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1 Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70–275, México, D.F. 04510, México
Received for publication February 16, 1999. Accepted for publication June 22, 1999.
ABSTRACT
Populations of Abies in southern Mexico and Guatemala (A. flinckii, A. guatemalensis, A. hickeli, and A. religiosa) have a patchy distribution. This pattern is particularly clear in A. guatemalensis. Genetic diversity within populations, measured by average heterozygosity at 16 isozyme loci, is lower than the range reported for most conifers (mean Ho ranging from 0.069 in A. guatemalensis to 0.113 in A. flinckii), while differentiation among populations is higher than that observed in most conifer species studied (
= Fst ranging from 0.073 in A. hickeli to 0.271 in A. flinckii). Estimated levels of gene flow are low (ranging from 0.672 in A. flinckii to 3.17 in A. hickeli). Populations in most cases had an excess of homozygosity over that expected under Hardy-Weinberg equilibrium, suggesting some inbreeding (Fis ranging from 0.074 in A. flinckii to 0.235 in A. guatemalensis). A significant relationship between gene flow and geographic distance was observed in A. religiosa, but not in the other three taxa studied. The patterns of genetic variation appear to have been influenced by the distributions and histories of these species. Paleoclimatic evidence suggests that the ranges of these species retreated upwards during the Pleistocene glaciation and became fragmented during the warming period that followed. The populations could have passed through genetic bottlenecks that reduced genetic variation and led to interpopulation differentiation.
Key Words: Abies flinckii • Abies guatemalensis • Abies hickeli • Abies religiosa • gene flow • genetic variation • genetic drift • inbreeding
Most conifers studied have high levels of genetic diversity, as measured by isozymes (Hamrick, Godt, and Sherman-Broyles, 1992
; Fady and Conkle, 1993
; Matusova, 1995
; El-Kassaby and Ritland, 1996
). It has also been found that the majority of forest tree species show low levels of isozyme differentiation among populations. These studies have mostly been conducted with economically important conifers of widespread and often continuous distributions. The high within-population genetic diversity and low among-population differentiation observed in conifers have been attributed to common life-history traits, such as longevity and extensive gene flow (Hamrick, Godt, and Sherman-Broyles, 1992
; Streiff et al., 1998
). However, the biogeographic history of a species should also contribute significantly to current patterns of genetic variation.
The Abies species of southern Mexico and Guatemala provide an excellent opportunity to study the effects of species distributions on patterns of genetic variation because the different species have contrasting geographical distributions. There are ~50 species of Abies (firs) in the world, distributed in the temperate regions of the Northern Hemisphere (Welch, 1991
). Of these, Martínez (1948)
listed eight species as occurring in Mexico, six of them endemic to the country. Liu (1971)
reduced this number to six species, four of them endemic to Mexico. Farjon (1990)
listed six species in Mexico, four of them endemic to the country. Rushforth (1989)
has since described two additional species from western Mexico, and Debreczy and Racz (1995)
have described three more.
We considered four species of Abies that occur in southern Mexico and Guatemala. Abies guatemalensis Rehder is the southernmost representative of the genus, distributed at altitudes between 2000 and 4000 m in El Salvador, Guatemala, Honduras, and the southern Mexican states of Chiapas, Oaxaca, Guerrero, Jalisco, Hidalgo, and San Luis Potosí (Martínez, 1948
; Donahue et al., 1985
). Although widely distributed, it has relatively few isolated populations and is considered a threatened species (FAO, 1986
). Abies religiosa (H. B. K.) Schl. et Cham. is distributed principally above 2000 m along the Transversal Volcanic Belt, in the states of Guerrero, Jalisco, Michoacán, México, Morelos, Hidalgo, Tlaxcala, Puebla, and Veracruz and the Distrito Federal (Martínez, 1948
). Its distribution is more continuous than that of A. guatemalensis. Abies religiosa var. emarginata Martínez is distributed in the states of Jalisco and Michoacán, and A. guatemalensis var. jaliscana Martínez has a very restricted distribution in western Jalisco. Martínez (1948)
described these two varieties, but Rushforth (1989)
subsequently combined them to form a new species, A. flinckii Rushforth. Our field observations suggest that these populations are indeed morphologically similar to each other and distinct from A. religiosa and A. guatemalensis. They differ even more strikingly in phenology from the latter two species, beginning vegetative growth as much as 3 mo earlier. The maintenance of very different morphologies and phenologies in trees of A. flinckii and A. religiosa intermixed at the same site in the Sierra de Manantlán strongly suggests that these are reproductively isolated separate species. Abies hickeli Flous et Gaussen has a limited range, with a few populations in the states of Oaxaca and Veracruz (Martínez, 1948
).
