Blackwell Science, LtdOxford, UKBOJBotanical Journal of the Linnean Society0024-4074The Linnean Society of London, 2004? 2004 1464 453467 Original Article THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) P. KORNHALL and B. BREMER Botanical Journal of the Linnean Society, 2004, 146, 453–467. With 5 figures New circumscription of the tribe Limoselleae (Scrophulariaceae) that includes the taxa of the tribe Manuleeae PER KORNHALL1* and BIRGITTA BREMER1,2 1 Department of Systematic Botany, Evolutionary Biology Centre, Uppsala University, Sweden The Bergius Foundation at the Royal Swedish Academy of Sciences, Sweden 2 Received September 2003; accepted for publication June 2004 Cladistic analyses by maximum parsimony and Bayesian inference methods of chloroplast and nuclear sequence data indicate a new position for Limosella (Scrophulariaceae). Following this result, a new circumscription of the tribe Limoselleae is presented where the tribe Manuleeae is included in Limoselleae. Further, the study discloses that the genus Sutera is paraphyletic in its present circumscription, but that the two sections of Sutera, Sutera and Chaenostoma, are monophyletic. To accommodate these findings the genus Chaenostoma is re-established. Furthermore, the genus Jamesbrittenia recently expanded by Hilliard is shown to be a highly supported monophyletic group in its current circumscription. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467. ADDITIONAL KEYWORDS: Bayesian inference – Chaenostoma – hybridization – ITS – Jamesbrittenia – Limosella – ndhF – parsimony – Sutera – trnT/F. INTRODUCTION Members of the tribe Manuleeae Bentham (Scrophulariaceae) are common elements of the Cape Flora. Recently, the tribe Selagineae Horan. (former family Selaginaceae Choisy) was included in the Manuleeae by Kornhall, Heidari & Bremer (2001). The tribe now comprises about 625 species in 28 genera and is nearly entirely southern African in its distribution. The only exceptions to this are one species of Jamesbrittenia Kuntze that extends as far as to India, one of Hebenstretia L. that extends northwards to Eritrea, and the monotypic genus Barthlottia Fischer that, together with a species of Selago L., occurs on Madagascar. The aforementioned study indicated that the current circumscriptions of the genera Sutera Roth and Manulea L. are incorrect. Fieldwork in the year 2001 (by the first author) provided material that made it possible to look more closely into these relationships and also to *Corresponding author. E-mail: per.kornhall@ebc.uu.se test more thoroughly the taxonomic status of Jamesbrittenia, a genus that encompasses many taxa formerly considered to belong to Sutera (Hilliard, 1994). New material of the cosmopolitan genus Limosella L. was also included in the study after an unpublished molecular analysis of Scrophulariaceae (B. Oxelman, P. Kornhall, R. G. Olmstead & B. Bremer, unpubl. data) pointed towards a connection between the tribe Manuleeae and the genus Limosella. Historically, the plants now belonging to Manulea, Sutera and Jamesbrittenia have an intertwined taxonomy. Manulea, today with 74 species, was erected by Linnaeus 1767, Sutera, erected by Roth 1807, now has 49 species, and Jamesbrittenia, now encompassing 83 species, was erected by Kuntze 1891. Many plants of Sutera have synonyms in Manulea, and Jamesbrittenia in Sutera. The genus Manulea fide Hilliard (1994) is characterized by indumentum with balloon-tipped eglandular hairs; inflorescence a thyrse, raceme or panicle; corolla tube cylindrical, often abruptly expanded below limb and bent; stamens inserted halfway up in the corolla tube or higher with anthers always © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 453 454 P. KORNHALL and B. BREMER included or the anterior pair just visible in the mouth of the corolla. The genus was divided by Hilliard (1994) into four sections: Dolichoglossa, Thyrsiflorae, Manulea and Medifixae. Sutera fide Hilliard (1994) is characterized by flowers usually alone in leaf-axils; corolla tube most often funnel-shaped, broad in the mouth; anticous pair of anthers exerted (in all but two species). Hilliard (1994) divided the genus into two sections; a small section Sutera encompassing three species and among them the type of the genus, S. foetida Roth, and a larger section Chaenostoma with 46 species. The latter section corresponds closely to Bentham’s (1846) description of the genus Chaenostoma. The name means gaping mouth, pointing to the funnel-formed corollas. One of the main differences between Sutera and Manulea, according to Hilliard (1994), is the inclusion or exertion of the anthers, but it should be noted that in the three species of section Sutera, two have included anthers and thus resemble the genus Manulea. According to Hilliard (1994), Sutera has semi-annular nectariferous glands at the base of the ovaries and Manulea has lateral glands. Other characters are shared by the two genera, indicating that they are closely related. The use of the name Jamesbrittenia was, until Hilliard’s (1994) monograph of the Manuleeae, more or less restricted to the species J. dissecta Kuntze. The genus, fide Hilliard, has 83 species, most of which were earlier considered parts of Sutera. They are characterized by a calyx divided almost to the base, an abruptly expanded corolla tube with a band of clavate hairs in the throat, the anthers usually included, the posterior filaments usually pubescent and decurrent down the corolla tube, and a short included and minutely bifid stigma. Most of our understanding