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Population Biology |
2Dipartimento di Biologia Vegetale, Università degli Studi di Napoli Federico II, I-80139 Naples, Italy; 3Dipartimento di Ecologia, Università della Calabria, Arcavacata di Rende, I-87036 Cosenza, Italy; 4Geobotanisches Institut, ETH Zürich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland
Received for publication March 7, 2003. Accepted for publication July 1, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: Anacamptis (Orchis) palustris • bottleneck • chloroplast minisatellite locus • conservation genetics • genetic diversity • haplotype variation • Mediterranean region • Orchidaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and by more recent historic, typically anthropogenic, processes. Glacial cycles during the Oligocene have indirectly affected the Mediterranean region by repeatedly changing climatic conditions and sea water levels. These factors have led to repeated range changes of species that presumably have affected their genetic diversity and population structure (Hewitt, 1996
).
Repeated range contractions and cycles of habitat colonization followed by extinction may lead to the loss of genetic diversity. At marker loci with low mutation rates, this signature from the Oligocene should still be visible in a species (Willis, 1996
). In contrast, genetic variability at more rapidly evolving markers, such as nuclear and chloroplast minisatellite and microsatellite repeats, may have recovered from ancient bottlenecks, provided that population sizes have been sufficiently large and stable since the end of the last glacial maximum to allow the accumulation of neutral genetic variation.
Such rapidly evolving markers may allow detection of the signature of more recent changes in population sizes (Schaal et al., 1998
), for example as a consequence of habitat alteration (e.g., through drainage, fragmentation, or the introduction of alien species). These anthropogenic activities may have reduced population sizes, extirpated local populations, and increased distances among remnant populations, which decrease the likelihood of gene exchange among populations.
Retrodunal marsh habitats along the Mediterranean coast represent one of the habitats most strongly affected by human activity. These marshes, typically on oligocenic soil that was generated after the last glacial period (Bell and Walker, 1992
), are home to highly diverse plant and animal communities that are adapted to the salinity import from the neighboring sea. Since Roman times, these marshes have been extensively drained to reduce breeding grounds of Anopheles mosquitoes, the vectors of malaria, and to recover land suitable for agriculture. Such habitats, formerly covering hundreds of kilometers along the Italian coasts, are now limited to a few patches that are often isolated from neighboring patches, thus forming an interrupted chain of habitat islands along the Mediterranean coast.
Orchids are a group of highly diverse plants that attract strong public interest because of their large, showy flowers and their often fascinating pollination biology. Because they often grow in species-rich habitats that are particularly affected by human change, such as marshes and calcareous grasslands, a large proportion of orchids are considered endangered (e.g., Conti et al., 1992
).
Anacamptis palustris (Orchidaceae), the bog orchid, occurs in retrodunal marsh habitats in Mediterranean regions of Europe. A comparison of the present distribution with that indicated by herbarium specimens collected during the last century has shown that A. palustris formerly was widespread in the Mediterranean area (P. Grünanger, personal observations), but has become rare in the recent past. Nowadays, A. palustris has a highly disjunct distribution along the Mediterranean coast (Grünanger, 2000
). The largest known populations exist in northern Italy and are the focus of the present study.
Despite the strong interest in orchid biology and conservation, however, little is known about orchid population structure and conservation genetics. This stems in part from the observation that allozyme diversity is low in many species, and especially in rare and endangered taxa, whereas many widely distributed species show appreciable amounts of allozyme variation (Arduino et al., 1995
; Soliva and Widmer, 1999
; Sun and Wong, 2001
; Brzosko et al., 2002
; Squirrel et al., 2002
). Only recently have more rapidly evolving markers, such as microsatellites and minisatellites, been developed and applied to orchids (Gustafsson, 2000
; Soliva et al., 2000
; Cafasso et al., 2001
; Pellegrino et al., 2001
; Cozzolino et al., 2003a
). It is these rapidly evolving markers that are most likely to be informative for studying the consequences of habitat fragmentation, population bottlenecks, and gene flow (Luikart et al., 1998
; Schaal et al., 1998
).
