Andrea C. Premoli

Genetic variation in Fitzroya cupressoides (alerce), a threatened South American conifer

Fitzroya cupressoides (alerce, Cupressaceae) is a large and exceptionally long‐lived conifer, endemic to a restricted area of southern Chile and neighbouring areas of Argentina. As a result of its high economic value, the species has been severely exploited for timber, and remnant populations are fragmented and often highly disturbed. The species is thought to have undergone a major range contraction during the last glaciation. In order to assess the extent of genetic variation using DNA markers within and between populations of this species, samples were obtained from throughout the natural range and analysed for random amplified polymorphic DNA (RAPD) variation. Eight 10‐mer and three 15‐mer primers were used to produce a total of 54 polymorphic bands. Shannons diversity estimates were calculated to provide an estimate of the degree of variation within each population. Values varied from 0.343 to 0.636 with only the lowest value differing significantly from the others (Spop= 0.547). This indicated that there is a significant degree of variation within each population, and did not provide evidence for genetic ‘bottleneck’ effects within the species. A pairwise distance measure calculated from the RAPD data was used as an input for principal coordinate (PCO) and amova analyses. The first three principal coordinates of RAPD distances described 8.3, 5.9 and 5.4% of the total variance, respectively, and a degree of clustering of samples according to their geographical origin was detectable. amova analysis indicated that although most of the variation (85.6%) was found within populations, a significant proportion (P < 0.002) was attributable to differences between populations. An upgma dendrogram constructed using ΦST values derived from amova produced a pattern broadly similar to that produced by the PCO, highlighting differences between three main groups of populations within Chile: those from the northern Coastal Range, the southern Coastal Range and Central Depression, and the Andes. Populations from Argentina also emerged as significantly different from those in Chile. These results are interpreted in the context of the postglacial history of the species, and their implications for the development of conservation strategies for Fitzroya are discussed.

Genetic variation in the vulnerable and endemic Monkey Puzzle tree, detected using RAPDs

Araucaria araucana (Monkey Puzzle), a southern South American tree species of exceptional cultural and economic importance, is of conservation concern owing to extensive historical clearance and current human pressures. Random amplified polymorphic DNA (RAPD) markers were used to characterise genetic heterogeneity within and among 13 populations of this species from throughout its natural range. Extensive genetic variability was detected and partitioned by analysis of molecular variance, with the majority of variation existing within populations (87.2%), but significant differentiation was recorded among populations (12.8%). Estimates of Shannons genetic diversity and percent polymorphism were relatively high for all populations and provide no evidence for a major reduction in genetic diversity from historical events, such as glaciation. All pairwise genetic distance values derived from analysis of molecular variance (ΦST) were significant when individual pairs of populations were compared. Although populations are geographically divided into Chilean Coastal, Chilean Andes and Argentinean regions, this grouping explained only 1.77% of the total variation. Within Andean groups there was evidence of a trend of genetic distance with increasing latitude, and clustering of populations across the Andes, suggesting postglacial migration routes from multiple refugia. Implications of these results for the conservation and use of the genetic resource of this species are discussed.

Genetic variation in the threatened South American conifer Pilgerodendron uviferum (Cupressaceae), detected using RAPD markers

Pilgerodendron uviferum (Cipres de las Guiatecas, Cupressaceae) is a long-lived conifer, endemic to southern Chile and Argentina, reaching a southern limit in Tierra del Fuego. Remnant populations are generally fragmented and highly disturbed because of exploitation for timber, grazing and fire. The extent of genetic variation within and between 16 populations of this species, distributed throughout its range, was assessed using random amplified polymorphic DNA (RAPD) markers. Eight 10-mer primers produced a total of 84 scorable markers, 30 of which (35.7%) were polymorphic. AMOVA indicated that 18.6% of the variation recorded was attributable to differences between populations, a relatively high value compared with other conifers from the region. Pair-wise Phist comparisons between populations were all significant at P<0.05, with one exception, highlighting a high degree of population differentiation. Values of Shannons diversity index (S) differed significantly among populations (P=0.002, ANOVA), values ranging from 0.337 to 0.716, suggesting that some populations are currently characterised by very low genetic variability. Current patterns of genetic variation were related to biogeographic history and human impact. The high degree of population differentiation recorded here highlights the need for additional conservation measures for this species, both in terms of incorporating further populations into the protected areas, and the restoration of severely degraded populations, to ensure their continued viability.

