Botanical Journal of the Linnean Society, 2009, 160, 402–417. With 4 figures Gnidia (Thymelaeaceae) is not monophyletic: taxonomic implications for Thymelaeoideae and a partial new generic taxonomy for Gnidia 1 School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3209, South Africa 2 Compton Herbarium, South African National Biodiversity Institute, Private Bag X7, Claremont 7735, South Africa 3 Molecular Systematics Laboratory, Department of Botany and Plant Biotechnology, University of Johannesburg, PO Box 524, Auckland Park, 2006, South Africa 4 Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Received 19 March 2009; accepted for publication 12 June 2009 We address the generic limits of Gnidia (Thymelaeaceae) through a phylogenetic analysis of nuclear ribosomal DNA internal transcribed spacer (ITS) and plastid rbcL, trnL intron and trnL-F intergenic spacer regions. Maximum parsimony and Bayesian inference were used to produce trees and assess internal support. The most significant conclusion drawn from the molecular analysis is that Gnidia is polyphyletic as currently circumscribed, comprising at least four distinct lineages that are each related to other genera within Thymelaeoideae. Gnidia pinifolia and G. racemosa are members of a clade within which Struthiola is embedded; a second group of species allies with Drapetes as sister to Passerina; and a third lineage corresponds to the previously recognized genus Lasiosiphon. The remaining species of Gnidia included in this study are allied with the Australian genus Pimelea. The taxonomic implications of these findings are discussed in relation to the principle of monophyly. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417. ADDITIONAL KEYWORDS: internal transcribed spacer (ITS) – Lasiosiphon – molecular systematics – Passerina – Pimelea – rbcL – Struthiola – trnL-F. INTRODUCTION Gnidia L. (Thymelaeaceae) is a genus of about 140 species of perennial herbs, shrubs and small trees. Species’ diversity is greatest in tropical and southern Africa, with about 20 species endemic to Madagascar. Domke (1934) recognized four subfamilies in Thymelaeaceae: Aquilarioideae, Gonystyloideae, Synandrodaphnoideae (= Gilgiodaphnoideae; Robyns, 1975) and Thymelaeoideae. This has been a generally popular classification and one that is also supported *Corresponding author. E-mail: mvdbank@uj.ac.za 402 by the molecular findings of Van der Bank, Fay & Chase (2002). Flowers with a single ovule are a distinguishing feature of the largest subfamily, Thymelaeoideae. Peddiea Harv. ex Hook., however, is the exception among Thymelaeoideae in having bilocular ovaries with a single ovule in each locule (Peterson, 1978). Gnidia and Pimelea Banks & Sol. ex Gaertn. (approximately 110 species from Australia and New Zealand) are the largest genera in the subfamily. Domke (1934) included Gnidia in his tribe Gnidieae, subtribe Gnidiinae, with other southern African genera, including Dais L., Lasiosiphon Fresen., Craspedostoma Domke, Struthiola L. and Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 ANGELA J. BEAUMONT1, TREVOR J. EDWARDS1, JOHN MANNING2, OLIVIER MAURIN3, MARLINE RAUTENBACH3, MOLEBOHENG C. MOTSI3, MICHAEL F. FAY4, MARK W. CHASE4 and MICHELLE VAN DER BANK3* PHYLOGENY OF GNIDIA (THYMELAEACEAE) MATERIAL AND METHODS TAXON SAMPLING In total, we analysed 106 species, representing 32 genera of Thymelaeaceae and including 35 species of Gnidia that represent the full range of floral diversity and habit within the genus. Representatives of Sphaerosepalaceae [Dialyceras coriaceum (R.Cap.) J.-F.Leroy, Rhopalocarpus sp.] and Neuradaceae (Grielum humifusum E.Mey. ex Harv. & Sond.) were selected as outgroups, because of their close relation- ship to Thymelaeaceae (Fay et al., 1998; Bayer et al., 1999). An earlier study by Van der Bank et al. (2002) concluded that Thymelaeaceae are monophyletic with four subfamilies, and Synandrodaphnoideae and Gonystyloideae are successively sister to Aquilarioideae/Thymelaeoideae. Therefore, representatives of Aquilarioideae, Gonystyloideae and Synandrodaphnoideae were also included. Voucher specimens for the taxa used in this study and GenBank accession numbers are listed in Appendix 1. DNA EXTRACTION, AMPLIFICATION AND SEQUENCING DNA was extracted using the 2X cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1987) from herbarium, fresh or silica-dried material. DNA was purified using either a QIAQuick polymerase chain reaction (PCR) purification kit (QIAgen, Inc., Hilden, Germany) or caesium chloride/ethidium bromide gradient centrifugation. PCR amplification and sequencing for rbcL and the trnL-F region (intron and spacer) were performed as in Van der Bank et al. (2002). The ITS nuclear ribosomal DNA region was amplified using the primers of White et al. (1990; ITS 2, 3, 4 and 5). For PCR amplification of the ITS region, the following programme was used: pre-melt at 94 °C for 120 s, denaturation at 94 °C for 60 s, annealing at 48 °C for 60 s, extension at 72 °C for 3 min, final extension at 72 °C for 7 min (30 cycles). For the editing and assembly of complementary electropherograms, Sequencher version 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA) was used. Each base position was checked for agreement of the complementary strands, and most sequences had nearly 100% of both strands available. The aligned matrices are available from MVDB and MWC: mvdbank@uj.ac.za; m.chase@kew.org. PHYLOGENETIC ANALYSIS OF MOLECULAR DATA Molecular data were analysed using maximum parsimony and Bayesian methods employing PAUP* version 4.0b10 (Swofford, 2003) and MrBayes version 3.1b2 (Ronquist & Huelsenbeck, 2003), respectively. Prior to Bayesian analysis, the best-fit model of evolution was determined for each molecular marker via the Akaike information criterion (Akaike, 1979), as implemented in MODELTEST version 3.06 (Posada & Crandall, 1998), which uses log-likelihood scores to estimate the model of DNA evolution best suited to a specific dataset (Posada & Crandall, 1998). TREE SEARCHES AND BRANCH SUPPORT We did not analyse each of the plastid regions separately because, individually, they exhibit low levels of Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Lachnaea L. (including Cryptadenia Meisn.; Beyers, 2001), and the remaining members of tribe Gnidieae, namely Thymelaea Miller, Passerina L., Kelleria Endl., Drapetes Banks ex Lam. and Pimelea, in individual subtribes. Linnaeus (1753) established Gnidia with three species, including the type species Gnidia pinifolia L. He distinguished Gnidia from related genera in the family by its tetramerous perianth and eight stamens, and remarked on the resemblance between Gnidia and Passerina, noting that only the presence of petal-like structures (his corollâ) in Gnidia and their absence in Passerina could distinguish them. The distinction between Gnidia and other African Thymelaeoideae has fluctuated considerably (Table 1). Floral characters previously used to distinguish genera are variable, resulting in the reduction of some groups to synonymy under Gnidia. For example, the pentamerous floral plan in Lasiosiphon is unstable, with inflorescences occasionally including flowers with parts in fours or sixes. In contrast, the tetramerous condition in flowers of Gnidia s.s. appears stable (A. J. Beaumont, pers. observ.). Peterson (1959) considered this slight instability of the pentamerous condition to justify the reduction of Lasiosiphon to synonymy under Gnidia. Ding Hou (1960), citing Peterson (1959), acknowledged the challenge of finding robust characters to separate genera in Thymelaeaceae. Peterson (1978) provided the most recent account of Thymelaeaceae in tropical and eastern Africa, and modern southern African flora accounts (for example, Bredenkamp & Beyers, 2000; Goldblatt & Manning, 2000) have followed his broad circumscription of Gnidia. To assess the relationships among genera of Thymelaeoideae, we performed a combined phylogenetic analysis of nuclear and plastid molecular datasets: nuclear internal transcribed spacer (ITS) ribosomal DNA and plastid rbcL, trnL intron and trnL-F intergenic spacer regions. Representatives of 32 of the 45 genera accepted in Thymelaeaceae were included in the study, among which are 23 of the 33 genera of Thymelaeoideae. 403 404 Gilg (1894) Tribe I Subtribe I Genus Gnidieae Gnidiinae Gnidia (including Lasiosiphon, Arthrosolen) Pearson (1913); Wright (1915); Burtt Davy (1926) Marloth (1925) Euthymelaeae Gnidieae Gnidia Gnidia Genus Genus Lasiosiphon Tribe II Dicranolepideae Subtribe II Linostomatinae Genus Englerodaphne Englerodaphne Tribe III Genus Arthrosolen Genus Genus Genus Domke (1934) Gnidieae Gnidiinae Gnidia (including Gnidia Arthrosolen, Englerodaphne, Epichroxantha) Craspedostoma Lasiosiphon Lasiosiphon Dicranolepideae Englerodaphne Dapneae Arthrosolen Phillips (1944, 1951) Lasiosiphon Hutchinson (1967) Peterson (1959, 1982) Gnidia (including Gnidia (including Gnidia (including Lasiosiphon, Pseudognidia, Lasiosiphon, Englerodaphne, Craspedostoma, Arthrosolen, Arthrosolen, Epichroxantha) Pseudoginidia, Pseudognidia, Basutica, Basutica, Struthiolopsis) Struthiolopsis, Craspedostoma) Lasiosiphon Englerodaphne Englerodaphne Arthrosolen Pseudognidia Basutica Struthiolopsis Dyer (1975) Englerodaphne Arthrosolen Basutica Struthiolopsis A. J. BEAUMONT ET AL. Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Table 1. Classification of Gnidia and associated genera PHYLOGENY OF GNIDIA (THYMELAEACEAE) 405 Table 2. Statistics from maximum parsimony analyses obtained from separate and combined datasets trnL-F Combined plastid 118 1378 1074 304 (22.1%) 193 (14%) 106 984 620 364 (37%) 226 (23%) 120 2362 1694 668 (28.3%) 419 (17.7%) 205 679 0.69 0.84 1.87 2610 1421 0.58 0.81 53 668 0.54 0.83 2.19 sequence divergence; in addition, there is no reason to suspect incongruence among different regions of the plastid genome as they cannot assort independently or recombine. To evaluate congruence, we analysed the ITS and plastid matrices separately before combining them. All matrices were analysed using heuristic searches with 1000 random sequence additions, but keeping only ten trees per replicate to reduce the time spent on branch swapping in each replicate. Tree bisection-reconnection (TBR) was performed with MulTrees on (keeping multiple equally parsimonious trees) and all character transformations treated as equally likely (Fitch parsimony; Fitch, 1971). The trees collected in the 1000 replicates were then used as starting trees for another search without a tree limit. For the illustration of branch lengths, DELTRAN (delayed transformation) character optimization was used instead of ACCTRAN (accelerated transformation) because of reported errors with the latter in PAUP* 4.0b10. Internal support was estimated by the bootstrap as implemented by PAUP* using 1000 bootstrap replicates performed with equal weights employing TBR branch swapping with 10 trees held at each step and simple taxon addition. As a result of the poor quality DNA of some taxa, we could not amplify all regions for all taxa, and thus the individual data matrices do not contain identical sets of taxa. We investigated the effects of these missing data on the patterns of relationships and support in the combined analysis by comparing results from matrices in which we included only taxa for which all data were available with results of the larger matrices with missing data. We found that neither was affected in any obvious way, and therefore illustrate the combined results with all taxa. Congruence between the ITS and plastid datasets Internal transcribed spacer (ITS) 86 651 293 358 (55%) 293 (45%) 651 1599 0.36 0.72 4.46 Combined plastid + ITS 120 3014 1988 1026 (34%) 712 (23.62%) 5360 3040 0.48 0.75 was addressed by comparison of bootstrap percentages from the separate analyses. Bootstrap trees were considered incongruent only when ‘hard’ (i.e. with high bootstrap support) instead of ‘soft’ (with low bootstrap support) incongruence was displayed (Seelanan, Schnabel & Wendel, 1997; Wiens, 1998). No ‘congruence tests’, such as the incongruence length difference test, were used, because of their reported unreliability (Reeves et al., 2001; Yoder, Irwin & Payseur, 2001). The following scale for support percentages was used: 50–74%, low; 75–84%, moderate; 85–100%, high. A Bayesian approach for inferring phylogenies was also used. For each matrix, the best model was selected using MODELTEST version 3.06 (Posada & Crandall, 1998). For all regions, GTR + I + G was the resulting model, with substitutions = 6, rates = gamma, base frequency = empirical, clock = unconstrained. Four parallel Markov chain Monte Carlo estimations were run for 3 000 000 generations with trees sampled every 200 generations. The resulting trees were plotted against their likelihoods to determine the point at which likelihoods converged on a maximum value, and all the trees before convergence were discarded as the ‘burn-in’. All remaining trees were imported into PAUP* 4.0b10, and a majority-rule consensus tree was produced showing frequencies (i.e. posterior probabilities or PP) of all observed bi-partitions. The following scale was used to evaluate PP: below 0.85, poor; 0.85–0.95, moderate; 0.95–1.0, high. RESULTS The characteristics of each partition and the statistics of each analysis are reported in Table 2. The alignment of ITS sequences between subfamilies of Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Number of taxa included Number of included characters Number of constant characters Number of variable sites Number of parsimony informative sites Number of trees (Fitch) Number of steps (tree length) Consistency index Retention index Average number of changes per variable site (number of steps/ number of variable sites) rbcL 406 A. J. BEAUMONT ET AL. COMBINED MOLECULAR ANALYSIS The parsimony analysis resulted in 5360 equally parsimonious trees (tree length, 3040 steps; CI = 0.48; RI = 0.75). Of the 3014 included characters, 1988 were constant, 1026 (34%) were variable and 712 (23.6%) were potentially parsimony informative. The combined maximum parsimony analysis is largely congruent with the Bayesian analysis, and therefore results can be displayed on the same tree (Fig. 3). Thymelaeaceae are strongly supported as monophyletic (99 bp/1.0 PP). Aquilarioideae (100 bp/1.0 PP) and Synandrodaphnoideae are moderately supported as successively sister (98 bp/1.0 PP and 78 bp/0.70 PP, respectively) to Thymelaeoideae (89 bp/1.0 PP). Gonystyloideae are paraphyletic to the rest of Thymelaeaceae, comprising two clades: (I) three taxa of Octolepis Oliv., namely O. dioica Capuron, O. dioica forma oblanceolata Capuron and Octolepis sp. (97 bp/ 1.0 PP); (II) representatives of Lethedon Spreng., Arnhemia Airy Shaw, Deltaria Steenis, Gonystylus Teijsm. & Binn. and Solmsia Baill. (75 bp/1.0 PP). Within Thymelaeoideae, three major clades are retrieved: clade I comprises tropical African and south-eastern Asian taxa; clade II includes exclusively non-African taxa; and clade III comprises southern and tropical African, south-eastern Asian, Australasian and New World taxa. Clades II and I are strongly supported as successively sister to clade III (89 bp/1.0 PP and 97 bp/1.0 PP, respectively). Clade I includes Craterosiphon Engl. & Gilg and Synaptolepis Oliv. grouped together, with Enkleia Griff. and Dicranolepis Planch. successively sister to them. Clade II includes two strongly supported sister clades comprising Wikstroemia plus Stelleria L. (98 bp/1.0 PP) and Diarthron Turcz. plus Thymelaea and Daphne (89 bp/ 1.0 PP). Edgeworthia Falc. is sister (97 bp/1.0 PP) to this pair of clades. Clade III includes all remaining genera in the analysis. Within clade III, there is strong support for the clade comprising Dais L. and Phaleria Jack (99 bp/1.0 PP) and moderate support for Ovidia Raf. and Dirca L. (68 bp/0.99 PP). Passerina (99 bp/1.0 PP), Struthiola (95 bp/0.98 PP) and Stephanodaphne Baill. (100 bp/1.0 PP) are strongly supported as monophyletic assemblages. The largest genus in this clade, Gnidia, is shown to be highly polyphyletic. Gnidia penicillata Lichtenst. ex Meisn. is strongly supported (73 bp/1.0 PP) as being embedded within Lachnaea, but even with the exclusion of G. penicillata, the remaining species of the genus are dispersed among four clades: clade 1 positions Drapetes muscosus Lam. sister to six Gnidia taxa (69 bp/1.0 PP); clade 2 allies Gnidia pinifolia L. and Gnidia racemosa Thunb. with Struthiola (100 bp/1.0 PP); clade 3 allies 14 species of Gnidia with Pimelea and Thecanthes (99 bp/1.0 PP); and clade 4 retrieves as monophyletic those Gnidia taxa previously recognized as Arthrosolen or Lasiosiphon (100 bp/1.0 PP). DISCUSSION Thymelaeaceae are strongly supported as monophyletic in both parsimony and Bayesian analyses Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Thymelaeaceae and the outgroup families was difficult, and there were many ambiguous regions. Thus, Edgeworthia Meisn., Wikstroemia Spreng., Thymelaea Mill. and Daphne were selected as outgroups for this data matrix. The aligned plastid matrix included the rbcL gene with 1378 base pairs (bp) and the trnL-F region (intron and spacer) with 984 bp. The aligned ITS dataset consisted of 651 bp. As a result of ambiguous alignments, portions of the trnL-F region had to be excluded (three regions, 300 bp in total). Some taxa in the trnL-F region had deletions of 397 bp or more (for example, G. coriacea Meisn., G. galpinii C.H.Wright, G. humilis Meisn., G. squarrosa L., G. subulata Lam., G. aff. viridis, Pimelea clavata Labill., P. decora Domin, P. gilgiana E.Pritz., P. graniticola Rye, P. haematostachya F.Muell., P. holroydii F.Muell., P. punicea R.Br., P. pygmaea Meisn., P. sanguinea F.Muell., P. spiculigera F.Muell., P. trichostachya Lindl., Thecanthes punicea Wikstr. and T. sanguinea (F.Muell.) Rye). The aligned region of ITS contained the most variable sites: 358 (55%) compared with trnL-F with 364 (37%) and rbcL with 304 (22%). The number of potentially informative characters was also higher for ITS (293; 45%) than for trnL-F (226; 23%) or rbcL (193; 14%). Variable positions changed more rapidly for ITS: 4.46 vs. 2.19 (rbcL) and 1.87 (trnL-F). The length of the combined plastid regions (rbcL + trnL-F) included in the analysis was 2362 positions, 28.3% of which were variable and 17.7% potentially informative. Analysis resulted in 2610 equally parsimonious trees with a consistency index (CI) of 0.58 and retention index (RI) of 0.81. The combined plastid analysis (Fig. 1) was largely congruent with the ITS analysis (Fig. 2), except for one moderately supported incongruence. In the plastid tree, Lachnaea was moderately supported as monophyletic (79 bp), whereas, in the ITS tree, monophyly received no support. For Gnidia, the phylogenetic trees did not conflict with each other, although many taxa were reduced to polytomies because of a lack of sufficient informative sequence variation. Where taxon placement differed slightly between the two topologies, bootstrap support was weak and did not provide credible evidence of conflict. We thus directly combined the plastid and nuclear datasets. 