Fine Roots in Hemiboreal Forest Stands and Clearcut Areas with Nutrient-Rich Organic Soils in Latvia: Morphological Traits, Production and Carbon Input

Abstract

Within this study, we evaluated the fine root (trees and understory vegetation combined) morphological traits, fine root production (FRP), and carbon (C) input with fine root litter in forest stands (dominated by either coniferous or deciduous trees) and clearcut areas (previously dominated by coniferous trees) with nutrient-rich organic soils. The study was conducted in 26 sites in hemiboreal forest land in Latvia and summarizes the results obtained in a two-year study (2020–2022) using the root ingrowth method. Traits and production of fine roots varied significantly depending on forest development stage (stand or clearcut area), dominant tree species type (coniferous or deciduous), and soil drainage status (drained or naturally wet). According to the results of the second study year, mean FRP among groups of study sites varied from 0.58 ± 0.13 to 1.38 ± 0.28 t ha⁻¹ yr⁻¹, while C input with fine root litter ranged from 0.28 ± 0.06 to 0.68 ± 0.14 t C ha⁻¹ yr⁻¹. More than half (59 ± 4%) of the total FRP occurred in the upper 0–20 cm soil layer. FRP tended to correlate positively with soil C/N ratio and negatively with soil pH and soil nutrient concentration. Incubating ingrowth cores for at least two years is strongly recommended to accurately estimate annual FRP and C input. This helps to avoid potential underestimation that may occur when using results of only one incubation year (12 months after ingrowth core installation). This study provided new insights into the dynamics and traits of fine roots and will help to improve the accuracy of C flow estimation in hemiboreal forests with nutrient-rich organic soils in Latvia.

1. Introduction

Fine roots, commonly defined as plant roots with a diameter of two millimeters or less, are essential for overall health, resilience, and functioning of forest ecosystems, despite their diminutive size [1,2]. These subterranean structures are crucial for water uptake, nutrient cycling, and soil stabilization [3]. While fine root biomass makes up only a small percentage (0.5%–10%) of the overall forest biomass, fine root production (FRP) is estimated to account for almost one-third of the world’s yearly net primary production [4,5,6]. Fine roots contribute significantly to litter inputs in forest ecosystems, making up 33% of the total litter input [7]. Fine root litter decomposition plays a crucial role in carbon (C) and nutrient cycling, as they mineralize and release nutrients essential for plant and microbial uptake [7,8]. Moreover, tree fine roots serve as the foundation for the development of the root network, facilitating the establishment of mycorrhizal associations. This symbiotic relationship enhances tree nutrition and stress tolerance, bolsters defense against diseases, and helps to maintain soil structure and stability [9,10].

FRP, as well as anatomical and morphological traits, exhibits considerable variation attributed to genetic diversity (plant functional type) and variations in forest site characteristics including soil resource availability and local environmental conditions [1,11,12]. Fine root morphological characteristics and production are predominantly influenced by the following environmental factors: soil temperature, soil moisture conditions (including groundwater level and precipitation patterns), nutrient availability, soil density and compaction, oxygen availability, and ecological relationships with other species [13,14,15,16,17,18]. The average fine root biomass is smaller in the cooler climatic conditions of boreal forests compared to the warmer conditions of temperate and tropical forests, which might be related to the availability of water and nutrients [6,19,20]. The fine root biomass in boreal and temperate forests has been reported to increase with higher amounts of precipitation [21,22,23]. The vertical distribution of fine root biomass demonstrates a trend characterized by elevated root densities within the uppermost soil layers, particularly within the 0–10 cm or 0–20 cm depth ranges, and a steady decrease in density as soil depth rises [24,25]. Fine root characteristics may vary among different tree species. In a study carried out in North-Western Slovakia, Norway spruce exhibited a lower overall standing stock of fine roots compared to European beech, and spruce roots were located closer to the soil surface than beech roots. Additionally, spruce showed higher seasonal variability and turnover rates of fine roots. Beech, on the other hand, demonstrated a higher ratio of root production to mortality, potentially due to better resistance to physiological stresses, such as drought episodes [26]. A study carried out in Canada shows that temperate tree species (e.g., Pinus strobus, Picea glauca, and Quercus rubra) exhibit varied acclimation responses in absorptive fine root biomass and rooting depth in response to varying water availability [27]. Additionally, a significant impact of stand age on fine root biomass has been found. It is generally considered that fine root biomass increases until canopy closure, after which it remains stable without increasing or decreasing [28,29,30]. In addition, variation in FRP and mortality from year to year is relatively large in young forest stands compared with closed-canopy forests where interannual changes in the fine root standing crop is generally small [31]. However, in a study carried out in a Norway spruce chronosequence in Norway, stand age did not influence the number of root tips or the frequency of branching [32]. Seasonality in fine root development has also been observed in northern forest ecosystems, where root growth typically peaks in late spring and early summer, followed by a rapid decline in the fall [32,33]. Several previous studies also show that higher soil acidity generally leads to lower fine root biomass, though there can be species-specific variations [34,35,36].

