DOI: 10.5061/dryad.8kprr4xm2
title: Evolution of multiple postzygotic barriers between species of the Mimulus tilingii complex

description: Species are often defined by their ability to interbreed (i.e., Biological
 Species Concept), but determining how and why reproductive isolation
 arises between new species can be challenging. In the Mimulus
 tilingii species complex, three species (M.
 caespitosa, M. minor, and M. tilingii) are largely
 allopatric and grow exclusively at high elevations (&gt;2000m). The
 extent to which geographic separation has shaped patterns of divergence
 among the species is not well understood. In this study, we determined
 that the three species are morphologically and genetically distinct, yet
 recently diverged. Additionally, we performed reciprocal crosses within
 and between the species and identified several strong postzygotic
 reproductive barriers, including hybrid seed inviability, F1 hybrid
 necrosis, and F1 hybrid male and female sterility. In this study, such
 postzygotic barriers are so strong that a cross between any species pair
 in the M. tilingii complex would cause nearly complete
 reproductive isolation. We consider how geographical and topographical
 patterns may have facilitated the evolution of several postzygotic
 barriers and contributed to speciation of closely related members within
 the M. tilingii species complex.
description: <i>Plant
 materials</i> When we began this study, we tentatively classified plants from 12 populations (17 maternal families) within the <em>M. tilingii</em><i style="font-style:normal;"> </i>species complex into three putative species: <em>M. caespitosa</em>, <em>M. minor</em>, <em>M. tilingii</em>. All maternal families were self-fertilized for one to eight generations. To generate experimental plants, seeds were sown onto wet paper towels in petri dishes, sealed with parafilm, and cold-stratified at 4°C for seven days to disrupt seed dormancy. After cold-stratification, petri dishes were transferred to a growth chamber that provided constant supplemental light at 26°C. After germination, seedlings were transplanted to 3.5” pots with moist Fafard 4P growing mix (Sun Gro Horticulture, Agawam, Massachusetts, USA) and transferred to a growth chamber with 16h days at 23°C and 8h nights at 16°C. For assessments of hybrid plant viability and fertility, seedlings were allowed to establish in the growth chamber then moved to a 16h, 23°C/8h, 16°C greenhouse. <em>Morphological assessment</em> To characterize genetically-based morphological differences among species within the <i>M. tilingii</i> complex, we grew 66 plants from 11 populations (16 maternal families) together in a growth chamber. We measured a suite of 16 floral and vegetative traits. First, we measured four leaf traits: when the third leaf pair was fully expanded, we used one leaf from the second leaf pair to measure leaf length and width, petiole length, and number of trichomes that exerted past the edge of the leaf (then standardized by leaf length). Next, we measured ten flower traits from one flower on the second flowering pair: corolla height and width, corolla tube length and width, stamen length, pistil length, pedicel length, capsule length, calyx length, and degree of flower nodding. When performing floral measurements, we also measured two stolon traits: number of stolons and stolon length. All traits were measured using calipers, except for the degree of flower nodding, which was measured on photographs using imageJ (Rasband 1997).  <em>Postmating reproductive isolating barriers</em> To investigate postmating reproductive isolating barriers among species in the <i>M. tilingii </i>complex, we performed a crossing experiment using plants from 13 maternal families across 10 populations (maternal families: <i>M. caespitosa</i> = 7, <i>M. minor</i> = 2, <i>M. tilingii</i> = 4). For this experiment, we used some of the same individuals as in the morphological analysis above but supplemented them with full siblings from each maternal family. Intraspecific crosses (CxC, MxM, and TxT, where C = <i>M. caespitosa,</i> M = <i>M. minor</i>, and T = <i>M. tilingii</i>) included two types: 1) crosses within maternal families (i.e., between full sibs), and 2) crosses between maternal families within species. Three days prior to each cross, we emasculated maternal parents to avoid contamination from self-pollination. For intraspecific crosses, we generated 62 unique maternal-family cross combinations and 160 total crosses (CxC = 44, MxM = 4, TxT = 14; 1-6 fruits per cross combination).  For interspecific crosses, we performed 86 unique and 210 total interspecific crosses (CxM =10, MxC =12, MxT = 8, TxM = 7, TxC = 25, CxT = 24; 1-8 fruits per cross combination). We used these crosses to assess the following sequentially-acting postmating reproductive isolating barriers: 1) postmating, prezygotic reproductive isolation, 2) hybrid seed inviability, 3) later-acting hybrid inviability, and 4) hybrid male and female sterility.  <i>Postmating, prezygotic isolation</i> To assess postmating, prezygotic reproductive isolation, we measured seed production per fruit from crosses within and between species.  <i>Seed viability</i>             We used two different methods as a proxy for measuring seed viability. First, we performed a visual seed assessment. Recent studies in <i>Mimulus </i>have shown that inviable hybrid seeds are often darkened and/or shriveled (Garner et al.<i> </i>2016, Oneal et al. 2016, Coughlan et al.