62 86 100 56 78 1 99 88 64 60 67 70 85 82 69 73 71 100 2 100 56 87 85 87 74 98 54 3a 52 89 56 57 58 85 76 4 97 3b 97 80 99 100 90 94 96 98 87 78 79 74 97 76 78 96 100 50 50 100 63 74 98 100 96 Aquilarioideae Synandrodaphnoideae Gonystyloideae Outgroups Figure 1. Strict consensus tree based on combined plastid data (rbcL and trnL-F). Bootstrap percentages above 50 are shown above the branches. The three clades indicated are as follows: (I) tropical African and south-eastern Asian taxa; (II) non-African taxa; and (III) southern and tropical African, south-eastern Asian and Australasian species plus two New World taxa. Lineages 1–4 indicate non-monophyletic Gnidia. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 69 62 Thymelaeoideae 79 Clade II 72 Clade I 81 Lachnaea pedicellata Lachnaea rupestris Lachnaea laniflora Lachnaea marlothii Lachnaea oliverorum Lachnaea penicillata Lachnaea axillaris Lachnaea filicaulis Lachnaea gracilis Lachnaea grandiflora Lachnaea pusilla Lachnaea aurea Lachnaea capitata Lachnaea densiflora Lachnaea laxa Lachnaea nervosa Lachnaea alpina Lachnaea macrantha Lachnaea pomposa Lachnaea filamentosa Gnidia penicillata Gnidia penicillata Gnidia cf. anomala Gnidia denudata Gnidia aff. renniana Gnidia geminiflora Gnidia fastigiata Gnidia renniana Drapetes muscosus Passerina ericoides Passerina montivaga Passerina burchellii Passerina falcifolia Passerina drakensbergensis Passerina obtusifolia Passerina nivicola Struthiola ciliata Struthiola striata Struthiola leptantha Struthiola dodecandra Struthiola salteri Struthiola tomentosa Gnidia pinifolia Gnidia racemosa Thecanthes punicea Thecanthes sanguinea Pimelea holroydii Pimelea decora Pimelea haematostachya Pimelea clavata Pimelea graniticola Pimelea argentea Pimelea pygmaea Pimelea spiculigera Pimelea trichostachya Pimelea gilgiana Pimelea forrestiana Gnidia pilosa Gnidia coriacea Gnidia galpinii Gnidia humilis Gnidia subulata Gnidia phaeotricha Gnidia squarrosa Gnidia bojeriana Gnidia dumetorum Gnidia danguyana Gnidia decaryana Gnidia bakeri Gnidia caffra Gnidia calocephala Gnidia gilbertae Gnidia glauca Gnidia kraussiana Gnidia madagascariensis Gnidia sericocephala Gnidia aberrans Gnidia caniflora Gnidia scabrida Gnidia setosa Gnidia singularis Gnidia wikstroemiana Stephanodaphne cuspidata Stephanodaphne cremastachya Stephanodaphne oblongifolia Stephanodaphne capitata Phaleria capitata Dais cotinifolia Peddiea involucrata Peddiea africana Ovidia andina Dirca palustris Gnidia aff. viridis Wikstroemia canescens Stelleria chamaejasme Wikstroemia gemmata Thymelaea hirsuta Daphne mezereum Diarthron vesiculosum Edgeworthia chrysantha Synaptolepis alternifolia Craterosiphon scandens Enkleia siamensis Dicranolepis disticha Gyrinops walla Aquilaria beccariana Synandrodaphne paradoxa Lethedon aff . salicifolia Lethedon balansae Lethedon cernua Gonystylus macrophyllus Deltaria brachyblastophora Solmsia calophyla Arnhemia cryptantha Octolepis dioica Octolepis dioica fo. oblanceolata Octolepis sp. Rhopalocarpus sp. Dialyceras coriaceum Grielum humifusum 407 Clade III PHYLOGENY OF GNIDIA (THYMELAEACEAE) 71 71 93 95 96 2 94 97 90 79 1 100 61 84 100 100 72 100 72 69 73 84 97 3 100 61 100 100 61 71 100 75 100 69 84 90 100 100 100 100 4 100 97 92 100 84 100 86 Figure 2. Strict consensus tree from the parsimony analysis of the nuclear ribosomal internal transcribed spacer (ITS) region. Bootstrap percentages above 50 are indicated above the branches. The two clades indicated are as follows: (II) the non-African taxa; and (III) the southern and tropical African, south-eastern Asian and Australasian species plus two New World taxa. Lineages 1–4 indicate non-monophyletic Gnidia. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 99 98 Lachnaea axillaris Lachnaea filicaulis Lachnaea pedicellata Lachnaea rupestris Lachnaea gracilis Lachnaea grandiflora Lachnaea laniflora Lachnaea marlothii Lachnaea pusilla Struthiola dodecandra Struthiola leptantha Struthiola ciliata Struthiola striata Struthiola tomentosa Gnidia pinifolia Gnidia racemosa Passerina ericoides Passerina montivaga Passerina nivicola Passerina obtusifolia Passerina falcifolia Passerina burchellii Lachnaea alpina Lachnaea filamentosa Lachnaea oliverorum Lachnaea pomposa Gnidia cf. anomala Gnidia denudata Gnidia renniana Lachnaea aurea Lachnaea laxa Drapetes muscosus Gnidia penicillata Lachnaea capitata Lachnaea densiflora Lachnaea nervosa Gnidia aberrans Gnidia scabrida Gnidia setosa Gnidia wikstroemiana Pimelea decora Pimelea haematostachya Thecanthes punicea Thecanthes sanguinea Pimelea argentea Pimelea clavata Pimelea trichostachya Pimelea forrestiana Pimelea spiculigera Pimelea gilgiana Pimelea graniticola Pimelea holroydii Pimelea pygmaea Gnidia phaeotricha Gnidia squarrosa Gnidia galpinii Gnidia humilis Gnidia coriacea Gnidia aff. viridis Stephanodaphne cuspidata Stephanodaphne cremastachya Stephanodaphne oblongifolia Stephanodaphne capitata Ovidia andina Dirca palustris Peddiea involucrata Peddiea africana Gnidia dumetorum Gnidia madagascariensis Gnidia gilbertae Gnidia bojeriana Gnidia caffra Gnidia calocephala Gnidia sericocephala Gnidia kraussiana Gnidia danguyana Gnidia decaryana Gnidia bakeri Dais cotinifolia Wikstroemia gemmata Wikstroemia canescens Thymelaea hirsuta Daphne mezereum Edgeworthia chrysantha Thymelaeoideae 67 Clade III A. J. BEAUMONT ET AL. Clade II 408 PHYLOGENY OF GNIDIA (THYMELAEACEAE) 27 204/1.00 99 Thymelaeoideae Clade III 5 14 Clade II 6/1.00 84 7 Clade I 26/1.00 98 198/0.77 78 6/1.00 66 Aquilarioideae Synandrodaphnoideae Gonystyloideae Outgroups Figure 3. One of the equally most parsimonious trees from the combined rbcL, trnL-F region and internal transcribed spacer (ITS) analysis (consistency index, 0.48; retention index, 0.75; tree length, 3040 steps). Numbers displayed above each branch are Fitch lengths (DELTRAN optimization)/PP > 0.5 from Bayesian analysis (in bold). Percentages below the branches are bootstrap percentages equal to or greater than 50. Full arrows indicate groups not present in the Fitch strict consensus tree. The three clades indicated are as follows: (I) the tropical African and south-eastern Asian taxa; (II) the non-African taxa; and (III) the southern and tropical African, south-eastern Asian and Australasian species plus two New World taxa. Lineages 1–4 indicate non-monophyletic Gnidia. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 12/1.00 89 7 6 Lachnaea pedicellata Lachnaea rupestris Lachnaea laniflora Lachnaea marlothii 3/1.00 Lachnaea oliverorum 8/0.93 51 1 Lachnaea penicillata 3/0.96 4 Lachnaea axillaris 6/1.00 4 Lachnaea filicaulis 6 67 5 Lachnaea grandiflora 4/0.76 5/1.00 4 Lachnaea pusilla 12/1.00 52 6 Lachnaea gracilis 78 2 Lachnaea laxa 4/0.76 11 4 Lachnaea nervosa 13 4/0.97 Lachnaea aurea 8/1.00 5 Lachnaea capitata 9/1.00 55 3 Lachnaea densiflora 71 1 Gnidia penicillata 28/1.00 7/1.00 Gnidia penicillata 100 73 7 Lachnaea alpina 4 2/0.98 Lachnaea pomposa 2 9/1.00 Lachnaea macrantha 50 4 Lachnaea filamentosa 87 23 Gnidia cf. anomala 1/0.75 6 4/0.76 Gnidia denudata 8/1.00 5 12/0.78 Gnidia aff. renniana 60 66 7 Gnidia geminiflora 81 30/1.00 2 Gnidia fastigiata 21/1.00 8 1 9/1.00 100 Gnidia renniana 100 69 43 Drapetes muscosus 4 Passerina drakensbergensis 8/1.00 5 8/1.00 5/0.91 Passerina obtusifolia 89 3 6 Passerina falcifolia 70 6 Passerina burchellii 3 26/1.00 8 Passerina ericoides 5/0.99 18/1.00 97 8 Passerina montivaga 91 99 18 Passerina nivicola 2 Struthiola salteri 13/0.84 8 Struthiola tomentosa 7/0.66 4 6/0.85 Struthiola dodecandra 7 Struthiola leptantha 13/0.98 9 Struthiola ciliata 95 2/0.93 28/1.00 8 Struthiola striata 72 100 31/1.00 25 Gnidia pinifolia 2 100 44 Gnidia racemosa 25 Pimelea forrestiana 8/1.00 12 Pimelea spiculigera 4 74 20 Pimelea graniticola 6 