Apiaceae) Based on Phylogenetic Analyses of Nuclear (ITS) and Plastid (Rps16 Intron) DNA Sequences
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South African Journal of Botany 2004, 70(3): 407–416 Copyright © NISC Pty Ltd Printed in South Africa — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY ISSN 0254–6299 A molecular systematic investigation of Cymopterus and its allies (Apiaceae) based on phylogenetic analyses of nuclear (ITS) and plastid (rps16 intron) DNA sequences F-J Sun and SR Downie* Department of Plant Biology, University of Illinois, Urbana 61801, United States of America * Corresponding author, email: [email protected] Received 5 September 2003, accepted in revised form 7 November 2003 Considerable controversy exists with regard to the maximum likelihood methods. For those trees exhibit- proper delimitation of Cymopterus (Apiaceae subfamily ing the greatest resolution, the results revealed that the Apioideae) and its relationship to the other perennial perennial, endemic apioid genera of North America endemic umbellifers of western North America. (north of Mexico) comprise a weakly supported mono- Previous molecular systematic studies using nuclear phyletic group, with Angelica constituting its sister line- (rDNA ITS) and, for subsets of taxa, plastid (rps16 intron age. Cymopterus is confirmed as highly polyphyletic. and trnF-trnL-trnT) DNA sequences have revealed that The rps16 intron yielded substantially fewer parsimony the genus Cymopterus is polyphyletic, with its species informative characters than those of the ITS region and inextricably linked with those of Aletes, Lomatium, when analysed, separately or in combination, with ITS Musineon, Oreoxis, Pseudocymopterus, Pteryxia, data, resulted in trees of poor resolution. Only one pre- Tauschia, and several other genera of the region. viously identified species group of Cymopterus is sup- Herein, in an effort to increase resolution of relation- ported as monophyletic; this group coincides with sec- ships, we procured rps16 intron sequence data from 74 tion Phellopterus, and is recognised by its showy, accessions of Cymopterus and its allies so that ITS and basally connate bractlets. Many other genera endemic intron matrices are available for the same set of 129 to western North America appear to be highly artificial taxa. These data matrices were analysed, separately and assemblages of species that will likely be abandoned in in combination, using maximum parsimony and (or) any revised system of umbellifer classification. Introduction The genus Cymopterus Raf., the spring-parsleys, comprises Podistera S. Watson, Pseudocymopterus J.M. Coult. & some 40 herbaceous perennial taxa restricted primarily to Rose, Pteryxia (Nutt. ex Torr. & A. Gray) J.M. Coult. & Rose, xeric habitats in western North America (Kartesz 1994, and Shoshonea Evert & Constance. Collectively, these Hartman 2000). These plants are low-growing, possess plants present such a confusing intergradation of similar thickened taproots, and have ternately cleft, compound or characteristics that generic delimitation is made exceeding- dissected leaves. Their most distinctive feature, however, is ly difficult and, in the absence of mature fruits, many species their fruit, for the two lateral and usually one or more of the are essentially indistinguishable (Jones 1908, Mathias 1930, three dorsal ribs are conspicuously winged and undulating. Cronquist 1997, Downie et al. 2002). With regard to the Rarely, the dorsal ribs may be wingless and, in such cases, delimitation of Cymopterus, extremes in treatment range the fruits resemble those of Lomatium (Cronquist 1997, from the recognition of many small, generically distinct ele- Downie et al. 2002). ments (such as Aulospermum J.M. Coult. & Rose, Glehnia, Much confusion exists with regard to the proper delimita- Oreoxis, Phellopterus (Nutt. ex Torr. & A. Gray) J.M. Coult. & tion of Cymopterus and its relationship to the other indige- Rose, Pseudocymopterus, Pteryxia and Rhysopterus J.M. nous umbellifers of western North America (Jones 1908, Coult. & Rose) to multiple sections and subgroups within a Mathias 1930, Theobald et al. 1963, Evert and Constance highly variable and expanded genus (Jones 1908, Mathias 1982, Weber 1984, Gilmartin and Simmons 1987, Cronquist 1930, Mathias and Constance 1944–1945). Cronquist 1997). These genera include Aletes J.M. Coult. & Rose, (1997) included Oreoxis, Phellopterus, Pseudocymopterus Glehnia F. Schmidt ex Miq., Harbouria J.M. Coult. & Rose, and Pteryxia within a broadly circumscribed Cymopterus. Lomatium Raf., Musineon Raf., Neoparrya Mathias, Recently, we have carried out molecular systematic inves- Oreonana Jeps., Oreoxis Raf., Orogenia S. Watson, tigations of Cymopterus and its allies using nuclear (rDNA 408 Sun and Downie ITS) and, for subsets of this group, plastid (rps16 intron and American genera (Downie et al. 2002, Sun et al. 2004). We trnF-trnL-trnT) sequences (Downie et al. 2002, Sun et al. rooted all trees with Aethusa cynapium L., based on the 2004). Our results revealed that the perennial endemic api- results of previous higher-level investigations where this oid genera of North America (with the inclusion of Polytaenia species was sister taxon, or closely related, to all aforemen- DC., Taenidia (Torr. & A. Gray) Drude, Tauschia Schltdl., tioned taxa (Downie et al. 2000, 2002). Thaspium Nutt. and Zizia W.D.J. Koch) comprise a weakly The experimental methods used to obtain these intron supported monophyletic group, with Angelica and members sequence data were the same as outlined previously of the meso-American Arracacia clade constituting two of (Downie et al. 2002, and references cited therein). The DNA several possible sister lineages (Sun et al. 2004). The lack sequences were aligned manually, facilitated by their con- of resolution and poor branch support obtained in the ITS servative nature. GenBank accession numbers for each of trees precluded unambiguous hypotheses of relationship, these newly acquired sequences are presented in Table 1, but did suggest that many of these western North American and the data matrix is available upon request. genera, where resolved, are not monophyletic. Cymopterus The resulting ITS and rps16 intron data matrices were first is polyphyletic, with its species inextricably linked with those analysed using maximum parsimony (MP), with gap states of many other genera of the region. Thus, it appears that the treated as missing data. Both separate and combined analy- traditional emphasis placed on fruit characters in circum- ses were performed. MP trees were sought using the heuris- scribing genera within the group has led to artificial assem- tic search strategies of PAUP* (Swofford 1998), as blages of species and, as such, a complete reassessment of described in Downie et al. (2002). The maximum number of the generic limits of these North American Apiaceae is in MP trees was set at 20 000. Bootstrap values were calculat- order. ed from 100 000 replicate analyses using ‘fast’ stepwise- To resolve relationships among the perennial endemic addition of taxa, and only those values compatible with the genera of North American Apiaceae and to better ascertain 50% majority-rule consensus tree were recorded. The num- the phylogenetic placements of those elements that current- ber of additional steps required to force particular taxa into a ly comprise Cymopterus within the group, herein we monophyletic group was examined using the constraint obtained additional sequence data from the chloroplast option of PAUP*. To examine the extent of conflict among rps16 intron to produce parallel ITS and intron data sets for the ITS and rps16 intron data sets, the incongruence length a comprehensive sampling of 129 taxa. Previous studies difference test of Farris et al. (1995) was implemented, using have demonstrated the utility of the rps16 intron for phy- PAUP*’s partition-homogeneity test. This test was performed logeny estimation in Apiaceae (Downie and Katz-Downie with 100 partition-homogeneity test replicates, using a 1999, Lee and Downie 2000, Downie et al. 2000, 2002), and heuristic search option with simple addition of taxa, Tree- it was hoped that this region could supply the information Bisection-Reconnection (TBR) branch swapping, and required to increase resolution and branch support of the MaxTrees set at 500. resulting phylogenetic estimate. A maximum likelihood (ML) analysis of combined ITS and rps16 intron data was then performed. The program Materials and Methods Modeltest version 3.06 (Posada and Crandall 1998) was used to select an appropriate model of nucleotide substitu- DNA sequence data from the chloroplast rps16 intron were tion that best fits these combined data. These settings were procured from 74 accessions of North American Apiaceae entered into PAUP* and a heuristic search carried out using subfamily Apioideae (Table 1) and combined with previously random addition sequence and TBR branch swapping. One published intron data for 55 taxa (Downie and Katz-Downie thousand bootstrap replicate analyses were conducted 1999, Downie et al. 2002), for a final tally of 129 accessions employing neighbour-joining searches with ML distance, included in the phylogenetic analyses. ITS sequence data using the ML parameters estimated by Modeltest. were already available for this same set of species (Downie et al. 2002, Sun et al. 2004). With the exception of the nar- Results and Discussion rowly endemic and rarely collected Cymopterus mega- cephalus M.E. Jones, we sampled all 40 taxa of Cymopterus Sequence analysis listed by Kartesz (1994). We also included the recently described C. constancei (Hartman 2000), and