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Genetic relationships of Mytilus galloprovincialis Lmk.
populations worldwide: evidence from nuclear -DNA
markers
Claire Daguin, Philippe Borsa
To cite this version:
Claire Daguin, Philippe Borsa. Genetic relationships of Mytilus galloprovincialis Lmk. populations
worldwide: evidence from nuclear -DNA markers. Crame A., Harper E., Taylor (eds). Bivalve
Sys-tematics and Evolution., Geological Society of London Special, pp.389-397, 2000, 177. �ird-00202216�
Genetic relationships of M y t i l u s g a l l o p r o v i n c i a l i s Lmk. populations worldwide: evidence from nuclear -DNA markers
Claire Daguin and Philippe Borsa
Laboratoire Génome Populations In t e r a c t i o n s a n d I R D , S t a t i o n M é d i t e r r a n é e n n e d e l'Environnement Littoral, 1 Quai de la Daurade, 34200 Sète, France
C o r r e s p o n d i n g a u t h o r : P . B o r s a (p h i l i p p e b o r s a@y a h o o .fr) 3724 words of text, 35 references, 2 tables, 3 figures
Abbreviated title: Geographical structure of M y t i l u s g a l l o p r o v i n c i a l i s
T o b e c i t e d a s :
Daguin C., Borsa P. 2000. – Genetic relationships of M y t i l u s g a l l o p r o v i n c i a l i s Lmk. populations worldwide: evidence from nuclear-DNA markers. In Crame A., Harper E., Taylor J. (eds.), B i v a l v e S y s t e m a t i c s a n d E v o l u t i o n . G e o l o g i c a l S o c i e t y o f L o n d o n S p e c i a l
Abstract: Allozyme surveys of genetic variation in M y t i l u s g a l l o p r o v i n c i a l i s Lmk.
t h r o u g h o u t t h e w o r l d h a v e i d e n t i f i e d t h r e e g r o u p s w i t h i n t h i s s p e c i e s : a n o r t h e a s t e rn (NE) A t l a n t i c g r o u p t h a t a l s o i n c l u d e s t h e M . g a l l o p r o v i n c i a l i s population of South Africa, a M e d i t e r r a n e a n g r o u p t h a t a l s o i n c l u d e s t h e M . g a l l o p r o v i n c i a l i s populations from the eastern a n d t h e w e s t e r n c o a s t s o f t h e N o r t h P a c i f i c , a n d a n A u s t r a l a s i a n g roup. Hypotheses that have b e e n p r o p o s e d t o a c c o u n t f o r t h e g e n e t i c d i f f e r e n t i a t i o n p a t t e r n s a n d d i s j u n c t , w o r l d w i d e distribution of M. g a l l o p r o v i n c i a l i s i n c l u d e t h e r e c e n t i n t r o d u c t i o n o f t h i s s p e c i e s i n t o t h e Southern Hemisphere and the North Pacific thro u g h h u m a n a g e n c y , a n d a n a l t e r n a t i v e h y p o t h e s i s t h a t e a c h o f t h e t h r e e g r o u p s i s e n d e m i c . W e u s e d t w o n u c l e a r-DNA markers (the p o l y p h e n o l i c a d h e s i v e p r o t e i n g e n e Glu-5 ’ a n d t h e f i r s t i n t r o n o f t h e a c t i n g e n e mac-1 ) t o i n v e s t i g a t e i n m o r e d e p t h t h e g e n e t i c r e l a t i o n s h i p s a m o n g M. g a l l o p r o v i n c i a l i s populations. Samples were taken between 1996 and 1999 from California, the NE Atlantic, the
Mediterranean Sea, South Africa, Korea, Western Australia, Tasmania, and New Zealand. NE Atlantic M . e d u l i s L. were used a s a n o u t g r o u p . W h i l e a l l M . g a l l o p r o v i n c i a l i s samples were fixed, or nearly so, for the diagnostic G allele at locus Glu-5 ’, c o r r e s p o n d e n c e a n a l y s i s o f
mac-1 allele -f r e q u e n c y d a t a h i g h l i g h t e d t h e g e n e t i c d i s t i n c t n e s s o f A u s t r a l a s i a n m u s s e l s
r e l a t i v e t o o t h er M . g a l l o p r o v i n c i a l i s populations. The latter consisted of two differentiated groups (NE Atlantic and Mediterranean) as formerly reported at allozyme loci. Another sample, from Chile, was nearly identical to Mediterranean M . g a l l o p r o v i n c i a l i s . Nuclear-DNA d a t a t h u s e n f o r c e t h e i d e a t h a t M . g a l l o p r o v i n c i a l i s have probably been introduced from the Mediterranean to the North Pacific (and now Chile), and from the NE Atlantic to South Africa. We argue that Australasian mussels derive from a proto - M . g a l l o p r o v i n c i a l i s p o p u l a t i o n i n t r o g r e s s e d b y M . e d u l i s -l i k e g e n e s , a n d s h o u l d b e c o n s i d e r e d a s a r e g i o n a l s u b s p e c i e s o f M . g a l l o p r o v i n c i a l i s .
Smooth-shelled M y t i l u s s p p . m u s s e l s a r e d i s t r i b u t e d o v e r t h e t e m p e r a t e a n d s u b-polar regions of all oceans. Global surveys of allozyme variation in M y t i l u s s p p . p o p u l a t i o n s h a v e f o c u s e d on the clarification of the systematics of the genus (McDonald & Koehn 1988; Varvio e t a l . 1988; Koehn 1991; McDonald e t a l . 1991). These unambiguously showed that all smooth-shelled M y t i l u s populations in the Southern and the Northern Hemispheres could be ascribed t o o n e o f t h e t h r e e s p e c i e s M . e d u l i s Linnaeus, 1758, M . g a l l o p r o v i n c i a l i s Lamarck, 1819, or
M . t r o s s u l u s Gould, 1850. Full species status is warranted by the fact that each of thes e
entities maintains its genetic integrity over broad geographical areas in spite of hybridization in areas of overlap, and in the face of potential for long-distance dispersal by planktotrophic larvae (Koehn 1991). Although the majority of the genetic dif f e r e n c e s w a s a c c o u n t e d f o r b y this systematic distinction, geographical differentiation was also detected within each species. For example, McDonald e t a l . (1991) recognized Australasian (Australia, Tasmania, New Zealand) M y t i l u s a s M . g a l l o p r o v i n c i a l i s , but noted some differences in allele frequency between the latter, and their Northern Hemisphere conspecifics.
