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Genetic variation in desiccation tolerance of Dendrobaena octaedra cocoons originating from different climatic regions
Soil Biology & Biochemistry 35 (2003) 119–124 www.elsevier.com/locate/soilbio
Genetic variation in desiccation tolerance of Dendrobaena octaedra cocoons originating from different climatic regions Martin Holmstrupa,*, Volker Loeschckeb,c a
Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark b Department of Ecology and Genetics, University of A˚rhus, Ny Munkegade, Building 540, DK-8000 A˚rhus C, Denmark c Institute of Advanced Study, La Trobe University, 3086 Bundoora, Vic., Australia Received 24 May 2002; received in revised form 16 October 2002; accepted 21 October 2002
Abstract The aim of this study was to examine genetic variation in desiccation tolerance in cocoons of the parthenogenetically reproducing earthworm Dendrobaena octaedra by comparing populations originating from different geographic regions (Denmark, Norway and Finland), representing large differences in precipitation and temperature. In one experiment, the tolerance of the three populations to increasing desiccation stress in the range from 100 to 91.6% relative humidity (RH) was examined, aiming to represent ecologically relevant RH values. In a second experiment, the effect of cocoon size on desiccation tolerance was investigated at 92.3% RH in the same three populations. There were highly significant differences in desiccation tolerance between populations, indicating a high genetic differentiation of this trait in D. octaedra. Cocoons from Denmark were much more sensitive (71 ^ 14% mortality at 91.6% RH) than cocoons from Norway (21 ^ 4% mortality) and Finland (4 ^ 5% mortality). Cocoons of worms from Finland and Norway were significantly larger than cocoons produced by worms from Denmark suggesting that cocoons from Denmark lost water at a higher rate when subjected to low humidity. Assuming that slow dehydration is necessary for physiologically based protection mechanisms it may be expected that desiccation tolerance is positively correlated with cocoon size. However, within each of the populations cocoon fresh weight did not have any significant impact on desiccation tolerance. When all populations were pooled there was a significant positive effect of cocoon fresh weight on desiccation tolerance, explaining about 20% of the total variation (linear regression). It seems therefore that genetic variation of desiccation tolerance in D. octaedra cocoons is related to variation in both cocoon size and other, physiologically based tolerance mechanisms. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Earthworm cocoons; Lumbricidae; Dehydration; Geographic differences; Genetic variation
1. Introduction The earthworm Dendrobaena octaedra (Savigny) is widespread in the Northern hemisphere where it is often the dominant earthworm species in coniferous forests and tundra soils (Sto¨p-Bowitz, 1969). It lives in the litter layer of the forest floor, in rotting tree stumps, and under moss or lichens. The species reproduces by apomictic (mitotic) parthenogenesis (Casellato and Rodighiero, 1972) and deposits its egg capsules (cocoons) close to the surface of the soil or litter. The life cycle duration in the field is probably 1 –2 years depending primarily on the temperature of the habitat. The cocoons of D. octaedra are often exposed to desiccating conditions during dry periods due to the * Corresponding author. Tel.: þ 45-89-20-14-00; fax: þ 45-89-20-14-14. E-mail address: [email protected] (M. Holmstrup).
shallow depth at which they are found. It is likely that the desiccation tolerance of cocoons is very important for recruitment in populations of this species (Bouche´, 1972). It has been shown that cocoons of D. octaedra are very tolerant to desiccation in comparison with other earthworm species. About 50% of cocoons from a Danish population tolerated exposure for 14 days to 93% relative humidity (RH) (Holmstrup and Westh, 1995). For a permeable soil organism like earthworm cocoons this value represents a severe level of desiccation. For example, the permanent wilting point of plants, a water potential of 2 15 bar, is equivalent to 98.9% RH. Because of their exposed location it is likely that cocoons may have to cope with humidities down to 90% RH, maybe sometimes even lower. Cocoons (or in fact the embryos) have evolved physiological adaptations to meet with desiccation stress. They are able to tolerate loss of practically all osmotically active water
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 2 4 3 - 2
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(approximately 85% water loss) whereby they come into vapour pressure equilibrium with their surroundings (Holmstrup and Westh, 1995). Concomitantly with dehydration they accumulate sorbitol and glucose (Holmstrup, 1995), which in turn will protect membranes and proteins against the dehydration, and reduce cellular water loss of the embryo (for review see Holmstrup and Zachariassen (1996)). Climatic stresses are environmental factors of paramount importance for biological systems. Numerous examples from terrestrial plants and animals show that in particular cold and drought are factors that dictate the distribution of species (for review see Hoffmann and Parsons (1991)). The global climatic changes we are facing are predicted to include an increased frequency and intensity of extreme weather conditions including drought (IPCC, 2001), which may result in altered distribution patterns of species both at local and regional scales (Hodkinson, 1999; Hodkinson et al., 1999). Earthworms, including D. octaedra, have a significant influence on the decomposition of dead plant material (Lee, 1985; McLean and Parkinson, 1997; Lavelle et al., 1997), and ingest large quantities of soil and thereby have a great influence on soil structure, water holding abilities, and soil aeration (Edwards and Shipitalo, 1998). Changes in the occurrence of engineering species such as earthworms could therefore potentially have implications for the bio-geochemistry of soils at a large scale. The presence of genetic variation in a species is a requirement for evolutionary adaptation to changing environmental conditions such as increasing occurrence of drought. Whereas genetic variation of desiccation tolerance has been intensively studied in insects (Hoffmann and Harshman, 1999), virtually nothing is known on genetic variation of stress tolerance in earthworms. The aim of the present study was therefore to examine the desiccation tolerance of three populations of D. octaedra originating from Denmark, Central Norway and Central Finland, representing regions of different climate. It is likely that D. octaedra has spread from southern and eastern populations into Fennoscandia after the last glaciation 10,000 years B.P. (Terhivuo et al., 1987; Terhivuo and Saura, 1990, 1997). The presence of large variation in desiccation tolerance, correlated with the harshness of the climate at the region of origin, may therefore indicate a potential for relatively rapid evolutionary adaptation to changing climatic conditions.
