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Population Biology |
2Institute of Forest Genetics, Pacific Southwest Research Station, USDA Forest Service, 2480 Carson Road, Placerville, California 95667 USA; 3Departamento Forestal, Universidad Autónoma Agraria Antonio Narro, Buenavista, Saltillo, Coahuila, México; 4Service de Génétique et d'Amélioration des Arbres Forestieres, Centre National de la Recherche Forestiere, Charia Omar Ibn El Khattab, B.P. 763 Rabat, Morocco; 5Programa Forestal, Instituto de Recursos Naturales, Colegio de Postgraduados en Ciencias Agricolas, Montecillo, México, C.P. 56230, México; and 6Centro de Genética Forestal, Universidad Autónoma Chapingo, Apartado Postal No. 37, Chapingo, México, C.P. 56230, México
Received for publication January 2, 2001. Accepted for publication March 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: endangered species • fitness • fragmentation • genetic distance • genetic structure • isozymes • pollen allele frequencies • selfing
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Saccheri et al., 1998
; Westemeier et al., 1998
). Many species in Mexico are represented by small, scattered populations, and also are presumed threatened or endangered. Of a total 22 000 plant species in Mexico, 52% are endemic (Rzedowski, 1993
), and many endemics are among those endangered. Among 16 endemics in a total of 42 pines that occur in Mexico (Farjon and Styles, 1997
), 7 (44%) are listed by the International Union for Conservation of Nature and Natural Resources (IUCN) as species of concern (Farjon and Page, 1999
). Nevertheless, in anemophilous species with high pollen output, such as pines, mechanisms to promote outcrossing might offset the effects of fragmentation, reducing the loss of genetic diversity and the danger of extinction (Ledig, 1998
). In Mexican pines, the net effect of fragmentation seems to have been speciation, since Mexico is a secondary center of diversity for the genus (Eguiluz-Piedra, 1988
; Styles, 1993
).
Weeping piñon (Pinus pinceana Gordon), a Mexican endemic, provides an excellent opportunity to study the effects of fragmentation and low density on patterns of genetic variation and inbreeding. Weeping piñon is a short tree or bush of the Sierra Madre Oriental, reaching from 4 m tall at maturity up to 10 or, perhaps, 12 m (Martínez, 1953
; Perry, 1991
; Farjon and Styles, 1997
). It ranges from Coahuila in the north to Hidalgo in the south (Fig. 1), a distance of 
750 km, and is distributed between 1400 and 2700 m in elevation on calcareous slopes or in barrancas (Farjon and Styles, 1997
). Of all the Mexican pines, it occupies the most arid sites, below or just overlapping the lower border of the true piñon–juniper zone (Passini, 1985
). Annual precipitation is only 300–400 mm, falling mostly in the summer months (Perry, 1991
). Trees may be scattered far apart among Mexican piñon (Pinus cembroides Zuccarini) and drooping juniper (Juniperus flaccida Schlechtendal), or, at lower elevations, with a variety of sclerophyllous and deciduous shrubs of the desert (Farjon and Styles, 1997
). Not only is weeping piñon scattered among other tree and shrub species where it occurs, but its range is highly fragmented, especially in the south, in the States of San Luis Potosí, Querétaro, and Hidalgo. It is considered a paleorelict (Perry, 1991
).
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). It was placed in the "lower risk" category in part because information about the species was lacking. However, Perry (1991)
claimed it was "clear that this species is now rare and endangered" because in all areas that he visited, "reproduction was extremely limited" and mature trees were scarce. Grazing by goats, burros, and range cattle reduces or eliminates seedling reproduction, and large branches are removed for firewood, limiting even the capacity for reproduction. Seed harvest and consumption by the campesinos may also be a factor.
The taxonomy of several rare piñons, including weeping piñon, is still in flux. Weeping piñon was provisionally included in subgenus Strobus, section Parryanae, subsection Nelsoniae by Farjon and Styles (1997)
. The only other member of this subsection is Nelson piñon (Pinus nelsonii G. R. Shaw). In the most recent taxonomic treatment of the pines, Price, Liston, and Strauss (1998)
included weeping piñon with several other piñons in subsection Cembroides, but it is certainly not closely related to other members of the group, such as the common Mexican piñon, and these authors call for further analysis of the species in their subsection Cembroides. Perry (1991)
placed weeping piñon in a new subsection, subsection Pinceana, which included Nelson piñon, maxipiñon (Pinus maximartinezii Rzedowski), and Rzedowski piñon (Pinus rzedowskii Madrigal Sánchez & Caballero Deloya).
