1. Castilleja levisecta (golden paintbrush) and Castilleja hispida (harsh paintbrush) are bee-pollinated perennials, native to the prairies of the Pacific Northwest. However, since the time of European settlement, the Pacific Northwest prairies have declined due to fire suppression, habitat destruction, and the introduction of invasive species. Only 3% of the historic extent of Pacific Northwest prairie remains, making it a critically endangered ecosystem. This prairie habitat destruction has caused C. levisecta to become endangered itself with only eleven wild populations remaining. C. hispida is widespread but is the primary host plant for the caterpillars of the endangered Taylor's checkerspot butterfly (Euphydryas editha taylori). To restore the Pacific Northwest prairie, C. hispida and C. levisecta are being planted together at many field sites in Washington. C. hispida usually has bright red flowers while C. levisecta has golden yellow flowers. However, Castilleja plants with intermediate flower colors have been found at the field sites, providing evidence that C. hispida and C. levisecta are hybridizing. The hybrids have lower pollen fertility and may threaten the genetic purity of C. levisecta. This study analyzes whether flower color can accurately predict hybridization history. (1) If the Castilleja plant has a flower that is intermediate in color between yellow and red, then the plant will have a genetic history that indicates it is a hybrid between Castilleja levisecta and Castilleja hispida. (2) If the plant has a flower that is yellow in color, then it will have a genetic history that indicates it is pure Castilleja levisecta. (3) If the plant has a flower that is red in color, then it will have a genetic history that indicates it is pure Castilleja hispida. Independent-flower color, source of plants Dependent- allelic frequency and presence (genetic history) Castilleja levisecta (golden paintbrush) and Castilleja hispida (harsh paintbrush) are bee-pollinated perennials, native to the prairies of the Pacific Northwest. However, since the time of European settlement, the Pacific Northwest prairies have declined due to fire suppression, habitat destruction, and the introduction of invasive species. Only 3% of the historic extent of Pacific Northwest prairie remains, making it a critically endangered ecosystem. This prairie habitat destruction has caused C. levisecta to become endangered itself with only eleven wild populations remaining. C. hispida is widespread but is the primary host plant for the caterpillars of the endangered Taylor's checkerspot butterfly (Euphydryas editha taylori). To restore the Pacific Northwest prairie, C. hispida and C. levisecta are being planted together at many field sites in Washington. C. hispida usually has bright red flowers while C. levisecta has golden yellow flowers. However, Castilleja plants with intermediate flower colors have been found at the field sites, providing evidence that C. hispida and C. levisecta are hybridizing. The hybrids have lower pollen fertility and may threaten the genetic purity of C. levisecta. This study analyzes whether flower color can accurately predict hybridization history. (1) If the Castilleja plant has a flower that is intermediate in color between yellow and red, then the plant will have a genetic history that indicates it is a hybrid between Castilleja levisecta and Castilleja hispida. (2) If the plant has a flower that is yellow in color, then it will have a genetic history that indicates it is pure Castilleja levisecta. (3) If the plant has a flower that is red in color, then it will have a genetic history that indicates it is pure Castilleja hispida. Independent-flower color, source of plants Dependent- allelic frequency and presence (genetic history) Pure C. hispida used was considered red while pure C. levisecta was considered yellow in flower color. Pure C. hispida and C. levisecta differed significantly in their genetic history (p<0.0001). Hybrids had significantly lower percent C. levisecta alleles than pure C. levisecta (p<0.0001) and significantly lower percent C. hispida alleles than C. hispida (p=0.0007). Hybrids tended to strongly favor one parent rather than have 50% of each parent in their DNA. (Figure 1) The was no overall pattern to hybrid flower color. (Figure 1). However, yellow, dark yellow and yellow with orange flowers together had a significantly different percent C. levisecta alleles when compared to all oranges, pinks, and reds in an unpaired t-test. (p=0.018). There was not a significant difference in genetic history among plants