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 ⁎ B. Bytebier a, , T. Van der Niet b,1, D.U. Bellstedt a, H.P. Linder c

a Biochemistry Department, Stellenbosch University, Private Bag X1, Stellenbosch 7602, South b School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, 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 that only during the first year after fire. Pachites has been considered closely related to 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 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 comprising ((+Coryciinae s.s.)+(Satyrium+ )). © 2008 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Cape flora; ; Molecular systematics; ; Orchideae; ; Satyriinae

1. Introduction which is often subcapitate. The are non-resupinate, subactinomorphic and not spurred. The two species of Pachites The orchid genus Pachites was established by John Lindley grow in dry, sandy or stony, oligotrophic soils derived from the in 1835 for Pachites appressa, a specimen of which was col- sandstones of the Cape Supergroup and are endemic to the Cape lected by William Burchell on the Langeberg near Swellendam Floristic Region (Goldblatt, 1978). Like many other Cape orchid on 15 January 1815. In his description Lindley remarked that it species, they flower only in the first year after fire. Moreover, they is “A very curious , with a rostellum so thick and large as are rare and consequently have been collected only sporadically, completely to cut off the anthers from the stigmatic processes or which has hampered detailed study. arms, which project forwards like two horns” (Lindley, 1830– Schlechter (1901) thought that Pachites was closely related to 1840). Thus he derived the name from the Greek word pachys, Satyrium Sw. and later formalised his observations by putting the which means “thick” and alludes to the thick column (Kurzweil two genera together in the subtribe Satyriinae Schltr. (Schlechter, and Linder, 2001). Harry Bolus, in the first volume of his 1926). Pachites shares with Satyrium a gynostemium with an monumental work on the South African orchids, described a elongate column-part, a pendent anther, non-resupinate flowers second species, Pachites bodkinii, in 1893, based on a single and undifferentiated and (Schlechter, 1901; Kurz- specimen collected by Alfred Bodkin on Muizenberg in 1890 weil, 1996). Pachites differs from Satyrium by its subactino- (Bolus, 1893–1896). morphic perianth, spurless flowers, presence of vestiges of adaxial Both species are slender to robust terrestrial herbs with root and structure of the rostellum (Kurzweil, 1996). tubers and cauline leaves. The is a terminal , The phylogenetic position of Pachites in the Diseae (sensu Kurzweil and Linder, 2001) is not clear (Linder and

⁎ Kurzweil, 1994; Kurzweil, 1996; Kurzweil and Linder, 2001). Corresponding author. Linder and Kurzweil (1994) performed a cladistic analysis of E-mail address: [email protected] (B. Bytebier). 1 Current address: School of Biological and Conservation Sciences, the morphological characters of all genera in the tribe Diseae University of KwaZulu Natal Pietermaritzburg, Private Bag X01, Scottsville and found only weak support for a monophyletic Satyriinae. Of 3209, South Africa. 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) 306–312 307 nerves frequently present and column-part well developed, observed was P. bodkinii (Fig. 1). This gave us the opportunity to the latter was certainly the most important character (Linder and analyse its phylogenetic relationships based on DNA sequence Kurzweil, 1994). Furthermore, the monophyly of Pachites has data, and to determine whether it is embedded in Satyrium (con- been in doubt. In the morphological analyses of Linder and sistent with the monophyly of Satyriinae, but not Satyrium), sister Kurzweil (1994), the two species do not group together, from to Satyrium (thus consistent with the monophyly of both Satyium which the authors concluded that Pachites is a paraphyletic and Satyriinae), or more distantly related (thus consistent with the assemblage relative to Satyrium. Although they share the same monophyly