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Genetic population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci.

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Genetic Population Structure and Gene

Flow

in

the Atlantic Cod Gadus morhua:

A

Comparison

of

AUozyme and Nuclear

RFLP

Loci

Grant H. Pogson, Kathryn A. Mesa' and Robert G. Boutilier

Department of Zoology, University of Cambridge, Cambridge GB2 3EJ England Manuscript received February 15, 1994

Accepted for publication September 10, 1994

ABSTRACT

High levels of gene flow have been implicated in producing uniform patterns of allozyme variation among populations of many marine fish species. We have examined whether gene flow is responsible for the limited population structure in the Atlantic cod, Gadus morhua L., by comparing the previously published patterns of variation at 10 allozyme loci to 17 nuclear restriction fragment length polymor- phism (RFLP) loci scored by 11 anonymous cDNA clones. Unlike the allozyme loci, highly significant differences were observed among all populations at the DNA markers in a pattern consistent with an isolation-by-distance model of population structure. The magnitude of allele frequency variation at the nuclear RFLP loci significantly exceeded that observed at the protein loci

(x'

= 24.6, d.f. = 5, P < 0.001). Estimates of gene flow from the private alleles method were similar for the allozymes and nuclear

RFLPs. From the infinite island model, however, estimates of gene flow from the DNA markers were fivefold lower than indicated by the proteins. The discrepancy between gene flow estimates, combined with the observation of a large excess of rare RFLP alleles, suggests that the Atlantic cod has undergone a recent expansion in population size and that populations are significantly displaced from equilibrium. Because gene flow is a process that affects all loci equally, the heterogeneity observed among populations at the DNA level eliminates gene flow as the explanation for the homogeneous allozyme patterns. Our results suggest that a recent origin of cod populations has acted to constrain the extent of population differentiation observed at weakly polymorphic loci and implicate a role for selection in affecting the distribution of protein variation among natural populations in this species.

G

ENE flow is a powerful homogenizing force that may act to prevent genetic divergence arising among populations by either drift or selection (re- viewed by FELSENSTEIN 1976; SLATKIN 1985a). Evidence for the importance of gene flow in affecting the distri- bution of genetic variation among natural populations is often inferred from a species' potential for dispersal. For example, species with high dispersal capabilities often exhibit low levels of population differentiation

(e.g., WAPLES 1987), whereas those with limited means of dispersal, or insurmountable barriers to movement, often display a significant degree of population struc- ture (e.g., LARSON et al. 1984). In these instances, indi- rect estimates of gene flow agree with migration rates expected to occur among populations. In other studies, however, indirect and direct estimates of gene flow have produced conflicting results ( . g . , EHIUICH et al. 1975; BAKER 1981) that have been interpreted as reflecting the highly stochastic nature of gene flow or the possible action of selection (see discussion in SLATKIN 1985a).

The spatial patterns of variation observed at electro-

Cmesponding author Grant H. Pogson, Department of Biology, A318 Earth and Marine Sciences Bldg., University of California, Santa Cruz, CA 95064. E-mail: pogson@orchid.ucsc.edu

'

Presmt address; Department of Biology, University of California, Santa Cruz, C A 95064.

Genetics 139: 375-385 (Janualy, 1995)

phoretic loci among freshwater, anadromous, and ma- rine fish species generally conforms with that expected from varying levels of gene flow (see reviews by GEL-EN- STEN 1985; WARD et al. 1994). In a recent review of electrophoretic data from 49 freshwater and 57 marine species of fish, WARD et al. (1994) showed that the mean level of population subdivision in the former (GST = 0.222) were significantly greater than observed in the latter ( G S T = 0.058) even though the mean heterozygos- ity levels were nearly identical in both groups (HT = 0.064 and 0.062, respectively). Despite the lack of stud- ies obtaining direct estimates of gene flow in fishes, the authors conclude that marine species apparently exchange between one and two orders of magnitude more migrants per generation than freshwater species. The degree of population subdivision shown by seven anadromous species was intermediate between freshwa- ter and marine species (GST = 0.108), but not signifi- cantly different from either group.

Despite these general results, the importance of gene flow among several widely distributed marine fishes re- mains controversial. In the case of the Atlantic cod,

Gadus morhua, the controversy arises from the ambigu-

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376 G. H. Pogson, K. A. Mesa and R. G. Boutilier

Norwegian coastal and arctic stocks at blood protein loci such as hemoglobin-1 (Hbl) and transferrin ( T j )

(FRYDENBERG et al. 1965; SICK 1965; MOLLER 1968;

DAHLE and JORSTAD 1993), but not among populations

sampled throughout the entire species' range and scored for a larger set of routine electrophoretic loci

(MOM et al. 1985). The heterogeneity observed among

protein-coding loci has been interpreted as reflecting the action of selection at a subset of loci, but this is incompatible with the continuing action of gene flow suggested by tagging studies (e.g., RASMUSSEN 1959; GULLAND and WILLIAMSON 1962; TEMPLEMAN 1974), which should eliminate differences among populations unless selection is particularly strong. Recent studies examining patterns of mtDNA variation in the Atlantic cod have corroborated the electrophoretic and tagging results in revealing limited population differences

(SMITH et al. 1989; CARR and MARSHALL 1991; ARNASON

and RAND 1992). Here again, however, exceptions have been reported (DAHLE 1991). The low levels of mtDNA polymorphism observed in the Atlantic cod relative to other marine fish species has suggested the possibility that its limited population structure may reflect a recent origin of populations, possibly involving a recent bottle- neck (see SMITH et al. 1989; ARNASON et d . 1992). De- termining whether gene flow has recently ceased among populations or is continuing at low levels is prob- lematic, however, since both would produce patterns of high genetic similarity.

