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Conservation genetics of Leucadendron argenteum (Silvertree) — A flag ship species of the Cape Peninsula
South African Journal of Botany 88 (2013) 361–366
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Conservation genetics of Leucadendron argenteum (Silvertree) — A flag ship species of the Cape Peninsula Steffen Heelemann a,⁎, Fahiema Daniels b, Anthony G. Rebelo b, Peter Poschlod a, Christoph Reisch a a b
University of Regensburg, Institute of Botany, 93040 Regensburg, Germany South African National Biodiversity Institute, Private Bag X7, Claremont, Cape Town 7735, South Africa
a r t i c l e
i n f o
Article history: Received 6 October 2012 Received in revised form 26 August 2013 Accepted 26 August 2013 Available online 1 October 2013 Edited by JS Boatwright Keywords: AFLP Cape Floristic Region Fynbos Proteaceae Silwerboom
a b s t r a c t The Silvertree (Leucadendron argenteum (L.) R.Br.) is an iconic tree to South Africans and tourists alike. This endangered species is endemic to the Cape Peninsula, the most southwestern part of Africa. Despite its visual presence, no population genetic data of L. argenteum are currently available, but such information is crucial for effective conservation management. A historical question is whether the inland populations are natural or planted? This study aimed to reveal the genetic structure and possible differences of L. argenteum populations 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 Peninsula gene pool. Furthermore, genetic differences between populations were expected to be higher at inland populations because they are further apart from each other, relative to the Peninsula populations. Plant leaf 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 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 populations. 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 Planta naturalis, in patria, argentea excellit fronde, inter arbores nitidissima omnium. [Linnaeus, 1753]
2. Introduction The conebushes, Leucadendron R.Br. (Proteaceae), have attracted much research interest for many reasons. Adapted to a fire-prone environment and low-nutrient soils (Cowling and Holmes, 1992), Leucadendron is unique in the Cape Flora Proteaceae in having a diversity of seed dispersal and storage strategies (including serotiny, myrmecochory, therophily) thus prompting ecological (Bond, 1985; Hattingh and Giliomee, 1989; Le Maitre, 1988a,b; Midgley, 1987; Thuiller et al., 2004), population genetic (Tansley and Brown, 2000) and phylogenetic research (Barker et al., 2002, 2004). It is also of ⁎ Corresponding author. Tel.: +49 17623590324. E-mail address: [email protected] (S. Heelemann). 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
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 (Littlejohn, 2001; Littlejohn and Robyn, 2000; Liu et al., 2006a, 2006b, 2007). Although an alternative classification approach was proposed by Salisbury in 1809, proposing four genera of conebushes to align the group with the rest of the family where genera are determined largely by seed type, this has not been adopted, although Salisbury's groups are recognized at the sub-section level of the genus (Williams, 1972). In Linnaeus's famous Species Plantarum (1753) the Silvertree is described as “the most shining and splendid of all plants” (in Ben-Jaacov and Silber, 2010; Williams, 1972), and as the type of the genus Protea and the Proteaceae. This Linnaeus reversed in 1771, a conserved decision, thus it is now Leucadendron argenteum (L.) R.Br. (Williams, 1972). Recent studies support a paraphyletic genus Leucadendron (Barker et al., 2004). Because of its charismatic and attractive appearance, it is of particular interest to locals and tourists, as well as the horticultural crop industry (Van Wyk, 1983). The Silvertree is a conspicuous member of the Cape Flora, easily distinguishable from other plants on Table Mountain region by its large, silver leaves and exceptional height among Fynbos shrubs (Williams, 1972). Its range is centred on the Table Mountain, extending
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from Lions Head to Tokai and Noordhoek on the Cape Peninsula, where it is associated with wetter communities on granite (and shale) fynbos, being absent from the sandstone substrates. Isolated inland populations occur in Helderberg, Simonsberg and Paarl Mountain, with very small populations at Riebeek Kasteel and Tygerberg (Rebelo, 2001). The Silvertree is one of many localized and rare Proteaceae (Tansley, 1988) with a current IUCN Red List threatened status of Endangered (Raimondo et al., 2009; Rebelo et al., 2006, http://redlist.sanbi.org/ species.php?species=794-8), due to habitat destruction by urban expansion of Cape Town and degradation of habitat by fire exclusion on the Peninsula, wine farming and alien plants on the inland populations. The species has an unusual growth form in the genus, with a single erect stem, from which branches radiate at annual increments. It is exceptionally fast growing, up to 1 m per year, attaining 10 m by 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 Phytophthora cinnamomi Rands (root rot), which kills 5–10% of populations per year (Van Wyk, 1973; Von Broembsen and Kruger, 1985), but most plants have set seed before succumbing to the obvious symptoms which result in death within hours. Although the flowerheads are large, pollination is by minute beetles, with no evidence of wind pollination. The Silvertree is partly serotinous, but its seeds are released under hot conditions. The fruit is a large rounded nut, and has a parachute-type dispersal mechanism, but dispersal distances are typically only a few metres (Notten and Van der Walt, 2008). Secondary caching by rodents also occurs. Anecdotal evidence suggests that fruit may stay dormant for at least 80 years. It is dioecious with a chromosome number of 26 (Liu et al., 2006b). Very little research has been conducted around L. argenteum and most references are of anecdotal character (Rebelo pers. obs.). Exceptions are early germination experiments by Herschel in 1836 (Rourke, 1994), economical interests (Ben-Jaacov and Silber, 2010) and biomimetic research (Koch et al., 2008, 2009). In the forest-scarce Cape region L. argenteum was an important firewood and plantings were established in the 1700s to 1800s (Notten and Van der Walt, 2008). Currently no data on the population genetic structure of L. argenteum are available. However, such data are important for an optimal conservation management plan of the species in order to preserve its entire genetic variation. Crucial to this issue is establishing whether the inland populations are natural or were established by early colonialists for fuel and building. Furthermore, we asked how large genetic variation within individual populations of L. argenteum is, and should any differences between populations exist how this might affect the ex-situ conservation management of the species. 