Assessment of Three Provenances of Juglans neotropica Diels to Identify Optimal Seed Sources in the Northern Ecuadorian Andes

Abstract

Identifying optimal seed sources is critical for the propagation and restoration of Juglans neotropica Diels in the northern Ecuadorian Andes, where populations are declining due to habitat loss and overexploitation. This study evaluated the seed quality and germination performance of Juglans neotropica from three ecologically distinct provenances: a natural regeneration site (Cuyuja), a pure plantation (Natabuela), and an agroforestry system (Pimampiro). Five phenotypically superior trees were selected from each site, and germination was assessed under controlled nursery conditions over a 150-day period using a completely randomized design. Initial viability tests confirmed the physiological integrity of the seeds across all provenances. Germination onset ranged from day 55 to day 73, with significant differences in germination percentage, speed, and uniformity. The agroforestry provenance showed the highest germination rate (69%) and superior performance in all physiological indices, while natural regeneration had the lowest (15%). Post-trial viability assessments indicated that a substantial proportion of non-germinated seeds from Cuyuja remained dormant or deteriorated. These findings underscore the role of agroforestry systems in enhancing seed physiological quality and support their prioritization for large-scale propagation and ecological restoration initiatives involving Juglans neotropica.

1. Introduction

Juglans neotropica Diels, commonly referred to as Andean walnut, is a multipurpose forest tree species native to the South American Andes, with notable ecological and socioeconomic significance in Ecuador [1,2]. Its timber is highly prized for its durability, grain quality, and aesthetic appeal, serving both national and international markets [3]. Additionally, its edible nuts are valued for their nutritional properties and potential in specialty food markets [4], while the species holds cultural value through its use in artisanal crafts and religious items, reflecting the identity and traditions of highland communities [5].

From an ecological standpoint, J. neotropica contributes to biodiversity conservation and the provision of ecosystem services such as watershed protection, climate regulation, and habitat connectivity [6]. Its presence in fragmented Andean landscapes supports the persistence of endemic and migratory fauna. However, the species has experienced a marked population decline due to deforestation, overharvesting, and habitat degradation. In the past three decades, more than 50% of natural populations have been lost, prompting their classification as endangered (EN A2cd) by the IUCN [2]. In Ecuador, the loss of over 30% of Andean forest cover has further threatened its long-term viability [7].

In response, various conservation and restoration strategies have been implemented in regions such as Imbabura and Napo. These include the management of natural regeneration areas, protection of remnant stands, and establishment of agroforestry systems and monoculture plantations incorporating Juglans neotropica [8,9]. Despite these efforts, the success of restoration initiatives has been inconsistent, partly due to the use of non-local or genetically unsuitable seed sources [10].

In the context of forest genetic improvement and assisted restoration, the identification and use of genetically superior provenances—defined as populations originating from specific ecological or geographic zones—are critical to optimizing plantation performance and resilience [11,12]. Juglans neotropica displays considerable genetic and phenotypic variation across its distributional range, influenced by altitudinal, climatic, and ecological gradients [13]. This inherent variability provides an opportunity to select source populations with advantageous traits for specific environments, thereby increasing the likelihood of restoration success.

Ecuador’s wide range of elevations and climates contributes to significant local adaptation within J. neotropica, as documented in several population genetic studies [14,15]. In forest tree breeding programs, early-stage selection of optimal provenances is particularly effective, as it capitalizes on natural variation and offers the highest potential genetic gains relative to cost and effort [16,17].

To date, no studies have directly evaluated and compared germination behavior and physiological seed quality of Juglans neotropica across contrasting provenance types. This work addresses that gap by introducing a novel approach focused on seed performance under controlled conditions.

Specifically, this study aims to (i) evaluate differences in seed quality among three distinct provenances; (ii) analyze their germination performance under controlled conditions; and (iii) identify the provenance with the highest viability and adaptive potential.

These results are expected to inform seed sourcing strategies in ecological restoration and genetic conservation programs across Andean forest landscapes.

2. Materials and Methods

2.1. Study Sites and Environmental Conditions

The study was conducted in three contrasting sites in the northern Ecuadorian Andes, representing distinct ecophysiological conditions relevant for the assessment of seed source performance in Juglans neotropica (Figure 1).

