Conserv Genet (2010) 11:2357–2368 DOI 10.1007/s10592-010-0122-8
RESEARCH ARTICLE
Conservation genetics of the endangered endemic Sambucus palmensis Link (Sambucaceae) from the Canary Islands Pedro A. Sosa • Miguel A. Gonza´lez-Pe´rez Claudio Moreno • Jake B. Clarke
•
Received: 25 September 2009 / Accepted: 10 August 2010 / Published online: 4 September 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Five polymorphic microsatellites (simple sequence repeat; SSR) markers were used to estimate the levels of genetic variation within and among natural populations from different islands of the endangered endemic from the Canary Islands Sambucus palmensis Link (Sambucaceae). Genetic data were used to infer potential evolutionary processes that could have led to present genetic differentiation among islands. The levels of genetic variability of S. palmensis were considerably high; proportion of polymorphic loci (P = 100%), mean number of alleles per locus (A = 6.8), average expected heterozygosity (He = 0.499). In spite of its small population size and endemic character, 58 different multilocus genotypes were detected within the 165 individuals analyzed. All samples located in different islands always presented different multilocus genotypes. Principal Coordinates Analysis, genetic differentiation analysis (FST and GST0 ) and Bayesian Cluster Analysis revealed significant genetic differences among populations located in different islands. However, this genetic differentiation was not recorded among Tenerife and La Gomera populations,
P. A. Sosa (&) M. A. Gonza´lez-Pe´rez Departamento de Biologı´a, Universidad de Las Palmas de Gran Canaria, Campus Universitario de Tafira, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain e-mail:
[email protected] C. Moreno Departamento de Geografı´a, Universidad de Las Palmas de Gran Canaria, C/. Pe´rez del Toro, 1, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain J. B. Clarke East Malling Research, New Road, East Malling, Kent ME19 6BJ, UK
possibly revealing the uncontrolled transfer of material between both islands. AMOVA analysis attributed 77% of the variance to differences within populations, whereas 8% was distributed between islands. The levels of genetic differentiation observed among populations, and the genetic diversity distribution within populations in S. palmensis, indicate that management should aim to conserve as many of the small populations as possible. Concentrating conservation efforts only on the few large populations would result in the likelihood of loss of genetic variability for the species. Keywords Bottleneck Conservation genetics Endangered species Plant conservation Relictual distribution SSR
Introduction The Canary Islands (Spain) are an archipelago in the Atlantic Ocean, belonging to the Macaronesian region. The islands have a rich endemic flora, with around 680 endemisms constituting at least 50% of their native plant species (Santos-Guerra 2001; Reyes-Betancort et al. 2008). It is estimated that around 20% of the endemics are endangered, with around 247 included in the Spanish Red Book of rare and endangered species (Ban˜ares et al. 2004; Moreno 2008). Sambucus palmensis Link, (Sambucus nigra subsp. palmensis), Sauco or Canary elderberry is one of these endangered endemic taxa (Ban˜ares 1990; Marrero-Go´mez et al. 1998; Beltra´n et al. 1999; Ban˜ares et al. 2004). This is due to the small number of naturally occurring individuals (approximately 380 individuals) distributed in four of the Canary Islands: La Palma, La Gomera, Tenerife and Gran Canaria. The extremely reduced distribution of this
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perennial, often with less than 10 individuals per population, has been interpreted as being a relictual distribution of a previously more widespread species, severely affected by habitat degradation, its use for medical purposes and its limited germination (Marrero-Go´mez et al. 1998; Beltra´n et al. 1999; Ban˜ares et al. 2003). Because of the low number of individuals and its reduced distribution, S. palmensis was included in the National Catalogue of Endangered Species and in the Canaries Catalogue of Endangered Species, Canarian Government Orden 20/2/91-(Annex I), under the category of ‘‘in danger of extinction’’. It has also been included as CR (critically endangered) under subcategory B1ab (IUCN 2006), in the Red List of Endangered National Plants (VVAA 2000), Bern Convention and European Community Habitat Directive (Ban˜ares et al. 2003). Sambucus palmensis is an allogamous perennial shrub, reproducing by seed and vegetatively, producing new stems from the base of the trunk (Marrero-Go´mez et al. 1998; Ban˜ares et al. 2003). Flowering is generally from May to July. Birds are the main dispersal agents of the species’ seeds, either regurgitating or defecating the seeds after ingesting the fruit (Beltra´n et al. 1999). Germination tests carried out by the Garajonay National Park, in La Gomera, showed a small percentage of fruit germination. Also, S. palmensis frequently produces seeds without embryos, producing fruits which are aborted in early development. In Tenerife, the species grows in isolated and dispersed populations, always in the potential zone of the Canarian Laurisilva forest. The main population is located in a protected area in the north east of Tenerife, (Reserva Integral del Pijaral, Anaga), and it comprises only 28 individuals across a 5 km2 area (Beltra´n et al. 1999; Ban˜ares et al. 2003). The remaining individuals are spread, isolated and dispersed, across the northern slopes of the island. The distances among these individuals are considerable, with several kilometres separating them, therefore none can be considered a true population (Beltra´n et al. 1999). On the island of La Palma only two locations of the species are known: the first in Barranco de Sabuquero (Garafı´a), which comprises around sixteen individuals, probably of natural origin, in an area of 4 km2; the second in Los Tiles, around 15 km from the first location, made up of more than 50 individuals, mostly reintroduced vegetatively (Beltra´n et al. 1999). In La Gomera, the highest number of individuals are found (up to 600 at the last census carried out by the Garajonay National Park, A. Ferna´ndez pers. comm.), dispersed in more than a dozen localities in and out of the National Park area, separated by less than 10 km. However, fewer than ten plants located here are considered as naturally occurring, while the rest
