South African Journal of 88 (2013) 361–366

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South African Journal of Botany

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Conservation genetics of argenteum (Silvertree) — A flag ship species of the

Steffen Heelemann a,⁎, Fahiema Daniels b, Anthony G. Rebelo b, Peter Poschlod a, Christoph Reisch a a University of Regensburg, Institute of Botany, 93040 Regensburg, Germany b South African National Biodiversity Institute, Private Bag X7, Claremont, 7735, article info abstract

Article history: The Silvertree ( (L.) R.Br.) is an iconic to South Africans and tourists alike. This Received 6 October 2012 endangered species is endemic to the Cape Peninsula, the most southwestern part of Africa. Despite its visual Received in revised form 26 August 2013 presence, no population genetic data of L. argenteum are currently available, but such information is crucial for Accepted 26 August 2013 effective conservation management. A historical question is whether the inland populations are natural or Available online 1 October 2013 planted? This study aimed to reveal the genetic structure and possible differences of L. argenteum populations Edited by JS Boatwright on the Cape Peninsula and inland at Helderberg, Paarl Mountain and Simonsberg. It was expected that inland populations would exhibit reduced genetic variation due to their isolation from each other and the main Cape Keywords: Peninsula gene pool. Furthermore, genetic differences between populations were expected to be higher at inland AFLP populations because they are further apart from each other, relative to the Peninsula populations. material was collected and AFLP was used to assess the genetic variation. In general, low genetic variation was present within all populations (mean Nei's gene diversity 0.11 ± 0.01) and no significant differences between Peninsula and inland populations were found. Minor differences in molecular variances were found between Silwerboom Peninsula and inland populations (PhiPt = 0.11), being double between Peninsula populations (PhiPt = 0.08) than between inland populations (PhiPt = 0.04). This supports a possible anthropogenic origin of inland popu- lations. Although the genetic variation of populations is very similar, they should not be managed as a single gene pool. Inland populations are more similar to each other compared to the Peninsula ones and therefore might be managed as one genetic entity. In contrast, Peninsula populations show a higher degree of differentiation and should be managed to maintain genetic integrity by minimizing further cross planting. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Preface horticultural importance, with an estimated 50 million cut stems of Leucadendron sold annually in Eurasia (Ben-Jaacov and Silber, 2010) and thus the focus of economical floricultural and breeding research Planta naturalis, in patria, argentea excellit fronde, inter arbores (Littlejohn, 2001; Littlejohn and Robyn, 2000; Liu et al., 2006a, 2006b, nitidissima omnium. 2007). Although an alternative classification approach was proposed [Linnaeus, 1753] by Salisbury in 1809, proposing four genera of conebushes to align the group with the rest of the family where genera are determined largely 2. Introduction by type, this has not been adopted, although Salisbury's groups are recognized at the sub-section level of the genus (Williams, 1972). The conebushes, Leucadendron R.Br. (Proteaceae), have attracted In Linnaeus's famous Species Plantarum (1753) the Silvertree is de- much research interest for many reasons. Adapted to a fire-prone en- scribed as “the most shining and splendid of all ” (in Ben-Jaacov vironment and low-nutrient soils (Cowling and Holmes, 1992), and Silber, 2010; Williams, 1972), and as the type of the genus Leucadendron is unique in the Cape Flora Proteaceae in having a di- and the Proteaceae. This Linnaeus reversed in 1771, a conserved decision, versity of seed dispersal and storage strategies (including serotiny, thus it is now Leucadendron argenteum (L.) R.Br. (Williams, 1972). Recent myrmecochory, therophily) thus prompting ecological (Bond, 1985; studies support a paraphyletic genus Leucadendron (Barker et al., 2004). Hattingh and Giliomee, 1989; Le Maitre, 1988a,b; Midgley, 1987; Because of its charismatic and attractive appearance, it is of particular in- Thuiller et al., 2004), population genetic (Tansley and Brown, 2000) terest to locals and tourists, as well as the horticultural crop industry and phylogenetic research (Barker et al., 2002, 2004). It is also of (Van Wyk, 1983). The Silvertree is a conspicuous member of the Cape Flora, easily distinguishable from other plants on region ⁎ Corresponding author. Tel.: +49 17623590324. by its large, silver and exceptional height among Fynbos E-mail address: [email protected] (S. Heelemann). (Williams, 1972). Its range is centred on the Table Mountain, extending

