DOI: 10.5061/dryad.wpzgmsbzg
title: Data from: The evolution of a placenta accelerates the evolution of post-copulatory reproductive isolation

description: The evolution of placentation is predicted to intensify intergenomic
 conflicts between mothers and offspring over optimal levels of maternal
 investment by providing offspring opportunities to manipulate mothers into
 allocating more resources. Parent-offspring conflicts can result in the
 evolution of reproductive isolation among populations when conflicts
 resolve in different ways. Postzygotic reproductive isolation is
 hypothesized to evolve more rapidly following the evolution of
 placentation due to the predicted increase in conflict. We tested
 this hypothesis by performing interpopulation crosses within
 placental and non-placental species of Poeciliopsis to determine if the
 relationship between genetic distance and measures of postzygotic
 reproductive success differed as function of reproductive mode. We did not
 observe any differences in offspring viability or sterility among crosses.
 Offspring size declined rapidly as a function of interpopulation genetic
 distance within the placental species, but not among
 our non-placental species. The decrease in offspring size in the
 placental species was beyond normal variation, likely representing a major
 fitness cost, consistent with the prediction that negative epistatic
 interactions are evolving more quickly among populations in our placental
 species than the non-placental species. We discuss how our results support
 the role parent-offspring conflicts play in the evolution of reproductive
 isolation and reproductive mode.
description: We assessed interfertility among populations from three species
 of Poeciliopsis. We selected <em>Poeciliopsis prolifica</em>
 and <em>Poeciliopsis infans</em> to represent a paired
 comparison of closely related placental and non-placental species from the
 same clade, respectively (Reznick, Mateos et al. 2002, Pollux, Meredith et
 al. 2014). Poeciliopsis gracilis, was included as an additional
 non-placental species from a separate clade (Reznick, Mateos et al. 2002).
 We established lab populations at the University of California Riverside
 with 15 wild-caught pregnant females and five adult males from 4
 populations of P. prolifica, 4 populations of P. nfans, and 2 populations
 of P. gracilis (Table S1) between 2014-2015. Fish from an
 additional population of P. gracilis collected in 2004 and maintained in 3
 stock tanks were also included (Table S1). To ensure allopatry, we
 collected each population from a different river (Figure 1; Table S1).
 Schrader et al. (2013) found in their crosses among populations of H.
 formosa that differences among populations in offspring size cause
 reductions in the viability of offspring from hybrid crosses. We exploited
 existing data for <em>P. infans</em> and <em>P.
 gracilis</em> (Frías-Alvarez, Macías Garcia et al. 2014) to choose
 sites with large differences in late-stage embryo size. We recorded water
 pH, water temperature, canopy cover, and elevation at each collection site
 (Table S1) and combined that with the annual rainfall data for each locale
 so that we could assess the potential influence of local adaptation as a
 contributor to our results. We do not have environmental data for the
 population of P. gracilis collected in 2004. Wild-caught adults were
 housed in 19 or 38 L aquaria with clumps of aquatic moss (Vesicularia
 dubyana) to provide cover for newborn offspring. We removed F1 offspring
 daily and reared them in group tanks. We used the metamorphosis of the
 male anal fin into the gonopodium, the intromittent organ, to identify the
 sex of individuals while they were immature, then moved them into
 single-sex aquaria to rear them to maturity. Lab-born male tanks
 were seeded with two females from the same population to stimulate sperm
 production. As lab-born females approached sexual maturity, we isolated
 them into individual 2-gallon tanks containing gravel and aquatic moss,
 and fed them ad libitum. Once individual lab-born females reached sexual
 maturity, we used them in a single intrapopulation or interpopulation
 cross. We performed three replicate crosses in all
 possible interpopulation cross directions, and six intrapopulation crosses
 within each population as a control. We placed lab-born adult males into
 the tanks of isolated virgin females for 7 days, after which time they
 were removed. We monitored the mated females daily in order to record the
 latency time (in days) between mating and producing their first brood.
 Once females gave birth to their first brood, daily monitoring continued
 for a period of 60 days, after which females were euthanized using MS-222
 and preserved in 95% ethanol. We removed newborn offspring from the female
 tanks daily to ensure individuals were less than 24-hours old at the time
 of collection. The first 10 offspring born to a given female were
 immediately euthanized in MS-222 and preserved in 95% ethanol.
 All remaining offspring of both sexes were placed in a 2-gallon stock tank
 and allowed to reach sexual maturity. We monitored these tanks for 6
 months to see if the offspring that resulted from interpopulation crosses
 produced offspring of their own as a measure of hybrid sterility,
 recorded as the presence of healthy newborn offspring within the tank.
