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The effect of glaciation on the levels and patterns of genetic variation has been well studied in the Northern Hemisphere. However, although glaciation has undoubtedly shaped the genetic structure of plants in the Southern Hemisphere, fewer studies have characterized the effect, and almost none of them using microsatellites. Particularly, complex patterns of genetic structure might be expected in areas such as the Andes, where both latitudinal and altitudinal glacial advance and retreat have molded modern plant communities. We therefore studied the population genetics of three closely related, hybridizing species of Nothofagus (N. obliqua, N. alpina, and N. glauca, all of subgenus Lophozonia; Nothofagaceae) from Chile. To estimate population genetic parameters and infer the influence of the last ice age on the spatial and genetic distribution of these species, we examined and analyzed genetic variability at seven polymorphic microsatellite DNA loci in 640 individuals from 40 populations covering most of the ranges of these species in Chile. Populations showed no significant inbreeding and exhibited relatively high levels of genetic diversity (HE = 0.502–0.662) and slight, but significant, genetic structure (RST = 8.7–16.0%). However, in N. obliqua, the small amount of genetic structure was spatially organized into three well-defined latitudinal groups. Our data may also suggest some introgression of N. alpina genes into N. obliqua in the northern populations. These results allowed us to reconstruct the influence of the last ice age on the genetic structure of these species, suggesting several centers of genetic diversity for N. obliqua and N. alpina, in agreement with the multiple refugia hypothesis.
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The current genetic structure and diversity of natural plant populations can be seen as the resulting product of the interaction between biology, geography, and climatic change (Hewitt 2000). Important biological factors include the breeding system, life form, seed dispersal, and pollination mechanism of the species (Hamrick 1982; Hamrick and Godt 1996), which together with the geographical range influence the level of isolation by distance (Wright 1943) and the effectiveness of geographical barriers against colonization and pollen flow. In addition, the dramatic climatic changes that occurred during the ice ages in the Quaternary had major effects on the patterns of genetic diversity not only in the boreal and temperate regions of the Northern Hemisphere (Soltis et al. 1997; Hewitt 2000), but also in austral and temperate regions in the Southern Hemisphere (Ogden 1989; Premoli et al. 2003; Marchelli and Gallo 2004; Azpilicueta et al. 2009; Worth et al. 2009; Mathiasen and Premoli 2010).
The west coast of southern South America in Chile and western Argentina between 33°00′S and 41°30′S is crossed longitudinally by two mountain ranges separated by the Central Valley. The Coastal Range has altitudes between 500 and 2000 m above sea level (m a.s.l.), and the Andes have altitudes between 3000 and 6000 m a.s.l., both becoming generally lower from north to south. The Mediterranean forests are found between 33°00′S and 36°30′S, and the temperate rainforests south of 37°30′S, with an ecotonal zone – the transitional forests – in between (Donoso 1982; Veblen and Schlegel 1982).
Nothofagus obliqua (Mirb.) Oerst., N. alpina (Poepp. et Endl.) Oerst. (= N. nervosa), and N. glauca (Phil.) Krasser. are sympatric South American endemics. Together with two species from Australia (N. cunninghamii (Hook.) Oerst. and N. moorei (Muell.) Krasser.) and one from New Zealand (N. menziessii (Hook.) Oerst.), these species constitute subgenus Lophozonia (Manos 1997). This group of deciduous species grows in both Mediterranean and temperate rainforest regions in Chile and adjacent areas in Argentina, occupying different elevations from the Central Valley to the Coastal and Andean mountain ranges (Ormazabal and Benoit 1987). Of the three species, N. obliqua has the most extensive geographical distribution, covering nearly 1000 km in longitude, N. alpina has an intermediate range of 700 km, and N. glauca has a narrower distribution covering approximately 400 km (Fig. 1).
Figure 1. Range of distribution for (A) Nothofagus obliqua, (B) N. alpina, and (C) N. glauca in gray, showing the location of the sampled populations used in our study. Last glacial maximum extent of the ice sheet obtained from Hollin and Schilling (1981).
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These three species are tall, long-lived trees easily reaching 30 m in height and 300 years of age. These monoecious species have anemophilous pollination and a largely outcrossing breeding system (Riveros et al. 1995; Gallo et al. 1997; Ipinza and Espejo 2000). An important difference in reproductive biology among the three species is seed dispersal, which is predominantly accomplished by gravity in N. glauca and by a combination of wind and gravity in N. obliqua and N. alpina (Donoso 1993).
The current habitat of these species was largely affected by repeated glaciations during the Quaternary, influencing the distribution of forests. During the last glacial maximum (LGM ≈ 20,000 year ago), glaciers covered a large proportion of the current forest, and additionally, palynological research has identified periglacial effects (Villagran et al. 1995).