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.08.014 362 S. Heelemann et al. / South African Journal of Botany 88 (2013) 361–366 from Lions Head to Tokai and Noordhoek on the Cape Peninsula, where Table 1 it is associated with wetter communities on granite (and shale) fynbos, Localities of Leucadendron argenteum populations sampled in the , South Africa. The geographic location of the studied populations (in parenthesis) is illustrated being absent from the sandstone substrates. Isolated inland populations in Fig. 1. Letters and numbers refer to the populations illustrated in Fig. 1. occur in Helderberg, Simonsberg and Paarl Mountain, with very small populations at Riebeek Kasteel and Tygerberg (Rebelo, 2001). The Localities South latitude East longitude Altitude (m) Silvertree is one of many localized and rare Proteaceae (Tansley, Cape Peninsula populations 1988) with a current IUCN Red List threatened status of Endangered Devils Peak (D) 33.95028 18.45725 210 (Raimondo et al., 2009; Rebelo et al., 2006, http://redlist.sanbi.org/ Kirstenbosch (E) 33.98924 18.42510 240 Lions Head (B) 33.94081 18.39323 267 species.php?species=794-8), due to habitat destruction by urban ex- Oranjekloof (A) 34.00417 18.38715 154 pansion of Cape Town and degradation of habitat by fire exclusion on Platteklip (C) 33.95497 18.42642 429 the Peninsula, wine farming and alien plants on the inland populations. Inland populations The species has an unusual growth form in the genus, with a single Helderberg (3) 34.05477 18.85200 238 erect stem, from which branches radiate at annual increments. It Paarlberg (1) 33.75406 18.93672 428 is exceptionally fast growing, up to 1 m per year, attaining 10 m by Simonsberg (2) 33.89653 18.90372 360 15 years. Plants may survive for up to 80 years, but typically are killed by fires at an average of 15 years of age. From 5 to 8 years of age, stands become infected with cinnamomi Rands (root rot), which and samples were standardised at a dilution of 7.8 ng/μl. For the ampli- kills 5–10% of populations per year (Van Wyk, 1973; Von Broembsen fied fragment length polymorphism (AFLP) procedure, genomic DNA and Kruger, 1985), but most plants have set seed before succumbing (approximately 50 ng) was used for restriction and ligation reactions to the obvious symptoms which result in death within hours. Although with MseI and EcoRI restriction enzymes and T4 DNA Ligase (both the flowerheads are large, pollination is by minute beetles, with no were from Fermentas) were conducted in a thermal cycler for 2 h at evidence of wind pollination. The Silvertree is partly serotinous, but its 37 °C. Polymerase chain reactions (PCRs) were run in a reaction volume are released under hot conditions. The is a large rounded of 5 ml. Preselective amplifications were performed using primer pairs nut, and has a parachute-type dispersal mechanism, but dispersal with a single selective nucleotide, MseI and EcoRI together with H2O, distances are typically only a few metres (Notten and Van der Walt, Puffer S, dNTPs and Taq-Polymerase (PeqLab). The PCR reaction param- 2008). Secondary caching by rodents also occurs. Anecdotal evidence eters were: 2 min at 94 °C, 30 cycles of 20 s of denaturing at 94 °C, 30 s suggests that fruit may stay dormant for at least 80 years. It is dioecious of annealing at 56 °C, and 2 min of extension at 72 °C, followed by with a chromosome number of 26 (Liu et al., 2006b). 2 min at 72 °C and ending with 30 min at 60 °C. After an extensive Very little research has been conducted around L. argenteum and screening of selective primer combinations with eight randomly select- most references are of anecdotal character (Rebelo pers. obs.). Excep- ed samples, selective amplifications were performed with the three tions are early germination experiments by Herschel in 1836 (Rourke, primer combinations (MseI + CTC/EcoRI + AAC, MseI + CTC/EcoRI +

