Winter diet of Japanese macaques from Chubu Sangaku National Park, Japan incorporates freshwater biota

The Japanese macaque (Macaca fuscata) is native to the main islands of Japan, except Hokkaido, and is the most northerly living non-human primate. In the Chubu Sangaku National Park of the Japanese Alps, macaques live in one of the coldest areas of the world, with snow cover limiting the availability of preferred food sources. Winter is typically a bottleneck for food availability potentially resulting in marked energy deficits, and mortality may result from famine. However, streams with groundwater upwelling flow during the winter with a constant water temperature of about 5 °C are easily accessible for Japanese macaques to search for riverine biota. We used metabarcoding (Cytochrome c oxidase I) of fecal samples from Japanese macaques to determine their wintertime diet. Here we provide the first robust evidence that Japanese macaques feed on freshwater biota, including brown trout, riverine insects and molluscs, in Chubu Sangaku National Park. These additional food sources likely aid their winter survival.

The freshwater fish Salmo trutta (brown trout) was definitively identified in 2018 and 2019 from the fecal samples ( Table 2). The sequences had 100% sequence similarity to other Salmo trutta entries in the genetic sequence database GenBank (https:// www. ncbi. nlm. nih. gov/ genba nk/). The freshwater mollusc species found in the diet (Potamopyrgus antipodarum, the New Zealand mud snail, and Semisulcospira dolorosa) were identified with > 99% sequence similarity in 2018 and 2019 but were not present in 2017. Freshwater arthropod insect sequences were found in all years with five genera detected. Two genera were Plecoptera, Nemoura fluva and Sweltsa sp., the other three were Conchapelopia sp. (Chironomidae), Tipula sp. (Tipulidae) and Dixa sp. (Dixidae). Nemoura fluva showed over 98% sequence similarity and the other four genera over 90% similarity to sequences in GenBank. One species of freshwater copepod was also found, Mesocyclops leuckrati, with over 99% sequence similarity.

Discussion
To the best of our knowledge, there are no previous published accounts of Japanese macaques definitively eating freshwater animals in streams, including brown trout. Feeding strategies of Japanese macaques change according to seasonal fluctuations in food resources 16,17 and typically winter is a bottleneck for food availability 18 thereby potentially resulting in a significant energy deficit 19 . Japanese macaques are at their upper elevation limit (1500 m) in Kamikochi and mortality may result from famine or hypothermia which tends to occur towards the end of winter in March and April 20 .
Consumption of vertebrates is rare 21 . Previously, Japanese macaques have been shown to opportunistically capture marine fish, either when being dried 22 or washed up on beaches 23 . Closely related species have been shown to feed on freshwater fish. For example, long tailed macaques actively capture fish from freshwater pools 24 , and wild chacma baboons from drying desert pools 25 . Presumably, the Japanese macaques follow a similar approach capturing brown trout in shallow pools along the stream margin although they may opportunistically eat dead fish although none were observed despite extensive wintertime sampling of the benthos. The capture of live fish has been suggested as probably rare in Japanese macaques 25 but brown trout were found in seven fecal samples from 2018 and 2019. Unfortunately, using metabarcoding, it is not possible to determine, whether the DNA signal came from different individual fish in each subsequent year.  Table 2. BLAST results from freshwater chordates, molluscs and aquatic arthropods (or arthropods having aquatic life stages) sequences identified in the fecal samples of Japanese macaques. % similarity = percentage of similarity between the sequences found in the fecal samples and the sequences in the GenBank database; % coverage = percentage of the sequence found in our sample aligned to a sequence in GenBank; number of reads = number of times that same sequence occurred in the samples. www.nature.com/scientificreports/ Animals attached to rocks require less energy expenditure to capture than fish. Although terrestrial insects are a major source of food for Japanese macaques, especially in summer, this is the first confirmed record of aquatic insect larvae and nymphs in their diet. Of the five genera that were found, three (N. fluva and Sweltsa sp., the two stoneflies, and the dipteran Tipula sp.) are relatively large (>1 cm) at the final instar stage. At the time of year the fecal samples were collected, these insects would not be present as adults, in their terrestrial life-cycle stage, so we conclude that they were captured from the freshwater environment. Japanese macaques collecting insects by turning stones over in the stream is a form of extractive foraging 26 .
Japanese macaques have previously been reported to eat marine mussels 27 and terrestrial snails and slugs 11,28 . The Burmese long-tailed macaque had been shown to use stone tools to crack open shellfish and this foraging task is time intensive 29,30 . One of the snails Potamopyrgus antipodarum, the New Zealand mud snail eaten at Kamikochi, is an invasive species first identified in Japan in 1990 31,32 with an average shell size of 0.5 cm. These molluscs are easily removed from the stones and we assume they are swallowed whole and feeding on them does not involve forceful extraction from the shell, as with marine mussels.
Due to their relatively small size, we speculate that the freshwater copepod, Mesocyclops leuckrati was consumed through the Japanese macaques drinking stream water.
In general, Japanese macaques have a wider home range in winter when food resources are scarce. However, the Kamikochi area lies in a deep valley where the surrounding mountain ranges are >2500 m and have steep inclines, so their home range cannot be expanded. As a result, the population density is exceptionally high, increasing from 90 individuals in two troops in 1990 to 205 individuals in four troops in 2018 6,33 . These Kamikochi macaques are thus forced to overwinter within an extremely harsh environment with high snowfall (mean max depth for 2017-2019 was 930 mm) and low winter temperature (minimums to -20 °C see Fig. 1). These larger populations create additional stress for overwintering survival. An abundance of groundwater upwellings and hot spring inputs from active volcanoes in the Kamikochi region ensures many streams are flowing over the winter without ice cover allowing easy access to the monkeys. Many streams have a stable water temperature year-round (5 to 6 °C) contributing to high biomass of fish and freshwater benthos 34 which would be accessible to the Japanese macaques. Hence, the Kamikochi area may be the only environment in Japan where the topographical, geological, and meteorological conditions facilitate this unique feeding of freshwater biota by Japanese macaques to supplement their winter diet.  www.nature.com/scientificreports/ Each step of the molecular analysis (DNA extraction, PCR setup, template addition, electrophoresis and PCR clean-up) was conducted in a separate sterile laboratory dedicated to that step with sequential workflow to ensure no contamination. Each room was equipped with ultra-violet sterilization which was switched on for a minimum of 15 min before and after each use. The PCR set-up and template addition were undertaken in laminar flow cabinets with HEPA filtration.

