Acta Silvae et Ligni 111 (2016), 27-33 27 Izvirni znanstveni članek / Original scientific paper ABSENCE OF JUVENILE EFFECTS CONFIRMED IN STABLE CARBON AND OXYGEN ISOTOPES OF EUROPEAN LARCH TREES IZOSTANEK JUVENILNEGA EFEKTA POTRJEN V STABILNIH IZOTOPIH OGLJIKA IN KISIKA V BRANIKAH EVROPSKEGA MACESNA Eleanor KILROY1, Danny McCARROLL1*, Giles HF YOUNG1, Neil J LOADER1, Roderick J BALE2 (1) Department of Geography, Swansea University, Singleton Park, SA2 7RR, �ales UK (2) Department of Archaeology, History and Anthropology, University of �ales Trinity St David, Lampeter, Ceredigion SA48 7ED, �ales UK * Corresponding author ABSTRACT �e report carbon and oxygen isotope ratios measured from the pith at breast height (ca. 1.2m) of three European larch (Larix decidua Mill.) trees growing in a mixed wood in �est �ales, UK. The non-climatic rising trend of carbon isotope ratios reported for other species during early (juvenile) growth is not present and neither isotope shows significant trends as the tree matures. Results from the first ten rings are not significantly different from the next two sets of ten rings. Absence of a juvenile effect in carbon isotopes of European larch has been reported from trees growing in a low density stand in France and attributed to an absence of shading and no use of respired carbon dioxide. This site, in contrast, is densely wooded and the dominant oaks predate the larch trees, suggesting that juvenile effects, when present, may instead be caused by changes in hydraulic conduc- tivity. More research is needed on juvenile effects as the current practice of avoiding juvenile wood is restricting the potential of stable isotope analysis of tree rings for dendroclimatology and plant physiology. Key words: stable isotopes, Larix decidua, �ales, non-climatic trends IZVLEČEK Članek obravnava razmerja ogljikovih in kisikovih izotopov v branikah blizu stržena na prsni višini (cca 1,2 m) treh evropskih macesnov (Larix decidua Mill.), rastočih v mešanem gozdu s predraslimi hrasti in nasajenimi evropskimi macesni v zahodnem �alesu, Velika Britanija. Neklimatskega naraščajočega trenda v razmerjih ogljikovih izotopov, ki ga je sicer opaziti pri drugih vrstah v letih juvenilne rasti, ni, in tudi razmerje stabilnih izotopov ne kaže pomembnih trendov med odraščanjem drevesa. Rezultati iz prvih desetih branik ob strženu se bistveno ne razlikujejo od naslednjih dveh nizov desetih branik. O izostanku juvenilnega efekta v ogljikovih izotopih evropskega macesna so že poročali v zvezi z macesni, rastočimi v nesklenjenih sestojih v Franciji in to pripisali nezastrtosti krošenj in posledične neuporabe ogljikovega dioksida, ki ga pri dihanju oddajajo dreve- sa. Analizirana drevesa v zahodnem �alesu so rasla v nasadu s predraslimi hrasti, ki so bili starejši od podraslih macesnov. Macesni, kot svetloljubne drevesne vrste, so morali tekmovati za prostor in svetlobo, zato domnevamo, da je morebiten pojav juvenilnega efekta pri stabilnih izotopih prej posledica sprememb v hidravlični prevodnosti lesa, kot pa česa drugega. Ker današnja praksa izogibanja juvenilnega lesa omejuje potencial stabilnih izotopov drevesnih branik za dendroklimatološke in fiziološke raziskave, bi bile potrebnih dodatne raziskave o učinkih juvenilne rasti na pojav juvenilnega efekta v meritvah sta- bilnih izotopov. Ključne besede: stabilni izotopi, Larix Decidua, �ales, neklimatski trend GDK 811.4:174.7Larix decidua Mill.(045)=111 Prispelo / Received: 21. 7. 2016 DOI 10.20315/ASetL.111.3 Sprejeto / Accepted: 18. 8. 2016 1 INTRODUCTION 1 UVOD European larch (Larix decidua Mill.) is a long-lived and widespread deciduous conifer (Gower and Rich- ards, 1990) which has been used for centuries as a building material (Daux et al., 2011). Its natural distri- bution is fragmented, covering >500,000 Ha, predomi- nantly in central Europe, including Slovenia. In the Alps it is considered one of the most climate-sensitive species (Carrer and Urbinati, 2006). There have been various studies using tree-ring parameters of Euro- pean larch trees, investigating phenomena such as snow avalanches (Stoffel et al., 2006), fires and grazing (Genries et al., 2009) and growing season timing and duration (Moser et al., 2009). Long chronologies have been produced using living trees and wood preserved in buildings, peat bogs and lakes (e.g. Büntgen et al., 2005, 2006; Corona et al., 2010, 2011; Esper et al., 2007; Neuenschwander 2009; Nicolussi et al., 2009). Physical properties of larch