Plant Wax N-Alkane Biomarkers in the Tropical Andes (Ecuador)

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Plant wax n-alkane biomarkers in the tropical Andes (Ecuador)

Teunissen van Manen, M.L.

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Citation for published version (APA): Teunissen van Manen, M. L. (2020). Plant wax n-alkane biomarkers in the tropical Andes (Ecuador).

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Download date:26 Sep 2021 “Leaf wax n-alkane patterns of six tropical 2 tree species show species-speci c environmental response” [published, 2019].

Ecology and Evolution (open access) | https://doi.org/10.1002/ece3.5458

Milan L. Teunissen van Manen1, Boris Jansen1, Francisco Cuesta2, Susana León-Yánez3, William D. Gosling1.

1 Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, The Netherlands. 2 Grupo de Investigación en Biodiversidad, Medio Ambiente y Salud (BIOMAS), Universidad de Las Américas (UDLA), Quito, Ecuador. 3 Escuela de las Ciencias Biológicas, Pontifi cia Universidad de Católica de Ecuador (PUCE), Quito, Ecuador AbsTrAcT There is a lack of knowledge on leaf wax n-alkane composition changes in response to the local environment. This knowledge gap inhibits the interpretation of plant responses to the environment at higher taxonomic levels and, by extension, inhibits the applicability of n-alkane patterns as a proxy for past environments. Here we studied the n-alkane patterns of five Miconia species and one Guarea species, in the Ecuadorian Andes (653 - 3507 m a.s.l.). Specifically, we tested for species-

specific responses in the average chain length (ACL), the C31/

(C31+C29) ratio (ratio) and individual odd n-alkane chain lengths across an altitudinally driven environmental gradient (mean annual temperature, mean annual relative air humidity and mean annual precipitation). We found significant correlations between the environmental gradients and species-specific ACL and ratio, but with varying magnitude and direction. Looking closer at the n-alkanes we found that the patterns are species-specific at the individual chain length level, which could explain the high variance in metrics like ACL and ratio. Although we find species-specific sensitivity and responses in leaf n-alkanes, we also find a general decrease in “shorter”

(C31) chain lengths with the environmental gradients, suggesting n-alkanes are useful for reconstructing past environments. 1 | Introduction

Fully understanding the way in which plant leaf waxes reflect their environment could give an insight into plant resilience to future climate change (Guo et al., 2015) and, is instru- mental for reconstructing past environmental conditions (Jansen and Wiesenberg, 2017).

The n-alkane fraction of the leaf wax, typically in the range of C20 and C37 (Eglinton and Hamilton, 1967), in particular has been suggested as a proxy for past environments due to their resistance to degradation (Jansen and Wiesenberg, 2017). Studies measuring n-alkane patterns across plant communities, functional groups, genera and species have shown that the n-alkane pattern seems to be influenced by several environmental factors, notably temperature and precipitation (Bush and McInerney, 2015, 2013; Feakins et al., 2016; Hoffmann et al., 2013; Sachse et al., 2006; Tipple and Pagani, 2013). However, it has been hard to identify a general trend in the n-alkane signal in response to the environmental factors. There have been numerous works exploring n-alkane patterns at the plant community or functional group level (for example, Bush and McInerney, 2015 and Feakins et al., 2016), but there are fewer studies at lower taxonomic levels (Hoffmann et al., 2013; Maffei et al., 1993; Sachse et al., 2006; Tipple and Pagani, 2013). Within the studies done at lower taxonomic levels the results are mixed; one study finds a genus specific response to hydrological factors but not to temperature (Hoffmann et al., 2013) and two other studies find species- specific response to increasing temperature (Sachse et al., 2006 and Tipple and Pagani, 2013). With limited knowledge on leaf wax n-alkanes at lower taxonomic levels, it remains unclear how prevalent these species-specific responses are and what characterizes them. In this study we aim to further the knowledge of species-specific responses in leaf wax n-alkane patterns. Specifically, we ask whether we observe species-specific responses in the n-alkane patterns from six tropical tree species, sampled along an altitudinally driven, environmental gradient in Ecuador.

1.1 | study region and species selection The area of study is the north western flank of the Andes (Pichincha province, Ecuador) (Fig. 1). This study took advantage of the Pichincha long-term forest development and carbon monitoring transect that was established in 2015 by the research non-profit Consorcio para

el Desarrollo Sostenible de la Ecorregión Andina (CONDESAN) (http://condesan-ecoandes. Production org/). CONDESAN catalogued the tree community composition and recorded environmen- 29 tal data at each site along the transect (Pinto et al., 2018). The transect captures environmental gradients in mean annual temperature (7.2- 21.6 °C/year), relative air humidity (96.1 - 99.8%) and annual precipitation (1580 - 2448 mm/year) (Karger et al., 2017). To investigate species-specific responses in leaf wax n-alkanes we targeted genera and species that were abundant and had a wide distribution along the Pichincha transect. We selected the most abundant and widely distributed species in the area; five species of Mico- nia and one species of Guarea (Appendix 1). The genus Miconia is one of the most diverse, widely distributed, genera found in the Ecuadorian Andes (Jørgensen and León-Yánez, 1999) and spans the entire transect (2854 m, 100% of transect length). Guarea kunthiana is an especially ubiquitous species in northern Ecuador (Jørgensen and León-Yánez, 1999) and spans most of the altitudinal transect (1559 m, 55% of transect length).

