AOBPreview originally published online on January 5, 2004
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Annals of Botany 93: 201-209, 2004
© 2004 Annals of Botany Company
Population Genetic Structure of Titanotrichum oldhamii (Gesneriaceae), a Subtropical Bulbiliferous Plant with Mixed Sexual and Asexual Reproduction
1 Royal Botanic Garden, 20A Inverleith Row, Edinburgh EH3 5LR, UK, 2 Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JH, UK and 3 Botanical Garden and Centre for Plant Research, University of British Columbia, Vancouver, Canada V6T 2TL
* For correspondence. Fax +1-604-822-2016, e-mail botwang{at}ntu.edu.tw
Received: 7 April 2003;; Returned for revision: 29 August 2003; Accepted: 4 November 2003 Published electronically: 5 January 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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• Background and Aims Titanotrichum oldhamii is a monotypic genus distributed in Taiwan, adjacent regions of China and the Ryukyu Isands of Japan. Its conservation status is vulnerable as most populations are small and widely scattered. Titanotrichum has a mixed system of reproduction with vegetative bulbils and seeds. The aim of this study was to understand the population genetic structure of Titanotrichum in relation to its specific reproductive behaviour and to determine possible implications for conservation strategies.
• Methods After an extensive inventory of most wild populations of Titanotrichum in East Asia, samples from 25 populations within its major distribution were carried out utilizing RAPD and inter-SSR molecular fingerprinting analysis.
• Key Results The findings support the conclusion that many populations reproduce predominantly asexually but that some genetic variation still exists within populations. However, significant amounts of variation exist between populations, perhaps reflecting population differentiation by drift. This partitioning of genetic diversity indicates that the level of inter-population gene exchange is extremely low. These findings are consistent with field observations of very limited seed production. The Chinese populations are similar to those of Northern Taiwan, while the Ryukyu populations fall within the range of variation of the north-central Taiwan populations. The Taiwanese populations are relatively variable and differentiation between north, east and south Taiwan is evident.
• Conclusions The distribution of Titanotrichum seems to be consistent with a former land connection between China, Taiwan and the Ryukyu Islands at a glacial maximum during the Quaternary, followed by progressive fragmentation of the populations. North-central Taiwan is the centre of genetic diversity, possibly due to the proximity of the former land bridge between the regions, together with the variety of suitable habitats in north Taiwan. The significance of these findings for conservation is discussed.
Key words: Facultative vegetative apomixis, sea-level change, Pleistocene biogeography, bulbil, vivipary, propagules, clonal plant.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Many plant species can reproduce clonally by creeping roots or stems, propagules such as bulbils and tubers, or agamous seeds. Bulbiliferous species have developed specialized vegetative organs for reproduction and the genetic and ecological significance of bulbiliferous reproduction is of evolutionary interest.
Bulbiliferous plants usually continue to produce flowers, thus retaining the potential for sexual reproduction (Arizaga and Ezcurra, 1995). Under extreme environmental conditions, as in Arctic or montane regions, clonal reproduction by pseudo-vivipary (sensu Kerner) may become dominant as suitable conditions for sexual reproduction are limited (Kerner, 1904; Klime
et al., 1997). Polygonum viviparum has been observed to initiate more bulbils in the lower part of the inflorescence when growing in Arctic-alpine regions as compared with warmer regions (Diggle, 1997). In other Arctic clonal plants, such as Saxifraga cernua and Poa alpina, seed set has only very rarely been observed (Briggs and Walters, 1997; Gabrielsen and Brochmann, 1998).
In tropical and subtropical plants, bulbils are fairly unusual (with a few exceptions such as Remusatia vivipara and Globba spp.), and bulbiliferous reproduction is seldom documented in tropical environments where pseudo-vivipary is not a reaction to extreme conditions (Moody et al., 1999). Clonal plants in the tropics, with certain notable exceptions such as Eichhornia paniculata (Glover and Barrett, 1987) and Aechmea magdalenae (Murawski and Hamrick, 1990), have rarely been investigated for genetic diversity and population structure.
