Simple Genetic Assay Distinguishes the Lamprey Genera Entosphenus and Lampetra and Further Supports Placement of the Kern Brook Lamprey in the Genus Lampetra

The lamprey genera, Lampetra and Entosphenus, often occur close in proximity to one another along the west coast of North America, and although morphological differences (particularly dentition) allow them to be distinguished easily as adults, identification of larvae is more difficult.  Differences in pigmentation can be used to distinguish large larvae, but larvae less than 60 mm total length are difficult to differentiate.  Genetic methods that have been used to distinguish between the genera include sequencing of short segments of the mitochondrial genome (e.g., cytochrome c oxidase subunit I, the DNA ‘barcoding’ gene) or restriction fragment length polymorphism (RFLP) assays for the mitochondrial cytochrome b gene.  However, DNA sequencing is time-consuming and expensive, and the recent discovery of several genetically divergent Lampetra populations in Oregon and California (with cytochrome b gene sequences differing by as much as 8% from other west coast Lampetra populations) have shown the RFLPs to no longer be diagnostic.  We have found that two microsatellite loci developed for the Pacific lamprey, Entosphenus tridentatus, can easily and reliably distinguish between Lampetra and Entosphenus.  We tested over 60 individuals from at least four Entosphenus species (distributed in WA, OR, and CA) and 130 individuals from at least three Lampetra species (from AK, BC, WA, OR, and CA), including all genetically-divergent populations (according to cytochrome b gene sequence) discovered to date.  One locus, Etr-1, was capable of differentiating these two genera in all cases; the other locus, Etr-6, was diagnostic in all but the two most genetically-divergent Lampetra populations (when it did not amplify at all).  Since the fragment size differences were sufficiently large to resolve on agarose gels (approximately 265 versus 230 base pairs and 175 versus 285 bp for Etr-1 and Etr-6, respectively), genus ID could be accomplished in a much less time-consuming and more cost-effective manner than either RFLPs or direct DNA sequencing.  We further tested these two loci on the Kern brook lamprey in California, which was described in 1976 as Entosphenus hubbsi but which shows dentition intermediate between Lampetra and Entosphenus.  All 16 individuals of this species were diagnosed as Lampetra with both microsatellite loci.  As early as 1999, mitochondrial DNA sequence data suggested that E. hubbsi should be recognized as Lampetra hubbsi; nuclear markers now support this.