Divergence between sea urchins and their microbiota following speciation
Data files
Apr 04, 2025 version files 167.95 MB
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Echinometra_sp_Egg_Amplicon.zip
167.94 MB
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README.md
16.85 KB
Abstract
Animals have a deep evolutionary relationship with microbial symbionts, such that individual microbes or an entire microbial community can diverge alongside the host. Here, we explore these host-microbe relationships in Echinometra, a sea urchin genus that speciated with the Isthmus of Panama and throughout the Indo-West Pacific. We find that the eggs from five Echinometra species generally associate with a species-specific bacterial community and that the relatedness of these communities is largely congruent with host phylogeny. Microbiome divergence per million years was higher in more recent speciation events than in older ones. We, however, did not find any bacterial groups that displayed co-phylogeny with Echinometra. Together, these findings suggest that the evolutionary relationship between Echinometra and their microbiota operates at the community level. We find no evidence suggesting that the associated microbiota is the evolutionary driver of Echinometra speciation. Instead, divergence between Echinometra and their microbiota is likely the byproduct of ecological, geographic, and reproductive isolations.
DOI: 10.5061/dryad.dz08kps3d
The data included here are of the sea urchins Echinometra sp. A and Echinometra sp. B (E. mathaei senso stricto) (i.e., those from Japan). Data for the species from Panama (E. lucunter, E. viridis, and E. vanbrunti) can be found at: doi.org/10.5061/dryad.2z34tmphq (https://datadryad.org/stash/dataset/doi:10.5061/dryad.2z34tmphq)
Description of the data and file structure
DNA_JAP_S181_L001_R1_001.fastq.gz DNA blank from extraction kit, forward
DNA_JAP_S181_L001_R2_001.fastq.gz DNA blank from extraction kit, reverse
EcA1_JAP_S34_L001_R1_001.fastq.gz Eggs from Echinometra sp A, forward
EcA1_JAP_S34_L001_R2_001.fastq.gz Eggs from Echinometra sp A, reverse
EcA2_JAP_S35_L001_R1_001.fastq.gz Eggs from Echinometra sp A, forward
EcA2_JAP_S35_L001_R2_001.fastq.gz Eggs from Echinometra sp A, reverse
EcA3_JAP_S36_L001_R1_001.fastq.gz Eggs from Echinometra sp A, forward
EcA3_JAP_S36_L001_R2_001.fastq.gz Eggs from Echinometra sp A, reverse
EcB1_JAP_S177_L001_R1_001.fastq.gz Eggs from Echinometra sp B, forward
EcB1_JAP_S177_L001_R2_001.fastq.gz Eggs from Echinometra sp B, reverse
EcB2_JAP_S178_L001_R1_001.fastq.gz Eggs from Echinometra sp B, forward
EcB2_JAP_S178_L001_R2_001.fastq.gz Eggs from Echinometra sp B, reverse
EcB3_JAP_S179_L001_R1_001.fastq.gz Eggs from Echinometra sp B, forward
EcB3_JAP_S179_L001_R2_001.fastq.gz Eggs from Echinometra sp B, reverse
EcB4_JAP_S180_L001_R1_001.fastq.gz Eggs from Echinometra sp B, forward
EcB4_JAP_S180_L001_R2_001.fastq.gz Eggs from Echinometra sp B, reverse
Code/Software
QUALITY CONTROL AND PRE-PROCESSING
IMPORT RAW FILES INTO QIIME2
NOTE: TO IMPORT MISEQ FILES, ALL RAW FILES HAVE TO BE IN ONE DIRECTORY… SELF-LABEL ‘all_files’ DIRECTORY
qiime tools import \
--type ‘SampleData[PairedEndSequencesWithQuality]’ \
--input-path /Echinometra_EggMicrob/rawreads_16S \
--input-format CasavaOneEightSingleLanePerSampleDirFmt \
--output-path EchinometraEggMbiome_import.qza
TRIM PRIMERS OFF READS
qiime cutadapt trim-paired \
--i-demultiplexed-sequences EchinometraEggMbiome_import.qza \
--p-front-f CCTACGGGNGGCWGCAG \
--p-front-r GACTACHVGGGTATCTAATCC \
--p-error-rate 0 \
--o-trimmed-sequences EchinometraEggMbiome_import_trim.qza
JOIN/MERGE PAIRED-ENDS
qiime vsearch merge-pairs \
--i-demultiplexed-seqs EchinometraEggMbiome_import_trim.qza \
--o-merged-sequences EchinometraEggMbiome_import_trim_paired.qza \
--p-minovlen 20 \
--p-maxdiffs 10 \
--p-minmergelen 350 \
--p-maxmergelen 550 \
--p-allowmergestagger \
--p-truncqual 10 \
--p-minlen 100
QUALITY CONTROL PAIRED-END BASED ON PHRED Q SCORES
qiime quality-filter q-score \
--i-demux EchinometraEggMbiome_import_trim_paired.qza \
--o-filtered-sequences EchinometraEggMbiome_import_trim_paired_QC25.qza \
--o-filter-stats EchinometraEggMbiome_import_trim_paired_QC25_stats.qza \
--p-quality-window 5 \
--p-min-quality 25
REIMPORT FILTERED FILES WITH MODIFIED NAMES
qiime tools export \
--input-path EchinometraEggMbiome_import_trim_paired_QC25.qza \
--output-path EchinometraEggMbiome_import_trim_paired_QC25
qiime tools import \
--type ‘SampleData[SequencesWithQuality]’ \
--input-path MANIFEST.txt \
--output-path EchinometraEggMbiome_import_trim_paired_QC25_NewNames.qza \
--input-format SingleEndFastqManifestPhred33
DENOISE PROCESSED READS
qiime deblur denoise-16S \
--i-demultiplexed-seqs EchinometraEggMbiome_import_trim_paired_QC25_NewNames.qza \
--p-trim-length 400 \
--o-representative-sequences EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq.qza \
--o-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table.qza \
--p-sample-stats \
--o-stats EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_stats.qza
CREATE ALIGNMENT OF REPRESENTATIVE SEQUENCES
qiime alignment mafft \
--i-sequences EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq.qza \
--o-alignment EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned.qza
