Can immature stages be ignored in studies of forest leaf litter arthropod diversity? A test using Oxford Nanopore DNA barcoding

Abstract

Forest soil and leaf litter support diverse arthropod mesofauna for which diversity dynamics is challenging to study due to the high number of species and specimens, small body size and limited taxonomic knowledge. Immature stages (larvae) are even harder to identify than adults, as their morphology is largely unknown. Therefore, larvae are often ignored, even though they may form a substantial proportion of specimens collected, are less mobile than adults and their exclusion may provide an incomplete diversity profile. Here, we use Oxford Nanopore DNA barcoding to investigate whether the inclusion of larvae provides a more complete taxonomic, phylogenetic and functional diversity profiles in leaf litter beetles (Coleoptera) from a subtropical forest in Taiwan. Our results indicate that larvae represent up to 38% of beetle specimens per sample, but most of them belong to 2–3 common species. Larvae of most beetle species are rarely collected repeatedly or in multiple specimens, possibly due to special habitat requirements or high seasonality. Taxonomic, phylogenetic and incidence‐based functional diversity measures were not affected by the exclusion of larvae in the Staphylinidae dataset but were strongly biased in all‐beetle dataset, especially when only common or abundant species were considered. Taxonomic beta diversity was not affected by the omission of larvae. Our results indicate that immature stages may be omitted in ecological studies of arthropods in case both adults and larvae co‐occur in the same habitat, and the sites are sampled repeatedly. Caution is needed (1) in groups in which larvae and adults do not inhabit the same environment or strongly differ in biology, (2) when rare species are omitted or (3) when functional diversity is calculated from abundance data.

Overcoming life stage‐centric biases illuminates arthropod diversity, systematics and biology

“…As has been reported elsewhere (e.g., Fikáček et al, 2023), a substantial number of higher Coleopteran taxa were represented almost entirely by larvae. Of 16 species of Cantharidae, only 3 were represented by a single adult each (two of which were female).…”

“…Similarly, males and females of many species may differ considerably in abundances, activity period, behaviour and longevity (Cordellier et al, 2020;Kirkendall, 1993;Sielezniew et al, 2020;Smith et al, 2019;Williams et al, 2022). By omitting unidentifiable immature stages or sexes in such cases, we lose much that is of interest to the community under study, including accurate estimates of species richness, abundance, biomass, community structure and functional diversity (Fikáček et al, 2023;Marcos-García et al, 2012;Silveira & de Araújo, 2021). At the same time, studies of arthropod phylogeny, taxonomy and evolution are greatly strengthened by inclusion of characters from all the forms a species and its genome may manifest (Faria et al, 2021;Lawrence et al, 2011;Meier et al, 2016;Norton & Ermilov, 2014;Yeo et al, 2018).…”

Forest leaf litter beetles of Taiwan: first DNA barcodes and first insight into the fauna

“…We were also able to sort larval specimens into species and associate part of them with co-occurring adults. This task would be impossible using morphology (see Fikáček et al (2023)). In several widespread species, we were also able to compare DNA barcodes from Taiwan with those published from other areas: some were found nearly identical (e.g., in Hypomedon de-bilicornis (Wollaston, 1857)), others indicate that the East Asian specimens form an isolated lineage (e.g., in Perigona cf.…”

“…We used 96 uniquely tagged reverse (HCO2198) primers to identify the position of the sample in the 96-well plate, and four unique forward primers to identify individual plates. For the list of tagged primers, see Fikáček et al (2023). For PCR reaction, we mixed 6.25 μl of GoTaqR Green Master Mix (Promega Corporation, USA), 1.75 μl of dH 2 O, 2.00 μl of bovine serum albumin (BSA, 1 mg/ ml), 1.00 μl of forward primer, 1.00 μl of reverse primer, and 2.00 μl of DNA extract.…”

“…The base calling was performed subsequently in Guppy v4.0.11 software (Oxford Nanopore Technologies). For detailed protocols used, see Fikáček et al (2023).…”