April 2021 44T FINAL STUDY REPORT Aberdeen Materials Center Aberdeen, Idaho

Seed Production and Propagation of Northern Bog Violet ( nephrhophylla) for Nokomis Fritillary (Speyeria nokomis) Butterfly Habitat Restoration Derek Tilley, James Spencer, Mary Wolf*

Nokomis fritillary butterfly (Speyeria nokomis Edwards [Nymphalidae]). Photo by Alan Myrup.

ABSTRACT The Nokomis fritillary (Speyeria nokomis Edwards [Nymphalidae]) is a butterfly with unique dietary and reproduction requirements. The only confirmed food source of the Nokomis fritillary larva is the northern bog violet (Viola nephrophylla Greene []), making and plant production of this plant critical to habitat restoration. This study was conducted to determine the feasibility of seed production and propagation of northern bog violet. Seed was hand collected from 50 Northern bog violet grown in the greenhouse over a three-month period. In that time, we estimate that we collected approximately 31,000 . None of the plants showed rhizomatous growth, and asexual division of the plants was unsuccessful. Eight treatments including cold stratification and oxygenated water bath treatments were investigated in a growth chamber. The highest level of germination (33%) was achieved with 60d of cold- moist stratification without the aid of oxygenation. Increasing stratification to 90d did not further increase germination percentage.

*Derek Tilley, PMC Manager, Aberdeen, Idaho ([email protected]); James Spencer, Area Biologist, Roosevelt, Utah; Mary Wolf, PMC Agronomist; 1691 A South 2700 West, Aberdeen, Idaho 83210 INTRODUCTION The Nokomis fritillary (Speyeria nokomis Edwards [Nymphalidae]) is a small butterfly with a wingspan of approximately 50 to 75 mm. Males are orange-brown and females are black and creamy white (Figure 1). This species is endemic to portions of the southwestern United States where its habitat is restricted to streamside meadows, seepage areas, and marshes in otherwise arid environments. Its known distribution includes isolated populations in Utah, Colorado, Arizona, and New Mexico (Selby, 2007). The subspecies Speyeria nokomis nokomis is rare and was petitioned for listing by the U.S. Fish and Wildlife Service as endangered or threatened under the Endangered Species Act in 2013 (WildEarth Guardians, 2013). Reasons cited for its decline include habitat loss and fragmentation due to development, altered hydrology, heavy grazing, mineral extraction, and other human activities (Hammond & McCorkle, 1983). However, it currently has no legal protection at either the state or federal level (U.S. Fish and Wildlife Service, 2016), with the exception of populations within the Navajo Nation (Navajo Fish and Wildlife Department, 2005). The Nokomis fritillary has unique dietary and reproduction requirements that deserve special attention when developing and restoring habitat. Northern bog violet (Viola nephrophylla Greene [Violaceae]) is the only confirmed food source for the Nokomis fritillary larva (Scott, 1986), making seed and plant production of this plant critical to habitat restoration.

Female Nokomis fritillary butterflies deposit their eggs near their larval host plant, northern bog violet (Scott, 1986), often choosing hard substrates like tree trunks, logs, or other woody vegetation (Ellis, personal communication, cited in NatureServe, 2021). Eggs hatch 17 to 18d after laying and are followed by six larval instars before the pupal stage (Scott & Mattoon, 1981). The first tiny instar must endure winter exposure, unfed, until it can find the new emerging violet leaves it needs in early spring (Selby, 2007). Nokomis fritillaries do not migrate, but they are strong flyers and can move between isolated colonies within a continuous riparian zone (Arnold, 1989; Fleishman et al., 2002). It is unlikely that they will disperse long distances between highly isolated riparian systems (Selby, 2007). Nokomis fritillary butterflies are univoltine (having a single generation per year) with an adult flight Figure 1. Female (above) and male (below) Nokomis fritillary from late July to mid-September (Scott, butterflies foraging for nectar on musk thistle (Carduus nutans). 1986; Tilden & Smith, 1986). Adults forage Photo by Alan Myrup. for nectar on a variety of plant species though thistles (Carduus, Cirsium, and Onopordon spp. [Asteraceae]) and blue- and yellow- flowered composites appear favored (Figure 1) (Scott, 1986; Ellis, 1989; Opler & Wright, 1999).