of Satyrium but not Satyriinae). general appearance, this may be due to symplesiomorphic char- acters, and they have different gynostemium structures (Kurz- 2. Materials and methods weil, 1993a, 1996). According to the analysis of Linder and Kurzweil (1994), the derived features of both species are all 2.1. Taxon sampling autapomorphic, which hampered their phylogenetic placement. A veld fire in early 2005 on the Muizenberg Plateau on the Four species of the genus Sw., two of Cape Peninsula caused a spectacular display of flowers in the Harv. ex Lindl., one each of Sw. and following spring and early summer. One of the many orchids Sw., and six each of 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: 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 2.2. Molecular procedures form part of the tribe Diseae (sensu Kurzweil and Linder, 2001). To represent the Orchideae, we included two members of the Fresh leaf material of Pachites bodkinii was collected on 20 Habenariinae, both belonging to the genus Willd., November 2005, dried in silica gel and stored at −20 °C. DNA and four members of the Orchidinae, one of Rich., was extracted using the CTAB procedure of Doyle and Doyle one of Rich., one of R. Br. and one of (1987). DNA was amplified using standard PCR as fully Neck. The South American Codonorchis Lindl. detailed in Bytebier et al. (2007). Primers c2 (Bellstedt et al., (tribe Codonorchideae) was used to root the tree, based on the 2001) and f (Taberlet et al., 1991) were used for amplification phylogenetic results of Freudenstein et al. (2004), which of trnL-F.ThematK region was amplified with the primers −19F showed that Codonorchis is sister to the Disperis- (Molvray et al., 2000) and R1 (Kocyan et al., 2004). Amplifica- clade, which includes Satyriinae. Sequence data of the plastid tion profiles were as follows: for trnL-F, 30 cycles with 1 min trnL intron and trnL-trnF intergenic spacer region (hereafter denaturation at 94 °C, 1 min annealing at 55 °C, 90 s extension at termed trnL-F) and part of the plastid matK gene and 3′trnK 72 °C, followed by a final extension step of 6 min at 72 °C; for the intron (hereafter termed the matK region) was taken from matK region, 35 cycles with 30 s denaturation at 95 °C, 1 min Bytebier et al. (2007), for Disperis, Brownleea and Disa and annealing at 52 °C, 100 s extension at 72 °C and a final extension from Van der Niet et al. (2005) and Van der Niet and Linder step of 7 min at 72 °C. PCR products were cleaned by using the (in press) for Satyrium, Habenaria, Holothrix, Gymnadenia, Wizard SV Gel and PCR Clean-Up System (Promega Corp., Platanthera and Dactylorhiza, or was downloaded from Madison, USA). For cycle sequencing, the same primers were GenBank (Codonorchis lessonii, Pterygodium catholicum and used as above, except for matK region, where the reverse primer Corycium carnosum). Table 1 lists the taxa included in this R1 was omitted and two additional forward primers, 580F and study with the name of the collector, collection number and 1082F (Kocyan et al., 2004), were used. Cycle sequencing was locality, herbaria where specimens are deposited, and GenBank done with the BigDye Terminator v3.1 Cycle Sequencing Kit accession numbers. (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 Voucher informationb Originc GenBank accession number trnL-F matK Brownleea macroceras Sond. Bytebier 2293 (NBG, BR, K, NU) NAT, Sani Pass DQ415138 DQ414995 Brownleea parviflora Harv. ex Lindl. Kurzweil 1972 (MAL, UZL, SRGH) MLW, Nyika National Park DQ415137 DQ414994 Codonorchis lessonii (d'Urv.) Lindl. Rudall (Ref.: O-1398, Kew DNA Bank) CLS DQ415136 DQ414993 Corycium carnosum (Lindl.) Rolfe Kurzweil 1886 (NBG) CPP-WC, Fernkloof Nature Reserve AJ409391 AJ310012 Dactylorhiza maculata (L.) Soo Van der Niet T218 (Z) SWI, Rossberg AY705045/AY705005 EF612529 Sw. Bytebier 2086 (NBG) CPP-WC, Rondeberg DQ415187 DQ415045 Disa glandulosa Burch. ex Lindl. Bytebier 2629 (no voucher) CPP-WC, Table Mountain DQ415158 DQ415016 Disa longicornu L.f. Bytebier 2434 (BR) CPP-WC, Table Mountain DQ415159 DQ415017 Disa rosea Lindl. Bytebier 2423 (BR) CPP-WC, Bain's