History1985 - a major effluent pipeline was about to begindischarging industrial effluent over a shallowcontinental shelf area in Richards Bay1985 - surface plankton samples were collected, overseveral years, to assess the diversity of fish speciesspawning in the area, and the intensity and seasonalityof spawning.1986 – a second study in Park Rynie was started inorder to collect alive specimens.1987 – cataloguing of eggs and hatched larvae started2004 – DNA Barcoding was added to the procedure(including sampling of adults for reference library)
Work flow• By collecting both offshore (5km) and inshore (0.5km) a reasonableassessment of location of spawning was obtained for all the commoneggs in the study area.• A simple “key” based on the physical features of pelagic fish eggs,was used to separate eggs into basic groups.• eggs were hatched and both eggs and larvae were photographed.• once larvae had fully pigmented eyes, theywere anaesthetised with MS222, prior tofixing in 98% alcohol for DNA Barcoding.• other larvae were reared to the pointwhere fin counts and juvenile featuresaided in identification.
Work flow• DNA extraction was done using standard protocols at the CCDB.• A reduced elution volume was used.• PCR used Fish Cocktail (Ivanova 2007).• Sequences were queried against BOLD using its IdentificationEngine (only 100% were considered).• reared larvae of the same batch were fixed in formalin and serve as„para-vouchers‟
Today• some 2100 larvae have been barcoded since 2005• the local adult reference library (assembled in parallel) containssome 900 species• some 1500 species of marine fishes from South Africa are barcoded• 9000 fish species have been barcoded world-wide
Are we done? 40 35Phylogenetic Diversity (PD) 30 25 20 • PD calculated using Conserve based on 15 NJ trees generated in MEGA 4.0 • Sample size progressively increased by 10 10 random sequences 5 0 0 500 1000 1500 2000 # barcodes
Results• 1638 specimens (78%) could be identified using BOLD• they represent 280 known species• 10 of those are new records for South Africa• the remaining 22% could not be matched to any barcode sequenceon BOLD or GenBank.
TrendsMean monthly eggs per sample, averaged over 24 years
Trends• large rainfall causing mud to be washed out from rivers• the high nutrient load of such a deluge caused massive increase in eggnumbers• three most prolific pelagic egg spawners: Sardinops sagax, Etrumeusteres, and Scomber japonicus
Thank you!Acknowledgements:Erin CorstorphineTyler ZemlakPhilip HeemstraBiodiversity Institute ofOntario www.fisheggsandlarvae.com