Phylogeny, Morphological Evolution, and Speciation of Endemic Brassicaceae Genera in the Cape Flora of Southern Africa Author(s): Klaus Mummenhoff, Ihsan A. Al-Shehbaz, Freek T. Bakker, H. Peter Linder, Andreas Mühlhausen Reviewed work(s): Source: Annals of the Missouri Botanical Garden, Vol. 92, No. 3 (Oct., 2005), pp. 400-424 Published by: Missouri Botanical Garden Press Stable URL: http://www.jstor.org/stable/40035479 . Accessed: 29/02/2012 04:58
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http://www.jstor.org PHYLOGENY, Klaus Mummenhoff,2 MORPHOLOGICAL Ihsan A. Al-Shehbaz,3 Freeh T. Bakkerf EVOLUTION,AND H. Peter hinderf SPECIATIONOF ENDEMIC and Andreas Miihlhausen6 BRASSICACEAEGENERA IN THE CAPE FLORA OF SOUTHERNAFRICA1
Abstract
Heliophila (ca. 73 spp.), the ditypic Cycloptychisand Thlaspeocarpa,and the monotypic Schlechteria, Silicularia, Brachycarpaea, and Chamira are endemic to the Cape region of South Africa, where they are the dominant genera of Brassicaceae. They may be regarded as the most diversified Brassicaceae lineage in every aspect of habit, leaf, flower, and fruit morphology.The characters used in the separation of these genera and their species, especially fruit type (silique vs. silicle), dehiscence (dehiscent vs. indehiscent), compression (latiseptate vs. angustiseptate),and cotyledonary type (spirolobal, diplecolobal, twice conduplicate), have been used extensively in the delimitation of tribes. The rela- tionship and taxonomic limits among these genera are unclear and controversial. The present ITS study demonstrates the monophyly of tribe Heliophileae, with Chamira as sister clade. The other five smaller genera above are nested within two of the three main lineages of Heliophila, to which they should be reduced to synonymy.The current study reveals parallel evolution of fruit characters often used heavily in the traditional classification schemes of the family. However, the arrangementof species into three main clades largely corresponds with the distribution of morphological characters (e.g., habit, leaf shape, seed structure, inflorescence type, and pres- ence/absence of basal appendages on the pedicels, petals, and staminal filaments) not adequately accounted for in previous studies. Estimation of divergence times of the main lineages of Heliophila is in agreement with recent esti- mations in other plant groups, all of which date the diversification against a backgroundof aridificationin the Pliocene and Pleistocene. Species of one main clade are perennial, microphyllous shrubs/subshrubstypically restricted to poor sandstone soils in the southwesternand western parts of the Cape Floristic Region of South Africa. Species of the other two clades are predominantlyannuals that grow in more arid regions of Namibia and Namaqualand,as well as in the above sandstone areas of the Cape Region. The adaptive significance of various floral structures is discussed in terms of their possible role in the rapid diversification within Heliophila. Key words: Brachycarpaea,Cape flora, Cape Floristic Region, Chamira, Cycloptychis,Heliophila, Heliophileae, ITS, phylogeny, radiation, Schlechteria, Silicularia, speciation, Thlaspeocarpa,trnL-F.
Seven genera of Brassicaceae, Heliophila (73 represent the dominant Brassicaceae. Although spp.), Cycloptychis(2 spp.), Schlechteria (1 sp.), Si- several classification systems (Table 1) have been licularia (1 sp.), Thlaspeocarpa (2 spp.), Brachy- proposed (e.g., Hayek, 1911; Schulz, 1936; Jan- carpaea (1 sp.), and Chamira (1 sp.), are endemic chen, 1942), the relationships among these genera to southern Africa (for authornames of these genera remain unresolved. In a recent re-evaluation of the and their species, see Table 1 and Appendix 1). group, Appel and Al-Shehbaz (1997) placed the Most species occur in the winter-rainfallarea of the first six genera in the tribe Heliophileae and re- western Cape Floristic Region (CFR), where they tained Chamira in the monotypic Chamireae.They
1 We are grateful to the directors, curators, and collectors of the institutions that provided the plant material. We thank Ulrike Coja for technical assistance, Andreas Franzke for fruitful discussions, and Adelaide Stork, Ashley Nicholas, and two anonymous reviewers for constructive criticisms that helped improve the final draft. 2 Departmentof Botany, Faculty of Biology/Chemistry,University of Osnabrtick,Barbarastrasse 11, 49069 Osnabriick, [email protected]. 3 Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri, U.S.A. 63166-0299. 4 [email protected]. National Herbarium Nederland, Wageningen branch & Biosystematics Group, Wageningen University, Generaal Foulkesweg 37, 6703 BL Wageningen,The Netherlands, [email protected]. 5 Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, CH-8008, Switzerland, plin- [email protected]. 6 Departmentof Botany,Faculty of Biology/Chemistry,University of Osnabriick,Barbarastrasse 11, 49069 Osnabriick, [email protected]. Ann. Missouri Bot. Gard. 92: 400-424. Published on 10 October 2005. Volume 92, Number 3 Mummenhoff et al. 401 2005 Brassicaceae In Cape Flora
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Figure 1. Strict consensus ITS tree with the distribution of morphological data in Heliophila and related genera. Cleome spinosa (Cleomaceae), Aethionema saxatile, Alliaria petiolata, Rorippa amphibia, and Cardamine matthioli (Brassicaceae) served as the outgroup. The consensus tree is based on 14,560 equally parsimonious reconstructions found after heuristic search; jackknife support > 50% (10,000 replicates) are indicated at the branches. Taxon abbre- viation: H. = Heliophila, Thl. = Thlaspeocarpa,Bra. = Brachycarpaea, Cyc. = Cycloptychis,Sch. = Schlechteria,Sil. = Silicularia, Cha. = Chamira. 1 Sectional classification of Sonder (1846): I = Orthoselis subsection 1: herbaceous species, II = Orthoselis subsection 2: shrubby species, III = Ormiscus, IV = Lanceolaria, V = Pachystylum, VI = Leptormus.Heliophila 2 species not characterized by Roman numerals were not recognized by Sonder (1846) or they represent other genera. Fruit types: relative fruit length: q = silique, s = silicle; orientation and degree of the fruit compression: lat = latiseptate, ang = angustiseptate, ter = terete, inf. = inflated; fruit margin: mon = moniliform (fruits deeply constricted between the seeds), str = fruits with straight margins;fruit opening: deh = dehiscent, ind = indehiscent. 3 Habit: a = annual herb, p = perennial herb, s = shrub, ss = subshrub, ssa = subshrub, but annual shoots arising 4 - 5 from a woody crown. Pedicel: + = with two small bracts, = without two small bracts. Leaf: p = pinnately - 6 - divided, e = entire; = leaves exstipulate, + = leaves subtended by two minute stipules. Petal: = without 7 - 8 basal appendages, + = with basal appendages. Anther: + = presence of apicula, = apicula absent. Filament: - 9 - + = with basal appendages, = without basal appendages. Seed: = wingless or with a very narrowmargin, + 10 = distinct wing; p = papillate, s = smooth. Inflorescence: r = raceme, i = intercalaryinflorescence. Data compiled from Marais (1970), Bean (1990), and Goldblatt and Manning (2000) and the authors' studies of specimens at MO. 404 Annals of the Missouri Botanical Garden
flora, Linder (2003) suggested that the high level onema saxatile, Alliaria petiolata, Rorippaamphib- of endemism might be the consequence of ecolog- ia, and Cardamine matthioli were chosen as out- ical and geographical isolation of the CFR. He also groups on the basis of previous molecular suggested that the species richness might be the phylogenetic studies (Koch et al., 2001). Methods consequence of radiation that started between 18 for DNA extraction, PCR amplificationof the rDNA and 8 million years ago, which might be accounted ITS and cpDNA trnL-F regions, BigDye terminator for by the diverse limitations to gene flow (e.g., dis- labeled sequencing, sequence assembly and align- sected landscapes, pollinator specialization), as ment are described in Bowman et al. (1999) and well as by a climatically and topographicallycom- Mummenhoffet al. (2001, 2004). plex environment (e.g., altitudinal variation, soil types) allowing numerous niches and resulting in DATA ANALYSIS highly fragmented distribution ranges. Studies are needed to provide (i) phylogenetic The aligned sequences were subjected to both data demonstrating the monophyly of diversifying parsimony and Bayesian analysis, using lineages in the CFR, (ii) more molecular clock es- PAUP*4.0bl0 (PPC/Altivec) and MrBayes 3 (Ron- timates to accurately date the radiation, and (iii) quist & Huelsenbeck, 2003) implemented on a species-level phytogenies to detect sister-species Macintosh G4 computer. Jackknife analysis was relationships to study speciation. The current study carried out using PAUP* with settings so as to em- addresses these needs by providing a robust phy- ulate Parsimony Jackknifer (Farris et al., 1996): logeny of the tribe Heliophileae based on the