around The National Environmental Research Institute, Silkeborg (568N, 98E). Finnish worms were collected from conifer forest in the vicinity of Jyva¨skyla¨, Central Finland (628N, 268E). Norwegian worms were collected from conifer forest about 30 km north-west of Røros, Central Norway (638N, 128E). Climatic data from weather stations close to the sampling locations are shown in Table 1. 2.2. Cocoon production Cocoons for the experiments were obtained by culturing the sampled earthworms in a mass culture using an agricultural sandy loam soil as a substrate (Holmstrup et al., 1991). Earthworms from each population (approximately 50 adults) were kept at 15 8C in 1 l plastic pots halffilled with moist soil, at a density of 8 –10 specimens per pot. A mixture of dried cow dung and soil subsequently moistened to 50% of dry weight was added as food for the worms. After 3 weeks the cocoons were collected from the culture soil by washing and sieving (Holmstrup et al., 1991) and then incubated on moist filter paper in Petri dishes at 20 8C. Cocoons were inspected every second day using a stereo microscope with light from below. Cocoons having an undifferentiated early embryo were removed and kept in Petri dishes at 3 8C until used in experiments (Holmstrup, 1992). 2.3. Desiccation tolerance Cocoons were surface dried with filter paper and then incubated for 14 days at 20 8C in sealed plastic beakers, the atmosphere of which was controlled by aqueous NaCl solutions as described by Holmstrup et al. (2001). By varying the NaCl concentration between 0 and 148.1 g l21 the RH of the air in the beakers was maintained at six levels between 100% (control) and 91.6% RH. After desiccation exposure the cocoons were rehydrated on moist filter paper and incubated at 20 8C until all viable cocoons had hatched. For each RH level, four replicates consisting of 12 cocoons were used. Mortality was calculated as a percentage. Mortality data were transformed to square root (mortality þ 1) before applying a 2-way ANOVA with Tukey’s pairwise comparison using the statistical software SAS (SAS Institute, Cary, NC, USA). 2.4. Influence of cocoon size on desiccation tolerance
2. Methods 2.1. Sampling locations Approximately 50 adult specimens were collected from plant litter and moss at each location during July and August 1999. Worms were collected from an approximately 1 ha area. Danish worms were collected from mixed forest
About 150 cocoons of each population were categorized by eye as belonging to size classes designated ‘small’, ‘medium’, or ‘large’ cocoons. The fresh weight of groups of five cocoons was determined with a precision of 0.1 mg. From this value an average cocoon fresh weight was calculated, i.e. five cocoons gave rise to one observation. Within each population and size class, four replicates of 12 cocoons were then exposed to 92.3% RH for 14 days.