1994), economical interests (Ben-Jaacov and Silber, 2010)andbiomi- AAG, MseI + CTG/EcoRI + ACT) and H2O, dNTPs and Taq-Polymerase metic research (Koch et al., 2008, 2009). In the forest-scarce Cape region (PeqLab). L. argenteum was an important firewood and plantings were established PCR reactions were performed with the touch-down profile: 2 min at in the 1700s to 1800s (Notten and Van der Walt, 2008). 94 °C, ten cycles of 20 s of denaturing at 94 °C, 30 s of annealing, which Currently no data on the population genetic structure of L. argenteum was initiated at 66 °C and then reduced by 1 °C for the next ten cycles, are available. However, such data are important for an optimal conser- 2 min of elongation at 72 °C, followed by 25 cycles of 20 s of denaturing vation management plan of the species in order to preserve its entire ge- at 94 °C, 30 s of annealing at 56 °C and 2 min of elongation at 72 °C, end- netic variation. Crucial to this issue is establishing whether the inland ing with a final extension for 30 min at 60 °C. After DNA precipitation, populations are natural or were established by early colonialists for DNA pellets were vacuum-dried and dissolved in a mixture of Sample fuel and building. Furthermore, we asked how large genetic variation Loading Solution and CEQ Size Standard 400 (both Beckman Coulter). within individual populations of L. argenteum is, and should any differ- The fluorescence-labelled selective amplification products were separat- ences between populations exist how this might affect the ex-situ con- ed by capillary gel electrophoresis on an automated sequencer (CEQ servation management of the species. 8000, Beckman Coulter). Raw data were collected and analysed with the CEQ Size Standard 400 using the CEQ 8000 software (Beckman 3. Material and methods Coulter). Data were exported as crv-files, showing synthetic gels with AFLP fragments for each primer combination separately from all studied 3.1. Sampling procedure individuals and analysed in BioNumerics (Applied Maths, v. 3.0). Files were examined for strong, clearly defined bands by eye. Each band was The study area comprises the Cape Peninsula and inland hills where scored across all individuals as either present or absent. Silvertree populations are present. Plant leaf material from up to twenty randomly chosen individuals per population was collected from five 3.3. Statistical analysis Peninsula populations and three inland populations (Table 1, Fig. 1). However, the recently discovered and much smaller populations of In the AFLP data matrix, the presence of a band was scored as 1, Tygerberg (5 plants, in 2000) and Riebeek Kasteel (8 plants, in 2010) whereas the absence of the band was coded as 0. Finally, basic data were not included. structure consisted of a binomial (0/1) matrix, representing the scored AFLP markers. Nei's gene diversity, Shannon's information index, and 3.2. DNA isolation and AFLP analysis number and percentage of polymorphic loci (PL) were calculated for each population. Genetic variation was calculated via POPGENE v. 1.32 For each population, leaf material was collected and cooled on ice. (Yeh et al., 1999). Genetic variation within populations was calculated

Later they were placed into filter bags and dehydrated in silica gel. based on polymorphic bands as Nei's gene diversity [GD = ∑ I hij / I] DNA was isolated from 10 mg of dried plant material of individual (Nei, 1978) and Shannon information index [SI = ∑ pi ln pi] plants using the CTAB method (cetyltrimethylammonium bromide, (Lewinton, 1972) for each population. The binomial matrix was Rogers and Bendich, 1994). Both the DNA isolation and AFLP method subjected to an analysis of molecular variance (AMOVA, Excoffier (Vos et al., 1995) were adapted as previously described (Reisch, 2008; et al., 1992) using GENALEX v. 6.2 (Peakall and Smouse, 2006). Variance Reisch et al., 2005). DNA concentration was estimated photometrically, components and their significance levels for variation among Peninsula S. Heelemann et al. / South African Journal of Botany 88 (2013) 361–366 363

