Kahmen et al. (2011) explored the mechanistic explanations for sensitivity of δ18O to climate in tropical tree rings. Using the mechanistic Péclet-modified Craig-Gordon model (PMGG) they reported that both air temperature and air humidity influenced tree that δ18O, integrated via VPD, along an aridity gradient of *Metrosideros polymorpha* in Hawaii. Xylem water δ18O decreased along the gradient whilst leaf and stem δ18O increased, suggesting that evaporative enrichment dominated the source water signal in Hawaii's coastal island environment.

In tropical rainforest environments of Central Guyana, Pons and Helle (2011) found δ18O cellulose patterns mimicked the source water annual isotope cycle well, with minima in both rainfall and cellulose at the start of the area's primary wet season. They tested their hypothesis with dual isotope measurements, which revealed that cellulose δ13C also showed low moisture stress at the same time as the minima in cellulose δ18O. Moreover, these revealing studies were successfully carried out in tropical trees with anatomically indistinct tree rings (Pons and Helle 2011).

In South America, in the western Amazon basin, Brienen and Helle (2012) reported a lower influence of temperature and vapor pressure variations on treering δ18O records from *Cedrela odorata*. Tree-ring δ18O correlated well with δ18O in precipitation, regional precipitation variability during the wet season and streamflow from Amazonian rivers. Non time stable relationships with ENSO parameters were also observed, as well as an increase in δ18O values over the twentieth century, shared with ice core records. Baker et al. (2015) reinforced the dominance of a source water signal in the Brienen and Helle (2012) δ18O *Cedrela* record via correlations with other δ18O tree-ring records from low altitude rainforest tree species, and with high altitude *Polylepis tarapacana* in the Bolivian Altiplano. The δ18O signature of *Polylepis tarapacana* chronologies along the South American Altiplano revealed a strong signal of December to March precipitation associated to the South American Summer Monsoon (Rodríguez-Catón et al. 2021). In Southern Ecuador, ENSO variability has also been shown to be recorded in δ18O tree-ring records of *Cedrela montana* (Volland et al. 2016). Good agreement with wet season precipitation was found in high-resolution δ18O records of *Tachigali myrmecophila* in Manaus, Brazil, in *Cedrela odorata* at 12 °S in the Amazonian and a 150-year *Polylepis tarapacana* tree-ring δ18O record from the Bolivian Altiplano at 22 °S (Ballantyne et al. 2011).

In Costa Rica variations in the tree-ring δ18O records from 16-year old *Hyeronima alcorneides* were found to be sensitive to tropical precipitation amount, especially during ENSO events (Evans et al. 2006). The δ18O cycles measured in two *Pouteria* trees, which did not have visible tree rings, and were assigned a calendar year via a radiocarbon age-depth model, showed enriched δ18O during warm ENSO wet seasons (Anchukaitis and Evans 2010). Annual cyclicity in δ18O has also been revealed in ring-less mangrove *Rhizophora mucronata* which also recorded sensitivity to the ENSO event in 1997 (Verheyden et al. 2004a).

In Central Java in Indonesia, Schollaen et al. (2013) reported that δ18O chronologies from rainforest teak (*Tectona grandis*) recorded precipitation signals during the dry and the rainy season. The δ18O teak tree-ring record also showed strong sensitivity to ENSO events driven by warmer sea surface temperatures over the central Pacific (Schollaen et al. 2015). In Indonesia and Thailand, high-resolution δ18O records from cross dated tree rings of *Tectona grandis,* and ring-less *Samanea saman* grown at the same locality, show high correlations with instrumental data, reflecting the seasonal cycles of rainfall and air humidity (Poussart et al. 2004).

Tropical oxygen isotope dendroclimatology, to date, has revealed coherent correlations between various hydroclimate and source water signals and δ18O in tree-rings, often in trees with indistinct growth rings (e.g. Sano et al. 2009). These examples highlight opportunities to extend our understanding of past climate from trees to many parts of the tropics, from remote to highly populated areas where climate change may have direct and serious implications. Despite these promising single site studies, the environmental and physiological mechanisms controlling tropical isotopic signals between the atmosphere and soil, through the stem and canopy to the wood and into the cellulose of the tree-ring are highly complex, and more so in the tropics. The interplay between isotopic signals carried in the source water and those produced at the leaf level and during downstream enrichment and photosynthetic processes are still not well understood (Gessler et al. 2014). The mechanistic interpretation of δ18O in tropical trees remains therefore often problematic. Additional relevant discussions can also be found in Chaps. 13 and 22.

### **19.4 Conclusions**

The contributions of stable isotope dendroclimatology have increased tenfold, over recent decades, thanks to better analytical systems allowing greater sample throughput and replication, improved understandings of the mechanistic controls over the isotope signal and increased geographical and species coverage in example studies. Contributions to our understanding of the carbon–water-climate relationship in trees are now available from long, well replicated stable isotope tree-ring series across the old world, with pockets of emerging studies in previously underrepresented geographical areas. As global average temperatures continue to rise and we move into a '*1.5-degree world*' with increasing climate extremes, the additional glimpses into our pre anthropogenic climate that we obtain from tree-ring isotope records becomes ever more important. Coupled with the ability to explore how trees and woodland respond to changing climate and CO2 levels in the atmosphere, the isotope dendro proxies are now a vital part of the palaeoenvironmental toolbox.

### **References**

Allen ST, Kirchner JW, Braun S, Siegwolf RTW, Goldsmith GR (2019) Seasonal origins of soil water used by trees. Hydrol Earth Syst Sci 23:1199–1210

Anchukaitis KJ et al (2008) Stable isotope chronology and climate signal calibration in neotropical montane cloud forest trees. J Geophys Res American Geophysical Union (AGU) 113(G3). https:// doi.org/10.1029/2007jg000613

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# **Chapter 20 Stable Isotopes in Tree Rings of Boreal Forests**

### **Olga V. Churakova, Trevor J. Porter, Alexander V. Kirdyanov, Vladimir S. Myglan, Marina V. Fonti, and Eugene A. Vaganov**

**Abstract** The boreal forests are widely expanded from subarctic forest to tundra, and from taiga to forest-steppe zone (from 50 °N to 70 °N). We reviewed available stable isotope chronologies in tree-ring cellulose (δ13C, δ18O and δ2H) from 16 sites located in the Russian Federation; 4 research sites from Fennoscandia (Finland, Sweden and Norway); 5 sites from Canada, and 1 site from Alaska (USA) to evaluate impact of climatic changes from seasonal to annual scale across boreal forest ecosystems. Results of our review of carbon isotope data showed that drought conditions (mainly high vapour pressure deficit) are prevalent for western and central regions of Eurasia, Alaska and Canada, while northeastern and eastern sites of Eurasian subarctic are showing water shortage developments resulting from decreasing precipitation. Oxygen isotope chronologies show increasing trends towards the end of the twentieth century mainly for all chronologies, except for the Siberian northern and southern sites. The application of the multiple stable isotope proxies (δ13C, δ18O, δ2H)

O. V. Churakova (B) · A. V. Kirdyanov · M. V. Fonti · E. A. Vaganov

Institute of Ecology and Geography, Siberian Federal University, Svobodny pr. 79, Krasnoyarsk 660041, Russian Federation e-mail<EMAIL_ADDRESS>
O. V. Churakova · M. V. Fonti Forest Dynamics, Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland

T. J. Porter Department of Geography, Geomatics and Environment, University of Toronto Mississauga, 3359 Mississauga Road, L5L 1C6 Mississauga, Canada

A. V. Kirdyanov · E. A. Vaganov

V. S. Myglan Siberian Dendrochronological Laboratory, Siberian Federal University, Svobodny pr. 82, Krasnoyarsk 660041, Russian Federation

**Supplementary Information** The online version contains supplementary material available at (https://doi.org/10.1007/978-3-030-92698-4\_20).

V. N. Sukachev Institute of Forest SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Akademgorodok 50/28, Krasnoyarsk 660036, Russian Federation

is beneficial to study responses of boreal forests to climate change in temperaturelimited environments. However, a deeper knowledge of hydrogen isotope fractionation processes at the tree-ring cellulose level is needed for a sound interpretation and application of δ2H for climate reconstructions, especially for the boreal forest zone where forest ecosystems are more sensitive to climatic and environmental changes.

### **20.1 Introduction**

The boreal forest, including areas classically known as taiga, is the largest biome on earth (Apps et al. 2006), representing 17% of the earth's terrestrial ecosystems. It holds an estimated ~30% of terrestrial carbon stocks (Pan et al. 2011), making it a significant variable in the global carbon cycle. The boreal forest encompasses a zonal band roughly defined by 50–70 °N and occupies 1.2 billion hectares of land area (Soja et al. 2007), with the Siberian taiga accounting for 70% of this area (Kasischke 2000). The typical boreal climate is subarctic (e.g., Köppen zones Dfc and Dwc). In contrast to tundra areas to the north, the relatively low albedo of the boreal forest plays an important role in regulating the surface energy balance and climate of the subarctic latitudes (Bonan 2008).

Large areas of Fennoscandia, central and eastern Siberia (Russian Federation), northern Canada and Alaska represent the most extensive remaining areas of natural forests on the planet. The boreal forests are globally important for their economic and environmental values. Extensive areas of the boreal forests of Finland, Sweden, and parts of Canada are intensively managed for timber production and contribute 10–30% of the export income of these nations (ACIA 2004).

The study of boreal forest ecosystems is important because of their high sensitivity to regional and global climate changes, and potential to influence ground temperatures and the stability of vast pools of carbon currently locked in permafrost in the subarctic regions (Fig. 20.1a). Permafrost plays an important role in stabilizing the climatic system and climate-albedo feedbacks that are unique to the northern range of the boreal forest (Bonan 2008). Due to climate warming, both vapor-pressure deficit (VPD) and evapotranspiration are expected to increase in the boreal region, which has implications for tree's water relations (Sugimoto et al. 2002; ACIA 2004; Churakova (Sidorova) et al. 2016, 2020).

Many impacts of climate change are already apparent in the boreal forest including:

(i) spatially complex patterns of reduced and increased rates of tree growth (Briffa et al. 1998; Briffa 2000; Lloyd and Bunn 2007); (ii) larger and more extensive fires and insect outbreaks (Soja et al. 2007); and (iii) a range of effects due to permafrost degradation, including new wetland development and subsidence of the ground surface (Turetsky et al. 2019), with the associated loss of trees and ecological succession toward wetland plant communities.

In boreal regions, the traditional tree-ring parameters like tree-ring width and maximum latewood density are typically positively correlated with June-July and June–August temperatures, and therefore, have been successfully used to reconstruct summer temperatures over the past millennium (Briffa 2000; Sidorova and Naurzbaev 2002; Naurzbaev et al. 2002; Hantemirov et al. 2011; Grudd 2008; Kononov et al. 2009; D'Arrigo et al. 2008; Myglan et al. 2008; Schneider et al. 2015; Büntgen et al. 2021). However, in some areas of Alaska and northwestern Canada where the rate of recent climate warming has been most rapid, the generally reliable association between ring width and summer climate has been demonstrated to break down, potentially linked to warming-induced drought stress (Briffa 2000; Wilmking et al. 2004; D'Arrigo et al. 2008; Porter and Pisaric 2011; Porter et al. 2013). This phenomenon is referred to the "Divergence Problem" (DP) in the dendrochronology literature (D'Arrigo et al. 2008; Camarero et al. 2021). The emergence of the DP has stimulated the search for a more reliable tree-ring climate proxy in affected regions, including tree-ring δ13C and δ18O (Barber et al. 2000; Sidorova et al. 2009, 2010; Porter et al. 2009; Zharkov et al. 2021).

### **20.2 Characteristic of Boreal Zone**

### *20.2.1 Study Sites and Tree Species*

Trees in the boreal zone have showed great potential for stable isotope studies due to long-term tree longevity (Sidorova et al. 2008, 2010) and good subfossil wood preservation due to severe climate conditions and permafrost availability (Sidorova et al. 2013a, b; Helama et al. 2018; Churakova (Sidorova) et al. 2019) (Fig. 20.1).

**Fig. 20.1** Location of the study sites (Table S20.1) with δ13C (yellow triangles), δ18O (light blue triangles) and both δ13C and δ18O (red triangles) isotope tree-ring cellulose chronologies (**a**). Monthly precipitation (**b**) and mean air temperature (**c**) climatologies for the common period (1961– 1990) for all published sites, calculated from the 10'-spatial resolution dataset by New et al. (2002). Permafrost distribution from continuous to sporadic is available in Obu et al. (2019)

Dominant tree species are white spruce (*Picea glauca*) and black spruce (*Picea mariana* Mill.) in Canada and the USA (Alaska), Scots pine (*Pinus sylvestris* L.) in Fennoscandia, and a variety of larch tree species (*Larix sibirica* Ledeb., *Larix gmelinii* Rupr., *Larix cajanderi* Mayr.) in central, eastern and northeastern Siberia. Previous tree-ring isotope studies in the boreal region have focused mainly on developing stable carbon (δ13C) and oxygen (δ18O) isotope chronologies from cellulose (Fig. 20.1, Table 20.1 and Supplementary Table 20.1). To date there have been only a few studies in the boreal region focusing on hydrogen (δ2H) in tree-ring cellulose (e.g., in Finland, see Hilasvuori 2011). Based on the available literature and knowledge, our review focusses primarily on stable carbon and oxygen isotope studies in tree rings from the boreal zone.