of the morphology of Manuleeae emanates from the detailed monographs written by Hilliard (1994, 1999). As her monographs are the most complete taxonomic treatments of taxa belonging to the tribe, we also follow her terminology in our descriptions and keys. The genus Limosella, the mudworts, has a worldwide distribution with 18 described species. They are small, aquatic to semi-aquatic herbs, typically growing in mud and/or shallow waters. The leaves are more or less rosulate, subulate or cylindrical in shape, but can be differentiated into a petiole, and with a spatulate to ovate blade. Many species are heteroblastic and form submerged forms as well as land forms, and forms with swimming leaves. The small flowers are usually pedicellate, seldom sessile, and are open when present in air, but half to wholly closed when submerged in mud or water. The calyx is campanulate, five- (rarely four-) lobed. The white, pink, blue or lilac corolla consists of a cylindrical tube and a limb with five (rarely four) lobes with an indumentum of eglan- dular and glandular hairs. The number of stamens is typically four and the anthers are unithecous. The two-locular ovary carries a lingulate style with a rounded, sometimes weakly bifid, stigma. The fruit is small and opens with two valves, with the number of yellowish to brown seeds varying from three to over a hundred. The genus was placed in the tribe Sibthorpiae by Bentham (1846) and in Gratioleae by Hallier (1903), and was last revised by Glück (1934). Dumortier (1827) established the tribe Limoselleae for Limosella. The aims of this study were to establish a phylogeny that could be used to answer the following questions: are Manulea, Jamesbrittenia and Sutera monophyletic in their current circumscriptions, do the diagnostic features used to circumscribe and distinguish these genera reflect evolutionary history, and what is the taxonomic position of Limosella? MATERIAL AND METHODS CHOICE OF TAXA AND GENES We generated molecular data sets of sequences from the chloroplast regions ndhF and trnT-F, and from the nuclear ITS region, using already published sequences as well as new sequencing. To achieve a sampling as representative as possible, we sought taxa from all subgeneric groups of Jamesbrittenia, Manulea and Sutera recognized by Hilliard (1994). We sampled major groups of the Lamiales sensu APGII (The Angiosperm Phylogeny Group, 2003) to ascertain the position of Limosella. Olea europea was chosen as outgroup for the analysis of ndhF as Oleaceae has been shown to occupy a basal position in the Lamiales (Oxelman, Backlund & Bremer, 1999). In the combined and in the ITS analyses, we used Buddleja as outgroup, since it has been shown to be closely related to the Scrophularia/Verbascum/Manuleeae clade (Kornhall et al., 2001; Olmstead et al., 2001; Bremer et al., 2002). We sequenced ndhF as the region has been shown to carry information at this level of phylogeny in related taxa (Oxelman et al., 1999). TrnT-F was chosen to enhance resolution in more closely related taxa as the introns seem to have a faster substitution rate and hence yield higher phylogenetic resolution. By trnT-F we mean the whole region between the trnT (UGU) and the trnF (GAA) genes, including exons and intron of the trnL (UAA) gene and the two intergenic spacers. After indication of a hybrid event we also sequenced the nuclear internal transcribed spacer, ITS, in order to reveal discrepancy between the evolutionary histories of the nuclear and the chloroplast genome. Sequences from the ITS region have been widely used for phylogenetic purposes especially in closely related taxa (Baldwin, 1992; Baldwin et al., © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) 1995; Andreasen & Bremer, 2000; Zimmer et al., 2002). All investigated species together with EMBL/ GenBank accession numbers are shown in Appendix 4. From the sequences achieved we produced three data sets for analysis, one of ndhF to obtain a broader overview of the position of Limosella and Jamesbrittenia, and two data sets, one with the nuclear ITS sequences, and a combined of all three genes in order to investigate the Manulea/Sutera complex. SEQUENCING AND ALIGNMENT Extraction and PCR amplification was carried out following the protocols described in Kornhall et al. (2001). The ITS primers used are shown in Appendix 1. We performed sequencing on a MegaBACE 1000 DNA analysis system (Amersham Biosciences) following the protocol of the manufacturer. Sequenced fragments were assembled and edited using the software SEQUENCHER 3.1.1 (Gene Codes Corporation, 1991), and were thereafter imported into SE-AL alignment software (Rambaut, 1995) for alignment by eye. We excluded from the analyses very variable parts of the trnT-F and ITS matrices when we could not ascertain homology. The aligned matrices are available from the correspondence author. PHYLOGENETIC METHODS Phylogenetic methods used were maximum parsimony (MP) and Bayesian posterior probabilities (PP). We performed parallel analyses with MP and PP on all data sets. All MP analyses were performed with the PAUP* ver. 4.0b2a software (Swofford, 1999). Since several authors (Källersjö, Albert & Farris, 1999; Sennblad & Bremer, 2000) have pointed out that there is no justification for a priori weighting of codon positions when using parsimony, we weighted all positions equally. For the PP analyses we used the program MRBAYES ver. 2.01 (Huelsenbeck & Ronquist, 2001). To evaluate the runs we ran three independent analyses from random prior trees on all data sets, each with four heated chains. We plotted the support values for important nodes to obtain a measure of how well the chains had reached stationary following Huelsenbeck et al. (2002) and Leache & Reeder (2002), and estimated the burn-in by plotting the logarithm of the likelihood. We made preliminary runs with different models and as choice of model did not apparently change the result of the runs, we chose a model that made it possible to evaluate different parameters and followed our previous study (Kornhall et al., 2001). The model chosen for PP analyses was the general time reversible (GTR) model. 