Effects of population disjunction on isozyme variation in the widespread Pilgerodendron uviferum

Geographical range is considered a good predictor of the levels of isozyme variation in plants. Widespread species, often consisting of historically larger and more continuous populations,

maintain higher polymorphism and are less affected by drift, which tends to erode genetic variation in more geographically restricted species. However, widespread species occurring in small

and disjunct populations may not fit this pattern. In this study we examined genetic variation in Pilgerodendron uviferum, a conifer endemic to temperate forests of southern South America,

and is such a widespread and habitat-restricted species. Twenty populations along the whole range of Pilgerodendron were analysed by isozyme electrophoresis to resolve 14 putative genetic

loci. Eleven were polymorphic in at least one population although only six of them were polymorphic in more than one population. We found reduced within-population levels of isozyme

variation, with only 11% polymorphic loci (0.95 criterion), 1.2 mean number of alleles per locus, and mean observed and expected heterozygosities of 0.024 and 0.033, respectively. Most

genetic diversity was found within populations (HT=0.039, HS=0.033, FST 15%). Greater polymorphism and lower divergence was estimated in the more geographically restricted and closely

related Fitzroya. Thus, total range, in combination with information on the degree of among-population isolation, may be a better predictor of the levels of polymorphism than range size

alone.

Throughout the history of a given species, evolutionary forces acting in combination with its particular life history traits, shape its genetic characteristics as measured by the degree of

polymorphism and population genetic divergence. Geographical range has been shown to be a good predictor of the levels of allozyme variation in plants (Karron, 1987; Hamrick & Godt, 1989;

Hamrick et al., 1992; Gitzendanner & Soltis, 2000). Thus, geographically restricted species, usually consisting of small, isolated populations, are more susceptible to losses of genetic

variation due to genetic drift and restricted gene flow (Hamrick & Godt, 1989). However, widespread species consisting of disjunct populations may have less polymorphism and greater

among-population genetic divergence than those having more continuous populations.

The purpose of this study was to examine amounts and distribution of genetic variation in Pilgerodendron uviferum (D. Don) Florin (Cupressaceae), a widespread and habitat-restricted species,

occurring in isolated populations. The hypothesis is tested that the total range of a given species, in combination with information on the degree of among-population isolation, is a better

predictor of the degree of isozyme variability than range size alone.

Biogeographical patterns of genetic diversity in Pilgerodendron were compared with those in the other two Cupressaceae from temperate South America: Fitzroya cupressoides (Molina) I. M.

Johnst. and Austrocedrus chilensis (D. Don) Florin & Boutelje. These are monotypic genera included in the monophyletic subfamily Callitroideae, which consists of genera with Southern

Hemisphere distributions (Gadek et al., 2000). The three genera characterize floristically the Subantarctic Province within the Antarctic region (Cabrera & Willink, 1980), which in turn is

considered to be a biogeographic island, given its isolation dating to the Tertiary, from other similar Southern Hemisphere floras (Armesto et al., 1995).

Pilgerodendron, Fitzroya, and Austrocedrus are ‘endemic species sensu lato, which means that they belong exclusively to one place or region’ (Rapoport, 1982; pp. 14–15), in this case, the

temperate forests of southern Argentina and Chile. In addition, given that they have very different patterns of geographical distributions, the distinction between widespread and

geographically restricted will be considered in relation to the overall range of the biogeographical region. Thus, the concepts of endemism and geographical range are used here in relative

terms. Pilgerodendron has the most extended latitudinal distribution of any tree species in temperate South America, whereas Fitzroya and Austrocedrus occur over more restricted geographical

ranges but consist of larger and more continuous populations, particularly in the case of Austrocedrus. It is predicted that Pilgerodendron will have lower genetic variation and greater

among-population differentiation than Fitzroya and Austrocedrus, despite the greater geographical range of Pilgerodendron.

The aim of this study is to analyse the following questions. (i) What are the levels of isozyme variation in populations of Pilgerodendron? (ii) What is the degree of among-population

differentiation in this species? (iii) How does this information relate to that measured in closely related species which have different patterns of geographical distributions?

Pilgerodendron (common names: ciprés de las Guaitecas, ciprés de las islas o Ten; indigenous mapuche name: Lahuán) is a long-lived conifer endemic to southern Chile and the adjacent parts of

Argentina. Although restricted to wet and poorly drained sites, its overall geographical range extends over 1600 km from 39°36′ to 54°20′S (Szeicz et al., 2000), making it the world’s

southern-most conifer and having the most extended natural distribution of any tree species in temperate South America. Whereas in Chile it covers a total area of 564 922 ha (CONAF et al.,

1999), in Argentina it is less abundant, and is only found at scattered sites from 41°00′S to 50°19′S and from 71°25′W to 73°13′W (Rovere et al., pers. comm.). At its northern-most limit it

occurs as isolated populations in the Chilean Coastal range and on both slopes of the Andes, becoming more abundant to the south, characterizing the Chilean Archipelagos south of 44°S.