### *20.2.2 Permafrost*

About 80% of the world's boreal forests are located in the circumpolar permafrost zone (Helbig et al. 2016) which makes permafrost a particularly important component of the boreal forest. Boreal forests respond to both the timing and magnitude of changes in soil moisture and soil temperature, nutrient availability, as well as permafrost distribution and dynamics, which themselves are directly affected by snow and vegetation cover, soil texture and geothermal heat flux (ACIA 2004; Cable et al. 2013; Boike et al. 2013). Water released from thawing ice-rich permafrost can be an important moisture source for trees growing in regions with severe temperature limitations and low amount of precipitation (Sidorova et al. 2010; Churakova (Sidorova) et al. 2016; Zhang et al. 2000; Sugimoto et al. 2002; Saurer et al. 2016). Due to low temperatures in the subarctic belt, water loss is not yet as large as observed in European forest ecosystems (Saurer et al. 2014). However, with a continued increase in temperature (Sidorova et al. 2010), drought stress may increase accordingly (Sidorova et al. 2009; Knorre et al. 2010; Bryukhanova et al. 2015; Saurer et al. 2016; Ohta et al. 2019) in the boreal forest regions.

Under the projected climate warming, permafrost is expected to degrade and initially wetland areas (thermokarst lakes) will increase in extent (IPCC 2014), which has the potential to shift the regional carbon balance from a net carbon sink (under productive boreal vegetation) to a carbon source (microbial emission of CO2 and CH4 driven by access to thawed permafrost carbon. Lake drainage, hydroclimatic change and ecological succession also have the potential to moderate thermokarstcarbon balance impacts. A number of studies have reported a pronounced increase in the seasonal thaw depth in Western Siberia (Melnikov et al. 2004; Pavlov et al. 2004; Fyodorov-Davydov et al. 2009). Fyodorov-Davydov et al. (2009) investigated the spatial and temporal trends in the active soil layer (ASL) depth in northern Yakutia, Russia (Table S20.1). The ASL is the top layer of soil with high activity of microbial processes and which thaws during summer and freezes back again in autumn.

Seasonal dynamics of the cryosphere also have implications for the phenology of tree growth, on carbon and oxygen isotope ratios in plants due to the influence

**Table 20.1** Summary of available stable isotope chronologies in tree rings from the boreal forest

**Table 20.1** (continued)

**Table 20.1** (continued)

of active layer thaw on soil water availability and plant gas exchange, and on the isotope composition of soil water. The freezing process itself induces a soil water fractionation during the autumn freeze-back period (Lacelle 2011). In a closed system with converging freezing fronts extending downward from the surface and upward from the permafrost table, soil water fractionation is expected to obey Rayleigh distillation principles, with the most enriched ice forming first (i.e., near the surface and at the permafrost table) and progressively more 18O-depleted ice as the two freezing fronts converge roughly at the mid-point of the active layer (Lacelle 2011). This process, therefore, can lead to isotopic stratification of soil water with depth, which has potential implications for the mean isotopic composition of soil water used by trees in the early growing season.

The oxygen isotopic signal in tree-rings of trees growing on permafrost is also masked by the supply of isotopically depleted water from melted frozen soil leading to 'inverse' climate to tree-ring isotope relationship, as dry and warm summer conditions result in lower soil, root and wood in δ18O values (Sugimoto et al. 2002; Saurer et al. 2002, 2016).

A further complication of the isotope composition in tree-rings within the permafrost zone is caused by the impact of forest fires. Wildfires lead to significant changes in active soil layer depth and seasonal dynamics, with potentially long-term consequences for carbon, nutrient and water balance of the ecosystem (Sidorova et al. 2009; Kirdyanov et al. 2020). As both water and nutrient supply for plants predominantly depend on the freeze–thaw processes in the active soil layer (Zhang et al. 2000; Prokushkin et al. 2018), the wildfire-induced changes in isotopic composition of the source water and water availability for trees as well as changes in photosynthesis rate are recorded in tree-ring carbon and oxygen isotopes.

### *20.2.3 Climate*

The major advantage in studying northern forests is their distance to populated regions, allowing the study of tree responses to environmental changes without anthropogenic disturbances. A major disadvantage is the difficult accessibility, especially in northeastern Siberia, and the scarcity of weather stations. Yet, seasonal continuous measurements of climatic parameters are needed for future ecophysiological studies. Gridded large-scale climate data (CRU TS 4.02, 0.5° × 0.5°) (New et al. 2002; Harris et al. 2014) can help filling the gaps in the climate data. Gridded temperature and precipitation data are an important source of information to quantify climate reconstructions during the last decade and further back in time (first half of twentieth century). Several studies on stable isotope tree-ring cellulose chronologies for the boreal zone showed good correspondence with temperature signals from both, local weather stations and gridded data (http://climexp.knmi.nl) back in time (>100 years) (Sidorova et al. 2010; Churakova (Sidorova) et al. 2019). However, precipitation signals are better recorded by the local weather stations at the local scale compared to the gridded averaged data at the regional scale.

Sunshine duration and cloud cover are distributed heterogeneously across boreal regions. In summer, light duration lasts longer at high-latitudes than at the southern taiga and forest steppe zone (Young et al. 2012; Gagen et al. 2016; Churakova (Sidorova) et al. 2019).

Depending on the site location and impact of environmental parameters, conifer trees in the boreal zone can adapt to extremely low annual temperatures (−19.2 °C in northeastern Yakutia, data from the local Chokurdach weather station for the period from 1961 to 1990). The climate data obtained from the local weather stations (direct measurements) represent a wide range of minimum and maximum temperature extremes (e.g., −60 °C in Yakutia to +45 °C in Khakassia, Russian Federation). The amount of annual precipitation varies from 236 to 310 mm in northeastern Siberia and Northern America, respectively (Fig. 20.1b) to 502 mm towards Baikalskii ridge (Russian Federation), and further double increases to 1353 mm towards Norway's northeastern coastline.

# **20.3 Stable Carbon (δ13C) Isotopes**

### *20.3.1 Isotope Ecophysiology*

Application of stable carbon isotopes in tree-ring studies for the boreal zone has increased over the past decades because these proxies record information not only about temperature (Knorre et al. 2010; Sidorova et al. 2008, 2009, 2013a, b), but also about moisture changes (Kirdyanov et al. 2008; Sidorova et al. 2010; Tartakovsky et al. 2012; Churakova (Sidorova) et al. 2019, 2020, 2021b), as well as changes in sunshine duration/cloudiness (Young et al. 2012; Loader et al. 2013; Helama et al. 2018) (Table 20.1). Moreover, carbon isotope chronologies in tree-rings also captured signals of atmospheric circulation patterns (Saurer et al. 2004; Sidorova et al. 2010; Gagen et al. 2016) and facilitated the reconstruction of the river flow in Siberia (Waterhouse et al. 2000).

Climatic parameters like temperature, water availability, air humidity and vapor pressure deficit, and the impact of changes in ambient CO2 concentration on photosynthetic CO2 assimilation and water balance are reflected in the δ13C values of plant organic matter and provide an isotopic fingerprint in the wood of tree rings (see Chap. 9). The analysis of tree physiological properties using carbon isotope ratios is particularly useful when combined with a photosynthesis model. This facilitates the functional attribution of meteorological impacts to plant responses, such as stomatal and substomatal conductance vs. ambient CO2 concentrations (ci/ca ratio) (Farquhar and Lloyd 1989). Detailed insight into physiology (see Chap. 9) and resource distribution during tree-ring formation (see Chaps. 3, 13 and 15) may also be obtained through 13C-labeling experiments (Kagawa et al. 2006a, b; Masyagina et al. 2016).

### *20.3.2 Seasonal Variability*

Short growing season (up to 90 days in far North) and harsh climatic conditions of the boreal zone result in low tree stem increment (Vaganov et al. 2006). Compared to temperate trees with wider rings (Leavitt 1993), boreal trees might show a slower carbon turnover rate (Kagawa et al. 2006b). The highly resolved intra-annual measurements of δ13C within tree rings (earlywood/latewood or laser ablation with the step of 80–200 μm, see Chap. 7) helped to link changes in physiological and metabolic processes and, as a result, tree-ring growth and xylem anatomical structure associated to seasonal climatic variability.

Deciduous and evergreen angiosperms and gymnosperms depend on stored carbohydrates during their first stages of leaves/wood development (Ericsson 1979).

Boreal deciduous (*Betula pubescens* Ehrh., *Populus tremula* L.), conifer deciduous (*Larix gmelinii* (Rupr.) Rupr.) and conifer evergreen species (*Pinus sylvestris* L., *Picea obovata* Ledeb., *Picea abies* (L.) H. Karst.) were used to identify the physiological principle of climate responses related to the phenology and structural– functional features of wood. Intra-annual δ13C tree-ring analysis of gymnosperm and angiosperm species in Scandinavia (Vaganov et al. 2009), central and eastern Siberia (Kagawa et al. 2006b; Bryukhanova et al. 2011; Rinne et al. 2015; Fonti et al. 2018) and southern Siberia (Voronin et al. 2012) have shown that not only the temperature, but also soil moisture and rainfall might affect the dynamics of δ13C in tree rings. In particular, δ13C in latewood of *L. gmelinii* in Yakutia was reported to show better correlations with the growing season precipitation and soil water conditions than δ13C in earlywood (Kagawa et al. 2003). Variability of tree-ring width and δ13C under climatic conditions of extreme years in central Siberia indicated that an increased spring temperature initially led to an increase of tree growth. However, due to an increased use of water through transpiration, tree growth could be progressively reduced from temperature to moisture limitation.

To determine the extent to which trees rely on stored carbohydrates from previous years for tree-ring formation and how strongly the current photosynthates were used, the intra-annual δ13C variability was measured. Samples from *Larix gmelinii* (Rupr.) from two Siberian sites with a different hydro-thermal regime of permafrost soils were analyzed using (a) δ13C-labeling (Kagawa et al. 2006b) and (b) laser ablation coupled to the Compound-Specific Isotope Analysis (CSIA) (Rinne et al. 2015). Kagawa et al. (2006b) showed that latewood in *Larix gmelinii* Rupr. was mainly formed from current-year photoassimilates with minimal carry-over effect of carbohydrates from the previous year, while the early wood is produced from a mixture of current-year photoassimilates and previous-year carbohydrates. In *P. sylvestris* from the same site, δ13C values of early wood were significantly correlated with the previous year late wood (*r* = 0.42; *P* < 0.01) in a 100-year δ13C chronology, which is evidence of a carry-over effect. In contrast, Rinne et al. (2015) provided the evidence of a minimal carry-over effect of photosynthates formed during the previous year(s). The combination of different methods, such as CSIA and intraannual tree-ring isotope analyses will enhance a further mechanistic understanding of the carbon–water relationships within ecosystems, in particular, for the interpretation of retrospective tree-ring analyses (Rinne et al. 2015).

# *20.3.3 Annual and Decadal Carbon Isotope Variability Over Past 100 Years*

The mean δ13C value in tree-ring cellulose chronologies from Fennoscandia, Yakutia, the high-elevated mountain range in Khibini (Kola Peninsula) and the Altai Mountain range, analysed for the period from 1900 to 1998, showed mean values of −24‰. These earlier published chronologies indicated wetter conditions for these sites in the boreal zone compared to the drier northeastern and central sites of Eurasia and Alaska. Based on the available δ13C tree-ring cellulose chronologies from the southern part

**Fig. 20.2** Annual (**a**) and smoothed by a 11-year Hamming window (**b**) δ13C tree-ring cellulose chronologies obtained from conifer tree species from the boreal zone (for details see supplementary Table S20.1)

(Khakasia, Russian Federation) of the boreal zone, reduced soil moisture availability is reflected by mean δ13C values of −20.4‰ (Fig. 20.2a).

The δ13C in tree-ring cellulose chronologies (standardised to z-score) smoothed by a 11-year Hamming window show a general significant increasing trend over the recent decades for all, except for a few sites in northern Eurasia: Davan Pass (Tartakovsky et al. 2012), Khakasia forest steppe (Knorre et al. 2010), Tura (Sidorova et al. 2009) as well as Alaska (Barber et al. 2000), Canada (Porter et al. 2009) and Sweden, Torneträsk (Loader et al. 2013) (Fig. 20.2b).

These decreasing δ13C trends in tree-ring cellulose chronologies towards recent century from permafrost sites over the past decades were explained as an earlier beginning of the vegetation period in spring and increased use of residual soil carried over from autumn of the previous year (Sidorova et al. 2009; Knorre et al. 2010). Another reason, e.g., physiological effect of increasing atmospheric CO2, is also responsible for lower δ13C values. Thus, an earlier start of the vegetation period could lead to tree-ring formation during a period with higher water availability, resulting in stronger isotopic fractionation and 13C depletion (Knorre et al. 2010).

# **20.4 Stable Oxygen (δ18O) Isotopes**

### *20.4.1 Isotope Ecophysiology*

Oxygen isotopes in organic matter are modified by variation in the isotopic composition of source water, which is closely related to that of precipitation and soil water (though modified by evaporation at the soil surface). The δ18O of meteoric water is directly related to cloud/atmosphere air temperatures (Dansgaard 1964) as well as evaporation and condensation processes in the global water cycle. This is especially true in northern high-latitudes, as has been demonstrated at broad spatial scales across the North American arctic and subarctic based on precipitation isotope data from the Global Network of Isotopes in Precipitation (Porter et al. 2016). An earlier study by Saurer et al. (2002) also showed that average isotope values of 130 trees of a widely distributed genus (*Larix, Picea, Pinus*) within the Eurasian subarctic from Norway to Siberia are highly correlated with the modeled isotope distribution of precipitation showing a large east-to-west gradient (see Chap. 18). Input waters are modified (enrichment in 18O) in the leaf during transpiration, which is imprinted on photosynthates and cellulose through biochemical fractionation and exchange processes. In Siberia, the inter-annual variability of winter precipitation δ18O is closely related to temperature variability and the North Atlantic Oscillation, while the variability of summer δ18O appears to be dominated by regional processes involving evaporation and convection (Butzin et al. 2014). Therefore, δ18O values of tree rings reflect, as a first approximation, average ambient temperatures and humidity. Progress has been made in understanding the fractionation processes, where H2 18O-molecule goes from soil water to tree-ring cellulose (Craig and Gordon 1965; Dongmann et al. 1974; Farquhar and Lloyd 1989; Roden et al. 2000). These models have been validated with experimental data from deciduous and coniferous tree species. A detailed description of the leaf water enrichment processes is given in Chap. 10.

