TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Molecular phylogenetic and morphological study of Kohautia (Spermacoceae, Rubiaceae), with the recognition of the new genus Cordylostigma Inge Groeninckx,1 Helga Ochoterena, 2 Erik Smets1,3 & Steven Dessein4 1 Laboratory of Plant Systematics, K.U. Leuven, Kasteelpark Arenberg 31, P.O. Box 2437, 3001 Leuven, Belgium 2 Instituto de Biología, Universidad Nacional Autónoma de México, Apdo. Postal 70-367, CP 04510, Mexico 3 National Herbarium of the Netherlands, Leiden University Branch, P.O. Box 9514, 2300 RA Leiden, The Netherlands 4 National Botanic Garden of Belgium, Domein van Bouchout, 1860 Meise, Belgium Author for correspondence: Inge Groeninckx, inge.groeninckx@bio.kuleuven.be Abstract Kohautia Cham. & Schltdl. belongs to the predominantly herbaceous Rubiaceae tribe Spermacoceae. Species of Kohautia can easily be distinguished from other Spermacoceae by their monomorphic short-styled flowers in which anthers and stigma are included in the corolla tube, with the stigma always positioned below the anthers. Mainly because of this unique floral morphology, Kohautia was considered to be a distinct genus. Molecular data (atpB-rbcL, petD, rps16, trnLtrnF, ETS, ITS) confirm that the genus is biphyletic. Two distantly related clades correspond to the subgenera Kohautia and Pachystigma Bremek. A similar type of floral organisation thus seems to have evolved twice independently, resulting in similar, but distantly related lineages. In order to translate the biphyletic nature of Kohautia into a formal classification, the two subgenera are recognized at generic level. A substitute name, Cordylostigma Groeninckx & Dessein is proposed for K. subg. Pachystigma because of the existence of Pachystigma Hochst. in the Rubiaceae tribe Vanguerieae. Floral, pollen and seed characters were studied to morphologically characterize Kohautia s.str. and Cordylostigma. By optimizing pollination syndromes and pollen characters onto the molecular phylogeny, we investigated pollination shifts and pollen evolution within the two genera. Detailed floral morphological studies show that the nectar guides in the psychophilous species of Kohautia s.str. and Cordylostigma evolved in different ways but result in the same visual effect. Keywords Cordylostigma; Kohautia; molecular phylogeny; morphology; Pachystigma; pollination syndromes; Rubiaceae; Spermacoceae IntroductIon Kohautia Cham. & Schltdl. was first described in 1829 by Chamisso and von Schlechtendal who named the genus after its collector Franz Kohaut. The genus belongs to the (predominantly) herbaceous tribe Spermacoceae of the asterid family Rubiaceae. In the current circumscription, Kohautia comprises 36 species (Govaerts & al., 2006; Table 1) distributed from India, Pakistan, Iran to the Arabian Peninsula, the Sinai, Eastern Egypt, most of Africa south of the Sahara (including Socotra and Cape Verde Islands), Madagascar and Australia. Approximately two thirds of the species are perennial herbs; the remaining third are annuals. The African species of Kohautia were first revised by Bremekamp in 1952. Later, Mantell (1985) provided a comprehensive revision of the genus, taking into account infraspecific variation, pollination biology, anatomy, and distribution. Both Bremekamp and Mantell divided Kohautia into two subgenera based on the number of stigma lobes; K. subg. Kohautia including all species with two thin filiform stigma lobes and K. subg. Pachystigma Bremek. including all species characterized by having a single, ovoid to cylindrical stigma lobe. In addition, Mantell (1985) divided K. subg. Kohautia into two series. Kohautia ser. Kohautia is the phalaenophilous (pollinated by moths) and K. ser. Diurnae Bremek. is the psychophilous/ micro-melittophilous (pollinated by butterflies or small bees). All representatives of K. subg. Pachystigma (with the exception of Kohautia virgata (Willd.) Bremek.) are described as being butterfly-pollinated. Species of genus Kohautia are easily distinguished from other Spermacoceae by their monomorphic short-styled flowers in which anthers and stigma are always included, with the stigma held well below the anthers or occasionally just touching them (Bremekamp, 1952; Mantell, 1985). Although recognized as a distinct group of species, there have been different opinions at the generic level. Some authors included Kohautia in Hedyotis L. (Wight & Arnott, 1834: 405–418), others in Oldenlandia L. (Hooker, 1873, 1882: 64–71; Schumann, 1891), whereas Bremekamp (1952) and all later authors (e.g., Verdcourt, 1976; Mantell, 1985) considered Kohautia to be distinct enough to deserve the generic rank. Recent molecular studies within Spermacoceae based on chloroplast (atpB-rbcL, petD, rps16, trnL-trnF) and nuclear (ITS, ETS) DNA have shed new light on the phylogeny of Kohautia and several other representatives of the tribe (Kårehed & al., 2008; Groeninckx & al., 2009). These molecular studies provide answers to many taxonomic debates within the tribe, but also evoke numerous new questions because detected relationships contradict previous taxonomic treatments. The new clades await morphological support before 1457 Groeninckx & al. • Molecular and morphological study of Kohautia a new classification can be proposed. For example, Kårehed & al. (2008) and Groeninckx & al. (2009) demonstrated that Kohautia is not monophyletic. Species of the genus fall in two unrelated clades, which correspond to the subgenera Kohautia and Pachystigma. Because of their limited sampling within Kohautia (respectively ten and nine Kohautia species), Kårehed & al. (2008) and Groeninckx & al. (2009) postponed proposing a new generic circumscription. Nevertheless, both studies suggest that similar growth and floral traits evolved independently in the common ancestor of both K. subg. Kohautia and K. subg. Pachystigma, resulting in two very similar but distantly related lineages (see Fig. 1). By increasing sampling density, the present study investigates the evolution of the species traditionally referred to Kohautia into greater detail. The phylogeny of the genus is reconstructed using four plastid markers (atpB-rbcL, petD, rps16, trnL-trnF) and two nuclear markers (ETS, ITS). Floral, pollen, and seed characters were studied to morphologically characterize the two lineages. By optimizing pollination syndromes and pollen characters onto the resulting molecular phylogenies, we traced the evolutionary path of pollen characters and pollinators within the two clades. MaterIals and Methods Taxon sampling. — Sequences from previous studies (Kårehed & al., 2008; Groeninckx & al., 2009) were used as a basis for the phylogenetic analysis presented in this paper. K. subg. Pachystigma a K. ser. Kohautia K. amatymbica, K. australiensis,a K. caespitosa, K. cynanchica, K. dolichostyla, K. euryantha,* K. gracilis,* K. gracillima,* K. kimuenzae,* K. microflora,* K. nagporensis,* K. pappii,* K. quartiniana,* K. ramosissima, K. retrorsa, K. socotrana,* K. subverticillata, K. tenuisb K. ser. Diurnae K. subg. Kohautia Table 1. Most recent classification of Kohautia according to Mantell (1985) and adapted to Govaerts & al. (2006). Taxa not included in our sampling are indicated with an asterisk. K. angolensis,* K. aspera, K. azurea, K. coccinea, K. confusa,* K. grandiflora, K. huilensis,* K. platyphylla, K. pleiocaulis* K. amboensis,* K. cicendioides,* K. cuspidata, K. microcala, K. longifolia, K. obtusiloba, K. prolixipes,* K. stellarioides,* K. virgata Kohautia australiensis was not yet described when Mantell wrote her revision in 1985. Based on the presence of a bifid stigma, we conclude that the species belongs to K. subg. Kohautia. According to Halford (1992) who described the species, K. australiensis resembles K. coccinea in inflorescence and capsule shape. However, based on floral morphology and general habit we believe K. australiensis to be closer related to K. caespitosa. Therefore, K. australiensis is placed within K. ser. Kohautia. b Kohautia senegalensis, type of Kohautia, is a synonym of K. tenuis (Brunel & al., 1984). 