Titanotrichum oldhamii has evolved a mixed reproductive strategy of bulbils, rhizomes and seeds in its habitat of temperate to subtropical monsoon rain forest. The rhizomes are important for winter survival. It produces showy, tubular flowers in summer and is able to set seed if pollinators are available (Wang, 2003). The family Gesneriaceae, to which Titanotrichum oldhamii belongs, exhibits a wide range of morphological variation (Jong and Burtt, 1975; Möller and Cronk, 2001). Variable meristem behaviour is well known in vegetative parts of the Gesneriaceae (Burtt, 1970; Tsukaya, 1997; Imaichi et al., 2000), but variable floral meristem behaviour is unusual. However, in late season, inflorescence-borne bulbils replace all the floral meristems at the top of the racemes of Titanotrichum, forming hundreds of bulbil clusters for vegetative reproduction (Wang and Cronk, 2003).
In Titanotrichum, the division between bulbil propagation and sexual reproduction appears to vary between populations, possibly due to environmental factors. Some populations set more seeds than others, suggesting that the availability of pollinators between populations may differ (Wang, 2003). In general, however, seed set in Titanotrichum is rare, as can be observed in the wild and from herbarium material. This suggests that bulbil propagation in Titanotrichum is the major mode of reproduction and spread in the wild. However, the plant still produces functional flowers, and pollination experiments in Titanotrichum have shown that artificial pollination can readily produce seeds (Wang, 2003).
Titanotrichum oldhamii grows in shady habitats along creeks, particularly on wet, dripping cliffs. This favours water-mediated dispersal by fragments of rhizomes, bulbils and, rarely, seeds or leaf pieces (which can root and produce plantlets; C.-N. Wang, pers. obs.). From field and glasshouse observations, it is evident that it can regenerate from its tiny bulbils into 10 cm high plants in 2 months. However, Titanotrichum is not common in the wild and continues to decline (Walker, 1976; Wang et al., 1998). It has a scattered distribution in Taiwan, adjacent areas of China and the south Ryukyu Islands of Japan. Populations are often small and isolated and population bottlenecks are likely to occur. In small populations (N < 100), genetic drift and inbreeding may cause a loss of genetic diversity (Ellstrand and Elam, 1993). Moreover, insufficient gene flow between and within populations due to fragmentation may affect its fitness to adapt to current and future environmental changes.
In this study, both inter-SSR and RAPD markers have been used to explore the genetic structure of Titanotrichum, as these markers require no prior genomic information. The ready availability of data and the ease of use make them highly suitable for surveying endangered species of clonal reproduction (e.g. Stiller and Denton, 1995; Hollingsworth and Bailey, 2000). Inter-SSR markers require high PCR annealing temperatures and consequently have good reproducibility. Although the assumptions of RAPD analysis are slightly different from those of inter-SSR analysis, recent studies suggest results from these two data types are consistent with each other (Hollingsworth et al., 1998).
The primary aim of this study was to investigate clonal diversity at different levels in Titanotrichum oldhamii (within and among populations), and to link these patterns to geographical distribution. A detailed field survey and assessment of genetic diversity was therefore carried out using RAPDs and inter-SSRs to underpin its future conservation. The results presented here may also be suggestive of general patterns and processes in the history and origin of the Taiwan flora.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Field survey
As no detailed field survey of Titanotrichum had previously been carried out, information from herbarium specimen sheets, regional floras and local botanists was assembled prior to two extensive field trips. During the summer of 1999 and 2000, 25 natural populations of Titanotrichum oldhamii (Hemsl.) Soler in Taiwan, China and the Ryukyu Islands of Japan were located and investigated. In the field, the phenology of each population, population size, associated species, vegetation type and possible pollinators were recorded and identified. The geographical location of each population is summarized in Table 1 and Fig. 1.
