sample collections : using a pair of sterilized , stainless tweezers , we collected immediately 
 after defecation fresh cecal feces of two male ( wl4 and wl5 ) and two female ( wl7 and wl8 ) wild japanese rock 
 ptarmigans off the snow surface in the murodo area ( 36 34n , 137 36e , 2,450 m , above sea level ) of the 
 tateyama mountains in toyama prefecture , japan , on may 2014 . research field was mostly covered by snow , and birds apparently fed leaves of crowberry 
 ( empetrum nigrum var . japonicum ) , rhododendron ( rhododendron 
 brachycarpum ) and japanese stone pine ( or hai - matsu ) ( pinus pumila ) . similarly , we 
 collected cecal feces of two male ( yl2 and yl4 ) and three female ( yl1 , yl3 and yl5 ) captive svalbard rock 
 ptarmigans artificially reared off the floor of individual pens in the preservation and research center , the 
 city of yokohama , japan . these birds were all one year old and maintained on pelleted food for rabbit ( rm-4 , 
 funabashi farm , funabashi , japan ) supplemented with fresh japanese mustard spinach ( brassica 
 rapa var . perviridis ) and blueberries ( vaccinium spp . ) purchased 
 from the local market . all birds were in normal condition , but yl5 experienced chronic diarrhea of unknown 
 causes and treated with a probiotic ( miyarisan , miyarisan pharmaceutical co. , ltd . , tokyo , japan ) 10 days before 
 the sample collection . in both cases , samples were immersed in 
 dna conservation solution , and bacterial dna was extracted within 24 
 hr . dna extractions and high - throughput , ultra - deep sequencing of 16s rrna gene : to obtain 
 bacterial pellets , samples were centrifuged at 20,000  g for 10 min with a tomy mx-307 
 centrifuge ( tomy seiko , tokyo , japan ) , and the pellets were washed with tris - edta several times to remove 
 residual plant metabolites , such as phenolic compounds . genomic dna was extracted from the pellets using a quick gene dna tissue kit ( kurabo , tokyo , japan ) as per the 
 manufacturer s instructions ( n=3 ) . dna concentration was determined with a quant - it dsdna hs assay kit using a 
 qubit fluorometer ( invitrogen , carlsbad , ca , u.s.a . ) . the microbial community structure was analyzed by 16s rrna gene amplicon sequencing carried out with a miseq 
 desktop sequencer ( illumina , hayward , ca , u.s.a . ) as per the manufacturer s instructions 
 ( https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf ) . partial 16s rrna gene sequences including v3 and v4 regions were amplified using primers bakt_341f and bakt_805r 
  with illumina overhang adaptor sequences attached to their 5 
 ends . the reaction mixture ( 25 l ) contained 1  kapa hifi hotstart readymix ( kapa biosystems , 
 wilmington , ma , u.s.a . ) , 0.2 m of each primer and 2 l of template dna . pcr 
 was performed with takara s thermal cycler dice ( takara bio inc . , kyoto , japan ) under the following cycling 
 conditions : initial annealing at 95c for 3 min , followed by 25 cycles at 95c for 30 sec , 55c for 30 sec and 
 72c for 30 sec and a final extension at 72c for 5 min . the illumina sequencing adaptor and index tag sequences 
 were added to the amplicons using a nextera xt index kit ( illumina ) as per the manufacturer s instructions . brea , ca , u.s.a . ) and determined 
 by a quant - it dsdna hs assay kit using the qubit fluorometer ( invitrogen ) . resulting dna was mixed with phi x control dna in a ratio of 80:20 and used as a 
 template for paired - end sequencing using a miseq reagent kit v3 ( 600 cycles ) and the miseq desktop sequencer 
 ( illumina ) . phylogenetic analyses and community comparisons : read files ( fastq.gz ) were generated using 
 miseq reporter software version 2.3.32 ( illumina ) . the assembled sequencing data were analyzed 
 using qiime open - source bioinformatics pipeline version 1.8.0 . quality - based filtering was carried out using the following settings : maximum number of ambiguous bases=1 , 
 minimum sequence length=380 bp , maximum sequence length=600 bp , no ambiguous bases allowed and maximum number of 
 homopolymers=8 . to check for chimeric sequences , the 
 sequences were clustered into operational taxonomic units ( otus ) with a criterion of 98% identity by usearch . taxonomic assignment was conducted using a blast search against the greengenes database ( version 13_8 ; 