In A. palustris allozyme variation is known to be low (Arduino et al., 1996
), which renders these markers useless for inferring recent population processes in this species. However, a highly polymorphic minisatellite repeat located in the trnL intron of the plastid genome has recently been characterized in A. palustris (Cafasso et al., 2001
), and the molecular evolution of the repeat has since been elucidated (Cozzolino et al., 2003a
). This marker offers interesting possibilities to study population level processes in A. palustris. Chloroplast DNA (cpDNA), maternally inherited in most angiosperms (Corriveau and Coleman, 1988
), has half the effective population size of biparentally inherited markers and thus is more sensitive to changes in population size (Birky et al., 1989
; Mitton, 1993
). Currently, cpDNA variation is most widely used in phylogeographic and population genetic studies (Demesure et al., 1996
; Schaal et al., 1998
). CpDNA variation has been used in two cases to infer population bottlenecks in pines (Pinus), where cpDNA is paternally inherited and thus can move among populations through both pollen and seeds (Echt et al., 1998
; Morgante et al., 1998
). Due to the high mutation rate of the A. palustris cpDNA minisatellite, this marker is suitable to study population level processes in this orchid.
In the present study, we use the chloroplast minisatellite variation in five of the largest known remaining populations of A. palustris to assess the relationship between population size and genetic diversity, to test whether population bottlenecks have shaped the pattern and amount of genetic diversity in these populations, and to compare cpDNA variation with allozyme variation reported previously by Arduino et al. (1996)
for the same populations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study focuses on the largest Mediterranean populations of A. palustris. Chloroplast DNA variation in these populations was recently presented in a range-wide phylogeographic analyis of A. palustris (Cozzolino et al., 2003b
). Here, we use these data to investigate whether variation at the chloroplast minisatellite locus reveals the signature of habitat fragmentation and genetic bottlenecks in these populations of A. palustris (Table 1). Details concerning DNA extraction, polymerase chain reaction (PCR) amplification, sequencing, and analysis of fragment lengths are given by Cozzolino et al. (2003a
, b
). Estimates of population sizes were obtained by counting flowering individuals during the peak of the flowering season in 2001.
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pi2), and the unbiased gene diversity (He) of Nei (1975)
, where He = (n/(n – 1)(1 – pi2)).
To assess the consequences of demographic changes on genetic diversity, we applied the "heterozygosity excess test" as implemented in the software Bottleneck (Cornuet and Luikart, 1996
). This test is based on the principle that populations with a recent reduction in effective population size typically exhibit a reduction in both allele number and heterozygosity, but with a faster reduction of allele number compared with heterozygosity (Watterson, 1984
; Maruyama and Fuerst, 1985
; Allendorf, 1986
). In the case of haploid chloroplast DNA variation, heterozygosity can be viewed as gene diversity (according to Nei, 1987
). If a recent population bottleneck has occurred, the observed gene diversity (Ho) will be larger than the expected gene diversity (He) based on the number of haplotypes present in the population. This transient excess of Ho will be maintained until a new mutation-drift equilibrium is reached (Waldick et al., 2002
). We tested for recent population bottlenecks assuming a stepwise mutation model (SMM) for the A. palustris chloroplast minisatellite evolution (Cozzolino et al., 2003a
).
Populations that have undergone severe size reductions for a long period typically show a reduction of low-frequency allele classes. To detect distorted allele frequency distributions in the investigated populations, we employed the graphical method proposed by Luikart et al. (1998)
. Reductions in low-frequency allele classes or shifts in allele frequencies are a consequence of the loss of rare alleles during population bottlenecks and may be detected by comparing the distributions of allele frequency classes observed in a bottlenecked population with those under mutation-drift equilibrium. We therefore use the allele frequency distribution of the Ravenna population, previously established to be at mutation-drift equilibrium (Cozzolino et al., 2003a
), as a reference for comparison with other populations.