1458 TAXON 59 (5) • October 2010: 1457–1471 The Appendix lists all taxa included in this study with author names, voucher information and GenBank accession numbers. Our sampling includes 41 ingroup taxa, which represent 34 species of Spermacoceae. This set of taxa covers the major evolutionary lineages within the tribe (following Groeninckx & al., 2009). Our dataset contains 26 Kohautia taxa, representing 19 species currently described within the genus. Kohautia subg. Kohautia is thereby represented by 14 species, and K. subg. Pachystigma by 5 species. Despite many efforts, we were not able to amplify and/or sequence DNA from herbarium specimens of the other Kohautia species listed in Table 1 (indicated with an asterisk). DNA isolations were either of poor quality or contaminated with fungi. Three species from the sister tribe Knoxieae (Batopedina pulvinellata Robbr., Carphalea madagascariensis Lam., Pentanisia parviflora Stapf ex Verdc.) were chosen as outgroup taxa. Molecular phylogenetic reconstruction. — Methods for DNA extraction, PCR amplification, sequencing, sequence assembly, alignment and gap coding are as described by Kårehed & al. (2008) and Groeninckx & al. (2009). Equally weighted parsimony analyses were performed using Nona v.2.0 (Goloboff, 1993) launched through WinClada v.1.0 (Nixon, 2002). The different DNA regions were first analyzed separately to check for potential incongruence, but since the results were compatible the matrices were combined using a simultaneous approach (Nixon & Carpenter, 1996a). Heuristic searches for the shortest trees were performed using the parsimony ratchet (Nixon, 1999). Traditional searches were repeatedly conducted using TBR on 1000 Wagner trees constructed with random addition of taxon sequences, holding 10 trees per search and expending the memory to conduct more thorough analyses holding up to 10,000 trees (10 times h 100,000 mu*1000 h/10 max*). Ratchet runs of 200 iterations each, holding one tree per iteration and randomly weighting 10% of the potentially informative characters were carried out until the most parsimonious trees (MPT) were repeatedly found. All trees were collected, unambiguously supported branches collapsed, duplicate trees were identified and removed and a (strict) consensus tree was calculated using WinClada. In order to evaluate the relative support of the clades, jackknife (JS) and bootstrap (BS) analyses were executed using the ‘new technology’ option of TNT (Goloboff & al., 2008). In both cases, 1000 replications were conducted combining Sectorial Searches and Tree Fusion (Goloboff, 1999) using 100 random initial sequences on each of the 1000 replications, saving the consensus for further calculation of frequencies using WinClada (Nixon, 1999). Frequency values above 64% were plotted onto the consensus of the MPT. Floral, pollen, and seed morphology. — Flowers of the psychophilous Kohautia coccinea Royle (Zambia: Dessein & al. 751, BR), the psychophilous K. microcala Bremek. (Zambia: Dessein & al. 1321, BR), the phalaenophilous K. subverticillata (K. Schum.) D. Mantell (Zambia: Dessein & al. 462, BR) and the micro-melittophilous K. virgata (Willd.) Bremek. (Madagascar: Groeninckx & al. 121, BR) preserved in 70% ethanol were studied with scanning electron microscopy (SEM). Flowers were dissected under a Wild M3 stereomicroscope (Wild Heerbrugg Ltd, Heerbrugg, Switzerland). The floral material TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Fig. 1. Flowers of Kohautia species from K. subg. Pachystigma (= Cordylostigma) (A–E) and K. subg. Kohautia (= Kohautia s.str.) (F–G). a, Kohautia microcala (Zambia: Dessein & al. 1149, BR); B, Kohautia longifolia (Zambia: Dessein & al. 1119, BR); c, Kohautia microcala (Zambia: Dessein & al. 1149, BR); d, Kohautia microcala (Zambia: Dessein & al. 1321, BR); e, Kohautia microcala (Zambia: Dessein & al. 1321, BR); F, Kohautia caespitosa subsp. brachyloba (Zambia: Dessein & al. 506, BR); G, Kohautia coccinea (Zambia: Dessein & al. 751, BR). Photographs by Steven Dessein. was washed repeatedly in 70% ethanol and dehydrated in a 1 : 1 ethanol-dimethoxymethan mixture (DMM or formaldehydedimethylacetal) for 5 minutes and in pure DMM for 20 minutes. After critical-point drying (CPD 030, BAL-TEC AG, Balzers, Liechtenstein), the dried material was mounted on aluminum stubs using Leit-C and coated with gold (SPI Module Sputter Coater, Spi Supplies, West Chester, Pennsylvania, U.S.A.) prior to observation with a JEOL JSM-6360 SEM (Jeol Ltd, Tokyo, Japan). A palynological investigation was carried out for 13 species of K. subg. Kohautia and 4 species of K. subg. Pachystigma. Pollen samples were collected from herbarium specimens of BR (Table 2). Pollen grains were acetolysed according to the ‘wetting agent’ method (Reitsma, 1969). Under the SEM, external features were observed on grains that had been suspended in 70% alcohol and left to dry. Glycerin jelly slides were observed under a light microscope. Polar axis length (P) and equatorial diameter (E) were measured on ten grains of each specimen using Carnoy (Schols & al., 2002), and P/E ratios were calculated. Pollen terminology follows Punt & al. (2007). Seeds from herbarium specimens were directly mounted on aluminium stubs, coated with gold and observed under the SEM as described above. We investigated the seed morphology of nine species of K. subg. Kohautia and five species of K. subg. Pachystigma (Table 2). Pollination shifts and pollen evolution. — We investigated pollination shifts within K. subg. Kohautia and K. subg. Pachystigma by unambiguously optimizing pollination syndromes onto the consensus tree from the combined parsimony analysis in WinClada v.1.0 (Nixon, 2002). We made sure that the polytomies resulting on the consensus did not create artefacts on the character evolution interpretation (Nixon & Carpenter, 1996b). Information on the pollination biology of genus Kohautia was gathered from own field observations and from Mantell (1985), who based her descriptions on field observations in Ethiopia and, to a more limited extent, on observations of greenhouse-cultivated plants and studies of herbarium and fixed material. The number of pollen apertures and the presence/absence of a secondary reticulum were also optimised onto the consensus tree. results Molecular evidence. — Table 3 lists the characteristics of each data matrix used in the phylogenetic analysis. All tree searches of the combined analysis found the same tree lengths (L = 1603). Only two equally parsimonious trees were found after collapsing the unambiguously supported branches, with a consistency index (CI) of 0.60 and a retention index (RI) of 0.79. 