 ftp://greengenes.microbio.me/greengenes_release/gg_13_5/gg_13_8_otus.tar.gz ) . good s coverage was calculated using the formula 
 ( 1n / n ) , where n is the number of singletons and n is the total number of sequences . weighted unifrac analyses were also performed to calculate the pairwise distances between 
 the bacterial communities , and principal coordinates analysis ( pca ) 
 was applied to visualize the results . to estimate the possible source of these bacterial otus , we identified the 16s rrna gene sequences of top 20 
 otus in each sample by conducting a blastn search   against the ncbi 
 non - redundant database with identity > 98% and e - value < 1e in the top 5,000 hits . we 
 categorized constructed otus into human - origin , other mammalian - origin , bird - origin or environmental origin with 
 a sub - classification of body part , such as the gut or skin , according to the reference information from the 
 database . ethics : sampling was conducted under the supervision of an officer of toyama research 
 association of rock ptarmigan ( toyama raicho kenkyukai ) , which holds a permission from the 
 japanese ministry of environment and the local forestry office for the access to wild japanese ptarmigans in 
 tateyama . sampling from svalbard rock ptarmigans in the yokohama city zoo was permitted by the zoo 
 administration . nucleotide sequence accession numbers : sequencing datasets have been submitted to the ddbj 
 sequence read archive ( dra ) under accession number prjdb3819 . 16s rrna gene sequences of top 20 otus were also 
 deposited to ddbj with accession numbers lc055731 to lc055757 . we performed 16s rrna gene sequencing analyses with an illumina miseq sequencer to probe and retrieve bacterial 
 community structures in the samples . after quality filtering , the miseq sequnece dataset consisted of 886,713 
 reads with an average of 98,524  76,499 reads ( mean  sd ) per sample and an average sequence length of 407 bp . good s coverage values ranged between 0.74 and 0.77 for wild rock ptarmigans and between 0.77 and 0.84 for rock 
 ptarmigans in captivity ( supplementary table 1 ) . in 
 total , 107,612 and 67,895 operational taxonomic units ( otus ) defined with 98% sequence similarity were found in 
 samples from wild and captive rock ptarmigans , respectively . although the rarefaction curves ( supplementary fig . 1 ) indicated that our sequencing 
 effort was not sufficiently large to comprehend the diversity of bacteria in wild and captive rock ptarmigans , 
 we detected about 10,00020,000 otus with the good s coverage as high as 74 to 84% . git microbiota of birds have 
 not been well explored by deep sequencing approach comparing to mammals including human . particularly , information about wild birds is still very limited ; from 560 to 2,000 otus 
 were identified from the feces of various wild penguins   and from 150 
 to 500 otus were identified in cecal contents of emu in captivity . the bacterial community structure of cecal microbiota in wild rock ptarmigans was remarkably different from 
 that in captured rock ptarmigans ( tables 1 , 2table 1.plylum level prokaryotic microbiome in cecal feces from rock ptarmigans ( % in total reads)wild japanese rock ptarmigans ( n=4)captive bred svalbard rock ptarmigans ( n=5)actinobacteria32.086.58firmicutes84.059.58firmicutes28.571.88actinobacteria6.214.33bacteroidetes17.612.54bacteroidetes5.577.64synergistetes11.502.27proteobacteria1.561.56proteobacteria4.530.89verrucomicrobia1.422.82spirochaetes1.540.66tenericutes0.430.32euryarchaeota0.640.32cyanobacteria0.140.19cyanobacteria0.040.03tm70.120.16tenericutes0.030.02synergistetes0.020.02acidobacteria0.020.01chloroflexi0.010.00data are shown with mean  standard deviations , a ) hugenholtz et al . . and fig . 1.the principal components analysis ( pca ) plot of all 16s rrna gene sequences found in the cecal bacteriome 
 samples in wild ( wl ) and artificially ( yl ) reared rock ptarmigans . cecal feces of two male ( wl4 and wl5 ) 
 and two female ( wl7 and wl8 ) wild japanese rock ptarmigans were subjected to the study . one - year old two male ( yl2 and yl4 ) and three female ( yl1 , yl3 and yl5 ) captive 
 svalbard rock ptarmigans in the preservation and research center , the city of yokohama , japan . all birds 
