Metabarcoding of zooplankton to derive indicators of pelagic ecosystem status

Zooplankton play a key role in marine food webs and carbon cycling and are useful indicators of climaterelated changes and ocean health in pelagic ecosystems. Zooplankton are traditionally identified to species through microscopy, but new molecular techniques have enabled the identification of individual specimens (DNA barcoding) or multiple species in the same sample (DNA metabarcoding). Metabarcoding has been tested and refined using zooplankton collected in South African waters for the first time. Challenges to the implementation of DNA-based methods to measure zooplankton biodiversity easily and routinely include an incomplete DNA barcode reference library, logistical complexity and uptake of the new technology by environmental management agencies. These challenges call for a national effort to intensify zooplankton barcoding initiatives and to effectively engage stakeholders in developing a roadmap towards application of DNA-based methods in marine environmental management.
Significance:

Metabarcoding has been successfully applied to marine zooplankton for the first time in South Africa, demonstrating its potential as a tool to generate ecosystem indicators during routine ocean observations.
National barcoding efforts must be intensified to provide a comprehensive reference library of zooplankton DNA.
Effective engagement with stakeholders is required to overcome logistical and policy challenges, and to provide a roadmap towards application of DNA-based methods in marine environmental management.


Zooplankton play a key role in marine food webs and carbon cycling and are useful indicators of climaterelated changes and ocean health in pelagic ecosystems. Zooplankton are traditionally identified to species through microscopy, but new molecular techniques have enabled the identification of individual specimens (DNA barcoding) or multiple species in the same sample (DNA metabarcoding). Metabarcoding has been tested and refined using zooplankton collected in South African waters for the first time. Challenges to the implementation of DNA-based methods to measure zooplankton biodiversity easily and routinely include an incomplete DNA barcode reference library, logistical complexity and uptake of the new technology by environmental management agencies. These challenges call for a national effort to intensify zooplankton barcoding initiatives and to effectively engage stakeholders in developing a roadmap towards application of DNA-based methods in marine environmental management.

Significance:
• Metabarcoding has been successfully applied to marine zooplankton for the first time in South Africa, demonstrating its potential as a tool to generate ecosystem indicators during routine ocean observations.
• National barcoding efforts must be intensified to provide a comprehensive reference library of zooplankton DNA.
• Effective engagement with stakeholders is required to overcome logistical and policy challenges, and to provide a roadmap towards application of DNA-based methods in marine environmental management.

The need for ocean indicators
Recent warming, acidification and deoxygenation associated with climate change have greatly affected physical and chemical conditions in oceans. 1 Altered marine habitats have led to shifts in the distribution and phenology of organisms, with major implications for biological productivity of marine ecosystems. 1 Yet, human reliance on the goods and services provided by oceans continues to grow. 2 Against a backdrop of climate change and increasing exploitation of marine resources, observations of key ocean indicators are critical to inform policy and support ocean governance and management. 2,3 The importance of ocean observation systems is recognised by the United Nations Decade of Ocean Science for Sustainable Development (2021-2030), with one of the ten Ocean Decade Challenges being to expand the ocean observing system globally. 4 The Global Ocean Observing System (GOOS) programme has identified a suite of priority physical, biogeochemical and biological ecosystem variables, known as essential ocean variables (EOVs; Supplementary table 1), for routine and sustained observation to assess ocean changes globally, in support of ocean governance. 3 A complementary set of essential biodiversity variables (EBVs) 5 , to monitor and reduce biodiversity loss, has been defined by the Group on Earth Observations -Biodiversity Observation Network (Supplementary table 1). Zooplankton biomass and diversity were included as biological EOVs that represent the base of marine food webs. 2 Here, we report on recent molecular methodology (DNA barcoding and metabarcoding) that allows for the rapid and accurate measurement of marine zooplankton biodiversity and relative abundance, and its potential application as a long-term indicator of pelagic ecosystem status in South African coastal and neritic waters. We comment on both methodological and logistical considerations.