In forest ecosystems, fine root dynamics (production, mortality, and standing crop) can be altered due to disturbance events including clearcutting and thinning, and forest stands may become a source of carbon dioxide (CO₂) emissions [31,37,38]. Clearcut harvesting has been noted to disrupt a key pathway for replenishing soil organic matter C stocks by severing the connection provided by living roots [37]. At the same time, the fine root standing crop increases rapidly in the early years after clearcutting, and then remains relatively stable for many years thereafter [31]. Considering the crucial role of fine roots in C cycling, studies on the impact of clearcutting are essential but remain scarce. A study conducted in Norway explored whether there are significant differences in the average diameter of fine roots, root biomass, and specific root length between natural forests and mature forests that were previously clearcut. The study also investigated whether historical clearcutting affects ectomycorrhizal colonization, particularly in terms of variations in the number of root tips based on forest type and soil layer. However, the results did not show significant differences between these types of forests [39]. In a study carried out in a managed temperate forest in Czech Republic, clearcutting of a Norway spruce stand led to significant changes in bacterial community composition within tree roots over time [40]. A study carried out in Canada, in a high elevation forest dominated by Engelmann spruce and subalpine fir, suggests that smaller cutblocks (<10 ha) may maintain greater fine root longevity compared to larger clearcuts [41]. However, studies on fine roots including exploration of their dynamics during forest stand development or their response to forestry practices, as well as estimation of their role in the C cycling in forest stands and clearcut areas, are insufficient, especially in the hemiboreal zone of Europe [31]. Thus, additional knowledge is required to improve understanding of C sequestration by forests.

There is a recognized gap in the understanding of fine root dynamics within the hemiboreal region of Europe, particularly in response to forestry practices. Within this study, we aimed to evaluate the morphological traits and production of fine roots, and consequently the C input with fine root litter in hemiboreal forest stands and clearcut areas with nutrient-rich organic soils in Latvia. Forest stands represented coniferous-tree- and deciduous-tree-dominated stands aged from 10 to 73 years, while clearcut areas were previously dominated by coniferous trees (stem-only harvesting with logging residues used to reinforce the skidding trails was done). In total, this study summarizes the results obtained in 26 study sites in a two-year study using the root ingrowth method. We hypothesized that in nutrient-rich organic soils of the hemiboreal zone, fine root morphological traits and FRP are significantly influenced by dominant tree species (deciduous vs. coniferous), soil drainage status (drained vs. naturally wet), and forest development stage (stands vs. clearcuts). In our previous study [42], we established the preliminary findings that form the basis for the current research. Building on these initial results, this study aimed to further investigate fine root dynamics by extending the observation period. This study will help to fill the knowledge gaps about fine root dynamics and traits and to accurately estimate the associated C flows from this pool in the hemiboreal zone. Fine root studies are critical for understanding key aspects of tree physiology, growth, ecosystem functioning, and, consequently, for implementing sustainable forest management practices.

2. Materials and Methods

2.1. Study Sites

The two-year long study was initiated in 2020 and conducted in 26 sites in hemiboreal forests (23 sites in forest stands and 3 sites in clearcut areas) with drained and naturally wet organic soils in Latvia (Figure 1). According to Latvia’s national forest site type classification system [43], study sites with drained organic soils corresponded to Myrtillosa turf. mel. and Oxalidosa turf. mel. forest site types, while study sites with naturally wet organic soils corresponded to Dryopterioso-caricosa and Filipendulosa forest site types. Thus, organic soils in all study sites corresponded to nutrient-rich status [43,44]. Study sites in forest stands with drained organic soils were dominated by both coniferous tree species (Norway spruce Picea abies (L.) H. Karst.) and deciduous tree species (silver birch Betula pendula Roth and black alder Alnus glutinosa (L.) Gaertn), while study sites in forest stands with naturally wet organic soils were dominated only by deciduous tree species (

Table 1. Summary of characteristics of the study sites in forest stands (forest stand parameters); n—number of study sites.

Dominant Tree Species	Soil Drainage Status	Value	Forest Stand Parameters, Unit
			Stand Age, years	Basal Area, m2 ha−1	Tree Density, Tree Number ha	Growing Stock, m3 ha−1	Tree Diameter at Breast Height, cm	Tree Height, m
Deciduous (n = 13) including silver birch (n = 7) and black alder (n = 6)	drained (n = 6)	mean	38	20.0	1523	172	16.6	16.4
		S.E.	7	3.3	353	38	1.7	1.7
		median	34	18.8	1780	141	17.2	16.5
		range	19–62	11.0–33.7	340–2660	58–307	10.3–21.8	10.0–22.0
	naturally wet (n = 7)	mean	44	24.1	3700	272	12.9	14.1
		S.E.	10	5.3	1456	104	2.9	2.8
		median	56	27.9	2000	247	13.0	15.0
		range	10–73	2.5–39.9	1160–11,700	8–809	3.3–23.0	4.6–25.6
Coniferous (n = 10), Norway spruce (n = 10)	drained (n = 10)	mean	48	24.1	912	264	19.2	18.3
		S.E.	6	3.3	120	40	1.8	1.8
		median	50	27.4	780	291	20.3	20.5
		range	10–74	2.0–38.5	362–1700	7–462	4.2–25.4	3.2–23.0

). A general description of the composition of understory vegetation in forest stands with drained and naturally wet organic soils is provided in Bardule et al. (2023) [42]. Study sites in clearcut areas (drained soil moisture conditions) were previously dominated by Norway spruce, and harvesting took place in 2018–2019. In each study site, one sample plot was established (area 500 m², radius 12.62 m).

In Latvia, the average annual precipitation was recorded as 641.5 mm, 676.3 mm, and 685.6 mm for the years 2020, 2021, and 2022, respectively, while mean annual air temperatures were +8.8 °C, +7.0 °C, and +7.3 °C for the same years [45].