We used rarefaction to correct diversity estimates for differences in sample sizes, employing the option Rarefaction in the software Contrib (Petit et al., 1998
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and were excluded from all analyses. Variation in minisatellite repeat number, ranging from one to 20, was extensive and found in all populations (Table 1). The highest number of haplotypes was found in Ravenna (17) and the lowest in S. Margherita (3). Haplotype frequencies differed between the two geographic areas (Po River delta and Triveneto) from where populations were sampled; haplotypes N1 to N20 were found in Po River delta populations, with haplotype N4 being the most common. In the Triveneto populations, haplotypes N1 to N15 were found, and N2 was the most common (Table 1, Fig. 1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), these bottlenecks are most likely a direct consequence of human activities leading to habitat alterations, fragmentation, and increased isolation of remnant populations.
The loss of genetic diversity in populations as a consequence of small population sizes and random genetic drift is of major interest in conservation biology. Although the variation surveyed in this and other studies is most likely selectively neutral, its loss may nevertheless indicate the correlated loss of potentially adaptive genetic variation. This loss may reduce the chance of population survival if populations remain small or if environmental conditions change in the foreseeable future. In the absence of knowledge about the loss of adaptive genetic variation, which is much more difficult to assess, neutral genetic variation may provide some information about the causes and timing of population bottlenecks and may allow us to infer whether populations have experienced a recent loss of genetic diversity or whether they have existed for extended periods of time with low genetic diversity (Petit et al., 1998
).
We observed the well-established positive correlation between genetic diversity and population size. Such a correlation has been found previously for nuclear markers in several plant species (e.g., Fischer et al., 2000
). To the best of our knowledge, we find this association for the first time with a maternally inherited plastid marker. The larger sample sizes obtained from larger populations did not lead to this association, because it remained highly significant after rarefaction to a common sample size. The high cpDNA diversity detected thus allowed us to study the consequences of demographic changes in A. palustris.
We used several approaches to test whether population bottlenecks may explain the differences in haplotype variation found among A. palustris populations and to infer whether these bottlenecks occurred relatively recently in the history of the populations.
In theory, populations that have experienced a recent bottleneck show a loss of allelic diversity and heterozygosity (Nei et al., 1975
). The loss of allelic diversity, however, occurs faster than the reduction of heterozygosity. As a consequence, the observed heterozygosity in such a population is larger than the heterozygosity expected from the observed number of alleles. In the case of haploid data, such as chloroplast DNA variation, heterozygosity can be viewed as gene diversity according to Nei (1975)
. The fact that no significant difference between observed and expected gene diversity under SMM was found for Ravenna and S. Margherita suggests that these populations are in mutation-drift equilibrium (Table 2). The low number of observed haplotypes in S. Margherita is a consequence of the small population size, which in turn seems to have been small for sufficient time to allow the population to reach an equilibrium. Similarly, an equilibrium situation has been inferred previously for Ravenna (Cozzolino et al., 2003a
), and this result is supported here. In contrast, the remaining three populations, Orsi Mangelli, Monfalcone, and Isola di Cona, revealed a significant difference between observed and expected gene diversity. Contrary to expectations, however, these populations exhibit a lower than expected gene diversity (i.e., a heterozygote deficiency). This result may indicate that a bottleneck occurred in the past, but not in the recent past. Even if allelic diversity is expected to be more sensitive to a demographic bottleneck than heterozygosity, a significant reduction in heterozygosity nevertheless occurs after a transient state of heterozygosity excess (Nei et al., 1975
; Maruyama and Fuerst, 1985
). Alternatively, the observed heterozygote deficiency may indicate a transient state of recovery after a bottleneck.
There may, however, be a nondemographic explanation of this result. Our analyis has been based on genetic variation assessed at a single, highly polymorphic minisatellite repeat locus. It is well established that some highly polymorphic loci that evolve according to an SMM may exhibit a heterozygote deficiency, even if a bottleneck has occurred recently (Cornuet and Luikart, 1996
).