Fig. 1. The distribution of the Silvertree Leucadendron argenteum in the Western Cape, South Africa, showing known populations (from Protea Atlas Project data) and sample sites on the Peninsula (arrows with letter) and inland (arrows with number). Letters and numbers refer to the populations mentioned in Table 1. and inland region, among populations and within populations were cal- Mean Nei's gene diversity was 0.11 ± 0.01, ranging from 0.08 to 0.14. culated. Fst approximately equals Gst and is used for comparisons of spe- Mean Shannon's information index was 0.17, ranging from 0.13 to 0.21. cies differentiation levels (Hartl and Clark, 1989). Lowest genetic variation was found at Devil's Peak (GD = 0.08, NL = A Mantel test, based on 999 permutations, was conducted to test 39, PL = 28.3%) and Lions Head and Platteklip (SI = 0.13), i.e. the north- whether the matrix of pair-wise genetic distances (ΦPT), taken from ernmost populations on the Peninsula. Highest genetic variation oc- the AMOVA between populations, was correlated with the matrix of curred in Paarlberg (GD = 0.14, SI = 0.21, NoL = 56, PL = 40.5%). geographical distances between populations (Mantel, 1967; Sokal and Regarding genetic indices and according to t-tests, no significant dif- Rohlf, 1995). ferences occurred between Peninsula and inland populations. Genetic relatedness between individuals, assorting populations and Analysis of molecular variances for the entire region showed low differences between Peninsula and inland populations was analysed genetic variation (10%, PhiPt = 0.10) between populations (Table 3). by principal coordinates analysis (PCoA). Calculations and plotting Although on a low level genetic variation between Peninsula popula- were based on inter-individual Bray–Curtis similarities and performed tions was double that between inland populations, 8%, PhiPt = 0.08 in MVSP v. 3.12 (Kovach, 1999). vs. 4%, PhiPt = 0.04, respectively. Low genetic variation was also appar- ent between Peninsula and inland group (3%). 4. Results PCoAs showed no particular grouping between Peninsula and inland populations (Fig. 2). Mantel test revealed a weak but positive correla- AFLP analyses revealed 138 fragments (MseI + CAC/EcoRI + AAC tion (r2 = 0.09, p = 0.083) of geographic and genetic distance be- [46 fragments], MseI + CAT/EcoRI + AAG [59 fragments], MseI + tween populations. The error rate was estimated at 2.28% following a CAA/EcoRI + ACA [33 fragments]) with 46.75 ± 2.45 mean number of protocol of Bonin et al. (2004).Nosignificant correlation of Nei's gene polymorphic loci, 33.88 ± 1.77 mean percentage of polymorphic loci, diversity (r = −0.15; p = 0.72; r2 = 0.02) and Shannon's index ranging from 36 to 56, and 26.1 to 40.6%, respectively (Table 2). (r = −0.31; p = 0.45; r2 = 0.10) with population sizes occurred. 364 S. Heelemann et al. / South African Journal of Botany 88 (2013) 361–366

Table 2 and this has to be considered as part of the gene pool. For exam- Genetic variation within populations of Leucadendron argenteum in the Western Cape, ple, fragmented populations of the endangered caleyi South Africa. R.Br. – with a similar ecological growth and seed traits and genetic Localities n Nei's gene Shannon's Number of Percentage of variation to Silvertrees – show little influence on genetic structure diversity index polymorphic polymorphic (SI = 0.18 ± 0.02 vs. 0.17 ± 0.01, respectively) and it has been sug- (GD ± S.E.) (SI ± S.E.) loci (NoL) loci (PL) gested that seed bank and plant longevity (15 years average life-span) Cape Peninsula populations buffer genetic erosion and effects of fragmentation on genetic variation Devils Peak 20 0.08 ± 0.04 0.13 ± 0.06 39 28.26 (Llorens et al., 2004). It has even been shown that the genetic variation Kirstenbosch 20 0.1 ± 0.04 0.15 ± 0.05 43 31.16 Lions Head 20 0.12 ± 0.04 0.19 ± 0.06 50 36.23 within the seed bank can be larger than within the above ground popu- Oranjekloof 20 0.1 ± 0.04 0.16 ± 0.05 53 38.41 lation and that the seed bank is slowing the process of differentiation Platteklip 18 0.09 ± 0.04 0.13 ± 0.05 36 26.09 between populations (McCue and Holtsford, 1998).

