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Preliminary Study Using ISSRs to Differentiate Imperata Taxa (Poaceae: Andropogoneae) Growing in the US
Rodrigo Vergara, Marc C. Minno, Maria Minno, Douglas E. Soltis, and Pamela S. Soltis

Southeastern Naturalist, Volume 7, Number 2 (2008): 267–276

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2008 SOUTHEASTERN NATURALIST 7(2):267–276 Preliminary Study Using ISSRs to Differentiate Imperata Taxa (Poaceae: Andropogoneae) Growing in the US Rodrigo Vergara1,2,*, Marc C. Minno3, Maria Minno3, Douglas E. Soltis1, and Pamela S. Soltis2 Abstract - Imperata cylindrica (cogongrass) is an invasive weed long established in the southeastern US, and considerable effort is devoted to its control. Two native species, I. brevifolia (California satintail) and I. brasiliensis (Brazilian satintail), also occur in the US, and the latter is sympatric to cogongrass. Certain Imperata morphotypes growing in the field are difficult to identify. To clarify their identity, inter-simple sequence repeats (ISSRs) were used to assess genetic differentiation among eight populations in the US representing Brazilian satintail, California satintail, three potential morphotypes of cogongrass, and three unknowns. Samples preserved in 95% ethyl alcohol and silica-gel did not produce repeatable band patterns, so DNA from fresh leaves was extracted and analyzed by polymerase chain reaction (PCR) amplification. Results indicate that California satintail (D = 0.67), a commercial cogongrass cultivar (D = 0.66), and a short-hairy morphotype of cogongrass (D = 0.65) were the most distinctive operational taxonomic units (OTUs) compared. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed two well-supported clusters of taxa containing Brazilian satintail (Bootstrap value = 96%) and the tall morphotypes of cogongrass (Bootstrap value = 83%), respectively. Among the morphotypes of cogongrass analyzed, the tall-hairy and tall-glabrous plants formed a cluster from which the short-hairy morphotype and the cultivar were genetically divergent. Our results refute taxonomic arrangements placing Brazilian satintail as a synonym of cogongrass. Introduction Imperata cylindrica (L.) (cogongrass) is an invasive weed from Asia that has been established in the southeastern US for nearly 100 years (Tabor 1952a, b). Considerable effort is devoted toward controlling cogongrass, not only in the US, but also throughout its range (Byrd and Bryson 1999, Coile and Shilling 1993, Dozier et al. 1998, Tanner and Werner 1986, Van Loan et al. 2002). Despite the invasiveness of cogongrass, plant nurseries in the US have been propagating and selling ornamental cultivars of the species, commonly called Japanese blood grass, throughout much of the US for many years. In addition, two species occur natively in the US (Hitchcock 1950): Imperata brevifolia Vasey (California satintail, in the southwestern US) and Imperata brasiliensis Trinius (Brazilian satintail, in South Florida), which 1Department of Botany, University of Florida, Gainesville, FL 32611. 2Florida Museum of Natural History, University of Florida, Gainesville, FL 32611. 3600 NW 35th Terrace, Gainesville, FL 32607. *Corresponding author - rodver@ufl .edu. 268 Southeastern Naturalist Vol.7, No. 2 also is invasive in central Florida and Louisiana (Allen et al. 1991; Wunderlin and Hansen 2003, 2006). We surveyed several populations of Imperata species growing wild in the US and observed differences in biological and morphological characteristics of these plants. Specifically, we found shorthairy, tall-hairy, and tall-glabrous morphotypes. We were uncertain if these variants were all cogongrass or if more than one species was represented. The purpose of this study was to preliminarily analyze and compare the species and morphotypes of Imperata present in the US using inter-simple sequence repeats (ISSRs) in order to clarify the identity and distribution of the various Imperata taxa. Methods ISSRs were chosen for this study because of their suitability in identifying cultivars, varieties, and hybrids of