T h e p r e s e n t s t u d y i s c o n c e r n e d w i t h w o r l d w i d e g e n e t i c v a r i a t i o n i n M y t i l u s
g a l l o p r o v i n c i a l i s . Our main objective was to test with novel nuclear-DNA markers the
biogeographical hypotheses arising from the analysis of allozyme loci.
Allozyme surveys of genetic variation in M y t i l u s g a l l o p r o v i n c i a l i s along the Iberian P e n i n s u l a h a v e d e m o n s t r a t e d c l e a r-c u t g e o g r a p h i c a l i s o l a t i o n b e t w e e n t h e n o r t h e a s tern (NE) A t l a n t i c a n d t h e M e d i t e r r a n e a n p o p u l a t i o n s o f t h i s s p e c i e s ( S a n j u a n e t a l . 1994; Quesada e t
a l . 1995). Compilation and analysis of the dataset generated by the above allozyme studies
has since clarified the worldwide picture of the geographic stru cture in M. galloprovincialis (Sanjuan e t a l . 1997). These authors examined the allele frequency data at five allozyme loci using multidimensional scaling analysis on the matrix of Nei’s genetic distance estimates b e t w e e n p o p u l a t i o n s . U n f o r t u n a t e l y , t h e y d i d n o t c o n s i d e r a n y o u t g r o u p , s o t h e i r g e n e t i c n e t w o r k s w e r e u n r o o t e d . T h e y f o u n d e v i d e n c e f o r t h r e e g e n e t i c a l l y d i f f e r e n t i a t e d g r o u p s o f
M . g a l l o p r o v i n c i a l i s : a NE Atlantic group that also included M . g a l l o p r o v i n c i a l i s from South
Africa, a Mediterranean g r o u p t h a t a l s o i n c l u d e d M . g a l l o p r o v i n c i a l i s from California and eastern Siberia, and a third group from Western Australia and Tasmania. New Zealand Mytilus appeared to be more closely related to North Pacific M . g a l l o p r o v i n c i a l i s t h a n t o t h e o t h e r A u s t r a la s i a n p o p u l a t i o n s .
Phylogenetic inference is desirable for more in -d e p t h u n d e r s t a n d i n g o f t h e e v o l u t i o n a r y p r o c e s s e s i n c l u d i n g c o l o n i z a t i o n a n d v i c a r i a n c e t h a t s h a p e t h e g e n e t i c c o m p o s i t i o n o f s p e c i e s . F i g . 1 i s a n e i g h b o u r-joining tree, rooted by M y t i l u s e d u l i s , inferring t h e p h y l o g e n y o f M . g a l l o p r o v i n c i a l i s populations from allele -f r e q u e n c y d a t a a t s e v e n allozyme loci (McDonald e t a l . 1991; Väinölä & Hvilsom 1991; Quesada e t a l . 1995). The seven loci considered here included the five loci examined by San j u a n e t a l . (1997). Jackknife resampling of loci, where each locus was omitted in turn from the dataset supported a principal separation between NE Atlantic / Mediterranean M . g a l l o p r o v i n c i a l i s (samples S e s i m b r a a n d P a l a v a s ) a n d A u s t r a l a s i a n M . g a l l o p r o v i n c i a l i s (samples Albany, Huon River, and Wellington). Fig. 1 also confirms that Californian M . g a l l o p r o v i n c i a l i s ( L o s A n g e l e s ) i s g e n e t i c a l l y c l o s e r t o t h e M e d i t e r r a n e a n t h a n t h e A t l a n t i c p o p u l a t i o n . T h e W e l l i n g t o n ( N e w Zealand) sample occurred within an A u s t r a l a s i a n c l a d e ( t h a t i s , t o g e t h e r w i t h A l b a n y a n d Huon River) 4/7 times, whereas in the remaining 3/7 pseudotrees it diverged slightly before the node separating the Australian (Albany + Huon River) clade from the NE Atlantic / Mediterranean clade.
Ke n c h i n g t o n e t a l . (1995) obtained the sequence of 18S rDNA for several Mytilus spp. s a m p l e s . T h e s e i n c l u d e d M . g a l l o p r o v i n c i a l i s from an introduced and farmed population in Puget Sound in the eastern North Pacific, M . e d u l i s p l a n u l a t u s Lamarck, 1819 from
s o u t h e a s t e r n T a s m a n i a [ M c D o n a l d e t a l . (1991) had concluded that M. edulis planulatus were
M. g a l l o p r o v i n c i a l i s according to both allozyme -frequency and morphometric data], and
several M . e d u l i s a n d M . t r o s s u l u s s a m p l e s . K e n c h i n g t o n a n d c o l l a b o r a t o r s a l s o a n alysed an " M . g a l l o p r o v i n c i a l i s " sample from Morgat (Britanny, France) but we disregarded this
identification because this location lies within the European hybrid zone between M . e d u l i s a n d M. g a l l o p r o v i n c i a l i s ( C o u s t a u e t a l . 1991; Gosling 1992) where the r e i s n o e v i d e n c e f o r t h e p r e s e n c e o f n o n -i n t r o g r e s s e d M . g a l l o p r o v i n c i a l i s ( C o u s t a u e t a l . 1991; C. Daguin, F. Bonhomme, P. Borsa, unpublished data). The phylogeny inferred from the 18S rDNA s e q u e n c e s ( K e n c h i n g t o n e t a l . 1 9 9 5 ) s t r o n g l y s u g g e s t s a n e a r l y s e p a r a t i o n o f N o r t h e r n Hemisphere M . g a l l o p r o v i n c i a l i s from the other smooth-shelled M y t i l u s spp., including M.
e d u l i s , M . t r o s s u l u s, a n d A u s t r a l a s i a n M . g a l l o p r o v i n c i a l i s .