1459 TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Figure 2 shows the (strict) consensus tree from the parsimony analysis of the combined matrix (L = 1615, CI = 0.60, RI = 0.79). Similar to the studies of Kårehed & al. (2008) and Groeninckx & al. (2009), species of genus Kohautia fall in two well supported, biphyletic clades, which correspond to the two described subgenera; K. subg. Kohautia (BS = 100, JS = 100) and K. subg. Pachystigma (BS = 100, JS = 100). Kohautia subg. Kohautia is sister to a clade that includes Pentanopsis fragrans Rendle and Oldenlandia herbacea (L.) Roxb. (BS = 90, JS = 96). Kohautia subg. Pachystigma is sister to a clade containing species of Oldenlandia including O. corymbosa L., the type of the generic name, and O. capensis L. f. (BS = 100, JS = 100). Kohautia ramosissima Bremek. and K. cynanchica DC. form a grade sharing a most recent common ancestor with the rest of K. subg. Kohautia (respectively BS = 91, JS = 97 and BS = 71, JS = 74). Kohautia aspera (B. Heyne ex Roth) Bremek. Table 3. Characteristics of each data matrix used in in phylogenetic analyses I, II and III, and the corresponding tree statistics. Chars = total number of characters, PI = potentially informative characters, MPT = number of most parsimonious tree(s), CI = consistency index (Kluge & Farris, 1969), RI = retention index (Farris, 1989). No. of taxa Chars 1030 81 15 9 0.64 0.81 petD 41 1339 171 27 27 0.69 0.87 rps16 39 614 110 12 12 0.71 0.86 trnL-trnF 42 628 106 23 4 0.72 0.90 ETS 32 266 88 3 9 0.49 0.68 ITS 33 815 150 11 3 0.54 0.70 Combined 44 4692 706 91 2 0.60 0.79 K. amatymbica South Africa, Schlieben 7910 (BR) p K. angolensis Angola, Bamps 527 (BR) p K. aspera Tanzania, Kayombo & Kitaba 4230 (BR) p, s K. azurea Namibia, Seydel 3491 (BR) p, s subsp. brachyloba Tanzania, Kuchar 25133 (BR) Zambia, Dessein & al. 432 (BR) K. coccinea p, s p, s p, s Zambia, Dessein & al. 790 (BR) p Congo, de Witte 430 (BR) p, s Zambia, Dessein & al. 751 (BR) p, s K. cuspidata Angola, Hess-Wyss 52/108 (BR) p, s K. cynanchica South Africa, Dessein & al. 469 (BR) p, s K. grandiflora Ethiopia, Friis & al. 6864 (BR) p, s K. longifolia Tanzania, Bidgood & al. 2595 (BR) p, s K. microcala Zambia, Dessein & al. 1149 (BR) p, s Zambia, Dessein & al. 1321 (BR) p, s Zambia, Dubois 1309 (BR) s K. obtusiloba Tanzania, Gobbo 307 (BR) s K. platyphylla Ethiopia, Friis & al. 439 (BR) p Cameroon, De Wilde & De Wilde-Duyfjes 8950 (BR) s K. ramosissima Namibia, Merxmüller & Giess 32496 (BR) p K. retrorsa Oman, Ghazanfar 1823 (BR) p K. subverticillata Zambia, Dessein & al. 462 (BR) p, s Botswana, Dessein & al. 470 (BR) p Zambia, Dessein & al. 489 (BR) p, s K. tenuis K. virgata 1460 RI 37 Voucher information K. caespitosa subsp. amaniensis Ethiopia, de Wilde 5976 (BR) PI indels MPTs CI atpB-rbcL Table 2. Herbarium specimens of which pollen (p) and/or seeds (s) were studied, and their voucher information. Taxon PI Cameroon, De Wilde & De Wilde-Duyfjes 4870 (BR) p Senegal, Vanden Berghen 6262 (BR) s Madagascar, De Block 539 (BR) p, s and K. azurea (Dinter & K. Krause) Bremek., originally described within K. ser. Diurnae, are resolved as sister taxa, but without significant support (JS and BS both <65). The two species are more closely related to K. subverticillata (K. Schum.) D. Mantell of K. ser. Kohautia (BS = 99, JS = 100) than to other species of K. ser. Diurnae. Further internal tree resolution reveals Kohautia caespitosa Schnizl. subsp. caespitosa sister to K. amatymbica Eckl. & Zeyh., and Kohautia dolichostyla Bremek. sister to K. australiensis Halford. Both clades lack significant support. Kohautia caespitosa Schnizl. subsp. brachyloba (Sond.) D. Mantell is sister to a highly supported clade including K. caespitosa Schnizl. subsp. amaniensis (K. Krause) Govaerts and K. retrorsa (Boiss.) Bremek. (BS = 99, JS = 99). Except for Kohautia aspera and K. azurea, all other species of K. ser. Diurnae included in our sampling fall within one clade together with K. tenuis Cham. & Schltdl. (BS = 95, JS = 96), the type of the generic name. Within this clade, K. grandiflora DC. is highly supported as sister to K. tenuis (BS = 97, JS = 98). Relationships between K. coccinea Royle, K. platyphylla (K. Schum.) Bremek. and the clade of K. grandiflora and K. tenuis remain unresolved. Within K. subg. Pachystigma, Kohautia obtusiloba (Hiern) Bremek. is sister to a clade including the remaining Pachystigma species in our sampling. In this clade, Kohautia virgata (Willd.) Bremek. is resolved as sister to K. cuspidata (K. Schum.) Bremek., K. longifolia Klotzsch and K. microcala Bremek. (BS = 99, JS = 99). Relationships between the latter three species remain unresolved. Pollen morphology. — In general, the pollen grains of genus Kohautia are very small. The smallest pollen grains are observed in K. subg. Kohautia; P 17.60–25.99 μm, E 13.77–18.84 μm. The hexaploid K. amatymbica is the only species within the subgenus with much larger pollen (P 26.81–30.49 μm, E 19.80–23.91 μm). Species of K. subg. Pachystigma TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Fig. 2. (Strict) consensus of the combined parsimony analysis using atpB-rbcL, petD, rps16, trnL-trnF, ETS and ITS sequences. Bootstrap (left) and jackknife (right) values are indicated above branches. Outgroup = Batopedina pulvinellata, Carphalea madagascariensis and Pentanisia parviflora. OUTGROUP 99/99 100/100 90/96 have distinctly larger pollen grains than species of K. subg. Kohautia (P 20.76–29.33 μm, E 18.98–26.96 μm). The pollen shape within both subgenera varies from (oblate/prolate) spheroidal to prolate. Most species are characterized by having prolate spheroidal to subprolate pollen. Both subgenera have compound apertures consisting of an ectocolpus, a mesoporus and an endocolpus. In K. subg. Kohautia, the endoaperture is a relatively short colpus often with fish tail endings (Fig. 3A). The mesoporus is surrounded K. subg. Kohautia K. subg. Pachystigma 99/100 Arcytophyllum thymifolium Houstonia caerulea Kadua degeneri 100/100 Oldenlandia mitrasacmoides Bouvardia glaberrima 100/100 Spermacoce erosa 99/99 Oldenlandia capensis var. capensis 100/100 Oldenlandia corymbosa 100/100 Kohautia obtusiloba 99/99 Kohautia obtusiloba 100/100 Kohautia virgata Kohautia cuspidata 99/99 Kohautia longifolia Kohautia microcala Dentella repens 100/100 Pentodon pentandrus Hedyotis fruticosa Agathisanthemum bojeri 100/100 Oldenlandia goreensis Oldenlandia herbacea var. herbacea 100/100 Pentanopsis fragrans Kohautia ramosissima Kohautia cynanchica 100/100 Kohautia subverticillata subsp. subverticillata 99/100 91/97 Kohautia aspera Kohautia azurea Kohautia caespitosa subsp. caespitosa 71/74 Kohautia amatymbica 99/99 Kohautia amatymbica Kohautia dolichostyla Kohautia australiensis 85/86 Kohautia australiensis Kohautia caespitosa subsp. brachyloba 100/99 Kohautia caespitosa subsp. brachyloba Kohautia caespitosa subsp. amaniensis 99/99 Kohautia retrorsa Kohautia platyphylla Kohautia grandiflora 97/98 95/96 Kohautia tenuis Kohautia coccinea Kohautia coccinea by an annulus (Fig. 3A, G, H). In K. subg. Pachystigma, on the other hand, the endocolpus is an endocingulum (Fig. 3I) and an annulus is absent. Pollen has three to six apertures. Eightcolporate pollen grains, as reported by Bremekamp (1952), were not observed. Within K. subg. Kohautia, K. cynanchica, K. ramosissima (Fig. 3B), K. angolensis Bremek. (not included in our molecular sampling), and K. azurea are characterized by having almost exclusively 3-colporate pollen grains. The remaining species are characterized by having a mixture of 1461 Groeninckx & al. • Molecular and morphological study of Kohautia TAXON 59 (5) • October 2010: 1457–1471 4- to 5- (rarely 6-)colporate pollen grains. Within K. subg. Pachystigma, all species have 4- to 6-aperturate pollen, except K. virgata with 3-colporate pollen. In both subgenera, the inner nexine surface is granular (Fig. 3A, I). The sexine of most species is heterobrochate varying from reticulate to micro-reticulate (Fig. 3B–F, K). In general, species of K. subg. Pachystigma have larger lumina (Fig. 5K) than species of K. subg. Kohautia (Fig. 3D–F). Moreover, the tectum of K. subg. Pachystigma is subtended by longer columellae than in K. subg. Kohautia creating larger intercolumellar spaces (Fig. 3I). Within K. subg. Kohautia, large lumina were observed in the earlier derived taxa K. angolensis, K. cynanchica, and K. ramosissima (Fig. 3B), and in the sister species K. coccinea, K. platyphylla, K. tenuis and K. grandiflora (Fig. 3C, F). The smallest lumina were found in K. amatymbica. This species has a micro-reticulate to perforate sexine (Fig. 3D). Most species of K. subg. Kohautia have a double reticulum (Fig. 3E), except for the earlier derived K. cynanchica and K. ramosissima (Fig. 3B), and K. coccinea (Fig. 3F), K. platyphylla (Fig. 5C), K. tenuis and K. grandiflora. All studied species within K. subg. Pachystigma, lack a secondary reticulum (Fig. 3K). Seed morphology. — According to Mantell (1985), the seed shape is quite different in the two subgenera. She describes the seeds of K. subg. Kohautia as being angular-conic to angular-subconic, whereas seeds of K. subg. Pachystigma are more rounded. In our study, we did not observe these differences (Fig. 4A, C, E, G). There is, however, a difference in the seed surface of the two subgenera. In K. subg. Pachystigma, the seed coat is always distinctly reticulate with prominent radial walls, which are strait, curved or undulating (Fig. 4F, H). The tangential walls are sparsely or densely punctate (Fig. 4F, H). Similar punctate micro-sculpturing is observed in a number of Oldenlandia species (sister group of K. subg. Pachystigma) but is lacking in K. subg. Kohautia. In most species of K. subg. Kohautia, the seed coat is alveolate to reticulate-alveolate with the radial walls only slightly raised (Fig. 4B). Some species (e.g., Kohautia coccinea and K. platyphylla; Fig. 4C–D) are exceptional in having prominent radial walls similar to species of K. subg. Pachystigma. The tangential walls in K. subg. Kohautia are, however, never punctate as in K. subg. Pachystigma but favulariate (Fig. 4B) or with a central protuberance which is either ridged (e.g., K. platyphylla) or tuberculate (e.g., K. coccinea; Fig. 4D). Fig. 3. Pollen morphology of K. subg. Kohautia (a–h) and K. subg. Pachystigma (I–K). a, Broken pollen grain of K. platyphylla; endocolpus with fish tail endings and mesocolpus surrounded by an annulus; B, polar view on 3-colporate pollen of K. ramosissima; c, polar view on 4-colporate pollen of K. platyphylla; d, detail of micro-reticulate/perforate apocolpium of K. amatymbica; e, detail of (micro-)reticulate apocolpium of K. subverticillata with secondary reticulum; F, detail of (micro-)reticulate apocolpium of K. coccinea without secondary reticulum; G, equatorial view on pollen of K. caespitosa subsp. brachyloba; h, detail of aperture of K. caespitosa subsp. brachyloba; I, broken pollen grain of K. microcala; view on endocingulum; J, polar view on 5-colporate pollen of K. longifolia; K, detail of reticulate apocolpium of K. longifolia without secondary reticulum. 1462 TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Floral morphology. — Psychophilous species of the two subgenera Kohautia and Pachystigma have flowers with long, narrowly cylindrical corolla tubes, mostly inconspicuously coloured, and with barrel-shaped, inflated apical parts where the anthers are included (Fig. 1A, D, G). The corolla lobes are broadly lanceolate-elliptic to roundly elliptic, usually brightly coloured above, and paler below (Fig. 1A–E, G). The colour of the corolla lobes usually falls into the red end of the colour spectrum, i.e., red, pink, orange or lilac. The nectar is hidden at the base of the corolla tube, around the style. In the psychophilous species of K. subg. Kohautia, the fused basal parts of the corolla lobes are turgid and swollen at anthesis (Figs. 1G and 5A–B). Seen from above, these swollen parts slightly project into and over the corolla throat, forming a distinct cross or ‘bullseye’ in the centre of the flower (Fig. 5A). These intrusions are characterized by epidermal cells with a different shape and orientation (Fig. 5A–B) and may function as nectar guides. In K. subg. Pachystigma, the fused basal parts of the corolla lobes are beset with hairs (Fig. 5C–D). These hairs are paler or darker than the rest of the corolla, or contrastingly coloured (Fig. 1A–C, E), and may also function as nectar guides. Most species of K. subg. Kohautia are phalaenophilous. These moth-pollinated flowers are similar to butterfly-pollinated flowers in having long and narrow corolla tubes with an upper inflated, barrel-shaped portion containing the anthers Fig. 4. Seed morphology of K. subg. Kohautia (a–d) and K. subg. Pachystigma (e–h). a–B, Seed and detail of the seed coat of K. subverticillata; c–d, seed and detail of the seed coat of K. coccinea; e–F, seed and detail of the seed coat of K. microcala; G–h, seed and detail of the seed coat of K. virgata. Fig. 5. Flower morphology of psychophilous (a–d), phalaenophilous (e) and micromelittophilous (F–G) species. a, top view on psychophilous flower of K. coccinea with swollen, fused basal parts of the corolla lobes (‘bullseye’); B, dissected psychophilous flower of Kohautia coccinea; c, top view on psychophilous flower of K. microcala with hairs on the fused basal parts of the corolla lobes; d, dissected psychophilous flower of K. microcala; e, phalaenophilous flower of K. subverticillata; F, micro-melittophilous flower of K. virgata; G, nectary disc above the ovary surrounding the base of the style in K. virgata. Note the nectarostomata. 1463 TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia A B OUTGROUP OUTGROUP Kohautia ramosissima Kohautia ramosissima Kohautia cynanchica Kohautia subverticillata subsp. subverticillata Kohautia cynanchica Kohautia subverticillata subsp. subverticillata Kohautia aspera Kohautia aspera Kohautia azurea Kohautia azurea Kohautia amatymbica Kohautia amatymbica Kohautia caespitosa subsp. caespitosa Kohautia dolichostyla Kohautia caespitosa subsp. caespitosa Kohautia dolichostyla Kohautia australiensis Kohautia caespitosa subsp. brachyloba Kohautia australiensis Kohautia caespitosa subsp. brachyloba . Kohautia caespitosa subsp. amaniensis . Kohautia caespitosa subsp. amaniensis Kohautia retrorsa Kohautia coccinea Kohautia retrorsa Kohautia coccinea Kohautia grandiflora Kohautia grandiflora Kohautia tenuis Kohautia tenuis Kohautia platyphylla Kohautia platyphylla OUTGROUP OUTGROUP Kohautia obtusiloba Kohautia obtusiloba Kohautia virgata Kohautia virgata Kohautia cuspidata Kohautia cuspidata Kohautia longifolia Kohautia microcala Kohautia longifolia Kohautia microcala moth pollination bee pollination butterfly pollination unknown 3 apertures more than 3 apertures unknown Fig. 6. Optimization of pollination syndromes (a), number of pollen apertures (B) and the presence/absence of a secondary reticulum (c) onto the (strict) consensus tree of the parsimony analysis for K. subg. Kohautia and Pachystigma. (Fig. 5E). The corolla lobes are white to greenish-white or yellowish above (Fig. 1F). Below they are darker, often olive-green or brownish-red just like the corolla tubes. As in phalaenophilous species, the nectar is hidden at the base of the corolla tubes. In contrast to psychophilous flowers nectar guides are absent. Micro-melittophilous flowers of Kohautia azurea, K. aspera and K. virgata are typically blue-violet, whiteblue or white with short narrowly cylindrical tubes (Fig. 5F). Micro-melittophilous flowers differ from ordinary melittophilous flowers in their small size and smaller amount of nectar production, sufficient for smaller pollinators. A nectariferous disc showing many nectarostomata is present above the ovary surrounding the base of the style (Fig. 5G). Nectar is thus hidden, not too deeply but within easy reach of a bee’s short proboscis. Nectar guides are similar to those of psychophilous flowers. Pollination shifts in K. subg. Kohautia and K. subg. Pachystigma. — Optimizing pollinators onto the molecular phylogenetic reconstruction of K. subg. Kohautia, indicates that ancestral flowers were visited by moths (Fig. 6A). During evolution three shifts occurred; one shift from moth to butterfly pollination, one reversal back to psychophily in K. tenuis, and one shift from moth to bee pollination for the clade of Kohautia aspera and K. azurea. Species of K. subg. Pachystigma 1464 are all psychophilous with one transition to melittophily for K. virgata. Pollen evolution in K. subg. Kohautia and K. subg. Pachystigma. — Figure 6B shows the evolution of aperture number within the two subgenera. Within K. subg. Kohautia, 3-colporate pollen is considered the ancestral condition. During evolution a shift occurred from 