Pycnanthemum Pilot Study

by Dr. William Mendez, PDC Research Associate

Above: The broad, leafy bracts of Pycnanthemum muticum. Photo by author

When I moved to Charlottesville in retirement, I began growing a native plant garden. At first, plant selection was mostly random, driven by availability and by the sunny dry conditions of my selected garden patch. Based on internet research, it became clear that I must have the pollinator-friendly mountain-mint (Pycnanthemum). I found one at a local nursery; it turned out to be Pycnanthemum muticum (clustered mountain-mint). It was a rapidly growing, broad-leafed variety with bow-tie-shaped “bracts” (specially adapted leaves just below the inflorescence). It smelled nice too. For good measure, I got a pair. Later in the summer I was surprised to see that one plant had formed a neat clump, while the other, in addition to its clump, had sent out a perfect ring of bright red runners (stolons) extending outward, up to a foot or more. It turns out that this is a commonly observed phenomenon in some Pycnanthemum species. Why it happened in one plant and not the other may have been related to “ploidy” (some P. muticums are diploid, others tetraploid, others possibly pentaploid). Alternatively, it could simply have been due to differences in growing conditions.

The next year I decided to “branch out”. Based on availability, I bought a P. tenuifolium (narrowleaf mountain-mint). It was shorter than P. muticum (about 18 inches tall, compared to about twice that) with interesting needlelike leaves and much smaller inflorescences. It also had a much milder and almost citrusy odor. I also got a P. flexuosum, which, true to the “keys” had distinctive hairy/spiky “awned” inflorescences.

Then, I went to buy a second specimen of P. tenuifolium, and was given a plant that had narrow, but not needle-narrow leaves. In retrospect, it was probably P. virginianum, or another narrow-leaved species, taller, with larger inflorescences and the usual minty/mouthwashy aroma. After purchasing several more specimens that did not fit keys or were clearly mislabeled, I started to understand why this genus has been giving botanists headaches for a long time.  

The deeper I looked in the literature, the more complex the problem of characterizing the interrelationships within the genus became. Two authoritative flora (Gleason and Cronquist 1991 and Weakley et al. 2022) had very different keys for some species, and Weakley, in Flora of the Southeastern United States (FSUS) famously characterized Pycnanthemum as “a complex and difficult group”.  A monumental survey by Grant and Epling (1943), based on the examination of over 6,000 herbarium and fresh specimens, resulted in the identification of 21 (not 19) species, grouped into two “phylads” (identified as closely related but with no precise phylogenetic relationship), plus several outliers and intermediate, possibly hybrid, species. In those days, there was no way to analyze genomics, so relationships were based solely on plant morphology.

Another major source on Pycnanthemum systematics is a series of papers by Henrietta Chambers. She wrote her Ph.D. thesis on the genus in 1961 and over her 40+ year career, published five more analyses of different aspects of mountain-mint taxonomy and reproductive biology. She was the first to tediously count chromosomes in all the known Pycnanthemum species to characterize ploidy, and to systematically study natural and laboratory hybrids. From 1988-1998, she was curator of the Menthae collection at the National Clonal Germplasm Repository in Corvallis, Oregon, which included many Pycnanthemum specimens. 

In 2008, she and her husband Kenton Chambers wrote a major paper reorganizing the genus, synthesizing their past research, and incorporating recently developed genomic and biochemical evidence (Chambers and Chambers, 2008). Their analysis identified 19 species, which they classified into seven “sections” (Table 1, see below). The rationale for their organizational scheme was to include a diploid species (a likely ancestor) in each section, along with closely related (presumed descendent) species (polyploid), based on “key features of leaf shape, habit, inflorescence, morphology, and chromosome number”. Their analysis discussed problems related to species variability, similarity, and intermediate forms. They concurred with Grant & Epling’s suggestion of reticulate genetic history, noted the likely importance of apomixis and introgression, and recognized the complexities introduced by multiple ploidys within and between species. Based on their analysis, P. muticum appears to be particularly promiscuous, contributing genetic material to a range of polyploid descendants. 

Table 1.  Pycnanthemum Classification According to Chapman and Chapman (2008)

Section	Species	Chromosome Number
1. Pycnanthemum	P. albescens	38,76
	P.curvipes	40
	P.loomisii	38
	P. clinopodioides	76
	P. incanum	76
	P. pycnanthemoides	72
2.  Aristatae	P. flexuosum	36
	P. setosum	76
3.  Brachystemum	P.muticum	40, 80, 108(?)
	P. beadlei	76
	P. floridanum	78
4. Capitellatae	P.tenuifolium	40, 80
	P. pilosum	78
	P. torreyii	80
	P. verticillatum	76-78
	P. virginianum	80
5.  Macrocephalae	P. montanum	40
6. Nudae	P. nudum	40
7. Californicae	P. californicum	40

When I started to learn about Pycnanthemum, one of my first questions was: What about genomic (DNA sequence) data? DNA sequences should provide a very useful tool to shed light on species interrelationships and evolutionary history. However, very little genomic research has been published on Pycnanthemum. The two studies available to the Chapmans (Williams 2005 and Yetter 1989) were limited in scope and never published in peer-reviewed journals; their data was not available for review.   Nonetheless, Yetter made a convincing case, based on allozyme analysis, for auto- and allopolyploidy in the “virginianum complex.”  

