Phenological Variation of Native and Reforested Juglans neotropica Diels in Response to Edaphic and Orographic Gradients in Southern Ecuador

Abstract

Juglans neotropica Diels, classified as endangered on the IUCN Red List, plays a crucial role in the resilience of Andean montane forests in southern Ecuador—a megadiverse region encompassing coastal, Andean, and Amazonian ecosystems. This study examines how climatic, edaphic, and topographic gradients influence the species’ phenotypic traits across six source localities—Tibio, Merced, Tundo, Victoria, Zañe, and Argelia—all of which are localities situated in the provinces of Loja and Zamora Chinchipe. By integrating long-term climate records, slope mapping, and soil characterization, we assessed the effects of temperature, precipitation, humidity, soil moisture, and terrain steepness on leaf presence, fruit maturation, and tree architecture. Over the past 20 years, temperature increased by 1.5 °C (p < 0.01), while precipitation decreased by 22%, disrupting local edaphoclimatic balances. More than 2000 individuals were measured in forest stands, with estimated ages ranging from 11 to 355 years. ANOVA results revealed that Tundo and Victoria exhibited significantly greater DBH, height, and volume (p ≤ 0.05), with Victoria showing a 30% larger DBH than Argelia, the lowest-performing provenance. Soils ranged from loam to sandy loam, with slopes exceeding 45% and pH levels from slightly acidic to neutral. These findings confirm the species’ pronounced phenotypic plasticity and ecological adaptability, directly informing site-specific conservation strategies and long-term forest management under shifting climatic conditions.

1. Introduction

The study of tree phenotypic traits is fundamental to understanding how environmental variables influence their development and behavior [1]. Multiple studies by prominent researchers [2,3,4,5,6] have demonstrated that climate, soil properties, and biological competition significantly influence tree growth, morphology, and health. Among these factors, climate, soil properties, and terrain slope stand out as critical determinants of phenotypic expression [7,8]. This multidimensional perspective offers a comprehensive view of the interaction between forest biology and environmental conditions, providing valuable insights for the sustainable management of forest resources [9].

Climate change has become a major global concern due to its direct impacts on biodiversity and ecosystem functioning [10]. Trees, in particular, face considerable challenges from climate fluctuations. Variations in temperature, rainfall patterns, and the increasing frequency of extreme weather events have triggered notable phenotypic responses [11]. Recent research has documented how climate change alters tree physiology and behavior, influencing spatial distribution and the timing of seasonal events [12,13,14]. Moreover, rising incidents of drought and wildfires have further exacerbated forest vulnerability and adaptive capacity [10]. According to a report by the NGO Manos Unidas, elevated temperatures, stronger storms, and more frequent droughts are among the key climate change impacts affecting tree health and development [15,16].

The global rise in temperature poses a significant threat to the survival and development of the planet’s flora, especially trees, which are vital components of terrestrial ecosystems. As sessile organisms, trees are highly sensitive to changing environmental conditions that directly affect their growth and reproductive success. Factors such as solar radiation, relative humidity, and ambient temperature are closely tied to tree productivity [17,18]. Thermal stress, intensified by climate change, and shifts in precipitation regimes—as discussed in a recent study [19]—present considerable challenges to tree vitality. These environmental stressors disrupt natural cycles of growth, flowering, and fruiting, thereby negatively impacting phenotypic expression [20,21].

Understanding the environmental factors that shape both genetic and phenotypic intraspecific diversity is essential for decoding the mechanisms that drive divergence in highly diverse biomes such as the Neotropical savannas [22].

Building on this relationship, recent studies underscore the importance of selecting trees based on phenotypic traits linked to soil properties, highlighting how nutrient availability, texture, and water retention capacity directly influence root development, trunk growth, and foliage production. This reinforces the close relationship between soil characteristics and tree physiology, supporting the need to study this interaction as a basis for sustainable forest management [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23].

Juglans neotropica Diels performs best in fertile, well-drained soils—particularly those with loam-clay or loam-silt textures and a pH ranging from slightly acidic to neutral. According to [24], these conditions are ideal for its optimal development. In fact, these soil attributes are crucial for the successful establishment and maintenance of plantations of this species [25].

Therefore, understanding and accurately selecting the appropriate soil type is essential to enhance productivity and ensure long-term viability.

Trees that are ecologically adapted to specific soil conditions not only exhibit enhanced growth but also contribute significantly to biodiversity conservation and resilience to environmental stress. The intrinsic relationship between soil properties and tree development has been demonstrated in studies emphasizing the importance of selecting trees based on phenotypic responses to edaphic factors [23].

Key attributes such as nutrient availability, soil texture, and moisture retention are central to the development of roots, trunks, leaves, and fruits [26,27].

Beyond soil-related factors, tree morphology and structure are intricately linked to geographical conditions, particularly terrain slope. Topographic variation, defined by the inclination of the terrain, plays a critical role in regulating water distribution, erosion dynamics, and the availability of essential nutrients for tree growth [23,26]. Research further suggests that terrain slope can modulate genetic expression and tree architecture, especially in mountainous regions [23]. On steep slopes, trees often develop stronger root systems and aerodynamic crown structures that enhance resilience to wind exposure and other environmental challenges.

Prior to detailing species-specific attributes, an exhaustive literature review was conducted to characterize the phenological patterns of J. neotropica in native forest habitats. These findings informed the selection of study sites and the interpretation of seasonal performance indicators.

In studies of tree phenology, much attention is typically given to environmental factors such as climate, soil, and topography. However, one crucial aspect is often overlooked: tree age. In species like J. neotropica Diels, age is not merely a measure of time but a key determinant of phenological behavior. Young trees, for instance, do not flower or fruit in the same way as mature individuals. Their responses to seasonal changes can be more variable or even absent altogether. This variation in the timing and intensity of phenological events has been documented in several studies [28,29,30], supporting the inclusion of age as a meaningful explanatory variable. Understanding how age interacts with edaphoclimatic and orographic factors can provide a more comprehensive view of phenological patterns in complex ecosystems such as those found in southern Ecuador.

J. neotropica, commonly referred to as Andean walnut, is endemic to the montane forests of South America and plays a pivotal role in ecosystem stability. Its presence on steep slopes contributes to soil conservation and water retention through a complex root system [25]. In addition to its ecological function, this robust tree—distinguished by its bark and edible fruits—enhances biodiversity and offers important resources to mountain communities reliant on high-altitude ecosystems.

Classified as Endangered by the IUCN, J. neotropica provides multiple ecological and societal benefits, including edible seeds, natural dyes, and medicinal properties. It is also valued in urban landscaping due to its environmental adaptability.

To prevent its extinction, conservation strategies that incorporate genetic, ecological, and evolutionary approaches are essential. In this context, the study of biological diversity allows for the evaluation and preservation of species, the understanding of ecological interactions and adaptations, and the advancement of genetic improvement initiatives. Altogether, such efforts help safeguard biodiversity and support the sustainability of forest ecosystems.

This study explores the ecological significance of J. neotropica in montane forest conservation and its interactions with local communities, emphasizing its relevance to both environmental sustainability and human well-being. Therefore, the objective of this research was to analyze the influence of climate, soil type, and terrain topography on the phenotypic traits of parent trees in natural and planted populations of J. neotropica (≥0.5 ha). A comprehensive assessment was undertaken to evaluate how these environmental variables affect dendrometric growth, health status, and reproductive performance.

The selected phenotypic traits—including dendrometric parameters, reproductive indicators, and overall health—were chosen for their relevance as adaptive responses to climatic, edaphic, and topographic variation. Their assessment provides technical insight into the species’ ecological performance and informs targeted conservation strategies.

The main objectives of the study were:

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To deepen the understanding of the ecophysiology of J. neotropica, providing a solid foundation for its scientific analysis and application through slope-adaptation assessment and soil–phenotype correlations.

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To generate technical evidence that contributes to the sustainable management and long-term conservation of both natural and cultivated populations, adapting to diverse environmental conditions.

2. Materials and Methods

2.1. Study Area

The research focuses on Zone 7 in southern Ecuador, located between latitudes 3°30′ and 5°0′ south, and longitudes 78°20′ and 80°30′ west. It borders Zones 5 and 6 to the north, Peru to the south and east, and Peru and the Pacific Ocean to the west [31]. This area, recognized for its biological diversity, is distinguished by steep slopes often exceeding 45%, altitudinal gradients ranging from 400 to 3000 m a.s.l., transitional climates with temperature fluctuations above 25 °C and relative humidity below 70%, and soils of loam-clay to sandy-loam texture with neutral to slightly acidic pH. These physical and environmental traits confer significant ecological importance, shaping both species distribution and phenotypic variation across forest localities [32,33].

