Biodiversity and Composition of Methanogenic Populations in The

RESEARCH ARTICLE Biodiversity and composition of methanogenic populations in the rumen of cows fed alfalfa hay or triticale straw Yunhong Kong1,2, Yun Xia2, Robert Seviour3, Robert Forster2 & Tim A. McAllister2

1Department of Biological Science and Technology, Kunming University, Kunming, China; 2Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, AB, Canada; and 3Biotechnology Research Centre, La Trobe University, Bendigo, Vic., Australia Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021

Correspondence: Tim A. McAllister, Abstract Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, It is clear that methanogens are responsible for ruminal methane emissions, Lethbridge, AB, Canada. but quantitative information about the composition of the methanogenic com- Tel.: +1 403 3172240; munity in the bovine rumen is still limited. The diversity and composition of fax: +1 403 3823156; rumen methanogens in cows fed either alfalfa hay or triticale straw were exam- e-mail: [email protected] ined using a full-cycle rRNA approach. Quantitative fluorescence in situ hybridization undertaken applying oligonucleotide probes designed here identi- Received 4 October 2012; revised 6 December 2012; accepted 12 December fied five major methanogenic populations or groups in these animals: the Met- 2012. hanobrevibacter TMS group (consisting of Methanobrevibacter thaueri, Final version published online 8 January Methanobrevibacter millerae and Methanobrevibacter smithii), Methanbrevibacter 2013. ruminantium-, Methanosphaera stadtmanae-, Methanomicrobium mobile-, and Methanimicrococcus-related methanogens. The TMS- and M. ruminantium- DOI: 10.1111/1574-6941.12062 related methanogens accounted for on average 46% and 41% of the total methanogenic cells in liquid (Liq) and solid (Sol) phases of the rumen contents, Editor: Julian Marchesi respectively. Other prominent methanogens in the Liq and Sol phases included members of M. stadtmanae (15% and 33%), M. mobile (17% and 12%), and Keywords full-cycle rRNA approach; FISH; Methanimicrococcus (23% and 9%). The relative abundances of these methano- methanogens; rumen ecology. gens in the community varied among individual animals and across diets. No clear differences in community composition could be observed with dietary change using cloning techniques. This study extends the known biodiversity levels of the methanogenic communities in the rumen of cows.

host (Ellis et al., 2007). Methanogenesis leads to a loss of Introduction dietary energy and contributes to climate change, which Archaea constitute about 3–4% of the total microbial together emphasize that a better understanding of the numbers in the rumen with the majority of this domain microbiology of methanogenesis is essential for green- being methanogens (Lin et al., 1997; Sharp et al., 1998; house gas mitigation and improvement of production Yanagita et al., 2000; Ziemer et al., 2000). Most ruminal efﬁciency in the livestock industry (Janssen & Kirs, 2008).

methanogens utilize H2 or formate as substrates and Eight methanogenic species have been isolated, cul- derive energy from the reduction of CO2 to CH4. In the tured, and identiﬁed from the bovine rumen. They are rumen, polymeric carbohydrates and proteins are Methanobrevibacter smithii, Methanbrevibacter ruminanti- þ degraded to volatile fatty acids, NH4 ,CO2, and H2. Dis- um, Methanobrevibacter thaueri, Methanosphaera stadtm- posal of H2 through the reduction of CO2 to CH4 is anae, Methanobacterium aarhusense, Methanobacterium essential to prevent the accumulation of reducing equiva- formicicum, Methanosarcina barkeri, and Methanomicrobi-

MICROBIOLOGY ECOLOGY MICROBIOLOGY lents and the overall impedance of ruminal fermentation. um mobile (Jarvis et al., 2000; Whitford et al., 2001; Methane is a potent greenhouse gas with a global warm- Nicholson et al., 2007; Wright et al., 2007). However,

ing potential that is 25 times that of CO2, and emissions many of the methanogens identiﬁed through sequencing can account for 2–12% of the gross energy intake of the techniques have not been cultured in the laboratory, but

