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Demonstration of the potential of environmental DNA as a tool for the detection of avian species

Demonstration of the potential of environmental DNA as a tool for the detection of avian species www.nature.com/scientificreports OPEN Demonstration of the potential of environmental DNA as a tool for the detection of avian species Received: 16 October 2017 1,2 3,4 5 6 7 Masayuki Ushio , Koichi Murata , Tetsuya Sado , Isao Nishiumi , Masamichi Takeshita , 7 5 Accepted: 1 March 2018 Wataru Iwasaki & Masaki Miya Published: xx xx xxxx Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed dispersal, and thus monitoring bird species diversity is a first step towards avoiding undesirable consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual census of bird species is difficult. Environmental DNA (eDNA) is genetic material that persists in an environment and is derived from organ- isms living there, and researchers have recently been using eDNA to detect the presence of macro-organisms, 1–5 particularly those living in aquatic/semiaquatic ecosys . Fo tr exa ems mple, several fish species inhabiting a river can be detected by amplifying and sequencing DNA fragments extracted from wat b er s y ua sin mg p les methodologies such as quantitative PCR and eDNA metabarcoding. Quantitative PCR requires the design of 3,4,7,8 species-specic fi PCR primers and enables quantitative measurements of eDNA of target s , p w eh ciil es e eDNA metabarcoding, which has been becoming a common methodology in eDNA studies, uses a universal primer set and high-throughput sequencer (e.g., Illumina MiSeq) to enable qualitative detection of eDNA of multiple species 1,2,9–11 12 belonging to a target taxon (but see ref.). Although earlier studies mainly focused on detecting fish/amphibian species (i.e., organisms that have close associations with water), recent studies have shown that eDNA can be used to detect a diverse group of animals, 9,13,14 15 10 including mammals , reptiles and arthropods . Detecting the presence of animals is possible even if their 9,10,13,14 habitats are terrestrial because animals must have, in general, frequent opportunities to contact water in order to live. The findings of these recent studies imply that any organism, regardless of its main habitat, can potentially be detected by using eDNA if we can design suitable primers that enable amplification and identifica- tion of DNA fragments of target organisms and if we can collect appropriate media that contain eDNA. Wild birds represent an important part of the biodiversity in ecosystems, and they play a unique role in the maintenance of ecosystem functions. For example, in forest ecosystems, birds can contribute to maintenance of the tree community by seed dispersal and pollination, and to the reduction of herbivory by predation upon insect 16–18 herbivores . However, recent increases in anthropogenic impacts on ecosystems, e.g., urbanization and habitat 1 2 PRESTO, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan. Center for Ecological Research, Kyoto University, Otsu, 520-2113, Japan. Yokohama Zoological Gardens ZOORASIA, Kanagawa, 241-0001, Japan. 4 5 College of Bioresource Sciences, Nihon University, Kanagawa, 252-0880, Japan. Natural History Museum and Institute, Chiba, 260-8682, Japan. Department of Zoology, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan. Department of Biological Sciences, The University of Tokyo, Tokyo, 113-0032, Japan. Correspondence and requests for materials should be addressed to M.U. (email: ong8181@gmail.com) or M.M. (email: miya@chiba-muse.or.jp) SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 1 www.nature.com/scientificreports/ Primer name Information a,b Primers for the first PCR (with MiSeq sequencing primer and six random bases) ACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNN MiBird-U-F (forward) GGGTTGGTAAATCTTGTGCCAGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT NNNNNN MiBird-U-R (reverse) CATAGTGGGGTATCTAATCCCAGTTTG c,d Primers for the second PCR AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX 2nd PCR-F ACACTCTTTCCCTACACGACGCTCTTCCGATCT CAAGCAGAAGACGGCATACGAGAT XXXXXXXX 2nd PCR-R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Table 1. Detailed information for MiBird prim Ier tas. lic characters indicate the MiSeq sequencing primers. b c Bold Ns indicate random bases to improve the quality of MiSeq sequen Bold Xs in cing. dicate index sequences to identify each sample U. nderlined characters indicate P5/P7 adapter sequences for MiSeq sequencing. 19,20 fragmentation, drive substantial declines in bird species div, er whic sity h could have impacts on the ecological functions of birds. Monitoring bird species diversity is required for detecting such declines, and such detection is necessary for avoiding undesirable consequences in ecosystem functions due to the loss of avian biodiversity. To monitor bird species diversity, visual census is one of the most common m , a et n h d co ods nsidering the higher visibility of birds than that of fish and forest mammals, visual census is generally a successful method. However, if an alternative method can overcome limitations of visual census, such as low visibility at night or in a dense forest, and eliminate the requirement for taxonomic identification skill under field conditions, that method could be complementarily used for monitoring bird species diversity. In the present study, we tested the potential of eDNA as a tool for the detection of avian species. Previous eDNA surveys performed in marine ecosystems detected some avian species along with diverse fish/mammal 22–25 species (2–4 avian species per study) , suggesting that more diverse avian species are potentially detectable using eDNA if suitable primers are designed. To this end, we modified a previously reported universal primer 1,9 set for fish/mammals (MiFish/MiMammal ), such that the primer set accommodated bird-specific variations, and conducted avian eDNA metabarcoding. During the primer design, we did not try to eliminate the capability of detecting mammalian and other vertebrate species, because simultaneous detection of mammals and other vertebrates along with birds may be advantageous, especially for ecologists who are interested in co-occurrence patterns and potential interactions among various animal species. We performed a series of analyses to test the versatility of the designed primers: In e sixa lico minations of the primers, amplification of extracted tissue DNAs of birds belonging to various taxa, and field tests by analyzing water samples from zoo cages containing birds of known species composition. Additionally, we briefly examined the usefulness of the new primer set using water samples from field samples with unknown bird species composition. Methods All of the critical information of our study is described below, but is also listed in Table S1 to faci - litate compar isons with other studies, following the recommendations of Gold.ber . A g le l exp t al eriments were performed without direct captures of avian species, and carried out in accordance with the relevant guidelines and regula- tions. Also, all experimental protocols in the zoo were approved by Yokohama Zoological Gardens ZOORASIA. Primer design. To facilitate design based on comparisons of diverse avian sequences, we first batch down- loaded 410 avian sequences from RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) on June 9, 2015. Then, a base composition for a selected position in the conservative region was sh esq owuite n in M . The base compositions in selected characters were manually recorded in a spreadsheet for the primer design. In the primer design pro- cess, we considered a number of technical tips that enhance the primer annealing to the template without the use of degenerate bases : primers include some G/C at th -en e 3d ′ s to strengthen primer-template annealing at this position, but a string of either Gs or Cs at t -en hd s e 3h ′ ould be avoided: considering the unconventional base pairing in the T/G bond, the designed primers use G rather than A when the template is variably C or T, and T rather than C when the template is A or G; G/C contents of the primers fall between 40 and 60%, with an almost identical melting temperatur ). e ( T T was calculated using a nearest-neighbour thermodynamic model imple- m m mented in O ligoCalc . 1,9 We designed our primers by modifying previously developed MiFish/MiMammal pr,i w mh eir csh corr-e sponded to regions in the mitochondriS rRN al 12 A gene (insert lengt = h ca . 171 bp), and we named our primers MiBird-U (“U” indicates “universal”). Primer sequences with MiSeq adaptors (for the first- and second-round PCR) are listed in Tab1 le  . In silico evaluation of interspecific variation of MiBird sequences. e Th binding capacity of MiBird-U primers was computationally evaluated using the batch-downloaded 410 avian sequences. Using custom Ruby and Python scripts, the number of mismatches between MiBird-U primers and the 410 avian sequences as well as other non-target animal sequences (i.e., 741 mammalian, 197 amphibian, and 245 reptilian sequences) was cal- culated. Positions of base match/mismatch between MiBird-U primers and avian sequences were also examined using the downloaded avian sequences. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 2 www.nature.com/scientificreports/ Common name Scientific name Order Family Accession No. Spot-billed duck Anas zonorhyncha Anseriformes Anatidae LC104767 Grey nightjar Caprimulgus indicus Caprimulgiformes Caprimulgidae LC104768 Ancient murrelet Synthliboramphus antiquus Charadriiformes Alcidae LC104769 Lesser sand plover Charadrius mongolus Charadriiformes Charadriidae LC104770 Oriental turtle dove Streptopelia orientalis stimpsoni Columbiformes Columbidae LC104771 Cuculus poliocephalus Lesser cuckoo Cuculiformes Cuculidae LC104772 poliocephalus Northern Goshawk Accipiter gentilis fujiyamae Accipitriformes Accipitridae LC104773 Common kestrel Falco tinnunculus Falconiformes Falconidae LC104774 Chinese bamboo partridge Bambusicola thoracicus Galliformes Phasianidae LC104775 Red-throated loon Gavia stellata Gaviiformes Gaviidae LC104776 Red-crowned crane Grus japonensis Gruiformes Gruidae LC104777 Jungle crow Corvus macrorhynchos Passeriformes Corvidae LC104778 Eurasian sparrow Passer montanus Passeriformes Passeridae LC104779 Black-crowned night heron Nycticorax nycticorax Pelecaniformes Ardeidae LC104780 Great white pelican Pelecanus onocrotalus Pelecaniformes Pelecanidae LC104781 Great cormorant Phalacrocorax carbo hanedae Suliformes Phalacrocoracidae LC104782 Japanese pygmy woodpecker Dendrocopos kizuki Piciformes Picidae LC104783 Little grebe Tachybaptus ruficollis Podicipediformes Podicipedidae LC104784, LC104785 White-chinned petrel Procellaria aequinoctialis Procellariiformes Procellariidae LC104786 Short-tailed shearwater Puffinus tenuirostris Procellariiformes Procellariidae LC104787 King penguin Aptenodytes patagonicus Sphenisciformes Spheniscidae LC104788 Humboldt penguin Spheniscus humboldti Sphenisciformes Spheniscidae LC327059 Table 2. Extract DNAs used to test the performance of the MiBird primer set. Interspecific differences within the amplified DNA sequences are required for assignment of taxonomic cat- egories. Levels of interspecific variation in the target region (hereae ft r called ‘MiBird sequence’) across die ff rent taxonomic groups of birds were computationally evaluated using the 410 downloaded avian sequences. Among the sequences of the 410 avian, species with the deletion of primer regions (Hemignathu , s L mu oxonr pso cioc- cineus and Arborophila rufipectus ) were excluded, and 407 MiBird sequences were extracted and subjected to calculation of pairwise edit distances using custom Python scripts. Pairwise inter-species edit distances were cal- culated for all species pairs, and pairwise inter-genus edit distances were calculated for pairs of species belonging to different genera. The edit distance quantifies dissimilarity of sequences in bioinformatics and is defined as the minimum number of single-nucleotide substitutions, insertions or deletions that are required to transform one sequence into the other. In addition, the binding capacity and the levels of interspecific variations of the target region were further 30 31 evaluated using ‘primerTree’ packa og f R v e ersion 3.3.1 . Briefly, primerTree performs the following analysis: (1) In silico PCR against sequences in the NCBI database; (2) retrieval of DNA sequences predicted to be ampli- e fi d; (3) taxonomic identic fi ation of these sequences; (4) multiple DNA sequence alignment; (5) reconstruction of a phylogenetic tree and (6) visualization of the tree with taxonomic annotation. Thus, by using primerTree pack- age, species whose sequences can be amplified, phylogenetic relationships among these amplified species, and interspecific variations in the amplified sequences are rapidly visualized. Further information and instructions for the primerTree package can be found in Canno.n .et al Primer testing with extracted DNA. We tested the versatility of MiBird-U (no adapter sequences) using DNA extracted from 22 species representing major groups of bird 2 s ( ). D Ta o b u le bl   e-stranded DNA concen -tra tions from those samples were measured with a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, −1 Wilmington, DE, USA) and the extracted DNA was diluted t ng o 15 µ l u sing Milli-Q water. PCR was carried out with 30 cycles of a 15 µ l r eaction volume containinµg 4.5 l st erile distilled H O, 7.5 µ l 2× Gflex PCR Buffer 2+ (Mg , dNTPs plus) (Takara, Otsu, Japan), µ 0.7 l o f each primer (5 μM), 0.3 µ l Taq polymerase (Tks Gflex DNA Polymerase; Takara) and 1.2 µl tem plate. The thermal cycle profile aer a ft n initiamin den l 1 aturation at 94 °C wa s as follows: denaturation a °C f t 98 o r 10s; a nnealing at 50 °C f or 10s; a nd extension at 68 °C f or 10s w ith a final extension at the same temperature f min. or 7 Study site and water sampling for primer testing with eDNA from zoo samples. To test the versati lity of the newly designed primers for metabarcoding avian eDNA, we sampled water from cages on 13 December 2016 in Yokohama Zoological Gardens ZOORASIA, Yokohama, Japan (35°29 42″ N, 139°31 ′ ′35″ E), where we previously tested the usefulness of a universal primer set targeting m . W a e c mm hos al e t s he zoo as a sampling site because the information about avian species in a cage is precisely known, and because the zoo rears diverse taxonomic groups of animals (i.e 100 a ., > nimal species, including many mammals and birds). Thirteen cages, in which diverse taxonomic groups of birds were reared, were selected as sampling pl3 aces (T ). Most o able  f the SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 3 www.nature.com/scientificreports/ Common name Species name Order Family Steller’s sea eagle Haliaeetus pelagicus Accipitriformes Accipitridae Black-tailed gull Larus crassirostris Charadriiformes Laridae Capercaillie Tetrao urogallus Galliformes Tetraonidae Lady Amherst’s pheasant Chrysolophus amherstiae Galliformes Phasianidae Ruddy shelduck Tadorna ferruginea Anseriformes Anatidae Temminck’s tragopan Tragopan temminckii Galliformes Phasianidae Victoria crowned pigeon Goura victoria Columbiformes Columbidae Mandarin duck Aix galericulata Anseriformes Anatidae Humboldt penguin Spheniscus humboldti Sphenisciformes Spheniscidae Snowy owl Bubo scandiacus Strigiformes Strigidae Oriental white stork Ciconia boyciana Ciconiiformes Ciconiidae White-naped crane Grus vipio Gruiformes Gruidae Common crane Grus grus Gruiformes Gruidae Southern ground hornbill Bucorvus leadbeateri Coraciiformes Bucerotidae Harris’s hawk Parabuteo unicinctus Accipitriformes Accipitridae Emu Dromaius novaehollandiae Struthioniformes Casuariidae Table 3. Classification of the target bird species in the Zoorasia exper Sp im ecies k ent. ept in a walk-through bird cage with other bird species. target species were kept separately, but ruddy shelduck (Tadorna fe) w rruer gin e k eaept in a walk through bird cage (hereafter, “the bird cage”) with other bird species (i.e., Lady Amherst’s pheasant [Chrysolophus amhers- tiae], Temminck’s tragopan Tr [ agopan temminckii], Victoria crowned pigeo G n ou [ ra victoria] and mandarin duck [Aix galericulata]). Note that different individuals of Lady Amherst’s pheasant, Temminck’s tragopan, Victoria crowned pigeon and mandarin duck from those in the bird cage were separately kept (i.e., in different cages from the bird cage), and that each water sample of the bird species was collected from each cage. TM Each 100–200m l water sample was collected through a s0.45- terile µ m S ϕ terivex filter (Merck Millipore, Darmstadt, Germany) using a sterile 50-mL syringe (TERUMO, Tokyo, Japan). Aer t ft he filtration, approximately 2 ml of RNAlater (ThermoFisher Scientific, Waltham, Massachusetts, USA) was injected into the Ster - ivex car tridge, and the filtered water samples were stor °C f ed a ot 4 r u p to one day until further processing. Three negative controls (distilled water) were taken to the zoo to monitor contaminations during water sampling, filtration and transport. In addition to the survey in the zoo, we collected water samples from a pond adjacent to the Natural History Museum and Institute, Chiba (35°35 59″ ′ N, 140°8′18″ E; Funada-ike Pond) to test the potential effectiveness of the MiBird primers under a field condition with unknown bird species composition. Water collections at the pond were performed in the same way as those performed in the zoo. DNA extraction. The Sterivex filter cartridges were taken back to the laboratory, and DNA was extracted from the filters using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following a protocol described and illustrated in Miya .et . B al riefly, the RNAlater-supplemented solution was removed under a vacuum using the QIAvac system (Qiagen, Hilden, Germany). Proteinase-K solutµ io l), p n (20 hos phate buffered saline (PBS) (220 µ l) and buffer AL (200 µ l) were mixed, and 440 µ l o f the mixture was added to each filter cartridge. The materials on the filter cartridges were subjected to cell-lysis conditions by incubating the filters on a rotary shaker (at a speed of 20 rp m) at 50 °C f or 20min. Th e incubated mixture was transferred into a new 2-ml tube, and the collected DNA was purified using a DNeasy Blood and Tissue Kit following the manufacturer’s protocol. Aer t ft he purification, DNA was eluted using 100 µl o f the elution buffer provided with the kit. Paired-end library preparation. Prior to the library preparation, work-spaces and equipment -were ster ilized. Filtered pipet tips were used, and separation of pre- and post-PCR samples was carried out to safeguard against cross-contamination. We also employed two negative controls (i.e., PCR negative controls) to monitor contamination during the experiments. e Th first-round PCR (first PCR) was carried out with a 12µ -l reaction volume containin µ gl 6 o .0 f 2 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, WA, USA), 0.7 µl of M iBird primer (5µM p rimer F/R, w/ adaptor and six random bases; Ta1 b), 2.6 les  µ l of sterilized distilleO a d Hnd 2.0µ l of template. The thermal cycle profile aer a ft n initial 3 min den aturation at 95 °C wa s as follows (35 cycles): denaturation a °C f t 98 or 20 s; annealing at 65 °C f or 15s; a nd extension at 72 °C f or 15s, w ith a final extension at the same temperaturmin. e for 5 We performed triplicate first-PCR, and these replicate products were pooled in order to mitigate the PCR drop- outs. The pooled first PCR products were purified using AMPure XP (PCR product: AMPure XP be = a d 1: s 0.8; Beckman Coulter, Brea, California, USA). The pooled, purified, and 10-fold diluted first PCR products were used as templates for the second-round PCR. The second-round PCR (second PCR) was carried out with a 24- l reac µ tion volume containinµ g 12 l of 2 × KAPA HiFi HotStart ReadyMix, 1.4 µ l o f each primer (5 µ M p rimer F/R; Table  1), 7.2 µ l of sterilized distilled H O and 2.0µ l of template. Different combinations of forward and reverse indices were used for different tem- plates (samples) for massively parallel sequencing with MiSeq. The thermal cycle profile after a n m iin ni tial 3 SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 4 www.nature.com/scientificreports/ Figure 1. In silico evaluations of MiBird-U primers. Binding capacity of MiBird-U primers (a). y-axis represents the proportion of avian species that showed 0, 1, 2, 3, 4, or >5 mismatches (indicated by different colours) with MiBird-U F/R primers. The total number of avian species evaluated was 410. The phylogenetic tree was constructed for species that can be amplified using MiBird-U primers (b). A total of 2,000 sequences were retrieved from the database to construct the phylogenetic tree. Different classes are represented by filled circles with different colours. Lengths of branches correspond to the differences in sequences. Bar indicates edit distance. denaturation at ° C 95 was as follows (12 cycles): denaturation °C a t fo 9 r 8 2 0 s; combined annealing and extension at 72 °C (s huttle PCR) for 15 s, w ith a final extension at 72 °C f or 5min. e Th indexed second PCR products were mixed at equimolar concentrations to produce equivalent sequencing depth from all samples and the pooled library was purified using AMPure XP. Target-sized DNA o - f the puri fied library (ca. 370 bp) was excised using E-Gel SizeSelect (ThermoFisher Scientific, Waltham, MA, USA). The double-stranded