455 For the ndhF data set, the MP analyses consisted of a heuristic and a jackknife analysis. The heuristic analysis was run with 15 addition sequence replicates and TBR branch swapping. The jackknife was run with 10 000 jackknife replicates, 37% of characters deleted, ‘jac’ resampling method used (Farris et al., 1996), and we used NNI branch-swapping. The PP analysis on the ndhF was made with site-specific rates and site partition by codon. The chains were run for 220 000 generations. For the ITS, a heuristic MP search was carried out with a restraint of no more than 1000 trees saved in every addition sequence replicate. This was done to shorten computing time. The analysis was run with ten addition sequence replicates. Otherwise, the MP analysis was carried out as on the ndhF data set with the exception that the jackknife was done with 20 000 replicates. In the Bayesian analysis we used a model with gamma distributed rates instead of site-specific rates, otherwise the settings were identical to the ndhF analysis. The setting of the analyses of the combined data set was identical to the ITS, with the exception that the MP heuristic search was done as on the ndhF data. MORPHOLOGY Morphological traits were investigated using herbarium material. Flowers were studied after rehydration in heated water with a little detergent added. Ovaries from selected taxa were studied under a dissecting microscope. Along with the voucher material for the sequencing, type material of all species of Sutera was studied. We checked especially characters used by Hilliard (1994) to discriminate between Manulea and Sutera. RESULTS This study presents 22 new trnT-F, 26 new ITS and 41 new ndhF sequences. The ndhF data set consists of 77 taxa and 2260 unordered equally weighted characters, 1337 of which are constant and 548 (24%) parsimony-informative. The heuristic MP search yielded 18 157 equally parsimonious trees of length 2374, with consistency index, CI = 0.57, and retention index, RI = 0.78. The topologies of the trees obtained in the MP jackknife analyses were the same as in the PP analysis and are not shown here. The tree obtained in the PP analysis is shown in Figure 1. All PP runs gave the same topology and none of the nodes plotted varied more than 3% (see Appendix 1). Parameter values obtained in the runs are shown in Appendix 1. The rate of substitution for the third position is higher, as expected, followed © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 456 P. KORNHALL and B. BREMER Figure 1. The majority rule consensus tree from the three Bayesian analyses of the ndhF data set. Only nodes with posterior probabilities above or equalling 95% are shown, with values above branches. The black vertical line shows the position of Jamesbrittenia, the white line shows the position of Limosella, and the dashed line shows the Manulea/Sutera complex. Numbers in italics below branches are used for evaluation of the runs (see Appendix 3). by the first position and then the second. The burnin was estimated to 50 000 generations. The likelihood was then stable for approximately 30 000 generations in all three runs. The genus Sutera is, in this analysis, paraphyletic. Sutera sect. Chaenostoma constitutes a monophyletic clade (100% PP) as does Manulea (also 100% PP). These two form a well supported clade (100% PP) that is a sister-clade to the representatives of Sutera sect. Sutera. The whole Manulea/Sutera complex is monophyletic with 100% posterior probability. Jamesbrittenia is, according to our results, a well-defined and highly supported © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) 457 Figure 2. The majority rule consensus tree from the three Bayesian analyses of the ITS data set. Only nodes with 70% posterior probability or more are shown, with values above branches. The black vertical line shows the position of Manulea, the white line shows the position of Sutera section Chaenostoma, and the dashed line, Sutera sect. Sutera. Numbers below branches were used for evaluation of the runs (see Appendix 3). (100% PP) monophyletic clade, and Limosella is clearly positioned inside the Manuleeae. The ITS data set consists of 41 taxa and 904 unordered equally weighted characters, 533 of which are constant and 205 (23%) parsimony-informative. The shortest trees produced in the heuristic MP search were 784 steps long. Only the result from the PP analysis is shown (Fig. 2). Parameter and node values are © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 458 P. KORNHALL and B. BREMER shown in Appendices 2 and 3. No nodes varied more than 3% from the average PP. Burn-in was estimated to 20 000 generations. All three runs were then stable for approximately 10 000 generations. In the analysis of the ITS data set, Sutera is monophyletic, though not significantly supported (80% PP). Manulea is monophyletic (100% PP) as is the Manulea/Sutera clade (100% PP). An anomaly exists in the analysis, namely the position