Anim. Blood Grps biochem. Genet. 6 (1975): 71-80

Population differences of aspartate aminotransferase and peptidase in the bay mussel Mytilus edulis Allyn G. Johnson and Fred M. Utter Northwest Fisheries Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, Washington 98112, USA Received: 17 December 1974

Summary This investigation has demonstrated considerable heterogeneity among populations and some heterogeneity within populations in the distribution of alleles at two variant loci of Mytiltrs edulis. Although the causes of this variation remain obscure, some speculations have been made on the basis of available data. A cline for aspartate aminotransferase (AAT) alleles has been observed on the Pacific Coast. An immigration model has been proposed to explain the atypical ecological and genetic characteristics of large mussels found on Amchitka Island, Alaska. Marked differences were found in the distribution of peptidase alleles among collections from Southern California, the North Pacific Ocean, and New Jersey. Deviations from random distribution of phenotypes observed in comparisons made between large and small mussels from the New Jersey collection may reflect selection operating on these loci in this population. Introduction

The bay mussel (Mytilus edulis) has a wide distribution in the tidal zone and shallow coastal waters along the West and East Coasts of North America and other parts of the world. Because of its habitat requirements and distribution, this species is a desirable subject for investigation of genetic-environmental interaction and colonization processes. Several papers have recently been presented on biochemical genetic differences of M . edulis and other pelecypods, giving evidence for genetic response to environmental factors (Milkman & Beaty, 1970; Koehn et al., 1973; Levinton, 1973; Mitton et al., 1973). This paper extends our previous studies of genetic variation in M . eduZis where a six allele system was described for variations of aspartate aminotransferase 71

ALLYN G. JOHNSON AND FRED M. UTTER

(AAT) (Johnson 81 Utter, 1973). Here we describe AAT and peptidase variation in M . edzdis over a wide geographic range. The data include (1) a cline in the distribution of AAT aIleles on the West Coast; (2) the possibility of coexistence of exotic and native populations on Amchitka Island, Alaska, based on the distribution of AAT alleles; (3) differences in the distribution of peptidase alleles between West and East Coast populations; (4) evidence for possible gene-environment interactions at both loci in one East Coast collection.

Methods and materials

For this study, collections of mussels were made from the West and East Coasts of North America from April 1972 through June 1974 (Table 1 and Fig. 1). The mussels were studied by the methods previously reported (Johnson & Utter, 1973).

Fig. 1. Map showing location of Mytilrrs edrilis collections and their allele frequencies.

%

-..'/&.a

Boranof lsiand

; i : A1 AAT, = .393 Island

Peptidase

Peptidase 0 1.906

B = .833

Puget Sound,Wa Peptidase 0 = .778

Ti1 A AI Tarnook c = . 8Bay, 6 4 Ore

.-T

Yaquino Bay, Ore A A T c = .850

L a Jolla, Ca Peptidase

72

B = ,407

Anirn. Blood Grps biorhem. Genet. 6 11975)

