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Low genetic variation for Dendrobaena octaedra from Greenland compared to populations from Europe and North America: Refuge or selection?
ARTICLE IN PRESS Pedobiologia 50 (2006) 225—234
www.elsevier.de/pedobi
Low genetic variation for Dendrobaena octaedra from Greenland compared to populations from Europe and North America: Refuge or selection? Pernille Liland Hansena, Martin Holmstrupa, Mark Bayleyb, Vibeke Simonsena, a
Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsoevej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark b Department of Zoophysiology, Institute of Biological Sciences Building 131, C.F. Møllers Alle´, University of Aarhus, DK-8000 Aarhus C, Denmark Received 29 August 2005; accepted 8 December 2005
KEYWORDS Dendrobaena octaedra; Isozymes; Genetic diversity; Northern Hemisphere
Summary The genetic relationship of 345 specimens of the parthenogenetic lumbricid Dendrobaena octaedra from Greenland, Canada and Europe were analysed by means of isozymes. The results showed that populations from Greenland were markedly different from Canadian and European populations, suggesting that dispersal between Greenland and the continents has been much more restricted in the past than dispersal between North America and Europe. This observation supports to the notion that Greenland populations have persisted for a long period and perhaps have survived the last glacial period in ice-free refugia. A highly significant positive correlation was seen between diversity measured either as mean haploid diversity or clonal diversity and mean temperature of the annual coldest month. These results indicate that temperature might cause selection in colder climates or that sexual processes in D. dendrobaena could have been active recently on an evolutionary time scale. & 2006 Elsevier GmbH. All rights reserved.
Introduction
Corresponding author. Tel.: +45 8920 1400;
fax: +45 8920 1413. E-mail address: [email protected] (V. Simonsen).
The nunatak hypothesis proposes that some icefree refuge existed north of the southern boundary of the Pleistocene glaciers, where plants and animals could survive during the last or previous glacial periods. These refuges could either be
0031-4056/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2005.12.001
ARTICLE IN PRESS 226 mountains rising above the inland ice (which in Inuit language are called nunataks) or coastal icefree refuges. When the ice melted, plants and animals colonised previously ice-covered land from the refuge and the unglaciated areas south and east of the inland ice. The alternative to the nunatak hypothesis is the tabula rasa theory stating that all higher land plants and animals were exterminated north of the southern boundary of the Pleistocene glaciers, and that the present biota immigrated from the south or east (Nordal 1987; Dahl 1989; Brochmann et al. 2003). There is now strong geological evidence suggesting that some nunataks and ice-free coastal shelves existed within the maximum limits of the last glacial period 25,000–10,000 years ago. During this period the ice cover in Greenland was larger than today. Along its western and south-eastern margins it terminated on the continental shelf (Funder and Hansen 1996). However, in eastern Greenland, there is strong evidence for ice-free lowlands on the Hochstetter Forland (Funder et al. 1998) and for extensive ice-free uplands penetrating the ice cover in Jameson Land (Funder et al. 1998) and Germania Land (Landvik 1994). In south-west Greenland large ice-free nunataks probably existed between 671N and 721N (Funder and Hansen 1996). However, these ice-free areas were most likely so climatically harsh that only very tolerant organism could have survived. Recent climatic reconstructions from ice core projects in central Greenland indicated that the mean annual decline in temperature during the last glacial period was as much as 25 1C compared to the present temperature (Dahl-Jensen et al. 1998). Isozymes have shown their versatility in providing markers for analysing genetic compositions of populations. By examining the distribution of genotypes or phenotypes the phenetic relationship between populations may be estimated (Avise 1994). Phenetic studies have reinforced the view that any attempt to interpret population differentiation in terms of evolutionary factors may lead to erroneous conclusions in the absence of historical data (Selander and Whittam 1983; Slatkin 1987). Electrophoresis of enzymes has been applied to the study of earthworm species since the early 1980s but such genetic studies have mainly focused on local populations, whereas only a few studies have covered a wider geographic area (Jaenike et al. 1980; Jaenike and Selander 1985). Recently genetic sequence analysis has been used for studying clonal diversity in parthenogenetic earthworms (Heethoff et al. 2004). Dendrobaena octaedra Savigny, 1826 (Oligochaeta: Lumbricidae) is widespread in the Northern Hemisphere, where it is often the dominant earth-
P.L. Hansen et al. worm species in coniferous forests and tundra soils. It is an epigeic species living in the litter layer of the forest floor, in rotting tree stumps and under moss or lichens (Sto ¨p-Bowitz 1969). Therefore, it is regularly exposed to climatic stresses such as drought and cold. D. octaedra reproduces by apomictic parthenogenesis (Omodeo 1957), i.e. the offspring are clones of the parents. The cocoons of D. octaedra have been found to be extremely tolerant to drought (Holmstrup and Westh 1995; Holmstrup and Loeschcke 2003) and freezing (Holmstrup 1994; Holmstrup and Zachariassen 1996). Hatched individuals can withstand extracellular freezing and are thus freeze-tolerant, as one of only two known earthworm species with this ability (Rasmussen and Holmstrup 2002; Bindesbøl et al. 2005). This documented hardiness of D. octaedra opens the possibility that it could have survived the last glacial period in ice-free refuge and therefore may have an ancient origin in Greenland. It is unlikely that D. octaedra has arrived by natural dispersal in Greenland since the last glacial period, because the sea is a natural barrier for dispersal of adult earthworms and cocoons. On the other hand, anthropochorous dispersal of earthworms has been of significant importance for some earthworm species (Edwards and Bohlen 1996), so D. octaedra could have arrived in Greenland by human activity in more recent times. Different numbers of polyploidy are seen in D. octaedra. In Greenland and Iceland 6n individuals are recorded and in southern and central parts of Europe 5n and 8n individuals (Omodeo 1955, 1962; Casellato and Rodighiero 1972; Vedovini, 1973 cited in Casellato 1987). Variation in polyploidy may cause differentiation among individuals, because more chromosomes may increase the chance for mutation and thereby differentiation. Allelic frequencies determined by electrophoresis of isozymes tend to be correlated directly or indirectly with geographical variations in temperature. Nevo et al. (1984) analysed correlations of abiotic and biotic factors with genetic diversity assessed electrophoretically, using published data on natural populations of 1111 species from a wide range of geographic locations. These authors concluded that 20% of the genetic variance that could be explained was mainly associated with ecological heterogeneity, and temperature emerged as one of the major variables. The aim of this study was to see if populations of D. octaedra in Greenland might have an ancient origin when comparing Greenland populations to populations from Canada and Europe by using isozymes. On the other hand, D. octaedra might
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere have been introduced by man from these areas to Greenland and in that case greater similarity between Greenlandic populations and populations from the source area was expected. Furthermore, correlation to winter temperatures was analysed to determine the importance of temperature in accounting for the variation in isozymes.
Materials and methods
227
the laboratory in plastic beakers with moist soil and vegetation from the localities. D. octaedra were collected from coniferous forest in Europe and Canada, and from moss covered rocks in Greenland. Specimens from Disko and Jyva ¨skyla were obtained from laboratory cultures, which had been held in culture for one generation following procedures described by Holmstrup et al. (1991). About 200 individuals were collected to establish these laboratory cultures. Specimens from the other populations were kept under similar conditions in the laboratory until used for analysis.
Samples Three hundred and forty-five specimens of D. octaedra were used for this study. The origin of the populations is shown in Fig. 1 and additional information on the sampling area and the number of individuals are listed in Table 1. The localities Disko and Narssarssuaq in Greenland were separated by about 800 km and the 3 localities in Canada and Sweden, respectively, by about 300 km. The 3 samples from Silkeborg were separated by 5 km and the 3 samples from Narssarssuaq by 1 km. Specimens from the different localities were collected in August 2003 (Canada), in September and October 2003 (Sweden), in September 2003 (Narssarssuaq) and in September and November 2003 (Silkeborg). All populations were brought to
Analysis of isozymes Earthworms were held in Petri dishes with moist filter-paper at 5 1C for 24 h to empty their guts and avoid enzymes from intestinal bacteria interfering with the analysis. The tissue used was the whole part from anterior to clitellum, which were removed from each worm and stored at 80 1C in a 0.5 ml microtube with 50 ml tris-citrate buffer pH 7.0 containing 1% polyvinylpyrrolidon until electrophoresis. No attempt to determine sex of the worms was done, as it was assumed that the worms have apomictic pathenogenesis. The samples were analysed by horizontal starch gel electrophoresis (e.g. Hillis et al. 1996). Several
Dunvegan Edmonton Letbridge
Disko Umeå
Jyväskyla
Uppsala Narssarssuaq Lund Silkeborg
Figure 1. Map on the sampling localities.
