An RFLP assay to determine if Mytilus galloprovincialis Lmk. (Mytilidae; Bivalvia) is of Northern or Southern hemisphere origin.
ABSTRACT Mytilus galloprovincialis is one of three smooth shelled blue mussel species belonging to the Mytilus edulis species complex. Naturally occurring and introduced populations of M. galloprovincialis are widely distributed throughout many regions of the globe. Mytilus galloprovincialis includes morphologically indistinguishable Northern and Southern hemisphere mtDNA lineages that have been separated for ∼1 my. To distinguish recently introduced Northern M. galloprovincialis from resident Southern M. galloprovincialis in New Zealand, we developed a 16s rRNA RFLP assay. We compared RFLP assignments of 178 mussels with those generated from a 16s rRNA sequence-estimated phylogeny. All mussels were correctly assigned by the RFLP to their sequence-based phylogenetic placement. This assay allows the rapid identification of Northern and Southern hemisphere M. galloprovincialis and will provide an important tool for monitoring human mediated introductions of otherwise cryptic lineages.
- Journal of Molluscan Studies - J MOLLUS STUD. 01/2000; 66(4):573-575.
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ABSTRACT: The non-native Mediterranean mussel Mytilus galloprovincialis is broadly established in the northeast Pacific. Until recently, the coast north of Humboldt Bay, California, USA, was not considered a major zone of sympatry and hybridization with the native sibling species M. trossulus. However, M. galloprovincialis has been introduced in Washington, USA, and British Columbia, Canada, for aquaculture, has been collected from ballast water in-bound to Oregon, USA, and is now reported widely in Puget Sound, Washington. Here I review published reports of M. galloprovincialis alleles in the Northeast Pacific, including recent data showing that these alleles are more widespread and abundant in Washington than previously known. These results indicate the presence of a major zone of sympatry and hybridization in Washington waters that may be contiguous with the California zone. Because M. galloprovin-cialis has likely been introduced to the region on multiple occasions via multiple routes, it is unlikely that a sole source can be identified. Factors influencing the success and impacts of this now widespread invader remain to be investigated.
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ABSTRACT: Smooth-shelled mussels, Mytilus spp., have an antitropical distribution. In the Northern Hemisphere, the M. edulis complex of species is composed of three genetically well delineated taxa: M. edulis, M. galloprovincialis and M. trossulus. In the Southern Hemisphere, morphological characters, allozymes and intron length polymorphisms suggest that Mytilus spp. populations from South America and Kerguelen Islands are related to M. edulis and those from Australasia to M. galloprovincialis. On the other hand, a phylogeny of the 16S rDNA mitochondrial locus demonstrates a clear distinctiveness of southern mussels and suggests that they are related to Mediterranean M. galloprovincialis. Here, we analysed the faster-evolving cytochrome oxidase subunit I locus. The divergence between haplotypes of populations from the two hemispheres was confirmed and was found to predate the divergence between haplotypes of northern M. edulis and M. galloprovincialis. In addition, strong genetic structure was detected among the southern samples, revealing three genetic entities that correspond to (1) South America and Kerguelen Island, (2) Tasmania, (3) New Zealand. Using the trans-Arctic interchange as a molecular clock calibration, we estimated the time since divergence of populations from the two hemispheres to be between 0.5 million years (MY) and 1.3 MY (average 0.84 MY). The contrasting patterns observed for the nuclear and the organelle genomes suggested two alternative, complex scenarios: two trans-equatorial migrations and the existence of differential barriers to mitochondrial and nuclear gene flow, or a single trans-equatorial migration and a view of the composition of the nuclear genome biased by taxonomic preconception.Molecular Phylogenetics and Evolution 08/2008; 49(1):84-91. · 4.07 Impact Factor
MOLECULAR DIAGNOSTICS AND DNA TAXONOMY
An RFLP assay todetermine if Mytilus galloprovincialis
Lmk. (Mytilidae; Bivalvia) is of Northern orSouthern
K. M. WESTFALL,* P. H. WIMBERGER† and J. P. A. GARDNER*
*Centre for Marine Environmental and Economic Research, School of Biological Sciences, Victoria University of Wellington,
Wellington 6140, New Zealand, †Department of Biology, and Slater Museum of Natural History, University of Puget Sound,
Tacoma, WA 98416, USA
Mytilus galloprovincialis is one of three smooth shelled blue mussel species belonging to
the Mytilus edulis species complex. Naturally occurring and introduced populations of
M. galloprovincialis are widely distributed throughout many regions of the globe. Mytilus
galloprovincialis includes morphologically indistinguishable Northern and Southern
hemisphere mtDNA lineages that have been separated for ?1 my. To distinguish recently
introduced Northern M. galloprovincialis from resident Southern M. galloprovincialis in
New Zealand, we developed a 16s rRNA RFLP assay. We compared RFLP assignments of
178 mussels with those generated from a 16s rRNA sequence-estimated phylogeny.