ASPARTATE AMINOTRANSFERASE AND PEPTIDASE VARIATION IN MUSSEL

Peptidase staining followed the methods of Johnson et al. (1972). Only live mussels were extracted for the AAT study, while both fresh and frozen mussels were extracted for investigation of peptidase. No differences were found between peptidase patterns of extracts from fresh and frozen portions of the same individuals. No significant differences in AAT allele frequencies or distributions were found between collections of mussels from different heights and depths from clusters on pilings at Mukilteo, Washington. Except for the Amchitka Island collection, the mussels of various sizes were found clustered at each location. On Amchitka Island, small mussels (0.3-0.6 cm) were in clusters, while large mussels (1.0-5.5 cm) were solitary or in groups composed of not more than five individuals. Results AAT The phenotypic distribution and allele frequencies for AAT from the various locations sampled are presented in Table 1. The less frequent alleles, A , B’, B, D, E and F , are combined under the category X in Table 2 for comparisons of the phenotypic distributions and Hardy-Weinberg expectations. All collections except those from New Jersey and Amchitka Island, Alaska, conformed to Hardy-Weinberg expectations. The New Jersey collection was separated into two groups based on she11 length; the group consisting of smaller individuals deviated significantly from the expected Hardy-Weinberg proportions, while the large mussel group conformed to expectations. Both groups had similar allelic frequencies ; however, the small mussel group showed an extreme deficiency of heterozygous individuals. The AAT phenotypes of the collections from Amchitka Island were compared to Hardy-Weinberg expectations on the basis of shell length. Both the small-mussel (0.3-0.6 cm) and the large-mussel (1.0-5.5 cm) collections conformed to expectations; however, they possessed extremely different allele frequencies - small (C = 0.200 and X = 0.800) and large (C = 0.910 and X = 0.090). The allele frequencies of AAT along the West Coast of North America form a distinct cline (excepting the large individuals from the Amchitka ,collection). An increase of the B allele (0.096 -+ 0.800) and a corresponding decrease of the C allele (0.844 0.197) is seen as sampling proceeds northward an westward toward Asia (Table 1).The phenotypic distributions and allele frequencies for AAT from the Puget Sound area, Washington, agree with those previously reported (Johnson & Utter, 1973) and are similar to those observed in the East Coast collections. --f

Peptidnse The phenotypic patterns for peptidase included two faint staining anodal zones and one faster migrating zone which stained more intensely. In the strong staining zone, 11 phenotypes were observed indicating a five-allele (A’, A , B, C, 0 ) dimeric Anim. Blood Grps biochem. Genet. 6 (1975)

73

ALLYK G. JOHNSON AND FRED hi. UTTER

Table 1. Phenotypic distribution of aspartate aminotransferase of Mytilus editlis from the coasts of North America, 1917-73.

L. oca t ion1

Sample AAT phenotypes

Date

~

size

AA

AB

-

-

AC

AD

~~

A€

AE

BB

BC

BD

BE

-

-

Ltest Coast

Amchitka Is., AK1 Large Small Total Kodiak Is., AK' Baranof Is., AK Puget Sound, WA La Push, WA Tillamook Bay, OR Yaquina Bay, OR La Jolla. CA Eust Coast Woods Hole, M A Belmar, NJ

10!72-4/73

72

6'17i74 7/12-9,12 7 172-3 '14 5/73

7172 8/72 8/12

190 262 124 339 1089

4 3

27 66 120 352

194 121

7173

I 1 3 2

-

-

1 2

1 5

1

-

-

-

2

-

-

-

-

2 3 12 3 1 6 - - -

I

-

-

4 12 33

-

4 2

-

-

-

-

-

-

-

-

1

-

124 124

-

-

9 55 64 38

9 22 8 1 2 1 7

112 5 9 16 49

4

16 6

-

92

2 2 -

-

1 8

-

--

-

-

-

-

-

-

Amchitka Is., AK: large = 1.CL5.5 cm shell length, small = 0.3-0.6 cm shell length. Kodiak Island samples contained another allele (B') which was not found in the other collections. B' and 5 are wmbined i n this presentation. 1

Qriqin- PhtenotypeS Schematic

i I

m

-

m I I

I

I I

Fig. 2. Peptidase phenotypes of M y t i h s edrilis in starch gel.