ARTICLE IN PRESS 228
P.L. Hansen et al.
Table 1. Origin of Dendrobaena octaedra populations, abbreviation for the samples, latitude and longitude for the sampling areas and number of individuals Country
Locality
Sample abbreviation
Latitude and longitude
Canada
Lethbridge Edmonton Dunvegan
Let Ed Du
49.61 N, 112.81 W 53.61 N, 113.51 W 55.91 N, 118.61 W
30 19 11
Sweden
Lund Uppsala Umea(
L Up Um
55.81 N, 13.31 E 59.91 N, 17.61 E 63.81 N, 20.21 E
17 21 18
Finland
Jyva ¨skyla
J
62.41 N, 25.61 E
30
Greenland
Disko Narssarssuaq A Narssarssuaq B Narssarssuaq C
D NA NB NC
69.21 61.21 61.21 61.21
30 21 30 30
Denmark
Silkeborg, DMU Silkeborg, Nordskoven Silkeborg, Aarhusbakken
SD SN SAA
56.11 N, 9.81 E 56.11 N, 9.81 E 56.11 N, 9.81 E
Total
enzymes were tested and only those with reliable zymograms were used. These were the following 7 enzymes: aspartate aminotransaminase (AAT, E.C. 2.6.1.1), cytosol aminopeptidase (AP, E.C. 3.4.11.1), esterase (EST, E.C. 3.1.1.X), glucose phosphate isomerase (GPI, E.C. 5.3.1.9), isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), malate dehydrogenase (MDH, E.C. 1.1.1.37) and phosphoglucomutase (PGM, E.C. 5.4.2.2). A morpholine-citrate buffer, pH 6.1, was used for AAT, GPI and IDH, a tris-citrate buffer, pH 8.0, for AP and EST, a lithium hydroxide-borate acid buffer, pH 8.1, for MDH, and a histidine-citrate buffer, pH 5.7, for PGM. Starch gel electrophoresis and enzyme staining were similar to the procedures described by Richardson et al. (1986) and Hillis et al. (1996).
Analysis of data By the fact that the apomictic parthenogenetic reproduction takes place in D. octaedra, the individuals are treated as haploids regardless of the chromosomal number and no attempt to interpret the zymograms as genotypes was carried out. A specific band configuration was considered as a specific phenotype. However, when it was obvious that the phenotype consisted of two parts or zones by the intensity of the stain and probably determined by two different loci, two phenotypes were considered for that enzyme. The different zones
N, N, N, N,
53.31 45.41 45.41 45.41
W W W W
Sample size
29 29 30 345
are probably reflecting different loci for the enzyme detected. The type 0 was given if there was no band in a zone, despite clear and distinct bands in other zones. The observed phenotypes was numbered arbitrarily. The data from the typing of zymograms was analysed by Popgene 1.32 (Yeh et al. 2001, http:// www.ualberta.ca/fyeh/) for estimating frequencies of the phenotypes and for constructing a dendrogram based on Nei’s (1978) distance and depicted by applying the programme Tree View (Page 1996). Homogeneity between the samples was tested with the G-test (Fowler et al. 1998). The mean haploid diversity was estimated by the software GeneAlex (Peakall and Smouse 2001, http://www.anu.edu.au/BoZo/GenAlEx) as ð1=nÞSð1 Sp2i Þ where n is the number of zones and pi the frequency of the phenotypes in each zone. The clonal diversity was estimated as D ¼ 1 Sp2i , where pi is the frequency of the ith phenotype, the composite phenotype incorporating all phenotypes of all zones (Parker 1979). The diversity was used for testing for correlation to latitude or to the coldest average temperature during the winter using Spearman Rank correlation rS (Fowler et al. 1998). Temperatures were obtained from WorldClimate except for Silkeborg and Disko where data were derived from the Danish Meteorological Institute (DMI). Temperatures were derived as monthly mean air temperatures at weather stations adjacent to sampling localities.
Pheno-type
1 2 3
1 2
1 2 3
1 2 3 4 5 6 7
1 2 3 5
1 2 3
1 2
0 1 2 3 4 5
1 2 3
AAT-1
AAT-2
AP
EST
GPI
IDH
MDH
PGM-1
PGM-2
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.62 0.38
0.29 0.71
0.90
0.67
1.00
0.10
0.76 0.24
0.29 0.71
1.00
0.33
0.43 0.57
1.00
1.00
1.00
1.00
1.00
0.70 0.30
0.17 0.83
0.93
0.07
0.57 0.43
0.37 0.63
1.00
1.00
1.00
0.97 0.03
0.30 0.70
0.07 0.93
0.97
0.03
0.60 0.40
0.40 0.60
1.00
NC
1.00
0.33 0.67
1.00
1.00
1.00
0.03
0.47 0.20 0.30
0.37 0.63
0.57 0.43
0.53 0.47
Let
NB
D
NA
Canada
Greenland
0.21 0.68 0.11
0.68
0.32
1.00
1.00
1.00
0.11 0.89
0.16 0.79 0.05
0.26 0.74
1.00
Ed
0.82 0.18
0.82 0.18
1.00
1.00
1.00
0.91
0.09
0.09 0.82 0.09
1.00
0.91 0.09
Du
0.73 0.27
0.43 0.50 0.03 0.03
1.00
0.90 0.10
1.00
0.50 0.03
0.47
0.23 0.67 0.10
0.87 0.13
0.37 0.57 0.07
J
Finland
1.00
0.12 0.12 0.18 0.24 0.35
1.00
0.71 0.29
0.76 0.06 0.18
0.53
0.41 0.06
0.12 0.71 0.18
0.82 0.18
0.76 0.24
L
Sweden
Frequencies of the phenotypes for nine zones with enzyme activity for 14 samples of Dendrobaena octaedra.
Enzyme zone
Table 2.
0.71 0.24 0.05
0.10 0.57 0.10 0.14 0.10
1.00
1.00
0.62 0.24 0.14
0.10 0.29 0.05
0.57
0.29 0.57 0.14
0.71 0.29
0.90 0.10
Up
0.94 0.06
0.33 0.67
1.00
0.89 0.11
0.94 0.06
0.06
0.22 0.33
0.39
0.44 0.44 0.11
0.89 0.11
1.00
Um
0.97 0.03
0.03 0.41 0.17 0.07 0.31
0.07 0.93
0.48 0.52
0.38 0.48 0.03 0.10
0.31 0.24
0.07 0.38
0.21 0.66 0.14
0.76 0.24
0.28 0.72
SD
Denmark
0.97 0.03
0.14 0.21 0.03 0.62
0.17 0.83
0.55 0.45
0.31
0.59 0.10
0.03
0.07 0.14
0.38 0.38
0.21 0.62 0.17
0.69 0.31
0.48 0.52
SN
0.80 0.17 0.03
0.13 0.17
0.47 0.23
0.03 0.97
0.67 0.13 0.20
0.57 0.20 0.20 0.03
0.27
0.20 0.53
0.10 0.67 0.23
0.73 0.27
0.27 0.73
SAA
ARTICLE IN PRESS
Variation among earthworms from Northern Hemisphere 229
ARTICLE IN PRESS 230
P.L. Hansen et al.
