A Seedling Key for Kentucky Clover (Trifolium kentuckiense)

Jonathan O.C. Kubesch*, Lindsey Barbini, Connor Doyle, John Fike, Virginia Polytechnic Institute and State University (Virginia Tech)

A technical publication for dual submission to the Office of Kentucky Nature Preserves and the Kentucky Native Plant Society with the permission of Tara R. Littlefield.

Introduction

Kentucky clover (Trifolium kentuckiense) is a recently identified addition to Kentucky’s flora (Chapel and Vincent, 2013). The plant is known to two sites in the Bluegrass region, and the small population sizes put the species at risk for extinction in the wild. The species initially had been considered buffalo clover (T. reflexum), but the species complex was revised after considering ecological and morphological discrepancies (Chapel and Vincent, 2013).

The Woodford County site suffers from poor grazing management (Kubesch, 2020). Cattle move from the adjacent pasture into the shaded habitat. In part, this semi-natural state supports the encroachment of red clover (T. pratense) into the habitat. Minimizing grazing at the seedling stage will be crucial for protecting this species, which looks very similar to T. pretense during early stages of development. Thus, discerning between the critically endangered Kentucky and common red clover at the earliest stage is critical.

The Franklin County site does not appear to have any nonnative clover species, though a nearby powerline right-of-way or adjacent farmland might facilitate encroachment over time, which further justifies early comparisons of these similar species. Red clover is a biennial to short-lived, crown-forming perennial forage species that is the agricultural analogue to buffalo and Kentucky clovers in the same way that previous work has compared white clover to running buffalo clover (Vincent, 2001; Ball et al., 2015; Kubesch, 2018).

As part of ongoing propagation efforts, seedlings of red, buffalo, and Kentucky clovers were compared in a common garden greenhouse experiment in order to ascertain potential distinguishing characteristics for the native clover seedlings. The chronological and developmental period at which plants could be identified was sought on the basis of visual keys and some growth measurements.

Materials and methods

All three species were started on germination paper in plastic germination boxes on a lab bench in Smyth Hall, Virginia Tech, Blacksburg, VA. Multiple sources of germplasm were available for the red and buffalo clover, though the Kentucky clover seed had a sole origin (Table 1).

Table 1. Clover accessions used for the common garden greenhouse experiment.

SpeciesAccession/varietyYearSource
Red cloverKenland2006John Fike
Red cloverCinnamon Plus2006John Fike
Red cloverSS-0303RCG2020Southern States
Buffalo cloverCincinnati2020Dan Boone
Buffalo cloverGeorgia1978USDA
Buffalo cloverTexas1956USDA
Kentucky cloverWoodford County2017*Julian Campbell
*seed was produced from plants 1 generation removed from wild population
Figure 1. Germination procedure for the first weeks of the experiment
Figure 1b. Buffalo clover (Cincinnati-West Fork Woods) at first trifoliate leaf.
Figure 1c. Buffalo clover (Georgia) at first trifoliate leaf.
Figure 1d. Kentucky clover (Woodford) at first trifoliate leaf.

An additional accession of buffalo clover from Clarks River, KY, was started but did not germinate until after the study period began and was thus excluded from the analysis. After the germination period, seedlings were moved to the greenhouse and potted up into 6-cell packs in trays. The growing medium was a 2:1 mixture of calcined clay and commercial potting mix (Miracle Gro). Plants were watered regularly using catch trays and liquid fertilizer (0.5-2-1; Buddha Bloom root organic, aurora innovations, Eugene, OR) was used to supply macro and micro nutrients. Plants were maintained between 21-27 ℃ under supplemented natural light.

Plants were inspected at approximately 2-d intervals to identify the emergence of distinct morphological characteristics. Observations occurred once plants developed the first trifoliate leaves. Based on a priori information, plants were specifically examined for differences in pubescence, growth in terms of leaf size and number, and crown of reproductive stems. Visual assessments of morphological differences among and within accessions were augmented with the use of a 14 X hand lens.

Pubescence, or hairiness, was assessed both in relative amount and position on the plant (stems, leaves, midveins). Leaf morphology was assessed in the appearance on venation on leaflets as well as subtle differences in leaflet shape. In addition to measuring the days required for each species to reach specific growth stages, comparisons were made between developmental stages (first true leaf, second true leaf, etc.).

Results and discussion

Table 2. Potential key characters and their utility at varying developmental stages in red, buffalo, and Kentucky clovers.

Life StageLeaf pubescenceChevronsStem pubescenceCrown developmentOther notes
1st true leaf (V1)Tops of leavesOn red clover but not on Cincinnati and KentuckyOnly between red and Kentucky clovers
Figure 2. Visual differences between red clover (left) and Kentucky clover (right), 64 days after planting. March 30, 2021.
Figure 3. Bud development in 2 plants of TX-400 accession. March 23, 2021.

