DOI: 10.17129/botsci.2406

Functional traits of tree saplings and adults in a tropical cloud forest restoration context

Atributos funcionales de árboles juveniles y adultos en el contexto de restauración del bosque de niebla



Theuse of tree species’ functional traits is a promising approach in forest restoration. However, some traits may change during ontogeny.


Doesintraspecific variation in functional traits occur between sapling and adult stages? Do groups of species can be delimited based on functional traits regardless of their ontogenetic stage?

Study sites and dates:

Cloud forest restoration, Veracruz, Mexico, 2016.


Saplingsand adults of eight native tree species in different age plantations were measured for leaf area (LA), specific leaf area (SLA), stomatal density (SD), foliar nutrient content (C, N, P) and relative growth rate(RGR). Wood density (WD) was measured for adults. Data were analyzed using linear mixed models and principal component analysis (PCA).


Overall,SLA was higher in saplings than in adults. A few species showed intraspecific variation for LA (three species), SD (three) and foliar N content (one). Species with high WD (Quercus spp.) and intermediate WD (e.g., Liquidambar styraciflua) tended to have lower LA and SLA, and higher SD. Species with low WD (e.g., Heliocarpus donnellsmithii)had high SLA, RGR, and N content. PCA highlighted that saplings and adults of a same species were close to each other within the ordination space.


Intraspecific variation between saplings and adults was small for most traits (except SLA) in comparison to differences across species. Therefore species trait values(measured in individuals of any age) could be a useful tool to characterize groups of species during the forest restoration trajectory.


Key words: 

Growth rate; leaf area; specific leaf area; stomatal density; wood density.



Usaratributos funcionales de especies arbóreas es un enfoque prometedor en restauración ecológica. Aunque algunos atributos pueden cambiar durante la ontogenia.


¿Existe variación intraespecífica en atributos funcionales entre árboles juveniles y adultos? ¿Se pueden delimitar grupos de especies basados en atributos funcionales independientemente de su ontogenia?

Sitios y fecha de estudio:

Restauración de bosque de niebla, Veracruz, México, 2016.


Ajuveniles y adultos de ocho especies nativas en plantaciones de diferente edad se les midió área foliar (AF), área foliar específica (AFE), densidad estomática (DE), nutrientes foliares (C, N, P), y tasa de crecimiento relativa (TCR). Densidad de la madera (DM) se midió en adultos. Los datos se analizaron utilizando modelos lineales mixtos y análisis de componentes principales (ACP).


Engeneral, AFE fue mayor en juveniles que en adultos. Algunas especies mostraron variación intraespecífica para AF (tres especies), DE (tres) yN (una). Especies con DM alta (Quercus spp.) e intermedia (e.g., Liquidambar styraciflua) tuvieron AF y AFE bajos y DE alta. Especies con DM baja (e.g., Heliocarpus donnellsmithii)tuvieron AFE, TCR y N altos. ACP destacó que juveniles y adultos de unamisma especie estaban cercanos dentro del espacio de ordenación.


Lasdiferencias intraespecíficas entre juveniles y adultos para atributos funcionales (excepto AFE) fueron menores que las diferencias entre especies. Consecuentemente, los valores de los atributos de cada especie(medidos en individuos de cualquier edad) podrían ser una herramienta útil para caracterizar grupos de especies durante la trayectoria de restauración forestal.


Palabras clave: 

Área foliar; área foliar específica; densidad de madera; densidad estomática; tasa de crecimiento.


Theactive ecological restoration of degraded forest areas has mainly been conducted through a process of planting native tree species and assessing growth and survival in response to environmental variables. The use of the functional traits of trees has increasingly been studied as a promising approach to select species for forest restoration (Martínez-Garza et al. 2005, Flores et al. 2014, Ostertag et al. 2015, Gustafsson et al. 2016, Toledo-Aceves et al. 2017).Functional traits have been described as the main characteristics related to the reproduction, survival, growth and fitness of plant species, based on physiological, morphological and phenological characteristics (Violle et al. 2007). Functional traits are already in use as indicators of success in ecological restoration practices (Martínez-Garza et al. 2013, Ostertag et al. 2015).