cultivated plants (Wolfe and Liston 1998). ISSRs are dominant molecular markers generated by polymerase chain reaction (PCR) amplification using primers developed from within simple sequence repeats (SSRs). Each primer can potentially produce multiple random fragments from across the entire genome, yielding highly polymorphic bands. Such bands are interpreted as diallelic loci considering only band presence or band absence (Wolfe and Liston 1998). Analyses were conducted at the Laboratory of Molecular Systematics and Evolutionary Genetics, FLMNH, University of Florida, Gainesville, FL. Table 1. Imperata operational taxonomic units (OTUs) growing in the southern US analyzed using ISSR fragments. Cogongrass = I. cylindrica, Brazilian satintail = I. brasiliensis, and California satintail = I. brevifolia. IDA Species Morphotype Origin State: County Code 088 1 Cogongrass Short, hairy Japan AL: Mobile cyl (sh) (pubescent over the whole leaf sheath) 048 2 Cogongrass Tall, hairy Philippines FL: Levy cyl (th) (pubescent over the whole leaf sheath) 047 3 Cogongrass Cultivar (red leaves) Japan NC: Iredell cyl (cv) 005-2 4 Brazilian Typical (tall, glabrous) Native FL: Miami-Dade bra satintail 028 5 I. sp.B Tall, glabrous Unknown MS: Pearl River unk (MS) 054 6 California Typical (glabrous) Native CA: Ventura bvf satintail 052 7 I. sp.B Wide blade Unknown FL: Collier unk (SFL) 097 8 I. sp.B Tall, glabrous Unknown FL: Hillsborough unk (NFL) ASample identification from original collection. BPutative Brazilian satintail population. 2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 269 Sampling We collected, analyzed, and compared samples of Imperata from eight populations typified as eight different operational taxonomic units (OTUs) found in Alabama, California, Florida, Mississippi, and North Carolina (Table 1). In working with Imperata taxa from throughout the range of the genus in the US, we observed plants growing wild at numerous sites, including the original sites of introduction of cogongrass in the Mobile area of Alabama, at the Mississippi Experiment Station in McNeil, around Gainesville, FL, and at the US Department of Agriculture station near Brooksville, FL. For most populations, inflorescences were not available at the time of our visit, and we focused on vegetative characteristics. Plants of cogongrass from along the Gulf Coast, including eastern Louisiana, southern Mississippi, southern Alabama, and the western Florida Panhandle had hairy leaf sheaths and were shorter in height than most populations from peninsular Florida. Populations of California satintail from California, Arizona, and Nevada, as well as Brazilian satintail from Homestead (Miami-Dade County), FL, and plants of the Japanese blood grass cultivar from California, Maryland, and North Carolina were glabrous, except for hairs on the margins of the leaf sheaths in the vicinity of the ligule. We hypothesized that an unidentified specimen from Picayune State Forest in South Florida (Collier County), that was tall with very wide leaves and bulbous culm bases, was Brazilian satintail. Lastly, a tallglabrous taxon that we thought may also be Brazilian satintail was found growing at the Mississippi Experiment Station in McNeil, at one site in DeSoto National Forest in Mississippi, near Romar Beach in Alabama, and at many locations in peninsular Florida. DNA extraction and purification Although fresh leaves are the best material for DNA extractions, silica-gel dried samples are, in general, considered a suitable alternative for extracting high-quality DNA (Chase and Hills 1991). In studies using ISSRs, RAPDs, and AFLPs with some other members of Andropogoneae, most of the extractions employed fresh leaf tissue (Hodkinson et al. 2002, Nair et al. 1999, Pan et al. 2000) and, in a few cases, freeze-dried tissue (Besse et al. 1998). Apparently, silica-gel or alcohol-dried tissue stored at room temperature is not frequently used to extract DNA for ISSRs and other similar genetic markers in this group of plants. The extraction of DNA from silica-dried material has been shown to be problematic