eDNA as a jawsome tool for ‘Seq’-ing sharks

Poor visibility in sedimented seawater, coupled with elusive behaviour of elasmobranchs, render their sightings to be extremely rare in urban city reef environments. Environmental DNA tools can help improve detection successes, greatly reducing the phantom diversity of sharks and rays in Singapore.
eDNA as a jawsome tool for ‘Seq’-ing sharks
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Seeing through sedimented waters: environmental DNA reduces the phantom diversity of sharks and rays in turbid marine habitats - BMC Ecology and Evolution

Background Sharks and rays are some of the most threatened marine taxa due to the high levels of bycatch and significant demand for meat and fin-related products in many Asian communities. At least 25% of shark and ray species are considered to be threatened with extinction. In particular, the density of reef sharks in the Pacific has declined to 3–10% of pre-human levels. Elasmobranchs are thought to be sparse in highly urbanised and turbid environments. Low visibility coupled with the highly elusive behaviour of sharks and rays pose a challenge to diversity estimation and biomonitoring efforts as sightings are limited to chance encounters or from carcasses ensnared in nets. Here we utilised an eDNA metabarcoding approach to enhance the precision of elasmobranch diversity estimates in urbanised marine environments. Results We applied eDNA metabarcoding on seawater samples to detect elasmobranch species in the hyper-urbanised waters off Singapore. Two genes—vertebrate 12S and elasmobranch COI—were targeted and amplicons subjected to Illumina high-throughput sequencing. With a total of 84 water samples collected from nine localities, we found 47 shark and ray molecular operational taxonomic units, of which 16 had species-level identities. When data were compared against historical collections and contemporary sightings, eDNA detected 14 locally known species as well as two potential new records. Conclusions Local elasmobranch richness uncovered by eDNA is greater than the seven species sighted over the last two decades, thereby reducing phantom diversity. Our findings demonstrate that eDNA metabarcoding is effective in detecting shark and ray species despite the challenges posed by the physical environment, granting a more consistent approach to monitor these highly elusive and threatened species.

Who would have imagined that scientific concepts showcased in Crime Scene Investigation can potentially detect species that were once thought to be locally extinct? However, did you know that these molecular technologies can really be used to rediscover animals that have seemingly vanished, but are in fact leading private lives amongst us? As unimaginable as it sounds, the transformation of television fantasy to reality is now possible with recent environmental DNA (eDNA) tools. Akin to a crime scene scattered with clues (e.g., hair, fingerprints, and bodily fluids), the natural environment is chocked-full of trace DNA, be it from faeces or skin that have been released or shed by animals and plants that live in it. This trace genetic material, or eDNA, is detectable with molecular tools from a wide variety of environmental samples, ranging from seawater, soil, snow and even air. In the last five years, eDNA methods have become exponentially popular due to its broad applicability to address a wide range of scientific questions—from being hot on the trail of invasive species, to beating the bushes for biomonitoring endangered and elusive species.

Just six years ago when I was an undergraduate, the technology was in its infancy with just a handful of ‘proof-of-concept’ studies from freshwater and temperate marine water samples. Having just taken the first few baby steps into research, this technology was completely new to me and it was then that I entertained the thought, “I wonder if eDNA works in Singapore’s highly sedimented and tropical marine waters? Perhaps we may find more than the eye can see.” I was amazed by the revolutionary possibility of just collecting and sequencing seawater to reveal the organismal composition without having to physically observe them. Fast forward three years later, I managed to develop the pioneering eDNA protocol for detecting marine animals in Singapore at the Reef Ecology Laboratory, National University of Singapore. One fine morning during breakfast, our research fellow, Benjamin Wainwright, approached Marc Chang and I to ask if we were interested in testing my protocol for evaluating the diversity of sharks here. As an avid recreational diver with a particular fascination with large pelagics, I found this proposal extremely intriguing, and saw this as a perfect opportunity to test our recently developed eDNA protocols on conservation-related applications. Funny how our lab conjures the most exciting project ideas over mealtimes!

