Using Molecular Tools to Track the Origins of Invasive Codium Fragile spp. tomentosoides Populations in the Northwest Atlantic
This is envisioned as a six-month project to initiate work on the project proposed in the most recent NH Sea Grant RFP, which has been placed on hold, with funding possible for 2004 or 2005. Outside reviewers had one major criticism for the project: molecular markers had not yet been developed for Codium fragile, markers which would provide resolution for the population and phylogeography questions that were among the key experimental objectives of the proposal. During a six-month development period, we would focus on one of the four objectives in the original proposal: Develop PCR-based genetic markers to trace source populations of the invasive Asiatic green alga, Codium fragile spp. tomentosoides that is rapidly expanding along the Northwest Atlantic coast.
Several methods will be used to isolate DNAs from freshly collected Codium and from dried herbarium specimens. These include standard CTAB extraction (Doyle and Doyle, 1990), various modifications of the PureGen extraction protocol (i.e. +/' Rnase A, +/- Proteinase K; Gentra Corporation), which our lab has successfully applied to extraction of red and brown macroalgae, and finally classic DNA isolation with final ultracentrifuge purification. The last will only be used to prepare DNA for microsatellite isolations. The extraction protocols will be evaluated based on ease (e.g., is the protocol simple enough to apply to dozens of samples in a day), the yield and quality of DNA recovered, and the stability of extracted DNA. All of these factors are important for population level comparisons.
We will develop two general categories of molecular markers to differentiate Codium fragile subspecies and/or geographic origins: microsatellite loci and single nucleotide polymorphisms (SNPs), that will be identified in relatively variable chloroplast, mitochondria and nuclear genes. There are advantages for both types of markers. Microsatellites are inherently hypervariable, because of the propensity for slippage by DNA polymerase during meiotic and mitotic cycles. Indeed several of the outside reviewers of our more recent Sea Grant proposal strongly recommended that we develop microsatellite markers for the proposed study.
Development of microsatellite markers is frequently time consuming, and for the macroalgae these efforts have met with variable results. As noted by Wattier and Maggs (2001), there have been difficulties developing microsatellites for macroalgae and relatively few published studies have resulted. The number of alleles per locus is somewhat lower than observed for land plants, with a higher proportion of monomorphic loci. Null alleles result from mutations in the flanking primer regions; a diploid individual heterozygous for a null allele would be inadvertently scored as a homozygous individual. In Klein's lab, Wallace successfully developed microsatellite markers that differentiate several Fucus species and which show population structure in an estuarine habitat (Wallace, Klein and Mathieson, in prep.). However, the microsatellite loci Teasdale developed during the same period were monomorphic for DNAs collected across a wide geographic range from the red alga Porphyra umbilicalis.
The other potential problem with applying microsatellites to phylogeographic analysis is that microsatellites inherently have a higher level of homoplasy than do other types of genetic markers (Mank and Avise, 2003). Recognizing these potential problems with microsatellites, during this development period we will undertake one-two rounds of microsatellite development with protocols that have been successfully implemented in the Klein lab.
Klein's lab has also worked extensively with SNPs to examine population structure in conifers. The target genes will include the intron in the plastid rbcL gene of Codium (M67453), the internal transcribed spacers for the nuclear ribosomal genes (ITS), any mitochondrial loci for which primers can be developed (based on related genome sequences in GenBank), etc. There are currently 622 sequences available for the order Caulerpales in GenBank, however many represent multiple sequences of the same genes (EF-TU, rbcL).
Klein's lab has used a systematic method of screening for SNPs based on a sampling strategy suggested by Braverstock and Moritz: 1) the gene or spacer is amplified for several individuals representing the geographic range of the organism, 2) the amplified DNAs are sequenced to identify potential SNPs, and 3) subsequently larger numbers of individuals would be screened by indirect methods (SSCP, DGGE) to collect population data. For the purpose of this developmental project, only steps one and two will be carried to identify loci that are promising targets to differentiate populations and geographic origins of Codium fragile in the Northwest Atlantic. For proof of concept, these loci will be screened for both Codium fragile spp. tomentosoides and C. fragile spp. atlanticum, both of which may contribute to the recent range expansion of C. fragile in New England.