DOI: 10.5061/dryad.bk3j9kdn0
title: Statistical methods and data for: Absence of heterosis for hypoxia tolerance in F1 hybrids of Tigriopus californicus

description: This data set includes generalized structural equation models (SEM) for
 the parental populations and hybrid populations in Stata 18, code for
 figures in R and data in CSV format.  We used SEM over linear
 regression in order to also generate estimates of the errors and
 unexplained variance of the midpoint value of the parents.
  First, we encoded sex, nuclear genome composition, collection
 cohort, days in lab after collection, and arena as variables in both
 models.  For the hybrid model, we also added terms for
 mitochondrial genome and interaction terms between sex and nuclear genome
 composition because we observed a different trend in sex differences for
 one population.  Separate SEM equations were necessary since
 parental populations were perfectly colinear with mitochondrial type,
 whereas F1 hybrids were not.  Since observations were grouped by
 arena and trial, we utilized a clustered standard error calculator when
 combining both models to allow for different variance between
 populations.  After, we tested the linear combinations of the
 means to determine if F1 hybrids performed significantly different from
 the midpoint value of the two parental populations. By using the estimates
 from the SEM, we were able to include the estimated error from the models
 into the equation as well.  Lastly, we tested the linear
 combinations of the means to determine if the reciprocal crosses of F1
 hybrids performed significantly differently from each other. 
description: Copepods were collected from four locations along the California
 coastline in December 2019 and August 2021 under collection permits
 S-192510001-19262-001 and S-192510001-21127-001. All copepods were then
 transported to the University of North Carolina at Chapel Hill where they
 were maintained for experiments.  Gravid females from each population were
 transferred to petri dishes with artificial sea water (ASW) and stored in
 a 12L:12D light cycle at 20°C for at least one month (equivalent to one
 generation) prior to testing.  Salinity was adjusted to 35 ppt, and ground
 commercial fish flakes, which served as food, were added ad libitum every
 other week. F1 hybrids were generated by placing virgin females of one
 population with virgin males from a second population. Copepods were
 grouped by sex and population then placed in aggregates of 5 into arenas
 of a water bath.  Temperature was maintained at 20°C by a cooling tower
 that circulated chilled water through the water bath, and the water bath
 was kept inside a custom-built glove box.  An air stone placed in the
 water bath was used to deliver gas into the water continuously.  Nitrogen
 gas was delivered to the water bath until the dissolved oxygen level
 reached 0.05 mg/L or less and maintained for 20 hours, then atmospheric
 air was delivered for 10 hours.  At the end of the assay, copepods were
 prodded gently with a needle probe to prompt a swimming response, and, if
 copepods did not swim away, they were assumed to be dead. After each
 assay, all copepods were sacrificed, individually lysed, and genotyped. 
 After lysis, a portion of the mitochondrial DNA was amplified via
 polymerase chain reaction (PCR) using population specific primers.  Any
 confirmed heteroplasmic individuals were excluded from further
 analysis.
description: # Statistical methods and data for Absence of heterosis for hypoxia
 tolerance in F1 hybrids of Tigriopus californicus
 [https://doi.org/10.5061/dryad.bk3j9kdn0](https://doi.org/10.5061/dryad.bk3j9kdn0) ## Description of the data and file structure The data set includes experimental information (day, trial, pH, dissolved oxygen, temperature, time, etc.) and observations (alive, heteroplasmic or homoplasmic status) of *Tigriopus californicus* collected from hypoxia assays and genotyping.  Statistical analysis was performed in Stata 18, and the .do file for recreating the statistics.  For convenience, the data output file from the statistical analysis is also included.  Figures were generated in R, and an .rmd file containing the code for recreating the figures is included. ### Files and variables #### File: Plasmy\_Hypoxia\_Tolerance\_-\_Sheet1.csv **Description:** Data file of measurements and observations from hypoxia assays and genotype analysis. ##### Variables * trial: Numeric value for all samples measured at the same time. * date.init: Date in MM/DD/YY format for the date that the hypoxia assay was started. * time.init: Time in 24-hour clock format for the time that the hypoxia assay was started. * DO.init: Dissolved oxygen level (in mg/L) of the water bath when the hypoxia assay was started. * temp.init: Temperature (in degrees celsius) of the water bath when the hypoxia assay was started. * pH.init: pH of the water bath when the hypoxia assay was started. * date.start: Date in MM/DD/YY format for the date that the dissolved oxygen level was below 0.05 and the anoxia portion of the hypoxia assay began. * time.start: Time in 24-hour clock format when the dissolved oxygen level was below 0.05 and the anoxia portion of the hypoxia assay began. * DO.start: Dissolved oxygen level (in mg/L) of the water bath the dissolved oxygen level was below 0.05 and the anoxia portion of the hypoxia assay began. * temp.start: Temperature (in degrees celsius) of the water bath when the dissolved oxygen level was below 0.05 and the anoxia portion of the hypoxia assay began. * pH.start: pH of the water bath when the dissolved oxygen level was below 0.05 and the anoxia portion of the hypoxia assay began. * date.reoxy: Date in MM/DD/YY format when the anoxia portion of the hypoxia assay ended and the reoxygenation period began. * time.reoxy: Time in 24-hour clock format when the anoxia portion of the hypoxia assay ended and the reoxygenation period began. * DO.reoxy: Dissolved oxygen level (in mg/L) of the water bath when the anoxia portion of the hypoxia assay ended and the reoxygenation period began. * temp.reoxy: Temperature (in degrees celsius) of the water bath when the anoxia portion of the hypoxia assay ended and the reoxygenation period began. * pH.reoxy: pH of the water bath when the anoxia portion of the hypoxia assay ended and the reoxygenation period began. date.end: Date in MM/DD/YY format that the hypoxia assay ended and copepods were sacrificed. * time.end: Time in 24-hour clock format that the hypoxia assay ended and copepods were sacrificed. * DO.end: Dissolved oxygen level (in mg/L) of the water bath that the hypoxia assay ended and copepods were sacrificed. * temp.end: Temperature (in degrees celsius) of the water bath when the hypoxia assay ended and copepods were sacrificed. * pH.end: pH of the water bath when the hypoxia assay ended and copepods were sacrificed. * arena: Location in the water bath that the sample was located.  There were 8 arenas in the water bath, and samples were assigned randomly. * sex: Sex of the copepod; factor variable of male or female. * generation: Generation of the copepod; factor variable of parent (if non-hybridized) or F1 (if hybridized). * maternal: Maternal lineage of the copepod. * paternal: Paternal lineage of the copepod. * nuclear: Shorthand for the nuclear composition of the copepod; does not distinguish between maternal and paternal lineages. * swim: True or false binomial observation of whether the copepod was swimming at the end of the hypoxia assay. * coll.cohort: Year of collection for the stock (or the parental stock). * date.coll: Date of collection for the stock (or the parental stock). * time.lab: Difference in time between the date collected (date.coll) and the start date of the experiment (date.init). * ID: Individual ID of copepod for tracking during PCR analysis. * plasmy: Factor variable indicating whether the individual was homoplasmic (homo), heteroplasmic (hetero), or unable to be determined (blank). * notes: Miscellaneous observation notes. #### File: data\_output.txt **Description:** Results of the F1_Hybrid_Hypoxia.do file printed to a .txt file for those without access to Stata 18. ## Code/software Stata 18 for the .do file. RStudio v. 2023.06.2+561 for the .rmd file.