nu- percentage of characters deleted in each replicate clear ITS region of 55 of its 80 species. We used = 37, "fast" stepwise addition, and "Jac" re-sam- nonparametricrate smoothing (NPRS) of Sanderson pling method. Heuristic searches involved TBR (1997), as well as a "forced" (global) clock ap- branch swapping, MULPARS,and collapse branch- proach to date the diversification of Heliophileae es when maximum length is zero. Starting trees by implementing fossil data and published calibra- were generated by 1000 cycles of random addition tion points (Wikstromet al., 2001) and a range of sequence holding 3 trees at each step, and keeping published rDNA ITS substitution rates. In addition, no more than 1000 trees > length 904 steps. Trees the pattern of species distribution in relation to resulting from this search were then used as start- their ecology and phylogeny was investigated in an ing trees in a subsequent search during which they attempt to get insights into speciation mode in this were swapped to completion as far as possible. lineage. Bayesian analysis was performed on the ITS alignment using settings derived from ModelTest Material and Methods analysis (Posada & Crandall, 1998): the maximum likelihood model employed 6 substitution types MORPHOLOGY ("nst = 6") and rate variation across sites was mod- eled a distribution = All species of Brachycarpaea, Cycloptychis,He- using gamma (rates "gam- while invariant sites were also assumed. The liophila, Silicularia, Schlechteria, and Thlaspeocar- ma"), Markov chain Monte Carlo search was run with 4 pa have been critically evaluated morphologically. one of which The characters studied, all considered to be taxo- chains, "cold," for 1,000,000 gener- with trees 100 nomically important (see Marais, 1970), include ations, being sampled every gener- ations. After the first 10% of trees as habit; presence vs. absence of staminal appendag- discarding search results were summarized es; ovule number per ovary; orientation of fruiting "burnin,"Bayesian 50% rule consensus and the pedicels; fruit shape, dehiscence, compression, by majority posterior values are indi- presence vs. absence of carpophore, development probability ("clade credibilities") cated at the branches of the septum, valve texture, and valve sculpture; (Fig. 2). Likelihood ratio was the seed shape and presence vs. absence of wing; and testing performedusing 50% rule consensus tree in order cotyledonary type. These are listed in Table 2 and Bayesian majority to check for a molecular clock. An ultrametrictree Figure 1 and need no further details. was then produced from the Bayesian consensus tree both NPRS as in the DNA EXTRACTION, GENE AMPLIFICATION, using implemented pro- "r8s" version 1.06 described San- AND SEQUENCING gram (beta) by derson (1997), as well as by "forcing" a clock in Plant material, locality information,voucher de- PAUP*. The Heliophileae ITS tree with branch tails, and GenBank accession numbers are given in lengths in six decimals was saved and input in r8s, Appendix 1. Cleome spinosa (Cleomaceae), Aethi- in which the following branch length formatsettings Volume 92, Number 3 Mummenhoff et al. 405 2005 Brassicaceae in Cape Flora
Figure 2. Bayesian consensus tree with mean branch length and a posteriori probabilities, and the distributionof fruit types among the main clades of Heliophila s.l. Taxon abbreviationfollows Figure 1. Cleomespinosa (Cleomaceae), Aethionema saxatile, Alliaria petiolata, Rorippa amphibia, and Cardamine matthioli (Brassicaceae) served as the out- group. Illustrated species written in larger bold font. 406 Annals of the Missouri Botanical Garden
were used: "length = persite, nsites = 498, ultra- ECOLOGICAL OPTIMIZATIONS metric = no, round = yes"; divergence times of We followed the of Ma- other than the fixed node (see below) were esti- species-level taxonomy rais because it is the mated using the settings: "method = NPRS, algo- (1970) only comprehensive account of the However, he did use rithm = POWELL,"and "set num_time_guesses= Heliophileae. a rather broad species concept and recognized 10" in order to ensure optimal exploration of so- many infraspecific taxa occupying ecologically het- lution space. erogeneous habitats. The ecological attributes of In order to estimate error around node ages, we the species, and their distributionranges, were tak- performed100 replications of constrainedjackknife en primarily from Marais (1970). In addition, the analysis of the data matrix, saving each tree under herbariumholdings of BOL and PRE were surveyed the constraints of the consen- topological Bayesian for collection data, though their identities were not sus tree. In this identical trees are way, topology verified because most of them were studied by Mar- produced with variation in branch lengths reflecting ais and are well curated. Altitudinal data were "substitutional noise" as the re-sam- picked up by checked against the ranges in Germishuizen and All hundred trees were then an- pling procedure. Meyer (2003) and Goldblatt and Manning (2000). alyzed in r8s with respect to their A, B, and C Species were scored for the following four envi- nodes, using the settings above. Age estimates ronmental parameters: around these nodes were summarized using the 1. Distribution ranges (Roggeveld, Drakensberg, "profile"command. Eastern Cape, Southern Cape, Swartruggens,cen- In addition, we prepared a "forced (global) tral Cape mountains, Cedarberg-Nieuwoudtville, clock" tree in PAUP*. In this approach an ultra- Kamiesberg, Gordonia, Richtersveld, Namib). metric tree is produced by assuming overall clock- These ranges follow a combination of topographical like behavior of the data, under otherwise the same features of southern Africa that often provide nat- likelihood model as the one arrived at in the Bayes- ural boundaries to species distribution, and this ian analysis described above. Forced clock trees theme is used in combination with actual climatic were made in- and excluding the outgroup Cleome zones (see Fig. 3). spinosa. 2. Vegetation type (woodland, forest, grassland, As an alternative to the calibration using the Karoo, thicket, Namaqua Broken Veld, succulent dates from Wikstrom et al. (2001), we also cali- Karoo, Renosterveld, Strandveld, Fynbos). These are structuralrather than floristic. Ka- brated our ultrametric trees by applying a range of types largely roo refers to the to semi-arid sum- published rDNA ITS substitution rates. We took the shrubby grassy mer-rainfall of the central of rates published for DendroserisD. Don (Asteraceae) vegetation uplands southern whereas succulent Karoo is a from Sang et al. (1994), i.e., 3.9 X 10"9 subst./site/ Africa, very succulent winter-rainfallsemidesert yr, and Soldanella L. (Primulaceae) from Zhang et shrubby vege- tation (Rutherford & Westfall, al. (2001), i.e., 8.3 X 10~9 subst./site/yr, to repre- 1986). Namaqua Broken Veld is a taller shrubland characteristic of sent the currently known range of substitution rate the granitic escarpment of Namaqualand (Acocks, for this region in angiosperm eudicots. Ultrametric 1975). Renosterveld, strandveld, and fynbos are tree node height H is the result of accumulated typical vegetation types of the Cape Floristic Re- substitutions per site along two evolutionary lines; gion. The first is a widespread shrubland on richer therefore height = substitution rate r X twice di- soils dominated by Elytropappusrhinocerotis (L. f.) vergence time T, and T can be calculated as HI2r. Less. (Asteraceae). Strandveld is a non-pyrophytic Rate calibration was performed in TreeEdit v. woody shrubland of coastal dunes, and fynbos is l.OalO (Rambaut & Charlston, 2001) by dividing the typical pyrophytic heath vegetation of oligotro- the ultrametric node twice the substi- heights by phic soils dominated by Restionaceae, Proteaceae, tution rate, and multiplying by the number of sites and Ericaceae. used = in order to arrive at node (n 498) heights 3. Bedrock/substrate types (sandstone, shale, in millions of years. granite, coastal sand, acidic sand, limestone, Karoo We used fruit fossil data of Rorippa Scop. (2.5- shale). These categories represent the substrate of 5 million years old; Mai, 1995) to constrain the a general area, rather than a specific microhabitat, clade containing Rorippa and CardamineL. at 2.5- and as such can be inferred from geological maps. 5 mya minimally. These genera have been shown Sandstone, shale, limestone, coastal sand, and to be sister taxa in several phylogenetic studies acidic sand are the major substrate types of the (e.g., Mummenhoffet al., 2001, 2004). Cape Floristic Region. Karoo shale refers to the Volume 92, Number 3 Mummenhoff et al. 407 2005 Brassicaceae in Cape Flora
