Southern hemisphere origin.
Westfall KM, Wimberger PH, Gardner JPA (2010) An RFLP assay to determine if Mytilus galloprovincialis Lmk. (Mytilidae; Bivalvia) is of Northern or Southern hemisphere origin. Molecular Ecology Resources 10: 573-575.
2.1A 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. M. 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 to 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.
2.2A Introduction
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, Gérard et al. 2008). M. 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 exhibited roughly equal frequencies of a third unique haplotype (D) and the A haplotype of M. edulis (Rawson & Hilbish 1995).
Phylogenetic analysis of the 16s rRNA fragment revealed four distinct lineages (Hilbish et al. 2000). M. 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 clade is composed of monophyletic Northern and Southern 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 (Figure 2.1). To test the effectiveness of this new assay we performed the triple digest and sequenced 135 mussels of all three taxa from locations around the world.
2.3A Materials and Methods
Total cellular DNA was extracted from posterior adductor muscle tissue (Sokolov 2000). A 527 bp fragment of the 16s rRNA gene was amplified using primers 16sAR/16sBR (Palumbi 1996) in a 25 µL reaction containing 100 ng genomic DNA, 10 pmol each primer, 10 nmol total dNTP, 1X reaction buffer, 1.5 mM MgCl2, and 1U Taq. The amplification conditions were 3 min at 95ºC, 30 cycles at 95ºC for 30 s, 52ºC for 30 s and 72ºC for 45 s, and final extension at 72ºC for 3 min. A 20 µL triple restriction endonuclease digest containing 10U EcoRV, 5U NheI, 5U SpeI, 1X NEB Buffer #2, 100 mg BSA and 10 µL PCR product was incubated overnight at 37ºC. PCR and restriction products were scored on 1.5% agarose gels stained with ethidium bromide. Please observe that bands less than 100 bp in length are very faint due to electrophoretic conditions (Figure 2.1), 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).
2.4A Results and Discussion
16s rRNA sequences (Genbank Accession #’s GQ455380 to GQ455405) from RFLP assayed mussels were aligned with ClustalW (Thompson 1994) and a maximum likelihood phylogeny was estimated in PAUP*4.0 (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; this band was ignored for genotyping.
Figure 2.1 - Agarose gel image of restriction fragment profiles.
(1) Southern hemisphere M. galloprovincialis (lanes 2, 3, 4) fragments at (342, 167, 28) bp, (2) Northern hemisphere M. galloprovincialis (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.
All 178 M. galloprovincialis and M. edulis RFLP assignments matched their placement in the 16s rRNA sequence-based phylogeny (33 Northern M. galloprovincialis, 80 Northern M. galloprovincialis/M. edulis, 65 Southern 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 Northern 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 fouling, ballast water exchange and aquaculture. Used together with the species-specific nuclear DNA Me15/16 marker, this RFLP assay is a powerful tool to rapidly assess the taxonomic status of members of the Mytilus edulis species complex.
2.5A References
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Barsotti G, Meluzzi C (1968) Osservazioni su Mytilus edulis L. e Mytilus galloprovincialis Lamarck (A proposito dei Mytilus di Viserbella, Rimini). Conchiglie 4: 50-58. Gérard K, Bierne N, Borsa P, Chenuil A, Feral J-P (2008) Pleistocene separation of
mitochondrial lineages of Mytilus spp. mussels from Northern and Southern hemispheres and strong genetic differentiation among southern populations. Molecular Phylogenetics and Evolution 49: 84-91.
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Inoue K, Waite JH, Matsouka M, Odo S, Harayama S (1995) Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus. Biological Bulletin 189: 370-375.
Palumbi SR (1996) Nucleic acids II: the polymerase chain reaction. In: Molecular Systematics 2nd edition (eds. Hillis DM, Morris C, Mabel BK), pp. 205-247.
Sinauer Associates, Sunderland, Massachusetts.
Rawson PD, Hilbish TJ (1995) Distribution of male and female mtDNA lineages in populations of blue mussels, Mytilus trossulus and M. galloprovincialis, along the Pacific coast of North America. Marine Biology 124: 245-250.
Riginos C, Hickerson MJ, Henzler CM, Cunningham CW (2004) Differential patterns of male and female mtDNA exchange across the Atlantic Ocean in the blue mussel, Mytilus edulis. Evolution 58: 2438-2451.
Sokolov EP (2000) An improved method for DNA isolation from mucopolysaccharide-rich molluscan tissues. Journal of Molluscan Studies 66: 573-575.
Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position- specific gap penalties and weight matrix choice. Nucleic Acids Research 22: 4673- 4680.
Wonham MJ (2004) Mini Review: Distribution of the Mediterranean mussel, Mytilus galloprovincialis (Bivalvia: Mytilidae) and hybrids in the Northeast Pacific. Journal of Shellfish Research 23: 535-543.