DNA concentration of the library was quantified using a Qubit dsDNA HS assay kit and a Qubit uo fl rometer (ThermoFisher Scientific, Waltham, MA, USA). The double-stranded DNA concentration of the library was then adjusted t nM u o 4 sing Milli-Q water and the DNA was applied to the MiSeq platform (Illumina, San Diego, CA, USA). The sequencing was performed using a MiSeq Reagent Kit Nano v2 f × o 150 r 2 bp PE (Illumina, San Diego, CA, USA). Data processing and taxonomic assignment. The overall quality of the MiSeq reads was evaluated using the programas s F tqc (available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and SUGAR . Aer co ft nfirming the lack of technical errors in the MiSeq sequencing, low-quality tails were trimmed from each read using DynamicTrim.pl from t oh lexa e S Qa software package with a cut-off threshold set at a −1 Phred score of 10 (=10 error rate). The tail-trimmed pair-end reads were assembled using the software FLASH with a minimum overlap of 10 bp. Th e assembled reads were further filtered by custom Perl scripts in order to remove reads with either ambiguous sites or those showing unusual lengths compared to the expected size of the PCR amplicons. Finally, the softwarag e T Cleaner was used to remove primer sequences with a maximum of three-base mismatches and to transform the FASTQ format into FASTA (see Table S2 for the numbers of reads remained aer t ft hese pre-processing). e Th pre-processed reads from the above custom pipeline were dereplicated using UCL , w US ith Tthe num- ber of identical reads added to the header line of the FASTA formatted data file. Those sequences represented by at least 10 identical reads were subjected to the downstream analyses, and the remaining under-represented sequences (with less than 10 identical reads) were subjected to pairwise alignment using UCLUST. If the latter sequences observed for less than 10 reads showed at least 99% identity with one of the former reads (one or two nucleotide differences), they were operationally considered as identical (owing to sequencing or PCR errors and/ or actual nucleotide variations in the populations). e p Th rocessed reads were subjected to local BLASTN searc a hga es inst a custom-made database. e c Th ustom database was generated by downloading all whole mitogenome sequences from Sarcopterygii deposited in NCBI Organelle Genome Resourc h e t st (p://www.ncbi.nlm.nih.gov/genomes/OrganelleResource.cg = i? 8t 2a 8x 7) i.d As of 15 March 2016, this database covered 1,881 species across a wide range of families and genera (including birds, mammals, reptiles and amphibians). In addition, the custom database was supplemented by all whole and partial fish mitogenome sequences deposited in MitoFi in o sh rder to cover fish detection (note that MiBird primers amplify fish sequences as well; see Fig 1)..  −5 e Th top BLAST hit with a sequence identity of at least 97% a E-va nd lue threshold of 10 was applied to spe- cies assignments of each representative sequence. Reliability of the species assignments was evaluated based on the ratio of total alignment length and number of mismatch bases between the query and reference sequences. For example, if a query sequence was aligned to the top BLAST hit sequence with an alignment len bp w gt it h o h f 150 one mismatch present, the ratio was calculated a+ s 150/(1 1). The va lue one was added to the denominator to SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 5 www.nature.com/scientificreports/ MiBird-U-F G G G T T G G T A A A T C T T G T G C C A G C A 1 4 1 1 0 1 0 0 397 407 407 0 0 0 0 0 0 0 0 0 407 0 0 C 0 0 0 61 129 0 0 0 2 0 0 7 407 155 0 0 0 0 407 407 0 0 407 G 406 403 406 0 0 406 407 0 1 0 0 0 0 0 0 407 0 407 0 0 0 407 0 T 0 0 0 345 278 0 0 407 7 0 0 400 0 252 407 0 407 0 0 0 0 0 0 MiBird-U-R C A T A G T G G G G T A T C T A A T C C C A G T T T G A 0 407 0 406 0 0 0 2 0 0 0 407 0 1 0 407 407 0 0 0 0 406 0 1 0 0 0 C 407 0 0 0 0 1 0 0 0 0 0 0 0 406 0 0 0 0 407 406 404 0 0 1 0 5 1 G 0 0 0 0 407 0 407 405 407 407 0 0 1 0 0 0 0 0 0 0 0 1 405 0 0 0 406 T 0 0 407 1 0 406 0 0 0 0 407 0 406 0 407 0 0 407 0 1 3 0 2 405 407 402 0 Table 4. Nucleotide sequences of the universal primers (MiBird-U) and base compositions of the selected 407 avian species. Among the downloaded sequences, 3 species with the deletion of primer regions were excluded, resulting in the sequences of 407 avian species. Italic bases indicate primer sequences. Numbers indicate the number of avian species of which base matches with A, C, G, or T listed in the left column. Bold numbers indicate the number of avian species of which base matches with that of the primer. Edit distance 0 1 2 3 4 ≥5 Total Frequency distributions of the inter-specific/genus edit distances of the insert sequence Species 27 50 67 113 187 82,177 82,621 combinations Genus 10 23 40 80 157 Table 5. Frequency distributions of the interspecific edit distances of the primer set against bird sequences. Pairwise inter-species edit distances were calculated for all species pairs, and pairwise inter-genus edit distances were calculated for pairs of species belonging to different genera. avoid zero-divisors. This value (e.g., 150/( 1 + 1)) was calculated for the top and second-highest BLAST hit species, and the ratio score between these values was used as a comparable indicator of the species assignment. Results from the BLAST searches were automatically tabulated, with scientific names, common names, total number of reads and representative sequences noted in an HTML format. The above bioinformatics pipeline from data pre-processing through taxonomic assignment is available in supplements in a pre. A vio lu so s s , t th ud e a y bove bioinformatic pipeline can be performed on a website. For more detailed information, please see http://mitos fi h. aori.u-tokyo.ac.jp/mifish. Please note that the pipeline implemented in the website currently uses the custom s fi h database and does not aim to detect avian species (confirmed on 20 September 2017). Data availability. DDBJ Accession numbers of the DNA sequences analyzed in the present study are DRA006196 (Submission ID), PRJDB4990 (BioProject ID) and SAMD00096837–SAMD00096858 (BioSample ID). Results and Discussion Tests of versatility of designed primers in silico and using extracted DNA. First, the performance of MiBird-U primers was tested in si (Fig lico . 1 and Tables  4 and 5). When G/T pairs were accepted, MiBird-U-F and -R perfectly matched 390 (95.1%) and 388 (94.6%) species among 410 species tested, respectively, and 99.5% and 96.8% of the 410 species showed at most 1 mismatch (Fig 1a). A .  mong the avian sequences tested, all species showed no mismatch at the 3 -en ′d of MiBird-U-F, and most species ( 98.7%) s > howed no mismatch at the 3 -en ′d of MiBird-U-R (Table  4). In addition, inter-specific differences in the edit distance were calculated and 82,177 out of 82,621 combinations (99.5%) showed edit distance larger than 5 (T 5). Tha es be a le nalyses suggested that the target region of most avian species can be amplified using MiBird-U primers, and that the amplified sequences contain sufficient information required for assignment of taxonomic categories. To examine the range of species that can be amplified using MiBird-U primers, we performed an analysis with the primerTree packag . The e results confirmed that the primers can amplify avian species 1b (Fig ). M .  iBird-U primers can also amplify a diverse group of mammalian species in addition to amphibian, reptilian and s fi h species (Fig. 1b), which is not surprising because MiBird-U primers were produced by modifying fish/mammal-targeting universal primers. The potential of MiBird-U primers to amplify mammalian, amphibian, and reptilian species was also confirmed by in silico test of the binding capacity of MiBird-U primers (Table S3). The capacity of MiBird-U primers to detect mammalian and other species might be useful when simultaneous detection of these animals is desired (e.g., when one tries to study co-occurrence patterns and potential interactions among animals). Second, the performance of MiBird-U primers was evaluated using 22 extracted avian DNA samples. All of the extracted DNA samples were successfully amplified, and the resultant sequences were deposited in the DDBJ/ EMBLE/GenBank databases (Tab2 le  ). Together, the results of in s t ilies cots and the amplification of extracted DNAs suggested that MiBird-U primers are capable of amplifying/identifying DNA fragments derived from diverse avian species. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 6 www.nature.com/scientificreports/ Primer testing with eDNA from el fi d water samples. MiSeq sequencing and data pre-processing gen- erated 656,472 sequences from 21 samples (including 3 field negative controls and 2 PCR negative controls) (Table  5). In general, the quality of sequences produced by our experiment was high (i.e., most raw reads passed the filtering process; Table S2). Among the 16 water samples from zoo cages examined here, all avian species were successfully detected (Table  6). Briefly, eDNA samples of the Steller’s sea eagle (Haliaeetus pela ), c gica up s ercaillie (Tetrao urogallus), white-naped crane (Grus vipi ), co o mmon crane (Grus grus ) and southern ground hornbill (Bucorvus leadbe) ateri generated high numbers of sequence reads, and 64.9–94.9% of total sequence reads were assigned to the target avian species. Samples from cages of the black-tailed gull (Larus cra ), H ssirum ostrb is oldt penguin (Spheniscus humboldti), snowy owl (Bubo scandiacu), Or s iental white stork (Ciconia boycia ), H naarris’s hawk (Parabuteo unicinctus) and emu (Dromaius novaehollandiae) generated fewer sequence reads, and 1.4–28.8% of total sequence reads were assigned to the target avian species. The reason for these variations in the proportions of sequence reads from target avian species is not known, but as discussed in the prev, t ioh ue o s st bs ud er yved levels of variations were not surprising because detection of animals’ sequences relies on contacts of animals with water and because opportunities for animals to contact water would depend on animals’ behaviour. These considera- tions imply that the proportion of sequence reads from a particular avian species would be inherently spatially and temporally stochastic to some extent (see also results of mammalian eDNA metabarcodin . g in U ). It shio et al is not surprising that sequences of the Lady Amherst’s pheasant, ruddy shelduck, Temminck’s tragopan, Victoria crowned pigeon and mandarin duck were detected in the ruddy shelduck samp6 le (T ) bec a a bu le  se all of these five species were kept in the bird cage where the ruddy shelduck sample was collected. In addition to the target avian species, we frequently detected many non-target sp 6 a ecies (T nd S4). F abo