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 302–315 Published by Blackwell Publishing Ltd. All rights reserved Composition of ruminal methanogenic communities 303 two genera, Methanobrevibacter and Methanomicrobium, samples were collected in the last week of this second and a clade that is distantly related to the genus Ther- period. Before the cattle were fed, rumen digesta was col- moplasma [also referred as the RCC cluster (Janssen & lected from three sites (reticulum, dorsal, and ventral sac) Kirs, 2008)] appear to account for the majority of rumen within the rumen, thoroughly mixed and a subsample methanogens (Wright et al., 2007; Hook et al., 2009; King was placed in 200-mL air-tight centrifuge tubes and et al., 2011; Tymensen et al., 2012). immediately transported to the laboratory. Before DNA Most studies examining changes in the composition of extraction, rumen samples from each animal were ruminal methanogenic communities have used 16S rRNA fractioned into liquid, feed particle loosely associated and gene clone library analyses. However, such data cannot be feed particle adherent fractions as described by assessed quantitatively (Head et al., 1998) and only a few Kong et al. (2010a). studies have attempted to quantify individual rumen methanogenic community members. Using reverse trans- Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021 FISH sample fixation and FISH probing criptase qPCR, Zhou et al. (2009) reported that the size of ruminal methanogenic community was not affected in Samples for FISH were prepared as described by Kong cattle in response to a low or high energy diet. With et al. (2010b). Briefly, digesta samples were collected from qPCR, Tymensen et al., (2012) showed that switching the the rumen as described above. On-site, the mixture diet from forage to high-grain resulted in a profound (100 mL) was then transferred to a heavy-wall 250-mL reduction in total RCC abundance and an increase in the beaker and squeezed using a Bodum (Bodum Inc., Trien- relative abundance of free-living Methanobrevibacter spp. gen, Switzerland) coffee maker plunger. The extruded However, all such studies have been carried out at the liquid samples were fixed in paraformaldehyde (PFA, 4% whole community or genus level, and the composition of final concentration) for FISH probing of planktonic the methanogenic community at the species level is less methanogens (i.e. those in the Liq phase). The remaining clear. liquid was discarded, and the squeezed particulate sam- In this study, the full-cycle rRNA approach (Amann ples containing particle-associated cells were also fixed in et al., 1995) was used to investigate the composition of 4% PFA. After 3 h fixation, these particulate samples methanogenic populations in the liquid (Liq) and solid together with 4% PFA solutions were ‘stomached’ (Stom- fraction (Sol) of the rumen digesta of cows fed a high- acher 400 Circulator, Seaward, UK) at 230 r.p.m. for quality forage in the form of alfalfa hay or a low-quality 6 min. Stomached samples were then transferred into forage in the form of triticale straw. 16S rRNA gene clone clean 250-mL beakers and squeezed again. Filtrates were libraries were constructed using pooled DNA templates centrifuged (5000 g) to recover cells, which were then from rumen DNA of individual animals fed the same or washed three times with 19 PBS buffer, resuspended in different diet(s). Oligonucleotide 16S rRNA gene-targeted 50% ethanol (ca. 8–10 g L 1 dry weight) and stored probes were designed against sequences retrieved from at 20 °C until FISH probing of methanogens associated clone libraries. Quantitative fluorescence in situ hybridiza- with particulate digesta was executed (i.e. methanogens in tion (qFISH) was used to estimate the relative abun- the Sol phase). dances of individual members of the methanogenic FISH was carried out according to Amann (1995) on populations to investigate changes among individual ani- glass cover slips coated with gelatin (Kong et al., 2010a) mals and diets. according the procedure described by Xia et al. (2012). Compositions of the methanogen communities in the Liq and Sol were screened with previously described oligonu- Materials and methods cleotide probes targeting methanogens at the family or order level. Those used were MB1174 (Raskin et al., Sample collection and fractionation 1994) for members of the Methanobacteriales, MS1414 Sample collection and fractionation of the rumen digesta (Raskin et al., 1994) for Methanosarcinaceae, MX825 were carried out as described previously (Kong et al., (Raskin et al., 1994) for Methanosaetaceae and MG1200b 2010a). Briefly, four mature rumen-cannulated dry Hol- (Crocetti et al., 2006) for Methanomicrobiales. The probe stein cows were used in this crossover study. In the first ARC915 (Stahl & Amann, 1991) was used to estimate the 1-month period, two cows were fed alfalfa hay plus 2 kg total numbers of Archaea in each sample. Hybridization of a dry cow protein/mineral supplement, while the other conditions and optimal formamide concentrations for two were fed triticale straw plus 2 kg of supplement. these probes are listed in probeBase (Loy et al., 2003). Rumen samples were obtained in the last week of this 4′,6-diamidino-2-phenylindole (DAPI) staining of biomass period and then the diets were switched at the start of samples to determine total cell numbers was carried out the second 1-month period. The second set of rumen after FISH probing. DAPI (100 lL, 0.003 mg mL 1) was

