1 Introduction
In the past decades, natural environments have been disturbed and destroyed worldwide at alarming rates, which results in a large loss of species (Barnosky et al., 2011; Stork, 2018). In hyperdiverse ecosystems, such as those in the Neotropical region, several species could go extinct before even being identified (Bradshaw et al., 2011; Laurance, 1999). This indicates that biodiversity evaluation needs to be accelerated by combining the strengths of molecular biology, sequencing technology, and bioinformatics to recognize previously known and described species (Gostel & Kress, 2022), and to allow new findings. In this context, DNA-based approaches have become increasingly useful and promising tools for estimating diversity and guaranteeing rapid and accurate identification of species. Since the proposal of the DNA barcoding technique, using a short standard genetic marker for species-level identification and cryptic species detection (Hebert et al., 2003; Hebert et al., 2004), the procedure has been becoming progressively popular among conservationists and taxonomists (Farooq et al., 2020; Pearlet al., 2022), and paved the way for biological monitoring using metabarcoding (e.g., Steinke et al., 2022).
Aquatic insects play a crucial role for the equilibrium of aquatic ecosystems because of their complex life cycle, which distinguishes them from exclusively aquatic or terrestrial life forms, and generates a differentiated potential for understanding biogeographical and ecological research. It is therefore paramount to invest in knowledge of the diversity of these organisms, as they are extremely rich both in functionality and species numbers. Non-biting midges (Diptera: Chironomidae) are true flies, and frequently dominate aquatic insect communities in both abundance and species richness. It is a cosmopolitan group, occurring in an enormous variety of aquatic ecosystems, in all biogeographical regions of the world, including Antarctica. Presumably, the great species and habitat diversity in this family is a product of its antiquity, relatively low vagility and evolutionary plasticity (Ferrington, 2008), which makes the family not only a valuable source of indicator species for lentic and lotic aquatic ecosystems, but also one of the most interesting groups for phylogenetic and biogeographical analyses (Silva & Ekrem, 2016).
The advantage of dealing with hyperdiverse taxa is that they surely exhibit several repeated patterns, which may provide evidence of underlying processes (Coscaron et al., 2009). Therefore, a genus such asPolypedilum , widespread and rich in species, may be suitable for biogeographical and ecological research, as the diversity of these insects is strongly linked with the conservation of aquatic habitats.Polypedilum is one of the largest chironomid genera containing about 440 described species (Sæther et al., 2010). Larvae ofPolypedilum occur in nearly all types of still and flowing waters. Knowledge of their community compositions is essential due to their potential as bioindicators, since natural or man-made shifts have impact on them, and consequently in ecosystem processes. However, whilst most studies rather focus on taxonomic or phylogenetic issues (e.g., Bidawid & Fittkau, 1995; Bidawid-Kafka, 1996; Sæther & Sundal, 1999; Vårdal et al., 2002; Sæther & Oyewo, 2008; Oyewo & Sæther, 2008; Shimabakuro, et al., 2019; Pinho & Silva, 2020), so far there have been only few detailed studies on the species richness and species turnover of the hyperdiverse Chironomidae (Lin et al., 2015; Song et al., 2018).
The biota of South America always has attracted the attention of naturalists because of the interesting distributional patterns exhibited by its flora and fauna (Hooker, 1844-47; Darwin, 1859; Wallace, 1876). For more than a century, biogeographers have proposed theories to explain the origin and relationships of the biodiversity found in South America and other southern temperate regions such as Australia, New Zealand and South Africa (Silva & Farrell, 2017). Moreover, the region is a preferred target for investigating the function of these components in the dynamic of diversification, both by harboring the majority of the Earth’s species and extending across temperate and tropical belts. The high number of species in South America, on a regional as well as on a continental scale, makes the region an important reference mark for estimation of biodiversity loss. However, for the Neotropical non-biting midge fauna, the knowledge of the actual species diversity is fragmentary and formal identifications often are unachievable (Spies & Reiss, 1996).
Usually, automated species delimitation approaches are considered particularly useful in organisms with uncertain species boundaries, due to fragmentary taxonomic knowledge or signals in phylogenetic inferences being obscured by lineage sorting or introgression (O’Meara, 2010 and references therein). In this sense, several methods for species delimitation have been developed and applied, for instance, the Automatic Barcode Gap Discovery – ABGD (Puillandre et al., 2012), the Barcode Index Number – BIN (Ratnasingham & Hebert, 2013), the Generalized Mixed Yule Coalescent – GMYC (Pons et al., 2006), the Poisson Tree Processes – PTP (Zhang et al., 2013). Despite these approaches being suitable to delimit species, they can occasionally lead to uncertainty in genetic diversity estimates due to either oversplitting or overlumping of the taxa. Therefore, the integration of different algorithms is needed for accurate species delimitation. In this study, we first compare the performance of different methods of species estimation and evaluate how much these different approaches affect estimates of putative species richness in South America. We then test the hypothesis that there will be substantial differences in community structure between the Polypedilum fauna in South America, considered more diverse, and neighboring regions, particularly the Nearctic.