Species-Level Taxonomic Characterization of Uncultured Core Gut Microbiota of Plateau Pika

ABSTRACT Rarely has the vast diversity of bacteria on Earth been profiled, particularly on inaccessible plateaus. These uncultured microbes, which are also known as “microbial dark matter,” may play crucial roles in maintaining the ecosystem and are linked to human health, regarding pathogenicity and prebioticity. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is a keystone species in the maintenance of ecological balance. We used a combination of full-length 16S rRNA amplicon sequencing, shotgun metagenomics, and metabolomics to elucidate the species-level community structure and the metabolic potential of the gut microbiota of the plateau pika. Using a full-length 16S rRNA metataxonomic approach, we clustered 618 (166 ± 35 per sample) operational phylogenetic units (OPUs) from 105 plateau pika samples and assigned them to 215 known species, 226 potentially new species, and 177 higher hierarchical taxa. Notably, 39 abundant OPUs (over 60% total relative abundance) are found in over 90% of the samples, thereby representing a “core microbiota.” They are all classified as novel microbial lineages, from the class to the species level. Using metagenomic reads, we independently assembled and binned 109 high-quality, species-level genome bins (SGBs). Then, a precise taxonomic assignment was performed to clarify the phylogenetic consistency of the SGBs and the 16S rRNA amplicons. Thus, the majority of the core microbes possess their genomes. SGBs belonging to the genus Treponema, the families Muribaculaceae, Lachnospiraceae, and Oscillospiraceae, and the order Eubacteriales are abundant in the metagenomic samples. In addition, multiple CAZymes are detected in these SGBs, indicating their efficient utilization of plant biomass. As the most widely connected metabolite with the core microbiota, tryptophan may relate to host environmental adaptation. Our investigation allows for a greater comprehension of the composition and functional capacity of the gut microbiota of the plateau pika. IMPORTANCE The great majority of microbial species remain uncultured, severely limiting their taxonomic characterization and biological understanding. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is considered to be the keystone species in the maintenance of ecological stability. We comprehensively investigated the gut microbiota of the plateau pika via a multiomics endeavor. Combining full-length 16S rRNA metataxonomics, shotgun metagenomics, and metabolomics, we elucidated the species-level taxonomic assignment of the core uncultured intestinal microbiota of the plateau pika and revealed their correlation to host nutritional metabolism and adaptation. Our findings provide insights into the microbial diversity and biological significance of alpine animals.

This is a good paper describing taxonomic structure of the gut microbiota of the plateau pika that endemic on Qinghai-Tibet Plateau. Remarkably, the authors combined metagenomic binning approach to provide an in-depth view of the core uncultured bacterial taxa inhabited in the pika intestinal tract. I was impressed by the amount of work the authors have put into this study, and their manuscript was carefully written up. Generally, results have been well explained and the figures are of high quality. So, all in all, I think this is a great manuscript with a very good fit for Microbiology Spectrum.
I have several minor points for clarifications that may improve the manuscript (see below). Minor comments: 1. If possible, please provide metadata information of 105 intestinal content samples, so that readers can better understand the plateau's living environment and population representation. 2. The "tax" in line 106 should be changed to "taxa". 3. It is recommended to rewrite lines 121-134 and put the OPU information in the supplementary table. 4. It is suggested to rephrase lines 162-177. The taxa with relative abundance greater than 1% are provided in the text, and the rest taxa are presented in the supplementary table. 5. Pikas are herbivorous, consuming high levels of moss, which is exceptionally high in fibre and low in protein.Therefore, the genes related to cellulose degradation and fermentation should be numerous and valuable. It is suggested that the author focus on the genes related to cellulose degradation and fermentation in subsequent research. 6. In some places, figure legends are inadequate, such as Fig 4. 7. There have some misleading descriptions in this paper. If being polished by a native English speaker, the paper would highly benefit.
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If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit ourwebsite. This is a good paper describing taxonomic structure of the gut microbiota of the plateau pika that endemic on Qinghai-Tibet Plateau. Remarkably, the authors combined metagenomic binning approach to provide an in-depth view of the core uncultured bacterial taxa inhabited in the pika intestinal tract. I was impressed by the amount of work the authors have put into this study, and their manuscript was carefully written up.
Generally, results have been well explained and the figures are of high quality. So, all in all, I think this is a great manuscript with a very good fit for Microbiology Spectrum.
I have several minor points for clarifications that may improve the manuscript (see below).
Minor comments: 1. If possible, please provide metadata information of 105 intestinal content samples, so that readers can better understand the plateau's living environment and population representation.
2. The "tax" in line 106 should be changed to "taxa".

