American Journal of Botany 101(9): 1403–1408, 2014.
COMMENTARY
A COOL EXPERIMENTAL APPROACH TO EXPLAIN ELEVATIONAL TREELINES, BUT CAN IT EXPLAIN THEM?1 MAAIKE Y. BADER2, HANNAH LORANGER, AND GERHARD ZOTZ University of Oldenburg, Department of Biology and Environmental Sciences, Functional Ecology of Plants, 26111 Oldenburg, Germany At alpine treeline, trees give way to low-stature alpine vegetation. The main reason may be that tree canopies warm up less in the sun and experience lower average temperatures than alpine vegetation. Low growth temperatures limit tissue formation more than carbon gain, but whether this mechanism universally determines potential treeline elevations is the subject of debate. To study low-temperature limitation in two contrasting treeline tree species, Fajardo and Piper (American Journal of Botany 101: 788–795) grew potted seedlings at ground level or suspended at tree-canopy height (2 m), introducing a promising experimental method for studying the effects of alpine-vegetation and tree-canopy microclimates on tree growth. On the basis of this experiment, the authors concluded that lower temperatures at 2 m caused carbon limitation in one of the species and that treeline-forming mechanisms may thus be taxon-dependent. Here we contest that this important conclusion can be drawn based on the presented experiment, because of confounding effects of extreme root-zone temperature fluctuations and potential drought conditions. To interpret the results of this elegant experiment without logistically challenging technical modifications and to better understand how low temperature leads to treeline formation, studies on effects of fluctuating vs. stable temperatures are badly needed. Other treeline research priorities are interactions between temperature and other climatic factors and differences in microclimate between tree canopies with contrasting morphology and physiology. In spite of our criticism of this particular study, we agree that the development of a universal treeline theory should include continuing explorations of taxon-specific treeline-forming mechanisms. Key words: alpine tree line; carbon balance; ecophysiology; Fuscospora; growth limitation; methodology; micrometeorology; Nothofagus; Pinus; timberline.
In a recent paper in the American Journal of Botany, Fajardo and Piper (2014) presented an experimental approach that compares tree seedling performance in conditions typical for low-stature alpine vegetation and conditions representing treecanopy conditions at treeline. The rationale of this approach was that treelines are assumed to form because tree canopies cannot warm up as much as low-stature alpine vegetation can, due to a stronger coupling to atmospheric conditions (Körner, 1998). The lower temperatures experienced by a tree canopy do not allow growth, either because carbon assimilation is insufficient (the source-limitation or carbon-limitation hypothesis), or because growth processes (e.g., cell division or lignification) are directly impaired (the sink-limitation or growth-limitation hypothesis). Although most results of recent research, in particular on elevational patterns of nonstructural carbohydrate contents in trees (summarized by Hoch and Körner, 2012) and on low-temperature limits to wood formation (e.g., Rossi et al., 2008), support the growth limitation hypothesis, some findings 1 Manuscript received 6 June 2014; revision accepted 13 August 2014. The authors thank three anonymous reviewers for constructive comments on an earlier version of this commentary. Funding for our experiments and for travel of M.Y.B. to New Zealand was provided by the German Research Foundation (BA 3843/4-1, BA 3843/5-1 and -2). 2 Author for correspondence (e-mail: [email protected]); present address: University of Marburg, Faculty of Geography, Ecological Plant Geography, Deutschhausstraße 10, 35032 Marburg, Germany
doi:10.3732/ajb.1400256
seem to point at carbon limitation (Wiley and Helliker, 2012; Dawes et al., 2013; though see Palacio et al., 2014). This issue is thus not generally resolved. The new approach used by Fajardo and Piper (2014) aimed to study low-temperature limitation in trees experimentally by placing individuals of similar size and developmental stage in temperature regimes usually experienced by individuals of very different sizes, thereby avoiding any confusion of microclimatic effects with ontogenetic and size effects. Even though tree seedlings were used, this approach was not aimed at studying seedling performance as such but used seedlings as a smallversion model of a taller tree to study responses of tree growth to microclimatic conditions. In the experiment, one of the two species tested, Nothofagus pumilio, showed a decrease in growth and nonstructural carbohydrate (NSC) reserves when suspended at 2 m above the ground, where mean temperatures were lower than at ground level. The other species, Pinus contorta, showed no response. The authors concluded that their study is the first unambiguous test of the mechanism behind growth limitations at treeline elevation, with evidence for differences between treeline-forming taxa. Such an important finding deserves thorough scrutiny. Here, we argue that due to their particular experimental method, the observed carbon limitation may be due to other factors than low mean temperature and does not represent strong and unambiguous evidence for a treeline-specific phenomenon. However, we do agree with the authors that potential taxonspecific, growth-limiting mechanisms at treeline should be seriously considered when further developing alpine-treeline
American Journal of Botany 101(9): 1403–1408, 2014; http://www.amjbot.org/ © 2014 Botanical Society of America
