DOI: 10.5061/dryad.dz08kprwz
title: Comparative analysis of phenotypic plasticity sheds light on the evolution and molecular underpinnings of locust phase polyphenism

description: Locusts exhibit one of nature’s most spectacular examples of complex
 phenotypic plasticity, in which changes in density cause solitary and
 cryptic individuals to transform into gregarious and conspicuous locusts
 forming large migrating swarms. We investigated how these coordinated
 alternative phenotypes might have evolved by studying the Central American
 locust and three closely related non-swarming grasshoppers in a
 comparative framework. By experimentally isolating and crowding during
 nymphal development, we induced density-dependent phenotypic plasticity
 and quantified the resulting behavioural, morphological, and molecular
 reaction norms. All four species exhibited clear plasticity, but the
 individual reaction norms varied among species and showed different
 magnitudes. Transcriptomic responses were species-specific, but
 density-responsive genes were functionally similar across species. There
 were modules of co-expressed genes that were highly correlated with
 plastic reaction norms, revealing a potential molecular basis of
 density-dependent phenotypic plasticity. These findings collectively
 highlight the importance of studying multiple reaction norms from a
 comparative perspective.
description: Details about the data collection and processing are found in
 the Supplementary Information file<em>.</em>
description: Files can be
 found here: https://doi.org/10.5061/dryad.dz08kprwz <b>Supplementary Data File
 1:</b> Differentially expressed genes for the head tissue of
 <i>piceifrons</i>, and their annotation.
 This table lists all differentially expressed genes for the head
 tissue of <i>piceifrons</i>, discovered by either DEseq2 or
 edgeR, and their annotation by Blast2GO. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p </i>value (padj) were given for each contig.
 Additionally, gene annotation was obtained with Blast2GO, and the
 following outputs were given: the gene description, based on BLAST hits
 (Gene description), contig length (Length), the Expect value associated
 with the best BLAST hit (E value), the mean similarity at the nucleotide
 level to its BLAST hits (Similarity), the Gene Ontology IDs (GO IDs) and
 Gene Ontology names (GO names), and finally the annotation confidence
 level, for which ‘interpro’ refers to sequences with a hit to the interpro
 database, ‘blasted’ refers to sequences with at least one BLAST hit to the
 arthropod nr database, ‘mapped’ was given to sequences for which gene
 ontology information could be retrieved from its BLAST hit(s), and
 ‘annotated’ was only given to sequences for which the mapped GO terms also
 scored above the default thresholds for annotation.
 <b>Supplementary Data File 2:</b> Differentially
 expressed genes for the thorax tissue of <i>piceifrons</i>,
 and their annotation. This table lists all
 differentially expressed genes for the thorax tissue of
 <i>piceifrons</i>, discovered by either DEseq2 or edgeR, and
 their annotation by Blast2GO. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were given for each contig.
 Additionally, gene annotation was obtained with Blast2GO, and the
 following outputs were given: the gene description, based on BLAST hits
 (Gene description), contig length (Length), the Expect value associated
 with the best BLAST hit (E value), the mean similarity at the nucleotide
 level to its BLAST hits (Similarity), the Gene Ontology IDs (GO IDs) and
 Gene Ontology names (GO names), and finally the annotation confidence
 level, for which ‘interpro’ refers to sequences with a hit to the interpro
 database, ‘blasted’ refers to sequences with at least one BLAST hit to the
 arthropod nr database, ‘mapped’ was given to sequences for which gene
 ontology information could be retrieved from its BLAST hit(s), and
 ‘annotated’ was only given to sequences for which the mapped GO terms also
 scored above the default thresholds for annotation.
 <b>Supplementary Data File 3:</b> Differentially
 expressed genes for the head tissue of <i>americana</i>, and
 their annotation. This table lists all differentially
 expressed genes for the head tissue of <i>americana</i>,
 discovered by either DEseq2 or edgeR, and their annotation by Blast2GO.