Kloof DQ415166 DQ415024 Disa tripetaloides (L.f.) N.E.Br. Bytebier 2460 (NBG, BR, K) CPP-WC, Outeniqua Mountains DQ415153 DQ415011 Disa versicolor Rchb.f. Bytebier 2257 (NBG, BR, K) TVL-MP, Verloren Vallei DQ415275 DQ415134 Disperis capensis (L.f.) Sw. Bytebier 2362 (NBG, BR, K) CPP-WC, Betty's Bay DQ415142 DQ414999 Disperis cucullata Sw. Bytebier 2032 (NBG, BR) CPP-WC, Sir Lowry's Pass DQ415141 DQ414998 Disperis dicerochila Summerh. Kurzweil 1983 (MAL, UZL) MLW, Nyika National Park DQ415139 DQ414996 Disperis stenoplectron Rchb.f. Edwards & Bellstedt 2308 (NU) CPP-EC, Ntsikeni DQ415140 DQ414997 Gymnadenia conopsea (L.) R.Br. Van der Niet T219 (Z) SWI, Rossberg AY705046/AY705006 EF612530 Habenaria keniensis Summerh. Van der Niet T274 (Z) KEN, Mount Elgon EF601376/EF601426 EF621492 Habenaria petitiana T.Durand & Schinz Van der Niet T276 (EA, Z) KEN, Timboroa-Tinderet EF601377/EF601427 EF621493 Holothrix sp. Van der Niet T7 (Z) CPP, WC, Walker Bay Reserve EF601378/EF601428 EF621494 Pachites bodkinii Bolus Bytebier 2675 (BR) CPP-WC, Muizenberg EF629541 EF629540 Platanthera chlorantha Custer ex Rchb. Van der Niet T222 (Z) SWI, Rossberg AY705047/AY705007 EF612531 Pterygodium catholicum (L.) Sw. Kurzweil 1882 (NBG) CPP-WC, Fernkloof Nature Reserve AJ409447 AJ310064 Satyrium acuminatum Lindl. Van der Niet 18b (Z) CPP-WC, Tradouws Pass AY705008/AY705048 AY708010 Satyrium bracteatum (L.f.) Sw. Bytebier 2191 (NBG, GRA, BR) CPP-EC, Mount Thomas AY705014/AY705054 AY708016 Satyrium buchananii Schltr. Kurzweil 2053 (MAL) MLW, Nyika National Park AY705016/AY705056 AY708018 Satyrium chlorocorys Rolfe Kurzweil 1969 (MAL, PRE, SRGH, UZL) MLW, Nyika National Park AY705018/AY705058 AY708020 Satyrium cristatum Sond. Bytebier 2297 (NBG, GRA) CPP-EC, Elands Heights AY705021/AY705061 AY708023 Satyrium trinerve Lindl. Bytebier 2255 (NBG, BR) TVL-MP, Verloren Vallei AY705043/AY705083 AY708045 aAuthor 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) 306–312 309 et al. (2007). The cycle sequencing products were then analysed constrained to be monophyletic (for a protocol to carry out on an ABI Prism 3100 or 3130 XL 16-capillary Genetic Analyser the simulations, see Van der Niet et al., 2005). These data sets (Applied Biosystems, Foster City, USA) in the Central Analytical were subsequently analyzed using parsimony implemented in Facility, Stellenbosch University. PAUP⁎4.0b10 (Swofford, 2003) with 50 random addition sequence replicates, holding three trees each step and saving no 2.3. Phylogenetic analysis more than five trees. The difference in steps between a parsimony analysis constraining Satyriinae to be monophyletic (i.e. the same Electropherograms were edited in Chromas v1.45 (Techne- topology that was used to simulate the data) and the most lysium Pty., Tewantin, ). Alignment was done by eye in parsimonious tree for each of these 100 data sets was recorded. Bioedit v7.0.1 (Hall, 1999). Indels were coded with the “simple The observed value was contrasted to this null distribution indel coding” method of Simmons and Ochoterena (2000). generated through simulation. If the observed value does not fall There is no reason to suspect incongruence from a uniparentally out of the 5% tail of the null distribution, the hypothesis that a inherited, non-recombining genome, therefore the different gene difference in steps between a tree with a monophyletic Satyriinae regions were immediately combined for phylogenetic analysis. and the most parsimonious tree is caused by the stochastic nature Parsimony analyses were conducted using PAUP⁎4.0b10 of the substitution process cannot be rejected. (Swofford, 2003). Heuristic searches were done on a combined dataset. One thousand replicates of random stepwise taxon 3. Results addition were performed, holding one tree at each step with TBR branch swapping saving no more than 100 trees per replicate. 