcium concentrationand vice versa, whereas no significant difference was observed between controls and animalstranslocated into water of comparable calcium concentration. Current results, supported bysuch a new synthetic experimental approach, indicate that variations in water calcium levelsmay generate important ecophysiological differentiation between populations, which maysuggest a possible influence of environmental calcium on the geographic distribution ofGammarus fossarum.

Key words: adaptive diversity; amphipods; calcium metabolism; experimental ecology; Gammarusfossarum; molt cycle.

INTRODUCTION

Knowledge of the contribution of the environmentto intraspecific differentiation between neighboringpopulations is fundamental to understanding the eco-logical significance of geographic variation (for recentreferences, see Niewiarowski and Roosenburg 1993).A common approach is to identify differences betweenpopulations and to examine possible correlations be-tween observed biological variation and environmentalvariation. In this respect, a synthetic approach that in-cludes both comparative and experimental techniquesplays an important role in identifying ecologicalsources of biological variation and their evolutionarysignificance (Niewiarowski and Roosenburg 1993).However, owing to the diversity of ambient parametersand their multiple interrelations, it is often difficult to

Manuscript received 11 January 1996; revised 10 July1996; accepted 25 July 1996; final version received 25 Sep-tember 1996.

identify a predominant factor responsible for pheno-typic variation (Berven and Gill 1983).

Among freshwater crustaceans, Gammarus fossarum(Amphipoda, Gammaridae) has been considered a goodmodel for studying intraspecific variation as it lives inclusters with less populated areas between (Siegismund1988). Morphological and morphometrical characterswere first used to differentiate neighboring populationsof Gammarus fossarum (Dusaugey 1955, Roux 1970),as reported for other gammarid species (Bulnheim andScholl 1981, Scheepmaker 1987, Kane et al. 1992).However, it soon became apparent that they were poordistinctive criteria because of their considerableamount of variability, not only between different pop-ulations, but also within the same population, going sofar as to make the characterization of the species dif-ficult (Pinkster 1983, Sheepmaker and Van Dalfsen1989, Sheepmaker 1990). Moreover, identifying en-vironmental sources of such morphological variationremained unsuccessful (Roux 1970). The diversity

1580 Ecology, Vol. 78, No. 6JEAN-CLAUDE MEYRAN

FIG. 1. Geographical distribution of the different studysites of Gammarus fossarum populations in the Grenoble re-gion (crystalline area of Belledonne: B3, B4; limestone areasof Chartreuse: C1, C2, and Vercors: V1, V3). The inset mapof France gives the location of the study site.

TABLE 1. Comparative analysis of ionic concentrations (Ca, Mg, Na, K, HCO3, Cl, SO4, andNO3 [mg/L]) and pH in water from the different sites (see Fig. 1).

Ioniccomposition

and pH

Site

B3 B4 C2 V3 V1 C1

CaMgNaKHCO3

ClSO4

NO3

pH

12.452.201.400.15

36.600.608.001.207.76

14.851.500.830.13

41.500.307.201.507.73

75.803.602.100.50

221.002.70

12.0008.23

78.54.303.000.80

206.001.40

11.000.808.24

83.501.104.900.80

261.008.206.004.208.25

95.703.703.500.90

293.004.409.802.708.17

among geographic populations has been then consid-ered at the ecophysiological level, leading to the con-cept of physiological races (Roux 1967). Of the eco-physiological traits investigated among different pop-ulations (Roux 1971), the calcium (Ca) requirementduring molting is of key interest (Ormerod et al. 1988).Ca metabolism is a central parameter in the biology ofgammarids (Graf 1974). As in other crustaceans, there

is an important periodic Ca turnover through the cuticleduring molt cycles throughout life (reviewed in Graf1978, Greenaway 1985). Moreover, since Gammarusfossarum cannot store Ca before exuviation, as is thecase for most gammarids (Vincent 1963, Graf 1965,Wright 1979, 1980), the Ca turnover during the moltcycle may depend upon the Ca availability in the water.Previous ecophysiological investigations at this levelled to conflicting interpretations and a precise corre-lation between observed ecophysiological differencesand clearly delimited environmental parameters wasgenerally unclear (Roux 1971, Vincent 1971).

