DOI: 10.5061/dryad.6t1g1jx6r
title: Floral phenotype data from Mimulus parishii x M. cardinalis hybrids

description: Pollination syndromes are a key component of flowering plant
 diversification, prompting questions about the architecture of single
 traits and genetic coordination among traits. Here, we investigate the
 genetics of extreme floral divergence between naturally hybridizing
 monkeyflowers Mimulus parishii (self-pollinated) and M.
 cardinalis (hummingbird-pollinated). We mapped quantitative trait loci
 (QTLs) for 18 pigment, pollinator reward/handling, and dimensional traits
 in parallel sets of F2 hybrids plus recombinant inbred lines and generated
 nearly isogenic lines (NILs) for two dimensional traits, pistil length,
 and corolla size. Our multi-population approach revealed a highly
 polygenic basis (n = 190 QTLs total) for pollination syndrome divergence,
 capturing minor QTLs even for pigment traits with leading major loci.
 There was significant QTL overlap within pigment and dimensional
 categories. Nectar volume QTLs clustered with those for floral dimensions,
 suggesting a partially shared module. The NILs refined two pistil length
 QTLs, only one of which has tightly correlated effects on other
 dimensional traits. An overall polygenic architecture of floral divergence
 is partially coordinated by genetic modules formed by linkage (pigments)
 and likely pleiotropy (dimensions plus nectar). This work illuminates
 pollinator syndrome diversification in a model radiation and generates a
 robust framework for molecular and ecological genomics.
description: All hybrids in this study were generated from two highly inbred
 parental lines: Sierran CE10 for <em>Mimulus cardinalis</em>
 (Yuan <em>et al.</em>, 2013) and PAR for
 <em>M</em><em>. </em><em>parishii</em>
 (Fishman <em>et al.</em>, 2015; Nelson <em>et
 al.</em>, 2021; Liang <em>et al.</em>, 2023).
 F<sub>1</sub> hybrids were generated with PAR as the seed
 parent and selfed to generate F<sub>2</sub> seeds, while
 recombinant inbred lines (RILs) were generated by single-seed-descent from
 F<sub>2</sub> individuals through 3-6 generations of
 self-fertilization (Sotola <em>et al.</em>, 2023).
 <strong>Plant growth conditions for QTL mapping -
 </strong>F<sub>2</sub> hybrids were grown in two
 separate greenhouse common gardens at the University of Connecticut (UC)
 and the University of Montana (UM). At UC, seeds (CE10, PAR,
 F<sub>1</sub> hybrids, F<sub>2</sub> hybrids, and
 NILs) were sown into 98 cell trays (1.25"sq x 2"H), and then
 after four weeks, all seedlings were transferred to 18 pocket trays
 (3.25" sq x 3.14"H) and grown to flowering.  At UM,
 F<sub>2</sub> seeds (and a few CE10 and PAR controls) were
 germinated on wet sand in Petri dishes, seedlings transplanted into 3”
 square pots filled with Sunshine #4 soil-less potting mix, and plant grown
 to first flower and beyond (life history and plant architecture traits
 presented elsewhere). The RIL mapping population was grown at the
 University of Georgia (UGA) with seeds sown into 2.5” pots filled with
 Metromix 830 soil. Seedlings were transplanted into 4” pots and grown to
 flowering. In all three greenhouses, plants were grown under natural light
 supplemented with sodium lamps to a 16‐h daylength and fertilized
 regularly (UC: 2-3 times per week, UM and UGA: weekly).
