The genus Medicago (Leguminosae, Papilionoideae) contains about 90 species including the important forage crop alfalfa Medicago sativa and the genomic model Medicago truncatula. Despite intensive research on the genus because of its agricultural importance, there is a relative lack of information about chromosome number and genome size in some Medicago species, especially those from section Buceras that were formerly placed in the sister genus Trigonella, and are paraphyletic to the remainder of the genus Medicago. Past studies revealed that previous species delimitations did not conform well with complex patterns of morphological or genetic variation. Some published chromosome numbers, e.g., 2n = 28 and 2n = 44, differ from those of the rest of the genus, which are mostly 2n = 16 or polyploids thereof, although some cases of aneuploid reduction or dysploidy (e.g., 2n = 14) do exist. Here we estimated phylogenetic relationships of 42 accessions corresponding to 14 currently recognized Medicago species that are paraphyletic to the remainder of Medicago with a focus on Medicago monantha; for a number of those accessions, we obtained estimates of genome size (39) and chromosome number (14). We can confirm the delimitation of two species within section Buceras, and our data suggest that there are at least two entities with distinct geographic distributions within the currently recognized species M. monantha, which differ in chromosome number and genome size. Our data also suggest that polyploidy and post-polyploid descending dysploidy played a significant role in genome evolution within section Buceras. Our data provide a strong foundation for whole-genome sequencing projects and further in-depth research of these paraphyletic lineages.

Taxon Sampling - Of the 42 accessions of Medicago, 39 were 158 sampled for genome size and 14 were sampled for chromosome number. Most of the species within sect. Buceras, particularly M. monantha, were represented by multiple accessions from various locations to sample across their geographic range.

Genome Size Estimation using Flow Cytometry—Twelve of the 14 Medicago species studied were sampled for genome size, six of which were sampled from multiple accessions. Seeds of the species Glycine max (L.) Merr. cv. ‘Polanka’ and Raphanus sativus L. cv. ‘Saxa’, used as known genome size standards, were obtained from J. Doležel (Institute for Experimental Botany, Olomouc, Czech Republic; Doležel and Bartos 2005) and seeds of a third species used as a standard, M. truncatula cv. ‘Jemalong’, were obtained from the Medicago Hapmap Project (http://www.medicagohapmap.org). Seedlings of all accessions were grown in a growth chamber at 25°C under identical light and humidity conditions. Nuclei from samples of fresh leaf tissue were prepared following the procedure of Doležel and Bartos (2005) using Galbraith’s buffer (Galbraith et al. 1983). Samples were analyzed on a BD FACSCalibur flow cytometer at the Biodesign Institute, Arizona State University (Tempe, Arizona). Mean DNA content was estimated on ca. 15,000 nuclei, with peak means identified using CellQuest software (Becton Dickinson). Calculation of genome size in picograms (pg) used the sample peak mean divided by the standard peak mean, multiplied by the known genome size of the standard. Each plant accession was sampled three or more times on different days to minimize the effect of instrument drift and other variables. For most accessions, three or more different plants were sampled, but occasionally only one or two plants were sampled (see Appendix 2). Values reported are averages of the values obtained from each individual accession. The known standard genome size values used were 1.15 pg for M. truncatula cv. Jemalong (Blondon et al. 1994), 1.11 pg for Raphanus sativus cv. Saxa, and 2.3 pg for the genome size of Glycine max cv. Polanka rather than the 2.5 pg indicated in Doležel and Bartos (2005) based on results obtained with other standards (Steele et al. in preparation).

Steier_genome_size_calculations.csv – Genome size calculation described in “Methods”. See "Steier_README.txt" for a detailed description of each variable.

Steier_genome_size_raw_data.tar.gz – This file contains the raw genome size data produced by flow cytometry for each sample. An example of how to interpret these visualizations can be found in "Steier_Figure_S1.tiff" (see below). Folders are named following the convention "sample_PI#". Files are named following the convention "MM_DD_YY.sample#.pdf". "Steier_flow_cytometry_raw_data_index.xlsx" provides the sample name, PI #, date, and sample # for each experiment.

Supplemental Figure S1. Representative example of raw genome size data produced by flow cytometry. Location of peaks along the x-axis is used to calculate genome size in picograms. Peak M1 represents the standard, Medicago truncatula (cv. Jemalong), and peak M2 represents the experimental sample. On the right is a M. monantha group 1 accession (35), showing a smaller genome size and on the left is accession monantha 250, from M. monantha group 2, which indicates a larger genome size.

Supplemental Figure S2. Phylogram of tree number 1 of 1,000 equally most parsimonious trees (241 steps) produced in a heuristic search analysis of 49 nrDNA ITS and plastid encoded 5’ trnK intron/matK gene sequences from the accessions within “Dataset 2”. The scale bar indicates the number of substitutions, and branch length is proportional to this value.

Supplemental Figure S3. Chromosomes of (A) Medicago biflora (2n=16; accession #70), (B) M. brachycarpa (2n=16; #72), (C) M. astroites (2n=16; #216), (D) M. monspeliaca (2n=16; #247), (E-G) M. fischeriana (2n=14; #88, 266 and 267), (H-I) M. polyceratia (2n=28; #194 and 259), and (J-N) M. monantha (2n=26, 30, 36, 44 and 44; #264, 35, 36, 249 and 250, respectively) counterstained by DAPI (inverted in Adobe Photoshop). Arrowheads mark the putative fusion chromosomes in M. fischeriana. Scale bars indicate 10 μm.

Supplemental Figure S4. Map of locations of Medicago monantha accessions sampled in this study. Collection locations are distinguished by the accession’s voucher number, see Appendix 2. Unboxed numbers indicate geographic locations where M. monantha group 1 accessions were collected. Boxed numbers indicate geographic locations where M. monantha group 2 accessions were collected. The dashed line represents the 45˚ longitudinal line that Small and Fawzy (1992) concluded was a boundary between morphologically distinct populations of M. monantha. Accession latitude and longitude information was obtained from the USDA National Plant Germplasm System. These data were plugged into the map-making application on easymapmaker.com.

Supplemental Table S1. Accessions of Medicago species analyzed in this study. Species and section names follow Small (2011); original identification in parenthesis where applicable. USDA National Plant Germplasm System accessions begin with PI or W6, any other accessions are from Ernest Small’s collection, with seeds donated to K. P. Steele. Location information obtained from the USDA National Plant Germplasm System, or Ernest Small’s notes as summarized in Steele et al. (2010). Accessions with “*” originated from a mixed USDA seed collection. Average genome sizes listed in pg; +/- standard deviation with number of samples analyzed for genome size estimation “N”. Chromosome numbers with “*” were obtained from literature (Small 2011). For all columns, — indicates data not available.