in certain Poaceae because of the reactivation of DNases after the tissues are re-hydrated in the DNA extraction process, and the buffers used there are not effective (Adams et al. 1999). These authors also indicate that alcohol-dried tissue seems to overcome this problem by irreversibly denaturing the DNases. In our study, we tried extracting DNA from samples preserved in both alcohol and silica gel. Leaves of living plants from each population were 270 Southeastern Naturalist Vol.7, No. 2 cut into small pieces and stored in coded plastic vials containing either of two kinds of preservatives for over a year before processing. One set of samples was preserved with 95% ethyl alcohol, and a second set was dried and stored in silica gel. Approximately 20 mg of leaf tissue from each sample was ground in a mortar and pestle with liquid nitrogen and sand and processed using both a CTAB DNA extraction protocol modified from Doyle and Doyle (1987) and Cullings (1992) and the Promega Wizard DNA Extraction Kit. Preliminary ISSR runs revealed that there were no differences in DNA quality between extractions from samples preserved in alcohol or silica gel; in both cases, DNA quality was poor. Therefore, fresh leaves from eight available populations were used for the final analysis. DNA was extracted from ground fresh leaves using the QIAGEN DNeasy® Plant Mini Kit, obtaining clean DNA. Electrophoresis was used to check DNA quality in 1.2% agarose gels, which were stained with ethidium bromide, exposed to ultraviolet light, and photographed using an EDAS 290 Kodak camera. ISSR amplification Five ISSR primers chosen at random were obtained from the University of British Columbia Biotechnology Laboratory: UBCBL-set #9: 810, 815, 825, 830, and 841 (Table 2). The primers were optimized and further tested using DNA samples extracted from fresh leaves. PCR reactions were carried out in an Eppendorf Mastercycler thermocycler using the cycle profile described by Huang and Sun (2000). Optimization was made for each primer, testing two concentrations of formamide (1% and 2%) and two concentrations of MgCl2 (1.7 and 2.5 mM) in a factorial test through a temperature gradient using an Eppendorf Mastercycler gradient thermocycler. The PCR reaction also included Taq buffer (Mg-free), dNTP, Taq polymerase, genomic DNA, and the ISSR primer, completing a 15-μl reaction volume. The five primers were tested using the respective optimized ISSR reactions. After optimization, we performed three replications of each PCR reaction, for each primer/sample combination. Table 2. ISSR primers optimized for annealing temperature, MgCl2 concentration, formamide concentration, and tested for polymorphism and repeatability. Primers were obtained randomly from primer set #9, UBCBL (University of British Columbia Biotechnology Laboratory). # of Annealing analyzed Size Polymorphism Primer Sequence (5’–3’) temperature (C°) fragments range (bp) (%) 810A GAGAGAGAGAGAGAGA-T 41.8 – – – 815A CTCTCTCTCTCTCTCT-G 41.8 – – – 825 ACACACACACACACAC-T 41.1 16 540–1370 94 830 TGTGTGTGTGTGTGTG-G 41.8 18 380–1250 72 841 GAGAGAGAGAGAGAGA-YC 42.8 19 300–1260 95 ADiscarded because of low repeatability. 2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 271 PCR products were separated by electrophoresis in 2% agarose gels using a Sigma 100-bp ladder to assess band size. Band scoring and data analysis Bands were scored on agarose gels by first using a Kodak camera system (1D 3.5.4 USB, DC290 Capture) to assign the size of the fragments and then counting and matching bands among samples and replicates by eye in order to account for uneven migration in the gels. Bands coinciding in molecular weight and mobility were regarded as equal fragments. Bands representing fragments greater than 1500 bp were ignored, because they were not repeatable and weak. The presence of a band was coded as “1” and the absence as “2.” After bands were scored, the data matrix was analyzed using the TFPGA 1.3 software (Miller 1997) to obtain modified Rogers’ genetic distances (Wright 1978) and dendrograms generated by unweighted pair