DOI: 10.5061/dryad.8gtht76vb
title: Data from: Response to MHC-based olfactory cues in a mate choice context in two species of darter (Percidae: Etheostoma)

description: Mate choice is hypothesized to play an important role in maintaining high
 diversity at major histocompatibility complex (MHC) genes in vertebrates.
 Many studies have revealed that females across taxa prefer the scent of
 males with MHC genotypes different to their own. In this study we tested
 the “opposites-attract” hypothesis in two species of darter with known
 differences in female criteria used in mate choice: in the fantail darters
 (a paternal-care species), females prefer males with visual traits related
 to nest guarding and egg tending, while in rainbow darters (not a
 paternal-care species) female mate choice criteria are unknown. In
 dichotomous mate-choice trials, we presented females of both species with
 the scents of conspecific males with MHC class IIb genotypes that were
 either similar or dissimilar to that of the focal female. We evaluated the
 proportion of time each female spent with each male and calculated the
 average strength of female preference for both species. Female fantail
 darters demonstrated a preference for the scent of males with similar
 (rather than dissimilar) MHC genotypes, but this result was not
 statistically significant. Rainbow darter females showed no preference for
 the scent of males with similar or dissimilar MHC genotypes. Our results
 do not support the “opposites-attract” hypothesis in darters.
description: <em>Animal care and use
 statement</em> This research was conducted with
 the approval and oversight of the Bloomington Institutional Animal Care
 and Use Committee (BIACUC protocol numbers 19-003, 17-016, and 20-015).
 Scientific collection permits were issued to KMM by the Indiana Department
 of Natural Resources. <em>2.1 Subject
 collection</em> Individuals of the rainbow darter
 (<em>Etheostoma caeruleum</em>) and the fantail darter
 (<em>Etheostoma flabellare</em>) were collected with a seine
 net from Clear Creek, Bloomington, Indiana, Monroe County
 (39.12118432195659, -86.53886585996088) on April 6, 2019. We identified
 individuals to species based on morphological characteristics (Page and
 Burr, 2011; Simon, 2011). Individuals were sexed based on breeding
 characteristics. In both species, we identified females as gravid by the
 presence of distended abdomens. We identified male rainbow darters in
 breeding condition by nuptial coloration, and male fantail darters in
 breeding condition by darkened body color and the presence of egg mimics
 on the first dorsal fin (Kuehne and Barbour, 1983; Page, 1983).
 Individuals were transported to Indiana University in aerated, covered
 buckets in an air-conditioned vehicle. <em>2.2
 Care and housing</em> Individuals were housed
 separately by sex and species. Males and females of the same species were
 housed such that they were unable to see conspecifics of the opposite sex.
 We housed individuals in 20-gallon tanks (30" x 12" x 12")
 equipped with power filters and air pumps mounted with large air stones.
 River rock aquarium gravel was provided as substrate. Small terra cotta
 pots were provided as hides. We conditioned the tank water with Amquel
 Plus and performed 20% water changes once per week. Individuals were fed
 frozen brine shrimp and bloodworms once per day, as much as could be
 consumed in 5 minutes. <em>2.3 Fin clipping and
 VIE tagging</em> Individuals were fasted
 overnight ahead of the fin clipping and tagging procedures. Individuals
 were anesthetized with MS-222 (25 mg/L) until gill movement was slow and
 steady and the subject did not respond to touch. Paper towels moistened
 with conditioned water were placed on the gill covers to maintain the
 subject’s breathing during the procedure. Each subject was measured for
 standard length (tip of the nose to the caudal peduncle). A small
 (3mm<sup>2</sup>) piece of fin tissue was removed from the
 anal fin of each subject. Iodine was used to sterilize the fin clip site
 during the procedure. Each individual was injected with three visible
 implant elastomer (VIE) tags (Northwest Marine Technology), one anterior
 to the first dorsal fin, one between the first and second dorsal fin, and
 one behind the second dorsal fin. Each VIE tag combination was a unique
 color pattern used to identify the individual. The total procedure from
 start to finish took approximately three minutes. Individuals were place
 in conditioned water and allowed to recover from the anesthesia. Recovery
 was pronounced when the subject resumed normal swimming and breathing. All
 individuals were monitored over the following 24 hours for full recovery.
