DOI: 10.5061/dryad.7wm37pvzc
title: Crossing data and genomic data for: Strong postmating reproductive isolation in Mimulus section Eunanus

description: Postmating reproductive isolation can help maintain species boundaries
 when premating barriers to reproduction are incomplete. The strength and
 identity of postmating reproductive barriers are highly variable among
 diverging species, leading to questions about their genetic basis and
 evolutionary drivers. These questions have been tackled in model systems
 but are less often addressed with broader phylogenetic resolution. In this
 study we analyze patterns of genetic divergence alongside direct measures
 of postmating reproductive barriers in an overlooked group of sympatric
 species within the model monkeyflower genus, Mimulus. Within this Mimulus
 brevipes species group, we find substantial divergence among species,
 including a cryptic genetic lineage. However, rampant gene discordance and
 ancient signals of introgression suggest a complex history of divergence.
 In addition, we find multiple strong postmating barriers, including
 postmating prezygotic isolation, hybrid seed inviability, and hybrid male
 sterility, leading to complete or substantial postmating isolation in all
 species pairs. Hybrid seed inviability appears linked to differences in
 seed size, providing a window into possible developmental mechanisms
 underlying this reproductive barrier. While geographic proximity and
 incomplete mating isolation may have allowed gene flow within this group
 in the distant past, strong postmating reproductive barriers today are
 likely to prevent any ongoing hybridization. By producing foundational
 information about reproductive isolation and genomic divergence in this
 understudied group, we add new diversity and phylogenetic resolution to
 our understanding of the mechanisms of plant speciation.
description: Crossing data files are provided in .csv format. Variant calls
 are provided in .vcf.gz (gzipped variant call format), which can be opened
 and processed using bcftools (https://samtools.github.io/bcftools/) or another open-source software. Phylogenetic tree files are provided in newick tree format, which can be opened as a text file or by various tree visualization softwares. 
description: # Crossing data for 'Strong postmating reproductive isolation in
 Mimulus section Eunanus.' --- The data in this repository represent
 data related to the assessment of genomic relationships within selected
 members of Mimulus (syn. Diplacus) section Eunanus, as well as postmating
 reproductive barriers between Mimulus brevipes, Mimulus fremontii, Mimulus
 johnstonii, and Mimulus sp. 'Sespe Creek.' Phenotypic data were
 collected using plants grown in growth chambers at the University of
 Georgia, using a combination of wild-collected seeds and seeds produced by
 hand-pollination of growth-chamber-grown individuals. Data were collected
 by Matthew Farnitano between 2019 and 2022. SNP data and phylogenetic
 trees were produced from raw illumina sequencing data (separately archived
 at the NCBI Sequence Read Archive, accession PRJNA922914), using a subset
 of the same individuals for which phenotypic data were collected. Data
 were sequenced at the Duke University Center for Genomic and Computational
 Biology and processed at the University of Georgia using the Georgia
 Advanced Computing Resource Center cluster. Data processing details are
 provided in the associated manuscript. ## Description of the data and file
 structure ### Raw data files included in this repository: 1.
 Crosses_seed_counts.csv 2. Seed_sizes_measured.csv 3. Pollen_counts.csv 4.
 Eunanus_allsamples.complete.vcf.gz 5. Eunanus_allsamples.SNPs.RAxML.tre 6.
