Plant Conservation Alliance Archives - Kentucky Native Plant Society

From the Lady Slipper Archives: The Slender Lip Fern in Kentucky

The Lady Slipper newsletter of the Kentucky Native Plant Society has been published since the Society’s founding in 1986. We occasionally feature an article from a past issue. This year’s Fall Meeting will include a hike at Pine Creek Barrens Nature Preserve in Bullitt county. This article, from the fall of 2014, is about a rare species of fern, the slender lip fern, Myriopteris gracilis, found only in Kentucky in Bullitt county. The location of this fern is along Cedar Creek, in similar habitat to Pine Creek Barrens, and is about a mile away as the crow flies. This article first appeared in the fall of 2014, Vol. 29, No. 3. If you would like to see other past issues, visit the Lady Slipper Archives, where all issues from Vol. 1, No. 1, February 1986 to Vol. 39, 2024, can be found.

The Slender Lip Fern in Kentucky

by James Beck

A single low dolomite ledge near Cedar Creek in Bullitt County harbors one of the most unique plant populations in Kentucky. At a distance this might appear to be a population of the hairy lip fern, Myriopteris lanosa (Michx.) Grusz & Windham, a species known from >20 Kentucky counties. Most will probably know M. lanosa as Cheilanthes lanosa (Michx.) D.C. Eaton, a species recently transferred (along with most North and Central American species of Cheilanthes) to Myriopteris (Grusz and Windham 2013). However, closer inspection will reveal that these Bullitt Co. ferns have smaller, nearly beadlike ultimate segments that are densely hairy underneath, keying clearly to the slender lip fern, Myriopteris gracilis Fée (Cheilanthes feeii T. Moore), in either Jones (2005) or Cranfill (1980).

As the only known M. gracilis locality in the state, this small population would warrant considerable attention. Further investigation would reveal, however, that it is also one of three highly disjunct populations of this species in the eastern United States. The slender lip fern is widespread in the western and central U.S., common on calcareous rock outcrops from British Columbia south to northern Mexico, from southern California east to the Ozark Plateau and the upper Midwest’s “Driftless Zone” (Windham and Rabe 1993). The Bullitt Co. population, discovered by Clyde Reed in the early 1950s, represents a ca. 200 km disjunction from the nearest populations in southern Illinois (Reed 1952). The other two eastern disjunct populations are in southwestern Virgina (Wieboldt and Bentley 1982) and Durham Co., NC (Rothfels et al.

2012). These Kentucky, Virginia, and North Carolina populations add to what is already a remarkably large geographic range, a surprising level of geographic success considering that M. gracilis is exclusively asexual. The slender lip fern undergoes a modified meiosis that produces unreduced spores, which germinate and produce free-living unreduced gametophytes that then develop into adult sporophytes through mitosis. Because they bypass both recombination and the fusion of gametes, asexual species like M. gracilis are essentially genetically “frozen” line-ages, with minimal opportunity to create new genetic variation. Sex and recombination are traditionally thought of as necessary for maintaining the variation needed for adaptation, and asexual species are generally considered incapable of long-term evolutionary success. However, M. gracilis is one of a number of asexual species that occupy wider ranges than their sexual relatives. Although these big ranges could perhaps indicate success over shorter evolutionary time scales, they could simply be biogeographic illusions. As a polyploid (triploid), M. gracilis could have been derived from a sexual ancestor repeatedly over time. As a result, its broad dis-tribution could represent a single, successful lineage or a composite of several geographically smaller lineages formed at different times.