 All fin tissue regenerated within 3 weeks, and all VIE tag wounds healed
 within one week with no sign of infection or long-term effects. Behavioral
 trials were not performed until all individuals had fully healed and
 recovered from fin clipping and VIE tagging. Fin clips were frozen and
 stored for genotyping. <em>2.4 MHC
 genotyping</em> All individuals were genotyped at
 a 164 base pair portion of the MHC class IIb gene (exon 2) according to
 deep-sequencing methods developed for fish (Lighten et al., 2014). We used
 custom primers and protocols we had developed for previous research on
 darters (Million and Lively, 2022). Our prior research indicated the
 presence of up to 5 MHC IIb loci in both our species of interest, meaning
 an individual genotype could contain 1 to 10 alleles. The number of
 alleles was recorded for each genotype as well as the identity of each
 allele in each genotype. <em>2.5 Breeding
 conditions in the lab</em>  To maintain the
 breeding condition of all individuals in the lab during the trials, we
 manipulated ambient light and temperature to mimic the conditions of the
 natural breeding season. We kept the day length cycle at 14L:10D and
 maintained the ambient room temperature at 28 degrees Celsius. We assessed
 the success of our efforts by observing female gravidity and male nuptial
 characteristics as described above. <em>2.6
 Construction of motorized visual stimuli</em> To
 control for possible differences in visual and behavioral cues between
 live males, we constructed motorized visual stimuli with which to pair the
 olfactory cues during the trials. These models allowed us to present
 identical visual stimuli to the focal females and provide a visual context
 in which to interpret the olfactory stimuli. Motorized, painted models
 have been successfully used by other researchers for dichotomous choice
 trials in darters (Williams, Gumm, and Mendelson, 2013, Williams and
 Mendelson, 2010). 3D models of a male fantail darter and a male rainbow
 darter were purchased from the website TurboSquid (model artist:
 “adicaza”) (Figure 1). Files were modified for compatibility with a 3D
 printer. Life-sized male models were 3D printed using a Formlabs Form 2
 printer and Formlabs SG dental resin. 3D printing was performed in the
 Lennon lab at Indiana University. High-resolution images of a male fantail
 darter and a male rainbow darter were obtained from the North American
 Native Fishes Association photo gallery, and color palettes were developed
 from colors isolated from the images. Models were painted with colors and
 patterns resembling a male of each species in breeding condition (Figure
 2). Models of the same species were painted to be visually identical.
 Small glass amber-colored beads (2 mm outside diameter) were glued to the
 first dorsal fin of the fantail darter models to resemble egg mimics.
 Models were mounted onto stepper motors using magnets and transparent
 plexiglass platforms. Stepper motors were attached to microcontrollers,
 which in turn were connected to an Arduino Uno REV3 unit. Model movements
 were programmed using the software Arduino IDE 1. The Arduino unit was
 programmed so that the models made synchronized movements that resemble
 the courtship displays of live breeding males. Two visually identical
 models of each species were created. <em>2.7
 Experimental setup</em> A 37.9-liter tank (50.8
 cm" x 25.4 cm x 30.5) was set up in a location where the subject
 would have no visual contact with other individuals during the trial
 (Figure 1). The focal tank was surrounded with sheets of panda paper
 (Vivasun) to block external light and visual stimuli. The bottom of the
 tank was blacked out using matte black aquarium decal material. The tank
 was separated into three zones of preference, marked by narrow white
 lines. The neutral zone was centered in the middle and took up 80% of the
 tank bottom surface area. Each preference zone on the left and right took
 up 10% of the tank bottom surface area. Ambient temperature in the trial
 area matched that of the housing area. The tank was lit using ambient
 fluorescent light and a Nicrew ClassicLED Plus full-spectrum aquarium
 light mounted above the tank. Two 10-gallon tanks were placed underneath
 the focal tank, completely out of visual range of the focal subject. All
 tanks were partially filled with conditioned, aerated water (3/4 full for
 the focal tank, ½ full for each olfactory stimulus tank). At the
 conclusion of each trial, all tanks were emptied, scrubbed with 95%
 ethanol, allowed to air dry, and refilled with the same volume of fresh
 water. An AKASO EK7000 Pro 4K Action Camera was mounted near the tank to
 record all trials. All motorized and electronic devices were connected to
 a remote control so that they could be turned on and off without the
 observer in the room during a trial. The trials were monitored outside the
 room in real time using a cell phone remotely connected to the
 camera.  We pre-determined that if a participant showed
 signs of severe stress (gasping, attempting to jump out of the tank) at
 any point during a trial, then the trial would have been immediately
 terminated and the subject returned to housing. However, during these
 trials no individuals displayed signs of extreme stress, so we did not
 terminate any of the trials. No individual participated in more than one
 trial per 24 hour period, either as a subject or as a stimulus individual.