Most authors have felt that maxipiñon, which Farjon and Styles (1997)
did not assign to any subsection, is closely related to weeping piñon (Rzedowski, 1964
; Zavarin and Snajberk, 1987
; Perry, 1991
; Farjon, 1999
). Maxipiñon is known from only a single location. Weeping piñon and maxipiñon are very similar in their monoterpene composition (Zavarin and Snajberk, 1987
). Phylogenetic trees, constructed from variation in cpDNA, group weeping piñon and maxipiñon, but place Nelson piñon and Rzedowski piñon on distinctly different branches (Pérez de la Rosa, Harris, and Farjon, 1995
). Cladograms based on morphology and phenology separate weeping piñon and maxipiñon from the true piñons of subsection Cembroides and place them on the same branch as Nelson piñon, Rzedowski piñon, and Asian nut pines of subsection Gerardianae; however, maxipiñon branches off first and is not closely associated with any of the other species (Malusa, 1992
). By contrast, weeping piñon clustered with singleleaf piñon (Pinus monophylla Torrey & Fremont) and four-needle piñon (Pinus quadrifolia Parlatore ex Sudworth) and not with maxipiñon, based on seed and cotyledon characteristics (Eguiluz-Piedra, Niembro-Rocas, and Pérez-Rodríguez, 1985
). The most recent phylogeny, using the sequence of an internal transcribed spacer region of nuclear ribosomal DNA, strongly supported a clade of weeping piñon, Rzedowski piñon, and maxipiñon as a sister clade of piñons in the Cembroides group and placed Nelson piñon as an outgroup to the subsection Cembroides (Liston et al., 1999
).
Very little has been published about the genetic structure of weeping piñon. In an analysis of the monoterpene composition in three populations of weeping piñon, Zavarin and Snajberk (1987)
declared that the variability within and between populations was "exceptionally small," a characteristic weeping piñon shared with maxipiñon, which is also considered a relict. The lack of monoterpene variability suggested that weeping piñon may lack genetic diversity. However, recent analysis of isozymes in five populations of weeping piñon suggested high variability (Molina-Freaner et al., 2001)
.
We undertook a survey of weeping piñon to determine its level of genetic diversity and its genetic structure as a step in planning conservation measures. Within stands, weeping piñon is so scattered that we felt inbreeding might be an important consideration in its conservation. Therefore, we investigated its mating system. We also compared gene frequencies in weeping piñon to those in maxipiñon to see how closely the two species were related and, specifically, whether weeping piñon could be a progenitor of maxipiñon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Alleles at a locus can be detected by segregation among megagametophytes from a heterozygote. The genotype of the seed parent can be determined by analyzing a number of megagametophytes. When two different alleles at a locus are detected, the seed parent is unequivocally a heterozygote. When only one allele is detected, the tree is classified as a homozygote, although the possibility remains that it is a heterozygote and that by chance the sample included only one allele. The probability of misclassification decreases with increases in sample size. We assayed four megagametophytes per tree (or nine in Cañon la Yesera). With a sample of four, there is a probability of 0.125 of misclassifying a heterozygote as a homozygote at any one locus. That is, the probability that all four megagametophytes in a sample from a heterozygous tree carry the same allele is 2(
)4 = 0.125.
Seeds were germinated in petri dishes, and when radicles emerged, megagametophytes and embryos were dissected, separated, and extracted. For estimating allele frequencies, the number of parent trees per population, N, varied from 7 to 30 (a mean, 
, of 15.3, or 2N = 14–60 genomes). Because cones were absent on most trees in some populations, the number of trees sampled was small. Therefore, we also estimated allele frequencies and variability statistics from the pollen contribution to the embryos. Because we know the contribution of the egg (the haploid genotype of the megagametophyte) to the embryo, the pollen contribution can be deduced by subtraction. Allele frequencies from the pollen genotypes were based on an N of 44–123 per locus per population ( = 70.3 genomes per population).
