Conservation Genetics 1: 217–229, 2000. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
217
Conservation genetics of an endangered rainforest tree (Fontainea oraria – Euphorbiaceae) and implications for closely related species Maurizio Rossetto1,∗ , Jody McNally1 , Robert J. Henry1 , John Hunter2 & Maria Matthes2 1 Centre
for Plant Conservation Genetics, Southern Cross University, P.O. Box 157, Lismore NSW, Australia; National Parks and Wildlife Service, Coffs Harbour NSW, Australia (∗ Author for correspondence: E-mail:
[email protected]) 2 NSW
Received 8 March 2000; accepted 7 Juner 2000
Key words: Australia, conservation strategy, cpDNA, Euphorbiaceae, Fontainea, nrDNA, RAPD
Abstract Four new eastern Australian Fontainea species have been recently described and all have a limited distribution. F. oraria is the rarest, being restricted to 10 adult individuals within a single site in regrowth littoral rainforest. In order to develop adequate management strategies, this study was aimed at surveying the genetic variability remaining within the species by using RAPD analysis. To assist with the correct interpretation of the results, a matching study was conducted on four populations of the closely related F. australis. Similar amounts of withinpopulation genetic diversity were recorded for both species. The RAPD-based study suggested that adult plants are contributing unevenly to successive generations. RAPD analysis also recognised a close evolutionary relationship between F. oraria and F. australis. Sequencing of cpDNA (trnL-F) and nrDNA (ITS2) regions, confirmed recent divergence and possibly some historical reticulation between these two species and two other members of the genus. Of particular interest was the recognition that one of the F. australis populations (Limpinwood) represented a novel genotypic combination in need of conservation attention. The implications of the RAPD and sequencing results are discussed in reference to their influence upon the development of adequate conservation strategies for all important conservation units.
Introduction The Coastal Fontainea (Fontainea oraria) is known from a single site. The only known population comprises only 10 adult plants and 52 seedlings and is confined to a 600 m strip of regrowth littoral rainforest on private land in north-eastern New South Wales (Australia – Figure 1). The geological and environmental components of littoral rainforests are restricted to disjunct sites along a narrow strip of the eastern Australian coast. Over 75% of this vegetation type has been cleared mainly for urban and agricultural development, with most remaining patches being under one hectare in size (Jay 1990). The F. oraria site was cleared and burned in the 1950s for agricultural purposes and native vegetation was only left as windbreaks on the steep rocky edges. Consequently, the current regrowth rainforest is even-aged, relatively
simple in structure and shows signs of damage from strong sea-breezes and weed competition. The Coastal Fontainea is the most threatened species within this plant community and its survival can only be ensured by the appropriate management of the remaining individuals and their habitat. As a result, information on the genetic diversity and ecology of F. oraria will be fundamental to the development of long-term conservation strategies. A better understanding of the extent and distribution of genetic diversity within this species, will help in defining the relationships between single individuals and estimate their potential contribution to future generations. Of the many molecular techniques available to the conservation geneticist, random amplified polymorphic DNA (RAPD – Williams et al. 1990) analysis is particularly advantageous when dealing with extremely rare species. High polymorphism and
218 the availability of a large number of loci make this technique particularly attractive to projects dealing with low genetic diversity. Furthermore, its simplicity, low cost and universality are invaluable when resources are limited and the species is poorly known. As a result, RAPD analysis is being increasingly used in rare flora research (Van Buren et al. 1994; Rossetto et al. 1995, 1997, 1999; Hogbin et al. 1997; Palacios and Gonzales-Candelas 1997; Martin et al. 1999; Maunder et al. 1999). Successful implementation of the findings derived from population studies is dependent on a clear understanding of the relationships between the species under investigation, particularly if there is some uncertainty regarding taxonomic status. For instance, the Coastal Fontainea is morphologically similar to F. australis, another vulnerable species from northeastern New South Wales known from four small populations only (Jessup and Guymer 1985). The resemblance between these two species, makes F. australis an ideal target for a comparative RAPDbased study, but it also implies that distinction between these two taxa may need to be based on more than morphological characters. Molecular phylogenetics can provide the information required on the evolutionary divergence between these and other closely related taxa. Long term and short term genomic evolution can be examined by the analysis of chloroplast DNA (cpDNA) and nuclear ribosomal DNA (nrDNA) respectively. The trnL-F (GAA) intergenic spacer is one of the most variable regions of the chloroplast genome and is particularly useful at the intra-generic level (Taberlet et al. 1991). This region is variable and short enough to allow for rapid screening of numerous taxa. The internal transcribed spacer (ITS) region of the 18S–26S nrDNA evolves more rapidly than cpDNA, thus showing greater sensitivity at lower taxonomic levels (Baldwin et al. 1995). The combination of the information provided by these two complementary regions has been frequently used to define taxonomic relationships. The aim of this study was to provide information useful for the development of adequate management strategies for F. oraria. RAPD analysis was used to quantify and compare genetic diversity within the Coastal Fontainea and within populations of the closely related F. australis. A molecular phylogenetic study based on cpDNA and nrDNA was also conducted in order to evaluate the evolutionary relationships between these and another two recently described fontaineas (F. rostrata and F. venosa). It was
hoped that a sequencing-based study would clarify the relationships between these four newly described species and possibly identify valuable, but previously unrecognised, conservation units.