2.2. Fine Root Sampling

To estimate annual fine root production and determine fine root morphological traits, the ingrowth core method (where roots grow into in situ incubations containing soil without pre-existing roots) was used [46,47,48,49,50]. In each study site, at least six cylindrical-shape ingrowth cores (diameter 40 mm, length 80 cm) made of polyester fabric mesh and filled with root-free soil collected from the study site were installed randomly into the soil (a distance between ingrowth cores of at least 1 m was ensured) in May 2020. It was ensured that soil density in ingrowth cores was similar to that of the surrounding soil. A detailed description of the installation of ingrowth cores is provided by Bardule et al. (2023) [42]. The first set of the installed ingrowth cores (at least three ingrowth cores per study site) was recovered in May 2021 (12 months after installation), while the second set (remaining ingrowth cores) was recovered in May 2022 (24 months after installation). Ingrowth cores were removed by cutting off the roots that had grown through the ingrowth cores, avoiding pulling out roots and damage of the ingrowth cores. The collected samples represented the fine roots of trees and understory vegetation.

The obtained samples were further processed at the Forest Environment Laboratory of the Latvian State Forest Research Institute (LSFRI) “Silava” (Salaspils, Latvia). Samples (cores) were divided into 10 cm intervals (0–10, 10–20, 20–30 … 70–80 cm; only the second set of cores recovered in 2022 was divided), roots were washed free of soil particles, and fine roots (diameter ≤ 2 mm) were separated.

2.3. Determination of Fine Root Morphological Traits

Traits of fine root morphology, combining both tree roots and understory vegetation roots collected in 2021 and 2022, were determined by scanning each sample using an EPSON Expression 12000XL (Seiko Epson Corp., Japan, EU Importer: Epson Europe B.V., Amsterdam, The Netherlands; resolution 1200 dpi). The obtained scans and fine root biomass data (determined by drying samples at 70 °C to constant mass) were used to calculate the following morphological traits: fine root length density (RLD, m m⁻³ soil), specific root length (SRL, m g⁻¹ dry fine root mass), root volume density (RVD, cm³ m⁻³ soil), root dry mass density (RDMD, g m⁻³ soil), root surface area (RSA, m² m⁻³ soil) and specific root surface area (SRSA, m² g⁻¹ dry fine root mass), mean root diameter (MRD, mm), and fine root tip frequency expressed as the number of tips per unit of dry mass (T, n mg⁻¹ dry fine root mass). WinRhizoTM 2019 software (Régent Instruments Inc., Quebec City, QC, Canada), an image analysis system specifically designed for washed root measurements, was used to calculate fine root morphological traits based on obtained scans.

2.4. Estimation of Fine Root Production and Carbon Input with Fine Root Litter

FRP in the first study year was estimated as the mass of the fine roots extracted from the ingrowth cores. FRP in the second study year was estimated using the ingrowth-dividing method by dividing the fine root biomass in the cores by the incubation time (2 years in our case). It has been demonstrated that a 2-year incubation period is optimal, and extending it by an additional year is not worthwhile [24].

Based on the results of other previous studies [51,52,53,54], we assumed a simplified assumption that annual FRP is equal to annual fine root litter amount. To calculate C input with fine root litter, we multiplied annual fine root litter amount with C concentration in fine root biomass. C and nitrogen (N) concentrations in root biomass samples were determined with the elementary analysis method (Elementar El Cube elemental analyzer, Elementar Analysensysteme GmbH, Langenselbold, Germany) according to the LVS ISO 10694:2006 [55]. In addition, C/N ratio in fine root biomass was calculated.

2.5. Soil Sampling and Analysis

In all study sites (sample plots), two replicates of soil were sampled at fixed soil layers (every 10 cm) having a depth between 0 and 80 cm. Soil samples were delivered to the Forest Environment Laboratory at the LSFRI “Silava” and underwent pre-treatment for physico-chemical analysis following the LVS ISO 11464:2005 standard [56]. Soil bulk density was determined using the mass and volume of soil samples according to LVS ISO 11272:2017 [57]. Soil pH was determined using a pH meter in a suspension of soil in 1 mol L⁻¹ potassium chloride (KCl) solution, following the guidelines outlined in LVS EN ISO 10390:2022 [58]. The concentrations of calcium (Ca), magnesium (Mg), phosphorus (P), and potassium (K) were determined through extraction with concentrated nitric acid (HNO₃) and analyzed using inductively coupled plasma-optical emission spectrometry (ICP-OES) method. Total C (TC) and total nitrogen (TN) concentrations were determined using dry combustion methods (elementary analysis) as stated in LVS ISO 10694:2006 [55] and LVS ISO 13878:1998 [59], respectively. Carbonate concentration reflecting inorganic C (IC) concentration was determined using a Digital Soil Calcimeter. Organic C (OC) concentration was calculated as the difference between TC and IC concentrations. In addition, soil C/N ratio was calculated.

2.6. Data Analysis

All data processing, statistical analyses, and visualization were conducted using the software environment R (version 4.3.3) and RStudio 2023.12.1 (R Core Team, 2024). Figure 1 (map) was prepared using QGIS 3.34.4. We used the Shapiro–Wilk test to assess normality, specifically due to the small sample size (<30). Our data were non-normally distributed; therefore, we proceeded with non-parametric methods for analysis. Statistically significant differences in morphological traits of fine roots and FRP between groups (study years, forest stand development stage, and dominant tree species) were estimated using the Wilcoxon rank sum exact test, with pairwise comparisons adjusted for multiple testing using the Bonferroni correction. Correlations between pairs of variables (between annual FRP and variables of soil general chemistry, between annual FRP and different stand variables, and between C input with fine root litter and different stand variables) were tested with Spearman’s correlation coefficient (ρ) to assess the degree of dependence. In addition, simple regression analysis was conducted to determine relationships between annual C input with fine root litter and forest stand parameters (Table 1). All statistical analyses were carried out with a significance level of 95% (α = 0.05).

