Mitochondrial DNA variation in Mytilus edulis L. And the padstow mussel

J. Exp. Mar. Biol. Ecol., 1985, Vol. 92, pp. 251-258 Elsevier

251

JEM 562

MITOCHONDRIAL

DNA VARIATION

IN MYTZLUS EDULZS L. AND THE

PADSTOW

Department

MUSSEL

D. 0. F. SKIBINSKI of Genetics, University College of Swansea, Singleton Park, Swansea. SA2 8PP, U.K.

(Received

17 May 1985; revision

received

8 July 1985; accepted

9 July 1985)

Abstract: Mitochondrial

DNA (mtDNA) variation was studied using restriction enzyme analysis in samples of mussels (Myth) from three British populations. The first sample obtained from Swansea Bay, South Wales consisted of Mytilus edulis L. The second sample from the Padstow estuary in Cornwall was of individuals of the M. galloprovincialisLamarck form. The third sample was from Croyde Bay in North Devon where on the basis of allozyme and morphological evidence hybrid individuals occur. Evidence was found of distinct mtDNA differences between the Swansea Bay and Padstow samples. The sample from Croyde Bay contained genotypes found at both Swansea Bay and Padstow, thus providing corroborative evidence for the hybrid nature of this population. Key words: Myths;

mitochondrial

DNA;

restriction

enzymes;

population

genetics

INTRODUCTION

Much effort has been devoted to the study of the population genetics of Mytilus using protein variation (e.g. Skibinski et al., 1983; Koehn et al., 1984, and references therein). Electrophoretic techniques have revealed considerable macro- and microgeographic differentiation in allele and genotype frequencies, but it is not yet clear to what extent this differentiation can be attributed to selection acting directly on allozyme or other loci or be due to the mixing of and intergradation

of subspecies

or distinct

species.

In western Europe A4. galloprovincialis Lamarck, (the Mediterranean mussel) has a southerly distribution but occurs sympatrically with M. edulis L. in parts of southwestern England, France and the western and eastern coasts of Ireland. On the basis of both morphological and allozyme data it appears to interbreed quite freely with M. edulis at some localities (see Seed, 1978; and Gosling, 1984, for reviews). The two forms can be crossed experimentally and hence there is probably more justification for regarding them as subspecies rather than distinct biological species (Gosling, 1984). Nevertheless, a study of the extent of genetic differentiation between the forms is relevant to the interpretation of experimental studies of adaptive differences among allozyme genotypes. In this study restriction enzyme analysis is used to examine mitochondrial DNA (mtDNA) variation in samples from three British populations of Mytilus representing A4. edulis, M. galloprovincialis, and their hybrids. 0022-0981/85/$03.30

0

1985 Elsevier

Science

Publishers

B.V. (Biomedical

Division)

252

D. 0. F. SKIBINSKI

Mitochondrial DNA possesses features which may make it useful in phylogenetic and taxonomic studies at the specific or subspecific level (Brown, 1983; Avise & Saunders, 1984). mtDNA genotypes are reproduced asexually in the maternal line and most have a unique mutational origin. Thus, all individuals showing a particular mtDNA genotype probably belong to the same maternal clone. mtDNA also seems to evolve more rapidly than single copy nuclear DNA (Brown et al., 1982). Until now the use of restriction enzyme analysis of mtDNA as a tool in taxonomy and population genetics has been largely confined to studies of vertebrates. Little attention has been given to use of the technique with marine invertebrates although recent work on lobsters (McLean et al., 1983) provides an exception. In the present study tentative predictions can be made. The absence of mtDNA differences between M. edulis and M. galloprovincialis would give more support to the view that minimal heritable differences in general occur between the forms. By contrast, the presence of distinct mtDNA genotypes in M. edulis and in M. galloprovincialis and the presence of both types in the hybrid sample would provide support for the view that differences between the forms are more substantial.There is, however, evidence that mtDNA genotypes may flow across a species boundary in the absence of nuclear gene flow (Ferris et al., 1983) and thus the possible complication that the geographic distribution of mtDNA genotypes, allozymes and morphological characters may not coincide. MATERIALS AND METHODS

Samples of adult mussels were collected from each site between March and June 1984. The locations of the sampling sites are shown in Fig. 1. The first sample consisting

I 6’W

I

4Ow

1oW

Fig. 1. Location of sampling sites: 1, Swansea Bay; 2, Padstow Estuary; 3, Croyde Bay

of M. edulis was obtained from Swansea Bay, South Wales. The second sample of individuals of the M. galloprovincialis type was obtained from the Padstow estuary in