The Central Valley of central Chile (33°00′–36°00′S) is characterized by warm and dry summers and supports sclerophyllous forests. However, with more precipitation at higher elevations, it is possible to find populations of Nothofagus spp. that form presumably relict forests in the northern limit of their distributions, such as the N. obliqua island forests in the highlands of the Coastal Range between 33°00′ and 34°00′S (Donoso 1993; Villagran 2001). During the LGM, glaciers reached altitudes as low as 1200–3000 m lower than today, creating climatic conditions that favored the colonization of valleys by Nothofagus (Heusser 1990) and probably eradicated Nothofagus from the mountains. The Central Valley then could have served as one large panmictic glacial refugium or perhaps as several isolated refugia. The end of the ice age (10,000 years ago) gradually brought drier and warmer conditions to the area, pushing Nothofagus forests to their current distributions in the mountains.
Similar events occurred in the middle portion of the ranges of the three species (36°00′–39°00′S). Today, isolated populations of N. alpina, which has its main distribution in the Andes, occur on the top of the Nahuelbuta Mountains in the Coastal Range. These forest islands have been interpreted as the remnants of glacial populations that were once growing in the Central Valley during the ice age (Villagran 2001). Unlike central Chile, this area is currently wet enough for some other Nothofagus species (e.g., N. obliqua) to live in the Central Valley. According to pollen records, recolonization of Nahuelbuta by Nothofagus spp. started about 6000 years ago in the Holocene (Villagran 2001).
Finally, the area south of 40°00′S experienced different periglacial effects. The proximity of the glaciers during the last ice age allowed only the most cold-resistant and hygrophilous forest elements to survive in discontinuous populations in lowland sites in the Central Valley and in the Coastal Range (Villagran 2001). Vegetation was dominated by nonarboreal taxa mixed with these cold-resistant and hygrophilous species resembling a parkland with varying degrees of openness (Villagran et al. 1995; Moreno et al. 1999). Gradually, after 14,200 years ago, more mesic taxa started arriving in the area. Thermophyllous forest taxa (e.g., N. obliqua, N. alpina) might have expanded slowly to this area in the Holocene (after 10,000 years ago) when climatic conditions started to be warm and moist enough to support that vegetation (Moreno et al. 1999), which suggests that the refugia for those taxa were probably localized north of 40°00′S, in the Coastal Range and the Central Valley.
The hypothesis of glacial refugia for N. obliqua and N. alpina localized north of 40°00′S in places including the valleys near Nahuelbuta (Villagran 2001) or Rucañancu (39°30′S) in the Andes piedmont (Villagran 1991) is supported by the extant pollen records. However, there is the possibility that these species survived in low numbers in multiple scattered refugia associated with favorable microclimates at higher altitudes and latitudes, without leaving any trace in the pollen record (Markgraf et al. 1996). This latter hypothesis is supported directly by genetic studies in N. obliqua (Azpilicueta et al. 2009) and N. alpina (Marchelli et al. 1998; Marchelli and Gallo 2006; Carrasco et al. 2009) and indirectly by genetic studies in other tree species in the area (Allnutt et al. 1999; Premoli et al. 2000, 2003; Bekessy et al. 2002; Nunez-Avila and Armesto 2006) and southward (Pastorino et al. 2009; Mathiasen and Premoli 2010).
In accordance with Nothofagus anemophilous pollination, genecological studies on N. obliqua (Donoso 1979a) and N. alpina (Donoso 1987) and population genetics studies conducted using nuclear markers in N. alpina (Pineda 2000; Carrasco and Eaton 2002; Carrasco et al. 2009) show a north-to-south clinal pattern of variation produced by large-scale pollen flow and a climatic cline between the northern and southern populations. This pollen flow also facilitates interbreeding among species, generating natural hybrids in specific environmental conditions (i.e., N. alpina × N. obliqua (Donoso et al. 1990; Gallo et al. 1997; Marchelli and Gallo 2001) and N. obliqua x N. glauca (= N. leonii Espinosa) (Donoso 1979b)). Thus, anywhere these species grow in close proximity, there is a potential for hybridization and introgression among them. Likewise, this extensive pollen flow has been shown to maintain relatively high levels of genetic variability and low population differentiation in N. alpina and in most Nothofagus species (Table 1).
