Reference: Bio/. Bull. 196: 80-93. (February, 1999) Molecular Determination of Species Boundaries in Corals: Genetic Analysis of the Montastraea annularis Complex Using Amplified Fragment Length Polymorphisms and a Microsatellite Marker JOSE V. LOPEZ', RALF KERSANACH, STEPHEN A. REHNER 2 , AND NANCY KNOWLTON 3 Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Republic of Panama Abstract. Analyses of DNA have not been widely used to distinguish coral sibling species. The three members of the Montastraea annularis complex represent an important test case: they are widely studied and dominate Caribbean reefs. yet their taxonomic status remains unclear. Analysis of amplified fragment length polymorphisms (AFLPs) and a microsatellite locus, using DNA from sperm, showed that Montastraea faveolata is genetically distinct. One AFLP primer yielded a diagnostic product (880 bp in M. faveolata, 920 bp in M. franksi and M. annularis) whose homology was established by DNA sequencing. A second primer revealed a 630 bp band that was fixed in M. faveolata. and rare in M. franksi and M. annularis: in this case homologies were confirmed by Southern hybridizations. A tetranucle-otide microsatellite locus with several alleles exhibited strong frequency differences between M. faveolata and the other two taxa. We did not detect comparable differences between M. annularis and M. franksi with either AFLPs (12 primers screened) or the microsatellite locus. Comparisons of AFLP patterns obtained from DNA from sperm, somatic tissues, and zooxanthellae suggest that the technique rou-tinely amplifies coral (animal) DNA. Thus analyses based Received 5 June 1998; accepted 1 December 1998. 1 Current address: Division of Biomedical Research, Harbor Branch Oceanographic Institution. 5600 U.S. 1 North. Ft. Pierce. FL 34946; E-mail:
[email protected] 2 Current address: Department of Biology, P.O. Box 23360, University of Puerto Rico, Rio Piedras, San Juan, Puerto Rico, 00931. 1 Also at Marine Biology Research Division 0202. Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202; E-mail:
[email protected] Abbreviations: AFLP -amplified fragment length polymorphism, a registered trademark of Keygene. on somatic tissues may be feasible, particularly after diag-nostic differences have been established using sperm DNA. Introduction The recognition of species boundaries in sympatry is straightforward in principle, because the absence of inter-breeding implies the existence of at least some fixed genetic differences between taxa (Avise and Ball, 1990). However, the number of such differences may be very small if the isolation of taxa is recent or the rate of evolution is slow. If in addition sporadic hybridization occurs, the problem of defining species becomes particularly difficult (e.g., Howard et al., 1997). Closely related coral species appear to be especially challenging in this regard (Veron. 1995; Knowlton and Weigt, 1997). Species boundaries are in flux for a number of well-studied groups (e.g., Miller and Babcock, 1997; Miller and Benzie, 1997; Odorico and Miller, 1997; Willis et al.. 1997; Knowlton and Budd, unpubl.), and it is unclear whether these controversies are due to the technical chal-lenge of finding diagnostic characters between generally similar but reproductively isolated taxa, or alternatively, to the blurring of species boundaries by hybridization (Veron, 1995; Knowlton and Weigt, 1997; Willis et al., 1997). Molecular methods have great potential to resolve the na-ture of species boundaries because of the large number of unambiguous characters they provide (Avise. 1994). A clear example of these issues is presented by the proposed members of the Montastraea annularis species complex: M. annularis (formerly morphotype I or columnar morph). M. faveolata (formerly morphotype II or massive morph). and M. franksi (formerly morphotype III or bumpy morph) (Knowlton et al., 1992; Van Veghel and Bak, 1993: 80
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