Schilling, E.E. and A. Floden. 2012. Barcoding the Asteraceae of Tennessee, tribes Gnaphalieae and Inuleae. Phytoneuron 2012-99: 1-6. Published 12 November 2012. ISSN 2153 733X BARCODING THE ASTERACEAE OF TENNESSEE, TRIBES GNAPHALIEAE AND INULEAE EDWARD E. SCHILLING and AARON FLODEN Herbarium TENN Department of Ecology & Evolutionary Biology University of Tennessee _ Snoxulle, Tennessee 31290 k.edu and afl kedu ABSTRACT Results from barcoding studies of tribes Gnaphalicae and Inuleae for the Tennessee flora using data from the nuclear ribosomal ITS marker region are presented, and include first complete reports of this marker for 10 of the 13 species of these tribes that occur in the state. Sequence data from the ITS region separated all genera and most species of Gnaphalicae from Tennessee. Species pairs that were not distinguished included Antennaria plantaginifolia/ A. parlinii and Pseudognaphalium helleri/ P. micradenium. The ITS sequence data provided supporting evidenee for recent changes in the classification of the group, most notably of G an. Pseudognaphalium from Gnaphalium, as well as the species-level taxonomy of Gamochaeta. Issues were noted with current GenBank records, including apparent poor quality of some sequence data and possible mistakes in species identifications, which must be taken into account in barcoding efforts and which highlight the need to expand this highly useful database. KEY WORDS: Asteraceae, Gnaphalieae, Inuleae, molecular barcoding The Gnaphalieae, generally referred to as ““cudweeds”, are relatively anonymous yet common and widespread plants. They can be easily be overlooked or passed up by collectors. The major diversification of the tribe has occurred in the southern hemisphere, and the species from southeastern North America have not been well sampled in broad phylogenetic surveys of the tribe (e.g. Ward et al. 2009; Galbany-Casals et al. 2010; Nie et al. 2012). The current study was undertaken as part of a general effort to increase surging for molecular markers of Ast from southeastern North America in general and the state of Tennessee in particular. The classification of Gnaphalieae has changed dramatically at several levels in recent years. Gnaphalieae were once included in Inuleae, but more recent studies have shown that the two tribes are clearly distinct (Bremer et al. 1994). Both tribes are characterized by having heads that are often discoid or disciform, but Gnaphalieae is the sister group to Astereae and Anthemidae whereas Inuleae isa lineage at the base of the clade that includes the Heliantheae alliance (Funk et al. 2009). The generic level classification has also changed, with a formerly large Gaaphalian retaining the Old World species but the basically New World G h and Pseud being (Nesom 1990). Species level recognition has also been sharpened through eoctul studies that have refined circumscriptions and even described several previously 1 species in G h and Pseudognaphalium (Nesom 1990, 2001a, 2004a). The relatively technical features required to recognize accurately species of the genera of Gnaphalicae make them a logical target that would benefit from a molecular barcoding approach (Kress et al. 2005) to verify identifications. Gnaphalieae are represented in the state of Tennessee by a total of 12 species (Table 1), most of which were traditionally placed (e.g. Cronquist 1980) in two genera, Gnaphalium and Antennaria, as well as the introduced Facelis. Current classification separates Gnaphalium in Tennessee into Schilling and Floden: Barcoding Gnaphalieae and Inuleae of Tennessee 2 1 three different genera, including G and Pseudognaphalium (Table 1). Inuleae is represented by a single species, Pluchea camphorata. Most of the species of Gnaphaliecae in Tennessee are currently considered to be native to the flora, although their ruderal nature makes it difficult to be certain in all cases (Nesom 2004b). A species that appears to be clearly introduced is Facelis retusa, a South American weedy species (Cronquist 1980). Gnaphalium uliginosum 1 : considered by Cronquist (1980) to be introduced from Europe, although its probable sister relati to G exilifolium A. Nelson suggests that it is likely also to be native, perhaps in part, to North America (Nesom 2001b). One species, Pseudognaphalium helleri, is listed as a species of special concern for Tennessee with a state ranking of S2, based on its rarity within the state, although its global ranking of G4/G5 indicates that it is common in other parts of its range (Crabtree 2012). Prior to initiation of this study, GenBank records for the complete nuclear ribosomal ITS region were reported for only three of the thirteen species of Gnaphalicae and Inuleae found in Tennessee. The GenBank records for Antennaria are relatively old and consist of separate ITS-1 and ITS-2 sequences rather than the entire ITS region as a single entity. The purpose of the