December 2012 Number 1 - Utah Native Plant Society

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Utah Native Plant Society Flow cytometry is a method that quantifies nuclear DNA content by measuring the relative fluorescence of isolated nuclei that have been stained with a fluorescent dye. This method offers several advantages over traditional cytological methods including: 1) sample preparation and processing is relatively easy and rapid (allowing for large sample sizes), 2) sample material does not need to be actively dividing (a variety of tissues can be used, including dried and frozen material), and 3) sampling is relatively non-destructive (enabling studies of sensitive plants; Kron et al. 2007; Suda et al. 2007). Thus, flow cytometry provides a practical means by which intraspecific population level cytotype data can be gathered. The genus Phlox has been an important system for plant evolutionary studies of polyploidy, hybridization, and ecology (Ferguson and Jansen 2002; Ferguson et al. 1999; Grant 1959; Levin and Schaal 1970; Levin and Smith 1966; Wherry 1955). Phlox comprises ca. 65 species of annual and perennial herbs distributed predominantly in North America with a center of diversity in the western United States. We are using flow cytometry to study cytotype variation across this genus. The present study focuses on a broad sample of western, upright perennial Phlox species with special emphasis on two endemic species of conservation concern found in coniferous forests in Arizona and New Mexico, P. amabilis and P. woodhousei. These two species are closely related (Ferguson et al. 2012) and very similar based on gross morphology, sharing a characteristic woody-based upright perennial growth form, thick linear leaves and notched petals. However, their geographic distributions do not overlap, and they are readily distinguished by differences in style length and stigma placement relative to anther position. These taxa have been variously classified as distinct species or conspecifics (Cronquist et al. 1984; Wherry 1955). Our objectives for the present study were to examine 1) taxonomic and large-scale spatial patterns of cytotype variation across a subset of western Phlox taxa and 2) fine-scale spatial patterns of cytotype variation within and among populations of P. amabilis and P. woodhousei. METHODS The present study includes samples from seven species of upright, perennial Phlox from Arizona, California, Colorado, Idaho, Montana, Nevada, Utah and Wyoming (Table 1). For determination of cytotype, several leaves were collected from one to five individual plants at each sampling location for each taxon and stored on ice until nuclear extraction. Voucher specimens for each population were deposited at the Kansas State University Herbarium (KSC). We determined the cytotype of each sample of Phlox using flow cytometry. Flow cytometry measures nuclear DNA content, which can then be interpreted in terms of ploidy level, especially when closely related taxa are studied and when knowledge of ploidy level is independently assessed through conventional chromosome counts (Suda et al. 2007; Halverson et al. 2008). For each sample of Phlox, we placed 100-300 mg of chilled leaf tissue into a petri dish with 1.5 ml of chopping buffer, modified from Bino and others (1993) as described by Davison and others (2007). We chopped the leaves finely with a new razor blade and filtered the resulting liquid through a 30 µm filter into a microcentrifuge tube. Tubes were centrifuged at 500 x g for 7 min, the supernatant removed, the pellet re-suspended in 700 µl propidium iodide staining solution (50 mg/ml; BioSure), and 2µl of chicken erythrocyte nuclei singlets added (CEN internal standard; BioSure). Samples were protected from light and stored on ice for at least 30 min before analysis on a Becton Dickinson FACS Calibur flow cytometer at the Kansas State University Flow Cytometry Facility. The amount of fluorescence was measured for ~10,000 nuclei per sample. Resulting histograms were visually inspected for the presence of clear nuclear populations from the Phlox sample and the CEN internal standard, and mean peak values were calculated using the program Cell Quest (Becton Dickinson). Nuclear DNA content was calculated as the Phlox sample mean peak value divided by the CEN internal standard mean peak value multiplied by the 2C-value of the CEN internal standard (2.5 pg; Dolezel and Bartos 2005). Ploidy level was inferred for each sample based on the calculated DNA content. Inferred ploidy level was linked with chromosome count data for several samples. RESULTS AND DISCUSSION A total of 140 samples from seven species of Phlox collected from 63 locations were assessed for cytotype variation (Table 1). When cytotype was measured in multiple individuals from a single location, average nuclear DNA content was calculated. We did not detect any cytotype variation within populations based on our limited sampling. The results from flow cytometry were interpreted as measures of ploidy level rather than absolute measures of DNA content (see Suda et al. 2007). Results from chromosome counts confirmed ploidy levels of 2x, 4x, and 6x (for five samples; Table 1). Cytotype varied throughout the species studied and appeared to reflect both taxonomy and geography (Table 1). Populations of P. caryophylla and P. cluteana were diploid, while populations of P. aculeata were tetraploid; all of these taxa are fairly narrow endemics. In general, populations of the wide-ranging P. longifolia and P. stansburyi were geographically structured, with 190
Calochortiana December 2012 Number 1 Table 1. Samples included in this study. Voucher information, collection locality, number of individuals cytotyped (N), DNA content and inferred cytotype are noted. Taxon recognition for P. stansburyi follows Ferguson and others (2012); intraspecific classification for P. longifolia (including P. longifolia and P. viridis sensu Wherry [1955]) is under ongoing investigation and is not proposed here. Taxon Voucher County State Location Detail N DNA content (pg) P. aculeata Sidells 116 Twin Falls Co. ID 1 14.65 4X P. aculeata CF 789 Butte Co. ID 3 15.43 4X P. aculeata CF 787 Bonneville Co. ID 2 18.67 4X P. caryophylla CF 653 Archuleta Co. CO 1 7.61 2X P. cluteana CF 651 Apache Co. AZ 1 8.14 2X P. cluteana SDF 51008-2 Apache Co. AZ 3 8.84 2X P. longifolia SDF 42908-3 Navajo Co. AZ 3 8.37 2X P. longifolia SDF 43008-1 Coconino Co. AZ 3 8.60 2X P. longifolia SDF 42908-1 Navajo Co. AZ 3 8.74 2X P. longifolia SDF 51008-1 Apache Co. AZ 3 8.88 2X P. longifolia SDF 50408-1 Coconino Co. AZ 3 8.89 2X P. longifolia SDF 50908-1 Coconino Co. AZ 3 9.17 2X P. longifolia SDF 50708-1 Mojave Co. AZ 3 9.27 2X P. longifolia SDF 50808-1 Mojave Co. AZ 3 16.79 4X P. longifolia SDF 50808-2 Mojave Co. AZ 4 17.28 4X P. longifolia CF 728 Eagle Co. CO 1 15.25 4X P. longifolia CF 727 Mesa Co. CO 1 16.71 4X P. longifolia CF 788 Butte Co. ID 3 7.60 2X P. longifolia CF 806 Lemhi Co. ID 1 14.27 4X P. longifolia CF 603 Clark Co. ID 1 14.67 4X P. longifolia CF 792 Butte Co. ID 3 15.25 4X P. longifolia CF 589 Custer Co. ID 1 15.41 4X P. longifolia CF 794 Custer Co. ID 3 15.67 4X P. longifolia CF 786 Jefferson Co. ID 2 18.67 4X P. longifolia CF 601 Beaverhead Co. MT 1 13.63 4X P. longifolia CF 710 White Pine Co. NV 1 17.11 4X P. longifolia CF 724 Beaver Co. UT 1 8.38 2X P. longifolia CF 702 Sevier Co. UT 1 15.00 4X P. longifolia CF 611 Summit Co. UT 1 21.90 4X P. longifolia CF 734 Carbon Co. WY 1 13.98 4X a P. longifolia CF 215 Sublette Co. WY 1 14.65 4X Cytotype 191
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December 2012 Number 1 - Utah Native Plant Society

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