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Recent advances in molecular biological techniques have had profound consequences for our understanding of genetics, population biology, phylogeny and evolution of the Mollusca. These new developments were discussed at a one-day meeting held at the Natural History Museum, London, on 7 January 1998.

The aims were twofold:

  • to introduce non-specialists to the potential of molecular methods for the study of molluscs, and
  • to provide an opportunity for molecular researchers to meet and discuss techniques, problems and future developments.

The meeting was well attended, with about 60 participants, from 9 countries, and a wine reception followed. Nine lectures were delivered, and posters were also displayed.


Papers

  • D. Skibinski

    Mitochondrial DNA evolution in Mytilus

  • T. Backeljau

    The relationships of molluscs within the Metazoa: molecules versus morphology

  • V. Koufopanou, D.G. Reid, S.A. Ridgway & R.H. Thomas :

    A phylogeny of the patellid limpets, from mitochondrial gene sequences.

  • C. Wade, B.J. Clarke & P.B. Mordan :

    Towards a molecular phylogeny for the pulmonate land snails.

  • C. Wilding, J. Grahame & P.J. Mill:

    Gene flow and speciation in Littorina: where are the useful sequences?

  • I.F. Wilson & E.M. Gosling :

    The use of SSCPs and allozymes to investigate speciation in the Littorina saxatilis species complex

  • D. Rollinson, J.R. Stothard, S. Hughes, A. Lockyer, C.S. Jones & L. R. Noble:

    Molecular markers for medically-important snails

  • P.W. Shaw :

    Stock structure in a squid: are molecular markers helpful?

  • R.I. Lewis, Q. Huang, Y.M. Mak & G.A. Williams:

    An application of genetic markers to questions of conservation in Littoraria ardouiniana



  • Mitochondrial DNA evolution in Mytilus

    D. Skibinski (University College of Swansea)

    Mussels of the genus Mytilus have two types of mitochondrial DNA (mtDNA). The M type is transmitted paternally and the F type is transmitted maternally. The two genomes are highly diverged, by up to 20% or more, in base sequence. This unusual type of mtDNA inheritance has also been reported in freshwater unionid mussels. Because sperm carry few mitochondria compared with eggs, the M genome must replicate faster in those individuals destined to become males and must also be partitioned preferentially into sperm. The precise mechanisms responsible remain unidentified. Taxa from the Mytilus edulis species-complex (M. edulis, M. trossulus and M. galloprovincialis) are closely related, frequently hybridise in contact zones and show introgression of nuclear genes. Work on North American populations has demonstrated that the three taxa have distinct mtDNA haplotypes and that there are barriers to mtDNA gene flow between the taxa, presumably mediated through nuclear-cytoplasmic interactions.

    Our studies on European Mytilus populations provide different results. Mytilus edulis and M. trossulus have a high frequency of haplotypes in common, indicative of extensive asymmetric gene flow between the taxa. Moreover it appears that the M genome in M. trossulus has been replaced by variant F genomes. Populations of M. galloprovincialis in the eastern and western Mediterranean have highly distinct mtDNA genotypes for both F and M genomes, suggesting divergence following ancient vicariant events. Thus in Europe there is no close correspondence between taxonomic boundaries and mtDNA differences. This highlights the danger of basing generalisations about the evolutionary forces influencing mtDNA evolution on studies of populations from a restricted geographic area.

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    The relationships of molluscs within the Metazoa: molecules versus morphology

    T. Backeljau (Royal Belgian Institute of Natural Sciences,Brussels)

    The phylogenetic position of the Mollusca within the framework of Metazoa is still a highly controversial issue, because hitherto there appear to be no straightforward synapomorphies that unambiguously place molluscs as a sister-group to any other single phylum. Yet, there is currently little doubt that the Mollusca is a monophyletic protostomian phylum. Recent new ultrastructural, molecular and palaeontological data have provided exciting (though controversial) evidence with respect to molluscan phylogeny, although without being able to definitely pinpoint sister-group relationships. This talk discussed a number of these recent studies, and in particular focussed on: (1) the Articulata versus Eutrochozoa debate; (2) the Lophotrochozoa concept and the possibility of Entoprocta (= Kamptozoa) being the sister-group of molluscs; (3) the molluscan affinities of the problematic `worm' Xenoturbella; and (4) the likelihood of molluscan monophyly. In relation to these topics, both morphology and molecules may suffer from the same restrictions and caveats, so that it may be more appropriate to speak of the `morphology plus molecules versus fossils' debate.