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Collection of plant material and genomic DNA extraction
To assess the genetic diversity of Titanotrichum, DNA material for RAPDs and inter-SSRs were collected during the field survey. Leaf samples from up to 20 randomly chosen individuals per population were collected, but in southern Taiwan and the Ryukyu Islands only between five and ten individuals were sampled, reflecting the small size of these populations. For each individual, two medium-sized healthy leaves were chosen and dried rapidly in a sealed plastic bag using a 20 times greater volume or more of silica gel. The leaf tissue was then stored at ambient temperature until DNA extraction. In addition, living plants, as well as bulbils and seeds, were brought back to the Royal Botanic Garden Edinburgh for pollination experiments and progeny analysis. Voucher specimens were deposited in the herbarium of the Royal Botanic Garden Edinburgh (E).
Genomic DNA was extracted using a modified CTAB-based protocol (Doyle and Doyle, 1987). Isolated DNA was quantified by electrophoresis on a 1 % agarose gel along with a 1-kb DNA molecular weight marker (NEB Biolabs, Herts, UK) as a standard. The DNA concentration was adjusted to 1 ng µl–1 for all samples. This quantification is essential as different amounts of starting template may cause template competition in PCR amplification of RAPD and inter-SSR analysis (Halldén et al., 1996).
Primer selection and PCR conditions
One hundred 10-mer RAPD primers (Operon Technologies, Surrey, UK) and 30 inter-SSR primers (NAPS Unit, University of British Columbia, Canada) were screened for performance and allelic polymorphism among populations. Only eight RAPD primers and three inter-SSR primers showed the required polymorphism, suggesting low levels of genetic diversity within Titanotrichum oldhamii. The primer sequences are listed in Table 2.
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The PCR conditions for RAPD and inter-SSR followed Hollingsworth et al. (1998). For RAPD, 5–10 ng template DNA was incorporated in 15 µl reactions, containing 0·5 µM primer, 100 µM each dNTPs (Roche, Indianapolis, USA), 2·5 mM MgCl2, and 0·5 U Taq polymerase (Bioline, London, UK) and 1x Taq buffer (16 mM (NH4)2SO4, 67 mM Tris–HCl (pH 8·8), 0·01 % Tween20). The PCR amplification profile was as follows: 2 min at 95 °C, two cycles of 30 s at 95 °C, 1 min at 37 °C, 2 min at 72 °C, and two cycles of 30 s at 95 °C, 1 min at 35 °C, 2 min at 72 °C, followed by 41 cycles of 30 s at 94 °C, 1 min at 35 °C, 2 min at 72 °C, then a final extension at 72 °C for 5 min. For inter-SSR analysis, PCR reactions were carried out as above except using 0·2 µM primer. The PCR conditions for inter-SSR were more stringent because the primers are longer: 95 °C for 3 min, then 40 cycles of 20 s at 93 °C, 1 min at 55 °C, 20s at 72 °C, with a final 6 min extension at 72 °C.
All PCR reactions for RAPD and inter-SSR were performed with a Perkin-Elmer GeneAmp 9600 Thermal Cycler or a DNA engine (Peltier Thermal Cycler PTC-100). PCR products were separated on 1·6 % agarose gels in 1x TBE buffer with medium field strength [voltage/gel length (cm) ratio equal to 4, i.e. 20 V on 80 cm gel] and visualized under UV after ethidium bromide staining (0·1 µg ml–1). Negative controls (no template DNA) were also included in each PCR. To ensure RAPD reproducibility, most PCR reactions were repeated for reliability in data scoring.