Potential key characters

During the extension of the first trifoliate leaf, some potential differences were seen in terms of vigor in a common garden between red and Kentucky clovers. Red clover had larger leaves on longer petioles than the native species. However, in the field these differences in growth might be confounded by factors such as soil fertility, available rhizobacteria, and seedling density.

Of the morphological traits considered, leaf pubescence was particularly distinct between species. Unlike red clover, Kentucky clover lacked pubescence on the top of the leaves. Red clover was much more pubescent than Kentucky clover both in terms of hair density and length. Breeding efforts for the removal of pubescence led to the creation of ‘Freedom!’ red clover. This low-hair variety was developed before the discovery of Kentucky clover, but its introduction to the Woodford County site is unlikely. Buffalo clover accessions were not consistent in terms of pubescence. Plants from the Cincinnati population were noticeably less pubescent than plants from the Georgia accession. The Georgia accession also expressed noticeable venation not present in the other accessions.

Stem pubescence was relatively unreliable as a distinguishing trait for the 3 species investigated. The northern accessions of buffalo clover gradually developed pubescence to levels expressed in southern accessions from early in development (V5-9). Individuals had similar levels of pubescence along petioles as plants grew. Similarly, crown development was quite similar for all species, with the first trifoliate leaf emerging from the same position near the cotyledons on all plants.

Buffalo clover pubescence has been suggested to change over the lifecycle, though to what degree it remains uncertain (Vincent, 2001). In a life history assessment of Texan plant material, the pubescence was relatively consistent over the lifespan, though further comparative studies are needed (J. Kubesch, unpublished data).

Petiole length differed noticeably between red and Kentucky clovers by about 60 days after planting. Petioles were ≤5 cm for Kentucky clover but well in excess of 5 cm for most of the red clover. Kentucky clover had fishbone venation in comparison to the Cincinnati buffalo clover accession, which had less notable venation.

Developmental timing

Until trifoliate leaves start developing, the genus Trifolium is extremely difficult to identify. Even in agricultural experiments the results can be challenging (Tracy et al., 2014). Grown under common environmental conditions, the native clovers reached developmental stages at a pace similar to red clover varieties.

Growth rates diverged over the period of March 10-23, 2021. At around 57 days after planting, red and Kentucky clover could be discerned by morphology. Red clover was visibly bigger and much more pubescent than Kentucky clover. A Texas accession in the comparison was producing a reproductive stem at 57 days after planting, highlighting the morphological and developmental variability within buffalo clover.

Figure 4. A preliminary seedling key for Kentucky and red clovers.

Seedling keys will only be useful within a certain period of development. Given that clover seedlings are susceptible to hoof traffic damage during establishment, preventing cattle access around high densities of clover seedlings and then screening these clusters for Kentucky clover might be more effective than waiting to exclude cattle until clusters are screened.

Conclusions

  1. A seedling key for red and Kentucky clovers is presented for field usage. The key complements ongoing recovery efforts to support recruitment from the seed bank at natural sites as well as planting the species where herbivory can be managed.
  2. Red clover and Kentucky clover are difficult to discern on the basis of morphology until approximately 60 days after planting.

Acknowledgements

Thanks to fiancé, Sarah Grace Holland for her encouragement in Kubesch’s clover research. Thanks to Joe Lacefield for discovering this species. Appreciation to Dr. Julian Campbell for contributing the Woodford County seed of Kentucky clover. Appreciation to Dan Boone for contributing the Cincinnati seed of buffalo clover. Posthumous appreciation to Norm Taylor for preserving the Georgia and Texas seed of buffalo clover. The present work was supported in part by the Bee-Friendly Beef team: Raven Larcom, Parry Kietzman, Velva Groover, and Dr. Ben Tracy (Virginia Tech).

References

Ball, D. M., Hoveland, C. S., & Lacefield, G. D. (2015). Southern Forages. Peachtree Corners, GA: International Plant Nutrition Institute.

Chapel, K. J., & Vincent, M. A. (2013). Trifolium kentuckiense (Fabaceae, Papilionoideae), a new species       from Franklin and Woodford counties, Kentucky. Phytoneuron 2013-63: 1–6.

Kubesch, J.O.C. (2018). Edaphic and morphological factors affecting running buffalo clover (Trifolium stoloniferum) ecology. Ohio State University. Columbus, OH.

Kubesch, J.O.C. (2020). Native Clover Conservation in the Bluegrass: An Agronomic Perspective.                   https://www.knps.org/2020/03/24/native-clover-conservation-in-the-bluegrass-an-agronomic-                perspective/.

Vincent, M.A. (2001). The genus Trifolium (Fabaceae) in Kentucky. J. Ky. Acad. Sci. 62(1):1-17.