3.1. Data matrix This was followed by a second round of TBR branch swapping using all trees retained in memory. Support for each node was Alignment was straightforward for the matK matrix. inferred using the bootstrap procedure (Felsenstein, 1985). Especially the coding region was easy to align and only few Bootstrapping was performed using NoNa (Goloboff, 1993), gaps needed to be inserted. Alignment proved to be more spawned as a daughter process from WinClada (Nixon, 2002). difficult for the trnL-F matrix due to extensive repeat regions One thousand bootstrap replicates were run with 100 TBR within the Disa trnL intron as described in detail by Bellstedt searches per replicate, holding 10 trees per replicate, followed by et al. (2001). These repeat sequences do not contribute any “max⁎” to swap to completion. phylogenetic information but make the matrix unwieldy in its MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist length. They were therefore deleted. and Huelsenbeck, 2003) was used to infer a phylogenetic hy- pothesis using a Bayesian approach. Two different ways to 3.2. Parsimony analysis 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 The combined data matrix had 2931 characters, 398 (13.6%) of matK was treated as a different partition from the coding part of of which were potentially parsimony informative. The analysis matK. For each partition, the optimal model of sequence evolu- retrieved only one most parsimonious tree which was com- tion was calculated on a randomly chosen MPT from the set of pletely resolved and of which 85% (22 out of 26) of nodes had obtained trees from parsimony analysis of the entire data set, BS over 75%. The tree resulting from the combined analysis is using the Akaike Information Criterion (AIC) criterion as im- presented in Fig. 2. plemented in Modeltest 3.7 (Posada and Crandall, 1998). Four chains were run for 5 million generations and sampled every 1000 3.3. Bayesian inference analysis generations. This was repeated twice, and these independent runs were compared to make sure that similar estimates of substitution The harmonic mean of several Bayesian runs was not con- model parameters, topology and branch lengths were obtained. sistently higher for any partitioning scheme. Here we present the The number of “burn-in” generations needed before the various results from a Bayesian analysis where all data were treated as a log-likelihood values reached stationarity was determined by a single partition using the GTR+gamma+I model of sequence graphical plot, and the first 1000 sampled generations were dis- evolution. The Posterior Probability (PP) values obtained from carded. Swapping among chains and acceptance of proposed this analysis differ only marginally from any of the other Bayesian changes to model parameters were monitored to ensure that ef- analyses (data not shown). Inspection of all estimated parameter ficient mixing had occurred. Results from the run with the highest values, including tree likelihood, plotted against generation, harmonic mean of the −ln likelihood are reported here. showed convergence well before our set burn-in. The tree re- sulting from the Bayesian analysis had 18 ingroup nodes with PP 2.4. Hypothesis testing equal to or greater than 95%. This tree is fully congruent with the single tree retrieved by the parsimony analysis. We tested whether not finding a monophyletic Satyriinae in our parsimony analysis could be caused by the stochastic nature 3.4. Phylogenetic position of Pachites of the substitution process, rather than historical factors, using the parametric bootstrap (Huelsenbeck et al., 1996; Van der Niet In both the parsimony and the Bayesian analysis, Pachites et al., 2005). We simulated 100 data sets on a tree with Satyriinae 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 ren- 3.5. Hypothesis testing ders Satyriinae (Pachites+Satyrium) non-monophyletic. The key nodes that need to be collapsed for a potentially The hypothesis that a monophyletic Satyriinae was not monophyletic Satyriinae (i.e. the node subtending ((Disinae+ observed due to stochasticity of the substitution process Coryciinae s.s.)+(Satyrium +Orchideae)), and the node sub- was rejected based on the parametric bootstrap. The observed tending Satyrium +Orchideae) receive 97% BS and a PP of 1.0, value of the difference in number of steps between a tree and 92% BS and a PP of 1.0 respectively. In addition, the with Satyriinae constrained to be monophyletic and the most phylogenetic placement of Pachites is unambiguous as sister to parsimious tree is 11. This value falls outside 99% of the values ((Disinae +Coryciinae s.s.)+(Satyrium+Orchideae)). The node that are obtained through parametric simulation of the null subtending this clade receives 95% BS and a PP of 1.0. distribution. B. Bytebier et al. / South African Journal of Botany 74 (2008) 306–312 311

