Invertebrate Community of Scots Pine Coarse Woody Debris in the Southwestern Pyrenees under Different Thinning Intensities and Tree Species †

: Background and Objectives: The forest in the Southwestern Pyrenees Mountains (North-ern Spain) is mainly composed of pure Pinus sylvestris L. or a mix of P. sylvestris and Fagus sylvatica L. The most common forest management technique to harvest pine is the application of forest thinning with different intensities. It promotes a change in the forest composition and structure. Taking into consideration this region as a site specific research about this topic, we aimed to understand the CWD invertebrate composition response to different thinning intensities and canopy type of these tree species. Materials and Methods: CWD samples were collected belonging to intermediate and advanced decay classes, approximately 10 cm long and 5 cm in diameter. Using a design of three thinning intensities (0%, 20%, and 40% of basal area removed), with three replica-tions per treatment (nine plots in total), four samples were taken per plot (two per canopy type) to reach 36 samples in total. Meso- and macrofauna were extracted from CWD samples with Berlese– Tullgren funnels, and individuals were counted and identified. Results: Most of the taxonomic groups belonged to mesofauna, mainly to Acari and Collembola orders. On the other hand, the macrofauna represented a minimum percentage of the community composition. Our results indicated that although thinning intensities did not significantly affect the invertebrate community, canopy type and CWD water content influenced significantly. It is imperative to consider in forest management the responses of canopy type and thinning intensities in CWD water content, this dis-turbance could also slow down the organic matter decomposition process in the soil, thus affecting in the long term the natural cycle of nutrients.


Introduction
Coarse woody debris (CWD) is very important for forest ecosystems due to its multiple ecological functions, which include regulating carbon and nutrient cycles and providing different structural properties to forest soils, such as through affecting sediment transport storage and water retention. Previous works have also emphasized the importance of CWD for species diversity conservation. CWD is usually divided into five different decay classes from fresh (class 1) to fully decomposed (class 5) [1]. Chronologically, freshly woody debris is colonized by fungi and xylophagous insects that disperse  these fungi [2], followed by Diptera larvae, mites, and centipedes and coleopteran predators [3]. Consequently, greater abundances of xylophages and their predators are found in the initial decomposition classes. As decomposition proceeds, social insects such as ants or termites excavate their nests [4], feeding on fungi and broken sapwood. Hence, saprophages and parasitoids are more abundant in intermediate stages [5,6]. In the most advanced stages, fungi, organisms that feed on rotten wood, and their predators become dominant [3,7,8].
CWD production rates can be influenced by forest management practices [9,10]. Forest thinning is commonly used to control stand density and productivity [5], as well as to manage competition and nutrient cycling in forests, which is particularly important in mixed forests [11,12]. Previous research conducted by our team in mixed forests of Scots pine (Pinus sylvestris L.) and European beech (Fagus sylvatica L.) in the Southwestern Pyrenees found that forest thinning reduces litter biomass and nutrient reserves, alters litter production and decomposition rates [13,14], decreases the volume and biomass of fallen woody debris [12,15]and changes the microclimate [11].
Based on this background, it is reasonable to think that if, in the forests that are the object of this study (temperate mixed Scots pine and European beech forests), litter invertebrates have been affected by forest management and canopy type [9], woody debris colonization by invertebrates may have been altered. The main objective of this research was to study the process of colonization by mesofauna in CWD after applying different thinning intensities, as well as to determine whether the type of canopy and the decomposition class of CWD could influence the abundance, richness, and diversity of invertebrate present. In this context, it was expected that certain abiotic variables, such as water content of woody debris and type of canopy cover under different intensities of thinning, could explain the results found.

Study Area
This research was carried out in a mixed natural forest in the Southwestern Pyrenees (Navarre Province, Northern Spain), the dominant tree species being Scots pine and the codominant species European beech. These two species are the most important in European silviculture and they share their distribution zones in large regions.

Experimental Design
The experimental design consisted of nine 40 × 30 m plots. Three treatments were replicated three times: control (0% of basal area removed); intermediate thinning (20% of the Scots pine basal area eliminated in 1999 and again in 2009), and intense thinning (30% of the Scots pine basal area was removed in 1999 and 40% in 2009). In 1999, the stumps were left on the ground and most of the trunks and cut branches were removed [15]. In 2009, the stumps were left on-site but the trunks and large branches were removed for commercial use, leaving the rest of the harvested material in the plots. Each plot was mapped into two subplots depending on the types of arboreal canopy: composed of pure pine crowns or composed of a mixture of pine and beech crowns (and other broadleaved species if present).