Recent advances in our knowledge of the molt cyclein gammarids (Graf 1986) and of the physiology of Caturnover during molting (Graf 1979, Wright 1979,1980, Meyran and Graf 1993) allow reconsideration ofthe impact of environmental Ca on the diversificationbetween populations, at both comparative and experi-mental levels. In this respect, the Grenoble region(France) may offer a useful study site. First, in theupper reaches of the alpine streams near Grenoble,Gammarus fossarum has a high density and a patchydistribution (Dusaugey 1955, Roux 1963), as is gen-erally observed in other countries (Roux 1982,Schrimpff and Foeckler 1985, Foeckler and Schrimpff1985, Siegismund and Muller 1991). Also, there is aninteresting geological contrast between the neighboringmountain ranges of the Belledonne (crystalline rock)and the Chartreuse-Vercors (limestone regions) (seeFig. 1) that leads to characteristic local differences inthe Ca concentration of water (see Table 1).

Preliminary investigations have suggested that thediversity of the alpine populations of Gammarus fos-sarum may be related to the Ca concentration of thewater (Meyran 1994). This hypothesis was further an-alyzed in this paper at both descriptive and experi-mental levels to examine the influence of environmen-tal Ca on the geographic variability of Gammarus fos-sarum.

MATERIALS AND METHODS

Animals

Gammarus fossarum Koch is one of the four speciesof gammarids of the pulex group (Karaman and Pink-

September 1997 1581PHENOTYPIC DIVERSITY IN GAMMARUS

ster 1977a, b, 1987), recognizable by the setation ofthe second antennae and the morphology of uropodsand pleopods (Roux 1963, Goedsmakers 1981b). Nev-ertheless, morphological and morphometrical charac-teristics have great variability among different popu-lations and also among individuals of the same pop-ulation (Dusaugey 1955, Roux 1970). In the Grenobleregion, Gammarus fossarum is the predominant speciesobserved in the mountain torrents above an altitude of800 m (Dusaugey 1955). It can reach high populationdensities, which makes collection of large samples pos-sible.

Study area

To avoid the variability of multiple ambient param-eters leading to difficult interpretations, study siteswere selected for their comparable environmental char-acteristics, except for the water Ca concentration. Allwere located at an altitude of 900 m and within a 30-kmradius of Grenoble. They had similar climatic condi-tions, to avoid temperature differences, low contrastedseasonal hydric flux, and comparable hydrodynamiccharacteristics, to allow field rearing. They also in-cluded numerous specimens in the populations for suf-ficient and uniform sampling.

For comparison between populations from the dif-ferent geographic areas, two sites were selected in eacharea (Fig. 1). Sites from the crystalline BelledonneMassif were located on the Vorz torrent at ‘‘La Gorge’’(B3), and on the Laval torrent at ‘‘La Boutiere’’ (B4).Sites in the limestone Chartreuse Massif were locatedon the Vence torrent at ‘‘Le Sappey’’(C1), and on theCouzon torrent at ‘‘Saint-Pierre’’ (C2). Sites in thelimestone Vercors Massif were located on the Furontorrent above ‘‘Engin’’(V1), and on the Bruyant torrentat ‘‘Chateau-Bernard’’ (V3). Animals were collectedeither by turning stones and sweeping over the sedi-ment with a fine-meshed hand net, or by shaking aquat-ic plants or decaying leaves in a bucket. Only adultmales of maximum size were used.

At the beginning of each experimental period, whenthe animals were collected, water samples were takenusing the technique of Foeckler and Schrimpff (1985),for ionic titration through atomic absorption spectro-photometry (Pinta 1971), after HNO3 1% addition. Thewater chemistry analysis (ionic titration and pH deter-mination) was performed using standard methods(Franson 1992).

Specimen determination and dating

Gammarus samples were identified individually us-ing morphological keys (Roux 1970, Kamaran andPinkster 1977a, b, 1987). Then, they were subjected toa fine determination of their molt cycle stage using theexact staging method of Graf (1986). This was basedupon the microscopic observation of the integumentalmorphogenesis of the dactylopodite and the propoditefrom a freshly cut pereiopod. This rapid method allows

in vivo determination of the precise phase of the moltcycle for any specimen, without affecting the survivalof the animal. It was thus possible to follow a givenspecimen during the whole sequence of the differentphases of the molt cycle and to determine their specificduration.

