Available online at www.sciencedirect.com South African Journal of Botany 74 (2008) 306 – 312 www.elsevier.com/locate/sajb The phylogenetic position of the enigmatic orchid genus Pachites B. Bytebier a,⁎, T. Van der Niet b,1 , D.U. Bellstedt a , H.P. Linder c a b Biochemistry Department, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South Africa School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa c Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, CH-8008, Switzerland Received 10 June 2007; received in revised form 19 December 2007; accepted 9 January 2008 Abstract The orchid genus Pachites is endemic to the Cape Floristic Region and consists of two rare species that only flower during the first year after fire. Pachites has been considered closely related to Satyrium and was for that reason grouped with it in the subtribe Satyriinae. In 2005, we managed to collect material of P. bodkinii and here test the monophyly of the subtribe based on plastid DNA sequence data. We conclusively show that Satyriinae is not monophyletic in its current circumscription and that Pachites is sister to a clade comprising ((Disinae + Coryciinae s.s.) + (Satyrium + Orchideae)). © 2008 SAAB. Published by Elsevier B.V. All rights reserved. Keywords: Cape flora; Diseae; Molecular systematics; Orchidaceae; Orchideae; Orchidoideae; Satyriinae 1. Introduction The orchid genus Pachites was established by John Lindley in 1835 for Pachites appressa, a specimen of which was collected by William Burchell on the Langeberg near Swellendam on 15 January 1815. In his description Lindley remarked that it is “A very curious plant, with a rostellum so thick and large as completely to cut off the anthers from the stigmatic processes or arms, which project forwards like two horns” (Lindley, 1830– 1840). Thus he derived the name from the Greek word pachys, which means “thick” and alludes to the thick column (Kurzweil and Linder, 2001). Harry Bolus, in the first volume of his monumental work on the South African orchids, described a second species, Pachites bodkinii, in 1893, based on a single specimen collected by Alfred Bodkin on Muizenberg in 1890 (Bolus, 1893–1896). Both species are slender to robust terrestrial herbs with root tubers and cauline leaves. The inflorescence is a terminal raceme, ⁎ Corresponding author. E-mail address: bytebier@sun.ac.za (B. Bytebier). 1 Current address: School of Biological and Conservation Sciences, University of KwaZulu Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa. which is often subcapitate. The flowers are non-resupinate, subactinomorphic and not spurred. The two species of Pachites grow in dry, sandy or stony, oligotrophic soils derived from the sandstones of the Cape Supergroup and are endemic to the Cape Floristic Region (Goldblatt, 1978). Like many other Cape orchid species, they flower only in the first year after fire. Moreover, they are rare and consequently have been collected only sporadically, which has hampered detailed study. Schlechter (1901) thought that Pachites was closely related to Satyrium Sw. and later formalised his observations by putting the two genera together in the subtribe Satyriinae Schltr. (Schlechter, 1926). Pachites shares with Satyrium a gynostemium with an elongate column-part, a pendent anther, non-resupinate flowers and undifferentiated petals and sepals (Schlechter, 1901; Kurzweil, 1996). Pachites differs from Satyrium by its subactinomorphic perianth, spurless flowers, presence of vestiges of adaxial stamens and structure of the rostellum (Kurzweil, 1996). The phylogenetic position of Pachites in the tribe Diseae (sensu Kurzweil and Linder, 2001) is not clear (Linder and Kurzweil, 1994; Kurzweil, 1996; Kurzweil and Linder, 2001). Linder and Kurzweil (1994) performed a cladistic analysis of the morphological characters of all genera in the tribe Diseae and