3-colporate pollen to pluri-colporate pollen, followed by one reversal back to the 3-colporate state in K. azurea. In K. subg. Pachystigma, evolutionary reconstruction of the number of apertures is ambiguous. Acctran optimization supports pluri-colporate pollen as the character state in the most recent common ancestor of the subgenus with one shift to 3-colporate pollen in K. virgata. Deltran optimization, on the other hand, favours the 3-colporate condition as ancestral state with two parallel gains of pluri-colporate pollen. Figure 6C shows the presence/absence of a secondary reticulum within both subgenera. In K. subg. Kohautia, absence is supported as the character state in the most recent common ancestor of the subgenus with one shift to presence of a secondary reticulum followed by another shift to absence of a secondary reticulum. Within K. subg. Pachystigma, all species studied lack a secondary reticulum. The absence of a secondary reticulum is considered the ancestral condition. TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia C OUTGROUP Kohautia ramosissima Kohautia cynanchica Kohautia subverticillata subsp. subverticillata Kohautia aspera Kohautia azurea Kohautia amatymbica Kohautia caespitosa subsp. caespitosa Kohautia dolichostyla Kohautia australiensis Kohautia caespitosa subsp. brachyloba . Kohautia caespitosa subsp. amaniensis Kohautia retrorsa Kohautia coccinea Kohautia grandiflora Kohautia tenuis Kohautia platyphylla OUTGROUP Kohautia obtusiloba Kohautia virgata Kohautia cuspidata Kohautia longifolia Kohautia microcala secondary reticulum present secondary reticulum absent unknown taxonoMIc treatMent Based on the morphological differences between K. subg. Kohautia and K. subg. Pachystigma, Mantell (1985) tentatively suggested that the two subgenera could also be treated as genera. Eventually, she decided to maintain a broad definition for the genus Kohautia. Our molecular data and previous molecular studies (Kårehed & al., 2008; Groeninckx & al., 2009) clearly show the need to recognize the two subgenera as genera. Despite the unifying floral architecture, there are numerous morphological differences between the two subgenera. The number of stigmatic lobes is probably the most striking diagnostic field character: two in K. subg. Kohautia and one in K. subg. Pachystigma. Other differences can be found in floral, seed, and pollen morphology, and in the pollination biology of the two subgenera. Table 4 summarizes the main morphological differences between K. subg. Kohautia and K. subg. Pachystigma. In order to translate our results into a formal classification, Kohautia is here restricted to comprise the species of K. subg. Kohautia only and the species of K. subg. Pachystigma are transferred to the new genus Cordylostigma. The name Pachystigma is not available at the rank of genus as Pachystigma Hochst. already exists in the Rubiaceae tribe Vanguerieae. The name Cordylostigma refers to the presence of a single stigma lobe characteristic of its representatives. We decided to no longer recognize K. ser. Diurnae and ser. Kohautia as they are not supported as monophyletic by our molecular data. For the same reason, we do no longer recognize Cordylostigma ser. Barbatae and ser. Imberbae. In the following paragraphs, a description is given for the restricted genus Kohautia s.str. and the new genus Cordylostigma and synonyms of these names are listed. Kohautia Cham. & Schltdl. in Linnaea 4: 156. 1829, nom. cons. ≡ Hedyotis sect. Kohautia (Cham. & Schltdl.) Wight & Arnott, Prodromus 1: 417. 1834 ≡ Oldenlandia subg. Kohautia (Cham. & Schltdl.) Benth. & Hook. f., Gen. Pl. 2: 59. 1877 ≡ Kohautia (subg. “Eu-kohautia”) ser. Noctiflorae Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 91. 1952 – Type: K. senegalensis Cham. & Schltdl. – “Kohautia subg. Eu-kohautia” Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 81. 1952, non rite publ. (Art. 21.3). = Kohautia (subg. Kohautia) ser. Diurnae Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 81. 1952 – Type: K. coccinea Royle. Table 4. Morphological differences between Kohautia s.str. and Cordylostigma. Kohautia s.str. Cordylostigma Stigma Two-lobed One-lobed Seed coat Alveolate to reticulate-alveolate and tangential walls never punctate Distinctly reticulate with prominent radial walls and punctate tangential walls Pollen 3-colporate, or 4- to 5- (rarely 6-)colporate 4- to 6-colporate (except K. virgata with 3-colporate pollen) Endocolpus Short colpus, often with fish tail endings Endocingulum Mesoporus Surrounded by an annulus Not surrounded by an annulus Double reticulum Present in most species Absent in all species Pollinators Moths, butterflies and small bees Butterflies (only K. virgata is pollinated by small bees) Nectar guides in psychophilous flowers Intrusions Hairs 1465 Groeninckx & al. • Molecular and morphological study of Kohautia = Duvaucellia Bowdich in Bowdich & Bowdich, Exc. Madeira: 259. 1825, nom. rejic. – Type: D. tenuis S. Bowd. Annual or perennial herbs or subshrubs, sometimes with short woody subterranean stems; stipular sheath in the midstem region mostly with one or two fimbriae; corolla lobes above and entrance to tube always glabrous, white or brightly coloured; stigma two-lobed; stigma lobes filiform; pollen 3-colporate, or 4-to 5- (rarely 6-)colporate, P 17.60–25.99(– 30.49) µm, E 13.77–18.84(–23.91) µm, with short endocolpus (often with fish tail endings), with an annulus surrounding mesoporus, in most cases with secondary reticulum; seed coat alveolate to reticulate-alveolate, sometimes distinctly reticulate, with tangential walls never punctate; pollinators moths, butterflies, or small bees. — Number of species: 27. — Distribution: India, Pakistan, Iran, northern East and sub-Saharan Africa, Cape Verde Islands and Socotra. Cordylostigma Groeninckx & Dessein, nom. et stat. nov. ≡ Kohautia subg. Pachystigma Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 66. 1952 ≡ Kohautia (subg. Pachystigma) ser. Barbatae Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 66. 1952 (non Pachystigma Hochst. in Flora 25: 234. 1842) – Type: Kohautia longifolia Klotzsch. (C. longifolia (Klotzsch) Groeninckx & Dessein, see below). = Kohautia (subg. Pachystigma) ser. Imberbae Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 77. 1952 – Type: K. virgata (Willd.) Bremek. Annual or perennial herbs, sometimes with short woody subterranean stems; stipular sheath in the mid-stem region mostly with 2–8(11) fimbriae; corolla tube hairy or papillate at the throat inside; corolla lobes broad, elliptic, not parted at the base, mostly brightly coloured, with hairs at the base inside; stigma one-lobed; stigma lobes ovoid or cylindrical; pollen 4- to 6-colporate, exceptional 3-colporate (i.e., in C. virgata), P 20.76–29.33 µm, E 18.98–26.96 µm, with endocingulum, without annulus around mesoporus, without secondary reticulum; seed coat distinctly reticulate, with radial walls strait, curved or undulating, and with tangential walls punctate; pollinators butterflies, in C. virgata small bees. — Number of species: nine. — Distribution: mainly in eastern and southern Africa, also in Madagascar; one species extending to western Africa and into Sudan. nomenclatural changes The following nomenclatural changes need to be made (only homotypic synonyms are given): Cordylostigma amboensis (Schinz) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia amboensis Schinz in Vierteljahrsschr. Naturf. Ges. Zürich 68: 429. 1923 ≡ Kohautia amboensis (Schinz) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 75. 1952 – Type: Namibia, Amboland, Olukonda, Ondonga, Rautanen 824 (Z!, holo; BM, G!, K!, P!, iso; BR!, photo). 1466 TAXON 59 (5) • October 2010: 1457–1471 Cordylostigma cicendioides (K. Schum.) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia cicendioides K. Schum. in Bot. Jahrb. Syst. 33: 333. 1903 ≡ Kohautia cicendioides (K. Schum.) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 75. 1952 – Type: Angola, auf feuchtem Boden bei Pallanca, sine col., sine num. (B†); Neotype (designated by Bremekamp, 1952): Angola, between Sambos Mission Station and Cabama, Pearson 2492 (K!, neo; BR!, photo). Cordylostigma cuspidata (K. Schum.) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia cuspidata K. Schum. in Bot. Jahrb. Syst. 23: 413. 1897 ≡ Kohautia cuspidata (K. Schum.) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 74. 1952 – Type: Angola, Huila Plateau, Lopollo, December 1859, Welwitsch 5342 (B†, holo; K, LISU, BM, G!, P!, PRE!, iso). Cordylostigma longifolia (Klotzsch) Groeninckx & Dessein, comb. nov. ≡ Kohautia longifolia Klotzsch in Peters, Naturw. Reise Mossambique 1: 297. 1862 – Type: Mozambique, Sena, Peters s.n. (B†, holo). Neotype (designated by Bremekamp, 1952): Mozambique: Gonubi Hill, Schlechter 12181 (K, neo; BM, BR!, E!, G!, W, isoneo). Cordylostigma microcala (Bremek.) Groeninckx & Dessein, comb. nov. ≡ Kohautia microcala Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 73. 1952 – Type: Zambia, near Kalungwizi R, Walter 5 (K!, holo; BR!, photo). Cordylostigma obtusiloba (Hiern) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia obtusiloba Hiern in Oliver, Fl. Trop. Afr. 3: 56. 1877 ≡ Kohautia obtusiloba (Hiern) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 66. 1952 – Types: Tanzania, Bagamoyo District, Kingani, Kirk s.n. & Mozambique, Forbes 358 (both K!, syn). Cordylostigma prolixipes (S. Moore) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia prolixipes S. Moore in J. Bot. 43: 351. 1905 ≡ Kohautia prolixipes (S. Moore) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 67. 1952 – Type: Kenya, Kwale District, near Avisana, Daruma, Kässner 442 (BM!, holo; K, iso). Cordylostigma stellarioides (Hiern) Groeninckx & Dessein, comb. nov. ≡ Oldenlandia stellarioides Hiern, Catalogue of African Plants 1: 447. 1898 ≡ Kohautia stellarioides (Hiern) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 76. 1952 – Type: Angola, Pungo Andongo, Welwitsch 3052 (BM!, lecto, designated by Bremekamp, 1952; K, isolecto). Cordylostigma virgata (Willd.) Groeninckx & Dessein, comb. nov. ≡ Hedyotis virgata Willd. in Sp. Pl. 1: 567. 1798 ≡ Oldenlandia virgata (Willd.) DC., Prodr. 4: 425. 1830 ≡ TAXON 59 (5) • October 2010: 1457–1471 Kohautia virgata (Willd.) Bremek. in Verh. Kon. Ned. Akad. Wetensch., Afd. Natuurk., Sect. 2, 48: 77. 1952 – Type: Ghana, s.l., probably Thonning s.n. (B†, holo; S, isoneo, designated by Bremekamp, 1952). dIscussIon Generic delimitation problems within Spermacoceae. — Generic delimitations within Spermacoceae are complicated by the strong habitual similarity between the representatives, which makes it difficult to find morphological characters to define genera. The subdivision in genera is especially problematic in the Hedyotis-Oldenlandia complex, part of Spermacoceae including most species of the former tribe Hedyotideae. For centuries the taxonomic status of Hedyotis L., Oldenlandia L. and their satellite genera (e.g., Amphiasma Bremek., Arcytophyllum Willd. ex Schult. & Schult. f., Houstonia L., Kohautia, and Kadua Cham. & Schltdl.) has been a subject of discussion. The main issue has been whether most species of the complex should be lumped into Hedyotis (advocated by inter alia Merrill & Metcalf, 1946; Wagner & al., 1989; Fosberg & Sachet, 1991; Dutta & Deb, 2004), or if many small genera should be recognized in addition to a narrow circumscription of Hedyotis (supported for the African taxa by Bremekamp, 1952, for the neotropical taxa by Terrell & al., 1986; Terrell, 1991, 2001a–c, and for the Asian taxa by Terrell & Robinson, 2003). In the past, the delimitation of genera has been a somewhat subjective activity; certain morphological features were considered important for some authors to recognize a species or a group of species at the generic level, while for other authors they were not convincing enough to segregate a new genus. This has resulted in numerous taxonomic debates, which in the absence of molecular data could not be solved. However, purely molecular studies of Spermacoceae (Kårehed & al., 2008; Groeninckx & al., 2009) answer some of these taxonomic debates, but at the same time they generate numerous new taxonomic problems. The present study of the genus Kohautia demonstrates that an integrative approach, combining molecular data with morphological observations, can further untangle the taxonomic webs in Spermacoceae. Moreover, integrative studies also allow describing new genera and new species with more certitude, as has been demonstrated by Groeninckx & al. (2010 and in press). Phylogenetic relationships within genus Kohautia s.str. — Kohautia ramosissima and K. cynanchica are the earliest diverging species within the genus Kohautia s.str. Both species are centred in southern West Africa, and are distinguished from the other members of Kohautia s.str. by their 3-colporate pollen grains without a secondary reticulum. The two species differ from each other in a number of significant characters. Kohautia ramosissima differs from K. cynanchica in the more distinctly pedicellate and smaller flowers, and the larger pollen grains. Subsequent branching produced a highly supported clade with the two melittophilous species, K. aspera and K. azurea Groeninckx & al. • Molecular and morphological study of Kohautia (previously described in K. ser. Diurnae), as sister to the phalaenophilous species K. subverticillata. A close relationship between K. aspera and K. subverticillata is not surprising; a great deal of confusion has always existed in separating the two species because of their similar paired small, subsessile flowers. Both species are widespread herbs, occurring in Africa, south-west Arabia and India. Kohautia azurea has small flowers just like K. aspera and K. subverticillata, but is restricted to Namibia and is characterized by having 3-colporate pollen and pedicellate flowers. One of the most morphologically particular species within the genus is Kohautia amatymbica. This hexaploid species can be distinguished from other representatives of Kohautia not only by its unique chromosome number, but also by its exclusively geoxylic suffrutescent habit (weedy habit but with a short, underground woody base), capitate inflorescences with large showy flowers, and its relatively large pollen and seeds. Its distinct morphology makes it very difficult to discuss its resemblance to other Kohautia species. The only species to which it shows a slight morphological resemblance is K. kimuenzae (De Willd.) Bremek. from South West Congo, which was not included in the present study. Our molecular data suggest Kohautia amatymbica to be related to K. caespitosa subsp. caespitosa, but this sister relationships lacks significant support. Morphologically and biogeographically this relationship is quite surprising. Kohautia caespitosa subsp. caespitosa is distributed from Egypt to Northeast Tropical Africa and occurs also in the Arabian Peninsula, whereas K. amatymbica occurs in Tropical Africa and South Africa. Unexpectedly, Kohautia caespitosa subsp. caespitosa is not closely related to the other two caespitosa subspecies included in our analysis. Further research will have to confirm if the three subspecies are better treated as distinct species. Molecular data strongly support a relationship between Kohautia caespitosa subsp. amaniensis and K. retrorsa. Mantell (1985) already discussed a close relationship between the two species. Kohautia retrorsa has a south-east Arabian and Iranian distribution, forming a link between the African and Indian Kohautia species. Surprisingly, it shows more genetic and morphologic similarities to the Somalia-Masai centred K. caespitosa subsp. amaniensis (i.e., fruit shape and fruit wall) than to the neighbouring subsp. caespitosa of southwestern