Project: Native Clovers Archives - Kentucky Native Plant Society

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.

Recovering the globally rare Kentucky Clover in the Inner Bluegrass Region

By: Tara Littlefield

Long term monitoring of Kentucky Clover in Franklin County @T. LIttlefield

Big news for native clover conservation in the Bluegrass State! Several years of conservation collaboration has resulted in the first transplants of the globally rare Kentucky Clover back into the Bluegrass Woodlands this fall! This endangered clover was discovered (2010) and described (2013) recently and was known from only two privately owned limestone woodlands in the Inner bluegrass of Kentucky. Since its discovery, the Kentucky clover had disappeared from both sites despite annual monitoring and management efforts conducted by Office of Kentucky Nature Preserves Botanist Tara Littlefield and KDFWR biologist Joe Lacefield. Luckily, seed collection and propagation efforts were successful due to our collaborative efforts with Valerie Pence, Kristine Lindsey, and Mairead Kennedy from the Cincinnati zoo CREWs plant program, among many other partners, and the first batch of Kentucky Clover plants was transferred to OKNP in order to transplant into high quality, managed limestone woodlands.

Mairead Kennedy and Valerie Pence present propagated Kentucky Clover Plants at CREW @T. Littlefield

After several years of managing transplant sites for removal of invasive species such as bush honeysuckle (Lonicera maackii), OKNP staff and partners transplanted 75 Kentucky clover plants back into the original Franklin county site as well as transplanted new populations into 3 additional protected high-quality natural areas in Franklin county. OKNP botanists and partners will be monitoring the success of these transplants over the next several years and conducting quantitative studies on how best to manage for this rare clover and its unique limestone woodland habitat.

The power of partnership is key to conserving rare plants and the collaborative approach of the Kentucky Plant Conservation Alliance has made these recovery efforts possible. if you would like to help with these or other plant conservation alliance projects, please contact tara.littlefield@ky.gov.

Delisting of Running Buffalo Clover

By: Heidi Braunreiter, Office of Kentucky Nature Preserves Plant Ecologist

OKNP botanist conducting running buffalo clover survey in 2019 along Boone Creek in Clark County. Photo by Heidi Braunreiter.

Running Buffalo Clover (Trifolium stoloniferum) is a perennial clover with showy white flowers and leaves divided into three leaflets, and was historically native to several eastern and midwestern U.S. states. Based on numerous references to clovers in historical literature, it is believed to have been abundant across its historical range in pre-settlement times, which included the Bluegrass Region of Kentucky (Campbell et al. 1988). However by 1900, its extent had drastically reduced, and only five populations in total remained (Brooks, 1983). Throughout the 20th century, there were numerous fruitless attempts to relocate populations, which lead scientists to believe the species was extirpated from states where it was historically common. Finally, in 1983 populations were relocated in West Virginia and Indiana, as well as in northern Kentucky in 1987 (Campbell et al. 1988). With so few populations known, the U.S. Fish and Wildlife Service (USFWS) opted to list the species in 1987 as ‘federally endangered,’ because it was at a high risk of extinction (USWFS 2007).

The decline of running buffalo clover is believed to have been caused by a combination of factors: as land was settled, habitat destruction ensued and competition from non-native invasive species became a problem (Campbell et al. 1988). Another factor was the loss of the American Bison on the landscape. Historical observations noted the clover’s preference for sites where American Bison congregated (Bartgis 1985). Bison likely provided a means of fertilizer, seed scarification, seed dispersal, and the periodic disturbance regime necessary for the running buffalo clover’s life cycle (Campbell et al. 1988). The loss of bison from the landscape would have dramatically affected the species’ propagation.

Running buffalo clover in a cemetery in Boone County, Kentucky. Photo by Heidi Braunreiter.

Today, you will find running buffalo clover in areas that have periodical disturbances mimicking bison; for example, along trails where plants get trampled, in old estate lawns or cemeteries that are regularly mowed, and on stream terraces with occasional flooding events. Running buffalo clover also prefers dappled sunlight in a forested setting. If the forest canopy becomes either too closed or too open, running buffalo clover populations tend to decline in number as it does not tolerate full shade or full sun (USFWS 2021).