According to Graf (1986), the molt cycle is dividedinto five periods from one exuviation (ecdysis 5 periodE) to the next. During the postmolt (postecdysis 5period A: early postmolt 1 period B: late postmolt),the new cuticle is progressively calcified. The cuticleis stable during the whole intermolt (period C), then isgradually decalcified during the premolt (preecdysis 5period D).

Rearing and translocation experiments

For a comparative study of the duration of the moltcycle among geographic populations, dated specimenswere reared individually, for at least 2 mo, in 6 3 43 4 cm transparent plastic perforated boxes containingaquatic plants or decaying leaves for food, and im-mersed in water. Animals were examined for molt cyclestage every 10 d during the period of experimentation.

In the field, the rearing boxes of control specimenswere immersed in water from the collecting site (nativewater). Boxes of translocated specimens were trans-planted to another site (alien water). To simplify theexperimental design, not all combinations were per-formed (see Table 4). The average temperature of thewater (118C) was determinated by periodic measure-ments in situ in all study sites during the period ofexperimentation.

At the laboratory, animals were reared in a climate-controlled room (constant temperature: 118C), with adaily cycle of 12 h of light. Control specimens wereplaced into water from their native site, whereas trans-located specimens were reared in water from an aliensite. The experimental design involved the same com-binations as in the field experimentation (see Table 4).Water was changed weekly.

To further investigate the impact of the Ca concen-tration of the water on the duration of the molt cycle,some specimens from B3 and B4 populations werereared in their native water artificially enriched in Caup to a concentration equivalent to that of C1 site byspectrophotometrically controlled addition of CaSO4.

Both field and laboratory experiments were system-atically performed on 600 animals divided into uniformsamples each including 8 specimens of the same size,from all collecting sites. Experimentation occurred re-peatedly and simultaneously in the field and in the lab-oratory from summer to autumn 1993 and from springto autumn 1994.

Calcium balance analysis

At the end of each experiment, the total body Cacontent of the dated specimens after 24 h of starvationwas determined through atomic absorption spectro-


TABLE 2. Variability in the animals’ maximum size, expressed as dry mass, in milligrams (means with standard errors inparentheses) and mean duration (mean no. days, with standard errors in parentheses) of the molt cycle in control populationsaccording to the water Ca concentration at the different sites (N 5 number of observations; a, b, . . . 5 Duncan grouping:means with the same letter are not significantly different).

Site

B3 B4 C2 V3 V1 C1

Water Ca concentration(mg/L)

12.45 14.85 75.80 78.50 83.50 95.70

Animal dry mass (mg) 5.57 (1.10)N 5 20e

6.21 (0.93)N 5 50e

8.08 (2.45)N 5 44bc

8.78 (1.57)N 5 45ab

7.66 (1.27)N 5 34cd

9.40 (2.17)N 5 35a

Duration of the molt cycle(d)

39.19 (1.63)N 5 10b

39.18 (1.96)N 5 28b

41.96 (1.10)N 5 14a

42.91 (1.96)N 5 9a

42.87 (1.35)N 5 18a

42.79 (1.81)N 5 17a

TABLE 3. Analysis of variance for the dry mass of animals and the mean duration of the moltcycle in reared control populations (NS 5 nonsignificant; * P , 0.05; ** P , 0.01; *** P, 0.001) (localization of experiments: field or laboratory).

Source df MS F

Animal’s dry massSite of origin (SO)

Error6

25663.73

2.6523.98***

Mean duration of the molt cycleSite of origin (SO)Localization of experiments (LOC)SO 3 LOC

Error

614

109

55.7413.71

5.184.95

11.25***2.77 NS

1.05 NS

photometry. After measurement of their dry mass (Graf1969), whole specimens were homogenized and thenmineralized with H2SO4 6N (Meyran et al. 1987). TheCa concentration in homogenates was measured witha Varian apparatus at 422.7 nm after dilution in 0.2%lanthanum chloride. In order to avoid individual vari-ation related to the animal’s size, results were stan-dardized to a constant dry mass of 10 mg, followingGraf (1964). In the same way, the Ca balance duringthe successive phases of the molt cycle was determinedon 400 field-collected specimens at all stages of themolt cycle, directly originating from all geographicpopulations.

Data were analyzed using the SAS statistical package(SAS 1988).