 Crosses were deemed unsuccessful if females showed no visible signs of
 pregnancy within 120 days following the end of their 7-day male exposure
 period. We took multiple measures from the outcome of each cross to serve
 as an index of fitness. The total number of offspring born to a given
 female over the 2-month monitoring period was recorded as a measure of
 fecundity. Preserved offspring were measured for body length (mm), wet
 weight (mg), and dry weight (mg) at birth. We dissected females and
 removed all embryonic tissue to record the total number of developing
 embryos and the wet weight (g) of all the reproductive tissue. We then
 treated the wet mass of all reproductive tissue, including embryos divided
 by the total wet mass as a dependent variable (reproductive allocation)
 which represents the proportion of total wet mass devoted to reproduction.
 All species in the genus Poeciliopsis have
 superfetation, which means that a reproducing female normally
 carries multiple litters of developing offspring, each in discretely
 different stages of development. A consequence of superfetation is that
 litters of offspring are produced more frequently. Reproductive allocation
 is thus a composite of number of offspring per litter, offspring size
 and number of developing litters. Reproductive allocation was analyzed as
 a proportion of female wet weight to account for differences in the size
 of individual females.<br>Embryos were independently scored as
 viable or inviable by a group of individuals unfamiliar with the working
 hypothesis, based on whether the embryos exhibited the typical phenotype
 of a normally developing embryo (Haynes 1995). Inviable embryos exhibited
 the characteristic markers of their developmental stage, but were
 typically smaller, duller in color, and more semisolid than viable
 embryos. Embryos were scored as ambiguous whenever there was disagreement
 between individual scorers or when they only exhibited a subset of
 the characteristics of inviability. We measured the focal females for body
 length (g) and wet weight (g) to control for the influence of female size
 on offspring size and number. All procedures were approved and done in
 compliance with the guidelines set by the Institutional Animal Care and
 Use Committee (IACUC) at the University of California Riverside (Animal
 use protocols #20110007 and #20140003). Sequencing and
 Bioinformatics<br>To assess genetic distance among the populations,
 we extracted DNA from the tail tissue of five male and five female
 wild-caught individuals from each population. Extractions were performed
 using a Qiagen® DNeasy Blood &amp; Tissue Kit, with two
 modifications to the spin-column protocol; we extended the duration of
 proteinase K treatment to 10 hours then incubated each sample in 8 μL of
 RNAse for 30-minutes at 37 O C. We quantified DNA concentrations with a
 Qubit 2.0 Fluorescence Reader, and each sample was adjusted to a
 concentration of 10ng/ μL using a Zymo Research DNA Clean &amp;
 Concentrator™ kit. ddRAD sequencing was performed by the University of
 Texas at Austin Genomics Sequencing and Analysis Facility (GSAF),
 including enzyme digestion, size selection, adaptor ligation, and
 sequencing. We selected the EcoRI-MspI enzyme pair for digestion, and
 200-300 base-pair fragments were retained for sequencing. 2x150bp reads of
 the digested samples were obtained using the Illumina HiSeq 2500 system.
 We performed all demultiplexing, de novo assembly, and genetic distance
 calculations using the STACKS pipeline (Catchen, Hohenlohe et al. 2013).
 Each species was processed independently and all individuals within a
 species were processed and analyzed simultaneously. We used the
 process_radtags program to filter out low quality reads with raw phred
 scores below 10 (&lt; 90% probability of being correct) and
 uncalled bases. The STACKS core modules (‘ustacks’, ‘cstacks’, ‘sstacks’,
 and ‘populations’) were executed through the denovomap.pl program with a
 minimum stack depth of 3 reads, a maximum number of mismatches allowed
 between loci within an individual (M) of 2, a maximum number of mismatches
 between loci in a catalog (m) of 1, and the deleveraging and highly
 repetitive stack removal algorithms enabled. We used the ‘populations’
 function to estimate intrapopulation heterozygosity and pairwise F ST
 between all population combinations (Table S2).