Twenty populations of Pilgerodendron (12 from Chile and eight from Argentina) were sampled across the species’ geographical range (Table 1). Thirty individuals, separated by a minimum of 50

m, were randomly selected from each population. In the case of the smallest sampled population, located in Punta Bandera within the Glaciares National Park (Argentina), more than one sample

(1, 2 or 4 twigs) was collected from each of the 14 clumps, to determine if each clump was clonal in origin. From each individual, approximately 20 cm of twig with fresh leaf tissue was

collected, and the samples were kept cool until they were taken to the laboratory, where they were stored at 0–4°C. Enzyme extracts were prepared by crushing approximately 500 mg of leaf

tissue in liquid nitrogen to which 1 mL of extraction buffer (Mitton et al., 1979) was added. Homogenates were centrifuged and stored at −80°C until they were absorbed onto Whatman No. 3

paper wicks that were loaded into 12% starch gels.

Two buffer systems, morpholine-citrate pH 7.5 (Ranker et al., 1989) and histidine-tris pH 7.0 (King & Dancik, 1983), were run at constant currents of 20 and 35 mA, respectively, for about 5

h or until the marker dye had migrated at least 8 cm from the origin. Anodal slices were cut horizontally and stained for enzyme activity using the agarose-staining methods of Mitton et al.

(1979) and Soltis et al. (1983). Aconitase (ACO, EC 4.2.1.3), isocitrate dehydrogenase (IDH, EC 1.1.1.42), malic enzyme (ME, EC 1.1.1.40), peroxidase (PER, EC 1.11.1.7), 6-phosphogluconate

dehydrogenase (6PGD, EC 1.1.1.44), phosphoglucose isomerase (PGI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 5.4.2.2), and shikimate dehydrogenase (SKDH, EC 1.1.1.25) were separated using

morpholine-citrate buffers, whereas malate dehydrogenase (MDH, EC 1.1.1.37) was separated using histidine-tris buffers. The scoring of isozymes genotypes consisted of assigning consecutive

numbers so that the most anodal locus and/or allele were designated with the lowest numeral. Loci are considered putative since no genetic analysis was performed, although gel banding

patterns and interpretation of results were similar to those obtained in other plant species (Murphy et al., 1996).

Within-population isozyme variation was described by standard gene diversity measures using POPGENE v. 1.31 (Yeh et al., 1999). These measures were the proportion of polymorphic loci using

0.95 and 0.99 criteria (P < 0.95 and P < 0.99), the mean number of alleles per locus (A), and the observed and expected heterozygosities (HO and HE, respectively).

Deviations of polymorphic loci from the Hardy–Weinberg equilibrium were analysed using chi-squared tests. Population genetic structure was measured by F-statistics (Wright, 1965) using FSTAT

v. 2.9.1. (Goudet, 2000) which computes unbiased estimates. Total genetic diversity at a locus (HT) and genetic diversity within populations (HS) were calculated using 11 polymorphic loci,

according to Nei (1973). These indices were compared to those of the geographically restricted Fitzroya, using the same loci from published data by Premoli et al. (2000a,b), which in turn

were obtained using similar sampling schedules as in Pilgerodendron. Significant differences in the levels of among-population divergence (FST ˜ GST) were analysed using 95% confidence

intervals generated by bootstrapping over loci. Between-species comparisons in the levels of within-population variation and genetic diversity were analysed using the nonparametric

Mann–Whitney Rank Sum Test, with populations and loci as factors, respectively. Data on Austrocedrus were available from only one population, so no statistical comparison was performed with

this species.

For Pilgerodendron, 78% (11/14) of the resolved putative isozyme loci were polymorphic (0.95 criterion) in at least one population. However, approximately half of the polymorphic loci were

polymorphic in only one population (Table 2). At the species level and for the 14 loci, A averaged 2.3 alleles per locus and the expected heterozygosity was 0.035. Population-level

polymorphism was on average only 11.4% and 17.1% with the 0.95 and 0.99 criteria, respectively, not exceeding 36% and 43% for each criterion in any population. Reduced within-population

isozyme variability was also measured by A with 1.2 alleles per locus, and mean heterozygosities were 0.024 and 0.033 for HO and HE, respectively (Table 3). Variable phenotypes were obtained

between samples within clumps at Punta Bandera, indicating that at least some individual clumps were not the result of vegetative spread.

Observed genotypic frequencies deviated significantly from Hardy–Weinberg expectations in 48% of possible comparisons, 87% of which (data not shown) gave positive fixation indices. Estimates

of F-statistics by jackknifing over loci yielded average values of 0.394 (SE=0.165) and 0.284 (SE=0.204) for FIT and FIS, respectively. The 95% confidence interval for FIS (−0.042 to 0.646)

indicated that, on average, observed heterozygotes did not differ significantly from expectation, suggesting no significant inbreeding effects in Pilgerodendron. The analysis of genetic

structure indicated that most of the genetic diversity is found within populations (HT=0.050, HS=0.042) and the degree of differentiation among populations measured by FST was 16% (Table 4).

