Isolation (and culture) of the ciliates
N. ovalis was isolated from the hindgut of the cockroaches Periplaneta americana strains Amsterdam (PA), Bayer (PB), Dar es Salaam (PD), Nijmegen(PN) and Blaberus sp. strains Düsseldorf (BD) and Amsterdam (BA) taking advantage of the unique anodic galvanotaxic behaviour of N. ovalis .Euplotes sp. was grown in Erlenmeyer flasks containing 500 ml artificial seawater (465 mM NaCl, 10 mM KCl, 53 mM MgCl2, 28 mM MgSO4, 1.0 mM CaCl2, and 0.23 mM NaHCO3). Since Euplotes sp. requires living bacteria for growth, E. coli XL1-blue was supplied at regular intervals. Alternatively, a small piece of beef-steak (approximately 1 cm3) was placed into the culture medium to allow the growth of food bacteria. Euplotes sp. cells were harvested 28 days after the start of a new culture by filtration through a 4 μm plankton gaze.
Rumen ciliates were isolated by electromigration from the rumen fluid of a grass-fed, fistulated Holstein-Friesian cow, and lysed immediately after the isolation in a 8 M solution of guanidinium chloride and stored at minus 25°C until use.
DNA isolation, total RNA isolation and cDNA synthesis
DNA of N. ovalis and Euplotes sp. was isolated according to van Hoek et al. . Total rumen ciliate DNA was prepared after purification on a hydroxyapatite column (BioRad) using standard methods. Total RNA of N. ovalis was isolated using the RNeasy Plant mini-kit (Qiagen). Adaptor-ligated cDNA was prepared according to the SMART™ RACE cDNA Amplification kit (Clontech).
Isolation of the H-cluster, the 24 kD (hoxF) and 51 kD (hoxU) modules of the hydrogenase gene, and mitochondrial-type 24 kDa and 51 kDa subunits of mitochondrial complex I
H-clusters of Fe-hydrogenases were amplified from total rumen ciliate DNA using PCR with primers described earlier . In addition, DNA from type-strain rumen ciliates, kept by the ERCULE consortium was used as template for PCR.
To isolate the (nuclear-encoded) 24 kD (hoxF) and 51 kD (hoxU) -like genes, the primer-design was based on the H-cluster and the 24 kD (hoxF) and 51 kD (hoxU) region of the hydrogenase of N. ovalis PN . Their sequences are 5'-gtnatggcntgyccngghgghtg-3' (H-cluster forward primer) and 5'-ccntcyctrcadggnacrcaytg-3' (51 kDa reverse primer 1). Sequence-specific internal primers were designed to isolate the termini of the gene-sized chromosomes in combination with a telomere-specific primer using the telomere suppression PCR method [38, 39].
To isolate the (nuclear) genes encoding the 24 kDa and 51 kDa subunits of mitochondrial complex I, respectively, primers were based on conserved amino-acid regions of mitochondrial complex I genes. Their sequences are 5'-tgyggwachachccwtg-3' (24 kDa forward primer), 5'-ccnarrcaytcdacytc-3' (24 kDa reverse primer), 5'-gmhgargghgarccwgghac-3' (51 kDa forward primer), and 5'-cangwcatytcytcytcnac-3' (51 kDa reverse primer). The ORFs were completed as described above.
The amino acid sequences of the H-cluster were aligned using Clustal × 1.81 . The program Gblocks  was used to identify regions of defined sequence conservation and exclude ambiguously aligned positions from the alignment. The phylogenetic analysis of the sequences were performed with the program MRBAYES version 3.1.2 . Markov chain Monte Carlo from a random starting tree was initiated and run for 2 million generations. In these analyses, the JTT model of amino acid substitution and four gamma distributed rates of evolution were applied. Trees were sampled every 1000th generation. The first 25% of the samples were discarded as 'burn-in', and the rest of the samples were used for inferring a Bayesian tree. Examination of the log-likelihood and the observed consistency with the similar likelihood values between the two independent runs suggest that the run reached stationarity and that these burn-in periods were sufficiently long.