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    A phylogeny of the patellid limpets, from mitochondrial gene sequences

    V. Koufopanou, D.G. Reid, S.A. Ridgway & R.H. Thomas (Natural History Museum, London)

    The geographical distribution of the limpet family Patellidae is essentially antitropical, with 18 species in southern Africa, 10 in the northeastern Atlantic, and only 11 species elsewhere (although 4 of these do occur in the tropics). One possible explanation for this is the suggestion of a recent, perhaps Early Pliocene, migration from southern Africa northward. This hypothesis has been tested by deriving a molecular phylogeny, constructed from partial sequences of the 12S and 16S mitochondrial genes, obtained from 34 of the 38 patellid species. Five species of Nacellidae and three of Lottiidae were included as potential outgroups. Analysis revealed that the typical European patellids (Patella sensu stricto) form a single clade within which there is little molecular divergence, but are distant from all other patellids, thus refuting the idea of recent southern ancestry. From the limited fossil record and estimated rates of molecular divergence, it is suggested that Patella s.s. may have originated at least as early as the Upper Cretaceous, and that its northern distribution may have been achieved at the same time. The molecular phylogeny is largely in agreement with a recent morphological cladistic analysis of the Patellidae, which defined four generic clades (Patella s.s., Cymbula, Helcion, Scutellastra), although in the molecular tree Scutellastra is a paraphyletic group.

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    Towards a molecular phylogeny for the pulmonate land snails

    C. Wade, B.J. Clarke (University of Nottingham) & P.B. Mordan (NHM, London)

     An approximately 1400bp fragment of the large subunit (28S) ribosomal RNA gene has been sequenced for over 30 species of pulmonate land snails belonging to 15 stylommatophoran families. Phylogenetic analyses based on this fragment have revealed that the Orthurethra appear to be a good taxonomic group, for taxa of 6 orthurethran families cluster as a single monophyletic clade. In addition, the analyses reveal a possible sister-group relationship of the Clausiliidae with the Orthurethra. The phylogeny nevertheless provides little resolution of inter-family relationships within the Orthurethra, other than the separation of the Partulidae from the other orthurethran taxa. The molecular data are, however, consistent with traditional taxonomic classification at the family level, since the Helicidae, Clausiliidae, Zonitidae and Arionidae are all clearly resolved as clades. The relationships between superfamilial/subordinal groups are not well resolved, which lends support to the idea that the stylommatophoran radiation was explosive. The phylogenetic analyses do not appear to be consistent with the idea that the Orthurethra are the primitive stem group from which the other Stylommatophora evolved.

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    Gene flow and speciation in Littorina: where are the useful sequences?

    C. Wilding, J. Grahame & P.J. Mill (University of Leeds)

    A number of genetic markers, both mitochondrial and nuclear, are being explored, to evaluate their utility for enhancing our understanding of the striking variation observed in the gastropod genus Littorina, and in particular L. saxatilis. Following the cloning and sequencing of approximately 8Kb of L. saxatilis mitochondrial DNA , primers have been designed to target the COOH-terminus of the CoI gene. Substantial variation has been revealed, and although there is a general separation of the recognized species of British Littorina, some mtDNA haplotypes are shared. This paraphyly may be a consequence of hybridization, or may indicate shared ancestral haplotypes pre-dating the speciation events. Within the most variable species (L. saxatilis), two groupings of CoI haplotypes have been found, but these are not restricted to particular locations or morphotypes. It is suggested that these haplotypes may have evolved in separate refugia during the last ice age. Sequence variation has also been examined in the introns of two calmodulin genes. These sequences do not seem useful for phylogeny estimation in the L. saxatilis species complex', since the observed variation is not partitioned between species; however, higher-level phylogeny estimation within the genus may be possible. These introns also provide a number of potentially useful markers for population genetics. Variation in RAPD markers has been assessed at three sites around Britain, and this is currently being extended. RAPD variation shows differences between animals at different heights on the shore, and this variation covaries with morphological Mahalanobis distances, thus suggesting that morphological differences may have some genetic basis. Certain RAPD markers have been cloned and sequenced. One such sequence containing a GTT repeat motif has been amplified from animals representing two different morphotypes and there appear to be consistent differences in the size of the repeat between these two morphological forms.

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    The use of SSCPs and allozymes to investigate speciation in the Littorina saxatilis species complex

    I.F. Wilson & E.M. Gosling (Regional Technical College, Galway, & National University of Ireland, Galway)

    Analysis of allozyme variation and mitochondrial DNA cytochrome-b sequence variation has been carried out on populations of the rough periwinkle (Littorina saxatilis) species-complex, in an attempt to identify genetic markers that will be useful in classifying individuals to species. Twenty-three samples of L. saxatilis, L. arcana, L. compressa and L. neglecta were collected from twelve locations in Ireland and Britain during 1996 and 1997. Twelve polymorphic enzyme loci were investigated.

    Maximum Likelihood Estimation analysis showed that the populations clustered by species. However, there is evidence that the AAT-1 locus may be under selection, and after removing this locus from the analysis the populations clustered mostly by geography, apart from L. compressa which clustered separately. Samples of L. compressa showed lower levels of genetic variation. Analysis of Single Stranded Conformational Polymorphism (SSCP) and of sequences from the cytochrome-b gene from some of these populations showed that L. arcana clustered separately, but L. compressa samples were interspersed among those of L. saxatilis and L. neglecta. Further analysis is being carried out to confirm these findings and other mitochondrial genes and nuclear loci will be investigated to find suitable genetic markers.