We used techniques of starch gel electrophoresis based on the laboratory manual of Conkle et al. (1982)
to assay enzyme systems. In the megagametophytes, we were able to consistently score 27 presumptive loci in 18 enzyme systems, and in the embryos, 24 loci in 16 enzyme systems. The enzymes MNR and TPI, scored in the megagametophytes, could not be consistently scored in embryos. The enzymes ADH, FEST, LAP, MNR, PGI, PGM, and TPI were run on a Tris citrate/lithium borate buffer (buffer "A" of Conkle et al., 1982
); CAT, FDH, GDH, and GOT were run on a Tris citrate/sodium borate buffer (buffer "B" of Conkle et al., 1982
); and ACO, ALD, CADH, IDH, MDH, 6PG, and SKD were run on a morpholine citrate buffer (buffer "E," a pH 8.0 version of buffer "D" of Conkle et al., 1982
). We interpreted the number of loci and alleles by drawing on the experience gained in our laboratory from studies of allozymes of other conifer species (Conkle, 1981
). Samples of red pine (Pinus resinosa Aiton), which has proven to be an almost invariably monomorphic species, were included on each gel to aid interpretation. Where several zones of activity were observed for a single enzyme, numerals following the enzyme abbreviation were used for identification.
To determine whether allele frequencies in the trees were similar to those in the pollen pool, we used a chi-square test:
 | (1) |
; p. 228). The variances are qT(1 – qT)/2NT and qP(1 – qP)/NP.
For trees and the pollen pool, we separately estimated percent polymorphic loci, alleles per locus, and heterozygosity for each population, and Nei's (1978)
unbiased genetic distance and Rogers' (1972)
distance between pairs of populations with BIOSYS (Swofford and Selander, 1981
). For small samples such as ours, BIOSYS calculates unbiased heterozygosity (Nei, 1978
). We tested the relationships between mean annual height and diameter increments and individual heterozygosity at Bajada al Arroyo de Orduña by regression. Individual heterozygosity was calculated as the number of loci heterozygous in a tree, divided by the number of polymorphic loci for which the tree was scored. All inferences apply to the mature, cone-bearing or pollen-producing trees in the populations.
For the trees, fixation indices (F) within populations were calculated as the mean of the deviations of observed homozygosity from Hardy-Weinberg expectations at each locus. It is not possible to calculate fixation indices from pollen allele frequencies. We also used BIOSYS to calculate Wright's (1965)
F statistics. BIOSYS calculates FIS and FIT, the fixation indices of trees relative to the population and the meta-population, respectively, as weighted averages across alleles. FST, the proportion of the total genic diversity among populations, is calculated from the relationship
 | (2) |
Because stands of weeping piñon are clustered within regions that are widely separated, we divided populations into a northern region with six populations, and central and southern regions with one population each. "Step Wright78" of BIOSYS was used to apportion variation among regions, among populations within the northern region, and among trees within populations for the megagametophytes.
The degree of genetic isolation among populations was estimated by Nm, the number of migrants per generation, calculated by two methods. From Wright (1951)
we have
 | (3) |
Nm also can be calculated from the frequency of private alleles (unique alleles found in only one population), using simulations developed by Slatkin (1985)
:
 | (4) |
(1) is the mean frequency of private alleles and 
and ß are constants determined by fitting simulated data developed for sample sizes of 10 and 25 (from Barton and Slatkin, 1986
). Estimates of Nm were corrected for actual mean sample size, 17.5, by factors of 10/17.5 and 25/17.5, respectively.
To test for a relationship of genetic distance to geographic distance between pairs of populations, we used Mantel's generalized regression procedure (Mantel, 1967
; Liedloff, 1999
). Geographic distance between populations was calculated from latitude and longitude using Kindred's (1997)
distance calculator.