6/1.00 29 Pimelea holroydii 28 Pimelea gilgiana 3/0.55 22 14/0.95 4 Pimelea pygmaea 23 Pimelea argentea 8/0.91 29 12/1.00 Pimelea clavata 71 8/1.00 17 Pimelea trichostachya 69 63 10 Pimelea decora 39/1.00 2 Pimelea haematostachya 6/0.99 14/0.99 100 13 Thecanthes punicea 49/1.00 79 10 Thecanthes sanguinea 100 10 Gnidia pilosa 2 9 Gnidia caniflora 1 1 6/0.86 54 Gnidia setosa 1 1 Gnidia singularis 4 58/1.00 19/1.00 Gnidia scabrida 3 42/1.00 98 Gnidia aberrans 81 14/0.98 5 100 Gnidia wikstroemiana 58 19 Gnidia phaeotricha 23/1.00 26 40/1.00 Gnidia squarrosa 3 64 99 Gnidia humilis 1 6 Gnidia subulata 2 15/1.00 7 Gnidia galpinii 20/1.00 15 96 Gnidia coriacea 18 87 Gnidia aff. viridis 5 Stephanodaphne cremastachya 1 6 Stephanodaphne oblongifolia 2 9 31/1.00 Stephanodaphne capitata 52 3 100 8/0.95 Stephanodaphne cuspidata 17/1.00 31 Ovidia andina 18/0.99 66 5 37 Dirca palustris 68 5 Peddiea involucrata 28/1.00 10 Peddiea africana 100 4 Gnidia caffra 24/1.00 8 22/1.00 Gnidia calocephala 100 24 3/1.00 99 Gnidia sericocephala 15 16 95 Gnidia kraussiana 4 14/1.00 Gnidia glauca 8 34/1.00 79 Gnidia dumetorum 3/0.78 6 100 9/0.78 Gnidia madagascariensis 57 20/1.00 6 73 Gnidia gilbertae 37/1.00 9 84 4 Gnidia bojeriana 100 Gnidia danguyana 30/1.00 40 26 13/0.96 Gnidia decaryana 100 49 Gnidia bakeri 8/1.00 84 12 97 Phaleria capitata 35/1.00 7 Dais cotinifolia 99 10 Wikstroemia canescens 12/0.98 13 Stelleria chamaejasme 23/1.00 79 31 Wikstroemia gemmata 98 56/1.00 44 Thymelaea hirsuta 2/0.71 97 56 34/1.00 25/1.00 Daphne mezereum 76 17 89 Diarthron vesiculosum 95 68 Edgeworthia chrysantha 6 Synaptolepis alternifolia 6/1.00 3 Craterosiphon scandens 6/0.85 96 8 15/1.00 Enkleia siamensis 76 19 96 Dicranolepis disticha 7 Gyrinops walla 36/1.00 24 Aquilaria beccariana 100 68 Synandrodaphne paradoxa 9 Gonystylus macrophyllus 8 6 Arnhemia cryptantha 2/1.00 5 Deltaria brachyblastophora 6 3 Solmsia calophyla 13/1.00 75 1/0.99 Lethedon aff. salicifolia Lethedon balansae 1 62 1 Lethedon cernua 51 9/1.00 Octolepis dioica 25/1.00 Octolepis dioica fo. oblanceolata 96 3 97 Octolepis sp. 12 Rhopalocarpus sp. 50/1.00 6 Dialyceras coriaceum 100 Grielum humifusum 3/1.00 409 410 A. J. BEAUMONT ET AL. RELATIONSHIPS WITHIN THYMELAEOIDEAE Domke (1934) recognized four tribes in Thymelaeoideae, namely Dicranolepideae, Phalerieae, Daphneae and Gnidieae. A previous molecular study (Van der Bank et al., 2002) supported the results obtained here: that Thymelaeoideae, as circumscribed by Domke (1934), are shown to be a monophyletic group that includes three highly supported clades. Clade I comprises tropical African taxa plus the tropical Asian genus Enkleia Griff., and partly corresponds to Domke’s (1934) tribe Dicranolepideae and subtribes Linostomatinae and Dicranolepidinae. Clade II comprises seven taxa from Asia and the Mediterranean region, including northern Africa, and represents tribes Daphneae (subtribes Wikstroemiinae, Dendrostellerinae and Daphninae) and Gnidieae (subtribe Thymelaeinae; Domke, 1934). Clade III, the largest clade, includes southern and tropical African, southeastern Asian and Australasian species plus New World taxa. The taxa in clade III collectively represent, in part, the tribes Phaleriae (subtribe Phaleriinae), Daphneae (subtribe Daphnopsinae) and Gnidiinae (subtribes Drapetinae, Gnidiinae and Passerininae; Domke, 1934). Clade III received moderate support in the parsimony analysis and high support in the Bayesian analysis and comprises several lineages with low resolution because of low levels of genetic variation. The southern African Dais cotinifolia L. and the southern Pacific Phaleria capitata Jack grouped together with high support in both the parsimony and Bayesian analyses (99 bp/1.0 PP) and are moderately supported as being sister to the rest of clade III (79 bp/1.00). Two species of Peddiea Harv. from Africa and Madagascar are weakly supported in the Bayesian analysis (0.57 PP) as sister to clades 1, 2 and 3. In the parsimony analysis, however, they form an unsupported clade with Dirca and Ovidia from North and South America, respectively (Nevling, 1964; Heads, 1990) and Stephanodaphne from Madagascar and Mayotte (Rogers, 2004). The bilocular ovaries in Peddiea are anomalous among the otherwise unilocular condition in the rest of Thymelaeoideae. Furthermore, fruits are drupes in contrast with the dry fruits of Gnidia and other taxa in clades 1–3 (Peterson, 1978). There was strong support for the monophyly of Stephanodaphne (100 bp/1.0 PP), with Ovidia and Dirca sister to it (0.95 PP). The molecular data presented here strongly indicate that Gnidia, in its broad, inclusive sense (i.e. that of Peterson, 1959), is not monophyletic, and comprises at least four moderately to strongly supported clades in the parsimony and Bayesian analyses (Fig. 3). Different groups of Gnidia species are embedded within various southern African and Australian genera. In addition, six Gnidia taxa are sister to the monotypic Drapetes. One group of Gnidia spp. was shown to be sister to Passerina. The monophyly of Passerina was highly supported, corresponding to the findings of Van der Bank et al. (2002). Bredenkamp & Van Wyk (1996) suggested the placement of Passerina as the sole member of subtribe Passerininae on the basis of pollen morphology. Passerina is separated morphologically from the rest of Thymelaeaceae by the extrorse dehiscence of its anthers (Beyers & Marais, 1998; Bredenkamp & Beyers, 2000) and is also the only genus in Thymelaeaceae adapted to wind pollination. Our analysis provided clear evidence that Passerina is embedded within subtribe Gnidiinae as currently circumscribed, which includes Lachnaea, Gnidia and Struthiola. Lachnaea is sister to the Passerina/Gnidia clade, a placement weakly supported in the parsimony analysis and strongly supported in the Bayesian analysis. Support was moderate in the parsimony analysis and high in the Bayesian analysis for the monophyly of a slightly expanded circumscription of Lachnaea to include G. penicillata (73 bp/1.0 PP). This placement was confirmed by the inclusion of two accessions of G. penicillata in the analysis. Gnidia penicillata is anomalous in Gnidia with several features more typical of Lachnaea: a slender, conical stigma (vs. the capitate stigma of Gnidia), clearly obconical style (vs. generally uniformly cylindrical styles or very slightly obconical styles of Gnidia) and royal blue calyx lobes. Flowers in shades of blue, mauve or pink occur in several species of Lachnaea, but are rare in Gnidia. In addition, the floral scales number four to eight per flower in G. penicillata, whereas the floral scales always number eight in Lachnaea (Beyers, 2001). Gnidia penicillata more closely resembles other Gnidia species in its floral disc, and its floral scales are inserted above the level of the lower series of Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 (99 bp/1.0 PP). The major clades found in our analyses support those identified in the molecular analysis of Van der Bank et al. (2002) and are broadly compatible with the four subfamilies [Synandrodaphnoideae (= Gilgiodaphnoideae), Aquilarioideae, Thymelaeoideae and Gonystyloideae] recognized by Domke (1934), although the circumscription of Gonystyloideae should be re-examined. This study shows Gonystyloideae to be paraphyletic with the inclusion of Octolepis. Furthermore, although Domke (1934) included Lethedon, Solmsia and Octolepis in Aquilarioideae, our results support Rye (1990) who moved Lethedon to Gonystyloideae. The inclusion of Solmsia in Gonystyloideae corresponds to the findings of Domke (1934) and Van der Bank et al. (2002). PHYLOGENY OF GNIDIA (THYMELAEACEAE) stamens. In Lachnaea, the floral scales all arise at the same level as the filaments or are all inserted below the stamens. The relative positions of the floral scales and stamens have traditionally been used to distinguish Gnidia from Lachnaea (Wright, 1915; Beyers, 2001). Our results challenge the usefulness of this character in delimiting these genera, and the position of G. penicillata in Lachnaea and its taxonomic implications will be considered in a separate paper. AMONG GNIDIA SPECIES AND OTHER GENERA Clade 1 In this clade, Drapetes muscosus is sister to six representatives of Gnidia: G. renniana Hilliard & B.L.Burtt, G. fastigiata Rendle, G. geminiflora E.Mey. ex Meisn., G. aff. renniana, G. denudata Lindl. and G. anomala Meisn. This grouping has weak support in the parsimony analysis and strong support in the Bayesian analysis. The position of D. muscosus in clade 1 supports Domke’s (1934) classification of this New World genus in the tribe Gnidieae, together with African, European/North African (Thymelaea) and Australasian (Pimelea) taxa, rather than with other New World taxa that are representative of his tribes Dicranolepideae and Daphneae. The distribution of D. muscosus in southern South America and islands in the southern Atlantic and Pacific Oceans represents a geographical disjunction with