https://doi.org/10.5061/dryad.ncjsxkt3g

Description of the data and file structure

We include in this submission all data required to run included code (see below for which order to run scripts):

1. bedtools_gene_counts.gff

   Tab delimited text file with rows corresponding to counts of genes for each sample. The first column is the Ensembl gene name.

2. design.csv

   Comma delimited text file with information for each female for which we have a transcriptome. It contains the following columns:

   1. female - female ID
   2. spawn - whether a female laid eggs (TRUE or FALSE)
   3. lake - female lake of origin (Paxton or Priest)
   4. species - female species (Limnetic or Benthic)
   5. treatment - experimental treatment (Het = heterospecific courtship, Con = conspecific courtship, Fem = social control)
   6. preference - courtship preference score (0-4); NA for Fem treatment because there was no courtship
   7. trial time - length of trial (MM:SS)
   8. batch - RNA extraction batch
   9. RIN - RNA RIN score from Agilent Bioanalyzer; NA if missing (e.g., due to instrument error)

3. Female choice behavior data.csv

   Comma delimited text file with behavioral information for each female that was in the behavioral experiments. It contains the following columns:

   1. File name - JWatcher file name
   2. Trial - trial number
   3. Trt Code - Concatenation of lake code, species code, and treatment code
   4. Lake - Lake code (Pa = Paxton, Pr = Priest)
   5. Species - Species code (L = Limnetic, B = Benthic)
   6. Trt - Treatment code (C = conspecific courtship, H = heterospecific courtship, F = social control)
   7. Sequenced - was the RNA sequenced? (Y/N)
   8. Date - date of behavioral trial (MDDYY)
   9. Obs - observer initials (all trials had two observers)
   10. Duration - duration of trial
   11. Follow - number of times female followed male when he did a lead
   12. Lead - number of times male tried to lead female
   13. Examine - number of times female examined a male’s nest
   14. Show - number of times a male showed a female his nest
   15. Male Approach - number of times a male approached a female
   16. Rub - number of times a male rubbed a female while she was in his nest
   17. Angle - number of times a female angled her body (a signal of interest)
   18. Female Approach - number of times a female approached a male
   19. Follow/Lead - proportion of leads where a female followed
   20. Examine/Show - proportion of shows where a female examined a nest
   21. Preference Score - preference score (0-4)
   22. Zigs - number of male zig-zag displays
   23. Bites - number of times male bit female
   24. Chases - number of times male chased female
   25. Enter - number of times female entered a male nest
   26. Spawn - number of times a male spawned in his nest
   27. Swims - number of movements that were greater than 1 body length
   28. Nesting - time (ms) male spent within one body length of his nest

4. Female trial data.csv

   Comma delimited text file with experimental trial information for each female for each trial in the behavioral experiments. It contains the following columns:

   1. Trial # - trial number
   2. Jwatcher file - JWatcher file name
   3. Date - date of behavioral trial (M/DD/YY)
   4. Time Start - time trial started (HH:MM)
   5. Trial Time - total trial time (MM:SS)
   6. Observer - observer initials
   7. Female Lake - female lake (Paxton or Priest)
   8. Female Species - female species (Limnetic or Benthic)
   9. Female Tank - female tank ID
   10. Male Lake - male lake (Paxton or Priest)
   11. Male Species - male species (Limnetic or Benthic)
   12. Nesting Tank - male nesting tank ID
   13. Treatment - treatment (Con Male = conspecific courtship, Het Male = heterospecific courtship, Female-Female = social control)
   14. Male Mark - male elastomer mark (R/L); 4 locations: h = head, d1 = front dorsal, d2 = back dorsal, t = tail; 2 colors: p = pink, w = white; in Female-Female trials no fish were marked so those cells are marked NA; null = mark should have been given but was not
   15. Comments - comments, if none the cell is left blank
   16. Surgery Time - time from fish decapitated to brain in tube of RNAlater (MM:SS); NA for mistrials where there was no surgery
   17. # Eggs - number of eggs; NA for mistrials where there was no surgery
   18. Female Picture Date - date female had photo taken; NA if missing
   19. Female Weight (g) - female weight (g); NA if missing

5. Gene Expression.TPS

   TPS file with landmark data used to generate size information.

6. Male data.csv

   Comma delimited text file with phenotypic information about each male for each trial in the behavioral experiments. It contains the following columns (NA indicates missing data):

   1. Trial Number - trial number
   2. Trial Date - date of behavioral trial
   3. Nesting Tank - male nesting tank ID
   4. Lake/ Species - lake of origin and species of male (Pax = Paxton, Mrs = Priest, B = Benthic, L = Limnetic)
   5. Home Tank - ID of tank male came from
   6. Mark - male elastomer mark (R/L); 4 locations: h = head, d1 = front dorsal, d2 = back dorsal, t = tail; 2 colors: p = pink, w = white
   7. Mark Date - date when male was marked
   8. Pheno Date - date when male was phenotypes
   9. Picture Date - date when male was photographed
   10. Weight - male weight (g)
   11. Throat Area - male throat area (0-5 with 1/2 point increments)
   12. Throat Int - male throat intensity (0-5 with 1/2 point increments)
   13. Eye Color - male eye color
   14. Eye Int - male eye color intensity (0-5 with 1/2 point increments)
   15. Body Color - male body color
   16. Body Int - male body color intensity (0-5 with 1/2 point increments)
   17. Body Drk - male body darkness (0-5 with 1/2 point increments)
   18. Spots - how many black spots did male have? (None, Few, Medium, Many, All over)
   19. Mottled? - did male have stripes? (Y/N)

Code/Software

To recreate our analyses and figures, execute the included code in the following order:

1. Behavior and Morphology script.R

   Script for quantifying and analyzing male and female behavior and morphology.

2. DESeq2 script.R

   Script for running gene expression analyses using DESeq2.

3. WGCNA script.R, WGCNAscript2a1.R, WGCNAscript2a2.R

   Scripts for running WGCNA.

4. gprofiler script.R

   Scripts for GO analyses of differentially expressed genes from #2 and modules from #3.