found only weak support for a monophyletic Satyriinae. Of the three synapomorphies i.e. petals similar to lateral sepals, 0254-6299/$ - see front matter © 2008 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2008.01.002 B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 petal nerves frequently present and column-part well developed, the latter was certainly the most important character (Linder and Kurzweil, 1994). Furthermore, the monophyly of Pachites has been in doubt. In the morphological analyses of Linder and Kurzweil (1994), the two species do not group together, from which the authors concluded that Pachites is a paraphyletic assemblage relative to Satyrium. Although they share the same general appearance, this may be due to symplesiomorphic characters, and they have different gynostemium structures (Kurzweil, 1993a, 1996). According to the analysis of Linder and Kurzweil (1994), the derived features of both species are all autapomorphic, which hampered their phylogenetic placement. A veld fire in early 2005 on the Muizenberg Plateau on the Cape Peninsula caused a spectacular display of flowers in the following spring and early summer. One of the many orchids 307 observed was P. bodkinii (Fig. 1). This gave us the opportunity to analyse its phylogenetic relationships based on DNA sequence data, and to determine whether it is embedded in Satyrium (consistent with the monophyly of Satyriinae, but not Satyrium), sister to Satyrium (thus consistent with the monophyly of both Satyium and Satyriinae), or more distantly related (thus consistent with the monophyly of Satyrium but not Satyriinae). 2. Materials and methods 2.1. Taxon sampling Four species of the genus Disperis Sw., two of Brownleea Harv. ex Lindl., one each of Corycium Sw. and Pterygodium Sw., and six each of Disa P.J.Bergius and Satyrium Sw. were Fig. 1. Pachites bodkinii Bolus. A: drawing by H. Bolus as part of the protologue (Bolus, 1893–1896); B–C–D: plants in situ (photographs by D.U. Bellstedt). 308 B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 selected as representatives and place holders for the genera that form part of the tribe Diseae (sensu Kurzweil and Linder, 2001). To represent the Orchideae, we included two members of the Habenariinae, both belonging to the genus Habenaria Willd., and four members of the Orchidinae, one of Holothrix Rich., one of Platanthera Rich., one of Gymnadenia R. Br. and one of Dactylorhiza Neck. The South American Codonorchis Lindl. (tribe Codonorchideae) was used to root the tree, based on the phylogenetic results of Freudenstein et al. (2004), which showed that Codonorchis is sister to the Disperis-Ophrys clade, which includes Satyriinae. Sequence data of the plastid trnL intron and trnL-trnF intergenic spacer region (hereafter termed trnL-F) and part of the plastid matK gene and 3′trnK intron (hereafter termed the matK region) was taken from Bytebier et al. (2007), for Disperis, Brownleea and Disa and from Van der Niet et al. (2005) and Van der Niet and Linder (in press) for Satyrium, Habenaria, Holothrix, Gymnadenia, Platanthera and Dactylorhiza, or was downloaded from GenBank (Codonorchis lessonii, Pterygodium catholicum and Corycium carnosum). Table 1 lists the taxa included in this study with the name of the collector, collection number and locality, herbaria where specimens are deposited, and GenBank accession numbers. 