Gnidia. Although the flowers of Drapetes resemble those of Gnidia, these genera differ in the terminal rather than the sublateral attachment of the style, and the absence of internal phloem in Drapetes. Furthermore, Drapetes lacks the tenacious (stripping) bark that otherwise typifies the family. Readily identifiable morphological synapomorphies are lacking for clade 1. All taxa in this clade have eight stamens, although the flowers of G. anomala (syn. Pseudognidia anomala; Phillips, 1944) are often four staminate through the reduction or loss of the upper series of stamens. Elsewhere in Gnidia, fourstaminate species include G. aberrans C.H.Wright [syn. Basutica aberrans (C.H.Wright) E.Phillips; Phillips, 1944] and G. propinqua Hilliard. Gnidia aberrans is not included in clade 1, but in clade 3 with the similarly four-staminate G. singularis Hilliard, plus eight-staminate Gnidia spp. and characteristically two-staminate Australian taxa. These placements suggest that a reduction in stamen number has occurred several times within Gnidia and that the number of stamens alone is not taxonomically or phylogenetically informative. Gnidia singularis and D. muscosus both have flowers with four stamens, and filaments longer than anthers. Generally, filaments are short in Gnidia. The topology suggests that both states are independently derived in these two species. A detailed analysis of morphological characters in this clade and sampling of more species may better define clade 1. Clade 2 Gnidia pinifolia and G. racemosa form a grade with Struthiola in both the parsimony and Bayesian analyses. Resolution within Struthiola is low in the parsimony but moderate in the Bayesian analysis. These results correspond to the findings of Van der Bank et al. (2002), in which G. racemosa was well supported as sister to three representatives of Struthiola. Inflorescences in spikes, flowers each with four, not eight, stamens and bracteoles distinguish Struthiola from most Gnidia spp. (Pearson, 1913; Wright, 1915; Peterson, 1978; Hilliard, 1993). Gnidia pinifolia and G. racemosa have dissimilar features and, furthermore, scarcely resemble Struthiola. Inflorescences are not spicate, and bracteoles are lacking in both species; instead, we find many-flowered, terminal bracteate clusters in G. pinifolia and scattered, single flowers or few-flowered pseudobracteate clusters in G. racemosa. Furthermore, flowers of both taxa have eight, not four, stamens. Morphological synapomorphies are lacking for an expanded generic circumscription of Struthiola to include G. pinifolia and G. racemosa, and generic limits will have to be reconsidered for these taxa. Clade 3 Gnidia pilosa from mainland Africa is placed as sister to 13 species of the Australasian genera Pimelea and Thecanthes included in our analyses. This result is similar to that obtained by Van der Bank et al. (2002), in which G. pilosa and G. subulata Lam. (as G. aff. viridis) were allied to Pimelea. The remainder of clade 3 comprises 13 Gnidia taxa. Our molecular findings support the conclusions of Gilg (1894), Bentham (1873), Threlfall (1982) and Motsi et al. (MC Mosti, University of Johannesburg, Auckland Park, South Africa, unpubl. data) that Thecanthes should be included within Pimelea and that subtribe Pimeleinae is therefore monogeneric. The position of G. pilosa as sister to Pimelea is morphologically incongruous. Gnidia pilosa instead resembles two other African species: G. leiosiphon Gilg (Domke) and G. ovalifolia Meisn. All three species have paired, flat, flimsy leaves, with long internodes and few-flowered, ebracteate umbels with primary floral axes that lengthen during fruit development. These features led Gilg (1894) to establish Englerodaphne; Phillips (1944) maintained Englerodaphne but conceded that there were no ‘outstanding structural differences’ between the flowers of Gnidia and Englerodaphne. Modern treatments, however (for example, Arnold & De Wet Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 RELATIONSHIPS 411 412 A. J. BEAUMONT ET AL. Clade 4 This strongly supported clade comprises three subclades, containing southern and tropical African and Madagascan taxa. Our results expand on those obtained by Van der Bank et al. (2002), in which G. kraussiana Meisn. (= Lasiosiphon kraussii Meisn.) was separated from the rest of Gnidia. These initial results provided some support for reinstating the genus Lasiosiphon. Fresenius (1838) established Lasiosiphon for species with pentamerous flowers, ten stamens, small floral scales, hairy floral tubes with long, silky basal hairs (in all species hairs are smooth), involucral leafy bracts surrounding heads of many yellow flowers and alternate leaves. Lasiosiphon was recognized by Endlicher (1847), Leandri (1950), Meissner (1857), Wright (1915) and Phillips (1944), but not by Gilg (1894), Staner (1935), Peterson (1959, 1978) or Robyns (1975), who considered it a synonym of Gnidia. The first subclade within clade 4 comprises four Madagascan endemics: G. dumetorum Leandri, G. madagascariensis (Lam.) Decne. ex Cambess., G. gilbertae Drake and G. bojeriana Baill. Gnidia gilbertae has tetramerous flowers, but the rest have pentamerous flowers and, as such, were previously classified under Lasiosiphon (Leandri, 1950). This subclade received high support in the Bayesian analysis (1.00 PP) and moderate support (BP 84) was obtained in parsimony analysis. The second subclade includes the tropical and southern African species G. caffra (Meisn.) Gilg, G. calocephala Gilg, G. sericocephala (Meisn.) Gilg ex Engl. and G. kraussiana, with the position of G. glauca (Fresen.) Gilg unresolved. Gnidia kraussiana and G. caffra were previously included in Lasiosiphon. Gnidia calocephala and G. sericocephala also have pentamerous flowers and ten stamens, but were previously classified under Arthrosolen not Lasiosiphon. Arthrosolen included Gnidia-like species with no floral scales and represented a diverse collection of species, morphologically at odds with each other (Wright, 1915). The last subclade of clade 4 is an exclusively Madagascan group, highly supported in both parsimony and Bayesian analyses, and comprising G. bakeri Gilg, G. danguyana Leandri and G. decaryana Leandri. All species in this subclade have tetramerous flowers. Gnidia decaryana, however, more closely resembles species once included in Englerodaphne with flowers arranged in dense terminal clusters on comparatively long, bare peduncles. As the fruits mature, the primary floral axis elongates and the internodes lengthen, which is also a feature of Englerodaphne. These morphological traits are thus interpreted as convergent. The third species in this subclade, G. bakeri, is morphologically more similar to both G. calocephala and G. sericocephala than to G. danguyana or G. decaryana with which it groups in our results. Again, morphological synapomorphies supporting the molecular association of G. bakeri, G. danguyana and G. decaryana are elusive. CONCLUSIONS Few morphological characters have previously been used to distinguish genera within Thymelaeaceae, and the literature is full of discussions on the relative merits of the various characters that have been employed to delimit genera. Recent phylogenetic studies, for example Van der Bank et al. (2002) and Galicia-Herbada (2006), have included representatives of Thymelaeaceae as place holders, in the hope that the resolution of relationships would aid in the development of an improved taxonomic scheme. This hope appears to be unfounded because of a lack of Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 (1993), follow Peterson (1959) and list Englerodaphne as a synonym of Gnidia. Gnidia aberrans, G. caniflora Meisn., G. setosa Wikstr., G. scabrida Meisn., G. singularis and G. wikstroemiana Meisn. form a well-supported subclade within clade 3 in both the parsimony and Bayesian analyses. All of these species have local distributions in South Africa and tetramerous flowers. These six species represent different degrees of reduction of the androecium from eight-staminate (G. caniflora and G. setosa) to the upper series of stamens being smaller than the lower series (G. scabrida) to four-staminate (G. aberrans and G. singularis) or gynodioecious and eight-staminate (G. wikstroemiana; Beaumont, Edwards & Smith, 2006). A small subclade in clade 3 comprises two morphologically dissimilar species: G. phaeotricha Gilg and G. squarrosa Druce. Gnidia phaeotricha plants are cryptic within their grassland habitats, producing annual stems from a woody perennial rootstock, spike-like inflorescences with small, non-colourful flowers with very short tubes and floral axes that lengthen in fruit. In contrast, G. squarrosa is shrubby with capitate inflorescences and floral axes that do not lengthen in fruit (Wright, 1915; AJB, pers. obs.) making synapomorphies with which to define this subclade elusive. However, sister to the rest of clade 3 is a morphologically identifiable subclade comprising