Do upper thermal tolerances differ in geographically separated populations of the beachflea Orchestia gammarellus (Crustacea: Amphipoda)?

LJournal of Experimental Marine Biology and Ecology,229 (1998) 265–276

Do upper thermal tolerances differ in geographicallyseparated populations of the beachflea Orchestia

gammarellus (Crustacea: Amphipoda)?

*Kevin J. Gaston , John I. SpicerDepartment of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

Received 9 April 1997; received in revised form 6 March 1998; accepted 21 March 1998

Abstract

Fundamental (as opposed to realised) between-population differences in maximum temperaturetolerances were examined for the common beachflea of European shores, Orchestia gammarellus.Individuals were collected from two distinct populations, one in S.E. England and one in N.Scotland, and were acclimated to a number of different thermal regimes (5, 10, 15, 20 and 258C).Temperature tolerances increased with body size for virtually all combinations of population andacclimation temperature tested. Such a relationship complicates comparison between populations,although when body size differences were controlled for at two acclimation temperatures,individuals of the southern population tended to have higher thermal tolerances. Controlling forbody size tends, however, to underestimate the difference in temperature tolerances of the twopopulations. This is because the southern population consisted of larger individuals than thenorthern, giving rise to markedly different frequency distributions of tolerances. Clearly upperthermal tolerance limits do vary between these two geographically widely separated populations ofO. gammarellus and this variance cannot be accounted for by acclimation. Such a finding, were itto have general application, would have important implications for ecologists seeking to explainlarge-scale distribution patterns in terms of a ‘fixed’ species physiology. 1998 ElsevierScience B.V. All rights reserved.

Keywords: Acclimation; Beachfleas; Orchestia gammarellus; Physiological diversity; Populationdifferences; Temperature tolerance

1. Introduction

Arguably, physiological diversity underpins many patterns in ecology. Yet attempts to

*Corresponding author.

0022-0981/98/$ – see front matter 1998 Elsevier Science B.V. All rights reserved.PI I : S0022-0981( 98 )00057-4

266 K.J. Gaston, J.I. Spicer / J. Exp. Mar. Biol. Ecol. 229 (1998) 265 –276

link the two have been only partially successful (Feder, 1986; Garland and Adolph,1991). Part of the problem is that this diversity exists as a nested hierarchy (individual,population, species, assemblage), and examination of the different levels of thishierarchy has been uneven, with most attention being directed at between-speciescomparisons. It is debateable exactly how much we understand the nature and extent ofbetween-population differences, even for relatively well-studied vertebrate species(Garland and Adolph, 1991). Some species exhibit marked between-population variationin physiological tolerances (e.g., Jones and Johnston, 1992). Others do not (e.g.,Matthews, 1986), and the relative frequency of these two outcomes is unclear. In majorpart, this is because of a paucity not of acclimation studies per se which are plentiful, butof strictly comparable studies which have sought to determine the potential foracclimation of expressed tolerances, i.e. a great deal is known about what animalsactually do, but not a lot about what they can do. In the absence of prerequisiteknowledge of relevant demographic processes, the ecological consequences of suchvariation as is (or is not) uncovered are very often only informed guesses. Typicallytolerances are expressed as mean, or median, values for relatively small numbers ofindividuals (often approximately standardised for confounding traits such as body size),from which it is not possible to infer to what extent, and how, the frequencydistributions of tolerances for two populations might overlap. This latter feature mayhave important implications in making between-population comparisons.

Consequently we have examined fundamental (as opposed to realised) between-population differences in a selected physiological tolerance (maximum temperaturetolerance), for two geographically separate populations. Chosen for study were between-population differences of the common beachflea of European shores (Lincoln, 1979)Orchestia gammarellus. This species belongs to the Talitridae, the only family ofamphipods to have truly terrestrial representatives (Spicer et al., 1987). As O.gammarellus is semi-terrestrial it is likely to be influenced both by prevailing air and seatemperatures. There have been a number of studies of the temperature tolerances ofvarious semi- and euterrestrial talitrid species (Marsden, 1980; Lazo-Wasem, 1984;Marsden, 1985), including O. gammarellus (Backlund, 1945; Moore and Francis, 1986;Moore and Weeks, 1995) but, as yet, there are no directly comparable studies ofdifferent populations of the same species. In the present study amphipods were collectedfrom S.E. England and from N. Scotland, and acclimated to a number of differentthermal regimes in the laboratory. The maximum temperature tolerance of individuals,often regarded as a convenient and ecologically relevant index of overall thermalsensitivity (Huey et al., 1992) for each treatment, was then measured to determinewhether or not there were irreversible population differences in temperature tolerance.The relationships between tolerances and body size were then used to estimate thecompositions of the two populations with regard to their temperature tolerances.

