View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by edoc Blattner et al. Parasites Vectors (2019) 12:492 https://doi.org/10.1186/s13071-019-3750-y Parasites & Vectors RESEARCH Open Access Hidden biodiversity revealed by integrated morphology and genetic species delimitation of spring dwelling water mite species (Acari, Parasitengona: Hydrachnidia) Lucas Blattner1* , Reinhard Gerecke2 and Stefanie von Fumetti1 Abstract Background: Water mites are among the most diverse organisms inhabiting freshwater habitats and are considered as substantial part of the species communities in springs. As parasites, Hydrachnidia infuence other invertebrates and play an important role in aquatic ecosystems. In Europe, 137 species are known to appear solely in or near spring- heads. New species are described frequently, especially with the help of molecular species identifcation and delimi- tation methods. The aim of this study was to verify the mainly morphology-based taxonomic knowledge of spring- inhabiting water mites of central Europe and to build a genetic species identifcation library. Methods: We sampled 65 crenobiontic species across the central Alps and tested the suitability of mitochondrial (cox1) and nuclear (28S) markers for species delimitation and identifcation purposes. To investigate both markers, distance- and phylogeny-based approaches were applied. The presence of a barcoding gap was tested by using the automated barcoding gap discovery tool and intra- and interspecifc genetic distances were investigated. Further- more, we analyzed phylogenetic relationships between diferent taxonomic levels. Results: A high degree of hidden diversity was observed. Seven taxa, morphologically identifed as Bandakia con- creta Thor, 1913, Hygrobates norvegicus (Thor, 1897), Ljania bipapillata Thor, 1898, Partnunia steinmanni Walter, 1906, Wandesia racovitzai Gledhill, 1970, Wandesia thori Schechtel, 1912 and Zschokkea oblonga Koenike, 1892, showed high intraspecifc cox1 distances and each consisted of more than one phylogenetic clade. A clear intraspecifc threshold between 5.6–6.0% K2P distance is suitable for species identifcation purposes. The monophyly of Hydrachnidia and the main superfamilies is evident with diferent species clearly separated into distinct clades. cox1 separates water mite species but is unsuitable for resolving higher taxonomic levels. Conclusions: Water mite species richness in springs is higher than has been suggested based on morphological species identifcation alone and further research is needed to evaluate the true diversity. The standard molecular species identifcation marker cox1 can be used to identify species but should be complemented by a nuclear marker, e.g. 28S, to resolve taxonomic relationships. Our results contribute to the taxonomical knowledge on spring inhabiting Hydrachnida, which is indispensable for the development and implementation of modern environment assessment methods, e.g. metabarcoding, in spring ecology. Keywords: cox1, Barcoding, Species delimitation, Crenobiosis, 28S, Springs, Biodiversity, Phylogeny, ABGD *Correspondence: [email protected] 1 Department of Environmental Sciences, Geoecology Research Group, University of Basel, St. Johanns-Vorstadt 10, 4056 Basel, Switzerland Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Blattner et al. Parasites Vectors (2019) 12:492 Page 2 of 13 Background Nonetheless, many studies have shown that genetic spe- Water mites (Hydrachnidia) are highly diverse in aquatic cies delimitation, frequently using cox1 barcoding [31], habitats [1]. Tey have a complex life-cycle that includes has a large potential to reveal new species, resolve taxo- a prelarva, a parasitic larval stage, an initial resting stage nomic questions and contribute to biodiversity baselines (protonymph), a free living deutonymph, a second resting and assessments (e.g. [29, 32–35]). Species identifcations stage (tritonymph) and the fnal adult stage [2]. Hydrach- and their ecological interactions are crucial for contri- nidia disperse predominantly through passive rather than butions to crenobiology, community ecology, develop- active pathways because water mite larvae parasitize ing reliable bioindicators and understanding population other invertebrate taxa, generally insect hosts that fy dynamics. Moreover, newly developed methods to moni- [2–5]. tor invertebrate assemblages in freshwater environments, Nearly all freshwater environments are