of Verbascum. This position inside Manuleeae of a rather distant basal taxon is probably caused by scarce sampling and/or alignment problems. The latter are possibly due to the fast evolving nature of the ITS. The combined data set consists of 34 taxa and 4862 unordered equally weighted characters, 3669 of which are constant and 778 (16%) parsimony-informative. The heuristic search gave 240 equally parsimonious trees of length 1924, with CI = 0.76 and RI = 0.88. The strict consensus tree of these (not shown) has the same topology as the consensus tree obtained in the jackknife (not shown) and PP analyses. One randomly chosen tree from the heuristic search is shown as a phylogram in Figure 3. The tree from the PP analysis is shown in Figure 4. Parameter and node values are shown in Appendices 2 and 3. Two nodes varied more than 3%, namely nodes 4 and 23 (see Appendix 2). The former node had a very low average PP (61.7%) and the latter had a nonsignificant PP support value, 84% in average and below 70% MP jackknife support. Burn-in was estimated to 50 000 generations. All three runs then had stable likelihoods for approximately 10 000 generations. The phylogeny resembles the ndhF tree. Sutera is paraphyletic. Manulea and Sutera sect. Chaenostoma constitute a clade with 100% PP, Sutera sect. Sutera is sister to that clade, and the whole Manulea/Sutera complex is monophyletic (100% PP). The position of Limosella is also in accordance with the results from the ndhF analysis. In the morphological study we found that Sutera foetida (All Batten 1164, S) had an evident semiannular gland, which contradicts the findings of Hilliard (1994). Other examined specimens of Sutera had lateral nectariferous glands, e.g. Sutera campanulata Kuntze (All Batten 998, S), Sutera patriotica Hiern (Bremer & Bremer 3818, UPS), Sutera floribunda Kuntze (Bremer & Bremer 4315, UPS) and Sutera calciphila Hilliard (Kornhall 52, UPS). Simon, 1999; Huelsenbeck et al., 2001; Wilcox et al., 2002; Zanis et al., 2002; Archibald, Mort & Crawford, 2003). New software for these statistics has made it possible to analyse phylogenetic data with modelbased methods and obtain support values within a reasonable time scale. In this study Bayesian inference was applied in parallel with parsimony as we think that comparisons from real data sets between measures of support from two methods are desirable. Our experience from this and from an earlier study (Kornhall et al., 2001) shows that results obtained with Bayesian statistics do not differ from parsimony in well supported nodes. This also seems to be the general picture emerging from other studies, e.g. Kårehed (2002), Schneider et al. (2002) and Wilcox et al. (2002). Bayesian probabilities generally tend to have a numerically higher value than bootstrap support. Bootstrap support of 70–80% roughly corresponds to 95% posterior probability in studies where both methods have been applied to the same data (Huelsenbeck DISCUSSION PARSIMONY AND BAYESIAN INFERENCE In the last ten years there has been a growing interest in the use of model-based methods in phylogenetic reconstruction, especially in view of the introduction of methods for Bayesian inference (e.g. Larget & Figure 3. Phylogram of one of the 240 most parsimonious trees obtained in the heuristic MP analysis of the combined data set. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) 459 Figure 4. The majority rule consensus tree from the three Bayesian analyses of the combined data set. Only nodes with 95% posterior probability or more are shown, with values above branches. The black vertical line shows the position of Manulea, the white line, the position of Sutera sect. Chaenostoma and the dashed line Sutera sect. Sutera. Numbers in italics below branches were used for evaluation of the runs (see Appendix 3). et al., 2002; Kauff & Lutzoni, 2002). There is criticism that Bayesian statistics overestimate the support from data (Suzuki, Glazko & Nei, 2002) and studies that point to a risk of overparameterization (Rannala, 2002; Rydin & Kallersjo, 2002). However, there is also a study by Wilcox et al. (2002) which claims that Bayesian inference gives more accurate support values. We think Bayesian statistics has an advantage in that the © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 460 P. KORNHALL and B. BREMER results are easily interpreted according to biological praxis, i.e. posterior probabilities can be treated as ‘normal’ statistical probabilities, and 5% significance level used to test hypotheses (Huelsenbeck et al., 2002; Leache & Reeder, 2002). In bootstrap or jackknife analyses, where to place confidence levels is more or less arbitrary as the statistical meaning of the support is difficult to interpret. TAXONOMY Our results have implications for the taxonomy of the group. We will give below a new broadened circumscription of the Limoselleae to encompass the taxa of the Manuleeae. We will also give a new circumscription of the genus Sutera and revive the name Chaenostoma for a genus consisting of Sutera sect. Chaenostoma. About Jamesbrittenia We conclude that Hilliard’s (1994) wide circumscription of Jamesbrittenia is strongly supported by molecular evidence. We note that ITS was comparably difficult to amplify from the genus. This, together with the rather high chromosome number of n = 24 compared with a normally lower number of 14–16 in the Manuleeae, could indicate polymorphism in the nuclear genome. This could be tested by cloning and subsequent sequencing. Limosella – Limoselleae The position