le  r example, sequences of the Steller’s sea eagle were frequently detected in other samples, e.g., the Victoria crowned pigeon, Oriental white stork, Humboldt penguin and so on (T 6). A ab s o le  ur field negative controls generated no target bird sequences (Ta 6b ), i le  t does not seem likely that the detection of the sea eagle in other samples was due to cross-contamination during sampling or experiments. One possible reason for the detection of non-target avian species include the spatial closeness of the eagle’s cage and the other cages. For instance, the cages of the Victoria crowned pigeon (i.e., the bird cage) and Humboldt penguin were located close to the eagle’s cage, and thus it is possible that the eagle’s feathers and other tissues could be transported (e.g., via wind) to other cages. Also, zoo staff frequently moved among cages, and they were possible transporters (e.g., through their shoe sole) of materials containing DNA of non-target species. Other frequently detected non-target species were falcated teal (M), co areca mm falc o an s ta helduck (Tadorna tadorna), common moorhen (Gallinula chloropu ), fi s shes and humans (Table S4). The falcated teal, shelduck and moorhen were not kept in cages, but wild common moorhens and close relatives of the duck and shelducks (i.e., Eurasian wigeon [Anas penelope ] and common pochard [Aythya ferina], respectively) are commonly observed in the regulating pond on-site of sampling region, and thus their DNA might have contaminated zoo cages (possibly via feathers or other tissues) and thus have been detected by the metabarcoding. The frequently detected fish species here are also species that are commonly observed in Japan, and the zoo uses waters from a natural lake and rivers. Therefore, the fish sequences might have been derived from water under natural conditions. Detection of many human sequences was not surprising considering that visitors to the zoo and staff members, who are potential sources of human sequences, are almost always near the cages. It is also be possible that contaminations of human and fish DNA happened under the laboratory conditions (Table S4), because in our lab fish DNAs were routinely processed and humans were oen w ft orking (i.e., carry-over contam- inations). Specifically, ocean fish sequences were detected from zoo samples despite the efforts for decontami- nation, and these contaminants are likely due to previous work in the same lab. The sequences of these obvious non-target taxa (i.e., humans, fish, and potential non-target carry-over contaminations) may be excluded from further statistical anal if o yses ne may be interested in ecological interpretations of the results. Lastly, in order to test the usefulness of MiBird primers under a natural field condition, we performed a metabarcoding study using a water sample from a pond adjacent to the Natural History Museum and Institute, Chiba (Funada-ike Pond). As a result of MiSeq sequencing, 14,873 reads of avian species were generated from three water samples, and five avian species (common shoveler [Anas clypeata], 883 reads; falcated teal, 3,246 reads; common moorhen, 9,260 reads; light-vented bulbul [Pycnonotus sinensis], 745 reads; and common shelduck, 739 reads) were detected. As a systematic monitoring of the bird community (e.g., frequent visual observation) has not been performed in the study site, rigorous validation of the metabarcoding study was not possible. Some avian species detected, i.e., light-vented bulbuls, common shelducks and falcated teals, are rare, or not reported, in this region, suggesting that these species were misidentified. These possible misidentifications are likely to be attributable to a lack of reference sequences and/or insufficient inter-species dier ff ences in the amplified DNA region (i.e., partial S 1 m 2 itochondrial region) (see also r ). L efi .ght-vented bulbuls, common shelducks and falcated teals are relatives of brown-eared bulbuls (Hypsipetes a ), co maumm rotio s n pochards (Aythya ferina) and Eurasian wigeons (Anas penelo), r pe espectively, and these relatives are indeed common inhabitants in the sampling region. Together, these results suggest that MiBird primers were capable of detecting bird species under a field condition, but at the same time, improvements of reference sequence databases, further validations of MiBird primers, and careful interpretations are necessary. Conclusion A proof-of-concept that eDNA metabarcoding can potentially detect avian species has been already demonstrated in 22–25 previous studies , and in the present study we explicitly demonstrated the potential and usefulness of avian eDNA metabarcoding using our new primer set and MiSeq platform. Describing and monitoring the diversity of bird spe- cies, as well as other animals, is one of the critical steps in ecosystem conservation and management, but it can be labo- rious, costly and incomplete if one relies on a few traditional survey methods. The eDNA metabarcoding approach SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 7 www.nature.com/scientificreports/ Bird species name detected from sequences Common name of bird living in cage Scientific name Haliaeetus Larus Tetrao Chrysolophus Tadorna Tragopan Goura Aix Spheniscus Bubo Steller’s sea eagle Haliaeetus pelagicus 28,448 0 0 0 0 0 0 0 0 0 Black-tailed gull Larus crassirostris 0 4,437 0 0 0 0 0 0 0 0 Capercaillie Tetrao urogallus 0 0 36,095 0 0 0 0 0 0 0 Lady Amherst’s pheasant Chrysolophus amherstiae 0 0 0 39,151 0 0 0 25 0 0 Ruddy shelduck Tadorna ferruginea 0 0 0 138 2,848 209 2,750 7,939 0 0 Temminck’s tragopan Tragopan temminckii 0 0 376 0 0 57,072 0 0 0 0 Victoria crowned pigeon Goura victoria 11,023 0 0 0 0 0 2,186 24 0 0 Mandarin duck Aix galericulata 254 0 0 0 65 51 34 13,465 0 0 Humboldt penguin Spheniscus humboldti 1,905 0 0 0 289 0 85 428 1,834 0 Snowy owl Bubo scandiacus 901 0 0 0 65 0 64 191 0 425 Oriental white stork Ciconia boyciana 7,258 63 0 0 0 0 103 258 0 0 White-naped crane Grus vipio 186 0 527 0 22 0 36 33 0 0 Common crane Grus grus 0 0 0 548 0 0 0 0 0 0 Southern ground hornbillBucorvus leadbeateri 148 0 0 0 0 15 0 0 0 0 Harris’s hawk Parabuteo unicinctus 1,227 0 0 0 77 0 0 332 0 0 Dromaius Emu 396 0 0 0 67 0 25 146 0 0 novaehollandiae Field NC 0 0 0 0 0 0 0 0 0 0 Field NC 0 0 0 0 0 0 0 0 0 0 Field NC 0 0 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 0 0 Total sequence 51,746 4,500 36,998 39,837 3,433 57,347 5,283 22,841 1,834 425 Bird species name detected from sequences Common name of bird Non-target Total b,c living in cage Scientific name Ciconia G. vipio G. grus Bucorvus Parabuteo Dromaius sequences sequences % target living in cage Steller’s sea eagle Haliaeetus pelagicus 0 0 0 0 0 0 3,238 31,686 89.8 Black-tailed gull Larus crassirostris 0 0 0 0 0 0 17,922 22,359 19.8 Capercaillie Tetrao urogallus 0 13 0 0 0 0 19,477 55,585 64.9 Lady Amherst’s pheasant Chrysolophus amherstiae 0 0 15 0 0 0 8,130 47,321 82.7 Ruddy shelduck Tadorna ferruginea 0 0 0 0 0 0 12,498 26,382 10.8 Temminck’s tragopan Tragopan temminckii 0 0 0 0 0 0 1,944 59,392 96.1 Victoria crowned pigeon Goura victoria 0 0 0 0 0 0 13,108 26,341 8.3 Mandarin duck Aix galericulata 0 0 0 0 0 0 12,272 26,141 51.5 Humboldt penguin Spheniscus humboldti 0 0 0 0 0 0 28,091 32,632 5.6 Snowy owl Bubo scandiacus 0 0 0 0 0 0 28,566 30,212 1.4 Oriental white stork Ciconia boyciana 3,072 0 0 0 0 0 22,815 33,569 9.2 White-naped crane Grus vipio 0 59,678 0 31 0 0 2,390 62,903 94.9 Common crane Grus grus 0 0 52,717 0 0 0 2,586 55,851 94.4 Southern ground hornbillBucorvus leadbeateri 0 0 0 36,955 0 0 4,900 42,018 88.0 Harris’s hawk Parabuteo unicinctus 0 0 0 0 306 0 20,338 22,280 1.4 Dromaius Emu 0 22 0 0 0 1,647 3,412 5,715 28.8 novaehollandiae Field NC 0 0 0 0 0 0 27,218 27,218 Field NC 0 0 0 0 0 0 7,977 7,977 Field NC 0 0 0 0 0 0 40,890 40,890 PCR NC 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 Total sequence 3,072 59,713 52,732 36,986 306 1,647 277,772 656,472 Table 6. Sequence reads of detected species from water samples collected in the zoo. Bold numbers indicate a b sequence reads of a target species. Species kept in a walk-through bird ca Sg ee T e. able S4 for the contents of non-target sequences. See Table S3 for the contents of non-target sequences. presented here is non-invasive and efficient. Moreover, as information of non-target organisms (e.g., invertebrates and microbes in our case) is also encoded in eDNA, analyzing eDNA of organisms from multiple taxa might be useful for studying co-occurrence patterns and even potential interactions among organisms (e.g., bird-insect interactions). In conclusion, we propose that the eDNA metabarcoding approach can serve as an efficient alternative for taking a snapshot of bird diversity and could potentially contribute to effective ecosystem conservation and management. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 8 www.nature.com/scientificreports/ References 1. 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Iwasaki, W.e t al. MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol. Biol. Evol. 30, 2531–40 (2013). Acknowledgements We would like to thank Noriya Saito, assistant manager of Yokohama Zoological Gardens ZOORASIA for help in sampling at the zoo, and Asako Kawai for assistance with experiments. We also thank Hiroki Yamanaka of e D Th epartment of Environmental Solution Technology/The Research Center for Satoyama Studies in Ryukoku University for providing the opportunity for us to use the Illumina MiSeq platform. This research was supported by PRESTO (JPMJPR16O2) from Japan Science and Technology Agency (JST), CREST (JPMJCR13A2) from Japan Science and Technology Agency (JST), and ERTDF (4–1602) The Environment Research and Technology Development Fund, Japan. This study was approved by Yokohama Zoological Gardens ZOORASIA. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 9 www.nature.com/scientificreports/ Author Contributions M.U. and M.M. conceived and designed research; M.U., I.N. and K.M. performed sampling; M.U., I.N., T.S. and M.M. performed experiments; M.U., M.T. and W.I. performed data analysis; M.U. and M.M. wrote the early dra ft and completed it with significant inputs from all authors. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-22817-5. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Demonstration of the potential of environmental DNA as a tool for the detection of avian species

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Abstract