 Submersible pumps (Homasy 300 L/H, 4 W) were placed into each olfactory
 stimulus tank. The pumps were connected to the focal tank via clear food
 grade vinyl tubing (5/16 ‘’ ID, 7/16 ‘’ OD). The pumps were primed with
 clean conditioned water before and after every trial. Plastic clamps were
 used to control the water flow and to ensure that the water flowed into
 the focal tank at the same rate on each side. This was visually confirmed
 prior to the start of every trial. <em>2.8
 MHC-based choice trials</em> We used dichotomous
 choice trials to assess the response of females of each species to
 MHC-based olfactory cues from conspecific males (Figure 3). The method is
 standard for evaluating mate choice preferences in darters (Mattson et
 al., 2020; Williams et al., 2013). For each trial, to control for visual
 and behavioral differences between the two live males, two visually
 identical male models of the same species as the focal female were placed
 on either side of the focal tank and mounted on stepper motors. Before the
 start of the trial, blinds were used to block the view outside the tank of
 the focal female during acclimation. One focal female was acclimated to
 the focal tank for a minimum of 10 minutes. A female was considered
 acclimated when she initiated normal free swimming around all sides of the
 tank and exhibited normal breathing patterns. One live stimulus male was
 acclimated to each of the olfactory stimulus tanks out of view of the
 focal female for 10 minutes, the same amount of time that the focal female
 was acclimated to the focal tank. After the acclimation
 period, the blinds were removed, and the focal female was allowed to
 explore the tank and visually inspect each model. The trial began after
 the female had crossed into both preference zones, seen both models, and
 returned to the neutral zone. A trial did not progress if the focal
 individual did not cross both sides and return to the middle. At the start
 of the trial, both male models were turned on to provide identical visual
 displays for the female, and water was pumped into the focal tank on
 either side from each of the stimulus tanks. During each trial the focal
 female was presented with the scent of a male with an MHC genotype similar
 (differing by no more than one allele) or identical to her own on one
 side, and one male with an MHC genotype completely dissimilar to her own
 (none of his alleles matched hers) on the other side. To control for
 heterozygosity, we matched stimulus males so that they either had the same
 number of alleles in their genotypes or only differed in number by one.
 Due to small sample sizes, some females participated in more than one
 trial. In these cases, a female was always presented with a completely
 novel pair of stimulus males. The trial period lasted for 10 minutes, and
 the entire trial was observed outside the room by camera and was recorded.
 To control for potential side bias in the focal females, we switched the
 sides on which the similar and dissimilar male scents were presented to
 the focal female between each trial, and we switched the sides of the two
 models between each trial. Sample sizes for the trials were N = 4 for
 rainbow darters and N = 5 for fantail darters. Total number of trials run
 were N = 9 for rainbow darters and N = <em>2.9
 Trial analyses</em> Videos were analyzed using
 the software Boris (Friard and Gamba, 2016). We constructed an ethogram in
 the software to use during analysis. All videos were scored for the amount
 of time the focal female spent in each of the three zones of the focal
 tank. If during a trial a female spent more than 80% of her time in the
 neutral zone, we concluded that she had not interacted enough with the
 stimuli for us to score her preferences, and that trial was set aside from
 further analysis.  A strength of preference (SOP)
 equation was used to evaluate the responses to the stimuli. The equation
 is SOP = (S – D)/(S + D), where S = the time spent in the preference zone
 with the male with a similar MHC genotype and D = the time spent in the
 preference zone with the male with a dissimilar MHC genotype. The values
 obtained range from a minimum of -1 (strong preference for dissimilar
 genotype) to a maximum of 1 (strong preference for a similar genotype). A
 value at or near 0 indicates no strong preference for either genotype.