2. Materials and methods

Orchestia gammarellus was collected from two localities; from Upper Upnor,Chatham on the Medway Estuary in the south-east of England (Ordnance Survey Grid

K.J. Gaston, J.I. Spicer / J. Exp. Mar. Biol. Ecol. 229 (1998) 265 –276 267

´ ` `Ref. TQ 757 704) and Kerrachar Bay, Loch a Chairn Bhain, in the far north of Scotland(Grid Ref. NC 179 348). These localities lie close to the most southerly and northerlypossible limits of the geographic distribution of the species in Britain, being separated bysome 78 of latitude. All individuals were collected randomly from beneath rocks, stonesand weed at, and around, the high water mark. Collection was by removing thesubstratum inhabited by beachfleas into large polythene bags. Amphipods weretransported to the laboratory within 48 h of capture. Here the populations were keptseparate and maintained in large plastic aquaria (50 l) on their native substratum, subjectto a temperature range of 9–128C, until required in the experiments described below (41days). During this time they were kept in constant darkness and fed ad libitum withchopped carrots. Relatively soon after collection, 150 individuals from each populationwere sampled at random and weighed (60.1 mg) using a microbalance (Mettler).

A preliminary experiment was done to gauge the extent of the population differencesin temperature tolerance that exist between freshly collected animals. Within 60 h ofcollection, 40 large males (body length 18–20 mm) from each population weretransferred to culture conditions identical to those described above, but maintained at238C. Mortality was noted, initially every 15 min for the first 4 h and thereafter at 2-hintervals for a further 8 h.

In order to measure the maximum temperature tolerance of individuals from bothpopulations, experimental cultures were constructed at five different acclimationtemperatures; 5, 10, 15, 20 and 258C. Each culture consisted of small (2-l), coveredplastic aquaria, quarter-filled with pebbles soaked in artificial sea water (Tropic Marin,S 5 34‰). To each aquarium 25 individuals were added, from either the Chatham or theKerrachar population, encompassing as wide a body size range as possible. Acclimationlasted for 10 days during which time beachfleas were fed on chopped carrot ad libitumand the substratum was re-wetted with artificial sea water every 2 days. After this periodbeachfleas were removed individually and their temperature tolerance assessed (seebelow).

Temperature tolerance was measured by removing approximately 25 individuals froma single culture unit to a high precision water bath (Grant, LTD 6G/20) maintained atthe appropriate culture temperature. Individuals were kept in air in a subdivided plasticcontainer that was almost completely immersed in the water contained in the water bath.In each subdivision of the container a small square of filter paper soaked in a solution(S 5 34‰) of artificial sea water was placed. Both the wetted filter paper and the factthat the plastic container was immersed in a relatively enclosed water bath ensured thatthe beachfleas experienced a very high relative humidity. This should prevent evapora-tive water loss that could potentially produce a cooling effect in individuals: lethaleffects of temperature are linked to desiccation resistance in arthropods (e.g. Block et al.,1994). The plastic container had been kept at the acclimation temperature for 2 h beforeuse in the experiment. The temperature in the water bath was then increased, using a

21computerised temperature programmer (Grant, PZ1) at a rate of 18C min andmortality was assessed visually (when no visible movement was observed, individualswere removed and checked for cardiac activity). A small, hand-held mercury thermome-ter was used to check that the lag time between equilibration of the water and the plasticexperimental chamber was acceptable.


3. Results

3.1. Survival of freshly collected individuals at 238C

There was no mortality observed in freshly collected Orchestia gammarellus from theChatham population after 24 h at 238C. However, none of the individuals from theKerrachar population survived the 24-h test period, with an LT of approximately 6 h.50

3.2. Body size distributions

The body size distributions of both the Chatham and Kerrachar populations ofOrchestia gammarellus, based on 150 individuals in each case, were strongly right-skewed. After logarithmic transformation (Fig. 1), the differences between the twopopulations could not be examined by Student’s t-test, because the test for homogeneityof variances was statistically significant (F-ratio 5 4.995, P , 0.001). However, the twodistributions were significantly different using the non-parametric Mann-Whitney U-test(U 5 8129, Z 5 2 4.15375, n 5 150, n 5 150, P , 0.001), with the Chatham popula-1 2

tion (mean61 S.D. 5 43.4618.4 mg) being larger-bodied than the Kerrachar population(mean61 S.D. 5 36.4630.8 mg).