Figure3. Distributionareas of Heliophilaspecies and relatedgenera in outlines.The distributionalcategories followa combinationof the topographicalfeatures of southernAfrica, which often provide natural boundaries to dis- tributionranges, used in combinationwith actual distributions and zonesof climaticchange (see Goldblatt& Manning, 2000; Cowling& Heijnis,2001). fine-grained sedimentary rock that is widespread signed to any ancestral node reconstruction. This throughoutthe Karoobasin and also forms the ped- impacted the reconstructionof the basal but not the iments of the Drakensberg. Much of the Namaqua- more terminal nodes. In order to use DIVA, a fully land escarpment is granitic. resolved tree was needed, and one of the maximally 4. Soil type (stony-gravel, gravel, sand, calcrete, parsimonious trees was arbitrarily chosen for this loam, boulders, clay). This indicates the substrate analysis. The environmental attribute information at the microhabitat scale, and is recorded largely for some species was incomplete; in these cases the from herbariumnotes. species were scored the same as their sister spe- The environmental attributes of the internal cies. cladogram nodes were inferred using DIVA opti- mization (Ronquist, 1997). This optimization was Results to track in the distributions of developed changes phylogenetic analysis clades, assuming that vicariance and sympatric speciation were the "normal"situation, by attach- The ITS alignment, including the outgroups(Cle- ing a cost to dispersal, but not to sympatric spe- ome spinosa (Cleomaceae),Aethionema saxatile, Al- ciation or vicariance. This method does not enforce liaria petiolata, Rorippa amphibia, and Cardamine monomorphyat internal nodes, and it allows an- matthioli (Brassicaceae)), contained 57 taxa and cestors to remain polymorphic for individual attri- 473 characters of which 195 were parsimony infor- butes. As such it is the ideal approach for inferring mative. Using the above-mentioned settings, par- the evolutionaryhistory of environmentalattributes simony searches yielded 14,560 MPTs 904 steps (Linder & Hardy, 2005; Hardy & Linder, 2005). It long (CI = 0.52, RI = 0.69) of which the strict is implemented in the software DIVA (Ronquist, consensus is shown in Figure 1. In the Bayesian 1996). In the biogeographical analysis the number analysis and after the MCMC had finished, 1000 of possible results obtained by DIVA was very out of the 10,000 trees sampled were discarded as large. Therefore, a maximum of four areas were as- burnin "trees." Model parameter values had con- 408 Annals of the Missouri Botanical Garden
Table 3. Date estimates of the Heliophileae clade and its comprising subclades A, B, and C based on different approaches;numbers indicate millions of years. See text for further details.
Node^
Calibration Heliophileae ABC Wikstromet al.1 NPRS2 4.2/4.6 3.4/3.7 2.9/3.3 3.9/4.3 NPRS jackknife3 3.7-5.4 2.8-4.5 2.2-3.9 n.a. Forced clock4 1.9 1.3 1.0 1.7 rDNA ITS rates5 non-ultrametricdistances 2.7-5.8 1.1-2.3 1.5-3.2 2.2-3.8 Forced clock4 2.3-4.9 1.6-3.3 1.3-2.7 2.1-4.4 1 The Brassica/Cleome clade was dated 22 mya (from Wikstromet al., 2001). 2 Tree made ultrametric using NPRS; additional calibration by constraining the Rorippa/Cardamineclade to be min- imally 2.5/5.0 mya. 3 Estimated on jackknife re-sampled branch lengths under NPRS. 4 Tree made ultrametricassuming a global clock. 5 (3.9-8.3) X 10~9 subst./site/year (from Sang et al., 1994; Zhang et al., 2001). verged at TL = 3.15 (± 2.67), r(C<->T)= 5.15 With all model parametersestimated and excluding (±0.82), r(C<->G)= 0.74 (± 0.03), r(A<->T)= 2.22 some of the outgroup taxa, i.e., Cleomespinosa and (± 0.21), r(A<->G)= 2.98 (± 0.31), r(A<->C)= Aethionema R. Br., a molecular clock was rejected = = 1.42 (± 0.11), pi(A) 0.27, pi(C) 0.23, pi(G) (P < 0.01); therefore, the ingroup tree was made = = = = 0.22, pi(T) 0.29, a 1.08, and pinvar ultrametric using the NPRS method as implement- 0.12. Parsimony and Bayesian tree topologies were ed in r8s, as well as by applying a global clock in largely congruent. PAUP* ("forcedclock," see below). Node ages were The cpDNA trnL intron region data set included then estimated in the following ways: the age of the 370 base pairs, of which 57 were variable charac- entire tree including Cleome spinosa and Aethione- ters and only 13 were phylogenetically informative. ma was fixed at 22 (± 2) mya, as this is the age Due to the lack of informative both characters, par- estimated by Wikstrom et al. (2001) for their rbcL- simony and Bayesian analysis of this data set re- based Brassica/Cleome clade. The dates in Wik- sulted in a resolved tree poorly topology. Eight strom et al. (2001) may be severe underestimates were found that were all cpDNA lineages present of clade ages, possibly due to the "thin"taxon sam- (except two in the ITS as well. species) toplogy pling in that study (Wikstromet al., 2003). There- However, their relationships and the main lineages fore, actual dates obtained here for the Heliophila A, B, and C could not be resolved. Thus, the dis- ITS tree may be too young as well, although it is cussion hereafter is based on the ITS data only. not clear by how much. Moreover, as no further In all phylogenetic trees, one robust main clade outgroup ITS sequences could be aligned to the with three sublineages A through C is recognized Heliophila matrix, the only option was to fix the (Figs. 1, 2). These three clades form a trichotomy entire tree rather than a maximumconstraint. in the strict consensus tree and the Bayesian tree, apply In we a minimum and they comprise all Heliophila species along with addition, simultaneously applied constraint of 2.5- 5 for the clade the genera Thlaspeocarpa(clade C), Brachycarpaea, age mya containing and Cardamine as this Cycloptychis, Schlechteria, and Silicularia (clade Rorippa amphibia matthioli, to the of fruit fossils B). Within clade B, Brachycarpaeajuncea, Cyclop- corresponds age Rorippa (range tychis marlothii, and C. virgata form a well-sup- 2.5-5 mya; Mai, 1995). The r8s analysis described ported monophyletic group (jackknife support above estimated the age of the Heliophileae clade 99%), whereas Schlechteriacapensis and Silicularia to be 4.2-4.6 mya, whereas the constrained jack- polygaloides form the other. Chamira is a sister to knife analyses yielded a range of 3.7-5.4 mya for the main lineage, referredto hereafter as Heliophila the same node, and subclades A and B were esti- s.l. mated to be 2.8-4.5 and 2.2-3.9 mya, respectively. The confidence analysis for clade C could not be TIME ESTIMATES completed because the clade was not present in a The Bayesian tree was tested for clocklike be- large proportion of jackknife trees (for all dating havior of the data using likelihood ratio testing. results, see Table 3). Volume 92, Number 3 Mummenhoff et al. 409 2005 Brassicaceae in Cape Flora
The "forced clock" approach yielded ultrametric boulders and ledges, and occasionally for gravelly trees with significantly shorter branches within the soils. Clade B is almost entirely perennial, with a Heliophileae s.l. lineage than those obtained with single annual species; clades A and C are largely NPRS. This phenomenon has been observed before annual, but perennial lineages evolved seven times (Barraclough & Reeves, 2005; Linder & Hardy, (Fig. 6). 2004; Linder et al., 2005), and it is not fully un- The vegetation type optimization (Fig. 7) shows derstood at the moment. In order to assess the pos- clades A and B centered in fynbos, with outliers in sible influence of the relatively long Cleome spi- renosterveld, strandveld, and also the surrounding nosa-ingroup branch, we repeated the forced clock arid vegetation types. Clade C is more common in analysis excluding this outgroupto prevent < 0.5% Karooid vegetation, but with a wider range of out- increase of branch length in the ingroup (not liers than clades A and B - these include grass- shown). "Forced clock" ultrametric tree was then land, succulent karoo, fynbos, renosterveld, Na- analyzed in TreeEdit using the "scale tree" option, maqua Broken Veld, among others. The rock types scaling the total "node height" to 22 mya. This (Fig. 8) are simple, with clades A and B primarily yielded 1.9 mya for the Heliophileae clade and be- found on sandstone (with outliers on granite, shale, tween 1.0 and 1.7 mya for the main three subclades coastal and acidic sand). Clade C is primarilyfound A, B, and C. on Karoo shale. An application of a range of published rDNA ITS substitution rates on the forced clock ultrametric Discussion branch lengths (see above) yielded the age 2.3-4.9 mya for the Heliophileae clade. An age estimate of TAXONOMIC CONSIDERATIONS 2.7-5.8 mya for the Heliophileae clade resulted from the application of substitution rates to the Tribal level (non-ultrametric) GTR+I+F distances observed within the Brassica- among the rDNA ITS sequences. This estimate is Phylogenetic relationships ceae have been a source of considerable contro- in agreementwith the "ultrametric"estimates (max- and the most are associ- imum distance D = 22.43% between Cycloptychis versy, frequent problems ated with the tribal classification and delimitation marlothii and Heliophila suavissima; D = substi- tution rate r X number of sites n X twice diver- of genera, including some larger ones such as Alys- sum L., Arabis L., Brassica L., Draba L., , gence time T, and therefore T can be calculated as Heliophila and L. et D/2rn; using either the 3.9 X 10~9 or 8.3 X 10"9 Lepidium, Sisymbrium (Koch al., 2003, and references These are subst./site/yr for rDNA ITS rate, and with n being therein). problems mostly the result of a reliance on ho- 498, this approach yields an age estimate for the heavy potentially fruit characters et tribe at 2.7-5.8 mya). moplastic (Koch al., 2003). The Heliophileae were considered by Appel and Al-Shehbaz as a natural, well-defined tribe ECOLOGICALOPTIMIZATIONS IN HELIOPHILA (1997) based on a single synapomorphy,diplecolobal cot- One of its has The optimizationto the basal node or even basal yledons. genera, Brachycarpaea, spi- coiled a feature three nodes using DIVA is generally poorly re- rally cotyledons, interpreted by solved, as no sensible outgroup could be used. them as derived from diplecolobal ones. The rather Therefore, an interpretationof the basal nodes was unexpected occurrence of diplecolobal cotyledons avoided in this study, and instead we report the in three Australian species of Lepidium sect. Mon- situation within the three main clades. oploca subsect. Diploploca (Hewson, 1981) was The distributional optimization (Fig. 4) shows shown by Mummenhoff (unpublished) to have that clades A and B are centered in the Cedarberg- evolved independently from that of the Heliophi- Nieuwoudtville area, with H. cornuta being the only leae. The monotypic Chamira has persistent, twice widespread species. Several species are found fur- longitudinally folded cotyledons that act as the ma- ther to the east in the central southern Cape moun- jor photosynthetic organ. These features were used tains, and this is especially evident in clade B. by Appel and Al-Shehbaz (1997) to maintain the Clade C is centered in the Richtersveld, with out- genus in a unigeneric tribe, Chamireae. The pres- liers reaching south to the Roggeveld, and east to ent ITS study supports the recognition of the He- the Drakensberg (H. carnosa-H. rigidiuscula). The liophileae as a monophyletic group, with Chamira soils optimization (Fig. 5) shows that sandy soils as sister, underlining the potential broad-scale phy- are generally preferred, especially in clades A and logenetic utility of molecular markers in the Bras- C. Clade B is remarkable for its preference for sicaceae. The evaluation of Chamireaeas a distinct 410 Annals of the Missouri Botanical Garden
Figure 4. Distribution optimizations. Where many possible optimizations were indicated at an internal node, only the most widespread (that includes all areas of the other, more limited optimizations) is indicated. This is the most conservative possible route. Where only two, different optimizations are indicated, they are separated by a backslash. Area codes are: a- Roggeveld; b- Drakensberg; c- Eastern Cape; d- Southern Cape; e- Swartruggens;f - central Cape mountains; g- Cedarberg-Nieuwoudtville;h - Kamiesberg; i- Gordonia;j - Richtersveld; k- Namib. H. = He- liophila. Volume 92, Number 3 Mummenhoff et al. 41 1 2005 Brassicaceae in Cape Flora
Figure 5. Soil optimizations. Where many possible optimizationswere indicated at an internal node, only the most polymorphic (that includes all soils of the other, more limited optimizations)is indicated. This is the most conservative possible route. Where only two, different optimizations are indicated, they are separated by a backslash. Soils codes are: a- stony-gravel;b - gravel; c- loam; d- calcrete; e- boulders; f - clay; g- sand. H. = Heliophila. 412 Annals of the Missouri Botanical Garden
Figure 6. Habit optimizations. Habit codes are: a- annual; p- perennial. H. = Heliophila. Volume 92, Number 3 Mummenhoff et al. 413 2005 Brassicaceae in Cape Flora
Figure 7. Vegetation optimizations. Where many possible optimizations were indicated at an internal node, only the most polymorphic (that includes all vegetation types of the other, more limited optimizations)is indicated. This is the most conservative possible route. Where only two, different optimizations are indicated, they are separated by a backslash. Vegetation codes are: a- woodland; b- forest; c- grassland; d- Karoo shrublands; f - Namaqua Broken Veld; g- succulent Karoo;h - Renosterveld; i- Strandveld;k - Fynbos. H. = Heliophila. 414 Annals of the Missouri Botanical Garden
Figure 8. Bedrock/substrateoptimizations. Where many possible optimizationswere indicated at an internal node, only the most polymorphic (that includes all bedrock types of the other, more limited optimizations)is indicated. This is the most conservative possible route. Where only two, different optimizations are indicated, they are separated by a backslash. Bedrock codes are: a- Karoo Shale; b- coastal sand; c- acidic sand; d- limestone; e- granite; f - shale; g- sandstone. H. = Heliophila. Volume 92, Number 3 Mummenhoff et al. 415 2005 Brassicaceae in Cape Flora
tribe must await a comprehensive family-wide phy- to some Heliophila species (e.g., H. crithmifolia,H. logenetic analysis. seselifolia), all of which are annual herbs with ap- pendaged staminal filaments. Genericlimits within Heliophileae Schlechteria and Silicularia are closely related, and they form a subclade in clade B, closest to H. Large genera of Brassicaceae (e.g., Lepidium)are ephemera,H. nubigena, and H. tricuspidata.Except usually well characterized by distinct fruit types for H. ephemera, these taxa are all perennial sub- but exhibit considerable variation in habit (Mum- shrubs. Bean (1990) stated that H. ephemeradiffers menhoff et al., 2001). By contrast, Heliophila s.l. from the rest of Heliophila by having inflated fruits, shows both extreme variation in habit and striking spongy-walled seeds, coarsely tuberculate leaves, multitude of fruit shapes (Figs. 1, 2, Table 2). As and intercalary racemes. Upon a critical study of many as nine smaller genera have been segregated the type material of H. ephemera,we conclude that from Heliophila (for synonymy, see Marais, 1970), the inflorescence is a typical ratherthan intercalary all of which represent morphologicalextremes con- raceme. Interestingly,some Heliophila species (e.g., nected by intermediates in almost every conceiv- H. cedarbergensis,H. esterhuyseniae,H. scoparia, H. able character combination (Appel & Al-Shehbaz, dregeana, H. tulbaghensis), Schlechteria, and H. 1997). Although different classification schemes of ephemera share in clade B the apiculate anthers the Heliophileae have been proposed, the tribal and one or a few seeds per locule. However, except disposition of and systematic relationships among for the presence of basal appendages on the fila- component genera are unknown, and no phyloge- ments and their absence on petals, species of lin- netic concept has been put forward so far. All five eage B are not characterized by a consistent char- smaller genera assigned by Appel and Al-Shehbaz acter pattern (Fig. 1). (1997) to this tribe (Table 1) are well nested in two The five smaller genera {Brachycarpaea,Cyclop- of the three clades of Heliophila in the molecular tychis, Schlechteria, Silicularia, Thlaspeocarpa) trees (Figs. 1, 2). Thlaspeocarpacapensis is includ- nested in Heliophila s. str. are distinguishable by a ed in clade C, whereas Brachycarpaeajuncea to- combination of (rather than unique) morphological gether with the two Cycloptychisspecies, and Sili- characters all found within the Heliophila clades B cularia polygaloides along with Schlechteria and C. In our opinion, the maintenance of these capensis form two monophyletic groups within clade genera as distinct from Heliophila would mean that B, respectively. The phylogenetic position of these a paraphyletic Heliophila has to be recognized, a taxa in the molecular tree is also supported mor- position we do not support. Therefore, these five phologically. The two species of Cycloptychisclear- genera have been reduced to synonymy of Helio- ly form a monophyletic assemblage characterized phila (Al-Shehbaz & Mummenhoff,2005). by the presence of a carpophore, a feature not in found elsewhere the Heliophileae. Cycloptychis Infrageneric classification and taxonomic status of from its schiz- is easily distinguished Heliophila by componentgenera of tribe Heliophileae ocarpic, erect, appressed fruits with carpophores, and sculptured, thick, leathery or woody fruit Previous infrageneric classification schemes in valves (Table 2). Brachycarpaea is well defined by Heliophila (Candolle, 1821; Sonder, 1846) relied its angustiseptate fruits and spirally coiled cotyle- heavily on fruit morphology.However, the most re- dons, but it strikingly resembles Cycloptychis in cent taxonomic treatment of the genus (Marais, habit, flower size and morphology, schizocarpic 1970) did not recognize any sections. Recent mo- fruits (though with a rudimentarycarpophore) with lecular studies in the Brassicaceae clearly demon- one-seeded mericarps, apiculate anthers, and strated convergence in almost every fruit character smooth seeds. Therefore,these remarkablemorpho- (Koch et al., 2003, and references therein), and the logical similarities are in complete agreement with current study further supports that conclusion. Al- the molecular analysis (Figs. 1, 2). though Sonder (1846) did not have knowledge of The remaining three genera, Thlaspeocarpa,Sil- all species included in this study, none of his sec- icularia, and Schlechteria, form a group character- tions represents a monophyletic group. Instead, ized by lacking the septum and by having indehis- species of his sections are uniformly distributed cent fruits on recurved pedicels, but the features among all three main clades in the molecular tree used in the separation of these three taxa (i.e., sta- (Figs. 1, 2). Thus, the fruit types used by Sonder minal appendages, habit) are also found in Helio- to distinguish sections are not synapomorphiesand phila and Cycloptychis.Therefore, it is not surpris- evidently evolved independently in the different ing to find Thlaspeocarpain clade C as a relative lineages of Heliophila. 416 Annals of the Missouri Botanical Garden