Results The seven enzymes studied produced distinct and reliable zymograms, which were used for the analysis. The zymograms for PGM and AAT were divided into 2 zones designated PGM-1 and PGM-2 and AAT-1 and AAT-2, respectively, whereas only one zone was seen in the zymograms for the other enzymes. The phenotype 0 was observed only in few individuals for the isozyme PGM-1. The distribution of the phenotypes are listed in Table 2. A dendrogram estimated under the assumption that D. octaedra is an apomictic parthogenetic organism is depicted in Fig. 2. An obvious grouping into two groups is found, where the four samples from Greenland represent one group and the remaining samples make up the other group. When testing for homogeneity among samples from the same area (Narssarssuaq and Silkeborg), the three samples from Narssarssuaq did reveal homogeneity when applying Bonferroni corrections
D, G
NC, G
NA, G
NB, G
Ed, Can
Du, Can
SD, DK
(G ¼ 19:16, d.f. ¼ 10, P40:05) whereas the three adjacent samples from Silkeborg revealed heterogeneity among the samples (G ¼ 62:45, d.f. ¼ 28, Po0:05). Further analyses for homogeneity within the countries when possible also revealed significant deviations from homogeneity. No correlation was seen between phenotypic variation measured as mean haploid diversity or clonal diversity and latitude (r S ¼ 0:32, n ¼ 14, P40:05, r S ¼ 0:13, n ¼ 14, P40:05). A significant positive correlation was seen between mean haploid diversity or clonal diversity and mean temperature of the annual coldest month (r S ¼ 0:85, n ¼ 14, Po0:001, r S ¼ 0:80, n ¼ 14, Po0:002, respectively) indicating that phenotypic variation was increasing with increasing mean temperature of the coldest month. Table 3 shows the mean temperatures during the coldest months, November–May, for all locations. The specimens from Disko and Jyva ¨skyla were F1-generations, whereas the other populations were parental generations. To see if that would have an influence in the Spearman Rank test, the samples representing a F1-generation were removed, but there was still a significant correlation between the mean coldest temperature and the diversity (r S ¼ 0:87, n ¼ 12, Po0:001, r S ¼ 0:86, n ¼ 12, P ¼ 0:001, respectively). When considering the clonal variation and the composite phenotypic variation, a total of 176 clones were found. Of these, 125 were unique clones, i.e. only present in one individual, and 26 clones were shared by more individuals within the sample, but only found in that sample (Table 4). Twenty-five clones were present in more than one sample and of these 13 were found in more than one country. This low level of overlapping clones prevented further analysis of the clonal distribution, e.g. Renkonen index.
SN, DK
Let, Can
Discussion
Um, S
Genetic relationship
J, SF
Two major clusters were observed in the dendrogram shown in Fig. 2. One cluster consisted of the Greenlandic samples and the other major cluster of all the other samples. The first cluster showed that earthworms from Disko were more distantly related to the Narssarssuaq samples than the samples from Narssarssuaq were to each other. This result could be due to the geographic distance between Disko and Narssarsuaq, about 800 km, but on the other hand some exchange between the two localities might happen. No clear pattern was seen in this second cluster, only that the two samples
SAA, DK
L, S
Up, S
Figure 2. Dendrogram on populations of Dendrobaena octaedra. The abbreviations for the sampling localities are listed in Table 1, followed by an abbreviation of the country (Can ¼ Canada, DK ¼ Denmark, G ¼ Greenland, S ¼ Sweden, SF ¼ Finland).
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere Table 3. Month
Nov Dec Jan Feb Mar Apr May
231
Average temperature for seven months over several years for the locations Location Disko
Narssarssuaq
Lethbridge
Edmonton
Dunvegan
Jyva ¨skyla
Lund
Uppsala
Umea(
Silkeborg
7.8 9.9 14.8 19.6 19.9 8.2 0.5
3.0 6.0 8.6 7.9 7.7 2.5 4.6
0.7 5.5 8.4 5.8 1.4 5.5 11.1
4.0 10.1 13.7 10.7 4.5 4.5 10.9
6.8 16.0 20.3 15.2 6.7 3.3 10.3
2.0 6.8 9.5 9.4 4.9 1.2 8.4
4.2 1.0 0.6 0.8 1.9 5.8 11.4
0.5 2.6 4.2 4.5 1.9 3.3 9.1
2.0 7.9 10.1 7.7 3.8 1.4 7.8
4.4 1.8 0.2 0.1 2.3 5.8 10.8
Disko: Data obtained from 1961 to 1990. Narssarssuaq: Data obtained in 110 months between 1981 and 1990. Lethbridge: Data obtained in 650 months between 1936 and 1990. Edmonton: Data obtained in 1294 months between 1883 and 1990. Dunvegan: Data obtained in 427 months between 1880 and 1943. Jyva ¨skyla: Data obtained in 477 months between 1951 and 1990. Lund: Data obtained in 118 months between 1981 and 1990. Nearest weather station in Malmo ¨. Uppsala: Data obtained in 1708 months between 1739 and 1970. Umea( : Data obtained in 116 months between 1981 and 1990. Silkeborg: Data obtained from 1961 to 1990. Nearest weather station in Tirstrup. Data from WorldClimate Web adr.: http://www.worldclimate.com/. Except for Disko and Silkeborg, where data were obtained from DMI Web adr.: http://www.dmi.dk/. The annual coldest months are seen in bold in the table.
Table 4. Number of clones, number of unique clones and frequency of unique clones within the locality Locality
No. of clones
No. of unique Frequency of clones unique clones
D, G NA, G NB, G NC, G Let, Can Ed, Can Du, Can J, SF L, S Up, S Um, S SD, DK SN, DK SAA, DK
4 8 11 11 11 9 6 21 17 19 13 28 25 26
0 0 2 3 3 4 5 10 14 15 7 23 19 20
0.00 0.00 0.18 0.27 0.27 0.44 0.83 0.48 0.82 0.79 0.54 0.82 0.76 0.77
The abbreviations for the sampling localities are listed in Table 1, followed by an abbreviation of the country (Can ¼ Canada, DK ¼ Denmark, G ¼ Greenland, S ¼ Sweden, SF ¼ Finland).
from Edmonton and Dunvegan were more distant related to each others and to the other samples in the group. Notably the Greenland populations were distantly related to all other populations, emphasising the uniqueness of these populations compared to all the other populations studied. The only way
genetic differentiation can occur in an apomictic parthenogenetic reproducing species, where the offspring are identical to the parent, is through mutation and selection. The rate of mutation must be assumed to be the same in all populations, whereas the direction of selection may vary. The question arises if selection and mutation can account for the great differentiation seen in this study among Greenland worms and the other populations, or if the worms colonised Greenland from either Canada or Europe during the relatively short evolutionary period since the last glacial period by human agency? If so, this could only have happened during the last millennium when the first Norwegian settlers arrived in Greenland. Fossil remains of earthworms are not available, so nothing is known with certainty about the evolution of this animal group (Sto ¨p-Bowitz 1969). However, attempts have been made to understand their evolution through the study of comparative anatomy. Omodeo (1957) concluded that D. octaedra is endemic to Greenland, based on morphological and cytological differences between Greenland and other European populations. He suggested that the oligochaete fauna of Greenland, including D. octaedra, once immigrated through a North Atlantic bridge from Europe to America, or vice versa, and survived in situ after the disappearance of this landbridge, through at least the last glacial period. Fossils of terrestrial flora and fauna which have been collected on either side of the
ARTICLE IN PRESS 232 Greenland–Scotland ridge suggest that a land bridge once connected Greenland–North America with northwest Europe (Thiede 1983). Likewise, Perel (1979) cited in Terhivuo (1988) suggested that the presence of D. octaedra on isolated islands such as Novaya Zemlya and Ostrov Kolguyev in the Barents Sea north of Russia may date back to some preglacial era, when these islands were part of the continent. Omodeo (1963) suggested that the adaptive potential of terrestrial Oligochaetes, makes it hard to imagine a climatic modification (such as a glacial period) capable of totally destroying the populations of a genus or a family of earthworms living on an entire continent. Julin (1949) and Sto ¨p-Bowitz (1969) have both discussed the possibility of some hardy Lumbricidae species passing the last glacial period in refugia free of ice in association with some arctic plants. D. octaedra is regarded as one of the most stress tolerant earthworm species. For instance, it can survive in remote and harsh places such as western Siberia, where it has spread farther north than other earthworm species (Berman et al. 2001), and it has been found to be extremely tolerant to freezing and drought, factors of paramount importance for the distribution of species (Hoffman and Parson 1991). Specimens of D. octaedra overwinter in frozen soils as either adults or cocoons. Cocoons are often laid near the surface and adults do not exhibit any vertical migration to avoid seasonal changes in the temperature of the environment, typical for many of the deep burrowing earthworm species (Nordstro ¨m and Rundgren 1974). This combined with the large genetic dissimilarity between populations in Greenland and all other populations examined in this study suggests that D. octaedra has survived the last glacial period at least, in ice-free refuge in Greenland. The results of the present study thus support to the nunatak hypothesis, but by the fact that no population from the western coastal area of Canada is collected, it cannot be omitted that the worms have immigrated from that area.