A n u m b e r o f h y p o t h e s e s h a v e b e e n s u g g e s t e d t o a c c ount for the differentiation p a t t e r n s a n d d i s j u n c t w o r l d w i d e d i s t r i b u t i o n o f M y t i l u s g a l l o p r o v i n c i a l i s .
(1) M y t i l u s g a l l o p r o v i n c i a l i s has been introduced to South Africa, the western North Pacific, the eastern North Pacific, and perhaps Australasia through human agency (Wilkins et
a l . 1983; Grant & Cherry 1985; McDonald & Koehn 1988; McDonald e t a l . 1991; Vermeij
1 9 9 2 ) . T h i s h y p o t h e s i s i s b a s e d o n t h e l a c k o f e v i d e n c e f o r M . g a l l o p r o v i n c i a l i s -like mussels in the fossil record and their absence from the lit erature or museum collections in South Africa and in the North Pacific until recently (Wilkins e t a l . 1983; Grant & Cherry 1985; McDonald & Koehn 1988, and references therein; Geller 1999). However, sub-fossil M y t i l u s are present in Aboriginal middens in Tasmania, southern Australia, and New Zealand (McDonald e t a l . 1991, and references therein). This leads to the following, modified h y p o t h e s i s f o r t h e g e n e t i c a f f i n i t i e s o f A u s t r a l a s i a n M . g a l l o p r o v i n c i a l i s .
(2) Introduced M y t i l u s g a l l o p r o v i n c i a l i s h a v e d is p l a c e d n a t i v e A u s t r a l a s i a n Mytilus s p . ( s u g g e s t e d , a l t h o u g h b e l i e v e d t o b e u n l i k e l y b y M c D o n a l d e t a l . 1991) or introgressed with the latter (Seed 1992). Sanjuan e t a l . (1997) also write that “a human introduction of North Pacific mussels into Australia is [...] possible (Carlton 1987) [and] may explain the g e n e t i c h e t e r o g e n e i t y o f A u s t r a l a s i a n s a m p l e s ” . B o t h d i s p l a c e m e n t a n d i n t r o g r e s s i o n b y p r e s u m a b l y i n t r o d u c e d M . g a l l o p r o v i n c i a l i s h a v e a f f e c t e d n a t i v e M . t r o s s u l u s in California (McDonald & Koehn 1988; Geller 1999).
(3) North Pacific M y t i l u s g a l l o p r o v i n c i a l i s h a v e b e e n r e c e n t l y i n t r o d u c e d f r o m t h e South Pacific. Quoting Koehn (1991): “As M . g a l l o p r o v i n c i a l i s i s p r o b a b l y n a t i v e t o l a r g e areas of the South Pacific, introductions into northern Pacific sites [...] may not have originated in Europe.” However, this is contradicted by the quite large genetic dissimilarity between North Pacific and South Pacific M . g a l l o p r o v i n c i a l i s , which is at variance with the close genetic similarities between North Pacific an d M e d i t e r r a n e a n M . g a l l o p r o v i n c i a l i s (Gosling 1992).
(4) Australasian, North Pacific, and Mediterranean M y t i l u s g a l l o p r o v i n c i a l i s all derive from an ancestral Pacific stock (Sanjuan e t a l . 1997). While hypotheses (1) and (2) h e r e a b o v e r e l y o n t h e p r e s u mp t i o n t h a t M . g a l l o p r o v i n c i a l i s is native to the NE Atlantic / Mediterranean, Sanjuan e t a l . (1997) consider the alternative hypothesis that either Australasia or the North Pacific be the geographic origin of M . g a l l o p r o v i n c i a l i s . According t o t h i s h y p o t h e s i s , p r o t o -M . g a l l o p r o v i n c i a l i s crossed the Equator in the Pacific Ocean; North
Pacific M . g a l l o p r o v i n c i a l i s subsequently crossed the Arctic to colonize the NE Atlantic and t h e M e d i t e r r a n e a n . T h i s a m o u n t s t o p r o p o s e t h a t p r e s e n t -day Australasian, North Pacific, and Mediterranean populations are endemic forms of M . g a l l o p r o v i n c i a l i s . The data provided by Sanjuan e t a l . (their Table 4; 1997) however failed to substantiate part of this scenario, since t h e g e n e t i c d i s t a n c e e s t i m a t e b e t w e e n t h e N o r t h P a c i f i c a n d t h e M e d i t e r r a n e a n [ D = 0.029 (0.011-0.057)] was not significantly larger than that among sub-p o p u l a t i o n s w i t h i n t h e Mediterranean [D = 0.028 (0.007-0.054)].