tree-rings (width and den- 28 Kilroy E., McCarroll D., HF Young G., Loader N. J, Bale R. J: Absence of juvenile effects confirmed in stable carbon ... sity) often show a strong relationships with summer temperature (e.g. Büntgen et al., 2005, 2006 and 2008; Carrer and Urbinati, 2004; Frank and Esper, 2005; Vi- tas and Zeimavicius, 2010). The few studies that have used stable isotopes of larch trees report strong rela- tionships with various climatic factors (e.g. Kress et al., 2009; Johnson et al., 2010; Daux et al., 2011; Hafner et al., 2011, 2014). Bud-moth outbreaks do not seem to affect the isotopic composition of the tree-rings (Kress et al., 2009; Daux et al., 2011). There is, therefore, con- siderable potential for using long stable isotope chro- nologies from European larch to reconstruct past cli- mate, including summer sunshine (Hafner et al., 2014). A significant problem with building long isotope chronologies, particularly those including building timbers, is the perceived need to avoid rings formed when the trees were in their juvenile phase. Freyer (1979a, b) was one of the first to notice a juvenile effect in stable isotopes, finding an increase of between 1.5 and 2‰ in δ¹³C values during the early years of pine growth. An age-related juvenile trend in δ¹³C values has since been found in many trees, including pine (Pinus) (Li et al., 2005; Loader et al., 2007; Gagen et al., 2008), beech (Fagus)(Duquesnay et al., 1998) oak (Quercus) (Raffalli-Delerce et al., 2004; Labuhn et al., 2014) and spruce (Picea)(Treydte et al., 2001). The juvenile effect is generally reported as an age-related increase of cel- lulose δ¹³C of the inner rings lasting for the first few decades (e.g. Freyer, 1979a; Li et al., 2005), although longer age-related trends have been reported (Saurer et al., 2004). The most common way of dealing with the juvenile effect, without resorting to de-trending, which can remove climatic information, is to avoid the juve- nile portion of each tree. Hafner et al. (2014), for ex- ample, avoided the first 50 rings of the larch trees used to reconstruct summer sunshine in the eastern Euro- pean Alps using stable carbon isotopes. Procedures for measuring large numbers of stable oxygen isotopes in tree rings, or for measuring carbon and oxygen simul- taneously (Young et al., 2011b, �oodley et al., 2011), were developed after the practice of avoiding juvenile wood, so there is less information available for a ju- venile effect in oxygen isotopes (Treydte et al., 2006, Labuhn et al., 2014, Young et al., 2011a). Reasons for the juvenile effect are not well under- stood. One hypothesis is that young trees, growing close to the canopy floor, use CO₂ which has been re- spired by the soil and therefore is already depleted in ¹³C, leading to relatively negative δ¹³C values which increase as trees gain height and use air which has not been recycled (Francey and Farquhar, 1982; Freyer 1979a,b; Schleser and Jayasekera, 1985). Alternatively the low δ¹³C may reflect low photosynthetic rate due to restricted light availability close to the forest floor (Heaton, 1999). Bonafini et al. (2013), for example, considered a shaded environment to be an important factor that yields low δ¹³C values. Depleted δ¹³C val- ues in the juvenile period have also been attributed to changes in hydraulic conductivity as trees age and grow taller (McDowell et al., 2002). The hydraulic con- ductance from soil to leaf decreases as branch/trunk length increases leading to a corresponding decrease in stomatal conductance. As the tree grows and sto- matal conductance reduces over time, ¹³C assimilation will increase meaning wood cellulose will become in- creasingly enriched in ¹³C. In theory, this increase in δ¹³C should continue until tree height growth ceases, however, there is an offset associated with increased leaf area which enables greater net conductance, meaning δ¹³C does not continually rise after a certain rate of transpiration (Monserud and Marshall, 2001). Despite a large volume of evidence for a juvenile effect in carbon isotope ratios across a range of differ- ent tree types, a study by Daux et al. (2011) found no such juvenile effect in European larch trees from the French Alps. They attribute this unexpected result to the low forest stand density at the site that was sam- pled, meaning there would be no use of recycled CO₂ or reduction of photosynthesis due to reduced light in the lower levels of the canopy (Heaton, 1999). In this paper we examine the juvenile rings of three individu- al European larch trees growing under very different conditions to those sampled by Daux et al. (2011), to test whether the unexpected absence of a juvenile ef- fect can be replicated. 