study was to make a survey of sequence variation for the ITS region across Gnaphalieae and Inuleae that have been collected in the state of Tennessee. The goals included expanding the database of available samples for both barcoding and phylogenetic studies and assessing the value of this marker in identifying members of these tribes to genus or species level. MATERIALS AND METHODS DNA was extracted from leaf samples either collected fresh or taken from herbarium specimens (Table 1). For most samples the DNeasy Plant Mini Kit (Qiagen, Valencia CA) was used, although some freshly collected samples were processed using the CTAB method (Doyle & Doyle 1987). PCR amplifications and sequencing of the ITS region followed protocols outlined by Schilling et al. (2007). Afew samples required the use of the internal primers, “5.85 79 for” and “ITS 5.8SR,” for sequencing to obtain clean sequence, likely because of fungal contamination (Schilling et al. 2007). GenBank accession numbers are provided in Table 1. Although this study was not designed to undertake a rigorous phylogenetic analysis, parsimony analysis using the PAUP* 4.0b10 program (Swofford 2003) was utilized to provide a convenient way to make a comparative visualization of the sequence results and incorporated sequences deposited at GenBank of conspecific or closely related samples. This included a sequence for Anaphalis margaritacea (L.) Benth., which though native to North America has not been clearly documented to occur in Tennessee outside of horticultural settings. RESULTS AND DISCUSSION The complete ITS sequence region (ITS-1, 5.88 ribosomal DNA, ITS-2) varied in length in the newly reported Gnaphalicae samples from 633-641 bp. Sequences of Pseudognaphalium were consistently 633 bp; those of Gamochaeta 636-637 bp, and Antennaria showed the most length variation, with three of the four sampled species having a different length (636-638 bp). The ITS sequences for Gnaphalium (641 bp) and Pluchea (642 bp) were the longest of those sampled. There was no evidence in the electropherograms that any sample had multiple polymorphisms, either length or at individual sequence sites, which would provide evidence of recent or fixed interspecific hybridization. Results of a phylogenetic analysis of the ITS sequence data for samples of Gnaphalieae are shown in Figure 1, with the single member of Inuleae used as the outgroup. Each genus was placed as monophyletic with moderate to strong bootstrap support. Gamochaeta, which was once considered to be part of Gnaphalium, was placed sister to Facelis and further sister to Antennaria (Fig. 1). Schilling and Floden: Barcoding Gnaphalieae and Inuleae of Tennessee 3 Pseudognaphalium, also formerly considered part of Gnaphalium, was placed as sister to Anaphalis with strong support. Table 1. Plant material used for ITS barcoding studies of Gnaphalieae and Inuleae. All specimens at TENN, collected in Tennessee. Species ANTENNARIA Gaertn. A. howellii subsp. neodioica (Greene) R. Bayer A, parlinii Fernald A, plantaginifolia (L.) Hook. A, solitaria Rydb. FACELIS Cass. E retusa (Lam.) Sch.Bip. GAMOCHAETA Wedd. G argyrinea GL. Nesom G pensylvanica (Willd.) Cabrera G purpurea (L.) Cabrera GNAPHALIUM L. G uliginosum L. PSEUDOGNAPHALIUM Kirp. P. helleri (Britton) Anderb. P. micradenium (Weath.) GL. Nesom P. obtusifolium (L.) Hilliard & B.L.Burtt. PLUCHEA Cass. P. camphorata (L.) DC. DNA# Genbank 3444 3446 2542 3445 3082 3083 2766 3084 3085 3443 3086 3087 3089 3442 2566 3088 JX524604 JX524605 JX524601 JX524602 JX524603 JX524606 JX524596 JX524597 JX524600 JX524598 JX524599 JX524592 JX524593 JX524594 JX524595 JX524607 Voucher info Sharp 26822, Sevier Co. Murrell 75, Polk Co. Schilling 07-2542, Knox Co. Sharp 26281, Sevier Co. Crabtree FSF-07-034, Marion Co. Beck 4671, Marion Co. Schilling 08-2766, Knox Co. Phillippe 35455, DeKalb Co. Browne 78, Shelby Co. Estes 7859, Moore Co. Bresowar 122, Knox Co. Henry et al. s.n., Cocke Co. DeSelm s.n., Franklin Co. Patrick 3813, Roane Co. Schilling CF-4, Unicoi Co. McNeilus 00-919, Fentress Co. Schilling and Floden: Barcoding Gnaphalieae and Inuleae of Tennessee 4 — 3087 6. utiginosumn Gnaphalium LL 6. wliginosum FN645823 a0 3086 G. purpurea Gamochaeta ay 78 3085 G. pensyivanica G. affine 1980346 84 . purpurea AY9A7AN1 isa 100 2542 A. plantaginifolia 3445 A. plantaginifolia 3446 A. partini A. solitari Last a. esti ssp. neocotea ez 3089 P hellert 54 Antennaria 46 Outgroup Figure 1. Single shortest tree from parsimony analysis of nuclear ribosomal ITS data showing relationships among species of Gnaphalieae from Tennessee. Bootstrap values shown above branches. Samples labeled by DNA number (Tennessee samples, Table 1) or GenBank accession number. The sample of Pluchea camphorata Cnuleae) was used as the outgroup Antennaria. Four species of Antennaria are recorded for Tennessee, of about 45 species total for the genus. The genus is clearly