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Abstract Environmental managers need a rapid and cost‐effective monitoring tool for tracking the spread of invasive species, particularly at the onset of introduction. The macroalgaeCaulerpa proliferais considered an invasive species outside its native range, colonizing large patches of seafloor, reducing native species, and altering ecosystem functioning. Here, we developed a droplet digital PCR assay for detection ofC. proliferafrom environmental DNA seawater samples using the internal transcribed spacer (ITS) region. While the assay itself was confirmed to be highly efficient, we discovered concentrations ofC. proliferaeDNA were present below detectable levels in the water column surrounding an outbreak. To understand why, we conducted tank‐based experiments for two California invasive algae species,Caulerpa proliferaandSargassum horneri. The steady‐state eDNA concentration (eDNA copies/ gram of biomass detected) ofC. proliferawas found to be two orders of magnitude lower thanS. horneri. A meta‐analysis of steady‐state concentrations reported in the literature showed a remarkable range from ~10⁴–10¹¹(copies/g), revealing C. proliferato have the lowest recorded steady‐state concentrations of eDNA of any known species. We attributeC. prolifera'slow steady‐state eDNA concentration to its unique biology as a unicellular macroscopic algae which reduces the possible modes of eDNA release compared to similarly sized multicellular organisms. Critically our results demonstrate the potential limits of eDNA approaches, the influence of shedding rates in the reliability of species detections, and the vital importance of benchmarking and validating eDNA assays in both field and laboratory settings. 

Seagrass beds are disappearing at a record pace despite their known value to our oceans and coastal communities. Simultaneously, our coastlines are under the constant pressure of climate change which is impacting their chemical, physical and biological characteristics. It is thus pertinent to evaluate and record habitat use so we can understand how these different environments contribute to local biodiversity. This study evaluates the assemblages of fish found at fiveZosterabeds in Southern California using environmental DNA (eDNA) metabarcoding. eDNA is a powerful biodiversity monitoring tool that offers key advantages to conventional monitoring. Results from our eDNA study found 78 species of fish that inhabit these five beds around Southern California representing embayment, open coastal mainland and open coastal island settings. While each bed had the same average number of species found throughout the year, the composition of these fish assemblages was strongly site dependent. There were 35 fish that were found at both open coast and embayment seagrass beds, while embayment seagrass sites had 20 unique fish and open coast sites had 23 unique fish. These results demonstrate that seagrass fish assemblages are heterogenous based on their geographic positioning and that marine managers must take this into account for holistic conservation and restoration efforts.