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    Molecular markers for medically-important snails

    D. Rollinson, J.R. Stothard, S. Hughes (NHM, London), A. Lockyer, C.S. Jones & L. R. Noble (University of Aberdeen)

    In many developing countries water resource development projects may pose a threat to human health by the introduction or enhancement of waterborne diseases. The most significant reported increases in disease prevalence attributable to water development schemes concern schistosomiasis. This is due, in part, to invasion of snails that form an essential component of the parasite life cycle, many species flourishing in the sites polluted and frequented by man. Malacologists now have many new molecular tools at their disposal for studying snails and this talk considered some of the advantages that might accrue from more detailed molecular studies on the snails responsible for transmission of schistosomiasis.

    The relationships between schistosomes and their intermediate hosts is an extremely intricate one, with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. It follows that the distribution of a suitable snail host is a limiting factor in the distribution of the parasite. In order to gain a better understanding of the epidemiology of schistosomiasis the use of molecular techniques for snail identification, and for studying host-parasite relationships, has been investigated. Examples from projects in East and West Africa show how PCR/restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region of ribosomal RNA genes, random amplified polymorphic DNA (RAPD), and DNA sequence analysis, can all contribute to the discrimination of Bulinus species within the B. forskalii and B. africanus groups and provide novel methods of identification. Molecular markers for resistance genes are being sought by the application of a bulk segregant analysis approach (BSA), using stocks of Biomphalaria glabrata which differ in their susceptibility to Schistosoma mansoni. Screening of 250 random decamer primers has identified 3 primers which gave RAPD profiles with one or more extra bands in the F2 resistant progeny.

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    Stock structure in a squid: are molecular markers helpful?

    P.W. Shaw (University of Hull)

    Studies of the structure and dynamics of cephalopod populations have been hampered by a lack of reliable and informative markers. Morphological characters are often plastic, subject to environmental variation and to errors in scoring. Attempts to apply genetic markers such as allozymes have in most cases revealed very low levels of variation, limiting the power of statistical tests of differentiation. This talk presented results of the parallel application of allozyme and mitochondrial DNA methods, plus a relatively new class of population genetic markers, microsatellite DNA, for the first time in a cephalopod, the northeastern Atlantic squid Loligo forbesi.

    Previous studies of morphology, physiology and reproduction in L. forbesi had indicated that the population of the Azores archipelago was distinctly different from European populations, and that squid from the Rockall Bank matured earlier than continental shelf populations. Results from allozymes, mtDNA and microsatellite DNA all confirmed that the squid around the Azores were genetically (and therefore reproductively) isolated from the rest of the L. forbesi in the northeastern Atlantic. However, allozyme and mtDNA methods uncovered very low levels of genetic variation across the rest of the range of L. forbesi, and indicated no significant genetic structuring. In contrast, all seven microsatellite loci tested revealed high levels of genetic variation in all populations (mean expected heterozygosity 78%), and subtle but significant differentiation between samples from the European shelf and those from the offshore banks around Rockall and the Faeroe Islands. The levels of genetic differentiation detected suggested a barrier to extensive gene flow between the shelf and the offshore banks, but were not enough to indicate complete reproductive isolation. The study confirmed that molecular genetic markers are useful, and potentially powerful, tools in the study of cephalopod population biology. But it also indicated some of the problems of using such methods and the need for careful choice of markers. The levels of variation seen, and the increased power of resolution thus provided, indicated that microsatellites will be the genetic marker of choice in future studies of cephalopod populations. These regions of simple DNA sequence repeats were found to be abundant in the genome of L. forbesi, and simple to isolate and develop. Recent results from this laboratory for ommastrephiid squid (Adcock, unpubl.) and sepiid cuttlefish (Shaw, unpubl.) suggest that this may also be the case in other cephalopods.

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    An application of genetic markers to questions of conservation in Littoraria ardouiniana

    R.I. Lewis, Q. Huang, Y.M. Mak & G.A. Williams (University of Hong Kong)

    The littorinid gastropod Littoraria ardouiniana predominantly inhabits the seaward edge of mangrove forests; it is assumed to be ovoviviparous and to release veliger larvae. Mangrove habitats have been drastically reduced throughout Southeast Asia, and many have been destroyed for land reclamation in Hong Kong. This raises the question of continuing recruitment to remaining mangroves, since destruction of these habitats effectively increases the distance between recruitment sources. This talk described the investigation of population genetic structure at the polymorphic MPI locus among six populations of L. ardouiniana from mangroves throughout Hong Kong. Measurement of various shell dimensions revealed that populations are quite different in size and shape between habitats. Significant Fst values indicated that gene flow is significantly constrained among the remaining habitats, suggesting that the scale of habitat destruction may threaten the recruitment viability of remaining populations of L. ardouiniana. Such data are valuable in habitat regeneration schemes, where managers must consider such questions as natural or artificial recruitment of associated organisms, and proximity to natural recruitment sources.

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