 <strong>Phenotypic measurements for QTL
 mapping</strong> In the
 UC_F<sub>2</sub> growout, we measured three pigment, two
 pollinator reward/handling and nine dimensional floral traits, plus
 flowering time, on F<sub>2</sub>s (n = 253) plus parental
 lines and F<sub>1 </sub>hybrids (n =8 each) (Fig. 1). We
 scanned the ventral petal of each flower to quantify petal lobe
 anthocyanin (PLA) and carotenoid (PLC) pigment intensity. The proportion
 of red (R), green (G), and blue (B) pixels in a square area of the same
 size of the adaxial surface of the petal were estimated from scanned
 images using Image J (http://rsbweb.nih.gov/ij/) and the relative petal lobe anthocyanin and carotenoid concentrations estimated by [(R + B)/2] − G and [(R + G)/2] – B, respectively. Nectar guide anthocyanin (NGA) and nectar guide trichome length (NGT), were visually scored in F<sub>1</sub> and F<sub>2</sub> hybrids on semi-quantitative scales defined by the parental extremes (CARD = 9 and 7, respectively, PAR = 1 for both). Nectar volume (NEV) was measured for two flowers per individual on their first day of opening, using a pipette accurate to 1.5μL. To reduce environmental effects, nectar was measured at 4:00 PM-7:00 PM after watering at 12:00-1:00PM each day. Floral dimensions (Fig. 1c) were measured on one of the second pair of open flowers using a digital caliper, and stigma-anther distance calculated as pistil length – stamen length.  In addition to pollen number and viability (Sotola <em>et al.</em> 2023) and life history traits to be presented elsewhere, the UM_F<sub>2</sub> grow-out measured 9 traits shared with the UC_F<sub>2</sub> growout: flowering time, two floral pigment traits (PLA and PLC), and five floral dimension traits (corolla length and width, corolla tube width, and pistil and stamen length) and calculated stigma-anther separation. PLA and PLC were measured and calculated as described above, except that we scanned one of the two lateral petals. The standard corolla dimension traits <strong>(Fishman <em>et al.</em>, 2002)</strong> were measured with an engineering ruler on one flower of the first pair on the day it opened. In the RIL growout at UGA (n = 145 RILs, 18 each of CE10 and PAR parents and F<sub>1 </sub>hybrids), we recorded day to first flower (flowering time) and measured two floral pigment traits (PLA and PLC), nectar volume, and five floral dimension traits (corolla length and width, corolla tube width, and pistil and stamen length) and calculated stigma-anther separation. We measured floral pigment traits on the ventral petal as described above and used an engineering ruler to measure the floral dimensional traits on one flower from the first pair. To estimate nectar volume, we used a capillary tube to collect nectar from two flowers on their first day of opening and took the average; nectar was measured between 1:00 PM - 3:00 PM after watering in the morning at 8:00 AM - 9:00 AM. Other than pistil length (PIL), which includes the stigma lobes in the UC_F<sub>2</sub> and UGA_RIL measurements but not in the UM_F<sub>2</sub> dataset, and thus stigma-anther separation (SAS), traits with a shared abbreviation represent the same floral dimension. <strong>Fishman L, Beardsley P, Stathos A, Williams CF, Hill JP</strong>. <strong>2015</strong>. The genetic architecture of traits associated with the evolution of self-pollination in Mimulus. <em>New Phytologist</em> <strong>205</strong>: 907–917. <strong>Fishman L, Kelly AJ, Willis JH</strong>. <strong>2002</strong>. Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus. <em>Evolution</em> <strong>56</strong>: 2138–2155. <strong>Liang M, Chen W, LaFountain AM, Liu Y, Peng F, Xia R, Bradshaw HD, Yuan Y-W</strong>. <strong>2023</strong>. Taxon-specific, phased siRNAs underlie a speciation locus in monkeyflowers. <em>Science</em> <strong>379</strong>: 576–582. <strong>Nelson TC, Stathos AM, Vanderpool DD, Finseth FR, Yuan Y-W, Fishman L</strong>. <strong>2021</strong>. Ancient and recent introgression shape the evolutionary history of pollinator adaptation and speciation in a model monkeyflower radiation (Mimulus section Erythranthe). <em>PLoS Genetics</em> <strong>17</strong>: e1009095. <strong>Sotola VA, Berg CS, Samuli M, Chen H, Mantel SJ, Beardsley PA, Yuan Y-W, Sweigart AL, Fishman L</strong>. <strong>2023</strong>. Genomic mechanisms and consequences of diverse postzygotic barriers between monkeyflower species. <em>Genetics</em> <strong>225</strong>: iyad156. <strong>Yuan Y-W, Sagawa JM, Young RC, Christensen BJ, Bradshaw HD</strong>. <strong>2013</strong>. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. <em>Genetics</em> <strong>194</strong>: 255–263.
description: # Floral phenotype data from Mimulus parishii x M. cardinalis hybrids
 [https://doi.org/10.5061/dryad.6t1g1jx6r](https://doi.org/10.5061/dryad.6t1g1jx6r) This dataset consists of floral phenotypes for three separate recombinant populations (UC_F2, UM_F2, RIL) used for QTL mapping of pollination syndrome divergence.  The first sheet defines the traits and the following sheets contain columns with the trait values measured for each individual (row) in each population. The corresponding genotypic data was previously archived.  ## Description of the data and file structure Because some traits require additional handling (e.g., nectar volume, carotenoid values) and sometimes focal flowers are obviously deformed for one of the dimensional traits, the datasets are not perfectly complete for all individuals in each growout. Missing values are indicated by -.