2 Documented flowers visited include native and introduced thistles, horsemint (Agastache spp. [Lamiaceae]) and spotted joe pye weed (Eutrochium maculatum [Asteraceae]) (NatureServe, 2021).

Northern bog violet is a widespread, low growing herbaceous forb occurring throughout North America with the exception of the southeastern states (Figure 2). In the Southwest it can be found in bogs, marshes, seeps, moist meadows and streambanks throughout a broad elevational range and can be found in all plant communities from desert scrub to alpine forests so long as localized moist conditions exist (Kearney & Peebles, 1951; Welsh et al., 2003). Plants are 4 to 10 cm tall with 10 to 20 mm purple or blueish flowers with a notable 2 to 5 mm long spur in the posterior. The scientific name comes from the rounded kidney-shaped leaves with cordate bases. Some sources note the presence of thickened rhizomes (Hitchcock & Cronquist, 1961; Welsh et al., 2003), while others indicate plants arising from a stout vertical rootstock (Davis, 1952; Figure 2. Northern bog violet, Viola nephrophylla Greene, exhibiting perfect Goodrich & Huber, 2014). flower. Photo by Derek Tilley.

Pollination in the Violaceae is rather unique. Perfect flowers in the genus Viola are formed in the spring, specifically evolved for insect pollination. One petal is large and spurred with nectar guides to draw insects (typically bees) under the pollen-bearing anthers above (Zomlefer, 1994). However, later in the season, in response to lengthening photoperiods, the plants produce cleistogamous flowers in which self-fertilization occurs within permanently closed petals (Russell, 1960).

No verifiable propagation information could be found by the authors for northern bog violet; however, it is described by some commercial nurseries as an “easy germinator”. Propagation protocols for other North American native members of the violet genus typically involve a 90 to 120d cold-moist stratification period to break seed (Schultz et al., 2002; Bartow, 2015). Northern bog violet individuals with creeping rhizomes and branching rootstocks could likely also be propagated from plant dividing or rhizome cuttings, though we have not confirmed this.

Seed production of native Viola can be difficult (Bartow, 2014). Viola capsules mature indeterminately and are explosively dehiscent causing the need for specialized seed collection methods. Bartow (2014) planted early blue violet (V. adunca Sm. [Violaceae]) on weed barrier

3 fabric and let capsules dehisce and shatter. Seed was then gathered by vacuuming it directly off the fabric. MATERIALS AND METHODS Seed Production We collected mature northern bog violet plants from a native stand in northeastern Utah by digging up the whole plant and roots in early summer when soil was saturated. The plants were then transplanted into 30x45x10 cm greenhouse flats filled with a standard greenhouse soil mixture. Greenhouse temperatures were maintained between 20 to 35° C, and plants were watered as deemed necessary by visual inspection. In late summer these plants produced cleistogamous flowers and fruit and dispersed seed into the greenhouse flat. Flats were placed outside during the winter months beginning in November, and the flats were brought back into greenhouse in early spring. New seedlings emerged and were transplanted into a 2.5 m2 (120x210x30 cm) galvanized steel tank filled with 15 cm of soil (Figure 3). The tank was watered weekly to keep the soil moist. From those transplants, 50 total plants established. In early summer these plants Figure 3. Northern bog violet plants were transplanted into a 120x210x30 cm tank in late April. Photo by Derek Tilley. produced perfect flowers (Figure 2), but due to lack of pollination produced very little fruit or seed.

In late summer the plants produced fruit and seed via cleistogamy. The capsules were determined to be ripe when they began losing their green color and the pedicel began to elongate lifting the capsule above the leaves (Figure 4). These capsules were harvested daily, but each harvest took

Figure 4. Ripening capsule of northern bog violet (left) and dehiscent capsules (right). Photos by Derek Tilley.

