HORTSCIENCE 53(5):620–623. 2018. https://doi.org/10.21273/HORTSCI12922-18 downsizing that can occur through recombination-based processes, such as un- equal recombination and illegitimate recom- Cytogenetics and Genome Size bination (Grover and Wendel, 2010; Soltis et al., 2015). There have only been limited Evolution in Illicium L. reports on chromosome numbers and relative 1,5 2 3 genome sizes for species and cultivars of Thomas G. Ranney , Connor F. Ryan , Lauren E. Deans , Illicium. A base chromosome number of x =14 4 and Nathan P. Lynch and diploidy has been reported for Illicium Mountain Crop Improvement Lab, Department of Horticultural Science, anisatum, Illicium parviflorum, Illicium tern- Mountain Horticultural Crops Research and Extension Center, stroemioides, and Illicium verum (Baolian, North Carolina State University, 455 Research Drive, Mills River, 1990; Lepper, 1982; Lin, 1989; Stone and Freeman, 1968; Whitaker, 1933). However, NC 28759-3423 conflicting chromosome counts for I. flori- Additional index words. ANA grade basal angiosperms, chromosome numbers, DNA content, danum exist, with different sources reporting flow cytometry, plant breeding, reciprocal translocation, star anise, star flower shrub a base chromosome number of either x =13 (Stone, 1965; Stone and Freeman, 1968) or Abstract. Illicium is an ancient genus and member of the earliest diverging angiosperms x = 14 (Whitaker, 1933). Reports of genome known as the Amborellales, Nymphaeales, and Austrobaileyales (ANA) grade. These sizes for Illicium are also limited and vari- adaptable, broadleaf evergreen shrubs, including ’40 species distributed throughout able. Nagl et al. (1977) reported a 2C genome Asia and North America, are valued for diverse culinary, medicinal, and ornamental size of 6.72 pg (determined with scanning applications. The study of cytogenetics of Illicium can clarify various discrepancies and densitometry of Feulgen-stained nuclei) further elucidate chromosome numbers, ploidy, and chromosome and genome size whereas Pellicer et al. (2013, Supplemental evolution in this basal angiosperm lineage and provide basic information to guide plant Table 2) reported genome sizes for Illicium breeding and improvement programs. The objectives of this study were to use flow henryi and Illicium simonsii to be 29.3 and cytometry and traditional cytology to determine chromosome numbers, ploidy levels, 29.2 pg, respectively (determined with flow and relative genome sizes of cultivated Illicium. Of the 29 taxa sampled, including ’11 cytometry). Additional study of cytogenetics species and one hybrid, 2C DNA contents ranged from 24.5 pg for Illicium lanceolatum to of Illicium can clarify various discrepancies 27.9 pg for Illicium aff. majus. The genome sizes of Illicium species are considerably and further elucidate chromosome and ge- higher than other ANA grade lineages indicating that Illicium went through considerable nome size evolution in this basal angiosperm genome expansion compared with sister lineages. The New World sect. Cymbostemon had lineage. a slightly lower mean 2C genome size of 25.1 pg compared with the Old World sect. After many millions of years of diver- Illicium at 25.9 pg, providing further support for recognizing these taxonomic sections. gence, there are now 40 extant species of All taxa appeared to be diploid and 2n =2x = 28, except for Illicium floridanum and shrubs and small trees within the genus Illicium mexicanum which were found to be 2n =2x = 26, most likely resulting from Illicium (Morris et al., 2007), including six dysploid reduction after divergence into North America. The base chromosome number found in the New World and the remaining of x = 14 for most Illicium species suggests that Illicium are ancient paleotetraploids that species distributed throughout Asia (Shu, underwent a whole genome duplication derived from an ancestral base of x =7. 2008; Vincent, 1997). Parsing the taxonomy Information on cytogenetics, coupled with phylogenetic analyses, identifies some and systematics of Illicium has been chal- limitations, but also considerable potential for the development of plant breeding and lenging because of the somewhat surprising improvement programs with this genus. morphological similarities between species despite the age and broad distribution of the genus (Morris et al., 2007). Molecular phy- Illicium, previously considered as the sole Amborellales, form the most basal branches logeny studies have helped to clarify some genus in Illiiaceae, has more recently been of the angiosperm phylogeny, cumulatively species relationships (Hao et al., 2000; placed in the Schisandraceae within the order referred to as the ANA grade (Vialette- Morris et al., 2007; Oh et al., 2003). Morris Austrobaileyales (The