 For both DEseq2 and edgeR, the Log<sub>2</sub>FoldChange
 (Log<sub>2</sub>FC) and adjusted <i>p</i> value
 (padj) were given for each contig. Additionally, gene annotation was
 obtained with Blast2GO, and the following outputs were given: the gene
 description, based on BLAST hits (Gene description), contig length
 (Length), the Expect value associated with the best BLAST hit (E value),
 the mean similarity at the nucleotide level to its BLAST hits
 (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names (GO
 names), and finally the annotation confidence level, for which ‘interpro’
 refers to sequences with a hit to the interpro database, ‘blasted’ refers
 to sequences with at least one BLAST hit to the arthropod nr database,
 ‘mapped’ was given to sequences for which gene ontology information could
 be retrieved from its BLAST hit(s), and ‘annotated’ was only given to
 sequences for which the mapped GO terms also scored above the default
 thresholds for annotation. <b>Supplementary Data
 File 4:</b> Differentially expressed genes for the thorax tissue of
 <i>americana</i>, and their annotation.
 This table lists all differentially expressed genes for the
 thorax tissue of <i>americana</i>, discovered by either DEseq2
 or edgeR, and their annotation by Blast2GO. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were given for each contig.
 Additionally, gene annotation was obtained with Blast2GO, and the
 following outputs were given: the gene description, based on BLAST hits
 (Gene description), contig length (Length), the Expect value associated
 with the best BLAST hit (E value), the mean similarity at the nucleotide
 level to its BLAST hits (Similarity), the Gene Ontology IDs (GO IDs) and
 Gene Ontology names (GO names), and finally the annotation confidence
 level, for which ‘interpro’ refers to sequences with a hit to the interpro
 database, ‘blasted’ refers to sequences with at least one BLAST hit to the
 arthropod nr database, ‘mapped’ was given to sequences for which gene
 ontology information could be retrieved from its BLAST hit(s), and
 ‘annotated’ was only given to sequences for which the mapped GO terms also
 scored above the default thresholds for annotation.
 <b>Supplementary Data File 5:</b> Differentially
 expressed genes for the head tissue of <i>cubense</i>, and
 their annotation. This table lists all differentially
 expressed genes for the head tissue of <i>cubense</i>,
 discovered by either DEseq2 or edgeR, and their annotation by Blast2GO.
 For both DEseq2 and edgeR, the Log<sub>2</sub>FoldChange
 (Log<sub>2</sub>FC) and adjusted <i>p</i> value
 (padj) were given for each contig. Additionally, gene annotation was
 obtained with Blast2GO, and the following outputs were given: the gene
 description, based on BLAST hits (Gene description), contig length
 (Length), the Expect value associated with the best BLAST hit (E value),
 the mean similarity at the nucleotide level to its BLAST hits
 (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names (GO
 names), and finally the annotation confidence level, for which ‘interpro’
 refers to sequences with a hit to the interpro database, ‘blasted’ refers
 to sequences with at least one BLAST hit to the arthropod nr database,
 ‘mapped’ was given to sequences for which gene ontology information could
 be retrieved from its BLAST hit(s), and ‘annotated’ was only given to
 sequences for which the mapped GO terms also scored above the default
 thresholds for annotation. <b>Supplementary Data
 File 6:</b> Differentially expressed genes for the thorax tissue of
 <i>cubense</i>, and their annotation. This
 table lists all differentially expressed genes for the thorax tissue of
 <i>cubense</i>, discovered by either DEseq2 or edgeR, and
 their annotation by Blast2GO. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were given for each contig.
 Additionally, gene annotation was obtained with Blast2GO, and the
 following outputs were given: the gene description, based on BLAST hits
 (Gene description), contig length (Length), the Expect value associated
 with the best BLAST hit (E value), the mean similarity at the nucleotide
 level to its BLAST hits (Similarity), the Gene Ontology IDs (GO IDs) and
 Gene Ontology names (GO names), and finally the annotation confidence
 level, for which ‘interpro’ refers to sequences with a hit to the interpro
 database, ‘blasted’ refers to sequences with at least one BLAST hit to the
 arthropod nr database, ‘mapped’ was given to sequences for which gene
 ontology information could be retrieved from its BLAST hit(s), and
 ‘annotated’ was only given to sequences for which the mapped GO terms also
 scored above the default thresholds for annotation.
