| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Article |
2Centro de Investigaciones en Ecosistemas, Universidad Nacional Autónoma de México (UNAM), Antigua Carretera a Pátzcuaro No. 8701, Col. Ex-Hacienda de San José de la Huerta, 58190 Morelia, Michoacán, México; 3Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo (UMSNH), Edif. R planta baja, Ciudad Universitaria, 58040 Morelia, Michoacán, México
Received for publication June 7, 2006. Accepted for publication November 21, 2006.
ABSTRACT
We used isozymes (16 loci in 11 enzymatic systems) from Laelia speciosa, an endemic and endangered epiphytic orchid of Mexico, to assess the genetic diversity and population genetic structure in nine populations distributed along its geographic range, as well as to detect those populations that are genetically unique and therefore deserve high-priority protection. On average, the genetic diversity was high (percentage of polymorphic loci, Pp = 76%, mean number of alleles per locus, A = 3.34, the average observed heterozygosity HO = 0.302, the average expected heterozygosity HE = 0.382). Moderate levels of inbreeding ( f = 0.216, 95% confidence interval = 0.029–0.381) were found. Low levels of genetic differentiation were observed among populations (
p = 0.040); however, there was a significant correlation between geographic and genetic distances among the populations (Mantel test: r2 = 0.43, P < 0.05). Populations located within the same mountain range were genetically more similar. Private alleles were found, so proper management requires protection and maintenance of genetic diversity throughout its range. In case of reintroduction, we suggest using individuals propagated from seeds from as many capsules as possible, from close populations. An ex situ conservation strategy also is proposed.
Key Words: conservation genetics • epiphytic orchids • isozymes • Laelia speciosa • Mexico • Orchidaceae
Studies on conservation genetics of endangered plant species are necessary to establish management plans to preserve biodiversity. The maintenance of genetic diversity within and among populations of plant species is a critical issue for a long-term conservation program (Oyama, 1993
; Haig, 1998
; Bowen, 1999
). In theory, a high level of population genetic diversity in a species allows it to better adapt to environmental changes and determines its evolutionary capacity (Hamrick et al., 1991
; Frankham, 1995
; Hamrick and Godt, 1996
). Theoretical and field studies have shown that a positive correlation exists among the levels of genetic diversity and fitness in both plants and animals (Barret and Kohn, 1991
; Oostermeijer et al., 1995
; Sun, 1996
; Fischer and Matthies, 1998
; Groom, 1998
; Schmidt and Jensen, 2000
). For endangered species, the identification of unique genotypes is an important step to define units of conservation and evolutionary importance (Qamaruz-Zaman et al., 1998
).
Destruction, modification, and fragmentation of natural forests, as well as illegal extraction of orchids, have had a strong influence in their local extinction (Salazar-Chávez, 1996
). In fact, the family Orchidaceae is one of the best examples in which species have been driven to extinction as a result of human activities (Salazar-Chávez, 1996
; Hágsater et al., 2005
).
In genetic studies of orchid species, genetic diversity has varied from very low to very high. Outcrossing species have a greater genetic diversity than selfers or apomictics (Scacchi et al., 1991
; Sun, 1996
, 1997
; Sun and Wong, 2001
; Wallace, 2004
), widespread species in general have higher levels of variation than endemic species with a narrow geographic range (Scacchi et al., 1991
; Case et al., 1998
; Borba et al., 2001a
), and usually, larger populations have more diversity (Scacchi and De Angelis, 1989
; Sun, 1996
; Gustafsson, 2000
; Cozzolino et al., 2003
). Composition and dynamics of founding populations also have been related with genetic diversity; if the founder population contains few individuals, then it will be genetically depauperate, but multiple founder populations and/or large, diverse founding populations will have high levels of diversity (Squirrel et al., 2001
).
Recently, there has been more emphasis on genetic studies to develop strategies for conservation of orchid species. These studies have been useful to resolve taxonomic uncertainties, to select candidate populations of wild species to be given priority for conservation, and to propose other in situ and ex situ management strategies (Case et al., 1998
; Wong and Sun, 1999
; Szalanski et al., 2001
; Sharma et al., 2003
). Also, researchers have evaluated the effects of human activities on the diversity and genetic structure of orchids, discovering in some cases a negative effect (Cozzolino et al., 2003
), but others have not been able to detect strong effects on genetic variation (Gustafsson, 2000
; Wallace, 2002
; Murren, 2003
; Sharma et al., 2003
).
Most of the genetic studies on orchids have focused on terrestrial plants. In contrast, despite the high species diversity and ecological importance of epiphytic species, they have received relatively less attention, probably because of difficulties in accessing the canopy and the metapopulational structure of canopy plants (Ackerman and Ward, 1999
; Murren, 2003
; Trapnell and Hamrick, 2004
, 2005
; Trapnell et al., 2004
). Epiphytic species might have a different pattern of genetic structure because of the particular ecological characteristics of this habitat. The few studies in epiphytic orchids have shown that these plants in general harbor high genetic diversity and low population structure. Probably a high genetic diversity can be an adaptive advantage for epiphytic plants that live in that discontinuous and changing habitat.