3. Material and methods 3.1. Sampling procedure The study area comprises the Cape Peninsula and inland hills where Silvertree populations are present. Plant leaf material from up to twenty randomly chosen individuals per population was collected from five Peninsula populations and three inland populations (Table 1, Fig. 1). However, the recently discovered and much smaller populations of Tygerberg (5 plants, in 2000) and Riebeek Kasteel (8 plants, in 2010) were not included. 3.2. DNA isolation and AFLP analysis For each population, leaf material was collected and cooled on ice. Later they were placed into filter bags and dehydrated in silica gel. DNA was isolated from 10 mg of dried plant material of individual plants using the CTAB method (cetyltrimethylammonium bromide, Rogers and Bendich, 1994). Both the DNA isolation and AFLP method (Vos et al., 1995) were adapted as previously described (Reisch, 2008; Reisch et al., 2005). DNA concentration was estimated photometrically,
Table 1 Localities of Leucadendron argenteum populations sampled in the Western Cape, South Africa. The geographic location of the studied populations (in parenthesis) is illustrated in Fig. 1. Letters and numbers refer to the populations illustrated in Fig. 1. Localities
South latitude
East longitude
Altitude (m)
Cape Peninsula populations Devils Peak (D) 33.95028 Kirstenbosch (E) 33.98924 Lions Head (B) 33.94081 Oranjekloof (A) 34.00417 Platteklip (C) 33.95497
18.45725 18.42510 18.39323 18.38715 18.42642
210 240 267 154 429
Inland populations Helderberg (3) Paarlberg (1) Simonsberg (2)
18.85200 18.93672 18.90372
238 428 360
34.05477 33.75406 33.89653
and samples were standardised at a dilution of 7.8 ng/μl. For the amplified fragment length polymorphism (AFLP) procedure, genomic DNA (approximately 50 ng) was used for restriction and ligation reactions with MseI and EcoRI restriction enzymes and T4 DNA Ligase (both were from Fermentas) were conducted in a thermal cycler for 2 h at 37 °C. Polymerase chain reactions (PCRs) were run in a reaction volume of 5 ml. Preselective amplifications were performed using primer pairs with a single selective nucleotide, MseI and EcoRI together with H2O, Puffer S, dNTPs and Taq-Polymerase (PeqLab). The PCR reaction parameters were: 2 min at 94 °C, 30 cycles of 20 s of denaturing at 94 °C, 30 s of annealing at 56 °C, and 2 min of extension at 72 °C, followed by 2 min at 72 °C and ending with 30 min at 60 °C. After an extensive screening of selective primer combinations with eight randomly selected samples, selective amplifications were performed with the three primer combinations (MseI + CTC/EcoRI + AAC, MseI + CTC/EcoRI + AAG, MseI + CTG/EcoRI + ACT) and H2O, dNTPs and Taq-Polymerase (PeqLab). PCR reactions were performed with the touch-down profile: 2 min at 94 °C, ten cycles of 20 s of denaturing at 94 °C, 30 s of annealing, which was initiated at 66 °C and then reduced by 1 °C for the next ten cycles, 2 min of elongation at 72 °C, followed by 25 cycles of 20 s of denaturing at 94 °C, 30 s of annealing at 56 °C and 2 min of elongation at 72 °C, ending with a final extension for 30 min at 60 °C. After DNA precipitation, DNA pellets were vacuum-dried and dissolved in a mixture of Sample Loading Solution and CEQ Size Standard 400 (both Beckman Coulter). The fluorescence-labelled selective amplification products were separated by capillary gel electrophoresis on an automated sequencer (CEQ 8000, Beckman Coulter). Raw data were collected and analysed with the CEQ Size Standard 400 using the CEQ 8000 software (Beckman Coulter). Data were exported as crv-files, showing synthetic gels with AFLP fragments for each primer combination separately from all studied individuals and analysed in BioNumerics (Applied Maths, v. 3.0). Files were examined for strong, clearly defined bands by eye. Each band was scored across all individuals as either present or absent. 3.3. Statistical analysis In the AFLP data matrix, the presence of a band was scored as 1, whereas the absence of the band was coded as 0. Finally, basic data structure consisted of a binomial (0/1) matrix, representing the scored AFLP markers. Nei's gene diversity, Shannon's information index, and number and percentage of polymorphic loci (PL) were calculated for each population. Genetic variation was calculated via POPGENE v. 1.32 (Yeh et al., 1999). Genetic variation within populations was calculated based on polymorphic bands as Nei's gene diversity [GD = ∑ I hij / I] (Nei, 1978) and Shannon information index [SI = ∑ pi ln pi] (Lewinton, 1972) for each population. The binomial matrix was subjected to an analysis of molecular variance (AMOVA, Excoffier et al., 1992) using GENALEX v. 6.2 (Peakall and Smouse, 2006). Variance components and their significance levels for variation among Peninsula
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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 calculated. Fst approximately equals Gst and is used for comparisons of species differentiation levels (Hartl and Clark, 1989). A Mantel test, based on 999 permutations, was conducted to test whether the matrix of pair-wise genetic distances (ΦPT), taken from the AMOVA between populations, was correlated with the matrix of geographical distances between populations (Mantel, 1967; Sokal and Rohlf, 1995). Genetic relatedness between individuals, assorting populations and differences between Peninsula and inland populations was analysed by principal coordinates analysis (PCoA). Calculations and plotting were based on inter-individual Bray–Curtis similarities and performed in MVSP v. 3.12 (Kovach, 1999). 4. Results AFLP analyses revealed 138 fragments (MseI + CAC/EcoRI + AAC [46 fragments], MseI + CAT/EcoRI + AAG [59 fragments], MseI + CAA/EcoRI + ACA [33 fragments]) with 46.75 ± 2.45 mean number of polymorphic loci, 33.88 ± 1.77 mean percentage of polymorphic loci, ranging from 36 to 56, and 26.1 to 40.6%, respectively (Table 2).