All mussels were correctly assigned by the RFLP to their sequence-based phylogenetic
placement. This assay allows the rapid identification of Northern and Southern hemisphere
M. galloprovincialis and will provide an important tool for monitoring human mediated
introductions of otherwise cryptic lineages.
Keywords: mitochondrial DNA lineage, Mytilus edulis species complex, Mytilus galloprovincialis,
Northern vs. Southern hemisphere, species identification
Received 25 June 2009; revision received 10 August 2009; accepted 22 August 2009
Mytilus galloprovincialis is a widely distributed intertidal
invertebrate species in many temperate and subtropical
regions of the world as the result of natural range
expansion and human-mediated transport. It is one of
three sibling species in the M. edulis species complex:
Mytilus edulis Linne, 1758, M. galloprovincialis Lmk, 1819,
M. trossulus Gould, 1850 (McDonald et al. 1991). The
phylogenetic history of these species suggests that
M. galloprovincialis diverged from the ancestral M. edulis
in the Mediterranean Sea approximately 1 to 1.5 mya
(Barsotti & Meluzzi 1968; Riginos et al. 2004), and then
migrated to the Southern hemisphere via the Atlantic
Ocean during the Pleistocene approximately 0.84 to 1.2
mya (Hilbish et al. 2000; Gerard et al. 2008). Mytilus
galloprovincialis is now widely established in marine
temperate and subtropical waters of both hemispheres
as the result of human-mediated introductions (Apte
et al. 2000; Wonham 2004).
Rawson & Hilbish (1995) developed an RFLP assay
based on a 16s rRNA mtDNA fragment that allows rapid
identification of Northern hemisphere lineages within
the M. edulis species complex. Their assay showed that
M. edulis and M. trossulus have fixed unique haplotypes
(A and B respectively), while M. galloprovincialis exhib-
ited roughly equal frequencies of a third unique haplo-
type (D) and the A haplotype of M. edulis (Rawson &
Phylogenetic analysis of the 16s rRNA fragment
revealed four distinct lineages (Hilbish et al. 2000).
Mytilus trossulus is the sister group to the other Mytilus
taxa (Hilbish et al. 2000). The M. edulis mitochondrial
lineage includes both M. edulis and M. galloprovincialis
individuals (as identified by nuclear markers – denoted
M. edulis⁄M. galloprovincialis). The M. galloprovincialis
Correspondence: Kristen M. Westfall, Fax: +064-04-463-5331;
? 2009 Blackwell Publishing Ltd
Molecular Ecology Resources (2010) 10, 573–575doi: 10.1111/j.1755-0998.2009.02779.x
clade is composed of monophyletic Northern and South-
ern hemisphere sub-clades (Hilbish et al. 2000). The RFLP
assay described here distinguishes the Northern and
Southern M. galloprovincialis sub-clades.