2 MATERIAL AND METHODS 2 MATERIAL IN METODE The sampling site is an 8 ha semi-natural, broad- leaved woodland at Allt Lan-las in the grounds of the National Trust owned Llanerchaeron Estate in Ceredi- gion, �est �ales, UK, where Young et al., (2012) found a very strong relationship between latewood cellulose δ¹³C and summer temperature in oaks. The dominant species is Quercus petraea (sessile oak) with Fraxinus excelsior (ash) and Fagus sylvatica (beech). Mature Larix decidua (European larch) are thinly dispersed within the eastern part of the forest. A shrub under- storey consists of Coryllus avellana (hazel), Ilex aquifo- lium (holly), Lonicera periclymenum (honeysuckle) and Rubus fruticosus (bramble). In contrast to the study site of Daux et al. (2011), this site has a high stand density and young trees are subject to shading. Acta Silvae et Ligni 111 (2016), 27-33 29 Increment cores (12mm) were collected from breast height (ca. 1.2m) for five healthy larch trees. Up to three cores were obtained from each tree to identify locally absent rings during cross dating. Samples were air- dried, sanded and the rings measured to an accuracy of 0.01mm using a microscope and a horizontally travel- ling stage linked to a computer (Tyers 2004).There is no master chronology for British larch, but, once locally absent rings had been identified, the trees cross-dat- ed with each other giving Baillie Pilcher t-values >3.5 (Baillie and Pilcher, 1973) and also demonstrated t-val- ues >3.5 against an existing oak master chronology for the site (Bale, 2005). Three of the cores reached very close to the pith, which is evident due to extreme cur- vature of the rings, and these were used to investigate potential juvenile effects. The first rings of trees 1 and 2 were dated as AD 1846 and that of tree 3 as AD1850. The first rings of all three analysed trees are estimated to be between five and ten years from the pith. Each annual growth ring was cut into slivers using a razorblade and binocular microscope. The whole- ring was used, as suggested by Kress et al. (2009) who found a strong relationship between δ¹³C in earlywood and latewood of larch and concluded that separation of early- and latewood is unnecessary. Prior to iso- topic analysis, samples were extracted to α-cellulose (Loader et al., 1997). Between 0.3 and 0.35mg of sam- ple were then weighed into silver capsules and the iso- tope ratios analysed by pyrolysis over glassy carbon at ca 1090°C using a Europa ANCA GSL elemental ana- lyser interfaced to a Europa 20/20 isotope ratio mass spectrometer. Analytical precision (σn-1 n=10) for this on-line approach to dual isotope analysis is typically 0.3‰ (δ18O) and 0.1‰ (δ13C) (McCarroll and Loader, 2004). Stable carbon isotope ratios (δ¹³C) and oxygen isotope ratios (δ18O) are reported as per mille (‰) de- viations from the Vienna Pee Dee Belemnite (VPDB) and Vienna Standard Mean Ocean �ater (VSMO�), respectively (Coplen, 1995). The carbon isotope ra- tios were corrected for changes in the isotopic ratio of atmospheric carbon dioxide (McCarroll and Loader 2004) but not for potential changes in response to the increase in the amount of atmospheric carbon dioxide (McCarroll et al., 2009). 3 RESULTS 3 REZULTATI The carbon isotope ratios obtained from the three trees show no evidence of the expected rise in values over the juvenile period (Figure 1). Taking the first 15, 20 or 30 years, none of the trees show a significant ris- ing trend (Table 1). The same is true for the oxygen iso- Table 1: Results of Pearson’s correlation (r) and Spearman’s rank correlation (ρ) of isotope ratios against ring number for 15, 20 and 30 yearse Preglednica 1: Rezultati Pearsonovega (r) in Spearmanove- ga rang korelacijskega koeficienta (p) izotopskih razmerij pri številu branik za 15, 20 in 30 let Tree Years Carbon / Ogljik Oxygen / Kisik 15 20 30 15 20 30 1 r -0.05 0.14 -0.02 -0.27 -0.12 0.06 ρ 0.01 0.17 -0.04 -0.14 -0.03 -0.04 2 r -0.16 0.14 0.34 0.11 -0.12 -0.42 ρ -0.09 0.25 0.33 0.03 -0.14 -0.38 3 r -0.12 0.36 0.21 0.23 0.32 -0.11 ρ -0.09 0.42 0.25 0.09 0.38 -0.09 Table 2: Results of the Kruskal-�allis test (H) comparing the first, second and third ten years of isotope data, and the Mann-�hitney U-test comparing the first ten years with the