FEMS Microbiol Ecol 84 (2013) 302–315 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 304 Y. Kong et al. added to air-dried FISH glass cover slips, which were then (http://evolution.genetics.washington.edu/phylip/phylip- kept at room temperature for 10 min before being gently web.html), and employing the model Kumura 2-Parame- washed by dipping each slide in distilled water 3–5 times. ter. Phylogenetic identification of OTUs was performed Slides were air-dried and examined microscopically. using the BLAST search engine against the NCBI nucleotide sequence database (Altschul et al., 1997). DNA extraction, PCR amplification, clone library construction, and sequencing Design and specification of oligonucleotide probes Total DNA was extracted from each of the three isolated fractions from rumen digesta for each individual animal All 16S rRNA gene-targeted probes were designed using and pooled in equal molar amounts for PCR amplifica- ARB software against a database consisting of 16S rRNA tion as described previously (Kong et al., 2010b). Clone gene sequences retrieved from the Silva database (Pruesse Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021 libraries were then constructed for each individual animal et al., 2007) and from the clone libraries constructed in for each of the two diets fed. The sequence of FISH probe this study (Figs 1–3). The names, specificities, and form- MB1174 (Raskin et al., 1994; Crocetti et al., 2006) was amide concentrations of the newly designed probes are used to make a reverse PCR primer (renamed as listed in Fig. 4 together with the clones used to validate Met1174r: 5′-CAG GCC TAA CAC ATG CAA GTC-3′), each of these probes. Their specificities were assessed and the universal methanogenic primer Met86f (5′-GCT using Clone-FISH, carried out according to Schramm CAG TAA CAC GTG G-3′) (Wright & Pimm, 2003) was et al. (2002). The plasmid and host cell pair used was used as a forward primer to amplify 16S rRNA genes of pGEM-T and JM103 (DE3), respectively (Promega, Fisher the methanogenic community using Amplitaq polymerase Scientific, Toronto, ON, Canada). Briefly, clones with Gold (Applied Biosystems, Carlsbad, CA). zero or one mismatch were inserted into the plasmid The PCR cycle used consisted of a 5 min denaturation pGEMT equipped with a T7 RNA polymerase, and cloned period at 95 °C followed by 25 cycles of 95 °C 1 min, into JM109 (DE3) competent cells. These cells were incu- 55 °C 1 min and then 72 °C 1.5 min. After 25 cycles, an bated at 37 °C with isopropylthio-b-galactoside (IPTG) 8 min extension period at 72 °C was added. Clone (1 mM) and chloramphenicol (170 mg L 1) before being libraries were built using a TOPO TA Cloning Kit (Invi- fixed in PFA (4%) for FISH probing. The highest form- trogen Canada Inc., Burlington, ON, Canada) with elec- amide concentration (tested in 5% step-wise increases) at trocompetent cells following the protocol provided by the which a clear fluorescent signal was observed after FISH manufacturer. Individual clones were picked with a Qpix probing with competent cells with zero mismatches, but II (Genetix USA Inc., MA) into 96-well plates (Genetix). not with competent cells with one mismatch was selected Sequencing of these was carried out by Polymorphic as the optimal PFA concentration. (Polymorphic DNA Technologies, Inc., CA) using an ABI3730XL Genetic Analyzer with a 50 cm capillary Microscopy and enumeration of methanogens array. hybridized in FISH FISH and DAPI images were captured with a Leica epi- Phylogenetic and biodiversity analyses fluorescence microscope (Leica DM6000B) equipped with Partial 16S rRNA gene sequences were assembled using a Leica DFC500 camera and visualized and captured SEQUENCHER 4.5 (Gene Code Cooperation) and screened using the Cy3 and DAPI filters, respectively, after samples using the Chimera check program provided in RDP-II had been mounted in CITIfluo (Citifluor Ltd, London, (Cole et al., 2005) and Bellerophon (Huber et al., 2004). UK). The percentages of cells hybridizing with each For phylogenetic analysis, 16S rRNA gene sequences were probe were calculated as percentages of the total number pooled and submitted to the CLASSIFIER tool provided in of cells hybridizing with the probe ARC915 (designed for RDP-II. Trees in Figs 1–3 were built with ARB (Ludwig total Archaea) or stained with DAPI (detecting all micro- et al., 2004) using the neighbor-joining technique. Boot- bial cells) in the same microscopic field. All enumera- strap values were calculated with MEGA 4 with the model tions were carried out on digital images using IMAGEJ Kumura 2-Parameter (Tamura et al., 2007). Biodiversity (Abramoff et al., 2004) following the procedures index calculations including OTU recognitions, Shannon described by Kong et al. (2010a). At least 60 sets (FISH index values, and Chao 1 and Libshuff estimations were image with Cy3 labeled probe and DAPI image) of carried out using MOTHUR (version 1.10.2 http://www.mo- images taken with the 1009 objective lens from three thur.org) following the procedures provided. The distance different glass cover slips (20 from each) were used to files used in all calculations were prepared with PHYLIP 4.0 estimate percentages of targeted Archaea in the Liq or

Fig. 1. Distance tree (neighbor-joining) built using Archaeal 16S rRNA gene sequences obtained in this study (names in bold) and their close relatives. The oligonucleotide probes designed to match target sites in these clones are also shown. The bootstrap values (only those > 50% are shown) were calculated based on 1000 re-samplings. Scale bar represents 1 substitution per 10 nucleotides.

(a) (b) Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021

Fig. 2. (a, b) Distance tree (neighbor-joining) subtrees of Fig. 1 built using Archaeal 16S rRNA gene sequences obtained in this study (names in bold) and their close relatives. The oligonucleotide probes designed to match target sites in these clones are also shown. The bootstrap values (only those > 50% are shown) were calculated based on 1000 re-samplings. The scale bars represent 1 substitution per 20 nucleotides.