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are the result of a reintroduction programme developed by the National Park in the last 20 years (Marrero-Go´mez et al. 1998; Ban˜ares et al. 2003). Most of these reintroduced individuals are clones obtained by the asexual reproduction of some of those individuals thought to be of natural origin (A. Ferna´ndez comm. pers.). Finally, in Gran Canaria only two naturally occurring individuals have been found, in the locality of Valleseco (Beltra´n et al. 1999). The monitoring of population dynamics, population reinforcements, and the maintenance of germplam in seed banks constitute the main conservation strategies currently adopted by the Garajonay National Park (La Gomera), aimed at preventing potential population decline and at reducing the risk of extinction of S. palmensis. Werner et al. (2007) found no genetic differences among 40 samples analysed from La Gomera and Tenerife, using ISSR, trnL intron sequences, and trnL-trnF intergenic spacer, which was interpreted as a consequence of clonal reproduction of the species. Aside from this study, the availability of genetic data for the species is null. An increasing number of studies have demonstrated the value of genetic data in addressing issues of plant conservation biology, especially in identifying populations where genetic issues are likely to affect their prospects of long-term survival, in reintroduction biology, or in resolving taxonomic uncertainties (Frankham et al. 2002; Ouborg et al. 2006; Desalle and Amato 2004; Segelbacher et al. 2010). Understanding the level and apportionment of genetic diversity within and among populations is especially important for the conservation of island endemics because being island plants may make them even more susceptible to extinction (Frankham 1998; Prohens et al. 2007). In this sense, there are growing conservation genetics studies on Canary endemic flora (Bouza et al. 2002; Batista et al. 2004; Kim et al. 2005; Gonza´lez-Pe´rez et al. 2004a, 2008, 2009a; Oliva-Tejera et al. 2006; MoraVicente et al. 2009; Suarez-Garcı´a et al. 2009). Also, neutral hypervariable markers are useful in estimating the relative evolutionary importance of genetic factors such as mutation rates, gene flow, and genetic drift (Gonza´lezPe´rez et al. 2004b, 2009b, 2009c; Segarra-Moragues et al. 2005; Van Geert et al. 2008). The main goals of this study are: (i) to assess the existing levels of genetic variability in the species using highly polymorphic markers (microsatellites); (ii) to analyse the distribution of this genetic diversity among the different populations; (iii) to infer potential evolutionary processes that could have led to present genetic differentiation among the islands; and (iv) to use this molecular information as a tool for assessing the current conservation management plan for this endangered species and for designing conservation strategies.
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Materials
Extraction, amplification and separation of DNA
Populations and sampling
DNA was extracted from silica-gel-dried young leaves using a modified 29 CTAB protocol (Doyle and Doyle 1987). 150 ll of each total DNA sample were purified using QIAquick PCR purification Kit (QIAGEN). Forward and reverse primers of eleven microsatellite loci described by Clarke and Tobutt (2006) for Sambucus nigra were tested for PCR amplification. PCR amplifications were carried out following the protocols in the aforementioned publication. Each 25 ll PCR reaction contained approximately 20 ng of DNA, 10 pmol of each primer, and PCR Master Mix (Reddy-Mix, ABgene, Surrey, UK) which included 0.625 units of Taq DNA polymerase, 75 mM Tris– HCl, 20 mM (NH4)2SO4, 0.01% Tween20, 1.5 mM of MgCl2, and 0.2 mM of each dNTP. Amplifications were carried out using the following thermal cycling conditions: 1.5 min denaturation at 94°C; 10 cycles of 30 s denaturation at 94°C; 45 s annealing at 55°C, and 1 min elongation at 72°C, followed by 25 cycles of 30 s denaturation at 94°C;
In order to estimate consistently the levels of genetic variation, the sampling design included all the known areas of population distribution in an attempt to sample all known naturally occurring individuals. A total of 40 individuals were sampled in Tenerife: 26 from El Pijaral, in the north east of the island where the species grows in a very limited area, with the remainder comprising 14 individuals distributed across the whole northern area of Tenerife (Fig. 1). Samples from 61 individuals were collected from La Gomera. They included both naturally occurring individuals and those specimens reintroduced by the Garajonay National Park. In La Palma, the only two populations were both sampled: Los Tiles (48) and Garafı´a (16). Finally, the two naturally occurring plants in Gran Canaria were analysed, but due to technical difficulties, related to the amplification process, the results were not included in the analyses.