from Glacial Heritage and plants from Violet Prairie for both percent C. hispida and percent C. levisecta. However, when just the Glacial Heritage greenhouse plants were compared to Violet Prairie Nursery Plants, the difference was significant (p=0.003). Pure C. hispida used was considered red while pure C. levisecta was considered yellow in flower color. Pure C. hispida and C. levisecta differed significantly in their genetic history (p<0.0001). Hybrids had significantly lower percent C. levisecta alleles than pure C. levisecta (p<0.0001) and significantly lower percent C. hispida alleles than C. hispida (p=0.0007). Hybrids tended to strongly favor one parent rather than have 50% of each parent in their DNA. (Figure 1) The was no overall pattern to hybrid flower color. (Figure 1). However, yellow, dark yellow and yellow with orange flowers together had a significantly different percent C. levisecta alleles when compared to all oranges, pinks, and reds in an unpaired t-test. (p=0.018). There was not a significant difference in genetic history among plants from Glacial Heritage and plants from Violet Prairie for both percent C. hispida and percent C. levisecta. However, when just the Glacial Heritage greenhouse plants were compared to Violet Prairie Nursery Plants, the difference was significant (p=0.003). (1) DNA was extracted from dried leaf or seed samples Castilleja plants from Violet Prairie, Glacial Heritage (Field and and Greenhouse), Bald Hill, Joint Base Lewis McCord and Scatter Creek South field sites using a CTAB protocol. The CTAB protocol is a series of steps in which the DNA is cleaned, suspended, and re-suspended using CTAB buffer, mercaptoethanol, 24:1 chloroform-isoamyl, 70% ethanol, 90-95% ethanol,TE buffer, and isoproponal. (2) The amount of DNA extracted was quantified using the Nanodrop computer program. (3) The DNA was amplified using PCR (Polymerase Chain Reaction) with eight microsatellite primers. (4) Gel electrophoresis was conducted to determine that the PCR was successful. (5) The PCR results were run and scored for allelic presence on a Beckman Coulter CEQ 8000 Genetic Analysis System. (6) The relationship between genetic history and flower color was analyzed with a structure program. The sample size used in the analysis was 26 pure C. levisecta, 25 pure C. hispida, and 28 putative hybrid individuals. An unpaired t-test was used to determine significance. (1) DNA was extracted from dried leaf or seed samples Castilleja plants from Violet Prairie, Glacial Heritage (Field and and Greenhouse), Bald Hill, Joint Base Lewis McCord and Scatter Creek South field sites using a CTAB protocol. The CTAB protocol is a series of steps in which the DNA is cleaned, suspended, and re-suspended using CTAB buffer, mercaptoethanol, 24:1 chloroform-isoamyl, 70% ethanol, 90-95% ethanol,TE buffer, and isoproponal. (2) The amount of DNA extracted was quantified using the Nanodrop computer program. (3) The DNA was amplified using PCR (Polymerase Chain Reaction) with eight microsatellite primers. (4) Gel electrophoresis was conducted to determine that the PCR was successful. (5) The PCR results were run and scored for allelic presence on a Beckman Coulter CEQ 8000 Genetic Analysis System. (6) The relationship between genetic history and flower color was analyzed with a structure program. The sample size used in the analysis was 26 pure C. levisecta, 25 pure C. hispida, and 28 putative hybrid individuals. An unpaired t-test was used to determine significance. Hypothesis 1 was rejected because although intermediate flowers did indicate hybridization in some instances, many of the putative hybrids were composed mostly of one parent’s alleles. This may be evidence that the hybrids are back-crossing with parent plants in the field. Hybrids as a whole had significantly different genetic histories than their parents. Hypotheses 2 and 3 were true in the instances of pure populations but not among hybrid individuals. Some putative hybrids whose flower resembled the pure form of C. levisecta were actually comprised mostly of C. hispida alleles. Potential sources of error include insufficient aspiration of pipette, and the DNA and primers not fusing properly during PCR. Flower color would not be an accurate predictor of hybridization history in the field. Further research should be conducted to determine whether other morphological traits of C. hispida and C. levisecta can accurately be used to predict their genetic history. For example, C. hispida and C. levisecta have differences in stigma length, leaf-stem angle, and the shape of the leaf