group method with arithmetic mean (UPGMA). Support of nodes was obtained using bootstrapping with 1000 replicates. Results and Discussion Quality of DNA extractions and ISSR reactions In this study, samples preserved in silica-gel and alcohol yielded poorquality DNA, and the amplification of ISSR products was low and highly unrepeatable, regardless of the extraction protocol used. In contrast, satisfactory genomic DNA was obtained from fresh material extracted with the QIAGEN DNeasy® Plant Mini Kit. This DNA was of high molecular weight and it provided repeatable results in the amplification of PCR products. The ISSR optimization experiments using the genomic DNA obtained from fresh material indicated that, for all five primers, the best concentrations for MgCl2 and formamide were 2.5 mM and 1%, respectively. The annealing temperature varied depending upon the primers (Table 2), and the final formula for the PCR reactions was: 5.05 μl H2O, 1.5 μl Taq buffer (Mg-free), 1.5 μl 25 mM MgCl2, 1.2 μl 2.5 mM dNTPs, 0.15 μl Formamide Table 3. Wright’s (1978) modification of Rogers’ genetic distances among Imperata operational taxonomic units (OTUs) based on 53 ISSR loci. D is the average genetic distance for each OTU. OTUA 1 2 3 4 5 6 7 8 D 1 cyl (sh) – 0.63 0.66 0.67 0.57 0.71 0.63 0.64 0.65 2 cyl (th) – 0.67 0.66 0.34 0.73 0.67 0.31 0.52 3 cyl (cv) – 0.66 0.67 0.61 0.64 0.69 0.66 4 bra – 0.63 0.60 0.24 0.64 0.59 5 unk (MS) – 0.70 0.64 0.36 0.56 6 bvf – 0.61 0.71 0.67 7 unk (SFL) – 0.66 0.59 8 unk (NFL) – 0.57 AFor details regarding OTUs see Table 1. 272 Southeastern Naturalist Vol.7, No. 2 (SLS), 0.5 μl 10 μM primer, 0.1 μl Taq polymerase (Promega), and 5 μl DNA (1/50 dilution). After optimization, only primers 825, 830, and 841 showed polymorphic, clear, and repeatable band patterns. Therefore, only these primers were scored and analyzed. Scoring them conservatively on the gels (i.e., bands from different samples that appear at similar migration distances were scored as having the same band), the three primers yielded a total of 53 fragments or loci. The size of the analyzed fragments ranged from 300 to 1370 bp, and 87% of the loci were polymorphic (Table 2). Similarity among OTUs The genetic distances obtained among OTUs (Table 3) show that California satintail (bvf) from California, Japanese blood grass (cogongrass cultivar, cyl [cv]) and the short-hairy morphotype of cogongrass (cyl [sh]) collected in Alabama near the site of first introduction of cogongrass into the US from Japan, were the most distinctive OTUs compared, with average genetic distances (D) of 0.67, 0.66, and 0.65, respectively, from all other samples. The UPGMA dendrogram obtained from the genetic distances (Fig. 1) shows the formation of two well-supported clusters (bootstrap values >75%), which do not include any of the three OTUs above. The first cluster includes a known Brazilian satintail population Figure 1. UPGMA dendrogram comparing eight Imperata operational taxonomic units (OTUs; see Table 1) based on Wright’s (1978) modification of Rogers’ genetic distances and using ISSR markers. Numbers above branches are bootstrap percentages. Thick lines indicate clusters strongly supported by bootstrapping (bootstrap values >75%). Relative branch lengths indicate relative genetic distances between taxa. 2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 273 (bra) and an unknown wide-bladed plant that we thought was Brazilian satintail from south Florida (unk [SFL]). The second cluster is composed of the tall-hairy morphotype of cogongrass (cyl [th]) as well as the two tall-glabrous unknowns from northern Florida (unk [NFL]) and Mississippi (unk [MS]). The two tall morphotypes (hairy and glabrous) are common throughout peninsular Florida, but were found at only a few places in Mississippi and Alabama, including the second site of introduction of cogongrass at the Mississippi Experiment Station in McNeil. Although it would be useful to examine phylogenetic relationships among species within the genus using gene sequence data, these two highly supported clusters suggest that the