Mean Nei's gene diversity was 0.11 ± 0.01, ranging from 0.08 to 0.14. Mean Shannon's information index was 0.17, ranging from 0.13 to 0.21. Lowest genetic variation was found at Devil's Peak (GD = 0.08, NL = 39, PL = 28.3%) and Lions Head and Platteklip (SI = 0.13), i.e. the northernmost populations on the Peninsula. Highest genetic variation occurred in Paarlberg (GD = 0.14, SI = 0.21, NoL = 56, PL = 40.5%). Regarding genetic indices and according to t-tests, no significant differences occurred between Peninsula and inland populations. Analysis of molecular variances for the entire region showed low genetic variation (10%, PhiPt = 0.10) between populations (Table 3). Although on a low level genetic variation between Peninsula populations was double that between inland populations, 8%, PhiPt = 0.08 vs. 4%, PhiPt = 0.04, respectively. Low genetic variation was also apparent between Peninsula and inland group (3%). PCoAs showed no particular grouping between Peninsula and inland populations (Fig. 2). Mantel test revealed a weak but positive correlation (r2 = 0.09, p = 0.083) of geographic and genetic distance between populations. The error rate was estimated at 2.28% following a protocol of Bonin et al. (2004). No significant correlation of Nei's gene diversity (r = −0.15; p = 0.72; r2 = 0.02) and Shannon's index (r = −0.31; p = 0.45; r2 = 0.10) with population sizes occurred.
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Table 2 Genetic variation within populations of Leucadendron argenteum in the Western Cape, South Africa. Localities
n
Nei's gene diversity (GD ± S.E.)
Cape Peninsula populations Devils Peak 20 0.08 ± Kirstenbosch 20 0.1 ± Lions Head 20 0.12 ± Oranjekloof 20 0.1 ± Platteklip 18 0.09 ±
0.04 0.04 0.04 0.04 0.04
Shannon's index (SI ± S.E.)
Number of polymorphic loci (NoL)
Percentage of polymorphic loci (PL)
0.13 0.15 0.19 0.16 0.13
0.06 0.05 0.06 0.05 0.05
39 43 50 53 36
28.26 31.16 36.23 38.41 26.09
± ± ± ± ±
Inland populations Helderberg 18 Paarlberg 20 Simonsberg 20
0.12 ± 0.04 0.14 ± 0.04 0.1 ± 0.04
0.19 ± 0.06 0.21 ± 0.06 0.16 ± 0.05
50 56 47
36.23 40.58 34.06
Peninsula Inland Pooled
0.10 ± 0.01a 0.12 ± 0.01a 0.11 ± 0.01
0.15 ± 0.01b 0.19 ± 0.01b 0.17 ± 0.01
44.20 ± 3.22c 51.00 ± 2.65c 46.75 ± 2.45
32.03 ± 2.33d 36.96 ± 1.92d 33.88 ± 1.77
Sample size (n). Bold numbers show mean values ± S.E. Different letters indicate significant differences within indices (t-test). 138 loci in total, one private locus.
5. Discussion Genetic indices showed low levels of genetic variation within L. argenteum populations with no significant differences between Peninsula and inland populations. Molecular variance was also low between populations and among both regions. Nevertheless, twice as much genetic variation between populations was found on the Peninsula compared to inland populations when applying analysis of molecular variance separately. Furthermore, a positive Mantel test indicated the presence of genetic differences and spatial structure. We do not anticipate the recently discovered populations to be any different, due to their extremely small size (strongly suggesting that they were relatively recently planted). The present genetic variation in (Nei's GD 0.11) and between populations (PhiST 0.10), is lower than expected for long-lived, endemic, outcrossing and gravity-dispersed plant species from meta-studies (Hpop 0.19–0.26, PhiST 0.20–0.44, Nybom and Bartish, 2000). For example, higher genetic variation was observed within and between populations of rare Leucadendron elimense (RAPDs = 0.35, AMOVA = 27%) and within populations of the common and widespread Leucadendron salignum (0.30, RAPDs, Tansley and Brown, 2000). In Australian Persoonia Sm., relatively low genetic variation in populations (SI = 0.18–0.22, cf. L. argenteum SI = 0.17) and between populations (AMOVA = 16–21%) are reported (Rymer and Ayre, 2006), with Persoonia mollis having low gene variation (HT = 0.14) within population and average levels between them (0.22%, Krauss, 1997). It is suspected that seed banks of L. argenteum are typically 10 to 1000 times larger than the adult population (increasing with veld age)
and this has to be considered as part of the gene pool. For example, fragmented populations of the endangered shrub Grevillea caleyi R.Br. – with a similar ecological growth and seed traits and genetic variation to Silvertrees – show little influence on genetic structure (SI = 0.18 ± 0.02 vs. 0.17 ± 0.01, respectively) and it has been suggested that seed bank and plant longevity (15 years average life-span) buffer genetic erosion and effects of fragmentation on genetic variation (Llorens et al., 2004). It has even been shown that the genetic variation within the seed bank can be larger than within the above ground population and that the seed bank is slowing the process of differentiation between populations (McCue and Holtsford, 1998). Pollinating beetles and/or dispersal by rodents may bridge Peninsula populations thereby enabling gene flow. Only one pollen or seed per generation is necessary to ensure sufficient gene flow and avoid population differentiation (Slatkin, 1985), thereby diminishing fragmentation effects. However, inland populations are isolated and at least 10 km apart and genetic exchange seems unlikely. Although differences are at a low magnitude, it is interesting that genetic variation between Peninsula populations was double than between inland populations, the reverse of that predicted. Thus it can be suspected that inland populations are derived from Peninsula plants. Also the fact that the Paarl population showed higher (but not significantly) genetic variation points towards this scenario and could be attributed to several planting events, or plants from different Peninsula locations or the population's larger size. Because no historical calibration is possible it cannot be assumed that Peninsula populations are unaltered by plantings for fire wood, building