• San Francisco de Natabuela (Plantation): Located in Antonio Ante canton, Imbabura Province (2255 m a.s.l.), this site features a pure plantation system with annual precipitation ranging from 750 to 1000 mm and an average temperature of 16 °C [18].
• Pimampiro (Agroforestry System): This site in Pimampiro canton (2165 m a.s.l.) integrates Juglans neotropica and Coffea arabica in an agroforestry matrix, with lower precipitation (500–750 mm) and comparable temperature (16 °C) [19].
• Cuyuja (Natural Regeneration): Located in Quijos canton, Napo Province (2310 m a.s.l.), this montane site has significantly higher rainfall (1750–2000 mm) and a cooler climate (10 °C), supporting natural forest regeneration [20].

Germination trials were conducted at the Yuyucocha Campus of Universidad Técnica del Norte (Ibarra, Imbabura), situated at 2256 m a.s.l. under relatively uniform climatic conditions (17 °C and 600 mm/year), as recorded by the INAMHI meteorological station M1240 [21].

2.2. Selection of Seed Trees

Seed tree selection was conducted through a purposive, non-probabilistic approach based on phenotypic superiority, following established procedures for identifying superior individuals [22]. Initial assessments covered 99 individuals in Natabuela, 145 in Pimampiro, and 40 in Cuyuja.

From each site, 10 trees were preselected based on qualitative traits (crown shape, phytosanitary condition, trunk form) and quantitative metrics (fruit yield, DBH, commercial height, total height). The five most outstanding trees per provenance were ultimately selected for seed collection.

2.3. Seed Quality Assessment

Seed quality was evaluated prior to germination. Parameters included physical purity, seed mass (seeds per kilogram), and moisture content, determined according to protocols of the International Seed Testing Association [23]. These assessments ensured the comparability and reliability of the germination data.

2.4. Germination Trials

The germination trial was conducted under nursery conditions between November 2022 and April 2023, following seed collection in October 2022. A completely randomized design was applied. From each of the 15 selected seed trees (five per provenance), 40 seeds were sown in four replicates of 10 seeds per tree, resulting in a total of 600 seeds.

Seeds were sown in 15 × 20 cm polyethylene bags filled with a standardized substrate composed of 60% black soil, 30% organic compost, and 10% pumice. Throughout the 150-day evaluation period, environmental conditions were rigorously controlled. Air temperature was maintained between 25 °C and 28 °C, while relative humidity was kept above 85% through the use of a transparent polyethylene enclosure and a timed misting system. Fine water sprays were applied for two minutes every three hours during the daytime period (08:00–17:00), creating a stable humid microenvironment that favored seed hydration and metabolic activation.

Seed viability was assessed both before and after the germination period. Prior to sowing, a random sample of 25 seeds per provenance was selected. Testa rupture was performed to expose the embryo, which was examined under a stereoscopic microscope. Seeds were classified as viable if they exhibited a well-defined embryonic axis, uniform coloration, and no signs of necrosis or malformation. At the end of the trial, the same procedure was applied to 25 non-germinated seeds from each provenance to determine residual viability.

Five germination parameters were used to evaluate the physiological response of seeds collected from different provenances. The germination percentage (PG) was calculated as shown in Equation (1) [23]. Additional indices related to germination rate and temporal dynamics were calculated following standard methodologies, as shown in Equations (2)–(5) [24]. A summary of these formulas is presented in

Table 1. Equations used to compute germination parameters in Juglans neotropica.

Parameter	Equation		Description
Germination Percentage (%)	PG =${\displaystyle \frac{ \sum n}{N}}$× 100	(1)	n: Number of seeds germinated on a given day; N: Total number of seeds sown.
Germination Speed Coefficient	$CV={\displaystyle \frac{\sum n_i}{\sum \left(n_i{t}_i\right)}}$	(2)	$n_i$: Number of seeds germinated on day i; $t_i$: Number of days since sowing.
Germination Index	$IG={\displaystyle \frac{\sum {(n}_{i }{t}_i)}{N}}$	(3)	$n_i$: Number of seeds germinated on day i; $t_i$: Day after sowing; N: Total number of seeds sown.
Germination Speed Index	$M=\sum \left({\displaystyle \frac{n_i}{t_i}}\right)$	(4)	$n_i$: Number of seeds germinated per day; $t_i$: Time from sowing to germination of each seed.
Germination Uniformity Coefficient	$CUG={\displaystyle \frac{\sum n_i}{\sum {\left[\left(g-t_i\right)\right]}^2{n}_i}}$	(5)	g: Mean germination time; $t_i$: Days after sowing; $n_i$: Number of seeds germinated on day i.

.

To illustrate the temporal dynamics of germination, cumulative germination curves were constructed for each provenance based on daily emergence records. The curves were plotted on standardized axes using the total number of seeds per provenance as the reference. Visualization was performed using the ggplot2 package in R version 4.1.0, applying a logistic smoothing function to improve interpretability while preserving the empirical structure of the data.