In this study, we used isozyme markers to examine the levels and patterns of genetic variation in the Abies of southern Mexico and Guatemala. In particular, we were interested in the effects of species distribution and historical factors, such as habitat fragmentation and isolation of populations due to climatic changes.
MATERIALS AND METHODS
Branch samples were collected from 33 populations of Abies in southern Mexico and Guatemala. Included were ten populations of A. guatemalensis, 11 of A. religiosa, six of A. flinckii, and six of A. hickeli (Fig. 1). These populations represent most of the geographic range covered by these species. Because of our need to collect vegetative buds, the short period in which Abies cones are intact before disintegrating, and the large geographic area we sampled, we were unable to sample cones from these populations in the field. The populations we sampled were, however, previously described based on vegetative and reproductive characteristics by Martínez (1948)
, Donahue et al. (1985)
, and/or samples in the Herbario Nacional (MEXU). Forty individuals were sampled in each population, when possible. In the laboratory, vegetative buds were removed from the branch samples and were stored at -80°C for further use in gel electrophoresis. We maintained branch samples from each population as vouchers.
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; Cheliak and Pitel, 1984
). Buds from each individual were ground with extraction buffer (3:1 [v:v] mixture of buffer YO from Yeh and O'Malley [1980
] and VegII from Pitel and Cheliak [1984
]). The extract was absorbed in 1.2 x 1.5 mm chromatographic paper wicks. The enzyme analysis was done using two systems of electrode and gel buffers. The H buffer (Cheliak and Pitel, 1984
) was used to assay shikimate dehydrogenase (SDH, Enzyme Commission number 1.1.1.25, two loci), isocitrate dehydrogenase (IDH, E.C. 1.1.1.41, one locus), phosphoglucomutase (PGM, E.C. 2.7.5.1, two loci), and menadione reductase (MNR, E.C. 1.6.99.2, one locus). The R buffer (Ridgeway, Sherburne, and Lewis, 1970
) was used to assay leucine amino-peptidase (LAP, E.C. 3.4.11.1, one locus), phosphoglucoisomerase (PGI, E.C. 5.3.1.9, two loci), glutamic-oxaloacetic transaminase (GOT, E.C. 2.6.1.1, one locus), peroxidase (PER, E.C. 1.11.1.7, one locus), peptidase (PEP, E.C. 3.4.11.11, three loci), glutamate dehydrogenase (GDH, E.C. 1.4.1.3, one locus), and ribulosebiphosphate carboxylase (RUB, E.C. 4.1.1.39, one locus). Starch gels (12%) were run at 60 mA for 6–7 h on the H system and 4 h on the R system. All genotypic data for all individuals in this study are in the databases of the Comisión para el Uso y Conocimiento de la Biodiversidad (CONABIO; http://www.conabio.gob.mx) in Mexico City.
Allelic frequencies were obtained for each locus in each population. Based on these data, levels of polymorphism (P), observed (Ho) and Hardy-Weinberg expected (He) heterozygosity, and mean number of alleles per locus (A) were estimated with the program BIOSYS (Swofford and Selander, 1981
). Wright's (1965)
F statistics, total inbreeding (Fit), subdivision among populations (Fst), and inbreeding within populations (Fis) were estimated for each species by the method described in Weir and Cockerham (1984)
(F = Fit, = Fst and f = Fis) , using the program FSTAT (version 1.2; Goudet, 1995
). Partitioning of variation within and among species was estimated using the WRIGHT78 procedure of the program BIOSYS (Swofford and Selander, 1981
).
An estimate of gene flow (Nm), the average effective number of migrants exchanged between populations in each generation, was calculated from , and also from the number and frequency of private alleles (Barton and Slatkin, 1986
). Wright (1951)
showed that in an island model, where equilibrium has been reached between genetic drift and migration, Nm = (1 - )/4, where is the proportion of total genetic diversity represented by differences among populations.
Nm can also be calculated from the number and frequency of private alleles, unique alleles found in only one population (Slatkin, 1985
), as follows:
 |
). This method of estimating gene flow is likely to be more sensitive to errors in data collection (Slatkin, 1994
).