3.3. Temperature tolerances

Temperature tolerances of Orchestia gammarellus increased with body size for all

Fig. 1. Frequency distributions of body masses of individuals from Chatham (open bars) and Kerrachar (closedbars) populations. Body mass (mg) is on a logarithmic (base 10) scale.


Table 1Pearson correlation coefficients for relationships between body mass (log transformed) and temperature10

tolerance for different combinations of population and acclimation temperature (8C)

Population Acclimation temperature n r

Chatham 5 23 0.644***10 23 0.836***15 21 0.713***

NS20 22 0.162Kerrachar 5 24 0.644***

10 23 0.723***15 18 0.847***20 18 0.805***

NS, not significant, ***P , 0.001.

combinations of population and acclimation temperature, with the exceptions of theChatham population at 208C, for which there was no significant relationship (Table 1),and both populations at 258C, at which temperature all individuals died. At acclimationtemperatures of 10 and 158C tests of homogeneity of slopes of relationships betweenbody mass and temperature tolerance were statistically significant (Table 2). Atacclimation temperatures of 5 and 208C, this was not the case (Table 2), allowing aformal comparison of the elevations of the relationships for Chatham and Kerrachar. Inboth cases the ANCOVAS were significant, with individuals of the Chatham populationhaving greater tolerances for a given body size than individuals of the Kerracharpopulation (Table 2).

The distributions of temperature tolerances for the 150 individuals from the Chathamand Kerrachar populations for which body masses were determined was predicted foreach acclimation temperature on the basis of the observed relationships betweentolerance and body mass (accepting that these relationships are not always strong). Forall acclimation temperatures, the predicted frequency distributions of maximum tempera-ture tolerances for the two populations overlapped considerably, but those for theChatham population apparently had a greater mean than those for the Kerracharpopulation (Fig. 2). Differences between the distributions of predicted temperaturetolerances between the Chatham and Kerrachar populations could not be tested by

Table 2Tests of homogeneity of slopes of relationships between body mass (log transformed) and temperature10

tolerance for Chatham and Kerrachar populations at different acclimation temperatures (8C), and, where theseare not statistically significantly different the results of an ANCOVA on temperature tolerance between the twopopulations, with body mass as a covariate

Acclimation temperature Homogeneity ofslopes ANCOVA

df F df FNS5 1,43 1.088 1,44 8.339**

10 1,42 5.969*15 1,35 4.615*

NS20 1,36 3.366 1,37 20.291***

NS, not significant, *P , 0.05, **P , 0.01, ***P , 0.001.


Fig. 2. Frequency distributions of predicted maximum temperature tolerances (8C) of individuals fromChatham (open bars) and Kerrachar (closed bars) populations, for acclimation temperatures of: (a) 5, (b) 10, (c)15 and (d) 208C. The distribution could not be estimated for the Chatham population at 208C, because therewas no significant relationship between body mass and temperature tolerance at this acclimation temperature.


Fig. 2. (continued)

Student’s t-test, because at acclimation temperatures of 5 and 108C, tests of homogeneityof variances were statistically significant (58C, F-ratio 5 2.262, P , 0.001; 108C, F-ratio 5 1.386, P , 0.05; 158C, F-ratio 5 1.352, P . 0.05). However, using the non-parametric Mann-Whitney U-test, the predicted tolerances were different at all threeacclimation temperatures (Table 3). Likewise, using a Kolmogorov–Smirnov two-


Table 3Results of Mann-Whitney U-tests, of differences in temperature tolerances between Chatham and Kerracharpopulations at different acclimation temperatures (8C)

Acclimation temperature Mann-Whitney U-test Group means (8C)

U Z Chatham Kerrachar

5 1991 2 12.325*** 34.094 32.88310 3693 2 10.059*** 35.145 33.67815 1832 2 12.537*** 37.661 35.210

n 5 150, n 5 150.1 2

***P , 0.001.

sample test, which is not only sensitive to differences in the location of distributions butalso to differences in their shapes, the predicted tolerances for the two populations weredifferent (P , 0.001) at all three acclimation temperatures.

4. Discussion

Taking both UK populations together (and including individuals of different weights)the beachflea Orchestia gammarellus showed a wide variance in maximum fundamentaltemperature tolerance ranging from 29.5 to 39.58C. Comparison can be made with valuesestablished for some other invertebrates, for which tolerances have also been determined

21using a temperature increase of 18C min . The range for the beachflea was considerablylower than has been recorded for (adult) Arctic or temperate species of mites (40–45 and37–448C, respectively: Madge, 1965; Hodkinson et al., 1996), but only slightly lowerthan for (adult) Arctic or temperate insects (35–40 and 38–408C; Hodkinson et al.,1996).