 Further data analysis was conducted using the software R version 4.1.0 (R
 Development Core Team, 2018). Mean SOP between the species was compared
 using a Wilcoxon rank-sum test. Proportion of time spent with the male
 with the similar MHC genotype (as opposed to the male with the dissimilar
 MHC genotype) was tested against the null hypothesis that the females
 spent equal time with both males using a one-sample Wilcoxon test. Power
 analyses on the data were conducted using the software G*Power.
description: The .csv files can be opened as text files in Notepad or any
 similar text editing software. They can also be opened in Microsoft Excel
 or open-source office software such as Apache OpenOffice.
 The fasta file can be opened as a .txt file in Notepad or any
 similar text editing software, or in any software that reads FASTA files
 (such as BioEdit).
description: # Response to MHC-based olfactory cues in a mate choice context in two
 species of darter (Percidae: Etheostoma) This data README.md file was
 generated on 2023-07-20 by Kara Million GENERAL INFORMATION 1. Response to
 MHC-based olfactory cues in a mate choice context in two species of darter
 (Percidae: Etheostoma) 2. Author Information A. Lead Investigator Contact
 Information Name: Kara Million (contact for questions) Institution:
 University of North Alabama Address: Email:
 B. Associate or Co-investigator Contact
 Information Name: Melissa Proffitt Institution: University of Tennessee
 Knoxville Address: Email: C: Associate or
 Co-investigator Contact Information Name: Sierra Reese Institution:
 University of California Davis Address: Email:
 3. Date of data collection: 2019-2020 4.
 Geographic location of data collection: Clear Creek, Bloomington, Indiana,
 Monroe County, USA (39.12118432195659, -86.53886585996088) 5. Information
 about funding sources that supported the collection of the data: KMM was
 supported 356 by an NIH Common Themes in Reproductive Diversity Training
 Grant during this research (T32 357 HD049336). SJR was supported by an NSF
 summer REU (grant number 1460949). SHARING/ACCESS INFORMATION 1.
 Licenses/restrictions placed on the data: This work is licensed under a
 CC0 1.0 Universal (CC0 1.0) Public Domain Dedication license. 2. Links to
 publications that cite or use the data: pending 3. Links to other publicly
 accessible locations of the data: NA 4. Links/relationships to ancillary
 data sets: 5. Was data
 derived from another source? A. If yes, list source(s):
 6. Recommended citation
 for this dataset: Million, Kara; Proffitt, Melissa; Reese, Sierra (2023),
 Response to MHC-based olfactory cues in a mate choice context in two
 species of darter (Percidae: Etheostoma), Dryad, Dataset (doi pending)
 DATA & FILE OVERVIEW 1. File List: MHC alleles.fasta A FASTA file
 containing the DNA sequences of all the MHC alleles included in this
 study. Pairings.csv A file containing the IDs of all focal females and
 stimulus males used in each trial in this study. IDs correspond to the VIE
 tag combinations used to distinguish each individual, where Y = yellow, O
 = orange, P = pink (example: YOP = Yellow Orange Pink). GenotypeList.csv A
 file containing the MHC genotypes of all individuals used in this study.
 Allele names correspond to the alleles included in the FASTA file.
 TrialDetails.csv A file containing the details of all the trials run in
 this study. 1. Relationship between files, if important: NA 2. Additional
 related data collected that was not included in the current data package:
 None 3. Are there multiple versions of the dataset? No METHODOLOGICAL
 INFORMATION Description of methods used for collection/generation of data:
 Animal care and use statement This research was conducted with the
 approval and oversight of the Bloomington Institutional Animal Care and
 Use Committee (BIACUC protocol numbers 19-003, 17-016, and 20-015).
 Scientific collection permits were issued to KMM by the Indiana Department
 of Natural Resources. Subject collection Individuals of the rainbow darter
 (Etheostoma caeruleum) and the fantail darter (Etheostoma flabellare) were
 collected with a seine net from Clear Creek, Bloomington, Indiana, Monroe
 County (39.12118432195659, -86.53886585996088) on April 6, 2019. We
 identified individuals to species based on morphological characteristics.
 Individuals were sexed based on breeding characteristics. In both species,
 we identified females as gravid by the presence of distended abdomens. We
 identified male rainbow darters in breeding condition by nuptial
 coloration, and male fantail darters in breeding condition by darkened
 body color and the presence of egg mimics on the first dorsal fin.