### *20.4.2 Seasonal Variability*

The highly complex hydrological regime of boreal forests is given by a strong sinusoidal seasonal course of δ18O imprinted in precipitation water, reflecting the cloud condensation temperatures. Winter precipitation uptake is only possible in the warming spring and summer months after snow melt and active layer thaw. This explains the often good correlation between tree-ring δ18O values and winter temperature, when this fraction of the annual precipitation becomes available for trees (Sidorova et al. 2010). Oxygen isotopes are then incorporated and become visible in the tree rings. As described in part Sect. 20.2.1 of this chapter the hydrology of forests growing under permafrost conditions, only a shallow layer of soil thaws in summer, each soil layer with its own δ18O of soil water. This variation of seasonal δ18O from permafrost water must be taken into account for the evaluation of tree-ring chronologies. It is therefore not surprising that only few studies about the seasonal δ18O fluctuations in wood are available for the boreal zone, i.e. southern Siberia (Voronin et al. 2012) and central Siberia (Saurer et al. 2016).

**Fig. 20.3** Annual (**a**) and smoothed by a 11-year Hamming window (**b**) δ18O tree-ring cellulose chronologies obtained from conifer tree species from the boreal zone

# *20.4.3 Annual and Decadal Oxygen Isotope Variability Over Past 100 Years*

Annual δ18O values in tree-ring cellulose chronologies (Fig. 20.3) showed clear isotopic differences from the coldest sites in northeastern Yakutia (19.0‰) (Sidorova et al. 2008) and Canada (19.1‰) (Porter et al. 2009) towards warmest sites in Russian Altai (up to 27.7‰) (Loader et al. 2010; Sidorova et al. 2012, 2013a, b) and Khakassia (26.4‰) (Knorre et al. 2010). A 5-year block δ18O tree-ring cellulose chronology of black spruce trees (*Picea mariana* [Mill] B.S.P) from the Québec–Labrador peninsula, northeastern Canada (Naulier et al. 2015) showed the lowest isotopic value (16‰) compared to all other reviewed sites (Fig. 20.3a).

A decreasing δ18O trend was detected in the early 1900's between 11-year smoothed δ18O tree-ring cellulose isotope chronologies from Tura site (TUR) (Sidorova et al. 2009) and Canadian site Mackenzie Delta (Porter et al. 2009, 2014). This discrepancy can be explained by cold conditions in Canada compared to warmer periods at Siberian Tura site. Almost all δ18O values in tree-ring cellulose chronologies showed increasing temperature trends towards the end of the twentieth century, except for the Siberian sites in Yakutsk (Spasskaya Pyad) (Tei et al. 2013) and Khakassia (Knorre et al. 2010) (Fig. 20.3). Decreasing δ18O values in tree-ring cellulose chronologies can be explained by 18O depleted water from autumn precipitation of the previous year absorbed by the tree rings (Sidorova et al. 2009; Knorre et al. 2010).

The combination of tree-ring and stable isotope parameters (e.g., tree-ring width, cell wall thickness, maximum late wood density, Sidorova et al. 2010, 2012; Churakova (Sidorova) et al. 2019) enhances the strength of our interpretations and need to be pursued where possible.

# **20.5 Stable Hydrogen (δ2H) Isotopes**

As the oxygen and hydrogen isotopic composition of precipitation was already recognized to be related to temperature (Dansgaard 1964), early work on oxygen and hydrogen in plant material was conducted with the aim of using tree rings as an isotopic thermometer. Although the fractionation mechanisms in leaf water are the same for δ2H as for δ18O, the incorporation of hydrogen follows a different metabolic pathway. Therefore, correlation analyses with tree-ring hydrogen isotope time series and annual temperature records have been less successful (Epstein et al. 1976; Waterhouse et al. 2002; Augusti et al. 2006). Most recent studies by Voelker et al. (2014) showed potential for reconstruction of relative humidity from plant δ2H and δ18O as deuterium deviations from the global meteoric water line (GMWL).

The analysis of δ2H in tree-rings is still in an explorative stage (Kimak and Leuenberger 2015; Cormier et al. 2019; Lehman et al. 2021; Churakova (Sidorova) et al. 2021a; Schuler et al. 2022), but the application of the dual stable isotope approach (δ18O and δ2H) in tree-ring analyses is promising and will strengthen our future isotope interpretation. Chapter 11 discusses the principles of δ2H in tree rings in detail.

### **20.6 Conclusion and Outlook**

Carbon isotopes proved to be a reliable proxy for spring and summer temperature, vapor pressure deficit, sunshine duration/cloud cover and soil moisture changes. Oxygen isotopes were not only a temperature proxy but also an indicator for air humidity and water origin, showing also teleconnection with Arctic Oscillation via precipitation patterns. A mixed temperature and precipitation signal is mainly recorded for subarctic regions, covered by permafrost (Chap. 18).

The dual carbon and oxygen isotope approach is highly recommended for the interpretation of stable isotope chronologies from the boreal forest due to often mixed signals recorded in tree rings and the complex hydrology, which can be analyzed best by using dual or even triple isotopes (δ13C, δ18O, δ2H).

Based on the available stable carbon and oxygen isotope data sets across the boreal forest zone, we conclude that trees from western and central regions of Eurasia, Alaska and Canada are exposed to drought conditions, while no strong evidence for drought is observed at the northeastern and eastern sites of the Eurasian subarctic.

There are only few nitrogen isotope measurements in tree tissues, mainly in needles of conifer trees (Mack et al. 2004; Prokushkin et al. 2018) and in soil samples, e.g., northern Sweden (Högberg et al. 2006). So far, no data is available for δ15N in tree-ring chronologies from the boreal zone, because no strong δ15N signal in tree rings was found.

Trees growing at the circumpolar zone are a valuable archive and monitoring system for information not only about temperature but also about currently ongoing hydrological changes. A multi-proxy approach as a combination of stable isotopes and tree-ring parameters, new approaches (intra annual tree-ring analyses, δ2H and CSIA), and eco-physiological modelling (δ13C, δ18O, δ2H) will strengthen our interpretations and improve the quality of available climate reconstructions with annual time resolution.

**Acknowledgements** This work was supported by the *Russian Science Foundation (RSF) (project 21-17-00006) granted to O.C. (S.)* and *(project* 19-14-00028) granted to V.M.

### **References**

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# **Chapter 21 Stable Isotopes in Tree Rings of Mediterranean Forests**

**Giovanna Battipaglia and Paolo Cherubini**

### **21.1 Introduction**

Tree-ring studies in the Mediterranean Basin and in regions characterized by Mediterranean climates are scarce because of a lack of old trees and difficulties related to the clear identification of individual rings. Old trees are lacking because most regions with a Mediterranean climate have long histories of human activity; logging, grazing, and human-induced fires have occurred over millennia. Annual rings are difficult to identify in some woody plant species because highly variable climatic conditions can lead to the formation of intra-annual density fluctuations, which hamper the cross-dating of tree-ring series (Cherubini et al. 2013). Stable isotopes may help in identifying intra-annual density fluctuations (De Micco et al. 2007) and in understanding the physiological processes behind tree-ring formation, carbon uptake, and water use (Battipaglia et al. 2010a, 2014a).

Most of the tree-ring stable isotope studies carried out on Mediterranean tree and shrub species include the use of both carbon and oxygen stable isotopes (Table 21.1), which help to reconstruct past climate and retrospectively assess tree responses to the environment (McCarrol and Loader 2004). The values of the isotopic ratios reflect the extent to which the heavier isotope is discriminated compared to the lighter one during the physical and chemical processes involved in the synthesis of plant organic matter (Farquhar et al. 1989).

Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Birmensdorf, Switzerland

G. Battipaglia (B)

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania «L. Vanvitelli», via Vivaldi 43, 81100 Caserta, Italy e-mail<EMAIL_ADDRESS>
P. Cherubini

Department of Forest and Conservation Sciences, The University of British Columbia, Main Mall, Vancouver, BC 3041-2424, Canada

**Table 21.1** Summary of the main aim and outcomes of studies measuring stable isotopes in Mediterranean tree rings. References given in chronological order

**Table 21.1** (continued)

**Table 21.1** (continued)

In this chapter, we highlight the importance of stable isotope research in Mediterranean ecosystems and explain the link between the morphological and functional characteristics of Mediterranean species and the climatic and environmental adaptations that have occurred over millennia.

### **21.2 Mediterranean Climates**

Mediterranean climates occur around the world: in California, central Chile, western and southwestern Australia, southwestern South Africa, and in the Mediterranean Basin. As most tree-ring studies have been carried out in the Mediterranean, we focus on this region in this chapter. In a typical Mediterranean climate, winters are wet and rather mild, although frost and cold stress can occur (Larcher 2000; Mitrakos 1980; Terradas and Save 1992). Rainfall starts in September and continues until April, with a total precipitation amount of 400–1200 mm (Pignatti 2008). Summers are hot and dry; rainfall events are rare, and the season is often characterized by an extremely dry period, the so-called *summer drought*. These general features can vary in different coastal areas of the Mediterranean Basin depending on their elevation and local geomorphological and soil features, but the summer drought is always present (see Fig. 21.1). The main factors leading to the formation of Mediterranean-type climates are air circulation, latitude, topography, and the surface temperature of the surrounding water.

**Fig. 21.1 a** Map of the Mediterranean Basin showing locations of stable isotope tree-ring studies. The main findings of these studies are summarized in Table 21.1. Background map showing Mediterranean climates according to Köppen classification. **b** Two Walter and Lieth climate diagrams depicting the climate of two typical Mediterranean areas. Lower diagram: Capri (an island in the Tirrenic sea); upper diagram: Tramonti, at a higher altitude (period 1960–2010). **c** A few examples of Mediterranean species. Clockwise from top left: *Pinus halepensis* Mill of southern Spain; *Quercus ilex* L from southern Italy; *Euphorbia dendroides* L.: winter *habitus* and *summer habitus*

### *21.2.1 Mediterranean Climate and Vegetation*

Mediterranean plants are adapted to this climate and cope with summer drought through several changes at the phenological, morphological, physiological,

or biochemical levels (Pignatti 2008). These adaptations influence all relevant ecophysiological processes, i.e., transpiration and carbon assimilation, as well as the isotopic signal imprinted in plant organic matter. Hence, the isotopic composition of the wood material of Mediterranean trees reflects their functionality and adaptation strategies. To cope with drought stress, two main strategies are observed in Mediterranean plants: avoidance and tolerance (Pignatti 2008).

Many species survive summer drought by avoiding it through summer dormancy. This strategy is adopted by annual and perennial herbs and by woody species. Annual herbs have a very short life cycle that starts in late winter so that fruits are mature at the beginning of summer. Seeds then remain dormant until conditions are optimal for successful growth. Perennial herbaceous species often have a geophyte life-form, in which the above-ground parts of the plants (leaves, stems) are shed at the beginning of summer and the individual remains dormant by means of a perennating below-ground organ (bulb, tuber, rhizome). Some woody species also have summer dormancy, shedding all their leaves (e.g. *Euphorbia dendroides* L.) at the end of spring or summer. Several species, such as *Cistus,* change their leaves according to the season and are characterised by the alternation of a winter and a summer *leaf habitus*.

Plants without summer dormancy present tolerance attributes, which help them to face summer drought. Many species have a low leaf surface area, e.g., the needlelike leaves of *Rosmarinus officinalis* L. or *Juniperus phoenicea* L. subsp. *phoenicea,* whose juvenile needle-like leaves turn into scale-like leaves that are densely arranged around the stem in older branches. Other species have schlerophyllous leaves, with very dense mesophyll and thick cuticles to constrain water loss. Regulation of stomatal activity is a good strategy to cope with summer drought: stomatal closure can reduce transpiration rate and water loss. Several *species* count on *stomata,* which are *sunken* into the leaf surface and protected by dense, short trichomes, to reduce water loss. The lower leaf blades of *R. officinalis*, *Quercus ilex* L., and *Olea europaea* L. are examples of this feature. Other plants without summer dormancy tolerate high solar radiation by means of a reflecting cuticle (e.g., *Myrtus communis* L. subsp*. communis*) or long (often white) hairs on the upper leaf blade (e.g., *Centaurea cineraria* L. subsp. *cineraria*) that protect inner tissues from excessive radiation.

The adaptation mechanisms of Mediterranean plants are often linked with human activity, since their long association with humans has left its mark across much of the landscape. Human activities have influenced not only the type of community and forest composition, but also their main characteristics.