The study focused on the provinces of Loja and Zamora Chinchipe, with a combined area of 21,625.3 km², which constitutes approximately 8.65% of the Ecuadorian territory [31]. Six localities were selected: The Tundo, The Victoria, The Tibio, The Merced, The Zañe, and The Argelia. Notably, The Argelia is a forest plantation that has naturalized over time, meaning it exhibits ecological characteristics typical of a self-sustaining native forest—such as spontaneous regeneration, stable canopy cover, and functional integration into the surrounding ecosystem. This naturalization process has been observed over a period of approximately 25 to 30 years, during which the planted stand developed traits consistent with locally adapted populations. These areas host populations of J. neotropica Diels within homogeneous disetaneous forests, primarily belonging to Evergreen Montane Forest and transitional ecosystems of Zone 7, southern Ecuador [32].

To complement the regional description and support ecological interpretation, we compiled a comparative summary of environmental attributes across the six sampling localities.

Table 1. Environmental Context of Juglans neotropica Diels Localities in Southern Ecuador.

Localities	Elevation (m a.s.l.)	Mean Temperature (°C)	Relative Humidity (%)	Ecological Context
The Tibio	2100–2600	~18	90	Southern Montane Evergreen Forest of the Eastern Cordillera of the Andes
The Merced	2000–2500	~20	90	Southern Montane Evergreen Forest of the Eastern Cordillera of the Andes
The Tundo	1200–2400	~14	77	Catamayo-Alamor Semideciduous Foot Montane Forest and Evergreen Seasonal Montane Forest
The Victoria	1000–1600	~23	75	Catamayo-Alamor Semideciduous Foot Montane Forest
The Zañe	2200–3000	~11	80	Southern Montane Evergreen Forest of the Eastern Cordillera of the Andes
The Argelia	2130–2200	~15	78	Southern Montane Evergreen Forest of the Eastern Cordillera of the Andes

Note: The six sampling localities—The Tibio, The Merced, The Tundo, The Victoria, The Zañe, and The Argelia—were selected based on the presence of J. neotropica populations and ecological representativeness. This table provides a general overview of site-level environmental conditions to support territorial characterization and conservation relevance.

presents elevation ranges, mean temperatures, relative humidity, and ecological classifications for each site. Precise coordinates were intentionally omitted due to the IUCN Red List status of J. neotropica as an endangered species; instead, these descriptors serve to spatially anchor the study while preserving location sensitivity.

Fieldwork was carried out in these localities due to their ecological representativeness and concentration of mature individuals of different age classes. The combination of steep slopes, altitudinal gradients, and heterogeneous soils makes this region a suitable setting for evaluating the influence of environmental factors on phenotypic traits, providing key data for medium- and long-term conservation planning [31].

2.2. Climate

To assess the influence of climatic and edaphic factors—including precipitation, temperature, relative humidity, and soil moisture at one-meter depth—data collection was carried out following standardized field protocols in areas where J. neotropica is present in southern Ecuador. To obtain reliable data, globally recognized sources such as POWER—Data Access were consulted, providing daily climatological records from 1981 to the present [34].

These data were verified by cross-referencing climatological information from databases maintained by municipal governments, provincial councils, and parish administrations, as well as reports from both public and private research entities. These complementary sources significantly contributed to illuminating relevant climatic aspects of the study areas [35].

The resulting records originate from within natural populations of J. neotropica monitored over four decades (1981–2023). Climatic data were retrieved using the POWER Data Access Viewer platform on a daily basis over a 12-month period, totaling 365 days per year. Across the 42-year span, this approach yielded 15,330 individual data points per variable, yielding annual mean values that provide a robust and longitudinal characterization of the climatic conditions experienced by J. neotropica across decades.

2.3. Soil

To obtain detailed information on soil properties in the study area, a technical-administrative approach was implemented using SIGTIERRAS 2022 and the geopedological unit. In the first stage, geopedological mapping at a scale of 1:25,000 from SIGTIERRAS was used to analyze soil fertility, as well as physical, chemical, and microbiological aspects of the soil. The methodology incorporated the mass valuation of rural lands, following specific guidelines published in the Geoportal del Agro Ecuatoriano in 2017. Additionally, soil and geomorphology data were integrated to conduct a land use capacity analysis, with the active participation of SIGTIERRAS-MAG-IEE. This comprehensive approach provided a complete understanding of soil characteristics, facilitating more effective planning and sustainable management for J. neotropica.

Data collection was carried out using the SIGTIERRAS platform, selecting relevant variables that helped interpret how they affected the phenotypic characteristics of J. neotropica populations in the study area in southern Ecuador; these variables included hydrogen potential (pH), cation exchange capacity (CEC), fertility, morphology, slope, taxonomic order, texture, drainage, depth, stoniness, salinity, temperature, humidity, and organic matter (OM).

To ensure comprehensive information in the selected locations with the presence of the native forest species J. neotropica, a rigorous and scientific methodology proposed by [33] was implemented. This comprehensive methodology supports the acquisition of precise data on the composition and properties of the forest soil, thus establishing a solid scientific basis for subsequent analysis and conclusions. The process is detailed below:

2.3.1. Identification of Representative Areas

Representative areas larger than 0.5 ha of forest were selected, classified into homogeneous zones based on the predominant vegetation specifically J. neotropica and topography, allowing for precise environmental mapping. Specialized tools, such as shovels and cylindrical corers, were employed to extract composite soil samples at three distinct depths: 0–10 cm, 10–20 cm, and 20–30 cm. For each forest plot larger than 0.5 ha, three subsamples were collected per depth and combined into a single composite sample, ensuring exhaustive and representative collection. All samples were labeled with geospatial and environmental metadata and stored in airtight containers under biosafety protocols.

As shown in (Figure 1), the sampling pit reached a depth of 30 cm, enabling stratified collection across three soil layers.

Representative excavation site illustrating vertical depth measurement during composite soil sampling in a disetaneous forest dominated by J. neotropica in southern Ecuador. The measuring tape marks a depth of approximately 30 cm. This procedure was applied to forest plots exceeding 0.5 ha, with samples collected at three defined strata (0–10 cm, 10–20 cm, 20–30 cm), enabling stratified analysis of soil characteristics across ecological layers.

Furthermore, multiple samples were taken from each locality of origin to capture soil heterogeneity, with each sample labeled with detailed information regarding location, vegetation type, altitude, and orientation, facilitating subsequent analysis and study replication. The samples were stored in airtight containers following biosafety practices, and meteorological conditions, such as temperature and humidity, were documented during the collection process to enrich the environmental context of the analyses.

2.3.2. Sample Conservation and Laboratory Shipment

The soil samples collected were preserved following the methodology proposed by [36]. It was ensured that the samples were precisely labeled and recorded, stored under conditions that prevented contamination, and documented with relevant climatic data. These practices not only ensured the acquisition of accurate data regarding the composition and properties of the soil but also provided a solid scientific basis for future analyses.

2.4. Slope

Topographic Digitization of Localities

To determine the spatial distribution of J. neotropica habitats, a detailed methodological strategy was implemented. A slope map was first generated using control points obtained via GPS. Field information was subsequently processed with ARCGIS 10.8 software to ensure accuracy and robustness of the results. This strategy combined technical precision with local expertise, resulting in a comprehensive assessment of the target areas (Table 1). Terrain slope was calculated as a percentage using a Digital Elevation Model (DEM), establishing a quantitative basis for spatial analysis. A reclassification was then applied using previously defined indices, following the methodology proposed by [37,38] (

Table 2. Indices for classifying slopes in micro-watersheds of different localities.

Slope (%)	Classification Indices
0–15	1
15–30	2
30–45	3
>45	4

). While Table 2 presents the classification indices, Table 1 was designed to identify and digitally map areas with slopes exceeding 30% inclination. Experts and local landowners indicated that surpassing this threshold poses a significant ecological risk, particularly to J. neotropica habitats when forested areas are cleared for anthropogenic activities. In addition, criteria provided by local professionals and producers from the study areas were incorporated—such as site accessibility, slope operability for manual sampling, dominance of J. neotropica, and recent ecological disturbances reported by local actors. These practical, context-specific inputs enriched the methodology by ensuring ecological representativeness and logistical feasibility across selected sampling localities.

The purpose of Table 1 was to identify and digitally map areas with slopes exceeding 30% inclination. Experts and local landowners indicated that surpassing this threshold poses a significant ecological risk, particularly to J. neotropica habitats when forested areas are cleared for anthropogenic activities.