74

Anim. Blood Grps biochem. Genet. 6 (19751

ASPARTATE AMINOTRANSFERASE AND PEPTIDASE VARIATION IN MUSSEL

Allele frequencies

CD CE

CF

DD DE DF EE

2 2 -

-

27 36

-

-

BF

CC

-

5 9 10 6 9 59 10 173 82615

-

-

-

-

-

20 5 0 8 7 25.5 -

1 2 -

-

1 -

-

-

166 106 -

6 3

-

2

-

FF

A

E

C

-

0.063 0.800 0.597 0.246 0.205 0.091 0.130 0.098 0.C79 0.096

0.910 0.197 0.393 0.682 0.704 0.869 0.852 0.864 0.850 0.844

0.041 0.058

0.923 0.922

-

-

-

- -

-

-

-

-

-

0.014 0.003 0.006 0.024 0.036 0.010 0.018 0.030 0.063 0.060

-

-

-

-

-

-

-

-

0.018 0.008

-

3

EF

-

_ _ _ _ _ _ _

'

D

E

F

0.004 - _- 0.055 0.020 0,001 0.013

-

-

0.008 0.008

-

0.018 0.012

-

-

system (Shaw, 1964; Fig. 2). The absence of four of the fifteen phenotypic patterns expected with a five-dele system is consistent with the low frequencies of the A', C and D alleles (Table 3). Variants of dimeric peptidases have been reported in other organisms including man (Giblett, 1969). We assume this variation is the same as that described by Mitton et al. (1973). The A allele is predominant (0.663) in the New Jersey collection. The B allele is predominant in all West Coast collections (0.597-0.923), except La Jolla, California, where the A and B alleles are about equal in frequency (A = 0.435 and B = 0.407; Table 3). Although the combined frequencies of the New Jersey samples conform closely to Hardy-Weinberg proportions, the subsamples of large and small individuals each approached significance due to deviations in opposite directions from expected values. A contingency test indicated that the phenotypes were not randomly distributed between the two size classes (0.05 > P > 0.01). Mitton et al. (1973) observed a non-random distribution of peptidase and leucine aminopeptidase variants in M . e d u h which was attributed to epistatic interaction between loci. No such interaction was evident for peptidase and AAT loci on the basis of 542 samples tested from Puget Sound (contingency test xs = 5.33; df = 4; 0.3 > P> 0.2). Anim. Blood Grps biochem. Genet. 6 (1975)

75

ALLYN G. JOHNSON AND FRED M. UTTER

2 A CL. i\

a! 0

-

z

Pi

r-

8 c-,

N

5

,-

c Y

9

9

9

9

r ? v , o v , I

? 0

9 -

l

l

? c ?o

m c

-E d

76

Anim. Blood Grps biochem. Genet. 6 (1975)

ASPARTATE AMINOTRANSFERASE AND PEPTIDASE VARIATION IN MUSSEL

Discussion The patterns of AAT distribution provide a basis for some interesting speculation. The cline observed on the Pacific Coast for the AAT variants was followed in the small individuals collected on Amchitka Island where the frequency of the B allele is the greatest of any of the collections (0.800). These small mussels are abundant and form dense clusters. Except for their size, they are typical of mussels collected elsewhere. On the other hand, the large mussels collected at Amchitka were atypical from mussels collected from other areas in that they grew separately from the clustered mussels and were rare and solitary; their size and gene frequency, however, corresponded to that found in areas from Puget southward. The size and gene frequencies of the large mussels are consistant with a hypothesis that these mussels represent immigrants from southern waters, perhaps due to intensive ship activity in this area during World War 11. This hypothesis is further supported if the small numbers and isolation are regarded as inadaptive characteristics. However, the alternative hypothesis that the extreme differences among these groups of mussels are reflections of selection on a single indigenous population cannot be excluded without further data. The difference in AAT distribution (discussed in greater detail below with the peptidase data), seen in the New Jersey collection where a significant deficit of heterozygotes was observed in only the small mussels, appears to be a different type of phenomenon because individual clusters contained mussels of all sizes. The distribution of the peptidase variants is dissimiliar from that of the AAT variants. No cline was evident in samples collected from Washington State through Alaska (although no peptidase patterns developed from the small mussels from Amchitka). A marked difference in the allelic distribution between the LaPush, Washington and La Jolla, California samples exists which was not seen for the AAT variants. The allelic frequencies of the New Jersey collection differs from all other collections. However, the gene frequencies described by Mitton et al. (1973) for peptidase variation in a Long Island population (A = 0.484, B = 0.314, C = 0.202; assuming that the same genetic system was observed) are similar to those observed in the La Jolla collection. The sipificant deviations from expected values of the AAT phenotypes in the small mussels and the nonrandom distribution of peptidase phenotypes between size classes in the large mussels of the New Jersey collections are of interest. Significant deviations of heterozygotes from expected ratios within subpopulations of aquatic animals have been reported. Fujino & Kang (1968) observed an excess of transferrin heterozygotes in smaller length groups of skipjack tuna (Katsuwonus pelamis) that was not observed in large individuals and postulated a balanced polymorphism with a shift of survival values with age of fish. Utter et al. (1970) described si,onificant deficits of esterase heterozygotes in male but not female Pacific hake (Merluccius productus). Koehn et al. (1971) observed deficits of esAnim. Blood Grps biochem. Genet. 6 (1975)

