American Journal of Botany 93(11): 1684–1698. 2006. PHYLOGENY AND EVOLUTION OF BURMANNIACEAE (DIOSCOREALES) BASED ON NUCLEAR AND MITOCHONDRIAL DATA1 VINCENT MERCKX,2,5 PETER SCHOLS,2 HILTJE MAAS-VAN DE KAMER,3 PAUL MAAS,3 SUZY HUYSMANS,2 AND ERIK SMETS2,4 2Laboratory of Plant Systematics, K.U. Leuven, Kasteelpark Arenberg 31, BE-3001 Leuven, Belgium; 3National Herbarium of the Netherlands, Utrecht University Branch, Heidelberglaan 2, NL-3584 CS Utrecht, the Netherlands; and 4National Herbarium of the Netherlands, Leiden University Branch, P.O. Box 9514, NL-2300 RA Leiden, the Netherlands The mycoheterotrophic Burmanniaceae are one of the three families currently recognized in the order Dioscoreales. Phylogenetic inference using nucleotide sequences of the nuclear 18S rDNA region and the mitochondrial nad1 b-c intron revealed two well-supported, major lineages within the family, corresponding to the two tribes recognized in the family: Burmannieae and Thismieae. All data supported a strong relationship between Thismieae and Tacca (Dioscoreaceae) making both Burmanniaceae and Dioscoreaceae polyphyletic. The three largest Burmanniaceae genera, Burmannia, Gymnosiphon, and Thismia, are paraphyletic. The splitting of Burmanniaceae into Burmannieae and Thismieae indicates two independent origins of mycoheterotrophy and correlated loss of chlorophyll in Dioscoreales. In the genus Burmannia, in which many species still contain chlorophyll, the achlorophyllous species are nested in between the autotrophic species, suggesting many independent changes from autotrophy to heterotrophy or vice versa. A Bayesian relative rates test on the 18S rDNA data showed considerable variation in substitution rates among Burmanniaceae. The substitution rates in all Thismieae and many Burmannieae are significantly faster than in Dioscoreaceae, but there seems to be no correlation between rate increases and the loss of photosynthesis. Key words: 18S rDNA; Burmanniaceae; Dioscoreales; molecular phylogeny; mycoheterotrophy; nad1 b-c intron; Thismiaceae. Nonphotosynthetic mycoheterotrophic plants are among the 2004; Franke, 2006). In all mycoheterotrophs, this lifestyle has most intriguing species of angiosperms, yet they remain poorly resulted in extreme vegetative reductions. understood. Mycoheterotrophic plants are found in several To study and understand the evolutionary history of the distinct lineages of angiosperms. Although there are many adaptations of mycoheterotrophic families to mycorrhizal fungi, eudicot mycoheterotrophs (Gentianaceae, Ericaceae, and we need to unravel their phylogenetic relationships and to Polygalaceae) most mycoheterotrophic species are monocots identify the most related photosynthetic lineages. Despite our (Leake, 1994). Monocot families with mycoheterotrophic expanding knowledge of the systematic relationships in most representatives are Petrosaviaceae, Orchidaceae, Iridaceae, plant families, very little is known about the evolutionary history Corsiaceae, Triuridaceae, and Burmanniaceae. In contrast to of the mycoheterotrophic groups. First, there is the difficulty of green plants that fix carbon dioxide through photosynthesis, including nonphotosynthetic mycoheterotrophs in plastid DNA mycoheterotrophic plants obtain their organic carbon, partly or analyses. Previous studies showed that some achlorophyllous even exclusively, by digestion of fungal hyphae (Leake, 1994, species still contain amplifiable plastid DNA, but the resulting 2004). The majority of mycoheterotrophs, including Burman- sequences have proved problematic in alignment and analyses niaceae, are associated with arbuscular mycorrhizal fungi and due to elevated substitution rates (Chase et al., 1993; Nickrent et ‘‘steal’’ carbon and other nutrients indirectly from green plants al., 1998; Caddick et al., 2002a). Furthermore, it seems highly probable that these high substitution rates are not restricted to the by hooking themselves into the mycorrhizal networks that link chloroplast genome but can also be expected in the nuclear and adjacent autotrophic plants (Imhof, 1999a, b, 2001; Leake, mitochondrial genome as shown for some parasitic lineages (Nickrent and Starr, 1994; Nickrent et al., 1998; Davis et al., 1 Manuscript received 28 February 2006; revision accepted 24 August 2006. 2004; Chase, 2004). The authors thank J. Moonen for his hospitality and constant support in the field and M. De Bont, Easyware N.V., and Kariboe for making The third and perhaps main problem concerning the an expedition to the Guianas possible; J. Anderson, S. Dessein, R. Ek, phylogenetics of mycoheterotrophic taxa is the scarcity of T. Franke, S. Graham, W. Hahn, R. Harwood, A. Iban˜ez, L. Janeway, study material. Most mycoheterotrophs are small herbs found A. Jaquemart, C. Jongkind, K.