Populations were grouped into phylogenetic trees with distance Wagner procedures (Farris, 1972
; Swofford and Selander, 1981
). The nodes of the Wagner tree represent hypothetical common ancestors (HTUs, hypothetical taxonomic units) for the branches above them. The sum of the branch lengths that connect any two populations (OTUs, operational taxonomic units) is the patristic difference, which is an approximation of the number of evolutionary steps occurring in the phyletic lines connecting the OTUs. The root of the tree is the closest common ancestor of all the populations, and total branch length from this root approximates the divergence from this common ancestor. The distance Wagner procedure is preferable to the unweighted pair-group (UPGMA) method because the latter depends on the assumption that the rate of divergence between lines is homogeneous (Farris, 1972
). When founder events or genetic drift are likely events in the divergence of OTUs, rates of divergence are probably heterogeneous, and phenetic-similarity clustering techniques to estimate evolutionary trees are likely to be misleading and inadvisable. Because Nei's (1978)
genetic distance does not obey the triangle inequality, it is inappropriate for distance Wagner procedures (Swofford and Selander, 1981
). Therefore, Rogers' (1972)
distance measure was used.
To compare weeping piñon to maxipiñon, we used data from Ledig et al. (1999)
, reducing the data set for each species to 24 common loci in 16 enzyme systems. We calculated Nei's (1978)
unbiased genetic distance between taxa based on the 24 loci in common.
For mating system analysis, we used Ritland's (1986, 1989, 1990a, b, 1994)
generalized multilocus estimation (MLTF) and multilocus mating system (MLTR) programs to calculate the single- and multiple-locus estimates of outcrossing rate, ts and tm, in the samples from Sierra Zapalinamé, Cañon la Yesera, and Bajada al Arroyo de Orduña. Sierra Zapalinamé and Bajada al Arroyo de Orduña were among the largest samples because they had either the greatest number of families or the greatest number of progeny (120 and 100 megagametophyte and embryo pairs, respectively), and they represent the northern and southern portions of the weeping piñon range. Cañon la Yesera represents the central portion of the range, and the number of megagametophyte–embryo pairs was 63. Generalized multilocus estimation was used to estimate tm because the probability of outcrossing becomes much more accurate when information on the egg genotype is used (Ritland, 1990a
). Furthermore, smaller progeny arrays are adequate and bias is reduced when the megagametophyte genotype is known. The Newton-Raphson method, as explained in Ritland (1990a)
, was used for iteration, and p, the allele frequency, and t were estimated jointly. Standard errors for t at some loci in some populations were taken from the estimated values in 100 bootstraps using MLTR because the Newton-Raphson iterations failed to converge to reasonable values, a common occurrence. MLTR also was used to estimate the correlation of outcrossed paternity, rp, among progeny within a family. Although adequate for estimation of population outcrossing rates, samples were too small to estimate family outcrossing rates; estimates failed to converge for most families.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.10. Because seed-tree and pollen allele frequencies were similar, only the frequencies for the seed-trees are presented (Table 2). A table of the pollen allele frequencies is available from the corresponding author upon request.
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Unbiased estimates of expected heterozygosity, He, were similar for the trees and the pollen pool (Table 3); the mean for the trees was 0.174 and ranged from 0.128 to 0.201. For both the seed trees and the pollen pool, the percentage polymorphic loci, Pp, averaged between 50 and 60%, and the number of alleles per locus, A, was 1.8–1.9. Observed heterozygosity was less than expected heterozygosity in every population, suggesting inbreeding (Table 3). The mean of the inbreeding coefficients, 0.116, was similar to Wright's FIS, 0.140 (Table 4). An F between 0.116 and 0.140 suggests that the parents of the present generation were related on average as half-sibs because the inbreeding coefficient is half the relationship coefficient of the parents, and the relationship coefficient of half-sibs is 0.250.
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38 yr. The tallest tree was only 4.5 m in height and 12.5 cm dbh, with 38 rings. The oldest tree was 69 yr old and only 3.0 m in height. For the seven trees sampled at Cañon la Yesera, mean height and dbh were even less than that at Bajada al Arroyo de Orduña, 2.6 m and 6.5 cm, respectively, and the trees were younger, 30 yr old on average. At Bajada al Arroyo de Orduña, where data were sufficient for regression analysis, mean annual basal area increment was not related to individual heterozygosity, but mean annual height increment was (r = 0.51, P < 0.05; Fig. 2).