Angiosperm Phylog- Guiraud et al., 2011), and have origins dating et al. (2007), in agreement with Hao et al. (2000), eny Group, 2016). The Austrobaileyales, back to the Late Jurassic to Early Cretaceous provided a revised sectional classification of along with the orders Nymphaeales and 160–130 million years ago (Soltis et al., the genus with two sections: sect. Illicium 2008). The ancient origin of ANA grade (including the Old World species) and sect. angiosperms (including Illicium), morpho- Cymbostemon (including the New World Received for publication 25 Jan. 2018. Accepted logical similarity with early fossils, and species). Within sect. Cymbostemon there for publication 25 Jan. 2018. limited molecular divergence suggests that was also strong support for separation be- This work was funded, in part, by the North these lineages may provide features and tween the I. floridanum + I. mexicanum clade Carolina Agricultural Research Service (NCARS), insights into the foundational traits of early and the I. parviflorum + Illicium hottense + Raleigh, NC, the North Carolina Biotechnology angiosperms (Morris et al., 2007; Soltis et al., Illicium cubense + Illicium ekmanii clade. Center, Research Triangle Park, NC, the North 2009). Illicium are also of interest because of Carolina Nursery and Landscape Association, Chromosome numbers and nuclear ge- Raleigh, NC, and the Kenan Institute, Raleigh, their unique and diverse plant metabolites NC. Plant material was graciously provided by the nome sizes vary widely among angiosperms. that have both medicinal and culinary uses. JC Raulston Arboretum, Raleigh, NC; Dan Hinkley, The original base chromosome number of the Certain species of Illicium have been used in Indianola, WA; Rick Crowder, Hickory, NC; and angiosperm linage was most likely some- traditional medicine for treating pain, rheu- Ron Miller, Pensacola, FL. where between x = 6 and 9 (Ehrendorfer matism, and skin inflammation (Liu et al., We express our sincere thanks to Tom Eaker, Joel et al., 1968; Raven, 1975; Stebbins, 1971) 2009). Most plant organs of Illicium are Mowrey, Andra Nus, and the staff at the Mountain and increased over time with repeated cycles noticeably pungent with a strong odor of Horticultural Crops Research and Extension Center of whole genome duplication events (Soltis anise/terpenes. Extensive studies have been for their technical assistance. 1 et al., 2003). Genomes can further expand conducted to identify these compounds that JC Raulston Distinguished Professor. through amplification of noncoding, repeti- 2Research Intern. include prenylated C6–C3 compounds, neo- 3Graduate Research Assistant. tive DNA including retrotransposons (Leitch lignans, and secoprezizaane-type sesquiter- 4Research Specialist. and Leitch, 2013). However, this ‘‘one-way penes that are found exclusively in Illicium 5Corresponding author. E-mail: tom_ranney@ncsu. ticket to genomic obesity’’ (Bennetzen and (Liu et al., 2009). Many of these compounds edu. Kellogg, 1997) is often tempered by genome and/or crude extracts from Illicium are 620 HORTSCIENCE VOL. 53(5) MAY 2018 biologically active and demonstrate antibac- to better enable plant breeding and improve- (1996-041), and I. verum (2007-063) were terial, anticancer, anti-inflammatory, anti- ment programs. stained with 1% acetocarmine stain for at oxidant, antiviral (antiHIV), insecticidal, The objectives of this study were to de- least 5 min after which the root tips were neurotoxic, neurotrophic, and phytotoxic ac- termine chromosome numbers, ploidy levels, sectioned onto a slide with a drop of stain, tivities (Liu et al., 2009). Illicium verum is of and relative genome sizes of cultivated Illic- overlaid with a cover slip, and heated warm importance as a crop and the seedpods are ium taxa to gain further insights into the to the touch (Singh, 2003). Root tips were well known in kitchens as the spice star anise evolution, systematics, and the potential for squashed underneath a cover slip on a micro- (not to be confused with other species, which interspecific hybridization. scope slide, and chromosomes were counted have a similar appearance and can be toxic). under oil immersion at ·1000 magnification. However, most I. verum grown as a crop is Materials and Methods Flow cytometry. Relative 2C genome used as a source of shikimic acid and is the sizes were determined using a flow cytometer primary ingredient in the antiflu medication Cytology. Chromosome counts were de- (Partec PA-II; Partec, Munster,€ Germany). oseltamivir phosphate, sold as TamifluÒ termined for six species representing major Leaf tissue from 29 Illicium taxa was ob- (Wang et al., 2011). phylogenetic clades and sub-clades (Morris tained from the JC Raulston Arboretum, Illicium have a suite of desirable orna- et