 were in normal condition , but yl5 experienced chronic diarrhea of unknown causes and treated with a 
 probiotic . ) . cecal microbiota in artificially reared rock ptarmigans were predominantly firmicutes ( 84.1% ) followed 
 by actinobacteria ( 6.2% ) and bacteroidetes ( 5.6% ) . 1.5% ) , and other phyla , such as tenericutes , cyanobacteria , synergistetes and tm7 , had only smaller contributions ( < 1.0% ) . in contrast , firmicutes were less abundant 
 ( 28.6% ) in cecal microbiota in wild individuals . actinobacteria ( 32.1% ) , bacteroidetes ( 17.6% ) and synergistetes 
 ( 11.5% ) were the most contributing communities after firmicutes ( table 
 1 ) . data are shown with mean  standard deviations , a ) hugenholtz et al . . the principal components analysis ( pca ) plot of all 16s rrna gene sequences found in the cecal bacteriome 
 samples in wild ( wl ) and artificially ( yl ) reared rock ptarmigans . cecal feces of two male ( wl4 and wl5 ) 
 and two female ( wl7 and wl8 ) wild japanese rock ptarmigans were subjected to the study . one - year old two male ( yl2 and yl4 ) and three female ( yl1 , yl3 and yl5 ) captive 
 svalbard rock ptarmigans in the preservation and research center , the city of yokohama , japan . all birds 
 were in normal condition , but yl5 experienced chronic diarrhea of unknown causes and treated with a 
 probiotic . the dominant genera in artificially reared individuals were ruminococcus ( 10.5% ) and 
 unclassified ruminococcaceae ( 24.7% ) ( table 2table 2.genus level prokaryote microbiome in cecal feces from rock ptarmigans ( % in total reads)taxonwild japanese rock ptarmigans ( n=4)taxoncaptive bred svalbard rock ptarmigans ( n=5)unidentified genus in coriobacteriaceae17.265.17unidentified genus in ruminococcaceae24.656.30unidentified genus in synergistaceae11.072.52unidentified genus in clostridiales18.434.82bacteroides8.050.72ruminococcus10.521.67actinomyces5.961.92faecalibacterium8.473.79megasphaera5.441.12unidentified genus in lachnospiraceae5.171.45unidenitified genus in clostridiales5.230.50bifidobacterium4.864.30bifidobacterium4.991.15oscillospira4.061.12unidentified genus in ruminococcaceae3.940.84blautia3.000.52dialister3.361.88bacteroides2.372.99asteroleplasma3.320.76coprobacillus2.080.78slackia3.122.12eubacterium1.721.41genus yrc22 in paraprevotellaceae2.510.48akkermansia1.470.67prevotella2.141.06odoribacter1.413.15unidentified genus in bacteroidales1.840.62coprococcus1.392.95unidentified genus in s24 - 7 [ bacteroidales]1.630.21unidentified genus in rikenellaceae1.380.53oscillospira1.580.42unidentified genus in coriobacteriaceae1.171.86treponema1.460.93unidentified genus in enterobacteriaceae1.080.54desulfovibrio1.400.68streptococcus0.981.18unidentified genus in lachnospiraceae1.360.40clostridium0.941.30genus p-75-a5 in erysipelotrichaceae1.320.89unidentified genus in erysipelotrichaceae0.710.76parabacteroides1.140.15dorea0.560.28genus rfn20 in erysipelotrichaceae0.960.26unidentified genus in family rf390.430.10sutterella0.830.76turicibacter0.400.37megamonas0.740.32unidentified genus in clostridiaceae0.370.65genus vadinca11 in methanomassiliicoccaceae0.720.50lactobacillus0.330.25unidentified genus of enterobacteriaceae0.620.37sutterella0.320.70unidentified genus of bifidobacteriaceae0.610.65rikenella0.320.25eubacterium0.580.12unidentified genus in barnesiellaceae0.260.55data are shown with mean  standard deviations . ) . higher abundance of lachnospiraceae ( i.e. the genus blautia 
 and unclassified lachnospiraceae ) and faecalibacterium of firmicutes , and 
 akkermansia of verrucomicrobia were also distinctive in artificially reared birds , but not 
 detected as major genera in wild individuals . instead , the dominant genera in wild birds were the unidentified 
 genera of coriobacteriaceae ( 17.3% ) and synergistaceae ( 11.1% ) . in addition , genera 
 bacteroides , actinomyces and megasphaera , and an unidentified 
 genus of clostridiales , none of which were detected as major contributors in artificially reared individuals , 
 significantly contributed to the cecal microbiome in wild individuals . there were only 1,174 otus shared between the samples from wild and captive birds , whereas the other majorities 
 of otu , 106,438 otus and 66,721 otus , were specific to the wild and captivity environments , respectively . obvious differences between samples in the pca plot of weighted unifrac can be seen in fig . 1 . although blastn search does not directly prove the 