Environmental filtering to restore a community may act differentially on seedlings, saplings and adult trees, and there are several studies highlighting the importance of incorporating ontogenetic trait variationinto approaches that use plant functional traits (Poorter 2007, Spasojevic et al. 2014).Functional traits known to be good predictors of demographic rates and fitness in the regeneration stage are also apparently good predictors ofplant performance in the post-regeneration stage across a wide range ofNeotropical forests (Poorter et al. 2008). Some studies show how traits may differ with plant age-size (Gibert et al. 2016). Other studies have found variation in functional leaf traits at different ontogenetic phases in the restoration context (e.g., Martínez-Garza & Howe 2005, Lapok et al. 2017).

Tropical montane cloud forest (TMCF) is remarkably diverse in terms of its physiognomy and tree species composition but is particularly threatened by habitat destruction and global change (Bruijnzeel et al. 2010, Williams-Linera et al. 2013).Ecological restoration is important for preserving forest biodiversity and ecosystem services; however, restoration takes decades and the existing reports are frequently based on seedlings and saplings in recently established plantations, and uncommonly on adults in old restoration sites (Wortley et al. 2013).In the TMCF region of central Veracruz, Mexico, several ecological restoration sites were established since 1998 in recently abandoned pastures using early and late successional native tree species (Williams-Linera et al. 2016).The microenvironmental conditions prevalent in young plantations are more similar to those of large forest gaps or open areas, with lower canopy cover, higher light levels and lower soil water content. In contrast, in middle-age plantations, conditions are more akin to those found in secondary forests (Alvarez-Aquino et al. 2004, Muñiz-Castro et al. 2015).

In this study, young and middle-aged plantations of native tree specieswere used to compare the functional traits of saplings and adults of several species with different wood density in relation to growth rates.Plant relative growth rate is an intrinsic response of particular species in an assemblage (Rüger et al. 2012, Gibert et al. 2016)and has been used to compare the performance of different species in restoration assays. Leaf characteristics are highly variable but have been related to the ability of the plant to survive and grow and competefor light (Bongers & Popma 1990, Wright et al. 2004, Poorter et al. 2008).Furthermore, leaf and wood traits appear to be good proxies for physiological rates and are correlated with relative growth rate (Poorter & Bongers 2006, Janse-ten Klooster et al. 2007, Poorter et al. 2008, Gibert et al. 2016).We selected easily measurable functional traits (leaf area (LA), specific leaf area (SLA), stomatal density (SD), and foliar nutrients (C, N, P)) that have been demonstrated to have predictable trends duringsuccession and have been suggested for use in monitoring forest restoration (Martínez-Garza et al. 2013, Ostertag et al. 2015, Brancalion & Holl 2016).

The objective of this study was to evaluate the functional traits of the same tree species at the sapling (2-3 years) and adult (13-17 years)stages in ecological restoration sites in a TMCF region. We hypothesized that 1) functional traits would vary between saplings and adults, and 2) functional traits display a range of values that cause grouping of tree species regardless of the ontogenetic variation.

Materials and methods

Study sites.The study sites are located in the tropical lower montane forest regionof Veracruz, Mexico (19° 30' N; 96° 57' W) between 1,280 and 1,450 m asl. The climate is mild and humid throughout the year, with three distinct seasons: a relatively dry-cool season from November to March, adry-warm season in April-May and a wet-warm season from June to October. Annual precipitation is 1,600-1,800 mm and the mean temperatureis 17-18 °C. The soil has been classified as Andosol. The dominant treespecies are Carpinus tropicalis (Betulaceae), Clethra macrophylla (Clethraceae), Liquidambar styraciflua (Altingiaceae), Quercus lancifolia (Fagaceae), Q. sartorii (Fagaceae), Q. xalapensis (Fagaceae) and Turpinia insignis (Staphyleaceae) (Williams-Linera et al. 2013). In this region, five restoration plantations with native tree species were chosen (Pedraza & Williams-Linera 2003, Muñiz-Castro et al. 2015, Williams-Linera et al. 2010, 2015, 2016).The sites included plantations that were categorized as recently established or young (average age = 2.5 yr) and middle-aged (average age= 14.2 yr) (Table 1). The average distance among sites was 4.6 km. All sites were situated on slopes of 11 to 36°. Soils are volcanic in origin and > 2.5 m depth, mildly acidic (pH 5.1to 5.6), low in extractable P (4.5 mg/kg), high in organic matter (10-20 %), with a bulk density of 0.9 to 1.1 g/cm3, and texture from clay to sandy-clay-loam. Because of the sites’ proximity toeach other, there are clear similarities in climate, physical and chemical soil characteristics and geomorphic environmental conditions. Thus, we were confident that the trait values of saplings and trees can be reliably compared.