Figure 1 | Map of the study area. Dots and numbers refer to sites in Appendix 1, ordered by altitude. The green shading indicates altitude (m above sea level). The black delineates the Pichincha project study area. Blue lines represent rivers, land codes as follows: COL = Colombia, ECU = Ecuador, PER = Perú.

2 | Materials & methods

2.1 | sampling and environmental data collection

Chapter 2 Chapter A total of 87 samples (one sample per individual) were collected along the Pichincha transect between 500 – 2500 m a.s.l, from 14 permanent vegetation study sites. We aimed to 30 sample at least three individuals per species per site, but this was not always possible as species were not always present, or abundant, at every site (Appendix 1). Each sample consists of 20 - 25 leaf(lets) collected from the canopy. We selected undam- aged leaf(lets) and a wide range of leaf sizes and ages (Koch and Ensikat, 2008). Samples were collected using gloves and were wrapped in aluminum foil in the field, making sure no contact was made with the skin to avoid lipid contamination. Samples were bagged and placed in a cold storage (5 oC) until further analysis at the University of Amsterdam. Temperature and relative air humidity were measured at each site using Onsetcomp HOBO U23 Pro v2 Temperature/Relative Humidity data loggers at an hourly basis from 2016-2018. We calculated mean annual temperature (hereafter ‘temperature’) and mean relative air humidity (hereafter ‘humidity’) per site based on the two-year dataset. Annual precipitation (Bioclim 12, hereafter ‘precipitation’) per site was obtained from the CHELSA dataset at 30S resolution (1km) (Karger et al., 2017).

2.2 | leaf wax alkane extraction & analysis Lipid extraction was done following the protocol described in Jansen et al. (2013) with minor modifications. Leaf samples were freeze dried and milled to powder prior to analysis, and 0.1 g of the milled sample was extracted by Accelerated Solvent Extraction (ASE) using a Dionex 200 ASE extractor. ASE was done at 75 °C and 1500 pKa employing a heating phase of 5 min and a static extraction time of 20 min. Dichloromethane/methanol (DCM/MeOH) (93:7 v/v) was used as the extractant. We added 60 µg of a mixture of Androstane, Androstan and Euric acid (0.3 µg/µL per compound) to each extraction to provide an internal standard. Fractionation was done using a 10 mL solid phase column with approximately 1.5 g of silica gel (5% deactivated H2O) previously conditioned with acetone, DCM and hexane. The n-alkane fraction was obtained by eluting the column with hexane. The n-alkane fraction was analysed by injecting 1 µL of the sample into a GC-MS with quadruplet MS detection in full scan mode (50-650 amu), conform the following protocol: the sample was injected in a DB5 column (30 m) under constant flow of gas helium 0.8 mL/min. First ramp 60 °C/ min to 80 °C (hold 2 min); second ramp 20 °C/min to 130 °C; 4 °C/min to 350 °C (hold 10 min). n-Alkanes were identified and quantified by comparing with a known mixture of n- alkanes in the range C25-C33, and the internal standard employing the Thermo Xcalibur® software. We identifiedn- alkanes between C23 and C33, all of which are known to contribute to the higher plant leaf wax n-alkane fraction (Eglinton and Hamilton, 1967). The resulting n-alkane dataset was standardized by sample weight (grams of dry leaf sample used for extraction) (hereafter, n-alkane data). Three samples from different species and altitudes were selected for replicate analysis

(two samples run in triplicate and one sample in duplicate) and three other samples were Production already split in the field. In total, 14 replicate measurements were done simultaneous with 31 the rest of the analysis in order to check measurement variability. sample into a GC-MS with quadruplet MS detection in full scan mode (50-650 amu), conform the following protocol: the sample was injected in a DB5 column (30 m) under constant flow of gas helium 0.8 mL/min. First ramp 60 °C/min to 80 °C (hold 2 min); second ramp 20 °C/min to 130 °C; 4 °C/min to 350 °C (hold 10 min). n-Alkanes were identified and quantified by comparing with a known mixture of n-alkanes in the range C25-C33, and the internal standard employing the Thermo Xcalibur® software. We identified n-alkanes range C25-C33, and the internal standard employing the Thermo Xcalibur® software. We identified n-alkanes between C23 and C33, all of which are known to contribute to the higher plant leaf wax n-alkane fraction between C23 and C33, all of which are known to contribute to the higher plant leaf wax n-alkane fraction (Eglinton and Hamilton, 1967). The resulting n-alkane dataset was standardized by sample weight (grams of dry leaf sample used for extraction) (hereafter, n-alkane data).

Three samples from different species and altitudes were selected for replicate analysis (two samples run in triplicate and one sample in duplicate) and three other samples were already split in the field. In total, 14 replicate measurements were done simultaneous with the rest of the analysis in order to check measurement variability.

2.3 | Overall n-alkane patterns, metrics and environmental correlations 2.3 || Overall overall n n-alkane-alkane patterns, patterns, metrics metrics and andenvironmental environmental correlatio ns To obtaincorrelations species overall n-alkane patterns along the transect, we standardized n-alkane data to total n-alkane concentrationTo obtain species (hereafter, overall relative n-alkane abundances) patterns andalong then the calculated transect, species we standardized means and standardn-alkane deviation fromdata tothe total mean. n- alkane concentration (hereafter, relative abundances) and then calculated species means and standard deviation from the mean.