In this study, an endemic and endangered epiphytic Mexican orchid, Laelia speciosa (HBK) Schltr. (Orchidaceae), was chosen to assess its population genetic diversity and structure throughout its geographic distribution. Additionally, we want to detect those populations that are genetically unique and therefore deserve high-priority protection. Thousands of plants of L. speciosa are harvested each year from their habitat to be sold in the streets and markets in Mexico City and several other cities and towns in Mexico. The plants are grown in home gardens for the beauty of flowers, which do not last when cut, and they are also used in religious ceremonies (Salazar-Chávez, 1996
; Halbinger and Soto, 1997
; I. Ávila-Díaz, personal observation). This massive harvesting has caused local extinctions, but fortunately, large populations can still be found in some localities (Halbinger and Soto, 1997
; I. Ávila-Díaz, personal observation). Demographic studies of L. speciosa in populations with different levels of disturbance have shown that disturbed populations are more prone to extinction than are less disturbed ones (Hérnández-Apolinar, 1992
; Pérez-Pérez, 2003
). Thus, if this illegal traffic continues, many populations will be exterminated in the near future (Halbinger and Soto, 1997
; Pérez-Pérez, 2003
).
This work is part of a larger project that involves the study of other aspects of the biology of L. speciosa, as well as work with local civilian communities to establish a sustainable management program for this orchid.
MATERIALS AND METHODS
Plant species and study sites
Laelia speciosa is a long-lived perennial epiphyte with globular or ovoid pseudobulbs (inflated stem tissues), which carry one stiff, terminal leaf. The inflorescence is 15–25 cm in length, which bears 1–3 large, pale to dark pink-lilac to purplish flowers, 10–16 cm in diameter (Halbinger and Soto, 1997
). The flowers are primarily outcrossing, but they are also capable of selfing (I. Ávila-Díaz and K. Oyama, unpublished data). Flowers are pollinated by bumblebee queens of Bombus pennsylvanicus sonorous Say and B. ephippiatus Say (Medina, 2004
).
The plants grow on oak species, particularly on Quercus deserticola Trel. in open, deciduous forests, from 1440 to 2500 m a.s.l. (Halbinger and Soto, 1997
). Laelia speciosa is an endemic species of the central part of Mexico that includes oak forests of the Sierra Madre Occidental, Sierra Madre Oriental, the southern part of the Altiplanicie Mexicana (Mexican Plateau), and the Eje Neovolcánico Transversal (Trans-Mexican Volcanic Belt) (Halbinger and Soto, 1997
).
Nine different populations of L. speciosa were sampled throughout its geographic range (Fig. 1). In each population, 50 reproductive individuals (each from a different host tree) were sampled, except for one site (Lobera, Jalisco; LOJ) in which only 17 individuals were collected because that was the total number of support trees found. From each individual cluster, 3–5 pseudobulbs were collected and transported to a shade house where they were cultivated and maintained for 2 years under similar conditions until used for electrophoresis. This was done to reduce the effects that different environments can have on differential protein expression (Wendel and Weeden, 1989
).
|
). The extracts were adsorbed onto Whatman no. 3 filter paper and then applied to a starch gel 12% w/v (Starch Art Co., Smithville, Texas, USA). We tested different buffer systems and obtained the best resolution for more enzymes with three systems: (1) C system modified from Stuber et al. (1988)
: electrode buffer pH 8.3 with 0.19 M boric acid, 0.04 M lithium hydroxide and gel buffer: 9:1 Trizma base (Sigma, St. Louis, Missouri, USA) buffer pH 8.3 and electrode buffer; (2) D system modified from Stuber et al. (1988)
: electrode buffer pH 6.5 with 0.070 M l-histidine, 0.007 M citric acid monohydrate and gel buffer: 1 : 4 electrode buffer and distilled water; (3) Mitton system (Mitton et al., 1979
): electrode buffer pH 7.5 with 0.031 M sodium hydroxide, 0.295 M boric acid and gel buffer: 0.015 M Trizma base, 0.295 M citric acid monohydrate. Standard electrophoresis was performed until the inner marker (bromophenol blue) reached 6 to 7 cm from the application site using the following running conditions: systems C and D: 30 mA; Mitton system: 225 V. Eleven enzymatic systems with 16 loci were well resolved with the following buffer systems:, glutamate oxaloacetate transaminase (Got2), leucine aminopeptidase (Lap1, Lap2), and esterase (Est2) with buffer C system; malate dehydrogenase (Mdh1, Mdh3), phosphoglucomutase (Pgm3, Pgm4), phosphoglucoisomerase (Pgi2, Pgi3), isocitrate dehydrogenase (Idh1, Idh2), and 6-phosphogluconate dehydrogenase (6Pgd2) with buffer D system; and cathodic peroxidase (Cpx1), diaphorase (Dia2), and menadione reductase (Mnr3) with the Mitton buffer system.
The staining recipes were modified from Wendel and Weeden (1989)
and Luna-Reyes and Oyama (2005)
. Enzymatic systems with more than one locus were numbered in ascending order from the locus with more mobility. The alleles were numbered according to their mobility relative to the alleles of standard individuals present in all gels in each system.