 original source of bacteria , it may give some clues about the origin of the detected bacterial otu . in this 
 context , we found that the predominant bacterial otu in the artificially reared birds suggested to have a origin 
 from chicken ( yl - otu-2 , 3 , 8 , 10 , 12 and 19 ) , human ( yl - otu-0 , 7 and 16 ) , pig ( yl - otu-4 , 13 ) or rodent ( yl - otu-5 
 and 6 ) , all of which showed high sequence identities ( > 99% ) with known sequences . in contrast to that , 
 predominant bacterial otus detected in wild rock ptarmigans showed lower sequence identities ( < 96% ) with 
 known sequences , except for the otus ( wl - otu-1 , 4 , 9 , 12 , 14 and 17 ) which showed high sequence identities 
 ( > 99% ) with those obtained from wild birds , such as tetrao urogallus . other possible 
 sources , such as human , pig , cat and squirrels , were also suggested for some of the top 20 otus , but most of 
 them showed low similarities as mentioned above , which suggested un - relatedness rather than relatedness to otus 
 detected in wild rock ptarmigans instead ( supplementary 
 table 2 ) . the present results clearly show fundamental differences between cecal bacteriome in wild and artificially 
 reared rock ptarmigans . interesting but not surprising was the fact that artificially reared birds harbored 
 poultry bacteria , most likely due to being fed on feed for poultry supplemented with fresh green vegetables . moreover , human - hosted bacteria were also detected , which may be due to close contact with keepers or 
 veternarians of the center . rearing without contact from mothers easily deprives rock ptarmigans from 
 species - specific bacteria , which seems to be vertically transferred from progenitors , although several common 
 bacterial otus ( 1,174 otus ) were detected in both types of birds . unlike most of all commonly shared otus , which 
 were detected in a one - sided manner ( table 3table 3.species level comparison between wild and captive bred rock ptarmigansseq_codesuggested speciesddbj accessionwl4wl5wl7wl8yl1yl2yl3yl4yl5ptarmigan - otu0unidentified sp . in coriobacteriaceae ( olsenella 
 sp.)lc05573129,7472,1817,7765,2031135965243ptarmigan - otu1actinomyces sp.lc05573211,1451,6842,0251,18610010ptarmigan-otu2unidentified species in clostridialeslc05573351202,0272,85310,9972,4474,583ptarmigan - otu3unidentified species in synergistaceae ( cloacibacillus 
 sp.)lc05573411,5073,2745,6014,66900120ptarmigan - otu4bacteroides sp.lc05573511,4791,9374,5693,05810020ptarmigan-otu5megasphaera sp.lc0557369,5011,3822,4201,27200000ptarmigan-otu6bifidobacterium sp.lc05573761003731,0909,0492,537231ptarmigan-otu7bifidobacterium sp.lc0557388,9349632,7361,42100010ptarmigan-otu8slackia sp . ( s. equolifaciens)lc0557395,4106231,16992910010ptarmigan - otu9faecalibacterim sp.lc05574052011,3863,2547,9055,4261,103ptarmigan-otu10unidentified species in ruminococcaceae ( sporobacter 
 termitidis)lc0557414,127618826664107125ptarmigan - otu11asteroleplasma sp.lc0557422,4378241,1932,53300010ptarmigan-otu12unidenfieid species in clostridiales 
 ( roseburia sp.)lc0557434,8855911,1615737301765425ptarmigan - otu13unidentified species in ruminococcaceaelc0557442771171,4842,6115,2662,6303,010ptarmigan - otu14dialister sp.lc0557458901,0462,4541,55000000a ) values are numbers of sequence read identified . yl1yl5 , captive bred svalbard rock 
 ptarmigans at the preservation and research center , the city of yokohama . details , see text . ) , a limited number of otus , as listed in table 
 4table 4.otus relatively evenly shared by wild and captive bred rock ptarmigans seq codesuggested speciesddbj accessionwl4wl5wl7wl8yl1yl2yl3yl4yl5ptarmigan - otu31unidentified species in clostridialeslc0557502,33618521013345113226121647ptarmigan - otu46unidentified species in clostridialeslc0557521,00327770154623059010ptarmigan - otu37unidentified species in clostridialeslc0557511,39115747421470667157465ptarmigan - otu76unidentified species in ruminococcaceaelc0557561,35010210658328311160ptarmigan - otu20unidentified species in enterobacteriaceaelc0557497774231026334371931891,907ptarmigan - otu88pseudoflavonifractor sp.lc05575763548126785201174136ptarmigan-otu56unidentified species in clostridialeslc055753415923224311,5002256549ptarmigan - otu58unidentified species in lachnospiraceaelc0557542282361271403071,473537680ptarmigan - otu69unidentified species in lachnospiraceaelc05575520612351479381275376247ptarmigan - otu153unidentified species in coriobacteriaceaelc055747104141281242711896ptarmigan - otu194unidentified species in clostridialeslc05574810713751417891172179a ) values are numbers of identified sequence reads . yl1-yl5 , 