 Individuals were transported to Indiana University in aerated, covered
 buckets in an air-conditioned vehicle. Care and housing Individuals were
 housed separately by sex and species. Males and females of the same
 species were housed such that they were unable to see conspecifics of the
 opposite sex. We housed individuals in 20-gallon tanks (30" x
 12" x 12") equipped with power filters and air pumps mounted
 with large air stones. River rock aquarium gravel was provided as
 substrate. Small terra cotta pots were provided as hides. We conditioned
 the tank water with Amquel Plus and performed 20% water changes once per
 week. Individuals were fed frozen brine shrimp and bloodworms once per
 day, as much as could be consumed in 5 minutes. Fin clipping and VIE
 tagging Individuals were fasted overnight ahead of the fin clipping and
 tagging procedures. Individuals were anesthetized with MS-222 (25 mg/L)
 until gill movement was slow and steady and the subject did not respond to
 touch. Paper towels moistened with conditioned water were placed on the
 gill covers to maintain the subjects breathing during the procedure. Each
 subject was measured for standard length (tip of the nose to the caudal
 peduncle). A small (3mm2) piece of fin tissue was removed from the anal
 fin of each subject. Iodine was used to sterilize the fin clip site during
 the procedure. Each individual was injected with three visible implant
 elastomer (VIE) tags (Northwest Marine Technology), one anterior to the
 first dorsal fin, one between the first and second dorsal fin, and one
 behind the second dorsal fin. Each VIE tag combination was a unique color
 pattern used to identify the individual. The total procedure from start to
 finish took approximately three minutes. Individuals were place in
 conditioned water and allowed to recover from the anesthesia. Recovery was
 pronounced when the subject resumed normal swimming and breathing. All
 individuals were monitored over the following 24 hours for full recovery.
 All fin tissue regenerated within 3 weeks, and all VIE tag wounds healed
 within one week with no sign of infection or long-term effects. Behavioral
 trials were not performed until all individuals had fully healed and
 recovered from fin clipping and VIE tagging. Fin clips were frozen and
 stored for genotyping. MHC genotyping All individuals were genotyped at a
 164 base pair portion of the MHC class IIb gene (exon 2) according to
 deep-sequencing methods developed for fish. We used custom primers and
 protocols we had developed for previous research on darters (Million and
 Lively, 2022). Our prior research indicated the presence of up to 5 MHC
 IIb loci in both our species of interest, meaning an individual genotype
 could contain 1 to 10 alleles. The number of alleles was recorded for each
 genotype as well as the identity of each allele in each genotype. Breeding
 conditions in the lab To maintain the breeding condition of all
 individuals in the lab during the trials, we manipulated ambient light and
 temperature to mimic the conditions of the natural breeding season. We
 kept the day length cycle at 14L:10D and maintained the ambient room
 temperature at 28 degrees Celsius. We assessed the success of our efforts
 by observing female gravidity and male nuptial characteristics as
 described above. Construction of motorized visual stimuli To control for
 possible differences in visual and behavioral cues between live males, we
 constructed motorized visual stimuli with which to pair the olfactory cues
 during the trials. These models allowed us to present identical visual
 stimuli to the focal females and provide a visual context in which to
 interpret the olfactory stimuli. 3D models of a male fantail darter and a
 male rainbow darter were purchased from the website TurboSquid (model
 artist: adicaza) (Figure 1). Files were modified for compatibility with a
 3D printer. Life-sized male models were 3D printed using a Formlabs Form 2
 printer and Formlabs SG dental resin. 3D printing was performed in the
 Lennon lab at Indiana University. High-resolution images of a male fantail
 darter and a male rainbow darter were obtained from the North American
 Native Fishes Association photo gallery, and color palettes were developed
 from colors isolated from the images. Models were painted with colors and
 patterns resembling a male of each species in breeding condition (Figure
 2). Models of the same species were painted to be visually identical.
 Small glass amber-colored beads (2 mm outside diameter) were glued to the
 first dorsal fin of the fantail darter models to resemble egg mimics.
 Models were mounted onto stepper motors using magnets and transparent
 plexiglass platforms. Stepper motors were attached to microcontrollers,
 which in turn were connected to an Arduino Uno REV3 unit. Model movements
 were programmed using the software Arduino IDE 1. The Arduino unit was
 programmed so that the models made synchronized movements that resemble
 the courtship displays of live breeding males. Two visually identical
 models of each species were created. Experimental setup A 10-gallon tank
 (20" x 10" x 12") was set up in a location where the
 subject would have no visual contact with other individuals during the
 trial. The focal tank was surrounded with sheets of panda paper (Vivasun)
 to block external light and visual stimuli. The bottom of the tank was
 blacked out using matte black aquarium decal material. The tank was
 separated into three zones of preference, marked by narrow white lines.