Assessing zooplankton biodiversity
Zooplankton play a vital role in the functioning of marine ecosystems. As grazers in pelagic food webs, they provide the main energy pathway from primary producers to higher trophic levels such as fish, squid, and marine mammals. Their excretions fuel the microbial food web and contribute significantly to carbon sequestration via the biological pump. 6,7 Zooplankton are physiologically sensitive to temperature and have short life spans, thus thermal changes are rapidly reflected in their population dynamics. 8 Because they are not fished commercially, changes in zooplankton communities reflect actual environmental or ecosystem-mediated changes, largely unaffected by exploitation trends. 8,9 Zooplankton are therefore excellent 'sentinels' or indicators of change in pelagic ecosystems, with applications extending to climate, fisheries, invasive species, ecosystem health, definition of pelagic ecoregions, biodiversity and ecosystem assessments. 8,9 Ongoing advancement of DNA barcoding reference data sets (e.g. BOLD, GenBank; Supplementary table 1) coupled with recent metabarcoding technologies now makes rapid and accurate processing of taxonomically complex zooplankton samples logistically feasible. This new technology prompted a shift from traditional microscopy methods to genes as a measure of marine diversity. 10 DNA barcodes can distinguish between visually similar or cryptic species, are independent of life stage, and reduce researcher bias through standardisation of reference systems. 10 12 with permission, ©NISC (Pty) Ltd.

Figure 1:
The relative percentages of DNA barcode records available for marine zooplankton taxa, globally (pale bars) and for South Africa (dark bars). Numbers next to the bars are the numbers of species known locally/globally. invertebrates) (Figure 1). Holoplanktonic species were grossly underrepresented, despite making up the bulk of zooplankton biomass and with high importance as ecological indicators. The paucity of holoplanktonic barcodes stemmed from too few specialist taxonomists, the so-called 'taxonomic impediment'. 13 Metabarcoding uses the same reference databases as barcoding but allows for identification of multiple taxa simultaneously from mixed samples by using high-throughput sequencing platforms. 14 Potential applications of metabarcoding are broad-ranging, including revolutionising biodiversity assessments in any environment for which DNA barcode reference databases are available 15 , establishing time-series of diversity 16 and developing biotic indices for routine biomonitoring 17 . The progression from barcoding of individual specimens to metabarcoding of entire communities is now well underway in South Africa, with studies published on diatom communities in the St Lucia estuary 18 , bacterial communities in waterholes in the Kruger Park 19 and biomonitoring of freshwater macroinvertebrates 20 , among others.

Towards routine biodiversity monitoring
The adoption of molecular approaches such as metabarcoding in marine environmental management requires an iterative 'translational molecular ecology' approach (constant two-way communication between scientists and stakeholders). 17 Recent applications that demonstrate this process internationally include routine monitoring of ichthyoplankton, biosecurity monitoring for non-indigenous species, and ecological status assessments. 17  the development of and adherence to standardised protocols to guarantee data comparison across spatial and temporal scales are crucial. 17 Metabarcoding of marine zooplankton in South Africa has been optimised to follow best-practice protocols set by the international Scientific Committee on Oceanic Research working group MetaZooGene (Supplementary table 1). Optimisation challenges were the design of taxon-specific mini-barcode primers to increase species detection rates 21 , experimental validation of primer cocktails to test their efficiency in detecting rare species 22 , and comparison of species identities obtained from metabarcoding and microscopy 12 figure 1). The pilot project is a collaboration between several academic institutions, which are in turn funded by the Department of Science and Innovation and the National Research Foundation. 12,21,22 Key findings were that costs, coordination and uptake will be the main challenges for a prospective long-term programme.
In addition to a successful 'proof of concept' exploratory project and an improved barcode reference database, progress towards the routine use of metabarcoding in monitoring of the marine environment in South Africa will require a 'translational molecular ecology' process to facilitate its uptake at environmental management and policy levels. 17 The role of national environmental observation agencies, primarily the South African Environmental Observation Network (SAEON) and the Department of Forestry, Fisheries and the Environment (DFFE), in providing operational budgets and coordination, and as long-term custodians of data and indices, will be critical for actionable progress beyond exploratory research. Our Research Letter describes the initial development of a DNA-based method for biodiversity assessments of South African pelagic ecoregions, including enabling conditions for its uptake in marine environmental management.