DOI: 10.5061/dryad.2m854
title: Data from: Unearthing carrion beetles’ microbiome: characterization of bacterial and fungal hindgut communities across the Silphidae

description: Carrion beetles (Coleoptera, Silphidae) are well known for their behaviour
 of exploiting vertebrate carcasses for nutrition. While species in the
 subfamily Silphinae feed on large carcasses and on larvae of competing
 scavengers, the Nicrophorinae are unique in monopolizing, burying and
 defending small carrion, and providing extensive biparental care. As a
 first step towards investigating whether microbial symbionts may aid in
 carcass utilization or defence, we characterized the microbial hindgut
 communities of six Nicrophorinae (Nicrophorus spp.) and two Silphinae
 species (Oiceoptoma noveboracense and Necrophila americana) by deep
 ribosomal RNA amplicon sequencing. Across all species, bacteria in the
 family Xanthomonadaceae, related to Ignatzschineriao larvae, were
 consistently common, and several other taxa were present in lower
 abundance (Enterobacteriales, Burkholderiales, Bacilli, Clostridiales and
 Bacteroidales). Additionally, the Nicrophorinae showed high numbers of
 unusual Clostridiales, while the Silphinae were characterized by
 Flavobacteriales and Rhizobiales (Bartonella sp.). In addition to the
 complex community of bacterial symbionts, each species of carrion beetle
 harboured a diversity of ascomycetous yeasts closely related to Yarrowia
 lipolytica. Despite the high degree of consistency in microbial
 communities across the Silphidae—specifically within the
 Nicrophorinae—both the fungal symbiont phylogeny and distance-based
 bacterial community clustering showed higher congruence with sampling
 locality than host phylogeny. Thus, despite the possibility for vertical
 transmission via anal secretions, the distinct hindgut microbiota of the
 Silphidae appears to be shaped by frequent horizontal exchange or
 environmental uptake of symbionts. The microbial community profiles,
 together with information on host ecology and the metabolic potential of
 related microorganisms, allow us to propose hypotheses on putative roles
 of the symbionts in carcass degradation, detoxification and defence.
description: Alignment of 16S rRNA
 sequences of the 50 most abundant bacterial OTUs associated with the
 hindgut of carrion beetles (Coleoptera, Silphidae)Genomic DNA was
 extracted from the hindguts of males and females of eight species of
 carrin beetles (Coleoptera, Silphidae), respectively, and subjected to
 bacterial tag-encoded FLX amplicon pyrosequencing. Reads were
 quality-trimmed and analyzed using QIIME (Caporaso et al. 2010). After a
 multi-step OTU picking strategy with cdhit (Li & Godzik 2006) and
 uclust (Edgar 2010), an OTU table was generated describing the occurrence
 of bacterial phylotypes within the samples, and all OTUs that amounted to
 less than 0.1% in all of the samples (=2166 OTUs) were excluded from
 further analysis. Of the remaining OTUs, 23 were removed due to their
 likely chimeric origin as identified by UCHIME, which left 522 OTUs for
 the final analysis. For phylogenetic reconstruction, the 50 most abundant
 OTUs across host taxa were selected, and their representative sequences
 (picked with –m longest using pick_rep_set.py in QIIME) were trimmed to
 300 bp in order to remove low-quality end regions. The resulting sequences
 as well as closely related sequences identified by BLASTn against the NCBI
 nr database were aligned to the SILVA SSU database (Pruesse et al. 2007)
 using the SINA aligner (Pruesse et al. 2012) and imported into ARB (Ludwig
 et al. 2004). Further representative relatives were included, and the
 final alignment was exported, and common gaps were
