DOI: 10.5061/dryad.dz08kps3d
title: Divergence between sea urchins and their microbiota following speciation

description: 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.
description: # Divergence between sea urchins and their microbiota following speciation
 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](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](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