The relationship of gene flow between pairs of populations (M) to geographic distance (Slatkin, 1993
) was examined. Values of M were obtained by computing (a measure of among-population differentiation; Weir and Cockerham, 1984
) for each pair of locations using a program provided by Slatkin (isolation by distance program). A regression of log10M on log10k (k = geographic distance) was then constructed to see if there was a linear relationship. The significance of the relationship was evaluated by Mantel's (1967)
test, since the usual statistical methods cannot be used because values of M from different pairs of populations are not independent. This was done for each taxon studied. Finally, we used PHYLIP (Felsenstein, 1995
) to construct a neighbor joining tree (Saitou and Nei, 1987
), based on Nei's unbiased genetic distances (Nei, 1978
). One hundred bootstrap samples over loci were used to estimate the confidence of the branch points.
RESULTS
Levels of polymorphism and expected average heterozygosity were lowest for A. guatemalensis, intermediate for A. hickeli, and highest for A. flinckii and A. religiosa (Table 1). The range of variation among populations in expected heterozygosity was particularly high in A. religiosa, which included the population with the lowest genetic diversity (R21, He = 0.008), as well as the population with the highest value (R54, He = 0.235).
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= Fst estimates for all species, particularly A. flinckii and A. religiosa (Table 2). The mean estimate of differentiation among populations (Fst = ) considering all species is 0.280, with 0.221 due to differences among populations within species and only 0.056 due to differences among species.
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for each species were 0.672 (95% confidence interval = 0.46–1.43) for A. flinckii, 1.8 (1.34–3.72) for A. guatemalensis, 0.75 (0.43–1.57) for A. religiosa, and 3.17 (1.83–11.65) for A. hickeli, with an overall mean of 1.6 (obtained with the mean , when we analyze all species together). Estimates of Nm calculated from the method of private alleles were 3.42 for A. flinckii, 2.88 for A. guatemalensis, 1.67 for A. religiosa, and 2.70 for A. hickeli. Estimates of Nm from private alleles were higher than estimates from . It has been shown that both methods can provide accurate estimates of Nm under several circumstances (Slatkin and Barton, 1989
), but the method of private alleles is more sensitive to errors in data collection and could be less accurate in practice (Slatkin, 1994
). Simulations done by Slatkin (1993)
show that in a stepping stone model, M (gene flow) and geographic distance should be inversely correlated, with a slope of -1 in linearly distributed populations. This pattern was observed in A. religiosa, but not in any of the other species (Fig. 2).
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) between populations of A. flinckii was 0.051 (SD = 0.035), between populations of A. guatemalensis 0.011 (0.009), between populations of A. religiosa 0.040 (0.033), and between populations of A. hickeli 0.010 (0.006). These estimates of genetic distances, particularly for A. religiosa and A. flinckii, are relatively high compared to other north-temperate conifers and show high differentiation between populations. This is in agreement with the estimates of . Considering all populations of all species together, the mean overall genetic distance was 0.040, similar to the mean genetic distances between populations within the same species. The phenogram constructed from these distances showed a monophyletic group for A. flinckii, within a group containing eight of the ten A. guatemalensis populations and distinct from the A. religiosa populations (Fig. 3). Abies hickeli populations from Oaxaca also formed a monophyletic group. Abies religiosa populations formed another monophyletic group that also included two populations of A. guatemalensis and the one population of A. hickeli from Veracruz. Within this group, there were two subgroups, one consisting of populations from the Mexico City area and west (G44, R12, R13, R15, R21, R22, R49, and R53) and the other consisting of populations principally from the Mexico City area and east (G52, H46, R45, R48, and R54, with R55 being the exception). Levels of confidence in the branch points of this phenogram are relatively low.
