Abstract
Kalmia is a highly ornamental genus of shrubs native to North America and Cuba and grown as a valuable nursery crop throughout much of the temperate world. Although most species of Kalmia have previously been found to be diploid with 2n = 2x = 24, one species, Kalmia polifolia, has been found to be tetraploid. However, sampling within the genus has been limited, and information on the ploidy levels of specific cultivars is lacking. The objective of this study was to determine the relative genome sizes and ploidy levels of species, hybrids, and cultivars of Kalmia. Flow cytometry was used to determine the relative genome sizes of 67 accessions representing species, interspecific hybrids, cultivars, and chemically induced polyploids. Traditional cytology was used to calibrate genome sizes with ploidy levels. Results showed that relative genome sizes were conserved with 1Cx values ranging from 0.57 pg for Kalmia carolina to 0.70 pg for Kalmia latifolia. Most species of Kalmia were diploid including K. buxifolia (Leiophyllum buxifolium), K. carolina, K. cuneata, K. hirsuta, K. latifolia, and K. microphylla. Although plants of K. carolina (Kalmia angustifolia var. carolina) were uniformly diploid, the closely related, but more northerly distributed, K. angustifolia was primarily tetraploid, providing additional justification for treating these as separate species. An unusual triploid of K. angustifolia f. candida was also documented. Kalmia polifolia included both tetraploid and potentially pentaploid individuals, indicating a ploidy series within this species. Kalmia latifolia cultivars also included one triploid, two cytochimeras, and two chemically induced tetraploids. Overall, polyploidy was more prevalent in Kalmia than previously reported and varied both within and among species. This broader survey of relative genome sizes and ploidy levels in Kalmia provides valuable information for plant breeders and new insights into the systematics and cytogenetics of the genus.
Kalmia L. is a small but diverse genus containing nine to 11 species (Ebinger, 1974; Southall, 1973; Weakley, 2012) native to North America and Cuba (Jaynes, 1997). Deciduous or evergreen woody shrubs, Kalmia spp. have a varied morphology and are each distinct. Some of their diverse characteristics include plants that are “… erect, or ascending, branching shrubs or rarely trees. The leaves are simple, alternate, opposite or whorled, coriaceous, dark green above, light green beneath, entire and often revolute (Southall, 1973).” Kalmia latifolia, and to a lesser degree, K. angustifolia L. and K. polifolia Wangenh. are valuable nursery crops and have been cultivated in North America and Europe since the early 1700s (Jaynes, 1997).
Cytology was performed on Kalmia species by Dr. Richard Jaynes of the Connecticut Agricultural Experiment Station in the late 1960s (Jaynes, 1969). His examinations found most species of Kalmia to be diploid (2n = 2x = 24), with the exception of K. polifolia that was tetraploid (2n = 4x = 48). However, that study only examined material from one or two plants per species to determine ploidy, and in the case of K. angustifolia, only samples from North Carolina were obtained as representatives of that species (Jaynes, 1969). Kalmia angustifolia and K. carolina Small [K. angustifolia var. carolina (Small) Fernald] are closely related with North Carolina provenances now generally classified as K. carolina (Gillespie and Kron, 2010; Southall and Hardin, 1974; Weakley, 2012). Cytology on Ericaceous plants, including Kalmia, is notoriously difficult, due in part to the small chromosomes (Jaynes, 1997). Flow cytometry provides a more efficient approach to broaden sampling within and among species and can provide accurate estimates of ploidy, particularly for Ericaceous plants (Jones et al., 2007).
The objective of this study was to survey genome sizes and ploidy levels of a broad range of species, hybrids, and cultivars of Kalmia from throughout North America.