This research question is the focus of my graduate student David Wickell’s M.S. thesis at Wichita State University, and we have spent the 2013 and 2014 field seasons collecting M. gracilis across its wide range. That is what brought me to Cedar Creek this July – the chance to visit the disjunct Kentucky population and add it to our growing genetic dataset. On the long drive east from Wichita I prepared myself for disappointment, however. Although Reed noted that plants were “quite frequent” in his original publication, by 1980 Ray Cranfill noted only “three or four adult individuals.” These plants were presumably the ones observed and photographed by Richard Cassell and the Kentucky State Nature Preserves Commission’s Deborah White in 1994, although subsequent visits failed to relocate this population. On my visit I had the good fortune of working with KSNPC’s Tara Littlefield, and within 10 minutes she led me right to the plants! The population was healthy and sporulating but still quite small (nine adult individuals), and thorough searches of numerous nearby ledges failed to locate additional plants. Photos, geographic coordinates, and habitat notes were taken, along with a tiny amount of leaf material from one plant. DNA extracted from this material will be analyzed along with 94 samples from 20 states collected by myself, my student David, and several collaborators. From each plant we will obtain a kind of genomic “fingerprint,” and the relative genomic distinctiveness of each plant will allow us to determine how many lineages are found across M. gracilis‘ range. The logic is straightforward; individuals from the same lineage are asexual clones of one another and should be essentially genetically identical. On the other hand, individuals from different lineages should exhibit considerably higher levels of genomic distinctiveness. Data from our 95-individual dataset should clearly distinguish between the two alternatives discussed earlier: that of a single successful asexual lineage, or that of many restricted, less successful lineages. The status of the KY and VA (also visited in July) populations will be particular interest. Do these two populations represent the same asexual lineage, suggesting a sort of “stepping” stone colonization? Or do they belong to different lineages, suggesting that M. gracilis was once more widespread and diverse in eastern North America?

Whatever secrets M. gracilis holds, the opportunity to visit a truly unique piece of the Kentucky flora was one this native Kentuckian will remember. Special thanks go to the KSNPC for permission to conduct sampling, to Tara Littlefield (KSNPC) for showing me the site, and to Richard Cassell, Ray Cranfill, Ron Jones, and Deborah White for insightful correspondences

Cranfill, R. 1980. Ferns and Fern Allies of Kentucky. Kentucky State Nature Preserves Commis-sion Scientific and Technical Series, no. 1. 284 pgs.

Grusz, A.L., and M.D. Windham. 2013. Toward a monophyletic Cheilanthes: the resurrection and recircumscription of Myriopteris (Pteridaceae). PhytoKeys 32: 49-64.

Jones, R.L. 2005. Plant Life of Kentucky. The University Press of Kentucky. 834 pgs.

Reed, C.F. 1952. Notes on the ferns of Kentucky, III. Cheilanthes feei on Silurian limestone in Kentucky. American Fern Journal 42: 53-56.

Rothfels, C.J., E.M. Sigel, and M.D. Windham. 2012. Cheilanthes feei T. Moore (Pteridaceae) and Dryopteris erythrosora (D.C. Eaton) Kunze (Dryopteridaceae) new for the flora of North Carolina. American Fern Journal 102: 184-186.

Wieboldt, T.F., and S. Bentley. 1982. Cheilanthes feei new to Virginia. American Fern Journal 72: 76-78.

Recovering America’s Wildlife Act Plant Conservationist Virtual Rally!

Calling all plant lovers!

We need everyone interested in plant conservation to rally together for the passage of the Recovering America’s Wildlife Act. With historic levels of funding and support from both sides of the aisle, this bipartisan bill is critical to protecting our nation’s plant life. This rally will feature leaders in plant conservation and give you an opportunity to take action to encourage your Members of Congress to vote YES on this historic bill. Bring your passion and excitement, because we need YOU to push Recovering America’s Wildlife Act over the finish line!

Special guest speakers from Atlanta and California Botanical Gardens, SE Plant Conservation Alliance, Eastern Band of Cherokee Indians, NatureServe, Center for Plant Conservation, Garden Club of America, Office of Kentucky Nature Preserves, & National Wildlife Federation will share the latest information and why we need your help!

The rally will occur November 13th, 2023 from 2-3pm EST. This is a virtual rally hosted on Zoom. Be sure to register here: Meeting Registration – Zoom

Could Native Clovers have High Nutritional Value for Beef Cattle in the Upper South? A new research project at Virginia Tech

Makayla Bryant¹, Jonathan Omar Cole Kubesch^1,2

Virginia Tech School of Plant and Environmental Sciences; Blacksburg, VA
Country Home Farms; Pembroke, VA

Eastern North America’s tall fescue grasslands produce a plethora of beef stockers, replacement heifers, and cow-calf pairs. These pastures replaced a variety of native woodlands and grasslands in the past 70 years. While tall fescue is a useful forage for much of the year, most tall fescue acreage consists of toxic endophyte Kentucky 31 tall fescue. This endophyte reduces animal performance in terms of average daily gain, reduced reproductive success, and decreased milk production. A lot of this tall fescue acreage cannot be easily converted to alternate forages, such as novel endophyte tall fescue or warm-season forages, in part due to soil conservation as well as economic constraints.

