Species in the faeces: DNA metabarcoding as a method to determine the diet of the endangered yellow-eyed penguin

Abstract

Context. Diet variability is a significant driver of seabird decline; however, data on seabird diet composition and trends have been affected by changes in precision and resolution owing to the evolution of different sampling methods over time. We investigated the effectiveness of applying a passive molecular diet method using faeces obtained from the endangered yellow-eyed penguin. Aims. To assess the feasibility of applying DNA metabarcoding methods to yellow-eyed penguin faeces to evaluate diet, and to compare the reliability of diet results derived from adults and chicks, and from latrine versus fresh faecal samples. Methods. We collected 313 faecal samples from yellow-eyed penguins resident on the Otago coast of New Zealand from October 2016 to August 2017. We used polymerase chain reaction (PCR) with mitochondrial 16S cephalopod and chordate primers to amplify prey DNA present in the faecal samples, and tested the completeness of our assembled reference databases based on previous diet research. Amplified prey DNA sequences were then assigned to taxa from our reference databases by using QIIME2. Key results. Mitochondrial 16S chordate PCR primers were effective at identifying 29 fish taxa, with 98.3% of amplified sequences being identified to species or genus level in 193 samples (61.7% collected). There was no significant difference in the number, occurrence or proportion of ray-finned fish prey DNA sequences derived from fresh samples or latrines. Mitochondrial 16S cephalopod PCR primers classified 1.98% of amplified DNA sequences as targets, with 96.5% of these target sequences being identified to species or genus level in 48 samples (15.3% collected), and five taxa identified. Conclusions. We recommend the collection of latrine samples to enable long-term monitoring of the diet of yellow-eyed penguins, which will optimise the trade-off between wildlife disturbance and dietary resolution. Further refinement is needed to identify cephalopod dietary components for yellow-eyed penguins, because our cephalopod primers were not as specific as those used for ray-finned fishes, amplifying a large number (>98%) of non-cephalopod species. Implications. DNA metabarcoding offers a robust and comprehensive alternative to other, more intrusive, seabird diet-assessment methods, but still requires parallel studies to provide critical information on prey size, true diet composition and diet quality.

Current applications and future promise of genetic/genomic data for conservation in an Aotearoa New Zealand context

“…Such non-invasive sampling minimises disturbance and physical harm, which could be critical in studies of threatened species such as hoiho (yellow-eyed penguin, Megadyptes antipodes; Ellenberg et al, 2007Ellenberg et al, , 2013Young et al, 2020).…”

“…to infer the diets of kekeno (New Zealand fur seal, Arctocephalus forsteri; Emami-Khoyi et al, 2016), kororā (little blue penguin, Eudyptula minor; Murray et al, 2011), hoiho (Young et al, 2020), and Bryde's whales (Balaenoptera edeni brydei; Carroll et al, 2019). eDNAbased diet analysis is especially useful for analysing soft-bodied prey, which are otherwise difficult to identify.…”

Diet plasticity and links to changing foraging behaviour in the conservation of subantarctic yellow‐eyed penguins (Megadyptes antipodes)

“…Stable isotope analysis offers advantages over other techniques such as stomach contents analysis, which often represent diet from only a single meal, and can be biased by differing prey digestibility (Duffy & Jackson, 1986; van Heezik & Seddon, 1989; van Heezik, 1990a; Pütz, 1995). Techniques such as faecal analysis, and particularly faecal DNA analysis, can be used to determine prey species over extended periods and multiple individuals to avoid the single meal constraint, and faecal DNA avoids differential digestion issues (Deagle et al, 2010; Young et al, 2020). However, DNA samples need to be stored at −20°C to prevent degradation (Young et al, 2020), so transport from the subantarctic is logistically difficult.…”

The relevance of pedigrees in the conservation genomics era.

“…The conservation genetics toolbox Since the late 1960's, conservation genetics has grown from a handful of techniques into a fully-fledged discipline that uses genetic information to inform the conservation management of threatened species worldwide (Avise 2008). This field has developed a substantive toolbox applied to understand phylogenetics and species delimitation (e.g., Yusefi et al, 2020;Coimbra et al, 2021), population structure and demographics (e.g., Coimbra et al, 2020), natural community profiling (e.g., Young et al, 2020), and the level of standing genetic variation within and among populations (e.g., Zhang, Luan, Ren, Hu, & Yin, 2020). Much discussion regarding the conservation genetic toolbox has been dedicated to the types of variants that are used for genetic inference, and for good reason: in a relatively short time frame, the field has experienced remarkable growth, from detecting variants using allozyme protein electrophoresis to detecting hundreds of thousands of variants through high throughput sequencing (HTS) approaches (Hohenlohe, Funk, & Rajora, 2021).…”