Data analysis
RAPD and inter-SSR amplified DNA bands were scored conservatively and, to avoid co-migrating band errors only positions of bright bands sharing similar intensity, fragment size above 0·4 kb and exact position were scored for presence/absence (1/0). A pair-wise distance matrix (Dist = 1 – J) based on RAPD and inter-SSR data was generated, using Jaccard’s similarity coefficient: J = Sij/Tij, where S is the total number of shared bands and T is the total number of bands between the ith and jth individuals (Sneath and Sokal, 1973). The computer package R (Legendre and Vaudor, 1991) and the statistical program NT-SYS (Rohlf, 1996) were used to perform a principal coordinate analysis (PCO). The same distance matrix was imported to the program package Arlequin (Schneider et al., 2000) for AMOVA (analysis of molecular variance; Excoffier et al., 1992), to analyse the population structure within and among populations (percentage of variance, Phist). For diversity measurement, two indices were used to estimate genetic diversity within and between populations. These are Shannon’s index (S) and Simpson’s index (D). Shannon’s index of diversity was computed using POPGENE (Yeh et al., 1999) using the formula corrected for binary RAPD and inter-SSR data: S = –
pi log2 pi, where pi is the frequency of presence of each RAPD or inter-SSR band to provide an estimate of degree of variation within each population. Although Shannon’s index provides less genetic information than other diversity indices, it is less biased simply because it does not rely on a Hardy–Weinberg equilibrium (Chalmers et al., 1992). Many recent studies favour this index for comparison across different species (Bussell, 1999; Allnutt et al., 2001). For comparison with other studies on clonal species, the proportion (G/N) of genets (G) in the total population (N) and the unbiased Simpson’s index of diversity (D) were also calculated, using the formula: D = 1 – [ni(ni – 1)]/[N(N – 1)], where ni is the number of individuals with RAPD and inter-SSR genotype in clone i, and N is the sample size. Finally a UPGMA tree connecting 25 populations was constructed to interpret population relationships. To test the possible correlation between geographic distance and genetic distance, a Mantel test provided by the R package was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Field observations
In this survey, 25 natural populations of Titanotrichum oldhamii were investigated (20 from Taiwan, three from China and two from Japan). Usually, Titanotrichum can be found around the headwaters of streams under subtropical broadleaved rain forest dominated by Fagaceae and Lauraceae. It is often associated with other herbaceous plants such as Rhynchotechum and Begonia species but has a far more restricted distribution. Titanotrichum favours habitats such as wet limestone areas where roots are semi-exposed to dripping water. This habitat type has been affected by habitat change and human disturbance, although some populations in Taiwan and Japan grow within the protection of national parks. Most populations of Titanotrichum are small, scattered and isolated from each other. Seed set was only observed in medium to large populations (more than 25 individuals) in open habitats. Pollinators like bumble-bees are more frequent visitors to these populations than to those in dense shade. It was also observed that many individuals remained in a vegetative phase during the flowering season. This is especially true for individuals growing in dense shade, where they seldom reach the reproductive phase before the above-ground parts die at the end of the flowering season, while the rhizome perennates.
Population differentiation from combined RAPD and inter-SSR data
A total of 118 reproducible bands were amplified from eight RAPD primers and 35 bands from three inter-SSR primers. Similar conclusions were drawn from each data set independently and therefore data were combined to further explore our findings. In the combined data set of 153 RAPD and inter-SSR amplified ‘loci’, 119 bands (77·78 %) were polymorphic (Table 2). Two hundred and seven RAPD and inter-SSR banding phenotypes were identified from 283 individuals. Individuals that shared the same phenotypes were only found within populations and not between populations. One small population (‘Urauchi’ from Japan) consisted entirely of individuals sharing the same banding phenotype (Table 3).
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PCO analysis
The first three coordinates of the PCO analysis for all genotypes describe 15·2 %, 8·4 % and 8·2 % of the total variance (31·8 % cumulative). Individuals from China, southern Taiwan and eastern Taiwan formed three distinctive groups, while populations from northern and ‘north-central’ Taiwan and Japan showed some degree of overlap (Fig. 2). Generally, Taiwanese populations covered most of the PCO space. Chinese populations were closest to northern Taiwan populations. The former grouped in two clusters: North Fujien Province versus south and west Fujien, but the two groups are placed together at one end of the PCO space (Fig. 2). The two Japanese populations exhibited considerable differences, despite their close proximity on the small island of Iriomote (arrow indicated in Fig. 2). From the UPGMA tree, considerable genetic differences can also be seen between populations in northern and north-central Taiwan (Fig. 3). Populations ‘Manueuan waterfall’ (K) and ‘Peichatien shan’ (L), which are situated at different altitudes on Peichatien mountain, were more dissimilar than north (A) and south Fujien (China) populations (B and C) in China (Fig. 3). Fujien Province is about the same size as Taiwan.
