Trophic rewilding is increasingly applied in restoration efforts, with the aim of reintroducing the ecological functions provided by large-bodied mammals and thereby promoting self-regulating, biodiverse ecosystems. However, empirical evidence for the effects of megafauna introductions on the abundance and richness of other organisms such as plants and invertebrates, and the mechanisms involved still need strengthening.

In this study, we use environmental DNA (eDNA) metabarcoding of dung from co-existing feral cattle and horses to assess the seasonal variation in plant diet and dung-associated arthropods and nematodes. We found consistently high diet richness of horses, with low seasonal variability, while the generally lower dietary diversity of cattle increased substantially during summer. Intriguingly, season-specific diets differed, with a greater proportion of trees in the horses’ diet during winter, when cattle relied more on shrubs. Graminoids were generally underrepresented in herbivore diets compared to previous studies, possibly due to the high prevalence of forbs in the study area.

Dung-associated arthropod richness was higher for cattle, largely due to a high richness of flies during summer. Several species of dung-associated arthropods were found primarily in dung from one of the two herbivores, and our data confirmed known patterns of seasonal activity. Nematode richness was constantly higher for horses, and nematode communities were markedly different between the two species.

Our results demonstrate complementary effects of cattle and horses through diet differences and dung-associated invertebrate communities, enhancing our understanding of large herbivore effects on vegetation and associated biodiversity. These results are directly applicable for decision-making in rewilding projects, suggesting biodiversity benefits by inclusion of functionally different herbivores.

Samples were collected each month through 2020 in a Danish rewilding site: "The Mols Laboratory".

From each month, 10 samples were collected from horses (Exmoor ponies), and 10 from cattle (Galloway cattle). For each species of herbivore, 5 samples were collected from open habitats, and 5 were collected from forest habitats.

DNA was extracted from each sample using the Fast DNA Stool Mini kit from Qiagen, and amplified by two primer sets - the ITS2-S2F/ITS4 (Fahner et al. 2016, doi: 10.1371/journal.pone.0157505) targeting a 370 bp fragment of the nuclear ITS region (for amplifying plant DNA), and the BF-1/BR-1 primers (Elbrecht & Leese 2017, doi: 10.3389/fenvs.2017.00011) targeting a 217 bp fragment of the mitochondrial COI gene, optimized for amplifying invertebrates.

See connected publication for additional details.

ITS2-S2F: ATGCGATACTTGGTGTGAAT

ITS4: TCCTCCGCTTATTGATATGC

BF-1: ACWGGWTGRACWGTNTAYCC

BR-1: ARYATDGTRATDGCHCCDGC

The amplicons were sequenced at a NovaSeq 6000 platform, using 250 bp paired end sequencing for ITS and 150 BP paired end sequencing for COI.

Data consists of 40 sequencing libraries, where 20 is from the ITS amplification (1_1 to 5_4) and 20 from the COI amplification (6_1 to 10_4). For each library, there is at least 2 files with the ending "_1" and "_2" containing forward and reverse reads respectively. For some libraries, there are additional files with the "add" or "add2" notation, which represent files from additional sequencing runs, which were done in some cases to increase the number of sequences obtained.

These files are merged before performing the demultiplexing (see README.md for details)

Each library represents a PCR replicate, meaning that the 5 sample groups were amplified 4 times for each primer set. Each library contains samples (e.g., JAN1, AUG3, DEC5), field controls (marked with a "K" in the sample name, e.g., JAN21K), extraction blanks (CNE), and PCR blanks (NTC).

The "tags" files contain a column with sample names followed by the PCR replicate number ("_1", "_2", "_3", "_4"), followed by two columns containing the tags used with the forward and reverse primer for each sample (the same tags were used for forward and reverse primer). 

To follow the exact filtering and data analysis steps, we refer to the connected publication, and you are always welcome to send an email with questions you might have.