American Journal of Botany 97(1): 156–173. 2010. M OLECULAR SYSTEMATICS OF THE NEOTROPICAL GENUS PSIGURIA (CUCURBITACEAE): IMPLICATIONS FOR PHYLOGENY AND SPECIES IDENTIFICATION 1 P. Roxanne Steele 2,3,4 , Lauren M. Friar 3 , Lawrence E. Gilbert2 , and Robert K. Jansen 2,3 2 Section of Integrative Biology, The University of Texas at Austin, 1 University Station C0930, Austin, Texas 78712 USA; and 3 Institute of Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station C0930, Austin, Texas 78712 USA Varying morphological features in many groups of tropical vines confound identifi cation, requiring molecular tools for distin- guishing species. Confusion is amplifi ed in Psiguria , a small genus found in Central and South America and the Caribbean, be- cause male and female fl owers of these monoecious plants are widely separated by time and position on a branch. We present the fi rst phylogeny of Psiguria utilizing a combination of eight chloroplast intergenic spacers, the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA repeat, and the intron of the low-copy nuclear gene serine/threonine phosphatase, for a total aligned length of 9456 base pairs. Analyses include multiple accessions of all species in the genus. The data support the mono- phyly of Psiguria and elucidate several species boundaries. Also presented are Psiguria -specifi c DNA barcodes, which include the chloroplast regions: ndhC-trnV , rps16-trnQ , rpoB-trnC , ndhF-rpl32 , and psbZ-trnM . For the fi rst time, systematists, ecologists, and evolutionary biologists will have the tools to confi dently identify species of Psiguria with DNA barcodes that may be useful in other genera of Cucurbitaceae. Key words: angiosperms; Caribbean; chloroplast; DNA barcoding; Gurania ; Guraniinae; Helmontia ; ITS; low-copy nuclear; phylogenetics. Vines and lianas are important components of tropical forest species identifi cations are confounded by several morphologi- ecosystems. They are found in both primary and secondary for- cal variants. ests, at all levels of vegetation from forest fl oor to canopy, and Psiguria is a neotropical genus of monoecious, perennial they are most commonly seen without fl owers. Adaptations vines with a geographic distribution extending from southern for defense, camoufl age, pollinator attraction, or increased Mexico to Paraguay and into the Caribbean islands. The genus exposure to light include leaf mimicry, changes in leaf shape, has been the focus of several ecological and evolutionary stud- texture, or size over the life span of an individual, as well ies on the sexual system and the interactions with various in- as variation in fl ower shape, size, and color. Such infraspecifi c sects in the community, but there has never been a phylogeny transformations make identifi cation based on morphology very proposed for the genus. Psiguria plays a central role in a suite challenging, if not impossible. One solution is to use molecular of plant – animal interactions, including host to several species data to defi ne species boundaries. An example is presented of Blepharoneura Loew fruit fl ies ( Condon, 1984 ; Condon and here with the genus Psiguria Arn. (Cucurbitaceae), whose Gilbert, 1990 ; Condon and Norrbom, 1994 ; Condon and Steck, 1997 ; Condon et al., 2008 ). Additionally, most species of Heli- conius Kluk butterfl ies rely heavily on nutrients obtained from 1 Manuscript received 2 July 2009; revision accepted 1 October 2009. The authors thank B. B. Simpson, D. R. Hansen, C. R. Linder, and two pollen of Psiguria fl owers. Unlike other lepidopterans that ob- anonymous reviewers for valuable comments on the manuscript. The tain nitrogenous compounds necessary for egg production as authors acknowledge the following research grants: NSF Doctoral larvae, Heliconius butterfl ies assimilate amino acids and pro- Dissertation Improvement Grant DEB 0808294, the Plant Biology Program teins from pollen gathered by adults, principally from Psiguria at The University of Texas at Austin, the American Society of Plant Biology, and a few members of the sister genus, Gurania Cogn. ( Gilbert, and the Botanical Society of America. They also thank MO, NY, F, GH, 1972, 1975, 1977 ; Boggs et al., 1981 ). Although most of the and TEX herbaria for approving the use of leaf material on loans for DNA Heliconius -collected pollen is destroyed by the butterfl ies, a studies; M. Condon (Cornell College), S. Swensen (Ithaca College), R. suffi cient portion is transferred to female fl owers (presumably Abbott (University of Florida), T. S. Quedensley (University of Texas at Austin), and H. Schaefer (University of Munich) for providing leaf mistaken for a pollen source [ Gilbert, 1975 ]), fulfi lling the but- material; R. Fern á ndez and P. Protti (Costa Rica), F. Axelrod (University of terfl y ’ s role as Psiguria ’ s primary pollen vector ( Condon, 1984 ; Puerto Rico-R í o Piedras), T. Clase (Jard í n Bot á nico Nacional Dominican