DNA metabarcoding of zooplankton enhances community-level analyses of connectivity in a marine pelagic environment.

Doctoral Degree. University of KwaZulu-Natal, Pietermaritzburg.Zooplankton are abundant and diverse marine organisms that form ecologically important communities in marine pelagic ecosystems. They are well-suited for biomonitoring of ecosystem health and changes in biodiversity because their community structure and biomass respond rapidly to environmental variation. Biomonitoring of zooplankton communities using traditional morphology-based species identification methods is labor-intensive due to their cryptic morphology, high diversity and small body size. Fast-developing molecular techniques such as DNA metabarcoding (large-scale, high-throughput DNA sequencing of targeted gene regions to simultaneously identify multiple species present in samples) may provide higher resolution, accurate, faster and more cost-effective biomonitoring tools. The primary objectives of this dissertation were to develop and test a novel DNA metabarcoding approach for biomonitoring of marine zooplankton over the continental shelf of eastern South Africa. Novel taxon-specific DNA mini-barcode primers were designed to increase species identification rates of selected taxa. Artificially assembled mock communities with known composition and relative abundances were then used in an experimental setup to test detection rates and the accuracy of designed and published primers. The DNA metabarcoding protocol was then used to assess connectivity among zooplankton communities over the narrow KwaZulu-Natal continental shelf. Plankton tow nets were used to sample cross-shelf transects at three sites (uThukela, Durban and Aliwal), which are strongly influenced by the Agulhas Current but differ in shelf width, seafloor substrate and benthic habitat structures. Connectivity network analysis detected distinct clustering of zooplankton communities associated with each transect. The hypothesis that a dynamic ocean current regime associated with the offshore Agulhas Current (nearby and flowing parallel to the shelf-edge) would result in similar well-mixed alongshore zooplankton communities was rejected. A strong benthicpelagic coupling effect was inferred based on the species composition of planktonic larvae and benthic adults occurring at the respective transects. This dissertation provides a refined and novel method for biomonitoring of marine pelagic environments in coastal waters, based on taxonspecific DNA metabarcoding of zooplankton communities. The approach is well-suited to measuring the long-term effects of climate change on marine pelagic ecosystems and ocean productivity

Testing the utility of DNA barcoding in South African Hemiptera : using eThekwini species as a case study.

Master of Science in Genetics. University of KwaZulu-Natal, Durban 2017.The eThekwini municipal region and surrounding areas (Durban, Kwazulu-Natal, South Africa) are situated within the globally important Maputaland-Pondoland-Albany biodiversity hotspot. The biodiversity present in this region is under significant pressure from urbanization and climate change. This highlights the need to provide tools that can assist in the discovery and identification of species at an accelerated pace, to create biodiversity inventories which can be used for appropriate conservation planning. The creation of species inventories is a difficult task, more especially for hyper-diverse groups such as terrestrial arthropods. These groups can be morphologically cryptic or difficult to identify using traditional morphology-based taxonomy. Therefore, molecular-based methods of species identification have been proposed to assist in traditional taxonomy. DNA barcoding has been suggested as a mechanism which enables biologists to “label” or “tag” species, using nucleotide variations in short sequences known as DNA barcodes. This study investigates the utility of DNA barcoding and the use of the mitochondrial cytochrome oxidase c subunit 1 (COI) marker to identify species of Hemiptera efficiently and accurately. This study presents a preliminary DNA barcode reference library for Hemiptera collected from 18 different localities within and around the eThekwini municipal region. To test the success of DNA barcoding and the COI marker, matches between morphospecies and barcode clusters (BINs) were analyzed and the presence of the DNA barcode gap in the data was examined. The DNA barcode gap is the gap between the intraspecific and interspecific genetic distances, the lack of the DNA barcode gap suggests that taxa cannot be reliably sorted into species based on the genetic data. Analyses revealed that DNA barcoding using the COI marker is a successful method of identifying Hemiptera species in this study. Thereafter, a case study was selected within the Buffelsdraai Landfill Site Community Reforestation Project, to test whether DNA barcoding could be used to assess the potential of Hemiptera as an indicator of ecological restoration success. The Hemiptera species composition and assembly were assessed by analyzing multiple diversity indices, ordination, UPGMA cluster analysis and phylogenetic analysis. Hemiptera was seen to be sensitive to changes in an ecosystem which make this order an effective environmental and biological indicator. With the help of DNA barcoding, specific families of Hemiptera were identified as habitat-specific and good biological indicators for future studies of ecological restoration and reforestation

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 climate-related 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 design and testing of mini-barcode markers in marine lobsters.

Full-length mitochondrial cytochrome c oxidase I (COI) sequence information from lobster phyllosoma larvae can be difficult to obtain when DNA is degraded or fragmented. Primers that amplify smaller fragments are also more useful in metabarcoding studies. In this study, we developed and tested a method to design a taxon-specific mini-barcode primer set for marine lobsters. The shortest, most informative portion of the COI gene region was identified in silico, and a DNA barcode gap analysis was performed to assess its reliability as species diagnostic marker. Primers were designed, and cross-species amplification success was tested on DNA extracted from a taxonomic range of spiny-, clawed-, slipper- and blind lobsters. The mini-barcode primers successfully amplified both adult and phyllosoma COI fragments, and were able to successfully delimit all species analyzed. Previously published universal primer sets were also tested and sometimes failed to amplify COI from phyllosoma samples. The newly designed taxon-specific mini-barcode primers will increase the success rate of species identification in bulk environmental samples and add to the growing DNA metabarcoding toolkit

Metabarcoding of ichthyoplankton communities associated with a highly dynamic shelf region of the southwest Indian Ocean.

Drifting fish eggs and larvae (ichthyoplankton) can be identified to species using DNA metabarcoding, thus allowing for post hoc community analyses at a high taxonomic resolution. We undertook a regional-scale study of ichthyoplankton distribution along the east coast of South Africa, focused on the contrasting environments of the tropical Delagoa and subtropical Natal Ecoregions, and on exposed and sheltered shelf areas. Zooplankton samples were collected with tow nets at discrete stations along cross-shelf transects (20-200 m depth) spaced along a latitudinal gradient that incorporates a known biogeographical boundary. Metabarcoding detected 67 fish species, of which 64 matched prior distribution records of fishes from South Africa, with the remaining three known from the Western Indian Ocean. Coastal, neritic and oceanic species were present, from epi- and mesopelagic to benthopelagic and benthic adult habitats. By family, Myctophidae (10 species), Carangidae, Clupeidae, Labridae (each with 4 species) and Haemulidae (3 species) were most speciose. Ichthyoplankton community composition varied significantly with latitude, distance to coast, and distance to the shelf edge. Small pelagic fishes had the highest frequency of occurrence: Engraulis capensis, Emmelichthys nitidus and Benthosema pterotum increased in frequency towards the north, whereas Etrumeus whiteheadi increased towards the south. Chub mackerel Scomber japonicus accounted for most variability related to distance from the coast, whilst African scad Trachurus delagoa correlated with distance to the shelf edge. Dissimilarity between communities in the Delagoa and Natal Ecoregions was 98-100%, whereas neighbouring transects located within the sheltered KwaZulu-Natal Bight had lower dissimilarity (56-86%). Onshore transport of ichthyoplankton by Agulhas Current intrusions plausibly explained the abundance of mesopelagic species over the shelf. Metabarcoding followed by community analysis revealed a latitudinal gradient in the ichthyoplankton, associations with coastal and shelf-edge processes, and evidence of a spawning area in the sheltered KwaZulu-Natal Bight

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