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The levels of genetic diversity we observed in these Abies species were lower than means reported from studies of other gymnosperms (P = 71.1, A = 1.83, He = 0.151), other temperate zone species (P = 63.5, A = 1.81, He = 0.166), and other species with outcrossing mating systems and wind-dispersed pollen (P = 53.0, A = 1.84, He = 0.173) (Hamrick, Godt, and Sherman-Broyles, 1992
). The estimates were also lower than those reported for other species of Abies (Table 3). There are relatively few reports of natural populations of tree species with very low levels of genetic variation. Such low levels of variation usually occur in species with very narrow geographic distributions, species that are inbreeding colonizers (Brown and Marshall, 1981
), or species with very small population sizes (Hartl and Clark, 1989
). In many populations we observed hundreds of individuals, enough to counteract the effects of genetic drift. In some of the populations, however, we observed relatively low total sizes, particularly for A. guatemalensis, a species in which we found lower levels of heterozygosity. Estimates of expected heterozygosity varied from almost twofold to almost 30-fold among populations within species (0.064–0.158 for A. flinckii, 0.044–0.094 for A. guatemalensis, 0.073–0.135 for A. hickeli, and 0.008–0.235 for A. religiosa), providing further evidence for the possible action of genetic drift on some of these populations.
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, indicating that a high portion of the total reduction in observed heterozygosity (Fit) is due to inbreeding within subpopulations, although interpopulational differentiation is also considerable, especially for A. guatemalensis. Fis was also high in A. religiosa (0.216), but lower than . While most conifers studied have displayed an excess of heterozygosity over that expected under Hardy-Weinberg equilibrium, heterozygote deficits have been reported for some predominantly outcrossing species (Parker and Hamrick, 1996
), including Abies (Shea, 1990
; Fady and Conkle, 1993
; Table 3).
Estimates of for the populations analyzed show higher amounts of genetic differentiation among populations within species than that found for most other conifers and other species of Abies. This should not be surprising given the general bias in the literature toward studies of widespread, economically important conifers of the Northern Hemisphere. Mean values were 0.271 for A. flinckii, 0.122 for A. guatemalensis, 0.073 for A. hickeli, and 0.250 for A. religiosa. The relatively lower value for A. hickeli may be due to its more restricted geographic distribution, with all but one of the populations occurring in central Oaxaca. Hamrick and Godt (1996a)
reported a mean Fst of 0.101 for a variety of species with wind-dispersed pollen, and Hamrick, Godt, and Sherman-Broyles (1992)
reported mean Fst values of 0.063 for seven species of Abies and 0.073 for a variety of gymnosperm species.
The relatively high values of for the Abies species of southern Mexico and Guatemala reflect spatial genetic structure and suggest that genetic drift or selection or both have been important forces in their evolution. These results also suggest restricted levels of gene flow. The estimated levels of gene flow (Nm) in these species are lower than those needed to counteract genetic drift (Nm > 4; Jorgensen and Hamrick, 1997
) and are lower than the mean values reported for other outcrossing species with wind-dispersed pollen (Hamrick, 1987
).
Gene flow in A. religiosa is somewhat geographically restricted, with long-distance dispersal not sufficiently common to prevent isolation by distance. The sampled populations have likely existed for a long time, sufficient to permit isolation by distance to become apparent (Slatkin, 1993
). The failure to detect an isolation by distance pattern in the other species could be due to continuing gene flow among populations, such as in the case of A. hickeli (Nm = 3.17), or due to the populations not being at drift-migration equilibrium because of historical events, such as recent range expansion or fragmentation of populations (Comes and Abbot, 1998
). Gene flow may have been so restricted between even the closest populations within A. flinckii and A. guatemalensis that the regression between M and distance was not sensitive enough to detect a pattern of isolation by distance. Also, the very scattered and isolated populations of A. guatemalensis may not meet the assumption of the isolation by distance model.
Our phenogram provisionally suggests the existence of three major groups, A. guatemalensis (including A. flinckii), A. hickeli, and A. religiosa. We must caution, however, that the bootstrap confidence levels in this tree are low, likely due to the low levels of variation in the species, and further data from more variable markers will be necessary to draw firm phylogenetic conclusions.
While the populations of Abies flinckii form a monophyletic group, they are embedded within the A. guatemalensis group, suggesting that these may comprise a single species, as suggested by Martínez (1948)
and Farjon (1990)
. This would not be entirely surprising, given the geographic distributions (Fig. 1) of the two species, but the great differences in phenology we observed between these species in the field suggest that they may be reproductively isolated. The clear difference between A. flinckii and A. religiosa is most strikingly apparent in populations F20 and R21, which represent trees mixed together on the same site in the Sierra de Manantlán. Although growing on the same site, their morphologies and phenologies are very different and no intermediates are found.
