A Nuclear Rdna Phylogeny of Smilax (Smilacaceae) Kenneth M
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Aliso: A Journal of Systematic and Evolutionary Botany Volume 22 | Issue 1 Article 47 2006 A Nuclear rDNA Phylogeny of Smilax (Smilacaceae) Kenneth M. Cameron New York Botanical Garden Chengxin Fu Zhejiang University Follow this and additional works at: http://scholarship.claremont.edu/aliso Part of the Botany Commons Recommended Citation Cameron, Kenneth M. and Fu, Chengxin (2006) "A Nuclear rDNA Phylogeny of Smilax (Smilacaceae)," Aliso: A Journal of Systematic and Evolutionary Botany: Vol. 22: Iss. 1, Article 47. Available at: http://scholarship.claremont.edu/aliso/vol22/iss1/47 Aliso 22, pp. 598-605 © 2006, Rancho Santa Ana Botanic Garden A NUCLEAR rDNA PHYLOGENY OF SMILAX (SMILACACEAE) KENNETH M. CAMERON 1 AND CHENGXIN Fu2.3 1The Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, The New York Botanical Garden, Bronx, New York 10458, USA ([email protected]); 2Laboratory of Systematic and Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University, Hangzhou 3/0029, China 3 Corresponding author ([email protected]) ABSTRACT Smilacaceae are a taxonomically confused, cosmopolitan family of Liliales characterized by climb ing habit, reticulate leaf venation, paired petiolar tendrils, unisexual flowers, and superior ovaries. Deviations from this generalized morphology have led to the division of Smilacaceae into at least seven different genera and five sections within the large genus Smilax. In particular, taxa with connate tepals (Heterosmilax), more than six stamens (Pleiosmilax, Oligosmilax), or herbaceous habit (He mexia) have been variously classified. Using DNA sequences of 96 taxa from the nuclear rDNA ITS gene region, parsimony analyses provide moderate resolution, but generally poor bootstrap support for phylogenetic relationships in the family. Rhipogonum and Lapageria are closely related to Smilax, but may be better classified in separate families. Within Smilax few of the sections are monophyletic, but three major lineages are evident. The first is a primarily Old World clade that contains species of Heterosmilax, the erect, woody species of Smilax from Asia, and the herbaceous Smilax species. Within this clade there is evidence that the S. herbacea complex represents a single eastward dispersal from Asia to North America. A primarily New World clade (or paraphyletic grade in some trees) is also present in the ITS cladogram. From within this group there is evidence of a single westward dispersal from North America to Asia. The third lineage is represented by S. aspera, which is the sole member of Smilax section Smilax, and is sister to the entire genus. Its position in the cladogram is intriguing and may help to shed light on the family's greater evolutionary and biogeographic history. Key words: DNA, evolution, Heterosmilax, Liliales, molecular, Philesiaceae, Rhipogonum, Smilaca ceae, Smilax, systematics. INTRODUCTION never completed. Recently published treatments of Smilax for specific geographic areas, however, have helped to sort Smilax L. (Smilacaceae) is well known to both temperate out some significant nomenclatural problems, and suggest and tropical field botanists because it is one of the most abundant and easily recognized climbing plants in many eco that the size estimate of the genus may be inflated. For ex systems of the Old and New Worlds. Within the Neotropics, ample, Andreata's (1995) revision of the Brazilian species Smilacaceae are ranked among the top 25 most diverse plant of Smilax reduced the total number from ca. 50 to 26 species. families with climbing species, and closely tied with Amar The treatment of Smilax for the Flora of China (Chen and yllidaceae s.l. as the second most diverse group of climbing Koyama 2000) considered a total of 79 species (39 of which monocots after Araceae (Gentry 1991). Morphologically, are endemic to that country), and the Flora of North America however, Smilax is unusual among monocots, and is fre North of Mexico (Holmes 2002) treated 20 species, eight of quently confused as a dicot by students of plant taxonomy which are considered by some taxonomists to be synonyms because of its "woody" stems and pronounced reticulate leaf of S. herbacea. Australia and Africa possess only two spe venation. In general, members of the genus are characterized cies each, whereas Europe has only one. There are probably as climbing, woody vines with prickles, alternate leaves ex at least 15 species in Mexico (Morton 1962), several of hibiting reticulate venation, and paired stipular tendrils. The which extend into the United States, Central America, and plants are dioecious, and produce inconspicuous flowers the West Indies, bringing the total number worldwide to no with either