Data analysis
The following indices of genetic variation were computed: percentage of polymorphic loci (Pp), mean number of alleles per locus (A), and the average observed (HO) and expected (HE) heterozygosities. Deviations from Hardy–Weinberg equilibrium (HWE) were tested using an exact test with a Monte Carlo method. F statistics according to Weir and Cockerham (1984)
were estimated. The fixation indices, f (equivalent to FIS) and F (equivalent to FIT) were calculated for each polymorphic locus for the total population (including all sites) and averaged over all loci. Excess of homozygosity is indicated by statistically significant positive values of f and F. Li and Horvitz's (1953)
2 statistic was used to test whether f and F values per locus were significantly different from zero. As an indicator of the degree of differentiation among populations, FST (unbiased estimate ) was calculated. The values may indicate from equal ( = 0) up to entirely different ( = 1) allele frequencies among populations. Significance of per locus was determined by 2 = 2 N (k – 1), df = (k – 1) (s – 1) where N is the sample size, k is the number of alleles, and s = number of subpopulations (Workman and Niswander, 1970
). Bootstrapping over loci was performed using 1000 replicates to generate 95% confidence intervals (CIs). Also Nei's (1978)
unbiased genetic distance was calculated, and genetic differentiation between pairwise populations was illustrated in an UPGMA (unweighted pair group method with arithmetic mean) dendrogram (Sneath and Sokal, 1973
). Heterogeneity in allele frequencies among populations was tested by exact test (Raymond and Rousset, 1995
). Populations were geo-referenced and pairwise geographic distances were calculated with the ArcView program (ESRI, 1996
). Correlation between genetic distance and Euclidean distance between population pairs was assessed and statistically evaluated by Mantel's test with 1000 permutations. Analyses were carried out by using the TFPGA computer program (Miller, 1997
). Distribution of private and rare alleles were illustrated with Venn diagrams.
RESULTS
Genetic variability
Using 11 enzymatic systems, we obtained 16 loci with a total of 77 alleles (Appendix). Several loci were monomorphic in some of the populations, while others were polymorphic in all populations, with at least six alleles present in six of the loci. Pgi2 and Pgi3 were the most polymorphic, with nine alleles each. The populations had moderate to high levels of genetic variability: percentage of polymorphic loci (Pp) ranged from 68.8% to 81.3%; the mean number of alleles per locus (A) was between 3.13 and 3.74; the observed heterozygosity (HO) varied from 0.263 to 0.358, and the mean expected heterozygosity (HE) ranged from 0.333 to 0.431 (Table 1).
|
|
= 0.040, P < 0.001), indicating detectable allelic frequency differences among sites. Nei's (1978)
unbiased genetic distance ranged from 0.007 between the OLM (Olvido, Michoacán) and INM (Indaparapeo, Michoacán) populations to 0.064 between the XIG (Xichú, Guanajuato) and AMD (Amole, Durango) populations (Table 3). The UPGMA dendrogram was partially congruent with the location of populations according to the mountain ranges in Mexico (Fig. 2). The AMD population, the northernmost population studied, was the most different from all populations, as clearly shown in Fig. 2. The two populations in the Sierra Madre Oriental (Tula, Tamaulipas [TUT] and Hualula, Hidalgo [HUH]) formed a group, while the populations from Eje Neovolcánico Transversal in the state of Michoacán (OLM, INM) formed another group, next to MAJ (Mazamitla, Jalisco) from the same mountain range.
|
|
There was a positive significant correlation between pairwise genetic distance and euclidean distance between sites (Fig. 3; Mantel's test, r2 = 0.43; P < 0.05).
|
|
Genetic variability
Our results on the genetic diversity of populations of L. speciosa suggest that this orchid has higher levels of genetic variation (Pp = 76.4%, A = 3.34, HE = 0.382) than those reported for monocots (Pp = 59.20%, A = 2.38, HE = 0.181), long-lived perennial herbaceous species (Pp = 39.60%, A = 1.42, HE = 0.205), plants with wide geographic range (Pp = 58.90%, A = 2.29, HE = 0.202), plants with seeds dispersed by wind (Pp = 55.4%, A = 2.10, HE = 0.144), plants with sexual reproduction (Pp = 51.60%, A = 2.00, HE = 0.151) (Hamrick and Godt, 1990
), and terrestrial orchids (Scacchi and De Agelis, 1989; Case, 1993
; Sun, 1996
; Sharma et al., 2003
). Our values, however, were within those found for epiphytic and rupicolous orchids, which in general have high genetic diversity (Ackerman and Ward, 1999
; Bush et al., 1999
; Borba et al., 2001a
; Murren, 2003
; Trapnell and Hamrick, 2004
; Trapnell et al., 2004
). This pattern is also observed in other common epiphytic plants such as bromeliads (Soltis et al., 1987
; González-Astorga et al., 2004
), ferns (Ranker, 1992
; Hooper and Haufler, 1997
), and bryophytes (Akiyama, 1994
; Snäll et al., 2004
).
