Systematic Botany (2014), 39(1): pp. 205–215 © Copyright 2014 by the American Society of Plant Taxonomists DOI 10.1600/036364414X678215 Date of publication 02/05/2014 Revisiting Luffa (Cucurbitaceae) 25 Years After C. Heiser: Species Boundaries and Application of Names Tested with Plastid and Nuclear DNA Sequences Natalia Filipowicz,1,4 Hanno Schaefer,2 and Susanne S. Renner3 1Medical University of Gdansk, Department of Biology and Pharmaceutical Botany, Hallera 107, 80-416 Gdansk, Poland. 2Plant Biodiversity Research, Technische Universita¨tMu¨ nchen, 85354 Freising, Germany. 3Systematic Botany and Mycology, University of Munich (LMU), Menzinger Strasse 67, 80638 Munich, Germany. 4Author for correspondence ([email protected]) Communicating Editor: Martin Wojciechowski Abstract—The circumscription of the five to eight species of Luffa, as well as their correct names, have long been problematic. Experts on the genus, most recently C. Heiser and C. Jeffrey, have disagreed on the number of species in the New World and the application of the name L. operculata, which in turn affected the names L. quinquefida and L. sepium. Heiser used classic biosystematic methods, including experimental crossing, to infer species boundaries, but neither researcher had today’s option of using DNA sequences for this purpose. We sequenced 51 accessions of Luffa, representing the geographic range of the genus and as much as possible topotypical or type material. Phylogenies from four non-coding plastid regions and the nuclear ribosomal DNA spacer region show that eight clades of specimens have geographic- morphological coherence. Heiser’s view that Luffa has three species in the New World is supported, and there are four species in tropical and subtropical Asia. Australia has an endemic species, differing from the Indian species with which it had long been lumped. Our vouchered ITS and plastid sequences from throughout species’ ranges are available in GenBank and can serve to identify Luffa material similar to standard DNA barcoding regions. We also provide new arguments for Heiser’s application of the name L. operculata to a South American species, countering Jeffrey’s arguments in favor of its use for a Central American species. Keywords—Australian endemic, barcoding, New World Luffa, nomenclature, species circumscription. Luffa is a genus of five to eight species of climbers distrib- living plants. They produced a Wagner tree of morphological uted in subtropical and tropical Southeast Asia, Australia, traits and performed parsimony analyses of either 21 or Polynesia, Africa and the New World (Jeffrey 1962, 1992; 26 characters, including sexual system (one species of Luffa Heiser and Schilling 1988, 1990; Heiser et al. 1988). Together is dioecious, the others monoecious), flowering times, mor- with 11 other genera it belongs to the Sicyoeae clade (Kocyan phology (including pollen), and chemical characters (Schilling et al. 2007; Schaefer et al. 2009) in the gourd family and Heiser 1981; Heiser and Schilling 1988, 1990; Heiser et al. (Cucurbitaceae). Two species are economically important, 1988). The material from their cultivation experiments was and it was probably this aspect that interested Charles carefully documented with herbarium vouchers deposited Heiser, a North American botanist focusing on processes of in the herbarium of the Indiana University in Bloomington. domestication (Pickersgill and Anderson 2011). The dried The results led to the recognition of three species in the fibrous fruits (Fig. 1F) serve as sponges and are sometimes New World: L. quinquefida occurring from Mexico (Gulf of described as vegetable sponge, luffa sponge, or dishrag California) to Nicaragua, L. operculata from Panama to south- gourd (Marr el al. 2005a). They are imported to Europe and ern Brazil, and L. astorii in coastal Venezuela, Ecuador, and North America from Asia and South America, where they are Peru. This contradicted earlier treatments that had accepted produced at varying scales, primarily from smooth loofah, just one variable species in that region or at most two (Jeffrey L. aegyptiaca Mill. (= L. cylindrica (L.) M. J. Roem.; Nicolson 1962, 1992). et al. 1988). The fruit matrix is also used to make tissue for The cladogram Heiser and Schilling (1990: Fig. 3) obtained engine filters, steel helmet linings, insulation, and for door with their 26 traits, including four flavonoid characters, and bath mats (Jansen et al. 1993), and lately as a carrier for partly matched an UPGMA dendrogram from plastid microbial cell immobilization in bioreactors (Phisalaphong microsatellites (Chung et al. 2003: Fig. 3), although the latter et al. 2007). Especially interesting is the mixing of loofah lacked L. echinata Roxb. and L. graveolens Roxb., and neither fibers with recycled plastic to produce strong, lightweight study assessed statistical support. Following Heiser’s work panels for furniture and low cost houses in Paraguay (Jeffrey on New World loofahs, Marr and colleagues focused on 2008). In India, tropical China and Southeast Asia, the imma- documenting the geographic range and uses of