DOI: 10.5061/dryad.ncjsxkt6n
title: Cryptic genetic variation in brain gene expression precedes the evolution of cannibalism in spadefoot toad tadpoles

description: The origins of novel behaviors are poorly understood, despite behavior’s
 hypothesized roles in evolution. One model, “genetic accommodation”,
 proposes that selection on ancestral phenotypic plasticity may precede the
 evolution of novel traits. A critical assumption of genetic accommodation
 is that ancestral lineages possess heritable genetic variation for trait
 plasticity that is revealed in novel environments, thereby providing the
 raw materials for subsequent refinement of the novel trait in derived
 lineages. Here, we use a combination of behavioral and RNA-seq approaches
 to test this assumption in the context of a novel tadpole behavior:
 predatory cannibalism. Cannibalism evolved in the spadefoot genus Spea,
 where an invertebrate diet induces a carnivorous tadpole morph capable of
 consuming live conspecific tadpoles. In contrast, closely related
 Scaphiopus tadpoles do not induce this carnivorous phenotype. Through
 species comparisons, we found that ancestral Spea likely expressed
 behavioral plasticity and harbored latent (i.e., “cryptic”) genetic
 variation in brain gene expression plasticity associated with
 cannibalism-inducing cues. Further, we found this cryptic genetic
 variation contains genes specifically associated with a dietary response
 and cannibalism in derived Spea. Our results suggest that novel behaviors,
 alongside novel morphologies, can evolve via the process of genetic
 accommodation. More generally, our results provide key evidence for the
 plausibility of genetic accommodation, revealing for the first time that
 cryptic genetic variation — the raw material for the evolution of novel
 traits — exists in natural populations at the level of gene expression.
description: # Cryptic genetic variation in brain gene expression precedes the
 evolution of cannibalism in spadefoot toad tadpoles This readme file was
 generated on 2024-02-24 by Dante J. Nesta GENERAL INFORMATION Title of
 Dataset: Cryptic genetic variation in brain gene expression precedes the
 evolution of cannibalism in spadefoot toad tadpoles 1st
 Author/Corresponding Author Information Name: Dante J. Nesta ORCID:
 0009-0007-8206-660X Institution: Indiana University Bloomington Address:
 915 E 3rd St, Bloomington, IN 47405 Institutional Email:
 [dnesta@iu.edu](mailto:dnesta@iu.edu) Alternative Email:
 [dantenesta1@gmail.com](mailto:dantenesta1@gmail.com) 2nd Author/Principal
 Investigator Information Name: Cristina C. Ledón-Rettig ORCID:
 0000-0002-1417-7486 Institution: Indiana University Bloomington Address:
 915 E 3rd St, Bloomington, IN 47405 Email:
 [crisledo@iu.edu](mailto:crisledo@iu.edu) Date of data (animal)
 collection: 2022-04-25 Geographic location of data collection:
 38°59'40.3"N, 87°01'04.9"W Information about funding
 sources that supported the collection of the data: NSF grant DEB-1754136
 ## DATA & FILE OVERVIEW File List: ScAg.txt - Trait/behavior data for
 Scaphiopus holbrookii samples survival.txt - Survival data for Scaphiopus
 holbrookii samples trinotate_annotation_report.withToadNames.tsv -
 Annotated transcriptome of Scaphiopus holbrookii
 trinotate_annotation_report.withToadSymbols_Spea.tsv - Annotated
 transcriptome of Spea bombifrons sc_di_counts.gene.tsv - RNA-seq raw
 counts of Scaphiopus holbrookii diencephalon tissue sc_tel_counts.gene.tsv
 - RNA-seq raw counts of Scaphiopus holbrookii telencephalon tissue
 sp_di_counts.gene.tsv - RNA-seq raw counts of Spea bombifrons diencephalon
 tissue sp_tel_counts.gene.tsv - RNA-seq raw counts of Spea bombifrons
 telencephalon tissue sc_diPhenoData_full.txt - Treatment/phenotype data
 for Scaphiopus holbrookii samples (for diencephalon tissue)
 sc_telPhenoData_full.txt - Treatment/phenotype data for Scaphiopus
 holbrookii samples (for diencephalon tissue) sp_diPhenoData.txt -
 Treatment/phenotype data for Spea bombifrons samples (for diencephalon
 tissue) sp_telPhenoData.txt - Treatment/phenotype data for Spea bombifrons
 samples (for telencephalon tissue) CGV_Behavior.R - R script to run trait