Materials and methods Study species The genus Fontainea Heckel (Euphorbiaceae – Cluytieae) comprises six recognised species of rainforest shrubs and trees. Five are endemic to eastern Australia and one is endemic to New Caledonia (Jessup and Guymer 1985). Until recently, only two Fontainea species were recognised within eastern Australia, F. pancheri Heckel and F. picrosperma C. White (Airy Shaw 1974). After a critical review of the genus, four new species were described by Jessup and Guymer (1985). F. rostrata and F. venosa were removed from the classification of F. pancheri while F. pancheri was found to be confined to New Caledonia. Two new species, F. oraria and F. australis, were also described in the revision (Jessup and Guymer 1985). These four new taxa are restricted to relatively small areas of sub-tropical eastern Australia. The Coastal Fontainea, F. oraria Jessup and Guymer, is a tree up to 5 m with glossy green, glabrous, elliptic or scarcely obovate leaves. The smooth, dark brown-grey bark exudes a reddish watery substance when damaged. A dioecious species, the terminal or axillary male inflorescences consist of small flowers with four or five whitish petals. The female inflorescences are terminal, often reduced to two or three flowers and the soft-fleshy, red drupes are depressed globular. F. oraria is found in a single location near the coast in north-eastern New South Wales, with all known individuals being located within a 600 m radius amongst regrowth littoral rainforest. The main study species is similar to F. australis Jessup and Guymer which differs by having smooth endocarp, slightly smaller leaves, longer floral axis and more distal gland placement. F. australis is only known from four populations, mostly within protected areas that are in some cases very small (for instance the Limpinwood and Wanganui populations number less than 50 individuals each). F. rostrata Jessup and Guymer and F. venosa Jessup and Guymer are also known from a limited number of populations in southern Queensland. F. venosa is a tree growing up to 18 m, with larger
219 Table 1. Collection details of the individuals used in this study. The codes used in the dendrograms in Fig. 2 and the number of individuals sampled (N) are also indicated. The F. oraria individuals represent all those known for this species Species
Location (state)
N
Code
F. oraria (mature plants) F. oraria (seedlings) F. oraria (propagated) F. australis F. australis F. australis F. australis F. venosa F. rosrata
Lennox Head (NSW) Lennox Head (NSW) – Wanganui (NSW) Limpinwood Res. (NSW) Numinbah Res. (NSW) Nightcap NP (NSW) Bahr’s Scrub (Qld) Gympie (Qld)
10 52 10 11 10 10 10 1 1
F S G W L N C – –
leaves than the other species. F. rostrata has the most northern distribution of these four taxa and is morphologically similar to F. oraria and F. australis.
Plant material and DNA extraction In this study, a total of 72 F. oraria individuals were sampled (Table 1). These comprise all known in situ plants (10 mature individuals and 52 densely clustered seedlings) and 10 nursery grown individuals. Within the natural population, the majority of the seedlings are located directly under the canopy or in the proximity of individual F4, the largest and most prolific female plant known. The nursery grown plants also originated from seed collected from that same individual. A potential difficulty with population studies on rare species based on molecular techniques, is the paucity of comparative data. As a result, a similar RAPD-based study on a closely related species, F. australis, was conducted in order to produce comparative information upon which to base future management strategies. A total of 41 F. australis individuals from four populations (Figure 1) were analysed for that purpose (Table 1). DNA extractions were performed on fresh leaf tissue using a modified CTAB method (Maguire et al. 1994). RAPD-PCR reactions All F. oraria mature plants were initially screened with 7 ninemer primers from Rossetto et al. (1999)
Figure 1. Distribution range for the F. oraria and F. australis populations sampled.
