Conservation Genetics 1: 57–66, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Conservation genetics of the endangered conifer Fitzroya cupressoides in Chile and Argentina A.C. Premoli1∗ , T. Kitzberger1 & T.T. Veblen2 1 Centro
Regional Universitario Bariloche, Universidad Nacional del Comahue, Quintral 1250, 8400 Bariloche, Argentina; 2 Department of Geography, University of Colorado, Boulder, CO 80309-0260, USA; ∗ author for correspondence (E-mail:
[email protected]) Received 22 December 1999; accepted 19 April 2000
Key words: gene pool, isozymes, Patagonia, protected areas, rare species
Abstract Intraspecific patterns of genetic variation can often be used to identify biogeographic divisions which can be especially useful in the design of conservation strategies. Although abundant empirical evidence exist on the genetic characteristics of plant species from the Northern Hemisphere as well as tropical endangered taxa, this information is particularly limited on threatened species from endemism-rich areas in the southern Andes of Argentina and Chile. The objective of the current study was to analyze the levels and distribution of the isozyme variation in Fitzroya cupressoides (Mol.) Johnst. (Cupressaceae), a rare conifer restricted to temperate rainforests of northern Patagonia, and to evaluate the role of current conservation areas protecting the gene pool of this valuable longlived conifer. Sampling schedules consisted of fresh foliage collected from 30 randomly selected trees at each of 24 different populations located along the geographic range of the species. Extraction of enzymes followed standard procedures and homogenates were loaded in 12% starch gels which were analyzed by horizontal electrophoresis. Eleven enzyme systems were resolved using a combination of four different buffer solutions which yielded information on 21 putative loci, 52% of them were polymorphic in at least one population. Relatively low levels of within-population genetic variability were scored in Fitzroya populations which were approximately half of the typical levels published for gymnosperms (percent of polymorphic loci, P = 23 vs. 53% and expected heterozygosity, HE = 0.077 vs. 0.155 for Fitzroya and other conifers respectively). Substantial between-population variation was detected, and certain individual populations stand out as much more genetically variable than nearby populations, which in turn are located outside protected areas. Our findings suggest that if the objective is to protect key species like Fitzroya, spatially explicit genetic information can be a useful tool to attain this goal.
Introduction Knowledge of distribution patterns of genetic variation in plants is important for informing conservation strategies. The theoretical basis of conservation genetics resides in the fact that preservation of genetic variability is essential to the maintenance of evolutionary potential of natural populations (Frankel and Soulé 1981). In recent years, the protection of genetic diversity within species has become a primary goal of biological conservation, and the use of genetic markers has been suggested as a valuable tool for achieving that goal, particularly of tree species endemic to
temperate forests in South America (Premoli 1998). However, some debate has arisen over the relative importance of ecological and genetic factors in survival of species and populations (Lande 1988; Falk and Holsinger 1991; Schmeske et al. 1994; Hamrick and Godt 1996). Although the persistence of most species over the short term depends upon demographic and environmental threats, genetic variability also needs to be considered in planning effective long-term conservation strategies (Mace et al. 1996). In the present study we analyze the amount and distribution of isozyme variation in Fitzroya cupressoides (alerce), a long-lived tree endemic to