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Nei's genetic distance (D) also expresses population differentiation. Nei's D was minimal among populations in the northern cluster of Coahuila and Zacatecas, but much greater between the northern populations and those representing the central and southern portions of the range (Table 5). A distance Wagner tree based on Rogers' (1972)
genetic distance graphically reflects the relationships among populations (Fig. 3). The association between geographic distance and Nei's genetic distance was weak and nonlinear; using Mantel's generalized regression procedure, r = 0.54 (Table 5, Fig. 4). The correlation was weak because Cañon la Yesera is genetically unique and is at intermediate distance from the other populations. Genetic distance between weeping piñon and maxipiñon was about twice as great as the maximum distance between populations of weeping piñon and nearly two orders of magnitude greater than distances between populations in Coahuila and Zacatecas.
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The multilocus outcrossing rate ranged from 0.836 (0.732 < tm < 0.940; P = 0.95) at Bajada al Arroyo de Orduña, the southernmost population sampled, to 0.897 (0.823 < tm < 0.971; P = 0.95) at Sierra Zapalinamé, in the north (Table 6). The lower estimate, at least, suggests significant selfing, in agreement with the inbreeding coefficient, FIS, of 0.140. In every case, the mean single-locus outcrossing rate was lower than the multilocus rate (Table 6), and the standard errors indicated that the difference approached statistical significance at Bajada al Arroyo de Orduña and Sierra Zapalinamé. The difference between tm and ts suggests that some inbreeding occurred by crosses among relatives, in addition to inbreeding by selfing. The difference might also be due to the Wahlund effect if our sampling scheme included different subpopulations.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and 0.142 for 57 outcrossing endemic plants (Hamrick and Godt, 1996
). For the weeping piñon mother-tree data (27 loci), unbiased He ranged from 0.128 to 0.201, and averaged 0.174. Percentage polymorphic loci (Pp) in weeping piñon was 44.4–66.7%, about the same as the mean for outcrossing endemics (54.4%) but twice as high as that for woody endemics (26.3%) reviewed by Hamrick and Godt (1996)
and Hamrick, Godt, and Sherman-Broyles (1992)
. Of course, the category "endemics" spans a range of geographic scales.
Genetic structure of weeping piñon was as expected for endemics with fragmented ranges; populations were strongly differentiated. About 15.2% of the observed diversity in weeping piñon was among populations compared to 17.9% for all outcrossing plant endemics (Hamrick and Godt, 1996
) and 14.1% for woody endemics (Hamrick, Godt, and Sherman-Broyles, 1992
).
The combination of high levels of diversity and high levels of population differentiation appears to be common in Mexican conifers. Molina-Freaner et al. (2001)
found high genetic diversity and high differentiation in weeping piñon, Laguna pine (Pinus lagunae Passini), and Mexican populations of bishop pine (Pinus muricata D. Don). In weeping piñon He was 0.373 and FST was 0.247, based on 13 isozyme loci and five populations (Molina-Freaner et al., 2001)
. In nine populations of the Mexican endemic Rzedowski piñon, He averaged 0.219, and FST was 0.175 (Delgado et al., 1999
), even though the geographic distance between the most widely separated populations was only 25 km. Other Mexican pines with similar genetic structure are Gregg's pine (Pinus greggii Engelmann ex Parlatore; Ramírez-Herrera and Vargas-Hernández, 1996
) and Apache pine (Pinus engelmannii Carrière; Bermejo-Velázquez, 1993
). Populations of some Mexican firs (Abies spp. Linnaeus) were also well differentiated (Aguirre-Planter, Furnier, and Eguiarte, 2000)
.
Low rates of gene flow may lead to differentiation among populations because of random genetic drift, and estimates of Nm over the range of weeping piñon were relatively low for pines, between 1.39 and 2.09. Nm in pines generally ranges between 4.6 and 17.2 in rangewide studies (reviewed in Ledig, 1998
). The value of 4.6 was recorded in Table Mountain pine (Pinus pungens Lambert), a species with scattered populations, like weeping piñon. Lower values of Nm have been found in another Mexican endemic, Chihuahua spruce (Picea chihuahuana Martínez), and in Coulter pine (Pinus coulteri D. Don), both species with highly fragmented ranges in which the evidence for genetic drift was unmistakable (Ledig et al., 1997
; Ledig, 2000
).
