Phlox (Polemoniaceae) is a phenotypically diverse genus with substantial interpopulation differentiation for numerous morphological characters and ploidy; it includes ≈65 species with centers of diversity in the eastern and western United States (Wherry, 1955). The most important horticultural forms of Phlox are found in the eastern species, which are taxonomically distributed among three sections (Annuae, Occidentales, and Phlox) and six subsections (Divaricatae, Subulatae, Phlox, Paniculatae, Stoloniferae, and Cluteanae) based on variations in the calyx, style length, and geographic distribution (Wherry, 1955). Subsections Paniculatae and Phlox include species having a style that exceeds 15 mm in length and that is as long as, or longer than, the corolla tube (Fig. 1) (Wherry, 1955). This feature differentiates these species from all other Phlox taxa that are collectively known as “long-styled” phloxes. Style length has been suggested as a possible barrier to interspecific hybridization among species of Phlox that differ for the trait, but this has not been rigorously tested and documented (Levin, 1966; Locklear, 2011; Symons-Jeune, 1953).
Among long-styled phloxes, P. amplifolia and P. paniculata comprise subsection Paniculatae, and are distinguished from taxa in subsection Phlox by their areolate leaf veins, white or cream-colored pollen, and pubescent calyces (Fig. 1; Table 1). Subsection Phlox contains up to six species, but the taxonomy of this group has been historically confused and revised several times; a well-resolved taxonomy or phylogeny does not yet exist. The taxonomic confusion is caused by extensive intraspecific variation prevalent in Phlox carolina and P. glaberrima, which has led some to refer to these as the P. carolina–P. glaberrima complex, but more recently referred to as the “P. glaberrima complex” (Ferguson and Jansen, 2002; Ferguson et al., 1999; Wherry, 1932a, 1932b, 1945, 1955). These species are phenotypically similar to those in subsection Paniculatae, but are differentiated by having obscure leaf veins, yellow pollen, and glabrous calyces (Fig. 1; Table 1). To some degree, the taxa can be further distinguished from each other on the basis of morphology, geographic distribution, and habitat preference, but the traditional morphological characters used to differentiate them overlap and render identification of wild-collected germplasm difficult, if not impossible (Table 1). Extensive phenotypic variation within these two subsections has resulted in the selection of cultivars and putative interspecific hybrids, but the potential for further breeding and selection in this group remains unrealized, and has likely been hindered by lack of phylogenetic and taxonomic resolution. Genome size and ploidy estimation provide an additional criterion for assessing phylogenetic relationships among these phloxes, and to identify potential breeding barriers to hybrid cultivar development.
Distinguishing morphological characteristics and habitat preferences of taxa from the “Phlox glaberrima complex” in subsection Phlox and from the two taxa in subsection Paniculatae, as described by Wherry (1955), used in this study to assign taxa to accessions in the germplasm collection.
The taxonomy of subsection Paniculatae has remained stable since formal recognition, but phenotypic variation in P. paniculata has resulted in selection of over 800 cultivars (Bendtsen, 2009; Fuchs, 1994; Lierval, 1866; Locklear, 2011; Pridham, 1934; Symons-Jeune, 1953; Wherry, 1933, 1935a). Far more cultivars have been selected from this species than any other in the genus. Many of these are still grown, but several have also been lost to cultivation, reintroduced under different names, or have not been introduced in some regions, like the United States (Bendtsen, 2009; Hawke, 2011, 2013; Locklear, 2011; Symons-Jeune, 1953; Wherry, 1955). Cultivars have been primarily selected for variations in flower color, duration of flowering period, and variegated foliage. One cultivar, David, was chosen as the 2002 “Perennial Plant of the Year” by the Perennial Plant Association because of its adaptability to different climatic regions, soil types, and disease resistance. Phlox paniculata continues to be the focus of breeding programs, and cultivars are still introduced annually. The closely related P. amplifolia has rarely been introduced into cultivation, although European nurseries have reputedly developed cultivars (Deam, 1940; Wherry, 1935a). However, because of gross morphological similarity of P. amplifolia to P. paniculata, the true identity of these cultivars has yet to be demonstrated, and the breeding potential of P. amplifolia remains largely unknown.
