December 2012 Number 1 - Utah Native Plant Society

Calochortiana December 2012 Number 1
MATERIALS AND METHODS
Sample Collection
Phacelia argillacea leaf tissue samples were collected
from Tucker in the 2006 and 2008 field seasons
and from Railroad in the 2006, 2007, and 2008 field
seasons. A single basal leaf was removed nondestructively
from each individual. The 2004 Tucker samples
represent half-sibling progeny from wild-collected seeds
of 15 maternal individuals (a total of 53 plants) in the
2004 field season. These individuals were grown for
seed production. Phacelia crenulata samples represented
individuals greenhouse-grown from seeds of a
bulk wild collection. Phacelia argylensis and some P.
glandulosa samples were collected from Brigham
Young University Herbarium; specimens were annotated
as sampled for this study. The remaining P. glandulosa
samples represent bulk population samples from
two closely adjacent populations collected by Frank
Smith in 2007 (Figure 1).
DNA Extraction and AFLP Analysis
Fresh leaf tissue samples for DNA extraction were
dried over silica gel, lypophilized, or frozen at -80C immediately
after collection. DNA was extracted from
tissue samples using a Qiagen Plant Mini Kit (QIAGEN,
Inc., Valencia, CA) with minor modifications in the protocol
to achieve a higher concentration of DNA.
AFLP analysis was carried out following Vos et al
(1995) with minor modifications. The enzymes EcoRI
and MseI were used for DNA digestion. Each plant
sample was fingerprinted with six primer combinations.
The primer extensions used were EcoAA/MseA,
EcoAA/MseG, EcoAA/MseT, EcoAC/MseA, EcoAC/
MseG, and EcoAC/MseT. Fragment separation and detection
was carried out on a LI-COR 4300 DNA Analysis
System (LI-COR Biosciences, Lincoln, NE) on a
6.5% polyacrylamide gel. Only unambiguous bands
(50 – 350 bp) were scored for presence or absence.
Bands that were monomorphic among all samples were
discarded from analysis of polymorphic bands. Principal
components analysis was performed on the complete
data set, on data from the three close congeners alone,
on data from P. argillacea alone, and on data from the
Tucker half-sib families alone. We used SAS software
(SAS Institute, Cary, NC) for the analysis.
RESULTS
AFLP analysis produced a total of 535 reliably reproducible
bands, 124 of which were polymorphic. Seventy-five
of these bands were polymorphic only between
P. crenulata and the other Phacelia species (P. glandulosa,
P. argylensis, and P. argillacea; Table 1). This
clearly demonstrated that the P. glandulosa group is
strongly genetically differentiated from P. crenulata, the
putative distant congener in the study. The three close
congeners were much more genetically similar. Phacelia
argillacea exhibited nine bands that were polymorphic
with P. argylensis, seven polymorphic bands
within P. glandulosa from herbarium material, and
seven polymorphic bands within P. glandulosa collected
by Frank Smith in western Colorado. An unexpected
result was that the Smith collections were even more
differentiated from other P. glandulosa than was P. argillacea,
with 15 bands polymorphic between the two
groups. In contrast, P. argylensis was closely similar to
the herbarium-collected P. glandulosa group, with only
3 polymorphic bands. Within P. argillacea, we observed
a total of 30 polymorphic bands, however no polymorphic
bands were found between the Tucker and
Railroad populations, suggesting low genetic differentiation.
When we analyzed data from all four Phacelia species
included in the study, the first principal component
represented 79% of the total variation and clearly separated
P. crenulata form the other three species, reflect-
Table 1. Number of polymorphisms identified using the AFLP (amplified fragment length polymorphism)
technique between or within pairs of species or, in the case of P. glandulosa, within-species groups.
P. argylensis P. glandulosa (H) P. glandulosa (F) P. crenulata
P. argillacea 9 7 7 78
P. argylensis 3 18 93
P. glandulosa (H) 15 87
P. glandulosa (F) 83
(H) Herbarium-collected samples from individual herbarium specimens.
(F) Field-collected bulk samples from two closely adjacent populations.
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