The high genetic diversity of L. speciosa can be explained in different ways. It is primarily an outcrossing plant, although it is also capable of selfing (I. Ávila-Díaz and K. Oyama, unpublished data) similarly to what has been reported for many other orchids (Borba et al., 2001b
; Wallace, 2003
; Trapnell and Hamrick, 2005
; Tremblay et al., 2005
; Torres, 2006
), favoring the increase of genetic variation in their populations. Orchids produce thousands of tiny seeds dispersed by wind that colonize new sites enhancing gene flow between populations (Dressler, 1981
; Murren and Ellison, 1998
; Trapnell and Hamrick, 2004
; Trapnell et al., 2004
). Additionally, its high level of genetic diversity could be related to its large geographic distribution and the fact that different generations can be found at a particular time due to its long-lived perennial habit, leading to the sampling of multi-aged populations.
Plants distributed in forest canopies follow different life history strategies compared with terrestrial plants, which might be reflected in their population structure. Recent studies considering epiphytic plants as metapopulations have shown that population substructure can occur at the level of individual trees (Trapnell et al., 2004
). At this level, inbreeding depression by mating between relatives and low distance gene flow can occur. The combination of both outcrossing and selfing reproduction in the same individual may offer an advantage to the individual by ensuring the production of offspring even under adverse environmental conditions or in colonization events when there is variable pollinator availability (Kalisz et al., 1999
, 2004
; Vogler and Kalisz, 2001
; Kalisz and Vogler, 2003
). The relative proportion of these two modes of reproduction is species dependent, and Benzing (1978)
has even suggested that obligate epiphytes would be characterized by increased autogamy to ensure high seed set. This mixed breeding system is present in L. speciosa (I. Ávila-Díaz and K. Oyama, unpublished data), a species in which even though outcrossing is the most common mechanism to produce seeds, autogamy and mating between relatives could exist, explaining the moderate levels of f (FIS) (0.216). The proportion of flowers successfully producing fruits in L. speciosa has been reported between 4.97% and 15.15% in populations with different degrees of disturbance (Medina, 2004
). These values correspond to those found in other orchid species in which, within a season, few flowering individuals produce fruits contributing to the seed pool, which is particularly true in populations of tropical orchids (Tremblay and Ackerman, 2001
; Tremblay et al., 2005
). The small number of individuals that successfully produce fruits per year and the fact that only some plants achieve reproductive success over many years (I. Ávila-Díaz, personal observation) translate into a greater chance of mating among relatives, as reported for Catasetum viridiflavum Hooker (Murren, 2003
).
Genetically variable outcrossing species whose population sizes have been recently reduced may be affected more severely by genetic drift and inbreeding than those species that historically have been considered rare (Kay, 1993
). Those effects would tend to accelerate the extinction of populations that have been strongly extracted from their habitat, as in the case of L. speciosa and many other epiphytic orchid species, which despite having high genetic variation could be endangered.
Genetic structure
In general, L. speciosa had a lower (FST) value among its populations than those reported for terrestrial orchids (Alexandersson and Ågren, 2000
; Tremblay et al., 2005
) and a slightly lower value than that reported for epiphytic orchids (Murren, 2003
; Trapnell and Hamrick, 2004
). It has been suggested that common epiphytic plants have low population structure in comparison to terrestrial plants, possibly related to the effective wind dispersal of spores and seeds from the canopy habitat (Akiyama, 1994
; Murren and Ellison, 1998
; Bush et al., 1999
; Trapnell and Hamrick, 2004
; Trapnell et al., 2004
) or to the behavior of the pollinator, leading to long-distance pollen movement (Alexandersson and Ågren, 2000
; Trapnell and Hamrick, 2005
). In the particular case of L. speciosa, the pollinators are bumblebee queens of Bombus pennsylvanicus sonorous and B. ephippiatus (Medina, 2004
).
Gene flow in L. speciosa is limited because of the great distance between the populations (in our study, the greatest Euclidean distance between two of the populations studied was 776 km) as shown by a positive linear relationship between genetic and geographic distances between sites. This event has been interpreted as a result of regional equilibrium between gene flow and drift (Hutchinson and Templeton, 1999
). Such isolation by distance also explains why the populations differ genetically, as shown by exact testing for population differentiation (Raymond and Rousset, 1995
). The isolation of the populations restricted by gene flow can be natural or a direct consequence of human activities such as forest fragmentation and massive extraction of plants leading to local extinction.
The UPGMA analysis shows an interesting pattern among the populations studied. Populations from the Eje Neovolcánico Transversal are the closest populations and the most similar genetically. It is possible those populations have had the same origin and still could maintain some levels of gene flow among them. Populations from Sierra Madre Oriental are different and isolated from those of Sierra Madre Occidental by the central Mexican Plateau, a very dry area, that restricts gene flow between populations from these mountain ranges. The most distinct population was the one from Durango (AMD), which is located in the limits of the northernmost distribution area of the species. The LOJ population, from the Sierra Madre Occidental, is more similar genetically to the populations from the Eje Neovolcánico Transversal. This population is the smallest sampled and is relatively close (approximately 5 km) to that mountain range, hence the possibility that its origin is due to the natural or human dispersion from the same populations native to the Eje Neovolcánico Transversal.
