Prey ration, temperature, and predator species influence digestion rates of prey DNA inferred from qPCR and metabarcoding.

Diet analysis is a vital tool for understanding trophic interactions and is frequently used to inform conservation and management. Molecular approaches can identify diet items that are impossible to distinguish using more traditional visual-based methods. Yet, our understanding of how different variables, such as predator species or prey ration size, influence molecular diet analysis is still incomplete. Here, we conducted a large feeding trial to assess the impact that ration size, predator species, and temperature had on digestion rates estimated with visual identification, qPCR, and metabarcoding. Our trial was conducted by feeding two rations of Chinook salmon (Oncorhynchus tshawytscha) to two piscivorous fish species (largemouth bass [Micropterus salmoides] and channel catfish [Ictalurus punctatus]) held at two different temperatures (15.5 and 18.5°C) and sacrificed at regular intervals up to 120 h from the time of ingestion to quantify the prey contents remaining in the digestive tract. We found that ration size, temperature, and predator species all influenced digestion rate, with some indication that ration size had the largest influence. DNA-based analyses were able to identify salmon smolt prey in predator gut samples for much longer than visual analysis (~12 h for visual analysis vs. ~72 h for molecular analyses). Our study provides evidence that modelling the persistence of prey DNA in predator guts for molecular diet analyses may be feasible using a small set of controlling variables for many fish systems.

Toward absolute abundance for conservation applications: Estimating the number of contributors via microhaplotype genotyping of mixed-DNA samples.

Molecular methods including metabarcoding and quantitative polymerase chain reaction have shown promise for estimating species abundance by quantifying the concentration of genetic material in field samples. However, the relationship between specimen abundance and detectable concentrations of genetic material is often variable in practice. DNA mixture analysis represents an alternative approach to quantify specimen abundance based on the presence of unique alleles in a sample. The DNA mixture approach provides novel opportunities to inform ecology and conservation by estimating the absolute abundance of target taxa through molecular methods; yet, the challenges associated with genotyping many highly variable markers in mixed-DNA samples have prevented its widespread use. To advance molecular approaches for abundance estimation, we explored the utility of microhaplotypes for DNA mixture analysis by applying a 125-marker panel to 1179 Chinook salmon (Oncorhynchus tshawytscha) smolts from the Sacramento-San Joaquin Delta, California, USA. We assessed the accuracy of DNA mixture analysis through a combination of mock mixtures containing DNA from up to 20 smolts and a trophic ecological application enumerating smolts in predator diets. Mock DNA mixtures of up to 10 smolts could reliably be resolved using microhaplotypes, and increasing the panel size would likely facilitate the identification of more individuals. However, while analysis of predator gastrointestinal tract contents indicated DNA mixture analysis could discern the presence of multiple prey items, poor and variable DNA quality prevented accurate genotyping and abundance estimation. Our results indicate that DNA mixture analysis can perform well with high-quality DNA, but methodological improvements in genotyping degraded DNA are necessary before this approach can be used on marginal-quality samples.

Parallel body shape divergence in the Neotropical fish genus Rhoadsia (Teleostei: Characidae) along elevational gradients of the western slopes of the Ecuadorian Andes.

Neotropical mountain streams are important contributors of biological diversity. Two species of the characid genus Rhoadsia differing for an ecologically important morphological trait, body depth, have been described from mountain streams of the western slopes of the Andes in Ecuador. Rhoadsia altipinna is a deeper-bodied species reported from low elevations in southwestern Ecuador and northern Peru, and Rhoadsia minor is a more streamlined species that was described from high elevations (>1200 m) in the Esmeraldas drainage in northwestern Ecuador. Little is known about these species and their validity as distinct species has been questioned. In this study, we examine how their body shape varies along replicated elevational gradients in different drainages of western Ecuador using geometric morphometrics and the fineness ratio. We also use sequences of the mitochondrial cytochrome oxidase c I gene and the second intron of the S7 nuclear gene to examine whether genetic data are consistent with the existence of two species. We found that body depth varies continuously among populations within drainages as a function of elevation, and that body shape overlaps among drainages, such that low elevation populations of R. minor in the Esmeraldas drainage have similar body depths to higher elevation R. altipinna in southern drainages. Although a common general trend of declining body depth with elevation is clear, the pattern and magnitude of body shape divergence differed among drainages. Sequencing of mitochondrial and nuclear genes failed to meet strict criteria for the recognition of two species (e.g., reciprocal monophyly and deep genetic structure). However, there was a large component of genetic variation for the COI gene that segregated among drainages, indicating significant genetic divergence associated with geographic isolation. Continued research on Rhoadsia in western Ecuador may yield significant insight into adaptation and speciation in Neotropical mountain streams.