 Eunanus_allsamples.SNPs.NJ.tre 7. Eunanus_allsamples.genes.ASTRAL.tre ###
 File descriptions 'Crosses_seed_counts.csv' contains counts of
 viable and inviable seeds from hand-pollination crosses. Seeds were scored
 by eye as viable or inviable based on shape (plumpness, filled vs. empty
 or shriveled). Each line represents an individual fruit which is the
 result of one attempted hand pollination. Fruits which failed to produce
 any seeds after pollination are included with counts of 0 for both viable
 and inviable seeds; these are analyzed under "crossing success"
 in the paper. Some very small particles were assumed to be unfertilized
 ovules and were not included in either inviable or viable seed counts;
 some of these may have been seeds that aborted very early. Variables
 included in 'Crosses_seed_counts.csv': * Batch: Which of three
 growth chamber grow-outs these seeds came from.  * Maternal: The ID of the
 maternal (seed) parent of the seeds counted. The format of the ID is
 Population_MaternalFamily_Individual. Population codes starting with BF
 indicate F1 hybrids between *M. brevipes* and *M. fremontii*; otherwise
 the starting letter indicates species, B=*M. brevipes, F*=M. fremontii,
 J=*M. johnstonii*, S=M. *'Sespe Creek'*. See supplemental
 information in the associated article for details about each Population
 and F1 line. An -X- in the ID indicates self-fertilization, with the
 following number indicating the offspring resulting from that
 self-fertilization. For example, S25_1_2-X-6 is the sixth offspring from a
 self-fertilization of individual S25_1_2. Population JX2 is derived from
 an intraspecific outcross between different families of population J31. *
 Paternal: The ID of the paternal (pollen) parent of the seeds counted. The
 format is the same is for Maternal. * Mat_family: The population or hybrid
 line that the maternal parent of the cross came from (see Maternal for
 details). * Pat_family: The population or hybrid line that the paternal
 parent of the cross came from (see Maternal for details). * Mat_Species:
 The species of the maternal parent of the cross. BxF indicates the
 maternal parent was an F1 hybrid between *M. brevipes* and *M.
 fremontii*.  * Pat_Species: The species of the paternal parent of the
 cross. BxF indicates the maternal parent was an F1 hybrid between *M.
 brevipes* and *M. fremontii*.  * Cross_type: categorization of the type
 and direction of the cross, indicating maternal and paternal species.
 Format is Maternal X Paternal. H indicates an F1 hybrid between *M.
 brevipes* and *M. fremontii*.  * Good seeds: the number of seeds that were
 counted and categorized as 'good' (e.g., assumed to be viable).
 Scoring is by eye based on plumpness, size, and shape.  * Shriveled seeds:
 the number of seeds that were counted and categorized as
 'shriveled' (e.g., assumed to be inviable). Again, scoring is by
 eye. All seeds were categorized as either 'good' or
 'shriveled'.  * Total seeds: the sum of good and shriveled
 seeds, equal to the total number of seeds produced by this fruit. 
 'Seed_sizes_measured.csv' contains seed length and width data
 for a subset of seeds from a subset of crosses. For majority
 'viable' crosses, only viable seeds were measured; for majority
 'inviable' crosses, only inviable seeds were measured. Therefore
 this data represents a comparison of inviable hybrid seeds to their viable
 parental counterparts. For fruits with many seeds, a random subset of
 seeds from the fruit were measured. Measurements were made by
 photographing seeds under a dissecting scope and measuring lengths in the
 software imageJ relative to a standard length. Length is the distance
 between the two furthest points of the seed (in mm) , while width is the
 largest distance perpendicular to the length measurement (in mm).
 Variables included in 'Seed_sizes_measured.csv': * Maternal: The
 ID of the maternal (seed) parent of the measured seeds. See description
 of 'Crosses_seed_counts.csv' for formatting details.  *
 Paternal: The ID of the paternal (pollen) parent of the measured seeds.
 The format is the same is for Maternal. * Mat_family: The population or
 hybrid line that the maternal parent of the cross came from (see Maternal
 for details). * Pat_family: The population or hybrid line that the
 paternal parent of the cross came from (see Maternal for details). *
 Mat_species: The species of the maternal parent of the cross, abbreviated.
 BREV=*M. brevipes*, FREM=*M. fremontii,* JOHN=*M. johnstonii,* SESP=*M.
 'Sespe Creek'* * Pat_species: The species of the paternal parent
 of the cross, abbreviated (same format as Mat_species).  *
 Cross_type: categorization of the type and direction of the cross,
 indicating maternal and paternal species. Format is Maternal X Paternal. *
 Fruit_ID: identifier of the fruit within a particular cross type.