Much of the differentiation among populations was associated with gaps between the northern cluster of populations and the central and southern fragments. For example, mean values of Nm between populations in the northern cluster and the central and southern populations were 0.99 and 2.13, respectively, while within the northern cluster, Nm was 4.21. The latter value approaches the typical range for pines. Cañon la Yesera may differ from the other populations because of its isolation, coupled with the small area occupied by weeping piñon within the central fragment, or it may be an artifact of our sample size. Because Nm takes many hundreds or thousands of generations to reach equilibria, our estimates probably reflect past gene exchange (Slatkin and Barton, 1989
). Thus, the estimates are mainly valuable by comparison to other species.
The mixed mating model for the estimation of outcrossing rate assumes loci are in linkage equilibrium. Although the number of progeny in our families was too small to estimate disequilibrium or even the recombination rate between loci, some of the loci that we used are almost certainly linked. Linkage arrangements are highly conserved in pine, and Conkle (1981)
reported tight linkage between Ald-1 and Got-3 and between an Idh locus and an Aco locus. He also reported tight to moderate linkage between Got-2 and Lap-2 in several pine species, and found moderate to weak linkage between Adh-1 and Pgi-2 and weak linkage between Adh-1 and Lap-2. At least some of Conkle's (1981)
loci must be homologous to the loci that we scored in weeping piñon, and because linkage is likely, our estimates of tm deserve closer scrutiny. Having said that, we feel the estimates are valid because the means of the single-locus estimates, ts, of outcrossing were similar to tm. The single-locus model does not, of course, depend on the assumption of linkage equilibrium between loci. Therefore, linkage probably had little effect on the estimate of tm reported here.
Despite the low density of the woodlands where weeping piñon is dispersed among Mexican piñon, juniper, sclerophyllous vegetation, or succulents, outcrossing is predominant. Although the data suggest a tendency toward higher rates of selfing in the southern fragments of the species, larger samples are needed to determine whether the trend is real. Based on the differences between single-locus and multilocus estimates of outcrossing, some crossing among relatives may be occurring. In the southern and central populations, the correlation of outcrossed paternity, rp, suggests that a high proportion of matings may occur between neighbors or pairs of synchronously flowering trees, reducing the opportunities for recombination.
Selfing and/or crossing between related trees was reflected in excess homozygosity. Inbreeding coefficients were positive in all of the eight populations sampled, and overall, FIS was 0.140. At Bajada al Arroyo de Orduña, we were able to test the relationship between homozygosity and growth, a fitness surrogate, to detect inbreeding depression. Results were equivocal. Mean annual height increment was correlated positively with individual heterozygosity (Fig. 2), but mean annual basal area increment was not. Inbreeding depression is most pronounced under competition and is cumulative over time (Ledig, Guries, and Bonefeld, 1983
). Neither factor was likely to accentuate the relationship in weeping piñon. The trees at Bajada al Arroyo de Orduña were widely spaced and were, on average, only 38 yr old. Further studies are needed.
The equilibrium inbreeding coefficient, Fe, is related to the outcrossing rate, t, as (Allard, Jain, and Workman, 1968
):
 | (5) |
Substituting our estimates of tm for t yields an estimated Fe of 0.088 for Cañon la Yesera, 0.066 for Bajada al Arroyo de Orduña, and 0.058 for Sierra Zapalinamé, compared to observed fixation indices of 0.076, 0.099, and 0.097 (Table 3), respectively. Thus, observed F approximates Fe well, suggesting that these populations may be at or near equilibrium with respect to inbreeding and selection against selfs.
The inbreeding depression of selfed progeny can be estimated from the inbreeding coefficient and the rate of selfing (Ritland, 1990c
):
 | (6) |
The distance Wagner tree suggests two clades, one of the six northern populations and the other of Cañon la Yesera and Bajada al Arroyo de Orduña. Bajada al Arroyo de Orduña, the southernmost population in our sample, is closest to the root of the tree, the hypothetical common ancestor. The central population and the northern populations all have diverged from the root to a similar degree. However, these relationships are tentative because of the limited sampling of populations from the southern and central fragments of the range. Increased sampling might demonstrate three clades.