3. Results

3.1. Morphological Traits of Fine Roots

All fine root morphological traits tended to increase (mostly significantly,

Table A1. Significance of differences in morphological traits between observations in cores in first and second study years (sampled 12 and 24 months after ingrowth cores installation, respectively); p-values according to the Wilcoxon rank sum exact test (p-values < 0.05 are shown in bold).

Morphological Traits	Coniferous-Tree-Dominated Stands, Drained	Deciduous-Tree-Dominated Stands, Drained	Deciduous-Tree-Dominated Stands, Naturally Wet	Clearcut Area, Drained
Root length density	<0.001	<0.001	<0.001	0.068
Specific root length	<0.001	<0.001	0.017	0.894
Root surface area	<0.001	<0.001	<0.001	0.010
Specific root surface area	<0.001	<0.001	<0.001	0.005
Average root diameter	0.078	0.068	0.001	<0.001
Root volume density	<0.001	<0.001	<0.001	0.002
Root dry mass density	<0.001	0.005	<0.001	0.077
Tips	0.641	0.030	0.182	0.035

) with incubation time (1–2 years) in all separated groups of study sites (coniferous-tree-dominated stands, deciduous-tree-dominated stands, and clearcut area, Figure 2, Table A1) excluding fine root tip number on a dry mass basis (T) in studied clearcut areas (a significant decrease in the second study year was observed). Among groups of different stand development stages and types of dominant tree species, the highest values of most of the studied fine root morphological traits (RLD, SRL, RSA, SRSA, RVD, RDMD, T) were observed in clearcut areas in both the first and second study year (Figure 2). An exception was MRD for which the highest values were found in coniferous-tree-dominated stands. When comparing coniferous- and deciduous-tree-dominated stands under drained conditions, significantly higher mean values of SRL (both study years), SRSA, and T (second study year) were observed in deciduous-tree-dominated stands, while significantly higher mean values of MRD (both study years) and RDMD (second study year) were observed in coniferous-tree-dominated stands (Figure 2). In the group of deciduous-tree-dominated stands where stands with different soil drainage status were separated, significantly higher mean values of SRL and SRSA (second study year) were observed under the drained soil condition, while a significantly higher mean value of RDMD (second study year) was observed under the naturally wet soil condition (Figure 2). No significant differences in other studied fine root morphological traits between drained and naturally wet soil conditions in deciduous-tree-dominated stands were observed.

3.2. Fine Root Biomass Production

In the first study year, estimated mean annual fine root biomass production (Figure 3) ranged from 0.49 ± 0.09 t ha⁻¹ yr⁻¹ in deciduous-tree-dominated stands (stands with drained and naturally wet soils were combined as no significant difference was observed, p = 0.200) to 1.13 ± 0.29 t ha⁻¹ yr⁻¹ in the clearcut area (drained). Furthermore, the difference between mean annual fine root biomass production in deciduous-tree-dominated stands and the clearcut area was statistically significant (p = 0.017).

In the second study year, estimated mean annual fine root biomass production ranged from 0.58 ± 0.13 t ha⁻¹ yr⁻¹ in deciduous-tree-dominated stands with drained soils to 1.38 ± 0.28 t ha⁻¹ yr⁻¹ in the clearcut area (drained). Thus, in the second study year, estimated mean annual fine root biomass production was higher in all separated groups of study sites than estimates of annual fine root biomass production in the first study year; a significant difference between first and second study years was observed for coniferous-tree-dominated stands (p = 0.010) and deciduous-tree-dominated stands with naturally wet soils (p = 0.001).

Results of the second study year showed that fine root biomass production peaked in the upper 0–20 cm soil layer and decreased with increased soil depth (Figure 4). In the upper 0–10 cm soil layer, mean fine root biomass production (mean from all separated groups of study sites) was 36 ± 4% of total fine root biomass production in the 0–80 cm soil layer, while more than half (59 ± 4%) of the total fine root biomass production occurred in the upper 0–20 cm soil layer (Figure 4).

Spearman’s correlation analysis between annual fine root biomass production and variables of soil general chemistry (

Table A2. Variables of soil general chemistry in different soil layers (0–20, 20–40, and 40–80 cm) in studied coniferous-tree-dominated stands (drained), deciduous-tree-dominated stands (drained and naturally wet), and clearcut area (drained); mean values ± S.E are shown.