Fig. 1 Distribution map of the populations of Sambucus palmensis sampled in the Canary Islands. Dots represent single populations, whereas greyscales represent the distribution of the individuals from the northern area of Tenerife and La Gomera
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45 s annealing at 50°C, and 1 min elongation at 72°C, and finally 5 min elongation at 72°C. The products were detected using an ABI 3130XL Genetic Analyzer and fragment sizes were determined using GENESCAN v. 2.02 and GENOTYPER v. 1.1 (Applied Biosystems, Inc.). We identified allele peak profiles at each locus and assigned a genotype to each individual. Data analysis Genetic diversity data analysis The alleles data matrix was entered into TRANSFORMER 3b.01 software (Caujape´-Castells and Baccarani-Rosas 2005) which allowed this data to be exported to different programs. Standard measures of genetic diversity calculated for this study included proportion of polymorphic loci (P), total number of alleles (NA), mean number of alleles per locus (A), observed heterozygosity (Ho), average expected heterozygosity (He). All these, plus allelic frequencies, were calculated for all populations using POPGENE vers. 1.31 (Yeh et al. 1997). Genetic structure data analysis To test genetic drift events in the natural populations, a bottleneck test was carried out using BOTTLENECK software (Piry et al. 1999), under the Infinite Allele Model (IAM), the Stepwise Mutation Model (SSM), and an intermediate Two-Phased Model (TPM). Of the three statistical methods used by the BOTTLENECK software, the Standardized differences test was not employed, because it requires at least 20 polymorphic loci to be reliable. In accordance with the hierarchical sampling design, the allelic frequencies data matrix was analyzed using a nested analysis of variance (AMOVA, Excoffier et al. 1992) to estimate the components of variance among and within islands and populations. AMOVA was applied to assess the partitioning of microsatellite variation in populations of Sambucus palmensis, and to test significance against the null hypothesis of no structure, using ARLEQUIN software (Schneider et al. 2000). Population structure was also inferred using a Bayesian clustering procedure (implemented in STRUCTURE; Pritchard et al. 2000) to identify the K (unknown) populations of origin of the sampled individuals and to assign the individuals simultaneously to the populations. The most likely value of K was assessed by comparing the likelihood of the data for different values of K. Populations and individuals were assigned to one cluster if their proportion of membership (qi) to that cluster was equal to or larger than 0.05. We assumed the model to be of population admixture and that the allele frequencies are independent.
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We conducted a series of independent runs for each value of K between 1 and 10. Choosing a value of K that maximizes the posterior probability of the data (PPD) can be difficult to apply for complex data sets including many groups (Rosenberg et al. 2002). In the case of highly structured data, as K is increased, the most divergent groups separate first into distinct clusters. Since the aim should be to find the smallest value of K that captures the major structure in the data, a second way to choose K is to consider the successive increase of the PPD for increasing values of K, which can be regarded as the increase in information at each addition of a set of allele frequencies (Evano et al. 2005). Due to the geographic spread of the population from the north of Tenerife (Tenerife North), a Bayesian cluster analysis was also carried out, only considering those individuals sampled from this area, in order to test the suitability of grouping them in a single population. Genetic differentiation data analysis In order to evaluate genetic differentiation among populations, pair-wise divergences among populations were analyzed using the FST approach (Weir and Cockerham 1984) using FSTAT version 1.2 (Goudet 1995). Because the magnitude of FST may depend on the variability of a locus, we also calculated a standardized measure of differentiation, G0 ST, which was developed by Hedrick (2005) to facilitate comparisons between loci that differ in variability. Standardized genetic differentiation measure (G0 ST) was reanalysed using RECODEDATA version 0.1 (Meirmans 2006) and calculated with FSTAT version 1.2 (Goudet 1995). In addition, the correlation between both genetic differentiation measures was estimated using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA). To visualize differences among populations, a Principal Coordinates Analysis (PCoA) based on pairwise Nei’s (1978) unbiased genetic distances among populations was performed using the program GENEALEX 6.1 (Peakall and Smouse 2006). Also, number and frequency of multilocus genotypes was determined by GENEALEX 6.1 (Peakall and Smouse 2006).