The genetic distance beween weeping piñon and maxipiñon (Table 5) is of a different order of magnitude than the distance between populations within weeping piñon, as it should be for two morphologically distinct and allopatric species. The important question is whether weeping piñon could be a progenitor of maxipiñon. Maxipiñon seems to have gone through an extreme bottleneck, and some of us (Ledig et al., 1999
) predicted that its progenitor must be polymorphic for at least the loci variable in maxipiñon. Maxipiñon is polymorphic for Mpi, but we could not score Mpi in weeping piñon. However, weeping piñon met the criterion for all other loci at which maxipiñon was polymorphic; i.e., Ald-2, Got-3, Lap-2, Mnr-1, 6Pg–1, Pgi-2, Skd-1, and Tpi-1 were polymorphic in maxipiñon and weeping piñon, although not every population of weeping piñon was polymorphic for all eight loci.
The genetic distance is least between maxipiñon and Cañon de la Laja in Zacatecas. Cañon la Yesera is the geographically closest population to maxipiñon, but the genetic distance between Cañon la Yesera and maxipiñon is among the highest. Maxipiñon is known from a single population near the southern end of the Sierra Madre Occidental. Cañon la Yesera and maxipiñon are in different cordillera, separated by the arid Meseta Central of México. A more direct migration route connects Cañon de la Laja to the southern Sierra Madre Occidental, because the morphotectonic provinces of the Sierra Madre Occidental and the Sierra Madre Oriental are in contact at the northern end of the Sierra Madre Oriental (Ferrusquía-Villafranca, 1993
).
The genetic structure of weeping piñon provides a hopeful note with regard to its conservation, which stands in contrast to the concerns expressed by IUCN (Farjon and Page, 1999
) and Perry (1991)
. Although it may be a paleorelict, weeping piñon does not seem to be in a genetic extinction vortex. Genetic diversity, based on 27 loci, is among the highest recorded in our laboratory. Nor is weeping piñon suffering from high rates of inbreeding. Although some selfing is indicated, it is not nearly as high as we expected in populations where trees were widely scattered. Furthermore, F seems to be close to equilibrium conditions, perhaps suggesting that the present population structure and mating system have existed for many generations.
Nevertheless, grazing by domestic animals is a relatively new phenomenon, dating no earlier than the Spanish conquest in 1519–1521. Weeping piñon now is threatened by population pressure, as are many species; the greater the human population, the more grazing animals. Grazing animals destroy seedlings, and the reproductive potential of mature trees is reduced because limbs are cut for firewood.
We recommend that areas be set aside for protection of weeping piñon, where grazing, cutting for firewood, and seed collection for consumption can be excluded. A minimum of three areas should be established, at least one each in the areas represented by (1) Coahuila and Zacatecas, (2) San Luis Potosí, and (3) Querétaro and Hidalgo, because of the strong genetic differentiation among these major fragments of the species' range. A program of ex situ conservation is also desirable. Weeping piñons could be planted for the value of their edible seeds in the market, and they would also be valuable in horticulture because of their attractive weeping form and their drought resistance. Ecological studies are needed to determine the timing of seed crops, seed longevity, the effect of seed predators, natural agents of seed dispersal, and the type of seed bed and climatic conditions conducive to the germination and survival of weeping piñon seedlings, to better manage the species for long-term survival. The role of humans in the ecology of weeping piñon should not be neglected, for it may be that pre-Hispanic people were an important component in sustaining the relict piñon ecosystem (Bye, 1993
).
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FOOTNOTES |
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7 Author for reprint requests (tledig{at}ucdavis.edu
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Allard R. W. S. K. Jain P. L. Workman 1968 The genetics of inbreeding populations. Advances in Genetics 14: 55-131
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) after Perry (1991)
) sampled for isozyme analysis. Abbreviations are: Cap = Ejido de Capulin, DdA = Dos de Abril, Zap = Sierra Zapalinamé, Fern = Jagüey de Ferniza, Mina = Sierra la Mina, Laja = Cañon de la Laja, Yes = Cañon la Yesera, Ord = Bajada al Arroyo de Orduña, Pmax = Pinus maximartínezii