Table 1. 

Characteristics of the restoration sites in the tropical montane cloud forest region of centralVeracruz, Mexico, where tree saplings or adults (stage) were sampled. MAP is mean annual precipitation, MAT is mean annual temperature. Plantation is the year of plantation establishment. Age (years) and stage (adult, sapling) of the plants.

SiteLatitude NLongitude WElevation (m asl)MAP (mm)MAT (°C)PlantationAgeStageReference
119° 35' 10''96° 57' 16.7''1,4501,83616.8200213adult1
219° 32' 10.1''96° 58' 4.9''1,4501,66917.9199817adult2
319° 30' 52.5''96° 59' 27.9''1,4051,92517.1200213adult1
419° 30' 57.5''96° 56' 51.5''1,3701,62118.520123sapling4
519° 30' 37.8''96° 56' 43.1''1,2801,62118.520132sapling3

Reference: 1, Muñiz-Castro et al. 2015; 2, Pedraza & Williams-Linera 2003; 3, Toledo-Aceves et al. 2017; 4, Williams-Linera et al. 2015.

Eight planted native tree species were selected from the five restoration sites (Table 2).All saplings were collected in young sites, and all adults were collected in middle-age sites. The sapling stage was < 10 cm diameterand < 10 m tall, and the adult stage included trees > ca. 10 cm diameter and > 10 m tall (Table 2).

Table 2. 

Tree species in tropical montane forest restoration sites in central Veracruz, Mexico. Wood density of adults (WD), mean diameter (cm) and height (m) of saplings and adults. Site numbers are given in Table 1.

SpeciesFamilyWDDiameter (cm)Height (m) Site
Carpinus tropicalis (Donn. Sm.) LundellBetulaceae0.553.814.74.69.52, 5
Heliocarpus donnellsmithii RoseMalvaceae0.348.812.34.910.31, 3, 5
Juglans pyriformis Liebm.Juglandaceae 0.513., 4
Liquidambar styraciflua L.Altingiaceae0.554.822.85.315.42, 5
Myrsine coriacea (Sw.) R. Br. ex Roem. & Schult.Primulaceae 0.513., 3, 5
Quercus germana Schltdl. & Cham.Fagaceae 0.743.112.51.810.61, 3, 5
Quercus xalapensis Bonpl.Fagaceae0.614.418.62.4141, 3, 5
Trema micrantha (L.) BlumeCannabaceae 0.518.426.45.618.51, 3, 5

Functional traits. Mature leaves withno herbivore damage that receive direct sunlight were collected from two branches on opposite sides of the stem or trunk with an oversized slingshot. The leaves of three saplings or three to six adults of each species were collected per site during the wet-warm season following standardized protocols (Cornelissen et al. 2003).The leaves were stored in black plastic bags and transported to the Laboratory of Functional Ecology at the Institute of Ecology, Xalapa, Mexico for subsequent analysis.

We selected 10 to 20 leaves from each individual. The area of each leaf (LA, cm2),excluding the petiole, was measured in a WinFOLIA Leaf Area Meter (Software program LA2400). The leaves were oven-dried at 60 °C for at least 72 hr and dry mass determined. Specific leaf area (SLA) was calculated as leaf area divided by leaf dry mass. The leaves were then ground for analysis to determine foliar carbon, nitrogen and phosphorus.Leaf C and N were determined using the TruSpec Micro, and leaf P by digestion with HNO3/ HClO4. The samples were analyzed using standard techniques (SEMARNAT 2002).

Stomatal density (SD) was determined in two fresh leaves per species and site. At the top, center and bottom of the abaxial surface of each leaf, we applied an imprinting paste followed by clear nail polish to produce stomatal imprints. Stomatal densities were determined using a compound microscope with 20x objective from photographs and expressed asstomata/mm2.

Wood density (WD) wasdetermined from a core extracted from each adult tree at a height of 1.3 m above ground level with a Pressler increment borer. In the laboratory, these cores were submerged in distilled water for three daysuntil fully hydrated. The volume of the cores was determined by the water displacement method, and dry weight was measured after the cores had been oven-dried at 60 °C for 72 hr.