In this study, we have examined the relative roles of gene flow and selection in determining population structure in the Atlantic cod, G. morhua, by comparing the patterns of variation at 10 polymorphic allozyme loci previously scored by MORK et al. (1985) to 1'7 cDNA- based nuclear restriction fragment length polymor- phisms (RFLPs) scored among 6 populations occupying similar geographic regions. If gene flow is responsible for the homogeneous population structure revealed by the allozymes, the nuclear RFLP loci are also expected to display low levels of population differentiation. If the RFLP loci exhibit significant heterogeneity among populations then gene flow can be eliminated as the explanation for the homogeneous allozyme patterns be- cause gene flow is a process that affects all loci equally. By showing significant differences between the distribu- tions of RFLP and allozyme polymorphism among cod populations, our results discount the importance of gene flow and implicate a role for selection in affecting the distribution of protein variation in this species.

MATERIALS AND METHODS

Animals: Six populations of the Atlantic cod (G. morhua

L.) were sampled throughout the geographic range of the species. The locations of these samples were selected to match a subset of the populations previously characterized at the

protein level by MORK et al. (1985). Figure 1 presents the positions of the following samples from Mork et aL's study: A, Gulf of Maine; B, Greenland; C, Iceland; D, North Sea; E, Malangen and F, Barents Sea. Sample locations, sizes, and collection dates of samples characterized in the present study are as follows: G, Nova Scotia (64"25'W, 44"33'N, n = 138, November 1991); H, Newfoundland (5Oo91'W, 46"96'N, n = 131, June-July, 1992); I, Iceland (2Oo50'W, 63"30'N, n = 84, April 1993); J, North Sea (2"91'E, 53"84'N, n = 81, November 1992); K, Balsfjord (18"57'E, 69"29'N, n = 87, January 1993) and L, Barents Sea (17"50'E, 70"29'N, n = 82, January 1993). The mean distance separating five of the six samples taken from similar geographic regions is conservatively estimated to be less than 350 km; the remaining pair (Newfoundland and Greenland) differ in location by -1600 km.

DNA extractions: Total DNA was isolated from EtOH-pre- served blood samples by a modification of the salt-extraction procedure of MILLER et al. (1988). Approximately 200 pl of

blood was pelleted, washed with high TE (100 mM Tris-HC1, 10 mM EDTA, pH 8.0) and resuspended in 700 pl of lysis buffer (10 mM Tris-HC1, 400 mM NaCl, 2 mM EDTA, pH 8.3) containing 0.8% SDS and 200 pg Proteinase K. After incubating the sample overnight at 55", protein was precipi- tated by the addition of 350 pl of saturated NaCl solution followed by vigorous shaking on a Vortex for 15 min. After centrifuging for 30 min at 14,000 rpm, the supernatant was transferred to a fresh microfuge tube. DNA was precipitated by the addition of 1 volume of isopropanol. Samples were pelleted, washed with 70% EtOH and resuspended in 100- 150 pl TE. DNA concentrations were determined by measur- ing absorbance at 260 nm on a Hewlett-Packard HP8452A Diode Array spectrophotometer.

cDNA probe isolation: The procedure for isolating clones and screening for polymorphism is similar to that described by POCSON and ZOUROS (1994). Approximately 2 g of liver was removed from an anesthetized adult cod, immediately frozen in liquid nitrogen, and used as a source of mRNA for the construction of a cDNA library in the phagemid vector lambda Bluemid (Clontech)

.

cDNA clones were isolated by plating out the library at low density and transferring individ- ual plaques to 100 p1 SM buffer (100 mM Tris-HC1, 10 mM MgC12, pH 7.4). Phage DNA was prepared for amplification by the polymerase chain reaction (PCR) by two cycles of freeze- thawing at -80". cDNA inserts were amplified by 30 cycles of denaturation (94" for 1 min), primer annealing (45" for 1 min) and extension (72" for 2.5 min) on a Perkin-Elmer Cetus DNA thermal cycler. Reactions were carried out in 10 mM Tris-HCI (pH 8.3 at 25"), 50 mM KCl, 1.5 mM MgC12, 200

p~ dATP, dGTP, dCTP and dTTP, 0.4 p~ SK primer (5'- TCTAGAACTAGTGCATC-3') ,0.4 p~ KS primer (5"CGAGG TCGACGGTATCG3'), 3 p1 phage DNA and one unit Taq

polymerase in a final volume of 50 pl. Amplified cDNA clones were visualized on 0.8% agarose gels stained with ethidium bromide.