six stamens (male) or three stigmas (female); the more than 150. This estimate excludes a handful of species inflorescence is typically an umbel or panicle of umbels, and endemic to the West Indies, Central, and South America (but the fruit is a berry. not present in Mexico or Brazil), Southeast Asia (absent in Estimates for the size of the genus are given as ca. 350 China and Australia), and the Pacific Islands. Nevertheless, species found worldwide (Takhtajan 1997). Species deter even if one estimates another 50 species for these three ar mination, however, is extremely difficult because plants are eas, the size of the genus is probably not much larger than dioecious, flower infrequently, and show considerable phe ca. 200 species. notypic variation within populations and even among leaves Controversy not only surrounds the delimitation of Smilax of the same individual plant. As a result, the taxonomy of species, but also of the entire Smilacaceae (formerly part of Smilax is highly problematic, and there has been no mono Liliaceae s.l.) and its constituent genera as well. Under the graph published for the entire genus. Koyama ( 1960) initi classification system of Takhtajan ( 1997), for example, three ated an attempt at such, in which he proposed that the genus genera: Smilax, Heterosmilax Kunth, and Pseudosmilax be divided into six sections (see Discussion), but this was Hayata are treated as members of Smilacaceae, which is one VOLUME 22 Nuclear rDNA Phylogeny of Smilax 599 of four families in the order Smilacales. The other families dition of 10% DMSO. Primers designated "ny43": are Philesiaceae (Philesia Comm. ex Judd, Lapageria Ruiz TATGCTTAAAYTCAGCGGGT and "ny47": AACAAGG & Pav.); Rhipogonaceae (Rhipogonum Forst.); and Luzur TTTCCGTAGGTGA were commonly used to both amplify iagaceae (Luzuriaga Ruiz & Pav., Behnia Didr., Drymophila and sequence the ITS region. An annealing temperature of R. Br., Eustrephus R. Br., and Geitonoplesium A. Cunn.). 55°C for 30 cycles was discovered to produce the greatest Cladistic analyses of molecular (Chase et al. 2000) and com quality amplification product. In all cases, resulting PCR bined molecular with morphological data (Rudall et al. 2000) products were purified using QIAquick® spin columns have shown that Luzuriagaceae and other climbing members (QIAGEN Inc., Valencia, California, USA) according to the of Liliales with .reticulate leaf venation (e.g., Petermannia manufacturer's protocols. Cycle sequencing reactions were Klotzsch) are only distantly related to Smilacaceae. On the performed using a combination of purified PCR template, other hand, these data show that Lapageria, Philesia, and primer, and BigDye Reaction mix (Applied Biosystems, Inc., Rhipogonum may be sufficiently close enough to Smilax to Foster City, California, USA) for 20 cycles. These reactions warrant their recognition within a more broadly defined Smi resulted in complete forward and reverse strands of the lacaceae, rather than treated as separate families (Rudall et genes for nearly all sequences. Centri-Sep® sephadex col al. 2000). The exact relationships among all these taxa are umns (Princeton Separations, Inc., Adelphia, New Jersey, still unclear, but studies with increased data and taxon sam USA) were used according to the manufacturer's instructions pling (see other papers in this volume) are beginning to sup to remove excess dye terminators and primer from the cycle port relationships in which these three lineages are not sequencing products. These were subsequently dehydrated, monophyletic. resuspended in a mixture of formamide and loading dye, and The objectives of this study were to produce a preliminary loaded onto a 5% denaturing polyacrylamide gel. Samples molecular phylogenetic analysis of Smilacaceae, so that a were analyzed on an Applied Biosystems ABI 377XL au number of fundamental questions related to the systematics tomated DNA sequencer, and resulting electropherograms of the family could be addressed. These include: (1) what is were edited using Sequencher vers. 3.0 software (Gene the natural generic circumscription of Smilacaceae? (i.e., are Codes Corporation, Ann Arbor, Michigan, USA). The com Smilax, Heterosmilax, and Pleiosmilax monophyletic gen plete matrix was initially aligned using CLUSTAL_X era?); (2) are Koyama's (1960) sections monophyletic or (Thompson et al. 1997) with default parameters, but then should the family be subdivided otherwise?; and (3) are adjusted manually using MacClade vers. 4.0 (Maddison and there clear patterns of biogeography within the family? We Maddison 2000). consider the study to be preliminary because it is based on a limited taxon sample for only a single nuclear gene region Phylogenetic Analyses (ITS), but we are actively working to add additional taxa The ITS