Natural foraging selection and gut microecology of two subterranean rodents from the Eurasian Steppe in China

Natural foraging selection and gut microecology of two subterranean rodents from the Eurasian Steppe in China | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Natural foraging selection and gut microecology of two subterranean rodents from the Eurasian Steppe in China Zhenghaoni Sz, Heping Fu, Shuai Yuan, Kai Chen, Tingting Han, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4293070/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract As the most abundant group of mammals, rodents possess a very rich ecotype, which makes them ideal for studying the relationship between diet and host gut microecology. Zokors are specialized herbivorous rodents adapted to living underground. Unlike more generalized herbivorous rodents, they feed on the underground parts of grassland plants. There are two species of the genus Myospalax in the Eurasian steppes in China: one is Myospalax psilurus , which inhabits meadow grasslands and forest edge areas, and the other is M. aspalax , which inhabits typical grassland areas. How are the dietary choices of the two species adapted to long-term subterranean life, and what is the relationship of this diet with gut microbes? Are there unique indicator genera for their gut microbial communities? Relevant factors such as the ability of both species to degrade cellulose are not yet clear. In this study, we analysed the gut bacterial communities and diet composition of two species of zokors using 16S amplicon technology combined with macro-barcoding technology. We found that the diversity of gut microbial bacterial communities in M. psilurus was significantly higher than that in M. aspalax and that the two species of zokors possessed different gut bacterial indicator genera. Based on the results of Mantel analyses, the gut bacterial community of M. aspalax showed a significant positive correlation with the creeping-rooted type food, and there was a complementary relationship between the axis root type food and the rhizome type food dominated (containing bulb types and tuberous root types) food groups. Functional prediction based on KEGG found that M. psilurus possessed a stronger degradation ability in the same cellulose degradation pathway. Neutral modelling results showed that the gut flora of the M. psilurus has a wider ecological niche compared to that of the M. aspalax . This provides a new perspective for understanding how rodents living underground in grassland areas respond to changes in food conditions. Myospalax Gut microbiota Diet Cellulose degradation Microbial nich Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Background Microbes, as resident populations that colonise the mammalian body (especially the gut)[ 1 ], far outnumber mammalian somatic cells, and unique genes encoded by microbes outnumber the host's genome 100-fold [ 2 ]. Most microorganisms residing in the gut have profound effects on host physiology and nutrition, which are critical for host health [ 3 ]. Thanks to the rise of human intestinal microbiome research, the emerging field of bacterial-dominated gut microecology attempts to answer a growing number of biological questions [ 4 – 5 ]. Recently, more and more research is shifting from the gut microbiomes of humans to those of other mammals in their natural environments in order to more deeply explore the mutual adaptations and selection of the host and gut bacteria [ 6 – 8 ]. Mutually beneficial symbiosis between a host and gut microbiota is thought to have arisen through a long period of co-evolution [ 9 ]. As an important component of the host’s metabolism, intestinal microbiota can provide substrates, enzymes, and energy to the host [ 10 ]. The composition of the gut microbiome changes with the host's physiological state, food, and habitat [ 11 ]. In addition to vertical inheritance caused by genetic factors, diet is a major determinant of gut microbiota composition [ 12 – 14 ]. Rodents, as the most abundant group of mammals, have a very rich ecotype [ 15 – 16 ], making them ideal for studying the relationship between diet and host gut microecology [ 17 ]. Food habits reflect the adaptability and ecological role of animals [ 18 – 19 ]. As r-strategists, rodents have evolved diverse food habits to adapt to various complex environments [ 20 – 21 ]. Some of these have evolved to be highly specialized, such as animals in the subfamily Myospalacinae (zokors). Unlike generalized herbivorous rodents, zokors are specialized herbivorous rodents adapted to living underground, which feed on the underground parts of grassland plants [ 21 ]. The subfamily Myospalax contains two genera, Myospalax and Eospalax [ 22 ]. There are two species of the genus Myospalax in China: one is the North China Zokor ( M. psilurus ), which inhabits meadow grasslands and forest edge areas, and the other is the Steppe Zokor ( M. aspalax ), which inhabits typical grassland areas. [ 23 – 24 ]. The two species diverged from their ancestors about 2.9 million years ago, and the two populations came into secondary contact about 1.7 million years ago, resulting in weak gene flow. Although M. aspalax experienced population shrinkage and M. aspalax experienced population expansion, the effective population size of M. aspalax is still larger than that of M. psilurus [ 25 ]. So far, research on these two species of zokors has mainly focused on morphology, genetics and other topics [ 26 – 28 ], and research on their microecology, especially research on the relationship between the dietary choices of two species and their intestinal microorganisms, is relatively rare. Based on the ecological differences described above, we hope to compare the gut microbial profiles of these two closely related species based on dietary composition and to describe the types of gut microbial communities of the two zokors through indicator genera. M. psilurus lives at the edges between forest margins and grasslands [ 29 ], where the vegetation types are more diverse, and there are more species of plants available for consumption than for M. aspalax . Based on the amount of plant diversity in the two zokor habitats and the expansion shown by M. psilurus population, we hypothesized that M. psilurus has higher dietary diversity and microbial diversity than M. aspalax . As specialized herbivorous rodents, their diets are rich in fibre, which is a potential carbon source [ 30 ] and the function of the gut microbiota may be enriched in cellulose degradation. Food digestion and absorption are key processes in animals’ adaptive evolution [ 29 ]. Population expansion is inseparable from the efficient use of food. We predict that the gut flora of M. psilurus will be slightly better at degrading cellulose. Indicator genera in the gut microbial communities of these two kinds of zokors and the relationship between food types and gut flora are also issues that this study aims to explore. Our results attempt to provide new insights into how rodents living underground in the Eurasian steppes respond to differences in diet. 2 Results Analysis of food composition and dietary differences Based on the results of macro-barcoding sequencing, after excluding species that could not be classified and those with ultra-low abundance, there were a total of 55 food species for M. psilurus and M. aspalax . Of these, 25 species were unique to M. psilurus , and 4 species were unique to M. aspalax (Additional file 1). From the macro-barcoding results, we determined the top ten species in the diets of each species of zokor. Sanguisorba officinalis was the main food species of M. psilurus , accounting for about 68% (Fig. 1 A). Allium tuberosum and Phlomis sp. were the main sources of food for M. aspalax , accounting for 38.1% and 14%, respectively (Fig. 1 B). The diversity of food composition of M. psilurus was higher than that of M. aspalax . The analysis of species differences in food habits shows that food components were significantly different between the two species of zokors (Fig. 1 C). S. officinalis , A. tuberosum , and Thalictrum minus were detected in the diets of both species of zokors, but at different abundances. S. officinalis was more abundant in the diet of M. psilurus than in that of M. aspalax , whereas A. tuberosum and T. minus were more commonly eaten by M. psilurus (Fig. 1 C). Pimpinella sp. , Potentilla sp. , Viola sp. , Bupleurum scorzonerifolium , Adenophora sp. , Plantago sp. , and Galium dahuricum were exclusively found in the samples from M. psilurus (Fig. 1 C). Sequencing information and gut microbial α- diversity We obtained a total of 933735 optimised sequences from 33 samples using 16S amplicon sequencing, resulting in 28295 valid sequences with an average sequence length of 413. As the number of sequenced samples increased, the dilution curve flattened out, and the coverage rate of all samples was over 99.90%. This indicates that the amount of sequenced sequences was reasonable, and the depth of sequencing was sufficient to cover all samples (Additional file 2). The 33 samples from the two species of zokors contained a total of 1280 OTUs, and the number of shared OTUs accounted for 69.14% (Fig. 2 A), namely norank_f_Muribaculaceae (37.62%), Lachnospiraceae_NK4A136_group (10.43%) and unclassified_f_Lachnospiraceae (9.98%), and other species in 108 genera (S3). The number of OUTs unique to the North China zokor was 251, and 114 for M. aspalax (Fig. 2 A). The gut bacterial communities of the two species of zokors showed highly significant differences in the abundance-based coverage estimators (ACE) index, Shannon index and Fischer phylogenetic diversity index, and the diversity of gut bacteria of M. psilurus was significantly higher than that of M. aspalax (Fig. 2 B-D). The analysis of community composition at the genus level showed that the top 10 genera in the gut bacterial communities were significantly different between the zokor species and also varied by individual within a species. The two zokor species’ gut bacterial communities were composed of the three phyla of Firmicutes, Bacteroidota, and Desulfobacterota (Fig. 2 E). The gut bacterial community of M. psilurus mainly contained species from 10 genera, including Akkermansia (82.32%) and Rikenella (9.05%), and that of the M. psilurus mainly contained species from the genera norank_f_norank_o_Izemoplasmatales (62.89%), Bacteroides_ pectinophilus_group (9.10%) and other species of 16 genera (Additional file 3). Analysis of the association between food types and gut microorganisms All plant species were classified into eight types according to the type of plant root system. Mantel analysis of the gut bacterial communities of the two species of zokors with food root types revealed a significant positive correlation between the gut bacterial communities of M. aspalax and creeping-Rooted plants (Fig. 3 ). The correlation between the gut bacterial community and food type (by root type) of the two species of zokors showed the same trend in terms of statistical significance, although the original hypothesis could not be rejected (Fig. 3 ). That is, the gut bacterial communities of both species of zokors showed a positive correlation with creeping-rooted, bulb type and axis root type foods, and a negative correlation with tuberous root type, sparse clump type, rhizome type, fibril root type and dense clump type foods (Fig. 3 ). Indicator