77

ALLYX G . JOHNSON AND FRED M. UTTER

Table 3 . Frequencies of peptidase phenotypes of M . eddis collected along the coasts of North America, 1972-73,

__ Location

Sample size

Lbi,Jf COflJI

~ m c h i t k aIs., AK

69

Peptidase phenotypes* A‘ A’

AA

AB

-

-

(10.3)

10 (10.3) 30 (28.1) 11 (9.7) 108 (117.6) 2 (4.4) 14 (18.9)

25 (23.0) 28 (30.0) 53 (52.8)

3 (7.7) 13 (15.8) 16 (24.0)

Kodiak Is., .4K

Bdranof Is., AK Puget Sound, W A

534

-

1.a Push, WA

29

-

LA Jolia, CA

54

1 (0.0)

Emr Coast Bzlmar, NJ (1.G5.0 cm) Bslrnar. NJ Hzlmar, NJ (Total)

45

-

75

-

120

-

(0.7) 6 (4.3) (0.0) 18 (10.8) 1 (0.3)

14

AC

3

BB

9 (10.7) 1 (0.3) 3 (4.9)

49 (47.6) 38 (41.3) I11 ( I 14 5) 328 (325.8) 24 (2 I .4 I I7 (9.2)

11 (10.8) 26 (18.7) 37 (30.0)

4 (0.5) 5 (7.3) 9 (2.41

(1.4)

I (5.6)

_.

(0.0)

* Expected values assuming a Hardy-Weinberg distribution are in parenthesis.

x? test of observed and expected values assuming a Hardy-Weinberg distribution and the West Coast A’A, C, and D phenotypes combined as X, while the East Coast X = A’, B, C phenotypes combined. d.f. = 1. **

terase heterozygotes which fluctuated seasonally in Notropis stramineus that suggested significant differential mortality. The deviations from random distribution reported here may also reflect some form of selection affecting these two loci in this population. Koehn & Mitton (1972) have pointed out that selective coefficients among individual loci probably vary both temporally and spatially in response to numerous external and internal factors and stated that loci responding to environmental heterogeneity cannot provide meaningful information about subpopulational structuring (i.e., different phenotypic distributions among various segments of a population-sexes, ages, etc.). If, indeed, the large mussels at Amchitka are southern migrants and the deficit of heterozygotes in the small mussels from the New Jersey collection reflects a response to the environment, then the AAT locus may be useful for both subpopulational structuring and estimating patterns of environ-

78

Anim. Blood Grps biochem. Gener. 6 (1975)

ASPARTATE AMINOTRANSFERASE AND PEPTIDASE VARIATION IN MUSSEL

Allele frequencies

CC

BC

7 (8.3) 31 (28.1)

I1 (13.8) 65 (62.6) (2.6) 4 (4.3) 0 (1.8)

7

(5.2) 2 (7.2)

=

y**

P

AD

BD

A'

A

B

C

D

-

-

-

0.094

0.833

0.073

-

0.91

0.52 > P > 0.3

3 (1.3) -

4 (4.0)