<i> </i>2020). Following these studies, we scored round, plump seeds as fully developed and seeds with irregular phenotypes (darkened, shriveled, or wrinkled) as underdeveloped. Second, for a subset of crosses, we also assessed seed viability by scoring seed germination. For intraspecific crosses, we measured seed germination rates for 48 unique and 76 total crosses (CxC = 36, MxM = 3, TxT = 9; 1-3 fruits per cross combination). For interspecific crosses, we scored germination for 72 unique and 133 total crosses (CxM = 8, MxC = 7, MxT = 7, TxM = 7, TxC = 20, CxT = 23; 1-4 fruits per cross combination). To determine germination rates, we sowed all seeds from each fruit onto wet paper towels in petri dishes (100 seeds per petri dish to avoid overcrowding). Petri dishes were sealed with parafilm, cold-stratified at 4°C for seven days, and then transferred to a growth chamber that provided constant light at 26°C. Ten days later, we scored germination rate as the number of seedlings that had germinated per total number of seeds planted per fruit. <i>F1 viability </i>             To investigate later-acting (post-seed) hybrid inviability, we tracked survival to flowering in a subset of the seedlings from the germination tests described in the previous section. We transplanted seedlings from petri dishes into flats with 6-cm cells and transferred them to a 16h, 23°C/8h, 16°C greenhouse. We transplanted 5-16 offspring from each of 27 unique intraspecific crosses (CxC = 17, MxM = 2, TxT = 8; total intraspecific offspring = 315) and 1-23 F1 hybrids from each of 29 unique interspecific crosses (F1s: CxM = 5, MxC = 4, TxM = 4, TxC = 9, CxT = 7; total interspecific offspring = 334). All interspecific cross combinations were represented in these analyses except for MxT, which did not produce viable offspring due to the severe seed inviability phenotype. For each individual, we scored the number of days to flowering as a proxy for viability.  <i>F1 sterility</i>              Finally, using a subset of the intraspecific and hybrid offspring grown to flowering, we investigated both male and female fertility. We assessed male fertility in 4-14 offspring from each of 27 unique intraspecific crosses (CxC = 17, MxM = 2, TxT = 8; total intraspecific offspring = 206) and 4-13 F1 hybrids from each of 28 interspecific crosses (F1s: CxM = 5, MxC = 4, TxM = 4, TxC = 9, CxT = 6; total interspecific offspring = 193). For each individual, we collected anthers from 1-3 of the first four flowers and suspended the pollen in a lacto-phenol aniline blue stain, which stains viable pollen a dark blue color. To estimate pollen viability for each individual, we determined the proportion of viable pollen grains from a haphazard sample of about 100 pollen grains per flower. In a few cases, flowers did not produce functional anthers or pollen; these flowers were excluded from further analyses.              To investigate female fertility, we performed supplemental hand-pollinations on intraspecific and hybrid offspring using one or both of their fertile parents as pollen donors. For each of these hand-pollinations, we counted the number of seeds produced per fruit. We used this approach to assess female fertility in 2-9 offspring from each of 27 unique intraspecific crosses (CxC = 17, MxM = 2, TxT = 8; total intraspecific offspring = 119, 1-3 fruits per individual) and 1-7 F1 hybrids from each of 27 interspecific crosses (F1s: CxM = 5, MxC = 4, TxM = 4, TxC = 9, CxT = 5; total interspecific offspring = 112, 1-4 fruits per individual).   
description: We grew maternal families belonging to three species in
 the <em>Mimulus tilingii </em>complex in a common garden and
 assessed for differences in morphology. Additionally, we performed intra-
 and interspecific crosses and tested for postmating reproductive barriers,
 including seed set differeces, seed viability, F1 viability, and F1
 fertility. Here, we include our file (tilingiiCH1_rawdata) containing
 four raw data sheets labeled: "raw_morphological_data",
 "normalized_morphological_data",
 "F0_postzygotic_barriers", and
 "F1_postzygotic_barriers".  For the first
 data sheet ("raw_morphological_data"), columns
 A-C include information on maternal families that were measured for 16
 floral and vegetative traits (raw values are listed in columns E-W). NAs
 denote measurements that were not taken (n=1) or structures that were not
 present (n=2).   To perform a linear discriminant
 analysis (LDA) with the morphological data, we transformed trait values to
 meet LDA assumptions (i.e., that values are normally distributed). The
 second data sheet ("normalized_morphological_data") lists the
 normalized trait values that were used for our LDA. To denote if trait
 values were transformed and how they were transformed, we included
 descriptive names in the column headers. For example, we performed a log
 transformation for petiole length; therefore, we named the column header:
 "petiole_length_log_transformed". We list our
 raw data for intra and interspecific total seed set and seed viability
 (morphological and germination rate) in the data sheet named:
 "F0_postzygotic_barriers". In this data sheet, columns A-F
 include details of the cross, including the maternal and paternal
 lines, their respective species names, and the date the cross was
 collected. Each row represents a single fruit. The following columns
 include raw values for fruits with seed viability assessed through a
 visual assessment and seeds in a fruit that were planted to
 score germination rate.  For our last data sheet
 ("F1_postzygotic_barriers"), we include details regarding F1
 inviability and sterility. In this sheet, each row is an individual plant,
 where individuals grouped by borders are offspring of the same
 unique cross.  Columns C-E list the specific maternal and paternal parents
 and the species cross name. Columns J-O describe female fertility, where
 the total seed per fruit is listed specifically in columns L-O. Columns
 P-Y describe pollen fertility, including pollen viability from a single
 flower, up to three flowers. Column Z describes hybrid
 inviability.