Arabia. Molecular data support a close relationship between the psychophilous species K. coccinea, K. grandiflora and K. platyphylla (originally described in K. ser. Diurnae), and the phalaenophilous species K. tenuis (originally described in K. ser. Kohautia and the type of the generic name Kohautia). The phalaenophilous K. tenuis is closely related to the psychophilous K. grandiflora. The sister relationship is well supported by molecular, morphological and biogeographical data. Both species are diploid and occur more or less sympatrically (centered in the Sudanian Regional Centre of Endemism) in open areas in dry fire-prone grassland and open woodland (K. grandiflora is also often found in seasonally waterlogged clay soils). They resemble each other in a number of morphological characters; both species have mucronate corolla lobe 1467 Groeninckx & al. • Molecular and morphological study of Kohautia TAXON 59 (5) • October 2010: 1457–1471 apices, subglobose to spherical capsules and similar testa cell sculpturing (testa cells with indistinctly reticulate radial walls and distinctly globular or alveolate periclinal walls). The main difference between K. grandiflora and K. tenuis lies in their inflorescence structure (compact, more or less corymbiform vs. staggered) and in the shape and colour of their corolla lobes (broad and brightly coloured above with distinct intrusions at the corolla throat vs. linear and white above without intrusion in the corolla throat). Pollination shifts within genus Kohautia s.str. — Optimisation of pollination syndromes onto the molecular trees suggests that pollination shifts might have triggered speciation in the genus Kohautia s.str. Three pollination shifts have occurred in Kohautia s.str.; one shift from the ancestral state of moth pollination to butterfly pollination, a second from moth to bee pollination, and the third was a reversal from butterfly to moth pollination (Fig. 6A). The occurrence of pollination shifts raises questions about the genetic mechanisms underlying such transitions. At first it might appear that a simple shift from ancestral white corolla lobes to brightly coloured ones may have caused a shift from moth to butterfly pollination. However, more complex genetic adaptations are necessary to induce a shift in the formation of nectar guides, in the timing of flower opening (night vs. day), in scent release and nectar physiology. The genetic understanding of complex traits like pollination syndromes is making progress (Bradshaw & Schemske, 2003; Galliot & al., 2006; Cronk & Ojeda, 2008). In monkeyflowers (genus Mimulus L.), for example, Bradshaw & Schemske (2003) demonstrated that a single allele substitution at a flower colour locus could produce a shift from bee to hummingbird pollination. In the genus Ipomoea L., on the other hand, changes in the control of the anthocyanin biosynthetic pathway are associated with shifts in flower colour from purple to red, and shifts from bee to hummingbird pollination (Zufall & Rausher, 2003, 2004). Natural selection for shifts in pollinators is often generated by “competitive” interactions with sympatric congeneric species (Armbruster, 1996). It is commonly concluded that competition for pollinators lowers reproductive success, and that evolution therefore selects for plant characteristics that reduce the extent of pollinator sharing. Differences in flowering time, but also in flower morphology and in flower colour are commonly attributed among sympatric species to selection for reducing competition for pollinators. As mentioned above, the phalaenophilous K. tenuis and the psychophilous K. grandiflora occur more or less sympatrically (Sudanian Regional Centre of Endemism). The different pollination syndromes in the two species may form an effective barrier preventing hybridization. Sympatric species with the same pollination syndrome, on the other hand, may be kept apart by mechanical isolation, i.e., extreme differences in flower size as in K. coccinea and K. platyphylla, or by differences in ploidy level, e.g., K. cynanchica (2n) and K. ramosissima (4n). Phylogenetic relationships within Cordylostigma. — Our molecular data support a close relationship between Cordylostigma cuspidata, C. longifolia and C. microcala. Based on morphological observations, Mantell (1985) already suggested a close relationship between C. longifolia and C. microcala. In her revision, she even postulated that C. microcala might be an extreme form of the variable C. longifolia. Besides resemblance in morphology, the distribution range of the two species overlaps in the Southeastern and central parts of the Zambezian Region, where they grow in seasonally waterlogged soils, in grassland and open woodland. Cordylostigma cuspidata differs from C. microcala and C. longifolia, by its capituliform inflorescences, with 10–15 flowers with red, purplish-red or bright pink corolla lobes. In contrast to C. microcala and C. longifolia, C. cuspidata occurs in the western Zambezian Regional Centre of Endemism and the Northwestern part of the Kalahari-Highveld Transition Zone. Sister to the clade of Cordylostigma cuspidata, C. longifolia and C. microcala is the micro-melittophylous C. virgata. Cordylostigma virgata is a widespread species with a disjunctive distribution (Tropical Africa, South Africa and the western Indian Ocean). The absence of hairs in the corolla throat, the lack of constrictions above and below the anthers, the often emergent sterile connectives on the anthers, and the seeds with wavy periclinal testa walls set C. virgata apart from all the other Cordylostigma species. Mantell (1985) suggested C. virgata to be closely related to C. longifolia because both species occur in Madagascar. Further research is needed to falsify this biogeographic speciation hypothesis. Cordylostigma obtusiloba is sister to the remaining Cordylostigma species in our analysis. Morphologically, C. obtusiloba is a distinct and easily recognizable species. It has very slender stems arising from a short, inconspicuous (sometimes woody) underground stem; slender, pedicellate, few-flowered inflorescences; and flowers with relatively long, funnel-shaped corolla tubes with an unusual dilated apical part. Floral evolution in genus Kohautia s.str. and genus Cordylostigma. — Molecular data reveal that the monomorphic short-styled flowers have evolved twice independently within Spermacoceae, i.e., in Kohautia s.str. and in Cordylostigma. Additional floral ontogenetic observations are needed to determine if the floral organization of Kohautia s.str. and Cordylostigma has common grounds. By studying the floral morphology of both genera into more detail, we noticed for example that the formation of nectar guides in the psychophilous species of Kohautia s.str. and Cordylostigma is accomplished in different ways but producing the same visual effect. In genus Kohautia s.str., nectar guides are formed by intrusions, whereas in genus Cordylostigma hairs function as nectar guides. Superficially the nectar guides look the same in the two genera, and could be incorrectly interpreted as homologous. Comparative floral morphology shows that the nectar guides of the two genera have a different structure. The nectar guides can be explained as an evolutionary response to similar selective pressures (i.e., adaptations to butterfly pollination) and they are thus treated as analogous. Floral morphology studies of the psychophilous species of Kohautia s.str. and Cordylostigma have not only demonstrated convergent evolutionary characters, but identified a discriminating character supporting each clade. Pollen and pollination. — Pollen morphology may be correlated with pollination vectors (Hesse, 2000), in particular 1468 TAXON 59 (5) • October 2010: 1457–1471 aperture and exine ornamentation characteristics are correlated with specific pollinators (Proctor & al., 1996; Tanaka & al., 2004). Our