Screening for RFLPs: Inserts falling within a certain size range (0.5-1 kb) were randomly selected as hybridization probes to screen for RFLPs. Clones were nonradioactively labeled with digoxegenin-11-dUTP (DIG1 1-dUTP) by 30 CY- cles of an asymmetric PCR reaction. Labeling was carried out in 10 mM Tris-HC1 (pH 8.30 at 25"), 50 mM KCl, 1.5 mM MgC12, 20 p~ each of dATP, dGTP, dCTP, and dTTP, 0.2 /.LM KS primer, 5 ,UM DIG11-dUTP, 4 pl template and one unit

Taq polymerase in a final reaction volume of 50 pl using the cycling conditions described above. Unincorporated D I G l 1- dUTP was removed by passing the reaction mix through a Sephadex G 5 0 column.

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GREENLAND

OH

.I

J

FIGURE 1.-Locations of cod populations sampled throughout the north Atlantic Ocean. 0, locations of samples previously characterized at the protein level by M o m et al. (1985); 0 , locations of samples scored for the nuclear RFLP loci in present study. Sample names are listed in MATERIALS AND METHODS and in Table 1.

seven unrelated individuals that had been digested with vari- ous restriction endonucleases according to the manufacturer

(Boehringer), separated on 0.8% agarose gels in 1 X TBE buffer, pH 8.3, for 18-20 hr and transferred to nylon mem- branes (Amersham Hybond N) by a Pharmacia VacuGene apparatus. DNA was fixed to the filters by baking for 2 hr at

80”. Membranes were prehybridized for 2 hr at 42“ in 55% formamide, 5X SSPE, 5 X Denhardt’s solution, 0.5% SDS, and 200 pg/ml RNA (from Torula yeast) in a Robbins hybridiza- tion incubator. Hybridizations were carried out overnight in 55% formamide, 5 X SSPE, 0.2% SDS and 200 pg/ml RNA at 42”. Filters were washed twice for 20 min in 2X SSC, 0.2% SDS at room temperature. Restriction fragments were detected by the chemiluminescent assay of TIZARD et al. (1990) by applica- tion of 0.125 mM AMPPD (Tropix) to the filters and subse- quent exposure to Kodak XK-1 X-ray film for 4-5 hr. The criteria set for identifying a polymorphism was the observation of at least one heterozygote among the seven individuals scored for at least one of the four restriction enzymes repre- sented on a screening blot. See POGSON (1994) and POGSON and ZOUROS (1994) for more details.

Scoring DNA polymorphism: Total DNA samples (7 pg) from all 603 individuals were digested with six restriction en- zymes (fiuII, DmI, TaqI, PstI, SacI, and BstEII). The digested samples were size-fractionated by electrophoresis, transferred to nylon filters, and probed as described above. Membranes were hybridized up to four times, after stripping the filters with boiling 0.2X SSC, 0.2% SDS immediately before each reprobing. Eleven cDNA clones were hybridized to filters in various combinations. PlruIIdigested samples were probed with cDNA clones GM309, GM867, GM307 and GM842; DraI digests with clones GM842, GM860, GM727 and GM798; TuqI

digests with clones GM738, GM865, GM777 and GM842; SacI digests with GM615 and GM842; BstEII digests with GM798 and GM727, and PstI digests with GM798.

A total of 17 RFLPs were scored. Each polymorphism is characterized by a specific cDNA probe-restriction enzyme

combination. For the eight clones used to score one polymor- phism (GM309, GM867, GM307, GM860, GM738, GM865, GM777 and GM615) the name of the cDNA probe is synony- mous with the name of the cDNA “locus.” cDNA clones GM727, GM798 and GM842 were used to scored two, three and four polymorphisms, respectively. Because the indepen- dence of polymorphisms resolved by clones GM727, GM798 and GM842 is likely to be violated by the tight linkage between the segregating restriction sites, “single locus” statistics for each presented throughout the paper (with the exception of allele numbers) represent means calculated over the multiple polymorphisms revealed by the same clone. If linkage disequi- librium between these variable sites is complete, this amounts to using information provided by only one polymorphism per cDNA clone. If the multiple sites are in linkage equilibrium, this averaging provides a summary statistic for each structural gene region. Because the detection of a polymorphic restric- tion site by a specific cDNA clone is unaffected by linkage, statistics on allele numbers are based on all 17 RFLPs. The treatment of linkage disequilibrium will be addressed in a future paper (G. H. POCSON, K. A. MESA and R. G. BOUTILIER, unpublished observations).

Restriction fragment sizes were estimated by unweighted linear regression relative to the positions of DNA size stan- dards run in two lanes of each gel (BRL, 1-kb ladder). At least one reference individual was included on all gels to facilitate the identification of common alleles. Rare and/or private variants were identified as distinct if their sizes differed from previously observed fragments by an a priori measurement error set at ?l.O% of the restriction fragment’s length.