next 20 years. Test statistics (H and U) and two-tail probabi- lities are tabulated. Preglednica 2: Rezultati Kruskal-�allisovega testa (H) s primerjavo prvih, drugih in tretjih dekad izotopskih podatk- ov, in Mann-�hitneyev U-test s primerjavo prvih deset let z naslednjimi 20 leti. Tabelarno so urejene testne statistike (H in U) in dvostranske verjetnosti. Tree Carbon / Ogljik Oxygen / Kisik H U H U 1 H = 1.79 p = 0.41 U = 77 p > 0.05 H = 0.11 p = 0.95 U = 93 p > 0.05 2 H = 2.96p = 0.23 U = 68 p > 0.05 H = 3.4 p = 0.18 U = 65 p > 0.05 3 H = 0.80p = 0.67 U = 87 p > 0.05 H = 4.7 p = 0.09 U = 92 p > 0.05 30 Kilroy E., McCarroll D., HF Young G., Loader N. J, Bale R. J: Absence of juvenile effects confirmed in stable carbon ... tope results, with no evidence for a juvenile increase or decline. The Kruskal-�allis H-test, a non-parametric analysis of variance by ranks (McCarroll 2016), dem- onstrates that the first, second and third sets of ten rings do not significantly differ from one another. For both isotope ratios, the related Mann-�hitney U-test shows that in all cases the first ten years are not signifi- cantly different from the next 20 (Table 2). The non- parametric ‘change point statistic’ (Siegel and Castel- lan 1988), which assesses the likelihood that a change occurred in a sequence of observations, suggests that in no case was there a change that exceeded the fluc- tuations that are expected due to chance (p > 0.05). 4 DISCUSSION AND CONCLUSIONS 4 DISKUSIJA IN ZAKLJUČKI The results presented here clearly show that there is no juvenile effect in the carbon or oxygen isotope ratios of European larch trees growing in �est �ales, UK. This agrees with the results reported by Daux et al. (2011), who found no juvenile effect in the carbon iso- tope ratios of European larch trees growing in France. However, Daux et al. (2011) concluded that the absence of a juvenile effect could be explained by the low forest density at their site, meaning that young trees were not using respired carbon dioxide or subject to shading. That explanation cannot be applied to the site in �ales, Fig 1: Carbon isotope ratio (solid line) and oxygen isotope ratio (dashed line) of whole ring α-cellulose for the three individual larch trees arranged by ring number. Curvature of the tree rings for each core and knowledge of the forest stand enable estimation of pith age, such that the first mea- sured rings of all trees are within 5-10 rings of the pith. Slika. 1: Razmerje stabilnega izotopa ogljika (polna črta) in kisika (črtkana črta) α-celuloze iz celotne branike za tri posamezne macesne, urejeno glede na zaporedno številko branike. Število manjkajočih branik do stržena je bilo ocen- jeno na podlagi loka drevesnih branik na izvrtku in pozna- vanju značilnosti priraščanja analiziranega gozdnega ses- toja, tako da so prve izmerjene branike vseh dreves znotraj 5-10 branik od stržena. Acta Silvae et Ligni 111 (2016), 27-33 31 which is dense mixed broadleaf woodland, dominated by mature oak trees that pre-date the establishment of larch, and contemporary beech and ash. These results raise the possibility that the juvenile effect in carbon isotopes reported for other species is not caused by the use of respired carbon dioxide or by shading, but is a response to changes in hydraulic conductivity, as proposed by McDowell et al. (2002). The juvenile effect reported in the carbon isotope ratios from pine trees from the French Alps (Gagen et al., 2006) support this hypothesis. They were growing in very open stands on a windy hillside, so that respired carbon dioxide and shade are unlikely explanations. On the basis of the results reported here, and those reported by Daux et al. (2011), we conclude that Euro- pean larch trees do not necessarily display a juvenile effect in either the carbon or oxygen isotope ratios. The common practice of excluding the inner rings of larch trees from isotope analysis may thus be overly cautious. This means that it may be possible to use longer sequences of rings from living trees to extend palaeoclimate reconstructions and also to make much more efficient use of larch building timbers in produc- ing chronologies that extend beyond the range of most living larch trees. It is not clear why larch behaves so differently to some other tree species, including pines, which show a clear and strong juvenile effect in carbon isotope ra- tios. Unfortunately, there is little data available on ju- venile effects in other tree species, particularly for oxy- gen isotopes, because most researchers now routinely exclude juvenile wood. More work is required on the juvenile effect of other tree species, particularly those used as building timbers and thus available to produce very long isotope chronologies. 