## 4 Discussion
For example, while potentially harmful cyanobacteria show reasonable equivalence between visual and molecular methods the same cannot be said of diatoms and dinoflagellates, where large discrepancies remain. Although internal consistency is generally high using visual and molecular methods for taxonomic assignment, the results of the two methods differ greatly from each other (Figure 5). There are several explanations for this that are inherent in the two methods. The likelihood of successful preservation is important for visual assignment of taxonomy with diatoms being generally easy to preserve and observe. Haptopbytes, cryptophytes, and cyanobacteria can be more difficult to preserve and may be underrepresented using visual methods. Conversely, cryptophytes may be overrepresented usingmolecular methods as environmental DNA resulting from frequent blooming can contaminate the sample and inflate estimates of extant members of the population. Likewise, high gene copy number in an organism can create errors in molecular estimates and are especially problematic with dinoflagellates given their very large and duplicated genomes [29]. This may explain why molecular estimates were consistently one or two orders of magnitude lower than visual estimates of dinoflagellates in this study. Another issue with molecular assignment is the sequence itself. Some taxonomic clades (descendants of a common ancestor) are difficult to sequence even with degenerate primers due to large numbers of pairwise differences between their sequence and other clades. Additionally, certain clades can be poorly represented in taxonomic databases and a conflation of these two issues can cause large underestimation of abundance. This is a likely explanation for the low abundances of diatoms observed by molecular methods. Combined with the ease of preservation and identification of this group of algae, this results in a large disparity in observed abundances using visual and molecular identification. This disparity is likely the dominant driver of differences observed between the methods (Figure 5), as samples with high abundances of diatoms had strongly negative eigen values on principal component axis 1. Likewise, relative abundances of green algae and cyanobacteria are likely drivers of axis 2 given the separation of the molecular assignments that are consistently low in the abundance of diatoms and dinoflagellates on this axis. In view of discrepancies in the size classes of organisms regulated under the Ballast Water Convention [6] and several reports showing that large numbers of planktonic organisms often fall outside regulated size categories [7; 8; 9; 10; 11], there remains a clear need for indicative measures of ballast water treatment that are independent of organism size and do not require a high degree of taxonomic expertise. While HTS can provide important information on the sources and transport of potentially harmful organisms that could form the basis for risk assessment, its use as a regulatory tool appears limited. Such an application would require the development of a reliable molecular'signature' based on the genetic integrity of samples following ballast water treatment. While the results reported here relate only to untreated ballast water, follow-up testing on treated water calibrated against microscopy would be needed to identify changes in nucleic acid profiles that reflect the elimination of harmful organisms. Disparity between HTS and microscopic counts of some microorganisms, as seen here, suggests that this approach may be limited in scope to specific taxonomic groups showing good correlation between microscopy and molecular profile. Current results indicate that cyanobacteria might fit this profile, although diatoms and dinoflagellates would not. Additionally, an important characteristic of a prognostic test for ballast water treatment efficacy is that sampling and analysis should ideally be completed during the period of a port visit.