 <b>Supplementary Data File 7:</b> Differentially
 expressed genes for the head tissue of <i>nitens</i>, and
 their annotation. This table lists all differentially
 expressed genes for the head tissue of <i>nitens</i>,
 discovered by either DEseq2 or edgeR, and their annotation by Blast2GO.
 For both DEseq2 and edgeR, the Log<sub>2</sub>FoldChange
 (Log<sub>2</sub>FC) and adjusted <i>p</i> value
 (padj) were given for each contig. Additionally, gene annotation was
 obtained with Blast2GO, and the following outputs were given: the gene
 description, based on BLAST hits (Gene description), contig length
 (Length), the Expect value associated with the best BLAST hit (E value),
 the mean similarity at the nucleotide level to its BLAST hits
 (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names (GO
 names), and finally the annotation confidence level, for which ‘interpro’
 refers to sequences with a hit to the interpro database, ‘blasted’ refers
 to sequences with at least one BLAST hit to the arthropod nr database,
 ‘mapped’ was given to sequences for which gene ontology information could
 be retrieved from its BLAST hit(s), and ‘annotated’ was only given to
 sequences for which the mapped GO terms also scored above the default
 thresholds for annotation. <b>Supplementary Data
 File 8:</b> Differentially expressed genes for the thorax tissue of
 <i>nitens</i>, and their annotation. This
 table lists all differentially expressed genes for the thorax tissue of
 <i>nitens</i>, discovered by either DEseq2 or edgeR, and their
 annotation by Blast2GO. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were given for each contig.
 Additionally, gene annotation was obtained with Blast2GO, and the
 following outputs were given: the gene description, based on BLAST hits
 (Gene description), contig length (Length), the Expect value associated
 with the best BLAST hit (E value), the mean similarity at the nucleotide
 level to its BLAST hits (Similarity), the Gene Ontology IDs (GO IDs) and
 Gene Ontology names (GO names), and finally the annotation confidence
 level, for which ‘interpro’ refers to sequences with a hit to the interpro
 database, ‘blasted’ refers to sequences with at least one BLAST hit to the
 arthropod nr database, ‘mapped’ was given to sequences for which gene
 ontology information could be retrieved from its BLAST hit(s), and
 ‘annotated’ was only given to sequences for which the mapped GO terms also
 scored above the default thresholds for annotation.
 <b>Supplementary Data File 9:</b> Differentially
 expressed genes for all head tissues using the
 <i>piceifrons</i> transcriptome as reference, and their
 annotation. This table lists differentially expressed
 genes for the head tissues of <i>piceifrons, americana,
 cubense</i> and <i>nitens</i>, using the
 <i>piceifrons</i> transcriptome as reference. After aligning
 all filtered reads for the different species to the
 <i>piceifrons</i> head transcriptome, differentially expressed
 genes were calculated with both DEseq2 and EdgeR for each species
 separately. Any gene that is differentially expressed in at least one
 species, was included in the table. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were listed. Additionally, gene
 annotation was obtained with Blast2GO, and the following outputs were
 given: the gene description, based on BLAST hits (Gene description),
 contig length (Length), the Expect value associated with the best BLAST
 hit (E value), the mean similarity at the nucleotide level to its BLAST
 hits (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names
 (GO names), and finally the annotation confidence level, for which
 ‘interpro’ refers to sequences with a hit to the interpro database,
 ‘blasted’ refers to sequences with at least one BLAST hit to the arthropod
 nr database, ‘mapped’ was given to sequences for which gene ontology
 information could be retrieved from its BLAST hit(s), and ‘annotated’ was
 only given to sequences for which the mapped GO terms also scored above
 the default thresholds for annotation.