Four major types of vegetation can be recognized in the Mediterranean-European region according to the bioclimate type and the elevation (Quézel and Médail 2003; Rivas-Martínez et al. 2007; Médail 2008). First, a thermo-Mediterranean vegetation rises from sea level to ~200**–**500/800 m a.s.l. and is dominated by sclerophyllous communities (e.g., *Olea europaea*, *Ceratonia siliqua*, *Chamaerops humilis*, *Pistacia lentiscus*, *Pinus halepensis, Pinus brutia*, etc.). A meso-Mediterranean vegetation takes over between ~100**–**500/1000 m a.s.l. and is dominated by sclerophyllous forests (*Quercus ilex*, *Quercus suber,* and *Q. coccifera*) or *Pinus halepensis*/*P. brutia* forests in drought-prone forest systems. This then transitions to a supra-Mediterranean vegetation between ~500**–**1500/1800 m a.s.l., with deciduous oaks forests (e.g. *Acer*, *Carpinus, Ostrya*, *Quercus,* and *Sorbus*) in the more humid areas. Finally, a mountain-Mediterranean vegetation belt between ~1500**–**2000 m a.s.l. includes *Fagus* and deciduous and semi-deciduous *Quercus*forests, as well as several coniferous forests with *Pinus nigra*, *Pinus sylvestris,* firs (*Abies alba* and most of the Mediterranean *Abies* spp.), and even cedar (*Cedrus libani* s.l.) on Cyprus (Médail et al. 2019).

# **21.3 Mediterranean Wood Formation in Mediterranean Tree Species**

The Mediterranean basin has an extension of more than 2 million square kilometers, and it is the largest of the world's five mediterraneanc-climate regions, as well as the world's second largest biodiversity hotspot (Myers et al. 2000).

The Mediterranean region is considered to be one of the climate-change hotspots, being a transition zone between temperate and arid and tropical regions (Diffenbaugh and Giorgi 2012). It has experienced an increase in drought frequency in the past three decades, and forecasts predict increased irregularity of the intra-annual precipitation pattern, and increasing temperature in the next decades (IPCC 2017). Water availability is considered the key factor driving ecophysiological processes in Mediterranean vegetation. It influences cambial activity, photosynthetic rates, dry matter production, phenology, allocation of carbon to roots and leaves, and overall growth activity of woody plants (Margaris and Papadogianni 1977; Lo Gullo and Salleo 1988; Orshan 1989; Gratani 1995; Salleo et al. 1997; Davis et al. 1999).

In Mediterranean ecosystems, tree species are adapted to cope with the so-called "double stress" of summer drought and winter low temperatures (Mitrakos 1980; Cherubini et al. 2003). These stressors trigger the formation of Intra Annual Density Fluctuations (IADFs) (Campelo et al. 2007; Battipaglia et al. 2016a; De Micco et al. 2016), which are caused by the interruption of the normal course of growth during the growing season. When favorable growth conditions resume, growth resumes, resulting in a change in wood density (Tingley 1937; Schulman 1938). In Mediterranean regions, this happens irregularly in space (at different sites at the same time), time (in different years at the same site), and among species (as well as individual trees). This ability to stop and restart growth within a growth season is indicative of the high plasticity of Mediterranean species, whose wood functional traits enable them to preserve hydraulic conductivity throughout the different seasons, achieving a trade-off between hydraulic efficiency and safety (see Beeckman 2016).

IADFs make it difficult to clearly identify individual annual rings; as a result, the tree rings of Mediterranean species are used less often for dendroecological purposes less than those of plants growing in temperate environments. However, a variety of recent research techniques have provided valuable information about the short-term growth variability of Mediterranean trees and shrubs. These include the intensive monitoring of stem radial variation using dendrometers (Sánchez-Costa et al. 2015), the assessment of cambial phenology (Camarero et al. 2010; Vieira et al. 2017), the measurement of xylem anatomical traits (De Micco et al. 2019; Carvalho et al. 2015; Pacheco et al. 2016), stable isotopes within tree rings (on tree-ring sub-sections, Klein et al. 2005; in continuum by laser ablation, Battipaglia et al. 2010a, b; 2014a) and a combination of methodologies (Zalloni et al. 2018, 2019; Balzano et al. 2018; Castagneri et al. 2018).

# **21.4 Carbon and Oxygen Stable Isotopes in Tree Rings of Mediterranean Species**

### *21.4.1 Carbon Isotopes*

Mediterranean species, which grow in climates characterized by summer drought and wet periods during the growing season, show wood structure adjustments that are mostly related to the need to maintain high conductivity when water is available, and to prevent xylem embolism when dry conditions occur (Cherubini et al. 2003; De Micco and Aronne 2012; Meinzer et al. 2010; Sperry et al. 2008). Tree-ring formation is therefore directly influenced by these adjustment processes and carbon stable isotopes can investigate the link between xylem hydraulic properties and the related physiological mechanisms.

Water shortage negatively influences carboxylation rates by reducing the CO2 concentration in the gaseous spaces within the plant (Ci) due to stomata closure, causing a decrease in carbon isotope discrimination (Farquhar et al. 1989; Scheidegger et al. 2000). Many studies conducted in Mediterranean climates have shown that plants growing under water stress (stress induced by low soil water content and high evaporative atmospheric demand) produce tree-rings with higher δ13C (Ferrio et al. 2003; Battipaglia et al. 2010a; Maseyk et al. 2011). However, leaf water availability ultimately influences tree-ring δ13C. This availability is a consequence not only of soil water content, but also of the physical structure and hydraulic resistance along the plant xylem and the way the plant transports and uses water (e.g. leaf phenology) (Masle and Farquhar 1988; Warren and Adams 2000). Further, tree δ13C is influenced by the leaf-to-air vapour pressure deficit (VPD), which is the driving force for transpiration. Studies on isotope-climate relationships have shown that mean annual precipitation controls carbon discrimination (*-*13C) in several forest ecosystem types (Schulze et al. 1998; Diefendorf et al. 2010; Kohn 2010), as well as in tree rings of a large range of Mediterranean species (Shestakova et al. 2019; del Castillo et al. 2013). These studies generally suggest a stronger influence of precipitation on *-*13C in drier environments (Warren et al. 2001).

### *21.4.2 Carbon Isotope Discrimination as a Proxy for iWUE*

Carbon isotope composition has been used to calculate intrinsic water-use efficiency (iWUE) (see Chap. 17) in Mediterranean species (Ehleringer et al. 1993; Altieri et al. 2015; Battipaglia et al. 2016b; Dawson and Ehleringer 1993; Moreno-Gutiérrez et al. 2012). iWUE is key to the survival of Mediterranean species under drought conditions (Farquar et al. 1989); according to Medrano et al. (2009), a high iWUE can be considered an adaptive trait of Mediterranean species. Inter- and intra-species interactions influence iWUE, and iWUE can vary with inter- and intra-annual climate variability, stand density, and tree size (Forrester 2015).Within and across tree species, variations in iWUE reveal a continuous ecophysiological gradient of plant water-use strategies ranging from "profligate/opportunistic" (low iWUE) to "conservative" (high iWUE) (Moreno-Gutierrez et al. 2012). Several factors can affect iWUE at individual and stand levels, such as tree age and height (Francey and Farquhar 1982; Farquhar et al. 1982, Bert et al. 1997; McDowell et al. 2011a, b; Brienen et al. 2017), site density and characteristics (Battipaglia et al. 2010a; Zalloni et al. 2018), forest management, climate, and increasing atmospheric CO2 (Silva and Horward 2013, De Micco et al. 2019).

Both network studies and case studies have highlighted a general increase in iWUE in Mediterranean species (Linares et al. 2009; Di Matteo et al. 2010; Linares et al. 2011; Peñuelas et al. 2011; González-Muñoz et al. 2015; Brito et al. 2016; Fernández-de-Uña et al. 2016; Paris et al. 2018; Shestakova et al. 2019). All studies agree that the increase in iWUE does not translate into growth enhancement in response to increasing atmospheric CO2. However, it is not yet clear why this occurs because it is extremely difficult to disentangle the influence of single factors on iWUE variation and to quantify the extent to which drought overrides a positive CO2 fertilization effect (see Peñuelas et al. 2011). Moreover, iWUE can vary due to both photosynthetic and stomatal conductance rates, since both affect the ratio between CO2 partial pressure in leaf intercellular space and in the atmosphere. To better understand the role of assimilation on iWUE, several authors have coupled tree-ring δ13C values with wood δ18O values (the dual isotope approach; Chap. 16, paragraph 5).

### *21.4.3 Oxygen Isotopes as a Proxy for Source Water*

Oxygen isotopes can provide valuable information about changes in source water, which is typically either precipitation or groundwater (Dansgaard, 1964). No fractionation occurs when water is taken up by the roots (Wershaw et al. 1966), so the δ18O of xylem water can be used to investigate plant water source use in a range of environmental conditions (Sternberg and Swart 1987; Ehleringer and Dawson 1992; Dawson et al. 1998a, b). Very few studies, however, have focused on Mediterranean environments (Sargeant and Singer 2016; Singer et al. 2013), where water sources are quite complex and subject to strong seasonal fluctuations. The interface between the influx of marine water and the freshwater table depends strongly on climatic factors and on the exploitation of groundwater for urban or rural purposes, both of which influence the quality of water available for plant communities.

The isotopic composition of rainfall is typically subject to seasonal patterns that are influenced by geographic and climatic factors. Generally, autumn–winter precipitation has a more negative isotopic signature (ranging between ∼−7.8 and −6.0 ‰) than spring–summer precipitation (ranging between ∼−4.0 and +0.8 ‰) (Alessio et al. 2004). Consequently, the freshwater table is labelled by meteoric water, and primarily by the abundant autumn–winter and spring precipitation events (Wu et al. 1996). In contrast, soil water in the shallow layers depends on spring–summer precipitation. Further, water occurring 10–50 cm below the dry soil surface is subject to evaporative enrichment and therefore attains less negative or even positive δ18O values (Craig and Gordon 1965; Allison and Leaney 1982; Yakir et al. 2000). A further source of water is the marine water table, which is typically characterized by enriched values of δ18O (∼0 ‰).

Thus, the δ18O composition of tree rings will reflect the isotopic signature of the tree's water source: rings will have a depleted δ18O signature if trees take up water from the groundwater table (Dawson and Ehleringer 1998), and an enriched δ18O signature if trees take up water from the vadose zone. Indeed, water in the vadose zone is generally linked to recent precipitation and is subject to high evaporative enrichment of δ18O (Dawson and Ehleringer 1998). These source-water signatures will be different for individual rings within a given species depending on the annual position of the water table and the degree of mixing between precipitation, surface flow, and groundwater during periods of xylem uptake (McCarroll and Loader 2004). It should also be noted that considerable fractionation occurs at the leaf/needle level, where lighter isotopes are preferentially lost via transpiration, leading to an enrichment in plant tissue δ18O of up to 20‰ compared to soil water (Craig and Gordon 1965; Saurer et al. 1998; McCarroll and Loader 2004; Barbour 2007; Cernusak et al. 2016). Indeed, it has been demonstrated that the source water signal can be modified by large changes in evaporative enrichment in drought-adapted Mediterranean species. These species tightly regulate transpiration through their stomata (Ferrio and Voltas, 2005), an ability that is thought to be a functional adaptation to drought (Pallary et al. 1995; Battipaglia et al. 2009).

Singer et al. (2013) and Sargeant and Singer (2016) used annual tree-ring δ18O analyses to interpret plant water use at various temporal and spatial scales as a function of regional hydrology and climate. Both studies underlined the great plasticity of plants as evinced by their ability to take up different sources of water and to modulate their root depth to take up water from different compartments in order to avoid competition. This promising approach needs to be exploited further, as it could provide important information regarding the ability of plants to survive drought events cooperatively (Altieri et al. 2015).

# **21.5 Application of Carbon and Oxygen Isotopes in Tree-Rings of Mediterranean Species**

The carbon and oxygen composition of tree-rings has been used for elucidating whether plant functional responses are related to stomata control of water losses (gs) or to varying assimilation rates (A), since δ18O shared a dependence on gs with δ13C, but is thought to be independent of variation in A (Scheidegger et al. 2000; Grams et al. 2007; Roden and Farquhar 2012).

Indeed, even if the interpretation of the double model δ13C—δ18O is not straightforward (Roden and Siegwolf 2012), and may sometimes be hampered by changes in the source water isotopes (Gessler et al. 2014), it can still provide important information when applied in strongly water-limited ecosystems, such as the Mediterranean (Ripullone et al. 2009; Moreno-Gutiérrez et al. 2012; Voltas et al. 2013; Gessler et al. 2014; Altieri et al. 2015; Battipaglia et al. 2016a). In the next paragraphs, we will illustrate specific examples of a double δ13C—δ18O model that is relevant for the Mediterranean region.

# *21.5.1 Carbon and Oxygen Isotopes and Forest Dieback in the Mediterranean Basin*

Forest vulnerability is reported to be increasing worldwide; forest dieback episodes have been recorded for different species in all biomes (Allen et al. 2015; Adams et al. 2017; Hartmann et al. 2018) and particularly for tree and shrub species in Mediterranean ecosystems. Manifested by a loss in tree vigour (leaf shedding, canopy and shoot dieback) and growth declines, these dieback cases reveal the high vulnerability of forest ecosystems. Increased tree mortality seems to be a response to the rapid rise in temperature and associated drying trends (Camarero et al. 2015; Colangelo et al. 2017). In the Mediterranean, trends of increasing temperature and altered precipitation patterns lead to higher probabilities of extreme events, such as heat waves and fires (IPPC 2014). Species-, age- and microsite-specific plant responses to changing climatic conditions are responsible for the degree of adaptation and survival under limiting conditions. These factors also determine interspecific competition, and thus vegetation dynamics.