 Individual seeds with the same fruit ID and the same Cross type are from
 the same fruit.  * Seed_ID: identifies the seed within a particular fruit
 and cross type.  * Length: distance in mm between the two furthest points
 of the seed, measured manually in imageJ from a microscopy photograph. *
 Width: largest distance (in mm) across the seed perpendicular to the
 length measurement, measured manually in imageJ from a microscopy
 photograph.  'Pollen_counts.csv' contains counts of viable and
 inviable pollen after staining with aniline blue. Pollen was counted using
 a hemocytometer under a dissecting scope. Squares_counted refers to the
 number of 1mm squares of the hemocytometer that were counted; each square
 represents a volume of 0.1mm^3 = 0.1uL out of a total sample volume of
 50uL for each flower. All anthers from a single flower were placed in a
 50uL sample. Variables included in 'Pollen_counts.csv': *
 Flower: unique identifier of the flower from which pollen was collected.
 If only a single flower was collected from an individual, this is just the
 individual ID. If multiple flowers were collected from one individual,
 \_flowerX or \_fX is used to distinguish each flower, where X is a unique
 number.  * Group: The species or hybrid type of the individual from which
 pollen was collected*.* BxF indicates an F1 hybrid between* M. brevipes*
 and* M. fremontii*.  * Population_Line: The population or hybrid line of
 the individual that pollen was collected from. BF2, BF3, and BF5 represent
 independent F1 hybrid crosses from which multiple seeds were derived.
 Details on the parentage of these crosses is in the associated article
 supplemental materials.  * Individual: The individual ID of the plant that
 pollen was collected from. Format is as in Maternal
 in 'Crosses_seed_counts.csv' above.  * Squares: the number of
 1mmx1mmx0.1mm grid squares on a hemocytometer that were counted. Entire
 squares were always counted. * n_viable: the number of blue-stained (i.e.
 viable) pollen grains counted within the hemocytometer squares.  *
 n_inviable: the number of unstained (i.e. inviable) pollen grains counted
 within the hemocytometer squares.  * Date_counted: the date that pollen
 was counted using the hemocytometer.
 'Eunanus_allsamples.complete.vcf.gz' is a variant call file
 containing SNP calls for 33 Eunanus section samples plus 5 outgroup
 samples in section Diplacus. The file contains both invariant sites and
 biallelic SNP variants. Sites have been filtered for various quality
 metrics, and to retain only sites for which at least 80% of samples have a
 called genotype. The file is in gzipped .vcf format and can be viewed
 using the bcftools utilities
 ([https://samtools.github.io/bcftools/](https://samtools.github.io/bcftools/)) or other open-source tools for reading vcf files. 'Eunanus_allsamples.SNPs.RAxML.tre' is a phylogenetic tree file in newick (text) format, giving the inferred relationships between samples. The tree was produced using the maximum-likelihood program RAxML ([https://raxml-ng.vital-it.ch/#/](https://raxml-ng.vital-it.ch/#/)) with model GTR+Gamma and an ascertainment bias correction. The dataset was an alignment constructed from the variant sites in 'Eunanus_allsamples.complete.vcf.gz', with heterozygous sites randomly called to one or the other allele. Bootstrap proportions (from 1000 rapid bootstraps in RAxML) are provided as branch labels. 'Eunanus_allsamples.SNPs.NJ.tre' is a phylogenetic tree file in newick (text) format, constructed using a neighbor-joining approach with the R package phangorn ([https://cran.r-project.org/web/packages/phangorn/index.html](https://cran.r-project.org/web/packages/phangorn/index.html)). The dataset was the same alignment as the above RAxML tree. Bootstrap proportions (from 1000 bootstrap replicates) are included in the tree. 'Eunanus_allsamples.genes.ASTRAL.tre' is a phylogenetic tree file in newick (text) format, constructed using ASTRAL from a set of gene trees. Each gene tree was constructed using RAxML. The tree is provided in ASTRAL's full annotation format, with quartet and posterior probability information embedded in a branch label string. ## Sharing/Access information ### These data are associated with the following publication: Farnitano, M.C. and Sweigart, A.L. Strong postmating reproductive isolation in Mimulus section Eunanus. *Journal of Evolutionary Biology*, Volume 36, Issue 10, 1 October 2023, Pages 1393–1410. [https://doi.org/10.1111/jeb.14219](https://doi.org/10.1111/jeb.14219) Corresponding Author: Matthew C. Farnitano [mattfarnitano@gmail.com](mailto:mattfarnitano@gmail.com) An earlier version of this manuscript was preprinted at: [https://doi.org/10.1101/2022.12.21.521469](https://doi.org/10.1101/2022.12.21.521469)