www.nature.com/scientificreports OPEN Demonstration of the potential of environmental DNA as a tool for the detection of avian species Received: 16 October 2017 1,2 3,4 5 6 7 Masayuki Ushio , Koichi Murata , Tetsuya Sado , Isao Nishiumi , Masamichi Takeshita , 7 5 Accepted: 1 March 2018 Wataru Iwasaki & Masaki Miya Published: xx xx xxxx Birds play unique functional roles in the maintenance of ecosystems, such as pollination and seed dispersal, and thus monitoring bird species diversity is a first step towards avoiding undesirable consequences of anthropogenic impacts on bird communities. In the present study, we hypothesized that birds, regardless of their main habitats, must have frequent contact with water and that tissues that contain their DNA that persists in the environment (environmental DNA; eDNA) could be used to detect the presence of avian species. To this end, we applied a set of universal PCR primers (MiBird, a modified version of fish/mammal universal primers) for metabarcoding avian eDNA. We confirmed the versatility of MiBird primers by performing in silico analyses and by amplifying DNAs extracted from bird tissues. Analyses of water samples from zoo cages of birds with known species composition suggested that the use of MiBird primers combined with Illumina MiSeq could successfully detect avian species from water samples. Additionally, analysis of water samples collected from a natural pond detected five avian species common to the sampling areas. The present findings suggest that avian eDNA metabarcoding would be a complementary detection/identification tool in cases where visual census of bird species is difficult. Environmental DNA (eDNA) is genetic material that persists in an environment and is derived from organ- isms living there, and researchers have recently been using eDNA to detect the presence of macro-organisms, 1–5 particularly those living in aquatic/semiaquatic ecosys . Fo tr exa ems mple, several fish species inhabiting a river can be detected by amplifying and sequencing DNA fragments extracted from wat b er s y ua sin mg p les methodologies such as quantitative PCR and eDNA metabarcoding. Quantitative PCR requires the design of 3,4,7,8 species-specic fi PCR primers and enables quantitative measurements of eDNA of target s , p w eh ciil es e eDNA metabarcoding, which has been becoming a common methodology in eDNA studies, uses a universal primer set and high-throughput sequencer (e.g., Illumina MiSeq) to enable qualitative detection of eDNA of multiple species 1,2,9–11 12 belonging to a target taxon (but see ref.). Although earlier studies mainly focused on detecting fish/amphibian species (i.e., organisms that have close associations with water), recent studies have shown that eDNA can be used to detect a diverse group of animals, 9,13,14 15 10 including mammals , reptiles and arthropods . Detecting the presence of animals is possible even if their 9,10,13,14 habitats are terrestrial because animals must have, in general, frequent opportunities to contact water in order to live. The findings of these recent studies imply that any organism, regardless of its main habitat, can potentially be detected by using eDNA if we can design suitable primers that enable amplification and identifica- tion of DNA fragments of target organisms and if we can collect appropriate media that contain eDNA. Wild birds represent an important part of the biodiversity in ecosystems, and they play a unique role in the maintenance of ecosystem functions. For example, in forest ecosystems, birds can contribute to maintenance of the tree community by seed dispersal and pollination, and to the reduction of herbivory by predation upon insect 16–18 herbivores . However, recent increases in anthropogenic impacts on ecosystems, e.g., urbanization and habitat 1 2 PRESTO, Japan Science and Technology Agency, Kawaguchi, 332-0012, Japan. Center for Ecological Research, Kyoto University, Otsu, 520-2113, Japan. Yokohama Zoological Gardens ZOORASIA, Kanagawa, 241-0001, Japan. 4 5 College of Bioresource Sciences, Nihon University, Kanagawa, 252-0880, Japan. Natural History Museum and Institute, Chiba, 260-8682, Japan. Department of Zoology, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan. Department of Biological Sciences, The University of Tokyo, Tokyo, 113-0032, Japan. Correspondence and requests for materials should be addressed to M.U. (email: ong8181@gmail.com) or M.M. (email: miya@chiba-muse.or.jp) SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 1 www.nature.com/scientificreports/ Primer name Information a,b Primers for the first PCR (with MiSeq sequencing primer and six random bases) ACACTCTTTCCCTACACGACGCTCTTCCGATCT NNNNNN MiBird-U-F (forward) GGGTTGGTAAATCTTGTGCCAGC GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT NNNNNN MiBird-U-R (reverse) CATAGTGGGGTATCTAATCCCAGTTTG c,d Primers for the second PCR AATGATACGGCGACCACCGAGATCTACAC XXXXXXXX 2nd PCR-F ACACTCTTTCCCTACACGACGCTCTTCCGATCT CAAGCAGAAGACGGCATACGAGAT XXXXXXXX 2nd PCR-R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT Table 1. Detailed information for MiBird prim Ier tas. lic characters indicate the MiSeq sequencing primers. b c Bold Ns indicate random bases to improve the quality of MiSeq sequen Bold Xs in cing. dicate index sequences to identify each sample U. nderlined characters indicate P5/P7 adapter sequences for MiSeq sequencing. 19,20 fragmentation, drive substantial declines in bird species div, er whic sity h could have impacts on the ecological functions of birds. Monitoring bird species diversity is required for detecting such declines, and such detection is necessary for avoiding undesirable consequences in ecosystem functions due to the loss of avian biodiversity. To monitor bird species diversity, visual census is one of the most common m , a et n h d co ods nsidering the higher visibility of birds than that of fish and forest mammals, visual census is generally a successful method. However, if an alternative method can overcome limitations of visual census, such as low visibility at night or in a dense forest, and eliminate the requirement for taxonomic identification skill under field conditions, that method could be complementarily used for monitoring bird species diversity. In the present study, we tested the potential of eDNA as a tool for the detection of avian species. Previous eDNA surveys performed in marine ecosystems detected some avian species along with diverse fish/mammal 22–25 species (2–4 avian species per study) , suggesting that more diverse avian species are potentially detectable using eDNA if suitable primers are designed. To this end, we modified a previously reported universal primer 1,9 set for fish/mammals (MiFish/MiMammal ), such that the primer set accommodated bird-specific variations, and conducted avian eDNA metabarcoding. During the primer design, we did not try to eliminate the capability of detecting mammalian and other vertebrate species, because simultaneous detection of mammals and other vertebrates along with birds may be advantageous, especially for ecologists who are interested in co-occurrence patterns and potential interactions among various animal species. We performed a series of analyses to test the versatility of the designed primers: In e sixa lico minations of the primers, amplification of extracted tissue DNAs of birds belonging to various taxa, and field tests by analyzing water samples from zoo cages containing birds of known species composition. Additionally, we briefly examined the usefulness of the new primer set using water samples from field samples with unknown bird species composition. Methods All of the critical information of our study is described below, but is also listed in Table S1 to faci - litate compar isons with other studies, following the recommendations of Gold.ber . A g le l exp t al eriments were performed without direct captures of avian species, and carried out in accordance with the relevant guidelines and regula- tions. Also, all experimental protocols in the zoo were approved by Yokohama Zoological Gardens ZOORASIA. Primer design. To facilitate design based on comparisons of diverse avian sequences, we first batch down- loaded 410 avian sequences from RefSeq (https://www.ncbi.nlm.nih.gov/refseq/) on June 9, 2015. Then, a base composition for a selected position in the conservative region was sh esq owuite n in M . The base compositions in selected characters were manually recorded in a spreadsheet for the primer design. In the primer design pro- cess, we considered a number of technical tips that enhance the primer annealing to the template without the use of degenerate bases : primers include some G/C at th -en e 3d ′ s to strengthen primer-template annealing at this position, but a string of either Gs or Cs at t -en hd s e 3h ′ ould be avoided: considering the unconventional base pairing in the T/G bond, the designed primers use G rather than A when the template is variably C or T, and T rather than C when the template is A or G; G/C contents of the primers fall between 40 and 60%, with an almost identical melting temperatur ). e ( T T was calculated using a nearest-neighbour thermodynamic model imple- m m mented in O ligoCalc . 1,9 We designed our primers by modifying previously developed MiFish/MiMammal pr,i w mh eir csh corr-e sponded to regions in the mitochondriS rRN al 12 A gene (insert lengt = h ca . 171 bp), and we named our primers MiBird-U (“U” indicates “universal”). Primer sequences with MiSeq adaptors (for the first- and second-round PCR) are listed in Tab1 le  . In silico evaluation of interspecific variation of MiBird sequences. e Th binding capacity of MiBird-U primers was computationally evaluated using the batch-downloaded 410 avian sequences. Using custom Ruby and Python scripts, the number of mismatches between MiBird-U primers and the 410 avian sequences as well as other non-target animal sequences (i.e., 741 mammalian, 197 amphibian, and 245 reptilian sequences) was cal- culated. Positions of base match/mismatch between MiBird-U primers and avian sequences were also examined using the downloaded avian sequences. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 2 www.nature.com/scientificreports/ Common name Scientific name Order Family Accession No. Spot-billed duck Anas zonorhyncha Anseriformes Anatidae LC104767 Grey nightjar Caprimulgus indicus Caprimulgiformes Caprimulgidae LC104768 Ancient murrelet Synthliboramphus antiquus Charadriiformes Alcidae LC104769 Lesser sand plover Charadrius mongolus Charadriiformes Charadriidae LC104770 Oriental turtle dove Streptopelia orientalis stimpsoni Columbiformes Columbidae LC104771 Cuculus poliocephalus Lesser cuckoo Cuculiformes Cuculidae LC104772 poliocephalus Northern Goshawk Accipiter gentilis fujiyamae Accipitriformes Accipitridae LC104773 Common kestrel Falco tinnunculus Falconiformes Falconidae LC104774 Chinese bamboo partridge Bambusicola thoracicus Galliformes Phasianidae LC104775 Red-throated loon Gavia stellata Gaviiformes Gaviidae LC104776 Red-crowned crane Grus japonensis Gruiformes Gruidae LC104777 Jungle crow Corvus macrorhynchos Passeriformes Corvidae LC104778 Eurasian sparrow Passer montanus Passeriformes Passeridae LC104779 Black-crowned night heron Nycticorax nycticorax Pelecaniformes Ardeidae LC104780 Great white pelican Pelecanus onocrotalus Pelecaniformes Pelecanidae LC104781 Great cormorant Phalacrocorax carbo hanedae Suliformes Phalacrocoracidae LC104782 Japanese pygmy woodpecker Dendrocopos kizuki Piciformes Picidae LC104783 Little grebe Tachybaptus ruficollis Podicipediformes Podicipedidae LC104784, LC104785 White-chinned petrel Procellaria aequinoctialis Procellariiformes Procellariidae LC104786 Short-tailed shearwater Puffinus tenuirostris Procellariiformes Procellariidae LC104787 King penguin Aptenodytes patagonicus Sphenisciformes Spheniscidae LC104788 Humboldt penguin Spheniscus humboldti Sphenisciformes Spheniscidae LC327059 Table 2. Extract DNAs used to test the performance of the MiBird primer set. Interspecific differences within the amplified DNA sequences are required for assignment of taxonomic cat- egories. Levels of interspecific variation in the target region (hereae ft r called ‘MiBird sequence’) across die ff rent taxonomic groups of birds were computationally evaluated using the 410 downloaded avian sequences. Among the sequences of the 410 avian, species with the deletion of primer regions (Hemignathu , s L mu oxonr pso cioc- cineus and Arborophila rufipectus ) were excluded, and 407 MiBird sequences were extracted and subjected to calculation of pairwise edit distances using custom Python scripts. Pairwise inter-species edit distances were cal- culated for all species pairs, and pairwise inter-genus edit distances were calculated for pairs of species belonging to different genera. The edit distance quantifies dissimilarity of sequences in bioinformatics and is defined as the minimum number of single-nucleotide substitutions, insertions or deletions that are required to transform one sequence into the other. In addition, the binding capacity and the levels of interspecific variations of the target region were further 30 31 evaluated using ‘primerTree’ packa og f R v e ersion 3.3.1 . Briefly, primerTree performs the following analysis: (1) In silico PCR against sequences in the NCBI database; (2) retrieval of DNA sequences predicted to be ampli- e fi d; (3) taxonomic identic fi ation of these sequences; (4) multiple DNA sequence alignment; (5) reconstruction of a phylogenetic tree and (6) visualization of the tree with taxonomic annotation. Thus, by using primerTree pack- age, species whose sequences can be amplified, phylogenetic relationships among these amplified species, and interspecific variations in the amplified sequences are rapidly visualized. Further information and instructions for the primerTree package can be found in Canno.n .et al Primer testing with extracted DNA. We tested the versatility of MiBird-U (no adapter sequences) using DNA extracted from 22 species representing major groups of bird 2 s ( ). D Ta o b u le bl   e-stranded DNA concen -tra tions from those samples were measured with a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, −1 Wilmington, DE, USA) and the extracted DNA was diluted t ng o 15 µ l u sing Milli-Q water. PCR was carried out with 30 cycles of a 15 µ l r eaction volume containinµg 4.5 l st erile distilled H O, 7.5 µ l 2× Gflex PCR Buffer 2+ (Mg , dNTPs plus) (Takara, Otsu, Japan), µ 0.7 l o f each primer (5 μM), 0.3 µ l Taq polymerase (Tks Gflex DNA Polymerase; Takara) and 1.2 µl tem plate. The thermal cycle profile aer a ft n initiamin den l 1 aturation at 94 °C wa s as follows: denaturation a °C f t 98 o r 10s; a nnealing at 50 °C f or 10s; a nd extension at 68 °C f or 10s w ith a final extension at the same temperature f min. or 7 Study site and water sampling for primer testing with eDNA from zoo samples. To test the versati lity of the newly designed primers for metabarcoding avian eDNA, we sampled water from cages on 13 December 2016 in Yokohama Zoological Gardens ZOORASIA, Yokohama, Japan (35°29 42″ N, 139°31 ′ ′35″ E), where we previously tested the usefulness of a universal primer set targeting m . W a e c mm hos al e t s he zoo as a sampling site because the information about avian species in a cage is precisely known, and because the zoo rears diverse taxonomic groups of animals (i.e 100 a ., > nimal species, including many mammals and birds). Thirteen cages, in which diverse taxonomic groups of birds were reared, were selected as sampling pl3 aces (T ). Most o able  f the SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 3 www.nature.com/scientificreports/ Common name Species name Order Family Steller’s sea eagle Haliaeetus pelagicus Accipitriformes Accipitridae Black-tailed gull Larus crassirostris Charadriiformes Laridae Capercaillie Tetrao urogallus Galliformes Tetraonidae Lady Amherst’s pheasant Chrysolophus amherstiae Galliformes Phasianidae Ruddy shelduck Tadorna ferruginea Anseriformes Anatidae Temminck’s tragopan Tragopan temminckii Galliformes Phasianidae Victoria crowned pigeon Goura victoria Columbiformes Columbidae Mandarin duck Aix galericulata Anseriformes Anatidae Humboldt penguin Spheniscus humboldti Sphenisciformes Spheniscidae Snowy owl Bubo scandiacus Strigiformes Strigidae Oriental white stork Ciconia boyciana Ciconiiformes Ciconiidae White-naped crane Grus vipio Gruiformes Gruidae Common crane Grus grus Gruiformes Gruidae Southern ground hornbill Bucorvus leadbeateri Coraciiformes Bucerotidae Harris’s hawk Parabuteo unicinctus Accipitriformes Accipitridae Emu Dromaius novaehollandiae Struthioniformes Casuariidae Table 3. Classification of the target bird species in the Zoorasia exper Sp im ecies k ent. ept in a walk-through bird cage with other bird species. target species were kept separately, but ruddy shelduck (Tadorna fe) w rruer gin e k eaept in a walk through bird cage (hereafter, “the bird cage”) with other bird species (i.e., Lady Amherst’s pheasant [Chrysolophus amhers- tiae], Temminck’s tragopan Tr [ agopan temminckii], Victoria crowned pigeo G n ou [ ra victoria] and mandarin duck [Aix galericulata]). Note that different individuals of Lady Amherst’s pheasant, Temminck’s tragopan, Victoria crowned pigeon and mandarin duck from those in the bird cage were separately kept (i.e., in different cages from the bird cage), and that each water sample of the bird species was collected from each cage. TM Each 100–200m l water sample was collected through a s0.45- terile µ m S ϕ terivex filter (Merck Millipore, Darmstadt, Germany) using a sterile 50-mL syringe (TERUMO, Tokyo, Japan). Aer t ft he filtration, approximately 2 ml of RNAlater (ThermoFisher Scientific, Waltham, Massachusetts, USA) was injected into the Ster - ivex car tridge, and the filtered water samples were stor °C f ed a ot 4 r u p to one day until further processing. Three negative controls (distilled water) were taken to the zoo to monitor contaminations during water sampling, filtration and transport. In addition to the survey in the zoo, we collected water samples from a pond adjacent to the Natural History Museum and Institute, Chiba (35°35 59″ ′ N, 140°8′18″ E; Funada-ike Pond) to test the potential effectiveness of the MiBird primers under a field condition with unknown bird species composition. Water collections at the pond were performed in the same way as those performed in the zoo. DNA extraction. The Sterivex filter cartridges were taken back to the laboratory, and DNA was extracted from the filters using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) following a protocol described and illustrated in Miya .et . B al riefly, the RNAlater-supplemented solution was removed under a vacuum using the QIAvac system (Qiagen, Hilden, Germany). Proteinase-K solutµ io l), p n (20 hos phate buffered saline (PBS) (220 µ l) and buffer AL (200 µ l) were mixed, and 440 µ l o f the mixture was added to each filter cartridge. The materials on the filter cartridges were subjected to cell-lysis conditions by incubating the filters on a rotary shaker (at a speed of 20 rp m) at 50 °C f or 20min. Th e incubated mixture was transferred into a new 2-ml tube, and the collected DNA was purified using a DNeasy Blood and Tissue Kit following the manufacturer’s protocol. Aer t ft he purification, DNA was eluted using 100 µl o f the elution buffer provided with the kit. Paired-end library preparation. Prior to the library preparation, work-spaces and equipment -were ster ilized. Filtered pipet tips were used, and separation of pre- and post-PCR samples was carried out to safeguard against cross-contamination. We also employed two negative controls (i.e., PCR negative controls) to monitor contamination during the experiments. e Th first-round PCR (first PCR) was carried out with a 12µ -l reaction volume containin µ gl 6 o .0 f 2 × KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, WA, USA), 0.7 µl of M iBird primer (5µM p rimer F/R, w/ adaptor and six random bases; Ta1 b), 2.6 les  µ l of sterilized distilleO a d Hnd 2.0µ l of template. The thermal cycle profile aer a ft n initial 3 min den aturation at 95 °C wa s as follows (35 cycles): denaturation a °C f t 98 or 20 s; annealing at 65 °C f or 15s; a nd extension at 72 °C f or 15s, w ith a final extension at the same temperaturmin. e for 5 We performed triplicate first-PCR, and these replicate products were pooled in order to mitigate the PCR drop- outs. The pooled first PCR products were purified using AMPure XP (PCR product: AMPure XP be = a d 1: s 0.8; Beckman Coulter, Brea, California, USA). The pooled, purified, and 10-fold diluted first PCR products were used as templates for the second-round PCR. The second-round PCR (second PCR) was carried out with a 24- l reac µ tion volume containinµ g 12 l of 2 × KAPA HiFi HotStart ReadyMix, 1.4 µ l o f each primer (5 µ M p rimer F/R; Table  1), 7.2 µ l of sterilized distilled H O and 2.0µ l of template. Different combinations of forward and reverse indices were used for different tem- plates (samples) for massively parallel sequencing with MiSeq. The thermal cycle profile after a n m iin ni tial 3 SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 4 www.nature.com/scientificreports/ Figure 1. In silico evaluations of MiBird-U primers. Binding capacity of MiBird-U primers (a). y-axis represents the proportion of avian species that showed 0, 1, 2, 3, 4, or >5 mismatches (indicated by different colours) with MiBird-U F/R primers. The total number of avian species evaluated was 410. The phylogenetic tree was constructed for species that can be amplified using MiBird-U primers (b). A total of 2,000 sequences were retrieved from the database to construct the phylogenetic tree. Different classes are represented by filled circles with different colours. Lengths of branches correspond to the differences in sequences. Bar indicates edit distance. denaturation at ° C 95 was as follows (12 cycles): denaturation °C a t fo 9 r 8 2 