2.5. Phenology

To study the phenology of J. neotropica in ecologically representative contexts, including both native forests and naturalized forest plantations such as The Argelia, a comprehensive methodology was implemented, encompassing various stages [39]. Information was gathered on seasonal patterns, life cycles, and environmental factors influencing the phenology of the selected species. Subsequently, the key species was identified through systematic sampling techniques, using study plots strategically distributed in the native forest.

The geographic layout of the studied localities—including native forests and The Argelia plantation—is illustrated in Figure 2, providing contextual reference for phenological monitoring.

Once the individuals of the species J. neotropica were identified, continuous monitoring protocols were established to record phenological events such as flowering, fruiting, and leaf fall. These records were maintained over a representative period of one calendar year to capture monthly variability. This temporal design follows current environmental sensitivity criteria that emphasize organ-specific phenological responses to climatic variation, as reported by [29]. To complement direct observation, advanced technologies such as state-of-the-art binoculars or prismatic devices were employed to obtain more detailed and precise data on the phenology of the forest species. In addition, visual inspection methods grounded in [40] were applied to assess desirable phenotypic attributes of J. neotropica, including vigor, dominance, structural conformation, apparent health, and foliar expression. This inclusive characterization covered approximately 30% of the individuals recorded in each provenance locality, without applying exclusion criteria, thereby contributing to a representative ecological description aligned with sustainable forest monitoring practices.

Phenological monitoring has been conducted continuously since 2019. In 2023, a partial analytical cutoff was made, linked to the completion of the associated doctoral studies. Nevertheless, monitoring efforts continue to the present day. Given the recurrent patterns observed between 2019 and 2023, representative phenological calendars were developed for each locality.

Data collection was complemented with environmental measurements, including climatic variables, soil properties, and topographic characteristics of the study area. This approach allowed for the analysis of the relationship between phenological events and environmental factors, providing a more comprehensive understanding of the processes governing the phenology in the J. neotropica forest. Finally, advanced statistical analyses were applied to identify significant patterns and establish correlations between the different variables collected.

Phenological monitoring has been conducted continuously since 2019 across ecologically distinct localities. In 2023, a partial analytical cutoff was applied, coinciding with the completion of the associated doctoral research. Despite this cutoff, monitoring efforts remain ongoing. Based on recurrent patterns observed between 2019 and 2023, representative phenological calendars were constructed for each locality. These calendars integrate key developmental events—flowering duration, fruiting onset, and leaf abscission timing—alongside environmental variables such as altitude, slope, temperature, and relative humidity, enabling correlation analyses and supporting the interpretation of phenophase variability.

The proposed methodology aimed not only to document the phenology of J. neotropica within native forest environments, but also to characterize its interactions with surrounding environmental factors across both natural and planted contexts, recognizing the ecological relevance of semi-natural stands in the study region.

This integrative methodology generated foundational insights for the conservation, sustainable management, and ecological restoration of J. neotropica, particularly by encompassing phenological patterns across both native and naturalized forest stands, in alignment with restoration-oriented forest monitoring practices recommended by the [41]. Its rigorous and systematic implementation ensured the acquisition of robust and contextually relevant data, thereby advancing scientific understanding of species dynamics and resilience in response to environmental fluctuations

2.6. Determination of the Age of Trees

2.6.1. The Calculation of Transit Time

In accordance with the mixed-method framework established for the ecological characterization of J. neotropica, transit time estimation was incorporated as a technically justified, non-invasive strategy to approximate tree age across the study area. This approach offered a means to harmonize indirect estimation techniques with empirical field observations, particularly in contexts where legally constrained sampling limited the availability of conventional dendrochronological data. The model’s application draws on regionally validated growth increments (CAI-A), ensuring methodological continuity while enabling diameter-class-based temporal reconstruction.

As part of the mixed-method approach applied to the ecological characterization of J. neotropica, the age of individual trees was estimated through indirect methods, grounded in field-based dendrometric data, archival validation, and ethically sourced plant material.

Given that J. neotropica is classified as “Endangered” by the International Union for Conservation of Nature (IUCN), and in accordance with current regulations from Ecuador’s Ministry of the Environment, Water and Ecological Transition (MAATE), destructive sampling techniques on live specimens are prohibited. This restriction limited the application of conventional dendrochronological methods across much of the study area, thereby necessitating indirect strategies for growth estimation.

Nevertheless, in locations characterized by steep slopes, high wind exposure, and geodynamic landslides, naturally fallen individuals were encountered. In such areas—including sites such as The Tundo, The Victoria, and montane sectors of the Loja Highlands such as The Zañe—material was ethically collected without compromising the integrity of remnant forest populations or violating environmental regulations. For these cases, the method proposed by Gonzaga (1997), cited in [42], was applied. This technique involves the extraction of cross-sections near ground level (Figure 3A), followed by the calculation of the Annual Current Increment (CAI-A) (Figure 3B).

While this study prioritized indirect approaches, it is acknowledged that ring-counting in cross-sections constitutes the most direct method for age determination, as highlighted by [43] in their evaluation of tropical species in India’s Western Ghats. However, in the case of J. neotropica, factors such as indistinct ring formation and legal limitations preclude the systematic use of such techniques.

A total of 2176 individuals were recorded across all surveyed forest localities. The technical procedure was carried out in five stages:

1.

Diameter-Based Classification

Trees were grouped into diameter classes using diameter at breast height (DBH) measurements, taken at 1.30 m above ground level. The class intervals were set at 10 cm, following the methodological criteria proposed by [44] in Silviculture in the Tropics: Tropical Forest Ecosystems and Their Tree Species—Possibilities and Methods for Their Long-Term Management. This segmentation is widely recognized as a practical silvicultural tool for characterizing forest structure and analyzing population dynamics in tropical ecosystems. It enabled the modeling of growth variability and facilitated the construction of class-specific adjustment curves for transit time estimation.

2.

Calculation of Annual Current Increment (CAI-A)

CAI-A was estimated for each diameter class by correlating observed growth with the approximate age of representative individuals.

3.

Adjustment of CAI-A vs. Diameter Class Curve

Values were corrected by class and fitted with a trend curve to model growth variability.

4.

Calculation of Transit Time by Class

Obtained by dividing the range of each diameter class by its corresponding CAI-A, representing the time required for an average tree to transition through the class.

5.

Estimation of Total Growth Duration

The cumulative transit times were summed to estimate age from seedling stage to the upper limit evaluated.

The transit time model served as a complementary adjustment mechanism, grounded in validated CAI-A references, without altering the underlying indirect estimation framework applied throughout the study.

Prior to consulting tropical growth models developed in Asia and South America, region-specific research conducted in Loja province was reviewed. The studies by [45,46] applied dendrochronological methods to Andean forest ecosystems, providing technically relevant parameters for age estimation and biological rotation periods of J. neotropica. These locally derived findings were considered as empirical reference points to support the growth ranges used in the present study.

In sites where direct sampling was not feasible, growth models developed for ecologically similar tropical species—such as Ocotea radiaei, Baikiaea plurijuga, and Mora excelsa—from Malaysia, Guyana, India, and Thailand [42] were consulted. This approach enabled reliable estimates within acceptable error margins, ensuring scientific robustness, legal compliance, and respect for forest conservation principles. It is worth noting that the age estimation model employed was developed with an empirically established margin of error of ±5%, operating at a 95% confidence level, as a methodological basis prior to inferential statistical analysis.

2.6.2. Dendrochronological Validation and Error Estimation

To validate transit-time estimates, a dendrochronological protocol was applied to 12 naturally fallen trees (one section per tree) in The Tundo, The Victoria, and The Zañe:

1.

Sample preparation

Twelve cross-sections were collected at 60 cm above the root collar. Sections were air-dried for 14 days and sanded sequentially with 180, 320, to 4000-µm grit sandpapers.

2.

Ring-width measurement

Under a 20× binocular microscope and using a digital caliper (0.01 mm precision), ring widths were measured and recorded in a structured datasheet.

3.

Visual crossdating

Width patterns (narrow vs. wide rings) across the 12 series were compared to assign each ring to its calendar year. Samples with highly eroded or indistinct rings were excluded.

4.

Correlation between CAI-A and dendrochronological age

Pearson’s correlation coefficient (r) was calculated between ages estimated by the transit-time method (CAI-A) and those obtained by ring counts, yielding r = 0.91 (p < 0.001; n = 12).

5.

Error estimation and confidence intervals

The standard error (SE) of the CAI-A age estimates was computed as:

$$SE={\displaystyle \frac{SD}{\sqrt{n}}}$$

Here SD is the standard deviation of age differences and n = 12.

The 95 % confidence interval was determined by:

$${IC}_{95\%}=t_{\mathrm{0,95},n-1}\ast SE$$

The average margin of error across all diameter classes was ±5%.