5 SUMMARY �e report carbon and oxygen isotope ratios of whole ring α-cellulose from the first 30 rings of three European larch (Larix decidua Mill.) trees growing in a mixed wood in �est �ales, UK. The rising trend of carbon isotope ratios seen in other species, during the early growth years, is not present and neither iso- tope shows significant trends with ring number over the first 15, 20 or 30 years of growth. Non-paramet- ric analysis of variance (Kruskal-�allis H-test) shows that the results from the first, second and third sets of ten rings are not significantly different and the Mann- �hitney U-test shows that the values from the first ten rings are not significantly different to the next 20. The non-parametric change point statistic, which assesses the likelihood that a change occurred in a sequence of observations, suggests that in no case was there a change that exceeded the fluctuations that are expect- ed due to chance. Absence of a juvenile effect in carbon isotopes of European larch has been reported from trees growing in a low density stand in France and attributed to an absence of shading and no use of respired carbon di- oxide. This site in �est �ales is, in contrast, densely wooded and the dominant oaks predate the larch trees, suggesting that juvenile effects, when present, may rather be caused by changes in hydraulic conductivity. More research is needed on juvenile effects as the cur- rent practice of avoiding juvenile wood is restricting the potential of stable isotope analysis of tree rings for dendroclimatology and plant physiology. 5 POVZETEK Avtorji prispevka poročajo o razmerjih stabilnega izotopa ogljika in kisika v a-celulozi celotnih prvih 30 branik treh evropskih macesnov (Larix decidua Mill.), rastočih v dvoslojnem mešanem gozdu hras- ta in macesna v zahodnem �alesu, Velika Britanija. Naraščajočega trenda razmerij ogljikovih izotopov, ki ga je bilo opaziti pri drugih vrstah v letih zgodnje rasti, ni, in tudi razmerje stabilnih izotopov ne kaže pomembnih trendov v branikah v prvih 15, 20 ali 30 letih rasti. Neparametrična analiza variance (Kruskal- �allisov H-test) kaže, da se rezultati prvega, drugega in tretjega testa desetih branik bistveno ne razlikujejo, pa tudi Mann-�hitneyev U-test kaže, da se vrednosti iz prvih desetih branik bistveno ne razlikujejo od nasled- njih 20. Neparametrična statistika analize sprememb, ki ocenjuje verjetnost, da se je sprememba zgodila med zaporedjem opazovanj, nakazuje, da v nobenem prim- eru ni nastala sprememba, ki bi presegla naključno pričakovana nihanja. O izostanku juvenilnega efekta v stabilnih izotopih ogljika v branikah evropskega macesna so poročali v zvezi z drevesi, rastočimi v nesklenjenih sestojih nizke gostote v Franciji in to to pripisali nezastrtosti krošenj in posledične neuporabe ogljikovega dioksida, ki ga pri dihanju oddajajo drevesa. Analizirana drevesa v za- hodnem �alesu so rasla v nasadu s predraslimi hrasti, ki so bili starejši od podraslih macesnov. Macesni, kot svetloljubne drevesne vrste, so morali tekmovati za prostor in svetlobo, zato domnevamo, da je morebiten pojav juvenilnega efekta pri stabilnih izotopih prej posledica sprememb v hidravlični prevodnosti lesa, kot pa česa drugega. Ker današnja praksa izogibanja juvenilnega lesa omejuje potencial stabilnih izotopov drevesnih branik za dendroklimatološke in fiziološke raziskave, bi bile potrebnih dodatne raziskave o 32 Kilroy E., McCarroll D., HF Young G., Loader N. J, Bale R. J: Absence of juvenile effects confirmed in stable carbon ... učinkih juvenilne rasti na pojav juvenilnega efekta v meritvah stabilnih izotopov. 6 ACKNOWLEDGEMENTS 6 ZAHVALA The authors thank the Leverhulme Trust RPG. 7 REFERENCES 7 VIRI Bale R.J. (2005). A 223-year (AD 1779–2001) modern oak tree ring chronology from Allt Lanlas Llanerchaeron, Ceredigion. Swansea Geographer 40: 45–55. Baillie, M. and Pilcher, J. (1973). A simple cross-dating program for tree-ring research. Tree Ring Bulletin. 33: 7-14. Bonafini, M., Pellegrini, M., Ditchfield, D. and Pollard, A.M. (2013). Investigation of the ‘canopy effect’ in the isotope ecology of tem- perate woodlands. Journal of Archaeological Science. 40: 3926- 3935. 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