genera and functional prediction of two zokor species’ gut bacteria Based on the results of the indicator species analysis, the gut flora of both species of zokors showed the presence of indicator genera. There were 19 indicator genera in the guts of M. psilurus and 8 indicator genera in M. aspalax . Among them, Cellulosilyticum , Tuzzerella , NK4A214_group, and Monoglobus were the main indicator genera in the gut of the North China zokor, and Blautia was the main indicator genera in the gut flora of M. aspalax (Fig. 4 ). Based on the results of the test for differences in KEGG functions in the OUT set of the indicated species, the abundance of KEGG functions in the tertiary pathways related to metabolism (primary pathway) were all higher in M. psilurus than in M. aspalax . The two species of zokors showed highly significant differences in 19 pathways (Fig. 5 ). The functions enriched to were related to amino acid metabolism, carbohydrate metabolism and glycogen biosynthesis and metabolism (Fig. 5 ). Eight of these were related to amino acid metabolism, namely 1) cysteine and methionine metabolism, 2) alanine, aspartate and glutamate metabolism, 3) glycine, serine and threonine metabolism, 4) phenylalanine, tyrosine and tryptophan biosynthesis, 5) valine, leucine and isoleucine biosynthesis, 6) histidine metabolism, 7) arginine and proline metabolism, and 8) lysine degradation (Fig. 5 ). There were 10 pathways associated with carbohydrate metabolism, including 1) amino sugar and nucleotide sugar metabolism, 2) pyruvate metabolism, 3) glycolysis/gluconeogenesis, 4) starch and sucrose metabolism, 5) pentose phosphate pathway, 6) butanoate metabolism, 7) glyoxylate and dicarboxylate metabolism, 8) propanoate metabolism, 9) fructose and mannose metabolism, and 10) galactose metabolism (Fig. 5 ). Only one pathway, peptidoglycan biosynthesis, was associated with glycan biosynthesis and metabolism (Fig. 5 ). Gut bacteria associated with cellulose degradation Because of the herbivory of the two species of zokors, we paid special attention to genes related to carbohydrate metabolism. In rodents, the most abundant gene associated with fibre metabolism was predicted to be beta-glucosidase (K05349). This happened to be present in both zokor gut bacterial communities and differed significantly between the two (Fig. 6 ). In addition to this, two genes, endoglucanase (K01179) and oligosaccharide reducing-end xylanase (K15531), were also significantly different between the two species of zokors, and the abundance of all three genes was greater in the gut bacterial community of M. psilurus than that of M. aspalax (Fig. 6 ). These results suggest that M. aspalax possesses a stronger degradation ability for the cellulose degradation pathway. Neutral Community Modelling (NCM) of gut flora The neutral community model successfully estimated most of the relationships between the frequency of occurrence of OTUs and their corresponding changes in abundance. The goodness of fit for the gut microbial communities of M. psilurus and M. aspalax were 66.62% (Fig. 7 A) and 51% (Fig. 7 B), respectively. The explanation rate of stochastic processes in the gut flora of M. psilurus was higher than that of M. psilurus , i.e., stochastic processes were more important in the gut bacteria of M. psilurus . The migration rate (Nm) shows that the microbial community spreads in the gut of M. psilurus (Nm = 5043) much more than that of M. aspalax . (Nm = 2423) (Fig. 7 A, B). These results suggest that the gut flora of M. psilurus has a wider ecological niche than that of M. aspalax . 3 Discussion In this study, we compared the gut microbiota and natural foraging preferences of two species of the genus Myospalax , distributed in China and living in the Eurasian steppe zone. We focused on the differences in the structure and function of gut bacterial communities between the two species and analysed the relationship between microbiota and food types. The results of the study showed that the dietary diversity and α-diversity of the bacterial community of M. psilurus were significantly higher than those of M. aspalax , i.e., the North China zokor consumed more food types than the steppe zokor, and the species richness, diversity and genealogical diversity of the gut bacterial community in M. psilurus were higher than those in M. aspalax . In general, diet-associated microbes have a wider range of sources, and along with a more diverse diet, hosts may be exposed to and carry a greater diversity of microbes [ 31 ]. On the other hand, the composition of the microbiota is also dependent on the nutrients available in the gut, so a varied diet may increase the α-diversity of the gut microbiota by providing a more diverse range of nutrients [ 32 ]. From this we infer that a richer food composition led to the higher diversity of the gut bacterial community in the North China zokor. We identified the genera found among the gut flora of each rodent species. The gut bacterial community of M. psilurus mainly contained species from 10 genera, including Akkermansia (82.32%) and Rikenella (9.05%), and that of the steppe zokor mainly contained species from 16 genera, including norank_f_norank_o_Izemoplasmatales (62.89%), Bacteroides_pectinophilus_group (9.10%) and other species in 16 genera. In studies of the human gut microbiome, bacteria of the genus Akkermansia have been found to be involved in mucin degradation [ 33 ] and are a probiotic associated with a reduced risk of obesity-related metabolic syndrome [ 34 ]. Rikenella was found to be the top taxon significantly and positively associated with BMI in a study based on patterns of intestinal flora and body weight changes in wild house mice ( Mus musculus ) at different latitudes [ 35 ]. norank__f__norank__o_o_Izemoplasmatales are considered to be DNA degraders [ 36 ]. The abundance of the Bacteroides_pectinophilus_group in enterobacteria was found to be significantly negatively correlated with the prevalence of non-alcoholic fatty liver disease (NAFLD) in a multi-ethnic cohort obesity phenotype study [ 37 ]. In conclusion, there were differences in the composition of the intestinal bacterial community between the two species of zokors, and the dominant genera were mainly probiotic-associated groups, with no significant presence of microorganisms that posed a threat to the health of the hosts. Two kinds of zokor had different indicator genera of intestinal bacteria. Cellulosilyticum, Tuzzerella, NK4A214 and Monoglobus were the main markers in the intestinal tract of North China zokor, and Blautia was the main indicator genus in the intestinal bacterial community of steppe zokor. It has been shown that Cellulosilyticum is associated with fibre and protein breakdown [ 38 ]. In preventive medicine studies with naringin regulating the microbiota and metabolome in mice, alterations in Tuzzerella were found to be most associated with host endogenous metabolites [ 39 ]. NK4A214 of the Ruminococcus family is one of the more abundant genera in the rabbit gut [ 40 ], capable of degrading plant polysaccharides to produce volatile fatty acids such as butyric acid [ 41 – 43 ], which in turn promotes apoptosis in colon cancer cells and reduces intestinal inflammation [ 44 ]. Monoglobus is considered to be a highly specialised group of pectin-degrading sugar biota in the human gut [ 45 ]. NK4A136 and Blutia are beneficial bacteria that produce short-chain fatty acid [ 46 ]. Blutia is a new genus of Lachnospiraceae that produces short-chain fatty acid (SCFA) through glucose metabolism and digests dietary cellulose [ 47 ], and it is widely found in mammalian faeces and intestines with potential probiotic properties [ 48 ]. The functions of the two zokor cecum bacterial community indicator species are involved in plant polysaccharide degradation, and production of short-chain fatty acids, which is in line with the findings of most studies on the function of the gut flora of herbivores [ 49 – 51 ]. Analysis of the food habits of the two species of zokors showed that M. psilurus mainly fed on S. officinalis (68%), while M. aspalax preferred a combination of A. tuberosum and Phlomis sp. (52.1%). In relation to the specialised root-feeding habits of zokors and the root morphology of these three plant species, it is clear that zokors preferred larger food items such as axis root type, bulb type and tuberous root plants, and given that their foraging behaviour occurs without the aid of visual searching, this is consistent with what is predicted in the theory of optimal foraging in animals [ 52 ]. Environmental heterogeneity shapes the heterogeneity of the distribution of resources available to foragers, and the community structure of natural grassland vegetation clearly follows the same heterogeneity. Optimal foraging theory predicts that an animal's ability to utilise patches of resources is key to foraging success [ 53 ]. As they live underground, zokors are inherently limited in their choice of food resources. Unlike the above-ground parts of plants, root systems are wrapped in a dense medium such as soil, and it is difficult for zokors to weigh the choice of such food resources from multiple perspectives (such as form and color), so a "large amount and easy to obtain" becomes the primary criterion for consideration. Therefore, in their foraging behaviour, zokors do not primarily target the dominant species or the established species of the corresponding grassland type to eat, but rather chose species with large root systems as their main food source. It is well known that herbivores rely on microorganisms living in their gastrointestinal tract to efficiently digest their fibre-rich diets [ 54 – 55 ]. The plant cell wall polysaccharides that make up the bulk of this fibre represent a potentially rich source of carbon [ 30 ] that is highly resistant to enzymatic breakdown [ 56 – 57 ], but certain microbial taxa have evolved mechanisms for degrading sugars from these structural polysaccharides in order to, through intestinal fermentation, gain more access to chemical energy from the diet [ 58 – 59 ]. Short-chain fatty acids (SCFA) are the end products of polysaccharide fermentation produced by gut flora [ 60 – 61 ]. Over a long evolutionary period, animals have optimised their digestive physiology by expanding the volume of either the foregut or hindgut [ 62 – 63 ]. While the microbial communities involved in fibre digestion in foregut fermentation have been well described by studies on ruminant animals [ 64 – 65 ], studies targeting the catabolic utilisation of plant polysaccharides have also focused more on foregut fermentation [ 66 – 67 ] or, to a lesser extent, economically viable monogastrics [ 68 – 69 ]. Little is known about the microbial taxa that perform this function in wild herbivores with hindgut fermentation. Plant cell walls are mainly composed of cellulose, hemicellulose, pectin, and lignin [ 70 – 71 ]. Cellulose is the most abundant of these plant cell wall polysaccharides [ 72 ], and its crystal structure makes it one of the most difficult substances to hydrolyse [ 73 ]. Therefore, microorganisms in the gut that can efficiently break down cellulose are critical for herbivores’ digestion. We know that host carbohydrate-active enzymes (CAZymes) are mainly produced in the cecum [ 74 ]. In the rumen, cellulose degradation is facilitated in part by the efforts of bacterial communities [ 75 ], including members of the