Relative growth rates (RGR) in height and diameter were estimated using published and unpublished databases from previous works conducted in different years in the same sites, species and permanently tagged individuals used in this study (Table 1, 2). We used the equation RGR = ln H2 - ln H1/(t2 - t1), where H2 and H1 are height/diameter, and t2 and t1 are time in years (Hunt 1990).RGR was calculated for one period between two censuses and expressed over an interval of a year. The time span used to calculate RGR was 3-4 years for saplings and 8-10 years for adults.

Statistical analysis.Differences in leaf traits between stages were analyzed using a linear mixed model with stage as fixed effect, and sites as a random effect. Toattain normality and homoscedasticity in the residuals of both models, we used a log10 transformation. Inspection of residuals was used to verify whether the model’s assumptions had been met. WD was compared with respect to species using generalized linear model. Post hoc tests were conducted using Tukey's HSD. Analyses were conducted using the statistical platform of R version 3.4.2 ( A principal component analysis (PCA) was run to summarize and tovisualize the main trends of sapling and adult tree species, and the relationships with foliar traits and RGR. The PCA was run in the PC-ORD software (McCune & Grace 2002).


Leafarea showed significant stage × species interaction, and differences among species, but LA was similar between saplings and adults (Figure 1A, Table 3). LA of H. donnellsmithii was higher for saplings than for adults, but LA of Q. xalapensis and T. micrantha was higher for adult trees. Also, LA was higher for Heliocarpus donnellsmithii, intermediate for four species (e.g., Q. xalapensis), and smaller for Trema micrantha, Carpinus tropicalis and Myrsine coriaceae (Table 4).

Figure 1. 

Boxplots of leaf functional traits measured in tree saplings and adults in tropical montane cloud forest restoration sites in Veracruz, Mexico. (A) leaf area, (B) specific leaf area, (C) stomatal density, (D) carbon, (E) nitrogen, and (F) phosphorus. The crossbar within the box indicates the median and the dotted line the mean; the length of the box represents the interquartile range of distribution, the lower and upper fences indicate the 10th and 90th percentiles, respectively, and dots represent outliers of the data.

Table 3. 

Results of linear mixed models used to evaluate the effect of stage (sapling, adult) on eight species in tropical montane cloud forest restoration sites in central Veracruz, Mexico. Significant effects are in boldface type.

 Stage Species Stage × Species
 dfF p dfF p dfF p
Leaf area1, 32.440.21647, 3928.87< 0.00017, 393.750.0034
Specific leaf area1, 323.970.01637, 3927.33< 0.00017, 391.780.1201
Stomatal density1, 30.480.53687, 11331.42< 0.00017, 11311.18< 0.0001
Foliar C1, 40.670.46007, 1227.02< 0.00017, 122.480.0797
Foliar N1, 40.60.48147, 1234.1< 0.00017, 124.160.0151
Foliar P1, 40.050.82997, 1214.480.00017, 120.410.8794

Table 4. 

Leaf functional traits ofsaplings and adults of tree species in tropical montane cloud forest restoration sites in Veracruz, Mexico. Traits are leaf area (LA), specific leaf area (SLA), stomata density (SD), foliar carbon (C), nitrogen (N) and phosphorous (P). Values are mean and standard error. Boldface type denotes difference (α = 0.05) between saplings and adults within a same species. Species trait in a same column accompanied by thesame letter did not differed significantly (α = 0.05).