Sol samples from each cow fed each speciﬁed diet. The of the 20 sets of images derived from one glass cover. mean values and standard deviation (Fig. 6) were calcu- Between 2000 and 4000 DAPI, positive cells were visual- lated based on the three mean values obtained from each ized for each image.

(a) (b) Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021

Fig. 3. (a, b) Distance tree (neighbor-joining) subtrees of Fig. 1 built using Archaeal 16S rRNA gene sequences obtained in this study (names in bold) and their close relatives. The oligonucleotide probes designed to match target sites in these clones are also shown. The bootstrap values (only those > 50% are shown) were calculated based on 1000 re-samplings. The scale bars represent 1 substitution per 20 nucleotides.

FEMS Microbiol Ecol 84 (2013) 302–315 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 308 Y. Kong et al. Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021

Fig. 4. Oligonucleotide probes, sequences, target sites, speciﬁcities, and formamide concentrations used in FISH. 16S rRNA sequences (at the probe target sites) of Archaeal clones used to determine the optimal formamide concentrations of the FISH probes are also shown. *Clone ADP63 was obtained from a diary manure digester by Xia et al. (2012).

Nucleotide sequence accession number Identification of methanogenic populations The 16S rRNA gene sequences obtained in this study have The 361 methanogenic clones were classified using CLAS- been deposited in the GenBank database under accession SIFIER provided in RDP-II. They were all members of numbers JQ179497–JQ179858. the phylum Euryarchaeota, belonging to genera Methano- brevibacter (314 clones) and Methanosphaera (19 clones) in the Methanobacteriaceae, Methanimicrococcus (one Results clone) in the Methanosarciaceae and Methanomicrobium (seven clones) in the Methanomicrobiaceae, and unclassi- 16S rRNA gene clone library analyses fied Methanobacteriaceae (two clones) and unclassified To examine the composition of methanogen communi- Euryarchaeota (18 clones). Mothur analysis revealed that ties in these rumen samples, a set of already available these clones comprised 23 OTUs at a distance of 0.03 oligonucleotide FISH probes targeting methanogens at (Table 1). Of these, 15 OTUs contained representatives of the family or order level (as described earlier) was first previously cultivated species: Methanobrevibacter strain applied. The MB1174 probe designed for Methanobacteri- NT7 (represented by OTUs 1, 13, and 16), M. stadtmanae ales hybridized to cells in all samples examined, and (represented by OTU 2), M. ruminantium (represented more than half (50–60%) of the Archaea hybridized with by OTU 3), M. thaueri (represented by OTU 5), Methan- the archaeal probe ARC915 (Fig. 5a). The MS1414, obrevibacter millerae (represented by OTUs 6 and 11), MX825, and MG1200b probes designed for members of Methanobrevibacter gottschalkii (represented by OTU 7), the Methanosarcinaceae, Methanosaetaceae, and most Methanobrevibacter strain Z8 (represented by OTUs 9 and Methanomicrobiales, respectively, each hybridized with a 14), Methanobrevibacter strain SM9 (represented by OTU small fraction (< 5%) of the cells that hybridized with 12), M. mobile (represented by OTU 15), and Methano- the ARC915 probe (data not shown). Therefore, the brevibacter strain Y1 (represented by OTU 17). Others sequence of probe MB1174 was chosen as a reverse pri- consisted largely of unclassifiable 16S rRNA gene mer in combination with the universal forward primer sequences of Euryarchaeota (represented by OTUs 4, 8, Met86f to amplify the 16S rRNA genes of the methano- 10, and 20), Methanobrevibacter (OTUs 18 and 19), and a gens using PCR. In total, eight clone libraries were con- few Methanosphaera (OTU 21) and Methanimicrococcus structed from the pooled templates from three fractions (OTU 23). (liquid, feed particle loosely associated and feed particle adherent fractions) for each of the individual animals Community biodiversity analysis fed the same or different diet(s). A total of 361 methanogenic clones (ca. 1100 bp) were successfully retrieved The differences in the composition of the ruminal metha- after excluding any bacterial clones (25 clones) and nogenic communities between any two cows fed the same archaeal clones closely related to Thermoplasmatales (75 diet or different diets were estimated statistically using clones). S-Libshuff in Mothur (Table 2). No clear pattern was

(a) (b)