genera Fibrobacter [ 50 ] and Ruminococcus [ 76 ]. Due to the phytophagous nature of both zokors, we paid particular attention to pathways related to carbohydrate metabolism in their gut bacterial communities. Glycosyl hydrolases (EC 3.2.1-) are genes mainly involved in cellulose and hemicellulose degradation [ 77 ]. We searched and screened the KO pathways in the OTU set consisting of the two zokor gut bacterial indicator genera based on the "EC 3.2.1-" search condition and found three pathways (K05349, K01179, and K15531) that were significantly different between the two zokor species. K05349 and K01179 are the most frequently mentioned pathways in the degradation of complex fibres [ 42 ]. K05349 is associated with β-glucosidase metabolism, K01179 is associated with endoglucanase metabolism, and K15531 is associated with the metabolism of oligosaccharide reduced-end xylanase. β-glucosidase genes are present in almost all bacterial phyla [ 78 – 79 ]. All organisms involved in cellulose degradation have a cellulase system consisting of a multi-enzyme complex of three enzymes: exoglucanase (also known as cellulose biohydrolase, EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and β-glucosidase (BGL, EC 3.2.1.21), which work synergistically for the complete hydrolysis of cellulose [ 80 ]. Therefore, the presence of K05349 and K01179 in the metabolic pathway of the two zokor species’ gut bacteria is not unexpected. In a study of five desert rodent gut bacterial communities, predictions of the cellulose degradation function showed that K05349 and K01179 were the two most abundant genes in these rodents’ gut bacterial communities [ 51 ]. The KEGG pathways predicted from the gut flora of those five different dietary species of desert rodents and this study’s two specialised subterranean rodents suggest that the cellulose degradation pathways of K05349 and K01179 act as 'generalists' in the rodent gut bacterial community. In addition to the two pathways mentioned above, the metabolic pathway associated with K15531 also differed between the two species of Myospalax , but its abundance was much lower than that of the two pathways mentioned above. Oligosaccharide reduced-end xylanase (K15531; EC3.2.1.156) is a high molecular mass xylanase that degrades xylan, a dietary fibre in plant cell walls [ 81 ]. Xylan is the most common hemicellulose component of grass, leaves, straw, and wood, the second most abundant renewable resource on earth [ 82 – 83 ], a major component of hemicellulose [ 84 ], and a polysaccharide component of up to 45% of ruminant feed [ 85 ]. Xylan is an abundant non-cellulosic polysaccharide found in plant biomass [ 86 ], and xylanases catalyse the β-bonding of xylan to release by-products that can be utilised by ruminants [ 87 ]. Of interest, this pathway has not been reported in intestinal bacterial studies in herbivorous monogastrics, but rather in a study based on obesity complications in US immigrants, which concluded that levels of oligosaccharide-reducing-end xylanase (K15531) increased with fibre intake, which was negatively correlated with the severity of obesity [ 88 ]. These results imply that the process of cellulose degradation by the bacterial community in the gut of zokor includes not only pathways that are prevalent in mammals, but also unique degradation pathways. Among these cellulose degradation pathways, M. psilurus possessed a stronger degradation capacity. Based on the estimation of the neutral community model of the intestinal bacterial communities of the two species of zokors, it was found that the importance of stochastic processes in the gut bacteria of M. psilurus exceeded that of the M. aspalax , and that the mobility of the intestinal bacterial community of M. psilurus was greater than that of M. aspalax , i.e., the microbial community was spreading farther in the intestines of M. psilurus than in those of M. aspalax . Combined with the differences in other ecological characteristics (including the number of OTUs, α-diversity, and KEGG function prediction) of the gut bacterial community of M. psilurus between the two as described above, we can conclude that the intestinal bacterial community of M. psilurus has a wider ecological niche compared with that of M. aspalax . 4 Conclusions Dietary diversity and gut microbial bacterial community diversity were significantly higher in M. psilurus than in M. aspalax , and the two zokor species possessed different gut bacterial indicator genera. Cellulosilyticum , Tuzzerella , NK4A214_group, and Monoglobus were the main indicator genera in the gut tract of M. psilurus , and Blautia was the main indicator genera in the gut flora of M. aspalax . There was a significant positive correlation between the gut bacterial community of M. aspalax and the rhizome type plants, and there was a complementary relationship between the axis root type food and rhizome type plant dominated (including bulb type and tuberous root food) food groups. In the cellulose degradation pathway, M. psilurus possessed a stronger degradation ability. The gut flora of M. psilurus has a wider ecological niche than that of M. aspalax . Gut microorganisms revealed the dietary choices of M. psilurus and M. aspalax . 5 Materials and methods To address the above questions, we conducted a preliminary investigation of the intestinal bacterial communities and dietary composition of two species of zokor, using the 16S amplicon technique in combination with the macro-barcoding technique. Sample collection Two species of zokors were captured using the circular tongs trap method from August-September 2021 (autumn) in the Hulunbeier meadow grassland (Chenbalhu Banner) and the typical grassland of Xilingol (Zhengxibai Banner) in Inner Mongolia, China. A total of 33 individuals were collected, including 18 M. psilurus and 15 M. aspalax (Fig. 8 ). Samples of cecum contents and stomach contents were collected and then immediately stored in liquid nitrogen. The samples were transported to the laboratory on dry ice and stored at -80℃. The above experimental protocol and procedures were approved by the Laboratory Animal Welfare and Ethics Committee of the Inner Mongolia Agricultural University (File No. NND2023081). DNA extraction and sequencing of cecum and stomach contents Microbiota from cecum contents and plant DNA from stomach contents were extracted using the TruSeq ™ DNA Sample Prep Kit according to the instructions. DNA quality was checked using a NanoDrop2000 ultra-trace spectrophotometer (Thermo Scientific, USA) and 1% agarose gel electrophoresis. The 16SrRNA V3-V4 region of the microbiota was amplified using universal primers (338F and 806R) [ 89 ], and plant root communities were assessed by amplifying trnL operons(trnL-F: 5'-CGAAATYGGTAGACGCTACG-3' and trnL-R: 5'- CCDTYGAGTCTCTGCACCTATC-3') [ 90 ]. Each sample was replicated three times, and the PCR products from the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were recovered by cutting the gel using the Axy Prep DNA Gel Recovery Kit (AXYGEN Inc.), eluted with Tris_HCl, and detected by 2% agarose electrophoresis. Referring to the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified by QuantiFluor™-ST Blue Fluorescence Quantification System (Promega), after which the PCR products were mixed according to the corresponding proportion of each sample according to the sequencing amount required. After constructing the clone library using the TruSeq ™ DNA kit, high-throughput sequencing (250 bp, double-end sequencing) was performed using the Illumina Nova Seq platform. Bioinformatics analysis The raw sequences were quality controlled using fastp software [ 91 ] and FLASH (version1.2.7) software for splicing [ 92 ]. Bases with quality value below 20 in the tails of the reads were filtered, and a window of 50 bp was set. If the average quality value within the window was below 20, then the back-end bases were truncated starting from the window, reads below 50 bp after quality control were filtered, and reads containing N bases were removed. Based on the overlap relationship between PEreads, pairs of reads were spliced (merged) into one sequence with a minimum overlap length of 10bp; the maximum mismatch ratio allowed in the overlap region of the spliced sequences was 0.2, and the non-compliant sequences were screened. The samples were distinguished according to the barcode and primers at the first and last ends of the sequences, and the sequences were adjusted based on the direction. The number of mismatches allowed by the barcode was 0, and the maximum number of primers mismatched was 2. Using UPARSE software (version 7.1), OTU clustering was performed on non-repetitive sequences (excluding single sequences) according to 97% similarity, and chimeras were removed during the clustering process to obtain OTU representative sequences [ 93 – 94 ]. All sample sequences were levelled by sequence pumping at the minimum number of sample sequences, and the average sequence coverage (Good's coverage) for each sample could still reach 99.09%. The RDPclassifier (version2.11) was used to compare to the Silva16SrRNA gene database (v138) for taxonomic annotation of intestinal microbial OTU species [ 95 ], with a confidence threshold of 70%, and to count the community composition of each sample at several different taxonomic levels. 16S functional prediction analyses were performed using PICRUSt2 (version2.2.0) software [ 96 ]. The ny_v20221012 species classification database and the RDP Classifier2.13 software were used to classify and annotate gastric capacitance DNA macro-barcode sequences. The Species 2000 Chinese Nodal Plant Group Database [ 97 ] was used to search for plant species that might be present in the habitats of both species of zokors using the "distribution area + classification system" filters. The distribution range of plants that could potentially be consumed by the North China zokor was limited to "Inner Mongolia Autonomous Region" and "Heilongjiang Province". The distribution of plants that could potentially be eaten by the steppe zokor was limited to "Inner Mongolia Autonomous Region" and "Hebei Province", and the search condition was "distribution in the above areas". The plant classification order was specified from Plantae to Magnoliopsida for screening and downloading information on plant species found in the two study habitats. The results of the downloads were cross-referenced with the macro-barcode annotations to match species with the same name and exclude species that are not reported in the habitat. Several alpha diversity indices (ACE, Shannon, and Pd indices) were calculated using mothur software [ 98 ], and the Wilcoxon rank-sum test was used for analyses of between-group differences in alpha diversity. Using the data table in the tax_summary_a folder, R statistical software (version3.3.1) was used to produce microbial community bar charts and plant community pie charts. The Wilcoxon rank-sum test was performed using R statistical software and the scipy package of python to analyse differences between plant species in the samples of the two zokor species’ stomach contents. The p-values were corrected using the Fdr method, and the confidence interval was calculated using the Welch’s t-test with a confidence level of 0.95. All plants were classified into 8 types by plant root type, and the gut bacterial communities of both zokor species were analysed by food root type with a Mantel analysis using the vegan package (vsesion 2.4.3). IndVal was calculated using the lndval function of the labdsv package [ 99 ] to find the indicator genera of the gut bacterial communities of the two zokors, and OTU clustering information was compared with the sequenced microbial genome databases using the PICRUSt2 software to obtain the corresponding species in the KEGG database ( https://www.kegg.jp/ ) for the functional type and abundance of the corresponding species. Differences in KEGG pathways between the two zokor species were analysed using Statistical Analysis of Macrogenome Mapping (STAMP) v2.1.3, and the false discovery rate was controlled using the Benjamini-Hochberg procedure [ 100 ]. Declarations Funding This research was financially supported by the National Natural Science Foundation of China, China (Grant numbers 32060256, 32060395), the Major Science and Technology Project of Inner Mongolia Autonomous Region (Grant number 2021ZD0006) and Basic scientific research business expenses of universities directly under Inner Mongolia Autonomous Region (Grant numbers BR221307, BR221037). Author Contribution Zhenghaoni Shang designed the experiment and wrote the manuscript. Kai Chen and Tingting Han analyzed the data. Fan Bu, Shanshan Sun, Na Zhu and Duhu Man collected samples. Shuai Yuan and Heping Fu revised the manuscript. All the authors participated in the review and editing and approved the final manuscript. Acknowledgement We are grateful for all those who helped with the field sampling. Data Availability The datasets presented in this study can be found in online repositories. 