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Genetic diversity indices
Shannon’s diversity index (S) and Simpson diversity index (D) both indicated that populations in northern Taiwan (S = 0·29 and D = 0·96) and north-central Taiwan (S = 0·26 and D = 0·97) were most diverse. This is confirmed by the percentages of RAPD and inter-SSR band polymorphisms (Table 3). Populations in Japan, China, central and southern Taiwan are comparatively low in diversity, i.e. their S values are below 0·17 and the percentage of polymorphic bands is below 33·33 % (Table 3). The overall diversity for Titanotrichum was not high (S'sp = 0·314) but was higher than expected for a clonal plant.
The Mantel test showed no significant correlation between genetic and geographic distance (r = 0·417, P = 0·12).
Composition of genetic variation
The partitioning of the genetic variation was further examined by analysis of molecular variance (AMOVA). The majority of variation was found among populations, with genetic differences among geographic regions (Taiwan, China and Japan) less marked (14·5 %, Table 4). Half of the variation (50·0 %) was found among all populations rather than their geographic locations (P < 0·001, Table 4). It is also notable that only 35·5 % of the variance occurred within populations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Population structure and genetic diversity
The PCO distribution of the 25 Titanotrichum populations suggests that the Chinese populations, populations in eastern Taiwan and populations from southern Taiwan form three distinct groups (Fig. 2). In contrast, individuals from north-central Taiwan are scattered across the PCO space and partly overlap with these three groups. The two Japanese populations were quite different from each other, one with affinity to north-central Taiwan populations and the other to central and southern Taiwan populations (Fig. 2). It is tempting to speculate that these results represent at least two independent migration events from Taiwan to the tiny island Iriomote of Japan.
The AMOVA showed that the majority of the genetic variation was among populations within regions rather than within populations or among geographic regions (Table 4). Genetic distances among all the populations, as indicated in the UPGMA tree (Fig. 3), were similar, with little correlation with geographic distance, possibly suggesting that the populations represent fragments from a wider distribution in the past (see discussion below). In no case was the same clonal genotype found within different populations, although they were sometimes observed within populations.
The populations in southern Taiwan and Japan were small and relatively clonal (i.e. G/N < 0·63 in Table 3), which could be due in part to a founder effect at the edge of the range. On the other hand, populations in northern and north Taiwan are quite diverse (Table 3 and Figs 2 and 3). Field observation and the diversity measures indicate that these populations may have a higher level of sexual reproduction (or a lower recruitment from bulbils). Considerable seed set, a high proportion of sexually mature individuals and pollinator visits were recorded in populations of northern and north-central Taiwan (C.-N. Wang, pers. obs.). These factors probably contribute to their higher diversity index than populations in China, Japan and south Taiwan (S = 0·26–0·29; Table 3). The relative high estimate of genotypic diversity given by the Simpson index (D = 0·933) is in line with the observation that there is relatively little redundancy of genotypes even within populations.
Population differentiation in relation to paleogeography and glaciation
Species history and former distribution can have a major effect on population genetic differentiation. In Titanotrichum, it was found that the genetic distance among populations did not fully correlate with the geographic distance between Taiwan, China and the Ryukyu Islands. The lack of congruence suggests that there is no simple ‘isolation by distance’ explanation for the population differentiation. Other factors, such as sea-level changes at the glacial maximum, are likely to be involved in structuring the populations, as the local flora (south- east China–Taiwan–Ryukyu) was greatly affected by Quaternary glacial periods (1·6 Myrs to present) (Sheng, 1995). The four most recent glaciations apparently facilitated a huge amount of lowland (altitude below 1000 m) species redistribution and migration between China, Taiwan and south Ryukyu (Sheng, 1995). Wang and Zhang (1994) note that 30–40 % of the extant flora of Taiwan is shared with south-east China, emphasizing the importance of the land bridge. Similarly, a common Ryukyu–Taiwan–south-east China fauna is also evident from reptile and amphibian assemblages (Ota, 1998). The depth of the sea between Taiwan, mainland China and the Ryukyu Islands (the Taiwan Strait) is about 100 m on average, and during a glacial maximum the sea level is likely to have been 120–140 m lower, thus connecting the islands to the mainland (Zeng, 1993). Taiwan has long been thought to be a refugium for the south-east Chinese flora as it contains a variety of habitats.