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theory. First, because we should continue to question whether there is really one mechanism explaining this low-temperature life-form boundary or whether equally valid representatives of this life-form are constrained differently. And second, because only by systematically exploring the variation beyond a universal treeline-forming mechanism can we hope to understand and predict treeline elevations in real landscapes. Confounding temperature conditions—It is an excellent idea to test differences in plant performance under low-vegetationand tree-canopy-temperature regimes independent of ontogeny and size. Growing seedlings at ground level and at canopy level (but outside an actual canopy) to mimic the different levels of atmospheric coupling is a highly promising approach. However, there is one fundamental problem: soil temperatures in suspended pots fluctuate very strongly. They fluctuate much more strongly than soil temperatures at ground level and also more strongly than air temperatures around the suspended pots (but less than air temperatures at ground level, see fig. 2 of Fajardo and Piper, 2014). In the experiment of Fajardo and Piper (2014), suspended seedlings thus experienced similar temperature fluctuations in roots (3–10°C) and shoots (3–8°C), whereas groundlevel seedlings experienced low root-zone fluctuations (6–8°C) and high shoot-zone fluctuations (2–14°C). Such strong temperature fluctuations in suspended pots clearly do not mimic root-zone conditions below a tree canopy, which are very stable because of the large soil volume and the shade provided by the canopy (Körner and Paulsen, 2004). In the experiment of Fajardo and Piper (2014), the average temperature in the suspended pots was about 1 K lower than in ground-level pots. In that sense, the suspended pots did mimic one aspect of soil conditions under a tree canopy, which are nearly always cooler during the growing season than under nearby alpine vegetation (Bendix and Rafiqpoor, 2001; Bader et al., 2007; Körner, 2012). The crucial question here is, however, what do these mean temperatures mean physiologically? Biological rates respond nonlinearly to temperature, so that at a given mean temperature, fluctuating temperatures should lead to different mean biological rates than constant temperatures. On the other hand, mean growing season temperatures are the most consistent thermal parameter at treelines worldwide (Körner and Paulsen, 2004; Cieraad et al., 2014; Paulsen and Körner, 2014) and experimental results from Hoch and Körner (2009) showed similar growth rates in two conifer species at constant and variable temperatures. Although these observations still await a physiological explanation, they suggest that mean temperature really has a biological meaning. The lower mean root-zone temperature in the suspended pots could thus rightfully be expected to slow seedling growth. However, as long as it cannot be excluded that the strong temperature fluctuations contributed to the observed differences in seedling performance, the observed effect cannot be unequivocally attributed to the different mean temperatures. How likely is it that these fluctuations were really a problem? The answer depends on how temperature differences between root and shoot and on how temperature fluctuations in general affect seedling physiology. Although these seem two very basic biological questions, there is surprisingly little information to guide an answer (Pregitzer et al., 2000). Opposite effects of root and shoot temperature on plant nutrient status (Weih and Karlsson, 2001) and biomass allocation patterns (Larigauderie et al., 1991) suggest that temperature differences between roots and shoots, aside from their absolute temperatures, can affect
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whole-plant performance. However, we did not find experiments focusing explicitly on this question. Temperature-fluctuation effects on plant growth are hardly studied either. In an experiment addressing this for trees at low mean temperatures, conifer seedlings grew similarly well under constant or variable (ca. 6 K amplitude around 6°C or 12°C) daily and seasonal temperature regimes, with only slight positive effects of fluctuations for Larix decidua but not for Pinus mugo (Hoch and Körner, 2009). In contrast, in the only other experiment with trees that we could find, temperature variability (5 or 10 K amplitude around 23°C, with a 5-d fluctuation) clearly affected growth and root to shoot ratios in poplar (Populus deltoides ×nigra) cuttings, with positive effects of fluctuations at the intermediate but not at the greater amplitude (Cerasoli et al., 2014). Obviously, this limited and ambiguous evidence does not allow generalizations. Additional experiments addressing temperature-fluctuation effects on tree growth, addressing differences between species or functional types, the mean temperature and the amplitude of the fluctuations, are clearly desirable. Apart from identifying potential artifacts in experiments like that of Fajardo and Piper (2014), such studies could greatly contribute to understanding the physiological meaning of different temperature parameters for treeline formation. Confounding moisture conditions— There is a second concern: how severe was drought stress in these suspended plots, and did this stress affect the results? Seedlings were only watered during the first month of the growing season. Even though precipitation during the growing season was ca. 500 mm at this Patagonian site (Fajardo and Piper, 2014), this statistic does not preclude that rainless periods were frequent and that the soil in the pots dried out repeatedly during these periods, especially in the suspended pots. If P. contorta is less sensitive to drought than N. pumilio, this sensitivity could explain the species-specific responses. The documented carbon limitation of N. pumilio (reduced growth associated with low NSC contents) could thus be caused by water stress and not by low temperature. The authors argued that water shortage should not have affected N. pumilio seedlings, based on results of earlier experiments where seedlings did not respond to watering in ambient and warmed conditions (Piper et al., 2013). However, not requiring extra watering in full-soil conditions does not imply drought tolerance in suspended pots. Playing devil’s advocate, one could even argue that this earlier experiment did show a trend, though not significant, to higher growth rates under watering (Piper et al., 2013). Similar negative effects of drought (as a result of experimental warming) have been observed at treeline in New Zealand for seedlings of Fuscospora cliffortioides (previously Nothofagus solandri var. cliffortioides, Heenan and Smissen, 2013) (Melanie Harsch, University of Washington, Seattle, personal communication) and in North America for recently germinated Pinus flexilis seedlings (Moyes et al., 2013). In the contested experiment by Fajardo and Piper (2014), the higher mortality (significant when combining both species) in the suspended pots also suggests a stress factor confounding or aggravating a potential temperature effect. To conclude, the study by Fajardo and Piper (2014) does not, because it could not, provide unambiguous support for lowtemperature-induced carbon limitation in N. pumilio. Because adult trees of the same species show no increase in NSC with elevation and thus do not appear carbon limited (Fajardo et al., 2011), the fundamentally different growth-limitation for Nothofagus compared with other treeline trees remains to be shown.