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Chen","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Chen","suffix":""},{"id":296396010,"identity":"907f6319-cd16-4c6a-b0f1-b291949e36bb","order_by":4,"name":"Tingting Han","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Han","suffix":""},{"id":296396011,"identity":"1191805f-a0cc-421c-a4c7-0f7832b78c05","order_by":5,"name":"Fan Bu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Bu","suffix":""},{"id":296396012,"identity":"db32d6e7-b77b-43b2-9920-b0ca5a2edea5","order_by":6,"name":"Shanshan Sun","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shanshan","middleName":"","lastName":"Sun","suffix":""},{"id":296396013,"identity":"3a92816c-7214-44ea-8ac1-24b75d5195be","order_by":7,"name":"Na Zhu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Na","middleName":"","lastName":"Zhu","suffix":""},{"id":296396014,"identity":"28dfda3a-b16e-4870-91bb-93ef1de2c81a","order_by":8,"name":"Duhu Man","email":"","orcid":"","institution":"Hulunbuir University","correspondingAuthor":false,"prefix":"","firstName":"Duhu","middleName":"","lastName":"Man","suffix":""}],"badges":[],"createdAt":"2024-04-19 12:12:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4293070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4293070/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55539528,"identity":"597cb462-da81-45a2-848b-cc983b5a651a","added_by":"auto","created_at":"2024-04-29 16:59:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199877,"visible":true,"origin":"","legend":"\u003cp\u003eFood composition of the two species of zokors. \u003cstrong\u003eA.\u003c/strong\u003e Top 10 species in the diet of\u003cem\u003e M. psilurus\u003c/em\u003e. \u003cstrong\u003eB.\u003c/strong\u003eTop 10 species in the diet of \u003cem\u003eM. aspalax\u003c/em\u003e. \u003cstrong\u003eC.\u003c/strong\u003e t-test for between-group differences in food composition between the two species of zokors, with p-values corrected using the Fdr method, and confidence intervals calculated using Welch's t-method, with a confidence level of 0.95. p-values after correction are shown on the right side, denoted as *0.01\u0026lt;p≤0.05, **0.001\u0026lt;p≤0.01, ***p≤0.001, and ***p≤0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/e500f1677849b815b576330d.png"},{"id":55539935,"identity":"25ef2cf2-3cc5-4366-a390-f4e701d60d64","added_by":"auto","created_at":"2024-04-29 17:07:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":436819,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial diversity and species composition in the guts of two species of zokors. \u003cstrong\u003eA.\u003c/strong\u003e Venn diagram showing the percentages of OTUs common and unique to the two species of zokors. \u003cstrong\u003eB.\u003c/strong\u003e Test of intergroup differences in ACE index of the gut bacterial community of the two species of zokors. \u003cstrong\u003eC.\u003c/strong\u003e Test of intergroup differences in the Shannon index of the intestinal bacterial communities of the two species of zokors. \u003cstrong\u003eD.\u003c/strong\u003e Test of intergroup differences in the Pd index of the intestinal bacterial communities of the two species of zokors. \u003cstrong\u003eE.\u003c/strong\u003e Composition of the two zokor species’ intestinal bacterial communities at the genus level. Significance levels were denoted as *0.01\u0026lt;p≤0.05, **0.001\u0026lt;p≤0.01, ***p≤0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/10805921ec833fc69382ff60.png"},{"id":55539933,"identity":"ea8832c8-5ea6-44d1-843e-490cb697d145","added_by":"auto","created_at":"2024-04-29 17:06:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68929,"visible":true,"origin":"","legend":"\u003cp\u003eMantel analyses of food types and gut bacterial communities of the two species of Myospalax. The colour of the line indicates the p-value of the correlation between the gut microbial community and the food composition distance matrix (Blue: p \u0026lt; 0.05; Gray: p ≥ 0.05). The thickness of the line indicates the strength of the correlation between the microbial community β-diversity distance matrix and the food composition distance matrix (Wide: r ≥ 0.4; Medium: 0.4 ≥ r ≥ 0.2; Thin: r \u0026lt; 0.2). Solid lines indicate positive correlations and dashed lines indicate negative correlations. The colours of the boxes reflect the strength and direction of correlation between food types, with blue representing a positive correlation and red representing a negative correlation.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/3d61c3cd4d26cb99691672a1.png"},{"id":55539934,"identity":"f69f738f-6ed2-43b5-aed4-3738f27cd9ff","added_by":"auto","created_at":"2024-04-29 17:06:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222065,"visible":true,"origin":"","legend":"\u003cp\u003eAbundance and indicative values of bacterial indicator genera in the gut flora of two species of \u003cem\u003eMyospalax\u003c/em\u003e. Indicator genera are listed on the y-axis, and their indicator values are represented on the x-axis. The colours of the circles and the background represent the two species of zokors, pink for \u003cem\u003eM. psilurus\u003c/em\u003e and blue for \u003cem\u003eM. aspalax,\u003c/em\u003e with the size of the \"○\" indicating the abundance of the indicator genera.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/0b37b9eecdbf54beabe643f7.png"},{"id":55539520,"identity":"99bd322f-d998-4c33-a5ae-9c1c465e0b6f","added_by":"auto","created_at":"2024-04-29 16:58:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":116058,"visible":true,"origin":"","legend":"\u003cp\u003eFunctions of metabolism-related intestinal bacterial communities, including differences between the two species of \u003cem\u003eMyospalax\u003c/em\u003e. The x-axis of the left bar graph indicates the average absolute abundance of the three-level pathways in different subgroups, and the y-axis lists the names of the pathways. The circles with letters on the right side of the bar graph represent the secondary pathway to which the corresponding functional pathway belongs: A for amino acid metabolism, C for carbohydrate metabolism, and G for glycan biosynthesis and metabolism. The middle area is the confidence interval, and the dots represent the average absolute abundance of the species in the two groups. The bars on the dots are the upper and lower limits of the confidence intervals for the difference, and the right-hand side is the corrected p-value, *0.01＜p≤0.05, **0.001＜p≤0.01, ***p≤0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/3d18805743c0c12f35a80947.png"},{"id":55539519,"identity":"f8f7710c-bbf8-416f-ac55-e1d49964af2c","added_by":"auto","created_at":"2024-04-29 16:58:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115655,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted abundance of cellulose degradation related genes. The y-axis represents the predicted values of absolute abundance of related genes. *0.01＜p≤0.05, **0.001＜p≤0.01, ***p≤0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/c46535712cb010b7bec06737.png"},{"id":55539525,"identity":"f7f28fdb-427f-46e7-b39c-d21c63123ec8","added_by":"auto","created_at":"2024-04-29 16:59:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":172031,"visible":true,"origin":"","legend":"\u003cp\u003eEstimation of the neutral community model for the gut bacterial communities of the two species of zokors. The x-axis represents the logarithm of the average relative abundance of species, and the y-axis depicts the frequency of occurrence. The solid line represents the fit of the neutral model, and the upper and lower dashed lines represent the 95% confidence of the model prediction. R\u003csup\u003e2\u003c/sup\u003e represents the overall goodness-of-fit of the neutral community model, and the higher R\u003csup\u003e2\u003c/sup\u003e indicates the closer the model is to the neutral model, which means that the construction of the community is affected by stochastic processes more, and less by deterministic processes. Nm is the product of metacommunity size (N) and migration rate (m) (Nm = N×m), which is used to assess the degree of dispersal among communities.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/e774463a3d75749949276999.png"},{"id":55539524,"identity":"78d6e87a-8c35-488b-957b-96700c7d9e15","added_by":"auto","created_at":"2024-04-29 16:59:00","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":401232,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the sampling points for two species of zokors.