material and conservation purposes over the last two centuries (see also http://protea.worldonline.co.za/silver.htm). However, comparison with other Proteaceae in the study region is possible. For example, Mimetes hirtus Salisbury – a proteoid shrub not commercially cultivated – has low inherit genetic variation (Nei's gene diversity = 0.15), as well as comparable 8% differentiation between Cape Peninsula populations (Reisch et al., 2010). This is a genetic pattern unlikely to be influenced by plantings and gives an indication that L. argenteum genetics were thus not significantly impacted by plantings (Nei's gene diversity = 0.10, 8% differentiation between Peninsula populations). Historical research might help resolve the issue of inland populations being planted, but the information is not readily available. From the data on genetic variation it is visible that inland populations are different, suggesting that they were established from the Peninsula. The expected but not observed lower genetic variation within inland populations might point to several planting events in the past. On the basis of our results we suggest the following guidelines that allow conservation authorities ex-situ conservation and population management. L. argenteum is an endemic, rare, endangered flagship species surviving in only few populations. Those that are threatened by land use change and commercial pine plantations. Therefore an exsitu 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. Entire region 156/8 Peninsula 98/5 Inland 58/3
Between Peninsula and inland 156/8
Genetic variation
D.f.
SS
MS
%
PhiPt
Between populations Within populations Between populations Within populations Between populations Within populations
7 148 4 93 2 55
151.32 1013.26 71.23 609.96 25.71 373.36
21.62 6.85 17.81 6.56 12.86 6.79
10% 90% 8% 92% 4% 96%
0.10
Among regions Between populations Within populations
1 6 148
34.27 117.04 1013.26
34.27 19.51 6.85
3% 8% 89%
0.08 0.04
0.11
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.
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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 conservation, however, populations at Kirstenbosch, Devil's Peak and Platteklip inherited less genetic variation and therefore future sampling campaigns should not only concentrate on these. Although populations are very similar in their genetic variation they should not be managed as one genetic entity. Inland populations are more similar to each other compared to the Peninsula ones, therefore inland populations can be managed as one gene pool. In contrast, Peninsula populations show a higher degree of differentiation and should be managed separately. Conservation should focus on restoring the extensive seed banks that should still exist under the pine plantations and invaded Afromontane forest. The plantations should be removed and the natural fynbos fire regimes restored which will allow the wild Leucadendron populations to proliferate. Horticultural conservation is not an option because the plants require too much space and are highly susceptible to P. cinnamomi infections under nursery and cultivated conditions. For example they no longer survive for long in the watered sections of Kirstenbosch garden areas despite extensive plantings, for this reason, and maintaining separate gene pools within local stands will not be feasible. At least 50% of the historical range can be restored between Rhodes Memorial and Constantia Neck on the lower, granite, east slopes of Table Mountain. It is recommended that seed banks be allowed to restore the populations and that plantings be minimized to maintain local genetic integrity. Where plantings are needed, they should be sourced from the nearest local population. Acknowledgements We would like to thank Petra Schitko and Virginia Babl for their technical assistance, as well as Table Mountain National Park and City of Cape Town and Cape Nature Reserves for the sampling permission. References Barker, N.P., Weston, P.H., Rourke, J.P., Reeves, G., 2002. The relationships of the southern African Proteaceae as elucidated by internal transcribed spacer (ITS) DNA sequence data. Kew Bulletin 57, 867–883. Barker, N.P., Vanderpoorten, A., Morton, C.M., Rourke, J.P., 2004. Phylogeny, biogeography, and the evolution of life-history traits in Leucadendron (Proteaceae). Molecular Phylogenetics and Evolution 33, 845–860. Ben-Jaacov, J., Silber, A., 2010. Leucadendron: a major proteaceous floricultural crop. In: Janick, J. (Ed.), Horticultural Reviews. John Wiley & Sons, Inc., Oxford, UK, pp. 167–228. http://dx.doi.org/10.1002/9780470767986.ch4. Bond, W.J., 1985. Canopy-stored seed reserves (serotiny) in Cape Proteaceae. South African Journal of Botany 51, 181–186. Bonin, A., Bellemain, E., Eidesen, P.B., Pompanon, F., Brochmann, C., Taberlet, P., 2004. How to track and assess genotyping errors in population genetics studies. Molecular Ecology 13, 3261–3273. Cowling, R.M., Holmes, P.M., 1992. Flora and vegetation. In: Cowling, R.M. (Ed.), The Ecology of Fynbos: Nutrients, Fire and Diversity. Oxford University Press, Cape Town, pp. 23–61.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Conservation genetics of Leucadendron argenteum (Silvertree) — A flag ship species of the Cape Peninsula Steffen Heelemann a,⁎, Fahiema Daniels b, Anthony G. Rebelo b, Peter Poschlod a, Christoph Reisch a a b
University of Regensburg, Institute of Botany, 93040 Regensburg, Germany South African National Biodiversity Institute, Private Bag X7, Claremont, Cape Town 7735, South Africa
a r t i c l e
i n f o
Article history: Received 6 October 2012 Received in revised form 26 August 2013 Accepted 26 August 2013 Available online 1 October 2013 Edited by JS Boatwright Keywords: AFLP Cape Floristic Region Fynbos Proteaceae Silwerboom