ISSR markers employed in this study are able to discriminate between cogongrass and Brazilian satintail, and that the two species are therefore genetically divergent. Based on morphological traits and interbreeding, Hall (1998) and Ward (2004) thought Brazilian satintail to be the same species as cogongrass, but the genetic divergence between them shown by our ISSR analysis does not agree with this concept, suggesting parallel morphological evolution. Figure 2 illustrates the differences in electrophoresis band patterns between the two well-supported clusters, as well as the consistency of band patterns among replicates. Figure 2. Gel illustrating PCR-amplified fragments from ISSR analysis using primer UBCBL 841. First lane (L) is the Sigma 100-bp ladder (standard band sizes are shown). The following lanes are the band patterns of the Imperata operational taxonomic units (OTUs) studied with three replications. 1 = cyl (sh), 2 = cyl (th), 3 = cyl (cv), 4 = bra, 5 = unk (MS), 6 = bvf, 7 = unk (SFL), 8 = unk (NFL) (see Table 1). Arrows show the two highly consistent clusters as indicated by bootstrapping (see Fig. 1). Arrows pointing upward indicate the cluster representing typical and wide-bladed morphotypes of Brazilian satintail (I. brasiliensis). Arrows pointing downward indicate the cluster representing tall-hairy and tall-glabrous morphotypes of cogongrass (I. cylindrica). 274 Southeastern Naturalist Vol.7, No. 2 Our results also indicate that the unknown wide-bladed population from south Florida (Picayune State Forest in Collier County) was Brazilian satintail and the other unknown populations from northern Florida and Mississippi were cogongrass. These tall-glabrous plants clustered strongly with tall-hairy cogongrass. The most common types of cogongrass in the US have hairs on the leaf sheaf, unlike California satintail and Brazilian satintail, and this character should help with the identification of non-fl owering Imperata plants. However, we demonstrate here that a plant lacking hairy leaf sheaths can still be cogongrass. Based on the literature, we believe that the shorthairy morphotype of cogongrass represents its original introduction from Japan, while one or both of the tall morphotypes are originally from the Philippines (Tabor 1949, 1952a, 1952b). Overlap of populations of Brazilian satintail with cogongrass in Florida and Louisiana as well as with different variants of cogongrass in Alabama, Mississippi, and Florida may set the stage for hybridization to occur. Interbreeding of different species and variants may potentially produce hardier, more aggressive plants that reproduce well from seed. Such hybrids could be very problematic to control. The tall-glabrous morphotype of cogongrass may represent such a hybrid because it shares characteristics of Brazilian satintail (e.g., lack of hairs on the leaf sheath). However, additional analysis of Imperata taxa using ISSRs or other genetic markers is needed, especially using more primers and comparing more populations in the US, to look for hybridization. Analysis of all ten species of Imperata using additional primers is also necessary in order to understand better the genetic similarities among these grasses. Acknowledgments We thank Richard Reardon (US Forest Service) for providing funding for this project, the US Park Service for granting permission to collect on federal lands, Dr. Ashley Morris (University of South Alabama) for her advice on ISSR methods, and Dr. Pablo Speranza (Universidad de la República, Uruguay) for his help with DNA extractions from grasses. Literature Cited Adams, R.P., M. Zhong, and Y. Fei. 1999. Preservation of DNA in plant specimens: Inactivation and re-activation of DNases in field specimens. Molecular Ecology 8:681–684. Allen, C.M., R.D. Thomas, and M.G. Lelong. 1991. Brachiaria plantaginea, Imperata cylindrica, and Panicum maximum: Three grasses (Poaceae) new to Louisiana and a range extension for Rottboellia chochinchinensis. Sida 14:613–615. Besse, P., G. Taylor , B. Carroll, N. Berding, D. Burner, and C.L. McIntyre. 