4 less than 5 min per day. The capsules were placed in a paper sack at room temperature for drying. Violet capsules are explosively dehiscent, so the bag needed to be kept closed (but not airtight) to prevent seed from shooting out of the bag. After drying, dried stems and capsule pieces were sieved by hand with a 2.1 mm screen, and dust was removed using a 0.7 mm screen. We removed immature and unfilled seed by running the seed over an air column set on a very low setting. The yields reported here are from harvests from 1 July through 1 October (92d). By October, photoperiods had shortened, and the plants returned to producing perfect flowers with no seed production.

Vegetative Propagation To see if plants could be divided or propagated vegetatively, we looked at the roots of all the field dug materials from the native stands. We also dug up 5 healthy mature greenhouse-reared plants after the first growing season and looked for rhizomes. We also attempted to divide the plants into sections with roots for transplanting.

Seed Germination To investigate and better define germination requirements of northern bog violet, we tested 8 seed treatments including 3 cold-moist stratification (strat) treatments of 30, 60, and 90d, a non- stratified control, and an oxygenated water treatment (oxy), a treatment that has shown promise in overcoming dormancy in wetland species and some forbs (Tilley, 2013; Tilley & Pickett, 2021). The treatments therefore were as follows: 1) 0d strat control, 2) 30d strat, 3) 60d strat, 4) 90d strat, 5) 0d strat +oxy, 6) 30d strat+oxy, 7) 60d strat+oxy, 8) 90d strat+oxy.

For the oxygenated water treatment, we placed 100 seeds in a fine mesh bag and submerged the seed into a mason jar filled with distilled water with an aquarium bubbler for aeration for the entire duration of the germination trial (35d) (Figure 5). Aeration was performed using a Profile 1500 aquarium air pump fitted with a 2.5 cm bubbling air stone. The cold-moist stratification was done by placing seed in a fine mesh bag surrounded by moist sand in a 2° C (35° F) cooler for the desired length of time, 30, 60 or 90d. For stratification and non-treated control treatments, 100 seeds were placed on blotter paper moistened with 5 ml distilled water in 90 mm petri dishes, and the dishes were put in Ziploc bags to retain moisture. All were placed in a Hoffman growth chamber (Hoffman Manufacturing, Inc., Corvallis, Oregon) with a 12-h light/dark cycle with 21° C day and 15° C night temperatures based on commonly used Viola Figure 5. Northern bog violet seeds in an propagation protocols (Schultz et al., 2002; Love & Akins, oxygenated water bath treatment. Photo by 2020). Germination was recorded at 7, 14, 21, 28, and 35d Derek Tilley. after initiation (DAI). Plants were considered germinated if they were observed to have an emerging root or shoot greater than 2 mm in length.

Each treatment was replicated four times in a randomized complete block design with each shelf of the growth chamber serving as a block. Final germination at 35-DAI was analyzed with the

5 analysis of variance procedure after the data was determined to be normally distributed with the Shapiro-Wilk normality test using Statistix 10 Analytical Software (Tallahassee, Florida). In order to obtain a normal distribution of data, null values obtained from the 0d stratification and 0d+oxy treatments were omitted. Means were separated using the least significant difference test at P<0.05.

Plant Production In addition to the above treatments, we sowed untreated seed into Jiffy Pellets® and naturally stratified the seed by leaving them outdoors for 90d (1 November-1 February) before bringing into the greenhouse.

RESULTS AND DISCUSSION Seed Production Total clean seed harvested for the season from the 2.5 m2 tank came to 28.6 g (Figure 6). We weighed 4 replications of 1,000 seeds and calculated an average of 1,100,000 seeds/kg (497,000 seeds/lb). We estimate that we collected therefore approximately 31,000 seeds over the 3-mo period. We stopped harvesting seed on 1 October. At this time the day length had shortened, and the plants returned to producing perfect flowers which required insect pollination for further seed production. We do not know how long plants will ultimately Figure 6. Seeds of northern bog violet. Scale at top of photo is survive in these greenhouse conditions, but 1 mm. Photo by Derek Tilley. periodic replacement of plants could ensure continuous production if desired.

Vegetative Propagation Despite being described by some in the taxonomic literature as having stout elongated rhizomes (Hitchcock & Cronquist, 1961; Welsh et al., 2003), we saw no evidence of rhizomes on any of the plants dug in the native stands or on the greenhouse produced plants. The root systems we saw were very fibrous (Figure 7). It may be possible to divide the plants vegetatively in a way that preserves enough roots around the swollen petiole bases, but we were Figure 7. Fibrous root systems of northern bog violet. Photo by Derek Tilley. unsuccessful at getting divisions to establish.