Sustar, E. 2017. The effects of scarification on germination rates of running buffalo clover. Ohio State University, Columbus, OH. Unpublished Research Report.

Tracy, B. F., Schlueter, D. H., & Flores, J. P. (2014). Conditions that favor clover establishment in permanent grass swards. Grassland Science, 61, 34-40. doi:10.1111/grs.12075.

Updated contact information and affiliations

jonathankubesch@vt.edu

School of Plant and Environmental Sciences; Virginia Agricultural Experiment Station, Clover Seedbank and Propagation Coordinator for the Kentucky Commonwealth.

Wildflower Week 2021—Something for Everyone

For over 30 years the Kentucky Native Plant Society, in partnership with Natural Bridge State Resort Park, has held a Wildflower Weekend at the park, offering wildflower hikes, exploring the region’s rich natural history and resources in the state park and the Red River Gorge, as well as evening presentations. Last year, due to COVID-19, we were forced to cancel Wildflower Weekend for the first time ever.

This year, with the pandemic still disrupting in-person activities, KNPS is hosting a virtual Wildflower Week! This will include virtual events from April 10th through April 17th, including a week-long, statewide BotanyBlitz on iNaturalist. This is an opportunity to broaden our spring wildflower scope to the entire state of Kentucky and allow us to highlight natural areas across the state! In addition to the BotanyBlitz we will have virtual field trips, led by naturalists from around the state, online workshops, a wildflower trivia contest, and more.

With so many activities, be sure to check out the Wildflower Week 2021 homepage for details and summaries of our events. In particular, refer to the WW Schedule at the bottom of the page.

BotanyBlitz: A Community Science Event
Wildflower Week 2021

Wildflower Week 2021 is shaping up to be an amazing series of native plant related activities. The centerpiece of the week will be our community science project, the 2021 BotanyBlitz. From Saturday, April 10, through Saturday, April 17, we are encouraging everyone to visit parks and natural areas throughout the Commonwealth, find and photograph plants, with an emphasis on natives in bloom, and upload them to the KNPS Wildflower Week 2021 BotanyBlitz project.

xxxxxxxxAn example of an iNaturalist observation

A botany blitz is an event where anyone who joins the project on iNaturalist can use a camera (or cellphone) to snap a picture of a plant, in a given area (in this case all of Kentucky), during a given time frame (in this case April 10-17), and then upload the image (called an Observation) to their iNaturalist account. At the end of the week, we will have a gallery, a map showing the locations, and totals by numbers, species, and by each person, of all the great plants we found! Expert botanists and people with a keen identifying eye will be able to add comments to your specimens on why they agree or disagree with a certain identification, so we can all learn better ways to ID plants in the future.

If you do not have an iNaturalist account, please consider setting one up and then joining the BotanyBlitz project. It’s easy and fun and each observation contributes to our knowledge of the plants of Kentucky. Nick Koenig recently wrote an article showing the simple steps to join iNaturalist and the BotanyBlitz project and a short video of how to make and upload an observation. Check it out HERE!.

If you are not already familiar with iNaturalist, take a look at the site and some of KNPS’ other projects, such as the Kentucky Botanists Big Year 2021.

We are hoping that we can get at least one observation from every county in Kentucky. Won’t you join us and show the rest of us what native plants are blooming in your county?

Botanical Drawing: Flower Portraits in Pencil
Wildflower Week 2021

When: Wednesday, April 14th from 5:30PM-8:30PM EDT
Location: Online Live Class via Zoom
Instructor: Amy Tipton

As part of KNPS’s Wildflower Week 2021 activities we are incredibly excited to be able to offer this botanical drawing class.

Plants offer an unending supply of interesting shapes, textures, and details for artists to discover and translate to the page. Botanists and artists alike must develop skills of seeing plants deeply. Amy will help introduce participants to critical exploration of plant forms. An understanding of basic plant parts and floral arrangement will be discussed briefly to assure accurate representations of the selected subject specimen(s). Once the participants choose a plant/flower to draw, they will learn how to select an interesting composition, capture the subject’s proportions, create a line drawing, and complete their plant portrait with a range of values created through an exploration of different types of mark. A live Zoom format will offer opportunities for students to ask questions.

Sign Up and Zoom Registration

You can sign up for the class and get other information (supplies needed, etc.) at Amy’s KNPS Wildflower Week Botanical Drawing (Flower Portraits in Pencil) page.

Instructor

Amy Tipton is the Assistant Director of the UNC Charlotte Botanical Gardens. She holds a Masters of Science degree from Eastern Kentucky University, where she studied native plant ID and plant ecology under the guidance of Dr. Ron Jones. Amy holds bachelor degrees in Drawing and Art History, as well as an MFA in Printmaking.  She enjoys botanical drawing and contributed over 50 pen and ink line drawings to Jones’ Plant Life of Kentucky. Amy has been a recipient of the Ophelia Anne Dowden Education Grant from the American Society of Botanical Artists.