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Table 1 Monthly mean air temperatures, average maximum air temperatures, average minimum air temperatures, and precipitation at meteorological stations adjacent to the sampling locations in Denmark1, Finland2 and Norway3. Data for Denmark and Finland originate from World Meteorological Organization (1996) and are based on observations in the period 1961–1990. Data for Norway originate from The Norwegian Meteorological Institute based on observations in the period 1957–1999 Station
Month I
Mean temperature (8C) Denmark 20.2 Finland 210.0 Norway 210.5
II
20.1 29.5 29.5
Average maximum temperature (8C)a Denmark 2.1 2.5 Finland 26.8 25.9 Norway 26.1 24.6 Average minimum temperature (8C)b Denmark 22.9 22.9 Finland 214.0 213.8 Norway 215.7 214.7 Amount of precipitation (mm) Denmark 69 46 Finland 43 30 Norway 33 29
III
IV
V
VI
VII
VIII
IX
2.1 24.7 25.4
5.7 1.3 20.6
10.8 8.7 5.3
14.1 14.1 9.8
15.4 15.7 11.7
15.3 13.6 10.5
12.3 8.3 6.3
8.9 3.4 1.5
4.4 22.2 24.9
1.3 27.2 29.1
7.5 2.6 0.4
5.3 20.3 20.3
10.1 5.7 3.9
15.7 14.3 10.6
19.0 19.5 15.5
20.0 20.8 17.2
20.2 18.2 16.0
16.4 12.1 11.0
12.2 6.2 5.1
7 0.1 21.5
3.6 24.2 24.7
11.2 6.6 5.2
20.9 29.5 210.8
1.5 23.3 24.9
5.8 2.6 0.4
9.1 7.9 4.8
10.9 10.1 6.8
10.6 8.8 5.9
8.4 4.4 2.5
5.6 0.4 21.7
20.1 24.9 28.7
1.5 210.8 213.9
3.9 21.8 24.2
56 35 28
43 37 25
70 78 70
71 91 65
90 56 40
94 59 36
79 47 38
52 41 30
58 56 57
88 67 49
X
XI
XII
I– XII
816 640 500
Karup airport (568 100 N, 98 300 E), 20 m. a. s. l.; 2Jyva¨skyla¨ airport (628N, 258 300 E), 20 m. a. s. l.; 3Røros (638N, 128E), 628 m. a. s. l. The mean of the daily maximum temperatures. The mean of the daily minimum temperatures. 1
a b
Assessment of mortality, data treatment and statistical analysis followed the description in Section 2.3.
3. Results Desiccation tolerance differed considerably between the three populations (Fig. 1). Cocoons from Finland tolerated all desiccation levels tested with low mortality even at 91.6% RH. Mortality of cocoons from Norway was about 20% at the lowest RH, whereas cocoons from Denmark had
Fig. 1. Percentage mortality (mean ^ SD, n ¼ 4) of Danish, Finnish and Norwegian D. octaedra cocoons exposed for 14 days to different desiccation stress levels (different relative humidities).
a mean mortality of 70% at 91.6% RH. The statistical analysis showed that the effect of geographic origin was highly significant (P , 0.0001), whereas the interaction between geographic origin and desiccation level was not statistically significant (P ¼ 0.22). Pairwise comparisons of population mortalities were all significantly different (Tukey’s studentized range, P , 0.05, n ¼ 20) when omitting control data. Average (grand mean ^ SD) cocoon fresh weight was lowest in the Danish population (3.86 ^ 0.44 mg, n ¼ 32), intermediate in the Norwegian population (4.49 ^ 0.67 mg, n ¼ 32) and highest in cocoons from Finland (5.20 ^ 0.88 mg, n ¼ 38). The effect of geographic origin on cocoon fresh weight was highly significant (P , 0.0001), and the three populations differed significantly from each other (Tukey’s studentized range, P , 0.05). The within population effect of cocoon fresh weight on mortality at 92.3% RH was not significant in populations originating from Denmark or Finland (P ¼ 0.34 and 0.62, respectively). For cocoons from Norway the effect of size was statistically significant (P ¼ 0.01), suggesting a beneficial effect of increasing size. Also when the three populations were pooled, the effect of fresh weight on mortality was highly significant (ANOVA, P , 0.0001) and positively correlated with survival (Fig. 2). A linear regression of the pooled data showed that 20% of the variation of mortality could be explained by cocoon fresh weight (P , 0.01). The mortality percentages of the three populations (grand mean ^ SD, n ¼ 12) in this experiment
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Fig. 2. Percentage mortality (mean ^ SD, n ¼ 4) of various size classes of D. octaedra cocoons from Denmark, Finland and Norway after exposure to 92.3% RH for 14 days. Horizontal bars indicate ^ SD of mean fresh weight in the various size classes.
were largely in agreement with the mortality percentage at the same RH shown in Fig. 1 (Denmark: 50.7 ^ 21.5%; Norway: 44.4 ^ 21.6%, Finland: 10.8 ^ 22.0%).