2.2. Molecular procedures Fresh leaf material of Pachites bodkinii was collected on 20 November 2005, dried in silica gel and stored at − 20 °C. DNA was extracted using the CTAB procedure of Doyle and Doyle (1987). DNA was amplified using standard PCR as fully detailed in Bytebier et al. (2007). Primers c2 (Bellstedt et al., 2001) and f (Taberlet et al., 1991) were used for amplification of trnL-F. The matK region was amplified with the primers −19F (Molvray et al., 2000) and R1 (Kocyan et al., 2004). Amplification profiles were as follows: for trnL-F, 30 cycles with 1 min denaturation at 94 °C, 1 min annealing at 55 °C, 90 s extension at 72 °C, followed by a final extension step of 6 min at 72 °C; for the matK region, 35 cycles with 30 s denaturation at 95 °C, 1 min annealing at 52 °C, 100 s extension at 72 °C and a final extension step of 7 min at 72 °C. PCR products were cleaned by using the Wizard SV Gel and PCR Clean-Up System (Promega Corp., Madison, USA). For cycle sequencing, the same primers were used as above, except for matK region, where the reverse primer R1 was omitted and two additional forward primers, 580F and 1082F (Kocyan et al., 2004), were used. Cycle sequencing was done with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) as detailed in Bytebier Table 1 Species used in this analysis together with voucher information, origin of the plant material and GenBank accession numbers Taxona Brownleea macroceras Sond. Brownleea parviflora Harv. ex Lindl. Codonorchis lessonii (d'Urv.) Lindl. Corycium carnosum (Lindl.) Rolfe Dactylorhiza maculata (L.) Soo Disa bracteata Sw. Disa glandulosa Burch. ex Lindl. Disa longicornu L.f. Disa rosea Lindl. Disa tripetaloides (L.f.) N.E.Br. Disa versicolor Rchb.f. Disperis capensis (L.f.) Sw. Disperis cucullata Sw. Disperis dicerochila Summerh. Disperis stenoplectron Rchb.f. Gymnadenia conopsea (L.) R.Br. Habenaria keniensis Summerh. Habenaria petitiana T.Durand & Schinz Holothrix sp. Pachites bodkinii Bolus Platanthera chlorantha Custer ex Rchb. Pterygodium catholicum (L.) Sw. Satyrium acuminatum Lindl. Satyrium bracteatum (L.f.) Sw. Satyrium buchananii Schltr. Satyrium chlorocorys Rolfe Satyrium cristatum Sond. Satyrium trinerve Lindl. a Voucher informationb Bytebier 2293 (NBG, BR, K, NU) Kurzweil 1972 (MAL, UZL, SRGH) Rudall (Ref.: O-1398, Kew DNA Bank) Kurzweil 1886 (NBG) Van der Niet T218 (Z) Bytebier 2086 (NBG) Bytebier 2629 (no voucher) Bytebier 2434 (BR) Bytebier 2423 (BR) Bytebier 2460 (NBG, BR, K) Bytebier 2257 (NBG, BR, K) Bytebier 2362 (NBG, BR, K) Bytebier 2032 (NBG, BR) Kurzweil 1983 (MAL, UZL) Edwards & Bellstedt 2308 (NU) Van der Niet T219 (Z) Van der Niet T274 (Z) Van der Niet T276 (EA, Z) Van der Niet T7 (Z) Bytebier 2675 (BR) Van der Niet T222 (Z) Kurzweil 1882 (NBG) Van der Niet 18b (Z) Bytebier 2191 (NBG, GRA, BR) Kurzweil 2053 (MAL) Kurzweil 1969 (MAL, PRE, SRGH, UZL) Bytebier 2297 (NBG, GRA) Bytebier 2255 (NBG, BR) Originc NAT, Sani Pass MLW, Nyika National Park CLS CPP-WC, Fernkloof Nature Reserve SWI, Rossberg CPP-WC, Rondeberg CPP-WC, Table Mountain CPP-WC, Table Mountain CPP-WC, Bain's Kloof CPP-WC, Outeniqua Mountains TVL-MP, Verloren Vallei CPP-WC, Betty's Bay CPP-WC, Sir Lowry's Pass MLW, Nyika National Park CPP-EC, Ntsikeni SWI, Rossberg KEN, Mount Elgon KEN, Timboroa-Tinderet CPP, WC, Walker Bay Reserve CPP-WC, Muizenberg SWI, Rossberg CPP-WC, Fernkloof Nature Reserve CPP-WC, Tradouws Pass CPP-EC, Mount Thomas MLW, Nyika National Park MLW, Nyika National Park CPP-EC, Elands Heights TVL-MP, Verloren Vallei GenBank accession number trnL-F matK DQ415138 DQ415137 DQ415136 AJ409391 AY705045/AY705005 DQ415187 DQ415158 DQ415159 DQ415166 DQ415153 DQ415275 DQ415142 DQ415141 DQ415139 DQ415140 AY705046/AY705006 EF601376/EF601426 EF601377/EF601427 EF601378/EF601428 EF629541 AY705047/AY705007 AJ409447 AY705008/AY705048 AY705014/AY705054 AY705016/AY705056 AY705018/AY705058 AY705021/AY705061 AY705043/AY705083 DQ414995 DQ414994 DQ414993 AJ310012 EF612529 DQ415045 DQ415016 DQ415017 DQ415024 DQ415011 DQ415134 DQ414999 DQ414998 DQ414996 DQ414997 EF612530 EF621492 EF621493 EF621494 EF629540 EF612531 AJ310064 AY708010 AY708016 AY708018 AY708020 AY708023 AY708045 Author abbreviations follow Brummitt and Powell (1992). b Herbarium acronyms are according to Holmgren et al. (1990). c Abbreviations for geographical regions follow Brummitt (2001). B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 et al. (2007). The cycle sequencing products were then analysed on an ABI Prism 3100 or 3130 XL 16-capillary Genetic Analyser (Applied Biosystems, Foster City, USA) in the Central Analytical Facility, Stellenbosch University. 