RESULTS

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378

-

D

FIGURE 2.-Examples of cDNA-based polymorphisms. (A) clone GM615 hybridized to DNA samples digested with S n d . (B) clone GMSOS hybridized to samples digested with RndI. (C) clone GM842 hybridized to samples digested with ILoztll. (D) Clone GM798 hvbridized to samples digested with Ii&II. Faint bands visible at loci GM.309 and GM842 arc from previous hybridizations of the same membrane bv different cDNA clones.

clones randomly selected from an adult cod liver cDNA library. Examples of cDNA-based polymorphisms are shown in Figure 2. Common alleles at all loci are restric- tion enzyme-dependent, suggesting that the genetic basis of the polymorphism is the gain o r loss of restric- tion sites. T h e mean sizes of restriction fragments de- tected by cDNA clones (weighted by their frequencies) ranged from a low of 1.78 kh at locus GM865 to a high of 12.4 kb at locus GMGl5 and exhibited overall mean of 5.48 kb. T h e Mendelian nature of common alleles at all loci was confirmed by performing pair crosses and resolving restriction fragments in DNA samples ex- tracted from 21day-old larvae (G. H. POGSON, unpub- lished data). With only two exceptions (both involving rare alleles) heterozygous individuals possessed two re- striction fragments of similar intensity (the fainter up- per bands seen in Figure 2 , A and D, are due to less efficient hybridization to higher molecular weight frag- ments). This banding pattern is expected if the polv- morphic restriction site falls outside the coding region represented by the cDNA clone. In the hvo exceptional

cases, three-banded heterozygotes were observed with the sizes of the two smallest fragments summing to that of the largest fragment. One of these alleles is shown in Figure 2C. This banding pattern is consistent with the polymorphic site occurring within the boundaries of the coding region hybridizing with the cDNA clone. In these instances it is not possible to determine if the variable site occurs within an exon of the structural gene probed, or within an intron, if present.

Comparison of levels of variability among popula-

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TABLE 1

Levels of genetic variation in six populations of Gadus morhua at 10 allozyme loci and 17 nuclear RFLP loci detected by 11 cDNA clones

Mean No. of alleles Mean no.

sample size of alleles

Population per locus Private Rare Total per locus Ho He D

Allozymes

Gulf of Maine 96.8 1 3 18 1

.so

0.131 0.129 0.016

Greenland 95.4 2 1 18 1.80 0.133 0.131 0.015

Iceland 99.9 1 3 19 1.90 0.129 0.135 -0.044

North Sea 99.1 0 3 18 1

.so

0.132 0.133 -0.008

Malangen 93.3 1 6 22 2.20 0.133 0.135 -0.015

Barents Sea 83.2 0 4 19 1.90 0.133 0.132 0.008

Nova Scotia 138 12 35 83 4.83 0.327 0.319 0.025

Newfoundland 131 17 33 86 5.02 0.374 0.367 0.019

Iceland 84 20 22 78 4.50 0.349 0.351 -0.006

North Sea 81 5 22 63 3.62 0.336 0.340 -0.012

Balsfjord 87 15 18 69 3.73 0.391 0.384 0.018

Barents Sea 82 13 18 67 3.98 0.366 0.349 0.049

Allozymes" 94.6 0.8 3.3 19.0 1.90 0.132 0.133 -0.008

Nuclear RFLPs" 100.5 13.7 24.7 74.3 4.28 0.357 0.352 0.014

Nuclear RFLPs

"Values are means.

= 0.640), approximately a quarter were observed in two to five populations ( n =

7, mean frequency

= 0. 012) and 19% were private alleles ( n = 5, mean frequency = 0.0060). In contrast, of the 172 alleles detected at the

17

RFLP loci nearly half were private variants unique to one population ( n = 82, mean frequency = 0.0059),

31% were rare alleles present within two to five of the populations ( n = 54, mean frequency = 0.010) and only 21% were common to all six populations ( n = 36, mean frequency = 0.475). The numbers of rare alleles observed within populations exhibited a correlation with sample size ( r = 0.960, P = 0.0024) but the num- bers of private alleles observed did not ( r = 0.144, P = 0.785). Private alleles were most common in the Ice- landic population where they represented 25% of the total and least common in the North Sea where they represented only 8% of the total. Due to the high pro- portion of rare and private RFLP variants, the distribu- tion of alleles belonging to these three classes differs significantly between the two sets of markers

(x2

= 15.7, d.f. = 2, P

<

0.001).

Observed and expected heterozygosities for both sets of markers are also presented in Table 1. Genotypic proportions at both sets of loci conformed closely to Hardy-Weinberg expectations. The

D

values listed in Table 1 suggest no overall tendency for heterozygote deficiencies to occur in any of the samples, suggesting that none represents a heterogeneous mixture of genet- ically discrete subpopulations. For both allozyme and RFLP loci, heterozygosity levels were lowest in the North American samples. At the RFLP loci, mean heterozygos-

ity was highest in the Norwegian coastal sample (Balsfj- ord) despite this population being monomorphic for 2 of the

17 polymorphisms scored.

No correlation was seen between allozyme and RFLP heterozygosities among populations from similar geographic regions ( r = 0.63, P = 0.176).