58 temperate forests in southern South America. Isozymes are functionally similar forms of enzymes produced by different gene loci which together with allozymes (subsets of isozymes that represent different allelic alternatives of the same locus) are the most cost-effective method for investigating genetic phenomena at the molecular level (Murphy et al. 1996), and thus they can be used as genetic markers. Fitzroya has been cited as one of the world’s most spectacular examples of the serious impoverishment of forest genetic resources (Veblen et al. 1976). This impoverishment is due not only to the extirpation, or near extirpation of Fitzroya from entire habitats, but also to its slow recovery following logging. Seedling establishment is often scarce or nil after logging, and tree growth rates are exceptionally slow. Thus, after logging removes all mature trees from a site a long time must pass before a new generation of trees reaches sexual maturity. Even in forests unaffected by logging, coarse-scale disturbance events that permit abundant regeneration occur at intervals that typically are much longer than a century. Consequently, an inherently low rate of population turnover in this species makes it vulnerable to anthropogenic genetic impoverishment. This vulnerability, plus continued economic and political pressures to relax logging prohibitions (Lara et al. 1996), means that spatially explicit knowledge of the genetic variability of this species is required for effective planning of conservation strategies. For example, in Chile’s Central Depression only five small stands and eight sites of scattered trees remain from a forest type that formerly covered thousands of hectares (Fraver et al. 1999), and restoration efforts need to be guided by knowledge of how genetically similar these surviving trees are in relation to other potential sources of seed and cuttings. Fitzroya has been recently the subject of a genetic study by Allnutt et al. (1999) using random amplified polymorphic DNA (RAPD) variation, a technique that generates individual fingerprints via the polymerase chain reaction using short, random sequence primers. That study reported certain degree of amongpopulation genetic divergence although a limited number of populations (only 12) and individuals (a total of 89 samples) were analyzed. Thus, we will not only provide information on a wider range of populations using a different marker to be used in conservation efforts, but also further evidence to elucidate the type of polyploidy of alerce which is a tetraploid with 2n = 44 (Hair 1968). Isozyme electrophoresis is used
to distinguish allopolyploids from autopolyploids by the presence of fixed heterozygosity in the former (e.g. Watson et al. 1991) whereas polysomic segregation is expected in the latter (e.g. Soltis and Rieseberg 1986).
The species Fitzroya is a large long-lived conifer (Cupressaceae) of a monotypic genus that grows in Chile and Argentina. It can reach up to 5 m in diameter and 50 m in height (Veblen et al. 1976; Lara 1991). It is the second oldest living tree in the world after bristlecone pine (Pinus longaeva), and tree-ring chronologies exceeding 3600 years have been developed from trees sampled in Chile (Lara and Villalba 1993). It grows as discontinuous populations in the coastal cordillera and the central depression of Chile and on the western and eastern slopes of the Andes in both Chile and Argentina from ca. 39◦500 to 42◦ 450 S. Generally, it occurs at elevations from ca. 100 to 1200 m, typically on nutrient-poor soils, and where mean annual precipitation ranges from 2000 to over 4000 mm (Veblen et al. 1995). Fitzroya stands have often been described as consisting almost exclusively of large, old trees with scarce or no younger individuals (Veblen et al. 1995 and references therein). This type of stand structure led earlier workers to conclude that the species is a climatic relict not capable of reproducing under current climatic conditions (Kalela 1941; Tortorelli 1956; Schmithusen 1960). However, more recent studies have elucidated the regeneration strategy of this long-lived conifer (Veblen et al. 1995; T.T. Veblen, unpublished data). Fitzroya is highly shade-intolerant and competes poorly with other tree species on sites that are edaphically and climatically favorable. Many of the stands of mature Fitzroya lacking regeneration are remnant populations which established following coarse-scale disturbance by fire, landslides, flood deposition, or volcanic ash deposition (Veblen and Ashton 1982; Lara 1991; Veblen et al. 1995; Fraver et al. 1999; Lara et al. 1999). During long intervals free of such coarse-scale disturbance, there typically is little or no regeneration of Fitzroya at that site. Recent studies have documented adequate Fitzroya regeneration following natural disturbances in various habitats in both Chile and Argentina as long as sites are not logged or subjected to browsing by livestock (Veblen and Ashton 1982; Lara 1991; Donoso et al. 1993; T.T. Veblen, unpublished data).