RESULTS

Environmental parameters: differences inwater Ca concentrations

Successive measurements indicated no obvious dif-ference in ionic concentration among water samples ofthe same study site from one period of experimentationto another. Data from Table 1, summarizing the firstanalysis, revealed that the most prominent differencesamong water samples from the different areas were Caconcentrations, which may be related to the geologicaldifferences of the leached substrata. High Ca titers werecharacteristic of waters from the limestone areas ofChartreuse and Vercors (from 75.8 mg/L in C2 to 95.7

mg/L in C1), whereas waters with low Ca concentrationwere localized within the crystalline area of the Bel-ledonne (from 12.4 mg/L in B3 to 14.8 mg/L in B4).Differences in concentrations were also noticed,2HCO3

which may be responsible for the differences in pHbetween waters from the two different geological areas.

Biological parameters

1. Differences between the populations in situ.—Measurement of the dry mass of samples of the dif-ferent populations at several times during the studyperiod revealed that specimens from the limestone ar-eas were on average heavier than those from the crys-talline sites, regardless of the timing of sampling (Table2). In the analysis of variance (Table 3, upper part),the effect ‘‘site of origin’’ (SO) is significant.

The comparative analysis of the duration of the moltcycle showed characteristic differences between pop-ulations (Tables 2–3). Whereas the duration of the moltcycle was not significantly different among populationsfrom a given geologic area, it was ø8.5% longer inthe populations from the limestone areas (C1, C2, V1,and V3) than in those from the crystalline sites (B3and B4) (Table 2). In the analysis of variance (Table3, lower part), the SO effect is significant. In fact, theduration of the molt cycle in smaller specimens fromlimestone populations was shorter than that observedfor animals of the same size living in crystalline areas(data not shown). No significant differences were ob-


FIG. 2. Calcium balance during the successive phases ofthe molt cycle (E; early B, late B; C; early D, late D) inanimals from the different study sites (B3, B4; C1, C2; V1,V3: see Fig. 1). The graphs proposed concern field animals.Dashed lines represent populations from crystalline areas (B3and B4); solid lines represent populations from limestoneareas (V1, V3, C1, and C2).

TABLE 4. Effects of both field and laboratory translocations of animals in natural water onthe mean duration (mean no. days, with standard errors in parentheses) of the molt cycle (N5 number of observations; A, B, C, D 5 Duncan grouping for each translocation site,comparison between sites of origin; a, b, g 5 Duncan grouping for each site of origin,comparison between translocation sites: means with the same letter are not significantlydifferent).

Source

Translocation to

B3 B4 C2 V3 V1 C1

B3 39.19 (2.63)N 5 10[C, a]

34.96 (3.19)N 5 9[C, b]

B4 39.18 (1.96)N 5 28[D, a]

35.09 (2.11)N 5 22[D, b]

35.80 (2.22)N 5 32[C, b]

C2 57.00 (4.34)N 5 8[B, b]

64.25 (3.6)N 5 4[A, a]

41.96 (3.1)N 5 14[B, g]

44.40 (1.51)N 5 5[B, g]

V3 58.89 (8.21)N 5 13[B, a]

61.89 (6.74)N 5 11[A, a]

42.91 (2.9)N 5 9[B, b]

43.30 (1.9)N 5 6[B, b]

V1 55.30 (4.7)N 5 8[BC, a]

42.87 (1.35)N 5 18[B, b]

41.42 (2.33)N 5 12[B, b]

C1 55.90 (11.8)N 5 4[B, a]

58.56 (11.05)N 5 12[AB, a]

41.11 (2.45)N 5 13[C, b]

42.79 (1.81)N 5 17[B, b]

served between field and laboratory measurements onall control specimens (LOC effect: Table 3, lower part).In the same way, the mortality rate was between 3 and12.7% in both natural and laboratory conditions, re-spectively.

Spectrophotometric analysis of the Ca balance in an-imals during the successive stages of the molt cyclerevealed characteristic differences among populationsaccording to habitat. These differences were observedin samples originating from the field and in animalsexposed to experimentation (Fig. 2). Whereas the totalbody Ca content was comparable in any specimen atexuviation (period E, when the old cuticle is lost) andduring intermolt (period C, when the calcified cuticleis stable), the Ca loss from the old cuticle to the waterduring premolt (period D) was more rapid in animalsfrom limestone sites than in those from crystalline ar-eas. In the same way, the Ca recovery from the waterinto the new exoskeleton during postmolt (period B)was faster in specimens from limestone areas than inthe others.