Fig. 5. Images of digesta samples from the (e) (f) rumen of cows fed alfalfa hay or triticale straw after color combination. Images from probes are labeled in red, and those from DAPI staining are in green. The yellow (combination of red and green) colored cells in (a–f) hybridized with probes ARC915, Mbr830, Mbr626, Msp541, Mmi1123, and Mmi1042, respectively. Bar, 10 lm. (b), (d–f) Share the same scale bar as (a). observed with regard to the effects of the individual ani- cocci of variable sizes (Fig. 5d), probe Mmi1123 with mals or diet on the composition of methanogenic com- large cocci (Fig. 5e), and probe Mmi1042 with small munities. At a distance of 0.03, between 7 and 13 cocci or coccobacilli of variable sizes (Fig. 5f). The rela- methanogenic OTUs could be detected in a single animal. tive abundances of these probe-defined methanogenic The species richness values of methanogenic populations populations were determined as shown in Fig. 6. in different animals using a Chao 1 estimation were The sums of the percentages of the methanogenic pop- between 7 and 18, and the Shannon index values calcu- ulations hybridizing with these probes varied among indi- lated for the methanogenic populations in different vidual animals and ranged between 1.9% and 3.7% of the animals fell between 1.23 and 2.18. total cell numbers (cells stained with DAPI) determined in the Liq and 3.1–4.8% in the Sol phases. These values are within the ranges detected with probe ARC915 in the qFISH of methanogenic populations in the same samples (i.e. 2–5% of the total cell numbers). The rumen of cows fed alfalfa hay or triticale straw average percentages of these probe-defined methanogenic Five FISH probes were designed against the methanogenic populations in the Liq and Sol phase of the rumen of cat- 16S rRNA gene sequences obtained in this study (Fig. 1) tle fed with alfalfa hay were 2.5 (0.6)% and 4.4 and their close relatives. The probe Mbr830 was designed (0.6)% respectively, which were similar to those [2.7 to target M. thaueri, M. millerae, M. smithii, and their (1.0)% and 3.8 (0.5)%, respectively] detected in the related clones, which are referred to here as the ‘thaueri- rumen of cattle fed triticale straw. millerae-smithii’ (TMS) group. The probes Mbr626, FISH probing was used to investigate the relative abun- Msp541, Mmi1123, and Mmi1042 were designed to target dances of these probe-defined methanogenic populations. M. ruminantium, M. stadtmanae-related, M. mobile, and Their percentages varied widely in the Liq or Sol phases Methanimicrococcus-related clones respectively. In all the in the rumen of the same cattle fed different diets, among samples examined, the probe Mbr830 hybridized with individual cattle fed the same diet and across individual large cocci (Fig. 5b), probe Mbr626 hybridized with small cattle fed different diets. Except cow #2 fed alfalfa hay, cocci or coccobacilli (Fig. 5c), probe Msp541 with large members of the TMS group were detected in the Liq and

Table 1. Comparison of ruminal methanogenic OTUs among cows five FISH probes)]. Methanbrevibacter ruminantium- fed alfalfa hay or triticale straw related methanogens were detected in the Liq and Sol No. of phases of all cows except those from cow #2 fed triticale sequences straw and the Liq of cow #4 fed triticale straw, Percentage of – – OTU AL-fed TR-fed Nearest taxon sequence identity and accounted for 16 53% and 6 53% of the total methanogens in the Liq and Sol phases, respectively, of 17 7Methanobrevibacter 97 other individual cows. Methanosphaera stadtmanae-related strain NT7 methanogens were detected in all samples except those in 2145 Methanosphaera 97 – stadtmanae the Liq phase of cows #1 3 fed triticale straw and 34163 Methanobrevibacter 99 accounted for 16–34% and 12–50% of total methanogens ruminantium in the Liq and Sol phases, respectively. Methanomicrobium 45 0Methanomassiliicoccus 89 mobile-related methanogens were detected in the Liq and Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021 luminyensis Sol phases of all cows except in cow 1 fed triticale straw 51625 Methanobrevibacter 98 and accounted for 6–33% and 6–41% of total methano- thaueri ZA10 gens in the Liq and Sol phases, respectively. Methanimi- 62024 Methanobrevibacter 98 millerae crococcus-related methanogens were detected in all 72430 Methanobrevibacter 98 samples except in the Sol phase of cows #2 and #3 fed gottschalkii triticale straw and accounted for 7–42% and 3–21% of 84 1Methanomassiliicoccus 89 the total methanogens in the Liq and Sol phase, respec- luminyensis tively. 93 1Methanobrevibacter 98 All the probe-defined methanogenic populations strain Z8 detected in the ruminal Liq phases were also detected in 10 2 1 Methanomassiliicoccus 88 luminyensis the Sol phases of the same animals except in cows #2 and 11 7 11 Methanobrevibacter 98 #3 where the M. stadtmanae-related methanogens were millerae ZA10 only detected in the Sol phases, and in cow #4 fed triti- 12 4 7 Methanobrevibacter 99 cale straw where M. ruminantium-related methanogens strain SM9 were only detected in the Sol phase. When the percent- 13 3 10 Methanobrevibacter 99 ages of the individual probe-defined methanogenic popu- strain NT7 lations in the Liq and Sol phases in the rumen of cows 14 1 2 Methanobrevibacter 98 strain Z8 fed alfalfa hay were compared with those in the Liq and 15 4 3 Methanomicrobium 98 Sol phases in the rumen of same animals fed triticale mobile straw, they were highly variable, and no clear trend was 16 2 1 Methanobrevibacter 97 observed with regard to the type of forage fed. The same strain NT7 results were found when the percentages of the individual 17 3 0 Methanobrevibacter 97 probe-defined methanogenic populations in the Liq and strain Y1 Sol phases in the rumen of cows fed alfalfa hay were 18 2 0 Methanobrevibacter 96 strain Y1 compared to those in the rumen of different cows fed 19 1 0 Methanobrevibacter 93 triticale straw. strain Y1 20 2 0 Methanomassiliicoccus 88 luminyensis Discussion 21 1 2 Methanosphaera 94 In this study, most of the methanogens in the rumen of stadtmanae the cows fed with two different diets were identified using 22 1 0 Methanobrevibacter 98 strain Y1 a full-cycle rRNA approach. They belonged to at least five 23 0 1 Methanimicrococcus 93 methanogenic genera or populations. Their relative abun- blotticola dances in the community varied among individual ani- Total 167 194 mals and across diets. The composition of these AL, alfalfa hay; TR, triticale straw. methanogenic communities is more diverse than known. This study describes the first published systematic high-resolution FISH study on the composition of the Sol phases of all other cows with a large range [18–58% ruminal methanogenic communities in cows fed with and 14–58%, respectively, of the total methanogens (total different diets. Most (up to 80%) of the Archaea in the sum of percentages of the methanogens detected using rumen of cows fed alfalfa hay or triticale straw could be