Branch lengths suggest that the populations of A. flinckii and the westernmost populations of A. religiosa (R21, R22, and R55) have undergone the highest degree of differentiation. These populations are quite isolated geographically and are not very large, conditions that may have permitted accelerated differentiation due to genetic drift. The relatively high value for A. religiosa may be due, in part, to the divergence between the western (e.g., R15, R21, R22, R49, and R53) and eastern (e.g., R45 and R48) populations that we observed in the tree.
All three of the populations that did not cluster with their conspecific populations (G44, G52, and H46) fall clearly within the reported geographic range of A. religiosa. The vegetative morphological differences between these populations and A. religiosa are not great and the species may have been misidentified by those who originally described these sites. In our field observations of vegetative characteristics, these three populations differed from A. religiosa principally in having leaves that were emarginate at the apex instead of acute, obtuse, or rounded. None of the populations showed evidence of being a mixture of species or hybrids. These populations are quite geographically isolated from their conspecific populations, and it is possible that they have undergone a very high degree of genetic drift and are now very different from the other populations in their species.
Population H4 corresponds to the type site for A. zapotekensis Debreczy, Rácz, and Ramírez, a new species described by Debreczy and Racz (1995)
. Our data suggest that it is a population of A. hickeli. Population R22 corresponds to the type site of A. colimensis Rushforth & Narave, a new species described by Rushforth (1989)
. Our data suggest that it is a population of A. religiosa, although the branch length suggests that it is a relatively more differentiated population. Population G10 corresponds to the type site of A. guatemalensis var. tacanensis (Lundell) Martínez. Our data suggest that it is a population of A. guatemalensis.
The short branch lengths separating the three major groups in the phenogram are consistent with the low amount of differentiation accounted for by differences among species (0.056). This low level of interspecific differentiation may be considered somewhat surprising, but high levels of genetic drift and low levels of gene flow among populations within species could generate high levels of intraspecific differentiation without necessarily leading to high levels of interspecific differentiation.
The effect of present geographic distribution on the patterns of genetic structure is not entirely clear. We found lower levels of genetic variation in A. guatemalensis than in the rest of the taxa, which was expected because of its smaller and more isolated populations. We also found indications of higher levels of inbreeding in this species. In contrast to what we expected, levels of interpopulational differentiation were higher in A. religiosa than in A. guatemalensis, although in both cases they were rather high.
In temperate species, cycles of glacial and interglacial periods likely have played an important role in shaping the present genetic variation patterns of plant populations (Konnert and Bergmann, 1995
). The last glacial period reached a peak between 25 000 and 18 000 yr ago (Roberts, 1998
). During this period, land temperatures dropped as much as 20°C and ocean temperatures ~5°C. These climatic changes led to changes in the distributions of many species, with species of the Northern Hemisphere displaced southwards. Afterwards, when warming began in the current interglacial period (~11 500 yr BP), species migrated northwards (Roberts, 1998
).
The genus Abies has a long history in Mexico, with pollen present in Mexico at least as far south as Veracruz (Paraje Solo Formation), in the middle Pliocene (five million years ago; Graham, 1993, 1999
). While we can estimate the current total sizes of these populations, we do not know their effective population sizes nor their historical sizes, both factors that can influence observed levels of variation. There are no detailed climatic data for Mexico during the glacial periods, but in general terms there is consistency in the data regarding the cold and dry climates during the glacial maximum (~18 000 yr BP; Lozano-García, 1993
). It is proposed that between 12 500 and 9000 yr BP, at the end of the Pleistocene, the climate was humid and colder than at present and forests reached their maximum development in Mexico (Lozano-García, 1993
). This suggests that during the last glaciation temperate species had a broader distribution than at present in Mexico, and populations of species that are now isolated could have been in contact. After 9000 yr BP, the climate became warmer (Lozano-García, 1993
), and populations could have been fragmented, becoming smaller and more isolated, with reduced effective population sizes (Ne).
The Abies species of southern Mexico and Guatemala may have experienced fragmentation of populations and range retractions with the warming trend that peaked ~6000 yr BP. Their populations could have been displaced to higher regions, possibly causing a greater degree of isolation between populations than we observe at present. These populations may have passed through a number of genetic bottlenecks that led to a loss of genetic diversity and interpopulational differentiation due to genetic drift. Small population sizes could also have led to inbreeding and the observed heterozygote deficiencies (Nei, Maruyama, and Chakraborty, 1975
). A number of the populations that we sampled have more than 100 individuals, which should be large enough to counteract genetic drift if all individuals reproduce, but Abies species are long-lived perennials with reproductive maturity at 20 yr and an average life span of 60 yr (Jacobs, Werth, and Guttman, 1984
). If new variation is accumulated in proportion to the number of generations (meiotic events) over time, then these species may not have had enough time to recover the variation that they lost due to genetic drift.