The main mechanisms triggering tree decline and mortality have been identified as carbon starvation and hydraulic failure, but the relative importance of the two processes and their link are not clear yet (McDowell et al. 2008, Sala 2009, Sala et al. 2010, Gruber et al. 2010, Sevanto et al. 2014, Gaylord et al. 2015, Hartmann 2015, Rowland et al. 2015, Salmon et al. 2015; Adams et al. 2017). Plants experience hydraulic failure when more water is lost by transpiration than a plant can take up through its root. This creates high xylem water tension and results in progressive cavitation and conductivity loss of the xylem (Sperry et al. 1998; McDowell et al. 2008; Sevanto et al. 2014; Salmon et al. 2015). This process may be most relevant during very severe, short-term droughts (McDowell et al. 2008). On the other hand, carbon starvation occurs when plants close their stomata to prevent desiccation under drought conditions (McDowell et al. 2008). Low water supply leads to a reduction of photosynthetic activity, which, when coupled with the depletion of non-structural carbohydrates (NSC), can induce a negative carbon balance, leading to so-called starvation (McDowell et al. 2008; McDowell and Sevanto 2010; Hartmann 2015; Salmon et al. 2015).

Gessler et al. (2018) developed a conceptual model, based on the Scheidegger model (Scheidegger et al. 2000, see Chap. 16), to assess the mechanisms of droughtinduced tree mortality. Using a synchronic approach, they investigated the tree-ring δ13C and δ18O data for different species and found that an increase in δ13C and δ18O in dying trees as compared to surviving ones indicates low photosynthetic activity and stomatal conductance. These conditions can lead to a slow or continuous decline in growth, possibly followed by carbon starvation-induced death. On the other hand, the long-term reduction of iWUE in dying trees seems to be associated with increased growth and higher stomatal conductance, inducing possible hydraulic failure in dying trees. However, those findings show a huge variability among species and sites. This variability is linked to the complex processes regulating growth and, in particular, the fractionation processes that take place during the mobilization of photosynthetic assimilates from the leaf to the wood (Gessler et al. 2014). Indeed, it has been demonstrated that trees may use remobilized carbohydrates (primarily starch) for the formation of tree rings. These stored carbohydrates may have been produced months or even years earlier (Gessler et al. 2014). This is especially possible in Mediterranean biomes, where the growing season is very long (Castagneri et al. 2018) and where xylogenesis can be interrupted more than once during the year (Balzano et al. 2019, 2018). In this case, a multidisciplinary approach is required to disentangle the different processes underpinning changes in growth and iWUE, and to better understand the causes of tree diebacks (Cailleret et al. 2017; Colangelo et al. 2017; Camarero et al. 2015, 2019).

### *21.5.2 Carbon and Oxygen Isotopes and IADF*

Intra-annual density fluctuations (IADFs) are abrupt changes in density within a tree ring, and are frequently found in Mediterranean species as a response to seasonal climate fluctuations between dry and wet periods (Bräuning 1999; Campelo et al. 2007; Cherubini et al. 2003; Rigling et al. 2001; Schulman 1938; Tingley 1937; De Micco et al. 2016). Although IADF frequency is related to tree age, size, and tree-ring width, as well as to genetic and site conditions, it mainly depends on drought. IADF frequency is the result of wood functional trait adjustments to preserve hydraulic conductivity under Mediterranean "double stress" conditions (De Micco et al. 2016). The ability to adapt to highly seasonal Mediterranean conditions may depend on a species´ capacity to adjust cambial activity in order to cope with the prevailing environmental conditions. Trees that are not able to quickly adjust their wood traits to respond to climate are more vulnerable to drought (Martinez-Meier et al. 2008). Nevertheless, the question of whether these adaptations result from a hydraulic structure adjustment to avoid stressful conditions or to take advantage of favorable ones is still open (Battipaglia et al. 2016a).

Carbon and oxygen stable isotopes have been used both separately and together to study IADF formation in *Pinus pinaster* (De Micco et al. 2007), *Arbutus unedo* (Battipaglia et al. 2010a; b), *Erica arborea* (Battipaglia et al. 2014a), *Quercus ilex,* and *Pinus pinea* (Zalloni et al. 2018, 2019), and to infer information about IADF functionality. In particular, the position of IADF within the rings is linked with several microclimatic factors and varies within the same species according to the soil water availability, reflects different δ13C signals (Battipaglia et al. 2010a). An increase in δ13C is almost always found in the first half of the ring (in the so-called E-IADF band of latewood-like cells in the earlywood; see De Micco et al. 2016) as a consequence of stomatal closure under drought stress, when the tree reduces its vulnerability to cavitation, with a low hydraulic conductivity (De Micco et al. 2007, Battipaglia et al. 2010a, 2014a). A decrease in δ13C is typically found in the second half of the ring in the so-called L-IADF (earlywood-like cells in the latewood) and corresponds with a reactivation of the cambium and of photosynthetic activity due to late-summer or early-autumn water availability following summer dormancy (Battipaglia et al. 2014a). The importance of the position of IADFs and their link to a common ecophysiological process was analysed using a network approach to examine δ13C and δ18O in several species (*P. pinea* from Italy, *Pinus halepensis* from Spain and Slovenia; *P. pinaster* from Portugal, *Larix decidua* from Poland, and *L. decidua xkaempferi* from Austria). For all the different sites and species, the types of IADF and their positions had the same isotopic signals in terms of carbon and oxygen. E-IADFs presented δ13C and δ18O in the range of −23 ± 0.6‰ and 32 ± 1‰, respectively; L-IADFs had δ13C and δ18O values of −27 ± 0.7‰ and 29 ± 0.5‰, respectively (Battipaglia et al. 2016a). These results should be extended to a larger database in order to assess how the isotopic composition of IADFs could help with dating problematic samples and support the interpretation of phenomena that trigger the formation of IADFs in the Mediterranean environment.

### *21.5.3 Carbon and Oxygen Isotopes and Fire*

Fire has been a frequent and important disturbance in Mediterranean forest ecosystems throughout the Holocene (Heinselman 1981; FAO, State of Mediterranean Forest 2018), shaping landscapes and determining vegetation distribution and dynamics. Tree rings have been used to reconstruct fire history and to determine the effects of fire on forest productivity. In recent years, carbon and oxygen isotopes measured in tree rings have proved to be a useful tool for understanding the complex ecophysiological processes that occur in Mediterranean tree species following a wildfire or prescribed burning (Beghin et al. 2011; Battipaglia et al. 2014b, c, 2016b, 2019; De Micco et al. 2014; Valor et al. 2018; Niccoli et al. 2019). Because conifers are widespread in the Mediterranean Basin and highly prone to fire damage, many studies have focused on conifer species, including *Pinus sylvestris* (Beghin et al., 2011), *Pinus halepensis* (Battipaglia et al. 2014b; c; Valor et al. 2018), *Pinus pinea* (Battipaglia et al. 2016b), and *Pinus pinaster* (Niccoli et al. 2019). Stable isotopes studies indicate that tree response and their potential to recover over the short- and long-term depends on fire severity. Strong wildfires produce a simultaneous increase in tree-ring δ13C and δ18O, suggesting a strong reduction in stomatal conductance and assimilation rate, which are often linked to crown damage (Battipaglia et al. 2014b; Niccoli et al. 2019). When fire severity is low to moderate, the effect on trees is less pronounced; often, the tree rings only report a small variation in δ13C, with no change of δ18O. In these cases, a reduction in tree growth is largely due to the reduced photosynthetic capacity of the burned trees (Battipaglia et al. 2016b). Following prescribed burning s, however, trees show a decrease in both δ13C and δ18O, indicating a possible favorable effect of reduced competition between the surviving plants. In addition to reducing competition, prescribed burning seems to lead to a release of nutrients, which can also stimulate the growth of surviving trees (Battipaglia et al. 2014c, 2016b; Valor et al. 2018; Niccoli et al. 2019).

### **21.6 Nitrogen Stable Isotopes**

Nitrogen availability is an important factor limiting productivity in Mediterranean ecosystems (Noy-Meir 1973; Gutierrez and Withford 1987; Lloret et al. 1999) and therefore influencing wood formation. Tree-ring δ15N is mainly influenced by the isotopic ratio of the available nitrogen sources (Nadelhoffer and Fry 1994; Peñuelas and Estiarte 1997; Evans 2001, Stewart et al. 2001). Thus, tree-ring δ15N allows for the reconstruction of the nitrogen source and the relative activity of the different biogeochemical processes that affect the δ15N of nitrogen compounds taken up by trees in the surrounding ecosystem (i.e., mineralization, nitrification, denitrification and, NO3 leaching; see Chap. 12). However, other fractionation events that occur in plants during assimilation processes can also influence tree-ring δ15N and complicate interpretation of the nitrogen sources (Evans 2001). Indeed, tree-ring δ15N may also depend on nitrogen reabsorption or re-translocation (Kolb and Evans 2002) or fractionation by different mycorrhizal associations (Michelsen et al. 1998; Craine et al. 2009). The incorporated tree-ring δ15N signature thus results from an integration of nitrogen sources and internal transformations. These complications may explain why only a limited number of studies have been carried out in Mediterranean ecosystems, most of which have focused on understanding the effects of climate change on carbon and nitrogen cycles (Peñuelas et al. 1996; Alfaro-Sanchez et al. 2016).

Tree-ring δ15N has also been used to assess changes in nitrogen availability due to (i) variations in atmospheric nitrogen deposition and (ii) their influence on ecosystem nitrogen dynamics, since the nitrogen isotope ratio in compounds produced as a result of anthropogenic activities may be significantly different from the natural background δ15N in the soil (Freyer 1991). Guerrieri et al. (2009, 2011) and Battipaglia et al. (2010b) showed that increases in nitrogen deposition from the atmosphere or from fertilization are reflected in tree-ring δ15N signals as a result of source δ<sup>15</sup> N (e.g., NOx, NHx forms, or NH4 <sup>+</sup> vs NO3 <sup>−</sup>) and the processes occurring during different phases of soil biogeochemical processes (i.e., losses caused by NO3<sup>−</sup> leaching, denitrification, and NH3 volatilization; see also Chap. 12). Changes in δ15N in annual tree-rings reflect the causes of variation in the tree's iWUE due to climatic or anthropogenic impacts, suggesting that an increase in nitrogen input from the atmosphere (Guerrieri et al. 2011) or from consociation from nitrogen-fixing species could led to an increase of trees iWUE under a scenario of reduction in precipitation, such as in Mediterranean area (Guerrieri et al. 2011; Battipaglia et al. 2017). Measurement of δ15N of the different possible sources, and a better understanding of the fractionation processes in the different species, is needed for a proper interpretation of tree-ring δ15N data.

### **21.7 Conclusions and Outlook**

Tree-ring isotope studies in the Mediterranean Basin have increased in the last years for two main reasons. First, there is general agreement regarding the importance of reliable information about how Mediterranean trees are responding to a changing climate. Such information informs assessments of the future state of such vulnerable forests, as well as their role in carbon sequestration and ecosystem services. Second, stable isotopes help to identify the ecological behavior and vulnerability of Mediterranean tree and shrub species, information that can improve management options. Further research is needed to improve our knowledge about the xylem plasticity of the different Mediterranean species. This will help pinpoint how cambial activity affects the functional processes of trees and their responses to climate change. The combination of multiple approaches, including stable isotopes, xylogenesis, wood anatomy, and long-term growth and isotope-based gas exchange data, may provide more insight on how plants optimize growth and minimize costs.

**Acknowledgements** The authors thank Francesco Niccoli for helping with Figure 21.1 and Erin Gleeson for English editing.

# **References**

density fluctuations. Trees—Struct Funct 26(2):513–524. https://doi.org/10.1007/s00468-011- 0612-4

in tree rings of Quercus ilex L. and Pinus pinea L. and. Tree Physiol 38(8):1213–1224. https:// doi.org/10.1093/treephys/tpy061

Zalloni E, Battipaglia G, Cherubini P, Saurer M, De Micco V (2019) Wood growth in pure and mixed Quercus ilex l. forests: drought influence depends on site conditions. Front Plant Sci 10. https://doi.org/10.3389/fpls.2019.00397

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# **Chapter 22 Stable Isotopes in Tree Rings of Tropical Forests**

**Peter van der Sleen, Pieter A. Zuidema, and Thijs L. Pons**

# **22.1 Introduction**

The analysis of growth rings in the stems of trees is a relatively new tool in tropical forests, as the existence of annual rings in tropical trees was not commonly recognized until recently. For a long time, the tropical environment was associated with yearround favorable growth conditions that were thought to prevent the formation of distinct annual growth rings. However, most tropical environments are seasonal to a various extent (Fig. 22.1), and the formation of annual tree rings in deciduous species growing in tropical climates with a pronounced dry season has been known for a long time (Coster 1927). Although ring boundaries of trees in the humid tropics are generally less clear than those in temperate trees, the formation of distinct annual growth rings has been shown for a large number of tropical tree species (Worbes 2002; Rozendaal and Zuidema 2011; Zuidema et al. 2012; Brienen et al. 2016; Schöngart et al. 2017). In addition to drought, other seasonally changing environmental factors, such as flooding and soil salinity, are known to induce the formation of annual ring boundaries (Schöngart et al. 2002; Chowdhury et al. 2008).

P. van der Sleen (B) · P. A. Zuidema

Forest Ecology and Forest Management Group, Wageningen University and Research, Droevendaalsesteeg 3, 6708 PB Wageningen, The Netherlands e-mail<EMAIL_ADDRESS>
P. van der Sleen

T. L. Pons

**Supplementary Information** The online version contains supplementary material available at (https://doi.org/10.1007/978-3-030-92698-4\_22).