In order to study shifts in the n-alkane pattern along the environmental gradient we cor- In order to study shifts in the n-alkane pattern along the environmental gradient we correlated two related two commonly used metrics (weighted average chain length (ACL) and the C31/C29 commonly used metrics (weighted average chain length (ACL) and the C31/C29 ratio) and the relative commonly used metrics (weighted average chain length (ACL) and the C31/C29 ratio) and the relative ratio) and the relative abundances to temperature, humidity and precipitation. abundances to temperature, humidity and precipitation. The ACL was calculated following Bush & McInerney (2013): The ACL was was calculatedcalculated following following Bush Bush & & McInerney McInerney (2013 (2013):):

= ( × )/( ) (1) (1) 21−33 𝑛𝑛 𝑛𝑛 𝐴𝐴𝐴𝐴𝐴𝐴 21−33 Σ 𝐶𝐶𝑛𝑛 𝑛𝑛 Σ𝐶𝐶𝑛𝑛 Where Cn isis thethe concentration of of n -n-alkanealkane per per gram gram of driedof dried sample sample and andn is then is number the number of carbon of atoms Where Cn is the concentration of n-alkane per gram of dried sample and n is the number of carbon atoms carbonof the measured atoms of n -thealkane measured (C21-C33 ).n- alkane (C21-C33). of the measured n-alkane (C21-C33). The ratio between C31 and C29 n-alkanes was calculated following Bush & McInerney (2013): The ratio between C31 and C29 n-alkanes was calculated following Bush & McInerney (2013): The ratio between C31 and C29 n-alkanes was calculated following Bush & McInerney (2013):

ratio = /( + ) (2) ratio = /( + ) (2) (2) 31 31 29 𝐶𝐶31 𝐶𝐶31 𝐶𝐶29 Where C31 and C29 stand for the concentrations of the C31 and C29 n-alkanes, respectively. Because the ratio Where CC3131 andand C C2929 stand stand for for the the concentrations concentrations of the of C the31 and C31 C and29 n- alkanes,C29 n-alkanes, respectively. respectively. Because the ratio asBecause a fraction the ofratio one as is a easy fraction to interpret of one and is easy it stan todardizes interpret variances, and it standardizes we chose it over variances, the simple we fraction thatchose is italso over reported the simple in literature fraction (Jansenthat is alsoand reportedNierop, 2009) in literature. Other (Jansenratios found and Nierop,in literature 2009). where also Other ratios found in literature where also calculated, but those ratios behaved similar to calculated, but those ratios behaved similar to the ratio presented here (Eq. 2) (Appendix 2) and thus are the ratio presented here (Eq. 2) (Appendix 2) and thus are not included in further analysis. Two ratio values of M. theaezans and M. clathrantha, were abnormally small compared to similar samples at the same site. We therefore removed these from all analysis (Fig. 3). CHAPTER TWO | 27 We correlated the ACL, the ratio and the relative abundances of the species sampled at three or more sites to temperature, humidity and precipitation using Spearman’s rank order correlation (i.e. ‘species response’). We also correlated the ACL, the ratio and relative abundances of the entire dataset (of all six species) to temperature, humidity and precipita-

Chapter 2 Chapter tion using Spearman’s rank order correlation (i.e. ‘site total response’). We chose to only show the correlations with the relative abundances of the odd chain 32 lengths because these dominate the higher plant n-alkane signal (Eglinton and Hamilton, 1967). We adopted a significance level of p-value < 0.01 because the bulk of our analysis are correlations (e.g. following Tipple and Pagani, 2013). Additionally, it should be noted that the environmental gradients are sampled across an elevational gradient, and therefore the environmental variables are not independent (Appendix 3). All statistical analyses were per- formed in R studio (R Core Team, 2017) using the base statistics functions and the tidyverse package (Wickham, 2017).

3 | rEsults

3.1 | overall n-alkane patterns Replicate measurements showed high coefficients of variation for absolute n-alkane concentration (most samples >10%, Appendix 4). However, the metrics had very low coefficients of variation (ACL below 0.5%, ratio below 9%, Appendix 4), giving us confidence that the relative measurements and metrics are accurate. The average species n-alkane pattern is typical of the leaf wax fraction of higher plants (Fig. 2). All species have an odd-over-even preference (OEP), typical of plants (Eglinton and

Hamilton, 1967). Only the C23 chain length deviated from the OEP in three of the six species, namely G. kunthiana, M. theaezans and M. bracteolata (Fig. 2). Production

Figure 2 | Species average n-alkane distributions, showing average relative abundances (average % of total concentration) across entire transect. Lines represent standard deviation from average, numbers in parentheses represent number of measurements (excluding outliers). 3.2 | Average chain length (ACL) The ACL of one species and the site total significantly correlated with the environmental variables (Fig. 3a-c,Fig. 4a, Appendix 5). Specifically, humidity and precipitation correlated positively with the ACL of G. kunthiana (rs = 0.5, p = 0.008 and rs = 0.5, p = 0.007, respectively). M. corymbiformis ACL was not significant for any of the environmental variables, but this is most likely due to the small sample size and short gradient over which it was sampled (Appendix 1). Site total ACL correlated positively with all environmental variables

(Fig. 3a-c, Fig. 4a, Appendix 5; temperature: rs = 0.66, p < 0.001, humidity: rs = 0.44, p < 0.001,

and precipitation: rs = 0.57, p < 0.001).