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Alternative experimental setups— Finding problems in an experiment is easy. More difficult and more useful is proposing solutions. Understanding how low temperatures limit tree growth is not only of fundamental biological interest but also affects model predictions of tree growth in a warmer and CO2richer future. Temperature effects can be studied in isolation in fully controlled conditions in growing chambers (e.g., Hoch and Körner, 2009). However, it is very difficult to mimic the typical treeline combination of high radiation and low air temperature. For the question of carbon vs. growth limitation, in particular, results from such experiments will be hard to translate to the real world. In the field, one option for studying temperature effects on treeline tree growth is to warm existing tree canopies. However, this technically and logistically very challenging manipulation is practicable only for small sections such as branches (Lenz et al., 2013) or buds (Petit et al., 2011). Such sectional studies can yield important information about local growth processes but cannot contrast carbon vs. growth limitation directly, because these involve microclimatic effects on whole-tree carbon gain and use. Using seedlings as model tissue for tree performance is a sensible alternative. Compared with adult trees, they allow for better replication and faster responses and a distinction of microclimatic from ontogenetic and size effects. Suspended seedlings experience tree canopy conditions, but the results may be confounded by soil temperature fluctuations not seen in natural soil and by uncontrolled soil moisture. The moisture issue is relatively easy to solve (disregarding logistic difficulties for maintaining an irrigation system at treeline). Whether temperature fluctuations are a problem remains to be investigated, as discussed above. As long as this is unclear, temperature needs to be controlled. Temperature fluctuations in suspended pots can be reduced by using reflective material, insulation, and ventilation layers around the pots. However, in trials related to a somewhat similar experiment, we were unable to design pots that can be suspended in air while maintaining the stable temperature conditions typical for full soil, let alone for full soil under a tree canopy. We tried this in northern Germany (ca. 53°N) in early spring, with relatively mild sunshine loads. At the alpine treeline in the middle of summer and at lower latitudes, we would expect the problem to be worse. Active temperature control using flowing water around the pots could work well from the physical point of view, though from the practical point of view such a system would be very challenging to install at most treeline sites. Assuming that root-zone temperatures can be controlled, the question remains which temperature regimes would allow an informative comparison between canopy-level seedlings and ground-level seedlings. Seedlings can be used as models to study tree growth in two distinct ways: as a model for tree tissue in general or as a small-version model of a tall tree. These approaches require explicit assumptions about temperature effects and require different temperature regimes. Using seedlings as a model for tree tissue in general seems reasonable for some questions because roots and shoots have similar temperature thresholds for growth (references in Rossi et al., 2007) and both need to be warm enough for a plant to grow (Körner and Hoch, 2006). For such a model, roots and shoots should ideally experience the same temperature regimes. Therefore, root-zone temperatures in suspended and ground-level pots should be regulated to follow the respective air temperatures (and irrigation adjusted accordingly).
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The alternative, using seedlings as a small-version model for a tree, assumes that whole-plant physiology is affected differently by root than by shoot temperature (Larigauderie et al., 1991; Weih and Karlsson, 2001) and that this is similar for seedlings and adult trees. Such a small-version tree model is what Fajardo and Piper (2014) had in mind for their experiment, which “aimed to mimic the low temperature effects on meristematic shoot and root tissues of a taller tree and, eventually, the temperature effects on the tree’s C balance as a whole.” For such a model, temperatures in the pots should follow the respective root-zone temperatures in alpine vegetation (groundlevel pots) and below trees (suspended pots). Thus, only suspended pots would need regulation (assuming the ground-level pots are buried in the ground and temperatures there are naturally representative), and this regulation could be based on measured soil temperatures under a nearby tree canopy. Alternatively, if the question is focused on aboveground temperatures only, root-zone temperatures could be similar in both treatments, i.e., could be regulated in suspended pots to follow the temperature in the ground-level pots. A common approach to manipulate temperature for seedlings at treeline is warming, either passively by using transparent roofs (Germino and Smith, 1999) or open-top chambers (e.g., Danby and Hik, 2007; Xu et al., 2012), or actively by using infrared lamps (Moyes et al., 2013). All these methods are useful for studying thermal constraints for seedlings and for mimicking future climate warming, keeping in mind restrictions inherent to the different types of warming. For mimicking tree canopy conditions, however, seedlings need to be cooled, which is more challenging than warming. Other than suspending seedlings in the air, options would be planting under the tree canopy or with artificial cover (but there will be confounding effects of shade), planting at higher elevations (but this option implies cooling both day and night, in contrast to tree canopies, which are warmer at night than low vegetation), or active cooling. Active cooling could be achieved via radiative cooling (e.g., using peltier or other electric cooling elements near the plants) or via convective cooling (using ventilators, supplying air from outside the soil–alpine vegetation boundary layer). As an imitation of convective cooling (and at night: warming) of the tree canopy, the ventilator option seems by far the better choice. This approach, on a larger scale using wind machines, is commonly used in horticulture to prevent radiation frosts (Perry, 1998). As in the suspended-pot experiment presented by Fajardo and Piper (2014), such a setup would have to control for soil moisture differences. Belowground temperatures could either be left to equilibrate with the air (the easier option), or aboveground ventilation could be accompanied by temperature regulation of the root zone to mimic soil conditions below a tree canopy, e.g., using electric temperature elements as is sometimes used, though again usually for heating only, in climate-change experiments (Melillo et al., 2002). In all of these setups, apart from accounting for potential microclimatic artifacts, the assumed role of the seedlings as models for tree growth should be made explicit and validated before translating the results obtained with the seedling models to adult trees. Comparing taxa regarding canopy–microclimate effects on tree growth is only one side of the story, however. To understand fully the role of microclimate in limiting tree growth, differences in canopy microclimates and tissue temperatures due to taxon-specific leaf and crown morphologies, transpiration rates and albedo also need to be considered (Leuzinger and