MASK HIGHLY VARIABLE NOISY POSITIONS IN ALIGNMENT
qiime alignment mask \
--i-alignment EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned.qza \
--o-masked-alignment EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask.qza
CREATE PHYLOGENY WITH FASTTREE
qiime phylogeny fasttree \
--i-alignment EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask.qza \
--o-tree EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask_tree.qza
ROOT PHYLOGENY AT MIDPOINT
qiime phylogeny midpoint-root \
--i-tree EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask_tree.qza \
--o-rooted-tree EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask_tree_rooted.qza
ASSIGN TAXONOMY WITH TRAINED CLASSIFIER
qiime feature-classifier classify-sklearn \
--i-classifier /Phototrophy_ArbaciaLarvae/qiime2/datasets/silva-138-99-classifier.qza \
--i-reads EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq.qza \
--o-classification EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_AssignTaxa.qza \
--p-confidence disable \
--p-read-orientation same
FILTER ARCHAEA
qiime taxa filter-table \
--i-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table.qza \
--i-taxonomy EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_AssignTaxa.qza \
--p-exclude Archaea \
--p-mode contains \
--o-filtered-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea.qza
FILTER FEATURES FROM DNA SAMPLES
qiime tools export \
--input-path EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea.qza \
--output-path EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea
biom convert \
-i EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea/feature-table.biom \
-o EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea/feature-table.txt \
--to-tsv
qiime feature-table filter-features \
--i-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea.qza \
--m-metadata-file FeaturesDNEchinometraEggMbiome_Akit \
--o-filtered-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_NoContam.qza \
--p-exclude-ids
FILTER SINGLETONS
qiime feature-table filter-features \
--i-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_NoContam.qza \
--p-min-frequency 2 \
--o-filtered-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_NoContam_No1.qza
REMOVE LOW
qiime feature-table filter-samples \
--i-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_No1.qza \
--m-metadata-file Samples2InEchinometraEggMbiome_clude2xt \
--o-filtered-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_No1_NoLow.qza \
--p-no-exclude-ids
RAREIFY TABLE
qiime feature-table rarefy \
--i-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_No1_NoLow.qza \
--p-sampling-depth 534 \
--p-with-replacement \
--o-rarefied-table EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_No1_NoLow_rare.qza
#rename ‘EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_table_NoArchaea_No1_NoLow_rare.qza’ to ‘EchinometraEggMbiome_rare.qza’
#rename ‘EchinometraEggMbiome_import_trim_paired_QC25_NewNames_deblur_RepSeq_aligned_mask_tree_rooted.qza’ to ‘EchinometraEggMbiome_TreeRooted’
#move data table and phylogeny into folder ‘analyses’
ANALYSES FOR FIGURES IN MANUSCRIPT
ALPHA RAREFACTION
qiime diversity alpha-rarefaction \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-max-depth 534 \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--output-dir /analyses/1_AlphaRare
qiime tools export \
--input-path /analyses/1_AlphaRare/visualization.qzv \
--output-path /analyses/1_AlphaRare/AlphaRare_visualization
BETA DIVERSITY
#unweighted
qiime diversity beta-phylogenetic \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--p-metric unweighted_unifrac \
--p-variance-adjusted \
--o-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac.qza
qiime diversity beta-group-significance \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--m-metadata-column Species \
--p-method permanova \
--p-pairwise \
--o-visualization /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_AB_unwUniFrac_permanoa.qzv
qiime diversity beta-group-significance \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--m-metadata-column Species \
--p-method permdisp \
--p-pairwise \
--o-visualization /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_AB_unwUniFrac_permdisp.qzv
qiime diversity pcoa \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac.qza \
--o-pcoa /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac_PCoA.qza
qiime tools export \
--input-path /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac_PCoA.qza \
--output-path /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_unwUniFrac_PCoA
#weighted
qiime diversity beta-phylogenetic \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--p-metric weighted_unifrac \
--p-variance-adjusted \
--o-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac.qza
qiime diversity beta-group-significance \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--m-metadata-column Species \
--p-method permanova \
--p-pairwise \
--o-visualization /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_AB_wUniFrac_permanoa.qzv
qiime diversity beta-group-significance \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--m-metadata-column Species \
--p-method permdisp \
--p-pairwise \
--o-visualization /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_AB_wUniFrac_permdisp.qzv