of the sampled Limosella species within a clade with members from the tribe Manuleeae is indisputable from our molecular analyses (Figs 1, 2, 4) and is also supported by B. Oxelman, P. Kornhall, R. G. Olmstead & B. Bremer (unpubl. data). However, only a few morphological traits support this position, one of which is the occurrence of synthecous anthers. On the other hand, there are no characters that contradict such a placement. The name Limoselleae, erected in 1827 by Dumortier, has priority over Manuleeae that was erected by Bentham & Hooker f. in 1876, and Selagineae and Hebenstretieae that were erected by Horaninov in 1847. We therefore propose below a new circumscription of the tribe Limoselleae that also includes the taxa of the Manuleeae. As implied by the geographical distribution of extant Limoselleae (in the above sense) taxa, the ancestors of the present day Limoselleae probably were confined to southern Africa. According to an earlier study of the tribe Manuleeae (Kornhall et al., 2001) the more basal taxa of the group have ovaries that contain many and hence smaller seeds. The taxa with larger seeds and fewer ovules appear further up in the cladograms. We could envisage that at some point, the lineage leading to Limosella evolved even smaller seeds and a mud-loving ecology. The traits for propagation in and through mud facilitated a wide distribution by migrating birds and thus made Limosella the first and only part of the Limoselleae to achieve a global distribution. That water plants with small seeds are widely dispersed is noted by, among others, Charles Darwin (1872). In The origin of species he writes: ‘. . . it has long been known what enormous ranges many fresh-water, and even marsh species, have, both over continents and to the most remote oceanic islands. . . . for the latter seem immediately to acquire, as if in consequence, a wide range’. A wide dispersion of aquatic plants is also noted by more recent authors such as Ridley (1930) and Gleason & Cronquist (1964). It is a fascinating thought that the cosmopolitan genus Limosella may have arisen from predecessors in southern Africa, especially when it is remembered that many of the Limoselleae are adapted to southern Africa’s rather dry conditions. Manulea/Sutera and Chaenostoma? The chloroplast and the combined analyses clearly show a paraphyletic Sutera, but the ITS analysis indicates monophyly of the genus. The latter result is weak (not significant 80% PP), but taken together with the morphological similarity of the sections Sutera and Chaenostoma, the result is noteworthy. The phylogram (Fig. 4) shows short branches in both Manulea and Sutera sect. Chaenostoma. The small sequence variation in Manulea and Sutera sect. Chaenostoma in all analyses indicates that they have either diverged very rapidly or have an extensive rate of hybridization and introgression. There is no support in our data for the subgeneric classification of Manulea by Hilliard (1994). None of the subgenera constitute monophyletic groups. The difference in nectaries, which is one of the characters Hilliard (1994: 2) used to discriminate between Manulea and Sutera, seems to be a misconception. We found a conspicuous semiannular gland in S. foetida from sect. Sutera (see Fig. 5), i.e. the character does not discriminate between Manulea and Sutera. There are three possible solutions to the taxonomic enigma of the Manulea/Sutera complex: i) keep the current taxonomy; ii) lump all taxa of the Manulea/ Sutera clade into an expanded Manulea (Manulea published 1767 has priority over Sutera and Chaenostoma); or iii) split Sutera into two genera. Of these solutions the first, though appealing for reasons of taxonomic stability, does not take into account the strong evolutionary evidence from the chloroplast genome. The second, to lump all taxa of the Manulea/ Sutera clade into an expanded Manulea, does not give recognition to the morphological diversity that in the first hand motivated the creation of the genera. There are characters, such as the non-decurrent pos- © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) B C A D Figure 5. Nectaries in Sutera taxa. Arrows indicate nectaries. A, ovary with semi-annular gland of S. foetida. B– D, pistils of S. patriotica, S. calciphila and S. floribunda, respectively, with lateral glands. (Drawings and photo by Per Kornhall.) ticous filaments, that could be used as diagnostic features for an expanded Manulea, but the morphological heterogeneity would be quite high in the genus. Manulea and Chaenostoma are, in almost all cases, easy to distinguish in the field. Manulea differs from Chaenostoma by having reddish or brownish flowers, abruptly inflated and often bent at the apex, in racemes, thyrses or panicles and with leaves more or less rosulate. This is in contrast to Chaenostoma that most often have white, solitary flowers on stems that are leafy throughout. This solution would 461 also require very many new combinations to be made in Manulea. We believe that the third alternative is the best, as it is supported by both molecular and morphological data. It also requires relatively few new combinations since many of the Sutera sect. Chaenostoma species already have synonyms in Chaenostoma. One or two species and hybrids of Sutera, e.g. S. hispida Druce and S. cordata (sometimes sold under the erroneous name ‘Bacopa’), have lately become quite popular as garden plants. These will now be transferred to Chaenostoma. To avoid this, the section Chaenostoma could possibly be conserved with a new type, but