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Hereditas 110: 149-158 (1989)
Morphological and genetic differences among recently founded poulations of noble crayfish (Astacusastacus) SVEIN ERIK FEVOLDEN and DAG 0. HESSEN Biological Institute, University of Oslo, Norway
FEVOLDEN, S. E. and HESSEN, D. 0.1989. Morphological and geneticdifferences among recently founded populations of noble crayfish (Astacus astacus). -Hereditas 110; 149-158. Lund, Sweden. ISSN 0018-0661. Received June 6, accepted October 10, 1988 A growing interest in noble crayfish, Astacus astacus, both for aquaculture and for stocking new ponds, has encouraged studies in morphometric and genetic variation among recently founded (last century) populations. Specimens from one lake in western Norway and three lakes in south-eastern Norway, all assumed to originate from the same ancestral population, were compared. There were significant differences between the one western and the three eastern populations in two morphometric characters: the ratios total length/ carapax width and total lengthllength of right chela. Activity in two specific esterase zones and one specific Lap locus was pronounced in the western population while rare and weak in the eastern populations. Intersample heterogeneity in allele frequencies at the polymorphic loci Lap1 and Xdh may be explained by random genetic drift, with some possible effect of natural selection.
Svein Erik Fevolden, Biological Institute, University of Oslo, P . 0. Box 1064, Blindem, N-0316 Oslo 3, Norway
At present a growing interest exists in Scandinavia for noble crayfish, Astacus astacus, both as an exploitable resource and for aquaculture purposes. Differences in mean individual size, size at maturity, and fecundity within and between natural populations have been documented (FJELDand T A U G B 0 L 1986; unpubl. data). These differences might be due to phenotypic responses to different environmental conditions, responses to fishing (or predation) pressure, or genetic differences. During the last century, native populations of Astacus astacus throughout Europe have been greatly reduced in number or become extinct due to infections by the fungus Aphanomyces astaci. Until summer 1987, Norway was the only country that had escaped the crayfish plague. Quite recently, however, the fungus has been recorded in eastern Norway, close to the Swedish border. During recent decades water pollution, in particular eutrophication and acidification, has also reduced population numbers. Several Norwegian watersheds are subject to severe acidification, which may strongly affect native crayfish populations (APPELBERG 1984; FJELD et al. 1988). An important task in future management of crayfish might be to reestablish such populations after water treatment such as liming, or to select more tolerant populations. Before stocking and restocking it is imperative to know of any genetic differences between popula-
tions. Local populations may be adapted to their local environment, and introduction of genetically different individuals could adversely alter the gene pool. The level of genetic variability within and between natural populations also indicates the potential for an active broodstock selection program towards animals most useful for aquaculture. Morphological features seem to be relatively stable within different species of crayfish; the few data available reveal a relatively low level of genetic variation (NEMETH and TRACEY1979; BROWN 1980; AITARDand PASTEUR 1984). BUSACK (1988) found very little interpopulation variability in Procambarus clarkii (Red swamp crayfish) while P. acutus (White river crayfish) exhibited considerably more genetic variability among geographic populations. To elucidate the level of such interpopulation variability in Astacus astacus, we selected individuals from four separate populations in different lakes. We hoped that the choice of lakes would make it possible to quantify morphological and genetic differences between populations subjected to different environmental conditions.
Study lakes
Three of the lakes, Lake Steinsfjorden, Lake Vreleren and Lake Harestuvann, are near Oslo, SE
150 s. E. FEVOLDEN
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Hereditas 110 (1989)
AND D. 0. HESSEN
, i ’
Fig. 1. Geographic location of the different lakes.
