DOI: 10.5061/dryad.9p8cz8wmb
title: Contrasting seasonal patterns in diet and dung-associated invertebrates of feral cattle and horses in a rewilding area

description: 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.
description: 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.
description: 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.