inhabited by such as the simultaneous identifcation of bulk sampled water mite species with a high degree of habitat speciali- individuals (metabarcoding) [36] or the indirect com- zation [6]. Mites in springs and other groundwater-infu- munity reconstruction by analyzing environmental DNA enced ecosystems occur in remarkably high diversity of (eDNA) [37], rely on previously established genetic spe- habitats [7–9]. Of the 970 recorded European water mite cies reference databases. Tus, a proper taxonomical species, 137 are found solely in or near springs and are knowledge and species description is greatly needed. adapted to several microhabitats, such as diferent sub- Several factors are important to account for when using strate types and environmental conditions [2, 9]. Due to genetic species identifcation methods [38], such as the the high degree of adaptation and their infuence on eco- presence of endosymbionts like the alpha-proteobacteria system functioning for other invertebrate taxa [10–14], Wolbachia sp. [39, 40] or the presence of pseudogenes these so called crenobiontic (occur exclusively in spring and nuclear copies of mitochondrial DNA (numts) [41– habitats) and crenophilous (tendency to be found in the 43], which compromise the suitability of mitochondrial spring brook) species play a critical role in spring species molecular markers to identify species. Standard barcod- communities. Considering that springs are island-like ing methods are mainly based on sequence similarity and habitats within an uninhabitable terrestrial matrix [15, the relation between intra- and interspecifc genetic dis- 16], spring dwelling water mite populations are assumed tance, which is commonly calculated by using the Kimura to be rather isolated. Tis would promote reproductive 2-parameter (K2P) [44] and uncorrected (p) distances isolation and therefore lead to an increased speciation [31, 45, 46]. Nonetheless, the usability of genetic markers rate [4, 17], which is among other things an explanation to identify species can vary between diferent taxonomic for the relatively high species diversity of water mites in groups, geographical origin and sampling strategy [31, springs. However, the degree of isolation of spring water 38, 47], which implies a taxon-specifc evaluation prior mite populations is highly dependent on the dispersal to a broad-scale application in environmental assessment abilities of their hosts and infuenced by taxon specifc and conservation. host spectra and specifcity [5, 18, 19]. Furthermore, In this study we use an integrative taxonomy approach the high microhabitat diversity in springs [15, 20], their to verify the species status and validity of the most com- relatively stable environmental conditions [21, 22] and mon spring related Hydrachnidia species in Europe. We absence of large predators, e.g. fsh [9], make them excep- tested the reliability of techniques commonly used to tionally favorable habitats for insect larvae and beneft identify and delimit species using fragments of the mito- their development. Terefore, the diversity and abun- chondrial cytochrome c oxidase subunit 1 gene (cox1) dance of water mite hosts is relatively high in springs [31, 48] and the D1-D2 region of the LSU rDNA gene compared to other freshwater habitats, which is likewise (28S) [49], or using both (e.g. [28, 50–52]). Tis study considered as precondition for the high number of creno- aims at improving knowledge and analytical techniques biont water mite species [9]. for assessing Hydrachnidia diversity in springs and Despite their importance for freshwater species com- explores the strength and weaknesses of standardized munities, the taxonomic knowledge about Hydrachnidia barcoding loci to identify water mite species. species is still limited today and new species are discov- ered frequently (e.g. [23–26]). Te intensity of re-exam- Methods ination of European Hydrachnidia has increased over Water mite sampling the past years with several taxonomic revisions pub- Te studied crenobiontic and crenophilous Hydrachnidia lished [9]. Most water mite species known today have species were sampled between 2008 and 2018 in 87 dif- been described based on morphology only and stud- ferent sampling sites across Europe during multiple sam- ies applying genetic methods to verify and complement pling occasions (Additional fle 1: Table S1). Most of the these descriptions are still relatively scarce (e.g. [27–30]).