The allozyme -b a s e d p h y l o g e n y p r o p o s e d i n F i g . 1 s u g g e s t s t h a t A u s t r a l a s i a n Mytilus
g a l l o p r o v i n c i a l i s differentiated early from all the other M . g a l l o p r o v i n c i a l i s , supporting the
h y p o t h e s i s t h a t A u s t r a l a s i a n M . g a l l o p r o v i n c i a l i s are endemic (Koehn 1991; McDonald et al. 1991). Fig. 1 f u r t h e r s h o w s t h a t t h e d i v e r g e n c e b e t w e e n N E A t la n t i c a n d M e d i t e r r a n e a n M.
g a l l o p r o v i n c i a l i s i s m o r e r e c e n t t h a n t h e s e p a r a t i o n b e t w e e n t h e A u s t r a l a s i a n a n d t h e N E
Atlantic / Mediterranean groups. The close genetic relationship of Californian with Mediterranean M . g a l l o p r o v i n c i a l i s (see above; Fig. 1) reflects an even more recent common origin, which is indeed compatible with the hypothesis that North Pacific M. galloprovincialis w e r e i n t r o d u c e d f r o m t h e M e d i t e r r a n e a n t h r o u g h h u m a n a g e n c y . Sanjuan e t a l . (1997) instead p r o p o s e t h a t “a genetic mixing between [M. g a l l o p r o v i n c i a l i s a n d M . e d u l i s ] , p e r h a p s combined with selective pressure, may explain the larger allozyme divergence between M.
g a l l o p r o v i n c i a l i s of Mediterranean vs Atlantic populations, than between Mediterranean vs
North Pacific populations”. If this hypothesis were true, allele frequencies in NE Atlantic M.
g a l l o p r o v i n c i a l i s w o u l d b e i n t e r m e d i a t e b e t w e e n t h o s e o f M . e d u l i s a n d M e d i t e r r a n e a n M. g a l l o p r o v i n c i a l i s . T h i s a p p e a r s t o b e t h e c a s e a t o n l y o n e ( A p ), perhaps two (a dding Gpi) out
of the six loci ( A p , Est-D, Gpi, L a p , M p i , O d h ) at which the most significant differences were o b s e r v e d b e t w e e n N E A t l a n t i c a n d M e d i t e r r a n e a n M . g a l l o p r o v i n c i a l i s ( Q u e s a d a et al. 1995) a n d w h i c h w e r e a l s o s c o r e d i n M . e d u l i s (Skibinski e t a l . 1980; Coustau e t a l . 1991; Sanjuan
e t a l . 1994). All these loci were taken into account for computing the neighbour-joining tree
of Fig. 1. The distinctness of the NE Atlantic vs Mediterranean M . g a l l o p r o v i n c i a l i s thus far appears to reflect vicariance more than differential introgression by M . e d u l i s allozyme genes.
T o w h a t e x t e n t d o n u c l e a r-DNA markers enforce and refine the conclusions drawn from allozyme data?
Methods
M y t i l u s g a l l o p r o v i n c i a l i s populations were sampled in California (sample BOD of Fig. 2), the
NE Atlantic (STB), the Mediterranean Sea (SET), South Africa (SAF), eastern Asia (KOR), Western Australia (AUS), Tasmania (TAS), and New Zealand (NZL) between 1996 and 1999. Reference M . e d u l i s L. samples were collected in the North Sea (GFP), the Skagerrak (FLO), and the Kattegat (GIL) in 1996 and 1997. A sample of uncertain taxonomic status was collected in southern central Chile (CHL) in 1998. All mussel shells in this sample were M.
g a l l o p r o v i n c i a l i s a c c o r d i n g t o m o r p h o l o g y . M y t i l u s samples from Chile examined so far at
allozyme loci and using morphometrics were M . e d u l i s -like (McDonald e t a l . 1991; Fig. 1), but a sample from southern Chile recently analysed at nuclear-DNA and mitochondrial-DNA loci exhibited alleles that are identical to those of New Zealand M . g a l l o p r o v i n c i a l i s (Toro 1998) raising the possibility that this species might also be present in southern Chile.
The total genomic DNA was extracted from each individual and used as template for p o l y m e r a s e-chain reactions (PCR) using primer pairs specific of a fragment of the
p o l y p h e n o l i c a d h e s i v e p r o t e i n g e n e Glu-5 ’, and a fragment of intron 1 o f t h e a c t i n g e n e
mac-1 . The Glu-5 ’ marker was developed by Inoue & Odo (mac-1994) and Rawson e t a l . (mac-1996). The mac-1 marker was developed by Ohre s s e r e t a l . (1997) and Daguin & Borsa (1999). Protocols
for DNA extraction, PCR, gel electrophoresis, and allele nomenclature at locus Glu-5 ’ h a v e b e e n r e p o r t e d i n B o r s a e t a l . (1999). Those for locus mac-1 h a v e b e e n d e t a i l e d i n D a g u i n & Borsa (1999). mac-1 i s t h e o n l y n o n -coding locus out of the ten nuclear loci (also including 7 allozyme loci, 18S rDNA, and Glu-5 ’) c o n s i d e r e d i n t h i s p a p e r .
Correspondence analysis (FCA: Benzécri 1982) was performed using the AFC
procedure implemented in BI O M E C O (Lebr e t o n e t a l . 1990) on the matrix of allelic frequencies p e r s a m p l e . T h i s m e t h o d , p r e v i o u s l y u s e d b y C o u s t a u e t a l . (1991) on allozyme data, is well-a d well-a p t e d t o e x p r e s s t h e g e n e t i c d i f f e r e n c e s p r e s e n t i n well-a d well-a t well-a s e t b e c well-a u s e t h e e i g e n v well-a l u e s o f each FCA’s axis a r e a n a l o g u e t o S . W r i g h t ’ s Fst (Guinand 1996).
Results and discussion
Table 1 gives the allele frequencies at locus Glu-5 ’. Allele G was fixed or nearly fixed in all
M y t i l u s g a l l o p r o v i n c i a l i s samples [thereby extending the preliminary findings of Raws o n et a l . (1996) and Borsa e t a l . (1999)] and in the M y t i l u s sp. sample from Chile. The Glu-5 ’
marker, being quasi-monomorphic, is therefore of little help to analyse the genetic r e l a t i o n s h i p s a m o n g M . g a l l o p r o v i n c i a l i s p o p u l a t i o n s .
