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.
| Species | Accession/variety | Year | Source |
| Red clover | Kenland | 2006 | John Fike |
| Red clover | Cinnamon Plus | 2006 | John Fike |
| Red clover | SS-0303RCG | 2020 | Southern States |
| Buffalo clover | Cincinnati | 2020 | Dan Boone |
| Buffalo clover | Georgia | 1978 | USDA |
| Buffalo clover | Texas | 1956 | USDA |
| Kentucky clover | Woodford County | 2017* | Julian Campbell |
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 Stage | Leaf pubescence | Chevrons | Stem pubescence | Crown development | Other notes |
| 1st true leaf (V1) | Tops of leaves | On red clover but not on Cincinnati and Kentucky | Only between red and Kentucky clovers |
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.
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
- 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.
- 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://sandbox.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
School of Plant and Environmental Sciences; Virginia Agricultural Experiment Station, Clover Seedbank and Propagation Coordinator for the Kentucky Commonwealth.