The molecular splitting of Heliophila, together studies of Bean (1990) revealed their presence in with the nested Brachycarpaea, Cycloptychis, Chamira and all other genera of the Heliophileae Schlechteria, Silicularia, and Thlaspeocarpa, in except Cycloptychis. Such "bracts" are typically three clades (A- C in Figs. 1, 2) correlates quite found in nearly all species of clades B and C, and well with the distribution of morphological char- their absence in a few species of these clades most acters. These character combinations (see below) likely represents parallel losses. It further appears were not adequately accounted for in previous tax- that the absence of "bracts" is diagnostic for all onomic treatments. species of clade A except Heliophila subulata. Species of clade A are typically annual herbs The recognition of monotypic and ditypic genera (rarely perennials, as in Heliophila subulata and H. in the Heliophileae mirrors several other cases in linearis) with "exstipulate," entire leaves (H. aren- the Brassicaceae where differences in fruit char- aria, H. digitata, and H. macowaniana have pin- acters often are overemphasized at the expense of nately lobed leaves), basally appendaged staminal taxonomically more useful other characters. Koch filaments and frequently also petals (H. subulata et al. (2003) listed several such examples, includ- has unappendaged filaments and petals), and ing Twisselmannia Al-Shehbaz versus Tropidocar- smooth, often wingless seeds (H. linearis and H. pum Hook., Thlaspi L. versus Alliaria Scop., and cornuta have winged seeds). Lepidium versus CoronopusZinn. Ideally, a critical Optimizationof different habits (Figs. 1, 6) shows evaluation of the taxonomic status of the Heliophila that species of clade B are shrubs/subshrubs (only s.l. clades (e.g., generic vs. subgeneric level) Heliophila ephemerais herbaceous) with exclusive- should await more sequence data from other mark- ly unappendaged staminal filaments and petals, ers and more extensive sampling of the ingroups simple leaves, wingless seeds (only H. scoparia with and outgroups. winged seeds), and apiculate anthers (H. macra and H. elongata with unapiculate anthers). Within clade Morphologicalcharacter evolution B, a terminal, well-supported (78% jackknife val- ue) subclade, which includes H. cedarbergensis,H. Despite the incompleteness of our study, one can esterhuysensiae,H. scoparia, H. dregeana, and H. safely make some generalizations regarding the tulbaghensis, is readily distinguished from the en- evolution of certain features (Fig. 1), For example, tire Heliophileae by having intercalary inflores- intercalary inflorescences and papillate seeds ap- cences (rather than typical racemes) and a gener- parently evolved only once within a terminal sub- ally papillate (vs. smooth) seed coat. clade of clade B that includes H. cedarbergensis,H. Species of clade C exhibit a mosaic of the char- esterhuyseniae, H. scoparia, H. dregeana, and H. acter states also occurring in clades A and B. All tulbaghensis. On the other hand, apiculate anthers species of this clade have smooth seeds, racemes, also probably evolved once at the base of clade B, and latiseptate fruits (Heliophila cornellsbergiahas with reversals in both H. macra and H. elongata. inflated fruits). The vast majorityof members of this By contrast, the evolution of indehiscent, one- or clade are annuals, but H. carnosa and H. suavissi- two-seeded fruits occurred independently in Thlas- ma are perennial subshrubs that produce annual peocarpa of clade C and at least twice in clade B: shoots from a woody crown. Optimization on the in the subclade containing Brachycarpaeaand Cy~ cladogram indicates that these morphological de- cloptychis as well as that including Schlechteria, viations represent secondary reversals. Further- Silicularia, and H, ephemera. Wingless seeds ap- more, the development of the seed wing seems to pear to be basal in each of clades A and B, and it be a primitive feature in the clade, and wingless is likely that winged seeds might be basal in clade seeds evolved independently in a subclade includ- C. It seems that the shift from one seed type to ing H. collina, H. pubescens, and H. pectinata, as another occurred several times within Heliophila well as in H. arenosa. The clade also shows a mo- s.l. Finally, taxa with entire leaves appear to be saic pattern in the presence of small "bracts"at the basal in the entire genus Heliophila s.l., and pec- base of pedicels, minute "stipules" at the leaf base, tinate or pinnately lobed leaves with filiformor nar- as well as in the development of basal appendages rowly linear segments evolved secondarily in clades on the petals and stamens. A and C (Fig. 1). The small appendages at the pedicel bases in the Fruit diversity within Heliophileae has no match Heliophileae were interpreted as "stipules" or oth- anywhere in the Brassicaceae. For example, fruit erwise completely reduced "bracts"(Marais, 1970; length ranges from long siliques to minute silicles, Appel & Al-Shehbaz, 2003), but functionally they fruit compression varies from latiseptate, terete, to probably represent extrafloral nectaries. Further angustiseptate, and upon maturity, the fruits may Volume 92, Number 3 Mummenhoff et al. 417 2005 Brassicaceae in Cape Flora
be dehiscent, indehiscent, or even schizocarpic Brassicaceae should be reduced to synonymy of (Marais, 1970; Appel & Al-Shehbaz, 1997, 2003). larger genera, and our present study strongly sup- The result of mapping fruit morphologies onto the ports that hypothesis. phylogenetic trees (Fig. 2) suggests that fruit char- In conclusion, we have demonstrated that the acters are quite variable even within each of the heavy reliance on fruit characters alone may well three main clades of Heliophila. On the basis of the lead to erroneous taxonomic results and that such present molecular studies, one can trace fruit evo- characters should be critically evaluated in light of lution from latiseptate and unconstricted (smooth) the molecular and other morphological data (Koch to moniliform siliques (e.g., H. dregeana and H. et al., 2003, and references therein). A critical tulbaghensis in clade B); from latiseptate silicles to evaluation of morphology in Heliophila and allies schizocarpic (Cycloptychis)and angustiseptate-did- reveals that the individual characters vary in their ymous (Brachycarpaea);and from latiseptate and value for systematic classification (see above). As dehiscent siliques to indehiscent silicles (Thlaspeo- demonstrated by Bailey et al. (2002) for the Hali- carpa). Furthermore, it appears that schizocarpic molobine Brassicaceae, it appears that a combina- fruits in the Heliophileae evolved once in the sub- tion of morphological characters is potentially use- clade including Brachycarpaea and Cycloptychis. ful in classification, and as demonstratedabove, we The occurrence in each of the three Heliophila sub- believe that this also holds within Heliophileae. clades of the same fruit types demonstrates con- vergent/parallel evolution (Fig. 2). For example, Cytology moniliformfruits evolved independently in each of Hardly anything is known about the chromosome clades A, B, and C, though they appear to have numbers in the Heliophileae. The four species evolved once within clade B in a well-supported studied, all with In = 20, are Heliophila africana (100% bootstrap) subclade that includes H. dre- (as H. pilosa), H. amplexicaulis, H crithmifolia, and geana and H. tulbaghensis (Fig. 1). H. linearis (Jaretzky, 1932; Manton, 1932). How- It is importantto note that fruit dehiscence and ever, these counts were based on material grown in relative length, both of which are major diagnostic botanical gardens, and no vouchers are