qiime diversity pcoa \
--i-distance-matrix /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac.qza \
--o-pcoa /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac_PCoA.qza
qiime tools export \
--input-path /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac_PCoA.qza \
--output-path /analyses/2_BetaDiversity/EchinometraEggMbiome_BetaPhyDiversity_wUniFrac_PCoA
ALPHA DIVERSITY
#faith
qiime diversity alpha-phylogenetic \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--p-metric faith_pd \
--output-dir /analyses/3_Alpha/EchinometraEggMbiome_AlphaPhylogenetic_faith_pd
qiime tools export \
--input-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaPhylogenetic_faith_pd/alpha_diversity.qza \
--output-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaPhylogenetic_faith_pd/alpha_diversity
#observed ASVs
qiime diversity alpha \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-metric observed_features \
--output-dir /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_observed_otus
qiime tools export \
--input-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_observed_otus/alpha_diversity.qza \
--output-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_observed_otus/alpha_diversity
#McintoshE
qiime diversity alpha \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-metric mcintosh_e \
--output-dir /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshE
qiime tools export \
--input-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshE/alpha_diversity.qza \
--output-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshE/alpha_diversity
#McintoshD
qiime diversity alpha \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-metric mcintosh_d \
--output-dir /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshD
qiime tools export \
--input-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshD/alpha_diversity.qza \
--output-path /analyses/3_Alpha/EchinometraEggMbiome_AlphaNonPhylogenetic_McintoshD/alpha_diversity
TAXA PLOTS
qiime feature-table group \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-axis sample \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--m-metadata-column Species \
--p-mode sum \
--o-grouped-table /analyses/EchinometraEggMbiome_rare.qza
qiime taxa barplot \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-taxonomy /analyses/EchinometraEggMbiome_AssignTaxa.qza \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--output-dir /analyses/4_TaxaplotsAB
qiime tools export \
--input-path /analyses/4_TaxaplotsAB/visualization.qzv \
--output-path /analyses/4_TaxaplotsAB/visualization
BETA PHYLOGENY
qiime diversity beta-rarefaction \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-metric weighted_unifrac \
--p-clustering-method upgma \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--p-sampling-depth 1602 \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--o-visualization /analyses/5_BetaDiversityAB/EchinometraEggMbiome_wBetaDiversity.qzv
qiime tools export \
--input-path /analyses/5_BetaDiversityAB/EchinometraEggMbiome_wBetaDiversity.qzv \
--output-path /analyses/5_BetaDiversityAB/EchinometraEggMbiome_wBetaDiversity
qiime diversity beta-rarefaction \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-metric unweighted_unifrac \
--p-clustering-method upgma \
--m-metadata-file /analyses/EchinometraEggMbiome_Metadata.txt \
--p-sampling-depth 1602 \
--i-phylogeny /analyses/EchinometraEggMbiome_TreeRooted.qza \
--o-visualization /analyses/5_BetaDiversityAB/EchinometraEggMbiome_unwBetaDiversity.qzv
qiime tools export \
--input-path /analyses/5_BetaDiversityAB/EchinometraEggMbiome_wBetaDiversity.qzv \
--output-path /analyses/5_BetaDiversityAB/EchinometraEggMbiome_unwBetaDiversity
PHYLOSYMBIOSIS
#make environment
git clone –recursive https://github.com/TreeCmp/TreeCmp.git
pip3 install ete3
mkdir /analyses/6_Phylosymbiosis
python topology_script.py
CO-PHYLOGENY
#define core
qiime feature-table core-features \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--p-min-fraction 0.2 \
--p-max-fraction 1.0 \
--p-steps 5 \
--o-visualization /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Core.qzv
qiime tools export \
--input-path /analyses/EchinometraEggMbiome_AssignTaxa.qza \
--output-path /analyses/EchinometraEggMbiome_AssignTaxa
#filter for candidates
qiime taxa filter-table \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-taxonomy /analyses/EchinometraEggMbiome_AssignTaxa.qza \
--p-include g__Photobacterium \
--p-mode contains \
--o-filtered-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Photobacterium.qza
qiime taxa filter-table \
--i-table /analyses/EchinometraEggMbiome_rare.qza \
--i-taxonomy /analyses/EchinometraEggMbiome_AssignTaxa.qza \
--p-include g__Vibrio \
--p-mode contains \
--o-filtered-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Vibrio.qza
#transform table to presence/absence
qiime feature-table presence-absence \
--i-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Photobacterium.qza \
--o-presence-absence-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Photobacterium_PresAbsen.qza
qiime feature-table presence-absence \
--i-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Vibrio.qza \
--o-presence-absence-table /analyses/7_CoPhylogeny/EchinometraEggMbiome_rare_Vibrio_PresAbsen.qza