Chapter 2 Chapter Figure 3 | Average chain length (ACL23-33) and the ratio (C31/(C31+C29)) distribution along three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual 34 precipitation (AP). Spearman’s rho correlation coefficients and significance are in Appendix 5. Colors and symbols correspond to species. Open symbols indicate outliers that were excluded from all analysis. Grey symbols (×,+) indicate species sampled at less than three sites. Site total includes data points of all species, including grey symbols. Numbers in parentheses indicate number of data points per correlation. 3.3 | ratio We observed four significant species-specific correlations with the environmental gradient (Fig. 3d-f, Fig. 4b, Appendix 5). Temperature positively correlated with the ratio of M. clathrantha and M. theaezans (rs = 0.56, p = 0.009, rs = 0.58, p = 0.003, respectively. Fig. 3d, Fig. 4b, Appendix 5). G. kunthiana correlated significantly with humidity and precipitation

(Fig. 3e-f, Table 1; humidity: rs = 0.55, p = 0.003, precipitation: rs= 0.57, p =0.002). The ratio of M. corymbiformis was not significant for any of the environmental variables; likely due to the limited sampling (Appendix 1). The site total ratio correlated positively with all environmental variables (Fig. 3d-f, Fig. 4b, Appendix 5; temperature: rs = 0.61, p < 0.001, humidity: rs

= 0.39, p <0.001 and precipitation: rs = 0.53, p < 0.001).

Figure 4 | Spearman’s rho correlation coefficients of average chain length (ACL23-33) and ratio (C31/ (C31+C29)) from individual species and site average against three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Colors correspond to species (blue and greens) and site total (grey). Numbers in parentheses indicate number of data points per correlation. Significance legend as follows: * p < 0.01, ** p <0.005, *** p < 0.001. Production

35 3.4 | odd chain species-specific n-alkane percentages Correlations between the environmental gradients and the percentages of odd chain species-specific n-alkanes varied between chain lengths and species (Fig. 5, Appendix 6).

Temperature and relative abundances of C33 significantly correlated for all species, except M. corymbiformis (due to limited sampling) (Fig. 5a, Appendix 6). Furthermore, the

C31 fraction of M. clathrantha correlated with temperature (rs = 0.63, p = 0.002 Fig. 5b). The Chapter 2 Chapter

Figure 5 | Spearman’s rho correlation coefficients of odd chain length species-specific n-alkanes from individual species and site average against three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Colors correspond to species (blue and greens) and site total (grey). Numbers in parentheses indicate number of data points per correlation. Significance legend as follows: * p < 0.01, ** p <0.005, *** p < 0.001. C29 fraction of M. theaezans correlated negatively with temperature (rs = -0.82, p < 0.001, Fig.

5c, Appendix 6). The C27 fraction of M. clathrantha and M. theaezans correlated negatively with temperature (rs = -0.61, p = 0.003 and rs = 0.51, p = 0.010, respectively. Fig. 5d, Appendix

6). Additionally, the C25 fraction of M. theaezans significantly correlated with temperature rs = 0.76, p < 0.001, Fig. 5e, Appendix 6). Humidity correlated significantly with the relative abundances of G. kunthiana. Spe- cifically, with increasing humidity the fraction of C33 increased, while the fraction of C29 decreased (rs = 0.58, p = 0.002 and rs = -0.54, p = 0.004, respectively. Fig. 5a,c, Appendix

6). Additionally, humidity and the C27 fraction of M. clathrantha also correlated negatively

(rs = -0.77, p < 0.001). Contrastingly, the C23 fraction of M. theaezans correlated positively with humidity (rs = 0.55, p = 0.004, Fig. 5f, Appendix 6). Precipitation significantly correlated with the fraction of C33 and C29 in G. kunthiana (rs = 0.58, p = 0.002 and rs = -0.52, p = 0.001, respectively. Fig. 5a,c, Appendix 6). At the site level, we observed that all environmental variables correlated positively with

“longer” chain lengths (C33 and C31) and negatively with “shorter” chain lengths (C29, C27, C25,

C23) (Fig. 5a-d, Appendix 6).

4 | dIscussion

4.1 | species-specific responses to environmental gradients We find that leaf wax n-alkane distributions from the taxa studied here vary with the environmental gradient (Fig. 2), and do so in species-specific ways (Fig. 3, Fig. 4, Fig. 5).

4.1.1 | Temperature Our results suggest that the n-alkane fraction of M. clathrantha and M. theaezans are sensitive to temperature. However, the metrics differ in reflecting the shift in n-alkane distributions (Fig. 3, Fig. 4). The underlying shifts in individual odd chain lengths indicate that the two Miconia species respond in distinct ways to the temperature gradient. In fact, the two species only overlap in the C33 and C27 chain lengths shifts with temperature (Fig.5). Shifts in the relative abundance of individual chain lengths has been observed before (Sachse