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Körner, 2007). Data addressing such differences for treeline trees are rare (Körner, 2012), though they would be relatively easy to obtain using dataloggers. Such data would be another valuable contribution toward understanding the mechanisms of treeline formation for different taxa and functional tree types. Alternative treeline-forming mechanisms in Nothofagus and other genera— Nothofagus treelines have long been regarded as unusual, occurring at higher temperatures than most northern-hemisphere treelines, which was explained by genusspecific limitations (Körner and Paulsen, 2004; Wardle, 2008). However, several recent studies suggest that mean temperatures in the growing season at these treelines are actually quite comparable to those at other treelines (Mark et al., 2008; Cieraad et al., 2014; Fajardo and Piper, 2014). Another argument against genus-specific limitations is presented in the study discussed here (Fajardo and Piper, 2014), where Pinus contorta seedlings did not outperform Nothofagus pumilio at 50 m above the treeline. The authors argue that because of the lower mean
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temperatures in the suspended pots, P. contorta should not outperform N. pumilio up to 330 m above the treeline, although this argument disregards temperature extremes. However, in an experiment in New Zealand started in the 1960s, Pinus contorta and other exotic conifers developed large stems up to 300 m above the local treeline, while saplings of the native treeline species Fuscospora cliffortioides (≡ Nothofagus solandri var. cliffortioides), though surviving ca. 150 m above the treeline, have still not emerged from the boxes that protected them as seedlings (Fig. 1). Clearly, Pinus is outperforming this close relative of Nothofagus in this case (Wardle, 1985). So the question remains: do the Nothofagaceae form treelines for different reasons than most other treeline tree families? Most treelines composed of Nothofagaceae are very abrupt boundaries from tall, closed forest to low alpine vegetation, suggesting that limitations to establishment outside the forest rather than limited growth determine the position of these treelines (Harsch and Bader, 2011). Above the natural treeline, Fuscospora cliffortioides seedlings depend on shade and/or
Fig. 1. (A) Overview of the treeline tree-establishment experiment installed by Peter Wardle in the 1960s in the Craigieburn Range, New Zealand (photos taken in March 2009 by M. Y. Bader). View down toward the treeline from the experimental garden at 1450 m, ca. 150 m above the current treeline. Exotic tree species, many of which have grown well at this elevation, have been cut down to prevent them from spreading into the native ecosystem. Note the shrubby Fuscospora cliffortioides (≡ Nothofagus solandri var. cliffortioides), the local treeline tree species that appears to have survived or established in the shelter of the now cut-down exotic trees. (B) Stump of Pinus contorta at 1450 m. The box is the remnant of an experimental 73% shade treatment. The rosette plant in the box is the alpine species Aciphylla cf. aurea, unrelated to the experiment. (C) Shade box with barely grown F. cliffortioides apparently unable to escape these sheltered conditions (at 1450 m) (experiment and results described in Wardle, 1985, 2008).
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frost protection, which also suggests that tree establishment and treeline advance are not limited by low growing temperatures alone but by the interaction with stressors like frost, excess radiation, and wind (Wardle, 1993, 2008). Such stressors cannot offer a universal explanation for treeline formation, as they vary strongly among treelines and can occur at any elevation (Körner, 2012). In the absence of such stressors and in the case of resistant species, treeline elevations can be controlled by low temperature limitations to growth, either via source or sink limitation. In all other cases, regional peculiarities and “taxon-specific” treelines emerge (Harsch and Bader, 2011). As these are arguably the rule rather than the exception (e.g., Piper et al., 2006; Wardle, 2008; Holtmeier, 2009; Malanson et al., 2011), we embrace the recommendation of Fajardo and Piper (2014) to keep developing a universal theory for treeline formation including variation between tree taxa and regional climates. Experiments to this end should address low-temperature limitations to growth as well as interactions with other climatic factors in all tree life stages. Preferably, such experiments should include several members of different functional tree types to allow generalizations beyond taxon-specificity. Conclusion— The presented experimental setup in Fajardo and Piper (2014) is a low-technology approach for studying a fundamental question in functional plant biology: what causes treelines? Though elegant, the experiment suffers from a potentially disqualifying practical problem: the strong fluctuations in soil temperature (and most probably soil moisture) in the suspended pots. As long as the effect of such fluctuations is unknown, they cannot be ignored and should be experimentally controlled. A promising alternative is to keep seedlings at ground level and ventilate them to mimic the atmospheric coupling found in tree canopies. These solutions require an infrastructure that is not usually available at treeline, though solar panels and local water sources could allow these setups even in remote sites. Apart from technical solutions, we discussed conceptual solutions: how can we interpret the data given the temperature and moisture fluctuations? To do this, soil moisture data would be needed as well as a much better understanding of the effects of temperature fluctuations on tree growth. At this stage, an unambiguous interpretation of the results of Fajardo and Piper (2014) seems impossible. Nothofagus pumilio seedlings are carbon-limited before they are growth-limited at the temperature and moisture conditions in the suspended pots, but it is unresolved whether the limitations are really due to the lower mean temperature or (1) to the strongly fluctuating rootzone temperature regime, or (2) to moisture stress. Although their experiment did not allow unambiguous conclusions about the causes of alpine treeline formation, it provides excellent food for thought on further experiments toward this goal. LITERATURE CITED BADER, M. Y., M. RIETKERK, AND A. K. BREGT. 2007. Vegetation structure and temperature regimes of tropical alpine treelines. Arctic, Antarctic, and Alpine Research 39: 353–364. BENDIX, J., AND M. D. RAFIQPOOR. 2001. Studies on the thermal conditions of soils at the upper tree line in the páramo of Papallacta (Eastern Cordillera of Ecuador). Erdkunde 55: 257–276. CERASOLI, S., T. WERTIN, M. A. MCGUIRE, A. RODRIGUES, D. P. AUBREY, J. S. PEREIRA, AND R. O. TESKEY. 2014. Poplar saplings exposed to recurring temperature shifts of different amplitude exhibit differences in leaf gas exchange and growth despite equal mean temperature. AoB Plants 6.