Materials and Methods
A diverse collection of Kalmia taxa was obtained from cooperators in the United States and Canada (Table 1). The Cuban species Kalmia ericoides C. Wright ex Griseb. was not available for analysis. Relative 2C genome sizes were determined using flow cytometry on recently expanded leaves (Greilhuber et al., 2007). Sample tissue was combined with an internal standard (Pisum sativum L. ‘Ctirad’, 2C DNA content = 8.76 pg, Greilhuber et al., 2007) and diced with a razor blade in a petri dish containing 400 µL of extraction buffer (CyStain Precise P; Partec, Münster, Germany). The nuclei suspension was poured through a 50-µm filter and stained with 1600 µL of a nucleotide staining buffer solution (CyStain ultraviolet Precise P Staining Buffer, Partec) containing 4’, 6-diamidino-2-phenylindole . Stained nuclei were analyzed using the PA-II flow cytometer (PA-II; Partec). A minimum of 3000 nuclei counts were analyzed for each sample with three samples for each species. Holoploid, 2C genome sizes for each sample were calculated as: 2C = DNA content of standard × (mean fluorescence value of sample ÷ mean fluorescence value of the standard). The relationship between genome sizes and ploidy levels was based on samples with confirmed chromosome numbers. Mean 1Cx monoploid genome size (i.e., genome size of the nonreplicated base set of chromosomes) was calculated as 2C genome size ÷ ploidy. Taxa were sampled in a completely randomized fashion. Data for 1Cx values were subjected to analysis of variance and means. Different taxa were separated using Tukey’s honestly significant difference, P ≤ 0.05 (SAS version 8.02; SAS Institute, Cary, NC).
Relative genome sizes and estimated ploidy levels for 67 accessions of Kalmia species, cultivars, and hybrids.
Cytology.
To confirm ploidy and calibrate with genome size the cultivar K. latifolia ‘Elf’ was selected and traditional cytology was conducted using a root squash technique following procedures outlined in Lattier et al. (2014). Root tips were collected from potted plants and placed in vials of prefixative solution (2 mm 8-hydroxyquinoline + 70 mg·L−1 cycloheximide). Vials were stored in the dark for 2 h at ambient room temperature after which they were placed in a dark refrigerator at ≈4 °C for an additional 2 h. Following the prefixative period, roots were removed and rinsed thoroughly in deionized water. The rinsed root tips were then placed in a 3:1 solution of ethanol and propionic acid and left overnight at ambient room temperature to complete the fixation process. The following day, the roots were rinsed with 70% ethanol three times and stored in 70% ethanol at ≈4 °C until root squashes could be performed. Root tips were removed from the ethanol storage solution and placed in a hydrolyzing solution made up of a 3:1 solution of 95% ethanol and hydrochloric acid (12.3 m) for 10 min. After hydrolyzing, the root tips were transferred into a modified carbol-fuchsin staining solution for 10 min. Once stained the terminal end of the root tip was excised under a dissecting stereo microscope and placed on a clean microscope slide in a drop of staining solution. A cover slip was applied, and the root tip tissue was squashed. Slides were observed using a light microscope (Nikon Eclipse 80i; Nikon, Melville, NY). Photographs, taken at multiple focal points, were layered (Photoshop CS6; Adobe, Mountain View, CA) to create an enhanced depth of field.
Results and Discussion
Cytology confirmed that K. latifolia ‘Elf’ is a diploid with 2n = 2x = 24 (Fig. 1) as has been reported previously for that species (Jaynes, 1969). Flow cytometry was also an efficient and consistent method for determining relative genome size and ploidy of Kalmia (Table 1). Values for multiple subsamples of each replicate were consistent with se for 2C values ranging from less than 0.01 to 0.1 pg demonstrating a high level of precision and repeatability and clearly distinguishing between ploidy levels. Results from flow cytometry revealed that genome sizes were relatively conserved with 1Cx values ranging from 0.57 pg for K. carolina to 0.70 pg for K. latifolia. These values are similar to other Ericaceous plants, including Rhododendron that had 1Cx values ranging from 0.63 to 0.83 pg (Jones et al., 2007). Cytometry results also showed there to be considerable variation in ploidy both between and within species (Table 1). Most species of Kalmia were predominantly diploid including K. buxifolia, K. carolina, K. cuneata, K. hirsuta, K. latifolia, and K. microphylla. In the case of K. latifolia, there were some exceptions. The cultivar K. latifolia Big Boy was a triploid, most likely the result of an unreduced gamete from one parent. There were also two mixoploid/cytochimeras found: K. latifolia ‘Showtime’ had both diploid and tetraploid tissues, whereas ‘Silver Dollar’ had a mixture of triploid and hexaploid tissues. These mixoploids most likely arose from mitotic irregularities (endoreduplication) in at least one of the histogenic layers resulting in stable cytochimeras (Joubès and Chevalier, 2000). Naturally occurring mixoploids have been documented in other Ericaceous plants including Rhododendron (De Schepper et al., 2001; Jones et al., 2007; Sakai et al., 2006). In the case of ‘Silver Dollar’, this apparently happened in a triploid plant that most likely resulted from the union of an unreduced gamete from one of the parents. Two additional plants of K. latifolia, H2014-222-002 and H2014-222-004, were confirmed to be homogeneous tetraploids that resulted from treatment of seedlings with the mitotic inhibitor oryzalin at North Carolina State University.