4. Discussion Associations between climatic conditions and patterns of genetic variation in stress resistance within a species have been shown for several plants and ectotherms (reviewed in Hoffmann and Parsons (1991)). These observations indicate that geographic variation in stress resistance can be genetically based. To our knowledge, no previous studies of earthworms have addressed this relation. The results from D. octaedra cocoons shown here seem to confirm the observations from many other ectothermic species. The population from Denmark was clearly the most sensitive to drought (Fig. 1). This is consistent with a larger precipitation in this region compared to Norway and Finland (Table 1), and therefore a prediction of lesser intensities of drought spells. It is interesting to note that desiccation tolerance in another Danish population located about 100 km away from the here studied population, under identical experimental conditions, was almost equal to that of the present study (Holmstrup and Westh, 1995), suggesting that the variation within regions is relatively small and rather constant through time. However, the much lower winter temperatures in Norway and Finland (Table 1) may also contribute to the differences in desiccation tolerance. It has been shown that D. octaedra cocoons are severely dehydrated when frost occurs in the soil or litter in which they are found (Holmstrup and Westh, 1994; Holmstrup et al., 2002). In fact, even at soil temperatures around 2 3 8C cocoons lose
about 60% of their fresh weight, equivalent to 80% of their original water content (Holmstrup, 1992). The dehydration occurring at sub-zero temperatures is an important mechanism for surviving frost in the soil by equilibrating the melting point of the cocoon fluids to the ambient temperature, thereby securing that freezing of the cocoon does not occur (Holmstrup et al., 2002). Danish forest soils rarely freeze whereas soils at the Finnish and Norwegian locations usually are frozen during winter even though snow cover will provide some insulation. Occasionally, hard frost periods in Finland and Norway may occur without a snow cover, causing low freezing temperatures even deep in the soil (Huhta, 1980). It seems therefore that the observed differences in drought tolerance between the populations match the climatic conditions in terms of both precipitation and temperature. Several authors have suggested that earthworm cocoons are much more tolerant to climatic stress than hatched individuals (Huhta, 1980; Satchell, 1980; Holmstrup and Westh, 1995), an observation also known from insects where immobile stages such as eggs and pupae are often much more stress tolerant than adult stages (Krebs and Loeschcke, 1995). Moreover, cocoon survival is probably important for persistence of earthworm (and enchytraeid) populations during adverse climatic conditions, with cocoons being the most desiccation tolerant life stage (Nielsen, 1955; Bouche´, 1972; Huhta, 1980; Parmelee and Crossley, 1988; Dymond et al., 1997; Doube and Auhl, 1998). Desiccation and frost may therefore inflict a hard selection pressure on earthworms in regions with a harsh climate, thereby favouring genotypes (clones) that are best suited to survive the specific climatic conditions of the particular site. Note that the drought levels used in this study mimic realistic water potentials that may be encountered by
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soil fauna in superficial soil layers or litter during summer drought or winter frosts at the sampling locations used in this study (Hillel, 1998; Holmstrup, 2001; Holmstrup et al., 2002). Having established that large variation for desiccation resistance exists between cocoons of D. octaedra from different geographic origin, with genetic variation between populations being the most likely explanation behind the differentiation, it is interesting to identify possible correlations with other traits that may have evolved with desiccation resistance. Cocoon fresh weight explained some but not all of the variation in desiccation tolerance (Fig. 2). Cocoon size is highly correlated to adult size in earthworms (Lofs-Holmin, 1983) indicating that adult worms from Finland and Norway were larger than Danish worms in this study. We have not investigated whether this size difference was genetically based but there are good reasons to believe it to be so in general. A large body of evidence shows that low temperature in most cases promotes large body size in ectothermic animals. This applies to both phenotypic development (Atkinson, 1994) and to the genetic evolution of body size in ectothermic animals (Partridge and French, 1996). There are good indications that this is also the case in D. octaedra as shown by Terhivuo et al. (1987) who reported increasing adult body size along a 1200 km south –north cline in Finland. However, as discussed earlier, other traits than cocoon size must also contribute to the observed variation in desiccation tolerance. We suggest that physiologically based adaptations such as the ability to accumulate sugars and polyols may differ between populations as the result of evolutionary adaptation to the specific climatic conditions. Adequate synthesis of sugars and polyols is a crucial factor in a variety of desiccation tolerant invertebrate species (Crowe et al., 1992; Ring and Danks, 1994). Membrane phospholipid composition is also important because the extreme water loss of the D. octaedra embryos (Holmstrup and Westh, 1995) may disrupt membrane integrity due to the removal of water molecules interacting with membrane molecules (Hazel and Williams, 1990). Further studies are needed to address these questions in more detail. D. octaedra reproduces by obligate apomictic parthenogenesis with offspring being genetically identical to mothers. Therefore a single individual may give rise to a new population. It should be expected that a hard selection pressure caused by, e.g. one summer with extreme drought could result in a rapid evolutionary change in favour of desiccation resistant genotypes (clones). On the other hand, occasional strong selection for desiccation resistance will decrease the number of genotypes in the population, which may have a negative effect on other fitness traits. Terhivuo et al. (1987) examined allozyme variation within and between D. octaedra populations in Finland and reported high genetic variation even within populations sampled from
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a small area, perhaps due to relatively rapid dispersal abilities or due to microhabitat differences favouring different genotypes. These observations, and the fact that we have shown high genetic variation in desiccation resistance in the present study, suggest that evolutionary adaptation to changing climatic conditions is possible in this earthworm species.