a b s t r a c t The Silvertree (Leucadendron argenteum (L.) R.Br.) is an iconic tree to South Africans and tourists alike. This endangered species is endemic to the Cape Peninsula, the most southwestern part of Africa. Despite its visual presence, no population genetic data of L. argenteum are currently available, but such information is crucial for effective conservation management. A historical question is whether the inland populations are natural or planted? This study aimed to reveal the genetic structure and possible differences of L. argenteum populations 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 Peninsula gene pool. Furthermore, genetic differences between populations were expected to be higher at inland populations because they are further apart from each other, relative to the Peninsula populations. Plant leaf 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 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 populations. 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 Planta naturalis, in patria, argentea excellit fronde, inter arbores nitidissima omnium. [Linnaeus, 1753]
2. Introduction The conebushes, Leucadendron R.Br. (Proteaceae), have attracted much research interest for many reasons. Adapted to a fire-prone environment and low-nutrient soils (Cowling and Holmes, 1992), Leucadendron is unique in the Cape Flora Proteaceae in having a diversity of seed dispersal and storage strategies (including serotiny, myrmecochory, therophily) thus prompting ecological (Bond, 1985; Hattingh and Giliomee, 1989; Le Maitre, 1988a,b; Midgley, 1987; Thuiller et al., 2004), population genetic (Tansley and Brown, 2000) and phylogenetic research (Barker et al., 2002, 2004). It is also of ⁎ Corresponding author. Tel.: +49 17623590324. E-mail address: [email protected] (S. Heelemann). 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
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 (Littlejohn, 2001; Littlejohn and Robyn, 2000; Liu et al., 2006a, 2006b, 2007). Although an alternative classification approach was proposed by Salisbury in 1809, proposing four genera of conebushes to align the group with the rest of the family where genera are determined largely by seed type, this has not been adopted, although Salisbury's groups are recognized at the sub-section level of the genus (Williams, 1972). In Linnaeus's famous Species Plantarum (1753) the Silvertree is described as “the most shining and splendid of all plants” (in Ben-Jaacov and Silber, 2010; Williams, 1972), and as the type of the genus Protea and the Proteaceae. This Linnaeus reversed in 1771, a conserved decision, thus it is now Leucadendron argenteum (L.) R.Br. (Williams, 1972). Recent studies support a paraphyletic genus Leucadendron (Barker et al., 2004). Because of its charismatic and attractive appearance, it is of particular interest to locals and tourists, as well as the horticultural crop industry (Van Wyk, 1983). The Silvertree is a conspicuous member of the Cape Flora, easily distinguishable from other plants on Table Mountain region by its large, silver leaves and exceptional height among Fynbos shrubs (Williams, 1972). Its range is centred on the Table Mountain, extending
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from Lions Head to Tokai and Noordhoek on the Cape Peninsula, where it is associated with wetter communities on granite (and shale) fynbos, being absent from the sandstone substrates. Isolated inland populations occur in Helderberg, Simonsberg and Paarl Mountain, with very small populations at Riebeek Kasteel and Tygerberg (Rebelo, 2001). The Silvertree is one of many localized and rare Proteaceae (Tansley, 1988) with a current IUCN Red List threatened status of Endangered (Raimondo et al., 2009; Rebelo et al., 2006, http://redlist.sanbi.org/ species.php?species=794-8), due to habitat destruction by urban expansion of Cape Town and degradation of habitat by fire exclusion on the Peninsula, wine farming and alien plants on the inland populations. The species has an unusual growth form in the genus, with a single erect stem, from which branches radiate at annual increments. It is exceptionally fast growing, up to 1 m per year, attaining 10 m by 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 Phytophthora cinnamomi Rands (root rot), which kills 5–10% of populations per year (Van Wyk, 1973; Von Broembsen and Kruger, 1985), but most plants have set seed before succumbing to the obvious symptoms which result in death within hours. Although the flowerheads are large, pollination is by minute beetles, with no evidence of wind pollination. The Silvertree is partly serotinous, but its seeds are released under hot conditions. The fruit is a large rounded nut, and has a parachute-type dispersal mechanism, but dispersal distances are typically only a few metres (Notten and Van der Walt, 2008). Secondary caching by rodents also occurs. Anecdotal evidence suggests that fruit may stay dormant for at least 80 years. It is dioecious with a chromosome number of 26 (Liu et al., 2006b). Very little research has been conducted around L. argenteum and most references are of anecdotal character (Rebelo pers. obs.). Exceptions are early germination experiments by Herschel in 1836 (Rourke, 1994), economical interests (Ben-Jaacov and Silber, 2010) and biomimetic research (Koch et al., 2008, 2009). In the forest-scarce Cape region L. argenteum was an important firewood and plantings were established in the 1700s to 1800s (Notten and Van der Walt, 2008). Currently no data on the population genetic structure of L. argenteum are available. However, such data are important for an optimal conservation management plan of the species in order to preserve its entire genetic variation. Crucial to this issue is establishing whether the inland populations are natural or were established by early colonialists for fuel and building. Furthermore, we asked how large genetic variation within individual populations of L. argenteum is, and should any differences between populations exist how this might affect the ex-situ conservation management of the species. 