et al., 2006), it is not known why species-specific n-alkane shifts would be species-specific Production at the individual chain length level. However, one reason for the diversity of distributions 37 and trends could be because the Miconia genus is genetically diverse (Goldenberg et al., 2013, 2008). Despite these species-specific differences, the results also suggest that both Miconia species increase the relative abundance of “longer” chain lengths with increasing temperature, as showcased by the ratio especially (Fig. 3, Fig. 5 b,c). This indicates that the relationship between increased biosynthesis of “longer” chain species-specific n-alkanes and temperature (Koch and Ensikat, 2008) is also at play here. 4.1.2 | Humidity and precipitation Our results of both the ACL and ratio metrics suggest that the leaf wax n-alkane fraction of G. kunthiana is sensitive to the hydrological factors (humidity and precipitation) (Fig. 3, Fig. 4, Appendix 5). Closer inspection of the n-alkane composition shows that this is due to a shift in relative abundance towards “longer” chain lengths; both “shorter” n-alkanes decrease and “longer” n-alkanes increase (Fig. 5a,c). The shifts in individual chain lengths with hydrological factors are pronounced in Guarea, but a less pronounced result is also seen in the Miconia species (Fig. 5d,f). This suggests that not all species are equally sensitive or respond similarly. This falls in line with work done by Hoffmann et al. 2013, who find opposite correlations between hydrological factors (including humidity and precipitation) and the ACL of two genera in Australia. The reason for the difference in sensitivity is unknown and understudied to this point. However, Hoffmann et al. 2013 suggest leaf morphological and evolutionary differences, which could also be applicable to the two genera studied here – Guarea has large compound leaves, composed of paripinnate leaflets, whereas Miconia has simple leaves (Pinto et al., 2018).

4.2 | site total responses Unlike the species metrics, all site total species-specific n-alkane metrics correlated positively with the environmental variables (Fig. 3, 4, Appendix 5). The results in our study, the uniformity in the site total species-specific n-alkane shifts

was due to the positive correlation of ≥C31 (“longer” chain lengths, Fig. 5 a,b) and negative

correlation of ≤C29 (“shorter” chain lengths, Fig. 5 c,d) with the environmental (temperature in particular). This suggests that, generally, the relative contribution of “longer” n-alkanes increased with temperature, which falls in line with findings on plant communities and soils (Bush and McInerney, 2015; Feakins et al., 2016; Tipple and Pagani, 2013). It is, however, important to that our data should not be viewed as a “community average” because it is based on a limited taxonomic sample and, as such, does not reflect the overall composition of the vegetation in our plots. Further research is required to scale up our findings to the community level and establish if the pattern is robust at this level. Specifically, by exploring the species-specific n-alkane pattern at the individual chain length level of plant communities and in soils. Chapter 2 Chapter 38 5 | conclusion

Our results indicate that the leaf wax species-specific n-alkane patterns in our study are determined by: 1) species-specific sensitivity to particular environmental gradients, and 2) species-specific responses at the individual chain length level. We also note these species- specific n-alkane shifts are part of complex multivariate patterns that likely introduce variance into summarizing metrics, such as ACL or a ratio of individual chain lengths. Our results suggest that there is no ‘one size fits all’ when it comes to characterizing leaf wax species-specific n-alkane responses. Alternative approaches, such as, looking at the relative abundances of individual chain lengths (for example, Fig. 5) or multivariate analysis (for example, Maffei et al., 1993) might be more informative to characterize the n-alkane fraction. Despite the evidence for species-specific responses at the chain length level, and the variance this likely introduces in summarizing metrics, we observe a general tendency of increasing relative abundances of “longer” n-alkanes with the environmental gradients. This aligns with what has been found in plant communities and soils (Bush and McInerney, 2015; Feakins et al., 2016; Tipple and Pagani, 2013), suggesting the metrics and n-alkane can be useful as a proxy for past environments. Further research should focus on whether the results found here are applicable elsewhere, and on deconstructing typical species-specific n-alkane metrics (such as ACL) in order to uncover the underlying patterns and understanding variance. Additionally, it should be further investigated if species-specific n-alkane patterns from leaves reflect those extracted from soils. The degradation processes from leaf to sedimentary record are understudied, having full understanding of this process is key to the application of species- specific n-alkanes as a proxy for past environments.

6 | Acknowledgements

Many thanks to Laurens Broeze, Esteban Pinto, Andrés Diaz and Gabriela López for their dedication and support in the field and lab in Ecuador. Financial support came the Institute for Biodiversity and Ecosystem Dynamics (University of Amsterdam). Additional funding came from the EcoAndes Project conducted by Consortium for the Sustainable Develop- ment of the Andean Ecoregion (CONDESAN) and United Nations Environment Programme (UNEP), funded by the Global Environmental Fund (GEF), cooperation agreement N° 4750.

7 | dATA Accessibility statement Production

39 Environmental and n-alkane data presented in this paper is accessible at Figshare DOI: 10.21942/uva.7610894 8 | conflicts of interest

None declared.

9 | Author contributions

MLTvM, WDG and BJ conceived and designed the study, MLTvM, FCC and SLY facilitated and conducted fieldwork. MLTvM did the lab analysis and analyzed the data. MLTvM, WDG and BJ wrote the manuscript. All authors contributed to the drafts of the manuscript and its final approval.

