DOI: 10.5061/dryad.w9ghx3fs4
title: Developmental evidence for parental conflict in driving Mimulus species barriers

description: The endosperm, a tissue that nourishes the embryo in the seeds of
 flowering plants, is often disrupted in inviable hybrid seeds between
 closely related species. A key question is whether parental conflict is a
 major driver of this common form of reproductive
 isolation.  Here, we performed reciprocal crosses
 between pairs of three monkeyflower species (Mimulus
 caespitosa, M. tilingii, and M. guttatus). The
 severity of hybrid seed inviability varies among these crosses, which we
 inferred to be due to species divergence in effective ploidy. By
 performing a detailed time series of seed development, we discovered
 parent-of-origin phenotypes that provide strong evidence for parental
 conflict in shaping endosperm evolution. We found that the
 chalazal haustorium, a tissue within the endosperm that occurs at the
 maternal-filial boundary, shows pronounced differences between reciprocal
 hybrid seeds formed from Mimulus species that differ in
 effective ploidy. These parent-of-origin effects suggest that the chalazal
 haustorium might act as a mediator of parental conflict, potentially by
 controlling sucrose movement from the maternal parent into the endosperm.
  Our study suggests that parental conflict in the endosperm may
 function as a driver of speciation by targeting regions and developmental
 stages critical for resource allocation and thus proper seed development.
description: Generation of Plant Material Here, we used one
 inbred line (formed from ≥8 generations of self-fertilization) for each
 focal species (M. caespitosa, M. tilingii, and M. guttatus). The same
 inbred lines were used in previous studies of hybrid seed inviability in
 M. tilingii and M. guttatus (Garner et al., 2016) and M.
 caespitosa (Sandstedt et al., 2021). The M. caespitosa inbred line, TWN36,
 originates from a high-alpine population at 1594m in Twin Lakes, WA.
 The M. tilingii inbred line, LVR1, is derived from a population at 2751m
 in Yosemite Park, CA. The M. guttatus inbred line, DUN10, originates from
 a population in the Oregon Dunes National Recreation Area. 
 In this study, we considered three intraspecific crosses (CxC,
 TxT, and GxG, where C = M. caespitosa, T = M. tilingii, and G = M.
 guttatus) and six interspecific crosses (CxT, TxC, TxG, GxT, CxG, GxC;
 maternal parent is always listed first). To generate diploid, experimental
 plants, we sowed 20-30 seeds for each inbred line on damp paper towels in
 petri dishes sealed with parafilm and cold-stratified them for 7 days to
 disrupt seed dormancy. After cold stratification, we transferred petri
 dishes to a growth chamber with 16-h days at 23°C and 8-h nights at 16°C.
 We transplanted seedlings into 3.5” pots with moist Fafard 4P growing mix
 (Sun Gro Horticulture, Agawam, MA) and placed the pots in the same growth
 chamber. Once plants began flowering, we randomly crossed within and
 between individuals (total plants: C = 22, T = 20, G = 16). For all
 crosses, we emasculated the maternal plant 1-3 days prior to each cross to
 prevent contamination from self-pollination. To
 investigate species divergence in effective ploidy, we performed several
 interspecific, interploidy crosses: C4xxT, TxC4x; T4xxG, GxT4x; C4xxG,
 GxC4x (4x subscript indicates tetraploid). To generate synthetic
 tetraploid individuals, we treated 100-200 seeds of TWN36 and LVR1 with
 0.1% or 0.2% colchicine and stored them in the dark for 24 hours (16 hours
 at 23°C and 8 hours at 16°C). The next day, we planted seeds onto Fafard
 4P potting soil using a pipette and placed pots inside the growth chamber
 under typical light and temperature conditions (16-h days at 23°C and 8-h
 nights at 16°C). Once seeds germinated, we transplanted seedlings into
 2.5” pots. After sufficient growth, we prepared samples for flow cytometry
 using a protocol adapted from Lu et al., 2017. Briefly, we extracted
 nuclei from one colchicine-treated sample and an internal control
 (2n Mimulus or Arabidopsis thaliana, Col-0) together in a single well. To
 extract nuclei, we chopped 100mg of leaf tissue (50mg colchicine-treated
 sample and 50 mg internal control) in 1mL of a pre-chilled lysis buffer
 (15mM Tris-HCl pH 7.5, 20mM NaCl, 80mM KCl, 0.5mM spermine, 5mM 2-ME, 0.2%