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ROSSI, S., A. DESLAURIERS, J. GRICAR, J. W. SEO, C. B. K. RATHGEBER, T. ANFODILLO, H. MORIN, ET AL. 2008. Critical temperatures for xylogenesis in conifers of cold climates. Global Ecology and Biogeography 17: 696–707. WARDLE, P. 1985. New Zealand timberlines. 1. Growth and survival of native and introduced tree species in the Craigieburn Range, Canterbury. New Zealand Journal of Botany 23: 219–234. WARDLE, P. 1993. Causes of alpine timberline: A review of the hypotheses. In J. Alden, J. L. Mastrantonio, and S. Odum [eds.], Forest development in cold climates, vol. 244, NATO ASI series A: Life sciences, 89–103. Plenum Press, New York, New York, USA. WARDLE, P. 2008. New Zealand forest to alpine transitions in global context. Arctic, Antarctic, and Alpine Research 40: 240–249. WEIH, M., AND P. S. KARLSSON. 2001. Growth response of mountain birch to air and soil temperature: Is increasing leaf-nitrogen content an acclimation to lower air temperature? New Phytologist 150: 147–155. WILEY, E., AND B. HELLIKER. 2012. A re-evaluation of carbon storage in trees lends greater support for carbon limitation to growth. New Phytologist 195: 285–289. XU, Z. F., T. X. HU, AND Y. B. ZHANG. 2012. Effects of experimental warming on phenology, growth and gas exchange of treeline birch (Betula utilis) saplings, Eastern Tibetan Plateau, China. European Journal of Forest Research 131: 811–819.
COMMENTARY
A COOL EXPERIMENTAL APPROACH TO EXPLAIN ELEVATIONAL TREELINES, BUT CAN IT EXPLAIN THEM?1 MAAIKE Y. BADER2, HANNAH LORANGER, AND GERHARD ZOTZ University of Oldenburg, Department of Biology and Environmental Sciences, Functional Ecology of Plants, 26111 Oldenburg, Germany At alpine treeline, trees give way to low-stature alpine vegetation. The main reason may be that tree canopies warm up less in the sun and experience lower average temperatures than alpine vegetation. Low growth temperatures limit tissue formation more than carbon gain, but whether this mechanism universally determines potential treeline elevations is the subject of debate. To study low-temperature limitation in two contrasting treeline tree species, Fajardo and Piper (American Journal of Botany 101: 788–795) grew potted seedlings at ground level or suspended at tree-canopy height (2 m), introducing a promising experimental method for studying the effects of alpine-vegetation and tree-canopy microclimates on tree growth. On the basis of this experiment, the authors concluded that lower temperatures at 2 m caused carbon limitation in one of the species and that treeline-forming mechanisms may thus be taxon-dependent. Here we contest that this important conclusion can be drawn based on the presented experiment, because of confounding effects of extreme root-zone temperature fluctuations and potential drought conditions. To interpret the results of this elegant experiment without logistically challenging technical modifications and to better understand how low temperature leads to treeline formation, studies on effects of fluctuating vs. stable temperatures are badly needed. Other treeline research priorities are interactions between temperature and other climatic factors and differences in microclimate between tree canopies with contrasting morphology and physiology. In spite of our criticism of this particular study, we agree that the development of a universal treeline theory should include continuing explorations of taxon-specific treeline-forming mechanisms. Key words: alpine tree line; carbon balance; ecophysiology; Fuscospora; growth limitation; methodology; micrometeorology; Nothofagus; Pinus; timberline.
In a recent paper in the American Journal of Botany, Fajardo and Piper (2014) presented an experimental approach that compares tree seedling performance in conditions typical for low-stature alpine vegetation and conditions representing treecanopy conditions at treeline. The rationale of this approach was that treelines are assumed to form because tree canopies cannot warm up as much as low-stature alpine vegetation can, due to a stronger coupling to atmospheric conditions (Körner, 1998). The lower temperatures experienced by a tree canopy do not allow growth, either because carbon assimilation is insufficient (the source-limitation or carbon-limitation hypothesis), or because growth processes (e.g., cell division or lignification) are directly impaired (the sink-limitation or growth-limitation hypothesis). Although most results of recent research, in particular on elevational patterns of nonstructural carbohydrate contents in trees (summarized by Hoch and Körner, 2012) and on low-temperature limits to wood formation (e.g., Rossi et al., 2008), support the growth limitation hypothesis, some findings 1 Manuscript received 6 June 2014; revision accepted 13 August 2014. The authors thank three anonymous reviewers for constructive comments on an earlier version of this commentary. Funding for our experiments and for travel of M.Y.B. to New Zealand was provided by the German Research Foundation (BA 3843/4-1, BA 3843/5-1 and -2). 2 Author for correspondence (e-mail: [email protected]); present address: University of Marburg, Faculty of Geography, Ecological Plant Geography, Deutschhausstraße 10, 35032 Marburg, Germany
doi:10.3732/ajb.1400256
seem to point at carbon limitation (Wiley and Helliker, 2012; Dawes et al., 2013; though see Palacio et al., 2014). This issue is thus not generally resolved. The new approach used by Fajardo and Piper (2014) aimed to study low-temperature limitation in trees experimentally by placing individuals of similar size and developmental stage in temperature regimes usually experienced by individuals of very different sizes, thereby avoiding any confusion of microclimatic effects with ontogenetic and size effects. Even though tree seedlings were used, this approach was not aimed at studying seedling performance as such but used seedlings as a smallversion model of a taller tree to study responses of tree growth to microclimatic conditions. In the experiment, one of the two species tested, Nothofagus pumilio, showed a decrease in growth and nonstructural carbohydrate (NSC) reserves when suspended at 2 m above the ground, where mean temperatures were lower than at ground level. The other species, Pinus contorta, showed no response. The authors concluded that their study is the first unambiguous test of the mechanism behind growth limitations at treeline elevation, with evidence for differences between treeline-forming taxa. Such an important finding deserves thorough scrutiny. Here, we argue that due to their particular experimental method, the observed carbon limitation may be due to other factors than low mean temperature and does not represent strong and unambiguous evidence for a treeline-specific phenomenon. However, we do agree with the authors that potential taxonspecific, growth-limiting mechanisms at treeline should be seriously considered when further developing alpine-treeline