Evidence of chloroplast capture in South American Nothofagus (subgenus Nothofagus, Nothofagaceae)

Subgenus Nothofagus, although geographically restricted at present to temperate areas of South America, has captured much attention in discussions of plant biogeography due to its widespread distribution through Gondwanan continents during the Tertiary. However, phylogenetic relationships within the subgenus Nothofagus have not yet been resolved. We examined geographic patterns of intraspecific and interspecific genetic variation to detect whether incongruences in nuclear or plastid DNA phylogenies occur, in order to better understand the evolutionary history of the subgenus Nothofagus. We conducted spatially-explicit sampling at 10 distinct locations throughout the range of austral South American forests and sampled all present Nothofagus species. We used ITS and chloroplast DNA sequences to estimate phylogenetic relationships. A phylogeny constructed from nuclear genes resolved the subgenus Nothofagus as monophyletic. We found that N. antarctica was a sister to a clade of evergreen species (N. betuloides, N. dombeyi, and N. nitida), while N. pumilio likely diverged earlier. Nine cpDNA haplotypes were distinguished in the subgenus Nothofagus which were associated to geographic locations rather than to taxonomic relationships. This species-independent cpDNA phylogeographic structures within the subgenus Nothofagus may be related to repeated chloroplast capture events over geological time in Patagonia.

Toward Integrated Analysis of Human Impacts on Forest Biodiversity: Lessons from Latin America

Although sustainable forest management (SFM) has been widely adopted as a policy and management goal, high rates of forest loss and degradation are still occurring in many areas. Human activities such as logging, livestock husbandry, crop cultivation, infrastructural development, and use of fire are causing widespread loss of biodiversity, restricting progress toward SFM. In such situations, there is an urgent need for tools that can provide an integrated assessment of human impacts on forest biodiversity and that can support decision making related to forest use. This paper summarizes the experience gained by an international collaborative research effort spanning more than a decade, focusing on the tropical montane forests of Mexico and the temperate rain forests of southern South America, both of which are global conservation priorities. The lessons learned from this research are identified, specifically in relation to developing an integrated modeling framework for achieving SFM. Experience has highlighted a number of challenges that need to be overcome in such areas, including the lack of information regarding ecological processes and species characteristics and a lack of forest inventory data, which hinders model parameterization. Quantitative models are poorly developed for some ecological phenomena, such as edge effects and genetic diversity, limiting model integration. Establishment of participatory approaches to forest management is difficult, as a supportive institutional and policy environment is often lacking. However, experience to date suggests that the modeling toolkit approach suggested by Sturvetant et al. (2008) could be of value in such areas. Suggestions are made regarding desirable elements of such a toolkit to support participatory-research approaches in domains characterized by high uncertainty, including Bayesian Belief Networks, spatial multi-criteria analysis, and scenario planning.

Regeneration mode affects spatial genetic structure of Nothofagus dombeyi forests

Disturbance may generate population bottlenecks by reducing population size and the number of founders establishing a new colony. We tested the hypothesis that the scale of disturbance affects the levels of genetic diversity and the spatial distribution of genotypes in naturally regenerating stands of Nothofagus dombeyi, an evergreen angiosperm tree, in northwestern Patagonia. At similar spatial scales, we predicted that old‐growth stands characterized by fine‐scale gap phase dynamics would be genetically diverse due to restricted gene flow among temporal and spatially isolated gaps. In contrast, young massively regenerated postfire cohorts resulting from coarse‐scale disturbances would be genetically more homogeneous. At each of three paired old‐growth and postfire stands a minimum of 50 trees were mapped and sampled within 1 ha. Fresh tissue was collected for isozyme analysis from a total of 361 trees along with tree cores and diameters. Tree age distributions reflected the dominant modes of regeneration. Six out of nine analysed loci were polymorphic. Mean genetic diversity parameters were greater but not significant in mature stands. Fixation indices suggested significant heterozygous deficit at two‐thirds of possible tests indicating a Wahlund effect due to local recruitment of related seeds. FST indicated moderate between‐stand divergence. Mature stands concentrated half of positively like joins and yielded significant (P < 0.05) autocorrelation coefficients at small distance classes (< 20 m). Fine‐scale patch dynamics within mature stands favours the maintenance of fine‐scale genetic structure as a result of shade intolerance and local seed dispersal. Conversely, postfire stands suffer the effects of genetic drift given that a few reproductive trees produce a somewhat impoverished and genetically uniform progeny. Bottleneck effects will depend upon the density of remnant trees which could also be a function of the severity of fire.