Over the past two decades, the Office of Kentucky Nature Preserves (OKNP) has conducted monitoring on running buffalo clover populations in Kentucky in order to look at population trends and manage for non-native invasive plants. Over this time, many new county records were discovered through targeted searches of historical estates, cemeteries, and stream restoration efforts in the Bluegrass Region. Several successful propagation and introductions efforts have also occurred over the years, for example, new populations were introduced and are now self-sufficient at Shaker Village at Pleasant Hill in Mercer County and Taylor Fork Ecological Area in Madison County.

In 2019, running buffalo clover was proposed for delisting by the USFWS, as many populations had been discovered since its initial listing over 30 years prior. In response to the 2019 proposed delisting, OKNP conducted a state-wide assessment of our running buffalo clover populations. Despite set-backs from the pandemic, we were able to survey 61 populations, which covered the majority of the known extant populations in Kentucky. Sites not included in the assessment were already determined to be extirpated, or inaccessible. Most populations in Kentucky occur on private lands, and although obtaining access to private lands can be difficult, we have been grateful for the curiosity, interest, and enthusiastic response from many property owners. Overall, population trends in Kentucky show approximately half of the populations as stable or increasing in size while the other half of populations are declining in size or extirpated (Table 1). Figure 1 shows an updated range map of extant running buffalo clover populations in Kentucky.

Table 1. Population trends of running buffalo clover in Kentucky during the 2019-2020 statewide survey.

Population Trends	Number of Populations
Relatively stable	11
Increased in size	18
Decreased in size	21
Extirpated**	9
Not applicable*	2
Total	61

**Majority previously believed extirpated; extirpation confirmed

* Previously uncounted; unable to compare trend

A huge success that came out of the 2019-2020 statewide survey was the discovery of the largest running buffalo clover population in Kentucky. On the Mt. Folly Farm in Clark County, a population of over 6,000 plants was counted along the Upper Howards Creek. Other large populations of running buffalo clover in the state top out around 1,000 to 2,000 plants. The significance of this population led to the creation of a new Registered Natural Area with OKNP at Mt. Folly Farm. The clover occurs in the floodplain of the creek in cattle pastures and riparian woods. This site holds potential for future research as it has running buffalo clover in areas with and without a cattle-grazing regime.

As of September 2021, running buffalo clover was delisted by USFWS as they believe there are a sufficient number of populations occurring on publicly owned and managed lands to sustain the species’ viability (USWFS 2021). As of 2020, 175 extant populations occur across its range in Indiana, Kentucky, Missouri, Ohio, Pennsylvania and West Virginia, and biologists in these states discover new populations every year. Within Kentucky, many populations occur on Wildlife Management Areas where stream restoration projects are ongoing. The key to the future success of this plant is the continued management of populations to keep invasive plants at bay and the continued implementation of appropriate disturbance regimes, be that mowing, grazing, or trampling.

If you are interested in adopting a running buffalo clover population in Kentucky to do yearly checkups to remove invasive species, please contact me! heidi.braunreiter@ky.gov.

Figure 1. Extant range of running buffalo clover in Kentucky as of February 2021.

Bibliography

Bartgis, R. 1985. Rediscovery of Trifolium stoloniferum Muhl. ex A. Eaton. Rhodora 87:425–429.

Brooks, R. 1983. Trifolium stoloniferum, Running buffalo clover: Description, distribution, and current status. Rhodora 85:343–354.

Campbell, J.J.N., M. Evans, M.E. Medley, and N.L. Taylor. 1988. Buffalo clovers in Kentucky (Trifolium stoloniferum and Trifolium reflexum) – Historical records, presettlement environment, rediscovery, endangered status, cultivation and chromosome number. Rhodora 90:399–418.

USFWS. 2007. U.S. Fish & Wildlife Service: Running Buffalo Clover (Trifolium stoloniferum) Recovery Plan: First Revision. Department of the Interior, U. S. Fish and Wildlife Service, Great Lakes-Big Rivers Region (Region 3), Fort Snelling, MN.

USFWS. 2021. U.S. Fish & Wildlife Service. Running Buffalo Clover Final Listing Determination Fact Sheet. https://www.fws.gov/midwest/endangered/plants/rbcl/runningb.html. Accessed 2 September 2021.