 and behavioral analyses CGV_GE.R - R script to run gene expression
 analyses Relationship between files, if important: “ScAg.txt” and
 “survival.txt” are to be used with “CGV_Behavior.R”
 “trinotate_annotation_report.withToadNames.tsv”,
 “trinotate_annotation_report.withToadSymbols_Spea.tsv”,
 “sc_di_counts.gene.tsv”, “sc_tel_counts.gene.tsv”,
 “sp_di_counts.gene.tsv”, “sp_tel_counts.gene.tsv”,
 “sc_diPhenoData_full.txt”, “sc_telPhenoData_full.txt”,
 “sp_diPhenoData.txt”, and “sp_telPhenoData.txt” are to be used with
 “CGV_GE.R” Additional related data collected that was not included in the
 current data package: N/A METHODOLOGICAL INFORMATION Description of
 methods used for collection/generation of data: See manuscript. Methods
 for processing the data: See manuscript. Software-specific information
 needed to interpret the data: R (v4.2.2) RStudio (v2022.12.0+353)
 BiocManager (v1.30.22) car (v3.1-2) DESeq2 (v1.38.3) edgeR (v3.40.2)
 glmmTMB (v1.1.9) lme4 (v1.1-35.1) lmerTest (v3.1-3) lmtest (v0.9-40)
 PCAtest (v0.0.2) RColorBrewer (v1.1-3) survival (v3.5-8) survminer
 (v0.4.9) tidyverse (v2.0.0) variancePartition (v1.28.9) Standards and
 calibration information, if appropriate: N/A Environmental/experimental
 conditions: Diet - Food items that individuals in experiment were
 exclusively fed during early life (e.g., detritus or brine shrimp).
 Density - Conspecific density that individuals in experiment experienced
 within rearing containers during early life (e.g., 3 individuals or 8
 individuals). Family - Sibship that individuals belonged to, determined by
 which egg clutches they hatched from. Cannibalism - Quantification of how
 many individuals disappeared from each rearing container overnight across
 the experiment. Describe any quality-assurance procedures performed on the
 data: N/A People involved with sample collection, processing, analysis
 and/or submission: Nathan J. Engbrecht - Locating ephemeral pools with Sc.
 holbrookii present Sarah R. Lagon - Collecting eggs and help rearing
 animals Katie M. Lo - Help rearing animals Brandon Olson - Gosner staging
 samples Daniel L. Woods - Quantifying behaviors Aaron M. Buechlein -
 Sequencing, transcriptome assembly, QC of expression data, preliminary
 interpretation of data Ram Podicheti - QC of expression data,
 interpretation of data Douglas Rusch - Sequencing, transcriptome assembly,
 QC of expression data, interpretation of data Alex Shepard - Manuscript
 feedback C. Hal Terry - Manuscript feedback Eleanor Shell - Manuscript
 feedback Erica Nadolski - Manuscript feedback DATA-SPECIFIC INFORMATION
 FOR: ScAg.txt Number of variables: 21 Number of cases/rows: 256 Variable
 List: ID - Sample identification number. Family - Sibship that each sample
 belonged to. Diet - Food items that animals were fed throughout the
 experiment; either shrimp or detritus. Density - Conspecific density that
 animals were subjected to during the experiment; either high (8 animals
 per container) or low (3 animals per container). Day_0 - Number of live
 animals counted in each container after placement into treatments. Day_1 -
 Number of live animals counted in each container on day 1. Day_2 - Number
 of live animals counted in each container on day 2. Day_3 - Number of live
 animals counted in each container on day 3. Day_4 - Number of live animals
 counted in each container on day 4. Day_5 - Number of live animals counted
 in each container on day 5. Day_6 - Number of live animals counted in each
 container on day 6. Day_7 - Number of live animals counted in each
 container on day 7. Cannibalism - Cumulative number of animals that
 disappeared per container over the course of 8 days. X24h_cannibalism -
 After behavioral assays, animals were kept unfed alongside their stimulus
 animal for 24 hours before being fed prior to sacrifice and dissection.
 This variable describes whether the focal animal consumed their paired
 stimulus animal during this 24-hour period. Dissection_day - Indicates the
 date that animal was sacrificed and dissected. Gosner_stage - Indicates
 the stage of development of each individual following dissection. Mass -
 Indicates the mass (in grams) of each individual prior to dissection.