Inland populations Pollinating beetles and/or dispersal by rodents may bridge Peninsula Helderberg 18 0.12 ± 0.04 0.19 ± 0.06 50 36.23 populations thereby enabling gene flow. Only one pollen or seed per Paarlberg 20 0.14 ± 0.04 0.21 ± 0.06 56 40.58 generation is necessary to ensure sufficient gene flow and avoid popula- Simonsberg 20 0.1 ± 0.04 0.16 ± 0.05 47 34.06 tion differentiation (Slatkin, 1985), thereby diminishing fragmentation Peninsula 0.10 ± 0.01a 0.15 ± 0.01b 44.20 ± 3.22c 32.03 ± 2.33d effects. However, inland populations are isolated and at least 10 km Inland 0.12 ± 0.01a 0.19 ± 0.01b 51.00 ± 2.65c 36.96 ± 1.92d apart and genetic exchange seems unlikely. Pooled 0.11 ± 0.01 0.17 ± 0.01 46.75 ± 2.45 33.88 ± 1.77 Although differences are at a low magnitude, it is interesting that Sample size (n). Bold numbers show mean values ± S.E. Different letters indicate genetic variation between Peninsula populations was double than be- significant differences within indices (t-test). 138 loci in total, one private locus. tween inland populations, the reverse of that predicted. Thus it can be suspected that inland populations are derived from Peninsula plants. 5. Discussion Also the fact that the Paarl population showed higher (but not sig- nificantly) genetic variation points towards this scenario and could be Genetic indices showed low levels of genetic variation within attributed to several planting events, or plants from different Peninsula L. argenteum populations with no significant differences between locations or the population's larger size. Peninsula and inland populations. Molecular variance was also low be- Because no historical calibration is possible it cannot be assumed tween populations and among both regions. that Peninsula populations are unaltered by plantings for fire wood, Nevertheless, twice as much genetic variation between populations building material and conservation purposes over the last two centuries was found on the Peninsula compared to inland populations when ap- (see also http://protea.worldonline.co.za/silver.htm). However, com- plying analysis of molecular variance separately. Furthermore, a positive parison with other Proteaceae in the study region is possible. For Mantel test indicated the presence of genetic differences and spatial example, hirtus Salisbury – a proteoid shrub not commercially structure. We do not anticipate the recently discovered populations to cultivated – has low inherit genetic variation (Nei's gene diversity = be any different, due to their extremely small size (strongly suggesting 0.15), as well as comparable 8% differentiation between Cape Peninsula that they were relatively recently planted). populations (Reisch et al., 2010). This is a genetic pattern unlikely to be The present genetic variation in (Nei's GD 0.11) and between popu- influenced by plantings and gives an indication that L. argenteum genet- lations (PhiST 0.10), is lower than expected for long-lived, endemic, ics were thus not significantly impacted by plantings (Nei's gene diver- outcrossing and gravity-dispersed plant species from meta-studies sity = 0.10, 8% differentiation between Peninsula populations). (Hpop 0.19–0.26, PhiST 0.20–0.44, Nybom and Bartish, 2000). For exam- Historical research might help resolve the issue of inland popula- ple, higher genetic variation was observed within and between popula- tions being planted, but the information is not readily available. From tions of rare Leucadendron elimense (RAPDs = 0.35, AMOVA = 27%) the data on genetic variation it is visible that inland populations are dif- and within populations of the common and widespread Leucadendron ferent, suggesting that they were established from the Peninsula. The salignum (0.30, RAPDs, Tansley and Brown, 2000). In Australian expected but not observed lower genetic variation within inland popu- Sm., relatively low genetic variation in populations (SI = lations might point to several planting events in the past. 0.18–0.22, cf. L. argenteum SI = 0.17) and between populations On the basis of our results we suggest the following guidelines that (AMOVA = 16–21%) are reported (Rymer and Ayre, 2006), with allow conservation authorities ex-situ conservation and population

Persoonia mollis having low gene variation (HT = 0.14) within popula- management. L. argenteum is an endemic, rare, endangered flagship tion and average levels between them (0.22%, Krauss, 1997). species surviving in only few populations. Those that are threatened It is suspected that seed banks of L. argenteum are typically 10 to by land use change and commercial plantations. Therefore an ex- 1000 times larger than the adult population (increasing with veld age) situ gene banking with seed bank conservation and management is

Table 3 Analysis of molecular variance of Leucadendron argenteum in the Western Cape, South Africa.