Acknowledgements Esko Martikainen is thanked for supplying D. octaedra from Jyva¨skyla¨. This study received financial support from the Danish Natural Science Research Council, Grant No. 9700177.
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Genetic variation in desiccation tolerance of Dendrobaena octaedra cocoons originating from different climatic regions Martin Holmstrupa,*, Volker Loeschckeb,c a
Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsøvej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark b Department of Ecology and Genetics, University of A˚rhus, Ny Munkegade, Building 540, DK-8000 A˚rhus C, Denmark c Institute of Advanced Study, La Trobe University, 3086 Bundoora, Vic., Australia Received 24 May 2002; received in revised form 16 October 2002; accepted 21 October 2002
Abstract The aim of this study was to examine genetic variation in desiccation tolerance in cocoons of the parthenogenetically reproducing earthworm Dendrobaena octaedra by comparing populations originating from different geographic regions (Denmark, Norway and Finland), representing large differences in precipitation and temperature. In one experiment, the tolerance of the three populations to increasing desiccation stress in the range from 100 to 91.6% relative humidity (RH) was examined, aiming to represent ecologically relevant RH values. In a second experiment, the effect of cocoon size on desiccation tolerance was investigated at 92.3% RH in the same three populations. There were highly significant differences in desiccation tolerance between populations, indicating a high genetic differentiation of this trait in D. octaedra. Cocoons from Denmark were much more sensitive (71 ^ 14% mortality at 91.6% RH) than cocoons from Norway (21 ^ 4% mortality) and Finland (4 ^ 5% mortality). Cocoons of worms from Finland and Norway were significantly larger than cocoons produced by worms from Denmark suggesting that cocoons from Denmark lost water at a higher rate when subjected to low humidity. Assuming that slow dehydration is necessary for physiologically based protection mechanisms it may be expected that desiccation tolerance is positively correlated with cocoon size. However, within each of the populations cocoon fresh weight did not have any significant impact on desiccation tolerance. When all populations were pooled there was a significant positive effect of cocoon fresh weight on desiccation tolerance, explaining about 20% of the total variation (linear regression). It seems therefore that genetic variation of desiccation tolerance in D. octaedra cocoons is related to variation in both cocoon size and other, physiologically based tolerance mechanisms. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Earthworm cocoons; Lumbricidae; Dehydration; Geographic differences; Genetic variation
1. Introduction The earthworm Dendrobaena octaedra (Savigny) is widespread in the Northern hemisphere where it is often the dominant earthworm species in coniferous forests and tundra soils (Sto¨p-Bowitz, 1969). It lives in the litter layer of the forest floor, in rotting tree stumps, and under moss or lichens. The species reproduces by apomictic (mitotic) parthenogenesis (Casellato and Rodighiero, 1972) and deposits its egg capsules (cocoons) close to the surface of the soil or litter. The life cycle duration in the field is probably 1 –2 years depending primarily on the temperature of the habitat. The cocoons of D. octaedra are often exposed to desiccating conditions during dry periods due to the * Corresponding author. Tel.: þ 45-89-20-14-00; fax: þ 45-89-20-14-14. E-mail address: [email protected] (M. Holmstrup).
shallow depth at which they are found. It is likely that the desiccation tolerance of cocoons is very important for recruitment in populations of this species (Bouche´, 1972). It has been shown that cocoons of D. octaedra are very tolerant to desiccation in comparison with other earthworm species. About 50% of cocoons from a Danish population tolerated exposure for 14 days to 93% relative humidity (RH) (Holmstrup and Westh, 1995). For a permeable soil organism like earthworm cocoons this value represents a severe level of desiccation. For example, the permanent wilting point of plants, a water potential of 2 15 bar, is equivalent to 98.9% RH. Because of their exposed location it is likely that cocoons may have to cope with humidities down to 90% RH, maybe sometimes even lower. Cocoons (or in fact the embryos) have evolved physiological adaptations to meet with desiccation stress. They are able to tolerate loss of practically all osmotically active water
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 2 4 3 - 2
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(approximately 85% water loss) whereby they come into vapour pressure equilibrium with their surroundings (Holmstrup and Westh, 1995). Concomitantly with dehydration they accumulate sorbitol and glucose (Holmstrup, 1995), which in turn will protect membranes and proteins against the dehydration, and reduce cellular water loss of the embryo (for review see Holmstrup and Zachariassen (1996)). Climatic stresses are environmental factors of paramount importance for biological systems. Numerous examples from terrestrial plants and animals show that in particular cold and drought are factors that dictate the distribution of species (for review see Hoffmann and Parsons (1991)). The global climatic changes we are facing are predicted to include an increased frequency and intensity of extreme weather conditions including drought (IPCC, 2001), which may result in altered distribution patterns of species both at local and regional scales (Hodkinson, 1999; Hodkinson et al., 1999). Earthworms, including D. octaedra, have a significant influence on the decomposition of dead plant material (Lee, 1985; McLean and Parkinson, 1997; Lavelle et al., 1997), and ingest large quantities of soil and thereby have a great influence on soil structure, water holding abilities, and soil aeration (Edwards and Shipitalo, 1998). Changes in the occurrence of engineering species such as earthworms could therefore potentially have implications for the bio-geochemistry of soils at a large scale. The presence of genetic variation in a species is a requirement for evolutionary adaptation to changing environmental conditions such as increasing occurrence of drought. Whereas genetic variation of desiccation tolerance has been intensively studied in insects (Hoffmann and Harshman, 1999), virtually nothing is known on genetic variation of stress tolerance in earthworms. The aim of the present study was therefore to examine the desiccation tolerance of three populations of D. octaedra originating from Denmark, Central Norway and Central Finland, representing regions of different climate. It is likely that D. octaedra has spread from southern and eastern populations into Fennoscandia after the last glaciation 10,000 years B.P. (Terhivuo et al., 1987; Terhivuo and Saura, 1990, 1997). The presence of large variation in desiccation tolerance, correlated with the harshness of the climate at the region of origin, may therefore indicate a potential for relatively rapid evolutionary adaptation to changing climatic conditions.