3. Material and methods 3.1. Sampling procedure The study area comprises the Cape Peninsula and inland hills where Silvertree populations are present. Plant leaf material from up to twenty randomly chosen individuals per population was collected from five Peninsula populations and three inland populations (Table 1, Fig. 1). However, the recently discovered and much smaller populations of Tygerberg (5 plants, in 2000) and Riebeek Kasteel (8 plants, in 2010) were not included. 3.2. DNA isolation and AFLP analysis For each population, leaf material was collected and cooled on ice. Later they were placed into filter bags and dehydrated in silica gel. DNA was isolated from 10 mg of dried plant material of individual plants using the CTAB method (cetyltrimethylammonium bromide, Rogers and Bendich, 1994). Both the DNA isolation and AFLP method (Vos et al., 1995) were adapted as previously described (Reisch, 2008; Reisch et al., 2005). DNA concentration was estimated photometrically,
Table 1 Localities of Leucadendron argenteum populations sampled in the Western Cape, South Africa. The geographic location of the studied populations (in parenthesis) is illustrated in Fig. 1. Letters and numbers refer to the populations illustrated in Fig. 1. Localities
South latitude
East longitude
Altitude (m)
Cape Peninsula populations Devils Peak (D) 33.95028 Kirstenbosch (E) 33.98924 Lions Head (B) 33.94081 Oranjekloof (A) 34.00417 Platteklip (C) 33.95497
18.45725 18.42510 18.39323 18.38715 18.42642
210 240 267 154 429
Inland populations Helderberg (3) Paarlberg (1) Simonsberg (2)
18.85200 18.93672 18.90372
238 428 360
34.05477 33.75406 33.89653
and samples were standardised at a dilution of 7.8 ng/μl. For the amplified fragment length polymorphism (AFLP) procedure, genomic DNA (approximately 50 ng) was used for restriction and ligation reactions with MseI and EcoRI restriction enzymes and T4 DNA Ligase (both were from Fermentas) were conducted in a thermal cycler for 2 h at 37 °C. Polymerase chain reactions (PCRs) were run in a reaction volume of 5 ml. Preselective amplifications were performed using primer pairs with a single selective nucleotide, MseI and EcoRI together with H2O, Puffer S, dNTPs and Taq-Polymerase (PeqLab). The PCR reaction parameters were: 2 min at 94 °C, 30 cycles of 20 s of denaturing at 94 °C, 30 s of annealing at 56 °C, and 2 min of extension at 72 °C, followed by 2 min at 72 °C and ending with 30 min at 60 °C. After an extensive screening of selective primer combinations with eight randomly selected samples, selective amplifications were performed with the three primer combinations (MseI + CTC/EcoRI + AAC, MseI + CTC/EcoRI + AAG, MseI + CTG/EcoRI + ACT) and H2O, dNTPs and Taq-Polymerase (PeqLab). PCR reactions were performed with the touch-down profile: 2 min at 94 °C, ten cycles of 20 s of denaturing at 94 °C, 30 s of annealing, which was initiated at 66 °C and then reduced by 1 °C for the next ten cycles, 2 min of elongation at 72 °C, followed by 25 cycles of 20 s of denaturing at 94 °C, 30 s of annealing at 56 °C and 2 min of elongation at 72 °C, ending with a final extension for 30 min at 60 °C. After DNA precipitation, DNA pellets were vacuum-dried and dissolved in a mixture of Sample Loading Solution and CEQ Size Standard 400 (both Beckman Coulter). The fluorescence-labelled selective amplification products were separated by capillary gel electrophoresis on an automated sequencer (CEQ 8000, Beckman Coulter). Raw data were collected and analysed with the CEQ Size Standard 400 using the CEQ 8000 software (Beckman Coulter). Data were exported as crv-files, showing synthetic gels with AFLP fragments for each primer combination separately from all studied individuals and analysed in BioNumerics (Applied Maths, v. 3.0). Files were examined for strong, clearly defined bands by eye. Each band was scored across all individuals as either present or absent. 3.3. Statistical analysis In the AFLP data matrix, the presence of a band was scored as 1, whereas the absence of the band was coded as 0. Finally, basic data structure consisted of a binomial (0/1) matrix, representing the scored AFLP markers. Nei's gene diversity, Shannon's information index, and number and percentage of polymorphic loci (PL) were calculated for each population. Genetic variation was calculated via POPGENE v. 1.32 (Yeh et al., 1999). Genetic variation within populations was calculated based on polymorphic bands as Nei's gene diversity [GD = ∑ I hij / I] (Nei, 1978) and Shannon information index [SI = ∑ pi ln pi] (Lewinton, 1972) for each population. The binomial matrix was subjected to an analysis of molecular variance (AMOVA, Excoffier et al., 1992) using GENALEX v. 6.2 (Peakall and Smouse, 2006). Variance components and their significance levels for variation among Peninsula
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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 calculated. Fst approximately equals Gst and is used for comparisons of species differentiation levels (Hartl and Clark, 1989). A Mantel test, based on 999 permutations, was conducted to test whether the matrix of pair-wise genetic distances (ΦPT), taken from the AMOVA between populations, was correlated with the matrix of geographical distances between populations (Mantel, 1967; Sokal and Rohlf, 1995). Genetic relatedness between individuals, assorting populations and differences between Peninsula and inland populations was analysed by principal coordinates analysis (PCoA). Calculations and plotting were based on inter-individual Bray–Curtis similarities and performed in MVSP v. 3.12 (Kovach, 1999). 4. Results AFLP analyses revealed 138 fragments (MseI + CAC/EcoRI + AAC [46 fragments], MseI + CAT/EcoRI + AAG [59 fragments], MseI + CAA/EcoRI + ACA [33 fragments]) with 46.75 ± 2.45 mean number of polymorphic loci, 33.88 ± 1.77 mean percentage of polymorphic loci, ranging from 36 to 56, and 26.1 to 40.6%, respectively (Table 2).