10 | rEferences

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Chapter 2 Chapter Hoffmann, B., Kahmen, A., Cernusak, L.A., Arndt, S.K., Sachse, D., 2013. Abundance and distribution of leaf wax n-alkanes in leaves of acacia and eucalyptus trees along a strong humidity gradient in Northern 40 Australia. Org. Geochem. 62, 62–67. https://doi.org/10.1016/j.orggeochem.2013.07.003 Jansen, B., Nierop, K.G.J., 2009. Methyl ketones in high altitude Ecuadorian Andosols confirm excellent con- servation of plant-specific n-alkane patterns. Org. Geochem. 40, 61–69. https://doi.org/10.1016/j. orggeochem.2008.09.006 Jansen, B., Wiesenberg, G.L.B., 2017. Opportunities and limitations related to the application of plant- derived lipid molecular proxies in soil science. SOIL 3, 211–234. https://doi.org/10.5194/soil-3-211-2017 Jørgensen, P.M., Leó n-Yánez, S., 1999. Catalogue of the vascular plants of Ecuador. Missouri Botanical Garden Press, St. Louis, Missouri , USA. Karger, D.N., Conrad, O., Böhner, J., Kawohl, T., Kreft, H., Soria-Auza, R.W., Zimmermann, N.E., Linder, H.P., Kessler, M., 2017. Climatologies at high resolution for the earth’s land surface areas. Sci. Data 4, 1–20. https://doi.org/10.1038/sdata.2017.122 Koch, K., Ensikat, H.J., 2008. The hydrophobic coatings of plant surfaces: Epicuticular wax crystals and their morphologies, crystallinity and molecular self-assembly. Micron 39, 759–772. https://doi. org/10.1016/j.micron.2007.11.010 Maffei, M., Mucciarelli, M., Scannerini, S., 1993. Environmental factors affecting the lipid metabolism in Ros- marinus officinalis L. Biochem. Syst. Ecol. 21, 765–784. https://doi.org/https://doi.org/10.1016/0305- 1978(93)90089-A Pinto, E., Pérez, Á.J., Ulloa Ulloa, C., Cuesta, F., 2018. Arboles representativos de los bosques montanos del noroccidente de Pichincha, Ecuador. CONDESAN, Quito, Ecuador. R Core Team, 2017. R: A language and environment for statistical computing. https://www.r-project.org/. Sachse, D., Radke, J., Gleixner, G., 2006. δD values of individual n-alkanes from terrestrial plants along a climatic gradient - Implications for the sedimentary biomarker record. Org. Geochem. 37, 469–483. https://doi.org/10.1016/j.orggeochem.2005.12.003 Tipple, B.J., Pagani, M., 2013. Environmental control on eastern broadleaf forest species’ leaf wax distributions and d/h ratios. Geochim. Cosmochim. Acta 111, 64–77. https://doi.org/10.1016/j.gca.2012.10.042 Production

41 11 | APPEndIcEs 4 4 4 4 4 3 1 4 4 4 4 3 3 1 1 individuals Triana ex Cogn. Monogr. Phan. 1891. Cogn. Monogr. ex Triana (Bonpl.) Cogn. Fl. Bras. 1888. (Bonpl.) Cogn. Fl. Bras. Geol. 1830. A. Juss. Bull. Sci. Nat. Phan. 1891. Cogn. Monogr. ex Triana 1888. (Bonpl.) Cogn. Fl. Bras. Geol. 1830. A. Juss. Bull. Sci. Nat. Triana ex Cogn. Monogr. Phan. 1891. Cogn. Monogr. ex Triana Triana ex Cogn. Monogr. Phan. 1891. Cogn. Monogr. ex Triana Phan. 1891. Cogn. Monogr. ex Triana 1888. (Bonpl.) Cogn. Fl. Bras. Geol. 1830. A. Juss. Bull. Sci. Nat. A. Juss. Bull. Sci. Nat. Geol. 1830. A. Juss. Bull. Sci. Nat. A. Juss. Bull. Sci. Nat. Geol. 1830. A. Juss. Bull. Sci. Nat. species author and reference Phan. 1891. Cogn. Monogr. ex Triana Geol. 1830. A. Juss. Bull. Sci. Nat. Miconia Miconia clathrantha Miconia theaezans Miconia kunthiana Guarea Miconia clathrantha theaezans Miconia kunthiana Guarea Miconia Miconia clathrantha Miconia Miconia clathrantha Miconia clathrantha theaezans Miconia kunthiana Guarea Guarea kunthiana Guarea Guarea kunthiana Guarea Miconia Miconia clathrantha kunthiana Guarea species sampled 8 8 1 4 3 3 2 11 12 total samples 1351 1595 1595 2347 2076 2704 2253 2255 2075 AP 98.6 99.3 99.6 98.8 98.8 98.9 99.7 99.6 99.8 HR 14.3 13.6 14.0 16.8 16.0 18.8 20.6 19.4 21.6 MAT 2212 2313 2203 1640 1879 1277 1018 827 653 altitude -78.5691 -78.6863 -78.6893 -78.7353 -78.7232 -78.8129 -78.8761 -78.8819 -78.9100 longi- tude 0.1132 -0.0153 -0.0116 -0.0820 0.0505 -0.0253 0.1685 0.1583 0.1873 latitude Chapter 2 Chapter Production

