Adam J. Dattilo

Development of microsatellite loci for the endangered species Pityopsis ruthii (Asteraceae)

PREMISE OF THE STUDY Microsatellite loci were developed for the endangered species Pityopsis ruthii and will permit genetic and conservation studies of the species. METHODS AND RESULTS A microsatellite-enriched library was used to develop 12 polymorphic microsatellite loci for P. ruthii. The loci amplified perfect and imperfect repeats with three to seven alleles per locus. Observed heterozygosity ranged from 0.05 to 0.80 and expected heterozygosity ranged from 0.23 to 0.75. CONCLUSIONS These microsatellite loci provide a sufficient set of markers for further investigation of population genetics of P. ruthii.

Restoration of the Endangered Ruth's Golden Aster (Pityopsis ruthii)

Abstract Pityopsis ruthii, Ruths Golden Aster, is an endangered herbaceous perennial that is endemic to small sections of the Hiwassee and Ocoee Rivers in the southeastern US. Our objective was to test the effect of bonded fiber matrix (BFM) on establishment and fecundity of Ruths Golden Aster in order to develop a robust restoration protocol. We augmented existing populations with plants grown from achenes collected at each restoration location. We monitored plantings through 3 growing seasons by measuring stem number, stem height, leaf number, flowering incidence, and number of flower heads per plant in the spring and fall of each season. We assessed survival at 1 month post-planting. We randomly assigned plants at each location to a treatment (BFM vs. no BFM) for analysis as a randomized complete-block design. Germination rate of filled seeds, number of acclimated seedlings, and percent of seedlings planted after 14 days of acclimatization differed significantly across sites. Survival was significantly higher at 1 month, fall year 1, spring/fall year 2, and spring year 3 for the plants mulched with BFM compared to the control. However, there were no significant differences between treatment for stem number, stem height, leaf number, flowering incidence, or final 3-year survival. The methods developed herein represent a major step towards meeting the recovery-plan objective of developing the ability to establish Ruths Golden Aster on suitable habitat. Herein, we provide a framework for augmentation or restoration of critical populations threatened by extirpation.

Current knowledge, threats, and future efforts to sustain populations of Pityopsis ruthii (Asteraceae), an endangered southern Appalachian species1

Abstract Pityopsis ruthii is a federally endangered plant, endemic to riparian areas of the Hiwassee and Ocoee rivers in southeastern Tennessee. The population size and spatial distribution of this species along the Ocoee River has been documented since 1985, yet about 90% of P. ruthii plants are recorded within a portion of the Hiwassee River. Our ongoing population census of these localities delineates 57 discrete site occurrences and constitutes the first population baseline for P. ruthii along the Hiwassee River. Evidence indicates that P. ruthii populations are either stable or increasing along the Ocoee River. Augmentation of natural flow and subsequent invasion of competing vegetation are frequently cited as a threat to the species; however, historical aerial photography suggests significant portions of P. ruthii habitat are resistant to succession. We discuss recent evidence that a number of natural and anthropogenic stressors are challenging population sustainability, including: invasive plants, insect pests, plant pathogens, genetic incompatibility, hybridization, inbreeding depression, and habitat disruption. It remains unclear how these stressors are currently affecting plant populations.

Population Structure and Genetic Diversity Within the Endangered Species Pityopsis ruthii (Asteraceae)

Pityopsis ruthii (Ruth’s golden aster) is a federally endangered herbaceous perennial endemic to the Hiwassee and Ocoee Rivers in southeastern Tennessee, United States. Comprehensive genetic studies providing novel information to conservationists for preservation of the species are lacking. Genetic variation and gene flow were evaluated for 814 individuals from 33 discrete locations using polymorphic microsatellites: seven chloroplast and twelve nuclear. A total of 198 alleles were detected with the nuclear loci and 79 alleles with the chloroplast loci. Gene flow was estimated, with the Hiwassee River (Nm = 2.16; FST = 0.15) showing higher levels of gene flow and lower levels of population differentiation than the Ocoee River (Nm = 1.28; FST = 0.19). Population structure was examined using Bayesian cluster analyses. Nuclear and chloroplast analyses were incongruent. From the chloroplast microsatellites, three clusters were identified; all were present in sampling sites at both rivers, indicating a lack of allele fixation along rivers. Nuclear markers revealed two clusters and separated by river. When the Hiwassee River locations were analyzed, four clusters were identified for both the chloroplast and nuclear microsatellites, though the individuals clustered differently. Analysis of the Ocoee River revealed two clusters for the chloroplast microsatellites and three for the nuclear microsatellites. We recommend P. ruthii be managed as four populations for the Hiwassee River and three populations for the Ocoee River. Our results provide critical genetic information for P. ruthii that can be used for species management decisions to drive future population augmentation/reintroduction and ex situ conservation efforts.

First report of powdery mildew on Ruth's golden aster (Pityopsis ruthii) caused by Golovinomyces cichoracearum (Erysiphe cichoracearum).

Ruths golden aster (Pityopsis ruthii (Small) Small: Asteraceae) is an endangered, herbaceous perennial that occurs only at a few sites along small reaches of the Hiwassee and Ocoee rivers in Polk County, Tennessee. As part of a planned restoration program, Ruths golden aster has been micropropagated in vitro and acclimatized to greenhouse conditions. In February 2011, several established plants in a greenhouse in Knoxville, TN exhibited signs and symptoms of powdery mildew including growth of white mycelium and conidiophores on the adaxial surface of leaves and slight curling upward of leaf margins. Mycelium was superficial and nipple-shaped appressoria were present. Mycelia, conidiophores, and conidia were removed from several leaves, mounted in water, and examined microscopically. Cylindrical to ovoid conidia (n = 100) lacking fibrosin bodies were borne in chains and had a mean length of 32.0 μm (19.2 to 38.7 μm) and width of 14.9 μm (6.3 to 21.2 μm). The description and dimension of the conidia agreed well with that provided for Golovinomyces cichoracearum (Erysiphe cichoracearum) reported on Coreopsis spp. (1,3) and Cirsium arvense (creeping thistle) (2). The teleomorph was not observed. Total genomic DNA was extracted from infected leaves, amplified with ITS1 and ITS4 primers for the 18S rRNA subunit (4), and visualized on a 2% ethidium bromide agarose gel. An amplicon of fungal origin, approximately 550 bp and smaller than the approximately 700-bp plant ITS amplicon, was excised, purified, and then sequenced. This sequence was deposited in GenBank (Accession No. JF779687) and was 99% identical to two G. cichoracearum accessions (Nos. AB77627 and AB77625). Infected leaves were rubbed on leaves of four healthy plants and healthy leaves were rubbed onto other healthy leaves of two additional plants as controls in the greenhouse. Signs of powdery mildew developed on those plants inoculated with infected leaves after 7 to 10 days and the morphology of the fungus was identical to our previous description. To our knowledge, this is the first report of G. cichoracearum (E. cichoracearum) infecting Ruths golden aster. We are not aware of the disease occurring in wild populations of the plant, but it does impact the production of micropropagated plants in the greenhouse. References: (1) D. A. Glawe et al. Online publication. doi:10.1094/PHP-2006-0405-01-BR. Plant Health Progress, 2006. (2) G. Newcombe and C. Nischwitz. Plant Dis. 88:312, 2004. (3) T. E. Seijo et al. Online publication. doi: 10.1094/PHP-2006-1214-01-BR. Plant Health Progress, 2006. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press Inc, New York, 1990.