Paraphyly in Tribe Onagreae: Insights into Phylogenetic Relationships of Onagraceae Based on Nuclear and Chloroplast Sequence Data

Onagraceae are a family of 17 genera in seven tribes, with the majority of species in tribes Onagreae and Epilobieae. Despite the species-richness of these two tribes, to date no phylogenetic study has been done with suf!cient taxon sampling to examine relationships between and within these tribes. In this study, we used DNA sequence data from one nuclear region (ITS) and two chloroplast regions (trnL-trnF and rps16) to infer phylogenetic relationships among 93 taxa across the family, with concentrated sampling in the large tribe Onagreae. Results strongly suggest that tribeGongylocarpeae is sister to tribes Epilobieae 1 Onagreae, both of which are monophyletic. Within Onagreae, Camissonia seems to be broadly paraphyletic, and Oenothera is also paraphyletic. In Oenothera there appear to be two lineages, one of which has Gaura 1 Stenosiphon nested within it. At the base of the Onagraceae phylogeny, we have clari!ed previous confusion regarding con"icting placements of Hauya and Lopezia based on nuclear versus chloroplast data. Results of these analyses are supported by morphology and suggest the need for new taxonomic delimitations, which are forthcoming. The plant family Onagraceae (Evening-primroses) comprises ca. 655 species across 17 genera (Levin et al. 2003), with at least two thirds of the species occurring in tribes Onagreae (8 genera, 262 spp.) and Epilobieae (2 genera, 172 spp.). Onagraceae have a world-wide distribution, with the majority of species concentrated in the New World, especially western North America. Over the past few decades, the family has developed as a model system for studying plant evolution. Comparative studies of cytology, embryology, palynology, anatomy, morphology, reproductive biology, and chemistry have all been completed for various groups within the family (reviewed in Raven 1988). Unfortunately, a limitation of these previous studies has been the absence of a robust phylogenetic framework within which to examine the evolution of these traits. To date there have been several molecular (Martin and Dowd 1986; Crisci et al. 1990; Sytsma et al. 1991b; Bult and Zimmer 1993; Conti et al. 1993) and morphological (Hoch et al. 1993) phylogenetic studies of the family, although only recently has there been a molecular study that included members of all Onagraceae genera (Levin et al. 2003). There have also been various phylogenetic studies of individual genera within the family, including Fuchsia (Sytsma and Smith 1988, 1992; Sytsma et al. 1991a; P. Berry et al., U. WisconsinMadison, in mss.), Lopezia (O’Kane and Schaal 1998), Clarkia (Sytsma and Smith 1988, 1992; Sytsma et al. 1990; Gottlieb and Ford 1996; Ford and Gottlieb 2003; W. J. Hahn et al., in mss.), Epilobium and Chamerion (Baum et al. 1994), and Gaura (Hoggard et al., 2004). However, no such study has focused on relationships among tribes Onagreae and Epilobieae. Furthermore, within Onagreae there have been no molecular phylogenetic studies of the species-rich genera Camissonia (62 spp.; western North America, 1 sp. in South America) and Oenothera (120 spp.; Americas, the majority of species in western North America). Using chloroplast rbcL and ndhF sequence data, Levin et al. (2003) showed that the small genus Gongylocarpus (2 spp.), previously included in tribe Onagreae (Raven 1964, 1979; Munz 1965), is strongly supported as sister to the rest of Onagreae 1 Epilobieae, and should be placed in its own tribe, Gongylocarpeae. That analysis also suggested that neither Camissonia nor Oenothera is monophyletic, although sampling within these genera was limited. Camissonia appears to lack any morphological synapomorphies (Raven 1969; Hoch et al. 1993), and the only character uniting Oenothera (stigma with 4 linear elongate non-commissural lobes) also characterizes Stenosiphon and Gaura (Hoch et al. 1993; Hoggard et al., 2004); however, Stenosiphon and Gaura differ because of the presence of an indusium at the base of the stigma lobes. 