that would leave section Sutera without any legitimate name. We therefore propose revival of the nomen conservandum Chaenostoma for a genus consisting of the morphologically distinct species in Sutera sect. Chaenostoma sensu Hilliard, leaving Sutera consisting of Sutera sect. Sutera (sensu Hilliard, 1994). The type of section Chaenostoma, Sutera aethiopicum Kuntze, is unfortunately not represented in our molecular sampling, but S. calciphila, S. patriotica and S. caerulea Hiern, represent its closest allies (Hilliard, 1994). We conclude that the characters used to subdivide the Manulea/Sutera complex into two genera do not demonstrate the evolutionary history of the species involved. We think that the division of the complex into three genera better reflects both the phylogeny and the morphological diversity of the species involved. A key to the genera Manulea, Chaenostoma and Sutera, and generic descriptions are given below. A list of the names and combinations in Chaenostoma can be found in Appendix 2. KEY TO THE GENERA MANULEA, CHAENOSTOMA AND SUTERA Aa. Posticous stamens included, inserted halfway up the tube or higher. Anticous pair sometimes visible in mouth but not exerted...................................................................................................... Manulea Ab. At least one pair of anthers exerted (in two species of Sutera all stamens included but then inserted near base of tube). ............................................................................................................................. B Ba. At least some cymules with 3–11 flowers in every inflorescence. Hairs present on upper surface of corolla lobes around mouth........................................................................................................ Sutera Bb. Flowers solitary in leaf axils. Corolla lobes glabrous on upper surface .................................................. Chaenostoma DESCRIPTIONS OF SUTERA AND CHAENOSTOMA Sutera Roth, Bot. Bemerk. 172 (1807). Syn. Sutera Roth section Sutera Hilliard 1994: 221. Bushy perennial or annual (S. foetida) herbs, glandular and sometimes foetid. Stems leafy throughout. Leaves opposite (S. cooperi), or alternate (S. griquensis), or opposite becoming alternate upwards (S. foetida), bases cordate, cuneate, abruptly contracted or tapering into a petiolar part, margins toothed or serrate. Inflorescence ± racemose, flowers in cymules or cymose racemes, rarely solitary, sometimes panicled. Bracts present or wanting, not adnate to base of pedicel. Calyx bilabiate, sometimes obscurely so, anticous lip 2-lobed, posticous lip 3-lobed, lobes ± linear-lanceolate, usually pubescent. Corolla tube cylindrical, or narrowly funnel-shaped (S. foetida), mouth round, limb nearly regular, lobes spreading, suborbicular to oblong, entire, glandularpubescent or glabrous (S. foetida) outside, often with glistening glands as well, inside with clavate hairs extending from throat out onto lower part of lobes. Sta- © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 462 P. KORNHALL and B. BREMER mens 4, didynamous, filaments not decurrent, anticous pair shortly exerted in one species (S. foetida), posticous pair included (in S. cooperi, S. griquensis deeply included and inserted in lower part of tube); all anthers synthecous. Stigma usually lingulate with 2 marginal bands of stigmatic papillae, included in two species and shortly exserted in S. foetida. Ovary ± elliptic in outline, ovules many in each loculus. Fruit a septicidal capsule with a short loculicidal split at tip of each valve, glabrous or with glistening glands. Seeds roughly elliptic in outline, sometimes angled by pressure, ambercoloured, testa thin, tightly investing the endosperm, which is alveolate, with several longitudinal rows of transversely elongated pits arranged in chequer-board fashion, under the SEM seen to be ornamented with ± oblong reticulations. Distribution: Southern Africa from the Orange Free State and Transvaal to the Cape. Three species: S. cooperi, S. griquensis and S. foetida. Chaenostoma Benth. in Hook., Comp. Bot. Mag. 1: 374 (1836), nom. cons. Syn. Sutera Roth section Chaenostoma (Benth.) Hilliard 1994: 221. SHRUBLETS, suffrutices or perennial herbs, rarely annual, mostly glandular, and sometimes aromatic or foetid. STEMS leafy throughout. LEAVES usually opposite, sometimes alternate upwards, bases either ± connate or decurrent in narrow wings or ridges, simple, entire to toothed, rarely more deeply lobed. INFLORESCENCE ± racemose, flowers mostly solitary in axils of leaves or bracts, sometimes in cymules or cymose racemes, sometimes panicled. BRACTS at most adnate to extreme base of pedicel. CALYX bilabiate, sometimes obscurely so, anticous lip 2-lobed, posticous lip 3-lobed, or rarely regularly divided into 6–9 lobes, lobes ± linear-lanceolate, usually pubescent. COROLLA tube funnel-shaped, mouth round, limb nearly regular, lobes spreading, suborbicular to oblong, entire, usually glandular-pubescent outside, often with glistening glands as well, inside usually with either 1– 5 longitudinal bands of clavate hairs in throat, or glabrous. STAMENS 4 (a 5th occasionally developed), didynamous, filaments usually inserted in upper part of corolla tube, not decurrent, anticous pair exserted, posticous pair either included or exserted; all anthers synthecous. STIGMA usually lingulate with 2 marginal bands of stigmatic papillae, exerted. OVARY ± elliptic in outline, often with glistening glands at least on the sutures, rarely glandular-pubescent as well, nectariferous gland semi-annular, ovules many in each loculus. FRUIT a septicidal capsule with a short loculicidal split at tip of each valve, glabrous or with glistening glands. SEEDS roughly elliptic in outline, sometimes angled by pressure, amber-coloured, pallid or grey- to violet-blue, testa thin, tightly investing the endosperm, which is alveolate, with several longitudinal rows of transversely elongated pits arranged in chequer-board fashion, under the SEM seen to be ornamented with irregular pustules. Distribution: Africa south of the Cunene and Zambezi rivers, mainly Cape, Natal, Transvaal. 