Whereas the mean number of alleles per locus for Pilgerodendron was only 1.2, for Austrocedrus it was 1.7 (Ferreyra et al., 1996), and for Fitzroya it was 1.5 (Premoli et al., 2000b). The

same pattern was found for expected heterozygosity and polymorphism (0.99 criterion), with population-level averages of 0.071 and 41% for Austrocedrus (Ferreyra et al., 1996), 0.077 and 33%

for Fitzroya (Premoli et al., 2000b), and only 0.033 and 17% for Pilgerodendron, respectively (all tests between Fitzroya and Pilgerodendron, P < 0.05, Mann–Whitney Rank Sum Test). Estimates

of genetic diversity indicated that total genetic diversity, as well as the within-population component, was significantly greater in Fitzroya, whereas Pilgerodendron had greater

among-population divergence (Table 4) (data for Austrocedrus not available). Mean FST for Pilgerodendron was twice as high as that for Fitzroya, and although they partially coincided in

their 95% confidence intervals, Pilgerodendron’s upper value exceeded that of Fitzroya.

Reduced isozyme variation was detected within populations of Pilgerodendron uviferum compared to the other two members of the Cupressaceae from southern Argentina and Chile, Austrocedrus

chilensis and Fitzroya cupressoides, which have more restricted geographical distributions. Pilgerodendron is the conifer with the most widespread overall geographical range in temperate

South America and thus, as suggested by reviews of isozyme data in plants (e.g. Hamrick & Godt, 1989; Hamrick et al., 1992), higher levels of isozyme variation would have been expected

relative to more geographically restricted species. However, our results were not surprising given that although Pilgerodendron has the most extended latitudinal range, it consists of

scattered and small populations, commonly restricted to particular habitats such as periglacial environments, lowland bogs, and wetlands. Austrocedrus, in contrast, is found in a wide array

of habitats, from temperate wet forests, to Mediterranean-type environments, as well as near the forest–steppe ecotone in Patagonia. Fitzroya, on the other hand, usually grows in humid areas

where annual precipitation ranges from 2000 mm to 4000 mm, occurs at different elevations from ≈100 m to 1200 m, and on different soil types, from poorly drained soils, to incipient soils

of volcanic origin, and well developed loamy soils (Veblen et al., 1995). Thus, in comparison to Pilgerodendron, Austrocedrus and Fitzroya generally consist of larger and more continuous

populations, and as a result, may maintain elevated polymorphism and among-population gene flow. Our results are consistent with Donoso’s (1995); p. 55 observations on Pilgerodendron, which

predicted that habitat-restricted species, occurring in isolated populations, would tend to be genetically monomorphic.

Although many woody species maintain relatively high levels of genetic variability (Hamrick & Godt, 1996), some examples exist of widespread conifers with limited genetic variation, such as

Pinus resinosa (Fowler & Morris, 1977) and Tsuga canadensis (Zabinski, 1992), with 0 and 10% polymorphism, respectively. The most common explanation for the reduction or absence of isozyme

variation is that each species has gone through one or more population bottlenecks. The reduced polymorphism and partial inbreeding measured in populations of Pilgerodendron, may be

explained in terms of locally surviving populations that were isolated throughout the last Glacial Maximum during the Pleistocene and which suffered the effects of past population

bottlenecks and reduced gene flow.

The results presented here are similar to those using RAPD variation for some of the same Pilgerodendron populations analysed here and for a subset of Fitzroya populations studied by Premoli

et al. (2000a,b). The analysis of 16 populations of Pilgerodendron yielded lower polymorphism (35.7%, Allnutt et al., pers. comm.) than that recorded in 12 populations of Fitzroya (72.4%,

Allnutt, pers. comm.). In addition, AMOVA analysis of RAPD variation indicated that a greater proportion of the total variation was found among different populations of Pilgerodendron

(18.55%, Allnutt et al., pers. comm.) than in Fitzroya (14.38%, Allnutt et al., 1999).

Data for species of Nothofagus (southern beech) from temperate South America in the subgenus Nothofagus, which in turn forms a monophyletic group (Manos, 1997), also show a similar pattern

to that found for the comparison between Pilgerodendron and Fitzroya. The widespread N. pumilio, which is distributed from 35° to 55°S latitude but is locally restricted to high-elevation

forests, showed reduced polymorphism and greater among-population divergence than N. dombeyi, which also has a wide range (35° to 48°S) but consists of more continuous populations. A

comparative analysis using isozyme loci indicated that N. dombeyi is significantly more variable than N. pumilio, with a mean number of alleles per locus of 1.6 vs. 1.2, a polymorphism (0.95

criterion) of 25 vs. 13%, observed heterozygosities of 0.082 vs. 0.019, and expected heterozygosities of 0.093 vs. 0.03 in N. dombeyi vs. N. pumilio, respectively (all tests P < 0.05,

Whitney Rank Sum Test). In addition, based on seven polymorphic loci, mean FST values were 0.180 for N. dombeyi and 0.312 for N. pumilio, suggesting greater among-population divergence in

the latter (calculated from Premoli, 1997, 1998).

Other studies using isozymes have yielded similar results in trees; for example, the rare Pacific yew (Taxus brevifolia) which occurs over a wide range but is sparsely distributed and never

abundant, indicated low polymorphism (