Mean Nei's gene diversity was 0.11 ± 0.01, ranging from 0.08 to 0.14. Mean Shannon's information index was 0.17, ranging from 0.13 to 0.21. Lowest genetic variation was found at Devil's Peak (GD = 0.08, NL = 39, PL = 28.3%) and Lions Head and Platteklip (SI = 0.13), i.e. the northernmost populations on the Peninsula. Highest genetic variation occurred in Paarlberg (GD = 0.14, SI = 0.21, NoL = 56, PL = 40.5%). Regarding genetic indices and according to t-tests, no significant differences occurred between Peninsula and inland populations. Analysis of molecular variances for the entire region showed low genetic variation (10%, PhiPt = 0.10) between populations (Table 3). Although on a low level genetic variation between Peninsula populations was double that between inland populations, 8%, PhiPt = 0.08 vs. 4%, PhiPt = 0.04, respectively. Low genetic variation was also apparent between Peninsula and inland group (3%). PCoAs showed no particular grouping between Peninsula and inland populations (Fig. 2). Mantel test revealed a weak but positive correlation (r2 = 0.09, p = 0.083) of geographic and genetic distance between populations. The error rate was estimated at 2.28% following a protocol of Bonin et al. (2004). No significant correlation of Nei's gene diversity (r = −0.15; p = 0.72; r2 = 0.02) and Shannon's index (r = −0.31; p = 0.45; r2 = 0.10) with population sizes occurred.
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Table 2 Genetic variation within populations of Leucadendron argenteum in the Western Cape, South Africa. Localities
n
Nei's gene diversity (GD ± S.E.)
Cape Peninsula populations Devils Peak 20 0.08 ± Kirstenbosch 20 0.1 ± Lions Head 20 0.12 ± Oranjekloof 20 0.1 ± Platteklip 18 0.09 ±
0.04 0.04 0.04 0.04 0.04
Shannon's index (SI ± S.E.)
Number of polymorphic loci (NoL)
Percentage of polymorphic loci (PL)
0.13 0.15 0.19 0.16 0.13
0.06 0.05 0.06 0.05 0.05
39 43 50 53 36
28.26 31.16 36.23 38.41 26.09
± ± ± ± ±
Inland populations Helderberg 18 Paarlberg 20 Simonsberg 20
0.12 ± 0.04 0.14 ± 0.04 0.1 ± 0.04
0.19 ± 0.06 0.21 ± 0.06 0.16 ± 0.05
50 56 47
36.23 40.58 34.06
Peninsula Inland Pooled
0.10 ± 0.01a 0.12 ± 0.01a 0.11 ± 0.01
0.15 ± 0.01b 0.19 ± 0.01b 0.17 ± 0.01
44.20 ± 3.22c 51.00 ± 2.65c 46.75 ± 2.45
32.03 ± 2.33d 36.96 ± 1.92d 33.88 ± 1.77
Sample size (n). Bold numbers show mean values ± S.E. Different letters indicate significant differences within indices (t-test). 138 loci in total, one private locus.
5. Discussion Genetic indices showed low levels of genetic variation within L. argenteum populations with no significant differences between Peninsula and inland populations. Molecular variance was also low between populations and among both regions. Nevertheless, twice as much genetic variation between populations was found on the Peninsula compared to inland populations when applying analysis of molecular variance separately. Furthermore, a positive Mantel test indicated the presence of genetic differences and spatial structure. We do not anticipate the recently discovered populations to be any different, due to their extremely small size (strongly suggesting that they were relatively recently planted). The present genetic variation in (Nei's GD 0.11) and between populations (PhiST 0.10), is lower than expected for long-lived, endemic, outcrossing and gravity-dispersed plant species from meta-studies (Hpop 0.19–0.26, PhiST 0.20–0.44, Nybom and Bartish, 2000). For example, higher genetic variation was observed within and between populations of rare Leucadendron elimense (RAPDs = 0.35, AMOVA = 27%) and within populations of the common and widespread Leucadendron salignum (0.30, RAPDs, Tansley and Brown, 2000). In Australian Persoonia Sm., relatively low genetic variation in populations (SI = 0.18–0.22, cf. L. argenteum SI = 0.17) and between populations (AMOVA = 16–21%) are reported (Rymer and Ayre, 2006), with Persoonia mollis having low gene variation (HT = 0.14) within population and average levels between them (0.22%, Krauss, 1997). It is suspected that seed banks of L. argenteum are typically 10 to 1000 times larger than the adult population (increasing with veld age)
and this has to be considered as part of the gene pool. For example, fragmented populations of the endangered shrub Grevillea caleyi R.Br. – with a similar ecological growth and seed traits and genetic variation to Silvertrees – show little influence on genetic structure (SI = 0.18 ± 0.02 vs. 0.17 ± 0.01, respectively) and it has been suggested that seed bank and plant longevity (15 years average life-span) buffer genetic erosion and effects of fragmentation on genetic variation (Llorens et al., 2004). It has even been shown that the genetic variation within the seed bank can be larger than within the above ground population and that the seed bank is slowing the process of differentiation between populations (McCue and Holtsford, 1998). Pollinating beetles and/or dispersal by rodents may bridge Peninsula populations thereby enabling gene flow. Only one pollen or seed per generation is necessary to ensure sufficient gene flow and avoid population differentiation (Slatkin, 1985), thereby diminishing fragmentation effects. However, inland populations are isolated and at least 10 km apart and genetic exchange seems unlikely. Although differences are at a low magnitude, it is interesting that genetic variation between Peninsula populations was double than between inland populations, the reverse of that predicted. Thus it can be suspected that inland populations are derived from Peninsula plants. Also the