Table 1. Genetic variability and differentiation assessed with nuclear genetic markers in other similar studies.
|From genus Nothofagus|
|N. alpina (= N. nervosa)||Reg||Allozymes||22||19||7||2.9||0.484||9.6||Pineda 2000|
|Allozymes||18||36||10||3.0||0.289||5.1||Carrasco and Eaton 2002|
|RAPDs||22||27||33||–||0.150||12.4||Carrasco et al. 2009|
|Allozymes||11||112||8||2.3||0.173||3.8||Marchelli and Gallo 2001|
|Allozymes||20||115||8||3.4||0.180||5.2||Marchelli and Gallo 2004|
|Allozymes||2||71||6||1.9||0.126||–||Milleron et al. 2008|
|Allozymes||2||30||6||1.6||0.163||–||Milleron et al. 2008|
|Microsats||2||71||3||4.3||0.474||–||Milleron et al. 2008|
|Microsats||2||30||3||3.8||0.474||–||Milleron et al. 2008|
|Microsats||14||35||7||4.4||0.452||6.1||Azpilicueta et al. 2013|
| N. obliqua ||Reg||Microsats||10||34||7||4.3||0.455||4.9||Azpilicueta et al. 2013|
|Allozymes||14||143||7||2.2||0.223||5.1||Azpilicueta and Gallo 2009|
| N. alessandrii ||Narr||Allozymes||7||27||7||1.8||0.182||25.7||Torres-Diaz et al. 2007|
| N. nitida ||Narr||Allozymes||4||42||15||1.3||0.045||4.7||Premoli 1997|
| N. betuloides ||Reg||Allozymes||4||28||15||1.5||0.116||12.0||Premoli 1997|
| N. dombeyi ||Reg||Allozymes||5||34||15||1.6||0.093||7.4||Premoli 1997|
| N. pumilio ||Reg||Allozymes||41||29||7||1.4||0.070||20.0||Mathiasen and Premoli 2010|
|Allozymes||6||90||5||2.0||0.084||–||Mathiasen and Premoli 2013|
|Microsats||6||50||5||3.7||0.496||–||Mathiasen and Premoli 2013|
| N. antarctica ||Reg||Allozymes||12||48||2||2.7||0.185||11.0||Pastorino et al. 2009|
|Allozymes||28||36||8||2.2||0.207||18.8||Acosta et al. 2012|
| N. truncata ||Reg||Allozymes||30||57||5||1.3||0.051||4.9||Haase 1992|
| N. menziesii ||Reg||Allozymes||5||52||15||1.5||0.116||–||Haase 1993|
| N. moorei ||Narr||ISSRs||20||7||42||1.8||0.168||10.4||Taylor et al. 2005|
|From other related genera|
| Fagus sylvatica ||Wide||Microsats||10||130||4||14.9||0.829||5.8||Buiteveld et al. 2007|
| F. japonica ||Reg||Microsats||16||34||13||8.6||0.659||2.3||Hiraoka and Tomaru 2009|
| Quercus glauca ||Wide||Microsats||10||19||4||6.5||0.741||4.2||Lee et al. 2006|
| Q. macrocarpa ||Wide||Microsats||14||34||5||11.2||0.864||2.7||Craft and Ashley 2007|
| Q. petraea ||Wide||Microsats||5||60||6||8.6||0.755||20.1||Bruschi et al. 2003|
|Microsats||7||30||13||7.0||0.797||0.8||Muir et al. 2004|
| Q. semiserrata ||Wide||Microsats||10||39||8||8.2||0.679||12.0||Pakkad et al. 2008|
| Q. garryana ||Reg||Microsats||22||15||7||4.9||0.597||4.9||Marsico et al. 2009|
Our goal is to evaluate, through the use of nuclear microsatellite markers, the levels of genetic diversity and structure of the three South American species of subgenus Lophozonia (Nothofagus) to infer how these genetic parameters are influenced by climatic change, geography, and biological factors. Thus, in this study we will compare results obtained here with studies using other biparentally inherited markers in N. alpina: allozymes (Pineda 2000; Carrasco and Eaton 2002) and RAPDs (Carrasco et al. 2009). Furthermore, our microsatellite data will also complement recent studies on N. obliqua (Azpilicueta et al. 2009) and N. alpina (Marchelli and Gallo 2006) based on chloroplast DNA, which is maternally inherited and traces exclusively colonization by seed dispersal. In contrast, microsatellite markers can detect both pollen flow and seed dispersal and therefore allow for fine-scale analysis of local and regional patterns of genetic diversity (Selkoe and Toonen 2006).
We hypothesize that populations of the three species growing in the Mediterranean forests between 33°00′S and 36°30′S may have reduced genetic diversity and higher levels of differentiation due to their proposed isolation since the end of the last glacial period (Heusser 1990; Villagran 2001). We further expect to see lower genetic diversity in the narrowly distributed N. glauca than in the more widespread N. obliqua and N. alpina. Finally, we do not expect to see a north-to-south pattern of diminishing variation due to founder effects in the colonization after the LGM as in the patterns often seen in plants in the Northern Hemisphere (Soltis et al. 1997, 2006; Taberlet et al. 1998; Hewitt 2000; Petit et al. 2003). Instead, we predict different centers of variation along the current distribution of the species in line with the multiple refugia hypothesis for South America (Markgraf et al. 1996; Premoli et al. 2000).