Private and rare alleles
The discovery of 10 private alleles in some populations of L. speciosa adds weight to the distinctness of populations, and the alleles may represent unique evolutionary trajectories, as proposed for other rare plants (Arft and Ranker, 1998
). Some authors have argued that private or rare alleles are of adaptive or evolutionary significance, perhaps representing possible loci of adaptive value, that is, a reservoir for adaptation to unusual conditions (Huenneke, 1991
; Torres et al., 2003
), so the evaluation of the significance of individual populations can be based on both levels of allelic richness and distinctiveness (Petit et al., 1998
).
A certain pattern of rare alleles is also observed in relation to the geographic distribution of the orchid, with populations growing in the same mountain range sharing one or more alleles between them. The populations from Sierra Madre Oriental and Sierra Madre Occidental are naturally isolated from each other because the Mexican Plateau between them is not favorable for the growing of Quercus deserticola, the main host of the orchid. The Eje Neovolcánico Transversal acts as a corridor connecting the populations from both mountain ranges, as shown by the alleles that are shared between the populations in this mountain range and the ones in the sierras.
Implications for conservation
We emphasize that populations of L. speciosa maintain high levels of genetic diversity throughout its range with low genetic differentiation. This is interesting, considering that the populations sampled have been affected by forest fragmentation and different levels of removal of individual plants and flowers by local people. Therefore, the impact of human activities on the genetic diversity is not quite clear yet, but perhaps it will become more evident in future generations.
When flowers are harvested, the recruitment of new individuals by seed is reduced, as shown by lower rates of population growth in intensively collected sites compared with those of pristine populations (Hernández-Apolinar, 1992
; Pérez-Pérez, 2003
). However, on many occasions the vegetative structures of individual orchids remain in place, perhaps serving as a genetic reservoir of the species. These populations could, in time, face problems associated with low recruitment.
Because of the high levels of genetic diversity and the genetic structure found, we suggest that when considering restoration strategies, particularly for reintroduction, it is important to restore with individuals propagated in vitro from seeds from as many capsules as possible and from populations close to the area where the reintroduction needs to take place to maintain the natural structure found in the field.
Because private and rare alleles are present in most of the populations, proper management involves protection and maintenance of genetic diversity throughout the range of L. speciosa for in situ conservation. We also suggest an ex situ conservation strategy with the collection of seeds and propagation of individuals from all populations.
A multidisciplinary project on L. speciosa that includes biological studies (e.g., demography, breeding system, in vitro propagation, seedling establishment, and relationship with mycorrhizal fungi) as well as work with people in local communities (e.g., environmental education programs, orchid propagation programs) is needed to reach a sustainable management of this orchid species. Other immediate actions are also necessary to preserve the habitat of this orchid throughout its geographic range and to enhance regulations and acts to reduce their illegal removal from the wild.
APPENDIX
Allelic frequencies in nine populations of Laelia speciosa. See Table 1 for the names of the populations. N = sample size
|
1 The authors thank A. Gónzález Rodríguez and A. C. Cortés-Palomec for comments on preliminary drafts of this manuscript; two anonymous reviewers for helpful suggestions; J. Serrato-R., O. Muníz-Serrato, and M.A. Pérez-Pérez for field assistance; R. Luna, N. Pérez, L. Herrera-Arroyo, G. Guerrero-Pacheco, and A. Valencia for technical assistance; and Monica McGloin for her help with English. This research was supported by Fondo Mexicano para la Conservación de la Naturaleza (FMCN, project A 1-99/130) and Consejo Nacional de Ciencia y Tecnología (SEMARNAT-CONACYT, project 2002-C01-0544); I.A.-D. was sponsored by doctoral scholarships from CONACYT (reg. 130249) and DGEP-UNAM (project 202392). 
4 Author for correspondence (iavila{at}oikos.unam.mx
)
LITERATURE CITED
Ackerman J. D. Ward S.. 1999. Genetic variation in a widespread, epiphytic orchid: where is the evolutionary potential?. Systematic Botany 24: 282-291.[CrossRef][ISI]
Akiyama H.. 1994. Allozyme variability within and among populations of the epiphytic moss Leucodon (Leucodontaceae: Musci). American Journal of Botany 81: 1280-1287.[CrossRef][ISI]
Alexandersson R. Ågren J.. 2000. Genetic structure in the nonrewarding, bumblebee-pollinated orchid Calypso bulbosa. Heredity 85: 401-409.
Arft A. M. Ranker T. A.. 1998. Allopolyploid origin and population genetics of the rare orchid Spiranthes diluvialis. American Journal of Botany 85: 110-122.[Abstract]
Barret S. C. H. Kohn J. K.. 1991. Genetic and evolutionary consequences of small population size in plants: implications for conservation. In D. A. Falk and K. E. Holsinger [eds.] Genetics and conservation of rare plants 3-30 Oxford University Press, Oxford, UK.
Benzing D. H.. 1978. The life history of Tillandsia circinnata (Bromeliaceae) and the rarity of extreme epiphytism among the angiosperms. Selbyana 2: 325-327.
Borba E. L. Felix J. M. Solferini V. N. Semir J.. 2001a. Fly-pollinated Pleurothallis (Orchidaceae) species have high genetic variability: evidence from isozyme markers. American Journal of Botany 88: 419-428.