4. Discussion form successive sister relationships with the Orchideae, and so far there seems to be no optimal way of synthesizing this taxonomic 4.1. Congruence with previously published phylogenetic analyses variation and keeping an informative classification i.e. one that does not merely consist of monogeneric subtribes. Such a low- Our tree is largely congruent with previous findings (e.g. information classification system is not particularly helpful and Douzery et al., 1999; Freudenstein et al., 2004), which is can be construed to be a challenge to hierarchical classification particularly interesting, as the topology retrieved by Douzery systems (De Queiroz and Gauthier, 1992; Brummitt, 2002; et al. (1999) was based on the nuclear maker ITS, where ours Nordal and Stedje, 2005; Ebach et al., 2006). and that of Freudenstein et al. (2004) was based on plastid markers. However, in our analysis Disinae is sister to Coryciinae 4.4. Character evolution 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 The main character used to group Pachites with Satyrium i.e. suggested by Douzery et al. (1999). Bootstrap support for this the elongated column-part, a feature otherwise unique to the tribe sister relationship is markedly higher in our analysis (79%) Orchideae, appears to be homoplasious according to our study. compared to that in Freudenstein et al. (55%). Bootstrap support Another unusual features used as a synapomorphy for Satyriinae for the alternative arrangement as suggested by Douzery et al. was the loss of resupination in the flowers, a character that has was below 50%. generally been shown to be highly homoplasious in Orchidaceae (Kurzweil et al., 1991; Dressler, 1993). The morphology-based 4.2. Monophyly of Satyriinae analysis of Linder and Kurzweil (1994, 1999) was based on minimizing the number of times these attributes evolved, yet the Based on DNA sequence data from the plastid genome, we molecular results (Douzery et al., 1999; Freudenstein et al., 2004; have shown conclusively that Satyriinae is not monophyletic in Bytebier et al., 2007) suggested that many of the peculiar its current circumscription i.e. including both the genera Satyr- morphological attributes evolved more than once. Thus morpho- ium and Pachites. Our finding corroborates those of Kurzweil logical evolution and features such as a spurred dorsal (1993a,b, 1996) and Kurzweil et al. (1995), who doubted that (Disperis, Brownleea, Disa), a developed solely from the Pachites was correctly placed in on the basis of median carpel (Brownleea, , Disperis, Coryciinae s.s.), morphology, seed ultrastructural characteristics and unusual a lip with bilobed appendices (Disperis, Pterygodium) and the anatomical features such as amphistomatic leaves with equally production of oil as reward (Disperis, Pterygodium, large leaf epidermal cells on both sides, irregularly thickened Corycium) are clearly much more complex than had been initially leaf cuticle, sclerenchymatous stem ground tissue, sclerenchy- anticipated and consist not only of the frequent occurrence of matous root pith and monostelic tubers. Kurzweil et al. (1995) these highly unusual morphologies, but also the apparently re- suggested that it might be more closely related to the Disinae current evolution of these features. and Corycinae on the basis of the equally large epidermal cells and polystelic tubers, which are common in the last two 4.5. Monophyly of Pachites subtribes. This suggestion is not supported by our data, but we concur with Kurzweil et al. (1995) that Pachites is peculiar and We included only one species of Pachites in this study. phylogenetically