2.5. Statistical Analysis

Statistical analyses were conducted using complementary methods to rigorously assess the effects of provenance on seed quality and germination performance. A one-way analysis of variance (ANOVA) was applied to compare physical seed quality parameters—namely physical purity, seed mass, and moisture content—across provenances.

To evaluate germination dynamics, a multivariate analysis of variance (MANOVA) was performed using five dependent variables: final germination percentage, germination speed coefficient, germination index, germination speed index, and uniformity coefficient. Prior to MANOVA, assumptions of multivariate normality and homogeneity of covariance matrices were tested using Mardia’s test and Box’s M test, respectively [25,26].

In addition, linear mixed-effects models (LMMs) were implemented to account for the hierarchical structure of the experimental design. In these models, provenance was treated as a fixed effect, and individual seed tree identity was included as a random effect to capture intra-provenance variability. Model diagnostics confirmed that residuals met the assumptions of normality and homoscedasticity [27].

All analyses were conducted in R version 4.1.0, using the packages stats (for ANOVA), MVN (for multivariate normality tests), and lme4 and lmerTest (for LMMs) [28].

3. Results

3.1. Dasometric Characteristics of Selected Trees

Trees from the Cuyuja provenance exhibited the highest mean values for diameter at breast height (DBH), total height (TH), and commercial height (CH) among the evaluated sites (Figure 2). These superior growth patterns are likely driven by favorable climatic conditions in Cuyuja, notably higher annual precipitation (1750–2000 mm) and lower mean temperatures (10 °C), which enhance water availability and reduce thermal stress, thereby supporting the vigorous growth of Juglans neotropica. Moreover, the natural regeneration ecosystem at Cuyuja likely maintains greater genetic heterogeneity and competitive interactions that promote adaptive vigor in selected individuals.

3.2. Phenotypic Traits of Seed Trees

Phenotypic assessments revealed consistent expression of qualitative traits across the three provenances (Figure 3). In Pimampiro and Natabuela, 60% of evaluated trees were classified as superior, while in Cuyuja the proportion reached 50%. These classifications were based on the cumulative scoring of crown morphology, trunk straightness, and phytosanitary condition, applied to all measured individuals within each site. Despite exhibiting the highest mean values in dasometric parameters, trees from Cuyuja more frequently presented bifurcated crowns and trunk irregularities, resulting in lower phenotypic scores for structural traits. Overall, traits such as crown form and phytosanitary condition showed limited intra-site variation, suggesting a stable phenotypic expression that may favor their use in early selection for seed improvement programs.

3.3. Seed Physical Quality

Seed purity showed statistically significant differences among provenances (ANOVA, p < 0.001), with Cuyuja presenting the highest physical purity (96.21% ± 2.59), exceeding values from Natabuela and Pimampiro (Figure 4). Despite this, purity was not strongly associated with germination success, suggesting that physiological dormancy may limit germination in natural stands.

3.4. Germination Performance

Initial viability assessments confirmed the physiological integrity of the seed lots used in the trial. None of the seeds analyzed exhibited signs of necrosis, structural malformation, or discoloration. Embryos were fully developed and morphologically intact across all provenances, indicating high initial viability and the absence of detectable deterioration prior to sowing.

Cumulative germination curves (Figure 5) reveal clear differences in germination timing and progression among provenances. Germination in Cuyuja began on day 55, followed by Pimampiro on day 57 and Natabuela on day 73. Although Cuyuja exhibited an earlier germination onset, its cumulative progression remained limited throughout the evaluation period. The agroforestry system (Pimampiro) demonstrated not only the earliest and most rapid germination but also the highest final accumulation.

At the end of the evaluation period, viability analysis of non-germinated seeds revealed patterns consistent with the cumulative germination profiles of each provenance. In Pimampiro, 30% of the non-germinated seeds showed signs of radicle emergence or testa rupture, 50% remained unchanged, and 20% exhibited tissue decomposition. In Natabuela, 40% showed evidence of germination activity, 40% showed no morphological changes, and 20% were deteriorated. In Cuyuja, only 20% of the non-germinated seeds exhibited signs of activation, while 50% remained unchanged and 30% showed signs of decomposition.

Multivariate analysis of variance (MANOVA) detected significant differences in germination variables among provenances (Pillai’s Trace = 1.07, F = 12.35, p < 0.001) (

Table 2. (a) MANOVA summary of germination variables of Juglans neotropica across three provenances; (b) Multivariate comparison of provenance means and grouping based on Hotelling’s test (Bonferroni-adjusted, α = 0.05).