The analysis of allele frequency distributions may present an alternative approach to the data. A population bottleneck is expected to cause a random loss of allelic variation as a consequence of random genetic drift. The effect is most dramatically seen in the rapid loss of low frequency alleles and a concomitant increase of intermediate and high-frequency alleles (Luikart et al., 1998
). Under equilibrium conditions, the allele frequency distribution depends primarily on the mutation rate and the mutational model. Generally, alleles at low frequency (lower than 0.1) are expected to be more common than alleles at intermediate or high frequency (Nei et al., 1976
; Chakraborty et al., 1980
). Such an allele frequency distribution is seen in Ravenna, for which mutation-drift equilibrium has been inferred. Here, all alleles have a frequency less than 0.13, with the exception of N4, which occurs at intermediate frequency. In contrast, the four smaller populations have a characteristic mode shift in the distribution of allele frequencies, with one allele in each population occurring at high frequency. In an immediate post-bottleneck scenario, the shift is expected to occur from low frequency to intermediate frequency (i.e., 0.1–0.2). The stronger shift observed in small A. palustris populations (with the most common allele frequencies at 0.4–0.7) may be an indication of the severity of the bottlenecks, but may also indicate that a considerable period of time has elapsed since the bottleneck (Fig. 3). These results are thus largely congruent with those obtained through the comparison of observed and expected gene diversities. Interestingly, however, the allele frequency distribution observed in S. Margherita suggests that this population has also experienced a severe bottleneck.
Inspection of haplotype number may provide an explanation of the observed heterozygote deficiency or, rather, of the observed haplotype excess (Fig. 1). Under equilibrium conditions, a locus evolving under SMM typically shows a contiguous allele distribution (i.e., all allele length variants), such as that seen in Ravenna. Under random genetic drift, alleles with intermediate lengths, which often occur at low frequencies, may be lost readily, leading to gaps in the distribution of allele sizes. Such a pattern is observed in all small populations. If new mutations occur according to an SMM, these gaps will be filled progressively with new alleles that evolved through mutation after the bottleneck. In post-bottleneck populations, this can lead to a transient excess of alleles, which translates into a transient heterozygote deficiency (Cornuet and Luikart, 1996
). The large number of low-frequency haplotypes with almost contiguous repeat numbers in Isola di Cona, Monfalcone, and Orsi Mangelli may be the result of such a restoration of genetic diversity after a bottleneck. The speed with which such restoration (i.e., the generation of new alleles) occurs depends largely on population size and mutation rate. Assuming that the mutation rate is equal for all populations, we find no evidence for restoration in S. Margherita, which is by far the smallest population of A. palustris studied.
The comparison of gene diversity with the effective number of alleles may also help elucidate the genetic consequences of demographic changes in populations. Gene diversity does not change immediately after a population bottleneck (Nei et al., 1975
), whereas the effective number of alleles, which is strongly influenced by rare alleles, is more sensitive to changes in population size (Shriver et al., 1993
; Petit et al., 1998
; Widmer and Lexer, 2001
). Accordingly, we found a more pronounced reduction of the effective number of alleles in small populations, compared with haplotype diversity (Fig. 4a). The peak in the effective number of alleles in Isola di Cona may indicate recovery after a bottleneck, because restoration of genetic diversity is expected to be seen most rapidly by examining the effective number of alleles (Nei et al., 1975
).
Although allele size (i.e., number of minisatellite repeats) is expected to be neutral, we find a positive and marginally significant correlation between population size and mean repeat length (Fig. 4b). In other words, the reduction in allele number in small populations is reflected in a reduction in allele size. At equilibrium, allele size distribution depends on the mutation rate and the mutational model (Shriver et al., 1993
). In Ravenna, which is in equilibrium, the distribution of allele sizes is skewed towards shorter alleles. These shorter alleles are more likely to be retained after a population bottleneck, simply because they are more common in large populations. Our results therefore support the observation that long alleles are lost as a consequence of random genetic drift (Ellegren, 2000
), and no selection needs to be invoked to account for the seemingly preferential maintenance of shorter alleles in small populations.