Group of Study Sites	Variables of Soil General Chemistry, Unit
	Bulk Density, kg m3	pH KCl	OC, g kg−1	TN, g kg−1	C/N ratio	P, g kg−1	K, g kg−1	Ca, g kg−1	Mg, g kg−1
0–20 cm soil layer (mean values were calculated from 0–10 cm and 10–20 cm soil layers)
Coniferous-tree-dominated stands, drained	240.9 ±29.0	4.2 ±0.1	478.8 ±19.9	23.6 ±1.0	20.5 ±0.6	0.93 ±0.08	0.35 ±0.03	17.1 ±1.4	1.56 ±0.17
Deciduous-tree-dominated stands, drained	285.4 ±47.1	5.5 ±0.2	365.8 ±40.6	24.9 ±2.9	14.8 ±0.4	1.31 ±0.14	0.81 ±0.13	27.5 ±3.7	2.52 ±0.32
Deciduous-tree-dominated stands, naturally wet	352.6 ±68.1	4.2 ±0.3	380.1 ±53.0	21.9 ±3.1	17.3 ±0.8	1.44 ±0.44	0.44 ±0.03	12.7 ±2.7	1.50 ±0.30
Clearcut area, drained	187.7 ±7.4	4.1 ±0.6	505.7 ±33.6	23.3 ±2.6	22.9 ±3.9	0.78 ±0.12	0.65 ±0.05	25.3 ±9.6	2.48 ±0.75
20–40 cm soil layer (mean values were calculated from 20–30 cm and 30–40 cm soil layers)
Coniferous-tree-dominated stands, drained	352.7 ±67.3	4.8 ±0.1	426.5 ±35.8	20.1 ±1.6	21.4 ±1.0	0.68 ±0.11	0.22 ±0.04	18.0 ±1.9	1.61 ±0.22
Deciduous-tree-dominated stands, drained	666.0 ±182.1	5.7 ±0.2	264.4 ±69.5	16.0 ±4.1	16.5 ±0.9	0.80 ±0.15	1.35 ±0.34	18.6 ±3.3	2.73 ±0.48
Deciduous-tree-dominated stands, naturally wet	516.4 ±152.0	4.8 ±0.6	351.8 ±71.6	17.8 ±3.6	18.7 ±1.4	1.19 ±0.39	0.29 ±0.04	15.0 ±3.0	1.54 ±0.25
Clearcut area, drained	189.9 ±7.5	4.3 ±0.6	477.7 ±33.4	27.2 ±8.5	22.9 ±5.9	0.47 ±0.05	0.34 ±0.02	24.3 ±9.4	2.51 ±0.75
40–80 cm soil layer (mean values were calculated from 40–50 cm, 50–60 cm, 60–70 cm, and 70–80 cm soil layers)
Coniferous-tree-dominated stands, drained	511.9 ±77.4	5.3 ±0.1	371.1 ±33.2	14.8 ±1.2	24.9 ±1.5	0.65 ±0.15	0.31 ±0.05	17.4 ±1.4	2.12 ±0.34
Deciduous-tree-dominated stands, drained	1032.8 ±151.1	6.1 ±0.2	159.2 ±49.8	8.9 ±2.8	22.8 ±3.2	0.53 ±0.09	1.35 ±0.24	14.5 ±2.2	4.57 ±0.81
Deciduous-tree-dominated stands, naturally wet	720.4 ±124.7	5.2 ±0.1	260.7 ±54.1	13.0 ±2.7	16.8 ±1.1	0.43 ±0.06	0.38 ±0.07	11.8 ±2.2	1.51 ±0.19
Clearcut area, drained	492.3 ±182.3	5.3 ±0.3	360.6 ±83.8	16.7 ±5.1	21.9 ±4.0	0.50 ±0.09	0.42 ±0.08	20.9 ±4.5	2.96 ±0.48

) was conducted for each soil layer separately (0–80 cm soil layer divided by 10 cm,

Table A3. Summary of Spearman’s correlation coefficients (ρ) between annual fine root biomass production (results of the second study year) and variables of soil general chemistry in different soil layers; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.

Soil Layer, cm	Variables of Soil General Chemistry
	Soil Bulk Density	pH KCl	OC	TN	C/N Ratio	P	K	Ca	Mg
0–10	0.066	−0.307	0.097	−0.208	0.424 *	−0.398	−0.254	−0.289	−0.243
10–20	0.234	−0.553 **	−0.114	−0.417 *	0.413 *	−0.338	−0.218	−0.479 *	−0.567 **
20–30	0.201	−0.743 ***	−0.040	−0.315	0.482 *	−0.210	−0.260	−0.510 *	−0.552 **
30–40	0.062	−0.705 ***	−0.003	−0.321	0.569 **	−0.236	−0.381	−0.417 *	−0.406
40–50	0.144	−0.157	−0.096	−0.312	0.096	−0.157	−0.073	−0.232	−0.117
50–60	0.086	−0.184	−0.039	−0.192	0.006	−0.159	−0.078	−0.151	−0.102
60–70	0.030	−0.059	0.036	−0.117	0.115	−0.140	−0.090	−0.045	−0.043
70–80	−0.001	−0.098	0.042	−0.099	−0.011	−0.025	−0.059	−0.043	−0.092

). In the upper soil layer (0–10 cm), a moderate positive correlation between annual fine root biomass production and soil C/N ratio was found (ρ = 0.424), while in 10–20 cm, 20–30 cm, and 30–40 cm soil layers, moderate and even strong negative correlations were found between annual fine root biomass production and soil pH (ρ in a range from −0.553 to −0.743), moderate positive correlations between annual fine root biomass production and soil C/N ratio (ρ in a range from 0.413 to 0.569), and moderate negative correlations between annual fine root biomass production and Ca and Mg concentrations in soil (ρ in a range from −0.406 to −0.567, Table A3). In deeper soil layers (40–50 cm, 50–60 cm, 60–70 cm, and 70–80 cm), only weak or no correlations between annual fine root biomass production and variables of soil general chemistry were found (Table A3).