fact that the Paarl population showed higher (but not significantly) genetic variation points towards this scenario and could be attributed to several planting events, or plants from different Peninsula locations or the population's larger size. Because no historical calibration is possible it cannot be assumed that Peninsula populations are unaltered by plantings for fire wood, building material and conservation purposes over the last two centuries (see also http://protea.worldonline.co.za/silver.htm). However, comparison with other Proteaceae in the study region is possible. For example, Mimetes hirtus Salisbury – a proteoid shrub not commercially cultivated – has low inherit genetic variation (Nei's gene diversity = 0.15), as well as comparable 8% differentiation between Cape Peninsula populations (Reisch et al., 2010). This is a genetic pattern unlikely to be influenced by plantings and gives an indication that L. argenteum genetics were thus not significantly impacted by plantings (Nei's gene diversity = 0.10, 8% differentiation between Peninsula populations). Historical research might help resolve the issue of inland populations being planted, but the information is not readily available. From the data on genetic variation it is visible that inland populations are different, suggesting that they were established from the Peninsula. The expected but not observed lower genetic variation within inland populations might point to several planting events in the past. On the basis of our results we suggest the following guidelines that allow conservation authorities ex-situ conservation and population management. L. argenteum is an endemic, rare, endangered flagship species surviving in only few populations. Those that are threatened by land use change and commercial pine plantations. Therefore an exsitu 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. Entire region 156/8 Peninsula 98/5 Inland 58/3
Between Peninsula and inland 156/8
Genetic variation
D.f.
SS
MS
%
PhiPt
Between populations Within populations Between populations Within populations Between populations Within populations
7 148 4 93 2 55
151.32 1013.26 71.23 609.96 25.71 373.36
21.62 6.85 17.81 6.56 12.86 6.79
10% 90% 8% 92% 4% 96%
0.10
Among regions Between populations Within populations
1 6 148
34.27 117.04 1013.26
34.27 19.51 6.85
3% 8% 89%
0.08 0.04
0.11
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.
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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 conservation, however, populations at Kirstenbosch, Devil's Peak and Platteklip inherited less genetic variation and therefore future sampling campaigns should not only concentrate on these. Although populations are very similar in their genetic variation they should not be managed as one genetic entity. Inland populations are more similar to each other compared to the Peninsula ones, therefore inland populations can be managed as one gene pool. In contrast, Peninsula populations show a higher degree of differentiation and should be managed separately. Conservation should focus on restoring the extensive seed banks that should still exist under the pine plantations and invaded Afromontane forest. The plantations should be removed and the natural fynbos fire regimes restored which will allow the wild Leucadendron populations to proliferate. Horticultural conservation is not an option because the plants require too much space and are highly susceptible to P. cinnamomi infections under nursery and cultivated conditions. For example they no longer survive for long in the watered sections of Kirstenbosch garden areas despite extensive plantings, for this reason, and maintaining separate gene pools within local stands will not be feasible. At least 50% of the historical range can be restored between Rhodes Memorial and Constantia Neck on the lower, granite, east slopes of Table Mountain. It is recommended that seed banks be allowed to restore the populations and that plantings be minimized to maintain local genetic integrity. Where plantings are needed, they should be sourced from the nearest local population. Acknowledgements We would like to thank Petra Schitko and Virginia Babl for their technical assistance, as well as Table Mountain National Park and City of Cape Town and Cape Nature Reserves for the sampling permission. References Barker, N.P., Weston, P.H., Rourke, J.P., Reeves, G., 2002. The relationships of the southern African Proteaceae as elucidated by internal transcribed spacer (ITS) DNA sequence data. Kew Bulletin 57, 867–883. Barker, N.P., Vanderpoorten, A., Morton, C.M., Rourke, J.P., 2004. Phylogeny, biogeography, and the evolution of life-history traits in Leucadendron (Proteaceae). Molecular Phylogenetics and Evolution 33, 845–860. Ben-Jaacov, J., Silber, A., 2010. Leucadendron: a major proteaceous floricultural crop. In: Janick, J. (Ed.), Horticultural Reviews. John Wiley & Sons, Inc., Oxford, UK, pp. 167–228. http://dx.doi.org/10.1002/9780470767986.ch4. Bond, W.J., 1985. Canopy-stored seed reserves (serotiny) in Cape Proteaceae. South African Journal of Botany 51, 181–186. Bonin, A., Bellemain, E., Eidesen, P.B., Pompanon, F., Brochmann, C., Taberlet, P., 2004. How to track and assess genotyping errors in population genetics studies. Molecular Ecology 13, 3261–3273. Cowling, R.M., Holmes, P.M., 1992. Flora and vegetation. In: Cowling, R.M. (Ed.), The Ecology of Fynbos: Nutrients, Fire and Diversity. Oxford University Press, Cape Town, pp. 23–61.
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