These validations confirm the reliability of CAI-A curves and transit-time estimates are supported by reliable dendrochronological ages, strengthening the indirect approach for estimating the age of J. neotropica.

To complement this validation, locality-specific margins of estimation error were calculated using the CAI-A approach across six sampling sites. These site-level averages ranged from ±1.21 years in The Argelia to ±3.08 years in The Tundo, with maximum deviations reaching ±19.23 years in large-diameter individuals (

Table 3. Estimated Margins of Error by Locality Based on CAI-A Transit-Time Methodology.

Locality	Mean ± SD (Years)	Min–Max (Years)
The Tibio	2.20 ± 1.50	0.53–9.61
The Merced	1.64 ± 0.72	0.55–4.06
The Tundo	3.08 ± 3.49	0.43–19.23
The Victoria	2.81 ± 1.76	0.85–8.52
The Zañe	2.07 ± 1.30	0.42–14.82
The Argelia	1.21 ± 0.66	0.42–3.84

Estimated margins of error by forest locality derived from CAI-A transit-time estimates. Dispersion reflects site-specific diameter distributions and ecological heterogeneity.

). This variability reflects local structural heterogeneity and supports a spatially contextualized application of the model. By explicitly reporting error margins, we address calibration concerns raised by reviewers and reinforce the reliability of age estimates within the current ecological framework.

2.7. Data Analysis

For data analysis, climatic variables were examined and graphs were generated using information collected over a 42-year period from the POWER-Data Access platform, covering precipitation, temperature, relative humidity, and Soil moisture at root level exceeded 60% in 1983, 1989, and 1998 in the localities of The Zañe, The Merced, The Tibio, and The Argelia. Time series analysis techniques were employed to identify trends and patterns over time. The data were preprocessed to eliminate outliers, and descriptive statistical methods were applied, visualizing the results using graphs created in Excel. For the soil variable, detailed information was collected using the SIGTIERRAS platform, including physical and chemical properties such as pH, cation exchange capacity (CEC), fertility, morphology, slope, taxonomic order, texture, drainage, depth, stoniness, salinity, temperature, moisture, and organic matter (O.M.) content. These properties were subjected to principal component analysis (PCA) to identify the main factors influencing soil characteristics, with the PCA results contrasted with physical and chemical laboratory analyses conducted for each study locality. Regarding the slope variable of the terrain, georeferenced information was collected and slope maps of the soil were constructed using ArcGIS 10.8, with a DATUM WGS84 projection and UTM coordinates, zone 17 South. The phenological records were analyzed to identify temporal trends in leaf production, flowering, and fruiting intensity across localities and months. Finally, for the age variable, descriptive statistical analysis was conducted, including mean, standard deviation, minimum, and maximum values. Regarding the dasometric variable (DBH), exploratory data analysis was performed to assess distributional assumptions. Given the non-normal distribution of DBH across localities, as evidenced by its skewed structure and high variability, the Kruskal–Wallis test was applied instead of ANOVA. This non-parametric test allowed for the comparison of median DBH values among localities without assuming normality. Post hoc comparisons were conducted to identify statistically homogeneous groups. This approach ensured methodological robustness in evaluating structural differences across provenances. All statistical analyses were conducted using InfoStat/Professional 2020 software.

3. Results

3.1. Climate

Long-term climate monitoring revealed consistent and statistically significant shifts in key ecological variables across natural populations of Juglans neotropica Diels. In The Zañe, The Tibio, and The Argelia, precipitation and soil moisture declined over time (τ = −0.263 and −0.414, respectively; p < 0.05), while temperature increased steadily (τ = +0.427, p < 0.001). These trends, confirmed by the Mann–Kendall test, align with broader regional warming scenarios and suggest a progressive reduction in water availability across mid-elevation zones. These patterns are visually summarized in Figure 4, which illustrates site-specific climatic variation from 1981 to 2023. Relative humidity also decreased significantly (τ = −0.345, p = 0.005), with notable drops in 2005 (68.7%) and 2010 (69.5%), disrupting previously stable microclimatic conditions in montane forests. In contrast, The Victoria and The Tundo exhibited more abrupt climatic shifts. Precipitation declined more steeply (τ = −0.379, p = 0.002), and temperature increases were more pronounced (τ = +0.590, p < 0.001), with values exceeding 30 °C across multiple periods from 1981 to 2023. Relative humidity in these sites fluctuated more sharply, falling below 60% in years such as 1985, 2005–2007, and 2022. Soil moisture remained relatively stable between 50–60%, yet by 2023, a decline to 55% was observed, suggesting a gradual depletion of subsurface reserves. Interannual records show that The Zañe, The Merced, The Tibio, and The Argelia received over 800 mm/year of precipitation during periods such as 1981–1984, 1986, 1989–1990, 1992–1994, 1997–2002, 2008, 2011–2012, 2021, and 2023. In these same sites, temperatures surpassed 25 °C in 2005 and 2010. Meanwhile, The Tundo and The Victoria recorded precipitation above 300 mm/year during 1981–1984, 1986–2004, 2008, 2011–2013, 2015–2017, and 2019–2023, while temperatures exceeded 30 °C consistently from 1981 to 2023. Relative humidity patterns further illustrate site-specific contrasts. In The Zañe, The Merced, The Tibio, and The Argelia, humidity remained above 70% during 1981–2004, 2006–2009, and 2011–2023. In The Tundo and The Victoria, values exceeded 60% during selected years but dropped below that threshold in periods such as 2005–2007, 2009–2011, and 2022. Soil moisture at root level also varied across sites. In The Zañe, The Merced, The Tibio, and The Argelia, values exceeded 60% in 1983, 1989, and 1998. In The Tundo and The Victoria, moisture levels remained between 50–60% throughout the study period, with peaks in 1983 (66%), 1989 (62%), and 1998 (70%), but declined to 55% by 2023. These climatic shifts, interpreted collectively, may compromise seedling recruitment, growth rates, and root-soil interactions essential to the resilience of J. neotropica in fragmented Andean forests.

3.2. Soil

The results present the physical and chemical properties of the soil recorded in natural and planted populations of J. neotropica (

Table 4. Physical, chemical, and morphological soil properties of J. neotropica sampling localities in zone 7 of Ecuador.

Localities	The Tibio	The Merced	The Tundo	The Victoria	The Zañe	The Argelia
pH	6.8	6.7	7.0	7.0	7.0	7.0
CEC meq/100 g	8.0	4.0	14	22	25	8.0
Fertility	Very low	Very low	Low	Medium	Medium	Low
Morphology	Heterogeneous slope	Rectilinear slope	Mountainous relief	Mountainous relief	Mountainous relief	Medium hilly relief
Slope	Steep > 40–70%	Steep > 40–70%	Very steep > 70%	Very steep > 70%	Very steep > 70%	Steep > 40–70%
Taxonomic Order	Inceptisols	Inceptisols	Alfisols	Alfisols	Entisols	Entisols
Texture	Clay loam	Silty clay loam	Clay loam	Clay loam	Loam	Sandy loam
Drainage	Good	Good	Moderate	Good	Good	Excessive
Depth	Shallow	Shallow	Shallow	Shallow	Superficial	Superficial
Stoniness	None	Frequent	Frequent	Few	Abundant	Abundant
Salinity	Non-saline	Non-saline	Non-saline	Non-saline	Non-saline	Non-saline
Temperature	Isothermal	Isothermal	Isothermal	Isothermal	Isothermal	Isothermal
Moisture	Udic	Udic	Ustic	Ustic	Udic	Ustic
O.M	High	Low	Low	Medium	Medium	Low

CODE: Site code; pH: Potential of Hydrogen; CEC: Cation Exchange Capacity; meq/100 g: milliequivalents per 100 gram; Fertility: Soil fertility level; Morphology: Relief morphology; Slope: Gradient as percentage; Taxonomic Order: Soil classification (USDA); Texture: Soil particle composition; Drainage: Water infiltration capacity; Depth: Effective soil depth; Stoniness: Presence of stones; Salinity: Salt content; Temperature: Thermal regime; Moisture: Soil moisture regime; O.M.: Organic Matter.

).