The accession numbers of the sequences used to calculate this tree are Nyctotherus ovalis BA AY608627; N. ovalis PN CAA76373; the sequences from rumen ciliates have been deposited in GenBank under accession numbers AM396939 – AM396957
For the 24kD/51kD domains less sequences had to be included to delineate the evolution, allowing a "manual" sequence alignment and phylogeny approach. Alignments of representative sequences from the 24kD/51kD domains were generated with MUSCLE . Sequences were edited and the most relevant parts from the alignments were selected manually using Seaview . Phylogenies were subsequently derived using the program PHYML  using the JTT model and an estimated number of invariable sites with four substitution rate categories. 100 bootstraps were performed; they are only indicated in the tree if they are ≥ 50.
The accession numbers of the used 24 kDa subunit/NuoE/hoxF sequences are [Agrobacter tumefaciens PIR:D97514, Bos taurus Swiss-Prot:P04394, Buchnera aphidicola Swiss-Prot:P57255, Burkholderia cenocepacia AU 1054 REFSEQ:YP_621511.1, Burkholderia xenovorans LB400 REFSEQ:YP_555778.1, Dechloromonas aromatica RCB GenBank:AAZ45735.1, Deinococcus radiodurans REFSEQ:NP_295224, Drosophila melanogaster GenBank:AAL68189, Escherichia coli Swiss-Prot:P33601, Hahella chejuensis KCTC 2396 REFSEQ:YP_431454.1, Heliobacillus mobilis EMBL:CAJ44288.1, Homo sapiens REFSEQ:NP_066552, Magnetospirillum magneticum AMB-1 REFSEQ:YP_422756.1, Methanospirillum hungatei JF-1 REFSEQ:YP_502735.1, Methanothermobacter thermautotrophicus str. Delta H GenBank:AAB86022.1, Methylococcus capsulatus str. Bath REFSEQ:YP_115124.1, Mus musculus Swiss-Prot:Q9D6J6, Mycobacterium vanbaalenii PYR-1 REFSEQ:ZP_01205720.1, N. ovalis BA (24 kDa) GenBank:AY628688, N. ovalis BD GenBank:AY608628, N. ovalis PA GenBank:AY608629, N. ovalis PB GenBank:AY608630, N. ovalis PD GenBank:AY608631, N. ovalis PN GenBank:CAA76373, Neurospora crassa Swiss-Prot:P40915, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_411790.1, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_412360.1, Nyctotherus ovalis BA GenBank:AY608627, Paracoccus denitrificans Swiss-Prot:P29914, Paramecium tetraurelia Swiss-Prot:Q6BFW6, Psychromonas ingrahamii 37 REFSEQ:ZP_01348563.1, Ralstonia eutropha GenBank:AAC06140, Ralstonia metallidurans CH34 REFSEQ:YP_583086.1, Ralstonia solanacearum UW551 REFSEQ:ZP_00943354.1, Rattus norvegicus Swiss-Prot:P19234, Rhodobacter sphaeroides ATCC 17029 REFSEQ:ZP_00918830.1, Rhodococcus opacus Swiss-Prot:P72304, Rhodoferax ferrireducens T118 REFSEQ:YP_525088.1, Streptomyces avermitilis MA-4680 REFSEQ:NP_823011.1, Syntrophus aciditrophicus SB REFSEQ:YP_461127.1, Tetrahymena thermophila Swiss-Prot:Q23LJ5, Thermoanaerobacter tengcongensis GenBank:AAM24146, Thermococcus kodakarensis KOD1 DDBJ:BAD85803.1, Thermotoga maritima REFSEQ:NP_227828, Thermus thermophilus Swiss-Prot:Q56221, Thiobacillus denitrificans ATCC 25259 REFSEQ:YP_314904.1, uncultured archaeon GZfos26D6 GenBank:AAU83055.1]
