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Evidence of Cellulose Metabolism by the Giant Panda Gut Microbiome

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Evidence of Cellulose Metabolism by the Giant Panda Gut Microbiome Evidence of cellulose metabolism by the giant panda gut microbiome Lifeng Zhua,1,QiWua,1, Jiayin Daia, Shanning Zhangb, and Fuwen Weia,2 aKey Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China and bChina Wildlife Conservation Association, Beijing 100714, China Edited by Rita R. Colwell, University of Maryland, College Park, MD, and approved September 7, 2011 (received for review December 2, 2010) The giant panda genome codes for all necessary enzymes associ- cellulose digestion (10). Although the giant panda can use non- ated with a carnivorous digestive system but lacks genes for cellulosic material from the bamboo diet using enzymes coded in enzymes needed to digest cellulose, the principal component of its own genome, digestion of cellulose and hemicellulose is im- their bamboo diet. It has been posited that this iconic species must possible based on the panda’s genetic composition, and must be therefore possess microbial symbionts capable of metabolizing dependent on gut microbiome. However, previous research using cellulose, but these symbionts have remained undetected. Here we culture methods and small-scale sequencing identified three examined 5,522 prokaryotic ribosomal RNA gene sequences in wild predominant bacteria from the panda gut—Escherichia coli, and captive giant panda fecal samples. We found lower species Streptococcus, and Enterobacteriaceae—none of which aids in richness of the panda microbiome than of mammalian microbiomes cellulose digestion (11–13). Thus, an incomplete understanding for herbivores and nonherbivorous carnivores. We detected 13 of the gut microbial ecosystem in this interesting and high-profile operational taxonomic units closely related to Clostridium groups I species remains because of restrictions in methodology and past and XIVa, both of which contain taxa known to digest cellulose. reliance on studies of captive animals. Seven of these 13 operational taxonomic units were unique to pandas compared with other mammals. Metagenomic analysis us- Results and Discussion ing ∼37-Mbp contig sequences from gut microbes recovered puta- We undertook a large-scale analysis of 16S rRNA gene sequen- tive genes coding two cellulose-digesting enzymes and one ces to profile microbial flora inhabiting the digestive system of β hemicellulose-digesting enzyme, cellulase, -glucosidase, and xy- giant pandas and used a metagenomic approach based on next- β Clostridium lan 1,4- -xylosidase, in group I. Comparing glycoside generation de novo sequencing to identify functional attributes fi hydrolase pro les of pandas with those of herbivores and omni- encoded in the gut microbiome. A total of 5,636 near–full-length vores, we found a moderate abundance of oligosaccharide-degrad- 16S rRNA gene segments were amplified from fecal samples of ing enzymes for pandas (36%), close to that for humans (37%), and seven wild and eight captive giant pandas. After exclusion of 74 the lowest abundance of cellulases and endohemicellulases (2%), putative chimeric and 30 chloroplast sequences, 5,522 sequences which may reflect low digestibility of cellulose and hemicellulose in were retained for analysis. Using a minimum identity of 97% as the panda’s unique bamboo diet. The presence of putative cellu- the threshold for any sequence pair, we identified 85 bacterial lose-digesting microbes, in combination with adaptations related operational taxonomic units (OTUs), 14 of which were pre- to feeding, physiology, and morphology, show that giant pandas viously undescribed (Fig. 1A and SI Appendix, Table S1). Cov- have evolved a number of traits to overcome the anatomical and physiological challenge of digesting a diet high in fibrous matter. erage of 99% was obtained across existing bacterial clone libraries, and we are confident that our dataset presents the most comprehensive assessment of gut microbes in this species based ccess to dietary resources shapes animal evolution (1). Early on the rarefaction method in DOTUR (14) (SI Appendix, Fig. Aon, animals lost the ability to synthesize many key com- S1A). The majority of microbes were members of the Firmicutes pounds, and instead this function is performed by symbionts (2). (62 OTUs, 4,633 sequences, 83.8% of the total of 5,522 For example, microbial symbionts assist with extracting nutrients sequences) and Proteobacteria (12 OTUs, 871 sequences, 15.8% from food and key compounds from the environment, and also of the total sequences), with the remainder belonging to the synthesize necessary metabolic compounds (1). Gut microbiota phyla Actinobacteria, Bacteroidetes, Cyanobacteria, and Acid- share specialized relationships with their