The estuarine pipefish, Syngnathus watermeyeri, is one of the rarest animals in Africa and occurs in only two South African estuaries. The species was declared provisionally extinct in 1994, but was later rediscovered and is currently listed by the IUCN as Critically Endangered. A conservation programme was launched in 2017, with the re-introduction of captive-bred individuals into estuaries where this species was recorded historically was the main aims. Successful captive breeding requires knowledge of the species’ dietary requirements. In the present study, we used metabarcoding of faecal DNA to identify prey species consumed by wild-captured S. watermeyeri from one of the two surviving populations. We compared the diet of the estuarine pipefish with that of the longsnout pipefish, S. temminckii, in the same estuary, to determine whether these two species compete for the same prey items. Both species occupy similar estuarine habitats, but S. temminckii has a much wider distribution and also occurs in the marine environment. Our results show that even though both pipefish species prey on three major invertebrate classes (Gastropoda, Malacostraca and Maxillopoda), the relative proportions differ. Syngnathus watermeyeri primarily targets Maxillopoda, with a single species of calanoid copepod constituting >95% of the Amplicon Sequence Variants (ASVs) identified from its faecal DNA, whereas the diet of S. temminckii mostly comprises snail and decapod crustacean larvae. Our finding supports the hypothesis that population declines and localised extirpations of S. watermeyeri during previous decades may have been the result of reductions in the abundance of calanoid copepods. Calanoids rely on freshwater pulses to thrive, but such events have become rare in the two estuaries inhabited by S. watermeyeri due to excessive freshwater abstraction for urban and agricultural use.

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Seahorses and pipefishes are heavily exploited for use in Traditional Chinese Medicine (TCM), and less frequently for curio markets or as aquarium fish. A number of recent studies have used DNA barcoding to identify species sold at TCM markets in East Asia, but the usefulness of this approach in determining the region of origin remains poorly explored. Here, we generated DNA barcodes of dried seahorses and pipefishes destined for TCM that were confiscated at South Africa’s largest airport because they lacked the export permits required for the CITES-listed seahorses. These were compared with published sequences and new sequences generated for Mozambican seahorses, with the aim of determining whether it is possible to identify their country of origin. All pipefishes were identified as Syngnathoides biaculeatus, a widespread Indo-Pacific species, but the published sequence data did not provide sufficient resolution to identify the region of origin. The same was true of the majority of seahorses, which could not even be identified to species level because they clustered among an unresolved species complex whose sequences were published under the names Hippocampus kuda, H. fuscus and H. capensis. The presence of a few specimens of a second seahorse, H. camelopardalis, suggests that the shipment originated from East Africa because the range of this seahorse is centred around this region, but again, it was not possible to determine their country of origin. Even though seahorses and pipefishes have high levels of genetic population structure because of their low dispersal potential, DNA barcoding was only suitable to tentatively identify species, but not their region of origin. DNA barcoding is increasingly used to identify illegally traded wildlife, but our results show that more sophisticated methods are needed to monitor and police the trade in seahorses and pipefishes.

Understanding the dietary preferences of endangered species can be useful in implementing conservation strategies, including habitat restoration, translocation, and captive breeding. Environmental DNA (eDNA) from feces provides a non-invasive method for analysing animal diets. Currently, metabarcoding, a PCR-based approach, is the method of choice for analysing such data. However, this method has limitations, specifically PCR bias, which can result in the overestimation of the importance of certain taxa and failure to detect other taxa because they do not amplify. The present study compared metabarcoding with metagenomics, a PCR-free method, to assess the diversity of prey items in the feces of a critically endangered South African estuarine pipefish, Syngnathus watermeyeri, and its widely distributed congener S. temminckii to investigate potential dietary competition. The metabarcoding results showed a distinct difference between the diets of S. watermeyeri and S. temminckii, with the former mainly consuming calanoid copepods and the latter preferring caridean shrimp. In each case, a single species dominated the sequences generated by metabarcoding. Metagenomics produced more species identifications, and although the same trend was found regarding the preference of S. watermeyeri for copepods and that of S. temminckii for shrimp, this approach identified additional, albeit yet unidentified, copepod species as being important in the diet of S. watermeyeri. We conclude that the lower number of species identified using metabarcoding was most likely a result of amplification bias, resulting in key copepod species missing from the dietary analysis. These findings suggest that metagenomics is not only a useful complementary method for molecular dietary analysis, but may in some cases outperform metabarcoding. However, metagenomics is even more strongly affected by the lack of reference sequences than is metabarcoding, as the majority of sequences originate from genomic regions that have not yet been sequenced for the putative prey species in question.