The extensive chloroplast DNA variation in populations of A. palustris is not reflected in the variation at nuclear allozyme loci. Nuclear loci are mostly fixed for the same subset of alleles (Arduino et al., 1996
). We interpret the discrepancy between cpDNA polymorphism and allozyme monomorphism as a consequence of the high minisatellite mutation rate, which has been estimated as 3.2 x 10–3 (Cozzolino et al., 2003a
). This value is considerably lower than the 1 x 10–6 estimated for allozymes (Wolfe et al., 1987
). Reanalysis of the allozyme data of Arduino et al. (1996)
revealed a strong heterozygote deficiency for nuclear allozymes in Ravenna, which contrasts with the equilibrium situation inferred based on cpDNA variation (Cozzolino et al., 2003a
). Interestingly, allozyme heterozygosity is similar between Ravenna and the small populations analyzed also in the present study. We take this as evidence for a historic population bottleneck that reduced both nuclear and cpDNA variation. However, the number of generations required for a new equilibrium value to be reached after a bottleneck depends on the mutation rate (Nei et al., 1975
). Given the much higher cpDNA minisatellite mutation rate, we infer that the historic population bottleneck occurred long enough ago to allow regeneration of cpDNA variation, but not of nuclear allozyme variation. Given that the effective population size of a maternally inherited cpDNA marker is half that of a biparentally inherited nuclear marker, we further infer that the population size in Ravenna has been large for several generations. Otherwise, the high number of haplotypes could not have evolved and been maintained.
Based on the history of the Mediterranean region from which the samples were collected, we hypothesize that A. palustris experienced a strong bottleneck during the last glacial maximum, which ended approximately 12 000 yr ago. At the end of the last glaciation, most nuclear genetic variation had probably been lost. Given the low allozyme mutation rate, it is reasonable to assume that nuclear genetic variation could not subsequently be restored, whereas there was sufficient time to restore cpDNA minisatellite variation. On the other hand, the loss of cpDNA variation in the four smaller A. palustris populations is most likely a more recent phenomenon and may be a result of anthropogenic changes in the habitat of the species. Even before Christ, the Romans had attempted to clear and drain swampy areas, such as those inhabited by A. palustris, to reduce malaria and other diseases. Later, these retrodunal marsh areas were converted into land suitable for agriculture or fell prey to rapidly growing cities and industrial areas.
The identification of a rapidly evolving cpDNA minisatellite repeat in A. palustris (Cafasso et al., 2001
) provided a useful marker to study genetic variation of this rare orchid, both within populations and between large and small populations. When studied in combination with more slowly evolving allozyme loci, it allowed us to infer bottlenecks that occurred at different times in the history of the species. A first bottleneck that presumably occurred as a consequence of climatic changes during the last glacial maximum affected all extant A. palustris populations and led most likely to a loss of allozyme and cpDNA variation. Chloroplast minisatellite variation apparently recovered after the historic bottleneck, unlike allozyme variation. At least one more recent bottleneck again reduced cpDNA minisatellite variation in the four small populations, most likely the consequence of anthropogenic changes to the orchid habitat. Evidence for the renewed recovery of cpDNA minisatellite variation in three of the smaller populations may indicate that recent conservation efforts, which attempt to protect habitats and stabilize population sizes, have achieved a degree of success.
Gene flow among populations could partly explain the evidence for a recovery of genetic diversity within populations and could reduce the risk of extirpation. This hypothesis is particularly appealing in orchids, as they have small, dust-like seeds that are easily dispersed by wind (Arditti and Ghani, 2000
). However, in the present study, gene flow through seeds seems to be of minor importance. Although the neighboring populations at Ravenna and Orsi Mangelli are not genetically differentiated, differentiation (as estimated by GST) is relatively high among the three other populations, suggesting that gene flow through seeds is not sufficient to save such small populations from extirpation (Cozzolino et al., 2003b
).
Despite the evidence for bottlenecks and a concomitant loss of genetic diversity, these populations represent the hotspot of genetic diversity for this species, because populations of A. palustris in other areas typically are even smaller and genetically more depauperate. It is therefore of primary importance to conserve the habitats in which the remaining large populations of this rare orchid are growing, in order to save them from extirpation.