2.3. Phylogenetic analysis Electropherograms were edited in Chromas v1.45 (Technelysium Pty., Tewantin, Australia). Alignment was done by eye in Bioedit v7.0.1 (Hall, 1999). Indels were coded with the “simple indel coding” method of Simmons and Ochoterena (2000). There is no reason to suspect incongruence from a uniparentally inherited, non-recombining genome, therefore the different gene regions were immediately combined for phylogenetic analysis. Parsimony analyses were conducted using PAUP⁎4.0b10 (Swofford, 2003). Heuristic searches were done on a combined dataset. One thousand replicates of random stepwise taxon addition were performed, holding one tree at each step with TBR branch swapping saving no more than 100 trees per replicate. This was followed by a second round of TBR branch swapping using all trees retained in memory. Support for each node was inferred using the bootstrap procedure (Felsenstein, 1985). Bootstrapping was performed using NoNa (Goloboff, 1993), spawned as a daughter process from WinClada (Nixon, 2002). One thousand bootstrap replicates were run with 100 TBR searches per replicate, holding 10 trees per replicate, followed by “max⁎” to swap to completion. MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) was used to infer a phylogenetic hypothesis using a Bayesian approach. Two different ways to partition the data were tried: one where all the data were allocated to one partition, and one where the trnL-F and the non-coding part of matK was treated as a different partition from the coding part of matK. For each partition, the optimal model of sequence evolution was calculated on a randomly chosen MPT from the set of obtained trees from parsimony analysis of the entire data set, using the Akaike Information Criterion (AIC) criterion as implemented in Modeltest 3.7 (Posada and Crandall, 1998). Four chains were run for 5 million generations and sampled every 1000 generations. This was repeated twice, and these independent runs were compared to make sure that similar estimates of substitution model parameters, topology and branch lengths were obtained. The number of “burn-in” generations needed before the various log-likelihood values reached stationarity was determined by a graphical plot, and the first 1000 sampled generations were discarded. Swapping among chains and acceptance of proposed changes to model parameters were monitored to ensure that efficient mixing had occurred. Results from the run with the highest harmonic mean of the −ln likelihood are reported here. 2.4. Hypothesis testing We tested whether not finding a monophyletic Satyriinae in our parsimony analysis could be caused by the stochastic nature of the substitution process, rather than historical factors, using the parametric bootstrap (Huelsenbeck et al., 1996; Van der Niet et al., 2005). We simulated 100 data sets on a tree with Satyriinae 309 constrained to be monophyletic (for a protocol to carry out the simulations, see Van der Niet et al., 2005). These data sets were subsequently analyzed using parsimony implemented in PAUP⁎4.0b10 (Swofford, 2003) with 50 random addition sequence replicates, holding three trees each step and saving no more than five trees. The difference in steps between a parsimony analysis constraining Satyriinae to be monophyletic (i.e. the same topology that was used to simulate the data) and the most parsimonious tree for each of these 100 data sets was recorded. The observed value was contrasted to this null distribution generated through simulation. If the observed value does not fall out of the 5% tail of the null distribution, the hypothesis that a difference in steps between a tree with a monophyletic Satyriinae and the most parsimonious tree is caused by the stochastic nature of the substitution process cannot be rejected. 