former members of Epichroxantha (Meissner, 1857), namely G. galpinii, G. humilis, G. coriacea, G. subulata and G. aff. viridis. These species resemble each other, sharing pungent, coriaceous leaves (except G. humilis in which leaves are more flimsy), tetramerous funnel-shaped flowers with four large membranous petals and eight stamens (Bond & Goldblatt, 1984; Levyns, 1950; A. J. Beaumont, pers. observ.). PHYLOGENY OF GNIDIA (THYMELAEACEAE) OPTION 1: Lachnaea including Gnidia penicillata 1 Gnidia cf. anomala Gnidia denudata Gnidia aff. renniana Gnidia geminiflora Gnidia fastigiata Gnidia renniana Drapetes muscoides Passerina 2 Section: Lachnaea Section: Drapetes Section: Drapetes Gnidia subg. Lachnaea Gnidia subg. Gnidia Section: Passerina Section: Gnidia Gnidia subg. Gnidia Gnidia pinifolia Gnidia racemosa Section: Pimelea Section: Pimelea Pimelea including Thecanthes Gnidia subg. Pimelea 3 4 Gnidia pilosa Gnidia caniflora Gnidia setosa Gnidia singularis Gnidia scabrida Gnidia aberrans Gnidia wikstroemiana Gnidia phaeotricha Gnidia squarrosa Gnidia humilis Gnidia subulata Gnidia galpinii Gnidia coriacea Gnidia aff. viridis Stephanodaphne cremastachya Stephanodaphne oblongifolia Stephanodaphne capitata Stephanodaphne cuspidata Ovidia andina Dirca palustris Peddiea involucrata Peddiea africana Gnidia caffra Gnidia calocephala Gnidia sericocephala Gnidia kraussiana Gnidia glauca Gnidia dumetorum Gnidia madagascariensis Gnidia gilbertae Gnidia bojeriana Gnidia danguyana Gnidia decaryana Gnidia bakeri Phaleria capitata Dais cotinifolia Gnidia subg. Epichroxantha Lasiosiphon Pimelea Section: Epichroxantha Lasiosiphon Figure 4. The two options proposed for a monophyletic circumscription of genera within Thymelaeoideae. obvious morphological synapomorphies observed for the clades found in the molecular studies. Many of the characters previously used to delimit genera in the family represent parallel adaptations. The results from the DNA studies indicate that the current generic limits are untenable, yet the way forward is unclear. The lumping of separate clades of Gnidia into other genera to which they are related would require paying no attention to the characters that have been used as the basis for these other genera, and it is not clear which other characters could replace them. If lumping is untenable, then perhaps splitting is a better option. Although additional species of this large genus should be included in the analysis pending a final classification of the subfamily, current results are sufficient to warrant a partial solution. We present two options towards a monophyletic circumscription of genera in the subfamily (Fig. 4). Option 1 proposes a very broadly circumscribed Gnidia, comprising clades 1, 2 and 3 and inclusive of all taxa between G. cf. anomala and G. aff. viridis. Within this large genus, it is possible to recognize three subgenera: subgenus Gnidia to include all Gnidia spp. in clades 1 and 2 plus the genera Lachnaea, Passerina and Struthiola; subgenus Pimelea for the genus Pimelea plus associated species from G. squarrosa to G. pilosa; and subgenus Epichroxantha for all taxa inclusive of G. galpinii to G. aff. viridis. The following monophyletic, morphologically diagnosable lineages within the large subgenus Gnidia can be recognized at sectional level: section Drapetes comprising D. muscosus plus associated species of Gnidia; section Passerina for species currently placed Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Struthiola OPTION 2: Section: Lachnaea Section: Passerina 413 414 A. J. BEAUMONT ET AL. DISCUSSION OF OPTIONS Option 1 proposes a synthetic circumscription of the genus Gnidia. As defined here, the genus encompasses well-known taxa from both Africa and Australia, which have clear monophyletic sublineages, including the African groups Lachnaea, Passerina and Struthiola, and the Australasian Pimelea (including Thecanthes). Such a circumscription is maximally stable. This is a distinct advantage given the weak correspondence between morphological discontinuities and monophyletic lineages. A significant disadvantage is the large number of nomenclatural changes that will be required. Flowers with two stamens distinguish Pimelea from other members of Thymelaeoideae, although our analysis suggests a closer link with some species of Gnidia than previously realized. Pimelea is large with ±110 species, and its synonymy under Gnidia would require extensive and unpopular name changes at this stage. Sampling of more Gnidia and Pimelea spp. would undoubtedly help to clarify the relationships among these groups. However, although the outcome of this may indeed make necessary extensive species name changes in Pimelea in future, we feel it prudent at this time to maintain the genus Pimelea (although aware of the obvious phylogenetic links with Gnidia) until we can offer more evidence in support of a concept of Gnidia that embraces Pimelea, either in the whole or in part. Some changes to the taxonomy of the family can be proposed without encountering too many problems (for example, Thecanthes should remain in Pimelea; MC Motsi, University of Johannesburg, Auckland Park, South Africa, submitted). We also formally propose the reinstatement of Lasiosiphon. Substantial changes in the circumscription of Gnidia are still required, but will have to await the results of much more detailed studies. ARGUMENTS FOR REINSTATING LASIOSIPHON All pentamerous Gnidia species sampled here group together in clade 4. A well-supported clade comprising Stephanodaphne, Peddiea, Dirca and Ovidia separates clade 4 from other Gnidia clades. We argue that this separation indicates a distinctive evolutionary route of specialization, although morphological synapomorphies for clade 4 as a whole are elusive. Clade 4 also contains two pentamerous Gnidia spp., G. calocephala and G. sericocephala, included previously in Arthrosolen because they lack floral glands. However, these species share the following characters with African species formerly classified as Lasiosiphon: flowers grouped in heads with an involucre of leafy bracts, hairy floral tubes, capitate stigmas and fleshy pedicels. We argue that the absence of floral glands should not exclude G. calocephala and G. sericocephala from Lasiosiphon in the same way that Struthiola anomala Hilliard is not excluded from Struthiola despite its lack of floral scales (Hilliard, 1993). The inclusion of tetramerous taxa and species with ebracteate few-flowered inflorescences expands on Fresenius’ (1838) original idea of Lasiosiphon as a solely pentamerous group with flowers always in heads with bracts. The diversity of morphological features represented among members of clade 4 may make it necessary to recognize subgeneric groups within Lasiosiphon in future. TAXONOMIC CHANGES We reinstate the genus Lasiosiphon as follows: Lasiosiphon Fresen. emend. A.J.Beaumont, Flora 21: 603 (1838). – Type: Lasiosiphon glaucus Fresen., Ethiopia: Rüppel s.n. (FR, holotype!). Synonyms, Gnidia L. pro parte; Arthrosolen C.A.Mey. pro parte. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 in this genus; section Lachnaea for species of Lachnaea plus G. penicillata and allied species; and section Gnidia for the remaining species of Gnidia plus Struthiola. The subdivision of subgenus Pimelea is less clear and should only be attempted following increased sampling within Gnidia. The remaining species of Gnidia comprise a single lineage from Africa and Madagascar. Most of the species in this lineage were previously classified under Lasiosiphon or Arthrosolen, and we propose that the genus Lasiosiphon be reinstated for the members of clade 4 (Fig. 4). Option 2 proposes a less extensive circumscription of the genus Gnidia, which should be restricted to include those taxa in clades 1 and 2, that is the genera Lachnaea, Passerina and Struthiola. Within this clade, two subgenera may be recognized: subgenus Gnidia to include Struthiola and related species of Gnidia (features that may help to define this group include anthers on short filaments, uniformly cylindrical styles and capitate stigmas); and subgenus Lachnaea for all remaining taxa, including Lachnaea and Passerina (features that may help to identify members in this subgenus include extrorse anthers, styles that widen towards the stigmas, often non-capitate stigmas and bracteoles). Within subgenus Gnidia, the eight lineages identified in option 1 are to be treated as sections, with an additional three sections in subgenus Lachnaea: section Drapetes, with the seven taxa outlined in option 1, section Passerina and section Lachnaea, all with the same taxa as in option 1. The genus Pimelea should be retained for members of clade 3, with subdivision into subgenera following more comprehensive analysis. The genus Lasiosiphon should be reinstated for the members of clade 4. PHYLOGENY OF GNIDIA (THYMELAEACEAE) ACKNOWLEDGEMENTS REFERENCES Akaike H. 1979. A Bayesian extension of the minimum AIC procedure of