Aden Ip of the Reef Ecology Lab, National University of Singapore, collecting seawater for eDNA in 2018. This seawater contains trace DNA from sharks, rays, and many other organisms unbeknownst to us. Image credit, Marc Chang.
Caption 1. Aden Ip of the Reef Ecology Lab, National University of Singapore, collecting seawater for eDNA in 2018. This seawater contains trace DNA from sharks, rays, and many other organisms unbeknownst to us. Image credit, Marc Chang.

We learnt from our collaborators, Zeehan Jaafar and Kelvin Lim, that there used to be a much higher diversity of sharks and rays, based on records kept at the Lee Kong Chian Natural History Museum, Singapore, as compared to recent citizen science sightings. In the past, there were large elasmobranchs like whale and bull sharks despite the tremendous amount of shipping traffic in our port waters. Encounters nowadays, are sadly rare and are mostly by chance. It really made me wonder if these big charismatic fishes were driven to local extinction because of our island-city’s rapid coastal urbanization, or if they are merely seeking refuge elsewhere? These constitute the dark diversity—species that have been historically reported and still exist in the greater surrounds of their known geographic ranges but are presently missing from a specific area. Or it could also be phantom diversity—extant species that are locally colonised but have become too rare to be detected by regular survey methods.

Caption 2. Conventional methods can most certainly allow observation of species that are common and/or abundant – as shown by the large shark or rays, depicted in or near the magnifying glass (top). However, for elusive species, dark or phantom diversity that are missed by visual observers (fish silhouettes), eDNA can help complement this by revealing their cryptic presence (bottom). These organisms could also be rare and endangered species, such as the hammerhead sharks, which urgently require tools for better monitoring and conservation. (Original artwork created by Aden Ip).

We decided to apply our established eDNA protocol to reassess the shark and ray diversity in Singapore, as detailed in our paper published in BMC Ecology and Evolution. The fundamental questions we asked were: Can eDNA reveal more elasmobranch diversity than visually observed? Will the sequencing data be able to help make better informed management decisions in the future? One of the critical components for eDNA’s success is the availability of a robust sequence database for matching of our eDNA sequences. These will allow meaningful inferences of species identities, and we chose two gene regions for this purpose – 12SV5 and COI, as these reference sequences were well-represented in global databases. Since elasmobranchs are expectedly rare, we collected almost 100 water samples from across 9 sites, so as to broaden our scope of detection.

Caption 3. Overlapping elasmobranch diversity in Singapore compiled from historical museum records (black), contemporary sightings (green), as well as species detected by eDNA metabarcoding of 12S (blue) and COI (red).

The most compelling result from our eDNA study was that we found up to 47 putative species, and managed to detect 16 sharks and rays with species-level identities. This is phenomenal, as eDNA found two times more species than the 7 (3 sharks and 4 rays) typically observed over the last twenty years. Comparing this updated species list from eDNA against the historical records, we found that the phantom diversity of sharks and rays have been considerably reduced by more than 20% after using eDNA. What is even more interesting is that our eDNA results uncovered two potential new species records! Of course, these records require further verification, possibly with evidence from sightings and proper identification of specimens by taxonomic experts. Nevertheless, the sensitivity of eDNA tools surpassing that of conventional sampling methods to uncover so much more elasmobranch diversity was extremely exciting for our team.

 Our study has thus led to a deeper understanding of elasmobranch diversity in Singapore. We learned that the blacktip reef shark is extremely abundant, and there are still large body-sized species like bull sharks roaming our waters, albeit secretly. Further, we were able to make coarse relative abundance inferences of these species with normalised sequence read counts. We believe these will have implications for future work in conservation and formulation of management strategies.

 It is increasingly apparent that eDNA metabarcoding methods hold enormous promise in revolutionizing the way we conduct species biomonitoring, community assessment and biodiversity conservation. With the rapid emergence of newer sequencing technologies, eDNA is now progressively applied to address a much wider array of different questions than ever before. Entering this exciting era of genomic research, we are highly encouraged by our study’s success in improved elasmobranch detection, and I feel extremely lucky to be able to conduct research work on a topic that I am passionate about. In the face of rising extinction rates, I look forward to continue using eDNA to complement conventional methods for improved species monitoring and to protect our natural heritage.

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