Evolutionary History and Taxonomy of the <Em>Cuscuta Umbellata</Em
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Wilfrid Laurier University Wilfrid Laurier University Scholars Commons @ Laurier Biology Faculty Publications Biology 10-2010 Evolutionary History and Taxonomy of the Cuscuta umbellata complex (Convolvulaceae): Evidence of Extensive Hybridization from Discordant Nuclear and Plastid Phylogenies Mihai Costea Wilfrid Laurier University, [email protected] Saša Stefanović University of Toronto Follow this and additional works at: https://scholars.wlu.ca/biol_faculty Part of the Biology Commons Recommended Citation Costea, Mihai and Stefanović, Saša, "Evolutionary History and Taxonomy of the Cuscuta umbellata complex (Convolvulaceae): Evidence of Extensive Hybridization from Discordant Nuclear and Plastid Phylogenies" (2010). Biology Faculty Publications. 73. https://scholars.wlu.ca/biol_faculty/73 This Article is brought to you for free and open access by the Biology at Scholars Commons @ Laurier. It has been accepted for inclusion in Biology Faculty Publications by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. Costea & Stefanović • Systematics of the Cuscuta umbellata complex TAXON 13 October 2010: 18 pp. in C. sect. Eugrammica subsect. Leptanthae; Yuncker, 1932). selected two species from the C. gracillima clade as outgroup In addition, C. lilliputana, a new species that is nested within (Appendix 1). this group, has been recently described (Stefanović & Costea, Morphology and micromorphology. — Descriptions 2008). In contrast, C. gracillima, C. sidarum (= C. saccharata), are based on herbarium material (Appendix 2). We examined C. deltoidea (= C. serruloba) do not belong to the C. umbellata the basic morphology of rehydrated flowers and capsules under clade as previously thought by Yuncker (1932, 1965), but form a Nikon SMZ1500 stereomicroscope equipped with a PaxCam a separate group (Stefanović & al., 2007; Costea & al., 2008). Arc digital camera and Pax-it 7.0 software (MIS Inc., Villa Most of the members of the C. umbellata complex are Park, Illinois, U.S.A.). Micromorphological measurements and known to occur in southwestern U.S.A. and Mexico, but three pictures were taken at 10 kV using a Hitachi SU1510 scanning species (C. umbellata, C. acuta, C. membranacea) grow in electron microscope. Herbarium samples (Appendix 2) were South America, and another one (C. hyalina) can be found coated with 30 nm gold using an Emitech K 550 sputter coater. in India, Pakistan and Africa. Recent phylogenetic studies The terminology regarding the micromorphology of flowers, have clearly indicated that the boundaries of some species seeds capsules, and pollen follows Costea & al. (2006a). Hun- (e.g., C. umbellata and C. desmouliniana) must be reconsid- dreds of photographs that illustrate details of the floral parts, ered (Stefanović & al., 2007; Stefanović & Costea, 2008). pollen and fruit morphology for all the species (including the These studies have also revealed discordant nuclear and plas- types) are available on the Digital Atlas of Cuscuta (Costea, tid phylogenies which suggested that at least two of the spe- 2007-onwards). The geographical distribution of taxa, phenol- cies, C. liliputana and C. desmouliniana, likely have a hybrid ogy, elevation and host ranges are based on observation made origin (Stefanović & Costea, 2008). These cases of reticula- from herbarium specimens. tion, together with the disjunct distribution of the taxa, have Molecular techniques and alignments. — To infer shown that the evolutionary history of the C. umbellata clade is phylogenetic relationships among species of the C. umbellata much more complex than previously thought. In view of these complex, sequences for the internal transcribed spacer (ITS) interesting preliminary findings based on relatively limited region of nuclear ribosomal DNA (nrDNA) as well as trnL-F sampling within the C. umbellata complex, we have studied intron/spacer region from the plastid genome (ptDNA) were numerous herbarium specimens that have accumulated world- obtained. In addition to the DNA samples used in previous wide since Yuncker’s monographs (Yuncker, 1932, 1965). The studies (Stefanović & al., 2007; Stefanović & Costea, 2008), aims of this study are to (1) recover the evolutionary history total genomic DNA was isolated from newly obtained speci- of the C. umbellata complex based on nuclear ITS and plastid mens as well. DNA extractions, polymerase chain reaction trnL-F sequences and further investigate the extent of reticu- (PCR) reagents and conditions, amplicon purifications, clon- late evolution known to occur in this group; (2) investigate the ing, as well as sequencing procedures followed the protocols morphology and