In contrast to the taxonomic stability of subsection Paniculatae, taxa within the morphologically divergent “P. glaberrima complex” of subsection Phlox have been the subject of debate, frequent change, and taxonomic confusion (Ferguson and Jansen, 2002; Ferguson et al., 1999; Locklear, 2011; Wherry, 1935b, 1945, 1955). The traditional morphological markers (calyx, vesture, and leaf size/shape) used to differentiate species and subspecies in related subsections, cannot be used to delineate taxa of this complex (Fig. 1; Table 1). In his final attempt to resolve historical taxonomic confusion, Wherry (1955) recognized two primary species, P. carolina and P. glaberrima, and six intraspecific taxa that were described on the basis of morphology, geographic distribution, and habitat preference. Molecular analysis of the “P. glaberrima complex” and related eastern taxa from other subsections using data from the internal transcribed spacer (ITS) region of ribosomal DNA and chloroplast DNA (cpDNA) restriction sites resulted in a paraphyletic grouping and included species from at least three subsections (Ferguson and Jansen, 2002; Ferguson et al., 1999). These studies underscore the concept of a species complex where a single, widespread, but polymorphic taxon is recognized. Previous data indicate that this polymorphism likely resulted from hybridization, but recent evolutionary divergence could produce similar results. More recently, Locklear (2011) recognized P. carolina and P. glaberrima as distinct, and described one subspecies for each species based on morphology, but also indicated P. carolina was “… an enigma … its distinctiveness as a species appears to have been more apparent in the past than it is today.” In our experience, Wherry’s intraspecific taxonomy based on morphological features does not consistently separate taxa, complicating germplasm collection and characterization efforts within these polymorphic taxa. Thus, the taxonomy or phylogeny is not resolved for morphologically similar taxa in this group. As these taxa reportedly share a close phylogenetic relationship with subsection Paniculatae, there is a need to clarify the relationships. This confusion may adversely affect breeding efforts, and highlights the need for a resolved molecular phylogeny and the use of additional markers, such as genome size and ploidy.
A potentially confounding factor in the taxonomic relationship of these species is the recent evidence for extensive cytotype variation in populations of other Phlox species, including tetraploids in Phlox pilosa; tetraploids, pentaploids, and hexaploids in Phlox amabilis and Phlox woodhousei; tetraploids and hexaploids in Phlox nana; and tetraploids in Phlox subulata (Fehlberg and Ferguson, 2012a; Flory, 1934; Smith and Levin, 1967; Worcester et al., 2012; Wright, 2014). We have also identified polyploid populations of P. pilosa and P. subulata (Zale, 2014). In all cases where polyploid Phlox have been identified, the plants did not exhibit obviously altered overall morphology (e.g., the “gigas” effect), and could thus be classified as cryptic polyploids (Fehlberg and Ferguson, 2012a; Worcester et al., 2012). Both the uncertain taxonomic status of P. carolina and P. glaberrima coupled with the likelihood that cytotype variation is more common in Phlox than previously thought, suggest that an analysis of genome size and inferred ploidy in other Phlox species could further enhance the understanding of the breeding potential in the genus.
Polyploidy has played an important role in the development of ornamental plant cultivars (Contreras et al., 2009; Parris et al., 2010; Ranney, 2006), but the extent to which it has been used in Phlox is uncertain. We could find no published reports about genome sizes for any Phlox cultivars and comparatively few for Phlox species although chromosome counts have been reported for the principal species (Flory, 1931, 1934; Meyer, 1944). A chromosome number survey of over 100 cultivars of P. paniculata using meristematic tissue isolated from branch tips determined that all had the diploid (2n = 2x = 14) number, but that certain cultivars exhibited varying numbers of chromosomal fragments (Flory, 1931, 1934; Meyer, 1944). The number of chromosomal fragments varied between squash preparations and was not repeatable, which makes the origin, confirmation, and significance of these fragments unclear. Polyploidy in P. paniculata is unknown, but this observation is based on a limited number of cultivars. Observations and comments by various growers and gardeners indicate that fruit and seed production in some cultivars is absent to very low, which could be a consequence of different factors including alterations in ploidy (Bendtsen, 2009; Wherry, 1935a). Determination of genome size and ploidy level in a wide range of cultivars would be of benefit to P. paniculata breeders and may have the potential to produce new breeding lines with novel, useful traits (Hawke, 2011; Locklear, 2011).
Differences in ploidy between species are a potent prezygotic barrier to hybridization and are known to affect speciation and evolution by contributing to ecotypic isolation, novel gene expression, and divergence (Eeckhaut et al., 2006; Hogenboom and Mather, 1975; Parris et al., 2010; Ranney, 2006). Knowledge of ploidy is crucial for characterization of germplasm collections and for directing interspecific hybridization efforts. Although genome size and ploidy has been characterized in some Phlox species, estimates for several taxa remain undocumented (Bennett and Leitch, 2012). The positive correlation generally established between genome size and ploidy in a number of genera has facilitated rapid and extensive study of cytotypic variation, not only between species but also between populations of a species (Greilhuber and Leitch, 2013). Flow cytometry provides a well-established, rapid method for estimating genome size in plants, and such estimates can be used to infer ploidy when chromosome counts are limited or difficult to generate (Doležel, 2009; Doležel et al., 1998; Parris et al., 2010). Analysis of genome size in populations of Dianthus broteri (Balao et al., 2009), P. pilosa (Worcester et al., 2012), Ranunculus parnassifolius (Cires et al., 2009), and others have demonstrated the existence of intraspecific cytotypic variation between populations that may have an adaptive value (Greilhuber and Leitch, 2013; Šmarda and Bureš, 2010). Thus, it cannot be assumed that individuals in different populations of a taxon necessarily have the same genome size or ploidy level.
The objective of this study was to survey the genome size in a diverse germplasm collection of Phlox that included the long-styled taxa in subsections Paniculatae and Phlox, to infer ploidy and assess cytotype variation both within and between species. Validation of inferred ploidy with chromosome counts would reinforce the characterization of the collection, and provide baseline information for use by Phlox breeders.
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