1. 1. Castilleja hispida: 4photo taken by Tom Kaye
a.Wolf Haven (WH)
b. Joint Base Lewis-McChord (JBLM)
c. Scatter Creek South (SCS)
d. Bald Hill (BH)
2.Assume hybrids [Glacial Heritage]:
a. Maternal Plants (F1)
b. Presumed F2
3. Castilleja levisecta: 4photo taken by Tom Kaye
a. Ebey’s Landing
b. Fort Lewis
c. Rocky Point
d. Naas
Molecular Markers Distinguish Hybridization Patterns in Castilleja
Lisa Cheung1; Jeremie Fant2; Andrea Kramer2; Adrienne Basey2
1Carleton College, Northfield, MN 55057; 2Chicago Botanic Garden, Glencoe, IL 60022
Introductions
Materials
Materials/Methods
Results
Discussion
References
Castilleja levisecta (golden paintbrush) is an endangered flowering
plant restricted in the Pacific Northwest United States (Figure 1).
This species is undergoing restoration in prairies native to another
Castilleja species, C. hispida, which is being used to restore habitat
to an endangered butterfly5. However, it has been observed that
hybridization of the two Castilleja species can occur2, which could
cause the genetic swamping of C. levisecta.
Hybridization between the two species can occur on the top and
bottom inflorescence.Although C. levisecta flowers first, C. hispida’s
flowering time overlaps with C. levisecta’s. Due to flowering
phenology, outcrossing between the two can result in hybridization
differences between the top and bottom [earlier and later,
respectively] inflorescence3. It is hypothesized that there will be a
distinguishable variance in the microsatellite regions between the
two species.Additionally, these variances will allow us to
understand the likelihood of hybridization related to the position
of the inflorescence.
To determine whether C. levisecta and C. hispida had detectable
differences in their microsatellite regions, I ran a Structure
analysis on 21 populations. Based on the restults, the 21
populations were assigned into two distinct clusters,
distinguished as levisecta (yellow) and hispida (red), respectively.
From Figure 2, there are notable differences between the two
Castilleja species, displaying a confidence over 90% as either C.
levisecta or C. hispida. Comparing these differences to the
Structure analysis from population 7 showed that the assumed
F1 hybrids from Glacial Heritage were a levisecta-hispida mix;
however, the analysis also indicated that some of the hybrids
were not F1.Assuming the hybrids were all F1, the Structure
analysis would display an approximately linear pattern at 50%
confidence across the population.Yet, some of the hybrids
displayed a 75% confidence towards levisecta and others a 25%,
suggesting that the hybrids are backcrossing to their pure species
(Figure 4).Additionally, these results suggest that not all hybrids
are distinguishable.
To determine whether hybrids displayed a noticeable
hybridization pattern, I compared the Maternal F1 to the F2.Yet,
comparison could only be analyzed for M1, M2, M15, M22 (see
Figure 3), and their respective offspring due to missing Maternal
F1 samples or polyploidy issues within the F2. However, the
Maternal F1 and F2 lines that were analyzed for both the top and
bottom inflorescence indicated that a directional hybridization
pattern was not observed (Figure 5). For example, M2 and M22
were backcrossing towards levisecta.Although the bottom
inflorescence for their respective offspring backcrossed towards
hispida, M2’s offspring’s top inflorescence backcrossed towards
levisecta; whereas, M22’s offspring’s backcrossed towards hispida.
This implies that there is no clear hybridization pattern.
Nevertheless, pollination is random and less specific, therefore,
there is no pattern for which inflorescence becomes pollinated
by time or position2.
1Basey,A. 2015. Genetic changes associated with native plant propagation: case study in
Castilleja levisecta. Master’s thesis, Northwestern University, Evanston, IL, and Chicago
Botanic Garden, Glencoe, IL.
2Clark, L.A. 2015. Bee-crossed lovers and a forbidden Castilleja romance: cross-breeding
between C. hispida and endangered C. levisecta in prairie restoration sites. Master’s
thesis, University of Washington, Seattle,WA.
3Fisher, L. L., Bakker, J. D., and Dunwiddie, P.W. 2015.An assessment of seed production and
viability of putative Castilleja levisecta x C. hispida hybrids. Report prepared fr the Center
for Natural Lands Management, University of Washington, Seattle,WA.
4Kaye,T. N., and Blakeley-Smith, M. 2008.An Evaluation of the Potential for Hybridization
Between Castilleja levisecta and C. hispida. Unpublished report. Institute of Applied
Ecology, Corvallis, OR.
5Lawrence, B.A., and T. N. Kaye. 2009. Reintroduction of Castilleja levisecta: Effects of
ecological similarity, source population genetics, and habitat quality. Restoration Ecology.
doi:10.1111/j.1526-100X.2009.00549.x.
DNA Extraction:
Extracted DNA from
leaf tissues and seed
capsules using a CTAB
protocol
Assessed extracted
DNA concentration
using a Nanodrop
2000.