 Visibility - Indicates (in %) frames in the animal was successfully
 tracked by ToxTrac (animal tracking software) throughout recorded behavior
 assays. Ave_speed - Mean average speed of each animal during behavioral
 assays (in mm/s). Ave_acceleration - Mean average acceleration of each
 animal during behavioral assays (in mm/s^2). Tot_dist - Total distance
 traveled by each animal during behavioral assays (in mm). Missing data
 codes: Missing data represented by NAs and blank cells. Specialized
 formats or other abbreviations used: N/A DATA-SPECIFIC INFORMATION FOR:
 survival.txt Number of variables: 7 Number of cases/rows: 1318 Variable
 List: ID - Sample identification number. Microcosm - Container that each
 animal was reared in. Family - Sibship that each sample belonged to. Diet
 - Food items that animals were fed throughout the experiment; either
 shrimp or detritus. Density - Conspecific density that animals were
 subjected to during the experiment; either high (8 animals per container)
 or low (3 animals per container). Time1 - Day (out of 8) that each animal
 was last recorded alive. Status1 - Alive/dead status of each individual; 1
 means animal survived, 0 means they did not. Missing data codes: None
 Specialized formats or other abbreviations used: N/A DATA-SPECIFIC
 INFORMATION FOR: trinotate_annotation_report.withToadNames.tsv Number of
 variables: 20 Number of cases/rows: 95118 Variable List: Gene_id - Gene
 identifier. Transcript_id - Transcript identifier. Sprot_Top_BLASTX_hit -
 SwissProt BLASTX hit accession number. RNAMMER - Predicted rRNAs. Prot_id
 - Protein identifier. Prot_coords - Mapped coordinates of amino acid
 sequence to DNA sequence. Sprot_Top_BLASTP_hit - SwissProt BLASTP hit
 accession number. Xenopus_proteome_BLASTX - BLASTX match to Xenopus
 tropicalis proteome. Spea_multiplicata_proteome_BLASTX - BLASTX match to
 Spea multiplicata proteome. Pfam - Pfam database protein family hits.
 SignalP - Predicted signal peptides. TmHMM - Predicted transmembrane
 proteins. Eggnog - EggNOG orthology annotation. Kegg - KEGG orthology
 annotation. Gene_ontology_BLASTP - Gene ontology results of BLASTP hits.
 Gene_ontology_Pfam - Gene ontology results of protein family hits.
 Xenopus_Gene_Symbol - Xenopus laevis gene symbol. Xenopus_Gene_Description
 - Xenopus laevis gene description. Spea_multiplicata_GeneSymbol - Spea
 multiplicata gene symbol. Missing data codes: Missing data represented by
 periods (.) DATA-SPECIFIC INFORMATION FOR:
 trinotate_annotation_report.withToadSymbols_Spea.tsv Number of variables:
 20 Number of cases/rows: 96139 Variable List: X.gene_id - Gene identifier.
 Transcript_id - Transcript identifier. Sprot_Top_BLASTX_hit - SwissProt
 BLASTX hit accession number. RNAMMER - Predicted rRNAs. Prot_id - Protein
 identifier. Prot_coords - Mapped coordinates of amino acid sequence to DNA
 sequence. Sprot_Top_BLASTP_hit - SwissProt BLASTP hit accession number.
 Xenopus_proteome_BLASTX - BLASTX match to Xenopus tropicalis proteome.
 Spea_multiplicata_proteome_BLASTX - BLASTX match to Spea multiplicata
 proteome. Pfam - Pfam database protein family hits. SignalP - Predicted
 signal peptides. TmHMM - Predicted transmembrane proteins. Eggnog - EggNOG
 orthology annotation. Kegg - KEGG orthology annotation.
 Gene_ontology_BLASTX - Gene ontology results of BLASTX hits.
 Gene_ontology_BLASTP - Gene ontology results of BLASTP hits.
 Gene_ontology_Pfam - Gene ontology results of protein family hits.
 Xenopus_Gene_Symbol - Xenopus laevis gene symbol. Xenopus_Gene_Description
 - Xenopus laevis gene description. Spea_multiplicata_GeneSymbol - Spea
 multiplicata gene symbol. Missing data codes: Missing data represented by
 periods (.) DATA-SPECIFIC INFORMATION FOR: sc_di_counts.gene.tsv Number of
 variables: 91 Number of cases/rows: 37660 Variable List: TranscriptID -
 Transcript identifier. XenopusBlastHit - Xenopus laevis BLASTN hit
 accession number. XenopusGeneSymbol - Xenopus laevis gene symbol.
 XenopusDescription - Xenopus laevis gene description.
 Spea_multiplicata_BlastHit - Spea multiplicata BLASTN hit accession
 number. Spea_multiplicataGeneName - Spea multiplicata gene name.
 SwissProtBlastAccession - SwissProt homology search hit accession number.
 SwissProtBlastHitTitle - SwissProt homology search hit identifier.
 Variables 9-91 - Sample identification numbers of Scaphiopus holbrookii
 diencephalon tissues. Missing data codes: Missing data represented by
 periods (.) DATA-SPECIFIC INFORMATION FOR: sc_tel_counts.gene.tsv Number
 of variables: 91 Number of cases/rows: 37660 Variable List: TranscriptID -
 Transcript identifier. XenopusBlastHit - Xenopus laevis BLASTN hit
 accession number. XenopusGeneSymbol - Xenopus laevis gene symbol.