Samples Collection and Laboratory Work
Thirty-six samples of pine woody debris were collected in late April 2015. The decay classes collected were classes 3 and 4 according to the Waddell's classification (2002). These samples were divided according to the definition provided by [4] and randomly collected under thinning treatments such as control, intermediate (20%) and intense (40%) thinning. A total of 18 samples were collected under pure pine canopy and another 18 under mixed canopy of pine, beech and other deciduous, with one sample of each decay class collected in each subplot. The collected samples were approximately shaped as wood cylinders, 10 cm long and 5 cm in diameter. In the laboratory, these samples were weighed (to ±0.1 g) as fresh weight and then placed in a Berlese-Tullgren funnel (a 40 W lightbulb on top of a sample container of 15 cm length × 8 cm diameter with a 2 mm nylon mesh at the bottom) for mesofauna extraction for six days, collecting the mesofauna in glass containers with 70% ethanol. After extracting the mesofauna, the woody samples were dried for 72 h in an oven at 70 °C and weighed. Water content percentage of coarse woody debris was determined by the difference between fresh and dry weights.
The contents of each container was analyzed with a stereoscope, the invertebrates identified with a key guidebook [16] and following a list of taxonomic groups of invertebrates previously found in leaf litter at the same plots by [9]. In this way, the invertebrates found were classified according to their order and, for some, even suborder. The total abundance of each taxonomic group was divided by sample weight.

Data Analysis
We estimated total abundance, richness, and the Shannon-Wiener diversity index per sample. The effects of thinning, canopy type, and CWD decay class on total abundance of macro-and mesofauna, species richness, Shannon-Wiener index, and CWD water content were analyzed using the nonparametric Kruskal-Wallis test, as data did not follow a normal distribution. Generalized mixed models (GLMs) with Poisson distribution were used to analyze the interactions between thinning intensity, canopy type, and CWD decay class for the variables of water content of CWD and total abundance of invertebrates per gram of decayed wood. Principal component analyses (PCA) were performed considering the variables that represented the variance in a greater percentage, such variables being: CWD water content, total abundance per gram of oribatid mites, other mites, Collembola, immature stages of macrofauna (larvae), immature mites, Coleoptera, and total abundance. The software used in this research was R Studio version 3.1.3 [17] and IBM SPSS Statistics version 25 [10].

Influence of Treatments in CWD Invertebrate Community
Total abundance and species richness decreased with thinning intensity, although no significant differences were found between treatments. No clear patterns for the Shannon-Wiener index were found (Table 2). Interestingly, in spite of not detecting direct significant effects of thinning, GLM results indicated a significant interaction between thinning intensity and decay class (Ddf2 = 3.557, p = 0.050), for which CWD in heavily thinned plots had the lowest invertebrate abundance.
Species richness was significantly higher in mixed patches than in pure pine patches (X 2 df1 = 4.016, p = 0.031). However, no significant differences between canopy types were found for total abundance and the Shannon-Wiener index ( Table 2). In addition, GLM results indicated that the interaction between pure pine canopy and decay class significantly affected total abundance, with the highest decomposed CWD (class 4) under pine canopy having the lowest abundance (Z = 2.148, p = 0.032).
Although the Shannon-Wiener index was not significantly different between decay classes, total abundance (X 2 df1 = 16.400, p < 0.001) and richness were significantly influenced by them (X 2 df1 = 7.123, p = 0.008). Water content (%) was significantly higher in class 4 than in class 3 (X 2 df1 = 8845, p = 0.003) ( Table 2). Table 2. Mean and Standard Error number of captured individuals per gram of coarse woody debris (CWD) in different treatments of thinning intensity, canopy type, and CWD decay class (significant differences at p < 0.05 in bold, n = 36).

Conclusions
Our results provide some of the first evidence of the interactive effects that overstory composition and thinning can have on CWD invertebrate taxonomic groups and richness in mixed forests of the two most widely distributed European tree species (Pinus sylvetris and Fagus sylvatica L.). As thinning and type of canopy cover modifies the moisture-radiation-wind balance in the forest soil, CWD moisture content seems to be reduced when tree density reduction crosses a threshold of around 20% of the initial basal area. Such change can affect differently invertebrate taxa, with some being more sensitive to moisture reduction than others are.