 removed.Bacteria_top50_OTUs_ARB_alignment_with_additional_seqs_renamed_trimmed.fastaAlignment of LSU rRNA sequences of fungi associated with the hindgut of carrion beetles (Coleoptera, Silphidae)Genomic DNA was extracted from the hindguts of males and females of eight species of carrin beetles (Coleoptera, Silphidae), respectively, and part of the fungal large subunit ribosomal RNA gene (LSU) was amplified for the different species and sexes, respectively, with primers LS1 and LR5 (Gibson & Hunter 2009). Amplicons were cloned into E. coli using the StrataClone PCR Cloning Kit (Agilent Technologies, USA) according to the manufacturer’s instructions. For Ni. humator, Ni. vespillo, Ni. orbicollis, Ni. puctulatus, Ni. tomentosus, Ne. americana, and O. noveboracense, inserts of 16 clones per species were amplified with the flanking primers M13fwd and M13rev and sequenced bidirectionally with M13fwd and M13rev. For Ni. vespilloides, 53 clones were sequenced unidirectionally with primer M13fwd. All sequences were blasted against the NCBI nr database for taxonomy assignment. Representative sequences were aligned with SINA (Pruesse et al. 2012) and imported into ARB (Ludwig et al. 2004). Closely related fungal reference sequences were selected from the SILVA LSU reference database and included in the alignment.Silphidae_fungal_LSU_ARB_alignment_renamed_trimmed_edited.fastaAlignment of two mitochondrial and two nuclear markers across eight species of carrion beetles (Coleoptera, Silphidae) and an outgroup taxon (Tribolium castaneum), in nexus format with MrBayes blockFor an analysis of the evolutionary history of microbial communities across the Silphidae, the host phylogeny was reconstructed based on two mitochondrial (cytochrome oxidase I and II, COI and COII hereafter) and two nuclear genes (28S rRNA gene and carbamoylphosphate synthetase, 28S and CAD hereafter). Most sequences for Nicrophorus spp., Ne. americana, and the outgroup species Tribolium castaneum were obtained from the NCBI database. The dataset was completed by sequencing CAD for Ne. americana, COI/COII for Ni. tomentosus, and CAD, COI, and 28S rRNA for O. noveboracense. COII could not successfully be amplified and sequenced for O. noveboracense, so it was coded as missing data for the analysis. Amplicons were sequenced bidirectionally with (CAD, some COI/II) or without (28S, most COI/II fragments) prior subcloning with the StrataClone PCR Cloning Kit (Agilent Technologies, USA). Curated protein-coding sequences (CAD, COI, and COII) were aligned based on their amino acid translation in Geneious Pro 5.4 (Drummond et al. 2011), and partial 28S rRNA gene sequences were aligned using the SINA aligner (Pruesse et al. 2012). The alignments were concatenated, and phylogenetic relationships were reconstructed using approximately-maximum-likelihood (ML, FastTree) and Bayesian algorithms (MrBayes 3.1.2, Huelsenbeck & Ronquist 2001; Huelsenbeck et al. 2001; Ronquist & Huelsenbeck 2003). For the ML analysis, the general time-reversible (GTR) model was used. For the Bayesian analysis, the dataset was partitioned into the four genes. For the three protein-coding genes, six substitution rates were allowed (GTR model), whereas all substitutions were restricted to the same rate (JC model) for the ribosomal gene. Due to saturation, third codon positions were excluded from the analysis for the two mitochondrial genes (COI and COII). We ran the analysis with four chains and a temperature of 0.2 for 10,000,000 generations (1,000,000 generations for the reduced dataset, see below) and confirmed that the standard deviation of split frequencies was consistently less than 0.01. Trees were sampled every 1000 generations, and a “burnin” of 1000 was used. We computed a 50% majority rule consensus tree with posterior probabilities for every node. We ran all analyses with the complete set of taxa as well as with a reduced set containing only the species analyzed in this study. In both cases, T. castaneum was included and defined as the outgroup to root the tree.Host_phylogeny_selected_Silphidae_concat.nexAlignment of two mitochondrial and two nuclear markers across 30 species of carrion beetles (Coleoptera, Silphidae) and an outgroup taxon (Tribolium castaneum), in nexus format with MrBayes blockFor an analysis of the evolutionary history of microbial communities across the Silphidae, the host phylogeny was reconstructed based on two mitochondrial (cytochrome oxidase I and II, COI and COII hereafter) and two nuclear genes (28S rRNA gene and carbamoylphosphate synthetase, 28S and CAD hereafter). Most sequences for Nicrophorus spp., Ne. americana, and the outgroup species Tribolium castaneum were obtained from the NCBI