42 43 El Cedral Ecolodge El Cedral Cloud Bellavista Forest Bellavista Cloud Bellavista Forest Reserva Rio Bravo Reserva Intillacta Reserva Mindo Lindo Mashpi Lodge Mashpi Lodge Mashpishungo/ Pambilina reserve Site Site metadata and sampling intensity. Site number with corresponds at Figure 1. each were recorded Three gradients environmental site: Mean an- CEDR 03 BECL 01 BECL 03 RIBR 01 INTI 01 MIND 01 MALO 02 MALO MALO 01 MALO MAPI 01 site 8 9 7 5 6 4 3 2 1 nr site site nual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Total samples = Total number of samples taken at each site. Species Species site. each at taken samples of number Total = samples Total (AP). precipitation annual mean and (RH) humidity air relative mean (MAT), temperature nual site. at each sampled per species individuals of = number and individuals reference taxonomic its site, including at each sampled the species = names of sampled Appendix Appendix 1 | 4 4 4 3 2 4 4 1 4 4 4 individuals (Bonpl.) DC. Prodr. 1828. (Bonpl.) DC. Prodr. Cogn. Bull. Acad. Roy. Sci. Belgique, Roy. Cogn. Bull. Acad. 3. 1887. sér. Cogn. Bull. Acad. Roy. Sci. Belgique, Roy. Cogn. Bull. Acad. 3. 1887. sér. Triana.Trans. Linn. Soc. London. Linn. Soc. London. Triana.Trans. 1871[1872]. Cogn. Bull. Acad. Roy. Sci. Belgique, Roy. Cogn. Bull. Acad. 3. 1887. sér. (Bonpl.) Cogn. Fl. Bras. 1888. (Bonpl.) Cogn. Fl. Bras. (Bonpl.) Cogn. Fl. Bras. 1888. (Bonpl.) Cogn. Fl. Bras. species author and reference Linn. Soc. London. Triana.Trans. 1871[1872]. (Bonpl.) Cogn. Fl. Bras. 1888. (Bonpl.) Cogn. Fl. Bras. (Bonpl.) Cogn. Fl. Bras. 1888. (Bonpl.) Cogn. Fl. Bras. Triana ex Cogn. Monogr. Phan. 1891. Cogn. Monogr. ex Triana Miconia Miconia bracteolata Miconia Miconia corymbiformis Miconia Miconia corymbiformis Miconia ochracea Miconia Miconia Miconia corymbiformis Miconia theaezans Miconia Miconia theaezans Miconia species sampled ochracea Miconia Miconia theaezans Miconia Miconia theaezans Miconia Miconia Miconia clathrantha 8 7 6 4 9 total samples 1337 1271 1251 1251 1471 AP 98.7 97.0 96.2 99.0 97.7 HR 7.2 8.3 9.9 10.2 13.0 MAT 3507 3421 3109 2932 2492 altitude -78.5907 -78.5958 -78.6008 -78.6004 -78.5705 longi- tude -0.1267 -0.1233 -0.1044 -0.1015 0.1195 latitude Chapter 2 Chapter Production

42 43 Reserva Yanacocha Reserva Reserva Verdecocha Reserva Reserva Verdecocha Reserva Reserva Verdecocha Reserva El Cedral Ecolodge El Cedral reserve YANA 01 YANA VERD 01 VERD 03 VERD 02 CEDR 01 site nr 14 13 12 11 10 site site ) 1 | ( continued Appendix Appendix 2 | Scatterplots showcasing similarity between four ratios. Colors indicate diff erent species. Chapter 2 Chapter

44 Appendix 3 | Correlation between environmental gradients and altitude. Blue shades indicate negative correlations, red shades indicate positive correlations. Color intensity indicates strength of correlation (Pearson’s correlation coeffi cient). ALT = Altitude, MAT = mean annual temperature, RH = relative humidity, AP = Annual precipitation. Production

45 Appendix 4 | Concentration and metrics variability of the replicate samples (Sample code). CONw = total n-alkane concentration (ng/g of dried sample); ACL = average chain length; RATIO = C31/(C31+C29); sd = standard deviation; CV(%) = coeffi cient of variation ; N(#) = number of replicates. Sample code CONw ±sd CV (%) ACL ±sd CV (%) RATIO ±sd CV (%) N(#) GK 03 141.2 72.9 51.6 30.9 0.1 0.4 0.9 0.0 2.8 2 GK 12 124.8 61.3 49.1 30.9 0.1 0.3 0.9 0.0 4.4 2 GK 13 78.1 6.10 7.90 29.3 0.1 0.3 0.6 0.0 1.9 2 GK 15 147.8 26.0 17.6 30.7 0.0 0.2 0.8 0.0 0.7 3 MCL 22 52.9 8.60 16.2 30.8 0.1 0.3 0.8 0.1 8.4 2 MTH 17 290.6 28.6 9.80 29.1 0.0 0.1 0.4 0.0 2.3 3 Chapter 2 Chapter

46 Appendix 5 | Spearman’s rank correlation coeffi cients s(r ) of the average chain length (ACL23-33) and the ratio

(C31/(C31+C29)) against three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Signifi cance legend as follows: * p < 0.01, ** p <0.005, *** p < 0.001. metric variable species rs p-value Guarea kunthiana 0.44 0.021 Miconia clathrantha 0.50 0.020 MAT Miconia corymbiformis 0.04 0.915 Miconia theaezans 0.37 0.068 Total 0.66 0.000 *** Guarea kunthiana 0.50 0.008 * Miconia clathrantha 0.06 0.805 ACL RH Miconia corymbiformis -0.04 0.915

C23-C33 Miconia theaezans 0.36 0.076 Total 0.44 0.000 *** Guarea kunthiana 0.50 0.007 * Miconia clathrantha -0.22 0.328 AP Miconia corymbiformis 0.04 0.915 Miconia theaezans 0.39 0.056 Total 0.58 0.000 ***

Guarea kunthiana 0.44 0.021 Miconia clathrantha 0.56 0.009 * MAT Miconia corymbiformis 0.21 0.565 Miconia theaezans 0.58 0.003 ** Total 0.61 0.000 *** Guarea kunthiana 0.55 0.003 ** Miconia clathrantha -0.02 0.930