(a) MANOVA Results
FV	Df	Pillai	approx F	num Df	den Df	Pr(>F)
Ecosystem	2	1.07	12.35	10.00	108.00	0.0000
Error	57
(b) Multivariate means and grouping
Ecosystem	PG	CV	IG	M	CUG	Group
Plantation	32.50	0.85	38.32	0.03	0.10	a
Natural Regeneration	15.00	0.71	15.10	0.02	0.00	b
Agroforestry System	69.00	0.96	72.76	0.07	0.01	c

FV: Source of variation; Df: Degrees of freedom; Pillai: Pillai–Bartlett statistic; approx F: Approximate F-statistic value; num Df: Numerator degrees of freedom; den Df: Denominator degrees of freedom; Pr(>F): p-value. Different letters in the “Group” column indicate statistically significant differences (p < 0.05) according to Hotelling’s test with Bonferroni correction.

). These differences reflect the influence of environmental conditions and silvicultural systems on seed physiological quality.

Canonical axis analysis (Figure 6) supported these findings, with germination percentage and germination index as the primary contributors to provenance differentiation. The agroforestry provenance showed stronger associations with faster and more uniform germination patterns.

Linear mixed models further confirmed these trends (Figure 7), revealing that the agroforestry provenance exerted a significant positive effect on PG and IG, with minimal intra-provenance variability, indicating consistent seed performance within that system.

4. Discussion

The dasometric analysis revealed notable differences among provenances. Trees from the natural regeneration site in Cuyuja exhibited greater diameter at breast height (DBH), total height, and commercial height, likely due to higher precipitation and cooler temperatures that optimize water availability and reduce thermal stress. Additionally, natural regeneration environments promote greater genetic diversity and structural heterogeneity, which enhance competitive adaptation and resilience, as documented in studies on Quercus ilex and Zelkova serrata [29,30,31].

In contrast, individuals in the Natabuela plantation and Pimampiro agroforestry system exhibited lower growth metrics. In Natabuela, high tree density may lead to intraspecific competition for light and nutrients, favoring height growth over diameter expansion [32,33]. In Pimampiro, interspecific competition between Juglans neotropica and coffee (Coffea arabica) likely restricts resource acquisition [34]. However, agroforestry systems provide long-term benefits by enhancing soil structure, promoting biodiversity, and sustaining ecosystem services [35,36].

Qualitative traits—such as crown morphology, trunk straightness, and phytosanitary status—were consistent across provenances, indicating high heritability and limited phenotypic plasticity. These characteristics have proven to be reliable indicators for the selection of seed trees in Juglans species [37,38]. However, significant quantitative trait variation among provenances underscores the need to conserve diverse seed sources to maintain adaptive capacity and ensure long-term genetic improvement [39,40].

Seed purity differed significantly among provenances, with Cuyuja showing the highest values. This may reflect reduced anthropogenic disturbance and the self-cleaning effect of high humidity and organic matter decomposition, as previously observed under similar ecological conditions [41,42]. Nonetheless, the higher purity did not translate into superior germination, indicating that purity alone is insufficient to predict seed performance.

Seed mass and moisture content did not differ significantly among provenances; however, both parameters followed environmental gradients. The larger seed size observed in this study (46.6 seeds/kg) compared to prior reports from Loja [41] and Colombia [43] may reflect local genotypic adaptation to higher elevation and lower temperature conditions [44,45]. Moisture content was highest in Cuyuja, consistent with its humid montane environment, although elevated seed moisture can compromise storage longevity if not properly managed [46].

From a physiological perspective, classifying the storage behavior of Juglans neotropica seeds is essential for interpreting their germination dynamics. The measured moisture contents (22.93% to 26.79%) fall within the range typical of recalcitrant seeds, which are highly desiccation-sensitive and cannot withstand the drying required for long-term storage [43]. This contrasts sharply with orthodox seeds—such as those of Juglans regia—which naturally exhibit moisture contents as low as 3.11–3.98% at maturity [47], are tolerant to desiccation levels typical of orthodox seeds (i.e., below 10%) [43], and frequently require cold stratification to break physiological dormancy [48]. The absence of documented storage or stratification protocols for Juglans neotropica, combined with its high initial moisture content, supports the hypothesis that its seeds exhibit recalcitrant or possibly intermediate storage behavior.