lobes. Putative hybrids may show intermediate phenotypes of these traits. Hypothesis 1 was rejected because although intermediate flowers did indicate hybridization in some instances, many of the putative hybrids were composed mostly of one parent’s alleles. This may be evidence that the hybrids are back-crossing with parent plants in the field. Hybrids as a whole had significantly different genetic histories than their parents. Hypotheses 2 and 3 were true in the instances of pure populations but not among hybrid individuals. Some putative hybrids whose flower resembled the pure form of C. levisecta were actually comprised mostly of C. hispida alleles. Potential sources of error include insufficient aspiration of pipette, and the DNA and primers not fusing properly during PCR. Flower color would not be an accurate predictor of hybridization history in the field. Further research should be conducted to determine whether other morphological traits of C. hispida and C. levisecta can accurately be used to predict their genetic history. For example, C. hispida and C. levisecta have differences in stigma length, leaf-stem angle, and the shape of the leaf lobes. Putative hybrids may show intermediate phenotypes of these traits. I would like to give many thanks to Drs. Jeremie Fant and Andrea Kramer for their advice and help as mentors. I would like to thank Lisa Cheung and Jennifer Fischer for collaboration in data collection, and Adrienne Basey for her data on C. levisecta. I would also like to thank Lauren Clark and Loretta Fisher for the Castilleja samples collected from the field sites and their documentation of their flower color. I thank Peter Dunwiddie for providing Images 2 and 3. Many thanks goes out to the Harris Family Foundation Plant Genetics Laboratory at the Chicago Botanic Garden for providing the resources and funding for this research. Clark, L. (2015). Bee-crossed Lovers and a Forbidden Castilleja Romance: Cross-breeding between C. hispida and endangered C. levisecta in prairie restoration sites. Fant, J., Weinberg-Wolf, H., Tank, D., & Skogen, K. (2013). CHARACTERIZATION OF MICROSATELLITE LOCI IN CASTILLEJA SESSILIFLORA AND TRANSFERABILITY TO 24 CASTILLEJA SPECIES (OROBANCHACEAE). Applications in Plant Sciences, 1-5. Fisher, L., Bakker, J., & Dunwiddie, P. (2015). An Assessment of Seed Production and Viability of Putative Castilleja levisecta x C. hispida Hybrids. Kaye, T., & Blakeley-Smith, M. (2008). An Evaluation of the Potential for Hybridization Between Castilleja levisecta and C. hispida. Lawrence, B., & Kaye, T. (2009). Reintroduction of Castilleja levisecta: Effects of Ecological Similarity, Source Population Genetics, and Habitat Quality. Restoration Ecology. I would like to give many thanks to Drs. Jeremie Fant and Andrea Kramer for their advice and help as mentors. I would like to thank Lisa Cheung and Jennifer Fischer for collaboration in data collection, and Adrienne Basey for her data on C. levisecta. I would also like to thank Lauren Clark and Loretta Fisher for the Castilleja samples collected from the field sites and their documentation of their flower color. I thank Peter Dunwiddie for providing Images 2 and 3. Many thanks goes out to the Harris Family Foundation Plant Genetics Laboratory at the Chicago Botanic Garden for providing the resources and funding for this research. Clark, L. (2015). Bee-crossed Lovers and a Forbidden Castilleja Romance: Cross-breeding between C. hispida and endangered C. levisecta in prairie restoration sites. Fant, J., Weinberg-Wolf, H., Tank, D., & Skogen, K. (2013). CHARACTERIZATION OF MICROSATELLITE LOCI IN CASTILLEJA SESSILIFLORA AND TRANSFERABILITY TO 24 CASTILLEJA SPECIES (OROBANCHACEAE). Applications in Plant Sciences, 1-5. Fisher, L., Bakker, J., & Dunwiddie, P. (2015). An Assessment of Seed Production and Viability of Putative Castilleja levisecta x C. hispida Hybrids. Kaye, T., & Blakeley-Smith, M. (2008). An Evaluation of the Potential for Hybridization Between Castilleja levisecta and C. hispida. Lawrence, B., & Kaye, T. (2009). Reintroduction of Castilleja levisecta: Effects of Ecological Similarity, Source Population Genetics, and Habitat Quality. Restoration Ecology. Background Hypothesis Materials and Methods (Procedure) Variables Results References Acknowledgements Conclusion Image 1: Two Putative Hybrids of C. levisecta and C. hispida with yellow flower color resembling that of pure C. levisecta. Image 2: pure C. hispida from Wolf Haven site. Image 3: Putative Hybrids of C. levisecta and C. hispida from Joint Base Lewis McCord Washington with a range of flower colors.