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theory. First, because we should continue to question whether there is really one mechanism explaining this low-temperature life-form boundary or whether equally valid representatives of this life-form are constrained differently. And second, because only by systematically exploring the variation beyond a universal treeline-forming mechanism can we hope to understand and predict treeline elevations in real landscapes. Confounding temperature conditions—It is an excellent idea to test differences in plant performance under low-vegetationand tree-canopy-temperature regimes independent of ontogeny and size. Growing seedlings at ground level and at canopy level (but outside an actual canopy) to mimic the different levels of atmospheric coupling is a highly promising approach. However, there is one fundamental problem: soil temperatures in suspended pots fluctuate very strongly. They fluctuate much more strongly than soil temperatures at ground level and also more strongly than air temperatures around the suspended pots (but less than air temperatures at ground level, see fig. 2 of Fajardo and Piper, 2014). In the experiment of Fajardo and Piper (2014), suspended seedlings thus experienced similar temperature fluctuations in roots (3–10°C) and shoots (3–8°C), whereas groundlevel seedlings experienced low root-zone fluctuations (6–8°C) and high shoot-zone fluctuations (2–14°C). Such strong temperature fluctuations in suspended pots clearly do not mimic root-zone conditions below a tree canopy, which are very stable because of the large soil volume and the shade provided by the canopy (Körner and Paulsen, 2004). In the experiment of Fajardo and Piper (2014), the average temperature in the suspended pots was about 1 K lower than in ground-level pots. In that sense, the suspended pots did mimic one aspect of soil conditions under a tree canopy, which are nearly always cooler during the growing season than under nearby alpine vegetation (Bendix and Rafiqpoor, 2001; Bader et al., 2007; Körner, 2012). The crucial question here is, however, what do these mean temperatures mean physiologically? Biological rates respond nonlinearly to temperature, so that at a given mean temperature, fluctuating temperatures should lead to different mean biological rates than constant temperatures. On the other hand, mean growing season temperatures are the most consistent thermal parameter at treelines worldwide (Körner and Paulsen, 2004; Cieraad et al., 2014; Paulsen and Körner, 2014) and experimental results from Hoch and Körner (2009) showed similar growth rates in two conifer species at constant and variable temperatures. Although these observations still await a physiological explanation, they suggest that mean temperature really has a biological meaning. The lower mean root-zone temperature in the suspended pots could thus rightfully be expected to slow seedling growth. However, as long as it cannot be excluded that the strong temperature fluctuations contributed to the observed differences in seedling performance, the observed effect cannot be unequivocally attributed to the different mean temperatures. How likely is it that these fluctuations were really a problem? The answer depends on how temperature differences between root and shoot and on how temperature fluctuations in general affect seedling physiology. Although these seem two very basic biological questions, there is surprisingly little information to guide an answer (Pregitzer et al., 2000). Opposite effects of root and shoot temperature on plant nutrient status (Weih and Karlsson, 2001) and biomass allocation patterns (Larigauderie et al., 1991) suggest that temperature differences between roots and shoots, aside from their absolute temperatures, can affect
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whole-plant performance. However, we did not find experiments focusing explicitly on this question. Temperature-fluctuation effects on plant growth are hardly studied either. In an experiment addressing this for trees at low mean temperatures, conifer seedlings grew similarly well under constant or variable (ca. 6 K amplitude around 6°C or 12°C) daily and seasonal temperature regimes, with only slight positive effects of fluctuations for Larix decidua but not for Pinus mugo (Hoch and Körner, 2009). In contrast, in the only other experiment with trees that we could find, temperature variability (5 or 10 K amplitude around 23°C, with a 5-d fluctuation) clearly affected growth and root to shoot ratios in poplar (Populus deltoides ×nigra) cuttings, with positive effects of fluctuations at the intermediate but not at the greater amplitude (Cerasoli et al., 2014). Obviously, this limited and ambiguous evidence does not allow generalizations. Additional experiments addressing temperature-fluctuation effects on tree growth, addressing differences between species or functional types, the mean temperature and the amplitude of the fluctuations, are clearly desirable. Apart from identifying potential artifacts in experiments like that of Fajardo and Piper (2014), such studies could greatly contribute to understanding the physiological meaning of different temperature parameters for treeline formation. Confounding moisture conditions— There is a second concern: how severe was drought stress in these suspended plots, and did this stress affect the results? Seedlings were only watered during the first month of the growing season. Even though precipitation during the growing season was ca. 500 mm at this Patagonian site (Fajardo and Piper, 2014), this statistic does not preclude that rainless periods were frequent and that the soil in the pots dried out repeatedly during these periods, especially in the suspended pots. If P. contorta is less sensitive to drought than N. pumilio, this sensitivity could explain the species-specific responses. The documented carbon limitation of N. pumilio (reduced growth associated with low NSC contents) could thus be caused by water stress and not by low temperature. The authors argued that water shortage should not have affected N. pumilio seedlings, based on results of earlier experiments where seedlings did not respond to watering in ambient and warmed conditions (Piper et al., 2013). However, not requiring extra watering in full-soil conditions does not imply drought tolerance in suspended pots. Playing devil’s advocate, one could even argue that this earlier experiment did show a trend, though not significant, to higher growth rates under watering (Piper et al., 2013). Similar negative effects of drought (as a result of experimental warming) have been observed at treeline in New Zealand for seedlings of Fuscospora cliffortioides (previously Nothofagus solandri var. cliffortioides, Heenan and Smissen, 2013) (Melanie Harsch, University of Washington, Seattle, personal communication) and in North America for recently germinated Pinus flexilis seedlings (Moyes et al., 2013). In the contested experiment by Fajardo and Piper (2014), the higher mortality (significant when combining both species) in the suspended pots also suggests a stress factor confounding or aggravating a potential temperature effect. To conclude, the study by Fajardo and Piper (2014) does not, because it could not, provide unambiguous support for lowtemperature-induced carbon limitation in N. pumilio. Because adult trees of the same species show no increase in NSC with elevation and thus do not appear carbon limited (Fajardo et al., 2011), the fundamentally different growth-limitation for Nothofagus compared with other treeline trees remains to be shown.