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FOOTNOTES |
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5 E-mail: alex.widmer{at}geobot.umnw.ethz.ch
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LITERATURE CITED |
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Arditti J. A. K. A. Ghani 2000 Tansley review No. 110—numerical and physical properties of orchid seeds and their biological implications. New Phytologist 145: 367-421.[CrossRef][ISI]
Arduino P. R. Cianchi W. Rossi B. Corrias L. Bullini 1995 Genetic variation in Orchis papilionacea (Orchidaceae) from the Central Mediteranean region: taxonomic inferences at the intraspecific level. Plant Systematics and Evolution 194: 9-23[CrossRef][ISI]
Arduino P. F. Verra R. Cianchi W. Rossi B. Corrias L. Bullini 1996 Genetic variation and natural hybridization between Orchis laxiflora and Orchis palustris (Orchidaceae). Plant Systematics and Evolution 202: 87-109[CrossRef][ISI]
Bell M. M. J. C. Walker 1992 Late Quaternary environmental change. Longman, Harlow, UK
Birky C. W. P. Fuerst T. Maruyama 1989 Organelle gene diversity under migration, mutation, and drift: equilibrium expectations, approach to equilibrium, effects of heteroplasmic cells, and comparison to nuclear genes. Genetics 121: 613-627
Brzosko E. A. Wroblewska M. Ratkiewicz 2002 Spatial genetic structure and clonal diversity of island populations of ladys slipper (Cypripedium calceolus) from the Biebrza National Park (northeast Poland). Molecular Ecology 11: 2499-2509[CrossRef][Medline]
Cafasso D. G. Pellegrino A. Musacchio A. Widmer S. Cozzolino 2001 Characterization of a minisatellite repeat locus in the chloroplast genome of Orchis palustris (Orchidaceae). Current Genetics 39: 394-398[CrossRef][ISI][Medline]
Chakraborty R. P. A. Fuerst M. Nei 1980 Statistical studies on protein polymorphism in natural populations. III. Distribution of allele frequencies and the number of alleles per locus. Genetics 94: 1039-1063
Conti F. A. Manzi F. Pedrotti 1992 Libro rosso delle piante d'Italia. Associazione italiana per il WWF e la Società Botanica Italiana, Roma, Italy
Cornuet J. M. G. Luikart 1996 Description and power analysis of two tests for detecting recent population bottlenecks from allele frequency data. Genetics 144: 2001-2014[Abstract]
Corriveau J. L. A. W. Coleman 1988 Rapid screening method to detect potential biparental inheritance of plastid DNA and results for over 200 angiosperm species. American Journal of Botany 75: 1443-1458[CrossRef][ISI]
Cozzolino S. D. Cafasso G. Pellegrino A. Musacchio A. Widmer 2003a Molecular evolution of a chloroplast tandem repeat locus in an orchid lineage. Journal of Molecular Evolution 57: 1-9[CrossRef][ISI][Medline]
Cozzolino S. D. Cafasso G. Pellegrino A. Musacchio A. Widmer 2003b Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation. Molecular Ecology 12: 2783-2792[CrossRef][Medline]
Demesure B. B. Comps R. J. Petit 1996 Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolution 50: 2515-2520.[CrossRef][ISI]
Echt C. S. L. L. De Verno M. Anzidei G. G. Vendramin 1998 Chloroplast microsatellites reveal population genetic diversity in red pine, Pinus resinosa Ait. Molecular Ecology 7: 307-316
Ellegren H. 2000 Heterogeneous mutation processes in human microsatellite DNA sequences. Nature Genetics 24: 400-402[CrossRef][ISI][Medline]
Fischer M. R. Husi D. Prati M. Peintinger M. van Kleunen B. Schmid 2000 RAPD variation among and within small and large populations of the rare clonal plant Ranunculus reptans (Ranunculaceae). American Journal of Botany 87: 1128-1137
Grünanger P. 2000 Orchidacee d'Italia. Quaderni di Botanica Applicata 11: 3-80
Gustafsson S. 2000 Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid. Molecular Ecology 9: 1863-1872[CrossRef][Medline]
Hewitt G. M. 1996 Some genetic consequences of ice ages and their role in divergence and speciation. Biological Journal of the Linnean Society 58: 247-276[CrossRef]