Borba E. L. Semir S. Shepherd G. J.. 2001b. Self-incompatibility, inbreeding depression and crossing potential in five Brazilian Pleurothallis (Orchidaceae) species. Annals of Botany 88: 89-99.
Bowen B. W.. 1999. Preserving genes, species, or ecosystems? Healing the fractured foundations of conservation policy. Molecular Ecology 8: S5-S10.[CrossRef][Medline]
Bush S. P. Kutz W. E. Anderton J. M.. 1999. RAPD variation in temperate populations of the epiphytic orchid Epidendrum conopseum and the epiphytic fern Pleopeltis polypodioides. Selbyana 20: 120-124.
Case M. A.. 1993. High levels of allozyme variation within Cypripedium calceolus (Orchidaceae) and low levels of divergence among its varieties. Systematic Botany 18: 663-677.[CrossRef][ISI]
Case M. A. Mlodozeniec H. T. Wallace L. E. Weldy T. W.. 1998. Conservation genetics and taxonomic status of the rare Kentucky lady's slipper: Cypripedium kentuckiense (Orchidaceae). American Journal of Botany 85: 1779-1786.
Cozzolino S. Cafasso D. Pellegrino G. Musacchio A. Widmer A.. 2003. Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation. Molecular Ecology 12: 2783-2792.[CrossRef][Medline]
Dressler R. L.. 1981. The orchids: natural history and classification Harvard University Press, Cambridge, Massachusetts, USA.
[ESRI] Environmental Systems Research Institute.. 1996. Arc View program, version 3.2a Website http://www.esri.com/.
Fischer M. Matthies D.. 1998. RAPD variation in relation to population size and plant fitness in the rare Gentianella germanica (Gentianaceae). American Journal of Botany 85: 811-819.[Abstract]
Frankham R.. 1995. Conservation genetics. Annual Reviews Genetics 29: 305-327.[CrossRef]
González-Astorga J. Cruz-Angón A. Flore-Palacios A. Vovides A. P.. 2004. Diversity and genetic structure of the Mexican endemic epiphyte Tillandsia achyrostachys E. Morr. ex Baker var. achyrostachys (Bromeliaceae). Annals of Botany 94: 545-551.
Groom M. J.. 1998. Allee effects limit population viability of an annual plant. American Naturalist 151: 487-496.[CrossRef][ISI]
Gustafsson S.. 2000. Patterns of genetic variation in Gymnadenia conopsea, the fragrant orchid. Molecular Ecology 9: 1863-1872.[CrossRef][Medline]
Hágsater E. Soto Arenas M. Á. Salazar Chávez G. A. Jimenez Machorro R. López Rosas M. A. Dressler R. L.. 2005. Las orquídeas de México Instituto Chinoín, Mexico City, Mexico.
Haig M. S.. 1998. Molecular contributions to conservation. Ecology 79: 413-425.[CrossRef][ISI]
Halbinger F. Soto M.. 1997. Laelia speciosa (H.B.K.) Schltr. In E. Hágsater, M. Soto, E. Greenwood, R. L. Dressler, P. J. Cribb, J. Rzedowski, P. M. Catling, C. J. Sheviak, and F. Chiang [eds.] Laelias of México. Orquídea (Méx.), vol. 15 133-142 Asociación Mexicana de Orquideología, Mexico City, Mexico.
Hamrick J. L. Godt M. J. W.. 1990. Allozyme diversity in plant species. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir [eds.] Plant population genetics, breeding, and genetic resources 43-63 Sinauer, Sunderland, Massachusetts, USA.
Hamrick J. L. Godt M. J. W.. 1996. Conservation genetics of endemic plant species. In J. C. Avise and J. L. Hamrick [eds.] Conservation genetics case histories from nature Chapman and Hall, New York, New York, USA.
Hamrick J. L. Godt M. J. W. Murawski D. A. Loveless M. D.. 1991. Correlations between species traits and allozyme diversity: implications for conservation biology. In D. A. Falk and K. E. Holsinger [eds.] Genetics and conservation of rare plants 75-86 Oxford University Press, Oxford, UK.
Hernández-Apolinar M.. 1992. Dinámica poblacional de Laelia speciosa (HBK) Schltr. (Orchidaceae) B.Sc. thesis, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Hooper E. A. Haufler C. H.. 1997. Genetic diversity and breeding system in a group of neotropical epiphytic ferns (Pleopeltis; Polypodiaceae). American Journal of Botany 84: 1664-1674.[Abstract]
Huenneke L. F.. 1991. Ecological implications of genetic variation in plant populations. In D. A. Falk and K. E. Holsinger [eds.] Genetics and conservation of rare plants 31-44 Oxford University Press, New York, New York, USA.