Producers might be able to reduce or eliminate toxic endophyte effects in cattle by overseeding this tall fescue with clovers. Clovers (Trifolium spp) are commonly overseeded into tall fescue stands. Red (T. pratense) and white (T. repens) clovers are currently recommended in Kentucky, Tennessee, and Virginia for pasture clover. There are native alternatives to red and white clover.

A suite of native clovers are present in the United States. Kentucky historically had at least three native species from the Mississippi River to the Appalachian Plateau. These species were present in native grasslands and woodlands throughout the region at the time of European settlement. Native clovers declined with land use change, overgrazing, and fire suppression, but they are still present in modest populations. Native clovers serve a similar nutritional role to wildlife as red and white clovers serve for livestock. Conservation crop science has led to some study as to whether native clovers are agronomically competitive with red and white clover.

Cattle nutrition can justify native clover conservation in pastures

Clovers provide crude protein to cattle in cool-season grasslands. This additional crude protein comes with no additional nitrogen (N) fertilization. Native clovers often serve as the protein concentrate for native ungulates such as bison and deer, as well as monogastric species such as turkeys. Secondary metabolites, such as isoflavones, can dilute or interfere with the alkaloids found in tall fescue pastures. Screening native clover species for traditional metrics of nutritive value can make subsequent analysis for secondary metabolites more effective. Ultimately, this project would provide publishable data regarding the nutritional value of native clovers in comparison to red and white clover under common conditions. This data would guide existing research at Virginia Tech, including Bee-friendly Beef (NIFA-funded). Native clovers are a prime candidate for native plants in livestock production. Including native plants in working grasslands could provide production and conservation opportunities, especially if native clovers offer improved animal nutrition over introduced species.

Native clovers can support cattle and co-exist with non-native grasses

Native clovers are an important protein source in the western rangelands. Species nutritional value has been reported for several of the species native to California, Wyoming, and Oregon range (Bentley & Green, 1954; Cooper, 1957; Cooper and Hunter, 1959; Hamilton, 1961; Hamilton & Gilbert, 1971). These western clovers establish in stands and grow similarly to their nonnative equivalents (Lulow, 2008). Clovers from the eastern United States have only recently been investigated for their agronomic potential. Native clovers have similar agronomic performance relative to traditional red and white clovers.

This is a picture of native clover seedlings in the greenhouse. — Image: Jonathan Kubesch

1990s research suggests that native and nonnative clovers cannot be easily crossed, but native species such as Carolina (T. carolinianum) and peanut (T. polymorphum) clovers in addition to the buffalo clovers have improved pest resistance relative to red and white clovers (Taylor et al., 1994; Quesenberry et al., 1997). Running buffalo clover (T. stoloniferum) is competitive in orchardgrass stands subject to hay and forage clipping schedules (Barker and Sparks, 2013). Running buffalo clover also appears to persist in regularly grazed pastures (e.g. Taylor Fork Ecological Area, Eastern Kentucky University; Good Enough Farm, Peru, Indiana). Buffalo clover (T. reflexum) has a similar seed weight and comparable establishment as red and white clovers (Sanne et al., 2023; Kubesch et al., 2023). This work is lacking in reporting the nutritional value of these native clovers.

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Successful buffalo clover establishment could require high seeding rates

By Jonathan O. C. Kubesch*,**, Frank Reith*, Dillon P. Golding*,***, Jake Sanne*, Forrest Brown*, Derek Hilfiker*, Joseph D. House****, Jenna Beville*, and Peter Arnold*,*****

*Virginia Tech School of Plant and Environmental Sciences, Blacksburg, VA

**Country Home Farms, Pembroke, VA

***Hoot Owl Hollow Farm, Woodlawn, VA

****Indiana National Guard, West Lafayette, IN

*****Arnold Classic Farms, Chestertown, MD

The public is familiar with red (Trifolium pratense) and white clover (Trifolium repens) growing throughout the Kentucky Commonwealth. However, North America, from Oregon to Florida, is home to a plethora of native clover species. Buffalo clover (Trifolium reflexum) is one of several clover species native to the eastern U.S.A. (Kubesch et al., 2022; Kubesch, 2020). This species demonstrates annual to short-lived perennial life histories, and has potential as a horticultural or agronomic crop (Quesenberry et al., 2003; Kubesch, 2020).