2. Effects of translocation on the duration of the moltcycle and the Ca balance.—Results of the transloca-tions performed are summarized in Table 4. In all cases,for a given experiment, laboratory results did not differsignificantly from field results (P . 0.05). No signif-icant differences (P . 0.05) were observed betweencontrols and specimens with comparable Ca concen-tration from neighboring sites (from C1 to V1; fromC2 to C1; from V1 to C1; from V3 to C1: Table 4).

Both field and laboratory data indicated a significantincrease in the duration of the molt cycle in specimenstranslocated to water with a lower Ca concentration.


TABLE 5. Analysis of variance for the mean duration of themolt cycle in animals translocated in natural water, per-formed on both field and laboratory data (NS 5 nonsignif-icant; * P , 0.05; ** P , 0.01; *** P , 0.001).

Source df MS F

Mean duration of the molt cycleSite of origin (SO)Site of translocation (ST)SO 3 ST

Error

66

18370

2 388.862 502.97

390.6920.52

116.40***121.97***19.04***

TABLE 6. Effects of translocation of B3 and B4 animals intheir native water, artificially Ca-enriched up to a concen-tration equivalent to that of C1 (equivalent C1) on the meanduration (mean no. days, with standard errors in parenthe-ses) of the molt cycle in laboratory experiments (N 5 num-ber of observations) vs. the mean duration of the molt cyclein B3, B4, and C1 control populations.

Originatingsite

Average duration of the molt cycle

Control specimensin native water

Specimens trans-located in

Ca-enriched water

B3

B4

C1

39.08 (1.84)N 5 23

38.78 (1.47)N 5 25

42.10 (1.02)N 5 5

34.92 (1.93)N 5 9

34.14 (1.41)N 5 11

TABLE 7. Analysis of variance for the mean duration of themolt cycle in B3 and B4 animals translocated in their nativewater, artificially Ca-enriched to a concentration equivalentto that of C1 (equivalent C1) (NS 5 nonsignificant; * P ,0.05; ** P , 0.01; *** P , 0.001).

Source df MS F

Mean duration of the molt cycleSite of origin (SO)Water Ca enrichment (DOP)SO 3 DOP

Error

221

68

49.73270.93

0.782.66

9.32***101.58***

0.30 NS

This ranges from 30.6% in the translocation from C1to B3 up to 53.1% in the translocation from C2 to B4.The in vivo chronological study of the phases of themolt cycle in those specimens revealed a typical in-crease in the duration of the early intermolt. In someextreme cases, from 20 to 80% of the population re-mained in intermolt before dying. Conversely, the du-ration of the molt cycle was significantly shortened inanimals translocated to water with a higher Ca con-centration (from 8.6% in the translocation from B4 toC1 up to 10.8% in the translocation from C2 to B3).In all cases, both effects SO (site of origin) and ST(site of translocation) are significant (Table 5).

A decrease in the duration of the molt cycle similarto that previously observed was seen in specimens fromB3 and B4 reared in their native water artificially en-riched with Ca up to a concentration equivalent to thatof C1 (from B3 to B3-equivalent C1; from B4 toB4-equivalent C1: Table 6). In the analysis of variance(Table 7), the effect ‘‘water Ca enrichment’’ (DOP) issignificant; the SO effect is due to a significant differ-ence between C1 and the other sites of experimentation.Spectrophotometric measurements comparing translo-cated and control specimens indicated no differencesin the Ca balance for each given stage of the molt cycle(see Fig. 2).

DISCUSSION

The present study clearly relates phenotypic differ-ences among alpine populations of Gammarus fossa-rum to the levels of environmental sources of Ca. Thepopulations from limestone areas of Chartreuse andVercors include specimens with a larger maximum sizeand a longer molt cycle than those from the crystallinesite of Belledonne. The Ca balance of animals fromlimestone areas was different during both pre- and post-molt from that of specimens from crystalline sites. Ex-perimental increasing of Ca in the environment canreduce the duration of the molt cycle and vice versa.