isolated. Based on a morphological analysis, Linder and Kurzweil (1994) and Kurzweil et al. (1995) suggested that Pachites might be 4.3. Taxonomic implications a paraphyletic assemblage relative to Satyrium:(P. bodkinii (P. appressa, Satyrium)). is a result of the many Removing Pachites from Satyriinae will leave this subtribe differences in the column structure between the two species monotypic like most of the other subtribes in Diseae such as of Pachites and the fact that some of the column features of Disinae (Bytebier et al., 2007), Brownleeinae and Huttonaeinae P. appressa resemble more those of Satyrium. Furthermore, an (Linder and Kurzweil, 1994; Kurzweil and Linder, 2001). elongated column was considered a synapomorphy for the sub- Furthermore, Douzery et al. (1999), Freudenstein et al. (2004) tribe and thus not evidence for monophyly of Pachites, whereas and Bytebier et al. (2007) have shown that Disperis Sw. does not the subactinomorphic flower were seen as primitive and thus form part of Coryciinae and should be segregated. The current also not as evidence. However, the new phylogenetic position of subtribal classification has therefore lost its usefulness, since it is Pachites suggests that both these features might be derived. no longer informative (Backlund and Bremer, 1998). A new Nevertheless, considering the extent of morphological conver- classification would be desirable, but to make any informed gence in this clade, this should be tested in further molecular decisions on this, molecular information (preferably also from a analyses, which will depend on collection of the second species nuclear gene region) on the phylogenetic position of Huttonaea of this rare and enigmatic genus. Harv., the relationships within the Corycinae s.s. and the de- limitation of the genera that form part of the Coryciinae will be Acknowledgments critical. Fortunately these analyses are in progress (K. Steiner, pers com.; R. Waterman, pers com.; T. Fulcher & M.W. Chase, We wish to thank T. Oliver for her help with the photographs pers com.). However, most of the genera of the Diseae appear to 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 Palumbi, S.R. (Eds.), Molecular Zoology: Advances, Strategies and – is gratefully acknowledged. 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 References sequences. Plant Systematics and Evolution 247, 203–213. Kurzweil, H., 1993a. The remarkable anther structure of Pachites bodkinii Backlund, A., Bremer, K., 1998. To be or not to be — principles of classification (Orchidaceae). Botanische Jahrbücher für Systematik, Pflanzengeschichte and monotypic plant families. Taxon 47, 391–400. und Pflanzengeographie 114, 561–569. Bellstedt, D.U., Linder, H.P., Harley, E.H., 2001. Phylogenetic relationships in Kurzweil, H., 1993b. Seed morphology in southern African Orchidoideae Disa based on non-coding trnL-trnF sequences: evidence of (Orchidaceae). Plant Systematics and Evolution 185, 229–247. numerous repeat regions. American Journal of Botany 88, 2088–2100. Kurzweil, H., 1996. Floral morphology and ontogeny in subtribe Satyriinae Bolus, H., 1893–1896. Icones Orchidearum Austro-africanarum Extratropi- (Fam Orchidaceae). Flora 191, 9–28. carum 1 (in 2 parts). Wesley and Son, London. Kurzweil, H., Linder, H.P., 2001. Tribe diseae. In: Pridgeon, A.M., Cribb, P.J., Brummitt, R.K., 2001. World geographical scheme for recording plant Chase, M.W., Rasmussen, F.N. (Eds.), Genera Orchidacearum. Orchidoi- distributions, International Working Group on Taxonomic Databases for deae (Part One), vol. 2. Oxford University Press, Oxford, pp. 11–58. Plants Sciences (TDWG)Edition 2. Hunt Institute for Botanical Documenta- Kurzweil, H., Linder, H.P., Chesselet, P., 1991. The phylogeny and evolution of tion, Pittsburgh. the Pterygodium-Corycium complex (Coryciinae, Orchidaceae). Plant Brummitt, R.K., 2002. How to chop up a tree. Taxon 51, 31–41. Systematics and Evolution 175, 161–223. Brummitt, R.K., Powell, C.E., 1992. Authors of Plant Names. Royal Botanic Kurzweil, H., Linder, H.P., Stern, W.L., Pridgeon, A.M., 1995. Comparative Gardens, Kew. vegetative anatomy