PCR Amplification:
Amplified 7
microsatellite regions
of the extracted DNA
using a Castilleja
polymerase chain
reaction (PCR)
program1.
Genetic Scoring:
Analyzed and scored
microsatellite products
with Beckman Coulter
CEQ 8000 Genetic
Analysis System to
determine allele size in
two species from six
populations total.
Analysis:
Ran data gathered
from the Beckman on a
Structure program to
assign individuals into
species and
populations.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
M1 1BA1 1BB1 1BB24 1BB30 1BB32 1BB4 1BB43 1TA1 1TA14 1TA27 1TA42 1TA44 1TB3
%Confidence
Sample
Levisecta
Hispida
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
M2 2BA23 2BA39 2BA5 2BA58 2BA70 2T13 2T16 2T18 2T5 2T6 2T7 2T8 2T9
%Confidence
Sample
Levisecta
Hispida
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
M15 15B11 15B12 15B13 15B3 15B4 15B5 15B6 15B8 15TA1 15TA2 15TA4 15TA6 15TA7 15TA8 15TB1 15TB2
*Confidence
Sample
Levisecta
Hispida
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
M22 22B1 22B10 22B2 22B3 22B5 22B6 22B7 22TB1 22TB1022TB1122TB15 22TB3 22TB4 22TB6 22TB8
%Confidence
Sample
Levisecta
Hispida
Popula'on
Species
Hispida
Levisecta
Individuals
Sample
ID
1
Levisecta
2%
98%
30
-­‐
2
Levisecta
-­‐
-­‐
-­‐
-­‐
3
Levisecta
2%
99%
35
-­‐
4
Levisecta
1%
99%
30
-­‐
5
Hispida
97%
3%
26
-­‐
6
Hispida
98%
2%
29
-­‐
7
F1
Hybrids
54%
46%
16
Maternal
F1
(M)
8
F2
Hybrids
62%
39%
7
1
BoDom
(1B)
9
F2
Hybrids
26%
74%
6
1
Top
(1T)
10
F2
Hybrids
13%
87%
5
2
BoDom
(2B)
11
F2
Hybrids
2%
99%
8
2
Top
(2T)
12
F2
Hybrids
29%
71%
2
3
BoDom
(3B)
13
F2
Hybrids
8%
92%
3
3
Top
(3T)
14
F2
Hybrids
23%
77%
8
14
BoDom
(14B)
15
F2
Hybrids
54%
46%
8
15
BoDom
(15B)
16
F2
Hybrids
19%
81%
8
15
Top
(15T)
17
F2
Hybrids
75%
25%
7
22
BoDom
(22B)
18
F2
Hybrids
89%
11%
8
22
Top
(22T)
19
F2
Hybrids
26%
74%
8
24
BoDom
(24B)
20
F2
Hybrids
6%
94%
5
25
BoDom
(25B)
21
F2
Hybrids
14%
86%
8
25
Top
(25T)
Acknowledgments
I’d like to thank Jeremie Fant and Andrea Kramer for their assistance and
mentorship throughout the research project, as well as Adrienne Basey for
providing the C. levisecta data. I would also like to thank Rebecca Nelson
and Jennifer Fischer for their help with DNA extraction and NSG-REU
grant DBI-1461007 for support funding this project.
Figure 3. Summary of % confidence, number of individuals, and population
for wild and reintroduced C. hispida, C. levisecta, and F1 and F2 hybrids.
Figure 1. Generalized map of wild and reintroduced populations of C.
hispida, C. levisecta, and F1 and F2 hybrids.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6
%Confidence
Population
Levisecta
Hispida
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
M1 M2 M4 M7 M11 M12 M13 M14 M15 M17 M18 M19 M20 M21 M22 M23
%Confidence
Sample
Levisecta
Hispida
Figure 5. Structure analysis of M1, M2, M15, M22, and their respective offspring.
Each graph represents genetic composition of maternal plants followed by the
genetic composition of 5-8 seeds collected from the top and bottom capsules.
Figure 4. Structure analysis for putative hybrid maternal lines assigned into
clusters of K=2.A straight black line at 50% represents F1 and dotted line
at 75% and 25% represents backcross to C. hispida and C. levisecta,
respectively
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 9 9 9 9 9 9 1010101010 1111111111111111 1212 131313 1414141414141414 1515151515151515 1616161616161616 17171717171717 1818181818181818 1919191919191919 2020202020 2121212121212121
%Confidence
Population
Levisecta
Hispida
Figure 2.Top: structure analysis for C. hispida and C. levisecta.Assigned into clusters of K=2, yellow corresponds to % confidence of C. levisecta and red corresponds to
% confidence of C. hispida. Bottom: structure analysis for F1 and F2 hybrids. Assigned into clusters of K=2, the mix of yellow and red determine the % confidence
towards C. hispida or C. levisecta.