Correlation with climate No correlation was found between latitude and the diversity either measured as the mean haploid diversity or clonal diversity. However, a positive correlation was seen between phenotypic variation and the mean temperature of the coldest month. It is important to emphasise that specimens from Disko and Jyva ¨skyla used in the study were F1-generations and that a greater diversity might have been observed, if specimens from the par-
P.L. Hansen et al. ental populations had been analysed. If these localities were omitted from the data set, the correlation remained highly significant emphasising this significant result. The temperature of the coldest month thus has a high impact on phenotypic variation maybe causing selection, expressed as lower phenotypic variation in colder climates. As the Greenland locations were representing cold climate the phenotypic variation was comparatively low. However, the distribution of Greenlandic phenotypes deviated from populations from all other localities studied as shown in Fig. 2. During glacial periods, the temperature is likely to have exerted a high selective pressure. This combined with habitat loss during the glacial period and the isolation of Greenland, hindering re-colonisation overseas, could explain the low phenotypic variation seen in Greenland populations compared to other populations. On the other hand, the sample from Dunvegan revealed more unique clones than the Greenlandic samples despite the fact that the average air temperature in winter is like the temperature in Greenland. Data for soil temperatures were not available, but it is likely that populations from Canada are living in a much more buffered habitat (coniferous forest litter layer) compared to populations from Greenland that are found under moss covering very exposed rocks. In effect, the winter temperatures experienced by D. octaedra in Greenland are considerably harsher than in Canada, which can explain why the lowest number of unique clones were found in the Greenlandic samples.
Clonal variation The result that 176 (51%) of the 345 individuals assayed represented a novel overall phenotype and that 125 (36.2%) were unique clones is consistent with results of Terhivuo et al. (1987) in Finland, where 147 out of 428 (34.3%) individuals of D. octaedra assayed represented a novel overall phenotype and 80 (18.6%) were unique clones. A high genetic variation is thus seen in D. octaedra in both studies. The extensive genetic variation seen in the populations of D. octaedra is surprising, when considering that it reproduces by apomictic (mitotic) parthenogenesis. The genotype is thus transmitted intact to the offspring from the mother without any recombination. One explanation for the genetic variation could be that sexual processes have been active in the not too distant past. Amphimictic reproduction secures a high level of genetic variation, because of recombination of
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere alleles or genes, and when reproduction shifts from amphimixis to apomictic parthenogenesis, the parental genome is preserved and its level of variation is only altered by mutation (White 1973). Neither evidence for automictic reproduction, in meiosis takes place, has been observed (Simonsen, pers. comm.). Different levels of suppression of male sexual organs and even male-fertile individuals of D. octaedra could indicate that recent sexual processes have been active in the past. Male organs are useless once parthenogenesis has arisen and selection for reduced investment in male organs is to be expected to save resources. Terhivuo et al. (1987) found various degrees of male suppression among clones of D. octaedra in eastern Fennoscandia, where rare male fertile individuals were occasionally found, too. Likewise, male fertile D. octaedra have been found in Iceland (Omodeo 1957). It is unknown, however, if the male fertile specimens found were actually functional males. Omodeo (1957) suggested the Icelandic populations of D. octaedra to be gynogenetic, making mechanical stimulation of the egg by a sperm-cell important for the development of the egg, and therefore biologically different from the parthenogenetic apomictic populations from Greenland. Conclusions on dispersal patterns cannot be drawn from this study, because only few mutual clones were seen among different localities. Two overlapping clones were observed in the samples from Disko and Narssarssuaq, separated by 800 km, Disko is an island separated from Greenland by the Disko Bay, so the shared clones could be a result of anthropochorous or other passive transportation in recent times internally in Greenland. A single individual may be sufficient for establishing a population, because of the parthenogenetic reproduction of D. octaedra. The two other clones found in Disko could thus have arisen by mutation. The mutual clones seen in the three samples from Narssarssuaq were probably due to the short distance of 1 km, which might facilitate active dispersal between sampling sites. Only two clones were found in all three samples from Silkeborg, though, separated by only 5 km. However, roads and the river Gudena( were separating the three sites from each other. When comparing the frequency of unique clones (Table 4), it was evident that the Silkeborg area had a much higher frequency and that the number of clones observed was more than twice compared to Narssarssuaq. This observation might indicate that clonal dispersal might be strongly dependent of the barriers in the landscape or strong directional selection among the samples from Narssarssuaq.
233
Acknowledgements We thank Jill Clapperton for sampling earthworms in Canada. This work was made possible through the EU Integrated Project ALARM (EU 6th Framework Programme No. GOCE-CT-2003506675).
References Avise, J.C., 1994. Molecular Markers, Natural History and Evolution. Chapman & Hall, New York. Berman, D.I., Meshcheryakova, A.N., Alfimov, A.V., Leirikh, A.N., 2001. Spread of the earthworm Dendrobaena octaedra (Lumbricidae: Oligochaeta) from Europe to Northern Asia is restricted by its insufficient freeze tolerance. Dokl. Biol. Sci. 377, 145–148. Bindesbøl, A.-M., Holmstrup, M., Damgaard, C., Bayley, M., 2005. Stress synergy between environmentally realistic levels of copper and frost in the earthworm Dendrobaena octaedra. Environ. Toxicol. Chem. 24, 1462–1467. Brochmann, C., Gabrielsen, T.M., Nordal, I., Landvik, J.Y., Elven, R., 2003. Glacial survival or tabula rasa? The history of North Atlantic biota revisited. Taxon 52, 417–450. Casellato, S., 1987. On polyploidy in Oligochaetes with particular reference to Lumbricids. In: Bonvicini, A.M., Omodeo, P. (Eds.), On Earthworms. Selected Symposia and Monographs U.Z.I., 2, Mucchi, Modena, pp. 75–87. Casellato, S., Rodighiero, R., 1972. Karyology of lumbricidae. III contribution. Caryologia 25, 513–524. Dahl, E., 1989. The nunatak theory reconsidered. Ecol. Bull. 38, 77–94. Dahl-Jensen, D., Mosegaard, K., Gundestrup, N., Clow, G.D., Johnsen, S.J., Hansen, A.W., Balling, N., 1998. Past temperatures directly from the Greenland Ice Sheet. Science 282, 268–271. Edwards, C., Bohlen, P., 1996. Biology and Ecology of Earthworms. Chapman & Hall, London. Fowler, J., Cohen, L., Jarvis, P., 1998. Practical Statistics for Field Biology. Wiley, Chichester. Funder, S., Hansen, L., 1996. The Greenland ice sheet— a model for its culmination and decay during and after the last glacial maximum. Bull. Geol. Soc. Denmark 42, 137–152. Funder, S., Hjort, C., Landvik, J.Y., Nam, S.I., Reeh, N., Stein, R., 1998. History of a stable ice margin in Greenland during the middle and upper Pleistocene. Quatern. Sci. Rev. 17, 77–123. Heethoff, M., Etzold, K., Scheu, S., 2004. Mitochondrial COII sequences indicate that the parthenogenetic earthworm Octolasion tyrtaeum (Savigny 1826) constitutes of two lineages differing in body size and genotype. Pedobiologia 48, 9–13. Hillis, D.M., Moritz, C., Mable, B.K., 1996. Molecular Systematics. Sinauer Associates, Sunderland.