Wildlife Ecology and Conservation Group, Wageningen University and Research, Droevendaalsesteeg 3, 6708 PB Wageningen, The Netherlands

Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

**Fig. 22.1 a** Map of the tropics showing locations of studies on stable isotopes in tropical treerings. Main findings of these studies summarized in Table 22.1. Background map showing tropical climates according to Köppen classification (map from Beck et al. 2018), with grey continental areas belonging to arid (mostly treeless) climate types. **b** Diagram showing general rainfall seasonality in the tropics, with a dry month defined as a month with <100 mm average precipitation. Figure adapted from Kricher (2011). **c** A few examples of tropical (woody) ecosystems. Clockwise from top left: evergreen rainforest close to the equator (no dry season) in Brazil, mountain cloud forest in Bolivia (moderate dry season; but moist year round), deciduous dry forest in Mexico (long dry season), and savanna vegetation in Tanzania (long dry season)

The "re-discovery" of annual growth-ring formation in tropical trees, and developments in stable isotope analyses has triggered studies on the variation of the natural abundance of isotopes in tropical trees, with the number of publications on stable isotopes in tropical tree rings increasing rapidly in recent years (Fig. 22.1). In this chapter, we provide an overview of research on stable isotopes in tropical tree rings, and the insights gained on the functioning of tropical forests and the impacts of global change. This chapter is a condensed and updated version of our earlier review (van der Sleen et al. 2017), with a main focus on lowland forests in the wet tropics between 23.5°N/S (Fig. 22.1).

### **22.2 Tropical climate**

The tropics are generally warm (mean annual temperature of >24 °C) and wet (except for dry grasslands and deserts). Most tropical ecosystems are seasonal to some extent, but in contrast to temperate regions, seasonal weather patterns are primarily driven by variation in precipitation amount and not temperature fluctuations. Across the tropics, rainfall seasonality is a result of large-scale atmospheric circulation patterns. At the equator, heat builds up, causing warm and moisture-laden air to rise. The ascending air diverges at the top of the troposphere and flows to more northern and southern latitudes. As the air cools, it loses its moisture as precipitation, becomes more dense, and ultimately descends as dry air in the subtropics, from where it returns equatorward near the surface, giving rise to the trade winds. These circulation patterns, on both the northern and southern hemisphere, are called the Hadley cells. The great equatorial convection zone, where the trade wind meet is known as the Intertropical Convergence Zone (ITCZ), and characterized by violent thunderstorms and high quantities of precipitation. The ITCZ moves north during the Northern hemisphere summer, and south during the austral summer, as a consequence of the Earth´s tilt. This seasonal movement induces wet and dry seasons in most tropical regions (Fig. 22.1). At locations lacking a true dry season, forests will remain evergreen throughout the year. At locations with a longer or drier dry season, the number of deciduous species increases. Further away from the Equator, forests give way to savanna, shrubland or grassland (Fig. 22.1). Although climate is an important factor determining forest type, it is also affected by other factors, such as soil characteristics and fire regime.

Another important climatic feature in tropical regions is the El Niño–Southern Oscillation (ENSO), which are irregular periodic variations in winds and sea surface temperatures over the tropical eastern Pacific Ocean. Although the factors responsible for the occurrence of ENSO events remain incompletely understood, the phenomenon occurs when the ITCZ migrates more southwards than normal. This raises sea surface temperature and disrupts the normal upwelling pattern along the west coast of South America. Eventually, this reduces the transport of warm surface water across the Pacific Ocean toward Asia, and as a result affects weather systems throughout the tropics and subtropics, causing heavy downpours and flooding in some regions and severe droughts in others. El Niño occurs every two to seven years and tends to alternate with another climatic phenomenon with opposite effects. This is called La Niña, and happens when trade winds gain abnormal strengths, increasing upwelling along the west Coast of South America and strengthening the flow of warm surface water westwards across the Pacific.

Local climate conditions, and its variability (including ENSO-driven anomalies), affect tree physiology and thus tree-ring isotope values. In addition, large-scale circulation patterns affect the δ18O signature of precipitation, which can be recorded in tree rings (Table 22.1).

**Table 22.1** Summary of the main usage and outcomes of studies measuring stable isotopes in tropical tree rings. References given in chronological order (detailed version of the table is available, see Supplementary Table S1)

**Table 22.1** (continued)

ENSO, el Niño Southern Oscillation; iWUE, intrinsic water-use efficiency; *<sup>g</sup>*<sup>s</sup> stomatal conductance \* Total number of species and those with non-distinct growth rings between brackets. Some species were involved in multiple studies, notable *Tectona grandis*, *Cedrela odorata*, and *Fokienia hodginsii*

### **22.3 Carbon Stable Isotopes**

### *22.3.1 Carbon Isotope Ecophysiology in the Tropics*

The stable carbon isotopic signature in the wood of tropical trees is influenced by a variety of factors related to rainfall, temperature, light conditions and species' responses to drought (for the basics of the 13C/12C isotope fractionation in plants see Chap. 9). The combined effects of these factors determine δ13C values measured in tropical tree rings. In some cases, contributions of individual environmental factors are hard to disentangle, as they can covary and exert similar effects on tree-ring δ13C.

Ontogenetic trends in tree-ring δ13C series can be substantial for tropical trees, as was illustrated for *Cedrela odorata* from Bolivia (Brienen et al. 2017). Juvenile trees growing under a forest canopy are exposed to reduced irradiance that is associated with a high ratio of intercellular over atmospheric CO2 concentration (*c*i/*c*a), and young trees possibly also absorb 13C-depleted CO2 near the forest floor (Medina and Minchin 1980; Medina et al. 1991; Buchmann et al. 1997). Both factors contribute to low δ13C values in the stem wood of small trees*.* Sub-canopy trees are exposed to higher light levels and slightly 13C-enriched CO2 due to high photosynthetic activity in the canopy, leading to higher δ13C values in their wood. Canopy and emergent trees are exposed to high light levels and CO2 with an atmospheric δ13C signature (Buchmann et al. 1997). In addition, ontogenetic trends in tree-ring δ13C may be driven by increased vapor pressure deficit (VPD) from understory to canopy and increases in hydraulic resistance with tree height. Therefore, changes in δ13C with tree size can provide valuable information in an ecological context (e.g. on light conditions; van der Sleen et al. 2014), but are an important confounding factor in a dendroclimatological context.

Decreasing water availability, or increasing VPD, results in stomatal closure, and therefore lowers the *C*i/*C*<sup>a</sup> ratio (e.g. Gebrekirstos et al. 2011). As a result, drought increases tree-ring δ13C values and intrinsic water-use efficiency (iWUE; the ratio of photosynthetic rate over stomatal conductance; *A*/*g*s) (e.g. Lambers et al. 2008; Cernusak et al. 2009; Craven et al. 2013). The available evidence suggests that the relationship between water availability and tree-ring δ13C also holds in regions with humid climatic conditions: δ13C of leaves collected in the rainy season was lower than in leaves collected in the dry season (French Guyana, Buchmann et al. 1997), and wood δ13C of trees growing near a creek was lower than that of trees growing on a comparatively drier ridge (Guyana, Pons et al. 2005).

Changes in light and nutrient conditions can also affect tree-ring δ13C values when photosynthetic activity increases (*A*) more that stomatal opening (*g*s) (e.g. Cernusak et al. 2009; van der Sleen et al. 2014). Interestingly, average tree-ring δ13C values and the impact of environmental conditions on δ13C ratios is species specific. Cooccurring tree species of similar DBH can exhibit large variations in δ13C values, which has been related to differences in successional status (Bonal et al. 2007), shade tolerance (Guehl et al. 1998; Bonal et al. 2000), leaf phenology patterns (Bonal et al. 2000), and drought tolerance (Craven et al. 2013).

# *22.3.2 Seasonal Variation in Tree-Ring δ13C*

Early studies on δ13C in tropical wood showed the presence of intra-annual variation, similar to what was found for other climatic regions (Leavitt and Long 1991). Subsequent studies confirmed this seasonality (e.g. Poussart et al. 2004; Verheyden et al. 2004; Ohashi et al. 2009), suggesting seasonal variation in water availability as underlying cause. Variation in δ13C within a single growth ring can however, not be unequivocally understood from current photosynthesis on the basis of the Farquhar et al. (1989) model. Other processes may interfere with patterns driven by climate seasonality, such as utilization of stored reserves early in the growing season, potential fractionation downstream from the carbon fixation in the leaves, and a varying fraction of C allocated to other processes than diameter increment in the growing season (Helle and Schleser 2004; Kagawa et al. 2006; Krepkowski et al. 2013; Fu et al. 2017). Although attempts have been made to identify annual rings in tropical trees that lack visible increment ring boundaries based on intra-annual δ13C patterns, δ18O proved to be more useful for that purpose.

# *22.3.3 Annual and Decadal Variation in Tree-Ring δ13C*

Several studies have investigated the inter-annual variation of δ13C and its correlation with precipitation amount (Table C). Strong negative correlations between tree-ring δ13C and annual precipitation were found for species from various sites in tropical America, Asia and Africa, whose growing conditions differed widely in annual precipitation (e.g. Fichtler et al. 2010; Rahman et al. 2020). Such climate sensitivity was further linked to ENSO variability in some studies (e.g. Brienen et al. 2011). In semi-arid Ethiopia, Gebrekirstos et al. (2009) also found a strong correlation of δ13C with precipitation for three *Acacia* species, but less so in *Balanites aegyptiaca*. Such differences may relate to water-use strategies, with drought-tolerant species showing a lower sensitivity to inter-annual variability in precipitation amount (Gebrekirstos et al. 2011; Craven et al. 2013).

Other studies combined δ13C sequences with measurements of δ18O. In some of these, δ13C showed correlation with other precipitation variables than those found for δ18O (Cullen and Grierson 2007; Schollaen et al. 2013). Nevertheless, δ18O series generally yielded stronger correlations with precipitation variables than δ13C series (Poussart and Schrag 2005; Ballantyne et al. 2011: and see discussion in the next section).

Studies using longer tree-ring sequences have consistently shown a declining δ13C trend over the last century in tropical trees (Hietz et al. 2005; Brienen et al. 2011; Loader et al. 2011), also when explicitly correcting for potential ontogenetic effects (Nock et al. 2011; van der Sleen et al. 2015a). After correcting for decreasing atmospheric δ13C over that period (the Suess effect), a rather constant 13C discrimination (-13C) generally remains. This leads to the conclusion that *C*i/*C*<sup>a</sup> remained constant over time and that, as a result of the increasing atmospheric CO2 concentration, iWUE has increased consistently over time (Silva and Anand 2013, see Chap. 17). A sustained increase of photosynthesis and/or a higher water-use efficiency under elevated CO2 are expected to stimulate tree growth if carbon and/or water are limiting factors (Körner 2009). A few studies also quantified temporal trends in tree growth using the same tree-ring sequences as used to determine δ13C trends (Nock et al. 2011; van der Sleen et al. 2015a). Interestingly, these studies found no indications for a growth stimulation over the past century. Stem diameter growth is not necessarily linearly linked to photosynthetic activity, because other aspects of the carbon balance of trees may have changed as well, such as phenology, leaf turnover, respiration and biomass allocation. However, if it is reasonable to assume that diameter growth can reflect changes in the total carbon balance of a tree, in particular on longer time scales, than the available tree-ring studies suggest that photosynthesis did not increase as a result of rising atmospheric CO2 concentration. This scenario could arise if tree growth is ultimately limited by nutrient availability in most tropical regions, or if a CO2-induced stimulation of photosynthesis has been compensated by an external climate-related stressor, such as increased temperature or decreased precipitation.

### **22.4 Oxygen Stable Isotopes**

### *22.4.1 Oxygen Stable Isotope Ecophysiology in the Tropics*

Water taken up by trees becomes enriched in 18O in leaves as a result of transpiration. The strength of this enrichment is mediated by environmental conditions (Barbour 2007; Chap. 10). CO2 taken up by the leaves exchanges its oxygen atoms with that of leaf water, causing a transfer of the isotopic signature of leaf water to sucrose. In addition, when cellulose is synthesized from sucrose in the stem, about 42% of the oxygen atoms are again exchanged with xylem water. This exchange causes the effect of enriched leaf water to be partly reverted, and results in a strong imprinting of the isotope signature of source water in the wood of trees. Unfortunately, the δ18O signature of absorbed source water is commonly unknown, especially over longer time scales. In addition, it is often unknown from what depth the root systems of tropical trees take up water, which further complicates the determination of the δ18O of source water, and thus the interpretation of intra- and inter-annual variation in δ18O in wood. Shallow-rooting trees, growing in dense canopies where isotopic enrichment at the soil surface is minimal, likely absorb water that consists mainly of recent precipitation, and hence tree-rings are imprinted with an 18O signal that is rather similar to that of rainwater. As rainwater resides only shortly at the soil surface before percolating down to the groundwater, the δ18O signature of groundwater likely integrates variation in δ18O in precipitation over several years (Chap. 18). The depth of water uptake has been estimated by measuring natural abundance of 18O in the soil profile and in xylem water (Jackson et al. 1995; Hasselquist et al. 2010; Ellsworth and Sternberg 2015) or by labelling soil water (Stahl et al. 2013). These studies show that deciduous trees tend to take up water from shallower depths than evergreen trees and that the depth of water uptake generally increases with tree age and size.