Out in the cold: genetic variation of Nothofagus pumilio (Nothofagaceae) provides evidence for latitudinally distinct evolutionary histories in austral South America

Nothofagus pumilio is the dominant and almost ubiquitous tree species in mountainous environments of temperate South America. We used two types of molecular markers (cpDNA and isozymes) to evaluate the effects of the Paleogene paleogeography of Patagonia and more recent climatic oscillations of the Neogene on such cold‐tolerant species’ genetic makeup. Phylogeographic analysis on sequences of three cpDNA non‐coding regions at 85 populations yielded two latitudinally disjunct monophyletic clades north and south of c. 42°S containing 11 and three haplotypes, respectively. This indicates a long‐lasting vicariant event due to the presence of an extended open paleobasin at mid latitudes of Patagonia. Also distribution patterns of cpDNA haplotypes suggest regional spread following stepping‐stone models using pre‐Cenozoic mountains as corridors. Comparable genetic diversity measured along 41 sampled populations using seven polymorphic isozyme loci provides evidence of local persistence and spread from multiple ice‐free locations. In addition, significantly higher heterozygosity and allelic richness at high latitudes, i.e. in areas of larger glacial extent, suggest survival in large and isolated refugia. While, higher cpDNA diversity in lower latitudes reflects the complex orogeny that historically isolated northern populations, lower isozyme diversity and reduced FST values provide evidence of local glacial survival in numerous small locales. Therefore, current genetic structure of N. pumilio is the result of regional processes which took place during the Tertiary that were enhanced by contemporary local effects of drift and isolation in response to Quaternary climatic cycles.

Phylogeographically concordant chloroplast DNA divergence in sympatric Nothofagus s.s. How deep can it be

• Here, we performed phylogenetic analyses and estimated the divergence times on mostly sympatric populations of five species within subgenus Nothofagus. We aimed to investigate whether phylogenetic relationships by nuclear internal transcribed spacer (ITS) and phylogeographic patterns by chloroplast DNA (cpDNA) mirror an ancient evolutionary history that was not erased by glacial eras. Extant species are restricted to Patagonia and share a pollen type that was formerly widespread in all southern land masses. Weak reproductive barriers exist among them. • Fifteen cpDNA haplotypes resulted from the analysis of three noncoding regions on 330 individuals with a total alignment of 1794 bp. Nuclear ITS data consisted of 822 bp. We found a deep cpDNA divergence dated 32 Ma at mid-latitudes of Patagonia that predates the phylogenetic divergence of extant taxa. Other more recent breaks by cpDNA occurred towards the north. • Complex paleogeographic features explain the genetic discontinuities. Long-lasting paleobasins and marine ingressions have impeded transoceanic dispersal during range expansion towards lower latitudes under cooler trends since the Oligocene. • Cycles of hybridization-introgression among extant and extinct taxa have resulted in widespread chloroplast capture events. Our data suggest that Nothofagus biogeography will be resolved only if thorough phylogeographic analyses and molecular dating methods are applied using distinct genetic markers.

Environmental v. genetically driven variation in ecophysiological traits of Nothofagus pumilio from contrasting elevations

Nothofagus pumilio (Poepp. et Endl.) Krasser is a broadleaved deciduous tree that dominates high-elevation forests in the southern Andes. We evaluated the degree to which differences in stomatal density and physiological traits (net assimilation, conductance and water use efficiency) were related to environmental and genetic influences with elevation by comparing plants growing under field and common garden conditions. Low-elevation leaves under field conditions had fewer stomata, although this pattern was not maintained in the common garden. Assimilation rates were >40% higher for high-elevation plants in the field, and 18% higher in the common garden, than those for low-elevation plants. In addition, under field conditions high-elevation plants tended to have higher stomatal conductance and lower instantaneous water use efficiency than did low-elevation plants; however, these differences were not significant in the common garden. Thus, assimilation seems to be under genetic control whereas ecophysiological traits related to the use of water appear to be more responsive to environmental cues. Our results suggest that plants growing along elevational gradients may show complex ecophysiological patterns. These patterns may be acquired by genetically driven responses to conditions that are fixed throughout the life span of individuals, such as soil nutrients. Also plastic adjustments may favour opportunistic use of available water during the dry season, particularly under Mediterranean-type climate regimes with summer drought.

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.