The majority of conifers studied by isozyme analysis display relatively high levels of genetic diversity and low levels of interpopulational differentiation compared to other groups of plants. They also generally display genotypic frequencies consistent with Hardy-Weinberg equilibrium or an excess of heterozygotes. These conifers are mostly species with relatively large ranges in the North Temperate zones of Asia, North America, north of Mexico, and Europe. These areas were heavily impacted by Pleistocene glaciation, with much of their areas covered by ice. The current distributions of many of these species are very recent, not giving them enough time to develop high levels of interpopulational differentiation. Many of them were able to maintain relatively large continuous populations south of the glacial front during the Pleistocene, avoiding drastic losses of variation due to genetic drift. Their current large, often continuous populations help to maintain variation and prevent interpopulational differentiation and inbreeding.
In contrast, we observed relatively low levels of genetic diversity and relatively high levels of heterozygote deficiency and interpopulational differentiation in the Abies species of southern Mexico and Guatemala, suggesting the action of genetic drift and relatively low levels of gene flow. These same patterns were also observed by Keiman-Freire (1997)
in a study of the Abies species of northern Mexico. Picea chihuahuana, another conifer with relatively small and isolated populations in northern Mexico, displayed the same pattern, with a low level of variation (He = 0.093), and high levels of heterozygote deficiency (Fis = 0.185) and interpopulational differentiation (Fst = 0.248) (Ledig et al., 1997
). Pinus rzedowski, a conifer with a small number of populations in a restricted area of west-central Mexico (western Michoacán) displays considerably higher within-population diversity (He = 0.219), but also has a significant deficiency of heterozygotes (Fis = 0.247) and a high level of interpopulational differentiation (Fst = 0.175; Delgado et al., 1999
). These data suggest that the levels and patterns of genetic variation observed for conifers in the North Temperate zone (Hamrick, Godt, and Sherman-Broyles, 1992
) are not consistent with those of the conifers of Mexico studied until now. Ge et al. (1998)
also reported very high levels of interpopulation differentiation in Cathaya argyophylla, a conifer with a restricted distribution in southern China. Hamrick and Godt (1996b)
pointed out that, not only life-history traits, such as those shared by conifers, influence the levels and distribution of genetic diversity among species, but also the phylogenetic, biogeographical, and evolutionary histories of the particular species are important.
From the standpoint of conservation, the high levels of interpopulation genetic differentiation we observed pose a real challenge. This may be the case for many Mexican conifers and much of the Mexican flora. Other isozyme studies of Mexican plant species have found relatively high levels of interpopulation differentiation (Izquierdo and Piñero, 1998
; Núñez-Farfán et al., 1996
; Cornejo-Romero, 1998
; Martínez-Palacios, Eguiarte, and Furnier, 1999
). This pattern of variation poses the challenge of preserving the many genetically distinct populations necessary to adequately capture the genetic variation of the species, within a country with strong social pressures to economically exploit the land (Carabias, Arriaga, and Cervantes, 1994
).
FOOTNOTES
1 The authors thank Andrés Keiman-Freire, Yolanda Nava-Cruz, Patricia Delgado-Valerio, Aldo Valera, Rodolfo Salas, and Elleli Huerta-Ocampo for assisting in the field collections, Rodrigo González-Chauvet, Eduardo Morales, Silvia Guzmán-Beltrán, and Oliva Godinez-Ibarra for assisting in the laboratory, Ariel Alvarez, Leigh Latta, Sue Ratner, and Eduardo Morales for assisting in the data analyses, and Francisco Molina-Freaner, Daniel Piñero, Eduardo Morales, Michael Lynch, and two anonymous reviewers for reading the manuscript. Financial support was provided by the Comisión Nacional para el Uso y Conocimiento de la Biodiversidad (CONABIO grant B138 to G.R.F. and L.E.E.) and by the Instituto de Ecología at the Universidad Nacional Autónoma de México. 
2 Author for correspondence (eaguirre{at}miranda.ecologia.unam.mx
).
3 Current address: TEAMS Program, College of Education, B203 Payne, Arizona State University, Tempe, Arizona 85287-0911 USA.
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