 The neutral zone was centered in the middle and took up 80% of the tank
 bottom surface area. Each preference zone on the left and right took up
 10% of the tank bottom surface area. Ambient temperature in the trial area
 matched that of the housing area. The tank was lit using ambient
 fluorescent light and a Nicrew ClassicLED Plus full-spectrum aquarium
 light mounted above the tank. Two 10-gallon tanks were placed underneath
 the focal tank, completely out of visual range of the focal subject. All
 tanks were partially filled with conditioned, aerated water (3/4 full for
 the focal tank, full for each olfactory stimulus tank). At the conclusion
 of each trial, all tanks were emptied, scrubbed with 95% ethanol, allowed
 to air dry, and refilled with the same volume of fresh water. An AKASO
 EK7000 Pro 4K Action Camera was mounted near the tank to record all
 trials. All motorized and electronic devices were connected to a remote
 control so that they could be turned on and off without the observer in
 the room during a trial. The trials were monitored outside the room in
 real time using a cell phone remotely connected to the camera. We
 pre-determined that if a participant showed signs of severe stress
 (gasping, attempting to jump out of the tank) at any point during a trial,
 then the trial would have been immediately terminated and the subject
 returned to housing. However, during these trials no individuals displayed
 signs of extreme stress, so we did not terminate any of the trials. No
 individual participated in more than one trial per 24 hour period, either
 as a subject or as a stimulus individual. Submersible pumps (Homasy 300
 L/H, 4 W) were placed into each olfactory stimulus tank. The pumps were
 connected to the focal tank via clear food grade vinyl tubing (5/16 ID,
 7/16 OD). The pumps were primed with clean conditioned water before and
 after every trial. Plastic clamps were used to control the water flow and
 to ensure that the water flowed into the focal tank at the same rate on
 each side. This was visually confirmed prior to the start of every trial.
 MHC-based choice trials We used dichotomous choice trials to assess the
 response of females of each species to MHC-based olfactory cues from
 conspecific males (Figure 3). The method is standard for evaluating mate
 choice preferences in darters. For each trial, to control for visual and
 behavioral differences between the two live males, two visually identical
 male models of the same species as the focal female were placed on either
 side of the focal tank and mounted on stepper motors. Before the start of
 the trial, blinds were used to block the view outside the tank of the
 focal female during acclimation. One focal female was acclimated to the
 focal tank for a minimum of 10 minutes. A female was considered acclimated
 when she initiated normal free swimming around all sides of the tank and
 exhibited normal breathing patterns. One live stimulus male was acclimated
 to each of the olfactory stimulus tanks out of view of the focal female
 for 10 minutes, the same amount of time that the focal female was
 acclimated to the focal tank. After the acclimation period, the blinds
 were removed, and the focal female was allowed to explore the tank and
 visually inspect each model. The trial began after the female had crossed
 into both preference zones, seen both models, and returned to the neutral
 zone. A trial did not progress if the focal individual did not cross both
 sides and return to the middle. At the start of the trial, both male
 models were turned on to provide identical visual displays for the female,
 and water was pumped into the focal tank on either side from each of the
 stimulus tanks. During each trial the focal female was presented with the
 scent of a male with an MHC genotype similar (differing by no more than
 one allele) or identical to her own on one side, and one male with an MHC
 genotype completely dissimilar to her own (none of his alleles matched
 hers) on the other side. To control for heterozygosity, we matched
 stimulus males so that they either had the same number of alleles in their
 genotypes or only differed in number by one. Due to small sample sizes,
 some females participated in more than one trial. In these cases, a female
 was always presented with a completely novel pair of stimulus males. The
 trial period lasted for 10 minutes, and the entire trial was observed
 outside the room by camera and was recorded. To control for potential side
 bias in the focal females, we switched the sides on which the similar and
 dissimilar male scents were presented to the focal female between each
 trial, and we switched the sides of the two models between each trial. 1.
 Description of methods/software used to analyze data Trial analyses Videos
 were analyzed using the software Boris. We constructed an ethogram in the
 software to use during analysis. All videos were scored for the amount of
 time the focal female spent in each of the three zones of the focal tank.