In The Tundo, The Victoria, The Zañe, and The Argelia, the soil presented a neutral pH of 7.0 (Table 4), while in The Merced and The Tibio, it was slightly acidic, with pH values of 6.7 and 6.8, respectively, averaging 6.75 (Table 3). The Victoria and The Zañe had a high Cation Exchange Capacity (CEC), with recorded values of 22 and 25 meq/100 g, respectively (Table 3), For The Tundo, the Cation Exchange Capacity (CEC) was medium, with a recorded value of 14 meq/100 g (Table 3); For The Tibio and The Argelia, the Cation Exchange Capacity (CEC) was low, with recorded values of 8 meq/100 g for both sites (Table 3); and for The Merced, the Cation Exchange Capacity (CEC) was very low, with a recorded value of 4 meq/100 g (Table 3). The Victoria, The Zañe had medium fertility; for The Tundo and The Argelia, it was low; and for The Merced and The Tibio, it was very low. The Tundo, The Victoria, and The Zañe presented a mountainous relief morphology; The Merced had a rectilinear slope; The Tibio had a heterogeneous slope; and The Argelia had a medium hilly relief. The Tundo, The Victoria, and The Zañe had a very steep slope > 70%; The Merced, The Tibio, and The Argelia had a steep slope of 40–70%. The Tundo and The Victoria had a taxonomic order of Alfisols; The Merced and The Tibio had Inceptisols; and The Argelia and The Zañe had Entisols. The Tundo, The Victoria, and The Tibio had a clay loam texture; The Merced had a silty clay texture; The Argelia had a sandy loam texture; and The Zañe had a loam texture. The Tundo had moderate drainage; The Victoria, The Merced, The Tibio, and The Zañe had good drainage, and The Argelia had excessive drainage. The Tundo, The Victoria, The Merced, and The Tibio had shallow depth; while The Argelia and The Zañe had very shallow depth. The Argelia and The Zañe had abundant stoniness; The Tundo and The Merced had frequent stoniness; The Victoria had few stones, and The Tibio had none. Regarding salinity, all locations shared non-saline soil. Regarding temperature, all locations shared isothermal soil. The Tundo, The Victoria, and The Argelia had ustic moisture; The Merced, The Tibio, and The Zañe had udic moisture. The Tibio had high Organic Matter (OM); The Victoria and The Zañe had medium OM; The Tundo, The Merced, and The Argelia had low OM.

3.3. Slope

Topographic slope emerged as a defining environmental variable across all J. neotropica localities, shaping hydrological dynamics, erosion susceptibility, and land-use constraints. All sampled populations were located on terrains with slopes exceeding 45%, a threshold that influences both root anchorage and water retention capacity.

Marked differences in steepness were observed among micro-watersheds, with certain fragments exhibiting abrupt elevation gradients that may affect phenotypic expression and regeneration success. These spatial patterns are visualized in the slope maps presented in Figure 5, which highlight the geomorphological context of each locality and its implications for forest structure and management.

3.4. Phenology

Phenological monitoring of J. neotropica between 2019 and 2023 revealed consistent seasonal rhythms and marked ecological differentiation across localities. Organ-specific responses to climatic variation were documented through continuous sampling protocols, complemented by visual inspection and precision instrumentation. This multifactorial approach—grounded in descriptive statistics and correlation analyses—enabled the identification of intra- and inter-provenance trends in flowering intensity, fruiting onset, and leaf abscission. Rather than offering a simple comparative overview, the analysis captured underlying ecological interactions and adaptive responses, highlighting how phenological rhythms are modulated by altitude, temperature, humidity, and slope. These findings reinforce the importance of geographic and environmental differentiation when designing conservation strategies tailored to the ecological profiles of J. neotropica populations. The high interannual consistency observed across the study period allowed for the construction of phenological calendars for each locality, summarizing the timing and duration of key developmental events. These calendars continue to be refined as monitoring progresses, ensuring that seasonal patterns remain validated and up to date. To deepen the understanding of seasonal dynamics, a multi-year analysis was conducted across four core localities—The Tundo, The Zañe, The Argelia, and The Victoria—coinciding with the completion of associated doctoral research in 2023. Flowering duration ranged from 4 to 9 weeks, with longer cycles in The Tundo and shorter ones in The Victoria. Fruiting onset occurred between weeks 28 and 36, with complete synchrony between The Argelia and The Zañe (r = 1.00), and pronounced asynchrony between The Victoria and The Tundo (r = −0.90). Pearson correlation analysis, based on 84 pairwise comparisons, revealed strong ecological affinities between high-altitude sites and divergent phenological rhythms in lower-elevation fragments. The Victoria consistently exhibited negative correlations across multiple traits, suggesting a distinct ecological profile shaped by thermal and hydric gradients. In contrast, The Argelia and The Zañe showed high synchrony, reinforcing their phenological alignment. These patterns are synthesized in

Table 5. Environmental and Phenological Parameters of Core Localities (2019–2023).

Locality	Altitude (m)	Slope (%)	Flowering Duration (Weeks)	Fruiting Onset (Week of Year)	Leaf Abscission Peak (Week of Year)	Mean Temperature (°C)	Relative Humidity (%)
The Tundo	1850	>55	12	26	17	~14	77
The Victoria	1500	>45	8	21	21	~23	75
The Zañe	2250	>60	8	30	22	~11	80
The Argelia	2150	>45	8	30	22	~15	78

Note: This table integrates environmental and phenological metrics from four core localities—The Tundo, The Victoria, The Zañe, and The Argelia—used for correlation analysis and seasonal development assessment. It complements the general environmental overview presented in Table 1 by linking site conditions to phenological synchrony and contrast.

, which presents a comparative summary of environmental and phenological parameters across the four core localities. The table highlights how altitude, slope, temperature, and humidity interact to influence developmental timing and synchrony, offering a robust framework for site-specific conservation planning.

Building upon the environmental and phenological parameters previously discussed, a correlation matrix was developed to examine pairwise relationships among the four core localities. This analysis encompassed 84 comparisons across seven variables, applying Pearson’s r coefficient to quantify degrees of ecological synchrony and contrast. The results revealed strong affinities between The Argelia and The Zañe, particularly in fruiting onset and leaf abscission (r = 1.00), while The Victoria consistently diverged, especially in thermal and phenological traits (e.g., r = −0.90 in fruiting onset; r = −0.87 in mean temperature). These patterns reinforce the ecological differentiation outlined earlier, underscoring the influence of altitudinal, thermal, and hydric gradients on seasonal development. The full correlation matrix, including technical interpretations for each locality pair, is provided in Supplementary Table S1. This resource offers a granular view of how environmental variables shape phenological rhythms and supports the broader ecological framework established in Table 5.

Seasonal calendars were constructed for four of the six monitored localities, revealing consistent developmental patterns with approximately one-month variations in key events. These patterns are visually summarized in Figure 6, which presents the phenological calendars for The Tundo, The Victoria, The Zañe, and The Argelia, based on continuous monitoring conducted between 2019 and 2023. These calendars synthesize long-term observations and reflect the species’ adaptive responses to site-specific conditions. The Tibio and The Merced were excluded from this synthesis due to insufficient or non-reproducible data—likely resulting from adverse environmental conditions or inconsistencies that compromised scientific validity. Despite these limitations, the selected localities provide robust and representative insights into the phenological dynamics of J. neotropica, highlighting the interplay of climatic, edaphic, and geographic factors in shaping seasonal development.

Taken together, these findings establish a robust foundation for site-specific conservation planning and adaptive management. In ecologically distinct localities such as The Victoria, the observed phenological asynchrony likely reflects unique adaptive pressures and habitat requirements. Recognizing these divergences is essential for tailoring interventions that align with local ecological dynamics, ensuring that conservation strategies remain responsive to both species-level traits and territorial gradients.

3.5. Age Structure

Age estimations of J. neotropica populations across six localities were derived from field-measured diameters and calibrated using previously established growth correlations. These estimations, represented in Figure 7, reveal distinct demographic profiles shaped by site-specific ecological histories.

In The Tibio, tree ages ranged from 14 to 187 years, distributed across nine diameter classes. The Merced exhibited a narrower age span, from 14.8 to 81.2 years, across six classes. The Tundo showed the broadest age distribution, with individuals ranging from 12.8 to 355 years, represented in eleven classes. In The Victoria, ages ranged from 20.7 to 170.4 years across eight classes, with the first diameter class unrecorded due to the absence of specimens. The Zañe presented ages from 12.8 to 296.5 years in ten classes, while The Argelia ranged from 11.8 to 76.8 years across six classes.

These age distributions reveal contrasting forest dynamics. Sites such as The Tundo and The Zañe exhibit wide age ranges, suggesting heterogeneous regeneration processes and possible historical disturbances or selective extraction. In contrast, The Argelia and The Merced display narrower age spans, potentially indicative of more recent establishment or uniform recruitment episodes. The absence of individuals in the smallest diameter class at The Victoria may reflect limited regeneration, sampling gaps, or site-specific constraints.

Such demographic contrasts underscore differentiated successional stages and reinforce the need for localized forest management strategies. Tailoring interventions to each site’s age structure is essential for promoting sustainable regeneration, conserving mature individuals, and maintaining long-term ecological resilience.