 <b>Supplementary Data File 10:</b> Differentially
 expressed genes for all thorax tissues using the
 <i>piceifrons</i> transcriptome as reference, and their
 annotation. This table lists differentially expressed
 genes for the thorax tissues of <i>piceifrons, americana,
 cubense</i> and <i>nitens</i>, using the
 <i>piceifrons</i> transcriptome as reference. After aligning
 all filtered reads for the different species to the
 <i>piceifrons</i> thorax transcriptome, differentially
 expressed genes were calculated with both DEseq2 and EdgeR for each
 species separately. Any gene that is differentially expressed in at least
 one species, was included in the table. For both DEseq2 and edgeR, the
 Log<sub>2</sub>FoldChange (Log<sub>2</sub>FC) and
 adjusted <i>p</i> value (padj) were listed. Additionally, gene
 annotation was obtained with Blast2GO, and the following outputs were
 given: the gene description, based on BLAST hits (Gene description),
 contig length (Length), the Expect value associated with the best BLAST
 hit (E value), the mean similarity at the nucleotide level to its BLAST
 hits (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names
 (GO names), and finally the annotation confidence level, for which
 ‘interpro’ refers to sequences with a hit to the interpro database,
 ‘blasted’ refers to sequences with at least one BLAST hit to the arthropod
 nr database, ‘mapped’ was given to sequences for which gene ontology
 information could be retrieved from its BLAST hit(s), and ‘annotated’ was
 only given to sequences for which the mapped GO terms also scored above
 the default thresholds for annotation.
 <b>Supplementary Data File 11:</b> Enrichment of Gene
 Ontology terms of differentially expressed genes, for each species and
 tissue combination. Fisher exact tests were performed
 in Blast2GO by comparing the Gene Ontology terms in the set of
 differentially expressed genes to the ones in the whole transcriptome.
 Results obtained for the four different species and two different tissues
 were all pooled together in a single table. The first three columns
 contain the full name (GO name), ID (GO ID) and category (GO category) of
 each GO term. For each species and tissue combination, the table contains
 the False Discovery Rate associated with a certain GO term (FDR), the
 amount present in the differentially expressed genes (# in DE genes) and
 in the whole transcriptome (# overall), and last the percentage of genes
 that were annotated with that specific annotation term within the set of
 all DE genes (% in DE genes) or within the full transcriptome (%
 overall). <b>Supplementary Data File
 12:</b> WGCNA module membership for each gene in the head
 tissue. Modules of co-expressed genes for the head
 tissue were obtained with WGCNA. For each contig, the module for which it
 obtained the highest membership value was listed in the column ‘Module’.
 As an estimate of the importance of the particular contig in explaining
 the variation of phenotypic traits, a gene significance (GS) value and an
 associated <i>p</i> value was calculated for each tested
 behavioural trait. Additionally, the module membership (MM) of each gene
 was calculated for each module, in addition to the associated
 <i>p</i> value. Finally, the table contains the gene
 annotation of Blast2GO for each gene. The following outputs were given:
 the gene description, based on BLAST hits (Gene description), contig
 length (Length), the Expect value associated with the best BLAST hit (E
 value), the mean similarity at the nucleotide level to its BLAST hits
 (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names (GO
 names), and finally the annotation confidence level, for which ‘interpro’
 refers to sequences with a hit to the interpro database, ‘blasted’ refers
 to sequences with at least one BLAST hit to the arthropod nr database,
 ‘mapped’ was given to sequences for which gene ontology information could
 be retrieved from its BLAST hit(s), and ‘annotated’ was only given to
 sequences for which the mapped GO terms also scored above the default
 thresholds for annotation. <b>Supplementary Data
 File 13:</b> WGCNA module membership for each gene in the thorax
 tissue. Modules of co-expressed genes for the thorax
 tissue were obtained with WGCNA. For each contig, the module for which it
 obtained the highest membership value was listed in the column ‘Module’.