study shows that the psychophilous species of both genera Kohautia s.str. and Cordylostigma have pollen grains without a secondary reticulum. The function of tectal columellae is undoubtedly complex, but one function of the intercolumellar spaces is the accommodation of pollen surface coatings (Heslop-Harrison, 1979). Tectal columellae increase the space for surface materials and there is evidence that ‘sticky’ pollen, with additional coating materials occurs frequently in flowers associated with bird-pollination (Muller, 1981) or insectpollination (Osborn & al., 1991). Unfortunately, these surface substances are removed by acetolysis and are soluble in ethanol. We could hypothesize that the absence of a secondary reticulum may increase the space for sticky coating materials, which in turn may increase the pollination effectiveness; ‘sticky’ pollen could be a mechanism to increase the pollen transfer by butterflies. Unfortunately, pollination studies within the tribe Spermacoceae are scarce, making it very difficult to confirm or refute this hypothesis. conclusIons Our study confirms that Kohautia as traditionally delimited is biphyletic. Both molecular and morphological data show the need to recognize K. subg. Kohautia as making up the entire genus Kohautia and K. subg. Pachystigma as the new genus Cordylostigma. Morphological similarity between species of Spermacoceae has generated systematic confusion. This paper demonstrates that detailed integrated studies, combining molecular and morphological data, can help to further solve these taxonomic problems. acKnoWledGMents We thank Dr. Rafaël Govaerts from the Royal Botanic Gardens of Kew and Bart Jacobs from the Laboratory of Plant Systematics of the K.U. Leuven for helpful discussions. We appreciate the comments by Dr. Charlotte Taylor and the two anonymous reviewers, which highly improved the text. 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Key to literature citations: (1) = Andersson & al., 2002; (2) = Groeninckx & al., 2009; (3) = Kårehed & al., 2008; (4) = Kårehed & Bremer, 2007. Agathisanthemum Klotzsch: A. bojeri Klotzsch, Zambia, Dessein & al. 671 (BR), EU542917(2), EU543018(2), EU543077(2), EU557678(3), –, AM939424(3). Arcytophyllum Willd. ex Schult. & Schult. f.: A. thymifolium (Ruiz & Pav.) Standl., Ecuador, Ståhl 4481 (GB), EU542923(2), AF333366(1), EU543082(2), EU557683(3), AM932921(3), AM939431(3). Bouvardia Salisb.: B. glaberrima Engelm., cult., Forbes s.n. (S), EU542925(2), EU543022(2), EU543084(2), EU557685(3), AM932922(3), AM939432(3). Dentella J.R. Forst & G. Forst.: D. repens (L.) J.R. Forst. & G. Forst., Australia, Andersson 2262 (GB), EU542932(2), AF333370(1), EU543091(2), EU557693(3), AM932930(3), AM939440(3). Hedyotis L.: H. fruticosa L., Sri Lanka, Larsson & Pyddoke 22 (S), EU542942(2), –, EU543098(4, EU557702(3), AM932941(3), AM939453(3). Houstonia L.: H. caerulea L., USA, Vincent & Lammers s.n. (GB), EU542953(2), AF333379(1), EU543109(2), EU557713(3), –, AM939464(3). Kadua Cham. & Schltdl.: K. degeneri (Fosberg) W.L. Wagner & Lorence, cult., Wood 5062 (PTGB), EU542958(2), AF333371(1), EU543113(2), EU557717(3), AM932953(3), AM939470(3). Kohautia Cham. & Schltdl.: K. amatymbica Eckl. & Zeyh., South Africa, Bremer & al. 4307 (UPS), EU542962(2), EU543035(2), EU543117(2), EU557721(3), AM932956(3), AM939484(3); South Africa, Venter & Venter 10287 (MO), GU951588, GU951636, GU951652, GU951621, GU951600, –; K. aspera (B. Heyne ex Roth) Bremek., Tanzania, Kayombo & Kitaba 4230 (BR), –, GU951640, GU951656, –, GU951604, –; K. australiensis Halford, Australia, Latz 17736 (BR), GU951589, GU951637, GU951653, GU951622, GU951601, GU951611; Australia, Albrecht & Latz 12201 (BR), GU951590, GU951638, GU951654, GU951623, GU951602, GU951612; K. azurea (Dinter & K. Krause) Bremek., Namibia, Seydel 3491 (BR), GU951591, GU951639, GU951655, GU951624, GU951603, GU951613; K. caespitosa Schnizl. subsp. amaniensis (K. Krause) Govaerts, Ethiopia, de Wilde 5976 (BR), GU951592, GU951641, GU951657, GU951625, –, GU951614; K. caespitosa Schnizl. subsp. brachyloba (Sond.) D. Mantell, Zambia, Dessein & al. 432 (BR), EU542963(2), EU543036(2), EU543118(2), EU557722(3), AM932957(3), AM939474(3); Tanzania, Kuchar 25133 (BR), GU951593, GU951642, GU951658, GU951626, GU951605, GU951615; K. caespitosa Schnizl. subsp. caespitosa, Zambia, de Wilde 4618 (BR), –, –, GU951659, –, –, –; K. coccinea Royle, Zambia, Dessein & al. 751 (BR), EU542964(2), EU543037(2), EU543119(2), EU557723(3), AM932959(3), AM939476(3); s.l., Wieringa 4916 (WAG), GU951594, GU951643, GU951660, GU951627, –, –; K. cuspidata (K. Schum.) Bremek., Malawi, La Croix 4501 (MO), GU951595, –, GU951661, GU951628, GU951606, GU951616; K. cynanchica DC., South Africa, Dessein & al. 469 (BR), EU542965(2), EU543038(2), EU543120(2), EU557724(3), AM932960(3), AM939477(3); K. dolichostyla Bremek., Somalia, Thulin 10819 (BR), GU951596, GU951644, GU951662, GU951629, GU951607, GU951617; K. grandiflora DC., Ethiopia, Friis & al. 6864 (BR), GU951597, GU951645, GU951663, GU951630, GU951608, GU951618; K. longifolia Klotzsch, Tanzania, Bidgood & al. 2595 (BR), –, GU951646, GU951664, GU951631, –, –; K. microcala Bremek., Zambia, Dessein & al. 1149 (BR), EU542966(2), EU543039(2), EU543121(2), EU557725(3), AM932962(3), AM939479(3); K. obtusiloba (Hiern) Bremek., Kenya, Luke 9035 (UPS), EU542967(2), EU543040(2), EU543122(2), EU557726(3), AM939481(3), –; Kenya, Luke & Robertson 2323 (MO), GU951598, GU951647, GU951665, –, –, –; K. platyphylla (K. Schum.) Bremek., Ethiopia, Friis & al. 439 (BR), –, GU951648, GU951666, GU951632, –, –; K. ramosissima Bremek., Namibia, Merxmüller & Giess 32496 (BR), –, GU951649, GU951667, 1470 TAXON 59 (5) • October 2010: 1457–1471 Groeninckx & al. • Molecular and morphological study of Kohautia Appendix. Continued. GU951633, GU951609, –; K. retrorsa (Boiss.) Bremek., Oman, Ghazanfar 1823 (BR), –, GU951650, GU951668, GU951634, –, –; K. subverticillata (K. Schum.) D. Mantell, Zambia, Dessein & al. 470 (BR), EU542968(2), EU543041(2), EU543123(2), –, EU557727(3), GU951619; K. tenuis Cham. & Schltdl., Burkina Faso, Madsen 5940 (MO), GU951599, GU951651, GU951669, GU951635, GU951610, GU951620; K. virgata (Willd.) Bremek., Madagascar, De Block & al. 539 (BR), EU542969(2), –, EU543124(2), EU557728(3), AM939483(3), AM932965(3). Oldenlandia L.: O. capensis L. f. var. capensis, Zambia, Dessein & al. 843 (BR), EU542980(2), EU543048(2), EU543133(2), EU557737(3), AM932974(3), AM939496(3); O. corymbosa L., Zambia, Dessein & al. 487 (BR), EU542982(2), EU543050(2), EU543135(2), EU557739(3), AM932979(3), AM939502(3); O. goreensis (DC.) Summerh., Zambia, Dessein & al. 1286 (BR), EU542988(2), EU543055(2), EU543141(2), EU557745(3), AM932985(3), AM939510(3); O. herbacea (L.) Roxb. var. herbacea, Zambia, Dessein & al. 463 (BR), EU542990(2), EU543057(2), EU543143(2), EU557747(3), AM932988(3), AM939552(3); O. mitrasacmoides F. Muell., Australia, Harwood 1516 (BR), EU542993(2), –, EU543146(2), EU557750(3), AM932992(3), AM939515(3). Pentanopsis Rendle: P. fragrans Rendle, Ethiopia, Gilbert & al. 7458 (UPS), –, EU543065(2), EU543153(2), EU557758(3), AM933002(3), AM939526(3). Pentodon Hochst.: P. pentandrus (K. Schum. & Thonn.) Vatke, Zambia, Dessein & al. 598 (BR), EU543002(2), EU543066(2), EU543154(2), EU557759(3), AM933003(3), AM939528(3). Spermacoce L.: S. erosa Harwood, Australia, Harwood 1148 (BR), EU543008(2), EU543070(2), EU543159(2), EU557765(3), AM933009(3), AM939537(3). Outgroup: Batopedina Verdc.: B. pulvinellata E. Robbr., Zambia, Dessein & al. 264 (BR), EU542924(2), EU543021(2), EU543083(2), EU557684(3), –, AM266989(4). Carphalea Juss.: C. madagascariensis Lam., Madagascar, De Block & al. 578 (BR), EU542926(2), EU543023(2), –, EU557686(3), –, AM267020(4). Pentanisia Harv.: P. parviflora Stapf ex Verdc., Zambia, Dessein & al. 678 (BR), EU543001(2), EU543064(2), EU543152(2), EU557757(3), –, AM266995(4). 1471