The accession numbers of the 51 kDa subunit/NuoF/hoxU sequences are [Agrobacterium tumefaciens Swiss-Prot:Q8U6U9, Aquifex aeolicus Swiss-Prot:O66841, Arabidopsis thaliana Swiss-Prot:Q8LAL7, Aspergillus niger Swiss-Prot:Q92406, Bos taurus GenBank:AF092131, Bradyrhizobium japonicum DDBJ:BAC48402, DDBJ:BAC50177, Burkholderia xenovorans LB400 REFSEQ:YP_555778.1, Candida tropicalis Swiss-Prot:Q96UX4, Caulobacter crescentus Swiss-Prot:Q9A6X9, Chromobacterium violaceum ATCC 12472 REFSEQ:NP_900616.1, Dechloromonas aromatica RCB GenBank:AAZ45735.1, Dictyostelium discoideum REFSEQ:XP_636489.1, Eubacterium acidaminophilum EMBL:CAC39230.1, Euplotes sp. GenBank:AY608636, Hahella chejuensis KCTC 2396 REFSEQ:YP_431454.1, Heliobacillus mobilis EMBL:CAJ44288.1, Leishmania major Swiss-Prot:Q9U4M2, Magnetospirillum magneticum AMB-1 REFSEQ:YP_422756.1, Methanospirillum hungatei JF-1 REFSEQ:YP_502736.1, Methanothermobacter thermautotrophicus str. Delta H GenBank:AAB86023.1, Methylococcus capsulatus str. Bath REFSEQ:YP_115124.1, Mycobacterium tuberculosis Swiss-Prot:P95176, Mycobacterium vanbaalenii PYR-1 REFSEQ:ZP_01205720.1, N. ovalis BA (51 kDa) GenBank:AY608632, N. ovalis BD GenBank:AY608628, N. ovalis PA (51 kDa) GenBank:AY608635, N. ovalis PB GenBank:AY608630, N. ovalis PD GenBank:AY608631, N. ovalis PN GenBank:CAA76373, Neisseria gonorrhoeae FA 1090 REFSEQ:YP_208779.1, Neisseria meningitidis Z2491 EMBL:CAB83334.1, Neurospora crassa Swiss-Prot:P24917, Nitrosococcus oceani ATCC 19707 GenBank:ABA59013.1, Nitrosomonas europaea ATCC 19718 EMBL:CAD85683.1, Nitrosomonas eutropha C71 REFSEQ:ZP_00669832.1, Nitrosospira multiformis ATCC 25196 REFSEQ:YP_411791.1, REFSEQ:YP_412360.1, Nyctotherus ovalis BA GenBank:AY608627, Paracoccus denitrificans Swiss-Prot:P29913, Psychromonas ingrahamii 37 REFSEQ:ZP_01348563.1, Ralstonia eutropha GenBank:AAC06140, Ralstonia eutropha JMP134 GenBank:AAZ60345.1, Ralstonia metallidurans CH34 REFSEQ:YP_583087.1, Ralstonia solanacearum EMBL:CAD15764.1, Ralstonia solanacearum UW551 REFSEQ:ZP_00943355.1, Rhizobium loti Swiss-Prot:Q98BW8, Swiss-Prot:Q98KR0, Rhizobium meliloti Swiss-Prot:P56912, Swiss-Prot:P56913, Rhodobacter capsulatus Swiss-Prot:O07948, Rhodococcus opacus Swiss-Prot:P72304, Rhodoferax ferrireducens T118 REFSEQ:YP_525088.1, Rickettsia prowazekii Swiss-Prot:Q9ZE33, Solanum tuberosum Swiss-Prot:Q43840, Streptomyces avermitilis MA-4680 REFSEQ:NP_823011.1, Synechococcus elongatus PCC 6301 EMBL:CAA73873.1, Syntrophus aciditrophicus SB REFSEQ:YP_461127.1, Tetrahymena thermophila SB210 GenBank:EAR96899.1, Thermococcus kodakarensis KOD1 DDBJ:BAD85803.1, Thermotoga maritima Swiss-Prot:O52682, Swiss-Prot:Q9WXM5, Swiss-Prot:Q9WY70, Thermus thermophilus Swiss-Prot:Q56222, Thiobacillus denitrificans ATCC 25259 REFSEQ:YP_314905.1, Trypanosoma brucei REFSEQ:XP_824451.1, Trypanosoma cruzi GenBank:EAN82122.1, uncultured archaeon GZfos26D6 GenBank:AAU83054.1, Yarrowia lipolytica Swiss-Prot:Q9UUU2]
Multivariate Comparative Analysis
The codon usage of the genes investigated in this study was subjected to a multivariate analysis by means of Principal Component Analysis (PCA) to visualise the genetic diversity of the ciliate species. PCA was performed using the GeneMaths XT software package (Applied Maths BVBA, Sint-Martens-Latem, Belgium ).