Norway, while the last, Lake Moensvatn, is situated on the Norwegian west coast close to Bergen (Fig. 1). Lake Steinsfjorden is the most productive crayfish locality in Norway, with average yields of 2.4-4.1 kg/ha (QVENILD and SKURDAL 1986). This lake is fully described in SKURDAL et al. (1986). Lake Vaeleren lies 15 km from Lake Steinsfjorden while Lake Harestuvann is approximately 20 km further east. Characteristics of the four lakes are given in Table 1. The crayfish populations in Lake Steinsfjorden and Lake Harestuvann are heavily exploited by local fishermen, in particular the former in which 90 % of individuals above the legal minimum size (90 mm) are removed each year (QVENILD and SKURDAL 1986). The Lake Vaeleren population is less heavily exploited, while the lake Moensvatn population is almost uninfluenced by human fishing. Predatory fishes, such as brown trout (Salmo trutta) and perch (Perca fluviatilis), inhabit all four lakes, but their impact on the crayfish populations is unknown. History of the populations Monks probably stocked a few Norwegian lakes with crayfish around the 16th century, but most of the present day populations were introduced
during the last century (HLIITFELDT-KAAS 1918). A few crayfish were accidentally introduced to Lake Steinsfjorden in the late 1880s (LUND1969) and soon established a dense population. The ancestors of this population are unknown. According to local knowledge the Lake Vaeleren population was probably stocked from Lake Steinsfjorden nearly 90 years ago and later restocked from Lake Tyrifjorden. The latter lake is connected to lake Steinsfjorden (Fig. l),and crayfish from Lake Steinsfjorden soon spread to it. The Lake Steinsfjorden population is thus ancestral to the Lake Vaeleren population, although we d o not know the exact date of stocking. The origin of the Lake Harestuvann population is unknown, but the lake definitely had a crayfish population early in this century (HUITFELDT-KAAS 1918). Although it is probable that this population also originated from Lake Steinsfjorden, the population may be older. In the west coast locality, Lake Moensvatn, 96 individuals were introduced from lake Steinsfjorden in 1938 (VIK1971). A condensed population history is presented in Table 2.
Materials and methods Random samples from the populations were taken from catches sampled by SCUBA-diving o r trap-
Table 1. Characteristics of the surveyed lakes. d = depth Lake
Steinsfjorden Vaeleren Harestuvann Moensvatn
Area km’
Max d
Mean d m
Trophy
Ca
PH
13.9 3.5 3.0 0.2
24 ? 15 ?
ca. 10
Mesa Oligo Oligolmeso Oligo
12-13
6.9-9.0 6.8-7.0 5.6-7.3 6.5-7.5
m
> 20 ca. 10 > 20
mgl-‘
6 5
6-8 6-7
Hereditas 110 (1989)
VARIATION I N NOBLE CRAYFISH
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Table 2. History of the four investigated populations of Astucus astacus. N = initial population size
Lake Vreleren Lake Moensvatn
ping. The specimens from Lake Valeren and Lake Harestuvann were captured in May-June 1985. Most specimens from Lake Steinsfjorden were sampled during early summer 1986, but for comparison some specimens sampled one year earlier (1/3 of the total) were included in the survey. The Lake Moensvatn population was sampled during early summer 1986. All populations were thus sampled at the same time of the year. The animals sampled were kept in separate enclosures in a temperaturecontrolled room with 12 h dark/light cycle. They were fed half-cooked potatoes and pieces of cooked prawns. From each population, 40-48 individuals were used for morphological measurements (numbers used for the electrophoretic assay are given in Table 6). We chose equal numbers of both sexes in the size range 65-105 mm (total length of mature individuals) in all populations. Total length, carapax length and width, and length of right chela were measured in all individuals as shown in Fig. 2. For the electrophoretic survey abdominal muscle tissue and liver tissue from individual specimens were homogenized in about an equal volume of chilled distilled water to which was added 100 pl Triton X-100, 100 pl 0-mercaptoethanol, plus 10 mg NADP per 100 ml. The homogenates were refrozen (-8O0C) and used the next day for the starch gel electrophoresis (methods as described by AYALA et al. 1973). Twenty-four additional specimens were sampled the first week of August 1987 from each of Lake Steinsfjorden and Lake Moensvatn. Homogenates from these were made from fresh animals and analyzed without prior freezing to check observed differences in zymogram activity between the western and eastern populations. Three buffer systems were used; (A) gel buffer: 30 mM Tris, 5 mM citric acid, pH 8.5, plus 1 volume-percent electrode buffer; electrode buffer: 60 mM lithium hydroxide, 30 mM boric acid, pH 8.1 (RIDGWAY et al. 1970); (B) gel and electrode buffer: 87 mM Tris, 8.7 mM boric acid, 1.2 mM EDTA (AYALA ct al. 1972); (C) gel buffer: 7.8 mM K,HP0,.3H20, 1.3 mM citric acid; electrode buffer: 214 mM K2HP0,.3H,0, 27.1 mMcitric acid (SELANDER and YANG1969). The buffer systems used for the different proteins are shown in Table 5.