\"□\" indicates the capture location of\u003cem\u003e M. psilurus\u003c/em\u003e and \"○\" indicates the capture location of the \u003cem\u003eM. aspalax\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/d374881fcd6d54d63e1d4ad2.jpg"},{"id":55956303,"identity":"cf35d82e-fae0-4486-b641-b7833166e3e4","added_by":"auto","created_at":"2024-05-06 20:20:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2214535,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/2883f0ee-1e52-4ea0-89fb-d4ac769e27b9.pdf"},{"id":55539522,"identity":"41044079-6f36-46dc-a12f-c79225ce49cc","added_by":"auto","created_at":"2024-04-29 16:58:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":654310,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4293070/v1/40482538d4aacfeb76f76aa1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Natural foraging selection and gut microecology of two subterranean rodents from the Eurasian Steppe in China","fulltext":[{"header":"1 Background","content":"\u003cp\u003eMicrobes, as resident populations that colonise the mammalian body (especially the gut)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], far outnumber mammalian somatic cells, and unique genes encoded by microbes outnumber the host's genome 100-fold [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Most microorganisms residing in the gut have profound effects on host physiology and nutrition, which are critical for host health [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thanks to the rise of human intestinal microbiome research, the emerging field of bacterial-dominated gut microecology attempts to answer a growing number of biological questions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Recently, more and more research is shifting from the gut microbiomes of humans to those of other mammals in their natural environments in order to more deeply explore the mutual adaptations and selection of the host and gut bacteria [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Mutually beneficial symbiosis between a host and gut microbiota is thought to have arisen through a long period of co-evolution [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As an important component of the host\u0026rsquo;s metabolism, intestinal microbiota can provide substrates, enzymes, and energy to the host [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The composition of the gut microbiome changes with the host's physiological state, food, and habitat [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition to vertical inheritance caused by genetic factors, diet is a major determinant of gut microbiota composition [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Rodents, as the most abundant group of mammals, have a very rich ecotype [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], making them ideal for studying the relationship between diet and host gut microecology [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Food habits reflect the adaptability and ecological role of animals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. As r-strategists, rodents have evolved diverse food habits to adapt to various complex environments [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Some of these have evolved to be highly specialized, such as animals in the subfamily Myospalacinae (zokors). Unlike generalized herbivorous rodents, zokors are specialized herbivorous rodents adapted to living underground, which feed on the underground parts of grassland plants [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The subfamily Myospalax contains two genera, \u003cem\u003eMyospalax\u003c/em\u003e and \u003cem\u003eEospalax\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. There are two species of the genus \u003cem\u003eMyospalax\u003c/em\u003e in China: one is the North China Zokor (\u003cem\u003eM. psilurus\u003c/em\u003e), which inhabits meadow grasslands and forest edge areas, and the other is the Steppe Zokor (\u003cem\u003eM. aspalax\u003c/em\u003e), which inhabits typical grassland areas. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The two species diverged from their ancestors about 2.9\u0026nbsp;million years ago, and the two populations came into secondary contact about 1.7\u0026nbsp;million years ago, resulting in weak gene flow. Although \u003cem\u003eM. aspalax\u003c/em\u003e experienced population shrinkage and \u003cem\u003eM. aspalax\u003c/em\u003e experienced population expansion, the effective population size of \u003cem\u003eM. aspalax\u003c/em\u003e is still larger than that of \u003cem\u003eM. psilurus\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. So far, research on these two species of zokors has mainly focused on morphology, genetics and other topics [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and research on their microecology, especially research on the relationship between the dietary choices of two species and their intestinal microorganisms, is relatively rare. Based on the ecological differences described above, we hope to compare the gut microbial profiles of these two closely related species based on dietary composition and to describe the types of gut microbial communities of the two zokors through indicator genera. \u003cem\u003eM. psilurus\u003c/em\u003e lives at the edges between forest margins and grasslands [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], where the vegetation types are more diverse, and there are more species of plants available for consumption than for \u003cem\u003eM. aspalax\u003c/em\u003e. Based on the amount of plant diversity in the two zokor habitats and the expansion shown by \u003cem\u003eM. psilurus\u003c/em\u003e population, we hypothesized that \u003cem\u003eM. psilurus\u003c/em\u003e has higher dietary diversity and microbial diversity than \u003cem\u003eM. aspalax\u003c/em\u003e. As specialized herbivorous rodents, their diets are rich in fibre, which is a potential carbon source [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and the function of the gut microbiota may be enriched in cellulose degradation. Food digestion and absorption are key processes in animals\u0026rsquo; adaptive evolution [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Population expansion is inseparable from the efficient use of food. We predict that the gut flora of \u003cem\u003eM. psilurus\u003c/em\u003e will be slightly better at degrading cellulose. Indicator genera in the gut microbial communities of these two kinds of zokors and the relationship between food types and gut flora are also issues that this study aims to explore. Our results attempt to provide new insights into how rodents living underground in the Eurasian steppes respond to differences in diet.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cp\u003e \u003cb\u003eAnalysis of food composition and dietary differences\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the results of macro-barcoding sequencing, after excluding species that could not be classified and those with ultra-low abundance, there were a total of 55 food species for \u003cem\u003eM. psilurus\u003c/em\u003e and \u003cem\u003eM. aspalax\u003c/em\u003e. Of these, 25 species were unique to \u003cem\u003eM. psilurus\u003c/em\u003e, and 4 species were unique to \u003cem\u003eM. aspalax\u003c/em\u003e (Additional file 1). From the macro-barcoding results, we determined the top ten species in the diets of each species of zokor. \u003cem\u003eSanguisorba officinalis\u003c/em\u003e was the main food species of \u003cem\u003eM. psilurus\u003c/em\u003e, accounting for about 68% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). \u003cem\u003eAllium tuberosum\u003c/em\u003e and \u003cem\u003ePhlomis sp.\u003c/em\u003e were the main sources of food for \u003cem\u003eM. aspalax\u003c/em\u003e, accounting for 38.1% and 14%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The diversity of food composition of \u003cem\u003eM. psilurus\u003c/em\u003e was higher than that of \u003cem\u003eM. aspalax\u003c/em\u003e. The analysis of species differences in food habits shows that food components were significantly different between the two species of zokors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). \u003cem\u003eS. officinalis\u003c/em\u003e, \u003cem\u003eA. tuberosum\u003c/em\u003e, and \u003cem\u003eThalictrum minus\u003c/em\u003e were detected in the diets of both species of zokors, but at different abundances. \u003cem\u003eS. officinalis\u003c/em\u003e was more abundant in the diet of \u003cem\u003eM. psilurus\u003c/em\u003e than in that of \u003cem\u003eM. aspalax\u003c/em\u003e, whereas \u003cem\u003eA. tuberosum\u003c/em\u003e and \u003cem\u003eT. minus\u003c/em\u003e were more commonly eaten by \u003cem\u003eM. psilurus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). \u003cem\u003ePimpinella sp.\u003c/em\u003e, \u003cem\u003ePotentilla sp.\u003c/em\u003e, \u003cem\u003eViola sp.\u003c/em\u003e, \u003cem\u003eBupleurum scorzonerifolium\u003c/em\u003e, \u003cem\u003eAdenophora sp.\u003c/em\u003e, \u003cem\u003ePlantago sp.\u003c/em\u003e, and \u003cem\u003eGalium dahuricum\u003c/em\u003e were exclusively found in the samples from \u003cem\u003eM. psilurus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSequencing information and gut microbial\u003c/b\u003e α-\u003cb\u003ediversity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe obtained a total of 933735 optimised sequences from 33 samples using 16S amplicon sequencing, resulting in 28295 valid sequences with an average sequence length of 413. As the number of sequenced samples increased, the dilution curve flattened out, and the coverage rate of all samples was over 99.90%. This indicates that the amount of sequenced sequences was reasonable, and the depth of sequencing was sufficient to cover all samples (Additional file 2). The 33 samples from the two species of zokors contained a total of 1280 OTUs, and the number of shared OTUs accounted for 69.14% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), namely norank_f_Muribaculaceae (37.62%), Lachnospiraceae_NK4A136_group (10.43%) and unclassified_f_Lachnospiraceae (9.98%), and other species in 108 genera (S3). The number of OUTs unique to the North China zokor was 251, and 114 for \u003cem\u003eM. aspalax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The gut bacterial communities of the two species of zokors showed highly significant differences in the abundance-based coverage estimators (ACE) index, Shannon index and Fischer phylogenetic diversity index, and the diversity of gut bacteria of \u003cem\u003eM. psilurus\u003c/em\u003e was significantly higher than that of \u003cem\u003eM. aspalax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-D). The analysis of community composition at the genus level showed that the top 10 genera in the gut bacterial communities were significantly different between the zokor species and also varied by individual within a species. The two zokor species\u0026rsquo; gut bacterial communities were composed of the three phyla of Firmicutes, Bacteroidota, and Desulfobacterota (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The gut bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e mainly contained species from 10 genera, including \u003cem\u003eAkkermansia\u003c/em\u003e (82.32%) and \u003cem\u003eRikenella\u003c/em\u003e (9.05%), and that of the \u003cem\u003eM. psilurus\u003c/em\u003e mainly contained species from the genera norank_f_norank_o_Izemoplasmatales (62.89%), Bacteroides_ pectinophilus_group (9.10%) and other species of 16 genera (Additional file 3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of the association between food types and gut microorganisms\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All plant species were classified into eight types according to the type of plant root system. Mantel analysis of the gut bacterial communities of the two species of zokors with food root types revealed a significant positive correlation between the gut bacterial communities of \u003cem\u003eM. aspalax\u003c/em\u003e and creeping-Rooted plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The correlation between the gut bacterial community and food type (by root type) of the two species of zokors showed the same trend in terms of statistical significance, although the original hypothesis could not be rejected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). That is, the gut bacterial communities of both species of zokors showed a positive correlation with creeping-rooted, bulb type and axis root type foods, and a negative correlation with tuberous root type, sparse clump type, rhizome type, fibril root type and