Condensed chromosomes of Kalmia latifolia ‘Elf’ viewed at 1000× using oil immersion.
Citation: HortScience horts 50, 10; 10.21273/HORTSCI.50.10.1426
Condensed chromosomes of Kalmia latifolia ‘Elf’ viewed at 1000× using oil immersion.
Citation: HortScience horts 50, 10; 10.21273/HORTSCI.50.10.1426
Condensed chromosomes of Kalmia latifolia ‘Elf’ viewed at 1000× using oil immersion.
Citation: HortScience horts 50, 10; 10.21273/HORTSCI.50.10.1426
As previously reported by Jaynes (1969), under the epitaph K. angustifolia var. carolina, the southern sheep laurel, K. carolina was confirmed to be diploid. However, samples of K. angustifolia, the northern sheep laurel, were all tetraploid, including ‘Hammonasett’, ‘Poke Logan’, ‘Royal Dwarf’, and ‘Wintergreen’. The cultivars Hammonasett and Poke Logan are known to be wild collected selections from Connecticut and Maine, respectively (Jaynes, 1997). In addition, a sample of K. angustifolia f. candida, a white-flowered form, was triploid. Most likely this particular plant is an oddity and not necessarily representative of the white-flowered form in general.
Kalmia polifolia also varied in ploidy.
A sample of K. polifolia obtained from Hidden Valley Nature Center in Jefferson, ME, was tetraploid. However, wild collected samples from Alburg, VT, had genome sizes consistent with pentaploids, though this could not be confirmed with cytology. The existence of putative pentaploid K. polifolia in the wild implies that there is a natural occurring ploidy series within the species, potentially including tetraploid, pentaploid and hexaploid individuals or populations. Additional sampling in this location would be warranted. Kron and King (1996) postulated that K. polifolia may be a hybrid between two lineages of Kalmia ancestors, which are no longer extant. It has often been found that interspecific hybrids have a greater propensity to form unreduced gametes leading to the formation of allopolyploids (Arnold, 2006; Ramsey and Schemske, 1998) and this may be the case for K. polifolia. The sample of the hybrid (K. polifolia × K. microphylla var. microphylla) ‘Rocky Top’ was a tetraploid (2C = 2.37 pg), suggesting that it formed from the union of an unreduced gamete from the K. microphylla parent.
This survey provides new information on the genome sizes and ploidy levels for species, cultivars, and hybrids of Kalmia. The discovery and confirmation of new natural and induced polyploids within the genus provides basic information for how to best use these plants in breeding programs. Although development of interspecific hybrids in Kalmia has been challenging (Jaynes, 1997), attempting these crosses at the tetraploid level may result in allopolyploids (amphidiploids) with greater fertility because of disomic chromosome pairing in meiosis (Ranney, 2006), allowing for advanced generations. Alternatively, the development of new triploid hybrids of K. angustifolia and K. carolina has the potential to help reduce fertility and invasiveness where they have become problematic in European forests (Inderjit and Mallik, 1996).
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