Comparison of levels of variability among loci: Table 2 lists a number of single locus statistics. The mean numbers of alleles and mean heterozygosity levels ob- served at the 10 allozyme loci were three to four times lower than seen at the DNA level. No correlation was seen, however, between allele numbers and heterozy- gosity levels for either set of markers. For the RFLP loci, this reflects the fact that the majority of alleles detected were either private or rare and thus had little effect on heterozygosity. The greater mean heterozygosity ob- served at the nuclear DNA markers resulted from the absence of loci exhibiting low levels of polymorphism and not from any tendency for the RFLP loci to possess greater individual heterozygosities. Table 2 shows that seven protein loci exhibit levels of polymorphism below that of the least variable RFLP locus (GM727). Frequen- cies of the most common allele at all 17 RFLPs are presented graphically in Figure 3. All loci were domi- nated by, at most, three common alleles whose com- bined frequency exceeded 0.940 in all populations.

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380 G. H. Pogson, K. A. Mesa and R. G. Boutilier

TABLE 2

Single locus statistics

Marker Locus N n H,

Fs7.

r t

Allozymes Cpkl 585 3 0.003 0.004 -0.263 -0.78

G 3 p l 494 3 0.036 0.003 0.143 0.61

Idh- 1 540 2 0.236 0.004 -0.259 -0.20

Idh-2 579 2 0.017 0.011* 0.001 0.001

Ldh-2 585 2 0.002 0.004 -0.380 - 1.06

Ldh-3 585 3 0.487 0.003 0.097 0.06

Mdlo3 585 2 0.007 0.009 0.043 0.02

Pgi-1 575 4 0.502 0.031*** 0.842 2.91**

Pgi-2 584 2 0.009 0.009 0.119 0.07

P P I 565 4 0.038 0.002 0.007 0.003

Nuclear RFLPs GM309 603 16 0.502 0.012** -0.386

GM867 603 14 0.486 0.031*** 0.626 2.28*

GM777 603 11 0.31 1 0.040*** 0.750 2.73**

GM865 603 6 0.456 0.018*** 0.632 2.55**

GM842 603 53 0.200 0.008 0.778 3.02**

GM860 603 10 0.508 0.013** 0.081 0.30

GM307 603 10 0.105 0.040*** 0.725 2.80**

GM738 603 5 0.445 0.142*** 0.875 3.27***

GM727 603 13 0.100 0.010* 0.624 2.40**

GM615 603 8 0.505 0.028*** 0.697 2.52**

GM798 603 26 0.480 0.309*** -0.089 -0.36

-0.48

Allozymes" 567.7 2.7 0.134 0.014** 0.822 2.97**

Nuclear FSLPs" 603 10.1 0.373 0.069*** 0.896 3.30***

Allele totals for GM727, GM798 and GM842 represent totals from two, three and four polymorphisms, respectively. See RESULTS for details. N, number of individuals; n, number of alleles; He, expected heterozygosity; FsT, Wright's fixation index; r, correlation between genetic distance and geographic distance; t, Mantel's nonparametric statistic.

"Values are means.

*

P < 0.05, ** P < 0.01,

***

P < 0.001.

8

0.6

cr

!'s0.5 O

0.1

OS2

t

0.0

I

I I I I I I

17 RFLP loci. 0, GM309; 0 , GM867; A, GM777;

A,

GM865; FIGURE 3.-Frequencies of the most common alleles at the

GM842 (Sad); D, GM860; b, GM307; 13, GM738; W, GM727 (DruI); 0 , GM727 (BstEII); +, GM615;

*,

GM798 (PstI); +,

GM798 (BstEII);

+,

GM798 (DraI).

V, GM842 ( T u @ ) ;

V,

GM842 (DruI); 4, GM842 (PVUII); 4,

10 allozyme loci exhibit significant FsT values ( I d h 2 and P p l ) . The mean FST value for the 10 protein loci is

significant, but due entirely to the strong effect of the P p l locus. In contrast, 10 of the 11 independent cDNA loci exhibit significant heterogeneity among popula- tions and the mean FST value for the RFLPs is highly significant. Although both sets of markers are character- ized by having one highly variable locus, the most diver- gent EWLP locus (GM798) exhibits allele frequencyvari- ation among populations that exceeds the most divergent allozyme locus (PPI) by a factor of 10.

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OS2O

I

0.18

1

0.16

8

3

0.14

8

0.12

G

y

0.10

0.08

3

g

0.06

d

t

0.04 -

0 0

e

0.02 - O ,%O 0 " "

0.00 I I I I

0 2000 4000 6Ooo SO00

Geographic Distance @XI)

FIGURE 4.-Correlations between Rogers' genetic distance and geographic distance for the two sets of markers. 0, allo- zyme loci; 0 , nuclear RFLP loci.

is highly significant

( x 2

= 24.6, d.f. = 5 , P

<

0.001), indicating that as a group the 10 allozyme loci are more homogeneous than expected from the variation exhib- ited by the RFLP loci. Removal of the most variable RFLP locus (GM798) does not alter this conclusion

( x 2

= 13.1, d.f. = 5, P = 0.023).