In contrast, preliminar y s u r v e y s o f g e n e t i c v a r i a t i o n a t t h e mac-1 l o c u s h a v e demonstrated considerable polymorphism, with up to 18 size -alleles in M y t i l u s
g a l l o p r o v i n c i a l i s samples from the NE Atlantic and the Mediterranean (Daguin & Borsa
1999). Here, allele -f r e q u e n c y d a t a a t l o c u s mac-1 s u g g e s t e d c l o s e r g e n e t i c a f f i n i t i e s o f A u s t r a l a s i a n M y t i l u s with M . e d u l i s r a t h e r t h a n M . g a l l o p r o v i n c i a l i s (Table 2): all three A u s t r a l a s i a n s a m p l e s p o s s e s s e d mac-1 allele a 2 at high frequency (this allele is found at moderate frequency in NE Atlantic M . e d u l i s , but at significantly lower frequency in M.
g a l l o p r o v i n c i a l i s : Daguin & Borsa 1999; Table 2) and the Tasmanian and New Zealand
s a m p l e s d i d n o t p o s s e s s a n y o f t h e a l l e l e s ( b 2, c 2) inferred to be characteristic of
Mediterranean and NE Atlantic M . g a l l o p r o v i n c i a l i s (Daguin & Borsa 1999; Table 2) although t h e s e w e r e p r e s e n t a t m o d e r a t e f r e q u e n c y i n W e s t e r n A u s t r a l i a . T h e t h r e e -dimensional p r o j e c t i o n o f t h e F C A o f M . g a l l o p r o v i n c i a l i s samples collected worldwide (Fig. 3) illustrates
t h e c l e a r d i s t i n c t n e s s o f A u s t r a l a s i a n M y t i l u s, with the first axis of FCA separating M. edulis and Australasian mussels from all the other samples. The second axis then clearly
d i s t i n g u i s h e d A u s t r a l a s i a n m u s s e l s f r o m M . e d u l i s . The third axis yielded evidence of c omparatively slighter, further distinction between NE Atlantic (STB) and Mediterranean (SET) M . g a l l o p r o v i n c i a l i s . The homogeneity of mac-1 allele frequencies within each of the two latter regions has been reported elsewhere (Daguin & Borsa 1999; unpublishe d ) . S o u t h African mussels clustered together with NE Atlantic M . g a l l o p r o v i n c i a l i s while mussel samples from California, Chile and Korea clustered together with Mediterranean M.
g a l l o p r o v i n c i a l i s . The estimate of θ, the equivalent of Wright’s Fst (Weir & Cockerham
1984), was ^θ = 0.027 between these two groups. This value compares with the value r e p o r t e d a t l o c u s mac-1 between NE Atlantic (northwestern African) and Western
Mediterranean M . g a l l o p r o v i n c i a l i s (^θ = 0.016; p = 0.02; Daguin & Borsa 1999) and with the mean Gst value reported for 13 allozyme loci between samples from the western and the e a s t e r n c o a s t s o f t h e I b e r i a n P e n i n s u l a ( Gst = 0.029; p < 0.001; Quesada e t a l . 1995).
T h u s , mac-1 data (Daguin & Borsa 1999; present results) are in accordance with the results of former allozyme -based surveys in (1) allowing a clear distinction between NE A t l a n t i c a n d M e d i t e r r a n e a n M y t i l u s g a l l o p r o v i n c i a l i s p o p ulations (Sanjuan e t a l . 1994; Q u e s a d a e t a l . 1995; Sanjuan e t a l . 1997); (2) demonstrating the genetic affinities of South African M . g a l l o p r o v i n c i a l i s with NE Atlantic M . g a l l o p r o v i n c i a l i s (Sanjuan e t a l . 1997); (3) demonstrating the genetic affinities o f M . g a l l o p r o v i n c i a l i s f r o m b o t h t h e e a s t e r n a n d t h e western coasts of the North Pacific with Mediterranean M . g a l l o p r o v i n c i a l i s (Sanjuan e t a l . 1997; Fig. 1); (4) arguing against the possibility that Australasian M . g a l l o p r o v i n c i a l i s were t r a n s p o r t e d t o t he North Pacific (Gosling 1992; Fig. 1).
A u s t r a l a s i a n M y t i l u s are M . g a l l o p r o v i n c i a l i s when considering allozyme data (McDonald e t a l . 1991), even though substantial differences with Northern Hemisphere M.
g a l l o p r o v i n c i a l i s were detected using multidimensional scaling (Sanjuan e t a l . 1997) and
n e i g h b o u r-joining phylogenetic inference (Fig. 1). Australasian M y t i l u s remained grouped with M . g a l l o p r o v i n c i a l i s w h e n c o n s i d e r i n g Glu-5 ’ data (Table 1). However, mac-1 d a t a (Table 2; Fig. 3) revealed strong differenc e s b e t w e e n A u s t r a l a s i a n M y t i l u s a n d N o r t h e r n Hemisphere M . g a l l o p r o v i n c i a l i s . The summing up of all data available regarding the genetic characterization of Australasian M y t i l u s (McDonald e t a l . 1991; Kenchington e t a l . 1995; this s t u d y ) s h o w s n o t i c e a b l e discrepancies among loci. For instance, let us examine allele frequency patterns at the four loci (allozyme loci Est-D a n d M p i ; n u c l e a r-DNA loci Glu-5 ’ a n d mac-1 ) t h a t c a n b e c o n s i d e r e d a s d i a g n o s t i c b e t w e e n M . g a l l o p r o v i n c i a l i s a n d M. edulis (Skibinski e t a l . 1983; Rawson e t a l . 1996; Daguin & Borsa 1999). Australasian M y t i l u s possess fixed or nearly fixed M . g a l l o p r o v i n c i a l i s alleles at M p i a n d Glu-5 ’, while at Est-D t h e f r e q u e n c y o f M . e d u l i s alleles was zero in sample Wellington, ≈0.25 in sample Albany, a n d ≈0.5 in sample Huon River (McDonald e t a l . 1991); at locus mac-1 , A u s t r a l a s i a n M y t i l u s
p o s s e s s a t h i g h f r e q u e n c y a s i z e -allele ( a 2) which is more characteristic of M . e d u l i s t h a n M.
