Molecular dietary analyses of western capercaillies (Tetrao urogallus) reveal a diverse diet

Conservation strategies centered around species habitat protection rely on species’ dietary information. One species at the focal point of conservation efforts is the herbivorous grouse, the western capercaillie (Tetrao urogallus), which is an indicator species for forest biodiversity conservation. Non-molecular means used to study their diet are time-consuming and at low taxonomic resolution. This delays the implementation of conservation strategies including resource protection due to uncertainty about its diet. Thus, limited knowledge on diet is hampering conservation efforts. Here, we use non-invasive environmental DNA (eDNA) metabarcoding on DNA extracted from faces to present the first large-scale molecular dietary analysis of capercaillies. Facal samples were collected from seven populations located in Norway (Finnmark, Troms, Trøndelag, Innlandet) and France (Vosges, Jura, Pyrenees) (n = 172). We detected 122 plant taxa belonging to 46 plant families of which 37.7% of the detected taxa could be identified at species level. The average dietary richness of each sample was 7 ± 5 SD taxa. The most frequently occurring plant groups with the highest relative read abundance (RRA) were trees and dwarf shrubs, in particular, Pinus and Vaccinium myrtillus, respectively. There was a difference in dietary composition (RRA) between samples collected from the different locations (adonis pseudo F5,86 = 11.01, r2 = 0.17, p = 0.001) and seasons (adonis pseudo F2,03 = 0.64, r2 = 0.01, p = 0.036). Dietary composition also differed between sexes at each location (adonis pseudo F1,47 = 2.77, r2 = 0.04, p = 0.024), although not significant for all data combined. In total, 35 taxa (36.8% of taxa recorded) were new capercaillie food items compared with existing knowledge from non-molecular means. The non-invasive molecular dietary analysis applied in this study provides new ecological information of capercaillies’ diet, improving our understanding of adequate habitat required for their conservation

Metagenomics: A viable tool for reconstructing herbivore diet

Metagenomics can generate data on the diet of herbivores, without the need for primer selection and PCR enrichment steps as is necessary in metabarcoding. Metagenomic approaches to diet analysis have remained relatively unexplored, requiring validation of bioinformatic steps. Currently, no metagenomic herbivore diet studies have utilized both chloroplast and nuclear markers as reference sequences for plant identification, which would increase the number of reads that could be taxonomically informative. Here, we explore how in silico simulation of metagenomic data sets resembling sequences obtained from faecal samples can be used to validate taxonomic assignment. Using a known list of sequences to create simulated data sets, we derived reliable identification parameters for taxonomic assignments of sequences. We applied these parameters to characterize the diet of western capercaillies (Tetrao urogallus) located in Norway, and compared the results with metabarcoding trnL P6 loop data generated from the same samples. Both methods performed similarly in the number of plant taxa identified (metagenomics 42 taxa, metabarcoding 43 taxa), with no significant difference in species resolution (metagenomics 24%, metabarcoding 23%). We further observed that while metagenomics was strongly affected by the age of faecal samples, with fresh samples outperforming old samples, metabarcoding was not affected by sample age. On the other hand, metagenomics allowed us to simultaneously obtain the mitochondrial genome of the western capercaillies, thereby providing additional ecological information. Our study demonstrates the potential of utilizing metagenomics for diet reconstruction but also highlights key considerations as compared to metabarcoding for future utilization of this technique

A two-step metagenomics approach for the identification and mitochondrial DNA contig assembly of vertebrate prey from the blood meals of common vampire bats (<i>Desmodus rotundus</i>)

The feeding behaviour of the sanguivorous common vampire bat (Desmodus rotundus) facilitates the transmission of pathogens that can impact both human and animal health. To formulate effective strategies in controlling the spread of diseases, there is a need to obtain information on which animals they feed on. One DNA-based approach, shotgun sequencing, can be used to obtain such information. Even though it is costly, shotgun sequencing can be used to simultaneously retrieve prey and vampire bat mitochondrial DNA for population studies within one round of sequencing. However, due to the challenges of analysing shotgun sequenced metagenomic data such as false negatives/positives and typically low proportion of reads mapped to diet items, shotgun sequencing has not been used for the identification of prey from common vampire bat blood meals. To overcome these challenges and generate longer mitochondrial contigs which could be useful for prey population studies, we shotgun sequenced common vampire bat blood meal samples (n = 8) and utilised a two-step metagenomic approach based on combining existing bioinformatic workflows (alignment and mtDNA contig assembly) to identify prey. After validating our results from detections made through metabarcoding, we accurately identified the common vampire bats' prey in six out of eight samples without any false positives. We also generated prey mitochondrial contig lengths between 138 bp to 3231 bp (median = 770 bp, Q1 = 262 bp, Q3 = 1766 bp). This opens the potential to conduct phylogenetic and phylogeographic monitoring of elusive prey species in future studies, through the analyses of blood meal metagenomic data

Supplementary material 1 from: Chua PYS, Carøe C, Crampton-Platt A, Reyes-Avila CS, Jones G, Streicker DG, Bohmann K (2022) A two-step metagenomics approach for the identification and mitochondrial DNA contig assembly of vertebrate prey from the blood meals of common vampire bats (Desmodus rotundus). Metabarcoding and Metagenomics 6: e78756. https://doi.org/10.3897/mbmg.6.78756

A two-step metagenomics approach for prey identification from the blood meals of common vampire bats (Desmodus rotundus

Supplementary material 1 from: Chua PYS, Carøe C, Crampton-Platt A, Reyes-Avila CS, Jones G, Streicker DG, Bohmann K (2022) A two-step metagenomics approach for the identification and mitochondrial DNA contig assembly of vertebrate prey from the blood meals of common vampire bats (Desmodus rotundus). Metabarcoding and Metagenomics 6: e78756. https://doi.org/10.3897/mbmg.6.78756

A two-step metagenomics approach for prey identification from the blood meals of common vampire bats (Desmodus rotundus