and classification of the Diseae (Orchidaceae). Botanical Bytebier, B., Bellstedt, D.U., Linder, H.P., 2007. A molecular phylogeny for the Journal of the Linnean Society 117, 171–220. large African orchid genus Disa. Molecular and Evolution 43, Linder, H.P., Kurzweil, H., 1994. The phylogeny and classification of the Diseae 75–90. (Orchidoideae: Orchidaceae). Annals of the Missouri Botanical Garden 81, De Queiroz, K., Gauthier, J., 1992. Phylogenetic . Annual Review of 687–713. Ecology and Systematics 23, 449–480. Linder, H.P., Kurzweil, H., 1999. Orchids of Southern Africa. A.A. Balkema, Douzery, E.J.P., Pridgeon, A.M., Kores, P., Linder, H.P., Kurzweil, H., Chase, Rotterdam. M.W., 1999. of Diseae (Orchidaceae): a contribu- Lindley, J., 1830–1840. The Genera and Species of Orchidaceous Plants. tion from nuclear ribosomal ITS sequences. American Journal of Botany 86, Ridgways, London. 887–899. Molvray, M., Kores, P., Chase, M.W., 2000. of mycoheterotrophic Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small orchids and functional influences on floral and molecular characters. In: quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11–15. Wilson, K.L., Morrison, D.A. (Eds.), Monocots: Systematics and Evolution. Dressler, R.L., 1993. Phylogeny and Classification of the Orchid Family. CSIRO, Melbourne, pp. 441–448. Cambridge University Press, London. Nixon, K.C., 2002. WinClada ver. 1.00.08. Published by the author, Ithaca, Ebach, M.C., Williams, D.M., Morrone, J.J., 2006. Paraphyly is bad taxonomy. New York. Taxon 55, 831–832. Nordal, I., Stedje, B., 2005. Paraphyletic taxa should be accepted. Taxon 54, 5–8. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the Posada, D., Crandall, K.A., 1998. MODELTEST: testing the model of DNA bootstrap. Evolution 39, 783–791. substitution. Bioinformatics 14, 817–818. Freudenstein, J.V., Van den Berg, C., Goldman, D.H., Kores, P.J., Molvray, M., Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic in- Chase, M.W., 2004. An expanded plastid DNA phylogeny of Orchidaceae ference under mixed models. Bioinformatics 19, 1572–1574. and analysis of jackknife support strategy. American Journal of Botany 91, Schlechter, R., 1901. Monographie der Diseae. Botanische Jahrbücher für 149–157. Systematik, Pflanzengeschichte und Pflanzengeographie 31, 134–313. Goldblatt, P., 1978. An analysis of the flora of Southern Africa; its charac- Schlechter, R., 1926. Das System der Orchideen. Notizblatt des Botanischen teristics, relationships and origins. Annals of the Missouri Botanical Garden Gartens und Museums zu Berlin 9, 563–591. 65, 369–436. Simmons, M.P., Ochoterena, H., 2000. Gaps as characters in sequence-based Goloboff, P., 1993. NONA, version 1.6. Distributed by the author, Buenos phylogenetic analyses. Systematic Biology 49, 369–381. Aires, Argentinia. Swofford, D.L., 2003. PAUP⁎. Phylogenetic Analysis Using Parsimony (⁎and Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor Other Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts. and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Taberlet, P.L., Gielly, P., Patou, G., Bouvet, J., 1991. Universal primers for the Series 41, 95–98. amplification of three non-coding regions of the chloroplast DNA. Plant Holmgren, P.K.,Holmgren, N.H., Barnett, L.C., 1990. Index Herbariorum. Part I: Molecular Biology 17, 1105–1109. The Herbaria of the World, Eighth Edition. . Regnum Vegetabile, vol. 120. Van der Niet, T., Linder, H.P., Bytebier, B., Bellstedt, D.U., 2005. Molecular New York Botanical Garden, New York. markers reject the monophyly of the subgenera of Satyrium (Orchidaceae). Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of Systematic Botany 30, 263–274. phylogeny. Bioinformatics 17, 754–755. Van der Niet, T., Linder, H.P., in press. Dealing with incongruence in the quest Huelsenbeck, J.P., Hillis, D.M., Jones, R., 1996. Parametric bootstrapping in for the species tree: a case study from the orchid genus Satyrium. Molecular molecular phylogenetics: applications and performance. In: Ferraris, J.D., Phylogenetics and Evolution. doi:10.1016/j.ympev.2007.12.008.

Edited by JC Manning