Correlation analyses between annual fine root biomass production and different stand variables including and excluding clearcut areas were conducted separately. Only weak or no correlations (ρ < 0.320, p > 0.110) were found between annual fine root biomass production in study sites (all study sites pooled including clearcut areas, results of the second study year were analyzed) and different stand variables (Table 1). Similarly, only weak or no correlations (ρ < 0.352, p > 0.099) were found between annual fine root biomass production in studied forest stands (clearcut areas were excluded from the analysis) and different stand variables (Table 1), excluding a moderate positive correlation found between annual fine root biomass production and growing stock (ρ = 0.410, p = 0.052).

3.3. Carbon Input through Fine Root Litter

Among groups of different forest stand development stages, the dominant tree species types and soil drainage status, the mean C concentration in fine root biomass ranged from 474.3 ± 2.1 g kg⁻¹ in coniferous-tree-dominated stands (drained) to 503.0 ± 4.0 g kg⁻¹ in deciduous-tree-dominated stands (naturally wet); furthermore, significant differences in C concentration between different groups were observed (

Table 2. Mean carbon (C) and nitrogen (N) concentration and C/N ratio in fine root biomass (tree and understory vegetation roots combined) in the studied coniferous-tree-dominated stands, deciduous-tree-dominated stands, and clearcut area (mean values ± S.E.). Different lowercase letters denote significant differences (p < 0.05) between different groups.

Group	C Concentration, g kg−1	N Concentration, g kg−1	C/N Ratio
Coniferous-tree-dominated stands, drained	474.3 ± 2.1 b	15.8 ± 0.6 a	30.7 ± 1.6 a
Deciduous-tree-dominated stands, drained	481.3 ± 2.3 b	18.9 ± 1.2 a	26.0 ± 1.8 a
Deciduous-tree-dominated stands, naturally wet	503.0 ± 4.0 a	19.0 ± 1.3 a	27.3 ± 1.9 a
Clearcut area, drained	490.5 ± 6.0 ab	19.0 ± 1.6 a	26.7 ± 2.6 a

). Mean C/N ratio in fine root biomass ranged from 26.0 ± 1.8 in deciduous-tree-dominated stands (drained) to 30.7 ± 1.6 in coniferous-tree-dominated stands (drained); however, no significant differences in C/N ratio between different groups were found (Table 2).

According to the second study year results, among studied forest stands, annual C input with fine root litter ranged up to 1.09 t C ha⁻¹ yr⁻¹ with a mean value of 0.51 ± 0.05 t C ha⁻¹ yr⁻¹ in coniferous-tree-dominated stands (drained), and 0.28 ± 0.06 and 0.53 ± 0.08 t C ha⁻¹ yr⁻¹ in deciduous-tree-dominated stands with drained and naturally wet soils, respectively. Mean annual C input with fine root litter in studied clearcut areas was 0.68 ± 0.14 t C ha⁻¹ yr⁻¹.

Correlation analyses between C input with fine root litter and different stand variables including and excluding clearcut areas were conducted separately. Only weak or no correlations (ρ < 0.315, p > 0.120) were found between annual C input with fine root litter in study sites (all study sites pooled including clearcut areas, results of the second study year were analyzed) and different stand variables (Table 1). Correlation analysis between annual C input with fine root litter in studied forest stands (clearcut areas were excluded from the analysis) and different stand variables (Table 1) revealed a moderate positive correlation between annual C input and growing stock (ρ = 0.413, p = 0.050), but only weak or no correlations (ρ < 0.350) were found between annual C input and other stand variables. The variation in annual C input with fine root litter (combining tree and understory vegetation roots) depending on stand basal area and growing stock (polynomial regressions) is shown in Figure 5 (clearcut areas included).

4. Discussion

Fine root traits indicate plant strategies for adapting to diverse environments and embody essential trade-offs between resource acquisition and associated costs. Our study summarizes the results of fine root morphological traits, production, and C input with fine root litter in hemiboreal forest stands (coniferous-tree- and deciduous-tree-dominated stands) and clearcut areas (previously dominated by coniferous trees) with nutrient-rich organic soils in Latvia. The study summarizes the results obtained in a two-year study using the root ingrowth method. Results of the first study year are partly summarized in Bardule et al. (2023) and highlighted the need for further research, as the results of the first year can provide an underestimation of FRP [42]. Results of the second study year summarized in our article (Figure 3) confirmed initial concerns and revealed that the estimated mean fine root biomass production in the second study year was higher than estimates of fine root biomass production in the first study year (annual FRP was compared). Thus, results of the first year of the study underestimated FRP by 22%–104% depending on the group of study sites (grouped depending on stand development stage, dominant tree species type, and soil drainage status). The highest underestimation of FRP (by 104%) was found for the group of deciduous-tree-dominated stands with naturally wet organic soil. According to the obtained results of the second study year (Figure 3), estimated mean fine root biomass production ranged from 0.58 ± 0.13 t ha⁻¹ yr⁻¹ in deciduous-tree-dominated stands with drained soils to 1.38 ± 0.28 t ha⁻¹ yr⁻¹ in clearcut areas (drained). Nevertheless, the results of the second year could still be slightly underestimated, as part of the fine roots grown in the first year could have already been decomposed after the incubation of 24 months. In addition, the limiting factor of the comparison of the results of the first and second study year in context of methodological aspects is natural interannual dynamics (variation) of FRP, which can reach a significant level [60].