Results For the eleven microsatellites identified for Sambucus nigra (Clarke and Tobutt 2006), five produced reproducible, and scorable polymorphic peaks: EMSn003, EMSn 016, EMSn017, EMSn021 and EMSn023, and were used in this research (Table 1). However, the Wilcoxon test under IAM model showed a significant heterozygotes excess (P \ 0.05), evidencing bottleneck signatures for Los Tiles (La Palma) and Pijaral (Tenerife) populations.
Conserv Genet (2010) 11:2357–2368 Table 1 Allelic frequencies at five polymorphic loci for Sambucus palmensis
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Island/population Locus EMSn003
La Palma Allele
Los Tiles
A
0.54
Tenerife Garafı´a
Pijaral
North of Tenerife 0.08
0.31
0.33
B
0.06
0.04
C
0.06*
D
0.08*
E
0.04
F 0.03 0.46
J
0.10 0.04
0.25
0.12
0.50
0.02
0.12
0.31
0.04
0.10*
M
0.06
A
0.02
0.28
B
0.02
0.28
0.96
0.38
0.60
E
0.03
0.37
F
0.03*
C
0.11*
B C
0.39
0.14* 0.54 0.04
0.02
0.56
0.11
0.22
0.02
0.07
0.49
0.50
D
0.03
E
0.03 0.51
0.44
0.76
0.04*
A
0.47
0.53
0.50
0.54
0.50
B
0.47
0.47
0.50
0.46
0.50
C
0.06* 1.000
1.000
A B
0.11* 0.52
C Asterisks signal the distribution of population-exclusive alleles
1.000
0.21*
G
EMSn023
0.79
A
F EMSn021
0.08
0.04*
G EMSn017
0.39
0.08*
L
D
0.26
0.15 0.23
0.03
K
EMSn016
Gomera
0.12*
G H I
La Gomera
D
0.89
0.98 0.02*
0.48*
For the five polymorphic loci surveyed, a total of 34 different alleles (from 3 to 13 per locus) were observed (Table 1). A total of 15 alleles were unique to some of the populations (private alleles): six private alleles were present in Tenerife North and three in Pijaral (both populations in Tenerife); two alleles were private to the Garafı´a population, and two different ones private to Los Tiles (both populations in La Palma); finally, two other alleles were unique to La Gomera. Most of these private alleles presented low allelic frequencies (ranging from 0.02 to 0.21). However, it is important to mention the high allelic frequencies (0.48) of allele D (locus EMSn023)
from the Los Tiles population (La Palma), because it means that nearly half the individuals from this locality hold this allele, which appears only in this population (Table 1). The Tenerife North population showed, on the whole, the highest levels of genetic variation, in spite of this population being the smallest (Table 2). Genetic diversity values for this population were P = 100%, He = 0.53, A = 4.2. Twenty-one different alleles were detected in this population. In contrast, La Gomera population showed the lowest levels of genetic diversity, with only 12 alleles (P = 80%, He = 0.32, A = 2.4; Table 2).