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Alternative experimental setups— Finding problems in an experiment is easy. More difficult and more useful is proposing solutions. Understanding how low temperatures limit tree growth is not only of fundamental biological interest but also affects model predictions of tree growth in a warmer and CO2richer future. Temperature effects can be studied in isolation in fully controlled conditions in growing chambers (e.g., Hoch and Körner, 2009). However, it is very difficult to mimic the typical treeline combination of high radiation and low air temperature. For the question of carbon vs. growth limitation, in particular, results from such experiments will be hard to translate to the real world. In the field, one option for studying temperature effects on treeline tree growth is to warm existing tree canopies. However, this technically and logistically very challenging manipulation is practicable only for small sections such as branches (Lenz et al., 2013) or buds (Petit et al., 2011). Such sectional studies can yield important information about local growth processes but cannot contrast carbon vs. growth limitation directly, because these involve microclimatic effects on whole-tree carbon gain and use. Using seedlings as model tissue for tree performance is a sensible alternative. Compared with adult trees, they allow for better replication and faster responses and a distinction of microclimatic from ontogenetic and size effects. Suspended seedlings experience tree canopy conditions, but the results may be confounded by soil temperature fluctuations not seen in natural soil and by uncontrolled soil moisture. The moisture issue is relatively easy to solve (disregarding logistic difficulties for maintaining an irrigation system at treeline). Whether temperature fluctuations are a problem remains to be investigated, as discussed above. As long as this is unclear, temperature needs to be controlled. Temperature fluctuations in suspended pots can be reduced by using reflective material, insulation, and ventilation layers around the pots. However, in trials related to a somewhat similar experiment, we were unable to design pots that can be suspended in air while maintaining the stable temperature conditions typical for full soil, let alone for full soil under a tree canopy. We tried this in northern Germany (ca. 53°N) in early spring, with relatively mild sunshine loads. At the alpine treeline in the middle of summer and at lower latitudes, we would expect the problem to be worse. Active temperature control using flowing water around the pots could work well from the physical point of view, though from the practical point of view such a system would be very challenging to install at most treeline sites. Assuming that root-zone temperatures can be controlled, the question remains which temperature regimes would allow an informative comparison between canopy-level seedlings and ground-level seedlings. Seedlings can be used as models to study tree growth in two distinct ways: as a model for tree tissue in general or as a small-version model of a tall tree. These approaches require explicit assumptions about temperature effects and require different temperature regimes. Using seedlings as a model for tree tissue in general seems reasonable for some questions because roots and shoots have similar temperature thresholds for growth (references in Rossi et al., 2007) and both need to be warm enough for a plant to grow (Körner and Hoch, 2006). For such a model, roots and shoots should ideally experience the same temperature regimes. Therefore, root-zone temperatures in suspended and ground-level pots should be regulated to follow the respective air temperatures (and irrigation adjusted accordingly).
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The alternative, using seedlings as a small-version model for a tree, assumes that whole-plant physiology is affected differently by root than by shoot temperature (Larigauderie et al., 1991; Weih and Karlsson, 2001) and that this is similar for seedlings and adult trees. Such a small-version tree model is what Fajardo and Piper (2014) had in mind for their experiment, which “aimed to mimic the low temperature effects on meristematic shoot and root tissues of a taller tree and, eventually, the temperature effects on the tree’s C balance as a whole.” For such a model, temperatures in the pots should follow the respective root-zone temperatures in alpine vegetation (groundlevel pots) and below trees (suspended pots). Thus, only suspended pots would need regulation (assuming the ground-level pots are buried in the ground and temperatures there are naturally representative), and this regulation could be based on measured soil temperatures under a nearby tree canopy. Alternatively, if the question is focused on aboveground temperatures only, root-zone temperatures could be similar in both treatments, i.e., could be regulated in suspended pots to follow the temperature in the ground-level pots. A common approach to manipulate temperature for seedlings at treeline is warming, either passively by using transparent roofs (Germino and Smith, 1999) or open-top chambers (e.g., Danby and Hik, 2007; Xu et al., 2012), or actively by using infrared lamps (Moyes et al., 2013). All these methods are useful for studying thermal constraints for seedlings and for mimicking future climate warming, keeping in mind restrictions inherent to the different types of warming. For mimicking tree canopy conditions, however, seedlings need to be cooled, which is more challenging than warming. Other than suspending seedlings in the air, options would be planting under the tree canopy or with artificial cover (but there will be confounding effects of shade), planting at higher elevations (but this option implies cooling both day and night, in contrast to tree canopies, which are warmer at night than low vegetation), or active cooling. Active cooling could be achieved via radiative cooling (e.g., using peltier or other electric cooling elements near the plants) or via convective cooling (using ventilators, supplying air from outside the soil–alpine vegetation boundary layer). As an imitation of convective cooling (and at night: warming) of the tree canopy, the ventilator option seems by far the better choice. This approach, on a larger scale using wind machines, is commonly used in horticulture to prevent radiation frosts (Perry, 1998). As in the suspended-pot experiment presented by Fajardo and Piper (2014), such a setup would have to control for soil moisture differences. Belowground temperatures could either be left to equilibrate with the air (the easier option), or aboveground ventilation could be accompanied by temperature regulation of the root zone to mimic soil conditions below a tree canopy, e.g., using electric temperature elements as is sometimes used, though again usually for heating only, in climate-change experiments (Melillo et al., 2002). In all of these setups, apart from accounting for potential microclimatic artifacts, the assumed role of the seedlings as models for tree growth should be made explicit and validated before translating the results obtained with the seedling models to adult trees. Comparing taxa regarding canopy–microclimate effects on tree growth is only one side of the story, however. To understand fully the role of microclimate in limiting tree growth, differences in canopy microclimates and tissue temperatures due to taxon-specific leaf and crown morphologies, transpiration rates and albedo also need to be considered (Leuzinger and