 TritonX-100). We stained nuclei with 4,6-Diamidino-2-phenylindole (DAPI),
 filtered nuclei for debris using a 40um Flowmi™ cell strainer, and
 aliquoted nuclei into a single well of a 96-well polypropylene plate. We
 assessed ploidy of each sample using a CytoFLEX (Beckman Coulter Life
 Sciences) flow cytometer. We calculated total DNA content using the
 following equation: We generated three synthetic
 polyploids for TWN36 and six for LVR1. For each synthetic polyploid, 2C
 DNA content was nearly doubled compared to corresponding diploid lines
 (TWN36, 2C = 1.38 pg; TWN364x, 2C = 2.69  0.09 pg; LVR1, 2C = 1.26 pg; LVR14x, 2C =
 2.64  0.05 pg). In
 some cases, we discovered that plants initially identified as tetraploid
 via flow cytometry were actually mixoploids. To ensure the crosses we
 performed were indeed interploidy, we determined the ploidy of the
 resulting progeny. From each interploidy cross, we planted 5-10 seeds per
 fruit, isolated nuclei from the resulting plants, and assessed 2C content
 using a flow cytometer for a few offspring as described above (3x
 TWN364xxLVR1, 2C = 1.92  0.04; 3x LVR1xTWN364x, 2C = 1.88  0.01 pg; 3x LVR14xxDUN10, 2C
 = 1.95  0.04 pg; 3x
 DUN10xLVR14x, 2C = 1.81  0.01 pg; 3x TWN364xxDUN10, 2C = 1.90  0.011 pg). We included data
 from interploidy crosses only when their progenies were confirmed to be
 triploids, or, in the case of 4x M. caespitosa, if we were using a
 confirmed stable polyploid line (i.e., self-fertilized at least one
 generation with polyploidy confirmed in the progeny). 
 Measuring seed size and seed viability  To
 measure seed size, we collected three replicate fruits per cross, with
 each fruit collected from a distinct plant. We imaged 50 seeds per fruit
 under a dissecting scope, for a total of 150 seeds per cross (except for
 one CxG fruit for which only 35 seeds were measured for a total of 135
 seeds). Seed area was measured using ImageJ (Rasband, 1997). 
 Using these same fruits, as well as fruits from interploidy
 crosses (2-5 fruits/cross, at least two fruits per cross from a distinct
 plant), we assessed seed viability using two different methods. First, we
 performed visual assessments of mature seeds, looking for irregular
 phenotypes (shriveled, wrinkled, or flat) known to be highly correlated
 with germination rates in these Mimulus species and their hybrids
 (Garner et al., 2016, Sandstedt et al., 2021). We scored the number of
 seeds that appeared round and plump (i.e., fully-developed) versus
 irregularly shaped (i.e., under-developed). Second, we performed
 Tetrazolium assays to assess seed viability on a subset of these same
 seeds (~100 seeds per fruit). For fruits generated from interploidy
 crosses and fruits that produced &lt;100 seeds, we stained 32-63
 seeds. We immersed seeds in a scarification solution (83.3% water, 16.6%
 commercial bleach, and 0.1% Triton X-100) and placed them on a shaker for
 15 minutes. After scarification, we washed seeds five times with water and
 incubated seeds with 1% Tetrazolium at 30°C. Two days later, we scored the
 number of seeds that stained dark red (viable) versus pink or white
 (inviable). As noted above, we also planted a subset of the seeds from
 interploidy crosses to assess ploidy and germination rates generally
 reflected both seed viability measurements (data not shown).
 Seed viability rescues To assess whether
 aberrant endosperm development contributes to seed defects in
 interspecific crosses, we attempted to rescue seed viability with a
 sucrose-rich medium. We collected three fruits 8 to 12 days after
 pollination (DAP) from each intra- and interspecific cross (not including
 interploidy crosses), with each fruit collected from a distinct plant. Of
 the three fruits per cross, at least one fruit was collected 8 DAP (to
 maximize the chance of rescue). On average, we dissected 40 whole immature
 seeds per fruit (range = 25-57) and placed them on petri dishes with MS
 media containing 4% sucrose. We sealed petri dishes with parafilm and
 placed them at 23°C with constant light for 14 days before scoring
 germination. Visualizing parent-of-origin effects
 during seed development  To compare trajectories of
 seed development, we performed intra- and interspecific crosses, and we
 collected fruits 3, 4, 5, 6, 8, and 10 DAP. For consistency, we performed
 crosses and collected fruits at the same time of day.