database. The dataset was completed by sequencing CAD for Ne. americana, COI/COII for Ni. tomentosus, and CAD, COI, and 28S rRNA for O. noveboracense. COII could not successfully be amplified and sequenced for O. noveboracense, so it was coded as missing data for the analysis. Amplicons were sequenced bidirectionally with (CAD, some COI/II) or without (28S, most COI/II fragments) prior subcloning with the StrataClone PCR Cloning Kit (Agilent Technologies, USA). Curated protein-coding sequences (CAD, COI, and COII) were aligned based on their amino acid translation in Geneious Pro 5.4 (Drummond et al. 2011), and partial 28S rRNA gene sequences were aligned using the SINA aligner (Pruesse et al. 2012). The alignments were concatenated, and phylogenetic relationships were reconstructed using approximately-maximum-likelihood (ML, FastTree) and Bayesian algorithms (MrBayes 3.1.2, Huelsenbeck & Ronquist 2001; Huelsenbeck et al. 2001; Ronquist & Huelsenbeck 2003). For the ML analysis, the general time-reversible (GTR) model was used. For the Bayesian analysis, the dataset was partitioned into the four genes. For the three protein-coding genes, six substitution rates were allowed (GTR model), whereas all substitutions were restricted to the same rate (JC model) for the ribosomal gene. Due to saturation, third codon positions were excluded from the analysis for the two mitochondrial genes (COI and COII). We ran the analysis with four chains and a temperature of 0.2 for 10,000,000 generations (1,000,000 generations for the reduced dataset, see below) and confirmed that the standard deviation of split frequencies was consistently less than 0.01. Trees were sampled every 1000 generations, and a “burnin” of 1000 was used. We computed a 50% majority rule consensus tree with posterior probabilities for every node. We ran all analyses with the complete set of taxa as well as with a reduced set containing only the species analyzed in this study. In both cases, T. castaneum was included and defined as the outgroup to root the tree.Host_phylogeny_Silphidae_concat.nexQuality-trimmed bTEFAP sequences of bacterial 16S rRNA from the hind-guts of eight species of carrion beetles (Coleoptera, Silphidae)DNA from adult hind-guts of 3-6 males and females per species was extracted individually using the SoilMasterTM DNA Extraction Kit (Epicentre, Madison, USA), according to the manufacturer’s instructions (including lysozyme treatment to break up Gram-positive bacterial cells). Identical volumes of the samples for each sex and species, respectively, were pooled for bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). DNA was sent to an external service provider (Research & Testing Laboratories, Lubbock, USA) for bTEFAP with 16S rRNA primers Gray28F and Gray519R (Sun et al. 2011) (Table S1). A sequencing library was generated through one-step PCR with 30 cycles, using a mixture of HotStar and HotStar HiFidelity Taq polymerases (Qiagen). Sequencing extended from Gray28F, using a Roche 454 FLX instrument with Titanium reagents and procedures at Research and Testing Laboratory (RTL, Lubbock, TX, USA, http://www.medicalbiofilm.org/). All low quality reads (quality cut-off = 25) and sequences &lt; 200 bp were removed following sequencing, which left 223,470 sequences for analysis (mean ± SD = 13,967 ± 5,946 per sample). Sample abbreviations are as follows: #SampleID Host species NvspFhind Nicrophorus vespillo NvspMhind Nicrophorus vespillo NhumFhind Nicrophorus humator NhumMhind Nicrophorus humator NvesFhind2 Nicrophorus vespilloides NvesMhind2 Nicrophorus vespilloides NorbF_ED Nicrophorus orbicollis NorbM_ED Nicrophorus orbicollis NpusF_ED Nicrophorus pustulatus NpusM_ED Nicrophorus pustulatus NtomF_ED Nicrophorus tomentosus NtomM_ED Nicrophorus tomentosus NameF_ED Necrophila americana NameM_ED Necrophila americana OnovF_ED Oiceoptoma noveboracense OnovM_ED Oiceoptoma noveboracenseSilphidae_16S_rRNA_sequences.fnaMapping file for the quality-trimmed bTEFAP sequences of bacterial 16S rRNA from the hind-guts of eight species of carrion beetles (Coleoptera, Silphidae)Mapping file containing sample IDs, barcode and primer sequences from 454 sequencing, and host species.Silphidae_16S_rRNA_sequences_mapping.txt