19(&1920 1938
?
96
TL
Fig. 2. Measurements used for calculating morphological differences between the different populations of Astacus astacus TL total length, CL carapax length, CW. carapax width, RC length of right chela
Twenty-six specific enzymes were examined, but only 19 for all four lakes (Table 5 ) . The staining procedures were as in FEVOLDEN and AYALA (1981). General protein stain (Coomassie brilliant blue) was used to show major muscle proteins. Genetic variation was determined by calculating frequencies of the different alleles ocurring at each locus, frequencies of the different genotypes, and by comparing observed genotype frequencies to Hardy-Weinberg expectations. The commonest allele at each locus was termed 100, and the other alleles were designated by their percentage mobility relatively to the 100 allele. Some loci could be scored only for a limited number of the animals available because of insufficient enzyme activity or inconsistent resolution. Levene's corrections for small sample sizes (LEVENE 1949) were therefore employed throughout. To show similarities
152 s. E. FEVOLDEN
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Table 3. Mean ratio between total length and carapax length (TLICL), total length and carapax width (TLICW), and total length and length of right chela (TURC) for the four different populations of Astacus asfacus. Males and females are separated in the TL/RC calculations. St = Lake Steinsfjorden, VZ = Lake Vzleren, Ha = Lake Harestuvann, Mo = Lake Moensvatn; F = females, M = males. Standard deviations in parenthesis St TLICL TLICW TLIRC F M
VZ
Ha
Mo
1.94 4.00
(0.0063) (0.0242)
1.93 3.99
(0.0101) (0.0330)
1.94 3.93
(0.005s) (0.0221)
1.92 3.74
(0.0089) (0.0269)
2.93 2.70
(0.0366 (0.0437)
3.13 2.65
(0.0777) (0.0648)
2.92 2.58
(0.0483) (0,0591)
2.88 2.40
(0.0581) (0.0465)
between samples, Nei’s unbiased genetic identity and distance factors (Na 1978) have been calculated for each individual locus and averaged across loci.
Results Morphological characteristics The morphological measurements (Table 3) revealed no significant difference in the ratio of total length/ carapax length (P>0.05, t-test), the ratio varying between 1.92 and 1.94 for all populations. Nor was any difference found between males and females with regard to this ratio. Inter-sample comparison of the ratio of total lengthkarapax width (sexes pooled) showed no significant differences between the populations of Lake Steinsfjorden and Lake Vaeleren (Table 4). A slight difference was recorded between the Lake Steinsfjordenand Lake Harestuvann populations (P0.25). Nevertheless. the rarer of two alleles in Vaeleren Fig. 4. Cluster dendrogram (unweighted pair group the initial population now dominates in all three method with arithmetic averaging) based on NEI’S(1978) founded populations. These observations may genetic identity formulas for comparison of 28 gene loci in seem to contradict the classical expectations of four populations of Astacus astacus founder effects (see MAYR 1963, and discussion and critics of the founder models in NEI et al. 1975; 1980;TEMPLETON 1980; BARTON and CHARLESLANDE servation would be that there are different “deWORTH 1984: CARSON and TEMPLETON 1984). mands” from the different environments as to The variation at the Xdh locus is less “dramatic” which loci must be kept active, that is, phenotypic and fits expectations from a founder model because plasticity at the enzyme-producing level. the rarer of two alleles in the ancestral Lake SteinsThere are environmental differences among the fjorden population has become even less common four lakes, e.g., calcium content, trophic level and in the founded populations. Moreover, signifipH, although no correlation seems obvious cantly lower heterozygosity at X d h , when combetween the observed gene-variation and lake pared to the ancestral population, is revealed both in characteristics of Table 1. Nevertheless, Est and Lake Harestuvann (x’ = 14.604, P
