Effects of Open Pollination, Selfing, Inbreeding, and Outbreeding Treatments on Seed Set and Viability in Spiraea virginiana Britton (Virginia Spiraea), an Endangered RoseKristin Emery, J. Rhode Ward, and H. D. ClarkeDepartment of Biology

IntroductionSpiraea virginiana (Britton)• Family Rosaceae

• Riparian shrub

• Range: OH, WV, VA, KY, TN, NC, GA

• Endangered in NC

IntroductionThreats• Dams

• Habitat disturbance

• Invasive plants

http://www.americanwhitewater.org/content/Photo/detail/photoid/14448/

IntroductionReproduction

• Generalist pollinators• Mostly asexual

o Rhizomes o Vegetative propagules

• Sexual reproduction is rareo No seedlings in the wild o High seed viability but low germinationo Higher seed viability when outbred

http://plants.usda.gov/core/profile?symbol=Spvi2

IntroductionObjectives• Identify possible barriers to the sexual reproduction of S. virginiana

• Test hand pollination methods to overcome potential barriers

Hypotheses• Stigma pollen loads would differ among study sites and would be

positively correlated with seed set

• Seed viability would be higher in the outbred treatments than in the open or self-pollinated treatments.

MethodsGenetics• DNA extraction from eight populations of S. virginiana in NC

• Multiplex PCR

• Sequencing

MethodsPollination• S. virginiana populations chosen from Ashe,

Graham, and Macon Counties, North Carolina

• Open, selfed, inbred, and outbred pollinations

MethodsPollen Loads

• Stigmas from open treatments collected, stored in a solution of 0.1% malachite green in 95% ethanol

• Brightfield microscopy used to count conspecific and foreign pollen grains on the stigmas

• Scanning electron microscopy used to confirm pollen shape

MethodsSeed Tests• 10+ from each pollination treatment at each subpopulation were dissected, and

seed counts were recorded

• 100 seeds from each pollination replicate were tested for viability using a 1% 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) solution

• Seeds planted in sterilized, moistened soil, stratified in the dark for 90 days @ 4 ⁰C, then placed in a growth chamber (light = 500 µmoles m -2 s-1 , temperature = 22 ⁰C)

Results

Both foreign and conspecific pollen loads on Graham County stigmas were significantly lower (P=0.0001) than in other populations.

Results

Mean (+ SE) seed set per flower by (A) population and (B) treatment. Mean seed set per flower in Ashe County was significantly lower than in Graham County (P=0.0022) and Franklin County (P<0.0001), but there was no significant difference between Graham and Macon County populations (P=0.2432). There was no significant difference among treatments (P=0.18).

Results

Number of (A) viable seeds and (B) germinants from each population. Twenty-eight out of 6,314 seeds tested from the Ashe County population were viable (0.44%). None of the tested seeds from the Graham and Macon County populations were viable. No seeds germinated after four weeks.

DiscussionPollen Load• Genera of floral visitors:

o Crabro (hornets) o Leptura (soldier beetles) o Mordella (tumblingflower beetles)o Popillia (scarab beetles) o Phymata (ambush bugs)

• Increased shade at the Graham County population could have contributed to the low pollen loads

DiscussionSeed Set• Seed set was not correlated with pollen

load, so pollen limitation is probably not the cause of low reproductive success

• Variation in seed set among different populations could be due to factors such as population size and resource availability

DiscussionSeed Viability and Germination• Resource competition could have contributed to the lack of viable seeds

from the Graham and Macon County populations

DiscussionFuture Studies• Ongoing molecular genetic research to

see if low genetic variation within and among populations could cause low reproductive output

• Compare light environment and physiological responses among different S. virginiana populations to see if these are correlated with seed viability and germination

Acknowledgements• Katherine Culatta, Sarah Farmer, Laudan

Nahavandi, Steve Jaslow, Kyanne Breden, Tanya Widen, Gwendolyn Casebeer, Marietta Shattelroe, Sarah Comito and students of the Botany research team helped with data collection

• J. Horton provided support for field work• T. Forrest identified pollinators• T. Meigs and J. Langille assisted with microscopy• Funding was provided by the University of North

Carolina at Asheville Undergraduate Research Program, the North Carolina Department of Transportation, the North Carolina Biotechnology Center, and the Botanical Gardens at Asheville

References[1] Britton, N. and A. Brown. 1913. An illustrated flora of the northern United States, Canada, and the British, possessions. 2 nd ed.

Vol. II. Charles Scribners’ Sons, New York, NY.[2] U.S. Fish and Wildlife Service. 1992. Virginia Spiraea (Spiraea virginiana Britton) Recovery Plan. Newton Corner, Massachusetts.

47 pp.[3] Ogle, D. W. 1991a. Spiraea virginiana Britton: I. Delineation and distribution. Castanea 56:287-296.[4] Glencoe, J. F. 1961. Spiraea virginiana Britton: A rare southern Appalachian endemic. MS thesis, West Virginia University,

Morgantown. 29 pp.[5] Anders, C. M. and Z. E. Murrell. 2001. Morphological and biogeographical variation within the imperiled Virginia Spiraea.

Castanea 66:24-41.[6] Brzyski, J. R., and T. M. Culley. 2013. Seed germination in the riparian zone: the case of the rare shrub, Spiraea virginiana

(Rosaceae). Castanea 78: 87-94. [7] Ogle, D. W. 1991b. Spiraea virginiana Britton: II. Ecology and species biology. Castanea 56:297-303.[8]Brzyski, J. R. 2010. Isolation and characterization of microsatellite markers in the rare clonal plant Spiraea virginiana

(Rosaceae). Am. J. Bot. 0:e20-e22.[9] Moles, A. T. and M. R. Leishman. 2008. The seedling as part of a plant’s life history. in Seedling ecology and evolution. M. A.

Leck, V. T. Parker, and R. L. Simpson, eds. Cambridge University Press, Cambridge, UK. p. 217-238.