around The National Environmental Research Institute, Silkeborg (568N, 98E). Finnish worms were collected from conifer forest in the vicinity of Jyva¨skyla¨, Central Finland (628N, 268E). Norwegian worms were collected from conifer forest about 30 km north-west of Røros, Central Norway (638N, 128E). Climatic data from weather stations close to the sampling locations are shown in Table 1. 2.2. Cocoon production Cocoons for the experiments were obtained by culturing the sampled earthworms in a mass culture using an agricultural sandy loam soil as a substrate (Holmstrup et al., 1991). Earthworms from each population (approximately 50 adults) were kept at 15 8C in 1 l plastic pots halffilled with moist soil, at a density of 8 –10 specimens per pot. A mixture of dried cow dung and soil subsequently moistened to 50% of dry weight was added as food for the worms. After 3 weeks the cocoons were collected from the culture soil by washing and sieving (Holmstrup et al., 1991) and then incubated on moist filter paper in Petri dishes at 20 8C. Cocoons were inspected every second day using a stereo microscope with light from below. Cocoons having an undifferentiated early embryo were removed and kept in Petri dishes at 3 8C until used in experiments (Holmstrup, 1992). 2.3. Desiccation tolerance Cocoons were surface dried with filter paper and then incubated for 14 days at 20 8C in sealed plastic beakers, the atmosphere of which was controlled by aqueous NaCl solutions as described by Holmstrup et al. (2001). By varying the NaCl concentration between 0 and 148.1 g l21 the RH of the air in the beakers was maintained at six levels between 100% (control) and 91.6% RH. After desiccation exposure the cocoons were rehydrated on moist filter paper and incubated at 20 8C until all viable cocoons had hatched. For each RH level, four replicates consisting of 12 cocoons were used. Mortality was calculated as a percentage. Mortality data were transformed to square root (mortality þ 1) before applying a 2-way ANOVA with Tukey’s pairwise comparison using the statistical software SAS (SAS Institute, Cary, NC, USA). 2.4. Influence of cocoon size on desiccation tolerance
2. Methods 2.1. Sampling locations Approximately 50 adult specimens were collected from plant litter and moss at each location during July and August 1999. Worms were collected from an approximately 1 ha area. Danish worms were collected from mixed forest
About 150 cocoons of each population were categorized by eye as belonging to size classes designated ‘small’, ‘medium’, or ‘large’ cocoons. The fresh weight of groups of five cocoons was determined with a precision of 0.1 mg. From this value an average cocoon fresh weight was calculated, i.e. five cocoons gave rise to one observation. Within each population and size class, four replicates of 12 cocoons were then exposed to 92.3% RH for 14 days.