dense clump type foods (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIndicator genera and functional prediction of two zokor species\u0026rsquo; gut bacteria\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the results of the indicator species analysis, the gut flora of both species of zokors showed the presence of indicator genera. There were 19 indicator genera in the guts of \u003cem\u003eM. psilurus\u003c/em\u003e and 8 indicator genera in \u003cem\u003eM. aspalax\u003c/em\u003e. Among them, \u003cem\u003eCellulosilyticum\u003c/em\u003e, \u003cem\u003eTuzzerella\u003c/em\u003e, NK4A214_group, and \u003cem\u003eMonoglobus\u003c/em\u003e were the main indicator genera in the gut of the North China zokor, and \u003cem\u003eBlautia\u003c/em\u003e was the main indicator genera in the gut flora of \u003cem\u003eM. aspalax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the results of the test for differences in KEGG functions in the OUT set of the indicated species, the abundance of KEGG functions in the tertiary pathways related to metabolism (primary pathway) were all higher in \u003cem\u003eM. psilurus\u003c/em\u003e than in \u003cem\u003eM. aspalax\u003c/em\u003e. The two species of zokors showed highly significant differences in 19 pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The functions enriched to were related to amino acid metabolism, carbohydrate metabolism and glycogen biosynthesis and metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Eight of these were related to amino acid metabolism, namely 1) cysteine and methionine metabolism, 2) alanine, aspartate and glutamate metabolism, 3) glycine, serine and threonine metabolism, 4) phenylalanine, tyrosine and tryptophan biosynthesis, 5) valine, leucine and isoleucine biosynthesis, 6) histidine metabolism, 7) arginine and proline metabolism, and 8) lysine degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). There were 10 pathways associated with carbohydrate metabolism, including 1) amino sugar and nucleotide sugar metabolism, 2) pyruvate metabolism, 3) glycolysis/gluconeogenesis, 4) starch and sucrose metabolism, 5) pentose phosphate pathway, 6) butanoate metabolism, 7) glyoxylate and dicarboxylate metabolism, 8) propanoate metabolism, 9) fructose and mannose metabolism, and 10) galactose metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Only one pathway, peptidoglycan biosynthesis, was associated with glycan biosynthesis and metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGut bacteria associated with cellulose degradation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBecause of the herbivory of the two species of zokors, we paid special attention to genes related to carbohydrate metabolism. In rodents, the most abundant gene associated with fibre metabolism was predicted to be beta-glucosidase (K05349). This happened to be present in both zokor gut bacterial communities and differed significantly between the two (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition to this, two genes, endoglucanase (K01179) and oligosaccharide reducing-end xylanase (K15531), were also significantly different between the two species of zokors, and the abundance of all three genes was greater in the gut bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e than that of \u003cem\u003eM. aspalax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results suggest that \u003cem\u003eM. aspalax\u003c/em\u003e possesses a stronger degradation ability for the cellulose degradation pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNeutral Community Modelling (NCM) of gut flora\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe neutral community model successfully estimated most of the relationships between the frequency of occurrence of OTUs and their corresponding changes in abundance. The goodness of fit for the gut microbial communities of \u003cem\u003eM. psilurus\u003c/em\u003e and \u003cem\u003eM. aspalax\u003c/em\u003e were 66.62% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and 51% (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), respectively. The explanation rate of stochastic processes in the gut flora of \u003cem\u003eM. psilurus\u003c/em\u003e was higher than that of \u003cem\u003eM. psilurus\u003c/em\u003e, i.e., stochastic processes were more important in the gut bacteria of \u003cem\u003eM. psilurus\u003c/em\u003e. The migration rate (Nm) shows that the microbial community spreads in the gut of \u003cem\u003eM. psilurus\u003c/em\u003e (Nm\u0026thinsp;=\u0026thinsp;5043) much more than that of \u003cem\u003eM. aspalax\u003c/em\u003e. (Nm\u0026thinsp;=\u0026thinsp;2423) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). These results suggest that the gut flora of \u003cem\u003eM. psilurus\u003c/em\u003e has a wider ecological niche than that of \u003cem\u003eM. aspalax\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"3 Discussion","content":"\u003cp\u003eIn this study, we compared the gut microbiota and natural foraging preferences of two species of the genus \u003cem\u003eMyospalax\u003c/em\u003e, distributed in China and living in the Eurasian steppe zone. We focused on the differences in the structure and function of gut bacterial communities between the two species and analysed the relationship between microbiota and food types. The results of the study showed that the dietary diversity and α-diversity of the bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e were significantly higher than those of \u003cem\u003eM. aspalax\u003c/em\u003e, i.e., the North China zokor consumed more food types than the steppe zokor, and the species richness, diversity and genealogical diversity of the gut bacterial community in \u003cem\u003eM. psilurus\u003c/em\u003e were higher than those in \u003cem\u003eM. aspalax\u003c/em\u003e. In general, diet-associated microbes have a wider range of sources, and along with a more diverse diet, hosts may be exposed to and carry a greater diversity of microbes [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. On the other hand, the composition of the microbiota is also dependent on the nutrients available in the gut, so a varied diet may increase the α-diversity of the gut microbiota by providing a more diverse range of nutrients [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. From this we infer that a richer food composition led to the higher diversity of the gut bacterial community in the North China zokor.\u003c/p\u003e \u003cp\u003eWe identified the genera found among the gut flora of each rodent species. The gut bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e mainly contained species from 10 genera, including \u003cem\u003eAkkermansia\u003c/em\u003e (82.32%) and \u003cem\u003eRikenella\u003c/em\u003e (9.05%), and that of the steppe zokor mainly contained species from 16 genera, including norank_f_norank_o_Izemoplasmatales (62.89%), Bacteroides_pectinophilus_group (9.10%) and other species in 16 genera. In studies of the human gut microbiome, bacteria of the genus \u003cem\u003eAkkermansia\u003c/em\u003e have been found to be involved in mucin degradation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and are a probiotic associated with a reduced risk of obesity-related metabolic syndrome [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eRikenella\u003c/em\u003e was found to be the top taxon significantly and positively associated with BMI in a study based on patterns of intestinal flora and body weight changes in wild house mice (\u003cem\u003eMus musculus\u003c/em\u003e) at different latitudes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. norank__f__norank__o_o_Izemoplasmatales are considered to be DNA degraders [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The abundance of the Bacteroides_pectinophilus_group in enterobacteria was found to be significantly negatively correlated with the prevalence of non-alcoholic fatty liver disease (NAFLD) in a multi-ethnic cohort obesity phenotype study [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In conclusion, there were differences in the composition of the intestinal bacterial community between the two species of zokors, and the dominant genera were mainly probiotic-associated groups, with no significant presence of microorganisms that posed a threat to the health of the hosts.\u003c/p\u003e \u003cp\u003eTwo kinds of zokor had different indicator genera of intestinal bacteria. Cellulosilyticum, Tuzzerella, NK4A214 and Monoglobus were the main markers in the intestinal tract of North China zokor, and Blautia was the main indicator genus in the intestinal bacterial community of steppe zokor. It has been shown that Cellulosilyticum is associated with fibre and protein breakdown [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In preventive medicine studies with naringin regulating the microbiota and metabolome in mice, alterations in Tuzzerella were found to be most associated with host endogenous metabolites [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. NK4A214 of the Ruminococcus family is one of the more abundant genera in the rabbit gut [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], capable of degrading plant polysaccharides to produce volatile fatty acids such as butyric acid [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which in turn promotes apoptosis in colon cancer cells and reduces intestinal inflammation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Monoglobus is considered to be a highly specialised group of pectin-degrading sugar biota in the human gut [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. NK4A136 and Blutia are beneficial bacteria that produce short-chain fatty acid [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Blutia is a new genus of Lachnospiraceae that produces short-chain fatty acid (SCFA) through glucose metabolism and digests dietary cellulose [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and it is widely found in mammalian faeces and intestines with potential probiotic properties [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The functions of the two zokor cecum bacterial community indicator species are involved in plant polysaccharide degradation, and production of short-chain fatty acids, which is in line with the findings of most studies on the function of the gut flora of herbivores [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of the food habits of the two species of zokors showed that \u003cem\u003eM. psilurus\u003c/em\u003e mainly fed on \u003cem\u003eS. officinalis\u003c/em\u003e (68%), while \u003cem\u003eM. aspalax\u003c/em\u003e preferred a combination of \u003cem\u003eA. tuberosum\u003c/em\u003e and \u003cem\u003ePhlomis sp.