ARTICLE IN PRESS 234 Hoffman, A., Parson, P., 1991. Evolutionary Genetics and Environmental Stress. Oxford University Press, Oxford. Holmstrup, M., 1994. Physiology of cold hardiness in cocoons of five earthworm taxa Lumbricidae: Oligochaeta. J. Comp. Physiol. B 154, 222–228. Holmstrup, M., Loeschcke, V., 2003. Genetic variation in desiccation tolerance of Dendrobaena octaedra cocoons originating from different climatic regions. Soil Biol. Biochem. 35, 119–124. Holmstrup, M., Westh, P., 1995. Effects of dehydration on water relations and survival of lumbricid earthworm egg capsules. J. Comp. Physiol. B 165, 377–383. Holmstrup, M., Zachariassen, K.E., 1996. Physiology of cold hardiness in earthworms. Comp. Biochem. Physiol. 115A, 91–101. Holmstrup, M., Østergaard, I.K., Nielsen, A., Hansen, B.T., 1991. The relationship between temperature and cocoon incubation time for some Lumbricid earthworm species. Pedobiologia 35, 179–184. Jaenike, J., Selander, R.K., 1985. On the coexistence of ecologically similar clones of parthenogenetic earthworms. Oikos 44, 512–514. Jaenike, J., Parker Jr., E.D., Selander, R.K., 1980. Clonal niche structure in the parthenogenetic earthworm Octolasion tyrtaeum. Am. Nat. 116, 196–205. Julin, E., 1949. De svenska daggmaskarterna. Ark. Zool. 42A, 1–58. Landvik, J.Y., 1994. The last glaciation of Germania Land and adjacent areas, northeast Greenland. J. Quart. Sci. 9, 81–92. Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89, 583–590. Nevo, E., Beiles, A., Ben-Shlomo, R., 1984. The evolutionary significance of genetic diversity: ecological, demographic and life-history correlates. In: Mani, G.S. (Ed.), Evolutionary Dynamics of Genetic Diversity. Springer, Berlin. Nordal, I., 1987. Tabula rasa after all? Botanical evidence for ice-free refugia in Scandinavia reviewed. J. Biogeogr. 14, 377–388. Nordstro ¨m, S., Rundgren, S., 1974. Environmental factors and lumbricid associations in southern Sweden. Pedobiologia 14, 1–27. Omodeo, P., 1955. Cariologia dei Lumbricidae, II Contributo. Caryologia 8, 135–178. Omodeo, P., 1957. Lumbricidae and Lumbriculidae of Greenland. Medd. Grønland 124, 1–27. Omodeo, P., 1962. Oligochaetes des Alpes. Mem. Mus. Civ. St. Nat. Verona 10, 71–96. Omodeo, P., 1963. Distribution of the terricolous oligochaetes on the two shores of the Atlantic. North
P.L. Hansen et al. Atlantic Biota. In: Lo ¨ve, Æ., Lo ¨ve, D. (Eds.), North Atlantic Biota and their History. Pergamon Press, Oxford, pp. 127–151. Page, R.D.M., 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358. Parker Jr., E.D., 1979. Ecological implications of clonal diversity in parthenogenetic morphospecies. Am. Zool. 19, 753–762. Peakall, R., Smouse, P.E., 2001. GenAlEx V5: Genetic Analysis in Excel. Population Genetic Software for Teaching and Research. Australian National University, Canberra. Perel, T.S., 1979. Ranges and Regularities in the Distribution of Earthworms of the USSR Fauna. Nauka, Moscow (in Russian with English summary). Rasmussen, L.M., Holmstrup, M., 2002. Geographic variation of freeze-tolerance in the earthworm Dendrobaena octaedra. J. Comp. Physiol. B 172, 691–698. Richardson, B.J., Baverstock, P.R., Adams, M., 1986. Allozyme Electrophoresis—A Handbook for Animal Systematics and Population Structure. Academic Press, Sydney. Selander, R.K., Whittam, T.S., 1983. Protein polymorphism and the genetic structure of populations. In: Nei, M., Koehn, R.K. (Eds.), Evolution of Genes and Proteins. Sinauer, Sunderland, pp. 89–114. Slatkin, M., 1987. Gene flow and the geographic structure of natural populations. Science 236, 787–792. Sto ¨p-Bowitz, C., 1969. A contribution to our knowledge of the systematics and zoogeography of Norwegian earthworms Annelida Oligochaeta: Lumbricidae. Nytt. Mag. Zool. 17, 169–280. Terhivuo, J., 1988. The Finnish Lumbricidae Oligochaete – fauna and its formation. Ann. Zool. Fennici 25, 229–247. Terhivuo, J., Saura, A., Hongell, K., Lokki, J., 1987. Enzymatic and morphometric variation of Dendrobaena octaedra Sav. Oligochaeta: Lumbricidae – in eastern Fennoscandia – a preliminary report. In: Bonvicini, A.M., Omodeo, P. (Eds.), On Earthworm. Selected Symposia and Monographs U.Z.I., 2, Mucchi, Modena, pp. 89–102. Thiede, J., 1983. Outstanding geological problems of the Greenland-Scotland ridge. In: Bott, et al. (Eds.), Structure and Development of the Greenland-Scotland Ridge. Plenum Press, London, pp. 313–317. White, M.J.D., 1973. Animal Cytology and Evolution. Cambridge University Press, Cambridge. Yeh, F., Boyle, T., Yeh, R., Ye, Z., 2001. Popgene 1.32.
www.elsevier.de/pedobi
Low genetic variation for Dendrobaena octaedra from Greenland compared to populations from Europe and North America: Refuge or selection? Pernille Liland Hansena, Martin Holmstrupa, Mark Bayleyb, Vibeke Simonsena, a
Department of Terrestrial Ecology, National Environmental Research Institute, Vejlsoevej 25, P.O. Box 314, DK-8600 Silkeborg, Denmark b Department of Zoophysiology, Institute of Biological Sciences Building 131, C.F. Møllers Alle´, University of Aarhus, DK-8000 Aarhus C, Denmark Received 29 August 2005; accepted 8 December 2005
KEYWORDS Dendrobaena octaedra; Isozymes; Genetic diversity; Northern Hemisphere
Summary The genetic relationship of 345 specimens of the parthenogenetic lumbricid Dendrobaena octaedra from Greenland, Canada and Europe were analysed by means of isozymes. The results showed that populations from Greenland were markedly different from Canadian and European populations, suggesting that dispersal between Greenland and the continents has been much more restricted in the past than dispersal between North America and Europe. This observation supports to the notion that Greenland populations have persisted for a long period and perhaps have survived the last glacial period in ice-free refugia. A highly significant positive correlation was seen between diversity measured either as mean haploid diversity or clonal diversity and mean temperature of the annual coldest month. These results indicate that temperature might cause selection in colder climates or that sexual processes in D. dendrobaena could have been active recently on an evolutionary time scale. & 2006 Elsevier GmbH. All rights reserved.
Introduction
Corresponding author. Tel.: +45 8920 1400;
fax: +45 8920 1413. E-mail address: [email protected] (V. Simonsen).
The nunatak hypothesis proposes that some icefree refuge existed north of the southern boundary of the Pleistocene glaciers, where plants and animals could survive during the last or previous glacial periods. These refuges could either be
0031-4056/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.pedobi.2005.12.001
ARTICLE IN PRESS 226 mountains rising above the inland ice (which in Inuit language are called nunataks) or coastal icefree refuges. When the ice melted, plants and animals colonised previously ice-covered land from the refuge and the unglaciated areas south and east of the inland ice. The alternative to the nunatak hypothesis is the tabula rasa theory stating that all higher land plants and animals were exterminated north of the southern boundary of the Pleistocene glaciers, and that the present biota immigrated from the south or east (Nordal 1987; Dahl 1989; Brochmann et al. 2003). There is now strong geological evidence suggesting that some nunataks and ice-free coastal shelves existed within the maximum limits of the last glacial period 25,000–10,000 years ago. During this period the ice cover in Greenland was larger than today. Along its western and south-eastern margins it terminated on the continental shelf (Funder and Hansen 1996). However, in eastern Greenland, there is strong evidence for ice-free lowlands on the Hochstetter Forland (Funder et al. 1998) and for extensive ice-free uplands penetrating the ice cover in Jameson Land (Funder et al. 1998) and Germania Land (Landvik 1994). In south-west Greenland large ice-free nunataks probably existed between 671N and 721N (Funder and Hansen 1996). However, these ice-free areas were most likely so climatically harsh that only very tolerant organism could have survived. Recent climatic reconstructions from ice core projects in central Greenland indicated that the mean annual decline in temperature during the last glacial period was as much as 25 1C compared to the present temperature (Dahl-Jensen et al. 1998). Isozymes have shown their versatility in providing markers for analysing genetic compositions of populations. By examining the distribution of genotypes or phenotypes the phenetic relationship between populations may be estimated (Avise 1994). Phenetic studies have reinforced the view that any attempt to interpret population differentiation in terms of evolutionary factors may lead to erroneous conclusions in the absence of historical data (Selander and Whittam 1983; Slatkin 1987). Electrophoresis of enzymes has been applied to the study of earthworm species since the early 1980s but such genetic studies have mainly focused on local populations, whereas only a few studies have covered a wider geographic area (Jaenike et al. 1980; Jaenike and Selander 1985). Recently genetic sequence analysis has been used for studying clonal diversity in parthenogenetic earthworms (Heethoff et al. 2004). Dendrobaena octaedra Savigny, 1826 (Oligochaeta: Lumbricidae) is widespread in the Northern Hemisphere, where it is often the dominant earth-
P.L. Hansen et al. worm species in coniferous forests and tundra soils. It is an epigeic species living in the litter layer of the forest floor, in rotting tree stumps and under moss or lichens (Sto ¨p-Bowitz 1969). Therefore, it is regularly exposed to climatic stresses such as drought and cold. D. octaedra reproduces by apomictic parthenogenesis (Omodeo 1957), i.e. the offspring are clones of the parents. The cocoons of D. octaedra have been found to be extremely tolerant to drought (Holmstrup and Westh 1995; Holmstrup and Loeschcke 2003) and freezing (Holmstrup 1994; Holmstrup and Zachariassen 1996). Hatched individuals can withstand extracellular freezing and are thus freeze-tolerant, as one of only two known earthworm species with this ability (Rasmussen and Holmstrup 2002; Bindesbøl et al. 2005). This documented hardiness of D. octaedra opens the possibility that it could have survived the last glacial period in ice-free refuge and therefore may have an ancient origin in Greenland. It is unlikely that D. octaedra has arrived by natural dispersal in Greenland since the last glacial period, because the sea is a natural barrier for dispersal of adult earthworms and cocoons. On the other hand, anthropochorous dispersal of earthworms has been of significant importance for some earthworm species (Edwards and Bohlen 1996), so D. octaedra could have arrived in Greenland by human activity in more recent times. Different numbers of polyploidy are seen in D. octaedra. In Greenland and Iceland 6n individuals are recorded and in southern and central parts of Europe 5n and 8n individuals (Omodeo 1955, 1962; Casellato and Rodighiero 1972; Vedovini, 1973 cited in Casellato 1987). Variation in polyploidy may cause differentiation among individuals, because more chromosomes may increase the chance for mutation and thereby differentiation. Allelic frequencies determined by electrophoresis of isozymes tend to be correlated directly or indirectly with geographical variations in temperature. Nevo et al. (1984) analysed correlations of abiotic and biotic factors with genetic diversity assessed electrophoretically, using published data on natural populations of 1111 species from a wide range of geographic locations. These authors concluded that 20% of the genetic variance that could be explained was mainly associated with ecological heterogeneity, and temperature emerged as one of the major variables. The aim of this study was to see if populations of D. octaedra in Greenland might have an ancient origin when comparing Greenland populations to populations from Canada and Europe by using isozymes. On the other hand, D. octaedra might
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere have been introduced by man from these areas to Greenland and in that case greater similarity between Greenlandic populations and populations from the source area was expected. Furthermore, correlation to winter temperatures was analysed to determine the importance of temperature in accounting for the variation in isozymes.