Table 2. Biodiversity index, distribution, and similarity of methanogenic populations in rumen digesta of animals fed either alfalfa hay or triticale straw

OTUs shared (%)†

Libraries* Number of sequences Number of sequences Species richness XY YX Shannon index P value‡ (XY, YX)

C1AL vs. C1TR 31 vs. 51 7 vs. 10 10 vs. 10 6/7 6/10 1.51 vs. 2.00 < 0.0001, 0.2976 C1AL vs. C2AL 31 vs. 49 7 vs. 13 10 vs. 18 7/7 7/13 1.51 vs. 2.18 0.6061, 0.0264 C1AL vs. C2TR 31 vs. 51 7 vs. 7 10 vs. 7 5/7 5/7 1.51 vs. 1.53 < 0.0001, 0.0497 C1AL vs. C3AL 31 vs. 43 7 vs. 8 10 vs. 13 5/7 5/8 1.51 vs. 1.23 0.7238, 0.0043 C1AL vs. C3TR 31 vs. 52 7 vs. 7 10 vs. 7 5/7 5/7 1.51 vs. 1.23 < 0.0267, 0.0263 C1AL vs. C4AL 31 vs. 44 7 vs. 7 10 vs. 7 4/7 4/7 1.51 vs. 1.40 < 0.0001, 0.0243 C1AL vs. C4TR 31 vs. 40 7 vs. 8 10 vs. 11 3/7 7/8 1.51 vs. 1.52 0.486, 0.0038 Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021 C1TR vs. C2AL 51 vs. 49 10 vs. 13 10 vs. 18 8/10 8/18 2.00 vs. 2.18 0.0211, 0.0084 C1TR vs. C2TR 51 vs. 51 10 vs. 7 10 vs. 7 5/10 5/7 2.00 vs. 1.73 0.0686, 0.3167 C1TR vs. C3AL 51 vs. 43 10 vs. 8 10 vs. 13 5/10 5/8 2.00 vs. 1.23 0.098, 0.0312 C1TR vs. C3TR 51 vs. 52 10 vs. 7 10 vs. 7 6/10 6/7 2.00 vs. 1.23 0.0002, 0.0009 C1TR vs. C4AL 51 vs. 44 10 vs. 7 10 vs. 7 7/10 7/7 2.00 vs. 1.40 0.0871, 0.0482 C1TR vs. C4TR 52 vs. 37 10 vs. 8 10 vs. 11 3/10 3/11 2.00 vs. 1.52 0.0195, 0.0009 C2AL vs. C2TR 49 vs. 51 13 vs. 7 18 vs. 7 6/13 6/7 2.18 vs. 1.53 < 0.0001, 0.0008 C2AL vs. C3AL 49 vs. 43 13 vs. 8 18 vs. 13 6/13 6/8 2.18 vs. 1.23 < 0.2966, 0.0029 C2AL vs. C3TR 49 vs. 52 13 vs. 7 18 vs. 7 6/13 6/7 2.18 vs. 1.23 0.0266, 0.1251 C2AL vs. C4AL 49 vs. 44 13 vs. 7 18 vs. 7 4/13 4/7 2.18 vs. 1.40 0.0036, 0.0009 C2AL vs. C4TR 49 vs. 40 13 vs. 8 18 vs. 11 4/13 4/8 2.18 vs. 1.52 0.0071, 0.1924 C3AL vs. C2TR 43 vs. 51 8 vs. 7 13 vs. 7 4/8 4/7 1.23 vs. 1.53 < 0.0001, 0.04 C2TR vs. C3TR 51 vs. 52 7 vs. 7 7 vs. 7 6/7 6/7 1.53 vs. 1.23 < 0.0001,< 0.0001 C4AL vs. C2TR 44 vs. 51 7 vs. 7 7 vs. 7 4/7 4/7 1.40 vs. 1.53 < 0.0001, 0.0029 C2TR vs. C4TR 51 vs. 40 7 vs. 8 7 vs. 11 2/7 2/11 1.53 vs. 1.52 0.0003, < 0.0001 C3AL vs. C3TR 43 vs. 52 8 vs. 7 13 vs. 7 5/8 5/7 1.23 vs. 1.23 0.0123, 0.7743 C3AL vs. C4AL 43 vs. 44 8 vs. 7 13 vs. 7 4/8 4/7 1.23 vs. 1.40 0.0497, 0.1951 C3AL vs. C4TR 43 vs. 40 8 vs. 8 13 vs. 11 6/8 6/11 1.23 vs. 1.52 0.1413, 0.1591 C4AL vs. C3TR 43 vs. 52 7 vs. 7 7 vs. 7 4/7 4/7 1.40 vs. 1.23 0.0083, 0.3038 C3TR vs. C4TR 52 vs. 40 7 vs. 8 7 vs. 11 3/7 3/11 1.23 vs. 1.52 0.1887, 0.1447 C4AL vs. C4TR 44 vs. 40 7 vs. 8 7 vs. 11 3/7 7/11 1.40 vs. 1.52 0.2064, 0.0963 AL vs. TR 167 vs. 194 22 vs. 17 25 vs. 19 16/22 16/17 3.18 vs. 2.89 0.0084, 0.0807