MITOCHONDRIAL

DNA VARIATION

IN MUSSELS

253

Cornwall. Mussels from this locality have been regarded as M. galloprovinciaiis representatives in several studies (e.g. Seed, 1971; Skibinski et al., 1978). The third sample was obtained from Croyde Bay. On the basis of a study of allozymes and morphology it has been suggested that both M. edulis and M. galloprovincialis and introgressed and hybrid individuals occur together at this locality (Skibinski et al., 1978). mtDNA was extracted from ripe mantle tissue stripped from female mussels between 3 and 6 cm in length. Developing eggs and oocytes are rich in mtDNA in many species (Billett, 1979), and in this study ripe mantle tissue was found to give higher yields of mtDNA than h~atop~~reas, gill or unripe mantle tissue. The methods of DNA preparation and solutions used were based closely on those described by Lansman et al. (1981). Mitochondrial preparations were made by differential centrifugation of homogenized mantle tissue or eggs. After lysis of the mitochondria with SDS, DNA was extracted with phenol and chloroform, precipitated with ethanol and redissolved in Tris-EDTA (TE) buffer. This gave preparations of mtDNA contaminated with RNA and nuclear DNA. Neither contaminant seriously interfered with visualization of mtDNA fragments greater in size than 500-1000 base pairs on ethidium bromide stained agarose gels. RNA contamination could be reduced by incubation with RNase. Nuclear DNA contamination could be reduced but never completely eliminated by a combination of sucrose step gradient cen~fugation (method as in Lansman et al., 1981) followed by incubation of mito~hond~a in MSB buffer ~ont~ning MgCl, and DNaseI prior to lysis. Attempts to purify mtDNA by caesium chloride ethidium bromide centrifugation have not so far proved successful. This is in part due to a low yield of supercoiled mtDNA and a wide nuclear DNA band in the gradient. Usually sufficient mtDNA could be obtained from individual mussels for six or more digests with the restriction enzymes BstEII, Pstl, HindIII, KpnZ, XbaZ, EcoRI and BamHZ. Agarose gels stained with ethidium bromide were photographed under UV illumination with Polaroid film, and negatives used to produce enlarged prints for mobility measurements. HindZII fragments of phage /z were used as size markers.

RESULTS

mtDNA preparations were made from 32 individual mussels, 8 from Swansea, 14 from Croyde, and 10 from Padstow. Morphologically all the Padstow and Swansea mussels resembled the British M. gaZioprovincialisand M. edulis forms, respectively. The majority of the Croyde mussels showed rather greater morphological resemblance to M. galloprovincialis. This is consistent with the observation that it4. galloprovincialis predominates amongst larger mussels in several mixed populations in southwestern England (Skibinski, 1983). One of the Croyde mussels was, however, typically M. edulis in appearance. All the restriction enzymes cut the mtDNA at least once. Polymo~hisms were

254

D. 0. F. SKIBINSKI

Fig. ,2. PIlotographs of mtDNA from individual mussels digested with several restriction enz ymes: the first and la St 1anes show L Iiindrirr marker fragments; two photographs of the same gel for diffe :rent expo sure and de :vellopment times are shown so that small and large fragments can be visualized against a backgrc mnd of smeared nuclear DNA; the banding patterns are interpreted diagr~matically in Fig. 3.

MITOCHONDRIAL

DNA VARIATION IN MUSSELS

255

observed for BstEII, XbaI, EcoRI, and Hind III but not for Pstl, &ml, and Raced. The last two enzymes appeared to have a single cut site while Hind III produced at least eight fragments. Some of the more frequently occurring digestion patterns are shown in Fig. 2 and interpreted diagrammatically in Fig. 3. Background smearing of nuclear

-

-

23.1

-

6.7

-

4.4

wm-

--2.3 m-

-2.0

Fig. 3. Diagrammatic interpretation of banding patterns shown in Fig. 2: Lanes 1 and 9, marker fragments (sizes in kilobases are shown); 2, BstEII genotype B; 3, BstEII genotype A; 4, EcaRI genotype A; 5, EcoRI genotype B; 6, Xbal genotype B; I, Xbal genotype A; 8, PsiI genotype A.