Materials and methods
227
the laboratory in plastic beakers with moist soil and vegetation from the localities. D. octaedra were collected from coniferous forest in Europe and Canada, and from moss covered rocks in Greenland. Specimens from Disko and Jyva ¨skyla were obtained from laboratory cultures, which had been held in culture for one generation following procedures described by Holmstrup et al. (1991). About 200 individuals were collected to establish these laboratory cultures. Specimens from the other populations were kept under similar conditions in the laboratory until used for analysis.
Samples Three hundred and forty-five specimens of D. octaedra were used for this study. The origin of the populations is shown in Fig. 1 and additional information on the sampling area and the number of individuals are listed in Table 1. The localities Disko and Narssarssuaq in Greenland were separated by about 800 km and the 3 localities in Canada and Sweden, respectively, by about 300 km. The 3 samples from Silkeborg were separated by 5 km and the 3 samples from Narssarssuaq by 1 km. Specimens from the different localities were collected in August 2003 (Canada), in September and October 2003 (Sweden), in September 2003 (Narssarssuaq) and in September and November 2003 (Silkeborg). All populations were brought to
Analysis of isozymes Earthworms were held in Petri dishes with moist filter-paper at 5 1C for 24 h to empty their guts and avoid enzymes from intestinal bacteria interfering with the analysis. The tissue used was the whole part from anterior to clitellum, which were removed from each worm and stored at 80 1C in a 0.5 ml microtube with 50 ml tris-citrate buffer pH 7.0 containing 1% polyvinylpyrrolidon until electrophoresis. No attempt to determine sex of the worms was done, as it was assumed that the worms have apomictic pathenogenesis. The samples were analysed by horizontal starch gel electrophoresis (e.g. Hillis et al. 1996). Several
Dunvegan Edmonton Letbridge
Disko Umeå
Jyväskyla
Uppsala Narssarssuaq Lund Silkeborg
Figure 1. Map on the sampling localities.
ARTICLE IN PRESS 228
P.L. Hansen et al.
Table 1. Origin of Dendrobaena octaedra populations, abbreviation for the samples, latitude and longitude for the sampling areas and number of individuals Country
Locality
Sample abbreviation
Latitude and longitude
Canada
Lethbridge Edmonton Dunvegan
Let Ed Du
49.61 N, 112.81 W 53.61 N, 113.51 W 55.91 N, 118.61 W
30 19 11
Sweden
Lund Uppsala Umea(
L Up Um
55.81 N, 13.31 E 59.91 N, 17.61 E 63.81 N, 20.21 E
17 21 18
Finland
Jyva ¨skyla
J
62.41 N, 25.61 E
30
Greenland
Disko Narssarssuaq A Narssarssuaq B Narssarssuaq C
D NA NB NC
69.21 61.21 61.21 61.21
30 21 30 30
Denmark
Silkeborg, DMU Silkeborg, Nordskoven Silkeborg, Aarhusbakken
SD SN SAA
56.11 N, 9.81 E 56.11 N, 9.81 E 56.11 N, 9.81 E
Total
enzymes were tested and only those with reliable zymograms were used. These were the following 7 enzymes: aspartate aminotransaminase (AAT, E.C. 2.6.1.1), cytosol aminopeptidase (AP, E.C. 3.4.11.1), esterase (EST, E.C. 3.1.1.X), glucose phosphate isomerase (GPI, E.C. 5.3.1.9), isocitrate dehydrogenase (IDH, E.C. 1.1.1.42), malate dehydrogenase (MDH, E.C. 1.1.1.37) and phosphoglucomutase (PGM, E.C. 5.4.2.2). A morpholine-citrate buffer, pH 6.1, was used for AAT, GPI and IDH, a tris-citrate buffer, pH 8.0, for AP and EST, a lithium hydroxide-borate acid buffer, pH 8.1, for MDH, and a histidine-citrate buffer, pH 5.7, for PGM. Starch gel electrophoresis and enzyme staining were similar to the procedures described by Richardson et al. (1986) and Hillis et al. (1996).
Analysis of data By the fact that the apomictic parthenogenetic reproduction takes place in D. octaedra, the individuals are treated as haploids regardless of the chromosomal number and no attempt to interpret the zymograms as genotypes was carried out. A specific band configuration was considered as a specific phenotype. However, when it was obvious that the phenotype consisted of two parts or zones by the intensity of the stain and probably determined by two different loci, two phenotypes were considered for that enzyme. The different zones
N, N, N, N,
53.31 45.41 45.41 45.41
W W W W
Sample size
29 29 30 345
are probably reflecting different loci for the enzyme detected. The type 0 was given if there was no band in a zone, despite clear and distinct bands in other zones. The observed phenotypes was numbered arbitrarily. The data from the typing of zymograms was analysed by Popgene 1.32 (Yeh et al. 2001, http:// www.ualberta.ca/fyeh/) for estimating frequencies of the phenotypes and for constructing a dendrogram based on Nei’s (1978) distance and depicted by applying the programme Tree View (Page 1996). Homogeneity between the samples was tested with the G-test (Fowler et al. 1998). The mean haploid diversity was estimated by the software GeneAlex (Peakall and Smouse 2001, http://www.anu.edu.au/BoZo/GenAlEx) as ð1=nÞSð1 Sp2i Þ where n is the number of zones and pi the frequency of the phenotypes in each zone. The clonal diversity was estimated as D ¼ 1 Sp2i , where pi is the frequency of the ith phenotype, the composite phenotype incorporating all phenotypes of all zones (Parker 1979). The diversity was used for testing for correlation to latitude or to the coldest average temperature during the winter using Spearman Rank correlation rS (Fowler et al. 1998). Temperatures were obtained from WorldClimate except for Silkeborg and Disko where data were derived from the Danish Meteorological Institute (DMI). Temperatures were derived as monthly mean air temperatures at weather stations adjacent to sampling localities.