 If during a trial a female spent more than 80% of her time in the neutral
 zone, we concluded that she had not interacted enough with the stimuli for
 us to score her preferences, and that trial was set aside from further
 analysis.  A strength of preference (SOP) equation was used to evaluate
 the responses to the stimuli. The equation is SOP = (S – D)/(S + D), where
 S = the time spent in the preference zone with the male with a similar MHC
 genotype and D = the time spent in the preference zone with the male with
 a dissimilar MHC genotype. The values obtained range from a minimum of -1
 (strong preference for dissimilar genotype) to a maximum of 1 (strong
 preference for a similar genotype). A value at or near 0 indicates no
 strong preference for either genotype. Further data analysis was conducted
 using the software R version 4.1.0 (R Development Core Team, 2018). Mean
 SOP between the species was compared using a Wilcoxon rank-sum test.
 Proportion of time spent with the male with the similar MHC genotype (as
 opposed to the male with the dissimilar MHC genotype) was tested against
 the null hypothesis that the females spent equal time with both males
 using a one-sample Wilcoxon test. Power analyses on the data were
 conducted using the software G*Power 1. Instrument- or software-specific
 information needed to interpret the data: See methods above for software
 information 2. Standards and calibration information, if appropriate: NA
 3. Environmental/experimental conditions: NA 4. Describe any
 quality-assurance procedures performed on the data: See data processing
 methods above 5. People involved with sample collection, processing,
 analysis and/or submission: Lead researcher: Kara Million Associated
 researchers: Melissa Proffitt, Sierra Reese Sequencing facility personnel
 (Center for Genomics and Bioinformatics, Indiana University): Jun Liu,
 Chris Hemmerich, Doug Rusch, Sumitha Nallu DATA-SPECIFIC INFORMATION FOR:
 \[MHC alleles.fasta] 1. Number of variables: 2 2. Number of cases/rows:
 126 3. Variable List: -sequence ID: a unique identifier for each allele,
 prededed by the symbol &gt; -DNA sequence: a sequence of DNA nucleotides.
 The DNA sequence immediately follows the sequence ID. 1. Missing data
 codes: "-" indicates a missing nucleotide 2. Specialized formats
 or other abbreviations used: NA \[Pairings.csv] 1. Number of variables: 3
 2. Number of cases/rows: NA 3. Variable List: Female: ID of focal female
 in trial Similar Male: ID of stimulus male with similar MHC genotype in
 trial Dissimilar Male: ID of stimulus male with dissic 1. Missing data
 codes: NA 2. Specialized formats or other abbreviations used: NA
 \[GenotypeList.csv] 1. Number of variables: 5 2. Number of cases/rows: 29
 3. Variable List: VIE ID: the VIE tag ID of the individual Sex: the sex of
 the individual species: the species of the individual Role: the role the
 individual played in the study, either as a focal subject or as a stimulus
 Genotype: the genotype of the individual, as indicated by the names of
 each allele. Names correspond to allele names in the FASTA file. 1.
 Missing data codes: NA 2. Specialized formats or other abbreviations used:
 NA \[TrialDetails.csv] 1. Number of variables: 14 2. Number of cases/rows:
 17 3. Variable List: Trial ID: unique identifier for each trial run in the
 study species: species of individuals used in the trial (both focal and
 stimulus) focal ID: ID of focal individual in the trial stim ID (similar):
 ID of stimulus individual with MHC genotype similar to the focal
 individual stim ID (dissimilar): ID of stimulus individual with MHC
 genotype dissimilar to the focal individual notes: any notes included
 about the trial Time L (s): time in seconds focal individual spent on left
 side of the tank Time R (s): time in seconds focal individual spent on
 right side of the tank Prop. L: proportion of time focal individual spent
 on left side of the tank Prop. R: proportion of time focal individual
 spent on right side of the tank Time NZ: time in seconds focal individual
 spent in the neutral (central) zone of the tank Prop. Time NZ: proportion
 of time focal individual spent in the neutral (central) zone of the tank
 total video length (s): total length of trial video in seconds dissimilar
 side: side on which the dissimilar stimulus male was placed 1. Missing
 data codes: NA 2. Specialized formats or other abbreviations used: NA
 References Million, K.M., Lively, C.M., 2022. Transspecific polymorphism
 and the convergent evolution of supertypes in major histocompatibility
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