3.6. Statistical Data Analysis and Its Influence on the Phenotypic Traits of J. neotropica in Southern Ecuador

3.6.1. ANOVA Climate

Statistical analysis using ANOVA revealed significant differences (p < 0.05) in climatic variables across the studied localities, confirming spatial heterogeneity in the environmental conditions that influence J. neotropica populations. These differences are illustrated in Figure 8, which compares temperature and precipitation patterns among the six monitored sites and highlights statistically significant contrasts. Mean annual temperature varied notably between mid-elevation and lowland sites, with potential implications for phenological timing, growth rates, and DBH–age correlations. Precipitation patterns also differed significantly, with southern localities exhibiting lower rainfall levels—conditions that may be linked to narrower age distributions and reduced recruitment potential.

These climatic gradients offer a coherent explanatory framework for the ecological contrasts described in Section 3.5 and Section 3.6. They underscore the role of abiotic factors in shaping demographic structure, regeneration dynamics, and phenotypic expression across populations. Understanding these spatial patterns is essential for interpreting site-specific responses and for guiding adaptive management strategies that align with local environmental realities.

3.6.2. Biplot Analysis of the Physical and Chemical Properties of Soil

The biplot analysis revealed distinct interactions between soil properties and localities, allowing for the identification of key ecological patterns. These relationships are visualized in Figure 9, which displays the distribution of physical and chemical soil variables across the six monitored sites. Component 2 (CP2) distinguished The Tibio and The Merced based on variables such as taxonomic order, depth, cation exchange capacity (CEC), moisture, fertility, and drainage. In contrast, The Tundo, The Victoria, The Argelia, and The Zañe grouped around the remaining properties, indicating shared edaphic profiles.

When physical and chemical properties were examined independently, specific affinities emerged: The Tibio and The Merced closely aligned in terms of CEC; The Argelia and The Zañe shared similarities in texture and stoniness; and The Tundo and The Victoria exhibited convergence in slope characteristics. Soil temperature and salinity showed minimal variation across all sites, suggesting the influence of uniform climatic conditions on these parameters.

The construction of CP1 was primarily weighted by organic matter (OM), pH, morphology, slope, and CEC, while CP2 was shaped by taxonomic order, depth, texture, drainage, and fertility. These components provide a robust framework for interpreting edaphic differentiation and its potential influence on phenotypic expression and site-specific adaptation.

3.6.3. Statistical Comparison of Phenotypic Traits

Phenotypic variation across J. neotropica populations was assessed using both parametric and non-parametric approaches, depending on the distributional properties of the data. These results are summarized in Figure 10, which compares DBH, height, volume, and phenological traits across the six monitored localities. Due to the lack of normality and homogeneity of variances in DBH measurements, a Kruskal–Wallis test was applied, revealing significant differences among the six localities (H = 157.69; p < 0.0001). The Victoria and The Tundo exhibited the highest median DBH values, while The Argelia recorded the lowest, suggesting structural heterogeneity shaped by ecological gradients and regeneration dynamics.

For traits such as Total Height (TH), Total Volume (TV), and Phenology (%), ANOVA was employed, as assumptions of normality and homoscedasticity were met. The Tundo and The Victoria consistently showed superior values across these traits, indicating favorable growth conditions and enhanced phenological performance. In contrast, The Argelia and The Zañe recorded the lowest values, reinforcing the ecological contrasts discussed in previous sections and highlighting the influence of site-specific environmental factors on phenotypic expression.

4. Discussion

4.1. Precipitation and Temperature

The results of this study confirm a sustained increase in temperature across the Loja–Zamora region, matching the trends reported by [47], who analyzed data from 40 meteorological stations from 1970 to 2000. While average temperatures remained below 18 °C in previous decades, recent records indicate a significant rise. Although no formal trend modeling was performed, temperature and precipitation trajectories were evaluated by contrasting historical records (1970–2000) with recent data (2001–2024) from study localities. Temporal shifts were interpreted using standardized annual means, consistent with regional methodologies [47,48]. This warming trend correlates with phenological changes observed in Juglans neotropica Diels, especially in earlier flowering and faster seed maturation, consistent with the responses described by [11,22,48].

Precipitation patterns display altitudinal divergence: higher zones show reduced rainfall, while lower elevations reveal an increasing trend, also supported by [47]. These dynamics affect phenological events such as reproductive synchrony, leaf expansion, and fruit drop timing. Data collected in different study localities reflect these hydrological sensitivities, particularly during extended drought periods.

Importantly, regional climate variability is heavily influenced by global phenomena—most notably the El Niño Southern Oscillation (ENSO)—which intensify temperature and rainfall anomalies in this transboundary region [25,49]. These interactions underscore the need to integrate global climatic variables into localized phenological models and adaptive forest management frameworks.

Study limitations include the use of interpolated climate data instead of in situ sensor readings, which may obscure microclimatic nuances. Additionally, phenological monitoring was partially limited in steep zones due to access constraints.

Practical applications of these findings include the need to adjust seed collection schedules, nursery cycles, and restoration interventions based on changing precipitation and temperature regimes. Further research should extend multi-year monitoring and incorporate genetic markers to assess the adaptive capacity of J. neotropica under evolving climatic scenarios.

4.2. Relative Humidity

The relative humidity patterns observed across the Loja–Zamora region reveal a complex interplay between altitude, seasonality, and long-term climatic shifts. Our data confirm that, while national averages remain above 70%, localized deviations—particularly in Sozoranga and parts of Loja and Zamora—suggest emerging microclimatic stressors. Notably, humidity values below 60% were recorded in multiple years (e.g., 1985, 2005, 2010, 2018), indicating episodic intensification of dry conditions that may reflect broader climatic transitions.

These findings align partially with the national trends reported by [50], yet diverge in their temporal and spatial granularity. For instance, while [50] describes stable humidity in the Oriente region (e.g., M008 Puyo station), our data show that inter-Andean zones such as Sozoranga exhibit greater variability and susceptibility to sub-threshold humidity events. This divergence underscores the need to disaggregate national datasets when modeling phenological responses at the landscape scale.

From a phenological standpoint, the observed declines in relative humidity coincide with shifts in reproductive synchrony and leaf phenophases in J. neotropica, particularly in sites with prolonged dry spells. Although our study did not isolate humidity as a sole driver, the correlation between low humidity years and delayed leaf expansion or fruit drop suggests a modulatory role, consistent with findings from [51,52]. These authors demonstrated that reduced atmospheric moisture can exacerbate water stress and disrupt phenological timing in broadleaf species, a dynamic we also observed in montane populations.

However, our study is limited by the use of interpolated humidity data and the absence of continuous in situ sensor readings, which may obscure short-term fluctuations and microclimatic effects. Microclimatic variability, modulated by factors such as slope orientation, canopy cover, and shading, can significantly influence the phenological expression of montane species like J. neotropica. Although this study relied on interpolated data, it is acknowledged that local microclimatic conditions may alter the synchrony of reproductive events, particularly in steep and ecologically fragmented zones. Ref. [53] demonstrated that forest canopy structure and terrain regulate microclimate through processes such as heat dispersion via evapotranspiration and solar radiation attenuation, directly affecting forest phenology. Moreover, the interaction between temperature, precipitation, and slope generates nonlinear effects on the onset and end of the growing season, with phenological divergences observed between shaded and sun-exposed slopes. Therefore, it is recommended to incorporate temperature and humidity sensors at both soil and canopy levels in future monitoring phases, and to evaluate slope orientation effects on phenological dynamics. This approach would better capture short-term fluctuations and strengthen predictive models of phenological response under climate change scenarios. Additionally, the lack of concurrent soil moisture measurements restricts our ability to disentangle atmospheric versus edaphic influences on phenology.

Despite these constraints, the practical implications are clear: restoration planning and nursery scheduling must account for humidity variability, especially in zones prone to episodic dryness. Incorporating humidity thresholds into phenological models could improve predictions of flowering and fruiting windows, thereby enhancing seed collection strategies and adaptive forest management.

Future research should prioritize multi-year monitoring with integrated humidity and soil moisture sensors, ideally coupled with genetic profiling to assess the adaptive capacity of J. neotropica under increasingly variable hydroclimatic regimes.

4.3. Soil Moisture at a Depth of One Meter (%)

The soil moisture data collected across the localities of Zañe, Merced, Tibio, and Argelia reveal consistently high root-zone water availability, with values exceeding 60% in the years 1983, 1989, and 1998. In contrast, the sites of Tundo and Victoria display a stable hydric profile ranging between 50–60%, with notable peaks in 1983 (66%), 1989 (62%), and 1998 (70%), followed by a slight decrease to 55% in 2023. These findings indicate prolonged subsurface hydric stability, punctuated by episodic moisture surges that may correspond to ecological thresholds relevant to the reproductive behavior of J. neotropica.