1998. Assessing genetic diversity in a sugarcane germplasm collection using an automated AFLP analysis. Genetica 104:143–153. Byrd, J.D., Jr., and C.T. Bryson. 1999. Biology, ecology, and control of cogongrass [Imperata cylindrica (L.) Beauv.]. Mississippi Department of Agriculture and Commerce-Bureau of Plant Industry, Fact Sheet 1999-01, Jackson, MS. 2 pp. 2008 R. Vergara, M.C. Minno, M. Minno, D.E. Soltis, and P.S. Soltis 275 Chase, M.W., and H.H. Hills. 1991. Silica gel: An ideal material for field preservation of leaf samples for DNA studies. Taxon 40:215–220. Coile, N.C., and D.G. Shilling. 1993. Cogongrass, Imperata cylindrica (L.) Beauv.: A good grass gone bad! Florida Department of Agriculture and Consumer Services, Botany Circular No. 28, Gainesville, FL. 3 pp. Cullings, K.W. 1992. Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1:233–240. Doyle, J.J., and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19:11–15. Dozier, H., J.F. Gaffney, S.K. McDonanld, E.R.L. Johnson, and D.G. Shilling. 1998. Cogongrass in the United States: History, ecology, impacts, and management. Weed Technology 12:737–743. Hall, D.W. 1998. Is cogon grass really an exotic? Wildland Weeds 1:14–15. Hitchcock, A.S. 1950. Manual of the Grasses of the United States, 2nd Edition, revised by Agnes Chase. United States Department of Agriculture, Miscellaneous Publication No. 200, US Government Printing Office, Washington, DC. 1971 reprint by Dover Publications. 1051 pp. Hodkinson, T.R., M.W. Chase, and S.A. Renvoize. 2002. Characterization of a genetic resource collection for Miscanthus (Saccharinae, Andropogoneae, Poaceae) using AFLP and ISSR PCR. Annals of Botany 89:627–636. Huang, J.C., and M. Sun. 2000. Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA. Theoretical and Applied Genetics 100:1050–1060. Miller, M.P. 1997. Tools for population genetic analysis (TFPGA) 1.3: A Windows program for the analysis of allozyme and molecular population genetic data. Computer software distributed by the author. Nair, N.V., S. Nair, T.V. Sreenivasan, and M. Mohan. 1999. Analysis of genetic diversity and phylogeny in Saccharum and related genera using RAPD markers. Genetic Resources and Crop Evolution 46:73–79. Pan, Y.B., D.M. Burner, and B.L. Legendre. 2000. An assessment of the phylogenetic relationship among sugarcane and related taxa based on the nucleotide sequence of 5S rRNA intergenic spacers. Genetica 108:285–295. Tabor, P. 1949. Cogon grass, Imperata cylindrica (L.) Beauv., in the southeastern United States. Agronomy Journal 41:270. Tabor, P. 1952a. Cogon grass in Mobil County, Alabama. Agronomy Journal 44:50. Tabor, P. 1952b. Comments on cogon and torpedo grasses: A challenge to weed workers. Weeds 1:374–375. Tanner, G.W., and M.R. Werner. 1986. Cogongrass in Florida: An encroaching problem. University of Florida, Wildlife and Range Sciences Publication WRS-5, Gainesville, FL. 4 pp. Van Loan, A.N., J.R. Meeker, and M.C. Minno. 2002. Chapter 28: Cogon grass, Pp. 353–364, In R. Van Driesche, S. Lyon, B. Blossey, M. Hoddle, and R. Reardon (Eds.). Biological Control of Invasive Plants in the Eastern United States. US Department of Agriculture, Forest Service Publication FHTET-2002-04, Government Printing Office, Washington, DC. 413 pp. 276 Southeastern Naturalist Vol.7, No. 2 Ward, D.B. 2004. New combinations in the Florida fl ora II. Novon 14:365–371. Wolfe, A.D., and A. Liston. 1998. Contributions of PCR-based methods to plant systematics and evolutionary biology. Pp. 43–86, In D.E. Soltis, P.S. Soltis, and J.J. Doyle (Eds). Molecular Systematics of Plants II: DNA Sequencing. Kluwer Academic Publishers, Dordrecht, The Netherlands. 574 pp. Wright, S. 1978. Evolution and the Genetics of Populations. Vol. 4: Variability Within and Among Natural Populations. University of Chicago Press, Chicago, IL. 580 pp. Wunderlin, R.P., and B.F. Hansen. 2003. Guide to the Vascular Plants of Florida. Second Edition. University Press of Florida, Gainesville, FL. 787 pp. Wunderlin, R.P., and B.F. Hansen. 2006. Atlas of Florida Vascular Plants. Available online at http://www.plantatlas.usf.edu. Accessed June 2, 2006.