CRYPTIC POPULATION DIVERSITY IN Heterosigma akashiwo
Megan Black, Chloe Deodato, Michael Jacobs, William Hardin, Michael Nishizaki, Anastasia Berg, and
Rose Ann Cattolico
University of Washington, Seattle, WA
A harmful algal bloom occurrence can actually represent a series of cascading blooms by several cryptic
strains of the same alga. Such diversity could account for the lack of a clear relationship between cell
number and toxicity in some algal populations. Population diversity can also present a confounding
challenge in developing models of bloom dynamics because strains can have varied excystment cues,
swimming abilities, growth rates, temperature robustness, predation rates, etc.
Unfortunately, population diversity within a species of bloom forming algae is difficult to determine.
Bar-coding strains via molecular sequencing addresses this problem. However, in order to be
ecologically meaningful, the amount of variability measured must reflect some degree of functional
change.
The nuclear encoded ribosomal ITS region has proven to be an excellent species level sequence marker in
raphidophyte, Heterosigma akashiwo. Unlike most organisms, H. akashiwo, has a short 576 base pair
ITS1-5.8S-ITS2 region, where nearly no base pair changes (SNPs) are observed among strains with vastly
different physiological characteristics.
Non-coding micro-satellites, in contrast, provide a level of detail that is so fine scale that it is useful to
discriminate at the individual/clonal level.
Intermediate to these sequence choices are five genes in the mitochondria that provide group-level
information. These mitochondrial genes code for essential proteins in the electron transport chain to
make ATP. By comparing mitochondrial genomes from vastly different Pacific and Atlantic strains, we
identified the cytochrome oxidase 1 (COX1), NADH dehydrogenase 2 (NAD2), NAD4, NAD5, and
NAD7 genes as having high variability of approximately one change per hundred base pairs.
Fifty H. akashiwo cultures from around the world were assembled to create a reference library. PCR
primers were developed and optimized for each of the five genes. Cells were harvested by centrifugation,
lysed, and DNA amplified. The PCR product was then enzymatically cleaned, and directly sequenced
with internal sequencing primers. The sequence reads were assembled, and then aligned. Individual gene
sequences were aligned to compare relatedness and look for single nucleotide polymorphisms (SNPs).
Relatedness was also compared by parsimony analysis of all five genes together. In all nearly 8000 base
pairs were sequenced per strain.
Twelve haplotypes have been identified in this ongoing work. These haplotypes show some geographic
affinity, although some regions, such as the Puget Sound, clearly have mixed populations. Also
noteworthy is that sub-cultures kept separate over thirty years in the laboratory have remained constant in
strain signature.
Although, most of the nucleotide changes encode synonymous amino acids, some changes are more
drastic and code for a different amino acid in the protein. Some substitutions (polar/non-polar) have the
potential to alter the shape and efficiency of the protein. These mitochondrial gene haplotypes also mirror
measurable functional changes in the cells.