Hybridization between levisecta and C. hispida Implications for prairie management Both of these rare species are endemic to Northwest prairies and share recovery sites.

We often don’t understand the reasons for a species rarity, much less their ecological interactions

Are these recovery efforts compatible? Federally Threatened Species Federally Castilleja levisecta Taylor’s checkerspot (Euphydryas editha taylori) Federally Threatened Species Common Native Species Federally Endangered Species Castilleja levisecta Castilleja hispida Taylor’s checkerspot (Euphydryas editha taylori) StudyStudy System System • In 2009, biologists reported Castilleja individuals of intermediate morphology on • In 2009,several biologists southwest reported Castilleja prairies where individualsboth paintbrush of intermediate species morphology have been planted. on several prairies where both paintbrush species have been planted. • The primary objective of this project is to evaluate hybrid fertility. • The primary objective of this project is to evaluate hybrid fertility. • If hybrids are fertile, C. levisecta could be threatened by genetic swamping. • If hybrids are fertile, C. levisecta could be threatened by genetic swamping. StudyStudy System System • Castilleja hispida has diploid, tetraploid and hexaploid races that vary with population. • In 2009, biologists reported Castilleja individuals of intermediate morphology on several• Castilleja prairies levisecta where bothis diploid. paintbrush species have been planted.

• The primary objective of this project is to evaluate hybrid fertility.

• If hybrids are fertile, C. levisecta could be threatened by genetic swamping. StudyStudy System System • Therefore, a secondary objective of this project is to evaluate how ploidy differences affect • In 2009,reproductive biologists isolation reported in this Castilleja system. individuals of intermediate morphology on several prairies where both paintbrush species have been planted. • In order for the concurrent reintroduction • Theefforts primary of C.objective levisecta of and this Taylor’s project checkerspot is to evaluateto proceed, hybrid fertility.managers MAY need to continue planting C. hispida. • If hybrids are fertile, C. levisecta could be threatened by genetic swamping. StudyStudy System System

• These objectives were addressed by making a series of F1 and F2 reciprocal crosses between C. levisecta and the three cytotypes of C. hispida.

• C. hispida seeds were from Washington populations.

• C. levisecta seeds were from the Willamette Valley reintroduced populations, which originated in Washington. Methods Overview 1. Estimate genome size of parental using flow cytometry (2016)

2. Create F1 hybrids (2016)

3. Estimate genome size of F1 hybrids using flow cytometry (2017)

4. Make F1 and F2 reciprocal crosses (2017) 5. Record and analyze three sequential post- fitness measures: fruit set, seed set, percent germination (2017) Nuts and Bolts

• We performed reciprocal crosses, with C. levisecta serving as both pollen donor and pollen recipient. • C. levisecta x C. levisecta crosses served as the reference group. Reference Group Fruit Set, Seed Set CALE x CALE and Germination 1.00 0.80

0.60

0.40

x 0.20 0.76 0.63 0.91

0.00 Prop. Fruit Set Prop. Seed Set Prop. Germination CALE x CALE F1 Fruit Set F1 Fruit Set, 1.00 0.80 Seed Set and 0.60 * 0.40 Germination Set Fruit Prop. 0.20 0.00 CALE x CALE CALE x CAHI(2x) x CALE x CAHI(4x) x CALE x CAHI(6x) x CAHI(2x) CALE CAHI(4x) CALE CAHI(6x) CALE

F1 Seed Set C. hispida (2x) 1.00 0.80 0.60 *** 0.40

Prop. Seed Set Seed Prop. 0.20 *** x C. hispida (4x) *** *** 0.00 CALE x CALE CALE x CAHI(2x) x CALE x CAHI(4x) x CALE x CAHI(6x) x CAHI(2x) CALE CAHI(4x) CALE CAHI(6x) CALE

C. hispida (6x) F1 Germination % C. levisecta (2x) 1.00

. 0.80 *** 0.60 *** Darker colors = higher ploidy levels 0.40

Prop. Germ Prop. 0.20 0.00 CALE x CALE CALE x CAHI(2x) x CALE x CAHI(4x) x CALE x CAHI(6x) x CAHI(2x) CALE CAHI(4x) CALE CAHI(6x) CALE F2 Fruit Set F2 Fruit Set, 1.00 0.80 Seed Set and 0.60 0.40 Germination Set Fruit Prop. 0.20 0.00 CALE x CALE CALE x Hybrid(2x) x CALE x Hybrid(3x) x Hybrid(2x) x Hybrid(2x) CALE Hybrid(3x) CALE Hybrid(2x)