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genotypes were found among individuals of different populations. In this sense, all individuals from the Pijaral population were genetically different from the Tenerife North population (Table 3, Fig. 2a), although both populations were located on the same island. Only in La Palma, a genotype was shared between more than one population: three individuals from Garafı´a were completely identical (for the 5 loci analysed) to 14 individuals from Los Tiles. The partitioning of genetic variance within Sambucus palmensis calculated through AMOVA (Table 4) attributed 77% of the variance to differences within populations, while 15% was attributable to variability among populations within islands. Only 8% of genetic diversity was found among islands. These results are in agreement with Principal Coordinates Analyses (Fig. 3), showing the genetic differences detected among populations located in different islands (Los Tiles and Garafı´a, in La Palma; Pijaral and Tenerife North in Tenerife). La Gomera population shows a close genetic relationship with the Tenerife North population (Fig. 3). It is important to note that the first three coordinates of the PCoA explain almost 92% of the detected variation. The standardized genetic differentiation measure (G0 ST) (Hedrick 2005), showed the same results as FST. Strong and significant correlations were found between both genetic differentiation measures (r = 0.96; P \ 0.01).Values of FST are significant among populations, especially among the populations located in La Palma and Tenerife, and in accordance with Principal Coordinates Analyses, La Gomera population is closely related to the Tenerife populations (FST = 0.062 between Gomera-Tenerife North and FST = 0.087 between Gomera-Pijaral) (Table 5). For the Bayesian analysis, using those individuals located in the north area of Tenerife (14 individuals, five microsatellite loci) and K = 1–10, the probability of the data was maximum, with K = 1, suggesting that the individuals analyzed can be grouped into a single genetic
Table 2 Mean genetic diversity indices obtained for five microsatellite loci in Sambucus palmensis in the Canary Islands Island
Population
La Palma
Los Tiles Garafı´a
16
Total La Palma
64
Tenerife
N 48
NA
A
Ho
He
P
12
2.4
0.62
0.43
100
18
3.6
0.60
0.50
80
20
4.0
0.61
0.48
100
Pijaral
26
19
3.8
0.65
0.47
80
North of Tenerife
14
21
4.2
0.45
0.53
100
Total Tenerife
40
26
5.2
0.58
0.52
100
61 165
12 34
2.4 6.8
0.45 0.55
0.32 0.50
80 100
La Gomera Gomera Total Sambucus palmensis
N Number of analysed individuals, NA total number of alleles, A average number of alleles per locus, HO observed heterozygosity, He expected heterozygosity, P proportion of polymorphic loci at 95%
These differences in the levels of genetic variability were also used to analyse the number of multilocus genotypes. All 165 individuals analysed were grouped in a total of 58 multilocus genotypes (Table 3). Of these, 129 individuals shared 22 genotypes and 36 samples showed a genotype combination which was not found in any other individual, and were therefore named as unique or single genotypes. The distribution of these genotypes was heterogeneous. In La Gomera 12 different genotypes were found and a substantial number of individuals analysed (22 of 61) showed the same set of multilocus genotypes (Genotype L; Fig. 2c). In La Palma, with 64 individuals analysed, 22 different genotypes were detected, seven of which were detected in more than one individual, and 15 genotypes were unique (Table 3, Fig. 2b). Finally, in Tenerife the highest number of different genotypes was found. Almost every individual from this island constitutes a genetically different specimen (Fig. 2a). Individuals located in different islands always presented different multilocus genotypes. So, the allelic combinations were exclusive to each island (Fig. 2a–c). In fact, no shared
Table 3 Number of genotypes multiloci detected by population Island
Population
La Palma
Los Tiles Garafı´a
48
5
7
12
58
16
3
8
11
73
Total La Palma
64
7
15
22
68
Pijaral
26
8
5
13
38
North of Tenerife
14
1
11
12
92
Total Tenerife
40
9
17
26
65
Gomera
61
7
5
12
42
165
22
36
58
62
Tenerife
La Gomera
Total Sambucus palmensis
N
N8 shared genotypes
N8 unique genotypes
Total N8 genotypes
% unique genotypes
Number of shared genotypes is referred to those genotypes shared by two individuals at least. Number of unique genotypes is referred to multiloci genotypes appeared in only one individual
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Fig. 3 Principal Coordinates Analysis based on pairwise Nei’s (1978) unbiased genetic distances among five Sambucus palmensis populations considered
Table 5 Genetic differentiation coefficient (FST) values (below the diagonal), and standardized gene differentiation G0 ST (Hedrick 2005) (above the diagonal) for all pairwise combinations of Sambucus palmensis populations La Palma Los Tiles Los Tiles Garafı´a
Fig. 2 Multilocus genotypes distribution within Sambucus palmensis populations. Each multilocus genotype based on variability of five microsatellites was named with a different letter. Note that each island contains different multilocus genotypes. Unique genotypes are referred to as those allelic combinations recorded in only one individual
Table 4 Values of AMOVA partitioning of microsatellite variation at the three hierarchical levels considered from Sambucus palmensis Source of variation
df
Percentage of variation
Fixation indices
Among islands
2
8%
FCT = 0.084ns
Among populations within island
2
15%
FSC = 0.159***
Within populations
325
77%
Total
329
ns not significant *** P \ 0.001
FST = 0.229***
Garafı´a 0.254
0.117
Tenerife Pijaral
La Gomera North Tenerife
Gomera
0.400
0.280
0.391
0.276
0.204
0.346
Pijaral
0.168
0.100
North Tenerife
0.108
0.074
0.066
0.168
Gomera
0.149
0.122
0.087
0.204 0.129
0.062
cluster. On the other hand, considering the total data set (165 individuals, five microsatellite loci, and five localities), and K = 1–10, the probability of the data was maximum with K = 9, suggesting the presence of additional levels of structure in the total sample. The increase of the PPD was high for K = 2, but for K [ 2 the increase in information became markedly less and showed gradually decreasing values. This result means that the information obtained by the third clusters (and the subsequent clusters) was much less important than the information obtained by the first two. Once two populations had been assigned to different clusters for K = 2, they never belonged to the same cluster for greater values of K. When two clusters (K = 2) were assumed, the individuals were assigned asymmetrically to each group. Predefined populations assigned to the first group included all the individuals from the Los Tiles (qI = 0.984) populations, and most individuals from the Garafı´a populations (qI = 0.820,
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qII = 0.180), both from La Palma. On the other hand, all individuals from el Pijaral (Tenerife) and La Gomera showed a higher proportion of mean individual membership of cluster II (qII = 0.965, qII = 0.988, respectively). Finally, Tenerife North showed a higher proportion of mean individual membership of cluster II (qI = 0.275, qII = 0.725).