Ind./Pop. Genetic variation D.f. SS MS % PhiPt

Entire region 156/8 Between populations 7 151.32 21.62 10% 0.10 Within populations 148 1013.26 6.85 90% Peninsula 98/5 Between populations 4 71.23 17.81 8% 0.08 Within populations 93 609.96 6.56 92% Inland 58/3 Between populations 2 25.71 12.86 4% 0.04 Within populations 55 373.36 6.79 96%

Between Peninsula and inland 156/8 Among regions 1 34.27 34.27 3% 0.11 Between populations 6 117.04 19.51 8% Within populations 148 1013.26 6.85 89%

Based on 138 AFLP fragments. Number of individuals and populations (Ind./Pop.). Degrees of freedom (D.f.). Sum of Squares (SS). Means squares (MS). Proportion of genetic variation (%). Significance level (p b 0.001) is based on 999 permutations. S. Heelemann et al. / South African Journal of Botany 88 (2013) 361–366 365

Fig. 2. Principal coordinates analysis of 156 Leucadendron argenteum individuals in the Western Cape, South Africa. Increment and Eigenvalues: Axis 1 (19.6%, 0.08), Axis 2 (14.4%, 0.06). Peninsula populations (empty markers), inland populations (full markers). Letters and numbers refer to the populations detailed in Table 1. highly recommended. All populations are suited for ex-situ gene bank Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA conservation, however, populations at Kirstenbosch, Devil's Peak and restriction data. Genetics 131, 479–491. Platteklip inherited less genetic variation and therefore future sampling Hartl, D.L., Clark, A.G., 1989. Principles of Population Genetics. Sinauer Associates, Sunderland, campaigns should not only concentrate on these. Massachusetts, USA. Hattingh, V., Giliomee, J., 1989. Pollination of certain Leucadendron species (Proteaceae). Although populations are very similar in their genetic variation South African Journal of Botany 55, 387–393. they should not be managed as one genetic entity. Inland populations Koch, K., Bhushan, B., Barthlott, W., 2008. Diversity of structure, morphology and wetting are more similar to each other compared to the Peninsula ones, there- of plant surfaces. Soft Matter 4, 1943–1963. fore inland populations can be managed as one gene pool. In contrast, Koch, K., Bhushan, B., Barthlott, W., 2009. Multifunctional surface structures of plants: an inspiration for biomimetics. Progress in Materials Science 54, 137–178. Peninsula populations show a higher degree of differentiation and Kovach, W.L., 1999. MVSP — A Multivariate Statistical Package for Windows, ver. 3.1. should be managed separately. Kovach Computing Services, Pentraeth, Wales, UK. fi Conservation should focus on restoring the extensive seed banks that Krauss, S.L., 1997. Low genetic diversity in (Proteaceae), a re-sensitive shrub occurring in a fire-prone habitat. Heredity 78, 41–49. should still exist under the pine plantations and invaded Le Maitre, D., 1988a. The effects of parent density and season of burn on the regeneration forest. The plantations should be removed and the natural fynbos fire re- of (Proteaceae) in the Kogelberg. South African Journal of gimes restored which will allow the wild Leucadendron populations to Botany 54, 581–584. Le Maitre, D., 1988b. Effects of season of burn on the regeneration of two Proteaceae with proliferate. soil-stored seed. South African Journal of Botany 54, 575–580. Horticultural conservation is not an option because the plants require Lewinton, R.C., 1972. The apportionment of human diversity. Evolutionary Biology 6, too much space and are highly susceptible to P. cinnamomi infections 381–398. Linnaeus, C., 1753. Species Plantarum, I. ed. Savlii, Estocolmo. under nursery and cultivated conditions. For example they no longer sur- Littlejohn, G.M., 2001. The challenges of breeding wild flowers cultivars for use in viveforlonginthewateredsectionsofKirstenboschgardenareasdespite commercial floriculture: African Proteaceae. Acta Horticulturae (ISHS) 552, 23–37. extensive plantings, for this reason, and maintaining separate gene pools Littlejohn, G.M., Robyn, A., 2000. Leucadendron: a multi-purpose crop. Acta Horticulturae (ISHS) 541, 171–174. within local stands will not be feasible. At least 50% of the historical range Liu, H., Yan, G., Sedgley, R., 2006a. InterspecifichybridizationinthegenusLeucadendron can be restored between and Constantia Neck on the through embryo rescue. South African Journal of Botany 72, 416–420. lower, granite, east slopes of Table Mountain. It is recommended that Liu, H., Yan, G., Shan, F., Sedgley, R., 2006b. 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