0.004

0.186

0.597

0.186

0.028

1.96

0.2 > P > 0.1

-

0.040

0.906

0.054

-

0.63

0.5 > P > 0.3

0.075

0.002

2.40

0.2

I

> P > 0.1

1 (0.6) -

1

0.001

0.14

0.778

(1.7) -

-

0.086

0.862

0.052

-

5.76

0.02 > P > 0.01

-

-

0.056

0.435

0.407

0.102

-

2.85

0.1 > P > 0.05

2 (1.3)

-

-

-

0.711

0.122

0.167

-

2.73

0.1 > P > 0.05

1

-

-

-

0.633

0.167

0.200

-

2.94

0.1 > P > 0.05

(3.0) 3 (3.6)

-

-

-

0.633

0.150

0.187

-

0.17

0.7 > P > 0.5

'

mental heterogeneity, depending on the environmental circumstances of a particular population. Acknowledgments

We are especially grateful to the following persons who provided samples and valuable information: James R. Dangel, John F. Palmisano, Paul A. Johnson Jr., Patricia J. Arasmith and Manny Etlinger. References Fujino, K. & T. Kang, 1968. Transferrin groups of tunas. Genetics 59: 79-91. Giblett, E. R., 1969. Genetic markers in human blood. F. A. Davis Co., Philadelphia. 629 pp. Johnson, A. G. & F. M. Utter, 1973. Electrophoretic variations of aspartate aminotransferase Anim. Blood Grps biochem. Genet. 6 (1975)

79

ALLYN- G. JOHNSON AND FRED M. UTTER

of the bay mussel, Mytiliis edulis (Linnaeus, 1758) Comp. Biochem. Physiol. 44B: 317-323. Johnson, A. G., F. hi. Utter & H. 0. Hodgins, 1972. Electrophoretic investigation of the family Scorpaenidae. U.S. Dep. Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service. Fish. Bull. 70: 403-413. Koehn, R. K. & J. B. Mitton, 1972. Population genetics of marine pelecypods. I. Ecological heterogeneity and evolutionary strategy at an enzyme locus. A m. Nnt. 106: 47-56. Koehn, R. K., J . E. Perez & R. B. Merritt, 1971. Esterase enzyme function and genetical structure of populations of the freshwater fish, Notropis straminens. Am. Nor. 105: 51-69. Koehn, R. K., F. J. Turano & J. B. Mitton, 1973. Population genetics of marine pelecypods. 11. Genetic differences in microhabitats of Modiolirs demissus. Evolririon 27: 100-105. Levinton, J., 1973. Genetic variation in a gradient of environmental variability: marine Bivnlvia (ikfollusco).Science 180: 75-76. Milkman, R. & L. D. Beaty, 1970. Large-scale electrophoretic studies of allelic variation in ' Mytilrrs ednlis. (Abstract.) Biol. Biill. 139: 430. Mitton, J. B., R. K. Koehn & T. Prout, 1973. Population genetics of marine pelecypods. 111. Epistasis between functionally related isoenzymes of Mytilus ednlis. Genetics 73: 437-496. Shaw, C. R., 1964. The use of genetic variation in the analysis of isozyme structure. In: Subunit structure of proteins, biochemical and genetic aspects. Brookhaven National Laboratories, Kew York. Brookhaven S y m p . Biof. 17: 117-130. Utter, F. M., C. J. Stormont & H. 0. Hodgins, 1970. Esterase polymorphism in vitreous fluid of Pacific hake, iMerlucciLrs productits. Anim. Blood Grps. biochem. Genet. 1: 69-82.

80

Anim. Blood Grps biochem. Genet. 6 (1975)

Population differences of aspartate aminotransferase and peptidase in the bay mussel Mytilus edulis.

This investigation has demonstrated considerable heterogeneity among populations and some heterogeneity within populations in the distribution of alle...
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