To assess the role played by the large number of rare RFLP variants in producing this result, FsT values for both sets of loci were recalculated after pooling geno- types into three classes: (i) homozygotes for the most common allele, (ii) heterozygotes for the most common allele, and (iii) all other genotypes. The mean FST values

determined for the 10 allozyme and the eleven inde- pendent RFLP loci were virtually unaffected by pooling (0.013 and 0.068, respectively), thus eliminating the possibility that the greater number of RFLP alleles led to a spurious elevation in the magnitude of population differentiation observed. N o correlation was observed between the degree of heterozygosity of a locus and its

FsT value for either the allozymes ( T = 0.495, P = 0.146) or the RFLPs ( T = 0.256, P = 0.447).

Comparison of spatial patterns of variation: Table 2 also lists for each locus the correlation between Rogers' genetic distance and geographic distance [calculated following MOW et al. (1985) as the shortest marine route between two samples]. Mantel's nonparametric test (MANTEL 1967) was used to determine the signifi- cance of the relationship between genetic and geo- graphic distance. Eight of the 11 independent RFLP loci exhibited significant correlations between genetic and geographic distance. For the allozymes, only the Pellocus exhibits a similar relationship. Although the mean genetic distances calculated for both sets of loci

0.15 0.12 0.09 0.06 0.03 0.0

A. Allozymes

E

,

I

North America

Greenland

Iceland

N o d Sea

Barents Sea

Mdangen

B. Nuclur RFLPs

Nova Scotia

Newfoundland

Iceland

North Sea

Barcnts Sea

Balsfjord

0.15

0.12 0.09 0.06 0.03 0.0 Rogers' Genetic Distance

FIGURE 5.-UPGMA dendograms of Rogers' genetic dis- tance among populations for the (A) allozyme and (B) nu- clear RFLP loci.

show highly significant correlations with geographic dis- tance, Figure 4 shows that the scaling of this relation- ship for the allozymes differs sharply from that observed for the nuclear RFLPs. Figure 5 compares the UPGMA dendograms constructed for the two sets of polymor- phisms by cluster analysis. Both dendograms show a similar population clustering and a clean break between the two samples from the western Atlantic and the four remaining samples. However, branch lengths for the RFLP loci are consistently five to six times those shown by the proteins, reflecting the detection of significant differences among populations within, as well as be- tween, clusters at the DNA level.

Comparison of gene flow estimates: Estimates of gene flow from the two sets of markers are presented in Table 3. The large number of private alleles observed at the RFLP loci enabled Nm to be estimated from Slat-

TABLE 3

Estimates of gene flow

No. of

Marker Method loci N m

Allozymes Private alleles 10 25.0

FST (all loci) 10 17.7

FST (excluding P e l ) 9 69.1

Nuclear RFLPs Private alleles 17 24.3

FST (all cDNA clones) 11 3.4

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382 G. H. Pogson, K. A. Mesa and R. G. Boutilier

kin’s (1985b) method [using constants provided in SLATKIN and BARTON (1989)l in addition to Wright’s island model. From the mean frequency of the 82 pri- vate RFLP alleles, -25 migrants are estimated to be exchanged among populations each generation. The five private allozyme alleles provide a nearly identical estimate. From the

FST

values of individual loci, esti- mates of Nm from the allozymes vary from 8 to 125; from the RFLPs they range from 0.6 to 31. Averaging over all loci, the proteins provide an estimate that is five times greater than the RFLP loci (Nm = 17.7 us. 3.4, respectively). If the most variable locus is excluded from both sets of markers, the protein loci produce a

10-fold higher estimate than the RFLPs. Despite the

reduced gene flow suggested by the RFLP loci, all esti- mates still exceed the level expected to homogenize allele frequencies under a neutral model ( i e . , Nm ex- ceeding unity).

DISCUSSION

Examination of the patterns of genetic variation among natural populations is capable of providing in- sights into the relative importance of selective us. sto- chastic evolutionary processes (CAVALLI-SFORZA 1966; LEWONTIN and KRAKAUER 1973). Unlike the effects of drift, mutation and migration, which affect all loci equally, selection is expected to act at different loci in a heterogeneous fashion. Obtaining evidence for selec- tion by examining spatial patterns of variation has proven difficult, however, due to the inability to accu- rately predict the variation expected by sampling error and breeding structure at a particular set of loci (see NEI and MARUYAMA 1975; ROBERTSON 1975; SLATKIN and ARTER 1991). Our study has attempted to circum- vent these difficulties by comparing the patterns of vari- ation at two distinct classes of polymorphism (allozymes and nuclear RFLPs) among six populations of the Atlan- tic cod, G. morhuu, sampled from similar geographic locations. Unlike the allozyme loci, highly significant differences were observed among all populations at the nuclear RFLP loci. Highly significant spatial patterns were also revealed by the DNA markers that were not reflected by the proteins. These differences eliminate gene flow as the explanation for the uniform frequen- cies of alleles observed at the allozyme loci and suggest that stochastic processes alone are incapable of account- ing for the differences observed between the electro- phoretic and nuclear RFLP loci.