As demonstrated by [54], soil moisture plays a critical role in vegetation phenology, particularly in low-altitude ecosystems affected by water limitations. Although the sites analyzed in this study belong to montane zones, the years with elevated root-zone moisture coincide with enhanced phenological synchrony in fruiting and leaf expansion across monitored populations. This correlation—though not examined through direct physiological tracking—suggests a modulatory role of stable subsoil water availability in phenological timing, reinforcing the arguments put forth by [55,56] regarding subsurface hydrodynamics and root system architecture.

The soil moisture values observed in this study also fall within or exceed those reported by [57], who measured elevated moisture at depths of 80–100 cm; by [58], who documented fluctuations between 0.49 and 0.54 m³/m³ under slopes of 14–20%; and by [59], whose findings further confirm the upper bounds of soil water retention in montane environments.

Nevertheless, the use of interpolated data limits the granularity of our interpretation. The absence of in situ sensor arrays and real-time measurements at multiple depths restricts precise correlation with microclimatic and edaphic variations. Moreover, the lack of phenological monitoring specific to the root zone prevents conclusive links between subsoil moisture and reproductive timing in J. neotropica.

Despite these limitations, the practical implications are clear. Sites with sustained subsoil moisture—particularly Tundo and Victoria—may offer favorable conditions for nursery establishment and seedling survival. Integrating soil moisture thresholds into restoration planning and phenological modeling could enhance the accuracy of flowering and fruiting predictions, improve seed collection strategies, and guide site-specific reforestation efforts.

Future research should incorporate multi-depth soil moisture sensors and pair them with phenophase-specific observations and genetic profiling. This approach would clarify the adaptive responses of J. neotropica under variable hydroclimatic regimes and contribute to the design of ecophysiologically sound restoration strategies across montane forest landscapes.

4.4. Soil

The physical and chemical attributes of the soil observed across the study sites reveal consistent patterns that appear to influence the viability and phenology of J. neotropica. In particular, sites such as The Victoria and The Zañe, which exhibit neutral pH (7.0), high cation exchange capacity (CEC: 22–25 meq/100 g), and medium fertility, coincide with mountainous morphology, good drainage, and a very steep slope—conditions that may contribute to stable phenophase expression and fruiting success. Conversely, The Merced and The Tibio show very low fertility, acidic pH (average 6.75), and lower CEC values (4–8 meq/100 g), associated with silty clay texture and heterogeneous relief, possibly indicating more restrictive conditions for root development and water retention.

Such findings support a localized edaphic influence on J. neotropica populations in Loja and Zamora, aligning with broader studies of forest ecology. For instance, [60] demonstrated that altitude, soil texture, and water content modulate species diversity, while [61] highlighted the role of organic matter and nitrogen in shaping phenological patterns—traits mirrored in our dataset where The Tibio, with higher OM content, may offer conditions for prolonged foliar expansion. Furthermore, [62,63] reinforce the relevance of slope and pH in distributional models, matching well with the alfisol-based profiles of The Victoria and The Tundo.

The consistency of traits such as isothermal regimes, neutral to slightly acidic pH, and steep slopes across several localities suggests a potential edaphic envelope conducive to J. neotropica establishment. However, the classification of soils into Inceptisols, Entisols, and Alfisols also implies varying degrees of horizon development and nutrient retention, which may differentially affect rooting depth and physiological resilience.

Nevertheless, the absence of longitudinal in situ physiological tracking and limited granularity in soil property measurements (e.g., no real-time monitoring of salinity fluctuations or root-zone temperature gradients) represent important limitations of this study. Additionally, the reliance on static soil profiles constrains temporal interpretation and inhibits direct causality claims regarding phenological modulation.

Despite these constraints, the practical implications are compelling. Identifying sites with high CEC, medium fertility, and stable drainage—such as The Victoria and The Zañe—could enhance the success of nursery operations and improve seedling establishment in restoration programs. Integrating these soil parameters into predictive phenological models may refine the timing of fruit collection and optimize planting strategies across montane landscapes.

Future research should incorporate multi-seasonal sampling protocols, including soil nutrient dynamics, moisture fluctuations, and below-ground phenological observations. Coupling these with genetic profiling and altitude-based climate modeling would strengthen the predictive power of site selection for J. neotropica, ultimately contributing to resilient forest management and conservation strategies.

4.5. Slope

Research has shown that the slope of the terrain plays a significant role in the phenology of broadleaf species, differing from the more direct impact of physical and chemical soil properties, which have also been widely studied. For instance, the study by [64] investigated how topographic variations affect plant community characteristics and soil factors in alpine grasslands, finding that both the orientation and gradient of the slope significantly influence species distribution and soil attributes. Similarly, [65] analyzed the use of satellite-based observations to monitor and predict phenology at landscape scale, concluding that slope is a critical determinant in the temporal dynamics of plant communities.

Our study contributes to this understanding by identifying consistent patterns in the location of J. neotropica populations on slopes exceeding 40%, with all sampled micro-watersheds exceeding 45% (Figure 5). These steep gradients appear to shape habitat suitability by influencing water drainage, erosion potential, and microclimatic variation. The spatial restriction of populations to these topographic conditions supports the notion of slope-driven niche limitation, as previously suggested by [37].

Nevertheless, the ecological stability of these zones is compromised by their inherent vulnerability to landslides and erosion, particularly under conditions of reduced vegetation cover. Field observations revealed slope destabilization in areas undergoing agricultural expansion—a trend also discussed by [66] in the context of ecosystem service degradation. Such pressure may undermine the ecosystem functions that steep-slope forests provide, generating tension between land use expansion and ecological resilience.

While the study provides topographic insights, it does not incorporate slope orientation analysis (e.g., north-facing vs. south-facing aspects) nor long-term erosion modeling, which may yield deeper understanding of soil–plant interactions and forest anchorage dynamics. Additionally, no phenological traits were directly correlated with slope gradient due to accessibility constraints in rugged terrain.

Given these findings, it is plausible that steep-slope zones where J. neotropica persists could represent priority areas for conservation, potentially serving as genetic reservoirs. The integration of slope-aware forest management and satellite monitoring could enhance land use planning and restoration strategies. Further research could explore links between slope gradient and reproductive timing, root stabilization, and runoff dynamics, shedding light on adaptive thresholds relevant to montane forest conservation under changing climatic conditions.

4.6. Phenology

The phenological patterns observed in J. neotropica in the The Argelia—The Tundo area reflect a marked sensitivity to the site’s topographic and edaphic conditions, consistent with previous studies highlighting intraspecific variability during nursery and plantation stages [33]. This phenotypic plasticity suggests adaptive potential that can be leveraged in restoration strategies tailored to elevation and slope gradients.

The predominance of budburst events prior to the onset of the rainy season indicates synchronization with increased soil moisture and surface nutrient availability, particularly in areas with residual vegetative cover. This trend aligns with findings by [67], who reported accelerated germination responses under optimized prehydration and mechanical scarification conditions.

Moreover, the flowering and fruiting patterns observed are consistent with the ecological fragmentation framework described by [25]. The reduction of continuous cloud forest areas generates selective pressures that shape reproductive phenology, resulting in more concentrated and less synchronized phenophases in isolated patches. This phenomenon translates into a phenological offset between localities, where differences in altitude, slope, and vegetation cover influence the timing of reproductive events. In this regard, the findings of this study underscore the urgent need to reestablish ecological corridors that facilitate genetic exchange and extend the reproductive window.

Thus, the phenological dynamics of J. neotropica reflect not only climatic adaptations but also functional responses to structural landscape degradation. Silvicultural management should incorporate these findings as criteria for differentiated planting, especially on exposed slopes and areas with high altitudinal variation, prioritizing provenances with greater phenological plasticity.

Although a partial cut was made in 2023 for academic purposes, phenological monitoring continues to this day. The consistency of the patterns recorded over five years supports the validity of the calendars developed, which provide a solid foundation for the ecological characterization and silvicultural planning of J. neotropica.

The statistical refinement applied in this study—particularly the use of Kruskal–Wallis for DBH and ANOVA for phenological traits—enhanced the resolution of inter-locality comparisons and reinforced the ecological validity of the observed patterns. The superior structural attributes recorded in The Tundo and The Victoria, such as higher DBH and total volume, coincided with more synchronized and extended phenophases, suggesting a functional linkage between growth performance and reproductive timing. In contrast, The Argelia and The Zañe exhibited both reduced structural metrics and fragmented phenological events, likely shaped by altitudinal stressors and landscape degradation. These findings validate the phenological calendars developed and support their use as tools for site-specific silvicultural planning, emphasizing the importance of selecting provenances with both structural vigor and phenological resilience.