and 10 tenmer primers from an Operon Technologies Inc. kit (OPC). Only primers exhibiting reproducible bands were used and the bands that could not be scored consistently were not included in the analysis. Three ninemers and four tenmers (OPC2, OPC8, OPC9 and OPC10) produced reliable banding patterns and were selected for this study (Table 2). PCR reactions were performed in a 25 µl total volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM of each dNTP, 0.5 units Taq DNA polymerase (Roche), 0.25 µM of primer, 10 ng DNA and DNAfree water. Reproducibility of banding patterns was tested by running duplicate reactions. All PCR reactions were prepared in sterile conditions and a negative control (in which DNA was omitted) was included with every PCR run. The PCR reaction was performed in a GeneAmpr PCR System 9700 (Perkin Elmer Biosystems) with an initial denaturation step at 94 ◦ C for 5 min, followed by 35 cycles each at 94 ◦ C for 15 sec, 34 ◦ C for 45 sec, 72 ◦ C for 1 min (annealing temperature was the same for all primers). This was followed by a final extension step at 72 ◦ C for 10 min.
220 Table 2. Summary of the results produced by RAPD analysis on F. oraria and F. australis. The sequence of each of the seven primers used, the number of bands and the size range are indicated for each primer. In this table F. australis is represented by all four putative populations
Primer
Approx. band size range (bp)
Number of bands per primer F. oraria F. australis
CCC TCC TCC GGG TGG TTG CCC ACC AAC GTG AGG CGT C TGG ACC GGT G CTC ACC GTC C TGT CTG GGT G
300–1600 600–1600 700–2800 200–2300 800–2100 600–3500 800–1500
12 7 9 10 5 10 4
15 7 7 11 8 10 5
Total
200–3500
57
63
1993). AMOVA is particularly useful for studies on outcrossing species, as these exhibit more of the assumptions on which the analysis is based upon (Bussell 1999). Arlequin ver. 2.000 (Schneider et al. 2000) was used to perform an analysis of molecular variance (AMOVA) on the F. oraria and F. australis RAPD data sets. Three-level hierarchical nested analysis was used to assess the amount and significance of the variance component associated with differences between and within groups. The two groups selected were F. oraria (represented by the single known population) and F. australis (further divided into four populations). For each analysis 1000 permutations were performed to obtain significance levels of variances. In order to better understand population differentiations within F. australis, Fst values for each pairwise population comparison were estimated using Arlequin.
RAPD data analysis
Sequencing reactions and analysis
Amplification products were resolved electrophoretically on 2% agarose gels run at 130 V in TBE for 3 hr and stained with ethidium bromide. A 100 bp size marker (Roche) was included with each gel in order to correctly size the bands produced. The RAPD patterns were visualised under UV light, digitised and scored using FragmeNT Analysis software (Molecular Dynamics). As the absence of RAPD fragments may not necessarily imply shared ancestry, Jaccard’s coefficient was used to analyse the presence/absence data as it calculates genetic similarity based on shared fragments only. Pairwise genetic similarities were calculated by S = Mxy /(Mt − Mxy0 ) where Mxy is the number of fragments shared between individuals, Mt represents the total number of fragments and Mxy0 the number of fragments not present in the individuals compared. Jaccard’s coefficient of similarity was estimated using NTSYS-pc 2.0 (Rohlf 1998) and genetic variability was calculated as V = 1 − S. Variability within populations and species was directly estimated from the matrix by averaging all pairwise results within the selected group. UPGMA clustering was performed from the SHAN option of NTSYS-pc and a dendrogram representing the relationship between all individuals tested was derived from the TREE option. Analysis of molecular variance (AMOVA) has become an important tool for investigating the partitioning of RAPD variation as it is not influenced by the dominant nature of the markers (Huff et al.
The trnL-F (GAA) intergenic spacer was amplified from total DNA using primers e and f of Taberlet et al. (1991) following standard protocol. The ITS2 region of the nrDNA transcript was amplified from total DNA using primers ITS3 and ITS4 from White et al. (1990) following standard protocol. Cycle-sequencing was carried out using the Big-Dye terminator following Perkin-Elmer’s protocol. Sequences were visualised on an ABI 377 sequencer at the Australian Genome Research Facility (Brisbane, Australia). Sequencing with the forward primers produced interpretable sequences for the entire product, however the reverse strand was also sequenced as a control. One individual of F. oraria was analysed, while for F. australis one individual was tested for each of the four populations (Limpinwood, Nightcap, Numinbah, Wanganui). Individuals of F. venosa and F. rostrata were also used in this comparative study (Table 1). Alignment of the sequences was performed using Clustal W (Thompson et al. 1994). The sequences obtained from the two DNA regions were analysed both separately and together. Phylogenetic relationships among the seven sequences were inferred with 100 random sequence additions and tree bisectionreconnection (TBR) branch swapping, permitting five trees to be held at each step. Tree searches were conducted via a Fitch parsimony approach (unordered characters state, Fitch 1971) using PAUP 4.0b4a (Swofford 2000). Internal support was evaluated by submitting the data to bootstrap analysis (100 repli-
221 Table 3. Genetic variability (within species and populations) calculated as V = 1 − S for F. oraria and F. australis based on RAPD data Genetic variability Species/population
V
F. oraria F. oraria (parental plants only) F. oraria (progeny only) F. australis F. australis Limpinwood F. australis Nightcap F. australis Numinbah F. australis Wanganui
43% 49% 41% 46% 40% 31% 40% 39%
cates). Neighbour-Joining analysis was performed using the Jukes-Cantor distance measure.