g a l l o p ro v i n c i a l i s (Table 2). At other allozyme loci, Australasian mussels can be considered as
either closer to M . g a l l o p r o v i n c i a l i s t h a n M . e d u l i s (A a p , A p , Gpi, a n d L a p ), or the contrary (P g m), or quite different from both (O d h ) (McDonald e t a l . 1991). Finally, 18S rDNAs of Tasmanian M y t i l u s showed higher sequence similarity with M . e d u l i s ( a n d M . t r o s s u l u s) than with M . g a l l o p r o v i n c i a l i s ( K e n c h i n g t o n e t a l . 1995).
T h e p r e s e n t r e s u l t s p r o v i d e d n e w i n s i g h t i n t o t h e b i o g e o g r a p h y o f M y t i l u s
g a l l o p r o v i n c i a l i s .
Fir s t , A u s t r a l a s i a n M y t i l u s now appear to be clearly distinct from all M.
g a l l o p r o v i n c i a l i s populations elsewhere in the world. We therefore reject the hypothesis that
these mussels were recently introduced by man (from, e.g. the North Pacific), or that they may h a v e u n d e r g o n e r e c e n t , s u b s t a n t i a l i n t r o g r e s s i o n b y a l i e n M . g a l l o p r o v i n c i a l i s (e.g. Seed 1992). Instead, Australasian M . g a l l o p r o v i n c i a l i s h a v e a p a t c h y g e n e t i c a r c h i t e c t u r e , w i t h h i g h f r e q u e n c y o f M . e d u l i s -like alleles at some loci and of M . g a l l o p r o v i n c i a l i s -like alleles at o t h e r l o c i . A s s u m i n g t h a t t h e c h o i c e o f M . e d u l i s a s a n o u t g r o u p o f M. galloprovincialis holds v a l i d , t h i s s u g g e s t s t h a t A u s t r a l a s i a n M y t i l u s originate from a proto-M . g a l l o p r o v i n c i a l i s p o p u l a t i o n t h a t u n d e r w e n t i n t r o g r e s s i o n b y p r o t o -M . e d u l i s . In other words, introgression by
M . e d u l i s -like alleles was detected i n A u s t r a l a s i a n m u s s e l s a t l o c i mac-1 a n d Est-D (and,
p e r h a p s , P g m) b u t t h i s m a y b e a n c i e n t s i n c e a t l o c u s mac-1 these alleles are now fixed or q u a s i-fixed. It is also possible that the M . e d u l i s -l i k e g e n e s o f A u s t r a l a s i a n m u s s e l s d o n o t derive from a proto-M . e d u l i s form, but originate from more modern Southern Hemisphere M.
e d u l i s (South America, Falklands, Kerguelen; McDonald e t a l . 1991). We expect that
sequencing analysis will allow us to tell whether the mac-1 a l l e l e s p r e s e n t i n A u s t r a l a s i a n mussels are phylogenetically closer to those from Northern Hemisphere, or Southern Hemisphere M . e d u l i s . On the basis of the close morp hological and allozymic resemblance b o r n e b y A u s t r a l a s i a n m u s s e l s t o M . g a l l o p r o v i n c i a l i s (McDonald e t a l . 1991), we propose t h a t t h e s e b e a t t r i b u t e d a s u b s p e c i f i c r a n k w i t h i n M . g a l l o p r o v i n c i a l i s .
Second, we reject the hypothesis, implicit in Sanjuan e t a l . (1997), that North Pacific and Mediterranean mussels are endemic forms of M . g a l l o p r o v i n c i a l i s b e c a u s e t h e r e w a s n o e v i d e n c e o f g e n e t i c d i f f e r e n t i a t i o n b e t w e e n p o p u l a t i o n s o f t h e s e t w o r e g i o n s . T h e c l o s e genetic similarity at all loci including mac-1 , o f p o p u l a t i o n s s e p a r a t e d b y s u c h g e o g r a p h i c a l d i s t a n c e s a n d b y g e o g r a p h i c a l b a r r i e r s ( c o n t i n e n t s ) i s c o n f o r m t o t h e h y p o t h e s i s o f r e c e n t introduction by man. Since fossil M . g a l l o p r o v i n c i a l i s have been found in the Mediterranean (Mars 1956) and have not been reported from the North Pacific (see Introduction), it is legitimate to presume that North Pacific M . g a l l o p r o v i n c i a l i s were introduced from the Mediterranean and not the contrary. Rejecting the present existence of endemic North Pacific
M . g a l l o p r o v i n c i a l i s d o e s n o t n e c e s s a r i l y i m p l y t h a t M . g a l l o p r o v i n c i a l i s is a Mediterranean
o f f s h o o t o f N o r t h A t l a n t i c M . e d u l i s as frequently assumed (Barsotti & Meluzzi 1968; Skibinski e t a l . 1983; Gosling 1984, 1992; Grant & Cherry 1985; Seed 1992; Rawson &
Hilbish 1998). As emphasized by Vermeij (1992), "It is possible that the original Tethyan
M y t i l u s p e r s i s t e d i n t h e M e d i t e r r a n e a n r e g i o n s t h r o u g h o u t t h e N e o g e n e , a n d t h a t t h e
P l e i s t o c e n e a n d R e c e n t M . g a l l o p r o v i n c i a l i s is derived from this purported stock rather than from the Pacific -derived t r o s s u l u s-e d u l i s g r o u p " , a l t h o u g h f o s s i l e v i d e n c e f o r t h i s s c e n a r i o remains to be found.