3. Results 3.1. Data matrix Alignment was straightforward for the matK matrix. Especially the coding region was easy to align and only few gaps needed to be inserted. Alignment proved to be more difficult for the trnL-F matrix due to extensive repeat regions within the Disa trnL intron as described in detail by Bellstedt et al. (2001). These repeat sequences do not contribute any phylogenetic information but make the matrix unwieldy in its length. They were therefore deleted. 3.2. Parsimony analysis The combined data matrix had 2931 characters, 398 (13.6%) of which were potentially parsimony informative. The analysis retrieved only one most parsimonious tree which was completely resolved and of which 85% (22 out of 26) of nodes had BS over 75%. The tree resulting from the combined analysis is presented in Fig. 2. 3.3. Bayesian inference analysis The harmonic mean of several Bayesian runs was not consistently higher for any partitioning scheme. Here we present the results from a Bayesian analysis where all data were treated as a single partition using the GTR + gamma+ I model of sequence evolution. The Posterior Probability (PP) values obtained from this analysis differ only marginally from any of the other Bayesian analyses (data not shown). Inspection of all estimated parameter values, including tree likelihood, plotted against generation, showed convergence well before our set burn-in. The tree resulting from the Bayesian analysis had 18 ingroup nodes with PP equal to or greater than 95%. This tree is fully congruent with the single tree retrieved by the parsimony analysis. 3.4. Phylogenetic position of Pachites In both the parsimony and the Bayesian analysis, Pachites is placed with strong support as sister to a clade comprising 310 B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 Fig. 2. The single most parsimonous tree from the analysis of the combined dataset. The numbers above the branches are the bootstrap percentages from the parsimony analysis, the numbers below the branches are the posterior probabilities from the Bayesian analysis. The nodes that need to be collapsed for a monophyletic Satyriinae are indicated with an arrow. ((Disinae + Coryciinae s.s.) + (Satyrium + Orchideae)). This renders Satyriinae (Pachites + Satyrium) non-monophyletic. The key nodes that need to be collapsed for a potentially monophyletic Satyriinae (i.e. the node subtending ((Disinae + Coryciinae s.s.) + (Satyrium + Orchideae)), and the node subtending Satyrium + Orchideae) receive 97% BS and a PP of 1.0, and 92% BS and a PP of 1.0 respectively. In addition, the phylogenetic placement of Pachites is unambiguous as sister to ((Disinae + Coryciinae s.s. )+ (Satyrium + Orchideae)). The node subtending this clade receives 95% BS and a PP of 1.0. 3.5. Hypothesis testing The hypothesis that a monophyletic Satyriinae was not observed due to stochasticity of the substitution process was rejected based on the parametric bootstrap. The observed value of the difference in number of steps between a tree with Satyriinae constrained to be monophyletic and the most parsimious tree is 11. This value falls outside 99% of the values that are obtained through parametric simulation of the null distribution. B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 4. Discussion 4.1. Congruence with previously published phylogenetic analyses Our tree is largely congruent with previous findings (e.g. Douzery et al., 1999; Freudenstein et al., 2004), which is particularly interesting, as the topology retrieved by Douzery et al. (1999) was based on the nuclear maker ITS, where ours and that of Freudenstein et al. (2004) was based on plastid markers. However, in our analysis Disinae is sister to Coryciinae s.s., as it is in the analysis of Freudenstein et al. (2004), instead of sister to a clade with Coryciinae s.s. + Satyium + Orchideae as suggested by Douzery et al. (1999). Bootstrap support for this sister relationship is markedly higher in our analysis (79%) compared to that in Freudenstein et al. (55%). Bootstrap support for the alternative arrangement as suggested by Douzery et al. was below 50%. 