L. acutangula ture fruits of L. aegyptiaca Mill. and L. acutangula (L.) Roxb. and L. aegyptiaca in China, India, Laos, and Nepal (Marr et al. (Fig. 1A) are eaten as vegetables, and extracts of the flowers, 2005a, 2005b). Linnaeus originally described L. acutangula as seeds, roots and leaves are used in ethno-medicine (Marr “habitat in Tataria, China,” but subsequent collecting et al. 2005a, 2005b). Because of its high fruit yield, a form of revealed the presence of the species (Fig. 1B) also in northern L. acutangula with bisexual in addition to unisexual flowers India. Further distinct populations occur in Yemen, where (self-compatible like all Cucurbitaceae) is cultivated in the they have been interpreted as feral introduced L. acutangula Indian state Bihar and the Bengal region near the Nepalese (Heiser and Schilling 1988), and in Australia (Fig. 1C), where border; it has been formally described as L. hermaphrodita they were described as L. cylindrica var. leiocarpa Naudin Singh & Bhandari (Marr et al. 2005b). (= L. aegyptiaca var. leiocarpa (Naudin) Heiser & Schilling, Over a period of about ten years, C. Heiser, E. Schilling, a synonym of L. sylvestris Miquel). and the Indian geneticist B. Dutt collaborated on Luffa, using Besides disagreeing on the number of Luffa species in the biosystematics approaches such as experimental crossing to New World, Jeffrey (1962; 1992) and Heiser et al. (1988) also infer reproductive barriers, chromosome counts, and mea- disagreed on the application of the name L. operculata. surements of vegetative and reproductive characters from Depending on the identity of its nomenclatural type, 205 206 SYSTEMATIC BOTANY [Volume 39 Fig. 1. Fruits and flowers of Luffa. The scale bar corresponds to 1 cm. A. Luffa acutangula, India, Rajasthan. B. Luffa acutangula var. amara, mature dehiscent fruit, India, Rajasthan. C. Luffa aegyptiaca immature fruit, Australia, Northern Territory. D. Luffa graveolens immature fruit, India, Uttar Pradesh. E. Luffa astorii immature fruit, Peru, Lambayeque. F. Luffa aegyptiaca dried fruit with the fibers that are used as a vegetable sponge, Africa, Tanzania G. Luffa operculata dried dehiscent fruit, Colombia, Rio Magdalana. H. Luffa astorii flower, Peru, Lambayeque. I. Luffa saccata male flower, Australia, Northern Territory. J. Luffa echinata male flower, Botanical Garden, Germany, Munich. K. Luffa quinquefida male flower. L. Luffa quinquefida leaf and immature fruit, both from Mexico, Sonora. M. Luffa astorii leaf, Peru, Lambayeque. Photographs by: T. Andres (G), T. R. Van Devender (K, L), N. Filipowicz (A, B), N. Holstein (F, J), H. Schaefer (C, I), A. Pandey (D), Maximilian Weigend (E, H, M). L. operculata would be an older name for L. quinquefida (which and introns, and the nuclear ribosomal DNA internal tran- would thus become a synonym), leaving L. sepium (G.F.W. scribed spacer region (ITS) from 51 accessions of Luffa selected Mey.) C. Jeffrey as the oldest name for the South American to represent the geographic range of the genus and as much as material. We take up these nomenclatural issues in the possible also topotypical or type material. Our goals were to Discussion, after resolving the main question how many spe- investigate (i) natural species boundaries in Luffa, (ii) whether cies to recognize in the Americas. To answer this question we New World Luffa have a single progenitor, and (iii) whether generated DNA sequences from non-coding plastid spacers L. graveolens indeed ranges from India to Australia, 2014] FILIPOWICZ ET AL.: PHYLOGENETICS OF LUFFA 207 L. acutangula from India to Yemen, and L. echinata from India Trichosanthes as outgroups and has 3,455 aligned nucleotides to Cameroon and Mali as traditionally assumed. (covering the trnL intron and the trnL-F, rpl20-rps12, ndhF- rpl32, and psbA-trnH intergenic spacers). The nuclear ITS Materials and Methods matrix consists of 48 Luffa accession, the same outgroups, and 684 aligned nucleotides. There was no statistically Taxon Sampling—The monophyly of Luffa has been supported in two supported (>75% ML bootstrap support) topological incon- family-wide analyses that included three and six of its species (Kocyan gruence between trees from the plastid and nuclear data et al. 2007; Schaefer et al. 2009) as well as in an analysis of Trichosanthes and relatives (de Boer et al. 2012: five species included). Based on these (trees shown in Figs. S1 and S2), and the matrices were studies, we selected two species from the genus Trichosanthes as an therefore combined. A tree from the combined data (Fig. 2) outgroup. Specimens from A, AAU, BRI, C, CANB, DUH, FTG, GH, shows L. acutangula as sister to the remaining species, IND, K, L, M, MICH, MO, U, and US (acronyms follow Thiers 2013) were a grade of Southeast Asian and Australasian species examined, and field observations were made by the second author in Australia, Brazil, Cambodia, China, Nigeria, Peru, Sierra Leone, and (L. aegyptiaca, L. echinata, L. graveolens, and L. saccata) and a Tanzania. In 2010, the first author visited the Indian herbaria BLAT, JCB, clade of all New World accessions.