# *22.4.2 Seasonal Variation in Tree-Ring δ18O*

The first δ18O studies on tropical trees quantified radial variation of δ18O in tree stems to reconstruct annual ring boundaries of tree species without anatomically distinct rings or to confirm the annual nature of ring formation (Evans and Schrag 2004; Poussart et al. 2004; Verheyden et al. 2004; Poussart and Schrag 2005; Evans 2007). In some of these studies, δ13C was also measured, but δ18O was found to be generally superior for this purpose. The suitability of δ18O for the identification of annual rings is based on its seasonal change in precipitation: rainwater δ18O is low during the rainy season and with heavy precipitation events (Dansgaard 1964; Villacıs et al. 2008; Kurita et al. 2009). High δ18O values of precipitation during the dry season can be further amplified in both soil and leaves due to higher evaporation at low humidity (Jackson et al. 1995; Cintra et al. 2019). This seasonality in δ18O was confirmed in tropical trees with distinct annual rings (Poussart et al. 2004; Managave et al. 2010; Ballantyne et al. 2011; Managave et al. 2011a; Schollaen et al. 2013). The identification of annual rings in homogeneous (ring-less) wood is most successfully done when intra-annual variation in source δ18O is large. This is the case in the western parts of the Amazon basin, where δ18O in precipitation is low in the rainy season due to rain-out of the heavy isotopes as water vapor travels from the Atlantic ocean across the basin (Sturm et al. 2007). Evidence for this effect is provided by the lower intra-annual variation in δ18O in evergreen trees from Guyana (1–4‰; Pons and Helle 2011) compared to trees sampled near Manaus, Brazil (3–6‰; Ohashi et al. 2016). A special case are trees in montane forests where the uptake of water during the rainy season is from precipitation, whereas moisture can also be directly absorbed from clouds in the dry season. These two water sources differ in δ18O values, which can result in large seasonal variation of tree-ring δ18O (Anchukaitis et al. 2008; Anchukaitis and Evans 2010). As the strength of intra-annual variation in δ18O varies across species (Poussart and Schrag 2005; Anchukaitis et al. 2008) and climatic conditions, selection of species and sites will determine the ability to identify annual rings in homogeneous wood.

# *22.4.3 Annual and Decadal Variation in Tree-Ring δ18O*

Because some trees incorporate the δ18O signature of rainwater in stem wood, time series of tree-ring δ18O can be used to quantify past variability in precipitation. Tree-ring δ18O has been correlated with basin-wide precipitation in the Amazon (Ballantyne et al. 2011; Brienen et al. 2012; Baker et al. 2015, 2016), and regional precipitation in Thailand (Poussart and Schrag 2005; Pumijumnong et al. 2020), Costa Rica (Anchukaitis and Evans 2010), India (Managave et al. 2011b), Indonesia (Schollaen et al. 2013, 2015), Laos and Vietnam (Xu et al. 2011), West Africa (van der Sleen et al. 2015b), and central Africa (Colombaroli et al. 2016). Particularly El Niño Southern Oscillation (ENSO) variability is often evident in δ18O sequences either from positive anomalies (Verheyden et al. 2004; Anchukaitis and Evans 2010; Zhu et al. 2012a) or negative ones (Evans and Schrag 2004; Brienen et al. 2012). The analysis of tropical tree-ring δ18O is developing into a powerful tool for reconstructing the variability of precipitation on regional scales.

Good synchronization of δ18O patterns among individual trees was found for several species (e.g. Poussart and Schrag 2005; Managave et al. 2011b; Brienen et al. 2012; van der Sleen et al. 2015b), sometimes over large spatial distances (Baker et al. 2015; Schwendenmann et al. 2015; Volland et al. 2016). Synchronous variability in δ18O can be higher than for ring-width, thus providing an alternative tool for cross dating (Baker et al. 2015; van der Sleen et al. 2015b; Volland et al. 2016) and identification of false and missing rings (Boysen et al. 2014). However, δ18O synchronization between individuals may be low for certain species or sites (e.g. Poussart and Schrag 2005; Baker et al. 2015). For δ18O in *Toona ciliata* from Thailand, low δ18O synchronization occurred (van der Sleen 2014), in spite of the ring-width series showing strong synchronization (Vlam et al. 2014). It is likely that trees that lack a common signal in tree-ring δ18O values exploit other water sources than recent precipitation (e.g. ground water). These results suggest that shallow rooting tree species on well-drained soils have the highest probability to record the δ18O variability of precipitation and thus have the highest potential as tools for climate reconstructions.

In several studies the two stable isotopes 18O and 13C were combined using a mechanistic interpretation, the so-called dual isotope approach (see Chap. 16), where *A/g*<sup>s</sup> obtained from -13C and *g*<sup>s</sup> derived from -18O could potentially provide an estimate of *A* (Scheidegger et al. 2000). This approach was used by Nock et al. (2011), who interpreted an increase of -18O over time as an indication of a decreasing *g*<sup>s</sup> in trees from Thailand. The observed decrease of -13C, and thus increasing *A*/*g*s, would then be the result of this decreasing *g*<sup>s</sup> and not an increasing *A*. However, the underlying assumptions in this approach are that the δ18O signature of source water is known and that the leaf to air vapor pressure difference (LAVPD) has remained constant over the period studied. In many tropical regions, this LAVPD may have increased as a result of global warming and/or decreased precipitation, leading to increased transpiration and δ18O over time. Even though *gs* is also partly controlled by LAVPD, this makes it nonetheless difficult to separate the *g*<sup>s</sup> effect from the LAVPD effect on transpiration.

A long-term increase of δ18O values has been encountered in several studies conducted on tropical tree species (Poussart and Schrag 2005; Xu et al. 2011; Brienen et al. 2012; van der Sleen 2014; van der Sleen et al. 2015b; Volland et al. 2016). Some of these trends could be caused by ontogenetic changes, but a consistent small trend over the past century was also found in studies that did correct for ontogenetic trends (van der Sleen 2014; van der Sleen et al. 2015b; Volland et al. 2016). For the Amazon region, these results are consistent with similar increases of δ18O in Andean ice cores (Thompson et al. 2006) and Andean lake sediments (Bird et al. 2011). Thus, the increasing trend in δ18O in tree rings likely reflects a pan-tropical phenomenon. The cause of this increase is yet unknown, and it is unclear whether it relates to climate change.

### **22.5 Nitrogen Stable Isotopes**

### *22.5.1 Nitrogen Stable Isotope Ecophysiology*

The value of plant δ15N depends on the δ15N of the N taken up and N losses in leaves, fruits, etc. Uptake can be in the form of nitrate, ammonium, organic N compounds or N2 in the case of nitrogen fixation. The δ15N of these sources varies in a complex manner (except for N2, which is used as standard and is thus 0‰ by definition; Chap. 12).

Higher soil and foliage δ15N are generally reported for tropical lowland forests, compared to temperate and boreal forests (Martinelli et al. 1999; Amundson et al. 2003), and tropical montane forest (Brearley 2013). This pattern is considered as evidence of more N losses and thus a more open N cycle in tropical lowland forests, because nitrate lost through leaching and/or denitrification is 15N-depleted. Temperate forests are generally more N-limited, whereas tropical forests tend to be more P-limited (Vitousek and Howarth 1991), which is consistent with their higher δ15N. Leguminous trees are abundant in tropical forests, although not all can form an effective symbiosis with Rhizobia. Yet, facultative leguminous N2-fixers can still be abundant (Menge and Chazdon 2016) and contribute to N-accumulation also in late successional stages of tropical forests (Roggy et al. 1999; Pons et al. 2007). This could be the reason for an alleviation of N-limitation, whereas N2-fixing trees are virtually lacking in temperate forest (except for early successional stages; Menge et al. 2009).

# *22.5.2 Annual and Decadal Variability in Tree-Ring δ15N*

So far, only three studies on temporal variation or trends δ15N in tree rings have been carried out in tropical forests (Hietz et al. 2010, 2011; van der Sleen et al. 2015c). Hietz et al. (2010) using two species in a Brazilian forest, found a gradual increase of tree-ring δ15N over time after statistical correction for tree age. The authors suggested that this result could be caused by an increase in tree turnover and thus gap formation that generates NO3 <sup>−</sup> losses and thereby increasing δ15N of the remaining soil N pool. In a subsequent study, Hietz et al. (2011) reported also an increase in δ15N in three species from a monsoon forest in Thailand. They also found a similar increase when comparing 40-year-old herbarium leaves with recent leaves from the same species and sample location in a Panamanian forest (BCI). The two forests are intensively monitored and there are no indications that the level of disturbance has increased over the past century. Such a consistent trend in δ15N in three widely separated tropical forests may therefore indicate an effect of increased anthropogenic N-deposition, which causes higher NO3 <sup>−</sup> losses (and thus δ15N enrichment of remaining soil nitrogen). However, in the most recent study, van der Sleen et al. (2015c) sampled annual rings in six species from three sites at different continents. They corrected for possible tree size effects by comparing wood sampled at a fixed diameter (20 cm) from different sized trees. Ten-year pooled samples were also collected between 1955 and 2005 from single trees, which showed increasing trends of δ15N in Bolivia and Cameroon. Surprisingly, the trends were absent in the fixed diameter sampling, showing evidence of potential ontogenetic effects. The discrepancy between the results of Hietz et al. (2011) and van der Sleen et al. (2015c) may also have been caused by a lower statistical power in the latter. Unfortunately, the interpretation of temporal changes in δ15N in the few available tropical treering studies remain strongly hampered by a limited understanding of the factors that influence soil- and tree δ15N values.

### **22.6 Conclusion and Perspective**

Tropical forests harbor an incredible biodiversity and provide ecosystem services on which millions of people depend. They are a major component of the global carbon cycle, storing some 25% of the total terrestrial carbon and accounting for a third of net primary production (Bonan 2008). Understanding the functioning of these forests and their responses to global change is therefore an urgent need for ecology, climate science and conservation. The study of stable isotopes in tropical tree rings offers unique opportunities to quantify how these trees respond to their environment, and can fill an important void in many tropical areas where the availability of climate data is limited or of short duration. In fact, stable isotopes in tree rings are essentially the only tools available to obtain cost-effective, high-resolution, long-term retrospective data on tree physiology and the environmental conditions affecting it.

Currently, stable isotopes research in the tropics has mainly focused on (i) quantifying the effects of rising atmospheric CO2 and climate change on tree physiology, (ii) identifying the drivers of growth variability and reconstruction of past climate, and (iii) the identification of annual rings in wood lacking anatomically distinct growth boundaries. The main findings of the available studies are shortly summarized for each isotope in Table 22.1. The application of stable isotopes continues to expand. New applications are numerous, and include the potential use of stable isotopes for timber tracing and to identify illegally logged wood (e.g. Vlam et al. 2018), to benchmark the predictions of dynamic global vegetation models (e.g. Zuidema et al. 2018), and for reconstructions of atmospheric CO2 and δ13C values (using trees with a C4 photosynthetic pathway; Ben et al. 2017).

Although tropical isotope research has centered on three stable isotopes (C, O, and to a lesser degree N), advances in analytical techniques and reductions in associated costs continue to broaden these analyses. This not only includes the analyses of other stable isotopes, but also the analyses of the intramolecular distribution of isotopes. For example, the position of 18O in the glucose moiety in cellulose can be used to separate source water from leaf water enrichment effects (Sternberg 2009; Waterhouse et al. 2013), and the position of 2H was related to the oxygenation/carboxylation ratio that depends on *C*<sup>i</sup> (Ehlers et al. 2015). These techniques can be used to infer more details about environmental effects on tropical trees than is possible with bulk isotopic ratios as done so far. Even though stable isotope research in the tropics still faces methodological and interpretation issues, we anticipate that it will continue to play a crucial role for our understanding of the functioning of tropical forests and their resilience to global change.

### **References**

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# **Chapter 23 Forest Management and Tree-Ring Isotopes**

### **John D. Marshall, J. Renée Brooks, and Alan F. Talhelm**

**Abstract** Forest management can be improved by the mechanistic understanding that tree-ring stable isotopes provide. Key management tools include genetic selection, competing vegetation control, thinning, and fertilization. These tools frequently change environmental conditions and physiological processes, such as photosynthesis, stomatal conductance, water uptake, and nitrogen cycling, which may leave isotopic signatures in tree-rings, providing detailed responses to management over decadal time periods. While data sets remain small, some trends have emerged from previous forest management studies using stable isotopes. Genotype selection sometimes shows isotopic evidence of maladaptation, especially in the presence of climate change. Competition control and thinning have different isotopic reactions depending on the dryness of the site; they generally obtain different responses depending on whether competition is primarily for aboveground (light) or belowground (water and nutrient) resources. Fertilization responses recorded in tree rings appear to be driven by initial increases in photosynthesis, and later by increases in leaf area index. Tree-ring isotopic applications can provide key insights to a much broader range of silvicultural objectives than included here, and we encourage their application in large-scale silvicultural experiments to reduce uncertainties and explain mechanisms of response. In future work, we suggest that management studies wishing to utilize tree-ring stable isotopic analysis include key ancillary measurements, especially leaf nitrogen concentrations, leaf-area index, xylem water sources, and canopy temperature, to help support interpretation of the isotopic data.

J. D. Marshall (B)

Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, 90183 Umeå, Sweden

e-mail<EMAIL_ADDRESS>
J. R. Brooks

#### A. F. Talhelm Department of Forest, Rangeland, and Fire Sciences, University of Idaho, Moscow, ID 83844, USA

Pacific Ecological Systems Division, Center for Public Health and Environmental Assessment, Office of Research and Development, U. S. Environmental Protection Agency, 200 SW 35th St. Corvallis, Corvallis, OR 97333, USA

### **23.1 Introduction**

Forest management activities are designed to alter stand and tree growth, composition, structure, and/or other ecosystem properties (Smith et al. 1997). Management activities such as thinning, prescribed burning, herbicide application, or fertilization change resource availability within forests, altering tree growth (Fox et al. 2007; Johnson and Curtis 2001; Jurgensen et al. 1997; Neary et al. 1999). However, because these activities often change the availability of multiple resources—including moisture, nutrients, and light (e.g. Jurgensen et al. 1997; Neary et al. 1999)—the physiological or ecological mechanisms responsible for changes in tree growth can be unclear. The forestry research community has long been interested in understanding how management activities affect both resource availability and tree physiology. Their wish has been for more mechanistic prescriptions to meet objectives including the optimization of forest productivity (e.g., Campoe et al. 2013; Forrester et al. 2013; Gauthier and Jacobs 2009; Gspaltl et al. 2013), adaptation to climate change (e.g. Chmura et al. 2011; Lindner et al. 2014; Martin-Benito et al. 2011), and the production and maintenance of ecosystem services (Deal et al. 2012; Ford et al. 2011; Luyssaert et al. 2018).