The highest mean value of FRP, particularly in drained clearcut areas, can be explained by the rapid distribution and growth of understory vegetation, including development of the root network after clearcutting. The removal of canopy trees reduces competition for light, water, and nutrients for ground vegetation. The growth ratios among leaves, shoots, and roots are influenced by the light availability. A study carried out in boreal forests clearly demonstrated that the structure of the forest canopy significantly affects the composition of the understory by altering the light environment [61]. Harper (1977) observed that under high light exposure, root growth was extensive while above-ground biomass development was limited [62]. Conversely, under the influence of the canopy shadow, the opposite pattern was noted [63,64]. In addition, in our study, FRP appeared to be higher in stands dominated by coniferous trees compared to those dominated by deciduous trees, while in the group of deciduous-tree-dominated stands, higher FRP was observed in stands with naturally wet organic soils than in stands with drained organic soils (Figure 3). In our case, naturally wet organic soils represent undrained soils which are periodically saturated, while the groundwater level periodically decreases as well. Butlers et al. (2024) recently reported that the mean groundwater level below the soil surface in undrained forest stands with organic soils in Latvia is 13 ± 4 cm [65]. Overall, tree FRP tends to be greater when the groundwater level is deeper. In a study carried out in a boreal bog in Finland, the water table drawdown led to a 740% increase in belowground tree FRP. However, this increase came at the expense of herb root production, which decreased by 38%, resulting in no significant overall change in total FRP [66]. In another study carried out in hemiboreal old-growth forest stands in Latvia, the FRP did not differ significantly between the stands with undrained or naturally wet (2.05 ± 0.31 t ha⁻¹ yr⁻¹) and drained (1.82 ± 0.26 t ha⁻¹ yr⁻¹) soils [67].

According to the results of a study using estimation models, European forests have an average FRP of 2.5 t ha⁻¹ yr⁻¹ [68]. According to the same study, the mean FRP in northern conifer forests was 2.96 t ha⁻¹ yr⁻¹ and that in northern broad-leaved forests was 2 t ha⁻¹ yr⁻¹. In a study carried out in Scots pine stands of different ages in Finland, FRP was 7.75 ± 3.39 and 8.6 ± 3.48 t ha⁻¹ yr⁻¹, in a pole stage and mature stand, respectively [69]. In hemiboreal coniferous forests in Estonia, the total FRP of the trees and the understory roots and rhizomes ranged from 2.11 to 10.4 t ha⁻¹ yr⁻¹, of which the understory comprised up to 28% [70]. Our FRP estimates are lower than those in the previously mentioned studies. As fine roots grow more rapidly in order to more efficiently exploit soil resources (nutrients) in poor soil to maintain growth and survival rates [71], lower FRP values we obtained compared to other studies, including mean estimates for European forests. These results could be explained by the comparatively high nutrient availability in our study sites, as stands with nutrient-rich organic soils were examined in this study. Furthermore, among our study sites, we found negative correlations between annual fine root biomass production and nutrient (N, Ca, Mg) concentrations in soil and positive correlation between annual fine root biomass production and soil C/N ratio (Table A3), which indirectly indicates soil mineralization and nitrate leaching [72]. However, the determined N, Ca, and Mg concentrations in soil only indirectly reflect nutrient availability in soil, as the total N and concentrated-HNO₃-extractable Ca and Mg concentrations were determined within the study. Many studies have explored the relationship between site nutrient availability and fine root dynamics through nutrient addition experiments, but the outcomes have been rather inconsistent. A recent extensive meta-analysis showed that nitrogen (N) addition often significantly increased FRP [73]. Conversely, several studies reported that N addition decreased FRP, possibly due to the input and profit principle [74,75]. In the meta-analysis conducted by Peng et al., results showed that FRP was inhibited by N addition in boreal forests, but was not significantly changed in temperate forests. Due to the rise in available soil N, it is more cost-effective for plant growth (through N absorption) to prolong the lifespan of fine roots rather than maintaining them at a state of metabolic exhaustion [75]. Consequently, extending fine root longevity reduces FRP, leading to declines in both FRP and turnover [73]. In another study carried out in Finland, in boreal peatland forest stands, production decreased with decreasing site fertility, but the lowest production (less than 0.5 t ha⁻¹ yr⁻¹) was observed at the most fertile site type. However, in this study, the lower production was more attributed to a higher water table and a potential bias [76]. Results of a study carried out in a Schrenk’s spruce (Picea schrenkiana) forest in China indicated that moderate N addition promotes fine root development, whereas excessive N can inhibit root system growth. In the same study, a significant positive correlation was also found between the fine root biomass and soil C [17]. Another meta-analysis indicated that, in temperate forests, external N input had a minor impact on FRP [77]. Input of P to soil is also often associated with an increase in FRP, but no such trend was observed in boreal forests separately [78].

At the same time, in another study carried out in Scots pine stands in Finland, individual site FRP values ranged from 0.30 to 4.73 t ha⁻¹ yr⁻¹, with an average of 1.20 t ha⁻¹ yr⁻¹ [76]. Our results for coniferous-tree-dominated stands were within this range.