available to charactersfor generic delimitation in the traditional verify the identity of the taxa involved. systems of Hayek (1911) and Schulz (1936), are controlled in thaliana the Arabidopsis by single DATING THE RADIATION MADS-box genes SHATTERPROOFand FRUIT- FUL (Liljegren et al., 2000; Ferrandizet al., 2000). Using different approaches to calibrate our trees Such a relatively simple control of fruit morphology (i.e., the 22 mya for the Brassica/Cleomeclade from (dehiscence/indehiscence, relative length) would Wikstrom et al. (2001), or a range of published an- easily explain the rapid and independent evolution giosperm rDNA ITS substitution rates (see Re- of multiple fruit types within various genera of the sults)), we arrive at age estimations of 2-5 mya for Brassicaceae, including Heliophila. One suspects the proliferation of the Heliophileae (see Table 3). that such simple inheritance controlling drastic The NPRS smoothing algorithm consistently esti- morphological differences might hold true for the mates older ages, but not more than around 5 mya. other characters addressed above. Given that the dates from Wikstrom et al. (2001) We think it likely that the number of genes con- may be far too young, our age estimation for the trolling the remarkable differences in fruit mor- Heliophileae proliferation could be up to 10 mya phology are relatively few, thus allowing rapid evo- maximally. On the other hand, the observation that lutionary changes independent of other aspects of our published rDNA ITS substitution rate-based es- morphology.The high degree of sequence similarity timate is remarkablysimilar to the "forced clock"- between several species of Heliophila and members derived estimation, combined with the notion that of the five smaller genera (Brachycarpaea,Cyclop- NPRS tends to overestimate ages, suggests a more tychis, Schlechteria,Silicularia, and Thlaspeocarpa) recent age of 2-5 mya. If accurate, the amazing strongly emphasizes the apparent rapidity with range of morphological variation observed in Cape which drastic changes in fruit morphology can Heliophila species would thus have been generated sometimes occur in the family, thus leading to clas- within a Pliocene-Pleistocene time frame. Such an sifications or generic delimitations that obscure estimate suggests a considerably faster rate com- rather than clarify evolutionary relationships. As pared with radiations in other Cape genera, such suggested by Koch et al. (2003), future molecular as Pelargonium L'Her. (Geraniaceae) and Phylica studies would most likely reveal that the vast ma- L. (Rhamnaceae), for both of which Miocene age jority of monotypic and oligotypic genera of the was suggested (Bakker et al., 2005; Richardson et 418 Annals of the Missouri Botanical Garden
al., 2001), and Restionaceae, for which an Oligo- cies, H. ephemera,is not ecologically differentfrom cene age was proposed (Linder & Hardy, 2004). the other members of clade B. Our estimate would also constitute the most re- Clade A, which consists largely of annuals (Fig. cent Cape radiation reported so far, which is much 6), is also centered in the CFR with an ancestral earlier than the 8-32 mya range given by Linder distribution optimized as restricted to the CFR (Fig. (2003) and Linder and Hardy (2004) for the Cape 4). It has a wider range of outliers than clade B Floral Region crown clade proliferations. Most of (three species reach the Kamiesberg and two the the Cape-clade radiation dates are based on NPRS- Roggeveld mountains);these are interpretedas dis- derived ultrametric trees, and several rely on the persal from the CFR. Although most species show Wikstromet al. (2001) dates for calibration. At the the typical CFR syndrome (i.e., occurring in fynbos on sand- moment there does not appear to be an indication heathy pyrophytic vegetation and growing the ancestral situ- of simultaneous radiation in these lineages. Clearly, stone-derived soils, Figs. 7, 8), is and the indicate a meta-analysis needs to be conducted in which ation uncertain, optimizations that the basal nodes are The two calibration and ultrametricity are arrived at in a polymorphic. coastal of to this concerted approach. species Heliophila belong clade, and they occur on limestone or coastal dunes, most- ly on alkaline sand, and in non-pyrophyticstrand- ECOGEOGRAPHICAL EVOLUTION IN HELIOPHILA veld. These represent two invasions of this habitat H. subulata and H. linearis. The three major clades of Heliophila s.l. can be by Clade C, also annuals (Fig. 6), largely characterized in terms of distribution pat- including mostly is centered on the Richtersveld in Karooid vege- terns and habit (Figs. 4, 6). Distribution patterns with an ancestral distribution to also reflect rainfall and tation, optimized gradients, vegetation type, be either the Richtersveld or the Richtersveld and bedrock types. Our crude data make it impossible the Roggeveld. Its species grow on sandy, loamy, or to achieve a finer resolution of the eco-geographical clayey soils derived from shale or granite (Figs. 5, patterns. 8). These areas are generally arid, have hot dry Clade B, which exclusively includes shrubs or summers and short winters, and receive less than subshrubs (except for the herbaceous Heliophila 400 mm of annual rainfall. They are generally rich ephemera), is almost completely restricted to the in annuals, possibly due to a combination of very CFR (Cedarberg-Nieuwoudtville, Swartruggens, harsh summers and relatively rich soils (Pienaar & central Cape mountains, southern Cape), with an Nicholas, 1988) that allow rapid growth in the ancestral distribution optimized as restricted to the short, winter-springfavorable season. CFR Its on soils de- (Fig. 4). species mostly grow The three Drakensberg species (Heliophila car- rived from Table Mountain sandstone (Fig. 8), very nosa, H. rigidiuscula, and H. suavissima) are nested often boulders or on rock among ledges (Fig. 5), in this group C, and placed in two subclades. These and in and renosterveld Fire is a fynbos (Fig. 7). species indicate two range extensions from the occurrence in with a fre- regular fynbos vegetation, Richtersveld to the east (Fig. 4), as well as a change of 5-50 and sev- quency years (van Wilgen, 1987), from annual to perennial habit (Fig. 6). In addition, eral show to survive after fire species adaptations they indicate a shift from sparse winter-rainfall et The associa- by resprouting (Schutte al., 1995). (less than 300 mm annually and without frost), to tions with rock also indicate habitats some- might wet summer-rainfall areas with frequent winter what from fire. The distribution protected pattern snow. It is a remarkable ecological range extension suggests a rainfall range of 300-2000 mm, with dry also matched by Pentaschistis airoides Stapf and summers. The optimizations indicate an ancestral several other grasses (Linder & Ellis, 1990). Pos- occurrence on sandstone, and in fynbos, with sev- sibly the harsh alpine environment of the Drakens- eral expansions on shale soils and into renosterveld berg has similarities to the harsh semidesert envi- vegetation. Furthermore, clade B can be divided ronment of the Namaqualand escarpment. The into an eastern subclade (clade Bl in Fig. 4), the other two Drakensberg Heliophila species, H. al- species of which grow in areas that receive more pina and H. formosa, were not included in this summer rain and contain several Eastern Cape spe- study, and it is unclear whether they also group cies. Clade B2 in Figure 4, which is centered more with the above three species. The only other spe- to the north, has no representatives in the summer- cies from KwaZulu-Natalincluded in this analysis wet Eastern Cape, but its species grow in the arid is the coastal H. elongata, a species that also grows Swartruggensthat receive no rain in the summer in the Eastern Cape and that groups basally into and very little in the winter. The only annual spe- Clade Bl in Figure 4. Volume 92, Number 3 Mummenhoff et al. 419 2005 Brassicaceae in Cape Flora