46 species. New description of the tribe Limoselleae Scrophulariaceae tribe Limoselleae Dumort., Florula Belgica p.52 (1827). Syn. Manuleae, Benth. & Hook. f., Genera plantarum 2: 915–919 (1876); Selagineae, Choisy in Memoires de la Société de Physique et d’Histoire Naturelle de Genève 2. 2 (1822); Hebenstretieae, Horaninov (1847); Selagineae, Horaninov (1847). Herbs or shrubs, often glandular. Leaves simple, without stipules, often opposite at the base of the plant and alternate upwards. Bracts (if present) often adnate to calyx. Flowers often solitary in leaf axils, often in racemes of cymes, or in panicles, occasionally corymbose, bisexual, zygomorphic to subactinomorphic. Calyx (3-)5(-9) lobed, obscurely to distinctly bilabiate. Corolla gamopetalous, tube cylindrical or funnelshaped, more or less bilabiate, posterior lip 2-lobed, anterior lip 3-lobed, sometimes lower lip seems wanting and posterior lip 4-lobed, with unicellular clavate hairs inside. Stamens, dorsifixed, synthecous (2-)4,(5), inserted in corolla tube. Stigma often lingulate with marginal bands of stigmatic papillae, rarely bifid, or entire with terminal papillae. Style solitary, terminal, and filiform. Ovary superior, 2-celled or rarely 1celled by abortion. Ovules one to many in each locule. Nectary often a small dorsal gland, sometimes annular. Fruit, when many-seeded a septicidal capsule, otherwise indehiscent. Seeds small, often with copious endosperm. Genera included: Barthlottia, Chaenostoma, Chenopodiopsis, Cromidon, Dischisma, Glekia, Globulariopsis, Glumicalyx, Gosela, Hebenstretia, Jamesbrittenia, Limosella, Lyperia, Manulea, Manuleopsis, Melanospermum, Microdon, Phyllopodium, Polycarena, Pseudoselago, Reyemia, Selago, Strobilopsis, Sutera, Tetraselago, Trieenea and Zaluzianskya. ACKNOWLEDGEMENTS We thank Mats Thulin and Bengt Oxelman for advice and support, Nahid Heidari for help in the laboratory, the Natural History Museum in Stockholm, Kew, Compton Herbarium, and the Muséum National d’Histoire Naturelle in Paris for sending material for study. Financial support has been © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) received from the Swedish Research Council, to Birgitta Bremer. The Royal Swedish Academy of Sciences and the Ax:n Jonsson foundation has granted support to Per Kornhall. 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Zimmer EA, Roalson EH, Skog LE, Boggan JK, Idnurm A. 2002. Phylogenetic relationships in the Gesnerioideae (Gesneriaceae) based on nrDNA and cpDNA trnL-F and trnE-T spacer region sequences. American Journal of Botany 89: 296–311. APPENDIX 1 ITS primers used for amplification and sequencing. *= used for amplification. Forward: P16 P17* P16,5 P16B ITS.LEU1 tca ctg aac ctt atc att tag agg a cta ccg att gaa tgg tcc ggt gaa gac gtc gcg aga agt yca ytg a cca ytg aac ctt atc att kag agg a gtc cac tga acc tta tca ttt ag Popp & Oxelman (2001) Popp & Oxelman (2001) B. Oxelman, unpubl. data B. Oxelman, unpubl. data Andreasen, Baldwin & Bremer (2000) Reverse: P25 P26sR ITS4 26S-82R* ggg tag tcc cgc ctg acc tg gat atg ctt aaa ytc ggc ggg t tcc tcc gct tat tga tat gc tcc cgg ttc gct cgc cgt tac ta Oxelman & Lidén (1995) B. Oxelman unpubl. data White et al. (1990) Popp & Oxelman (2001) APPENDIX 2 Evaluation of the different PP runs for the different data sets (see text). Node = chosen nodes, see Figs 2, 3, 5. Mean = mean values from the three runs in the PP-analysis. % = maximum deviation from the mean values in percent. Pars. = value from MP Jackknife analyses on comparable nodes. NdhF ITS Node Mean % Pars. Node Mean % Pars. 1 2 3 4 5 6 7 8 9 10 100 100 100 97.3 100 100 100 100 100 100 0 0 0 1.7 0 0 0 0 0 0 100 94 91 80 100 100 100 100 98 74 1 2 3 4 5 6 7 8 9 10 99.7 100 100 99.7 100 100 100 100 100 98.7 0.7 0 0 0.7 0 0 0 0 0 1.7 75 71 97 82 99 100 100 71 100 96 © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) 465 APPENDIX 2 Continued NdhF ITS Node Mean % Pars. Node Mean % Pars. 11 12 13 14 15 16 17 100 100 100 100 100 100 100 0 0 0 0 0 0 0 100 100 93 100 79 100 96 11 12 13 100 100 98.7 0 0 1.7 100 100 94 Comb. Node Mean % Pars. Node Mean % 1 2 3 4 5 6 7 8 9 10 11 12 13 100 98.7 100 61.7 100 100 100 100 98 100 100 100 100 0 0.7 0 10.3 0 0 0 0 1 0 0 0 0 97 84 100 Missing Missing 99 100 100 76 100 100 99 100 14 15 16 17 18 19 20 21 22 23 24 25 26 100 100 99 94 100 100 99.7 96.3 100 83.7 100 92 93 0 0 0 0 0 0 0.7 3.5 0 4.4 0 2.2 2.2 Pars. 100 100 94 74 97 95 Missing 77 100 Missing 97 Missing Missing APPENDIX 3 Posterior distributions for the parameters from the PP runs. Mean = the mean values from the three runs/PP analysis. % dev = the greatest deviation from the mean values in per cent. r(x–y) = the substitution rate for the transition/transversion from ¥ to y, p(x) = stationary frequency of nucleotide x, alpha = the shape parameter for the gamma distribution, ss(z) = the substitution rate for the codon position z. ndhF ITS Total Parameter Mean % dev. Mean % dev. Mean % dev. r(g–t) r(c–t) r(c–g) r(a–t) r(a–g) r(a–c) p(a) p(c) p(g) p(t) alpha ss1 ss2 ss3 1.00 3.37 2.53 0.25 3.12 2.26 0.30 0.13 0.16 0.42 0.39 0.68 0.48 1.84 0.00 1.76 1.67 4.12 1.50 0.68 0.18 0.66 0.09 0.36 0.03 0.21 1.15 0.22 1.00 1.65 0.66 1.98 3.30 0.88 0.21 0.29 0.28 0.22 0.36 0.00 0.05 0.47 1.50 0.25 1.21 0.11 0.32 0.10 0.44 0.23 1.00 1.81 1.35 0.52 2.07 1.08 0.29 0.18 0.18 0.34 0.00 0.35 0.11 1.13 0.21 0.43 0.01 0.19 0.15 0.19 © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 466 P. KORNHALL and B. BREMER APPENDIX 4 Species list with voucher information and/or accession numbers in GenBank/EMBL. * = sequences new in this publication. Numbers in parentheses after species names are for identification of sequences from the same species. Species Voucher ndhF Androya decaryi Perrier Anterothamnus pearsonii N.E. Br. Hansen 3472 (UPS) AF027276 AJ401392 Antirrhinum majus L. Barthlottia madagascariensis Fischer Guillaumet 3861 (P) L36392 AJ401438 Buddleja asiatica Lour. Buddleja indica = Nicodemia diversifolia Tenore Buddleja polystachya Fresen. Buddleja saligna Willd. Thulin 9405 (UPS) Bayliss 8158 (S) Buddleja thyrsoides Lam. Camptoloma canariense Hilliard Bengt Oxelman pers. comm. Jonsell 5558 (UPS) Camptoloma lyperiiflorum Hilliard Thulin, Beier & Hussein 9655 (UPS) Nordenstam & Lundgren 869 (S) Camptoloma rotundifolium Benth. Chenopodiopsis retrorsa Hilliard Cromidon decumbens Hilliard Dischisma ciliatum Choisy Freylinia tropica Moore Glekia krebsiana Hilliard Globularia cordifolia L. Glumicalyx flanaganii Hilliard & Burtt Halleria lucida L. Hebenstretia cordata L. Jacaranda sparrei Gentry Jamesbrittenia atropurpurea Hilliard Jamesbrittenia dissecta Kuntze Jamesbrittenia Jamesbrittenia Jamesbrittenia Jamesbrittenia filicaulis Hilliard foliolosa Hilliard megadenia Hilliard microphylla Hilliard Justicia carnea Lindl. Lamium purpureum L. Limosella aquatica L. Limosella grandiflora Benth. Limosella macrantha Fries Limosella major Diels Lyperia tristis Benth. (1) Lyperia tristis Benth. (2) Manulea annua Hilliard Manulea bellidifolia Benth. Manulea calciphila Hilliard Manulea caledonica Hilliard trnL ITS AJ401442 AJ296509 AJ550575 AJ401443 AJ401444 AJ550576 AJ551271 AJ550577 AJ550578 AF027277 L36405 Bremer 3765 (UPS) Bremer 3692 (UPS) Skarpe 372 (UPS) Kornhall 96 (UPS) Örtendahl 691 (UPS) Kornhall 58 (UPS) Lohammar 29.10. 1971 (UPS) Kornhall 112 (UPS) Hedberg 5640 (UPS) Hedberg & Aweke 5475 (UPS) Vlok 2488 (S) Bremer 3717 (UPS) Kornhall 6 (UPS) Hedberg 82011 (UPS) Kornhall 63 (UPS) Bremer 3714 (UPS) AJ401396, AJ401397 AJ401398, AJ401399 AJ401401 AJ401445, AJ401449 AJ296514 AJ401431, AJ401432 AJ401421 AJ401403 AJ401412 AJ401402 AJ401422 AF027282 AJ401413 AJ550569 AJ401414 AF102631 AJ550570 AJ401435, AJ401436 AJ401439 AJ550571 AJ401404 AJ550572, AJ550573 AF130155 U78694 AJ550547 AJ401450, AJ296515 AJ550579 AJ550580 AJ550581 AJ550582 AJ550583 AJ296511 AJ550584 AJ550588 AJ550552 AJ550553 AJ550548 AJ550525 AJ550526 AJ550587 AJ550586 AJ550585 AJ401406 AJ550554 AJ550555 AJ550556 AJ550557 AJ550558 AJ550527 AJ550528 AJ550529 AJ550530 AJ550533 AJ550531 AJ550589 AJ550614 AJ550590 AJ550591 AJ550592 AJ550593 © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467 THE TRIBE LIMOSELLEAE (SCROPHULARIACEAE) 467 APPENDIX 4 Continued Species Voucher ndhF trnL ITS Manulea cheiranthus L. Acock 4777 (S) Vlok 2514 (S) Hilliard & Burtt 12073 (S) AJ401446, AJ401452 AJ550532 AJ550594 Manulea chrysantha Hilliard Manulea crassifolia Benth. AJ401418, AJ401419 AJ550559 AJ401428, AJ401429 Manulea dubia Roessler Manulea exigua Hilliard Manulea glandulosa Phillips Manulea rubra L. f. Manulea schaeferi Pilger Manulea tomentosa L. Manuleopsis dinterii Thell. Melanospermum foliosum Hilliard Microdon lucidus Choisy Myoporum mauritianum A. DC. Olea europaea L. Phyllopodium cuneifolium Benth. Plantago lanceolata L. Polycarena formosa Benth. Pseudoselago ascendens Hilliard Reyemia chasmantiiflora Hilliard Scrophularia sp. Selago corymbosa L. Nordenstam 330 (S) Kornhall 82 (UPS) Bremer 3519 Kornhall 5 (UPS) Örtendahl 64 (S) Bremer 3781 (UPS) AJ550560 AJ550549 AJ550561 AJ550562 AJ401394 AJ401410 AJ401415 AJ401416 L36403 AF027288 AJ401430 L36408 AJ401423 AJ401433 AJ401425 L36411 AJ401434 AJ550534 AJ296520 AJ550535 AJ550536 AJ550537 Selago myrtifolia E. Mey. Sesamum indicum L. Stemodia glabra Oerst. Stilbe albiflora E. Mey. Sutera caerulea Hiern Sutera calciphila Hilliard Sutera campanulata Kuntze Sutera cordata Kuntze Sutera floribunda Kuntze Sutera foetida Roth (1) Vlok 2514 (S) Nordenstam et al. 967 (S) Vlok 00421a (S) Kornhall 52 (UPS) Batten 998 (S) Kornhall 106 (UPS) Batten 1065 (S) Batten 1107 (S) Sutera foetida Roth Sutera hispida Druce Batten 1164 (S) Kornhall 91 (UPS) Sutera patriotica Hilliard Sutera revoluta Kuntze Tetraselago longituba Hilliard & Burtt Trieenea glutinosa Hilliard Verbascum arcturus L. Bremer 3818 (UPS) Kornhall 98 (UPS) Verbascum thapsus L. Verbena bracteata Cav. Zaluzianskya glareosa Hilliard & Burtt Zaluzianskya minima Hilliard Gustafsson 134 (UPS) Bremer 3542 (UPS) AJ401420 L36413 AJ550574 AF027287 AJ550563 AJ550550 AJ550564 AJ550565 AJ401407, AJ401408 AJ550551 AJ550566, AJ550567 AJ401393 AJ550568 AJ401417 AJ401400 AJ401460, AJ296522 L36417 L36418 AJ401424 AJ401437 AJ550595 AJ550596 AJ550597 AJ550598 AJ550599 AJ550600 AJ550601 AJ550602 AJ401458, AJ296494 AJ550603 AJ550538 AJ550539 AJ550540 AJ550541 AJ296510 AJ550604 AJ550605 AJ550606 AJ550607 AJ550608 AJ550609 AJ550542 AJ550543 AJ550611 AJ550610 AJ551261 AJ551262 AJ550612 AJ550613 AJ550615 © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 453–467