Examination of 16s rRNA DNA sequences (Hilbish
et al. 2000) indicated that a triple digest using enzymes
EcoRV, NheI and SpeI distinguishes between Northern
and Southern M. galloprovincialis (Fig. 1). To test the effec-
tiveness of this new assay, we performed the triple digest
and sequenced 135 mussels of all three taxa from loca-
tions around the world.
Total cellular DNA was extracted from posterior
adductor muscle tissue (Sokolov 2000). A 527 bp frag-
ment of the 16s rRNA gene was amplified using primers
16sAR⁄16sBR (Palumbi 1996) in a 25 lL reaction contain-
ing 100 ng genomic DNA, 10 pmol each primer, 10 nmol
total dNTP, 1X reaction buffer, 1.5 mM MgCl2and 1U
Taq. The amplification conditions were 3 min at 95 ?C, 30
cycles at 95 ?C for 30 s, 52 ?C for 30 s and 72 ?C for 45 s,
and final extension at 72 ?C for 3 min. A 20 lL triple
restriction endonuclease digest containing 10 U EcoRV,
5 U NheI, 5 U SpeI, 1X NEB Buffer #2, 100 mg BSA and
10 lL PCR product was incubated overnight at 37 ?C.
PCR and restriction products were scored on 1.5% aga-
rose gels stained with ethidium bromide. Please observe
that bands less than 100 bp in length are very faint
because of electrophoretic conditions (Fig. 1), but this
does not affect the scoring capabilities as the sizes of the
other bands produced are diagnostic for the haplotype
groups described here. Nuclear DNA species identities
were obtained using the species-specific nuclear-DNA
PCR assay Me 15⁄16 (Inoue et al. 1995).
to GQ455405) from RFLP assayed mussels were aligned
with ClustalW (Thompson et al. 1994) and a maximum
likelihood phylogeny was
(Swofford 2003; model with 500 bootstrap iterations
available from K Westfall). The topology is not reported
here, but the major clades corresponded to those
reported by Hilbish et al. (2000). Hybrids as determined
by Me15⁄16 were not used in the analysis. Some
profiles included a faint band at ?600 bp representing
amplification of the male mitotype 16s rRNA gene;
All 178 M. galloprovincialis and M. edulis RFLP assign-
ments matched their placement in the 16s rRNA
sequence-based phylogeny (33 Northern M. galloprovin-
cialis, 80 Northern M. galloprovincialis⁄M. edulis, 65 South-
ern M. galloprovincialis). The assay also correctly assigned
all 12 M. trossulus that were sequenced. Thus, this RFLP
assay is a robust and rapid method to distinguish North-
ern hemisphere M. galloprovincialis and M. edulis from
Southern hemisphere M. galloprovincialis. The assay will
be useful for monitoring the introduction and possible
spread of Northern hemisphere M. galloprovincialis in the
Southern hemisphere, which can occur through hull foul-
ing, ballast water exchange and aquaculture. Used
together with the species-specific nuclear DNA Me15⁄16
marker, this RFLP assay is a powerful tool to assess rap-
idly the taxonomic status of members of the Mytilus edulis
Thanks to the University of Puget Sound for funding PHW’s
sabbatical leave. Partial support from Biosecurity New Zealand
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Interspecific variations in adhesive protein sequences of
for: (1) Southern hemisphere M. galloprovincialis (lanes 2, 3, 4)
fragments at (342, 167, 28) bp, (2) Northern hemisphere M. gallo-
provincialis (lanes 6, 7, 8) fragments at (342, 195) bp and (3)
Northern hemisphere M. edulis⁄M. galloprovincialis (lanes 10, 11,
12) fragments at (342, 85, 82, 28) bp. Note fragments under 100bp
are difficult to visualize, this does not affect scoring. Lanes 1, 5
and 9 are 100bp size ladder. M. trossulus profile not visualized,
fragments are at (370, 85, 82) bp.
Agarose gel image showing restriction fragment profiles
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574 MOLECULAR DIAGNOSTICS AND DNA TAXONOMY
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