DNA be~nning at a position on the gel slightly slower than the 23.1 kbase 2 fra~ent can be seen in Fig. 2. This does not, however, prevent direct visualization of any of the mtDNA bands. The change from BstEIZ genotype A to BstEZI genotype B and from EcoRZ genotype A to EcoRI genotype B can be most easily explained by the addition of a single new cut site. EcoRZ A has two fragments of similar size which are not distinguishable in mobility on the gel, but which are revealed in multiple digests. The difference between XbaI genotypes A and B might reflect a length polymorphism or, more likely, genotype A has an additions small fra~ent not visible on the gel. The fragments corresponding to the intense bands in Fig. 2 sum to 17,400 f 250 base pairs averaged over lanes, well within the size range expected for mtDNA (Brown, 1983). Table I shows genotype frequencies for the three populations for individual enzymes and for the B&II-Xbal-EcoRI multiple genotypes. This table includes data on rarer restriction genotypes not shown in Fig. 2. All these genotypes can be explained by

256

D.O.F. SKIBINSKI

changes which involve the addition or subtraction of one or two new restriction sites. The bands of the ~i~~~~~ patterns sum to substantially less than 17,400 base pairs possibly because different fragments have similar mobility as with EcoRI genotype B. TABLE I

Numbers of individual mussels showing particular restriction enzyme genotypes in samples from Swansea, Croyde and Padstow: the final seven rows of the table are for the BstEII-X&i-EeoIU multiple genotypes. Swansea Bay B&I1

Psti HindIII

A B c A A

B KpnI XbaI

EcoRl

A A B c A B

c BamHI ABE BBB BBC BCD BAA BBA CBB

D A

4 3 3

Croyde Bay 3

10

1 12

1 1

1

2 5

10 5 7 t 9 4

1 -

6 1 1 3 3

Padstow Estuary

1 11 3

1 1

f

I 5 3

For the enzymes BstEII, Xbd and EcoRI there is an indication of frequency differences between the Swansea (M e&&s) and Padstow (M. gal~o~~ovincialis) samples. Considering only the more frequent A and B genotypes and using Fishers exact test for 2 x 2 tables the difference is, however, only significant for EcoRI (P -e 0.01). For this enzyme the A genotype occurs in 9 out of the 10 Padstow mussels but is absent from the Swansea sample. A significant difference (P < 0.01) also occurs between Padstow and Swansea in the multiple genotype frequencies. The genotypes BAA and BBA predominate at Padstow while the genotypes ABB and BBB predominate at Swansea. These four genotypes can be interrelated in the sequence ABB + BBB t BBA -+ BAA where each arrow represents the addition of a new restriction site by base substitution. Thus three changes are required to inter-convert the genotypes at highest frequency at Swansea (ABB) and Padstow (BAA). In the sample from Croyde Bay one individual possessed a genotype (CBB) that was not found at Padstow or Swansea. Apart from this the Croyde sample appears to be

MITOCHONDRIAL

DNA VARIATION IN MUSSELS

257

intermediate genetically between the Padstow and Swansea samples. For example, for out of 14 individuals had the A genotype predominating at Padstow and 4 had the B genotype predominating at Swansea. The Croyde sample was rather more similar genetically to the Padstow sample which is consistent with the predominance of individuals with M. gulfoprovinciulisfeatures amongst larger mussels at Croyde. One of the Croyde mussels was identified a priori as M. edulis in appearance. This mussel in fact had the ‘~galloprovincialis”genotype BAA. Also, retrospective examination of the shell of the single mussel from Padstow having the “edu~is” genotype ABB revealed no M. edulis features. The genetic similarity between a pair of organisms may be computed from the total fraction of shared fragments over the restriction enzymes for which they are compared (Lansman et al., 1981). From this the proportion (p) of nucleotide sites showing base differences between the organisms can be estimated (e.g. Upholt, 1977). Average values of p have been calculated over 10 pairs of mussels taken at random with replacement from within each of the 3 samples to provide within population estimates of genetic similarity. The values obtained with standard errors are 0.0096 + 0.0016 for Swansea, 0.0049 + 0.0005 for Padstow and 0.013 4 0.0012 for Croyde. The genetic distance computed in a similar fashion between the Swansea and Padstow samples is 0.0193 & 0.0096. All the above values are within the range commonly found in conspecific comparisons, but, lower than those found in comparisons involving different species (see tables in Lansman et al., 198 1; and Avise & Lansman, 1983). As might be expected from the data of Table I the values for the Swansea (M. edulis) and Padstow (h&.ga~~o~rovin~iaiis~ samples are lower than those obtained for the comparison between these two populations. The Croyde Bay sample containing both “edulis” and “galioprovincialis”mtDNA genotypes has, as expected, a higher value of p than the other two samples.