Luikart G. W. B. Sherwin B. M. Steele F. W. Allendorf 1998 Usefulness of molecular markers for detecting population bottlenecks via monitoring genetic change. Molecular Ecology 7: 963-974[CrossRef][Medline]
Maruyama T. P. A. Fuerst 1985 Population bottlenecks and non-equilibrium models in population genetics. II. Number of alleles in a small population that was formed by a recent bottleneck. Genetics 111: 675-689
Mitton J. B. 1993 Molecular approaches to population biology. Annual Review of Ecology and Systematics 25: 45-69
Morgante M. N. Felice G. G. Vendramin 1998 Analysis of hypervariable chloroplast microsatellites in Pinus halepensis reveals a dramatic bottleneck. In A. Karp, P. G. Isaac, and D. S. Ingram [eds.], Molecular tools for screening biodiversity: plants and animals, 407–412. Chapman and Hall, London, UK
Nei M. 1975 Molecular population genetics and evolution. North-Holland, Amsterdam, Netherlands
Nei M. 1987 Molecular evolutionary genetics. Columbia University Press, New York, New York, USA
Nei M. R. Chakraborty P. A. Fuerst 1976 Infinite allele model with varying mutation rate. Proceedings of the National Academy of Sciences, USA 73: 4164-4168
Nei M. T. Maruyama R. Chakraborty 1975 The bottleneck effect and genetic variability in populations. Evolution 29: 1-10
Pellegrino G. D. Cafasso A. Widmer M. Soliva A. Musacchio 2001 Isolation of microsatellite loci from the orchid Serapias vomeracea (Orchidaceae) and cross-priming to the other Serapias species. Molecular Ecology Notes 1: 279-280[CrossRef][ISI]
Petit R. J. A. El Mousadik O. Pons 1998 Identifying populations for conservation on the basis of genetic markers. Conservation Biology 12: 844-855[CrossRef][ISI]
Schaal B. A. D. A. Hayworth K. M. Olsen J. T. Rauscher W. A. Smith 1998 Phylogeographic studies in plants: problems and prospects. Molecular Ecology 7: 465-474[CrossRef]
Shriver M. D. L. Jin R. Chakraborty E. Boerwinkle 1993 VNTR allele frequency distribution under the stepwise mutation model: a computer simulation approach. Genetics 134: 983-993[Abstract]
Soliva M. B. Gautschi C. Salzmann I. Tenzer A. Widmer 2000 Isolation and characterization of microsatellite loci in the orchid Ophrys araneola (Orchidaceae) and a test of cross-species amplification. Molecular Ecology 9: 2178-2179[Medline]
Soliva M. A. Widmer 1999 Genetic and floral divergence among sympatric populations of Gymnadenia conopsea s. l. (Orchidaceae) with different flowering phenology. International Journal of Plant Science 160: 897-905[CrossRef][ISI][Medline]
Squirrell J. P. M. Hollingsworth R. M. Bateman M. C. Tebbitt M. L. Hollingsworth 2002 Taxonomic complexity and breeding system transitions: conservation genetics of the Epipactis leptochila complex (Orchidaceae). Molecular Ecology 11: 1957-1964[CrossRef][Medline]
Sun M. K. C. Wong 2001 Genetic structure of three orchid species with contrasting breeding systems using RAPD and allozyme markers. American Journal of Botany 88: 2180-2188
Waldick R. C. S. Kraus M. Brown B. N. White 2002 Evaluating the effects of historic bottleneck events: an assessment of microsatellite variability in the endangered North Atlantic right whale. Molecular Ecology 11: 2241-2249[CrossRef][Medline]
Watterson G. A. 1984 Allele frequencies after a bottleneck. Theoretical Population Biology 26: 387-407[CrossRef][ISI]
Widmer A. C. Lexer 2001 Glacial refugia: sanctuaries of allelic richness, but not for gene diversity. Trends in Ecology and Evolution 16: 267-269
Willis K. J. 1996 Where did all the flowers go? The fate of temperate European flora during glacial periods. Endeavour 20: 110-114[CrossRef][ISI]
Wolfe K. H. W. H. Li P. M. Sharp 1987 Rates of nucleotide substitution vary greatly among plant mitochondrial, chloroplast and nuclear DNA. Proceedings of the National Academy of Sciences, USA 84: 9054-9058
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