DOI: 10.5061/dryad.j3tx95xg3
title: Insights into species delimitation of selected species in the flowering plant genus Medicago section Buceras (Leguminosae)

description: 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.
description: <strong><em>Taxon Sampling -
 </em></strong>Of the 42 accessions of
 <em>Medicago</em>, 39 were 158 sampled for genome size and 14
 were sampled for chromosome number. Most of the species within sect.
 <em>Buceras</em>, particularly <em>M.
 monantha</em>, were represented by multiple accessions from various
 locations to sample across their geographic range.
 <strong><em>Genome Size Estimation using Flow
 Cytometry—</em></strong>Twelve of the 14
 <em>Medicago</em> species studied were sampled for genome
 size, six of which were sampled from multiple accessions. Seeds of the
 species <em>Glycine max</em> (L.) Merr. cv. ‘Polanka’ and
 <em>Raphanus sativus</em> 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, <em>M. truncatula</em> 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 <em>M. truncatula</em> cv. Jemalong (Blondon et al. 1994), 1.11 pg for <em>Raphanus sativus</em> cv. Saxa, and 2.3 pg for the genome size of <em>Glycine max</em> 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).
description: <strong>Steier_genome_size_calculations.csv</strong>
 – Genome size calculation described in “Methods”. See
 "Steier_README.txt" for a detailed description of each
 variable.
 <strong>Steier_genome_size_raw_data.tar.gz
 </strong>– 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.
 <strong>Supplemental Figure S1</strong>.
 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, <em>Medicago
 truncatula</em> (cv. Jemalong), and peak M2 represents the
 experimental sample. On the right is a <em>M. monantha</em>
 group 1 accession (35), showing a smaller genome size and on the left is
 accession <em>monantha</em> 250, from <em>M.
 monantha</em> group 2, which indicates a larger genome
 size. <strong>Supplemental Figure
 S2</strong>. 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.
 <strong>Supplemental Figure S3</strong>. Chromosomes
 of (A) <em>Medicago biflora</em> (2n=16; accession #70), (B)
 M. <em>brachycarpa</em> (2n=16; #72), (C) <em>M.
 astroites</em> (2n=16; #216), (D) <em>M.
 monspeliaca</em> (2n=16; #247), (E-G) <em>M.
 fischeriana</em> (2n=14; #88, 266 and 267), (H-I) <em>M.
 polyceratia</em> (2n=28; #194 and 259), and (J-N) <em>M.
 monantha</em> (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 <em>M.
 fischeriana</em>. Scale bars indicate 10 μm.
 <strong>Supplemental Figure S4</strong>. Map of
 locations of <em>Medicago monantha </em>accessions sampled in
 this study. Collection locations are distinguished by the accession’s
 voucher number, see Appendix 2. Unboxed numbers indicate geographic
 locations where <em>M. monantha</em> group 1 accessions were
 collected. Boxed numbers indicate geographic locations where <em>M.
 monantha</em> 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
 <em>M. monantha. </em>Accession latitude and longitude
 information was obtained from the USDA National Plant<em>
 </em>Germplasm System. These data were plugged into the map-making
 application on easymapmaker.com.
 <strong>Supplemental Table S1</strong>. Accessions of
 <em>Medicago</em> 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.