0 s; combined annealing and extension at 72 °C (s huttle PCR) for 15 s, w ith a final extension at 72 °C f or 5min. e Th indexed second PCR products were mixed at equimolar concentrations to produce equivalent sequencing depth from all samples and the pooled library was purified using AMPure XP. Target-sized DNA o - f the puri fied library (ca. 370 bp) was excised using E-Gel SizeSelect (ThermoFisher Scientific, Waltham, MA, USA). The double-stranded DNA concentration of the library was quantified using a Qubit dsDNA HS assay kit and a Qubit uo fl rometer (ThermoFisher Scientific, Waltham, MA, USA). The double-stranded DNA concentration of the library was then adjusted t nM u o 4 sing Milli-Q water and the DNA was applied to the MiSeq platform (Illumina, San Diego, CA, USA). The sequencing was performed using a MiSeq Reagent Kit Nano v2 f × o 150 r 2 bp PE (Illumina, San Diego, CA, USA). Data processing and taxonomic assignment. The overall quality of the MiSeq reads was evaluated using the programas s F tqc (available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and SUGAR . Aer co ft nfirming the lack of technical errors in the MiSeq sequencing, low-quality tails were trimmed from each read using DynamicTrim.pl from t oh lexa e S Qa software package with a cut-off threshold set at a −1 Phred score of 10 (=10 error rate). The tail-trimmed pair-end reads were assembled using the software FLASH with a minimum overlap of 10 bp. Th e assembled reads were further filtered by custom Perl scripts in order to remove reads with either ambiguous sites or those showing unusual lengths compared to the expected size of the PCR amplicons. Finally, the softwarag e T Cleaner was used to remove primer sequences with a maximum of three-base mismatches and to transform the FASTQ format into FASTA (see Table S2 for the numbers of reads remained aer t ft hese pre-processing). e Th pre-processed reads from the above custom pipeline were dereplicated using UCL , w US ith Tthe num- ber of identical reads added to the header line of the FASTA formatted data file. Those sequences represented by at least 10 identical reads were subjected to the downstream analyses, and the remaining under-represented sequences (with less than 10 identical reads) were subjected to pairwise alignment using UCLUST. If the latter sequences observed for less than 10 reads showed at least 99% identity with one of the former reads (one or two nucleotide differences), they were operationally considered as identical (owing to sequencing or PCR errors and/ or actual nucleotide variations in the populations). e p Th rocessed reads were subjected to local BLASTN searc a hga es inst a custom-made database. e c Th ustom database was generated by downloading all whole mitogenome sequences from Sarcopterygii deposited in NCBI Organelle Genome Resourc h e t st (p://www.ncbi.nlm.nih.gov/genomes/OrganelleResource.cg = i? 8t 2a 8x 7) i.d As of 15 March 2016, this database covered 1,881 species across a wide range of families and genera (including birds, mammals, reptiles and amphibians). In addition, the custom database was supplemented by all whole and partial fish mitogenome sequences deposited in MitoFi in o sh rder to cover fish detection (note that MiBird primers amplify fish sequences as well; see Fig 1)..  −5 e Th top BLAST hit with a sequence identity of at least 97% a E-va nd lue threshold of 10 was applied to spe- cies assignments of each representative sequence. Reliability of the species assignments was evaluated based on the ratio of total alignment length and number of mismatch bases between the query and reference sequences. For example, if a query sequence was aligned to the top BLAST hit sequence with an alignment len bp w gt it h o h f 150 one mismatch present, the ratio was calculated a+ s 150/(1 1). The va lue one was added to the denominator to SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 5 www.nature.com/scientificreports/ MiBird-U-F G G G T T G G T A A A T C T T G T G C C A G C A 1 4 1 1 0 1 0 0 397 407 407 0 0 0 0 0 0 0 0 0 407 0 0 C 0 0 0 61 129 0 0 0 2 0 0 7 407 155 0 0 0 0 407 407 0 0 407 G 406 403 406 0 0 406 407 0 1 0 0 0 0 0 0 407 0 407 0 0 0 407 0 T 0 0 0 345 278 0 0 407 7 0 0 400 0 252 407 0 407 0 0 0 0 0 0 MiBird-U-R C A T A G T G G G G T A T C T A A T C C C A G T T T G A 0 407 0 406 0 0 0 2 0 0 0 407 0 1 0 407 407 0 0 0 0 406 0 1 0 0 0 C 407 0 0 0 0 1 0 0 0 0 0 0 0 406 0 0 0 0 407 406 404 0 0 1 0 5 1 G 0 0 0 0 407 0 407 405 407 407 0 0 1 0 0 0 0 0 0 0 0 1 405 0 0 0 406 T 0 0 407 1 0 406 0 0 0 0 407 0 406 0 407 0 0 407 0 1 3 0 2 405 407 402 0 Table 4. Nucleotide sequences of the universal primers (MiBird-U) and base compositions of the selected 407 avian species. Among the downloaded sequences, 3 species with the deletion of primer regions were excluded, resulting in the sequences of 407 avian species. Italic bases indicate primer sequences. Numbers indicate the number of avian species of which base matches with A, C, G, or T listed in the left column. Bold numbers indicate the number of avian species of which base matches with that of the primer. Edit distance 0 1 2 3 4 ≥5 Total Frequency distributions of the inter-specific/genus edit distances of the insert sequence Species 27 50 67 113 187 82,177 82,621 combinations Genus 10 23 40 80 157 Table 5. Frequency distributions of the interspecific edit distances of the primer set against bird sequences. Pairwise inter-species edit distances were calculated for all species pairs, and pairwise inter-genus edit distances were calculated for pairs of species belonging to different genera. avoid zero-divisors. This value (e.g., 150/( 1 + 1)) was calculated for the top and second-highest BLAST hit species, and the ratio score between these values was used as a comparable indicator of the species assignment. Results from the BLAST searches were automatically tabulated, with scientific names, common names, total number of reads and representative sequences noted in an HTML format. The above bioinformatics pipeline from data pre-processing through taxonomic assignment is available in supplements in a pre. A vio lu so s s , t th ud e a y bove bioinformatic pipeline can be performed on a website. For more detailed information, please see http://mitos fi h. aori.u-tokyo.ac.jp/mifish. Please note that the pipeline implemented in the website currently uses the custom s fi h database and does not aim to detect avian species (confirmed on 20 September 2017). Data availability. DDBJ Accession numbers of the DNA sequences analyzed in the present study are DRA006196 (Submission ID), PRJDB4990 (BioProject ID) and SAMD00096837–SAMD00096858 (BioSample ID). Results and Discussion Tests of versatility of designed primers in silico and using extracted DNA. First, the performance of MiBird-U primers was tested in si (Fig lico . 1 and Tables  4 and 5). When G/T pairs were accepted, MiBird-U-F and -R perfectly matched 390 (95.1%) and 388 (94.6%) species among 410 species tested, respectively, and 99.5% and 96.8% of the 410 species showed at most 1 mismatch (Fig 1a). A .  mong the avian sequences tested, all species showed no mismatch at the 3 -en ′d of MiBird-U-F, and most species ( 98.7%) s > howed no mismatch at the 3 -en ′d of MiBird-U-R (Table  4). In addition, inter-specific differences in the edit distance were calculated and 82,177 out of 82,621 combinations (99.5%) showed edit distance larger than 5 (T 5). Tha es be a le nalyses suggested that the target region of most avian species can be amplified using MiBird-U primers, and that the amplified sequences contain sufficient information required for assignment of taxonomic categories. To examine the range of species that can be amplified using MiBird-U primers, we performed an analysis with the primerTree packag . The e results confirmed that the primers can amplify avian species 1b (Fig ). M .  iBird-U primers can also amplify a diverse group of mammalian species in addition to amphibian, reptilian and s fi h species (Fig. 1b), which is not surprising because MiBird-U primers were produced by modifying fish/mammal-targeting universal primers. The potential of MiBird-U primers to amplify mammalian, amphibian, and reptilian species was also confirmed by in silico test of the binding capacity of MiBird-U primers (Table S3). The capacity of MiBird-U primers to detect mammalian and other species might be useful when simultaneous detection of these animals is desired (e.g., when one tries to study co-occurrence patterns and potential interactions among animals). Second, the performance of MiBird-U primers was evaluated using 22 extracted avian DNA samples. All of the extracted DNA samples were successfully amplified, and the resultant sequences were deposited in the DDBJ/ EMBLE/GenBank databases (Tab2 le  ). Together, the results of in s t ilies cots and the amplification of extracted DNAs suggested that MiBird-U primers are capable of amplifying/identifying DNA fragments derived from diverse avian species. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 6 www.nature.com/scientificreports/ Primer testing with eDNA from el fi d water samples. MiSeq sequencing and data pre-processing gen- erated 656,472 sequences from 21 samples (including 3 field negative controls and 2 PCR negative controls) (Table  5). In general, the quality of sequences produced by our experiment was high (i.e., most raw reads passed the filtering process; Table S2). Among the 16 water samples from zoo cages examined here, all avian species were successfully detected (Table  6). Briefly, eDNA samples of the Steller’s sea eagle (Haliaeetus pela ), c gica up s ercaillie (Tetrao urogallus), white-naped crane (Grus vipi ), co o mmon crane (Grus grus ) and southern ground hornbill (Bucorvus leadbe) ateri generated high numbers of sequence reads, and 64.9–94.9% of total sequence reads were assigned to the target avian species. Samples from cages of the black-tailed gull (Larus cra ), H ssirum ostrb is oldt penguin (Spheniscus humboldti), snowy owl (Bubo scandiacu), Or s iental white stork (Ciconia boycia ), H naarris’s hawk (Parabuteo unicinctus) and emu (Dromaius novaehollandiae) generated fewer sequence reads, and 1.4–28.8% of total sequence reads were assigned to the target avian species. The reason for these variations in the proportions of sequence reads from target avian species is not known, but as discussed in the prev, t ioh ue o s st bs ud er yved levels of variations were not surprising because detection of animals’ sequences relies on contacts of animals with water and because opportunities for animals to contact water would depend on animals’ behaviour. These considera- tions imply that the proportion of sequence reads from a particular avian species would be inherently spatially and temporally stochastic to some extent (see also results of mammalian eDNA metabarcodin . g in U ). It shio et al is not surprising that sequences of the Lady Amherst’s pheasant, ruddy shelduck, Temminck’s tragopan, Victoria crowned pigeon and mandarin duck were detected in the ruddy shelduck samp6 le (T ) bec a a bu le  se all of these five species were kept in the bird cage where the ruddy shelduck sample was collected. In addition to the target avian species, we frequently detected many non-target sp 6 a ecies (T nd S4). F abo le  r example, sequences of the Steller’s sea eagle were frequently detected in other samples, e.g., the Victoria crowned pigeon, Oriental white stork, Humboldt penguin and so on (T 6). A ab s o le  ur field negative controls generated no target bird sequences (Ta 6b ), i le  t does not seem likely that the detection of the sea eagle in other samples was due to cross-contamination during sampling or experiments. One possible reason for the detection of non-target avian species include the spatial closeness of the eagle’s cage and the other cages. For instance, the cages of the Victoria crowned pigeon (i.e., the bird cage) and Humboldt penguin were located close to the eagle’s cage, and thus it is possible that the eagle’s feathers and other tissues could be transported (e.g., via wind) to other cages. Also, zoo staff frequently moved among cages, and they were possible transporters (e.g., through their shoe sole) of materials containing DNA of non-target species. Other frequently detected non-target species were falcated teal (M), co areca mm falc o an s ta helduck (Tadorna tadorna), common moorhen (Gallinula chloropu ), fi s shes and humans (Table S4). The falcated teal, shelduck and moorhen were not kept in cages, but wild common moorhens and close relatives of the duck and shelducks (i.e., Eurasian wigeon [Anas penelope ] and common pochard [Aythya ferina], respectively) are commonly observed in the regulating pond on-site of sampling region, and thus their DNA might have contaminated zoo cages (possibly via feathers or other tissues) and thus have been detected by the metabarcoding. The frequently detected fish species here are also species that are commonly observed in Japan, and the zoo uses waters from a natural lake and rivers. Therefore, the fish sequences might have been derived from water under natural conditions. Detection of many human sequences was not surprising considering that visitors to the zoo and staff members, who are potential sources of human sequences, are almost always near the cages. It is also be possible that contaminations of human and fish DNA happened under the laboratory conditions (Table S4), because in our lab fish DNAs were routinely processed and humans were oen w ft orking (i.e., carry-over contam- inations). Specifically, ocean fish sequences were detected from zoo samples despite the efforts for decontami- nation, and these contaminants are likely due to previous work in the same lab. The sequences of these obvious non-target taxa (i.e., humans, fish, and potential non-target carry-over contaminations) may be excluded from further statistical anal if o yses ne may be interested in ecological interpretations of the results. Lastly, in order to test the usefulness of MiBird primers under a natural field condition, we performed a metabarcoding study using a water sample from a pond adjacent to the Natural History Museum and Institute, Chiba (Funada-ike Pond). As a result of MiSeq sequencing, 14,873 reads of avian species were generated from three water samples, and five avian species (common shoveler [Anas clypeata], 883 reads; falcated teal, 3,246 reads; common moorhen, 9,260 reads; light-vented bulbul [Pycnonotus sinensis], 745 reads; and common shelduck, 739 reads) were detected. As a systematic monitoring of the bird community (e.g., frequent visual observation) has not been performed in the study site, rigorous validation of the metabarcoding study was not possible. Some avian species detected, i.e., light-vented bulbuls, common shelducks and falcated teals, are rare, or not reported, in this region, suggesting that these species were misidentified. These possible misidentifications are likely to be attributable to a lack of reference sequences and/or insufficient inter-species dier ff ences in the amplified DNA region (i.e., partial S 1 m 2 itochondrial region) (see also r ). L efi .ght-vented bulbuls, common shelducks and falcated teals are relatives of brown-eared bulbuls (Hypsipetes a ), co maumm rotio s n pochards (Aythya ferina) and Eurasian wigeons (Anas penelo), r pe espectively, and these relatives are indeed common inhabitants in the sampling region. Together, these results suggest that MiBird primers were capable of detecting bird species under a field condition, but at the same time, improvements of reference sequence databases, further validations of MiBird primers, and careful interpretations are necessary. Conclusion A proof-of-concept that eDNA metabarcoding can potentially detect avian species has been already demonstrated in 22–25 previous studies , and in the present study we explicitly demonstrated the potential and usefulness of avian eDNA metabarcoding using our new primer set and MiSeq platform. Describing and monitoring the diversity of bird spe- cies, as well as other animals, is one of the critical steps in ecosystem conservation and management, but it can be labo- rious, costly and incomplete if one relies on a few traditional survey methods. The eDNA metabarcoding approach SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 7 www.nature.com/scientificreports/ Bird species name detected from sequences Common name of bird living in cage Scientific name Haliaeetus Larus Tetrao Chrysolophus Tadorna Tragopan Goura Aix Spheniscus Bubo Steller’s sea eagle Haliaeetus pelagicus 28,448 0 0 0 0 0 0 0 0 0 Black-tailed gull Larus crassirostris 0 4,437 0 0 0 0 0 0 0 0 Capercaillie Tetrao urogallus 0 0 36,095 0 0 0 0 0 0 0 Lady Amherst’s pheasant Chrysolophus amherstiae 0 0 0 39,151 0 0 0 25 0 0 Ruddy shelduck Tadorna ferruginea 0 0 0 138 2,848 209 2,750 7,939 0 0 Temminck’s tragopan Tragopan temminckii 0 0 376 0 0 57,072 0 0 0 0 Victoria crowned pigeon Goura victoria 11,023 0 0 0 0 0 2,186 24 0 0 Mandarin duck Aix galericulata 254 0 0 0 65 51 34 13,465 0 0 Humboldt penguin Spheniscus humboldti 1,905 0 0 0 289 0 85 428 1,834 0 Snowy owl Bubo scandiacus 901 0 0 0 65 0 64 191 0 425 Oriental white stork Ciconia boyciana 7,258 63 0 0 0 0 103 258 0 0 White-naped crane Grus vipio 186 0 527 0 22 0 36 33 0 0 Common crane Grus grus 0 0 0 548 0 0 0 0 0 0 Southern ground hornbillBucorvus leadbeateri 148 0 0 0 0 15 0 0 0 0 Harris’s hawk Parabuteo unicinctus 1,227 0 0 0 77 0 0 332 0 0 Dromaius Emu 396 0 0 0 67 0 25 146 0 0 novaehollandiae Field NC 0 0 0 0 0 0 0 0 0 0 Field NC 0 0 0 0 0 0 0 0 0 0 Field NC 0 0 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 0 0 Total sequence 51,746 4,500 36,998 39,837 3,433 57,347 5,283 22,841 1,834 425 Bird species name detected from sequences Common name of bird Non-target Total b,c living in cage Scientific name Ciconia G. vipio G. grus Bucorvus Parabuteo Dromaius sequences sequences % target living in cage Steller’s sea eagle Haliaeetus pelagicus 0 0 0 0 0 0 3,238 31,686 89.8 Black-tailed gull Larus crassirostris 0 0 0 0 0 0 17,922 22,359 19.8 Capercaillie Tetrao urogallus 0 13 0 0 0 0 19,477 55,585 64.9 Lady Amherst’s pheasant Chrysolophus amherstiae 0 0 15 0 0 0 8,130 47,321 82.7 Ruddy shelduck Tadorna ferruginea 0 0 0 0 0 0 12,498 26,382 10.8 Temminck’s tragopan Tragopan temminckii 0 0 0 0 0 0 1,944 59,392 96.1 Victoria crowned pigeon Goura victoria 0 0 0 0 0 0 13,108 26,341 8.3 Mandarin duck Aix galericulata 0 0 0 0 0 0 12,272 26,141 51.5 Humboldt penguin Spheniscus humboldti 0 0 0 0 0 0 28,091 32,632 5.6 Snowy owl Bubo scandiacus 0 0 0 0 0 0 28,566 30,212 1.4 Oriental white stork Ciconia boyciana 3,072 0 0 0 0 0 22,815 33,569 9.2 White-naped crane Grus vipio 0 59,678 0 31 0 0 2,390 62,903 94.9 Common crane Grus grus 0 0 52,717 0 0 0 2,586 55,851 94.4 Southern ground hornbillBucorvus leadbeateri 0 0 0 36,955 0 0 4,900 42,018 88.0 Harris’s hawk Parabuteo unicinctus 0 0 0 0 306 0 20,338 22,280 1.4 Dromaius Emu 0 22 0 0 0 1,647 3,412 5,715 28.8 novaehollandiae Field NC 0 0 0 0 0 0 27,218 27,218 Field NC 0 0 0 0 0 0 7,977 7,977 Field NC 0 0 0 0 0 0 40,890 40,890 PCR NC 0 0 0 0 0 0 0 0 PCR NC 0 0 0 0 0 0 0 0 Total sequence 3,072 59,713 52,732 36,986 306 1,647 277,772 656,472 Table 6. Sequence reads of detected species from water samples collected in the zoo. Bold numbers indicate a b sequence reads of a target species. Species kept in a walk-through bird ca Sg ee T e. able S4 for the contents of non-target sequences. See Table S3 for the contents of non-target sequences. presented here is non-invasive and efficient. Moreover, as information of non-target organisms (e.g., invertebrates and microbes in our case) is also encoded in eDNA, analyzing eDNA of organisms from multiple taxa might be useful for studying co-occurrence patterns and even potential interactions among organisms (e.g., bird-insect interactions). In conclusion, we propose that the eDNA metabarcoding approach can serve as an efficient alternative for taking a snapshot of bird diversity and could potentially contribute to effective ecosystem conservation and management. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 8 www.nature.com/scientificreports/ References 1. 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Iwasaki, W.e t al. MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline. Mol. Biol. Evol. 30, 2531–40 (2013). Acknowledgements We would like to thank Noriya Saito, assistant manager of Yokohama Zoological Gardens ZOORASIA for help in sampling at the zoo, and Asako Kawai for assistance with experiments. We also thank Hiroki Yamanaka of e D Th epartment of Environmental Solution Technology/The Research Center for Satoyama Studies in Ryukoku University for providing the opportunity for us to use the Illumina MiSeq platform. This research was supported by PRESTO (JPMJPR16O2) from Japan Science and Technology Agency (JST), CREST (JPMJCR13A2) from Japan Science and Technology Agency (JST), and ERTDF (4–1602) The Environment Research and Technology Development Fund, Japan. This study was approved by Yokohama Zoological Gardens ZOORASIA. SCiENTifiC Repo R tS | (2018) 8:4493 | DOI:10.1038/s41598-018-22817-5 9 www.nature.com/scientificreports/ Author Contributions M.U. and M.M. conceived and designed research; M.U., I.N. and K.M. performed sampling; M.U., I.N., T.S. and M.M. performed experiments; M.U., M.T. and W.I. performed data analysis; M.U. and M.M. wrote the early dra ft and completed it with significant inputs from all authors. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-22817-5. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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