C31/ RH Miconia corymbiformis -0.21 0.565

(C31+C29) Miconia theaezans 0.28 0.180 Total 0.39 0.000 *** Guarea kunthiana 0.57 0.002 ** Miconia clathrantha -0.26 0.248

AP Miconia corymbiformis 0.21 0.565 Production Miconia theaezans 0.23 0.274 47 Total 0.53 0.000 *** Appendix 6 (part I of II) | Spearman’s rank correlation coeffi cients (rs) of odd chain length n-alkanes from individual species and site total against three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Signifi cance legend as follows: * p < 0.01, ** p <0.005, *** p < 0.001

n-alkane variable species rs p-value Guarea kunthiana 0.55 0.003 ** Miconia clathrantha 0.57 0.007 * MAT Miconia corymbiformis 0.10 0.775 Miconia theaezans 0.53 0.007 * Total 0.61 0.000 *** Guarea kunthiana 0.58 0.002 ** Miconia clathrantha 0.05 0.841

C33 RH Miconia corymbiformis -0.10 0.775 Miconia theaezans 0.31 0.129 Total 0.38 0.000 *** Guarea kunthiana 0.58 0.002 ** Miconia clathrantha -0.18 0.448 AP Miconia corymbiformis 0.10 0.775 Miconia theaezans 0.23 0.264 Total 0.47 0.000 ***

Guarea kunthiana 0.05 0.798 Miconia clathrantha 0.63 0.002 ** MAT Miconia corymbiformis 0.18 0.616 Miconia theaezans 0.32 0.118 Total 0.56 0.000 *** Guarea kunthiana 0.22 0.271 Miconia clathrantha -0.03 0.883

C31 RH Miconia corymbiformis -0.18 0.616 Miconia theaezans 0.39 0.051 Total 0.35 0.000 *** Guarea kunthiana 0.31 0.114 Miconia clathrantha -0.31 0.169

Chapter 2 Chapter AP Miconia corymbiformis 0.18 0.616 Miconia theaezans 0.36 0.075 48 Total 0.49 0.000 *** Appendix 6 (part I of II) | (continued)

n-alkane variable species rs p-value Guarea kunthiana -0.44 0.022 Miconia clathrantha -0.45 0.042 MAT Miconia corymbiformis -0.42 0.233 Miconia theaezans -0.82 0.000 *** Total -0.47 0.000 *** Guarea kunthiana -0.54 0.004 ** Miconia clathrantha 0.05 0.839

C29 RH Miconia corymbiformis 0.42 0.233 Miconia theaezans -0.17 0.419 Total -0.38 0.000 *** Guarea kunthiana -0.55 0.003 ** Miconia clathrantha 0.28 0.212 AP Miconia corymbiformis -0.42 0.233 Miconia theaezans -0.15 0.480 Total -0.39 0.000 *** <0.01 * ; <0.005**; <0.001*** Production

49 Appendix 6 (part II of II) | Spearman’s rank correlation coeffi cients (rs) of odd chain length n-alkanes from individual species and site total against three environmental gradients: mean annual temperature (MAT), mean relative air humidity (RH) and mean annual precipitation (AP). Signifi cance legend as follows: * p < 0.01, ** p <0.005, *** p < 0.001

n-alkane variable species rs p-value Guarea kunthiana -0.40 0.041 Miconia clathrantha -0.61 0.003 ** MAT Miconia corymbiformis 0.14 0.694 Miconia theaezans -0.51 0.010 * Total -0.67 0.000 *** Guarea kunthiana -0.45 0.018 Miconia clathrantha -0.77 0.000 ***

C27 RH Miconia corymbiformis -0.14 0.694 Miconia theaezans -0.53 0.006 * Total -0.47 0.000 *** Guarea kunthiana -0.45 0.018 Miconia clathrantha -0.45 0.041 AP Miconia corymbiformis 0.14 0.694 Miconia theaezans -0.51 0.010 * Total -0.58 0.000 ***

Guarea kunthiana -0.40 0.037 Miconia clathrantha -0.40 0.070 MAT Miconia corymbiformis 0.47 0.174 Miconia theaezans 0.76 0.000 *** Total -0.56 0.000 *** Guarea kunthiana -0.27 0.179 Miconia clathrantha -0.45 0.041

C25 RH Miconia corymbiformis -0.47 0.174 Miconia theaezans 0.31 0.138 Total -0.44 0.000 *** Guarea kunthiana -0.27 0.172 Miconia clathrantha -0.18 0.424

Chapter 2 Chapter AP Miconia corymbiformis 0.47 0.174 Miconia theaezans 0.21 0.314 50 Total -0.58 0.000 *** Appendix 6 (part II of II) | (continued)

n-alkane variable species rs p-value Guarea kunthiana -0.24 0.231 Miconia clathrantha 0.10 0.667 MAT Miconia corymbiformis -0.32 0.361 Miconia theaezans 0.14 0.516 Total -0.53 0.000 *** Guarea kunthiana -0.20 0.319 Miconia clathrantha 0.09 0.692

C23 RH Miconia corymbiformis 0.32 0.361 Miconia theaezans 0.55 0.004 ** Total -0.31 0.000 *** Guarea kunthiana -0.29 0.145 Miconia clathrantha 0.27 0.234 AP Miconia corymbiformis -0.32 0.361 Miconia theaezans 0.17 0.423 Total -0.52 0.000 *** <0.01 * ; <0.005**; <0.001*** Production