micromorphology of the taxa involved; and detailed in Stefanović & al. (2007). Initial sequencing of nu- (3) provide a new classification of the C. umbellata complex clear and plastid amplicons was done directly. However, in the with the description of a new species, C. legitima. cases where significant polymorphism was detected, the PCR product was cloned and multiple clones per individual were sequenced. Sequences generated in this study are deposited MATERIALS AND METHODS in GenBank (accession numbers HM748863–HM748905; see Appendix 1). Sequences were aligned manually using Se-Al Taxon sampling. — We have studied specimens from v.2.0a11 (Rambaut, 2002). over 100 herbaria in connection with the upcoming treat- Phylogenetic analyses. — Phylogenetic analyses were ments of Cuscuta for Flora of North America Project, Flora conducted under parsimony and Bayesian optimality criteria; Neotropica, and a future monograph of the genus. A subset summary descriptions of these analyses, for individual as well of 34 accessions, representing eleven ingroup species of the as combined datasets, are provided in Table 1. C. umbellata complex, was used for the molecular phylogenetic Under the parsimony criterion, nucleotide characters were analyses (Appendix 1). Efforts were made to sample multiple treated as unordered and all changes were equally weighted. accessions, particularly for those species spanning large bio- Depending on the number of operational taxonomic units geographical ranges (e.g., C. hyalina) and/or those with vari- (OTUs) included, different search strategies were employed for able morphology (e.g., C. umbellata). As a result, two to nine the different matrices, using PAUP* v.4.0b10 (Swofford, 2002). individuals are included in the molecular analyses for all but For the ITS matrix, searches for most parsimonious (MP) trees one rare species, C. membranacea, known only from its type were performed using a two-stage strategy. First, the analyses locality. In addition, several taxa (C. lacerata, C. fasciculata, involved 10,000 replicates with stepwise random taxon ad- C. hyalina var. nubiana, C. umbellata var. dubia) are known dition, tree bisection-reconnection (TBR) branch swapping only from their type specimens (Yuncker, 1921, 1932), and saving no more than 10 trees per replicate, and MULTREES C. umbellata var. desertorum from two historical collections off. The second round of analyses was performed on all trees (Engelmann, 1859; Yuncker, 1932). Hence, these taxa could not in memory with the same settings except with MULTREES be sampled for the molecular analyses. Based on our previous, on. Both stages were conducted to completion or until one mil- more inclusive phylogenetic analyses of Cuscuta subg. Gram- lion trees were found. For the trnL-F matrix, a full heuristic mica (Stefanović & al., 2007; Stefanović & Costea, 2008), we search was performed, involving 1000 replicates with stepwise 2 TAXON 13 October 2010: 18 pp. Costea & Stefanović • Systematics of the Cuscuta umbellata complex random taxon addition, TBR branch swapping, and MUL- sampled every 100 generations. If needed, the run lengths were TREES option on. Given the relatively moderate number of increased until the standard deviation of split frequencies be- terminal units included in the combined dataset, we performed tween two runs was well below 0.01 (see Table 1 for details on a Branch-and-Bound search, therefore ensuring recovery of all MrBayes settings and number of generations used for each of MP trees. In all three cases, support for clades was inferred three analyses). Convergence of the chains was determined by by nonparametric bootstrapping (Felsenstein, 1985), using 500 examining the plot of all parameter values and the –lnL against heuristic bootstrap replicates, each with 20 random addition generation using Tracer v.1.3 (Rambaut & Drummond, 2004). cycles, TBR branch swapping, and MULTREES option off Stationarity was assumed when all parameter values and –lnL (DeBry & Olmstead, 2000). Nodes receiving bootstrap (BS) had stabilized. Burn-in trees were discarded and the remaining values < 60%, 60%–75%, and >75% were considered weakly, trees and their associated parameters were saved. Because no moderately, and strongly supported, respectively. significant differences between two runs were detected (for Bayesian phylogenetic inferences were performed using each of the three separate Bayesian analyses; Table 1), the re- MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003). Model- ported topologies and posterior probabilities (PP) are based Test v.3.7 (Posada & Crandall, 1998) was used to deter mine on trees from pooled runs. Only the nodes receiving ≥0.95 PP the model of sequence evolution that best fit the data by the were