Discussion Given their small population sizes and endemic character, as well as their very restricted geographic distribution (restricted to only four islands, and often with less than 10 individuals per population), we expected Sambucus palmensis populations to be genetically impoverished (Hamrick and Godt 1989; Ouborg et al. 2006). Very few studies have been performed with microsatellite markers in Canarian flora, and therefore it is not feasible to compare the genetic results of S. palmensis to other Canarian species. In spite of this, genetic diversity values detected in natural populations of S. palmensis (He = 0.50) were, in general, similar to those found in other endangered endemic species from the Canary Islands. Gonza´lez-Pe´rez et al. (2009c) recorded a He value of 0.56 for the endemic and endangered Myrica rivas-martinezii, using microsatellites. In addition, the same authors detected similar values of expected heterozygosity for the endemic endangered and tetraploid species Bencomia exstipulata, using the same markers (He = 0.44, Gonza´lez-Pe´rez et al. 2009b). However, the genetic diversity detected in S. palmensis is higher than that described for other endangered non-native species of the Canaries found in the literature (e.g. Borderea chouardii; He = 0.17, Segarra-Moragues et al. 2005), as well as for other endemic species (e.g. Dubautia arborea; He = 0.22, and D. ciliolata; He = 0.31; Friar et al. 2007). Reproductive systems and the history of a species have often been regarded as the main factors affecting levels of genetic diversity, genetic divergence, and genetic structure within and between plant populations (Loveless and Hamrick 1984; Hamrick and Godt 1989, 1996; SegarraMoragues et al. 2005; Gonza´lez-Pe´rez et al. 2008). In this sense, outcrossing perennials generally exhibit higher levels of genetic variability and lower levels of population differentiation, indicating the influence of the species traits on these parameters (Hamrick and Godt 1989, 1996). So, according to Hamrick and Godt (1989), reproductive biology is the most important factor in determining the genetic structure of plant populations. A previous study (Marrero-Go´mez et al. 1998) indicates that S. palmensis appears to display an autoincompatibility mechanism. In fact, one of the most important factors in explaining such a
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scarce number of individuals in the natural populations is the low production of viable seeds (E. Carque´, pers. comm.). Some preliminary results show that there is a tendency for fruit abortion. In a field experience realised in 1999 (Gonza´lez-Martı´n M, pers. comm.), only 7.1% of fruits produced seeds, suggesting any autoincompatibiliy system. In accordance with this, AMOVA analysis showed a percentage variation within populations of 77%, similar to those expected for outcrossing species (Hamrick and Godt 1989). On the other hand, the high level of diversity in species with a small number of populations and few individuals within each population may be an indication that the number of individuals of these species was larger in the recent past (Ellstrand and Elam 1993; Crawford et al. 2001; Prohens et al. 2007). In this sense, the Wilcoxon test under IAM model evidences significant bottleneck signatures (P \ 0.05) for the Los Tiles and Pijaral populations (data not shown). In accordance with this, both populations showed low genetic diversity in regard to population size, as well as a high genetic differentiation between them, suggesting that the populations were larger in the past. However, the precise historical distribution and population sizes of S. palmensis are very difficult to establish due to its rarity in the wild (Ban˜ares et al. 2004). In addition, reintroductions carried out in La Palma and La Gomera could explain part of the genetic pattern detected (genetic diversity, population differentiation, population structure, etc.) in these islands. Since the reinforcement programme carried out on both islands was based on individuals propagated by asexual means, this would explain the low genetic variability levels detected. On the other hand, the transfer of material between Tenerife and La Gomera could explain the genetic relationship between populations located in these islands. However, the number of individuals of this species was probably higher in the past (Beltra´n et al. 1999). As is the case with many other plant species from the Canary Islands, the introduction of rabbits, goats and other herbivores has had a relevant impact on the natural plant populations (Garcı´a-Casanova et al. 2001; Prohens et al. 2007). In addition, S. palmensis has been collected extensively for medicinal purposes during the last century (Beltra´n et al. 1999). However the main causes of the current fragmented and isolated distribution has been the loss of habitat and the destruction of laurel forest. Therefore, the distribution of the species could therefore have been much wider than it is currently, which could explain the high levels of genetic diversity detected in this endemic species. Considering that natural populations are almost completely isolated from each other, outcrossing and gene flow should be very low or zero between populations. According