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Table 1 Monthly mean air temperatures, average maximum air temperatures, average minimum air temperatures, and precipitation at meteorological stations adjacent to the sampling locations in Denmark1, Finland2 and Norway3. Data for Denmark and Finland originate from World Meteorological Organization (1996) and are based on observations in the period 1961–1990. Data for Norway originate from The Norwegian Meteorological Institute based on observations in the period 1957–1999 Station
Month I
Mean temperature (8C) Denmark 20.2 Finland 210.0 Norway 210.5
II
20.1 29.5 29.5
Average maximum temperature (8C)a Denmark 2.1 2.5 Finland 26.8 25.9 Norway 26.1 24.6 Average minimum temperature (8C)b Denmark 22.9 22.9 Finland 214.0 213.8 Norway 215.7 214.7 Amount of precipitation (mm) Denmark 69 46 Finland 43 30 Norway 33 29
III
IV
V
VI
VII
VIII
IX
2.1 24.7 25.4
5.7 1.3 20.6
10.8 8.7 5.3
14.1 14.1 9.8
15.4 15.7 11.7
15.3 13.6 10.5
12.3 8.3 6.3
8.9 3.4 1.5
4.4 22.2 24.9
1.3 27.2 29.1
7.5 2.6 0.4
5.3 20.3 20.3
10.1 5.7 3.9
15.7 14.3 10.6
19.0 19.5 15.5
20.0 20.8 17.2
20.2 18.2 16.0
16.4 12.1 11.0
12.2 6.2 5.1
7 0.1 21.5
3.6 24.2 24.7
11.2 6.6 5.2
20.9 29.5 210.8
1.5 23.3 24.9
5.8 2.6 0.4
9.1 7.9 4.8
10.9 10.1 6.8
10.6 8.8 5.9
8.4 4.4 2.5
5.6 0.4 21.7
20.1 24.9 28.7
1.5 210.8 213.9
3.9 21.8 24.2
56 35 28
43 37 25
70 78 70
71 91 65
90 56 40
94 59 36
79 47 38
52 41 30
58 56 57
88 67 49
X
XI
XII
I– XII
816 640 500
Karup airport (568 100 N, 98 300 E), 20 m. a. s. l.; 2Jyva¨skyla¨ airport (628N, 258 300 E), 20 m. a. s. l.; 3Røros (638N, 128E), 628 m. a. s. l. The mean of the daily maximum temperatures. The mean of the daily minimum temperatures. 1
a b
Assessment of mortality, data treatment and statistical analysis followed the description in Section 2.3.
3. Results Desiccation tolerance differed considerably between the three populations (Fig. 1). Cocoons from Finland tolerated all desiccation levels tested with low mortality even at 91.6% RH. Mortality of cocoons from Norway was about 20% at the lowest RH, whereas cocoons from Denmark had
Fig. 1. Percentage mortality (mean ^ SD, n ¼ 4) of Danish, Finnish and Norwegian D. octaedra cocoons exposed for 14 days to different desiccation stress levels (different relative humidities).
a mean mortality of 70% at 91.6% RH. The statistical analysis showed that the effect of geographic origin was highly significant (P , 0.0001), whereas the interaction between geographic origin and desiccation level was not statistically significant (P ¼ 0.22). Pairwise comparisons of population mortalities were all significantly different (Tukey’s studentized range, P , 0.05, n ¼ 20) when omitting control data. Average (grand mean ^ SD) cocoon fresh weight was lowest in the Danish population (3.86 ^ 0.44 mg, n ¼ 32), intermediate in the Norwegian population (4.49 ^ 0.67 mg, n ¼ 32) and highest in cocoons from Finland (5.20 ^ 0.88 mg, n ¼ 38). The effect of geographic origin on cocoon fresh weight was highly significant (P , 0.0001), and the three populations differed significantly from each other (Tukey’s studentized range, P , 0.05). The within population effect of cocoon fresh weight on mortality at 92.3% RH was not significant in populations originating from Denmark or Finland (P ¼ 0.34 and 0.62, respectively). For cocoons from Norway the effect of size was statistically significant (P ¼ 0.01), suggesting a beneficial effect of increasing size. Also when the three populations were pooled, the effect of fresh weight on mortality was highly significant (ANOVA, P , 0.0001) and positively correlated with survival (Fig. 2). A linear regression of the pooled data showed that 20% of the variation of mortality could be explained by cocoon fresh weight (P , 0.01). The mortality percentages of the three populations (grand mean ^ SD, n ¼ 12) in this experiment
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Fig. 2. Percentage mortality (mean ^ SD, n ¼ 4) of various size classes of D. octaedra cocoons from Denmark, Finland and Norway after exposure to 92.3% RH for 14 days. Horizontal bars indicate ^ SD of mean fresh weight in the various size classes.
were largely in agreement with the mortality percentage at the same RH shown in Fig. 1 (Denmark: 50.7 ^ 21.5%; Norway: 44.4 ^ 21.6%, Finland: 10.8 ^ 22.0%).