Current efforts to increase native clover populations involve laudable efforts regarding site management, as well as conservation horticulture (e.g Littlefield, 2022). After a site is prepared for planting, plugs are produced. Conservation horticulture work currently executes the following procedure:

Germinate seeds on filter paper in petri dishes (Figure 1)

Transfer seedlings to cell pack trays

Pot up plants into small pots (Figure 2)

Plug individuals into spaced nurseries or maintain on benches for seed production

**Figure 1. Running buffalo clover (*Trifolium stoloniferum*) germinating on filter paper under laboratory conditions. Smyth Hall, Virginia Tech, Blacksburg, VA January 30, 2023.**

**Figure 2. Running buffalo clover (*Trifolium stoloniferum*) growing in the greenhouse. University Greenhouses Bay 7A, Virginia Tech, Blacksburg, VA February 3, 2023.**

In restoration and agronomic contexts, seeding clover has a logistic and resource advantage over plugging clovers. Seeding clover can reduce the need for intensive planting efforts, reduce soil disturbance, and ease transportation of unique plant material. Seeding approaches require a basis for setting a seeding rate and dates. Often, clovers are timed for planting between Valentines’ Day and St. Patrick’s Day in the Upper South. Introduced red and white clovers are commonly frost seeded every several years into cool-season pastures (Kubesch et al., 2020).

Seeding clovers can also take advantage of physiological mechanisms that improve seed establishment. In the field, frost seeding involves defoliation of an existing grassland stand, broadcasting clover seed onto the stand, and letting freeze-thaw cycles incorporate the seed into the soil surface. Compared to many native and introduced grasses, clover seed coats allow the seed to survive freeze-thaw incorporation into the soil surface. Quesenberry et al (2003) reports that buffalo clover has a similar seed weight to introduced clovers. A common rate of pasture frost seeding is 4 lb/A red clover and 2 lb/A white clover (Kubesch et al., 2020).

Optimizing rather than maximizing seeding rate is desirable given the limited seed availability of buffalo clover as well as the desire to increase planting area in restoration attempts. Managers want to get a good stand with as little seed as necessary. In addition to generating stand densities that justify direct seeding over plugging, an optimal seeding rate should generate ground cover that conserves soil as well as meets existing criteria for composition.

The present experiment sought to determine whether a 2 lb/A or a 4lb/A seeding rate can optimize buffalo clover establishment relative to white and red clover. This objective was measured through emergence as well as cover assessments. The hypothesis of this study was that the higher seeding rate will achieve the aforementioned targets comparable to, or greater than, red clover and white clover.

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Buffalo clover has moderate seed trait diversity across geographic range

Jake Sanne*, Dillon P. Golding**, Peter Arnold*, Jenna Beville*, Derek Hilfiker*, Forrest Brown*, and Jonathan O. C. Kubesch*, ***

*Virginia Tech School of Plant and Environmental Sciences

**Hoot Owl Hollow Farm, Woodlawn, VA

***Country Home Farms, Pembroke, VA

Introduction

Buffalo clover (Trifolium reflexum), is a rare native clover present in the eastern United States. In Kentucky, this species occurs in the vicinity of Mammoth Cave, as well as further west. Conservation efforts in the eastern United States have maintained many of these native populations, though there is interest in using horticulture and agriculture to increase buffalo clover populations (Quesenberry et al., 2003; Kubesch, 2020; Kubesch et al., 2022).

A limitation of these alternative approaches is the lack of data regarding the establishment of native clovers. For many rare plants, plugs are grown in nurseries and then planted to field sites (Littlefield, 2022). Even in the only published horticultural research, seed was grown into plugs for field plantings (Quesenberry et al., 2003). Buffalo clover has a fair degree of phenotypic variability in growth form, life history, and flower color across the geographic range. These differences in plant material might also suggest diversity in seed characteristics.

Seed weight is an important seed characteristic for increasing buffalo clover populations. Seed weights are associated with increased establishment success (Catano et al., 2022; Westoby, 1998). Published seed weights of red (Trifolium pratense), white (Trifolium repens), and buffalo clover come from a publication using buffalo clover accessions from the Coastal Plain (Quesenberry et al., 2003). Coastal Plain accessions appear to have annual life histories whereas Ohio River Valley accessions appear to be short-lived biennials or perennials (Kubesch, unpublished observations). The present study sought to map and determine seed weights for buffalo clover. The authors hypothesized that Coastal Plain accessions would have different seed weights than the Ohio River Valley accessions.