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to Hamrick and Godt (1989), outcrossing plant species tend to display between 10% and 20% of genetic variation between populations. FST values for Sambucus palmensis populations are in this range (FST average = 0.193). At present, it is feasible that gene flow may have been interrupted by the shrinking area occupied by the populations and by the disappearance of populations that acted as a bridge between those in distant locations. Therefore, a decrease in population size or even the extinction of some populations that once served as a connection between extant populations could also be contributing to the FST values detected in S. palmensis. The role played by time and ecological stability must have been to amplify the action of drift and mutation in the genetic differentiation of Sambucus palmensis. In this sense, genetic drift may be a important factor acting on the species, since percentages among populations within islands (15%) were even higher than genetic variance among islands. Drift is one of the paramount factors explaining genetic heterogeneity in oceanic islands (Crawford et al. 1987; Witter and Carr 1988; Bouza et al. 2002; Sa´nchez-Doreste et al. 2006; Gonza´lez-Pe´rez et al. 2009a, b) and, in the face of the hypothesized of a long diversification time, mutation enhanced inter-island genetic differentiation. Thus a longisolated population could accumulate private alleles, reflecting their genetic differences due to isolation by distance (Prentice et al. 2003; Segarra-Moragues et al. 2005). This seems to be the case for S. palmensis populations, especially for those from Tenerife and La Palma which have private alleles and unique multilocus genotypes (Fig. 2). The number of island-specific alleles (19) represents more than half of the total alleles detected (34), indicating the importance of isolation as a factor in genetic differentiation for most present populations. On the other hand, it is feasible that deliberate but undocumented transfer of material among populations, particularly between Tenerife and La Gomera, has occurred. This conjecture would explain the significant relationship among populations located in these islands, especially with Tenerife North. The number of island-specific alleles for La Gomera is very low (only two), and from the total of 12 alleles detected in this island, 10 are located in Tenerife also. Moreover, the reinforcement programme carried out in La Gomera and at Los Tiles, in La Palma, which has been based on individuals propagated by asexual means, would explain the low genetic variability levels and the high number of identical multilocus genotypes observed in these two populations (Table 3). In fact, all multilocus genotypes observed in more than 10 individuals (named D, E, F, L and T in Fig. 2) belong to these populations, which reflects the fact that the reintroductions are clonal in origin, therefore decreasing the genetic variability levels in them.
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Implications for conservation Meta-analyses of the correlation between molecular and quantitative measures of genetic variation (Ouborg et al. 2006) conclude that molecular measures alone do not accurately reflect the evolutionary potential of populations. However, the documentation of genetic diversity and differentiation between populations using molecular markers provides initial guidance for conservation and can contribute to setting conservation priorities between populations (Neel and Ellstrand 2003; Desalle and Amato 2004; Tallmon et al. 2004; Segelbacher et al. 2010). Microsatellites and other molecular marker analysis has provided useful information for conservation purposes for other endangered Canarian species such as Bencomia exstipulata (Gonza´lez-Pe´rez et al. 2004a, 2009b), Myrica rivas-martinezii (Batista et al. 2004; Gonza´lez-Pe´rez et al. 2009c), Anagyris latifolia (Gonza´lez-Pe´rez et al. 2009a), Gnaphalium teydeum (Gonza´lez-Pe´rez et al. 2008), Sideritis discolor (Batista et al. 2004) and Dorycnium spectabile and Isoplexis chalcantha (Bouza et al. 2002). These data can provide valuable information for conservation biologists because they allow for the estimation of population genetic differentiation between and within populations and support the design of sampling strategies for ex situ collections (Francisco-Ortega et al. 2000; Sosa 2001; Frankham et al. 2002; Ouborg et al. 2006; Prohens et al. 2007). In this sense, and after applied molecular analysis in Bencomia exstipulata (Gonza´lez-Pe´rez et al. 2009b), reinforcement of the populations have been carried out following the maternal genotypes in order to maintain or increase the genetic diversity of the new populations. From the conservation point of view, initially each island population should be conserved separately because their populations are genetically differentiated, in order to avoid breaking eventual coadapted gene complexes (outbreeding depression; Storfer 1999; Hedrick and Kalinowski 2000). The presence of island-specific alleles and multilocus genotypes may have resulted from the separate evolution of the plants of different islands caused by the restriction of gene flow between islands. In addition, Bayesian cluster analyses identify two genetic pools, one corresponding to those individuals from La Palma, while individuals from Tenerife and La Gomera were clustered in the second population inferred. Therefore, we recommend a different management strategy for each population and each island, especially for the La Palma populations. Genetic diversity within populations is considered of great importance for possible adaptations to environmental changes and, consequently, for the long-term survival of a species (Frankel and Soule´ 1981; Simberloff 1988; Barrett and Kohn 1991; Sosa et al. 2002). However, in La Gomera, where only a few naturally occurring individuals have been