 As an estimate of the importance of the particular contig in explaining
 the variation of phenotypic traits, a gene significance (GS) value and an
 associated <i>p</i> value was calculated for each tested
 behavioural trait. Additionally, the module membership (MM) of each gene
 was calculated for each module, in addition to the associated
 <i>p</i> value. Finally, the table contains the gene
 annotation of Blast2GO for each gene. The following outputs were given:
 the gene description, based on BLAST hits (Gene description), contig
 length (Length), the Expect value associated with the best BLAST hit (E
 value), the mean similarity at the nucleotide level to its BLAST hits
 (Similarity), the Gene Ontology IDs (GO IDs) and Gene Ontology names (GO
 names), and finally the annotation confidence level, for which ‘interpro’
 refers to sequences with a hit to the interpro database, ‘blasted’ refers
 to sequences with at least one BLAST hit to the arthropod nr database,
 ‘mapped’ was given to sequences for which gene ontology information could
 be retrieved from its BLAST hit(s), and ‘annotated’ was only given to
 sequences for which the mapped GO terms also scored above the default
 thresholds for annotation. <b>Supplementary Data
 File 14</b>: Archive (zip) file containing raw data from behavioural
 assays for all four species of <i>Schistocerca</i>
 (Behaviour_dataset.csv) <b>Supplementary Data
 File 15</b>: Archive (zip) file containing raw colour data (jpg
 images and coordinates) for all four species of
 <i>Schistocerca</i> for running the program patternize
 (folder: Raw_colour_data_and_coordinates)
 <b>Supplementary Data File 16</b>: Archive (zip) file
 containing raw data from morphological measurements for all four species
 of <i>Schistocerca</i> (Morphology_dataset.csv)
 <b>Supplementary Data File 17</b>: Archive (zip) file
 containing: (1) Text file describing R scripts for running edgeR and
 DESeq2 using SARTools (Code_DE_analyses.txt) [raw reads used for these
 analyses are included in Supplementary Data File 18]
 <b>Supplementary Data File 18</b>: Archive (zip) file
 containing: (a) Raw data (raw count data and phenotypic trait data) for
 all four species of <i>Schistocerca</i> for running the
 program WGCNA (folder: Raw_data_for_DE_WGCNA)
 <b>Supplementary Data File 19:</b> Archive (zip) file
 containing: (a) FASTA file containing the alignment used for building the
 phylogeny of hexamerins (hexamerin.fasta); (b) Phylip file containing the
 alignment used for building the phylogeny of hexamerins (hexamerin.phy);
 (c) Newick file containing the maximum likelihood tree from the
 phylogenetic analysis of hexamerins (hexamerin.tre)
 <b>Supplementary Software File 1:</b> Archive (zip)
 file containing RMarkdown file used for analysing behavioural reaction
 norm data (Rmarkdown_behaviour.Rmd)
 <b>Supplementary Software File 2:</b> Archive (zip)
 file containing Html file describing codes used for analysing behavioural
 reaction norm data (Rmarkdown_behaviour.html)
 <b>Supplementary Software File 3:</b> Archive (zip)
 file containing RMarkdown file used for running patternize to analyse
 colour reaction norm data (Rmarkdown_colour.Rmd)
 <b>Supplementary Software File 4</b>: Archive (zip)
 file containing Html file describing codes used for running patternize to
 analyse colour reaction norm data (Rmarkdown_colour.html)
 <b>Supplementary Software File 5:</b> Archive (zip)
 file containing RMarkdown file used for analysing morphological reaction
 norm data (Rmarkdown_morphology.Rmd)
 <b>Supplementary Software File 6:</b> Archive (zip)
 file containing Html file describing codes used for analysing
 morphological reaction norm data (Rmarkdown_morphology.html)
 <b>Supplementary Software File 7:</b> Archive (zip)
 file containing RMarkdown file used for running WGCNA
 (Rmarkdown_WGCNA.Rmd) <b>Supplementary Software
 File 8:</b> Archive (zip) file containing Html file describing codes
 used for running WGCNA (Rmarkdown_WGCNA.html)