Pheno-type
1 2 3
1 2
1 2 3
1 2 3 4 5 6 7
1 2 3 5
1 2 3
1 2
0 1 2 3 4 5
1 2 3
AAT-1
AAT-2
AP
EST
GPI
IDH
MDH
PGM-1
PGM-2
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.62 0.38
0.29 0.71
0.90
0.67
1.00
0.10
0.76 0.24
0.29 0.71
1.00
0.33
0.43 0.57
1.00
1.00
1.00
1.00
1.00
0.70 0.30
0.17 0.83
0.93
0.07
0.57 0.43
0.37 0.63
1.00
1.00
1.00
0.97 0.03
0.30 0.70
0.07 0.93
0.97
0.03
0.60 0.40
0.40 0.60
1.00
NC
1.00
0.33 0.67
1.00
1.00
1.00
0.03
0.47 0.20 0.30
0.37 0.63
0.57 0.43
0.53 0.47
Let
NB
D
NA
Canada
Greenland
0.21 0.68 0.11
0.68
0.32
1.00
1.00
1.00
0.11 0.89
0.16 0.79 0.05
0.26 0.74
1.00
Ed
0.82 0.18
0.82 0.18
1.00
1.00
1.00
0.91
0.09
0.09 0.82 0.09
1.00
0.91 0.09
Du
0.73 0.27
0.43 0.50 0.03 0.03
1.00
0.90 0.10
1.00
0.50 0.03
0.47
0.23 0.67 0.10
0.87 0.13
0.37 0.57 0.07
J
Finland
1.00
0.12 0.12 0.18 0.24 0.35
1.00
0.71 0.29
0.76 0.06 0.18
0.53
0.41 0.06
0.12 0.71 0.18
0.82 0.18
0.76 0.24
L
Sweden
Frequencies of the phenotypes for nine zones with enzyme activity for 14 samples of Dendrobaena octaedra.
Enzyme zone
Table 2.
0.71 0.24 0.05
0.10 0.57 0.10 0.14 0.10
1.00
1.00
0.62 0.24 0.14
0.10 0.29 0.05
0.57
0.29 0.57 0.14
0.71 0.29
0.90 0.10
Up
0.94 0.06
0.33 0.67
1.00
0.89 0.11
0.94 0.06
0.06
0.22 0.33
0.39
0.44 0.44 0.11
0.89 0.11
1.00
Um
0.97 0.03
0.03 0.41 0.17 0.07 0.31
0.07 0.93
0.48 0.52
0.38 0.48 0.03 0.10
0.31 0.24
0.07 0.38
0.21 0.66 0.14
0.76 0.24
0.28 0.72
SD
Denmark
0.97 0.03
0.14 0.21 0.03 0.62
0.17 0.83
0.55 0.45
0.31
0.59 0.10
0.03
0.07 0.14
0.38 0.38
0.21 0.62 0.17
0.69 0.31
0.48 0.52
SN
0.80 0.17 0.03
0.13 0.17
0.47 0.23
0.03 0.97
0.67 0.13 0.20
0.57 0.20 0.20 0.03
0.27
0.20 0.53
0.10 0.67 0.23
0.73 0.27
0.27 0.73
SAA
ARTICLE IN PRESS
Variation among earthworms from Northern Hemisphere 229
ARTICLE IN PRESS 230
P.L. Hansen et al.
Results The seven enzymes studied produced distinct and reliable zymograms, which were used for the analysis. The zymograms for PGM and AAT were divided into 2 zones designated PGM-1 and PGM-2 and AAT-1 and AAT-2, respectively, whereas only one zone was seen in the zymograms for the other enzymes. The phenotype 0 was observed only in few individuals for the isozyme PGM-1. The distribution of the phenotypes are listed in Table 2. A dendrogram estimated under the assumption that D. octaedra is an apomictic parthogenetic organism is depicted in Fig. 2. An obvious grouping into two groups is found, where the four samples from Greenland represent one group and the remaining samples make up the other group. When testing for homogeneity among samples from the same area (Narssarssuaq and Silkeborg), the three samples from Narssarssuaq did reveal homogeneity when applying Bonferroni corrections
D, G
NC, G
NA, G
NB, G
Ed, Can
Du, Can
SD, DK
(G ¼ 19:16, d.f. ¼ 10, P40:05) whereas the three adjacent samples from Silkeborg revealed heterogeneity among the samples (G ¼ 62:45, d.f. ¼ 28, Po0:05). Further analyses for homogeneity within the countries when possible also revealed significant deviations from homogeneity. No correlation was seen between phenotypic variation measured as mean haploid diversity or clonal diversity and latitude (r S ¼ 0:32, n ¼ 14, P40:05, r S ¼ 0:13, n ¼ 14, P40:05). A significant positive correlation was seen between mean haploid diversity or clonal diversity and mean temperature of the annual coldest month (r S ¼ 0:85, n ¼ 14, Po0:001, r S ¼ 0:80, n ¼ 14, Po0:002, respectively) indicating that phenotypic variation was increasing with increasing mean temperature of the coldest month. Table 3 shows the mean temperatures during the coldest months, November–May, for all locations. The specimens from Disko and Jyva ¨skyla were F1-generations, whereas the other populations were parental generations. To see if that would have an influence in the Spearman Rank test, the samples representing a F1-generation were removed, but there was still a significant correlation between the mean coldest temperature and the diversity (r S ¼ 0:87, n ¼ 12, Po0:001, r S ¼ 0:86, n ¼ 12, P ¼ 0:001, respectively). When considering the clonal variation and the composite phenotypic variation, a total of 176 clones were found. Of these, 125 were unique clones, i.e. only present in one individual, and 26 clones were shared by more individuals within the sample, but only found in that sample (Table 4). Twenty-five clones were present in more than one sample and of these 13 were found in more than one country. This low level of overlapping clones prevented further analysis of the clonal distribution, e.g. Renkonen index.
SN, DK
Let, Can
Discussion
Um, S
Genetic relationship
J, SF
Two major clusters were observed in the dendrogram shown in Fig. 2. One cluster consisted of the Greenlandic samples and the other major cluster of all the other samples. The first cluster showed that earthworms from Disko were more distantly related to the Narssarssuaq samples than the samples from Narssarssuaq were to each other. This result could be due to the geographic distance between Disko and Narssarsuaq, about 800 km, but on the other hand some exchange between the two localities might happen. No clear pattern was seen in this second cluster, only that the two samples
SAA, DK
L, S
Up, S
Figure 2. Dendrogram on populations of Dendrobaena octaedra. The abbreviations for the sampling localities are listed in Table 1, followed by an abbreviation of the country (Can ¼ Canada, DK ¼ Denmark, G ¼ Greenland, S ¼ Sweden, SF ¼ Finland).
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere Table 3. Month
Nov Dec Jan Feb Mar Apr May
231
Average temperature for seven months over several years for the locations Location Disko
Narssarssuaq
Lethbridge
Edmonton
Dunvegan
Jyva ¨skyla
Lund
Uppsala
Umea(
Silkeborg
7.8 9.9 14.8 19.6 19.9 8.2 0.5
3.0 6.0 8.6 7.9 7.7 2.5 4.6
0.7 5.5 8.4 5.8 1.4 5.5 11.1
4.0 10.1 13.7 10.7 4.5 4.5 10.9
6.8 16.0 20.3 15.2 6.7 3.3 10.3
2.0 6.8 9.5 9.4 4.9 1.2 8.4
4.2 1.0 0.6 0.8 1.9 5.8 11.4
0.5 2.6 4.2 4.5 1.9 3.3 9.1
2.0 7.9 10.1 7.7 3.8 1.4 7.8
4.4 1.8 0.2 0.1 2.3 5.8 10.8
Disko: Data obtained from 1961 to 1990. Narssarssuaq: Data obtained in 110 months between 1981 and 1990. Lethbridge: Data obtained in 650 months between 1936 and 1990. Edmonton: Data obtained in 1294 months between 1883 and 1990. Dunvegan: Data obtained in 427 months between 1880 and 1943. Jyva ¨skyla: Data obtained in 477 months between 1951 and 1990. Lund: Data obtained in 118 months between 1981 and 1990. Nearest weather station in Malmo ¨. Uppsala: Data obtained in 1708 months between 1739 and 1970. Umea( : Data obtained in 116 months between 1981 and 1990. Silkeborg: Data obtained from 1961 to 1990. Nearest weather station in Tirstrup. Data from WorldClimate Web adr.: http://www.worldclimate.com/. Except for Disko and Silkeborg, where data were obtained from DMI Web adr.: http://www.dmi.dk/. The annual coldest months are seen in bold in the table.