6 Seed Germination The highest level of germination (33%) was achieved with 60d of cold-moist stratification without the aid of oxygenation (Figure 8). This was significantly higher germination than all other results (p=0.0014). The 90d cold-moist stratification, and 30d and 90d cold-moist stratification with oxygenated water treatment resulted in 23, 20 and 25% germination respectively. The oxygenated water treatment without stratification had no effect on the dormant seed and produced 0 germinants, as did the non-treated control and 30d cold-moist stratification treatment.

Effects of the oxygenated water treatments varied with the duration of stratification. Germination increased significantly with 30d of stratification raising germination from 0% to 20% with the addition of the oxygenated treatment; however, germination decreased significantly among seed stratified for 60d with the addition of the oxygenated water treatment (33% to 8%). There was no significant difference in germination caused by oxygenated water on seed stratified for 90d (23 and 20%).

Figure 8. Percentage of germinated seed after 35d following 8 germination treatments: non-treated control (control), 30d, 60d and 90d cold-moist stratification (Strat) with and without oxygenated water bath (Oxy). Error bars represent +/- 1 SD. Treatment bars with the same letter do not differ at the P<0.05 level.

Plant Production Naturally stratified seed was subjected to freeze-thaw cycles during 90d outdoors. This treatment produced excellent germination and establishment of seedlings when brought into the greenhouse (Figure 9).

CONCLUSION In one growing season we collected over 31,000 seeds with minimal effort and space. This technique may not be suitable for larger production due to the needed daily harvests, and the method used by Bartow (2014) might be easier and more economical for larger quantities.

7 Maximum germination achieved in our tests was 33%, so we estimate Pure Live Seed (PLS) collected for the season to be approximately 10,000. A more vigorous use of air to separate light (unfilled) from filled seed during the cleaning process would likely have raised the percentage of viable seed. It is difficult to extrapolate these data to larger-scale production; however, we do not foresee large quantities in demand for the species. Because northern bog violet habitats are typically small isolated springs and moist areas, this technique may be adequate to supply enough seeds for localized projects as needed.

None of the plants we grew showed signs of rhizomatous growth, but this might be specific to the one ecotype we used. However, we were unsuccessful at dividing the plants asexually. We saw some mortality of plants under the Figure 9. Northern bog violet seedlings emerging in Jiffy Pellets® after greenhouse conditions, and the plants we having been naturally stratified outdoors from 1 November through 1 had are probably best described as short- February (90d) in fluctuating freeze/thaw conditions. Photo by Derek lived perennials that died after expending Tilley their reproductive potential.

Maximum germination was achieved by stratifying the seed for 60d of cold-moist stratification without the aid of oxygenation. Increasing stratification to 90d did not further increase germination percentage. The decrease in total germination from the 60d+oxy treatment seems peculiar, and we could find no obvious reasons for the decline. Further testing may clarify these results. Seed requires a minimum of 30d stratification but 60d appears to be ideal.

The easiest method for producing plants for restoration projects may be to use natural outdoor stratification. Seed sown in Jiffy Pellets® and left outdoors for 90d before being brought into a greenhouse produced excellent germination and establishment of northern bog violet seedlings. These seedlings can be transplanted to pots of desired size and allowed to grow prior to onsite planting later in early summer.

LITERATURE CITED Arnold, R.A. (1989). Unaweep Seep butterfly study. Submitted to U.S. Bureau of Land Management, Grand Junction, CO. Bartow, A. (2014). Propagation protocol for early blue violet (Viola adunca Sm. [Violaceae]). Native Plants Journal 15(2):125-128. Bartow, A. (2015). Propagation protocol for production of Container (plug) Viola adunca plants.

