DNA barcoding for biodiversity assessment: Croatian stoneflies (Insecta: Plecoptera)

Abstract

Background The hemi-metabolous aquatic order Plecoptera (stoneflies) constitutes an indispensable part of terrestrial and aquatic food webs due to their specific life cycle and habitat requirements. Stoneflies are considered one of the most sensitive groups to environmental changes in freshwater ecosystems and anthropogenic changes have caused range contraction of many species. Given the critical threat to stoneflies, the study of their distribution, morphological variability and genetic diversity should be one of the priorities in conservation biology. However, some aspects about stoneflies, especially a fully resolved phylogeny and their patterns of distribution are not well known. A study that includes comprehensive field research and combines morphological and molecular identification of stoneflies has not been conducted in Croatia so far. Thus, the major aim of this study was to regenerate a comprehensive and taxonomically well-curated DNA barcode database for Croatian stoneflies, to highlight the morphological variability obtained for several species and to elucidate results in light of recent taxonomy. Methods A morphological examination of adult specimens was made using basic characteristics for distinguishing species: terminalia in males and females, head and pronotum patterns, penial morphology, and egg structures. DNA barcoding was applied to many specimens to help circumscribe known species, identify cryptic or yet undescribed species, and to construct a preliminary phylogeny for Croatian stoneflies. Results Sequences (658 bp in length) of 74 morphospecies from all families present in Croatia were recovered from 87% of the analysed specimens (355 of 410), with one partial sequence of 605 bp in length for Capnopsis schilleri balcanica Zwick, 1984. A total of 84% morphological species could be unambiguously identified using COI sequences. Species delineation methods confirmed the existence of five deeply divergent genetic lineages, with monophyletic origin, which also differ morphologically from their congeners and represent distinct entities. BIN (Barcode Index Number) assignment and species delineation methods clustered COI sequences into different numbers of operational taxonomic units (OTUs). ASAP delimited 76 putative species and achieved a maximum match score with morphology (97%). ABGD resulted in 62 and mPTP in 61 OTUs, indicating a more conservative approach. Most BINs were congruent with traditionally recognized species. Deep intraspecific genetic divergences in some clades highlighted the need for taxonomic revision in several species-complexes and species-groups. Research has yielded the first molecular characterization of nine species, with most having restricted distributions and confirmed the existence of several species which had been declared extinct regionally.

Detailed DNA barcoding of mayflies in a small European country proved how far we are from having comprehensive barcode reference libraries

“…Justifying the presence of multiple cryptic species is beyond the scope of this study, but in general, cryptic diversity was mostly discovered in widely distributed euryvalent species, similarly as in other European freshwater taxa (Csapó et al, 2020;Galimberti et al, 2020;Hlebec et al, 2022;Morinière et al, 2017;Sworobowicz et al, 2015). Our data confirmed the more frequent occurrence of tions (Csapó et al, 2020;Gonzalez-Castellano et al, 2020;Mamos et al, 2021;Múrria et al, 2017).…”

“…Justifying the presence of multiple cryptic species is beyond the scope of this study, but in general, cryptic diversity was mostly discovered in widely distributed euryvalent species, similarly as in other European freshwater taxa (Csapó et al., 2020; Galimberti et al., 2020; Hlebec et al., 2022; Morinière et al., 2017; Sworobowicz et al., 2015). Our data confirmed the more frequent occurrence of cryptic taxa in eudominant species, inhabiting various lotic habitats within the Western Palearctic ( Baetis rhodani and Alainites muticus ), Palearctic ( Baetis fuscatus , B. vernus , Caenis luctuosa , C. pseudorivulorum and Seratella ignita ) or Holarctic ( Cloeon dipterum , Centroptilum luteolum and Procloeon pennulatum ) region (Bauernfeind & Soldán, 2012).…”

“…Furthermore, the presence of endemic species and distinct genetic lineages, reflected by unique BINs, emphasizes the need to explore and analyse the local fauna, especially in ecologically diverse areas (e.g. Hlebec et al., 2022; Huemer et al., 2020; Pešić et al., 2021). An excellent proof of the above is Slovakia and the data presented here from the research on its mayflies.…”

“…Our data confirmed cryptic diversity in more than 20% of species (Figure 2b) and exceptionally high mean and maximum intraspecific variability for many of them. RESL delimitation analyses in BOLD assigned sequences to BINs, but it is also true that applying different delimitation approaches can result in different numbers of putative species or MOTUs (Hlebec et al., 2022; Ranasinghe et al., 2022). In our study, the number of MOTUs defined by four methods (bPTP, RESL, ASAP and ABGD) varied from 76 to 107 (Appendix S14; Figures 6 and 7), with the concordant result only in 59 MOTUs.…”

Assessing arthropod biodiversity with DNA barcoding in Jinnah Garden, Lahore, Pakistan

“…To get around these issues and increase precision and dependability, researchers commonly combine morphological identification with molecular methods like DNA barcoding ( Seberg et al, 2003 ). DNA barcoding is a widely used method in many different fields, such as: phylogenetic studies ( Hajibabaei et al, 2007 ), taxonomic analysis ( Dewalt, 2011 ), looking at biodiversity of insect communities ( Hlebec et al, 2022 ), examining genetic patterns ( Zhou et al, 2010 ), phylogenetic analysis ( Huang, Yang & Ke, 2016 ), and food authentication and safety ( Dawan & Ahn, 2022 ). Through the examination of particular genetic markers, such as the cytochrome oxidase I (COI) gene, which is highly conserved among species and less susceptible to homoplasy, DNA barcoding has become an effective tool in species identification ( Gonzalez et al, 2024 ; Antil et al, 2023 ).…”