Plant genome size measurements
Plant species were first identified by a group of plant taxonomists,
then all the species names were further standardized into the accepted
names according to The Plant List (version 1.1; www.theplantlist.org).
We recorded 286 herbaceous species in the transect (our study sites
contained mainly herbaceous vegetation); of these we were able to obtain
measurements of genome size from 169 species during subsequent visits to
representative study sites belonging to the Chinese Grassland Long-term
Research Stations (at least one site for each grassland type: alpine
grassland, desert steppe, typical steppe, and meadow steppe). These
stations represented the typical vegetation and species pool for each
grassland type and were convenient to re-visit. Samples were collected
to measure genome sizes during the growing seasons of 2017-2019 (from
July to September). We sampled plant species that occurred at each
grassland type of the transect, focusing primarily on (but not limited
to) the more common ones (details for the common species and species
richness of each study site are supplied in Supplementary Dataset 1 and
2).
For each species, three to twelve individuals were selected and leaf
samples from each individual plant were measured for the genome size and
the averaged value was used. For each individual, leaves from at least
three fresh samples were analysed by flow cytometer (BD LSRFortessa,
USA) (Doležel et al. 2007). Samples were chopped with a sharp
razor blade for ca. 15-25s in a petri dish containing 1mL ice-cold
nuclear LB01 isolation buffer (15 mmol/L Tris, 20 mmol/L EDTA-Na2, 0.5
mmol Spermine tetra-hydrochloride, 80 mmol/L KCl, 20 mmol/L NaCl,
0.1%volume percentage Triton X-100, 2 mmol/L DTT, pH=7.5). The
homogenate was gently sucked up by pipette and filtered through a 50 μm
nylon mesh. A volume of 50 μL of RNase (1 mg mL-1;
Sigma, St Louis, MO, USA) and 50 μL of Propidium iodide (PI, 1 mg
mL-1; CyStain PI Absolute P, Sysmex Partec GmbH
Görlitz, Germany) were then added and mixed by gentle shaking. The
mixture was incubated for 4 min in the dark at 4 °C and loaded onto the
flow cytometer to measure genome size. The pg of DNA per nucleus in each
peak is estimated by comparing measurements to a known internal
reference standard. We had five species as our primary reference
standard and two species as our secondary reference standard. For the
primary reference standard, seeds were obtained from Centre of Plant
Structural and Functional Genomics of the Institute of Experimental
Botany, Czech Academy of Sciences, in 2017, including Solanum
lycopersicum cv. Stupicke (1C = 0.98 pg) (Doležel et al. 1992),Glycine max cv. Polanká (1C = 1.25 pg) (Doležel et al.1994), Zea mays CE-777 (1C = 2.72 pg) (Lysak & Dolezel 1998),Pisum sativum cv. Ctirad (1C = 4.55 pg) (Doležel et al.1998), Secale cereale cv. Dankovske (1C = 8.10 pg) (Doleželet al. 1998). Based on the primary reference standard, genome
size of two local species: Kerria japonica (L.) DC (1C = 0.50
pg), Hosta plantaginea (Lam.) Aschers. (1C = 11.33 pg) were
further calibrated against S. lycopersicum and V. faba and
chosen as a secondary reference standard. The mean, standard deviation,
and individual measurements of the studied species and the used internal
standards are reported in Table S1. Note that we originally measured
genome size for 169 species but we excluded eight species with high
coefficients of variation in peak quantification (e.g. > 5
%) given their unprecise measurement (Doležel et al. 2007).
Among the 161 species sampled for genome size measurements, 23 species
occurred more than once across the resampling sites, where we could
check intraspecific variation of ploidy levels (Table S2). The
resampling sites refer to the sites that have been investigated during
the transect study but were revisited for measuring genome sizes. Most
of these species showed quite stable 1C DNA content values across the
transect, except Artemisia frigida and Agropyro cristatum .
Previous studies had reported the 1C value for diploid A. frigida(2.63 pg) and A. cristatum (6.49 pg) (Garcia et al. 2008;
Said et al. 2018), with their polyploid species being identified
in nature (Wan et al. 2011; Zhao et al. 2017). In our
case, the measured 1C values indicated that A. frigida andA. cristatum occurred more often as diploid (1C = 3.25 and 6.84
pg) in meadow steppe while as tetraploid (1C = 5.26 and 13.30 pg) in the
other three grassland types. We report the diploid and tetraploid 1C
values for those two species according to their occurring grassland type
(Table S1) and used the mean value in our data analysis to handle the
intraspecific ploidy variation.
For the 161 species, we assessed their contributions to biomass and
species richness of the total plant community at each site. Sites were
included in further analyses if, for that specific site, the species
that had genome size values contributed to more than 80% of biomass and
richness. Thus, for the final analysis, we used data for plant
communities containing 161 species from 52 sites along the transect.
Overall, the plant 1C values varied 260-fold from the smallestAstragalus scaberrimus (0.12 pg) to the largest Allium
ramosum (31.5 pg), with a median and mean of 1.59 and 3.19 pg,
respectively (Fig. S1; Table S1).