8 USDA NRCS-Corvallis Plant Materials Center Corvallis, Oregon. In: Native Plant Network. US Department of Agriculture, Forest Service, National Center for Reforestation, Nurseries, and Genetic Resources. http://NativePlantNetwork.org Davis, R.J. (1952). Flora of Idaho. Brigham Young University Press. Ellis, S. (1989). Preserve design for the Nokomis fritillary butterfly colonies, Unaweep Canyon, Mesa County, Colorado. Unpublished report submitted to the Nature Conservancy, Colorado Field Office, and U.S. Bureau of Land Management, Grand Junction, CO. Fleishman, E., Ray, C., Sjögren-Gulve, P., Boggs, C.L., & Murphy, D.D. (2002). Assessing the relative roles of patch quality, area, and isolation in predicting metapopulation dynamics. Conservation 16:706-716. Goodrich, S., & Huber, H. (2014). Uinta Flora. U.S. Department of Agriculture Forest Service- Intermountain Region. Hammond, P.C, & McCorkle, D.V. (1983). The decline and extinction of Speyeria populations resulting from human environmental disturbances (Nymphalidae: Arginninae). Journal of Research on the Lepidoptera 22(4):217-224. Hitchcock, C.L. & Cronquist, A. (1961). Vascular Plants of the Pacific Northwest. Part 3: Saxifragaceae to Ericaceae. University of Washington Press. Kearney, T.H., & Peebles, R.H. (1951). Arizona Flora. University of California Press. Love, S.L., & Akins, C.J. (2020). Sixth summary of the native seed germination studies of Norman C. Deno: species with names beginning with letters R through Z. Native Plants Journal 21(2):151-186. NatureServe. (2021). NatureServe Explorer: An online encyclopedia of life. NatureServe, Arlington, VA. https://explorer.natureserve.org/Taxon/ELEMENT_GLOBAL.2.120745/Speyeria_nokom is Navajo Fish & Wildlife Department. (2005). Navajo endangered species list: Resources Committee Resolution No. RCAU-103-05 (Effective 9 August 2005). Navajo Fish & Wildlife Department. Opler, P.A., & Wright, A.B. (1999). Peterson field guide to western butterflies. (Revised ed.) Houghton Mifflin Co. Russell, N.H. (1960). Studies in the photoperiodic responses of violets (Viola). South W. Naturalist 5:177-186. Schultz, J., Beyer, P., & Williams, J. (2002). Propagation protocol for production of Container (plug) Viola canadensis L. plants USDA FS - Hiawatha National Forest Marquette, Michigan. In: Native Plant Network. US Department of Agriculture, Forest Service, National Center for Reforestation, Nurseries, and Genetic Resources. http://NativePlantNetwork.org Scott, J.A., & Mattoon, S.A. (1981). Early stages of Speyeria nokomis (Nymphalidae). Journal of research on the Lepidoptera 20(1):12-15. Scott, J.A. (1986). The butterflies of North America. Stanford University Press. Selby, G. (2007). Great Basin Silverspot Butterfly (Speyeria nokomis [W.H. Edwards]): A technical conservation assessment. USDA Forest Service, Rocky Mountain Region. http://www.fs.fed.us/r2/projects/scp/assessments/greatbasinsilverspotbutterfly.pdf Tilden, J.W. & Smith, A.C. (1986). A field guide to Western butterflies. Houghton-Mifflin Co. Tilley, D.J. (2013). Soaking Nebraska sedge seeds in warm, aerated water improves germination. Native Plants Journal 14(1):55-58.

9 Tilley, D., & Pickett, T. (2021). Germination response of curlycup gumweed to oxygenated water treatment. Native Plants Journal 22(1):4-12. U.S. Fish and Wildlife Service. (2016). Endangered and threatened wildlife and plants; 90-day petition findings on 17 species. Federal Register 81: 1368-1375 Welsh, S.L., Atwood, N.D., Goodrich, S., & Higgins, L.C. (2003). A Utah Flora (3rd ed., revised). Brigham Young University. WildEarth Guardians. (2013). Petition to list the Great Basin silverspot butterfly (Speyeria nokomis nokomis) under the Endangered Species Act. https://pdf.wildearthguardians.org/site/DocServer/Great_Basin_Silverspot_Petition.pdf?d ocID=9363&AddInterest=1103 Zomlefer, W.B. (1994). Guide to families. The University of North Carolina Press.

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