Stable isotopes are an important tool to understand the response to trees, stands and ecosystems to management because they integrate and trace physical and physiological processes over time and space (Dawson et al. 2002; West et al. 2006; Sulzman 2008; Werner et al. 2012). Forestry researchers were early adopters of some stable isotope techniques (e.g., Heilman et al. 1982; Högberg et al. 1993; Nambiar and Bowen 1986). The use of these techniques has expanded rapidly in the last decade to encompass a variety of management practices in ecosystems from the African savanna to giant sequoia forests in California (e.g., Cramer et al. 2010; York et al. 2010). However, although stable isotopes can be powerful analytical tools, the application and interpretation of stable isotope methods can be complex because numerous abiotic and biotic processes alter the relative abundance of individual isotopes (Dawson et al. 2002; Sulzman 2008; Werner et al. 2012; West et al. 2006).

In this chapter, we describe isotopic consequences of management effects and discuss their mechanistic interpretation. We first present, in chronological order, several of the traditional management tools used in even-aged stands from planting to harvest, largely because much of the isotopic literature examining forest management occurred in such stands. This organization is intended to clarify, not to suggest that forest managers are limited to these tools and applications. In fact, these management tools can be used to foster a broad range of ecosystem services, which isotopic data can help inform. We provide both illustrative examples and, in the cases of nutrient additions (fertilization) and thinning, meta-analyses. We present the results of a new meta-analysis here, but we also summarize an earlier meta-analysis (Sohn et al. 2016) of thinning effects on drought responses of tree growth.

We focus here on carbon isotope discrimination (-13C, Chap. 9) and oxygen isotopic ratios (δ18O, Chap. 10), with brief mention of nitrogen isotope ratios (δ<sup>15</sup> N, Chap. 12). We neglect hydrogen isotope ratios (δ2H) because they are often so correlated with δ18O and because hydrogen exchanges so readily with atmospheric water vapor that the samples must undergo complex preparation (Chap. 11). The method is therefore not prevalent in forest management applications. Intense work is going on with δ2H preparation techniques and it seems likely that they will become more feasible in the near future.

### **23.2 Choice of Species and Genotype**

One of the most powerful tools of forest management is the ability to select the species and genotypes comprising the stand. Foresters can plant favored trees, cull unwanted species or phenotypes, or create conditions that are favorable for particular trees. Tree species differ substantially in -13C, which is correlated with intrinsic water-use efficiency (iWUE, Chap. 17) defined as the ratio of net photosynthetic rate to stomatal conductance (A/gs). Numerous studies have evaluated interspecific differences in -13C in order to evaluate traits such as drought tolerance and water-use efficiency (e.g., Bonhomme et al. 2008; Jansen et al. 2013; Voltas et al. 2006). Broadly, in interspecific studies, deciduous trees show more positive -13C and lower iWUE (intrinsic water-use efficiency) than co-occurring needle-leaved evergreens, which themselves have more positive -13C and lower iWUE than scale-leaved evergreens (Marshall and Zhang 1994). Long-lived leaves tend to have greater leaf mass per area (g/m2) (Wright et al. 2004), which is associated with lower mesophyll conductance (Flexas et al. 2014; Niinemets et al. 2009). This relationship is thought (Hultine and Marshall 2000) to be the primary reason why a negative correlation between leaf -13C and leaf mass per area has been frequently observed (Araus et al. 1997; Hanba et al. 1999; Hultine and Marshall 2000; Körner et al. 1991; Takahashi and Miyajima 2008; Talhelm et al. 2011). In any case, if a forest is likely to deplete the soil water supply during the growing season, then efficient use of that water generally enhances forest growth (Zhang et al. 1995; Zhang and Marshall 1995) and possibly survival (Timofeeva et al. 2017).

Species differences in water access have also been detected in δ18O of xylem water or wood. These differences in water source may be caused by differences in vertical root distribution, where water deeper in the soil tends to be more depleted in heavy isotopes (18O, 2H) than water found near the soil surface (Hartl-Meier et al. 2015; see Chap. 18). This isotopic difference within the soil profile has been used to understand how species partition water resources (Flanagan et al. 1992) and how water-use patterns change seasonally (Dawson 1998; Meinzer et al. 1999). When species are mixed, the root systems may be deployed at different depths, leading to differences in vertical water-uptake. The resulting niche partitioning may give rise to overcompensation, the tendency of a species mixture to be more productive than monospecific stands of either species alone (Pretzsch et al. 2013). However, species mixtures may also lead to growth reductions and mortality for some of the species (Granda et al. 2014). For example, shallow-rooted spruce trees were more negatively influenced by drought than more deeply rooted beech trees in a mixed stand (Brinkmann et al. 2018). Alternatively, differences in tissue or leaf water δ18O may also reflect differences in transpiration timing or rate (Chap. 10). In either case, δ18O differences provide an index of water sources and water use (Marshall and Monserud 2006). When combined with δ13C, δ18O in cellulose may allow one to distinguish whether water-use efficiency has been modified by changes in photosynthesis or transpiration (Scheidegger et al. 2000; see Chap. 16). However, using δ18O to draw insight into leaf gas exchange requires knowledge or assumptions about the δ18O of xylem water (Gessler et al. 2018; Roden and Siegwolf 2012; Chap. 18).

Seedling planting also offers the opportunity to control genetic composition (e.g., species, genotype, and clone) and initial stand structure. In intensive forestry, "tree improvement" is almost always focused on increased timber yield through rapid growth, wood quality, or increased pest resistance. Careless matching of genotype to environment can be deleterious, causing frost damage when coastal Douglas-fir (*Pseudotsuga menziesii*) was moved inland (Rehfeldt 1977), or suboptimal growth when cold-tolerant lodgepole pine (*Pinus contorta*; McLane et al. 2011) or Douglasfir (Leites et al. 2012) were moved to warmer climates. Planting can also be used to assist the migration of warm climate genotypes toward areas that were once too cold for them, but are no longer (Savolainen et al. 2004). Isotopic data have been used as a screening criterion to describe the populations within a species, often from young trees planted in common gardens (Gornall and Guy 2007; Guerra et al. 2016; Monclus et al. 2006). This approach has also identified provenance differences in rooting depth using δ8O (Voltas et al. 2015). Although these studies have mostly focused on ecological questions related to species adaptations to a particular climate, the observed correlation of -13C with height growth or biomass production has also been used to justify genotype selections (Marguerit et al. 2014; Zhang et al. 1995; Zhang and Marshall 1994, 1995). In addition, quantitative trait loci have been applied in native populations to determine whether WUE can be increased without decreasing productivity (Brendel et al. 2002, 2008; Marguerit et al. 2014). In a recent paper, Isaac-Renton et al. (2014) provided a compelling assessment of the climatechange consequences of climate maladaptation in lodgepole pine (Fig. 23.1). The northern populations were defined as "leading edge" because the species is presumably moving northward. These northern populations lacked some of the drought adaptations found in the southern "trailing edge" populations, which may reduce their ability to deal with future droughts. Lodgepole pine has been well studied in this respect, but these same questions can and should be asked about other species as well.

### **23.3 Control of Competing Vegetation**

Managers often prepare sites for planting or improve growing conditions for young trees by removing competing vegetation with herbicides or mechanical treatments. These treatments increase the availability of light, water, and nutrients and improve

**Fig. 23.1** Variations in growth and physiology of lodgepole pine (Pinus contorta) in relation to climate and provenance. **a** serial changes in several growth and tree-ring traits, including stable carbon isotope ratios emphasizing the excursion from the long-term mean in the drought year, 2002. **b** correlations among traits. **c** map of the seed sources and the planting sites. From Isaac-Renton et al. (2014)

tree growth. Because competition control increases the availability of several important resources simultaneously, stable isotopes can be useful for identifying the primary mechanism creating the growth response, particularly when complemented by supporting observations.

Competition control often increases -13C by releasing water to the trees, which would increase stomatal conductance. For instance, Ares and colleagues (2007) observed that five years of herbicide treatments reduced canopy cover of competing vegetation from 95 to 7% among newly established Douglas-fir saplings on the western slope of the Coast Range in the northwestern United States. For the Douglasfir, competition control increased tree growth, foliar N concentrations, and soil moisture availability. In analysis of tree ring -13C of the Douglas-fir trees, earlywood -13C was unaffected by the herbicide treatments, but the latewood was higher in -13C, which led Ares et al. (2008) to conclude that increased soil moisture availability in the late summer was the primary driver of increased tree growth. Likewise, in young ponderosa pine seedlings, reduced competition reduced water stress and increased -13C (Pinto et al. 2011, 2012). Similarly, removal of understory shrubs from stands of *Pinus densiflora* in western Japan increased foliar -13C and both stomatal conductance and foliar N. Increased foliar -13C suggests that the observed increases in growth were caused primarily by increased water availability (Kume et al. 2003).

In contrast, competition control sometimes improves light or nutrient status, which increases photosynthesis, decreasing -13C. For instance, mechanical and herbicide treatments to control grass growing beneath 10-year old white spruce (*Picea glauca*) trees in boreal Canada also increased tree growth, foliar N, and soil moisture, but reduced foliar -13C (Matsushima et al. 2012). This reduction in foliar -13C, which was correlated with foliar N concentration and coupled with a concomitant increase in foliar δ<sup>15</sup> N, led the authors to conclude that the increase in N availability was the primary mechanism by which competition control increased growth in their study. Similarly, competition control decreased red pine (*Pinus resinosa*) tree ring -13C in the north-central United States (Powers et al. 2010). Clearly, the physiological effects of competition control depend on which resources are released and the ability of trees to utilize these resources to acquire C.

### **23.4 Thinning**

Managers thin forests primarily to increase growth of the remaining trees or to create stands that are more resilient to climate stress, insects, disease, and wildfire. Like the removal of competing vegetation, decreasing tree density within a stand reduces competition for resources that limit tree productivity. Thinning can increase the availability of water, nutrients, and light; trees respond physiologically to an increase in each of those resources with changes in the isotopic signatures recorded in tree rings.

We assessed the -13C response across all years and all studies in a meta-analysis (Fig. 23.2) and found that the direction of the discrimination change depended on the mean annual precipitation at the site. While we note that this analysis contains data from only a relative handful of studies, we presume that this reflects differences in physiological responses to thinning. At low precipitation sites, discrimination increases due to the water released to the remaining trees when the thinned trees are removed. The released water increases conductance more than it does photosynthesis, increasing -13C relative to the control. In contrast, at sites with high precipitation, -13C decreases after thinning, presumably due to improvements in the light or nitrogen status of the remaining trees, either of which would tend to increase rates of net photosynthesis. The water released by thinning sites must have less influence on -13C than the light and nutrient increases on these high-precipitation sites. These responses are unlikely to be species-specific responses as nearly all species respond to water stress by decreasing -13C (Granda et al. 2014; Grossiord et al. 2014).

Under dry conditions, thinning can mitigate drought stress and tree ring stable isotopes have helped illuminate the physiological mechanisms of drought resistance

**Fig. 23.2** Normalized change in discrimination ± SE Versus mean annual precipitation at the study site. -13C values are mean responses from each study from the period up to 10 years following the thinning treatment. 1. Brooks and Mitchell (2011) (Thin); 2. Brooks and Mitchell (2011) (Thin x Fertilization); 3. Powers et al. (2010); 4. Sohn et al. (2014); 5. Martin-Benito et al. (2011); 6. Leavitt and Long (1986); 7. Navarro-Cerrillo et al. (2019) (Pinus nigra); 8. Navarro-Cerrillo et al. (2019) (Pinus sylvestris); 9. McDowell et al. (2003) Stand A; 10. McDowell et al. (2003) Stand B; 11. McDowell et al. (2003)) Stand C; 12. York et al. (2010)

(Sohn et al. 2012, 2014, 2016). In an earlier meta-analysis of thinning effects on drought response, tree ring isotopic data revealed that in conifers, heavy thinning influenced -13C in a drought year more strongly than in the other years (Sohn et al. 2016). The heavy thinning treatments also changed δ18O more strongly in the drought year. However, thinning influenced the isotopic data both before and after the drought year as well. An analysis of ring-width data in the same paper showed clearer patterns, in part because it included broadleaf species. For the broadleaves, thinning appeared to increase soil moisture available to the trees during drought, allowing them to resist changes in growth during the drought year. Voelker et al. (2019) also found that stand basal area influenced drought sensitivity; stands at lower basal areas demonstrated decreasing trends in A/gs, and greater resilience to droughts. However, they found stands with high basal area had increasing trends in A/gs over the same period of time, which they attributed to greater water limitations and lower drought resilience. Kruse et al. (2012) also noted that stand density influenced the degree of stomatal control over A/gs.

In more mesic environments, thinning can increase exposure of the remaining canopy to both greater light and higher vapor pressure deficits, as well as increase nutrient availability (Fig. 23.2). For example, *Pinus pinaster, Pinus radiata* and *Pinus resinosa* all decreased -13C after thinning, presumably from an increase in A relative to gs from greater light exposure and/or greater leaf nitrogen content (Warren et al. 2001; Powers et al. 2010). However, for *Pinus nigra, Pinus halepensis*, and *Pseudotusga menziesii* growing in mesic environments, tree ring -13C did not change significantly with thinning while growth increased, implying that both A and gs increased in response to increased canopy light exposure and water resources.