It should also be noted that the north and central Taiwan Strait is shallower (only 40 m deep) but the south Taiwan Strait is deeper, up to 400 m. Therefore any land bridge was likely to have first formed between north-central Taiwan, the southern Ryukyu Islands and China (Zeng, 1993). This is consistent with our finding that north and central Taiwan appears to be the centre of Titanotrichum’s genetic diversity, possibly because a Pleistocene land bridge formed here between China and Ryukyu (Fig. 1). The extreme rarity of Titanotrichum in south-east China may be due to extinction during glacial periods and human activity, as south-east China is highly populated. This is evident in our UPGMA analysis as the Chinese samples are not as deeply separated as those in northern Taiwan (Fig. 3). It is even possible that they have migrated relatively recently from Taiwan.
Diversity measures in mixed reproduction plants
The diversity measures in Titanotrichum indicate considerable total variation (S'sp = 0·314 and D = 0·933). This is similar to other findings for mixed-reproduction plants, which indicates that their diversity is comparable to that of outcrossing plants (Nybom and Bartish, 2000). From separate pollination experiments conducted both in the field and glasshouse, it has been shown that Titanotrichum benefits from outbreeding, as outcrossing between populations results in significantly higher seed set (Wang, 2003). Thus, it is likely that sexual outcrossing still plays an important role in maintaining the genetic diversity of Titanotrichum. However, in Titanotrichum most of the variation was found among all populations (64·5 % of AMOVA) rather than within (35·5 %, Table 4). Therefore, it is possible that sexual reproduction has mainly happened in the past whilst vegetative reproduction via bulbils has become increasingly dominant within their isolated habitat. The small population sizes and scattered distribution indicate the gene flow between populations is infrequent or even absent altogether. The high estimate of Phist in Titanotrichum oldhamii perhaps indicates genetic drift, as has been reported from Allium vineale (Ceplitis, 2001).
The continuing isolation of populations and limited mating could lead to the loss of sexual reproduction. A study of the clonal plant Decodon verticillatus indicates that complex traits like sex can be degraded by mutation when they no longer increase fitness (Eckert et al., 1999). A pilot study on fertility in individuals from different populations of Titanotrichum oldhamii also found different levels of fertility among individuals and populations (Wang, 2003).
Mixed-reproduction bulbiliferous plants seem to have higher genetic variation than obligate apomicts due to the contribution of more frequent sexual reproduction (Diggle et al., 1998). Although several papers have attempted to summarize the diversity measures of clonal plant species, general conclusions are extremely difficult to draw because of different sampling strategies and variation in plant life history (Ellstrand and Roose, 1987; Widén et al., 1994; Diggle et al., 1998). Some species show high levels of clonal diversity, e.g. Populus tremuloides (Jelinski and Cheliak, 1992; Yeh et al., 1995), while others show low levels, e.g. Saxifraga cernua (Bauert et al., 1998).
One general phenomenon in clonal species, and particularly in bulbiliferous plants, is polyploidy. Titanotrichum has a high chromosome number (2n = 40) for the Gesneriaceae. Duplicated chromosomes buffer polyploids from somatic point mutations, deletions and translocations. Polyploidy may also arise from natural hybridization, which facilitates fixed heterozygosity (Stebbins, 1984). It is possible that polyploid clonal plants contain a high amount of marker polymorphism compared with diploid species, simply because of their large genome size and hybrid origin.