The real interest in our own data does not, however, lie with interspecific comparisonsbut from the fact that there were important non-reversible differences in temperaturetolerance between the two geographically separate populations of Orchestia gammarel-lus. Furthermore, we were able to resolve such differences over a comparatively smallgeographic range. Discerning these differences was made more complex by the sizedependency of maximum temperature tolerance (see below for more detailed discussionof the effect of body size). This relationship complicated comparison between thepopulations, although when controlled for at two quite different acclimation tempera-tures (5 and 208C) it was clear that for any given body size a beachflea from the moresoutherly population exhibited a greater tolerance. In this sense, O. gammarellus exhibitsa labile temperature tolerance, which would appear responsive to local selectionpressures; Chatham experiencing as it does a generally warmer climate than Kerrachar.While it is true that O. gammarellus may be more influenced by the prevailingmicro-climate in wrack beds (Backlund, 1945; Moore and Francis, 1985), it is notunreasonable to assume that microclimate changes, although they may be buffered,roughly follow macroclimate changes.

Interestingly, using a method very similar to that used here, Backlund (1945) found


that adult individuals from a Swedish population of Orchestia gammarellus had anupper thermal death point of 378C, which it could be argued is perhaps higher than wemight have expected on the basis of our own data, although he provides no informationon the effect of acclimation. However, individuals from populations of O. gammarellusin the Azores have been noted to survive temperatures rising from 31 to 508C over 5 minand were found active beneath leaf litter in situ at a temperature of 42.38C (Moore andWeeks, 1995) which is greatly in excess of the highest temperature tolerance measuredfor individuals of either of our two UK populations. Moore and Francis (1985) havesuggested plausibly that the upper lethal temperature of O. gammarellus may be relatedto the melting point of epicuticular waxy material (36–448C). In the light of our ownfindings it would be of considerable interest to see if acclimation temperature influencesthe composition, and hence the melting points of these waxes. Were this so, it maysupply a mechanism for potentially increasing temperature tolerance both directly andindirectly (via restriction of water loss at high temperatures).

The contents of the preceding discussion run contrary to the view that the upperthermal lethal limits of a widely distributed species, such as Orchestia gammarellus, areapproximately the same throughout its geographic range (see the frequently cited reviewby Ushakov, 1964, for example). It is true that the sea urchin Strongylocentrotuspurpuratus, which has a range extending from Alaska to Mexico, has a set upper thermallimit of 23.58C (Farmanfarmaian and Giese, 1963), but its distribution on the shore isnot the same throughout its geographic range, with individuals occurring intertidally athigh latitudes and subtidally at low latitudes. The fiddler crab Uca lives in the supratidalzone, throughout its geographical range, as does O. gammarellus, and Vernberg andVernberg (1972) have commented that the upper thermal lethal limit of Uca, over arange extending from Massachussetts, USA, to Brazil, did not vary significantly.However, if we extract data on upper thermal tolerances of two geographically separatedpopulations (Cuba and Brazil) of Uca rapax from their figure 2 (p. 68) we can seeclearly that individuals from populations inhabiting lower latitudes showed a greatertolerance of high temperatures than did individuals from populations from higherlatitudes (LD 5 40 and 17 min, respectively, all ‘warm acclimated’ individuals tested50

at 428C). Such (irreversible) population differences in upper temperature tolerance havealso been demonstrated between Mediterranean and Atlantic populations of two speciesof bivalve mollusc (Ansell et al., 1986). We also have some good evidence, though, thattemperature (and salinity) tolerances of two populations of hermit crab, one fromMassachusetts and the other from S. Carolina, USA, were near identical when tested inthe laboratory (Young, 1991). In conclusion, although not invariant, it would appear thatlatitudinally separate populations of the same species, including O. gammarellus, can becharacterised by different upper thermal tolerances and that these differences cannotfully be accounted for by acclimation.