F2 Seed Set 1.00 0.80

Hybrid(2x) 0.60 0.40

Prop. Seed Set Seed Prop. 0.20 * 0.00 x CALE x CALE CALE x Hybrid(2x) x CALE x Hybrid(3x) x Hybrid(2x) x Hybrid(2x) CALE Hybrid(3x) CALE Hybrid(2x) Hybrid (3x) F2 Germination % 1.00 C. levisecta (2x) 0.80 ** ** 0.60 0.40

Prop. Germ. Prop. 0.20 0.00 CALE x CALE CALE x Hybrid(2x) x CALE x Hybrid(3x) x Hybrid(2x) x Hybrid(2x) CALE Hybrid(3x) CALE Hybrid(2x) Reproductive Isolation

• The fitness measures we used, fruit set, seed set, and percent germination, are also considered to be post-pollination barriers to reproduction 푓𝑖푡푛푒푠푠 표푓 ℎ푒푡푒푟표푠푝푒푐𝑖푓𝑖푐 푚푎푡𝑖푛푔푠 푅푒푝푟표푑푢푐푡𝑖푣푒 퐼푠표푙푎푡𝑖표푛 = 1 − 푓𝑖푡푛푒푠푠 표푓 푐표푛푠푝푒푐𝑖푓𝑖푐 푚푎푡𝑖푛푔푠

푝푟표푝표푟푡𝑖표푛 푓푟푢𝑖푡 푠푒푡 𝑖푛 ℎ푒푡푟표푠푝푒푐𝑖푓𝑖푐 푐푟표푠푠 푅퐼 = 1 − 푓푟푢푖푡 푠푒푡 푝푟표푝표푟푡𝑖표푛 푓푟푢𝑖푡 푠푒푡 𝑖푛 퐶퐴퐿퐸 푥 퐶퐴퐿퐸 푐푟표푠푠

n−1 AC = Absolute Contribution ACn = RIn 1 − ෍ ACi i=1

Ramsey (2003); Husband and Sabara (2004) F1 Reproductive Isolation

C. levisecta x diploid C. C. levisecta x tetraploid C. C. levisecta x hexaploid C. hispida hispida hispida RI CALE(2x) x RI CAHI(2x) x RI CALE(2x) x RI CAHI(4x) x RI CALE(2x) x RI CAHI(6x) x Barrier CAHI(2x) CALE(2x) CAHI(4x) CALE(2x) CAHI(6x) CALE(2x) Fruit Set -5.62% 57.75% 15.50% -26.74% -26.74% -5.62% Seed Set -1.16% 8.61% 84.31% 40.29% 98.61% 99.60% Germination % 36.34% -1.93% 20.52% 100.00% -20.48% -20.48% Cumulative 31.98% 60.65% 89.47% 100.00% 97.88% 99.49% Average 41.31% 94.74% 98.68%

100% = total reproductive isolation 0% = no reproductive isolation Negative values = higher hybrid fitness. F2 Reproductive Isolation

diploid F1 Hybrid x C. levisecta x diploid F1 Hybrid C. levisecta x triploid F1 Hybrid diploid F1 Hybrid RI CALE(2x) x RI Hybrid(2x) x RI CALE(2x) x RI Hybrid(3x) x RI Hybrid(2x) x Hybrid(2x) Barrier Hybrid(2x) CALE(2x) Hybrid(3x) CALE(2x) Fruit Set 0.42% -26.74% 36.63% -26.74% -26.74% Seed Set -18.70% 6.08% 96.33% 100.00% -0.94% Germination % -2.90% 16.01% 43.37% 14.72% Cumulative -21.62% 0.01% 98.68% 100.00% -9.10% Average -10.81% 99.34%

100% = total reproductive isolation 0% = no reproductive isolation Negative values = higher hybrid fitness. Conclusions

• Diploid CALE x CAHI F1 hybrids are fertile.

• Diploid F1 hybrids also display higher post-pollination fitness relative to C. levisecta parent plants. • Some triploid hybrids were fertile, but displayed substantially lower fitness relative to C. levisecta parent plants. Management Implications

• The diploid form of C. hispida crosses freely with C. levisecta. Diploid C. hispida should not be co-planted with C. levisecta under any circumstances. • The risk to C. levisecta comes in the form of genetic swamping, which can alter a threatened species’ genetic distinctiveness. • Gene-flow between these two species is substantially curtailed when polyploid C. hispida is crossed with C. levisecta. • Land managers who must C. levisecta at the same site as C. hispida could theoretically use a polyploid cytotype to mitigate the genetic risk to C. levisecta. Thank You

Peter Dunwiddie and the staff of The Center for Natural Lands Management

University of Washington Jon Bakker Loretta Fisher Lauren Clark

Chicago Botanic Garden Andrea Kramer Jeremy Fant

The Department of Botany and Plant Pathology - OSU