\u003c/em\u003e (52.1%). In relation to the specialised root-feeding habits of zokors and the root morphology of these three plant species, it is clear that zokors preferred larger food items such as axis root type, bulb type and tuberous root plants, and given that their foraging behaviour occurs without the aid of visual searching, this is consistent with what is predicted in the theory of optimal foraging in animals [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Environmental heterogeneity shapes the heterogeneity of the distribution of resources available to foragers, and the community structure of natural grassland vegetation clearly follows the same heterogeneity. Optimal foraging theory predicts that an animal's ability to utilise patches of resources is key to foraging success [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. As they live underground, zokors are inherently limited in their choice of food resources. Unlike the above-ground parts of plants, root systems are wrapped in a dense medium such as soil, and it is difficult for zokors to weigh the choice of such food resources from multiple perspectives (such as form and color), so a \"large amount and easy to obtain\" becomes the primary criterion for consideration. Therefore, in their foraging behaviour, zokors do not primarily target the dominant species or the established species of the corresponding grassland type to eat, but rather chose species with large root systems as their main food source.\u003c/p\u003e \u003cp\u003eIt is well known that herbivores rely on microorganisms living in their gastrointestinal tract to efficiently digest their fibre-rich diets [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The plant cell wall polysaccharides that make up the bulk of this fibre represent a potentially rich source of carbon [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] that is highly resistant to enzymatic breakdown [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], but certain microbial taxa have evolved mechanisms for degrading sugars from these structural polysaccharides in order to, through intestinal fermentation, gain more access to chemical energy from the diet [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Short-chain fatty acids (SCFA) are the end products of polysaccharide fermentation produced by gut flora [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Over a long evolutionary period, animals have optimised their digestive physiology by expanding the volume of either the foregut or hindgut [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. While the microbial communities involved in fibre digestion in foregut fermentation have been well described by studies on ruminant animals [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], studies targeting the catabolic utilisation of plant polysaccharides have also focused more on foregut fermentation [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] or, to a lesser extent, economically viable monogastrics [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Little is known about the microbial taxa that perform this function in wild herbivores with hindgut fermentation.\u003c/p\u003e \u003cp\u003ePlant cell walls are mainly composed of cellulose, hemicellulose, pectin, and lignin [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Cellulose is the most abundant of these plant cell wall polysaccharides [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], and its crystal structure makes it one of the most difficult substances to hydrolyse [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Therefore, microorganisms in the gut that can efficiently break down cellulose are critical for herbivores\u0026rsquo; digestion. We know that host carbohydrate-active enzymes (CAZymes) are mainly produced in the cecum [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In the rumen, cellulose degradation is facilitated in part by the efforts of bacterial communities [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e], including members of the genera \u003cem\u003eFibrobacter\u003c/em\u003e [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and \u003cem\u003eRuminococcus\u003c/em\u003e [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Due to the phytophagous nature of both zokors, we paid particular attention to pathways related to carbohydrate metabolism in their gut bacterial communities. Glycosyl hydrolases (EC 3.2.1-) are genes mainly involved in cellulose and hemicellulose degradation [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. We searched and screened the KO pathways in the OTU set consisting of the two zokor gut bacterial indicator genera based on the \"EC 3.2.1-\" search condition and found three pathways (K05349, K01179, and K15531) that were significantly different between the two zokor species. K05349 and K01179 are the most frequently mentioned pathways in the degradation of complex fibres [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. K05349 is associated with β-glucosidase metabolism, K01179 is associated with endoglucanase metabolism, and K15531 is associated with the metabolism of oligosaccharide reduced-end xylanase. β-glucosidase genes are present in almost all bacterial phyla [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. All organisms involved in cellulose degradation have a cellulase system consisting of a multi-enzyme complex of three enzymes: exoglucanase (also known as cellulose biohydrolase, EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and β-glucosidase (BGL, EC 3.2.1.21), which work synergistically for the complete hydrolysis of cellulose [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Therefore, the presence of K05349 and K01179 in the metabolic pathway of the two zokor species\u0026rsquo; gut bacteria is not unexpected. In a study of five desert rodent gut bacterial communities, predictions of the cellulose degradation function showed that K05349 and K01179 were the two most abundant genes in these rodents\u0026rsquo; gut bacterial communities [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The KEGG pathways predicted from the gut flora of those five different dietary species of desert rodents and this study\u0026rsquo;s two specialised subterranean rodents suggest that the cellulose degradation pathways of K05349 and K01179 act as 'generalists' in the rodent gut bacterial community. In addition to the two pathways mentioned above, the metabolic pathway associated with K15531 also differed between the two species of \u003cem\u003eMyospalax\u003c/em\u003e, but its abundance was much lower than that of the two pathways mentioned above. Oligosaccharide reduced-end xylanase (K15531; EC3.2.1.156) is a high molecular mass xylanase that degrades xylan, a dietary fibre in plant cell walls [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. Xylan is the most common hemicellulose component of grass, leaves, straw, and wood, the second most abundant renewable resource on earth [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e], a major component of hemicellulose [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], and a polysaccharide component of up to 45% of ruminant feed [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Xylan is an abundant non-cellulosic polysaccharide found in plant biomass [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e], and xylanases catalyse the β-bonding of xylan to release by-products that can be utilised by ruminants [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Of interest, this pathway has not been reported in intestinal bacterial studies in herbivorous monogastrics, but rather in a study based on obesity complications in US immigrants, which concluded that levels of oligosaccharide-reducing-end xylanase (K15531) increased with fibre intake, which was negatively correlated with the severity of obesity [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. These results imply that the process of cellulose degradation by the bacterial community in the gut of zokor includes not only pathways that are prevalent in mammals, but also unique degradation pathways. Among these cellulose degradation pathways, \u003cem\u003eM. psilurus\u003c/em\u003e possessed a stronger degradation capacity.\u003c/p\u003e \u003cp\u003eBased on the estimation of the neutral community model of the intestinal bacterial communities of the two species of zokors, it was found that the importance of stochastic processes in the gut bacteria of \u003cem\u003eM. psilurus\u003c/em\u003e exceeded that of the \u003cem\u003eM. aspalax\u003c/em\u003e, and that the mobility of the intestinal bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e was greater than that of \u003cem\u003eM. aspalax\u003c/em\u003e, i.e., the microbial community was spreading farther in the intestines of \u003cem\u003eM. psilurus\u003c/em\u003e than in those of \u003cem\u003eM. aspalax\u003c/em\u003e. Combined with the differences in other ecological characteristics (including the number of OTUs, α-diversity, and KEGG function prediction) of the gut bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e between the two as described above, we can conclude that the intestinal bacterial community of \u003cem\u003eM. psilurus\u003c/em\u003e has a wider ecological niche compared with that of \u003cem\u003eM. aspalax\u003c/em\u003e.\u003c/p\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eDietary diversity and gut microbial bacterial community diversity were significantly higher in \u003cem\u003eM. psilurus\u003c/em\u003e than in \u003cem\u003eM. aspalax\u003c/em\u003e, and the two zokor species possessed different gut bacterial indicator genera. \u003cem\u003eCellulosilyticum\u003c/em\u003e, \u003cem\u003eTuzzerella\u003c/em\u003e, NK4A214_group, and \u003cem\u003eMonoglobus\u003c/em\u003e were the main indicator genera in the gut tract of \u003cem\u003eM. psilurus\u003c/em\u003e, and \u003cem\u003eBlautia\u003c/em\u003e was the main indicator genera in the gut flora of \u003cem\u003eM. aspalax\u003c/em\u003e. There was a significant positive correlation between the gut bacterial community of \u003cem\u003eM. aspalax\u003c/em\u003e and the rhizome type plants, and there was a complementary relationship between the axis root type food and rhizome type plant dominated (including bulb type and tuberous root food) food groups. In the cellulose degradation pathway, \u003cem\u003eM. psilurus\u003c/em\u003e possessed a stronger degradation ability. The gut flora of \u003cem\u003eM. psilurus\u003c/em\u003e has a wider ecological niche than that of \u003cem\u003eM. aspalax\u003c/em\u003e. Gut microorganisms revealed the dietary choices of \u003cem\u003eM. psilurus\u003c/em\u003e and \u003cem\u003eM. aspalax\u003c/em\u003e.\u003c/p\u003e"},{"header":"5 Materials and methods","content":"\u003cp\u003eTo address the above questions, we conducted a preliminary investigation of the intestinal bacterial communities and dietary composition of two species of zokor, using the 16S amplicon technique in combination with the macro-barcoding technique.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample collection\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo species of zokors were captured using the circular tongs trap method from August-September 2021 (autumn) in the Hulunbeier meadow grassland (Chenbalhu Banner) and the typical grassland of Xilingol (Zhengxibai Banner) in Inner Mongolia, China. A total of 33 individuals were collected, including 18 \u003cem\u003eM. psilurus\u003c/em\u003e and 15 \u003cem\u003eM. aspalax\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Samples of cecum contents and stomach contents were collected and then immediately stored in liquid nitrogen. The samples were transported to the laboratory on dry ice and stored at -80℃. The above experimental protocol and procedures were approved by the Laboratory Animal Welfare and Ethics Committee of the Inner Mongolia Agricultural University (File No. NND2023081).