Materials and Methods

This study consisted of a mapping exercise, seed weight data collection, and then analysis. The mapping exercise discerned the positions of Coastal Plain and Ohio River Valley accessions using the geographic Fall Line as the demarcation between Coastal Plain and Ohio River Valley accessions. Accessions found on the corresponding side of the Fall Line were assigned to a Coastal Plain or Ohio River Valley. This mapping was conducted in ARCGIS software.

Seed weight data for a handful of accessions was accessible from the USDA GRIN system. Notably, these seed weight data overrepresented Coastal accessions rather than Ohio River Valley accessions. Additional data came from Quesenberry et al (2003) as well as manual measurements. Quesenberry et al (2003) selected accessions from TX, GA, MS, and FL. One accession in this study came from the Ohio River Valley. Seed from single plant selections of Cincinnati and Clarks River accessions were measured on a lab balance (Bonvoisin scale).

Data Analysis

Seed weight was treated as a completely randomized design. Initial analysis at the state-level was considered to address potential accession grouping at local scales. Differences in seed weight between accessions from the Coastal Plain and Ohio River Valley were also considered to test other known differences in accessions (e.g. annual or biennial life history). All analyses were conducted in SAS v9.4 (SAS Institute, Cary, NC). PROC GLIMMIX coded for a simple one-way ANOVA.

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**Clarks Rivers NWR Buffalo Clover (Trifolium reflexum) evaluation and conservation**

Jonathan O.C. Kubesch*, Kelly Winklelpleck*, Connor Doyle*, Lindsey Barbini*, John H. Fike*, and Michael Johnson**

*Virginia Tech School of Plant and Environmental Sciences

**Clarks River National Wildlife Refuge

Summary

Buffalo clover (Trifolium reflexum) is a true native clover of eastern U.S. provenance. Previous research suggests that buffalo clover has seed yields and weights similar to introduced clover species (making it suitable for increase). However, such research is limited, with little comparison of plant material from across the northern range of the species. In fact, there is overrepresentation of southern accessions from Georgia, Texas, Mississippi, and Florida. This overrepresentation stems from historical seed collections coming from Coastal Plain collections. In order to better represent accessions from the northern range of the species (both in current research and for future studies), this project sought to 1) conduct a comparison of buffalo clover accessions from across the range against red clover (T. pretense); and 2) bank seed from the Clarks River population for use in conservation and research. Plants were grown from seed to seed and studied in a common garden experiment with efforts taken to maintain genetic purity. Seed was then banked with the Southeastern Grasslands Initiative. Variability in phenotypes across the lifecycle were observed among northern and southern buffalo clover accessions, which suggests that the species complex has further structural variance. Southern accessions required less time to bloom and flowered without vernalization. Clarks River plant material has not yet flowered and appears to require vernalization.

Introduction

The native clovers of eastern North America are relics of the complex mosaic landscapes present prior to European settlement (Gillett and Taylor, 2001). Of these species, buffalo clover is an annual to biennial species with known morphological and reproductive variability (Vincent, 2001).

Kentucky clover (T. kentuckiense) is a recent addition to the flora (Chapel and Vincent, 2013). Species delimitation between Kentucky and buffalo clover has been determined using primarily floral characteristics; however, vegetative characters in a common garden experiment might further resolve the relationships within the species complex.

Previous work with Kentucky clover and running buffalo clover in greenhouse and field research suggest that these native species face fewer propagation challenges in comparison to other rare plants (Kubesch, 2018; Kubesch 2020). Historically, Dr. Norman Taylor at the University of Kentucky maintained all 3 clovers native to Kentucky in greenhouse collections (Kubesch, 2018; Daniel Boone, personal communication).

The objectives of this project were to collect seed from the northern edge of the buffalo clover range and compare buffalo to Kentucky clover. It was hypothesized that Kentucky clover would be more morphologically similar to buffalo clover accessions from Kentucky and Ohio than to accessions from the Coastal Plain.

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Winter Creeper Pull at Ashland- The Henry Clay Estate

Join us April 2nd at 11am EDT as we pull winter-creeper from populations of running buffalo clover at Ashland, The Henry Clay Estate in Lexington following the iNaturalist tutorial hike. To learn more about running buffalo clover, click here. You do not need to partake in the iNaturalist tutorial hike to help pull winter creeper, but you are welcome to! Bring a knee pad and gardening gloves if you have them. We will meet next to the Gingko Cafe. If you wish to join in, please email Heidi Braunreiter, heidi.braunreiter@ky.gov.