December 2012 Number 1 - Utah Native Plant Society

Utah Native Plant Society
Flow cytometry is a method that quantifies nuclear
DNA content by measuring the relative fluorescence of
isolated nuclei that have been stained with a fluorescent
dye. This method offers several advantages over traditional
cytological methods including: 1) sample preparation
and processing is relatively easy and rapid (allowing
for large sample sizes), 2) sample material does not
need to be actively dividing (a variety of tissues can be
used, including dried and frozen material), and 3) sampling
is relatively non-destructive (enabling studies of
sensitive plants; Kron et al. 2007; Suda et al. 2007).
Thus, flow cytometry provides a practical means by
which intraspecific population level cytotype data can
be gathered.
The genus Phlox has been an important system for
plant evolutionary studies of polyploidy, hybridization,
and ecology (Ferguson and Jansen 2002; Ferguson et al.
1999; Grant 1959; Levin and Schaal 1970; Levin and
Smith 1966; Wherry 1955). Phlox comprises ca. 65 species
of annual and perennial herbs distributed predominantly
in North America with a center of diversity in the
western United States. We are using flow cytometry to
study cytotype variation across this genus. The present
study focuses on a broad sample of western, upright perennial
Phlox species with special emphasis on two endemic
species of conservation concern found in coniferous
forests in Arizona and New Mexico, P. amabilis and
P. woodhousei. These two species are closely related
(Ferguson et al. 2012) and very similar based on gross
morphology, sharing a characteristic woody-based upright
perennial growth form, thick linear leaves and
notched petals. However, their geographic distributions
do not overlap, and they are readily distinguished by
differences in style length and stigma placement relative
to anther position. These taxa have been variously classified
as distinct species or conspecifics (Cronquist et al.
1984; Wherry 1955). Our objectives for the present
study were to examine 1) taxonomic and large-scale
spatial patterns of cytotype variation across a subset of
western Phlox taxa and 2) fine-scale spatial patterns of
cytotype variation within and among populations of P.
amabilis and P. woodhousei.
METHODS
The present study includes samples from seven species
of upright, perennial Phlox from Arizona, California,
Colorado, Idaho, Montana, Nevada, Utah and Wyoming
(Table 1). For determination of cytotype, several
leaves were collected from one to five individual plants
at each sampling location for each taxon and stored on
ice until nuclear extraction. Voucher specimens for each
population were deposited at the Kansas State University
Herbarium (KSC).
We determined the cytotype of each sample of Phlox
using flow cytometry. Flow cytometry measures nuclear
DNA content, which can then be interpreted in terms of
ploidy level, especially when closely related taxa are
studied and when knowledge of ploidy level is independently
assessed through conventional chromosome
counts (Suda et al. 2007; Halverson et al. 2008). For
each sample of Phlox, we placed 100-300 mg of chilled
leaf tissue into a petri dish with 1.5 ml of chopping
buffer, modified from Bino and others (1993) as described
by Davison and others (2007). We chopped the
leaves finely with a new razor blade and filtered the resulting
liquid through a 30 µm filter into a microcentrifuge
tube. Tubes were centrifuged at 500 x g for 7 min,
the supernatant removed, the pellet re-suspended in 700
µl propidium iodide staining solution (50 mg/ml;
BioSure), and 2µl of chicken erythrocyte nuclei singlets
added (CEN internal standard; BioSure). Samples were
protected from light and stored on ice for at least 30 min
before analysis on a Becton Dickinson FACS Calibur
flow cytometer at the Kansas State University Flow Cytometry
Facility. The amount of fluorescence was measured
for ~10,000 nuclei per sample. Resulting histograms
were visually inspected for the presence of clear
nuclear populations from the Phlox sample and the CEN
internal standard, and mean peak values were calculated
using the program Cell Quest (Becton Dickinson). Nuclear
DNA content was calculated as the Phlox sample
mean peak value divided by the CEN internal standard
mean peak value multiplied by the 2C-value of the CEN
internal standard (2.5 pg; Dolezel and Bartos 2005).
Ploidy level was inferred for each sample based on the
calculated DNA content. Inferred ploidy level was
linked with chromosome count data for several samples.
RESULTS AND DISCUSSION
A total of 140 samples from seven species of Phlox
collected from 63 locations were assessed for cytotype
variation (Table 1). When cytotype was measured in
multiple individuals from a single location, average nuclear
DNA content was calculated. We did not detect
any cytotype variation within populations based on our
limited sampling. The results from flow cytometry were
interpreted as measures of ploidy level rather than absolute
measures of DNA content (see Suda et al. 2007).
Results from chromosome counts confirmed ploidy levels
of 2x, 4x, and 6x (for five samples; Table 1).
Cytotype varied throughout the species studied and
appeared to reflect both taxonomy and geography
(Table 1). Populations of P. caryophylla and P. cluteana
were diploid, while populations of P. aculeata were
tetraploid; all of these taxa are fairly narrow endemics.
In general, populations of the wide-ranging P. longifolia
and P. stansburyi were geographically structured, with
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