4.2. Monophyly of Satyriinae Based on DNA sequence data from the plastid genome, we have shown conclusively that Satyriinae is not monophyletic in its current circumscription i.e. including both the genera Satyrium and Pachites. Our finding corroborates those of Kurzweil (1993a,b, 1996) and Kurzweil et al. (1995), who doubted that Pachites was correctly placed in Satyrinae on the basis of morphology, seed ultrastructural characteristics and unusual anatomical features such as amphistomatic leaves with equally large leaf epidermal cells on both sides, irregularly thickened leaf cuticle, sclerenchymatous stem ground tissue, sclerenchymatous root pith and monostelic tubers. Kurzweil et al. (1995) suggested that it might be more closely related to the Disinae and Corycinae on the basis of the equally large epidermal cells and polystelic tubers, which are common in the last two subtribes. This suggestion is not supported by our data, but we concur with Kurzweil et al. (1995) that Pachites is peculiar and phylogenetically isolated. 4.3. Taxonomic implications Removing Pachites from Satyriinae will leave this subtribe monotypic like most of the other subtribes in Diseae such as Disinae (Bytebier et al., 2007), Brownleeinae and Huttonaeinae (Linder and Kurzweil, 1994; Kurzweil and Linder, 2001). Furthermore, Douzery et al. (1999), Freudenstein et al. (2004) and Bytebier et al. (2007) have shown that Disperis Sw. does not form part of Coryciinae and should be segregated. The current subtribal classification has therefore lost its usefulness, since it is no longer informative (Backlund and Bremer, 1998). A new classification would be desirable, but to make any informed decisions on this, molecular information (preferably also from a nuclear gene region) on the phylogenetic position of Huttonaea Harv., the relationships within the Corycinae s.s. and the delimitation of the genera that form part of the Coryciinae will be critical. Fortunately these analyses are in progress (K. Steiner, pers com.; R. Waterman, pers com.; T. Fulcher & M.W. Chase, pers com.). However, most of the genera of the Diseae appear to 311 form successive sister relationships with the Orchideae, and so far there seems to be no optimal way of synthesizing this taxonomic variation and keeping an informative classification i.e. one that does not merely consist of monogeneric subtribes. Such a lowinformation classification system is not particularly helpful and can be construed to be a challenge to hierarchical classification systems (De Queiroz and Gauthier, 1992; Brummitt, 2002; Nordal and Stedje, 2005; Ebach et al., 2006). 4.4. Character evolution The main character used to group Pachites with Satyrium i.e. the elongated column-part, a feature otherwise unique to the tribe Orchideae, appears to be homoplasious according to our study. Another unusual features used as a synapomorphy for Satyriinae was the loss of resupination in the flowers, a character that has generally been shown to be highly homoplasious in Orchidaceae (Kurzweil et al., 1991; Dressler, 1993). The morphology-based analysis of Linder and Kurzweil (1994, 1999) was based on minimizing the number of times these attributes evolved, yet the molecular results (Douzery et al., 1999; Freudenstein et al., 2004; Bytebier et al., 2007) suggested that many of the peculiar morphological attributes evolved more than once. Thus morphological evolution and features such as a spurred dorsal sepal (Disperis, Brownleea, Disa), a stigma developed solely from the median carpel (Brownleea, Huttonaea, Disperis, Coryciinae s.s.), a lip with bilobed appendices (Disperis, Pterygodium) and the production of oil as pollinator reward (Disperis, Pterygodium, Corycium) are clearly much more complex than had been initially anticipated and consist not only of the frequent occurrence of these highly unusual morphologies, but also the apparently recurrent evolution of these features. 