Methods
Each sample was homogenized using sterile beads (2 min, 1,500 RPM; 1600 MiniG Automated Tissue Homogenizer and Cell Lyser, SPEX SamplePrep, NJ, USA) and a subsample (ca. 0.2 g) weighted directly into the first tube of a DNeasy PowerSoil DNA Isolation Kit (QIAGEN, CA, USA). DNA was extracted following manufacturer's instructions using a robotic workstation (QIAcube, QIAGEN). Samples were frozen (−20 °C) until further processing and negative extraction controls were included in each run to monitor contamination.
As Japanese macaque DNA was potentially more prevalent than dietary organisms during high-throughput sequencing (HTS), a blocking primer approach was used to inhibit DNA amplification of the Japanese macaque during the PCR step 35 . A blocking primer was designed to overlap with the 3′-end of the forward PCR primer and modified with a C3 spacer. The blocking primer was included at 10 times the concentration of PCR primers during amplification.
PCR products were visualized on 1.5% agarose gel with Red Safe™ DNA Loading Dye (Herogen Biotech, USA) and UV illumination. PCR negatives were run to assess for contamination during the PCR steps. The PCR products were purified, cleaned of primer dimers and normalized using SequalPrep Normalisation plate (Ther-moFisher, MA, USA), and submitted to Auckland Genomics (University of Auckland, New Zealand) for library preparation. Sequencing adapters and sample-specific indices were added to each amplicon via a second round of PCR using the Nextera™ Index kit (Illumina Inc., USA). Quality control was undertaken using a bioanalyzer before the library was diluted to 4 nM and denatured. A 15% PhiX spike was used and the final loading concentration was 7 rM. Sequence data were automatically demultiplexed using MiSeq® Reporter (version 2, Illumina Inc.), and forward and reverse reads assigned to samples. Raw sequence reads were deposited in the National Center for Biotechnology Information (NCBI) short read archive under the accession number PRJNA706701.
Raw reads were processed, subsequent to primers being removed with cutadapt 37 using the DADA2 package 38 within R. Reads were truncated to 221 and 223 bp and filtered with a maxEE (maximum number of "expected errors") of 2 and 2 for forward and reverse reads respectively (reads not reaching this threshold were discarded). DADA2 constructs a parametric error matrix (based on the first 10 8 bps in the dataset), the samples are dereplicated and sequence variants for the forward and reverse reads are inferred based on the derived error profiles from the samples. Singletons observed in the inference step are discarded. Subsequently, paired-end reads were merged with a maximum mismatch of 1 bp and a required minimum overlap of 10 bp. Forward and reverse reads, which did not merge were not included in further analysis. Chimeras were removed using the function removeBimeraDenovo. The resulting chimera-checked, merged Amplicon Sequence Variants (ASVs) were used for taxonomic classification using a redundant BOLD database 39 supplemented with COI sequences from NCBI within the DADA2 package, which is based on the rdp classifier 40 with a bootstrap of 50. The results were parsed into a table using the phyloseq package 41 , and reads assigned as primates were removed. The differentiation of the sequences from the annelids present in the Japanese macaque's intestinal tract already from those in the diet was not feasible and thus removed. Negative controls were assessed and the sum of reads from contaminating ASVs was subtracted from the samples.
Phylogenetically annotated COI sequences were used to characterize eukaryotic composition for each fecal sample using the package ggplot2 42 in R. Sequences of interest (freshwater arthropods, molluscs and fish) were selected and blasted using the BOLD and the NCBI databases for a more accurate taxonomy identification.

Data availability
Raw sequence reads were deposited in the National Center for Biotechnology Information (NCBI) short read archive under the accession number PRJNA706701. www.nature.com/scientificreports/