In our experiments on O. gammarellus, upper lethal temperatures increased withincreasing size, from two populations and at all temperature acclimations with theexception of the Chatham population at 208C. Although it is difficult to construct validcomparisons because of the large number of different experimental protocols used tostudy temperature tolerances, there do appear to be some fairly serious discrepancies inthe literature concerning the effect of body size on thermal tolerances of crustaceans. A


similar pattern of upper temperature tolerance increasing with size has been found forthe freshwater amphipod Gammarus pulex (Sutcliffe et al., 1981) and the crayfishOrconectes rusticus (Mundahl and Benton, 1990). Bovee (1949) also found a similarrelationship for the freshwater amphipod Hyalella azteca. However, Sprague (1963) onrepeating Bovee’s study on H. azteca found no such relationship and, on recalculation ofBovee’s data, revealed LT values that were not significantly different from his own.50

Indeed the absence of any intraspecific size effect on upper temperature tolerance hasbeen reported for various amphipod species (Lazo-Wasem, 1984; Agnew and Taylor,1986; Buchanan et al., 1988) as well as lobsters (McLeese, 1956) and terrestrial isopods(Edney, 1964), and appears to be at variance with our data. Sprague (1963) also found,using the same technique as he had to examine H. azteca (see above), that in thefreshwater amphipod Gammarus fasciaticus the smallest individuals were most resistantto high temperatures, a result which is the opposite of that which we found for O.gammarellus. This was also the case for the brackish water amphipod G. duebeni(Kinne, 1954) and, under some (but not all) experimental conditions, for two semi-terrestrial talitrids that are relatively close relatives of O. gammarellus, Transorchestia(Orchestia) chiliensis and Chroestia lota (Marsden, 1980, 1985). Clearly the problem ofthe influence of animal size on temperature tolerance needs to be examined in a numberof different species using an identical experimental protocol. Fortunately, in terms of thelikely impact of changing environmental temperatures on the two populations of O.gammarellus examined by us, the body size dependency of temperature tolerance islargely irrelevant, as what is important is the frequency distributions of these tolerances.

Taking cognisance of what has gone before, it is possible that the direction of theeffect of body size on the upper thermal tolerance of individuals may, in large part, bedue to the experimental design employed. Individuals of the Chatham population were,on average, larger than those of the Kerrachar population. Consequently our finding, thatthe greater maximum temperature tolerance of the former is under-estimated bycontrolling for body size, should at present be treated with some caution, until therelationship between body size and upper thermal tolerance becomes clearer.

In common with many other crustacean species, thermal acclimation affected theupper temperature tolerances recorded for Orchestia gammarellus with a temperaturedifference in median upper thermal tolerance of approximately 3–48C, betweenacclimation temperatures of 5 and 208C. This was in the same range as the difference inthermal tolerance between the two populations, but as discussed above, did not accountfor the difference between the two populations. The degree to which increasing theacclimation temperature resulted in an increase in upper temperature tolerance was morepronounced in the Chatham population compared with the Kerrachar population (Table3). The increase in thermal tolerance of O. gammarellus due to acclimation appears tobe in the same range, if not a little greater, than for two other amphipod species forwhich we have (roughly) comparable data: the freshwater Paramelita nigroculus(acclimation temperatures, 8.5 and 208C; difference in median upper thermal tolerance5 28C; Buchanan et al., 1988) and the euterrestrial talitrid Arcitalitrus sylvaticus(acclimation temperatures, 10 and 208C; difference in median upper thermal tolerance 5

2.68C; Lazo-Wasem, 1984).


5. Conclusion

Geographically separate populations of Orchestia gammarellus, both freshly collectedand acclimated to a number of different thermal regimes in the laboratory, differedsignificantly in their temperature tolerances. The upper thermal limit of this widelydistributed species was not even the same when comparing two British populations,never mind comparing populations from throughout its geographical range (Iceland toNorth Africa). Given this potential for large between-population variation in physiologi-cal tolerances, ecologists should proceed with caution when attempting to makegeneralisations about the physiology of a species based on studies of a single population.Whether such physiological variation has any wide generality remains to be seen,although it should be noted that this needs to be established urgently given many of thesimplistic assumptions about an animal’s physiology that underpin some of theexplanations of large-scale ecological processes. Significant between-population vari-ation in physiological tolerances will have important implications for ecologists seekingto explain large-scale distribution patterns in terms of a ‘fixed’ species physiology.

Acknowledgements

K.J.G. is a Royal Society University Research Fellow. We are grateful to P. and C.Brooker, and to P. and A. Koehn for accommodation, hospitality and transport, to C.Paradine for technical assistance, and to S. Chown and P.G. Moore for critical commentson earlier drafts of the manuscript.

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Do upper thermal tolerances differ in geographically separated populations of the beachflea Orchestia gammarellus (Crustacea: Amphipoda)?