EcoRl9

Drscussror\r The results of this study provide evidence of significant differences in the frequency of mtDNA genotypes between h4. edulis and M. galloprovinciuiis from Padstow. Moreover the morphological and allozymically intermediate population at Croyde Bay has intermediate genotype frequencies as expected for a hybrid population. These findings possibly provide further support for a taxonomic interpretation of the edul~-galI~provinciulis situation, that is that genetic differences between the forms are quite substantial. Nevertheless, compared with differences at the species level in other studies, the mtDNA differences between the Swansea and Padstow samples are relatively small as judged by the computed P values. Also none of the genotypes are perfectly diagnostic. Hopefully an analysis of larger numbers of individu~s from more sites using greater numbers of restriction enzymes wilI be more informative. At present an attempt is being made to clone purified mussel mtDNA for use as a

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D. 0. F. SKIBINSKI

probe. If this can be done the problem of smearing of contaminating nuclear DNA will be by-passed. The expe~mental techniques described in this paper only allow the study of mtDNA from fairly large reproductively mature female mussels. The use of a cloned probe will release this constraint, and thus remove any bias resulting from non-random sampling of individuals for analysis. ACKNOWLEDGEMENTS

I would like to thank the following for help and advice: Professors J. A. Beardmore and W. M. Brown, Drs D. Dixon, J. M. Parry, G. P. Wallis and R. Waters and Messrs C. A. Edwards, and D. Ellis. This work was supported in part by grant GR3/5563 from the N.E.R.C. to the author. REFERENCES AVISE,

J. C. & R.A. LANSMAN, 1983. Polymorphism of mitochondrial DNA in populations of higher animals. In, Evolution of genes and proteins, edited by M. Nei & R.K. Koehn, Sinauer Associates, Sunderland, Massachusetts, pp. 147-164. AVISE,J. C. & N. C. SAUNDERS,1984. Hybridisation and introgression among species of sunfish (Lepomis): analysis by mitochond~ai DNA and aliozyme markers. Genetics, Vol. 108, pp. 237-255. BILLETT,F. S., 1979. Oocyte mitochondria. In, Maternal effects in development, edited by D. R. Newth & M. Bails, Cambridge University Press, Cambridge, pp. 147-166. BROWN,W. M., 1983. Evolution of animal mitochondrial DNA. In, Evolution of genes and proteins, edited by M. Nei & R. K. Koehn, Sinauer Associates, Sunderland, Massachusetts, pp. 62-88. BROWN,W.M., E.M. PRAGER,A. WANG % A.C. WILSON, 1982. mtDNA sequences of primates: tempo and mode of evolution. J. Mol. EvoL, Vol. 18, pp. 225-239. FERRIS, S. D., R. D. SAGE, CM. HUANG, J.T. NIELSON& V. RITTE, 1983. Flow of mitochondrial DNA across a species boundary. Proc. Nat. Acad. Sci. U.S.A., Vol. 80, pp. 2290-2294. GOSLING, E., 1984. The systematic status of Mytilus galloprovincialis in western Europe: a review. Malacologia, Vol. 25, pp. 551-568. KOEHN, R.K., J.G. HALL & A.J. ZERA, 1984. Genetic differentiation of Mytilus edulis in eastern North America. Mar. Biol., Vol. 19, pp. 117-126. LANSMAN,R. A., R. 0. SHADE,J. F. SHAPIRO& J. C. AVISE, 1981. The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations III: techniques and potential applications. J. Mol. Evof., Vol. 17, pp. 214-226. MCLEAN, M., C.K. OKUBO& M.L. TRACEY,1983. mtDNA heterogeneity in Panutirus argus. Experienria, Vol. 39, pp. 536-538. SEED, R., 1971. A physiological and biochemical approach to the taxonomy of Mytilus edulis (L.) and M. galloprovincialis(Lmk.) from S. W. England. Cah. Biol. Mar., Vol. 12, pp. 291-322. SEED, R., 1978. The systematics and evolution of M.vtilus galloprovincia~~s(Lmk). In, Marine organisms: genetics, ecology and evolution, edited by B. Battagha & 3.A. Beardmore, Plenum Press, New York, pp. 447-468. SKIBINSKI,D. 0. F., 1983. Natural selection in hybrid mussel populations. In, Systematics association special Vol. No. 1, 24. Protein polymorphisms-adaptive and taxonomic significance, edited by G. S. Oxford & D. Rollinson, Academic Press, London, pp. 283-298. SKIBINSKI, D.O.F., M. AHMAD & J.A. BEARDMORE,1978. Genetic evidence for naturally occurring hybrids between Myiilus edulis and Mytilus galloprovincialis. Evolution, Vol. 32, pp. 354-364. SKIBINSKI,D. 0. F., J. A. BEARDMORE& T. F. CROSS, 1983. Aspects of the population genetics of ~yti~us (Mytilidae : Mollusca) in the British Isles. Biof. J. Linn. Sac., Vol. 19, pp. 137-183. UPHOLT,W. B., 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acids Res., Vol. 4, pp. 1257-1265.