The community structure of Methanomassiliicoccales in the rumen of Chinese goats and its response to a high-grain diet

Abstract

Background: The newly proposed methanogenic order 'Methanomassiliicoccales' is the second largest archaeal population in the rumen, second only to the Methanobrevibacter population. However, information is limited regarding the community of this new order in the rumen.

Methods: This study used real-time PCR and 454 pyrosequencing to explore the abundance and community composition of Methanomassiliicoccales in the rumen of Chinese goats fed a hay (0% grain, n = 5) or a high grain (65% grain, n = 5) diet.

Results: Real-time PCR analysis showed that the relative abundance of Methanomassiliicoccales (% of total archaea) in the goat rumen was significantly lower in the high-grain-diet group (0.5% ± 0.2%) than that in the hay-diet group (8.2% ± 1.1%, P < 0.05). The pyrosequencing results showed that a total of 208 operational taxonomic units (OTUs) were formed from ten samples at 99% sequence identity. All the sequences were identified as Methanomassiliicoccaceae at the family level, and most of the sequences (96.82% ± 1.64%) were further classified as Group 8, 9, and 10 at the Methanomassiliicoccales genus level in each sample based on the RIM-DB database. No significant differences were observed in the number of OTUs or Chao1's, Shannon's or Pielou's evenness indexes between the hay- and high-grain-diet groups (P ≥ 0.05). PCoA analysis showed that diet altered the community of Methanomassiliicoccales. At the genus level, the relative abundances of Group 10 (67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4 (2.07 ± 1.30 vs. 0.27 ± 0.30, P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group 9 was significantly higher in the hay-diet group (18.82 ± 6.20 vs. 47.14 ± 17.72, P = 0.020). At the species level, the relative abundance of Group 10 sp. (67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4 sp. MpT1 (2.07 ± 1.30 vs. 0.27 ± 0.30, P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group 9 sp. ISO4-G1 was significantly higher in the hay-diet group (12.83 ± 3.87 vs. 42.44 ± 18.47, P = 0.022).

Conclusions: Only a few highly abundant phylogenetic groups dominated within the Methanomassiliicoccales community in the rumens of Chinese goats, and these were easily depressed by high-grain-diet feeding. The relatively low abundance suggests a small contribution on the part of Methanomassiliicoccales to the rumen methanogenesis of Chinese goats.

Keywords: Community structure; Diet; Goat; Methanomassiliicoccales; Pyrosequencing.

Fig. 1

The relative abundance of Methanomassiliicoccales at the genus level based on the RIM-DB. N1 ~ 10, Name of each animal; Mmceae, Methanomassiliicoccaceae

Fig. 2

The relative abundance of Methanomassiliicoccales at the species level based on the RIM-DB. N1 ~ 10, Name of each animal; Mmceae, Methanomassiliicoccaceae

Fig. 3

Hierarchical clustering of samples based on the relative abundance of each OTU in each sample. Only OTUs with a relative abundance of at least 5% in one of the ten samples were included. The names of animals are shown below the heat map (N1 ~ 10). Heat map colors represent the relative abundance of OTUs (%)

Fig. 4

Effects of diet on Methanomassiliicoccales community structure. A principal coordinate analysis (PcoA) plot of unweighted unifrac dissimilarities is shown

Fig. 5

Phylogeny of Methanomassiliicoccales sequences and highly abundant OTUs. Representative sequences of highly abundant OTUs (bold, ≥5% of the relative abundance in one of the ten samples) were aligned by MEGA 7. The GenBank accession numbers of these highly abundant OTUs are KX525649–61. Sixty-three sequences from Groups 8, 9, and 10 and isolates and enrichment cultures were selected in the RIM-DB. Phylogenetic analysis was performed on the 762 bp-to-1099 bp region of the 16S rRNA genes via the Maximum Likelihood method based on the Kimura 2-parameter model. The tree was resampled 500 times, and only bootstrap values of ≥70% are shown. The dendrogram was rooted with four Methanopyrus sequences. The scale bar indicates 0.02 inferred nucleotide substitutions per position. Mmc., Methanomassiliicoccus; Mmp., Methanomethylophilus