4.5. Monophyly of Pachites We included only one species of Pachites in this study. Based on a morphological analysis, Linder and Kurzweil (1994) and Kurzweil et al. (1995) suggested that Pachites might be a paraphyletic assemblage relative to Satyrium: (P. bodkinii (P. appressa, Satyrium)). Paraphyly is a result of the many differences in the column structure between the two species of Pachites and the fact that some of the column features of P. appressa resemble more those of Satyrium. Furthermore, an elongated column was considered a synapomorphy for the subtribe and thus not evidence for monophyly of Pachites, whereas the subactinomorphic flower were seen as primitive and thus also not as evidence. However, the new phylogenetic position of Pachites suggests that both these features might be derived. Nevertheless, considering the extent of morphological convergence in this clade, this should be tested in further molecular analyses, which will depend on collection of the second species of this rare and enigmatic genus. Acknowledgments We wish to thank T. Oliver for her help with the photographs and the reviewers for their constructive criticism. Financial 312 B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 support from the National Research Foundation of South Africa is gratefully acknowledged. References Backlund, A., Bremer, K., 1998. To be or not to be — principles of classification and monotypic plant families. Taxon 47, 391–400. Bellstedt, D.U., Linder, H.P., Harley, E.H., 2001. Phylogenetic relationships in Disa based on non-coding trnL-trnF chloroplast sequences: evidence of numerous repeat regions. American Journal of Botany 88, 2088–2100. Bolus, H., 1893–1896. Icones Orchidearum Austro-africanarum Extratropicarum 1 (in 2 parts). Wesley and Son, London. Brummitt, R.K., 2001. World geographical scheme for recording plant distributions, International Working Group on Taxonomic Databases for Plants Sciences (TDWG)Edition 2. Hunt Institute for Botanical Documentation, Pittsburgh. Brummitt, R.K., 2002. How to chop up a tree. Taxon 51, 31–41. Brummitt, R.K., Powell, C.E., 1992. Authors of Plant Names. Royal Botanic Gardens, Kew. Bytebier, B., Bellstedt, D.U., Linder, H.P., 2007. A molecular phylogeny for the large African orchid genus Disa. Molecular Phylogenetics and Evolution 43, 75–90. De Queiroz, K., Gauthier, J., 1992. Phylogenetic taxonomy. Annual Review of Ecology and Systematics 23, 449–480. Douzery, E.J.P., Pridgeon, A.M., Kores, P., Linder, H.P., Kurzweil, H., Chase, M.W., 1999. Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences. American Journal of Botany 86, 887–899. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11–15. Dressler, R.L., 1993. Phylogeny and Classification of the Orchid Family. Cambridge University Press, London. Ebach, M.C., Williams, D.M., Morrone, J.J., 2006. Paraphyly is bad taxonomy. Taxon 55, 831–832. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Freudenstein, J.V., Van den Berg, C., Goldman, D.H., Kores, P.J., Molvray, M., Chase, M.W., 2004. An expanded plastid DNA phylogeny of Orchidaceae and analysis of jackknife support strategy. American Journal of Botany 91, 149–157. Goldblatt, P., 1978. An analysis of the flora of Southern Africa; its characteristics, relationships and origins. Annals of the Missouri Botanical Garden 65, 369–436. Goloboff, P., 1993. NONA, version 1.6. Distributed by the author, Buenos Aires, Argentinia. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41, 95–98. Holmgren, P.K., Holmgren, N.H., Barnett, L.C., 1990. Index Herbariorum. Part I: The Herbaria of the World, Eighth Edition. . Regnum Vegetabile, vol. 120. New York Botanical Garden, New York. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755. Huelsenbeck, J.P., Hillis, D.M., Jones, R., 1996. Parametric bootstrapping in molecular phylogenetics: applications and performance. In: Ferraris, J.D., Edited by JC Manning Palumbi, S.R. (Eds.), Molecular Zoology: Advances, Strategies and Protocols. Wiley-Liss Inc., New York, pp. 19–46. Kocyan, A., Qiu, Y.-L., Endress, P.K., Conti, E., 2004. A phylogenetic analysis of the Apostasioideae (Orchidaceae) based on ITS, trnL-F and matK sequences. Plant Systematics and Evolution 247, 203–213. Kurzweil, H., 1993a. The remarkable anther structure of Pachites bodkinii (Orchidaceae). Botanische Jahrbücher für Systematik, Pflanzengeschichte und Pflanzengeographie 114, 561–569. Kurzweil, H., 1993b. Seed morphology in southern African Orchidoideae (Orchidaceae). Plant Systematics and Evolution 185, 229–247. Kurzweil, H., 1996. Floral morphology and ontogeny in subtribe Satyriinae (Fam Orchidaceae). Flora 191, 9–28. Kurzweil, H., Linder, H.P., 2001. Tribe diseae. In: Pridgeon, A.M., Cribb, P.J., Chase, M.W., Rasmussen, F.N. (Eds.), Genera Orchidacearum. Orchidoideae (Part One), vol. 2. Oxford University Press, Oxford, pp. 11–58. Kurzweil, H., Linder, H.P., Chesselet, P., 1991. The phylogeny and evolution of the Pterygodium-Corycium complex (Coryciinae, Orchidaceae). Plant Systematics and Evolution 175, 161–223. Kurzweil, H., Linder, H.P., Stern, W.L., Pridgeon, A.M., 1995. Comparative vegetative anatomy and classification of the Diseae (Orchidaceae). Botanical Journal of the Linnean Society 117, 171–220. Linder, H.P., Kurzweil, H., 1994. The phylogeny and classification of the Diseae (Orchidoideae: Orchidaceae). Annals of the Missouri Botanical Garden 81, 687–713. Linder, H.P., Kurzweil, H., 1999. Orchids of Southern Africa. A.A. Balkema, Rotterdam. Lindley, J., 1830–1840. The Genera and Species of Orchidaceous Plants. Ridgways, London. Molvray, M., Kores, P., Chase, M.W., 2000. Polyphyly of mycoheterotrophic orchids and functional influences on floral and molecular characters. In: Wilson, K.L., Morrison, D.A. (Eds.), Monocots: Systematics and Evolution. CSIRO, Melbourne, pp. 441–448. Nixon, K.C., 2002. WinClada ver. 1.00.08. Published by the author, Ithaca, New York. Nordal, I., Stedje, B., 2005. Paraphyletic taxa should be accepted. Taxon 54, 5–8. Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14, 817–818. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Schlechter, R., 1901. Monographie der Diseae. Botanische Jahrbücher für Systematik, Pflanzengeschichte und Pflanzengeographie 31, 134–313. Schlechter, R., 1926. Das System der Orchideen. Notizblatt des Botanischen Gartens und Museums zu Berlin 9, 563–591. Simmons, M.P., Ochoterena, H., 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49, 369–381. Swofford, D.L., 2003. PAUP⁎. Phylogenetic Analysis Using Parsimony (⁎and Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. Taberlet, P.L., Gielly, P., Patou, G., Bouvet, J., 1991. Universal primers for the amplification of three non-coding regions of the chloroplast DNA. Plant Molecular Biology 17, 1105–1109. Van der Niet, T., Linder, H.P., Bytebier, B., Bellstedt, D.U., 2005. Molecular markers reject the monophyly of the subgenera of Satyrium (Orchidaceae). Systematic Botany 30, 263–274. Van der Niet, T., Linder, H.P., in press. Dealing with incongruence in the quest for the species tree: a case study from the orchid genus Satyrium. Molecular Phylogenetics and Evolution. doi:10.1016/j.ympev.2007.12.008.