Integrating ecomorphological groups into compound-specific isotope analysis of amino acids refines trophic niches of forest soil macrofauna

Soil macrofauna detritivores and predators play central roles in decomposition, nutrient cycling and predator – prey interactions, but resolving their trophic positions (TPs) and basal resource use in different forest types has long been challenging due to methodological constraints. Compound-specific isotope analysis of amino acids (CSIA-AA) overcomes these challenges by quantifying the contributions of bacteria, fungi and plants to consumer diets, and by providing baseline-independent trophic position estimates that account for intermediate microbial trophic levels. However, most recent CSIA-AA studies of soil animals analyzed trophic niches only at high-rank taxonomic levels. This approach reveals robust general patterns but may obscure trophic variations among species within these groups that differ in morphology and microhabitat use. Here, we collected 20 species from near-natural beech forests and spruce monocultures in Germany, representing major high-rank taxonomic groups of detritivores (Lumbricidae, Diplopoda, Isopoda) and predators (Chilopoda). Using CSIA-AA we quantified trophic positions and basal resource contributions from bacteria, fungi and plants, and compared patterns across high-rank taxonomic groups, ecomorphological groups (high-rank taxonomic groups subdivided by body size, body shape and vertical distribution in soil) and species. Ecomorphological groups included Epigeic, Endogeic and Anecic Lumbricidae, Slender and Globular Diplopoda, Small and Large Isopoda, and Litter and Soil Predators in Chilopoda. Detritivores obtained most essential amino acids (eAAs) from fungi, while Chilopoda showed similar contributions of bacteria, fungi and plants. TPs and basal resources varied little between forests but differed strongly among ecomorphological groups. In Lumbricidae, TPs increased from Anecic (1.7) to Epigeic (2.1) to Endogeic (2.5), accompanied by shifts in bacterial and plant contributions. Slender Diplopods occupied higher TPs than Globular Diplopods (2.8 vs. 2.5), while Small Isopods reached TPs similar to predators (3.5). Among Chilopoda, Litter Predators had higher TPs and incorporated more bacterial resources (41%) than Soil Predators (17%), suggesting differences in prey use between litter and soil. Hierarchical models confirmed that ecomorphological groups explained substantially more variation in trophic positions and resource use than high-rank taxonomic groups, while species identity added little explanatory power, except for Lumbricidae and Chilopoda. Our findings indicate that trophic niches of soil macrofauna are more linked to morphology and microhabitat preferences than to forest type. Assigning soil macrofauna taxa to ecomorphological groups captures major variation in trophic niches and thus represents a powerful approach for future CSIA-AA studies compensating for information loss when analyzing high-rank taxonomic groups rather than species due to biomass restrictions. • Trophic niches of forest soil macrofauna resolved using CSIA of amino acids • Detritivores mainly relied on fungal resources across beech and spruce forests • Ecomorphological groups showed distinct trophic positions and resource use • Trophic niches are determined by morphology and microhabitat rather than by forest type • Ecogroup-based CSIA-AA proposed as a robust tool for soil food web studies