Murawski and Gilbert, 1986 ). The historical relationship be- Republic), O. Plata (Herbario Nacional de Bolivia), and K. Meza (Per ú ) for tween these two evolving groups of organisms may have infl u- fi eld assistance and collecting plants; M. Timan á (Pontifi cia Universidad enced the reproductive strategy of the butterfl ies and the Cat ó lica del Per ú ) for help with plant-collecting permits; and fi nally UT separation of genera in the plant subtribe ( Gilbert, 1993 ). Austin ’ s Brackenridge Field Laboratory for providing facilities and staff Psiguria species occur in low densities in neotropical rain- assistance that maintained living collections used in the work. forests, commonly growing over shrubs and trees along edges 4 Author for correspondence (e-mail: [email protected] ); present address: University of Missouri-Columbia, 1201 Rollins St.-Bond LSC of gaps and streams ( Condon, 1984 ; Murawski and Gilbert, 311, Columbia, Missouri 65211 USA 1986 ; Condon and Gilbert, 1990 ). The genus is generally char- acterized by large, glabrous, palmately 3- to 5-lobed or com- doi:10.3732/ajb.0900192 pound (3- to 5-foliolate), leathery leaves. However, these leaf 156 January 2010] 157 characteristics vary over the life of an individual. For example, triphylla and P. warscewiczii have been used most often (to- fi rst leaves of P. pedata (L.) R. A. Howard, found in the Greater gether making up approximately 72% of annotated specimens). Antilles, are simple, shallowly trilobed, and relatively membra- It has not been clear whether this large percentage is due to their nous. Subsequent, or even adjacent, leaves may be more deeply having broader geographic distributions, being more common, lobed or completely separated into two or three leafl ets. Leaves or being used as catchall names. The remainder of the names on mature P. pedata are coriaceous and show further division, has been used for less than 8% of herbarium specimens, and typically pedate — three distinct leafl ets with side leafl ets that some names have not been used on any specimen other than the are further divided into two subleafl ets. Psiguria are serially type. The question has remained as to whether any of the other monoecious; i.e., male fl owers emerge before the plants switch 11 names listed in IPNI pertain to species distinct from those sex to produce female fl owers ( Gilbert, 1980 ; Condon, 1984 ). six. Flowers develop in clusters on long, pedunculate, indetermi- Past taxonomic treatments of Psiguria listed two species en- nate racemes or spikes. Both male and female fl owers are sal- demic to Caribbean islands: P. trifoliata (L.) Alain and P. pe- verform with tubular calyx and rotate petals; a similarity that, data ( Wunderlin, 1978 ; Howard, 1973 ). If it were confi rmed along with the stamen-like structure of the pistil, aids in “ train- that these are distinct species, many interesting biogeographical ing ” pollinator butterfl ies to visit both sexes. Sepals of both questions could be asked. These include where the genus origi- male and female fl owers are green and short (relative to the nated, how many times and in which direction it moved be- sister genus Gurania ). Petals are typically red with yellow tween Latin America and the islands, and whether it migrated bases, pink, or orange; however, this is another characteristic, by dispersal or vicariance. The divergence of the subtribe Gu- along with fl ower diameter, that can change drastically over the raniinae from sister genus Wilbrandia is estimated to be 11 ± 3 life of an individual. The fi rst male fl ower of P. ternata (M. J. million years ago (mya), and the split between Psiguria and Roem.) C. Jeffrey from Bolivia, for example, is typically light Gurania is estimated to be 6 ± 3 mya ( Schaefer et al., 2009 ). pink or salmon and very large and showy (12 – 13 cm in diame- Because of the much greater age of the Caribbean islands (45 – ter). However, subsequent fl owers are progressively smaller 46 my) ( Inturralde-Vinent and MacPhee, 1999 ; Graham, 2003 ), (down to 0.5 – 1.0 cm in diameter) with much darker, almost red, this timing suggests that dispersal played the major role in the petals. Additionally, the calyx tubes of P. ternata fl owers distribution pattern of Psiguria , and the only question is from change from fl ask-shaped to cylindrical in consecutive fl owers where dispersal occurred. in an infl orescence. Furthermore, plants are not always in Psiguria is placed in the tribe Coniandreae, subtribe Guranii- bloom, and sterile collections can make up approximately 15% nae along with only two other genera, Gurania and Helmontia of herbarium sheets. For these reasons and the fact that incom- Cogn. ( Jeffrey, 2005 ). Jeffrey (1978) contended that the integ- plete individuals of Psiguria are usually found on a given her- rity and distinctness of the subtribe is supported by pollen and barium sheet, questions have been raised as to how many fl ower morphology.