Analysis of genetic variation using dominant markers
One criticism of RAPDs is that they are prone to PCR artefacts. During DNA amplification, nested primer annealing and intrastrand interactions at the first few PCR cycles could accumulate a significant amount of rearranged DNA PCR products (Rabouam et al., 1999; Caetano-Anollés, 2001). Using inter-SSR data in parallel to RAPDs seems to be a good way for testing the reliability of RAPD data. The results presented here showed that the banding patterns obtained from inter-SSR primers are congruent with the results obtained from the RAPDs data, and therefore it can be assumed that RAPD artefacts in our data are not marked. The first two cycles of our RAPD profile used a higher annealing temperature (37 °C) than the remaining cycles to eliminate false amplification. Also fragments shorter than 400 base pairs were excluded, as short fragments are preferentially falsely amplified in PCR.
Co-migration is another potential problem in RAPD and inter-SSR analysis. However, for intraspecific comparison (as in our study), the co-migration error is likely to be low (Wu et al., 1999; Nybom and Bartish, 2000). Several studies have used techniques such as Southern blot and restriction digestion for checking RAPD band homology, and come to the conclusion that more than 91 % of RAPD bands are homologous, especially within species (Rieseberg, 1996; Wu et al., 1999). Although we did not check our RAPD band homology, due to the large data set, we have no evidence that our results are biased.
For rare or endangered species, a small sample size is unavoidable. This could lead to a significant bias in the population genetic estimates (Fischer et al., 2000). However, simulation results show that a sample size of 10–15 individuals is adequate for largely unbiased results (Isabel et al., 1999). In the study reported here 10–20 individuals from each population included in the analysis unless the population size was less than ten.
Conservation implications
The genetic differences between populations of Titanotrichum oldhamii are significant, which is reflected by its high Phist value. This may be due in part to genetic drift in small populations. As Titanotrichum populations undergo year-to-year fluctuations in numbers of mature individuals (populations in southern Taiwan and Ryukyu Islands have less than 15 mature individuals in each population) and there are an estimated total of fewer than 10 000 mature individuals, it is categorized as ‘vulnerable’ according to IUCN red list criteria (IUCN, 2001). Moreover, as further decline is likely with increasing human disturbance, it may become endangered in the future. The red list of the Japan flora and the flora of China includes Titanotrichum oldhamii as endangered (Walker, 1976; Wang et al., 1998).
The Japanese populations and those in eastern Taiwan are protected as they are located within the range of national parks or natural reserves. However, there is no protection as yet for the populations in north Taiwan, which is the centre of diversity. These populations are often found along highways where regular human disturbance may cause a significant reduction in population size. Low seed set in the wild, together with observations of infrequent visits of non-specialized pollinators, indicated that sexual reproduction has been impaired. Despite the numerous bulbils they produce, the recruitment rate is low and they are restricted by their limestone habitat requirement. Artificial transplanting of individuals in different populations may be advantageous to promote gene flow, since most populations are so isolated. Monitoring of populations to determine the effects of inbreeding depression and genetic drift is advisable in the future (Ellstrand and Elam, 1993). The results presented in this paper form a baseline from which future changes can be monitored.
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ACKNOWLEDGEMENTS |
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We thank Zhen-yu Li, from the Institutum Botanicum, Academiae Sinicae of China and Tokushiro Takaso from the Tropical Biosphere Research Center of Ryukyu University, Japan for their kind assistance on fieldwork, and Goro Kokubugata from Tsukuba Botanical Garden, Tokyo for providing DNA samples. Staff at the Royal Botanic Gardens Edinburgh, particularly Dr Michelle Hollingsworth and Will Goodall-Copestake, are greatly thanked for their assistance with molecular work. Financial support for the fieldwork was kindly provided by the Ministry of Education, Taiwan, the A. G. Side Fund of the Linnean Society and the Davis Expedition Fund of the University of Edinburgh.
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