*C1–C4 represent the four cows used in this study. The AL and TR represent the rumen microbial communities fed with alfalfa hay (AL) and triticale straw (TR), respectively. †XY represents the number of OTUs in the first library (as indicated in column 1) shared with the second library. YX represents the number of OTUs in the second library shared with the first library. ‡XY represents the P values obtained when the first library (as indicated in column 1) is compared with the second library. YX represents P values obtained when the second library is compared with the first library. identified using the five probes designed here (data not to M. stadtmanae and Methanimicrococcus blatticola are shown). In total, members of genera Methanobrevibacter also important members of these communities, and and Methanomicrobium constituted on average 63% and account on average for 37% and 47% of the total metha- 53% of the total methanogens in the Liq and Sol phases, nogens in the Liq and Sol phases, respectively. respectively, a result which is in agreement with results Methanobrevibacter-related methanogens comprised from cloning studies conducted by others (Wright et al., about half of the total methanogen cells in the rumen 2007; Hook et al., 2009; King et al., 2011; Tymensen communities examined here, lower than the percentages et al., 2012), where members of these two genera and the reported by Sharp et al. (1998) in a Holstein cow RCC clade accounted for the majority of rumen metha- (89.3%) and a model rumen broth (99.2%). On the other nogens identified. Moreover, half of each of total Methan- hand, percentages of M. stadtmanae-related methanogens obrevibacter cells in the Liq and Sol phases emerged as detected here (16–34% and 12–50% of total methanogens M. ruminantium-related methanogens, a finding that sug- in the Liq and Sol, respectively) are generally higher than gests that this group plays an important role in ruminal those (6–12% of total methanogens) detected by Zhou methanogenesis. However, in addition to the Methano- et al. (2009) using qRT-PCR in the Liq phase of the brevibacter and Methanomicrobium methanogens related rumen of steers fed a mixture of oat grain (74%) and

FEMS Microbiol Ecol 84 (2013) 302–315 ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 312 Y. Kong et al. Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021