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDNA extraction and sequencing of cecum and stomach contents\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMicrobiota from cecum contents and plant DNA from stomach contents were extracted using the TruSeq\u003csup\u003e\u0026trade;\u003c/sup\u003e DNA Sample Prep Kit according to the instructions. DNA quality was checked using a NanoDrop2000 ultra-trace spectrophotometer (Thermo Scientific, USA) and 1% agarose gel electrophoresis. The 16SrRNA V3-V4 region of the microbiota was amplified using universal primers (338F and 806R) [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e], and plant root communities were assessed by amplifying trnL operons(trnL-F: 5'-CGAAATYGGTAGACGCTACG-3' and trnL-R: 5'- CCDTYGAGTCTCTGCACCTATC-3') [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Each sample was replicated three times, and the PCR products from the same sample were mixed and detected by 2% agarose gel electrophoresis. The PCR products were recovered by cutting the gel using the Axy Prep DNA Gel Recovery Kit (AXYGEN Inc.), eluted with Tris_HCl, and detected by 2% agarose electrophoresis. Referring to the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified by QuantiFluor\u0026trade;-ST Blue Fluorescence Quantification System (Promega), after which the PCR products were mixed according to the corresponding proportion of each sample according to the sequencing amount required. After constructing the clone library using the TruSeq\u003csup\u003e\u0026trade;\u003c/sup\u003e DNA kit, high-throughput sequencing (250 bp, double-end sequencing) was performed using the Illumina Nova Seq platform.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatics analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe raw sequences were quality controlled using fastp software [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e] and FLASH (version1.2.7) software for splicing [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Bases with quality value below 20 in the tails of the reads were filtered, and a window of 50 bp was set. If the average quality value within the window was below 20, then the back-end bases were truncated starting from the window, reads below 50 bp after quality control were filtered, and reads containing N bases were removed. Based on the overlap relationship between PEreads, pairs of reads were spliced (merged) into one sequence with a minimum overlap length of 10bp; the maximum mismatch ratio allowed in the overlap region of the spliced sequences was 0.2, and the non-compliant sequences were screened. The samples were distinguished according to the barcode and primers at the first and last ends of the sequences, and the sequences were adjusted based on the direction. The number of mismatches allowed by the barcode was 0, and the maximum number of primers mismatched was 2. Using UPARSE software (version 7.1), OTU clustering was performed on non-repetitive sequences (excluding single sequences) according to 97% similarity, and chimeras were removed during the clustering process to obtain OTU representative sequences [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. All sample sequences were levelled by sequence pumping at the minimum number of sample sequences, and the average sequence coverage (Good's coverage) for each sample could still reach 99.09%. The RDPclassifier (version2.11) was used to compare to the Silva16SrRNA gene database (v138) for taxonomic annotation of intestinal microbial OTU species [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e], with a confidence threshold of 70%, and to count the community composition of each sample at several different taxonomic levels. 16S functional prediction analyses were performed using PICRUSt2 (version2.2.0) software [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. The ny_v20221012 species classification database and the RDP Classifier2.13 software were used to classify and annotate gastric capacitance DNA macro-barcode sequences. The Species 2000 Chinese Nodal Plant Group Database [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e] was used to search for plant species that might be present in the habitats of both species of zokors using the \"distribution area\u0026thinsp;+\u0026thinsp;classification system\" filters. The distribution range of plants that could potentially be consumed by the North China zokor was limited to \"Inner Mongolia Autonomous Region\" and \"Heilongjiang Province\". The distribution of plants that could potentially be eaten by the steppe zokor was limited to \"Inner Mongolia Autonomous Region\" and \"Hebei Province\", and the search condition was \"distribution in the above areas\". The plant classification order was specified from Plantae to Magnoliopsida for screening and downloading information on plant species found in the two study habitats. The results of the downloads were cross-referenced with the macro-barcode annotations to match species with the same name and exclude species that are not reported in the habitat.\u003c/p\u003e \u003cp\u003eSeveral alpha diversity indices (ACE, Shannon, and Pd indices) were calculated using mothur software [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e], and the Wilcoxon rank-sum test was used for analyses of between-group differences in alpha diversity. Using the data table in the tax_summary_a folder, R statistical software (version3.3.1) was used to produce microbial community bar charts and plant community pie charts. The Wilcoxon rank-sum test was performed using R statistical software and the scipy package of python to analyse differences between plant species in the samples of the two zokor species\u0026rsquo; stomach contents. The p-values were corrected using the Fdr method, and the confidence interval was calculated using the Welch\u0026rsquo;s t-test with a confidence level of 0.95. All plants were classified into 8 types by plant root type, and the gut bacterial communities of both zokor species were analysed by food root type with a Mantel analysis using the vegan package (vsesion 2.4.3). IndVal was calculated using the lndval function of the labdsv package [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e] to find the indicator genera of the gut bacterial communities of the two zokors, and OTU clustering information was compared with the sequenced microbial genome databases using the PICRUSt2 software to obtain the corresponding species in the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the functional type and abundance of the corresponding species. Differences in KEGG pathways between the two zokor species were analysed using Statistical Analysis of Macrogenome Mapping (STAMP) v2.1.3, and the false discovery rate was controlled using the Benjamini-Hochberg procedure [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was financially supported by the National Natural Science Foundation of China, China (Grant numbers 32060256, 32060395), the Major Science and Technology Project of Inner Mongolia Autonomous Region (Grant number 2021ZD0006) and Basic scientific research business expenses of universities directly under Inner Mongolia Autonomous Region (Grant numbers BR221307, BR221037).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZhenghaoni Shang designed the experiment and wrote the manuscript. Kai Chen and Tingting Han analyzed the data. Fan Bu, Shanshan Sun, Na Zhu and Duhu Man collected samples. Shuai Yuan and Heping Fu revised the manuscript. All the authors participated in the review and editing and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are grateful for all those who helped with the field sampling.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets presented in this study can be found in online repositories. The names of the repository and accession number(s) can be found below: Sequence Read Archive (NCBI, USA), PRJNA1089538.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVan Best N, Rolle-Kampczyk U, Schaap FG, Basic M, Olde Damink SWM, Bleich A et al. Bile acids drive the newborn\u0026rsquo;s gut microbiota maturation. Nat Commun. 2020;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLey RE, Peterson DA, Gordon JI. 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Nutrients. 2019;11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Myospalax, Gut microbiota, Diet, Cellulose degradation, Microbial nich","lastPublishedDoi":"10.21203/rs.3.rs-4293070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4293070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs the most abundant group of mammals, rodents possess a very rich ecotype, which makes them ideal for studying the relationship between diet and host gut microecology. Zokors are specialized herbivorous rodents adapted to living underground. Unlike more generalized herbivorous rodents, they feed on the underground parts of grassland plants. There are two species of the genus \u003cem\u003eMyospalax\u003c/em\u003e in the Eurasian steppes in China: one is \u003cem\u003eMyospalax psilurus\u003c/em\u003e, which inhabits meadow grasslands and forest edge areas, and the other is \u003cem\u003eM. aspalax\u003c/em\u003e, which inhabits typical grassland areas. How are the dietary choices of the two species adapted to long-term subterranean life, and what is the relationship of this diet with gut microbes? Are there unique indicator genera for their gut microbial communities? Relevant factors such as the ability of both species to degrade cellulose are not yet clear. In this study, we analysed the gut bacterial communities and diet composition of two species of zokors using 16S amplicon technology combined with macro-barcoding technology. We found that the diversity of gut microbial bacterial communities in \u003cem\u003eM. psilurus\u003c/em\u003e was significantly higher than that in \u003cem\u003eM. aspalax\u003c/em\u003e and that the two species of zokors possessed different gut bacterial indicator genera. Based on the results of Mantel analyses, the gut bacterial community of \u003cem\u003eM. aspalax\u003c/em\u003e showed a significant positive correlation with the creeping-rooted type food, and there was a complementary relationship between the axis root type food and the rhizome type food dominated (containing bulb types and tuberous root types) food groups. Functional prediction based on KEGG found that \u003cem\u003eM. psilurus\u003c/em\u003e possessed a stronger degradation ability in the same cellulose degradation pathway. Neutral modelling results showed that the gut flora of the \u003cem\u003eM. psilurus\u003c/em\u003e has a wider ecological niche compared to that of the \u003cem\u003eM. aspalax\u003c/em\u003e. This provides a new perspective for understanding how rodents living underground in grassland areas respond to changes in food conditions.\u003c/p\u003e","manuscriptTitle":"Natural foraging selection and gut microecology of two subterranean rodents from the Eurasian Steppe in China","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-29 16:58:54","doi":"10.21203/rs.3.rs-4293070/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b5539f50-5a01-405b-99f0-4fd1f4670fb5","owner":[],"postedDate":"April 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-06T20:11:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-29 16:58:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4293070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4293070","identity":"rs-4293070","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}