hosts, and advances in obacteria (Fig. 1 and SI Appendix, Fig. S2 and Table S1). Within genomics are revealing the dynamics of these relationships (3). the Firmicutes, 33 OTUs (60.8% of the total of 5,522 sequences) Recent developments in culture-independent methodologies were members of the class Clostridia and 29 OTUs (23.0% of based on large-scale comparative analyses of microbial small- total sequences) belonged to the class Bacilli. The high pro- subunit ribosomal RNA genes (16S ribosomal RNA) and met- portion of Firmicutes OTUs found in the gut of giant pandas agenomics have revealed the extent of microbial diversity and differs from the findings in previous studies attempting to char- – metabolic potential in greater detail (2 7). These techniques can acterize giant panda gut microbes (11–13) and is notably similar now be applied to animals that have acquired a profoundly new diet, presenting an opportunity to investigate host physiological and microbial systems in an evolutionary context. Author contributions: F.W. designed research; L.Z. and S.Z. performed research; L.Z., Q.W., The giant panda (Ailuropoda melanoleuca) is well known for J.D., S.Z., and F.W. analyzed data; and L.Z., Q.W. and F.W. wrote the paper. dietary oddities: a bamboo specialist within the mammalian or- The authors declare no conflict of interest. der Carnivora possessing a gastrointestinal tract typical of car- ∼ fi This article is a PNAS Direct Submission. nivores. It consumes 12.5 kg of this highly brous plant each Freely available online through the PNAS open access option. day (8), but because it lacks the long intestinal tract character- Data deposition: The sequences reported in this paper have been deposited in the Gen- istic of other herbivores, extensive fermentation is not possible Bank database. For a list of accession numbers, see SI Appendix. The metagenomic data (9). Giant pandas digest only ∼17% of dry matter consumed (8), project ID is 5936 in Integrated Microbial Genomes with Microbiome Samples (IMG/M). and have low digestion coefficients for bamboo hemicelluloses 1L.Z. and Q.W. contributed equally to this work. (27%) and celluloses (8%) (9). Indeed, the giant panda genome 2To whom correspondence should be addressed. E-mail: [email protected]. codes for all necessary enzymes associated with a carnivorous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. digestive system, but lacks the enzyme homologs needed for 1073/pnas.1017956108/-/DCSupplemental. 17714–17719 | PNAS | October 25, 2011 | vol. 108 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1017956108 Downloaded by guest on September 30, 2021 Fig. 1. Microbial flora of giant pandas. (A) Neighbor-joining tree containing one representative of each of 85 OTUs. Colors represent different phyla: yellow, Clostridia class of Firmicutes; blue, Bacilli class of Firmicutes; purple, Proteobacteria; gray, Actinobacteria, Bacteroidetes, Cyanobacteria, and Acidobacteria. seqs, sequences. All bootstrap values >80% are shown in this tree. (B) Relative abundance of sequences from wild and captive giant panda fecal samples; other Firmicutes belong to the Bacilli class. (C) Percentage of sequences from each fecal sample assigned to different phyla. W1–W7, wild giant panda samples; C1–C8, captive giant panda samples. Numbers below the sample number are total sequences from each individual. to that found in herbivorous mammals (2, 6). However, the based algorithm implemented in MEGAN (24). The results species richness in the giant panda was lower than that seen in again showed that the majority of microbes (71%) belong to the previous work on mammalian herbivores and nonherbivorous phylum Firmicutes (Fig. 2A). We also compared the catalog with carnivores (SI Appendix, Fig. S1B), perhaps due to the panda’s the Kyoto Encyclopedia of Genes and Genomes (KEGG) da- unique bamboo diet and simple digestive system. tabase and the Clusters of Orthologous Groups (COG) data- The proportion of 16S rRNA gene sequences belonging to the bases to assess the functional capacities present in bacterial class Clostridia of Firmicutes were high for both wild (73% of the metagenome (Fig. 2 B–D and SI Appendix, Figs. S4 and S5). Our total of 3361 sequences) and captive (42% of the total of 2161 analysis revealed that half of these predicted genes coding for sequences) giant pandas (Fig. 1 B and C). In Clostridia, we cellulose and hemicellulose-digesting enzymes are found in detected 13 OTUs closely related to Clostridium group I (10 species within the Clostridium genus (SI Appendix, Table S7). For OTUs, 1,457 sequences, 26.4% of the total sequences) and XIVa example, 2 of 12 putative cellulase genes in this study are ho- (3 OTUs, 185 sequences, 3.3% of the total sequences) (SI Ap- mologous (E-value: 2.00E-115 and 6.00E-29, respectively) to pendix,
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