description: # Poeciliopsis Population Cross Data
 [https://doi.org/10.5061/dryad.wpzgmsbzg](https://doi.org/10.5061/dryad.wpzgmsbzg) ## Description of the data and file structure Lab stocks were established from 11 populations of wild caught fish. Environmental measures (i.e. site data) was recorded at time of collection for each population. F1 individuals found in lab established stocks were raised to sexual maturity and used in intra- and interpopulation mating crosses (i.e. cross data).  Instances where F1 females did not make the first time were recorded (i.e. binary_recrosses).  Tissues from wild caught females were used for ddRADseq genomic sequencing processed using stacks pipeline. ### Files and variables #### File: binary\_recrosses.csv **Description:** Contains all outcomes for intra- and interspecific crosses of Poeciliopsis ##### Variables * cross.type: Denotes populations of origin for female and male (i.e. OA female x OA male) * success: 0 denotes unsuccessful mating from cross, 1 denotes successful mating from cross * mode: denotes reproductive mode of the species, whether non-placental (lec) or placental (mat) * species: denotes species of fish in cross * cross.status: Denotes if cross was within (intra) or between (inter) populations #### File: female\_dissections.csv **Description:** Contains data on the traits of females used in inter and intraspecific crosses of Poecilopsis ##### Variables * Female: unique identifier for an individual female * Cross: Denotes populations of origin for female and male (i.e. OA female x OA male) * pop mother: Denotes population of origin for female * Fem Length (mm): Denotes length of female * Fem Weight (g): Denotes wet weight of female * Reproductive Tissue Mass (g): Denotes total wet mass of reproductive tissue of female * Viable: number of viable embryos * Inviable: number of inviable embryos #### File: cross\_data.csv **Description:** Contains all cross outcomes, offspring measures, and predictor variables used in analyzing intra- and interpopulation crosses for 3 species of Poeciliopsis. ##### Variables * number: unique identifier of cross * mode: denotes reproductive mode of the species, whether non-placental (lecithotrophic) or placental (matrotrophic) * species: species of cross * maternal.pop: population of origin for mother * paternal.pop: population of origin for father * female: unique identifier for female used in cross * cross: denotes populations of origin for female and male (i.e. OA female x OA male) * cross.type: Denotes if cross was within (intra) or between (inter) populations * fst: Denotes pairwise genetic distance between populations of origins of mother and father * OSD: offspring size difference (difference in mean offspring size between populations of origins of mother and father) * ABSO: absolute offspring size difference * off.length (mm): average length of offspring resulting from cross * off.number: number of offspring resulting from cross * total.dry.mass (g): total dry mass of offspring resulting from cross * off.dry.mass (g): average dry mass of offspring resulting from cross * days.til.birth: days between start of cross mating period and when first offspring was birthed * maternal.mass (g): wet mass of mother used in cross * number.weighed: number of offspring weighed and averaged when estimating dry mass * number.viable: number of viable embryos counted during dissection * number.inviable: number of inviable embryos counted during dissection * number.ambiguous: number of ambiguous (in terms of viability) embryos counted during dissection * embryo.total: total number of embryos counted during dissection * inviable.ambiguous: total number of ambigious and inviable embryos counted durring dissection * reproductive.tissue.mass (g): total mass of all embryos and female reproductive tissue recovered during dissection * maternal.pop.offspring.size (g): average size of offspring in maternal population, estimated from intraspecific crosses  * paternal.pop.offspring.size (g): average size of offspring in paternal population, estimated from intraspecific crosses  * offdrymass1K: offspring dry mass (in dataset) * 1000 * maternalmass1K: maternal dry mass (in dataset) * 1000 * species.avg.odm1k: species average offspring dry mass (in dataset) * 1000 * species.avg.mm1k: species average offspring length in mm (in dataset)* 1000 * species.avg.len: species average offspring length (in dataset) * species.avg.off.num: species average offspring number (in dataset) * species.avg.days.til.birth: species average days until first birth (in dataset) * species.avg.viable: species average number of viable embryos (in dataset) * species.avg.inviable: species average number of inviable embryos (in dataset) * species.avg.repro.tissue: species average reproductive tissue mass (in dataset) * DiffOffMass: Difference in observed offspring mass of cross and species average mass * DiffMatMass: Difference in observed maternal mass of cross and species average maternal mass * DiffOffLen: Difference in observed offspring length of cross and species average length * DiffOffNum: Difference in observed offspring number of cross and species average number * DiffDaysTilBirth: Difference in observed days until birth of cross and species average days until birth * DiffViable: Difference in observed number of viable offspring of cross and species average number of viable offspring * DiffInviable: Difference in observed number of inviable offspring of cross and species average number of inviable offspring * DiffReproTissue: Difference in observed total reproductive tissue of cross and species average total reproductive tissue * log.off.dry.mass: log(offspring dry mass) * log.off.length: log(offspring length) * log.days.til.birth: log(days until first birth) #### File: site\_data.csv **Description:** Environmental measures recorded at collection sites used to establish lab stocks for fish used in study.  Null values indicate environmental measures that were not measured at time of fish collection. ##### Variables * site: unique identifier of site * population: population name (often town name, river name or river basin name) * species: species of Poecilopsis collected * elevation (m): elevation of site * ph: water ph of stream  * temp (C): water temperature of stream * canopy_cover (%): % of collection site that was covered by canopy * rainfall (mm): average annual rainfall at collection site