These physiological traits emphasize the need to maintain optimal moisture conditions in the germination substrate. In our trials, the agroforestry provenance (Pimampiro) consistently outperformed the others across all germination metrics: germination percentage (PG), germination index (IG), germination speed index (M), and speed coefficient (CV). These results suggest that agroforestry systems create favorable microclimatic conditions—such as moderated temperature, partial shade, enhanced soil structure, and higher organic matter content—that promote seed metabolic activity, mitigate dormancy, and enhance germination performance [49,50,51].

Despite these favorable conditions, germination was not fully synchronous. Marked asynchrony was particularly evident in seeds from the natural regeneration provenance, and to a lesser extent in the agroforestry system. Although germination in Cuyuja began slightly earlier (day 55), its cumulative curve plateaued rapidly, reflecting a limited physiological response. In Pimampiro, which initiated germination on day 57, the progression was sustained and consistent, yielding the highest cumulative values. Natabuela exhibited a delayed onset (day 73) but maintained a moderate and progressive increase, stabilizing between the other two provenances.

This pattern aligns with previous findings in Juglans neotropica, where delayed germination is associated with both physical barriers—such as a hard endocarp—and physiological dormancy regulated by endogenous inhibitors [52]. In our study, the low uniformity coefficient (CUG), particularly in Cuyuja, supports the presence of dormancy-related mechanisms. Additionally, the high initial moisture content may have interfered with the partial dehydration required to trigger germination, contributing to the observed temporal dispersion.

This mechanism may explain the paradox observed in Cuyuja, where the highest seed purity coincided with the lowest germination rates. In montane ecosystems, seed dormancy often functions as a bet-hedging strategy, delaying germination until environmental conditions are predictably favorable [53]. These findings highlight the importance of incorporating pre-germination treatments when using seeds from natural stands in restoration efforts.

Canonical axis analysis indicated that final germination percentage (PG) and germination index (IG) were the most discriminating variables among provenances, particularly favoring the Pimampiro agroforestry system. These indices are robust indicators of seed vigor and viability [24]. Meanwhile, the germination uniformity coefficient (CUG) was most strongly associated with Cuyuja, reflecting synchronized yet constrained germination, which aligns with dormancy-regulated behavior in montane species.

Linear mixed models further confirmed the significant effect of provenance on germination dynamics, with Pimampiro exhibiting superior and more consistent performance. This statistical robustness highlights its reliability for large-scale nursery propagation and forest restoration under variable environmental conditions.

To date, most research on Juglans neotropica has emphasized population structure, distribution patterns, or conservation status [13,14,41], with minimal attention to the physiological seed traits that underpin propagation success. This study is the first to integrate phenotypic selection of seed trees with multivariate germination analysis across ecologically contrasted provenances. The use of advanced statistical methods (MANOVA and LMMs) to assess germination behavior provides a methodological innovation, offering a more predictive approach for selecting seed sources with adaptive potential.

The superior performance of seeds from the Pimampiro agroforestry system supports its prioritization as a source for propagation and ecological restoration. Agroecological management appears to enhance seed physiological quality, possibly through improved soil conditions, microclimate buffering, and maternal effects—factors especially relevant under climate uncertainty [11,15].

Future research should explore the genetic basis of these differences using genomic tools such as SNP genotyping or GWAS, to identify markers associated with germination traits [13,14]. Parallel assessments of phenotypic plasticity across elevation and management gradients could elucidate the stability and adaptability of selected provenances in the face of environmental change [54,55].

5. Conclusions

The results of this study demonstrate that provenance exerts a significant influence on the seed quality and germination performance of Juglans neotropica in the northern Ecuadorian Andes. While the natural regeneration site in Cuyuja exhibited the most vigorous tree growth and highest seed purity, these advantages did not translate into germination success, likely due to dormancy-related constraints.

In contrast, seeds collected from the Pimampiro agroforestry system exhibited the highest values for germination percentage, index, and speed, along with reduced intra-provenance variability. These outcomes highlight the effectiveness of agroforestry management in enhancing seed physiological performance through favorable microclimatic and edaphic conditions.

The uniform expression of qualitative phenotypic traits across all provenances supports their reliability as selection criteria for seed tree identification. However, the observed variation in quantitative traits and germination behavior underscores the importance of maintaining genetic diversity through the conservation and integration of multiple seed sources.

Given its superior and consistent germination performance, the Pimampiro agroforestry system is identified as a priority seed source for propagation and restoration initiatives involving Juglans neotropica. Future research should incorporate genomic tools and phenotypic plasticity assessments to strengthen seed sourcing strategies and guide climate-resilient reforestation efforts in Andean landscapes.