Third, M . g a l l o p r o v i n c i a l i s was here identified for the first time in Chile. Its high genetic similarity, at locus mac-1 , with Mediterranean and North Pacific M. galloprovincialis suggests it has been recently introduced to Chile from either of these regions by maritime t r a n s p o r t o r p e r h a p s b y u n r e p o r t e d , i n t e n t i o n a l t r a n s p l a n t a t i o n . T h e p o p u l a t i o n f r o m w h i c h our Chilean sample originates thus a p p e a r s t o b e d i f f e r e n t f r o m a n y o f t h e M . e d u l i s -like C h i l e a n p o p u l a t i o n s s a m p l e d b y M c D o n a l d e t a l . (1991), but it is perhaps the same as the one s a m p l e d b y T o r o ( 1 9 9 8 ) . I n t h i s c a s e , t h e t a x o n o m i c s t a t u s p r o p o s e d b y T o r o f o r t h e s e m u s s e l s ( a s u b s p e c i e s o f M . e d u l i s ) s h o u l d b e d i s r e g a r d e d .
Note added in proof: A recent mitochondrial-DNA survey aimed at elucidating the origin of
the antitropical distribution pattern of M y t i l u s spp. [Hilbish e t a l ., M a r . B i o l . 136, 69-77 (2000)] yielded results that conf i r m t h e m a i n c o n c l u s i o n s o f t h e p r e s e n t s t u d y . M o s t A u s t r a l a s i a n M y t i l u s spp. female mitochondrial lineages clustered into a single clade (D2 ) w h o s e c l o s e s t r e l a t i v e w a s t h e D c l a d e f o u n d i n n o r t h e r n M . g a l l o p r o v i n c i a l i s (D1 ), d e m o n s t r a t i n g b o t h t h e o r i g i n a l i t y o f A u s t r a l a s i a n M y t i l u s s p . a n d t h e i r c l o s e r e l a t e d n e s s t o
M . g a l l o p r o v i n c i a l i s . A f e w A u s t r a l a s i a n m u s s e l s h o w e v e r p o s s e s s e d m i t o c h o n d r i a o f t h e A
t y p e c h a r a c t e r i s t i c o f M . e d u l i s : t h i s i s c o n s i s t e n t w i t h o u r h y p o t h e s is that these Australasian mussels derive from a proto-M . g a l l o p r o v i n c i a l i s p o p u l a t i o n i n t r o g r e s s e d b y M . e d u l i s – like g e n e s . M i t o c h o n d r i a l-D N A d a t a w e r e a l s o c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t N o r t h P a c i f i c
M . g a l l o p r o v i n c i a l i s originate from the Mediterra n e a n t h r o u g h h u m a n a g e n c y ( a l l m u s s e l s
from San Diego, California possessing mitochondria of type D1 or A a s d o M e d i t e r r a n e a n mussels), but Hilbish e t a l . (2000) failed to detect D1 haplotypes in their Chilean samples.
We are grateful to N. Bierne, F. Bonhomme, B. Delay, M. Raymond and R.D. Ward for discussions; to J. Taylor, R.D. Ward, and an anonymous referee for comments on an earlier manuscript; to J. Beesley, W. Borgeson, P. Boudry, P. Fréon, A. Leitao, C. Lemaire, D. Moraga, M. Ohresser, J. Panfili, C. Perrin, M. Raymond and C. Riquelme for providing samples; to V. Rolland for carefully examining the Chilean M y t i l u s g a l l o p r o v i n c i a l i s s h e l l s ; to S. Ramos Caetano for assistance in the laboratory; to IFREMER URM 16 for research funds; to MENRT and IRD for our salaries.
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Table 1. A l l e l e f r e q u e n c i e s a t l o c u s Glu-5’ i n s a m p l e s o f M y t i l u s galloprovincialis from the
N o r t h e r n a n d t h e S o u t h e r n H e m i s p h e r e s , a n d i n t w o M. edulis reference samples
Allele* Sample†
BOD CHL STB SET SAF KOR AUS TAS NZL FLO GIL
E − − − 0.01 0.05 − − − − 0.89 0.50 E' − − − − − − − − − 0.11 0.47 E'' − − − − − − − − − − 0.03 G 0.98 1.00 1.00 0.99 0.95 1.00 1.00 1.00 1.00 − − T 0.02 − − − − − − − − − − (N)‡ (23) (48) (19) (56) (65) (19) (46) (25) (77) (35) (16) * nomenclature of Borsa e t a l . (1999)
† abbreviations for samples as in legend to Fig. 2; data for GIL, BOD, and SET from Borsa e t
a l . (1999); additional data for SET from Rawson e t a l . (1996)
Table 2. A l l e l e f r e q u e n c i e s a t l o c u s mac-1 i n s a m p l e s o f M y t i l u s galloprovincialis from the
N o r t h e r n a n d t h e S o u t h e r n H e m i s p h e r e s , a n d i n t h r e e M. edulis reference samples
Allele* Sample† BOD CHL STB SET SAF KOR AUS TAS NZL FLO GIL GFP