Four species, Heliophila eximia, H. deserticola, is typically a raceme, but it is intercalary in five H. trifurca, and H. crithmifolia, show an expansion species of clade B (Fig. 1), which means that the into the arid Namib desert in which none is en- terminal flower is overtopped by new leafy growth demic (Fig. 4). Of these, H. eximia is a perennial, of the central axis (Marais, 1970; Bean, 1990). In a habit that goes against the general trend of the these intercalary inflorescences the growth recom- annual Heliophila species found in seasonally arid mences after a first flush of flowers, thus potentially areas. However, this species is found only in south- lengthening flowering time and allowing speciali- ern Namibia, and it might be considered as a mem- zation. The majority of Brassicaceae have flowers ber of the Richtersveld biogeographical region. with visible nectaries and easily accessible nectar The Roggeveld is biogeographically interesting for the visiting insects, such as short-proboscid because it forms the border between the CFR and flies, wasps, or beetles (Knuth, 1898; Schultze-Mo- the Gordoniaregion. There are no species of clade tel, 1986). Species of many genera have tubular B in this region, while clade A is represented by (e.g., Matthiola R. Br.) or internally appendaged its most widespread species, Heliophila cornuta, as flowers {Heliophila) in which the nectar is hidden, well as H. acuminata, which is also found in the and their flowers are pollinated by long-proboscid adjacent Cedarberg. Clade C is represented by 8 insects such as bees, bumblebees, moths, and but- of its 17 species in the region, which can be re- terflies (Knuth, 1898; Kugler, 1955; Schultze-Mo- garded as the center for clade C. tel, 1986; Procter et al., 1996). Both appendaged Evidently, the perennial species are associated and unappendaged flowers are found in Heliophila, with wetter or at least summer-wetareas, while the and species of clades A and C are typically char- annuals occupy the more arid western half of the acterized by basal appendages on the petals and subcontinent. If the annual habit is assumed to be staminal filaments (Fig. 1). primitive in Heliophila s.L, then the perennial habit Schulz (1931) suggested that the floral basal ap- (Fig. 6) evolved independently at least seven times pendages in Heliophila are associated with the nec- and was lost once (H. ephemera).The habit seems taries and often hide them. We suggest that the bas- to be tightly correlated with macro-ecology and al appendages of petals and filaments, which allow phylogeny, but it is unclear whether or not a change insects with longer rather than shorter probosces to of habit allowed the species to be established in access nectar, play a selective role that may pro- the Drakensberg region. mote interspecific reproductive isolation and there- fore may increase speciation rates and diversifica- tion. is known about ADAPTIVE SHIFTS/FACTORS EXPLAINING Unfortunately,hardly anything the floral of and exten- THE RADIATION IN HELIOPHILA biology Heliophila species, sive field studies would most likely provide polli- to the Goldblattand Manning(2000) and Linder (2003) nation data valuable understanding adaptive radiation of the in South Africa. suggested that the remarkable species richness in genus The leaves and of of the CFR might be the consequence of diverse lim- pedicels many species clades B and C are subtended small itations to gene flow (e.g., dissected landscapes, basally by ap- as or "bracts" pollinator specialization, long flowering times al- pendages interpreted "stipules" & but lowing much phenological specialization) and a (Marais, 1970; Appel Al-Shehbaz, 2003) extrafloralnectaries. If complex environment providing a diversity of se- functionally may represent attract insects and lective forces (e.g., geographical climatic variation, nectaries, they might prevent between flowers of the same altitudinal variation, different soil types, regular pollination neighboring inflorescence. Ants are and fires). Due to the lack of experimental and obser- poor cross-pollinators are attracted to extrafloral nectaries vational studies on the adaptive significance of var- (Chauhan, but it is unknown if such role ious structures, morphology will be used to infer 1979), they play any in In some of clade A H. the various functions of these structures. This ap- Heliophila. species (e.g., linearis, H. H. Schulz proach should result in a predictive frameworkon namaquana, africana), described swollen clavate in the which experimental studies can be based. (1931) styles ripe fruits. the of these In addition to the diversity in flower color and Although adaptive significance structures is unknown, Schulz size (see the introductory paragraphs), the differ- (1931) suggested a food offer to herbivores ences in inflorescence and flower structure obvi- that they might represent to prevent seed damage when snipped off. ously are linked to different pollinators or pollina- tion strategies (Johnson, 1996; Linder, 2003, and LiteratureCited references therein; Perret et al., 2003; Bakker et Acocks,J. P. H. 1975. VeldTypes of SouthAfrica, ed. al., 2005). The inflorescence in Heliophila species 2. Mem.Bot. Surv.South Africa 40: 1-128. 420 Annals of the Missouri Botanical Garden
Al-Shehbaz, I. A. 1984. The tribes of Cruciferae (Bras- Hewson, H. 1981. The genus LepidiumL. (Brassicaceae) sicaceae) in the Southeastern United States. J. Arnold in Australia. Brunonia 4: 217-308. Arbor. 65: 343-373. Janchen, E. 1942. Das System der Cruciferen. Oesterr. & G. Yang. 2001. A revision of Solms-laubachia Bot. Z. 91: 1-28. (Brassicaceae). HarvardPap. Bot. 5: 371-381. Jaretzky,R. 1932. Beziehungen zwischen Chromosomen- & K. Mummenhoff. 2005. Transferof the South zahl und Systematik bei den Cruciferen. Jahrb. Wiss. African genera Brachycarpaea,Cycloptychis, Schlechter- Bot. 76: 485-527. ia, Silicularia, and Thlaspeocarpato Heliophila (Bras- Johnson, S. D. 1996. Pollination, adaptation and speci- sicaceae). Novon 15: 385-389. ation models in the Cape flora of South Africa. Taxon Appel, 0. & I. A. Al-Shehbaz. 1997. Re-evaluation of 45: 59-66. tribe Heliophileae (Brassicaceae). Mitt. Inst. Allg. Bot. Jordaan, M. 1993. Brassicaceae. In: T. H. Arnold & B. Hamburg27: 85-92. C. 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Appendix 1. Collectiondata and GeneBankaccession numbersof South Africanendemic Brassicaceaespecies studied.
Gen Bank accession number Species1 Provenance2/ Herbarium-source3/ Collector4 ITS I ITS II Heliophila acuminata South Africa, SW, Malmesburydistrict, Klein Swartfontein, AJ863603 AJ864807 (Eckl. & Zeyh.) near Moorreesburg,33°08'S, 18°39'E / PRE / Acocks Steud. 24401 H. africana 1 (L.) Mar- South Africa, SW, Cape Town, 33°52'S, 18°31'E / NBG / AJ863602 AJ864808 ais Steiner 1975 H. africana 2 (L.) Mar- South Africa, Cape Peninsula, Olifantbos Bay / OSBU / AJ863601 AJ864809 ais Neuffer 9402 H. amplexicaulis L.f. South Africa, NW, SW, KM / cultivated, Botanic Garden AJ863611 AJ864810 Paris, France H. arenaria Sond. South Africa, NW, Clanwilliam Distr., between Nardouws AJ863600 AJ864811 Pass and Brakvlei, 33°10'S, 18°55'E / PRE / Marais 1436 H. carnosa (Thunb.) South Africa, N, Ceres Div., Baviaansberg,33°08'S, AJ863599 AJ864805 Steud. 19°37'E / BOL / Esterhuysen29796 H. cedarbergensisMarais South Africa, NW, Clanwilliam Distr., Langeberg, Central AJ863607 AJ864812 CedarbergMountains / BOL / Esterhuysen35055 H. collina 0. E. Schulz South Africa, NW, Calvinia, Nieuwoudtville Reserve, AJ863598 AJ864813 31°22'S, 19°07'E / NBG / Perry & Snijman 2199 H. cornellsbergiaB. J. South Africa, NW, Richtersveld, Cornellsbergin Stinkfon- AJ863576 AJ864803 Pienaar & Nicholas tein Mts. Southern slopes to a neck S of top / PRE / Oli- ver, Tolken & Venter715 H. cornuta Sond. South Africa, Ceres Div., Zwartruggens,Groenfontein, near AJ863597 AJ864804 Stompiesfontein, 33°05'S, 19°19'E / BOL / Bean & Trin- der-Smith2686 H. coronopifolia 1 L. South Africa, SW, Caledon, foot of Hoys Koppie, Hermanus, AJ863596 AJ864814 34°25'S, 19°14'E / MO / Williams982 H. coronopifolia2 L. Cultivated / Botanical Garden Halle, Germany AJ863592 AJ864817 H. coronopifolia3 L. Cultivated / Botanical Garden Aarhus, Denmark AJ863588 AJ864824 H. crithmifolia 1 Willd. South Africa, NW, in between Clanwilliam and Wuppertal, AJ863577 AJ864799 road side, 32°13'S, 19°10'E / OSBU / Neuffer 9258 H. crithmifolia 2 Willd. South Africa, LB, KM, South Cape district, Oudtshoorn, AJ863595 AJ864806 next to turnoff to Blossoms, 33°33'S, 22°11'E / BOL / Vlok 1048 H. descurva Schltr. South Africa, CedarbergMts., near Wuppertal,32°16'S, AJ863575 AJ864815 19°13'E / OSBU / Neuffer 9253 H. deserticola Schltr. Namibia, Oranjemund,5 km N of Oranje near Sendelings- AJ863594 AJ864798 drif, 28°09'S, 16°53'E / MO / Giess & Muller 14375 H. digitata L.f. South Africa, Clanwilliam, Farm Suurfontein/ NBG / White- AJ863593 AJ864816 head s.n. H. dregeana Sond. South Africa, Cold Bokkeveld, Elands Kloof at Twee Rivi- AJ863606 AJ864818 eren / MO / Goldblatt2578 H. elongata (Thunb.) South Africa, forests near Knysna, 34°01'S, 23°03'E / MO / AJ628255 AJ628256 DC. Lavranos6203 H. ephemeraP. A. Bean South Africa, OudtshoornProv., Swartberg,northern slopes, AJ628257 AJ628258 4 km E of Blouberg on Botha's track / MO / Viviers,Vlok & Bean 1551 H. esterhuyseniaeMarais South Africa, Caledon Prov., Franschhoek Mountains, Roes- AJ628259 AJ628260 bos Peak, 33°54'S, 19°08'E / BOL / Esterhuysen29422 H. eximia Marais South Africa, N, Richtersveld, Kodaspiek, main ridge SE of AJ863591 AJ864819 Beacon and up to summit, 28°32'S, 17°05'E / PRE / Oli- ver, Tolken & Venter390 H. gariepina Schltr. South Africa, N, Richtersveld, Kodaspiek, main ridge SE of AJ863590 AJ864820 Beacon and up to summit, 28°32'S, 17°05'E / PRE / Oli- ver, Tolken & Venter476 Volume 92, Number 3 Mummenhoff et al. 423 2005 Brassicaceae in Cape Flora
Appendix 1. Continued.