Since 2008, there has been very little (published) genomic analysis of Pycnanthemum. Being a small, agriculturally unimportant species meant that there was relatively little interest in mountain-mint when sequencing was expensive and techniques for characterizing phylogenetic relationships were not well-developed. While technology has greatly improved and costs have declined, still only a few genetic markers have been sequenced for a minority of species; far too few to address the complex issues of mountain-mint phylogeny.

Botanists are still trying to clarify the evolutionary history of Pycnanthemum, and different flora provide different keys for identification, many based on subtle differences in calyx conformation, degrees of stem pubescence, etc. Recent analyses (Buck & Flores-Renteria, 2022) have suggested that at least some of mountain-mint species form a “syngameon”, a group of species that maintain their identity despite frequent hybridization events.

In spring of 2025, I approached Piedmont Discovery Center’s Executive Director, Devin Floyd, to see if we could start to fill the gap in Pycnanthemum phylogenomics. He agreed, and our simple, inexpensive “Pilot Study” involved the collection of six specimens of the ubiquitous and distinctive P. tenuifolium from around Virginia-in locations ranging from Arlington to Christiansburg (thanks, PDC staff and Prof. Leighton Reid of VA Tech!). The samples were sent for sequencing to the Virginia Tech Fralin Life Sciences Institute. The pilot study looked primarily at the variation in chloroplast DNA sequences (see box) across the commonwealth. 

Chloroplast sequences were successfully assembled for the six samples. All assemblies took the expected circular form, with sequence lengths from 151,785 to 151,876 base pairs. This level of variation, carefully confirmed base-by-base of about 0.1 percent, is typical of infra-specific variation in Lamiaceae species. Sequences varied from one another by about one base per 2000; again, this is typical for Lamiaceae. No geographic patterns or outliers were found.  

The sequences were then “annotated” to identify genes for specific functions along the genome (colored boxes in figure above). Annotation software identified 130 genes plus several “pseudogenes” which represented non-functional fragments of DNA. The complement of genes was again constant across the samples, and very similar to that reported for closely related genera. 

While the pilot study found nothing dramatic, the chloroplast data for P. tenuifolium can serve as a baseline for further study of Pycnanthemum phylogenetics, being the first chloroplast of any mountain-mint species to be fully sequenced. The sequence has been accepted for inclusion in the National Center for Biotechnology Information Genbank database (Sayers et al. 2025) and can also be used as a reference by other researchers. In addition, we have developed and tested a cost-effective protocol for Pycnanthemum sampling, preservation, and sequencing. The Pilot Study Report is available for review if readers are interested.

This year we hope to continue the investigation, extending our research to other Pycnanthemum species. During the proposed study, we plan to determine chloroplast DNA sequences for Virginia native species, in addition to P. tenuifolium, and sequence many nuclear DNA markers from all collected species. Having this data will enable us to address a large number of basic questions about Pycnanthemum phylogenetics. Comparison of chloroplast (maternally inherited) and nuclear (biparentally inherited) phylogenies should be very informative, and analysis of nuclear gene markers could help to elucidate the extent to which Pycnanthemum evolutionary history is truly “reticulate” and how species are related within the evolutionary network.    

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References

Buck R, and L. Flores-Rentería (2022) The Syngameon Enigma. Plants (Basel). 11(7):895.

Chambers, K. and H. Chambers (2008). Infrageneric Classification and Nomenclatural Notes for Pycnanthemum (Lamiaceae). Journal of the Botanical Research Institute of Texas. 2(1): 193-199

Grant, E. and C. Epling. 1943. A study of Pycnanthemum (Labiatae). Univ. Calif. Publ. Bot. 20:195-240.

Gleason, H.  and A. Cronquist. (2004) Manual of Vascular Plants of Northeastern United States and Adjacent Canada, Second Edition

Sayers, E.W. et al. (2025) Database resources of the National Center for Biotechnology Information in 2025, Nucleic Acids Research, Volume 53, D20–D29,  https://doi.org/10.1093/nar/gkae979

Weakley et al. (2022) Flora of the Southeastern United States. Chapel Hill, NC.

Williams, R. 2005. The phylogeny of Pycnanthemum: hybridization and polyploidy in a taxonomically confusing genus. Herbarist 71:35-41.

Wetter, T.C. 1989. Systematic and phylogenetic relationships in the virginianum species complex of the genus Pycnanthemum (Labiatae). Ph.D. thesis, Miami University, Oxford, OH. Pp. 1-122.