 captive bred svalbard rock ptarmigans at the preservation and research center , the city of yokohama . , were detected in both groups of birds at relatively similar abundance . however , as shown in table 3 , completely undetectable otus were rare even within the otus 
 detected in the one - sided manner . this may indicate that bacteria in artificially reared birds would grow , if 
 wild - like conditions , especially feeding , were established . however , it may prove difficult for wild 
 condition - associated bacteria from such a small source to grow and rapidly replace bacteria associated with 
 captivity conditions , and hence , artificial reconstitution of rock ptarmigan - specific cecal microbiota is most 
 likely necessary . yl1yl5 , captive bred svalbard rock 
 ptarmigans at the preservation and research center , the city of yokohama . yl1-yl5 , 
 captive bred svalbard rock ptarmigans at the preservation and research center , the city of yokohama . olsenella sp . which , as shown above ( table 3 ) , 
 suggested the existence of wild rock ptarmigan - specific bacteria and , which belongs to the order actinomycetales , could be possibly regarded as 
 health - promoting elements within indigenous git bacteriome in ptarmigans , because olsenella was 
 previously classified as lactobacilli , and lactic acid bacteria are 
 usually regarded as health promoting in various animals hosts . . belongs to the family synergistaceae , which has been recognized as a 
 toxin - degrading agent in hawaiian native goats and enables goats to forage alkaloids - rich plants . as mentioned above , food selected by wild japanese rock ptarmigans 
 contains plant toxins , such as cyclic diterpenes and alkaloids . we 
 foresee that these particular bacteria will be major targets of future isolation work to test their ability to 
 degrade plant toxins . since the majority of wild japanese rock ptarmigans are infected with 
 eimeria protozoa , it is very likely that direct 
 inoculation of fecal slurry from wild birds to captive reared nestlings may not be safe . in this context , a sort 
 of bacterial cocktail containing both rock ptarmigan - specific and health - promoting bacteria would be a safer 
 option for the ex situ conservation program .
preservation of indigenous gastrointestinal microbiota is deemed to be critical for successful captive 
 breeding of endangered wild animals , yet its biology is poorly understood . here , we investigated cecal 
 bacterial communities in wild japanese rock ptarmigans ( lagopus muta japonica ) and compared 
 them with those in svalbard rock ptarmigans ( l. m. hyperborea ) in captivity . 
 ultra - deep 
 sequencing of 16s rrna gene indicated that the community structure of cecal microbiota in wild rock ptarmigans 
 was remarkably different from that in captive svalbard rock ptarmigans . 
 fundamental differences between 
 bacterial communities in the two groups of birds were detected at the phylum level . 
 firmicutes , 
 actinobacteria , bacteroidetes and synergistetes were the major phyla detected in wild japanese rock 
 ptarmigans , whereas firmicutes alone occupied more than 80% of abundance in captive svalbard rock ptarmigans . 
 furthermore , unclassified genera of coriobacteriaceae , synergistaceae , bacteroidaceae , actinomycetaceae , 
 veillonellaceae and clostridiales were the major taxa detected in wild individuals , whereas in zoo - reared 
 birds , major genera were ruminococcus , blautia , faecalibacterium and 
 akkermansia . 
 zoo - reared birds seemed to lack almost all rock ptarmigan - specific bacteria in 
 their intestine , which may explain the relatively high rate of pathogenic infections affecting them . 
 we show 
 evidence that preservation and reconstitution of indigenous cecal microflora are critical for successful 
 ex situ conservation and future re - introduction plan for the japanese rock ptarmigan .