Dear Editor and Reviewer
We deeply appreciate the effort that you and the reviewers put into carefully reviewing our work; the manuscript (ID: Spectrum03495-22) has now been revised according to your thoughtful and helpful comments. Response: Thank you for your suggestion. We reconstructed the phylogenetic tree using the full-length 16S rRNA sequences to provide comprehensive information.
The three categories of 618 OPUs were marked in one tree. Moreover, we highlighted the relative abundance and prevalence rate of each 618 OPUs to clarify that the highly prevalent OPUs were uncultured bacteria. We have uploaded the Q4: Is there a relationship between the sampling time (July, 2016) with species-level core microbiota? There would be some auxiliary microbiota in so-called "core microbiota" based on the samples in each time period.

Response:
We agree with your comments. There were some reports about the seasons' factors influencing the microbiota of plateau pika (1). In the present research, we investigate the "core microbiota" in a large number of plateau pika only at one time point. We chose July as the sampling time because the population density of pika reached its peak during this period. We also have been making an effort to keep surveillance of the gut microbiota of plateau pika through a wide range of time.
However, the sequencing data were still in curation. We hope to bring a more comprehensive study of the "core microbiota" of plateau pika in the future.
Q5: Based on the metabolic reconstruction of SGBs assigned uncultured taxonomic groups, supplying more discussions about how to isolate uncultured resources would be helpful to expatiate the importance of studying the species-level core microbiota.
Response: Thank you for your comments. We have added the relevant content in the discussion section.
We made the following revision (Lines 270 -287 in the revised manuscript): "Although the metagenomic-driven investigation has expanded our views on the diversity of microbial life, it is crucial to isolate and culture species from these uncultured lineages to test genome-based predictions of their metabolic functions and physiology and properly elucidate their ecological roles. Such as, some novel enzymatic reactions and pathways could only be discovered through experimental testing of live bacteria and not be detected by genomic sequencing alone. Plateau pika's species-level core gut microbiota is all uncultured bacteria affiliated with the anaerobic lineages. To enrich these hard-to-cultivate bacteria, we must design selective nutrient media and set up proper physicochemical conditions (for example, temperature, pH, and gas-phase composition). Through the function capacity analysis, we realized that the core microbiota of plateau pika could efficiently digest the plant polysaccharide and live in a unique metabolic microenvironment (for example, with a high-level oleamide, tryptophan, and hypoxanthine). Based on the current knowledge, the low-nutrient media with plant polysaccharide as the sole carbon source, and complement with metabolites as growth factors, would help the isolation of the uncultured core microbiota of plateau pika. In addition, some innovative techniques (for example, reverse genomics, live-FISH, and SlipChip) are also worth trying." Q6: Lines 92-94: it would be confusing that the sequences of OTU were picked out for phylogenetic inference with LTP and SILVA databases. The phylogenetic inference concerned with the sequence alignment; however, the main difference between LTP and SILVA databases is taxonomy (https://www.arbsilva.de/documentation/release-132/).

Response:
Sorry for the confusion caused by OTU phylogenetic inference. Our phylogenetic inference and taxonomic assignment were mainly based on the LTP database. We selected the relative reference sequences from the SILVA databases to supplement when there is no affiliated relative of OTU sequences in the LTP database. A detailed description can be found in the "Operational Phylogenetic Units (OPU) Assignment" section (lines in 356-383) of material and methods.
To avoid confusion, we made the following revision: "The representative sequences of each OTU were picked out for phylogenetic inference" (lines 107-108 in the revised manuscript).

Response:
We have changed "Akkermansiaceae" to "Akkermansiaceae" in italic face format (line 140 in the revised manuscript).
GHs families with GH43, GH2, and GH28 as the most abundant. Lineages of the Prevotella genus contained common member species of the human or non-human gut microbiota and have been profoundly implicated in health and disease (22).
Prevotella copri is among the essential members prevalent in over 40% of the human population and is associated with high-fiber diets (5, 23). While in the plateau pika, the SGBs of Prevotella accounted for 2.23% relative abundance and were affiliated with P. ruminicola and P. brevis, which are the predominant features of ruminants' gut microbiota (22). These Prevotella SGBs could be a particular feature in carbohydrate utilization for hindgut-fermenting herbivores. SGBs annotated to the family of Muribaculaceae are the second abundant taxa that accounted for 6.81%  (24). Consistently with the previous study proposed by Lagkouvardos (25), the species of Muribaculaceae isolated from mouse also observed a high occurrence of GH13 that implicated alpha-glucan degradation.
Although the SGBs of Treponema belonging to the Spirochaetota were characterized by the highest relative abundance of 9.48%, they carried less mean GHs number with 28.00±8.68 and were involved in 31 different GHs families. Treponema SGBs also mainly contain the GH13 family highly similar to the glycogen-debranching enzyme GlgX (Table S9). Collectively, CAZymes of plateau pika gut microbiota, especially the GHs family, have high diversity and metabolic versatility, and SGBs belonging to the genus Prevotella and the family Muribaculaceae may play central roles."