However, our experimental results appear to contra-dict the descriptive data. The comparison is biased atthe descriptive level as the measurements were madeonly on adults of maximum size, which were largestin limestone populations. As previously stated, the du-ration of the molt cycle in smaller specimens fromlimestone 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 asthey are not representative for a rigorous comparisonamong different populations.

The fact that populations of Gammarus fossarummay adapt to a large range of water Ca concentrationsis not surprising since this has been observed in mostgammarids (review in Roux 1971, Vincent 1971). Wa-ter Ca levels are not considered to be a limiting factorfor the distribution of those populations (Okland andOkland 1985). More generally, it is assumed that pop-ulations of native species are not limited by minerals(Fielder 1986). However, as variations in water Ca lev-els may generate morphological and physiological dif-ferences between animals, this environmental param-eter may be significant at least in the phenotypic dif-ferentiation of the populations.

Microgeographic variation in growth rates and max-imum sizes has commonly been observed among gam-marid populations (Goedmakers 1981a, b). The differ-ences in gammarid size from site to site were usuallycorrelated to size selective predation or to differencesin maturity from one population to another (Goedmak-ers 1981a, b). To avoid this biasing factor in our com-parative study, sampling was limited to mature malesof maximum body size. Thus, the variation in maxi-mum body size arising from our measurements mayreflect a genuine difference between populations from


limestone and crystalline areas. This is supported bythe congruence of the results obtained from all exper-iments. Possible artifacts caused by the sampling maybe avoided by the random periodic sampling proceduremade at all study sites during the whole period of ex-perimentation. Interestingly, such a correlation be-tween the Ca content of the water and the maximumgrowth of Gammarus fossarum has also been reportedfor other gammarid species (Vincent 1971). Additionalobservations reveal that the differences in maximumbody size observed among males are also true for thefemales (data not shown). As a positive correlation hasbeen found between fecundity and body size by Goed-makers (1981a), the impact of such differences may beimportant at the level of population dynamics. More-over, our field observations periodically performed dur-ing the entire experiment showed that the populationswith the maximum density were located in waters thatflow over limestone (J. C. Meyran, personal observa-tions). Therefore, the populations living on limestonenot only reach a maximum size but also have a max-imum growth rate. However, other factors such as mi-gration and predation rates may also be affecting thedifferences in density between low and high Ca sites.Since growth rates were correlated with the durationof the molt cycles in gammarids, as in other crustaceans(see references in Graf 1969), it is not surprising thatour descriptive data indicated that the molt cycle waslonger in control specimens from the limestone sites.

Previous data have only indicated inter- or intraspe-cific differences in water Ca tolerance among gam-marids, with conflicting interpretations reviewed byRoux (1971). More recently, Roux (1971) concludedan indirect influence of water Ca levels on the geo-graphical and ecological distribution of Gammarus fos-sarum. However, the existence of ‘‘physiological rac-es’’ was not excluded. Comparable findings were re-ported by Vincent (1963, 1971, 1972) on other gam-marid species, using experimental methods focused onthe intermolt cuticular calcification and the postmoltCa recovery.

The current investigations confirm the existence ofsignificant ecophysiological variations in Ca metabo-lism during the molt cycle among different populationsof Gammarus fossarum. These differences have beenshown through a synthetic approach using both themeasurement of the duration of the molt cycle by thestaging method of Graf (1986) and the spectrophoto-metric determination of the Ca balance during the moltcycle. Moreover, it is important to compare results ob-tained under laboratory conditions with those obtainedfrom animals in the field, since the situation in naturalbiotopes is usually characterized by various fluctuatingfactors (Koch-Kallnbach and Meijering 1977). Thus,our experimentation was conducted jointly in the fieldand in the laboratory, leading to congruent results.

Our measurements clearly show that, during molting,the environmental Ca availability appears to interfere

directly with the dynamics of the Ca shifts between theanimal and its surrounding medium. In this respect, thelow Ca levels in the water may be responsible for in-creasing the duration of the premolt period in speci-mens from the Belledonne, contrary to that observedin animals from waters with a high Ca concentrationfrom the Chartreuse and Vercors. However, the relativeduration of the whole molt cycle may not be affectedif we consider that the duration of the intermolt periodis different in both types of populations, because of thedifferent size of the specimens studied. This is sup-ported by the results of our translocation experiments,which indicated changes in the duration of the intermoltperiod. Comparison of the duration of the premolt pe-riod from one population to another suggests that an-imals from limestone sites may be considered as fa-vored compared to those from crystalline areas as theyreach their cuticular Ca equilibrium more rapidly,which is of key interest for survival. Interestingly,specimens from crystalline sites show a higher mor-tality rate after exuviation than those living on lime-stone (data not shown).