148 [Volume 29 SYSTEMATIC BOTANY Thus, a major goal of the present study is to understand relationships between and within tribes Onagreae and Epilobieae, with a particular emphasis on evaluating the monophyly of the large and diverse genera Camissonia and Oenothera. A phylogenetic framework will facilitate comparative analyses of chromosomal evolution and pollination biology of these diverse groups, as well as biogeographical studies of the radiation of these tribes in southwestern North America (Katinas et al. 2004). While the main focus of this study is on Onagreae and Epilobieae, we have included sampling from members of all Onagraceae genera. This strategy is not only important for examining relationships among tribes Onagreae and Epilobieae, but inclusion of DNA sequence data from both nuclear and chloroplast regions allows examination of previous con"ict among evolutionary reconstructions based on these two genomes and on morphology, especially as pertains to the placement of Hauya and Lopezia (Bult and Zimmer 1993; Conti et al. 1993; Hoch et al. 1993; Levin et al. 2003). The recently described genus Megacorax (González Elizondo et al. 2002) may be vital to discerning relationships of Hauya and Lopezia to the rest of the family, as Levin et al. (2003) found that Megacorax is sister to Lopezia. Because sampling of Lopezia species was limited in that study, it was unclear whether Megacorax should be placed within Lopezia. Thus, the present study includes additional sampling from various sections of Lopezia (Plitmann et al. 1973; O’Kane and Schaal 1998). In this paper we endeavor to: 1) examine relationships between and within tribes Onagreae and Epilobieae, 2) test the monophyly of Camissonia, Oenothera, and Gaura, 3) compare signal from nuclear vs. chloroplast data, especially as it relates to earlier con"ict regarding relationships of Hauya and Lopezia, and 4) further examine the sister taxon relationship previously reported between Megacorax and Lopezia. To accomplish these goals, we used DNA sequence data from one nuclear region (ITS) and two chloroplast regions, the trnL-trnF region (Taberlet et al. 1991) and the rps16 intron (Oxelman et al. 1997; Popp and Oxelman 2001). These gene regions evolve more rapidly than the protein-coding ndhF and rbcL genes used in our earlier study (Levin et al. 2003). MATERIALS AND METHODS Taxon Sampling. This study includes sampling from all eight tribes and 17 genera of Onagraceae, with a concentration on Onagreae and Epilobieae (Table 1). Within these two tribes we included at least one individual per section, subsection, or series, depending on current circumscriptions (Table 1). However, we did not sample from Chamerion sect. Rosmarinifolium, as Chamerion has previously been shown to be strongly monophyletic (Baum et al. 1994). We also did not include all of the subsections of Clarkia, as they are the subject of another analysis (W. J. Hahn et al., in mss.), and we discovered late in the analysis that our only sample of Gaura sect. Campogaura was misidenti!ed. Thus, that section is not in our study, and instead we included both subspecies of G. hexandra (sect. Pterogaura). In the other six tribes, two taxa were sampled from Ludwigia (tribe Jussiaeeae) to serve as a monophyletic outgroup for phylogenetic analyses, given previous studies that unambiguously place this genus sister to the rest of Onagraceae (e.g., Levin et al. 2003). One species each from tribes Hauyeae, Fuchsieae, Circaeeae, and Gongylocarpeae was also included. In order to more precisely determine the relationship of the newly described monotypic genus Megacorax to Lopezia (tribe Lopezieae), we sampled four Lopezia species from various sections plus Megacorax gracielanus. The cp trnL-trnF and nuclear ITS regions were sequenced from a total of 93 taxa. The cp rps16 region was sequenced from a subset of 75 species focused mainly in Onagreae, in order to improve resolution within this species-rich tribe. All taxa included in this study are listed in Table 1 with voucher information. DNA Extraction, Ampli!cation, and Sequencing. Total genomic DNA for the majority of taxa was provided by KJS (see protocols in Conti et al. 1996; Sytsma et al. 2002). However, several taxa were extracted by the senior author from either silica gel-dried or herbarium material using the Qiagen Dneasyy kit (Qiagen Inc., Valencia, CA). DNAs of Lopezia lopezioides, L. racemosa, and L. langmaniae were provided by S. O’Kane (Univ. Northern Iowa), and DNAs of Oenothera deltoides and O. pallida were provided by M. Evans (Univ. Arizona). ITS. Ampli!cation of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, composed of ITS1, the 5.8S gene, and ITS2 (Baldwin 1992; Baldwin et al. 1995) was mainly conducted by WJH using primers ITS4 (59-TCC TCC GCT TAT TGA TAT GC-39; White et al. 1990) and ITS5HP (59-GGA AGG AGA AGT CGT AAC AAG G-39; Hershkovitz and Zimmer 1996); these primers were also used for those ampli!cations done by the senior author. Standard PCR conditions were used, although Ready-togo PCR beads (Amersham Pharmacia Biotech Inc.) were employed for a few taxa that were dif!cult to amplify. PCR products were cleaned using PEG precipitation and ethanol cleaning (Morgan and Soltis 1993). Cycle sequencing used ABI Big Dye chemistry (Applied Biosystems, Foster City, CA), and was done in both directions using the same primers as for ampli!cation. Additional sequencing primers were used by WJH, including ITS2 (59-CGT AGC TAC TTC TTG CAT CG-39; White et al. 1990), ITS3B (59-GCA TCG ATG AAG AAC GTA GC-39; White et al. 1990), and C5.8S (59-TGC GTT CAA AGA CTC GAT-39; Suh et al. 1993). ITS sequences for Lopezia lopezioides, L. racemosa, and L. langmaniae were provided by S. O’Kane (Univ. Northern Iowa), and the sequences for Chamerion angustifolium, all Epilobium species, and Clarkia bottae were previously published by Baum et al. (1994) (see GenBank accession numbers in Table 1). TRNL-TRNF. Ampli!cation of the trnL intron, trnL 39 exon, and trnL-trnF intergenic spacer used primers ‘‘c’’ (59-CGA AAT CGG TAG ACG CTA CG-39) and ‘‘f’’ (59-ATT TGA ACT GGT GAC ACG AG-39) of Taberlet et al. (1991). PCR products were cleaned as described above. Cycle sequencing used ABI Big Dye chemistry, and was done in both directions using the same primers as for ampli!cation. A few taxa have sequences with long repeats, resulting in incomplete cycle sequence products. For these taxa, cycle sequencing was conducted with additional internal primers (d: 59GGG GAT AGA GGG ACT TGA AC-39, e: 59-GGT TCA AGT CCC TCT ATC CC-39; Taberlet et al. 1991). RPS16. Ampli!cation of the rps16 group II intron used the following primers adapted from Oxelman et al. (1997) and Popp and Oxelman (2001): forward primer P1840 (59-GTG GTA AAA AGC AAC GCG CGA CTT-39; similar to rpsF) and reverse primer P1839 (59-TCG GGA TCG CAC ATC AAT TGC AAC-39; similar to rpsR2). PCR products were cleaned as previously described. Sequencing used ABI Big Dye chemistry, and was done in both directions using the same primers as for ampli!cation. Due to the same cycle sequencing problem with long repeats mentioned above, two additional internal primers were used in sequencing some taxa: forward primer P1895 (59-GTG TAT CGT GCG GGA A-39) and reverse primer P1896 (59-GTA TTC TCA TAA CTC A-39). Cycle sequence products for all regions were precipitated and 2004] 149 LEVIN ET AL.: RELATIONSHIPS IN ONAGRACEAE TABLE 1. Taxa, vouchers, localities, and Genbank accession numbers for all sequences included in this study (*Megacorax is currently not placed in any tribe; best af!nity with Lopezieae). All tribes except Epilobieae and Onagreae contain a single genus; for these tribes, listed are the total number of species in that tribe and the number of sections (if relevant) currently circumscribed for that tribe’s genus. For tribes Epilobieae and Onagreae, total number of species per genus and sections per genus are indicated, as are total number of species per section [except Chamerion sect. Rosmarinifolium (4 spp.) and Gaura sect. Campogaura (1 sp.)]. Sectional information and total species numbers are based on Raven (1969); Raven and Gregory (1972); Tobe et al. (1987); Baum et al. (1994); O’Kane and Schaal (1998); Levin et al. (2003); and Wagner et al. (in mss.).