SpeciesLA (cm2)  SLA (cm2/g)  SD (no./mm2) 
SaplingAdult SaplingAdult SaplingAdult 
Carpinus tropicalis 9.0±0.5 22.2±0.9 d 280.9±11.6 261.2±12.5 b 102±13 90±11 b
Heliocarpus donnellsmithii197.0±26.893.0±7.1 a 375.9±934.7 280.5±6.5 a 58±7 60±4 de
Juglans pyriformis 55.2±2.0 59.0±4.1 b 127.6±5.4 90.6±4.1 e 52±4 57±7 de
Liquidambar styraciflua 32.4±1.7 48.3±3.4 c 169.5±5.2 154.3±10.7 d70±536±3 de
Myrsine coriacea 16.0±0.8 15.0±0.8 d 245.5±2.9 157.2±4.1 c 42±4 50±4 e
Quercus germana 36.4±1.1 50.0±2.4 c 117.1±2.5 121.0±2.4 e94±651±6 cd
Quercus xalapensis28.3±4.455.4±3.4 c 113.0±22.7 136.4±1.8 e 112±6 134±7 a
Trema micrantha16.1±1.138.3±1.7 d 253.9±6.1 131.7±3.0 c57±7111±5 bc
SpeciesC (%)  N (%)  P (cmol/kg)  
SaplingAdult  SaplingAdult  SaplingAdult  
Carpinus tropicalis 47.7±0.1 49.7±0.3 bc 1.8±0.0 2.9±0.2 d 4.7±0.1 4.3±0.7 ab
Heliocarpus donnellsmithii 48.5±0.4 48.1±0.3 bc 4.6±0.0 4.4±0.1 a 4.2±0.0 4.5±0.0 b
Juglans pyriformis 46.9±0.2 49.0±0.7 cd 2.9±0.0 2.6±0.0 bc 2.5±0.0 2.2±0.3 b
Liquidambar styraciflua 49.4±0.8 49.1±0.2 ab 1.7±0.5 2.1±0.1 d 4.5±0.6 3.2±0.2 ab
Myrsine coriacea 50.9±0.2 51.5±0.8 a 2.6±0.2 2.8±0.3 bcd 3.8±0.1 3.2±0.3 b
Quercus germana 49.4±0.2 47.5±0.2 ab 2.1±0.0 1.8±0.0 cd 3.9±0.3 4.5±0.1 b
Quercus xalapensis 49.6±0.0 50.3±0.5 ab1.6±0.02.8±0.5 d 6.7±0.3 6.4±1.3 a
Trema micrantha 45.1±0.1 46.0±1.2 d 3.2±0.0 4.3±0.3 b 3.4±0.8 4.1±0.0 b

Overall, SLA was higher in saplings than in adults (Figure 1B, Table 3), and there were differences among species (Table 3). SLA was higher in H. donnellsmithii, and lower in Q. xalapensis, Q. germana and J. pyriformis (Table 4).

Stomatal density displayed significant stage × species interaction and differed among species, but SD was similar in saplings and adults (Figure 1C, Table 3). L. styraciflua and Q. germana had higher SD in saplings, whereas T. micrantha had higher SD in adults (Table 4). Foliar C, N and P content were similar in both stages, but species differed in their foliar nutrient content (Figure 1D - F, Table 3, 4).

Wood density differed among tree species (F = 6.39, p = 0.019), with Q. germana and Q. xalapensis having the highest WD. The other species had intermediate values (L. styraciflua, C. tropicalis, J. pyriformis, M. coriacea, T. micrantha), while H. donnellsmithii had the lowest WD (Table 2).

Relative growth rate was higher for H. donnellsmithii, M. coriacea and T. micrantha than for the other species (Figure 2). Also, RGR was higher in saplings than in adults of those three species. RGR in height and diameter were correlated (r = 0.94, p < 0.0001), so we used RGR in height for further analysis.

Figure 2. 

Relative growth rate (RGR)in (A) diameter and (B) height for the saplings (circle) and adults (square) of eight tree species in tropical montane cloud forest restoration sites in Veracruz, Mexico. Filled symbols represent pioneer species; open symbols represent non-pioneer species. myr, Myrsine coriacea; hel, Heliocarpus donnellsmithii; tre, Trema micrantha; qxa, Quercus xalapensis; qge, Quercus germana; car, Carpinus tropicalis; jug, Juglans pyriformis; liq, Liquidambar styraciflua.

The PCA based on leaf traits and RGR of tree species in the sapling and adult stages is shown in Figure 3.The first three components of PCA explained 73.3 % of the total variation. LA, SLA and leaf N content had positive loading on axis 1; leaf P content and stomata were positively related to axis 2, and RGR had positive loading on axis 3 (Table 5). Q. xalapensis had the highest SD and foliar P content. H. donnellsmithii had high LA, SLA and foliar N. The clearest pattern was more separation of saplings and adults of species (H. donnellsmithii, M. coriacea and T. micrantha)in the right side of axis 1 (Euclidean distance, 141, 87, 136, respectively, mean = 121.8), and less separation between stages in species towards the central part (C. tropicalis; J. pyriformis, L. styraciflua) and left side of axis 1 (Q. germana and Q. xalapensis) (Euclidean distance, 27, 38, 40, 46, 42, respectively; mean = 38.6) within the ordination space.