Results RAPD data Overall, the RAPD data clearly distinguished between F. oraria and F. australis. A total of 67 reliable fragments were obtained, with fragment sizes ranging from 200 bp to 3500 bp and the number of fragments scored per primer varying from four to 15 (Table 2). Overall, F. australis individuals amplified a greater total number of fragments (63) than did F. oraria individuals (57, Table 2), with 10 and four exclusive bands (i.e. species specific) respectively being exhibited. Uniqueness of genotypes is likely to be due to evolutionary divergence between the two species, but could also be the result of drift which can potentially be particularly influential for neutral markers (especially when assessing small isolated populations). RAPD analysis differentiated every single F. oraria individual. Genetic variability was higher in parental plants, 49%, than in offsprings, 41% (Table 3). Only a few parental plants exhibited exclusive bands (i.e. individual specific); three were identified for individual F7 and two for F1. No bands exclusive to the offspring were recorded. Fragments exclusive to F1 were detected in 12 seedlings suggesting that F1 is successfully contributing to the seedling generation. In the dendrogram obtained by UPGMA analysis (Figure 2a) the majority of the offsprings cluster with plant F4. This is not surprising as most of the seedlings are located in close proximity, or directly under
the canopy of F4. Three of the nursery grown seedlings collected from F4 (G6, G7, G8) clustered with F6, thus suggesting that F6 may also have been a pollen donor. Since 49 of the 62 seedlings representing the most recent F. oraria generation, cluster with F4 (Figure 2a), it appears that this individual is playing a dominant role in contributing to successive generations. Conversely, no offspring cluster with some of the other mature individuals (FX, F2, F5 and F7), suggesting that these may not have contributed to the seedling generation (Figure 2a). Overall, genetic variability within F. oraria was lower (43%) than within F. australis (46% – Table 3). However, when considering single populations the variability detected within the sole F. oraria population (in particular when only mature plants were considered – 49%) was much higher than that measured from the same number of individuals within single F. australis populations (31% to 40% – Table 3). The dendrogram in Figure 2b shows that, with the exception of a few individuals from Wanganui and Numinbah, the F. australis populations grouped as distinct clusters. In particular, Limpinwood forms a cluster marginally separated from the other three F. australis populations. As expected, the analysis of molecular variance partitioned a significant amount of variation between the two species (14.8% – Table 4). Of even greater interest was the fact that a larger component of the total variance was due to differences between populations within groups (23.3% – Table 4). As only the F. australis group was represented by multiple populations, it can be concluded that the within-group differentiation mainly represents structure amongst the F. australis populations. As suggested by the dendrogram in Figure 2b and by the pairwise Fst values summarised in Table 5, most of such differentiation originates from the Limpinwood population. The average Fst value between Limpinwood and the other three population is 0.34, whereas the average Fst value between the Wanganui, Numinbah and Nightcap populations is 0.20. Sequencing data Despite distinguishing successfully between the different taxa and their populations (Figure 2b), RAPD analysis is not an appropriate technique to use to resolve inter-specific evolutionary relationships. As a result, direct sequencing of selected cpDNA and nrDNA regions was conducted in order to clarify taxo-
222
Figure 2. a) Dendrogram showing the genetic divergence between the F. oraria individuals as estimated by RAPD analysis. As indicated by the bar on the right side, the F4 individual clusters with a group of offsprings and no other parental genotype. b) Dendrogram showing the relationship between the two different Fontainea species. Four F. australis populations and the mature F. oraria individuals have been included. The codes for the four F. australis population are summarised in Table 1.
223
Figure 2. Continued.
224 Table 4. Analysis of molecular variance (AMOVA) on the RAPD data. The three-level analysis was conducted among and within two groups representing the two species. Degrees of freedom, sum of squares, variance components, percentual contribution of the variance components, corresponding fixation indices and P-values are indicated within the table Source of variation
d.f.
SS
Variance components
% Total
Fixation indices
P-value
F. oraria vs. F. australis Among populations within groups Within populations
1 3 109
218.0 122.8 918.6
2.01 3.17 8.43
14.76 23.31 61.93
0.14 0.27 0.38