Based on a dataset of FRB encompassing 95 forest stands in Finland, Lehtonen et al. (2016) elaborated models to estimate FRB in boreal forests. It was found that stand basal area outperformed other stand variables as a predictor of FRB. However, the applicability of this predictor in estimating FRP remained uncertain [79]. Another more recent study carried out in Finland indicated a positive correlation of FRP and stand basal area, indicating this as the best single predictor of FRP variation [76]. In our study, no significant correlation was found between stand basal area and FRP or C input with fine root litter. Instead, a moderate positive correlation was found between growing stock and both FRP and annual C input. Given that growing stock, which encompasses both stand basal area and tree volume, integrates multiple aspects of forest productivity and biomass accumulation, it appears to be a more comprehensive indicator of C cycling through fine root litter. This suggests that overall forest productivity, rather than basal area alone, plays a more significant role in driving FRP and C inputs via fine root litter. A limitation of our study was that fine roots of trees and ground vegetation were not evaluated separately. Distinguishing between these would provide more detailed insights into the specific contributions of different vegetation layers to fine root production and C input.

The key obstacles to accurately quantifying belowground C allocation are the uncertainty surrounding fine root turnover and the variability in fine root distribution. In a study carried out in southern Finland, higher belowground C inputs were found in silver birch stands (1.16 t C ha⁻¹ yr⁻¹) compared to sites where Scots pine dominated [68]. In our study, the annual C input with fine root litter was higher in conifer stands. In another study carried out in two Norway spruce stands in Finland, C input was 1.18 t C ha⁻¹ yr⁻¹ in the northern site, which is poorer in nutrients, and 0.94 t C ha⁻¹ yr⁻¹ in the southern site [45]. In another study carried out in Scots pine stands in Finland, annual belowground C input was 0.7–0.91 t ha⁻¹ yr⁻¹ [80]. Our obtained C input values are lower than the estimates of these studies. C input values were derived from FRP estimates, which were also lower than in several other studies carried out in Europe. This was explained by the comparatively high nutrient availability in our study sites.

Morphological traits of fine roots varied significantly among our study sites grouped depending on forest development stage, dominant tree species type, and soil drainage status (Figure 2). It is noteworthy that the highest values of most of the studied fine root morphological traits (RLD, SRL, RSA, SRSA, RVD, RDMD, T) were observed in clearcut areas. In a study carried out in Estonia and Finland, the mean SRA and SRL values were larger for silver birch, compared to Norway spruce and Scots pine [81], which is in accordance with our results. In another study carried out in Estonia, in drained peatland forests dominated by Downy birch and Norway spruce, the dominant tree species significantly influenced the variability of absorptive root morphological traits. Birch absorptive roots exhibited thinner diameters along with higher SRA and SRL, greater branching intensity (BI, also known as root tip frequency or specific root tip density [82,83], and lower root tissue density (RTD, related to RDMD) compared to spruce [84]. In a study carried out in Switzerland, Scots pine forest, soil moisture was found to have varying effects on fine root morphological traits. Irrigation slightly increased the SRL while reducing the root tissue density. However, the irrigation treatment had no effect on the MRD and numbers of tips and forks per root length (n cm⁻¹) [85]. In a study conducted in north-eastern Germany, in drained and rewetted black alder stands on peatland, the water regime had a considerable effect on SRA, with higher values observed in drained compared to rewetted sites. However, this effect was statistically significant only for root diameter classes of 1–2 mm and 2–5 mm, but not for those less than 1 mm in diameter [86]. In a study conducted in Central Poland, in deciduous forest stands and floodplains with periodic flooding and naturally occurring mature white poplar trees, soil moisture in the 10–20 cm layer was positively correlated with RSA, SRL, and SRSA, as well as fine root length and biomass. However, the correlation with root diameter was negative [87]. In contrast, our study did not directly correlate fine root morphological traits with soil moisture. Instead, we compared drained and naturally wet conditions and found no significant differences in the majority of fine root traits. However, in deciduous stands, significantly higher mean values of SRL and SRSA were observed under drained soil conditions during the second study year. Thus, our results support the findings of previous studies and highlight the complex relationship between fine root morphology, environmental conditions, and forest development stage. The results of our study will help to improve knowledge about fine root dynamics and traits as well as the accuracy of C flow estimation in hemiboreal forests in Latvia.

5. Conclusions

Traits and production of fine roots of trees and understory vegetation (fine roots combined) in hemiboreal forest land with nutrient-rich organic soils varied significantly depending on both forest development stage (stand or clearcut area), type of dominant tree species (coniferous or deciduous), and soil drainage status (drained or naturally wet). According to the results of the second study year, mean FRP among groups of study sites ranged from 0.58 ± 0.13 to 1.38 ± 0.28 t ha⁻¹ yr⁻¹, while C input with fine root litter ranged from 0.28 ± 0.06 to 0.68 ± 0.14 t C ha⁻¹ yr⁻¹. More than half (59 ± 4%) of the total FRP occurred in the upper 0–20 cm soil layer. FRP tended to correlate positively with soil C/N ratio, while negatively with soil pH and nutrient concentration in soil.

First-year-study results of FRP and, consequently, also C input, with fine root litter estimated using the root ingrowth method (ingrowth cores sampled 12 months after installation) potentially could significantly underestimate annual FRP and C input in forest land including both forest stands and clearcut areas when compared with the results of annual FRP and C input of the second study year (ingrowth cores sampled 24 months after installation). Thus, a study of at least two years (24-month incubation of ingrowth cores) is strongly recommended to estimate annual FRP and C input using the root ingrowth method.

This study addressed knowledge gaps regarding fine root dynamics and traits in the hemiboreal zone, emphasizing the importance of forest development stage, dominant tree species, and soil moisture conditions. These factors are crucial for accurately estimating C flows associated with fine root litter, which significantly contribute to C input in these ecosystems.