Ideally, the allozyme and nuclear DNA markers would have been scored in the same individuals. In our study, comparisons among markers were made from populations sampled from similar geographic locations (see Figure 1) but separated in time by up to 12 years (corresponding to -2-3 generations). Relating the patterns of allozyme variation from Mork et d ’ s (1985)

study to the RFLP loci scored in the present study is valid only if cod populations have undergone little tem- poral changes in genetic structure within the regions sampled. Several large electrophoretic studies in which populations have been sampled in consecutive years

(e.g.,

MOLLER 1967; DAHLE and JORSTAD 1993) or in which age classes have been compared (e.g., JAMIESON 1975) provide support for this requirement, although samples from some areas exhibit substantial Wahlund effects (e.g., JAMIESON and BIRLEY 1989). The highly similar UPGMA trees produced by the two sets of mark- ers (Figure 5) further suggest that our comparisons are valid by demonstrating that the extent of variation among populations from different regions is greater than the extent of variation present within regions.

A comparison of the mean FsT values of the two classes of polymorphism showed that the allozyme loci are significantly more homogeneous than the DNA markers (Table 2). Random sources of variation appear unable to account for these differences. Low levels of polymorphism combined with sampling error can lead to inflated FST values but this is opposite to the pattern

observed for the allozymes. Five allozyme loci with het- erozygosities

<

0.02 are monomorphic in at least one population, but only one (Idh-2) exhibits significant al- lele frequency variation. Three of the 17 RFLPs scored were monomorphic in at least one population (GM307 and one of the polymorphisms detected by clones GM842 and GM727), yet all three exhibited significant

FST values. A bias may have been introduced by the

characterization of one (or more) highly divergent pop- ulation(s) at the DNA level, but there there is no evi- dence to suggest that this occurred (see Table l ) .

A higher rate of mutation at the DNA level may ac- count for the increased resolution provided by the RFLPs in detecting population differences. However, differences in mutation rates between the protein and RFLP loci are unlikely to be great (see POGSON and ZOUROS 1994), and, providing u m, are known to have little effect on FsT values (CROW and AOKI 1984).

If mutation is driving population divergence at the DNA level, a correlation may be expected between the magni- tude of heterozygosity at an RFLP locus and its FST value, as suggested by recent studies examining patterns of variation at highly variable human microsatellite loci (BOWCOCK et ul. 1994). There is no evidence, however, for a similar relationship at the RFLPs scored in the present study.

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netic and geographic distance were observed at the RFLP loci (Table

2

and Figure 4), which is consistent with the outcome of random drift and limited gene flow in producing isolation-by-distance

(GJ:

WRIGHT 1943). Three of the RFLP loci revealed no spatial structure among populations but exhibited significant allele fre- quency heterogeneity. Two of these loci show weak dif- ferentiation that is attributable in both cases to the sig- nificant divergence of only one of the six populations

(GM309 in Iceland and GM860 in Nova Scotia). This pattern is likely to reflect the action of drift. In contrast, RFLP locus GM798 detected highly significant allele frequency differences among all populations at a level exceeding that expected by chance alone. The FST val-

ues calculated for the 11 independent cDNA loci are expected to be distributed as a chi-square with 10 de- grees of freedom. The mean FST value observed for

GM798 (0.309) exceeds the 95% confidence interval of this distribution, thus making drift an unlikely explana- tion for the heterogeneity observed. This suggests the possibility that the three polymorphisms scored by this cDNA clone may be tightly linked to a site undergoing selection. Further indirect evidence for the operation of selection at this locus based on geographic patterns of linkage disequilibrium will be addressed in a separate publication (G. H. POCSON, K. A. MESA and R. G. BOUTI- LIER, unpublished results).

Only 1 of the 10 protein loci (Pel) displays charac- teristics similar to the DNA markers. The divergent be- havior of the Pel locus has been previously attributed to selection by MORK et al. (1985), but its strong resem- blance to the majority of RFLP loci suggest that it may in fact be neutral. The significant differences among populations revealed by the DNA markers suggests that some of the remaining allozyme loci, previously as- sumed to be neutral, may actually be the selected set. The dilemma created by the patterns of DNA polymor- phism is why allele frequencies at the protein loci have not drifted to a similar extent. Balancing selection may be invoked to explain the uniform frequencies of alleles at moderately and highly polymorphic allozyme loci (G3p1, Zdh-1, Ldh-3 and Pgml)

,

but is unlikely to ac- count for the invariant patterns of alleles at weakly poly- morphic loci. The proposed maintenance of such uni- form frequencies by selection contrasts sharply with the strong associations of several well characterized allo- zyme polymorphisms with varying environmental fac- tors such as temperature (e.g., VICUE and JOHNSON 1973; WATT 1977; PLACE and POWERS 1979) or salinity (e.g., KOEHN et al. 1980).

