ABSTRACT 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

and 0.033, respectively. Most genetic diversity was found within populations (_H_T=0.039, _H_S=0.033, _F_ST 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. SIMILAR CONTENT BEING VIEWED BY OTHERS GENETIC DIVERSITY AND CONSERVATION OF SIBERIAN APRICOT (_PRUNUS SIBIRICA_ L.) BASED ON MICROSATELLITE

MARKERS Article Open access 11 July 2023 GENETIC ANALYSIS OF _BROMUS TECTORUM_ (POACEAE) IN THE MEDITERRANEAN REGION: BIOGEOGRAPHICAL PATTERN OF NATIVE POPULATIONS Article 19 August 2020

STRONGER GENETIC DIFFERENTIATION AMONG WITHIN-POPULATION GENETIC GROUPS THAN AMONG POPULATIONS IN SCOTS PINE PROVIDES NEW INSIGHTS INTO WITHIN-POPULATION GENETIC STRUCTURING Article Open

access 01 February 2024 INTRODUCTION 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? THE SPECIES _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. MATERIALS AND METHODS

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 (_H_O and _H_E, 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 (_H_T) and genetic diversity within populations (_H_S) 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 (_F_ST ˜ _G_ST) 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. RESULTS 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 _H_O and _H_E, 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 _F_IT and _F_IS, respectively. The 95% confidence interval for _F_IS (−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 (_H_T=0.050, _H_S=0.042) and the degree of differentiation among populations measured by _F_ST 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 _F_ST 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_. DISCUSSION 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 _F_ST 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

(<50%) and moderate among-population divergence (_G_ST ≈ 10%) (Scher, 1996). In contrast, _Pinus echinata_, with a wide range and wide habitat requirements, had high polymorphism (54%)

and lower divergence among populations (_G_ST=9%; Rajiv et al., 1997). In addition, species with restricted range but consisting of disjunct populations, also had elevated levels of total

diversity found among different populations, such as _Pinus pungens_ (_G_ST=13.5%; Gibson & Hamrick, 1991), _P. radiata_ (_G_ST=16%; Moran et al., 1988), _P. halepensis_ (_G_ST=30%;

Schiller et al., 1986), and _P. cembra_ (_G_ST=32%; Szmidt, 1982). Overall, it was suggested that the level of genetic heterogeneity among populations of pine species is greater in species

with geographically disjunct populations than in those with more continuous distributions (Hamrick & Godt, 1996). However, the relationship between divergence and polymorphism with

geographical range and continuity of their populations was not explored. Geographical range has been considered a good predictor of the levels of genetic variation in plant populations

(Hamrick & Godt, 1989). However, and particularly in the case of woody species, the geographical range only accounted for 44.5% of the total variation (34%) that was explained by a

multiple regression model, which included other life-history traits (Hamrick et al., 1992). One potential limitation of the explanatory power of these models may be related to the fact that

pooling information from unrelated taxa with dissimilar life histories makes generalizations difficult (Karron, 1987). It has been suggested that paired comparisons between congeners may

increase the probability that restricted and widespread species have similar ecological features and phylogenetic histories (Karron, 1987, 1997; Gitzendanner & Soltis, 2000). Therefore,

measured genetic differences can be attributed to the characteristics (such as geographical range) in which they differ. From the analysis of 11 pairs of congener species, rare species had

significantly lower levels of genetic variation than their widespread congeners (Karron, 1987). In addition, for the 34 pairs of congeners examined by the review of Gitzendanner & Soltis

(2000), different indicators of isozyme variation were significantly lower in the rare than in the widespread species. However, although some rare species maintained reduced genetic

variation, they had a large range of values, exceeding those of widespread congeners in some cases. Furthermore, the available species’ pairs did not differ significantly, as in the analysis

done by Hamrick & Godt (1989), in the estimates of total genetic diversity (_H_T) or in the degree of among-population differentiation measured by _F_ST ˜ _G_ST. All this information

suggests that geographical range alone may not be adequate in predicting levels of polymorphism and divergence between populations. It has been suggested that species which are considered

rare may exhibit a diverse array of total ranges, population sizes, and habitat specificity resulting in seven different types of rarity (Rabinowitz, 1981). As a result, rare species with

different attributes may maintain different levels and patterns of genetic diversity (Premoli et al., 1999; Gitzendanner & Soltis, 2000). For example, based on overall geographical

distribution alone, _Pilgerodendron_ can be considered widespread, whereas _sensu_ Rabinowitz it could be classified as a rare species with a wide range but occurring in disjunct habitats

along the Andes, which is expected to affect the levels of variability and the degree of among-population divergence. Thus, we suggest that available genetic information on species should be

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Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to the National Parks in both Chile and Argentina for facilitating our work in protected areas. We thank R.

Vergara and J.C. Aravena for sample collections of Chilean populations, A. Rovere for sample collections of Argentinean populations, and M. Caldiz for field and laboratory assistance.

Discussions with J. Armesto, R. Ennos, and A. Lara greatly improved the formulation of the concepts included in our manuscript. We thank John Brookfield and two anonymous reviewers for

insightful comments and suggestions. This research was supported by the European Commission-funded project SUCRE (Sustainable Use, Conservation and Restoration of Native Forest in southern

Chile and Argentina and south-central Mexico), Framework IV of DGXII, contract number ERBIC18CT970146. A.P. is supported by CONICET (Consejo Nacional de Investigaciones Científicas y

Técnicas de Argentina). AUTHOR INFORMATION Author notes * A C Newton Present address: UNEP-World Conservation Monitoring Centre, 219 Huntington Road, Cambridge, CB3 0DL, U.K. AUTHORS AND

AFFILIATIONS * Centro Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral 1250, Bariloche, 8400, Argentina A C Premoli & C P Souto * Applied Genetics, John Innes

Centre, Norwich, NR4 7UH, UK T R Allnutt * Institute of Ecology and Resource Management, University of Edinburgh, Darwin Building, Kings Buildings, Mayfield Rd, Edinburgh, EH9 3JU, UK A C

Newton Authors * A C Premoli View author publications You can also search for this author inPubMed Google Scholar * C P Souto View author publications You can also search for this author

inPubMed Google Scholar * T R Allnutt View author publications You can also search for this author inPubMed Google Scholar * A C Newton View author publications You can also search for this

author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to A C Premoli. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Premoli, A., Souto,

C., Allnutt, T. _et al._ Effects of population disjunction on isozyme variation in the widespread _Pilgerodendron uviferum_. _Heredity_ 87, 337–343 (2001).

https://doi.org/10.1046/j.1365-2540.2001.00906.x Download citation * Received: 01 November 2000 * Accepted: 28 March 2001 * Published: 01 September 2001 * Issue Date: 01 September 2001 *

DOI: https://doi.org/10.1046/j.1365-2540.2001.00906.x SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable

link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * ciprés de las Guaitecas * conifer *

Cupressaceae * Patagonia * _Pilgerodendron_