4.7. Age

The age structure of J. neotropica populations across six localities in southern Ecuador reveals marked variability, with estimated ages ranging from 11.8 to 355 years, distributed across distinct diameter classes (Figure 7). The Tundo locality stands out for hosting the oldest individuals, while The Merced and The Argelia exhibit comparatively younger populations. These differences likely reflect site-specific edaphic conditions, elevation gradients, and varying disturbance and management regimes.

Such variation is not merely demographic—it directly influences forest phenology. As noted by [68], age-class structure exerts a significant indirect influence on intra-annual phenology, mediated by species composition. This interaction suggests that forests with diverse age distributions, such as Tundo and Zañe, may exhibit more resilient phenological patterns under regional climatic variability, compared to younger or more homogeneous stands.

Growth variability is also evident in the Current Annual Increment (CAI) values reported by various sources: 1.2 cm according to [69], 0.37 cm by [45], ranges between 0.648 and 0.834 cm by [46], and specific values from [32] for Tundo (0.602 cm), Shucos (0.652 cm), Saraguro (0.828 cm), and The Argelia (0.65 cm). Ref. [70] reported a lower value of 0.12 cm, underscoring the influence of local ecological conditions. The correlation between these increments and diameter-based age estimates supports the reliability of the approach used for J. neotropica.

Recent studies confirm the dendrochronological potential of J. neotropica in tropical montane environments. Ref. [71] demonstrated the presence of annual rings and the species’ sensitivity to El Niño-related precipitation anomalies through isotopic and radiocarbon analyses in Piura, northern Peru. Likewise, [33] documented significant phenotypic variability among provenances during nursery and plantation stages, reinforcing the ecological influence of origin on growth and age structure.

Although systematic ring counting was not applied to all individuals in this study, direct ring counts were conducted on 12 reference trees, allowing for empirical calculation of CAI-A. These values, together with those reported by other researchers—[33,45,46,69,70,72]—formed the basis for estimating the age of all individuals across the surveyed localities. The consistency between these data and the diameter-class estimates validates the model employed and demonstrates its utility for accurately characterizing the age structure of J. neotropica under contrasting ecological conditions.

Furthermore, regional silvicultural guidelines and biological cutting cycles proposed by [73,74] reinforce the ecological and management relevance of age-based classifications, especially for endangered montane species such as J. neotropica.

Although the methodological design was robust and achieved adequate temporal and territorial coverage, two relevant limitations were identified. First, the selective harvesting of adult individuals in certain sectors of the study area may have altered the natural population structure, reducing the presence of specimens with larger diameters and advanced phenological maturity. This condition could have influenced the expression of the reproductive events observed.

Second, there was a notable lack of previous studies on the phenology of J. neotropica in natural forests of southern Ecuador. This gap limited the possibility of making direct comparisons and establishing regional references, which necessitated the development of interpretative criteria based on empirical observation and contextual analysis. Nevertheless, both factors were considered in the analysis, and field validation strategies were applied to ensure the reliability of the results. In this context, the few available studies—such as [39]—provided foundational insights into the species’ reproductive timing and ecological responses, which we used to frame its role in Andean montane forest resilience. By integrating these references with our empirical findings, we highlight J. neotropica as a structurally and functionally relevant species in high-altitude forest dynamics, particularly under scenarios of climatic stress and habitat fragmentation.

Based on the results obtained and the phenological variations recorded across localities, three key aspects are identified to support the adaptive management of J. neotropica under conditions of high environmental variability. First, sites with high cation exchange capacity (CEC), such as The Victoria (22 meq/100 g) and The Zañe (25 meq/100 g), exhibited superior structural and phenological performance. This suggests a mechanistic link whereby soils with higher CEC retain essential macronutrients (e.g., N, P, K) more effectively, enhancing nutrient availability during critical phenophases such as flowering and fruiting. This nutrient retention likely supports reproductive synchrony and biomass accumulation, consistent with findings in tropical montane forests that associate high CEC with increased plant productivity and niche specialization [75].

Second, the diverse age-class structure observed in The Tundo (12.8–355 years) and The Zañe (12.8–296.5 years) appears to modulate phenological resilience under climatic variability. Older trees may buffer environmental fluctuations through deeper root systems, stable carbon allocation, and genetic memory, acting as ecological stabilizers and reservoirs of adaptive traits [76].

Third, to address limitations associated with interpolated climate data and to strengthen ecological modeling, it is proposed to integrate in situ microclimate sensor networks (temperature, relative humidity, soil moisture) with remote sensing tools and local meteorological stations. This approach, validated in humid montane forests using low-cost wireless systems [77], would enable high-resolution monitoring of short-term variability and microclimatic heterogeneity, facilitating the development of robust predictive models and the identification of patterns relevant to silvicultural planning. In parallel, evaluating the response of J. neotropica to climate change scenarios—particularly in altitudinal transition zones and highly fragmented landscapes—through eco-physiological studies, multi-scenario simulations, and long-term monitoring could help establish tolerance thresholds, project potential altitudinal shifts, and define adaptive restoration strategies.

Finally, the design of silvicultural trials with contrasting provenances is recommended, based on their structural and phenological performance. This approach would allow validation of proposed phenological calendars, identification of genotypes with greater adaptive plasticity, and optimization of plant material selection for reforestation and conservation programs in climate-vulnerable territories.

4.8. Methodological Limitations

This study faced methodological constraints due to current environmental regulations. Since J. neotropica is classified as “Endangered” by the IUCN and Ecuador’s Ministry of the Environment (MAATE) prohibits destructive sampling of live specimens, dendrochronological validation was limited to 12 naturally fallen trees found in areas with steep slopes and high wind exposure. This condition is acknowledged as a limitation of the study, although rigorous protocols were applied to ensure the reliability of the data obtained.

Additionally, while the calculation of the Adjusted Current Annual Increment (CAI-A) is described in detail within the methodological framework, the absence of a simplified formula may hinder understanding for some readers. For clarity, CAI-A can be expressed as:

CAI-A = ΔD/Δt

where ΔD represents the observed diameter increment and Δt the estimated transit time per diameter class. This formula summarizes the principle applied in the growth modeling and may facilitate replication in future studies.

5. Conclusions

The results reveal marked climatic, edaphic, and phenological variability among the studied localities, directly influencing the population dynamics of Juglans neotropica Diels in fragmented Andean forests.

Significant trends of increasing temperature and decreasing relative humidity and precipitation, especially in sites such as The Argelia and The Victoria, suggest growing environmental pressure that may compromise the species’ natural regeneration. Cation exchange capacity (CEC) was highest in The Victoria and The Zañe (>20 meq/100 g), indicating greater potential for nutrient retention and vegetative development. Steep slopes (>45%) present in all localities represent a critical factor for reforestation planning, affecting erosion, drainage, and nutrient distribution.

Phenology showed intra- and inter-locality variations, with consistent patterns in four sites, while The Argelia exhibited lower phenological expression, possibly associated with less favorable edaphic and climatic conditions. Notably, The Argelia corresponds to a naturalized planted forest with over 70 years of establishment, suggesting that despite its structural maturity, factors such as low fertility, excessive drainage, and low CEC may limit its phenological performance.

Age structure revealed important contrasts among localities. Sites such as The Tundo and The Zañe exhibited wide age ranges, suggesting heterogeneous regeneration processes and possible historical disturbance or selective extraction events. In contrast, localities like The Argelia and The Merced showed narrower age distributions, potentially reflecting recent establishment or uniform recruitment. This age variability directly influences phenological expression, as younger individuals tend to exhibit less defined or incomplete patterns.

For reforestation, efforts should focus on provenances with CEC > 20 meq/100 g and slopes > 45%, while monitoring phenological changes in younger stands such as The Argelia to adjust adaptive management strategies. These findings provide a solid foundation for designing ecological restoration strategies tailored to local edaphic and climatic conditions, contributing to the effective conservation of J. neotropica in the Ecuadorian Andes.

Moreover, these findings align with Ecuador’s national forest restoration policies, particularly those aimed at enhancing ecosystem resilience and promoting native species recovery in degraded Andean landscapes. By identifying edaphic and climatic conditions favorable for J. neotropica, this study provides actionable insights for site-specific reforestation planning, contributing to the strategic goals of national programs such as Plan Nacional de Restauración de Ecosistemas Forestales (National Plan for Forest Ecosystem Restoration) and Programa Nacional de Reforestación (National Reforestation Program). Integrating these results into regional restoration frameworks can improve the effectiveness of conservation efforts and support long-term forest sustainability.