59 Conservation status of Fitzroya Because of its valuable wood, Fitzroya has been heavily logged since the early European settlement in the 1500s, and logging has eliminated most populations from accessible areas. In many cases post-logging regeneration has been insufficient to maintain the populations (Veblen et al. 1976). Extensive logging of this valuable timber species has extirpated many local populations, and extant stands are restricted to remote areas of difficult access (Veblen et al. 1976, 1995; Lara et al. 1996). Due to the heavy exploitation of alerce, concern about its conservation status led Argentinean authorities to list Fitzroya since 1941 on the Annex to the Convention on Nature Protection and Wildlife Preservation in the Western Hemisphere. In 1969 Chile enacted some protective legislation requiring management measures to assure regeneration after logging (Corporación Nacional Forestal 1974) and in 1976 logging was prohibited (Ministerio de Agricultura 1976). In 1973, Fitzroya was placed on Appendix I of CITES, Convention on International Trade in Endangered Species of Wild Fauna and Flora (1984) which prohibits its commercialization and international trade. Fitzroya’s conservation status under CITES was relaxed in the mid-1980s, but in 1987 it was returned to Appendix I (Lara et al. 1991). In addition, Fitzroya is included under the US Endangered Species Act and the importation of its wood into the United States is prohibited (Threatened Species Conservation Act 1979). Since the late-1980s, there has been very little illegal exploitation of the relatively small Fitzroya populations in Argentina where well over 80% of Fitzroya forests occur within the protected area system (Kitzberger et al., in press). However, enforcement of the logging ban in the much larger Chilean populations, including extensive private holdings, has often been lax, and illegal cutting of Fitzroya continued into the 1990s (Lara et al. 1996). Fitzroya is listed as vulnerable on the International Union for the Conservation of Nature’s (IUCN) Red List (Farjon et al. 1993), which means that although it is considered endangered it is regarded as facing a high risk of extinction in the wild in the medium-term future (IUCN 1994). Fitzroya is protected in several national parks. In Chile, the most extensive of these parks is Alerce Andino National Park at ca. 43◦ S in the Andes, but large areas of Fitzroya forest are also privately owned. In Argentina, most of the extant population of Fitzroya is protected by location in one of three
National Parks (Nahuel Huapi, Lago Puelo, and Los Alerces). Much smaller areas of Fitzroya forests occur in provincial reserves and on privately owned land in Argentina (Kitzberger et al., in press). Although large-scale logging of Fitzroya has been stopped in both Chile and Argentina, small-scale illegal logging activities continue, especially in Chile (Lara et al. 1996). In this study we address the following questions: What are the levels of within-population isozyme variation in a tree species which has suffered dramatic range reduction such as Fitzroya? What is the relative importance to the conservation of the entire gene pool of the species of geographically isolated populations, especially those located outside protected areas? What are the levels of between-population differentiation that could be utilized in restoring degraded populations?
Materials and methods Twenty-four naturally occurring populations were sampled throughout the species’ range (Table 1). Populations 1 through 12 are in Argentina on the eastern slopes of the Andes, and populations 13 through 24 are in Chile on the western slopes of the Andes or the coastal cordillera (Figure 1, Table 1). These two groups are subsequently referred to as eastern and western populations. Sampling consisted of collecting fresh leaf material from 30 randomly selected individuals in each population. Approximately 20 cm of twig with needle tissue was collected from randomly scattered trees separated by a minimum of 10 m in order to get a representative sample of each stand. Given that alerce is known to reproduce vegetatively (Veblen and Ashton 1982; Lara 1991), special care was given at the time of leaf collection to avoid sampling of the same individual. In cases of small populations all the individuals in the stand were sampled (e.g. Coastal Cordillera Eastern), whereas more than one population was sampled from locations with relative extensive and continuous stands (e.g. Lago Menéndez). Labeled samples were transported in a portable cooler and in the laboratory stored at 0–5 ◦ C. Fresh foliage was grounded in mortars using liquid nitrogen, and enzymes were extracted with the extraction buffer of Mitton et al. (1979). Homogenates were frozen at –80 ◦ C until electrophoresis was performed. The tissue homogenate was absorbed onto Whatman No. 3 paper wicks,
60 Table 1. Genetic variability measures in populations of Fitzroya cupressoides; 1–12 western and 13–24 eastern populations. N = average number of individuals analyzed per locus and population; A = mean number of alleles per locus; P
