This document summarizes a study on marine fish eggs and larvae from the East coast of South Africa. Samples were collected over several years starting in 1985 to assess fish diversity and spawning patterns. Eggs and larvae were identified using physical features and DNA barcoding. Over 2100 larvae have been barcoded representing 280 known species, with 10 being new records. Location of spawning was estimated by collecting offshore and inshore. Three species were found to be the most prolific spawners. Large rainfall events increased egg numbers likely due to increased nutrients.

1. Marine fish eggs and larvae from the
East coast of South Africa
Dirk Steinke, Allan Connell, Tyler Zemlak, Paul Hebert

2. History
Allan Connell

3. History
1985 - a major effluent pipeline was about to begin
discharging industrial effluent over a shallow
continental shelf area in Richards Bay
1985 - surface plankton samples were collected, over
several years, to assess the diversity of fish species
spawning in the area, and the intensity and seasonality
of spawning.
1986 – a second study in Park Rynie was started in
order to collect alive specimens.
1987 – cataloguing of eggs and hatched larvae started
2004 – DNA Barcoding was added to the procedure
(including sampling of adults for reference library)

4. Work flow
• By collecting both offshore (5km) and inshore (0.5km) a reasonable
assessment of location of spawning was obtained for all the common
eggs 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, they
were anaesthetised with MS222, prior to
fixing in 98% alcohol for DNA Barcoding.
• other larvae were reared to the point
where fin counts and juvenile features
aided in identification.

5. 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 Identification
Engine (only 100% were considered).
• reared larvae of the same batch were fixed in formalin and serve as
„para-vouchers‟

6. Today
• some 2100 larvae have been barcoded since 2005
• the local adult reference library (assembled in parallel) contains
some 900 species
• some 1500 species of marine fishes from South Africa are barcoded
• 9000 fish species have been barcoded world-wide

7. Are we done?
40
35
Phylogenetic 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

8. 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 sequence
on BOLD or GenBank.

9. Some observations
100
90
80
70
60
% Offshore
50
% Inshore
40
30
20
10
0
Monodactylus falciformis Pomadasys olivaceus Cubiceps pauciradiatus

10. Trends
Mean monthly eggs per sample, averaged over 24 years

11. Trends
• large rainfall causing mud to be washed out from rivers
• the high nutrient load of such a deluge caused massive increase in egg
numbers
• three most prolific pelagic egg spawners: Sardinops sagax, Etrumeus
teres, and Scomber japonicus

12. Thank you!
Acknowledgements:
Erin Corstorphine
Tyler Zemlak
Philip Heemstra
Biodiversity Institute of
Ontario
www.fisheggsandlarvae.com