Fig. 6. Relative abundances of Archaea hybridizing with different FISH probes in the ruminal liquid (Liq) and solid (Sol) phases of cows fed with alfalfa hay (AL) or triticale straw (TR). Error bars represent standard deviation from 3 9 10 countings. grass hay (20%). While members of Methanobrevibacter of ruminal methanogens (Skillman et al., 2006; Zhou sp. strain AbM4 were detected in rumen communities of et al., 2011). However, the coverage of these is limited, these steers (4–8% of total methanogens), this strain was and different primer pairs will amplify fragments from not identified in clone libraries retrieved from our sam- different methanogenic populations in the same sample ples. Moreover, M. aarhusense, M. formicicum, and (Skillman et al., 2006; Zhou et al., 2011). In this study, M. barkeri, all of which have been isolated from bovine the FISH probe MB1174 (designed for Methanobacteri- rumen samples, could not be detected in our study. It is ales) hybridized with more than half the methanogens in still not clear whether this is because they are not present all rumen samples examined. When its sequence was used in these cattle or that they were not recovered during as a reverse primer together with the universal methano- cloning because of the relatively small number of cattle gen forward primer Met87f, a high-resolution cloning sampled, the clones sequenced or biases of DNA extrac- analysis of members of the Methanobacteriales was achiev- tion and/or PCR amplification procedures. able. Methanobrevibacter clones constituted the majority To our knowledge, this is the first report that M. blatti- (87%) of the clones retrieved (314 of 361 clones), and 16 cola-related methanogens are present in high relative Methanobrevibacter OTUs were recognized. Fourteen of abundances in the bovine rumen. However, in this study, these shared > 97% sequence similarity to nine Methano- only one such clone (C4TRD08) was retrieved with the brevibacter isolates cultivated previously in the laboratory MB1174 reverse PCR primer designed to target the Met- (Table 1). Of these, four have been validly described and hanobacteriales, FISH performed showed that on average, others share < 97% sequence similarity with all other 23% and 9% of the methanogens in the Liq and Sol available methanogen clones, suggesting they represent phases, respectively, were members closely related to putative new species. The Methanobrevibacter detection M. blatticola. Whitford et al. (2001) and Sundset et al. rate is markedly higher than that suggested by the four (2009) also retrieved M. blatticola-related 16S rRNA gene OTUs (97% cutoff) generated by Wright et al. (2007) clones from cattle and reindeer rumen, but made no from the rumen of cattle (241 clones sequenced) with the effort to quantify them in situ. This would suggest that Met86f and Met1340r primer pair, or the eight OTUs the role and importance of M. blatticola-related methano- (98% cutoff) reported by Hook et al. (2009) from 166 gens in ruminal methanogenesis have been underesti- clones from dairy cows using the same primer pair. Fur- mated in past studies. thermore, the OTU detection rate is substantially higher The primer pair MB1174r and Met87f was applied suc- than the four OTUs (97% cutoff) reported by Tymensen cessfully for high-resolution cloning analysis of members et al. (2012) after analyzing 100 clones of free- of the Methanobacteriales. Many universal primer pairs living methanogens and those associated with protozoa have been used for PCR amplification of 16S rRNA genes from the rumen of black Angus heifers with the primer

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Ecol 84 (2013) 302–315 Published by Blackwell Publishing Ltd. All rights reserved Composition of ruminal methanogenic communities 313 pair Met86f and Met915r. In all these studies, Mothur flow of reducing equivalents would be expected to be was used to estimate the number of OTUs. The high Met- dramatic. hanobrevibacter species detection rate obtained in the In conclusion, the diversity and population composi- present study is considered to arise from using the novel tion of methanogen communities, in particular, the Met- Met86f and Met1174r primer pair together with fraction- hanobrevibacter populations, in the rumen of cattle fed ating the rumen digesta samples before preparation of alfalfa hay or triticale straw have been revealed here at a DNA templates for PCR amplification. high-resolution level. Populations related to the Methano- Seventy-five clones belonging to the RCC clade were brevibacter TMS group and M. ruminantium accounted retrieved in the clone libraries constructed in this study. for approximately half of the total methanogens identified, They were not included in our phylogenetic analysis, and while the remaining members consisted of M. stadtmanae, no FISH probes were designed for them because their M. mobile, and M. blatticola. The composition of the roles in rumen are still not clear. Paul et al. (2012) methanogenic community and the relative abundances of Downloaded from https://academic.oup.com/femsec/article/84/2/302/478402 by guest on 01 October 2021 reported (after this manuscript was submitted) that Ther- individual methanogen populations were not affected moplasmatales-related Archaea in termite guts and other markedly by changing the diet from alfalfa hay to triticale environments are methanogens. Their relative abundance straw and vice versa. The composition of the methanogen in the methanogen communities will be examined in community is shown by methodological improvements to future research. be more diverse than previously suspected. No clear pattern was apparent regarding the effect of diet or individual host cow on the composition of metha- Acknowledgements nogenic communities after cloning analysis, and the similarity in methanogen biodiversity in all cows regardless of Financial support for Yun Xia was provided by the Agri- diet suggests that neither had a marked effect on ruminal culture Crop Industry Development Fund of Alberta. We methanogen community composition. This is an outcome thank Lyn Paterson for excellent technical assistance. in sharp contrast to that of our earlier study using only clone library analyses, where population compositions of Authors’ contribution the bacterial communities in these ruminal digesta samples changed markedly among different hosts and diets Y.H.K. and Y.X. contributed equally to this study. (Kong et al., 2010a). It supports the view that the diverse chemoorganoheterotrophic bacterial populations in the References rumen are more sensitive to the nature of the organic substrates presented for fermentation than are the metha- Abramoff MD, Magalhaes PJ & Ram SJ (2004) Image nogens that utilize a relatively narrow spectrum of energy processing with imageJ. Biophotonics Int 11:36–41. sources (Janssen & Kirs, 2008). 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