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found, and which exhibited the lowest genetic diversity detected in the species, population reinforcement by artificially increasing the number of individuals from the natural population of the less genetically differentiated population of Tenerife North, might be justified. However, given that populations have different multilocus genotypes, conservation measures are aimed at preserving the diversity not only of alleles, but also the characteristic genetic composition of specific populations (Francisco-Ortega et al. 2000; Prohens et al. 2007), and the transfer of individuals from one island to another should be carried out with caution. To date, programmes to transplant rare Canarian endemics are limited, and some authors (Francisco-Ortega et al. 2000; Ban˜ares et al. 2001; Prohens et al. 2007) consider that reinforcement should be carried out with individuals from the same population conserved in seed banks. The authors argue that re-establishment via this method avoids introducing individuals from other populations, which could result in a change in the fitness and a reduction in local variation (Prohens et al. 2007). In the specific case of Sambucus palmensis, this could be true for the Tenerife and La Palma populations, which hold high genetic variability levels, and whose populations are considerably genetically differentiated. For these particular cases, it would be essential to increase the number of individuals through the use of seed banks, crossing individuals with rare alleles. On the other hand, in La Gomera, it is necessary to increase genetic variability through the propagation of new individuals by sexual means (outcrossing programme). The levels of genetic differentiation observed among populations, and the genetic diversity distribution within populations in S. palmensis, indicate that management should aim to conserve as many of the small populations as possible, because concentrating conservation efforts only on the few large populations would very likely result in substantial loss of genetic variability for the species. Management should aim to increase the size of small populations to minimize further loss of genetic variation. We know that genetic, demographic and reproductive studies complement each other in regard to the conservation of species and populations (Sosa 2001; Sosa et al. 2002). From the point of view of conservation genetics, it would be advisable to promote sexual reproduction among individuals with different genotypes, and therefore to establish an outcrossing programme. S. palmensis has been described as allogamous, and it reproduces both by seed and vegetatively (Marrero-Go´mez et al. 1998; Ban˜ares et al. 2004). However, one of the principal difficulties is that the germination rate is generally very low, and a low number of seedlings have been observed in natural populations.
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Finally, although acting on the above proposals is likely to increase these populations’ chances of survival, we must bear in mind that these indications are solely based on an assessment of genetic variation through only five polymorphic microsatellite loci. We should consider the probability that there is genetic diversity and differentiation that could not be recorded. Therefore, genetic differentiation among the populations studied could be higher than those reported in this research. However, conservation targets and priorities which depend solely on markers must be applied cautiously, and should be interpreted as a low estimate of what needs to be conserved. It is therefore necessary to investigate other important factors in the species’ Biology which may be crucial for the long-term survival of the populations. The data presented here provide guidance about which populations may be valuable from a genetic perspective and could also serve as a valuable baseline for monitoring the effectiveness of establishing protected areas, and restoring and maintaining genetic diversity. In this sense, the Canarian Government is realising a recovery plan considering the genetic analysis carried out. While in Tenerife, seeds have been collected from all mother plants. In La Gomera, collection has focused on those individuals with unique genotypes. Acknowledgments The authors would like to thank Angel Ferna´ndez (Garajonay National Park), Vicente Garcı´a Lopez, Julio Leal Pe´rez (Cabildo de La Palma) and Marcos Salas for their assistance in collecting samples. We also thank Elizabeth Ojeda (Viceconsejerı´a de Medio Ambiente, Gobierno de Canarias), Angel Palomares (Taburiente National Park), Manuel Marrero, Eduardo Carque´ and Angel Ban˜ares (Teide National Park) for their comments and observations. Javier Sosa and Andrew Stephens corrected the English. This research was supported by the Ministerio de Educacio´n y Ciencia, Direccio´n General de Investigacio´n (CGL2004-03839) of the Spanish Government.
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