Amy has taught for over 20 years.  Some of her past teaching posts have included instructing University-level drawing, art appreciation, and ecology, and 7th grade science as a National Science Foundation Fellow.  She often presents talks for those interested in native plants.  Since her arrival at UNC Charlotte Botanical Gardens in July of 2018, Amy has taught Wildflower ID, Native Tree ID, Native Shrub ID, Botanical Drawing I and II, Pressing Plants for Science and Art, and Botanical Watercolor for the Certificate of Native Plant Studies program. Amy has previously served on the board and strategic planning committee for the KNPS, and currently is active with the North Carolina Native Plant Society.

Opening & Closing Sessions
Wildflower Week 2021

Opening and Closing Sessions

Wildflower Week 2021 is going to kick off with an opening session on Saturday, April 10, and end with a closing session on Saturday, April 17. Both sessions will be virtual, and presented on Zoom. In order to participate in either or both sessions, fill out the registration form below.

Opening Session, April 10, 2021, 4PM-6PM EDT

Closing Session, April 17, 2021, 4PM-6PM EDT

    • Winners of BotanyBlitz 2021
    • Highlights of finds
    • KNPS activities for the remainder of 2021

Wildflower Trivia Night on Zoom
Wildflower Week 2021

“What is the Kentucky state flower? One point for the general name and one point for the common name of the specific species and one point for the full scientific name. There about 30 different species of this type in Kentucky.” (*answer below)

Join with other native plant enthusiasts for a Wildflower Trivia Night on Zoom, organized and presented by the staff of The Arboretum, State Botanical Garden of Kentucky.

As a part of Wildflower Week, The Arboretum will host a wildflower-themed trivia night on Tuesday April 13 at 7:00 p.m. EDT. This “ephemeral” event will take place on Zoom so you can join us from the comfort of your own couch! You and your team will be quizzed on Kentucky native wildflowers, botany, and flower-inspired pop culture. Don’t be intimidated if you are not an expert! All plant lovers, regardless of their knowledge level, are guaranteed to have fun and be challenged.

Pre-registration is required as space is limited! Please call (859) 257-9339 or send an email to Arboretum@uky.edu with your name, email address and phone number. Register with a friend to be on the same team or register by yourself and meet some new friends who also love plants!

*Answer to trivia question: Giant goldenrod (Solidago gigantea)

Virtual Field Trips During
Wildflower Week

Besides all of the other great events during Wildflower Week 2021 , we have scheduled a virtual field trip for every day of the Week. Each day the video of the virtual hike for that day will be posted on our website at 8:30AM. The video will be available for viewing from then on. You can see all of the posted videos at our Virtual Field Trips page, so bookmark that page and check in each day.

Sat. 10thFerns of Anglin Falls – Join Office of Kentucky Nature Preserves botanist Rachel Cook on a virtual hike through John B. Stephenson Memorial Forest State Nature Preserve. While this Preserve is known for its beauty, Anglin Falls, it also has a great diversity of ferns with 32 species!

Sun 11thWildflowers of Cove Springs Park

Mon 12thSpring Flora of Glades and Barrens at Embry Lay – Join Office of Kentucky Nature Preserves botanist Tony Romano for a tour of spring flowers and flora in a rare glade and barrens habitat complex. We’ll explore plants adapted to thin soils, fires, and grassland openings.

Tues 13thTrilliums of Kentucky – Trilliums are an unforgettable genus of spring wildflowers with their three leaves, petals and sepals, habitat preferences, interesting floriferous smells, and pollination and seed dispersal strategies. Join Tara Littlefield and family as they explore Kentucky’s Trillium diversity across Kentucky

Wed 14thWildflowers of Crooked Creek State Nature Preserve

Thur 15thWildflowers of an Heritage Land Conservation Fund site TBD.

Fri 16thNeat Flora of Dry Outcrops and Woodlands in the Kentucky River Valley – Cedar, ash, oaks, and honeysuckle aren’t the only things inhabiting the dry, rocky, steep habitats above the Kentucky River. Join Office of Kentucky Nature Preserves botanist Devin Rodgers for a tour of some of the neat flora adapted to these sunny, erodible, rugged, beautiful places.

Sat 17thEarly flowering plants of upland seeps, grasslands and woodlands in the Cumberland Plateau – Join Office of Kentucky Nature Preserves staff Tara Littlefield and Forest Service staff David Taylor and Claudia Cotton on a hike to see what is blooming in the upland grasslands, woodlands and seeps of the Cumberland Plateau.