4. Discussion Associations between climatic conditions and patterns of genetic variation in stress resistance within a species have been shown for several plants and ectotherms (reviewed in Hoffmann and Parsons (1991)). These observations indicate that geographic variation in stress resistance can be genetically based. To our knowledge, no previous studies of earthworms have addressed this relation. The results from D. octaedra cocoons shown here seem to confirm the observations from many other ectothermic species. The population from Denmark was clearly the most sensitive to drought (Fig. 1). This is consistent with a larger precipitation in this region compared to Norway and Finland (Table 1), and therefore a prediction of lesser intensities of drought spells. It is interesting to note that desiccation tolerance in another Danish population located about 100 km away from the here studied population, under identical experimental conditions, was almost equal to that of the present study (Holmstrup and Westh, 1995), suggesting that the variation within regions is relatively small and rather constant through time. However, the much lower winter temperatures in Norway and Finland (Table 1) may also contribute to the differences in desiccation tolerance. It has been shown that D. octaedra cocoons are severely dehydrated when frost occurs in the soil or litter in which they are found (Holmstrup and Westh, 1994; Holmstrup et al., 2002). In fact, even at soil temperatures around 2 3 8C cocoons lose
about 60% of their fresh weight, equivalent to 80% of their original water content (Holmstrup, 1992). The dehydration occurring at sub-zero temperatures is an important mechanism for surviving frost in the soil by equilibrating the melting point of the cocoon fluids to the ambient temperature, thereby securing that freezing of the cocoon does not occur (Holmstrup et al., 2002). Danish forest soils rarely freeze whereas soils at the Finnish and Norwegian locations usually are frozen during winter even though snow cover will provide some insulation. Occasionally, hard frost periods in Finland and Norway may occur without a snow cover, causing low freezing temperatures even deep in the soil (Huhta, 1980). It seems therefore that the observed differences in drought tolerance between the populations match the climatic conditions in terms of both precipitation and temperature. Several authors have suggested that earthworm cocoons are much more tolerant to climatic stress than hatched individuals (Huhta, 1980; Satchell, 1980; Holmstrup and Westh, 1995), an observation also known from insects where immobile stages such as eggs and pupae are often much more stress tolerant than adult stages (Krebs and Loeschcke, 1995). Moreover, cocoon survival is probably important for persistence of earthworm (and enchytraeid) populations during adverse climatic conditions, with cocoons being the most desiccation tolerant life stage (Nielsen, 1955; Bouche´, 1972; Huhta, 1980; Parmelee and Crossley, 1988; Dymond et al., 1997; Doube and Auhl, 1998). Desiccation and frost may therefore inflict a hard selection pressure on earthworms in regions with a harsh climate, thereby favouring genotypes (clones) that are best suited to survive the specific climatic conditions of the particular site. Note that the drought levels used in this study mimic realistic water potentials that may be encountered by
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soil fauna in superficial soil layers or litter during summer drought or winter frosts at the sampling locations used in this study (Hillel, 1998; Holmstrup, 2001; Holmstrup et al., 2002). Having established that large variation for desiccation resistance exists between cocoons of D. octaedra from different geographic origin, with genetic variation between populations being the most likely explanation behind the differentiation, it is interesting to identify possible correlations with other traits that may have evolved with desiccation resistance. Cocoon fresh weight explained some but not all of the variation in desiccation tolerance (Fig. 2). Cocoon size is highly correlated to adult size in earthworms (Lofs-Holmin, 1983) indicating that adult worms from Finland and Norway were larger than Danish worms in this study. We have not investigated whether this size difference was genetically based but there are good reasons to believe it to be so in general. A large body of evidence shows that low temperature in most cases promotes large body size in ectothermic animals. This applies to both phenotypic development (Atkinson, 1994) and to the genetic evolution of body size in ectothermic animals (Partridge and French, 1996). There are good indications that this is also the case in D. octaedra as shown by Terhivuo et al. (1987) who reported increasing adult body size along a 1200 km south –north cline in Finland. However, as discussed earlier, other traits than cocoon size must also contribute to the observed variation in desiccation tolerance. We suggest that physiologically based adaptations such as the ability to accumulate sugars and polyols may differ between populations as the result of evolutionary adaptation to the specific climatic conditions. Adequate synthesis of sugars and polyols is a crucial factor in a variety of desiccation tolerant invertebrate species (Crowe et al., 1992; Ring and Danks, 1994). Membrane phospholipid composition is also important because the extreme water loss of the D. octaedra embryos (Holmstrup and Westh, 1995) may disrupt membrane integrity due to the removal of water molecules interacting with membrane molecules (Hazel and Williams, 1990). Further studies are needed to address these questions in more detail. D. octaedra reproduces by obligate apomictic parthenogenesis with offspring being genetically identical to mothers. Therefore a single individual may give rise to a new population. It should be expected that a hard selection pressure caused by, e.g. one summer with extreme drought could result in a rapid evolutionary change in favour of desiccation resistant genotypes (clones). On the other hand, occasional strong selection for desiccation resistance will decrease the number of genotypes in the population, which may have a negative effect on other fitness traits. Terhivuo et al. (1987) examined allozyme variation within and between D. octaedra populations in Finland and reported high genetic variation even within populations sampled from
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a small area, perhaps due to relatively rapid dispersal abilities or due to microhabitat differences favouring different genotypes. These observations, and the fact that we have shown high genetic variation in desiccation resistance in the present study, suggest that evolutionary adaptation to changing climatic conditions is possible in this earthworm species.
Acknowledgements Esko Martikainen is thanked for supplying D. octaedra from Jyva¨skyla¨. This study received financial support from the Danish Natural Science Research Council, Grant No. 9700177.
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