Hutchinson D. W. Templeton A. R.. 1999. Correlation of pairwise genetic and geographic distance measures: inferring the relative influences of gene flow and drift on the distribution of genetic variability. Evolution 53: 1898-1914.[CrossRef][ISI]
Izquierdo L. Y.. 1995. Estructura y variación genética en cuatro especies de Aechmea (Bromeliaceae) en México: A. mexicana (Baker), A. lueddemanniana (K. Koch) Brong. ex Mez in Engl, Pflanzenr., A. macvaughii, L. B. Smith y A. tuitensis (P. Magaña y E. Lott) Ph.D. thesis, Universidad Nacional Autonóma de México, Mexico City, Mexico.
Kalisz S. Vogler D. Fails B. Finer M. Shepard E. Herman T. Gonzales R.. 1999. The mechanism of delayed selfing in Collinsia verna (Scrophulariaceae). American Journal of Botany 86: 1239-1247.
Kalisz S. Vogler D. W.. 2003. Benefits of autonomous selfing under unpredictable pollinator environments. Ecology 84: 2928-2942.[CrossRef][ISI]
Kalisz S. Vogler D. W. Hanley K. M.. 2004. Context-dependent autonomous self-fertilization yields reproductive assurance and mixed mating. Nature 430: 884-886.[CrossRef][Medline]
Kay Q.. 1993. Genetic differences between populations of rare plants: implications for recovery progammes. Botanical Society of the British Isles News 64: 54-56.
Li C. C. Horvitz D. G.. 1953. Some methods of estimating the inbreeding coefficient. American Journal of Human Genetics 5: 107-117.[ISI][Medline]
Luna-Reyes R. Epperson B. K. Oyama K.. 2005. Spatial genetic structure of two sympatric Neotropical palms with contrasting life histories. Heredity 95: 298-305.[CrossRef][ISI][Medline]
Medina N. D.. 2004. Éxito reproductivo en dos poblaciones de Laelia speciosa (HBK) Schltr. (Orchidaceae), en Michoacán, México B. Sc. thesis, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, Mexico.
Miller M. P.. 1997. Tools for population genetic analysis (TFPGA), version 1.3: a windows program for the analysis of allozyme and molecular population genetic data Computer program available at website http://www.marksgeneticsoftware.net.
Mitton J. B. Linhart Y. B. Sturgeon K. B. Hamrick J. L.. 1979. Allozyme polymorphisms detected in mature needle tissue of ponderosa pine. Journal of Heredity 70: 86-89.
Murren C. J.. 2003. Spatial and demographic population genetic structure in Catasetum viridiflavum across a human-disturbed habitat. Journal of Evolutionary Biology 16: 333-342.[CrossRef][ISI][Medline]
Murren C. J. Ellison A. M.. 1998. Seed dispersal characteristics of Brassavola nodosa (Orchidaceae). American Journal of Botany 85: 675-680.[Abstract]
Nei M.. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 9: 583-590.
Oostermeijer J. G. B. van Ejick M. W. van Leeuwen N. C. den Nijs H. C. M.. 1995. Analysis of the relationship between allozyme heterozygosity and fitness in the rare Gentiana pneumonanthe L. Journal of Evolutionary Biology 8: 739-757.
Oyama K.. 1993. Conservation biology of tropical trees: demographic and genetic considerations. Environment Update 1: 17-32.
Pérez-Pérez M. A.. 2003. Demografía de Laelia speciosa (HBK) Schltr. (Orchidaceae) bajo diferentes condiciones de manejo en la zona centro-norte del estado de Michoacán M.Sc. thesis, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, Mexico.
Petit R. J. Mousadik A. E. Ponds O.. 1998. Identifying populations for conservation on the basis of genetic markers. Conservation Biology 12: 844-855.[CrossRef][ISI]
Qamaruz-Zaman F. Fay M. F. Parker J. S. Chase M. W.. 1998. Molecular techniques employed in the assessment of genetic diversity: a review focusing on orchid conservation. Lindleyana 13: 259-283.
Ranker T. A.. 1992. Genetic diversity of endemic Hawaiian epiphytic ferns: implications for conservation. Selbyana 13: 131-137.
Raymond M. L. Rousset F.. 1995. An exact test for population differentiation. Evolution 49: 1280-1283.[CrossRef][ISI]
Salazar-Chávez G. A.. 1996. Conservation threats. In IUCN/SSC Orchid Specialist Group [eds.] Orchids: status survey and conservation action plan, 6–10. International Union for the Conservation of Nature/Species Survival Commission Cambridge, UK.
Scacchi R. De Angelis G.. 1989. Isoenzyme polymorphisms in Gymnaedenia conopsea and its inferences for systematics within this species. Biochemical Systematics and Ecology 17: 25-33.
Scacchi R. De Angelis G. Corbo R. M.. 1991. Effect of the breeding system on the genetic structure in three Cephalanthera spp. (Orchidaceae). Plant Systematics and Evolution 176: 53-61.[CrossRef][ISI]
Schmidt K. Jensen K.. 2000. Genetic structure and AFLP variation of remnant populations in the rare plant Pedicularis palustris (Scrophulariaceae) and its relation to population size and reproductive components. American Journal of Botany 87: 678-689.