 To visualize early seed development, we collected fruits 3 and 4
 DAP (N = 1 to 2 fruits per DAP per cross) and prepared them for clearing
 with Hoyer’s solution. We placed developing fruits in a 9 EtOH: 1 acetic
 acid fixative overnight. The following day, we washed fruits twice in 90%
 EtOH for 30 min per wash. We dissected immature seeds directly from the
 fruit onto a microscope slide with 100uL of 3 parts Hoyer’s solution (70%
 chloral hydrate, 4% glycerol, 5% gum arabic): 1 part 10% Gum Arabic and
 sealed the slide with a glass cover slip. We stored the microscope slides
 containing cleared, immature seeds at 4°C overnight. The next day, we
 imaged slides using the differential interference contrast (DIC) setting
 with the 20x objective on a Leica DMRB microscope. For each fruit, we
 scored the number of developing seeds with and without an intact chalazal
 haustorium (15-56 seeds per fruit; 32-111 seeds per cross per DAP); only
 seeds with visible embryos were scored. Additionally, we imaged an average
 of 11 seeds per fruit (3-15 seeds per fruit, 10-27 seeds per cross per
 DAP) to assess size differences in the endosperm and chalazal haustorium
 at 3 and 4 DAP. For the interploidy T4xxG cross, we imaged on average 18
 seeds per fruit (14-26 seeds per fruit, 29-40 seeds per cross per DAP). We
 outlined and measured the endosperm in all seeds and the chalazal
 haustorium when present using ImageJ (Rasband, 1997). Because the chalazal
 haustorium was not present for all imaged seed, sample sizes for its
 measurements were lower. We selected and measured images that represented
 typical seed development at each time point.   We
 defined the chalazal haustorium as two uninucleate cells that, together,
 form a continuous structure that penetrates toward the ovule hypostase
 cells (a group of tightly packed cells at the base of the ovule). To
 measure the chalazal haustorium, we began the outline near the epidermis
 of the seed (not including the hypostase cells) and extended it toward the
 micropylar region following Guilford & Fisk, 1952 (see their
 Figure 27). In addition, when measuring the endosperm, we started the
 outline at the same position near the epidermis of the ovule and extended
 it toward the opening of the micropylar haustorium. To
 visualize later seed development (after 4 DAP when the seed coat is too
 thick to clear with Hoyer’s solution), we collected whole fruits at 5, 6,
 8, and 10 DAP and stored them in a Formaldehyde Alcohol Acetic Acid
 fixative (10%:50%:5% + 35% water) for a minimum of 48 hours. After
 fixation, we dehydrated developing fruits with increasing concentrations
 of Tert Butyl Alcohol. Next, we washed fruits three times for two hours
 each with paraffin wax at 65°C before embedding them into a wax block. We
 sectioned wax blocks containing whole fruits into ribbons using a LIPSHAW
 Rotary Microtome (Model 45). Fruits collected at 5 and 6 DAP were
 sectioned into 12-um ribbons for better visualization of micropylar and
 chalazal domains, and fruits collected at 8 and 12 DAP were sectioned into
 8-um ribbons. Next, we gently placed ribbons in a warm (~40°C) water bath
 and positioned them onto a microscope slide. We placed slides on a slide
 warmer overnight to adhere sections completely to the glass. In a staining
 series, we first used Xylene as a clearing agent and performed several
 washes with increasing concentrations of EtOH to effectively stain nuclei
 and cytoplasm (1% Safranin-O and 0.5% Fast Green, respectively). We
 further washed stained slides with EtOH and finished the series with
 Xylene. We sealed slides with a glass coverslip using Acrytol as the
 mounting medium. We visualized slides using a Zeiss
 Axioskop 2 microscope with a 10x objective. For each fruit, we imaged at
 least 10 seeds with a developing embryo per fruit (except for severe
 embryo-lethal crosses: 10 DAP TxG, 8 seeds imaged; 10 DAP CxG, 1 seed
 imaged). We imaged at least five consecutive sections of each seed through
 the embryo. For all seeds imaged at 5 and 6 DAP, we scored the presence of
 the chalazal haustorium. Additionally, we categorized embryo development
 at 6, 8, and 10 DAP into four different stages: before globular to
 globular, late-globular to transition, early-heart to late-heart, and
 torpedo.