distinguished from other Gnaphalieae by at least 32 bp changes in the ITS sequences. The two samples of A. plantaginifolia were identical — the second sample tested was collected at the exact same site as the sample of A. howellii subsp. neodioica — and also identical to the sample of A. parlinii. All of the other species were separated by multiple changes, with 11 differences between A. solitaria and A. plantaginifolia and 25-27 differences between A. howellii subsp. neodioica and the other species. Complete ITS sequences (e.g., ITS-1, 5.8S rDNA, ITS-2) have not been previously deposited in GenBank for A. parlinii, A. plantaginifolia, or A. solitaria, Older records include individual ITS-1 and ITS-2 sequences for A. plantaginifolia and A. solitaria, but these were not retrieved in a BLAST search with standard parameters using newly obtained complete ITS sequences. The older sequences required several small gaps for alignment, which likely reflects the lower accuracy that was achieved using older technology. Facelis. Facelis is represented in Tennessee by a single introduced species, F retusa. The current report is the first record for the genus, which has 3-4 species total, in GenBank. Gamochaeta. The three species of Gamochaeta documented from Tennessee were each distinctive for ITS sequence, with pairwise differences as follows: G argyrinea-G: pensylvanica, 9 differences; G argyrinea-G. purpurea, 4 differences, G pensylvanica-G purpurea, 5 differences. No intraspecific variation was encountered in the two samples each of G argyrinea and G purpurea (Table 1). Samples from GenBank labeled G purpurea and G affine were identical to our sample of G pensylvanica, raising questions about whether they were accurately identified. Also distinct based ona GenBank sequence deposited for it was G coarctata (Willd.) Kerguélen, which may be expected Schilling and Floden: Barcoding Gnaphalieae and Inuleae of Tennessee 5 to be found naturalized in western Tennessee (Guy Nesom, pers. comm.) The name for another GenBank sample, G spicata (Lam.) Cabrera, is now considered to be a synonym for G coarctata (Pruski & Nesom 2004), and sequences for the two GenBank samples were identical and differed at 3 positions from those of G argyrinea. The distinctiveness of individual species of Gamochaeta is notable, especially considering that G argyrinea was only recently recognized to be distinct from G purpurea (Nesom 2004a). Similar results were obtained with a broader sampling of both species and samples of Gamochaeta by Cameron (2010), and the molecular results are further supported by detailed morphological analysis (Mac Alford and Kree Cameron, pers. comm.) Thus, a barcoding approach can be utilized to confirm the identity of samples of Gamochaeta collected in Tennessee. Gnaphalium. Gnaphalium as currently interpreted is represented by a single recent collection of the apparently non-native G uliginosum from eastern Tennessee. The ITS sequence from this sample was almost identical with a GenBank record for the species. It is clearly distinct from all other Gnaphalicae of Tennessee, differing by at least 51 bp changes. Pseudognaphalium. The amount of interspecific variation for Pseudognaphalium was the least observed for any of the genera of Gnaphalieae in Tennessee. Samples of P. helleri and P. micradenium were identical to one another and differed by two changes from a sample of P obtusifolium. A GenBank sequence for P. obtusifoliwn differed at a single position from our sample, and two other Pseudognaphalium sequences at GenBank (P. macounii and P. canescens, identification of the latter determined by G. Nesom) differed at three positions. These results suggested that species level diversification in Pseudognaphalium might be relatively recent, similar to what has been observed for other Asteraceae genera in eastern North America, such as Helianthus and Solidago. Pluchea. A single species of P/uchea out of the 40 or more species of the genus is native to Tennessee. The ITS sequence is the first record for the species in GenBank, and it is significantly different (minimum of 45 bp differences and 6 gaps) from other species of the genus represented in GenBank. ACKNOWLEDGEMENTS We thank S. Robertson, J. Schilling, and S. Yoon for laboratory assistance, J. May and V. Brown of the University of Tennessee MBRF for help with DNA sequencing, G. Nesom for helpful comments and for examining images of GenBank vouchers of Pseudognaphalium, M. Alford and K. Cameron for sharing unpublished results on Gamochaeta, and the numerous collectors, notably the late H. DeSelm and V. McNeilus, who have helped to assemble a broad and useful herbarium at TENN. Financial support provided by the Hesler Fund of the University of Tennessee Herbarium. LITERATURE CITED Bremer, K., A.A. Anderberg, P.O. Karis, B. Nordenstam, J. Lundberg, and O. Ryding. 1994. 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