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Körner, 2007). Data addressing such differences for treeline trees are rare (Körner, 2012), though they would be relatively easy to obtain using dataloggers. Such data would be another valuable contribution toward understanding the mechanisms of treeline formation for different taxa and functional tree types. Alternative treeline-forming mechanisms in Nothofagus and other genera— Nothofagus treelines have long been regarded as unusual, occurring at higher temperatures than most northern-hemisphere treelines, which was explained by genusspecific limitations (Körner and Paulsen, 2004; Wardle, 2008). However, several recent studies suggest that mean temperatures in the growing season at these treelines are actually quite comparable to those at other treelines (Mark et al., 2008; Cieraad et al., 2014; Fajardo and Piper, 2014). Another argument against genus-specific limitations is presented in the study discussed here (Fajardo and Piper, 2014), where Pinus contorta seedlings did not outperform Nothofagus pumilio at 50 m above the treeline. The authors argue that because of the lower mean
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temperatures in the suspended pots, P. contorta should not outperform N. pumilio up to 330 m above the treeline, although this argument disregards temperature extremes. However, in an experiment in New Zealand started in the 1960s, Pinus contorta and other exotic conifers developed large stems up to 300 m above the local treeline, while saplings of the native treeline species Fuscospora cliffortioides (≡ Nothofagus solandri var. cliffortioides), though surviving ca. 150 m above the treeline, have still not emerged from the boxes that protected them as seedlings (Fig. 1). Clearly, Pinus is outperforming this close relative of Nothofagus in this case (Wardle, 1985). So the question remains: do the Nothofagaceae form treelines for different reasons than most other treeline tree families? Most treelines composed of Nothofagaceae are very abrupt boundaries from tall, closed forest to low alpine vegetation, suggesting that limitations to establishment outside the forest rather than limited growth determine the position of these treelines (Harsch and Bader, 2011). Above the natural treeline, Fuscospora cliffortioides seedlings depend on shade and/or
Fig. 1. (A) Overview of the treeline tree-establishment experiment installed by Peter Wardle in the 1960s in the Craigieburn Range, New Zealand (photos taken in March 2009 by M. Y. Bader). View down toward the treeline from the experimental garden at 1450 m, ca. 150 m above the current treeline. Exotic tree species, many of which have grown well at this elevation, have been cut down to prevent them from spreading into the native ecosystem. Note the shrubby Fuscospora cliffortioides (≡ Nothofagus solandri var. cliffortioides), the local treeline tree species that appears to have survived or established in the shelter of the now cut-down exotic trees. (B) Stump of Pinus contorta at 1450 m. The box is the remnant of an experimental 73% shade treatment. The rosette plant in the box is the alpine species Aciphylla cf. aurea, unrelated to the experiment. (C) Shade box with barely grown F. cliffortioides apparently unable to escape these sheltered conditions (at 1450 m) (experiment and results described in Wardle, 1985, 2008).
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frost protection, which also suggests that tree establishment and treeline advance are not limited by low growing temperatures alone but by the interaction with stressors like frost, excess radiation, and wind (Wardle, 1993, 2008). Such stressors cannot offer a universal explanation for treeline formation, as they vary strongly among treelines and can occur at any elevation (Körner, 2012). In the absence of such stressors and in the case of resistant species, treeline elevations can be controlled by low temperature limitations to growth, either via source or sink limitation. In all other cases, regional peculiarities and “taxon-specific” treelines emerge (Harsch and Bader, 2011). As these are arguably the rule rather than the exception (e.g., Piper et al., 2006; Wardle, 2008; Holtmeier, 2009; Malanson et al., 2011), we embrace the recommendation of Fajardo and Piper (2014) to keep developing a universal theory for treeline formation including variation between tree taxa and regional climates. Experiments to this end should address low-temperature limitations to growth as well as interactions with other climatic factors in all tree life stages. Preferably, such experiments should include several members of different functional tree types to allow generalizations beyond taxon-specificity. Conclusion— The presented experimental setup in Fajardo and Piper (2014) is a low-technology approach for studying a fundamental question in functional plant biology: what causes treelines? Though elegant, the experiment suffers from a potentially disqualifying practical problem: the strong fluctuations in soil temperature (and most probably soil moisture) in the suspended pots. As long as the effect of such fluctuations is unknown, they cannot be ignored and should be experimentally controlled. A promising alternative is to keep seedlings at ground level and ventilate them to mimic the atmospheric coupling found in tree canopies. These solutions require an infrastructure that is not usually available at treeline, though solar panels and local water sources could allow these setups even in remote sites. Apart from technical solutions, we discussed conceptual solutions: how can we interpret the data given the temperature and moisture fluctuations? To do this, soil moisture data would be needed as well as a much better understanding of the effects of temperature fluctuations on tree growth. At this stage, an unambiguous interpretation of the results of Fajardo and Piper (2014) seems impossible. Nothofagus pumilio seedlings are carbon-limited before they are growth-limited at the temperature and moisture conditions in the suspended pots, but it is unresolved whether the limitations are really due to the lower mean temperature or (1) to the strongly fluctuating rootzone temperature regime, or (2) to moisture stress. Although their experiment did not allow unambiguous conclusions about the causes of alpine treeline formation, it provides excellent food for thought on further experiments toward this goal. LITERATURE CITED BADER, M. Y., M. RIETKERK, AND A. K. BREGT. 2007. Vegetation structure and temperature regimes of tropical alpine treelines. Arctic, Antarctic, and Alpine Research 39: 353–364. BENDIX, J., AND M. D. RAFIQPOOR. 2001. Studies on the thermal conditions of soils at the upper tree line in the páramo of Papallacta (Eastern Cordillera of Ecuador). Erdkunde 55: 257–276. CERASOLI, S., T. WERTIN, M. A. MCGUIRE, A. RODRIGUES, D. P. AUBREY, J. S. PEREIRA, AND R. O. TESKEY. 2014. Poplar saplings exposed to recurring temperature shifts of different amplitude exhibit differences in leaf gas exchange and growth despite equal mean temperature. AoB Plants 6.
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