-P. Lo, R. Newell, R. Neyland, D. Nickrent, in the ground layer of dense, humid primary forest. They do not A. Poulsen, P. Ruijters, S. Ruysschaert, N. Sasidharan, I. Van Der Linden, tolerate habitat disturbance and thus are largely restricted to M. Wapstra, and S.-Z. Yang for sending plant material or DNA more inaccessible locations. Collecting is further limited extractions; and D. Zhang, T. Franke, P. Berry, and two anonymous because ephemeral aboveground organs are produced only in reviewers for critical comments that greatly improved the manuscript. the rainy season (Leake, 1994; Bidartondo, 2005). As a result, Processing of Haplothismia was done by N. Bhaskaran. The research was supported by the Institute for the Promotion of Innovation through Science many mycoheterotrophic species are only known from the type and Technology in Flanders (IWT Vlaanderen, no. 31536 to V.M.). collection, and some are presumed to be extinct. On the other General financial support was provided by the K.U.Leuven (OT/05/35). hand, new species representing novel lineages are still being 5 Author for correspondence (e-mail: [email protected]. discovered (Zhang et al., 1999; Cheek et al., 2003; Woodward be) et al., in press). 1684 November 2006] MERCKX ET AL.—PHYLOGENY OF BURMANNIACEAE 1685 are regarded by most authors as different tribes, Burmannieae and Thismieae (Jonker, 1938; Stevenson and Laconte, 1995; Maas-van de Kamer, 1998; Cheek and Williams, 1999; Kiew, 1999; Caddick et al., 2002a, b; Yang et al., 2002; APG, 2003; Cheek, 2003; Sainge and Franke, 2005; Woodward et al., in press), but in some classifications Thismieae are considered as a separate family, the Thismiaceae (Chase et al., 1995, 2000; Takhtajan, 1997; APG, 1998; Caddick et al., 2000a, b; Neyland, 2002; Thiele and Jordan, 2002; Neyland and Hennigan, 2003). Burmannieae (or Burmanniaceae sensu stricto) are charac- terized by a persistent perianth and three stamens and contain two pantropical genera: Burmannia L. (about 60 species) and Gymnosiphon Bl. (about 20 species). It is noteworthy that all species in both genera are limited to one continent, and they are often difficult to distinguish (Jonker, 1938). The remaining seven Burmannieae genera (Apteria Nutt., Hexapterella Urb., Campylosiphon Benth., Cymbocarpa Miers, Dictyostega Miers, Marthella Urb., and Miersiella Urb.) are restricted to the neotropics and contain only one or two species each (Maas- van de Kamer, 1998). The tribe Thismieae (or Thismiaceae) consists of highly reduced mycoheterotrophic herbs and differs from Burman- nieae by a circumscissile perianth (Fig. 1B) and the presence of six stamens (except for Oxygyne with three stamens). All species of this tribe are remarkably rare, and some extreme and unexplained biogeographic disjunctions can be found within some genera. Five Thismieae genera are currently known. The largest genus, Thismia Griff. (about 35 species), inhabits the rain forests of South America and Asia, although a few species extend into more temperate regions in Australia, New Zealand, Fig. 1. Two species of Burmanniaceae that represent the two different Japan, and the upper midwestern USA. Afrothismia Schltr. tribes. (A) Burmannia bicolor Mart., tribe Burmannieae (Maas et al. 9673, (nine species) is restricted to the tropical rain forests of Africa Guyana). Note the reduced rosulate leaves. Although still green, this and the monospecific genus Haplothismia Airy Shaw is only species is probably partly dependent on mycorrhizal fungi for its organic known from a few collections at the type locality in the carbon. (B) Thismia panamensis (Standley) Jonk., tribe Thismieae Western Ghats (India; Airy Shaw, 1952; Sasidharan and (Aizprua 2946, Panama). After flowering, the perianth (with stamens Sujanapal, 2000). Two species of the intriguing genus Oxygyne and interstaminal lobes) drops off, leaving behind a cup-shaped fruit Schltr. are known from Cameroon and two from Japan (shown on the right). Bar 1 cm. ¼ (Schlechter, 1906; Abe and Akasawa, 1989; Maas-van de Kamer, 1998). In April 2005 a single specimen of a new In this study, we focused on the phylogeny of the Thismieae genus, Tiputinia, was collected in Amazonian mycoheterotrophic Burmanniaceae, a rather small family in Ecuador (Woodward et al., in press). This discovery stresses which 14 genera and about 130 species have been described, once again our incomplete knowledge of this group. although new species are discovered almost every year (e.g., Because the mycoheterotrophic habit of Burmanniaceae has Kiew, 1999; Zhang and Saunders, 1999, 2000; Liu et al., 2001; resulted in extreme vegetative reductions, subdivision of the Thiele and Jordan, 2002; Yang et al., 2002; Maas-van de family has always been problematic.