f1 – – 0.02 – 0.02 – – – – – – – f2 – – – – 0.01 – – – – – – – f3 – 0.01 – – 0.01 – – – – – – – b0 – – – – 0.01 – – – – – – – b05 – – – – – – – – 0.02 – – – b2 0.04 0.05 0.04 0.05 0.04 0.02 – – – – – – b1 0.32 0.32 0.15 0.21 0.09 0.42 0.04 – – – – 0.01 b3 – 0.01 – – – 0.02 – – – – – – b4 – – 0.02 – – – – – – – – – b5 – – 0.02 – – – – – – – – – c1 0.04 0.08 0.10 0.07 0.10 0.05 – – – – – 0.01 c12 – 0.01 – – – – – – – – – – c15 – – – – 0.01 – – – – – – – c2 0.41 0.39 0.50 0.54 0.53 0.43 0.16 – – – – – c3 – 0.01 0.02 – 0.04 – – – – – – – c4 – – – – – – – – – 0.05 – 0.02 c6 0.01 – – 0.01 0.01 – – – – – – – a0 – – – – – – – – – 0.02 – – a05 – – – – – – – – 0.04 – – – a1 – – 0.02 – – – – – – 0.06 0.02 0.01 a15 – – – – – – 0.01 – 0.01 – – – a2 – 0.03 0.02 – 0.02 0.02 0.62 0.99 0.92 0.10 0.15 0.20 a3 0.03 – 0.04 0.01 0.03 – 0.16 0.01 0.02 0.29 0.31 0.24 a4 – 0.01 – – – – – – – 0.07 0.17 0.18 a5 0.10 0.01 – – 0.02 – – – – 0.38 0.27 0.29 a6 – 0.01 – 0.01 0.02 – 0.01 – – – 0.08 0.04 a7 0.01 0.03 0.02 0.04 0.04 0.03 – – – – – – a8 0.01 0.05 0.04 0.06 0.01 0.02 – – – – – – a9 – – – – – – – – – 0.01 – – d – – – – – – – – – 0.01 – – (N)‡ (34) (76) (26) (68) (62) (30) (38) (40) (79) (47) (26) (42)
* nomenclature of Daguin & Borsa (1999); alleles are presented from the slower migrating (f1) to the faster migrating (d )
† abbreviations for samples as in legend to Fig. 2; data for GIL and SET from Daguin & Borsa (1999)
Fig. 1. M y t i l u s g a l l o p r o v i n c i a l i s . N e i g h b o u r-joining tree (Saitou & Nei 1987; NEIGHBOR procedure of PH Y L I P: Felsenstein 1993) constructed from the matrix of absolute genetic distance estimates (Gregorius 1984), based on electromorph frequency data at 7 allozyme loci (A p , Est-D, Gpi, L a p ,
M p i , O d h , P g m) in samples ‘Los Angeles, California’ (McDonald e t a l . 1991), ‘Palavas’
(Mediterranean) and ‘Sesimbra’ (NE Atlantic) (Quesada e t a l . 1995), ‘Yaldad Bay, Chile’, ‘Albany, W e s t e r n A u s t r a l i a ’, ‘Huon River Estuary, Tasmania’ and ‘Wellington, New Zealand’ (McDonald et
a l . 1991). Numbers at a node are scores of across-locus jacknife resampling. Electromorph identities
w e r e d e d u c e d f r o m c r o s s -comparisons of electromorph frequencies in McDonald & Koehn (1988), M c D o n a l d e t a l . (1991), Väinölä & Hvilsom (1991) and Quesada e t a l . (1995). M . e d u l i s sample ‘Skagerrak’ of Väinölä & Hvilsom (1991) which is also ‘SWE’ of Varvio e t a l . (1988) was used as o u t g r o u p t o r o o t t h e t r e e . T h e s e s a m p l e s w e r e c h o s e n because of their geographical proximity ( =) with samples BOD (= ‘Los Angeles’), CHL (= ‘Yaldad Bay’), STB (= ‘Sesimbra’), SET (=
‘Palavas’), AUS (= ‘Albany’), TAS (= ‘Huon River’), NZL (= ‘Wellington’), and FLO (=
‘Skagerrak’) of the present study (see Fig . 2). Rooting the entire tree with M . t r o s s u l u s sample ‘Tillamook, Oregon’ of McDonald e t a l . ( 1 9 9 1 ) d i d n o t c h a n g e i t s t o p o l o g y ( d a t a n o t s h o w n ) , confirming that the choice of M . e d u l i s a s o u t g r o u p f o r a l l M . g a l l o p r o v i n c i a l i s was appropriate. Scale bar = 0 . 1 u n i t a b s o l u t e g e n e t i c d i s t a n c e
Fig. 2. Sampling sites for M y t i l u s g a l l o p r o v i n c i a l i s . Abbreviations for samples: BOD Bodega Bay,
California; CHL Dichato, southern Central Chile; STB Setubal, Portugal; SET Sète, France [= sample SETE of Daguin & Borsa (1999)]; S A F Bloubergstrand, South Africa; KOR w e s t e r n c o a s t o f S o u t h Korea; AUS N e d l a n d s , W e s t e r n A u s t r a l i a ; TAS d’Entrecasteaux Channel, Tasmania; NZL Dunedin, New Zealand. Symbols refer to the present nuclear-DNA characterization of samples as
" M e d i t e r r a n e a n " M . g a l l o p r o v i n c i a l i s ( s q u a r e s ) , " A t l a n t i c " M. g a l l o p r o v i n c i a l i s (circles), and " A u s t r a l a s i a n " M . g a l l o p r o v i n c i a l i s (diamonds). Triangles symbolize M . e d u l i s reference samples:
FLO Flodevigen, Skagerrak; GFP Grand Fort Philippe, North France; GIL Gilleleje, Kattegat [=
sample GILL of Daguin & Borsa (1999)]
Fig. 3. M y t i l u s g a l l o p r o v i n c a l i s a n d M . e d u l i s . Three-dimensional representation of the outcome of
correspondence analysis (Benzécri 1982) on the matrix of mac-1 allelic frequencies per sample. Abbreviation for samples as in legend to Fig. 2
0.1 Skagerrak Yaldad Bay Sesimbra Palavas Los Angeles Wellington Huon River Albany 7 7 7 6 3 4
SET KOR BOD CHL STB SAF AUS NZL TAS FLO GIL GFP
Axis 3 (5.89%)