 XenopusDescription - Xenopus laevis gene description.
 Spea_multiplicata_BlastHit - Spea multiplicata BLASTN hit accession
 number. Spea_multiplicataGeneName - Spea multiplicata gene name.
 SwissProtBlastAccession - SwissProt homology search hit accession number.
 SwissProtBlastHitTitle - SwissProt homology search hit identifier.
 Variables 9-91 - Sample identification numbers of Scaphiopus holbrookii
 telencephalon tissues. Missing data codes: Missing data represented by
 periods (.) DATA-SPECIFIC INFORMATION FOR: sp_di_counts.gene.tsv Number of
 variables: 15 Number of cases/rows: 76784 Variable List: TranscriptID -
 Transcript identifier. Variables 2-15 - Sample identification numbers of
 Spea bombifrons diencephalon tissues. Missing data codes: Missing data
 represented by periods (.) DATA-SPECIFIC INFORMATION FOR:
 sp_tel_counts.gene.tsv Number of variables: 15 Number of cases/rows: 76784
 Variable List: TranscriptID - Transcript identifier. Variables 2-15 -
 Sample identification numbers of Spea bombifrons telencephalon tissues.
 Missing data codes: Missing data represented by periods (.) DATA-SPECIFIC
 INFORMATION FOR: sc_diPhenoData_full.txt Number of variables: 8 Number of
 cases/rows: 83 Variable List: Sample - Complete sample identifier (Sample
 number, tissue, and species). Food - Food items that animals were fed
 throughout the experiment; either shrimp or detritus. Synonymous with
 ‘Diet’ in other datasets. Density - Conspecific density that animals were
 subjected to during the experiment; either high (8 animals per container)
 or low (3 animals per container). Family - Sibship that each sample
 belonged to. Cannibalism - Cumulative number of animals that disappeared
 per container over the course of 8 days. Gosner_stage - Indicates the
 stage of development of each individual following dissection. Region -
 Brain region information (All diencephalon). ID - Sample identification
 number. Missing data codes: Missing data represented by blank cells
 DATA-SPECIFIC INFORMATION FOR: sc_telPhenoData_full.txt Number of
 variables: 8 Number of cases/rows: 83 Variable List: Sample - Complete
 sample identifier (Sample number, tissue, and species). Food - Food items
 that animals were fed throughout the experiment; either shrimp or
 detritus. Synonymous with ‘Diet’ in other datasets. Density - Conspecific
 density that animals were subjected to during the experiment; either high
 (8 animals per container) or low (3 animals per container). Family -
 Sibship that each sample belonged to. Cannibalism - Cumulative number of
 animals that disappeared per container over the course of 8 days.
 Gosner_stage - Indicates the stage of development of each individual
 following dissection. Region - Brain region information (All
 telencephalon). ID - Sample identification number. Missing data codes:
 Missing data represented by blank cells DATA-SPECIFIC INFORMATION FOR:
 sp_diPhenoData.txt Number of variables: 4 Number of cases/rows: 14
 Variable List: Sample - Complete sample identifier (Sample number, tissue,
 and species). Food - Food items that animals were fed throughout the
 experiment; either shrimp or detritus. Synonymous with ‘Diet’ in other
 datasets. Density - Conspecific density that animals were subjected to
 during the experiment; either high (8 animals per container) or low (3
 animals per container). Morph - Resulting ecomorph induced by treatment
 conditions following conclusion of the experiment (Either omnivore morph
 or carnivore morph). Missing data codes: None DATA-SPECIFIC INFORMATION
 FOR: sp_telPhenoData.txt Number of variables: 4 Number of cases/rows: 14
 Variable List: Sample - Complete sample identifier (Sample number, tissue,
 and species). Food - Food items that animals were fed throughout the
 experiment; either shrimp or detritus. Synonymous with ‘Diet’ in other
 datasets. Density - Conspecific density that animals were subjected to
 during the experiment; either high (8 animals per container) or low (3
 animals per container). Morph - Resulting ecomorph induced by treatment
 conditions following conclusion of the experiment (Either omnivore morph
 or carnivore morph). Missing data codes: None ## SHARING/ACCESS
 INFORMATION Licenses/restrictions placed on the data: N/A Links to
 publications that cite or use the data: TBD Links to other publicly
 accessible locations of the data: N/A Links/relationships to ancillary
 data sets: N/A Was data derived from another source? If yes, list
 source(s): No Recommended citation for this dataset: D. J. Nesta, C. C.
 Ledón-Rettig, Data from "Cryptic genetic variation in brain gene
 expression precedes the evolution of cannibalism in spadefoot toad
 tadpoles." Dryad. Available at 10.5061/dryad.ncjsxkt6n.