Gen Bank accession number Species1 Provenance2/ Herbarium-source3/ Collector4 ITS I ITS II H. glauca Burch. ex DC. South Africa, Ladismith Div., Anysberg, N slopes, 33°30'S, AJ863610 AJ864821 20°46'E / MO / Esterhuysen32859 H. linearis (Thunb.) DC. South Africa, Mossel Bay, between Outeniquastrandand AJ863589 AJ864822 Tergniet, 34°03'S, 22°12'E / BOL / Vlok 1949 H. macowaniana Schltr. South Africa, road from Clanwilliam and Cape Town, 20.5 AJ863587 AJ864825 km from Clanwilliam, road side, 32°21'S, 18°56'E / PRE I Marais 1447 H. macra Schltr. South Africa, Caledon Div., Hermanus, Fernkloof Nature AJ863609 AJ864826 Reserve, above Northcliff, 34°25'S, 19°14'E / MO / Rob- ertson 134 H. macrospermaBurch. South Africa, Albany, Fish River Pass, 33°17'S, 26°47'E / AJ863608 AJ864827 ex DC. MO / Bayliss 2257 H. minima (Stephens) South Africa, Richmond Distr., 31 km NW of MerrimanSta- AJ863586 AJ864828 Marais tion, 30°59'S, 23°33'E / PRE / Acocks24430 H. namaquana Bolus South Africa, Clanwilliam Dist., Pienaarsvlakte between AJ863585 AJ864802 Krom River and Matjiesriver/ BOL / Bean 1345 H. nubigena Schltr. South Africa, WorcesterDist., Keeromsberg/ BOL / Ester- AJ863613 AJ864829 huysen 33671 H. pectinata Burch. ex South Africa, Ceres-Clanwilliamroad via Groot, turnoff to AJ863584 AJ864830 DC. farm Kleinveld, S of Blinkberg Pass / PRE / Marais 1416 H. pubescens Burch. ex South Africa, Calvinia, Kleinfontein, Agterkop, Pk. Middel- AJ863583 AJ864831 DC. pos, 32°01'S, 20°04'E / PRE / Hanekom 2138 H. pusilla L.f. South Africa, Caledon, Hermanus, Die Duine, 34°25'S, AJ863582 AJ864832 19°14'E / PRE / Williams 1264 H. rigidiuscula Sond. South Africa, Mkambati,road to Lupatana, 31°19'S, AJ863572 AJ864797 29°57'E / MO / Hutchings 727 H. scoparia Burch ex South Africa, WorcesterDist., Hex Rivier Mts., Audensberg, AJ863605 AJ864833 DC. 33°28'S, 19°34'E / BOL / Esterhuysen28193 H. seselifolia Burch. ex South Africa, Williston Dist., Snyderspoortthrough the Bas- AJ863581 AJ864801 DC. terberge, 31°20'S, 20°55'E / STE / Thompson3161 H. suavissima Burch. ex South Africa, Orange Free State, Glen Landboukollege/ AJ863574 AJ864834 DC. PRE I PC &L Zietsman s.n. H. subulata Burch. ex South Africa, Riversdale, Stillbaai, ridge below reservoir, AJ863580 AJ864835 DC. 34°22'S, 21°26'E / STE / Bohnen 5223 H. tricuspidataSchltr. South Africa, Caledon Dist., Jonas Kop, Langeberg, NW of AJ863612 AJ864836 Genadendal, 34°01'S, 19°36'E / BOL / Esterhuysen 32705 H. trifurca Burch. ex South Africa, Namaqualand,road from Garies via eastern AJ863604 AJ864800 DC. mountains to Springbok, 30°07'S 17°59'E / OSBU / Neuf- fer 9280 H. tulbaghensis Schinz South Africa, Paarl Dist., path from Fransch Hoek Pass to- AJ863579 AJ864837 wards Paardekoop,near head of valley, 33°54'S, 19°09'E / BOL / Esterhuysen35692 H. variabilis Burch. ex South Africa, Richtersveld, top of Hellskloof, road going to AJ863578 AJ864838 DC. Springbokvlakte/ STE / Nicholas 2511 Brachycarpaeajuncea South Africa, Clanwilliam Dist., Berg Road / NBG / Barker AJ862707 AJ862708 (Bergius) Marais 10426 Chamira circaeoides South Africa, MalmesburyDist., Bokbaai, 33°34'S, 18°21'E AJ862719 AJ862720 (L.f.) Zahlbr. / BOL / Bean & Viviers1901 Cycloptychismarlothii South Africa, Ceres Dist., Zwartruggens,Groenfontein, near AJ862709 AJ862710 0. E. Schulz Stompiesfonteinin rugged refugium in TMS ridge, 32°59'S, 19°01'E / BOL / Bean & Trinder-Smith2687 Cycloptychisvirgata South Africa, Piketberg Dist., Piketberg Mts. near Piket- AJ862711 AJ862712 (Thunb.) E. Mey. ex berg, 32°53'S, 18°43'E / NBG / Barker 10341 Sond. 424 Annals of the Missouri Botanical Garden
Appendix 1. Continued.
Gen Bank accession number
Species1 Provenance2/ Herbarium-source3/ Collector4 ITS I ITS II Schlechteriacapensis Bo- South Africa, Clanwilliam Dist., Wolfberg,N Cedarberg, AJ862715 AJ862716 lus 32°28'S, 19°08'E / BOL / Esterhuysen29984 Silicularia polygaloides South Africa, Ceres Dist. / BOL / Leighton 2288 AJ862713 AJ862714 (Schltr.) Marais Thlaspeocarpacapensis South Africa, Sutherland, Voelfontein, valley S of farm AJ862717 AJ862718 (Sond.) C. A. Sm. house on road to mountain, 32°23'S, 20°39'E / MO / Goldblatt6319 Alliaria petiolata Campus area University of Osnabriick, Germany,52°16'N, AJ862703 AJ862704 (M. Bieb.) Cavara& 7°59'E Grande Aethionemasaxatile (L.) Cultivated, Botanical Garden of the University of Osna- AJ862697 AJ862698 R.