A striking feature of the nuclear RFLP loci is their possession of large numbers of rare and private alleles. This may reflect the increased ability to detect muta- tional events at the DNA level, a greater neutral muta- tion rate at the RFLP loci, or simply that a larger num- ber of nucleotides are being screened per RFLP

“locus” compared with a typical allozyme locus. In the present study, 129 of the 172 RFLP alleles detected had frequencies

<

0.01. Under the infinite alleles model (KIMURA 1983, p. 208), the expected number of alleles in this frequency class is only 22.9. A similar excess is observed at the 10 polymorphic protein loci scored in Mork et aL’s (1985) study (11 alleles observed with fre- quencies

<

0.01 but only 3.7 expected). Excess num- bers of rare alleles, significantly exceeding that ex- pected under the infinite alleles model, have been reported in previous electrophoretic surveys of protein polymorphism (see discussions in OHTA 1976; K”RA 1983). This feature of allele frequency distributions led OHTA (1975) to propose that many electrophoretic al- leles may not be strictly neutral but subject to weak negative selection. Ohta’s model of slightly deleterious alleles appears unable to account for the large excess of rare RFLP alleles since these are unlikely to experi- ence the same level of negative selection as electropho- retic alleles.

Alternatively, the significant excess of rare RFLP al- leles may indicate that the Atlantic cod has undergone a relatively recent expansion in population size in colo- nizing its present geographic range. This would imply that the populations sampled are significantly displaced from genetic equilibrium. This conclusion differs from that reached by GRANT and STAHL (1988) on the basis of allele frequency distributions at 41 electrophoretic loci in the Atlantic cod. These authors reported a sig- nificant excess of rare alleles in populations of the Pa- cific cod, Gadus macrocqbhalus, but not in G. morhua. An important difference between the present study and that of GRANT and STAHL (1988) is the omission (by choice) of monomorphic RFLP loci. If expected num- bers of rare allozyme alleles are recalculated from 18 loci that exhibit polymorphism in Grant and Stahl’s study, a significant excess is observed (12 present with frequencies

<

0.01 but only 6.7 expected), similar in magnitude to that in Mork et al.’s (1985) study.

If Atlantic cod have experienced a recent expansion in population size, it is likely that alleles common to all populations and present in moderate frequencies predated this expansion. Although this may also hold for some of the rare alleles, the majority have probably arisen after the radiation event. Different ages of com- mon us. rare alleles may account for marked differences in gene flow estimates provided by the

F,,

and private alleles methods (Table 3). In populations that are not at equilibrium gene flow will be overestimated by both indirect methods employed in this study. SLATKIN

(10)

384 G. H. Pogson, K. A. Mesa and R. G. Boutilier

to exchange migrants at a rate m per generation. The estimate of gene flow provided by the RFLP loci from the infinite island model ( N m = 3.4) approaches that required for differentiation to occur among popula- tions by random drift. Since this is likely to be an overes- timate, gene flow may actually be low enough to allow divergence to occur by drift alone, but a suitable num- ber of generations have not elapsed for this to be detect- able (particularly among populations from adjacent re- gions). This conclusion is similar to that reached in previous studies examining mitochondrial DNA se- quence variation in this species (SMITH et al. 1989;

h-

NASON et al. 1992).

In summary, our results have demonstrated signifi- cant differences between the patterns of allozyme and nuclear DNA polymorphisms among populations of a widely distributed marine fish species known to un- dergo large-scale migrations (e.g., GULLAND and WIL-

LIAMSON 1962). These differences eliminate gene flow as the explanation for the homogeneous allozyme pat- terns observed in G. morhua. The absence of population structure at weakly polymorphic loci in this species (in- cluding mtDNA variation) may be more indicative of a recent origin of populations. The extent of variability observed at moderately polymorphic allozyme loci such as Ldh3 or Idh-1 appears to be less than expected by chance. At other loci, such as RFLP locus GM798, the magnitude of allele frequency variation appears greater than expected by drift alone. Our interpretation of the differences between the two sets of loci as reflecting the prevalence of random drift acting at the DNA level and natural selection acting at the protein level is simi- lar to that reached in a comparable study on the Ameri- can oyster (KARL and AVISE 1992). In suggesting that selection rather than gene flow may be responsible for the homogeneous distributions of a subset of allozyme loci, our study questions the presumed neutrality of protein polymorphism and suggests that the invariant allozyme distributions observed in other marine fishes, or in other species, may have a similar basis.

We thank I. HUNTVON HERBING, M. TUPPER, J. A. NELSON, Y. TANG,

C. T. TAGC~ART, A. K. DANIEI.SDO?TIR, V. THORSTEINSSON, A. R. CHILD,

S. E. FEVOLDEN and T. PEDERSON for collection of samples and two

anonymous reviewers for providing helpful comments ou an earlier draft of this paper. Funding was provided by the Ocean Production Enhancement Network (Natural Sciences and Engineering Research Council, Canada) and the Northeru Cod Science Program (Depart- meut of Fisheries and Oceans, Canada).

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Figure

FIGURE 1.-Locations of cod populations  sampled throughout  the  north Atlantic Ocean
FIGURE 2.-Examples of the  same  membrane clone GM798 of cDNA-based polymorphisms. (A) clone GM615 hybridized to DNA samples digested with Snd
TABLE 1
TABLE 2
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References

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