autoregressive model fitting. Biometrika 66: 237–242. Arnold TH, De Wet BC. 1993. Plants of southern Africa: names and distribution. Memoirs of the Botanical Survey of South Africa 62: 1–825. Bayer C, Fay MF, De Bruijn AY, Savolainen V, Morton CM, Kubitzki K, Alverson WS, Chase MW. 1999. Support for an expanded family concept of Malvaceae within a recircumscribed order Malvales: a combined analysis of plastid atpB and rbcL DNA sequences. Botanical Journal of the Linnean Society 129: 267–303. Beaumont AJ, Edwards TJ, Smith FR. 2006. The first record of gynodioecy in a species of Gnidia (Thymelaeaceae) from South Africa. Botanical Journal of the Linnean Society 152: 219–233. Bentham G. 1873. Flora Australiensis: a description of the plants of the Australian territory, Vol. VI. London: L. Reeve & Co. Beyers JBP. 2001. The Cape genus Lachnaea (Thymelaeaceae): a monograph. Pretoria: National Botanical Institute, Strelitzia 11. Beyers JBP, Marais EM. 1998. Palynological studies of the Thymelaeaceae of the Cape Flora. Grana 37: 193–202. Bond P, Goldblatt P. 1984. Thymelaeaceae: Plants of the Cape Flora. Journal of South African Botany Supplementary 13: 429–434. Bredenkamp CL, Beyers JBP. 2000. Thymelaeaceae (Dilleniidae-Euphorbiales). In: Leistner OA, ed. Seed plants of southern Africa: families and genera. Pretoria: National Botanical Institute, Strelitzia 10, 544–546. Bredenkamp CL, Van Wyk AE. 1996. Palynology of the genus Passerina (Thymelaeaceae): relationships form and function. Grana 35: 335–346. Burtt Davy BJ. 1926. A manual of the flowering plants and ferns of the Transvaal with Swaziland, South Africa. New York: Longmans, Green and Co. Ltd. Ding Hou L. 1960. Thymelaeaceae. Flora Malesiana 6: 1–48. Domke W. 1934. Untersuchungen über die systematische und geographische Gliederung der Thymelaeaceen. Bibliotheca Botanica 27: 1–151. Doyle JJ, Doyle JS. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, Botanical Society of America 19: 11–15. Dyer RA. 1975. The genera of southern African flowering plants. Volume I, Dicotyledons. Pretoria: Department of Agricultural Technical Services. Endlicher S. 1847. Synopsis coniferarum. Sankt-Gallen: Scheitlin and Zollikofer. Fay MF, Bayer C, Alverson WS, De Bruijn A, Chase MW. 1998. Plastid rbcL sequence data indicate a close affinity between Diegodendron and Bixa. Taxon 47: 43–50. Fitch WM. 1971. Towards defining the course of evolution: minimum change for a specific tree topology. Systematic Zoology 20: 406–416. Fresenius JBGW. 1838. Lasiosiphon. Flora 21: 602. Galicia-Herbada D. 2006. Origin and diversification of Thymelaea (Thymelaeaceae): inferences from a phylogenetic study based on ITS (rDNA) sequences. Plant Systematics and Evolution 257: 159–187. Gilg E. 1894. Thymelaeaceae. In: Engler A, Prantl K, eds. Die natürlichen Pflanzenfamilien III, 6a. Leipzig: Engelman, 216–245. Goldblatt P, Manning JC. 2000. Cape plants: a conspectus of the Cape flora of South Africa. Pretoria: National Botanical Institute, Strelitzia 9. Heads MJ. 1990. A revision of the genera Kelleria and Drapetes (Thymelaeaceae). Australian Systematic Botany 3: 595–652. Hilliard OM. 1993. New species in Petalacte (Compositae) and Struthiola (Thymelaeaceae). Edinburgh Journal of Botany. 50: 181–184. Hutchinson J. 1967. The genera of flowering plants. Volume II, Dicotyledons. Oxford: Clarendon Press. Leandri J. 1950. Thymeleacées. Flora de Madagascar et des Comores 146: 1–38. Levyns MR. 1950. Thymelaeaceae Reichb. In: Adamson RS, Salter TM, eds. Flora of the Cape Peninsula. Cape Town: Juta, 117–164. Linnaeus C. 1753. Species plantarum. A facsimile of the first edition 1753. Volume 1. London: The Ray Society. Marloth R. 1925. The flora of South Africa: with synoptical tables of the genera of the higher plants. Cape Town: Darter Bros. & Co. Meissner CF. 1857. Thymelaeaceae. In: de Candolle AP, ed. Prodromus systematis naturalis regni vegetabilis 14. Paris: V. Masson, 493–605. Nevling LI Jr. 1964. Notes on the genus Ovidia. Darwiniana 13: 72–86. Pearson HHW. 1913. Order CXVIII: Thymelaeaceae. In: Thiselton-Dyer WT, ed. Flora of tropical Africa 4. London: L. Reeve & Co, 212–238. Peterson B. 1959. Some interesting species of Gnidia. Botaniska Notiser 112: 465–480. Peterson B. 1978. Thymelaeaceae. In: Polhill RM, ed. Flora of tropical East Africa. London: Crown Agents for Overseas Governments and Administrators, 1–37. Peterson B. 1982. Thymelaeaceae: Notes on Gnidia. Notes on African plants. Bothalia 14: 81–82. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 The authors thank A. Johns, M. Johns and Z. Rogers for plant samples; the Moss Herbarium (J), University of the Witwatersrand and the Bews Herbarium (NU) for material from herbarium specimens; Colette Robinson and Annemari van Niekerk who helped to compile the data matrix, and Stefan Dressler and Rainer Döring, Herbarium Senckenbergianum (FR), for the digital loan of the type of Gnidia glauca (syn. Lasiosiphon glaucus). This work was supported by the Thuthuka Programme (National Research Foundation, South Africa) and the University of Johannesburg, Faculty of Sciences. 415 416 A. J. BEAUMONT ET AL. and consensus in the cotton tribe (Malvaceae). Systematic Botany 22: 259–290. Staner P. 1935. Tribu Gnidieae. – Gnidia L. Bulletin du Jardin Botanique de l’Etat à Bruxelles. 13: 340–367. Swofford DL. 2003. PAUP*: phylogenetic analysis using parsimony, version 4.0. Sunderland, MA: Sinauer Associates. Threlfall S. 1982. The genus Pimelea (Thymelaeaceae) in eastern mainland Australia. Brunonia 5: 113–201. Van der Bank M, Fay MF, Chase MW. 2002. Molecular phylogenetics of Thymelaeaceae with particular reference to African and Australian genera. Taxon 51: 329–339. White TJ, Burns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenies. In: Innis M, Gelfand D, Snisky J, White T, eds. PCR protocols: a guide to methods and applications. San Diego, CA: Academic Press, 315–322. Wiens JJ. 1998. Combining data sets with different phylogenetic histories. Systematic Biology 47: 568–581. Wright CH. 1915. Order CXVIII: Thymelaeaceae. In: Thiselton-Dyer WT, ed. Flora Capensis 5. London: L. Reeve & Co, 25–79. Yoder AD, Irwin JA, Payseur BA. 2001. Failure of the ILD to determine data combinability for slow loris phylogeny. Systematic Biology 50: 408–424. APPENDIX 1 List of taxa with voucher information and GenBank accession numbers for each DNA region. Species, voucher specimen, herbarium acronym, trnL-F GenBank accession, rbcL GenBank accession, ITS GenBank accession. Neuradaceae: Grielum humifusum Thunb., Chase 5711, (K), —, AJ402955†, —. Sphaerosepalaceae: Dialyceras coriaceum (Capuron) J.-F.Leroy, Schatz & al. 3848, MO, —, AJ29723†, —. Rhopalocarpus sp., Chase 906, K, AJ30864†, Y15148*, —. Thymelaeaceae: Aquilaria beccariana Tiegh., Chase 1380, K, AJ308643†, Y15149*, —. Arnhemia cryptantha Airy Shaw, Lazarides 7870, K, AJ308678†, AJ297236†, —. Craterosiphon scandens Engl. & Gilg, Lock 84/84, K, —, AJ297235†, —. Dais cotinifolia L., Chase 1381, K, AJ308644†, AJ297234†, AJ744928. Daphne mezereum L., Chase 6357, K, AJ308645†, AJ297233†, AJ744931. Deltaria brachyblastophora Steenis, McPherson 4965, K, AM404304, AM398174, —. Diarthron vesiculosum C.A.Mey, Merton 3960, K, AJ308646†, AM39818, —. Dicranolepis disticha Planch., Gereau et al. 5626, MO, AM40435, AM39818, —. Dirca palustris L., Horn 12584, NBYC, AJ308647†, U26322*, AM159528. Drapetes muscosus Banks ex Sol., Kubitzki & Feuerer 99—34, HBG, AJ308648†, AJ297237†, AM159529. Edgeworthia chrysantha Lindl., Chase 6338, K, AJ308649b, AJ297920b, AJ744932. Enkleia siamensis (Kurz) Nevling, Von Beusekam 4060, K, —, AJ297921b, —. Gnidia aberrans C.H.Wright, Hilliard & Burtt 6898, NU, AM404222, AM162523, AM159508. Gnidia cf. anomala Meisn., Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM400982, AM162539, AM158940. Gnidia bakeri Gilg, Rogers et al. 126, MO, —, AM162506, AM159510. Gnidia bojeriana (Decne.) Baill., Rogers et al. 183, MO, AM404224, AM162507, AM159511. Gnidia caffra (Meisn.) Gilg, Burrows & Burrows 7754, J, —, AM398170, AM396520. Gnidia calocephala (C.A.Mey.) Gilg, Reid 885, PRE, AM404225, —, AM396521. Gnidia caniflora Meisn., Fourcade 5580, PRE, AM404223, AM396993, —. Gnidia coriacea Meisn., Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404227, AM162516, AM159512. Gnidia danguyana Leandri, Rogers et al. 76, MO, AM404226, AM162515, AM159513. Gnidia decaryana Leandri, Rogers et al. 108, MO, AJ745153, AJ745179, AJ744926. Gnidia denudata Lindl., Beaumont s.n., NU, AJ308670†, AJ295266†, AM159514. Gnidia dumetorum Leandri, Rogers et al. 109, MO, AM404228, AM162514, AM159515. Gnidia fastigiata Rendle, Hilliard & Burtt 6142, NU, AJ308650†, AM162513, —. Gnidia galpinii C.H.Wright, Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404230, AM396994, AM159516. Gnidia geminiflora E.Mey. ex Meisn., Goldblatt 3799, GB, AM404231, AM397275, —. Gnidia gilbertae Drake, Randrianasolo 529, MO, AJ745154, AJ745180, AJ744927. Gnidia glauca Gilg, J. Adanson 6156, K, AM404232, AM162511, —. Gnidia humilis Meisn., Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404236, AM162510, AM159517. Gnidia kraussiana Meisn., Beaumont s.n., NU, AJ308674†, AJ295267†, AM159518. Gnidia madagascariensis Baill., Rogers et al. 133, MO, AM404237, AM162509, AM159519. Gnidia penicillata —, —, —. Gnidia penicillata, —, —, —. Gnidia phaeotricha Gilg, Balkwill 10316, J, —, AM162517, AM159520. Gnidia pilosa Burtt Davy, Beaumont s.n., NU, AJ308651†, AJ295264†, —. Gnidia pinifolia L., I. Kruger 399, NBG, AM404240, AM162518, AM159521. Gnidia racemosa Thunb., Beaumont s.n., NU, AJ308665†, AJ295268†, AM159522. Gnidia renniana Hilliard & B.L.Burtt, Beaumont s.n., NU, AM404233, AM162519, AM396522. Gnidia aff. renniana Hilliard & B.L.Burtt, Edwards 1492, NU, AJ308666†, AJ295265†, —. Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Phillips EP. 1944. Notes on some genera of the Thymelaeaceae. Journal of South African Botany 10: 61–67. Phillips EP. 1951. The genera of South African flowering plants, 2nd edition. Botanical Survey Memoir 25: 526–531. Posada D, Crandall KA. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics Application Note 14: 817–818. Reeves G, Chase MW, Goldblatt P, de Chies T, Lejeune B, Fay MF, Cox AV, Rudall PJ. 2001. A phylogenetic analysis of Iridaceae based on four plastid sequences: trnL intron trnL-F spacer, rps4 and rbcL. American Journal of Botany 88: 2074–2087. Robyns A. 1975. Thymelaeaceae. In: Bamps P, ed. Flore d’Afrique Centrale (Zaïre-Rwanda-Burundi). Meise: Jardin Botanique National de Belgique, 1–681. Rogers Z. 2004. A revision of Stephanodaphne Baill. (Thymelaeaceae). Adansonia, sér. 3 26: 7–35. Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Rye BL. 1990. Thymelaeaceae. In: George AS, ed. Podostemaceae to Combretaceae. Canberra: Australian Government Publishing Service, Flora of Australia 18: 122–215. Seelanan T, Schnabel A, Wendel JF. 1997. Congruence PHYLOGENY OF GNIDIA (THYMELAEACEAE) 417 APPENDIX 1 Continued *Fay et al. (1998). †Van der Bank et al. (2002). ‡Van der Bank et al. (unpubl. data). Journal compilation © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160, 402–417 No claim to original government works Downloaded from https://academic.oup.com/botlinnean/article/160/4/402/2418424 by guest on 23 May 2022 Gnidia scabrida Meisn., Juli, Ecklon & Zeyher 53.7, S, AM404238, AM397277, AM396987. Gnidia sericocephala (Meisn.) Gilg ex Engl., Dehning & Dehning 108, J, AM404241, AM408173, AM159523. Gnidia setosa Wickstr., J. Hutchinson 519, GRA, AM404296, AM162520, AM159524. Gnidia singularis Hilliard, Manning 554, NU, AM404297, AM162521, —. Gnidia squarrosa (L.) A.P.Druce, Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404235, AM162522, AM159525. Gnidia subulata Lam., Beaumont s.n., NU, AJ308652†, AM162508, AM159509. Gnidia wikstroemiana Meisn., Beaumont & Smith SRFe9, NU, AM404299, AM162524, AM159526. Gonystylus macrophyllus (Miq.) Airy Shaw, Chase 1382, K, AJ308653†, AJ308677†, Y15150*, —. Gnidia aff. viridis Berg., Beaumont s.n., NU, AJ308652, AM162508, AM159509. Gyrinops walla Gaertn., Chase 10511, K, AM40430, AM39817, —. Lachnaea alpina (Eckl. & Zeyh.) Meisn., Beyers 258, NBG, AJ697829, AJ697771, AJ745754. Lachnaea aurea Eckl. & Zeyh., Aggenbach s.n., NBG, AJ697828‡, AJ697781‡, AJ745737‡. Lachnaea axillaris Meisn., Snijman 1871, NBG, AJ308671†, AJ297219†, AJ745742‡. Lachnaea capitata (L.) Crantz., Bean 2603, NBG, AJ697811‡, AJ697798‡, AJ745744‡. Lachnaea densiflora Meisn., Beyers 145, NBG, AM404353‡, —, AJ745738‡. Lachnaea filamentosa Meisn., Beyers 245, NBG, AJ697833‡, AJ697801‡, AJ745755‡. Lachnaea filicaulis (Meisn.) Beyers, Oliver 1108, NBG, AJ308672‡, AJ297221‡, AJ745729‡. Lachnaea glomerata Fourc., Beyers 192; NBG, AJ697832‡, AJ697765‡, AJ745736‡. Lachnaea gracilis Meisn., Beyers 254, NBG, AJ697819‡, AJ697767‡, AJ745722‡. Lachnaea grandiflora (L.f.) Baill., Handsford 7, NBG, AJ697820‡, AJ697768‡, AJ745730‡. Lachnaea laniflora (C.H.Wright) Bond, Oliver 10679, NBG, AJ697831‡, AJ697802‡, AJ745739‡. Lachnaea laxa (C.H.Wright) Beyers, Oliver & Oliver 11977, NBG, AJ697821‡, AJ697769‡, AJ745733‡. Lachnaea macrantha Meisn., Oliver 11017, NBG, AJ697822, AJ697784/5, —. Lachnaea marlothii Schltr., Oliver & Oliver 11304, NBG, AJ697823‡, AJ697776‡, AJ745726‡. Lachnaea nervosa (Thunb.) Meisn., Hansford & Hansford 103, NBG, —, AJ697793/4, AJ745747. Lachnaea oliverorum Beyers, Viviers & Vlok 430, NBG, AJ697817‡, AJ697786‡, AJ745752‡. Lachnaea pedicellata Beyers, Beyers 260, NBG, AM404354‡, AJ697778‡, AJ745724‡. Lachnaea penicillata Meisn., McDonald 1980, NBG, AJ697826, AJ697791/2, —. Lachnaea pomposa (= Lachnaea buxifolia) Beyers, Oliver 10767, NBG, AJ697835‡, AJ697796‡/AJ697797‡, AJ745753‡. Lachnaea pusilla Beyers, de Villiers 45, NBG, —, AJ697788, AJ745749. Lachnaea rupestris Beyers, Oliver 11262, NBG, AJ697807‡, AJ697779‡, AJ745731‡. Lethedon aff. salicifolia (Labill.) Aymonin, McPherson & Munzinger 18055, MO, AM404306‡, AM398175‡, —. Lethedon balansae (Baill.) Kosterm., McPherson & Munzinger 610, P/MO, AM404307‡, AM398176‡, —. Lethedon cernua (Baill.) Kosterm., McPherson & Munzinger 18025, MO, AM404305‡, AM398177‡, —. Octolepis dioica Capuron, Rogers et al. 46, MO, AM404350, AM398178, —. Octolepis dioica Capuron f. oblanceolata Capuron, Rogers et al. 102, MO, —, AM398179, —. Octolepis sp., Rogers et al. 165, MO, AM404349, AM398180, —. Ovidia andina Meisn., Kubitzki & Fewerer 99–42, NBG, AJ308675, AJ297222, AM159530. Passerina burchellii Thoday, Bredenkamp 1546, PRE, AM404356, AM162526, AM158925. Passerina drakensbergensis Hilliard & B.L.Burtt, Bredenkamp 1020, PRE, AM404358, AM162528, —. Passerina ericoides L., Bredenkamp 962, PRE, AM404359, AM162529, AM158927. Passerina falcifolia C.H.Wright, Bredenkamp 915, PRE, AJ745150, AJ297224†, AJ744917. Passerina montivaga Bredenkamp & A.E.van Wyk, P. van Wyk 2586, PRE, AM404361, AM162531, AM158930. Passerina nivicola Bredenkamp, Bredenkamp 1046, PRE, AJ308655†, AJ297226†, AJ744916. Passerina obtusifolia Thoday, Meyer 1505, PRE, AM404367, AM162532, AM158931. Peddiea africana Harv., Chase 6330, K, AJ308662†, AJ297227†, AJ744921. Peddiea involucrata (Barker) Baill., Rogers & al. 121, MO, AJ745151, AJ745176, AJ744920. Phaleria capitata Jack, Chase 1383, K, AJ308661†, AJ297228†, —. Pimelea argentea R.Br., M. Hislop & M. Griffiths WW 111.39, PERTH, AM406675, AM167530, AM162490. Pimelea clavata Labill., R. J. Cranfield 19510, PERTH, AM407408, AM167532, AM162492. Pimelea decora Domin, Purdie R.W. 5905, CANB, FJ572694, FJ572826, FJ572732. Pimelea forrestiana F.Muell., K. Coate 695, PERTH, AM407407, AM167533, AM162493. Pimelea gilgiana E.Pritz, I. B. Shepherd 269, PERTH, AM406678, AM167534, —. Pimelea graniticola Rye, B. Archer 1664, PERTH, AM406679, —, —. Pimelea haematostachya F. Muell., Lepschi BJ 1202, CANB, FJ572695, FJ572827, FJ572733. Pimelea holroydii F.Muell., S. van Leeuwen 3769, PERTH, AM406687, AM167539, AM162496. Pimelea pygmaea F.Muell., Chase 6360, K, AJ308669†, AJ297230†, AJ744922. Pimelea spiculigera var. thesioides (S.Moore) Rye, R. Davis 10390, PERTH, —, AM398183, AM162499. Pimelea spiculigera var. thesioides (S.Moore) Rye, J. Docherty 130, PERTH, AM406681, —, AM162500. Pimelea trichostachya Lindl., K. F. Kenneally 12623 & D. J. Edinger 3822, PERTH, AM406682, AM167537, AM162501. Solmsia calophylla Baill., Guillaumin s.n., K, AJ308656†, AJ295261†, —. Stellera chamaejasme L., Chase 5530, K, AJ308657†, AJ295262†, —. Stephanodaphne capitata (Leandri) Leandri, Rogers et al. 139, MO, AM407411, AM398184, AM159531. Stephanodaphne cremostachya Baill., Tolaria 13.01.1990, K, AJ308658, AJ295263, AM159532. Stephanodaphne cuspidata (Leandri) Leandri, Rogers et al. 68, MO, AM406683, AM398185, AM159533. Stephanodaphne oblongifolia Leandri, Rogers et al. 127, MO, AJ745152, AJ745177, AJ744924. Struthiola ciliata (L.) Lam., Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404300, AM397279, AM396986. Struthiola dodecandra (L.) A.P.Druce, Mark Johns 004, Kogelberg Reserve Field Herbarium, AM404298, AM398171, AM396988/AM396989. Struthiola leptantha Bolus, Beyers 265, NBG, AJ308639†, AJ297243†, AJ745757. Struthiola salteri Levyns, Mark Johns s.n., Kogelberg Reserve Field Herbarium, AM404301, AM397280, —. Struthiola striata Lam., Mark Johns 005, Kogelberg Reserve Field Herbarium, AM404302, AM398172, AM396990, AM396991. Struthiola tomentosa Andrews, Mark Johns s.n., Kogelberg Reserve Field Herbarium, —, AM162540, AM158946. Synandrodaphne paradoxa Gilg, Lisowski 46609, K, AJ308676†, AJ297240†, —. Synaptolepis alternifolia Oliver, Vollesen 4043, K, AJ308663†, AJ297239†, —. Thecanthes punicea (R.Br.) Wickstr., T. Handasyde TH99 488, PERTH, AM406684, AM167540, AM162502. Thecanthes sanguinea (F.Muell.) Rye, A. A. Mitchell 3945, PERTH, AM406685, —, AM162503. Thymelaea hirsuta Endl., Chase 1883, K, AJ308640†, Y152151*, AJ744930. Wikstroemia canescens Meisn., E 82170, AM406686, AM398186, AJ549496. Wikstroemia gemmata (E.Pritz.) Domke, Chase 3955, K, AJ308641†, AJ295269†/AJ297223†, AJ744929.