Sharma I. K. Jones D. L. French C. J.. 2003. Unusually high genetic variability revealed through allozymic polymorphism of an endemic and endangered Australian orchid, Pterostylis aff. picta (Orchidaceae). Biochemical Systematics and Ecology 31: 513-526.[CrossRef]
Snäll T. Fogelqvist J. Ribeiro P. J. Lascoux M.. 2004. Spatial genetic structure in two congeneric epiphytes with different dispersal strategies analysed by three different methods. Molecular Ecology 13: 2109-2119.[CrossRef][Medline]
Sneath P. H. Sokal R. R.. 1973. Numerical taxonomy. The principles and practice of numerical classification Freeman, San Francisco, California, USA.
Soltis D. E. Gilmartin A. J. Rieseberg L. Gardner S.. 1987. Genetic variation in the epiphytes Tillandsia ionantha and T. recurvata (Bromeliaceae). American Journal of Botany 74: 531-537.[CrossRef][ISI]
Squirrell J. Hollingsworth P. M. Bateman R. M. Dickson J. H. Light M. H. S. MacConaill M. Tebbitt M. C.. 2001. Partitioning and diversity of nuclear and organelle markers in native and introduced populations of Epipactis helleborine (Orchidaceae). American Journal of Botany 88: 1409-1418.
Stuber C. W. Wendel J. M. Goodman M. M.. 1988. Techniques and scoring procedures for starch gel electrophoresis of enzymes from maize (Zea mays). Technical Bulletin 286.: North Carolina State University, Raleigh, North Carolina, USA.
Sun M.. 1996. Effects of population size, mating system, and evolutionary origin on genetic diversity in Spiranthes sinensis and S. hongkongensis. Conservation Biology 10: 785-795.[CrossRef][ISI]
Sun M.. 1997. Genetic diversity in three colonizing orchids with contrasting mating systems. American Journal of Botany 84: 224-232.[Abstract]
Sun M. Wong K. C.. 2001. Genetic structure of three orchid species with contrasting breeding systems using RAPD and allozyme markers. American Journal of Botany 88: 2180-2188.
Szalanski A. L. Steinauer G. Bischof R. Petersen J.. 2001. Origin and conservation genetics of the threatened Ute ladies'-tresses, Spiranthes diluvialis (Orchidaceae). American Journal of Botany 88: 177-180.
Torres E. Iriondo J. M. Pérez C.. 2003. Genetic structure of an endangered plant, Antirrhinum microphyllum (Scrophulariaceae): allozyme and RAPD analysis. American Journal of Botany 90: 85-92.
Torres G. K. I.. 2006. Sistema y éxito reproductivo de Cuitlauzinia pendula La Llave y Lex. (Orchidaceae) en San Andrés Corú, Michoacán, México B.Sc. thesis, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacan, Mexico.
Trapnell D. W. Hamrick J. L.. 2004. Partitioning nuclear and chloroplast variation at multiple spatial scales in the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 13: 2655-2666.[CrossRef][Medline]
Trapnell D. W. Hamrick J. L.. 2005. Mating patterns and gene flow in the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 14: 75-84.[CrossRef][Medline]
Trapnell D. W. Hamrick J. L. Nason J. D.. 2004. Three-dimensional fine-scale genetic structure of the neotropical epiphytic orchid, Laelia rubescens. Molecular Ecology 13: 1111-1118.[CrossRef][Medline]
Tremblay R. L. Ackerman J. D.. 2001. Gene flow and effective population size in Lepanthes (Orchidaceae): a case for genetic drift. Biological Journal of the Linnean Society 72: 47-62.[CrossRef]
Tremblay R. L. Ackerman J. D. Zimmerman J. K. Calvo R. N.. 2005. Variation in sexual reproduction in orchids and its evolutionary consequences: a spasmodic journey to diversification. Biological Journal of the Linnean Society 84: 1-54.[CrossRef]
Vogler D. W. Kalisz S.. 2001. Sex among the flowers: the distribution of plant mating systems. Evolution 55: 202-204.[CrossRef][ISI][Medline]
Wallace L. E.. 2002. Examining the effects of fragmentation on genetic variation in Platanthera leucophaea (Orchidaceae): inferences from allozyme and random amplified polymorphic DNA markers. Plant Species Biology 17: 37-49.
Wallace L. E.. 2003. The cost of inbreeding in Platanthera leucophaea (Orchidaceae). American Journal of Botany 90: 235-242.
Wallace L. E.. 2004. A comparison of genetic variation and structure in the allopolyploid Platanthera huronensis and its diploid progenitors, Platanthera aquilonis and Platanthera dilatata (Orchidaceae). Canadian Journal of Botany 82: 244-252.[CrossRef]
Weir B. S. Cockerham C. C.. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38: 1358-1370.[CrossRef][ISI]
Wendel J. F. Weeden N. F.. 1989. Visualization and interpretation of plant isozymes. In D. E. Soltis and P. S. Soltis [eds.] Isozymes in plant biology 5-45 Dioscorides Press, Portland, Oregon, USA.
Wong K. C. Sun M.. 1999. Reproductive biology and conservation genetics of Goodyera procera (Orchidaceae). American Journal of Botany 86: 1406-1413.
Workman P. L. Niswander J. D.. 1970. Population studies on southwestern Indian tribes. II. Local genetic differentiation in the Papago. American Journal of Human Genetics 22: 24-49.[ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