Table 4. Number of clones, number of unique clones and frequency of unique clones within the locality Locality
No. of clones
No. of unique Frequency of clones unique clones
D, G NA, G NB, G NC, G Let, Can Ed, Can Du, Can J, SF L, S Up, S Um, S SD, DK SN, DK SAA, DK
4 8 11 11 11 9 6 21 17 19 13 28 25 26
0 0 2 3 3 4 5 10 14 15 7 23 19 20
0.00 0.00 0.18 0.27 0.27 0.44 0.83 0.48 0.82 0.79 0.54 0.82 0.76 0.77
The abbreviations for the sampling localities are listed in Table 1, followed by an abbreviation of the country (Can ¼ Canada, DK ¼ Denmark, G ¼ Greenland, S ¼ Sweden, SF ¼ Finland).
from Edmonton and Dunvegan were more distant related to each others and to the other samples in the group. Notably the Greenland populations were distantly related to all other populations, emphasising the uniqueness of these populations compared to all the other populations studied. The only way
genetic differentiation can occur in an apomictic parthenogenetic reproducing species, where the offspring are identical to the parent, is through mutation and selection. The rate of mutation must be assumed to be the same in all populations, whereas the direction of selection may vary. The question arises if selection and mutation can account for the great differentiation seen in this study among Greenland worms and the other populations, or if the worms colonised Greenland from either Canada or Europe during the relatively short evolutionary period since the last glacial period by human agency? If so, this could only have happened during the last millennium when the first Norwegian settlers arrived in Greenland. Fossil remains of earthworms are not available, so nothing is known with certainty about the evolution of this animal group (Sto ¨p-Bowitz 1969). However, attempts have been made to understand their evolution through the study of comparative anatomy. Omodeo (1957) concluded that D. octaedra is endemic to Greenland, based on morphological and cytological differences between Greenland and other European populations. He suggested that the oligochaete fauna of Greenland, including D. octaedra, once immigrated through a North Atlantic bridge from Europe to America, or vice versa, and survived in situ after the disappearance of this landbridge, through at least the last glacial period. Fossils of terrestrial flora and fauna which have been collected on either side of the
ARTICLE IN PRESS 232 Greenland–Scotland ridge suggest that a land bridge once connected Greenland–North America with northwest Europe (Thiede 1983). Likewise, Perel (1979) cited in Terhivuo (1988) suggested that the presence of D. octaedra on isolated islands such as Novaya Zemlya and Ostrov Kolguyev in the Barents Sea north of Russia may date back to some preglacial era, when these islands were part of the continent. Omodeo (1963) suggested that the adaptive potential of terrestrial Oligochaetes, makes it hard to imagine a climatic modification (such as a glacial period) capable of totally destroying the populations of a genus or a family of earthworms living on an entire continent. Julin (1949) and Sto ¨p-Bowitz (1969) have both discussed the possibility of some hardy Lumbricidae species passing the last glacial period in refugia free of ice in association with some arctic plants. D. octaedra is regarded as one of the most stress tolerant earthworm species. For instance, it can survive in remote and harsh places such as western Siberia, where it has spread farther north than other earthworm species (Berman et al. 2001), and it has been found to be extremely tolerant to freezing and drought, factors of paramount importance for the distribution of species (Hoffman and Parson 1991). Specimens of D. octaedra overwinter in frozen soils as either adults or cocoons. Cocoons are often laid near the surface and adults do not exhibit any vertical migration to avoid seasonal changes in the temperature of the environment, typical for many of the deep burrowing earthworm species (Nordstro ¨m and Rundgren 1974). This combined with the large genetic dissimilarity between populations in Greenland and all other populations examined in this study suggests that D. octaedra has survived the last glacial period at least, in ice-free refuge in Greenland. The results of the present study thus support to the nunatak hypothesis, but by the fact that no population from the western coastal area of Canada is collected, it cannot be omitted that the worms have immigrated from that area.
Correlation with climate No correlation was found between latitude and the diversity either measured as the mean haploid diversity or clonal diversity. However, a positive correlation was seen between phenotypic variation and the mean temperature of the coldest month. It is important to emphasise that specimens from Disko and Jyva ¨skyla used in the study were F1-generations and that a greater diversity might have been observed, if specimens from the par-
P.L. Hansen et al. ental populations had been analysed. If these localities were omitted from the data set, the correlation remained highly significant emphasising this significant result. The temperature of the coldest month thus has a high impact on phenotypic variation maybe causing selection, expressed as lower phenotypic variation in colder climates. As the Greenland locations were representing cold climate the phenotypic variation was comparatively low. However, the distribution of Greenlandic phenotypes deviated from populations from all other localities studied as shown in Fig. 2. During glacial periods, the temperature is likely to have exerted a high selective pressure. This combined with habitat loss during the glacial period and the isolation of Greenland, hindering re-colonisation overseas, could explain the low phenotypic variation seen in Greenland populations compared to other populations. On the other hand, the sample from Dunvegan revealed more unique clones than the Greenlandic samples despite the fact that the average air temperature in winter is like the temperature in Greenland. Data for soil temperatures were not available, but it is likely that populations from Canada are living in a much more buffered habitat (coniferous forest litter layer) compared to populations from Greenland that are found under moss covering very exposed rocks. In effect, the winter temperatures experienced by D. octaedra in Greenland are considerably harsher than in Canada, which can explain why the lowest number of unique clones were found in the Greenlandic samples.
Clonal variation The result that 176 (51%) of the 345 individuals assayed represented a novel overall phenotype and that 125 (36.2%) were unique clones is consistent with results of Terhivuo et al. (1987) in Finland, where 147 out of 428 (34.3%) individuals of D. octaedra assayed represented a novel overall phenotype and 80 (18.6%) were unique clones. A high genetic variation is thus seen in D. octaedra in both studies. The extensive genetic variation seen in the populations of D. octaedra is surprising, when considering that it reproduces by apomictic (mitotic) parthenogenesis. The genotype is thus transmitted intact to the offspring from the mother without any recombination. One explanation for the genetic variation could be that sexual processes have been active in the not too distant past. Amphimictic reproduction secures a high level of genetic variation, because of recombination of
ARTICLE IN PRESS Variation among earthworms from Northern Hemisphere alleles or genes, and when reproduction shifts from amphimixis to apomictic parthenogenesis, the parental genome is preserved and its level of variation is only altered by mutation (White 1973). Neither evidence for automictic reproduction, in meiosis takes place, has been observed (Simonsen, pers. comm.). Different levels of suppression of male sexual organs and even male-fertile individuals of D. octaedra could indicate that recent sexual processes have been active in the past. Male organs are useless once parthenogenesis has arisen and selection for reduced investment in male organs is to be expected to save resources. Terhivuo et al. (1987) found various degrees of male suppression among clones of D. octaedra in eastern Fennoscandia, where rare male fertile individuals were occasionally found, too. Likewise, male fertile D. octaedra have been found in Iceland (Omodeo 1957). It is unknown, however, if the male fertile specimens found were actually functional males. Omodeo (1957) suggested the Icelandic populations of D. octaedra to be gynogenetic, making mechanical stimulation of the egg by a sperm-cell important for the development of the egg, and therefore biologically different from the parthenogenetic apomictic populations from Greenland. Conclusions on dispersal patterns cannot be drawn from this study, because only few mutual clones were seen among different localities. Two overlapping clones were observed in the samples from Disko and Narssarssuaq, separated by 800 km, Disko is an island separated from Greenland by the Disko Bay, so the shared clones could be a result of anthropochorous or other passive transportation in recent times internally in Greenland. A single individual may be sufficient for establishing a population, because of the parthenogenetic reproduction of D. octaedra. The two other clones found in Disko could thus have arisen by mutation. The mutual clones seen in the three samples from Narssarssuaq were probably due to the short distance of 1 km, which might facilitate active dispersal between sampling sites. Only two clones were found in all three samples from Silkeborg, though, separated by only 5 km. However, roads and the river Gudena( were separating the three sites from each other. When comparing the frequency of unique clones (Table 4), it was evident that the Silkeborg area had a much higher frequency and that the number of clones observed was more than twice compared to Narssarssuaq. This observation might indicate that clonal dispersal might be strongly dependent of the barriers in the landscape or strong directional selection among the samples from Narssarssuaq.
233
Acknowledgements We thank Jill Clapperton for sampling earthworms in Canada. This work was made possible through the EU Integrated Project ALARM (EU 6th Framework Programme No. GOCE-CT-2003506675).
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