In the same way, it appears from translocation ex-periments that increasing Ca in water can reduce theduration of the molt cycle, probably through an im-provement of Ca recovery into the animal’s new cuticleduring postmolt (Vincent 1971). As an increase in thenumber of molt cycles for a given time may directlyinterfere with the growth rate, this environmental factormay be at least partly responsible for the differencesin adult maximum size observed between populationsfrom limestone and crystalline areas. Conversely, thepoor Ca availability in water may be related to thesmaller maximum body size of specimens from crys-talline areas.

A clear demonstration of the genuine impact of theenvironmental Ca in the ecophysiological differentia-tion among geographic populations appears when an-imals from B3 and B4 are reared in their native waterafter artificial addition of Ca to a concentration equiv-alent to that of C1. The results indicate similar effectson the duration of the molt cycle for a given Ca con-centration whether the specimens are translocated intoalien or native water.

The ecophysiological plasticity of Ca metabolismcurrently observed among alpine populations of Gam-marus fossarum may be a determining factor in thedynamics of colonization of this species. Migrationconstitutes the central parameter of colonization pro-cesses in gammarids (review in Goedmakers and Pink-ster 1981) but its relation with the environmental Caavailability is still controversial. Previous authors haveconsidered Gammarus fossarum to be a preglacial in-habitant of the Alps (review in Scheepmaker 1990),largely represented in this area (Dusaugey 1955, Sie-gismund and Muller 1991), and rather independentfrom the Ca content of the water (Roux 1971). Suchan independence may be limited, as suggested by our


ecophysiological results and our field observations,which showed that the most flourishing populationswere located in high Ca concentration areas. Moreover,current experimental data reveal that mortality ratesincreased in animals translocated to waters lower in Caconcentration. This relatively greater difficulty inadapting to lower environmental Ca levels, earlier ob-served in other gammarid species by Vincent (1971),allows us to suggest that the primitive populations ofGammarus fossarum probably lived in limestone areasand that colonization of the crystalline sites may havebeen secondary. This hypothesis agrees with the mi-gration theory earlier proposed by Pacaud (1945a, b),which was based only upon biogeographic considera-tions.

The phenotypic variation among geographic popu-lations of Gammarus fossarum observed in this studymay not only reflect environmental diversity, as pres-ently suggested, but may also have a possible geneticbasis. In this respect, the concept of microgeographicraces was proposed (Goedmakers 1980a, b, 1981a, b,Goedmakers and Pinkster 1981). Using enzymaticpolymorphism analysis, Siegismund and Muller (1991)found that local populations of Gammarus fossarumshow strong genetic differentiation compared to othergammarid populations. Such genetic differentiation isnow being further considered at the molecular levelthrough the comparison of mitochondrial DNA (Meyr-an and Taberlet 1996), as this genetic marker appearsto evolve rapidly at the sequence level in crustaceans(Palumbi and Benzi 1991).

Finally, the present study clearly attests to the valueof a synthetic approach including both comparative andexperimental techniques in identifying environmentalparameters responsible for biological variation amongneighboring populations. Moreover, investigation of Cametabolism during the molt cycle may provide a newpowerful ecophysiological tool to supplement the cri-teria generally used to test the phenotypic variabilityamong geographic populations of gammarids.

ACKNOWLEDGMENTS

The author thanks A. Zganic and the Laboratoire Regionald’Analyse des Eaux, Universite J. Fourier, Grenoble, for thewater chemistry analysis and the atomic absorption spectro-photometric dosage of calcium; Dr. I. Till-Bottraud for helpin statistics, Dr. B. Serra-Tosio for helpful advice in the choiceof the study sites, and L. Waits for help with the English.This work was funded by the Centre National de la RechercheScientifique and the University J. Fourier (Grenoble, France).

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Documents

IMPACT OF WATER CALCIUM ON THE PHENOTYPIC DIVERSITY OF ALPINE POPULATIONS OF GAMMARUS FOSSARUM