Hygrobates calabricus , a new species of water mite (Acariformes, Hydrachnidia, Hygrobatidae) from Italy, based on morphological and molecular evidence

In the present study we used an integrative taxonomic approach that combines morphological and DNA barcoding data to describe a new species of the H. nigromaculatus -complex, Hygrobates calabricus sp. nov . from Calabria, Italy. The average K2P-distance between DNA-barcode sequences of H. calabricus sp. nov. and its closest relative H . setosus Besseling, 1942 was estimated at 8.7±1.2%.


Introduction
Water mites of the genus Hygrobates Koch, 1837 have been found in all biogeographical regions except Antarctica (Pešić et al. 2017). Members of this genus are often one of the most abundant water mite species in all kind of running and standing waters all over Europe (Pešić et al. 2017). Recent integrative taxonomic studies applying DNA barcodes have revealed that most of the widely distributed European Hygrobates species which were considered to be morphologically well-defined species represent complexes of distinct (semi-cryptic) species that appear to have a more restricted distribution (e.g., Hygrobates nigromaculatus Lebert, 1879-Martin et al. 2010, Pešić et al. 2020H. fluviatilis (Ström, 1768) -Pešić et al. 2017, 2019bH. longipalpis (Hermann, 1804) -Pešić et al. 2019aH. longiporus Thor, 1898-Pešić et al. 2021H. calliger Piersig, 1896-Pešić et al. 2021.
The H. nigromaculatus species-complex includes four species: H. nigromaculatus Lebert, 1879 andH. setosus Besseling, 1942, both widely distributed in Northern and Central Europe (see Martin et al. 2010 for an overview) as well as H. lacrima Pešić, 2020 andH. limnocrenicus Pešić, 2020, both originally described from the Western Balkans (Pešić et al. 2020). The record of H. limnocrenicus from Goldersbach stream in South-West Germany in this study, confirmed by DNA barcode evidence, suggests that the latter species is more widely distributed.
As a result of an integrative taxonomic approach, we discovered one new species of the H. nigromaculatus species-complex from South Italy, which will be described in the present study.

Material and Methods
Water mites were collected by hand netting, sorted alive in the field, and immediately preserved in 96% ethanol. Specimens for molecular analysis were examined without dissecting under a compound microscope in ethanol, using a cavity slide with a central depression. Two specimens of the Hygrobates nigromaculatus complex from Italy, collected by the junior author, were sent for molecular analysis (see below). After DNA extraction, the specimen vouchers were stored in 96% EtOH and returned to the first author for morphological examination. The voucher of the successfully barcoded specimen was dissected and slide mounted in Faure's medium (gum arabic (15 g), distilled water (25 ml), glycerine (10 ml) and chloral hydrate (25 g)), while the second specimen was transferred to Koenike's fluid.
Morphological nomenclature follows Pešić et al. (2017; for explanations concerning morphology and measurements of Hygrobates species see there Figs. 1B-D). The holotype of the new species will be deposited in Naturalis Biodiversity Center in Leiden (RMNH).
All measurements are given in µm. The genital acetabula were measured on both sides, and therefore their dimensions were given as a range. The following abbreviations are used: Ac-1 to -3 = acetabula (numbered from anterior to posterior); Cx-I to -IV = coxae (numbered from anterior to posterior); dL = dorsal length; H = height; I-L-4-6 = fourth to sixth segments of first leg; L = length; mL = median length; P-1 to -5 = palp segments 1 to 5; W = width.

Molecular analysis
Molecular analyses was conducted at the Canadian Centre for DNA Barcoding (Guelph, Ontario, Canada; (CCDB; http://ccdb.ca/)). In the latter institution the specimens were sequenced for the barcode region of COI using standard invertebrate DNA extraction, amplification and sequencing protocols (Ivanova et al. 2007, Ivanova andGrainger 2007a, b).
Sequence comparisons were performed using MUSCLE alignment (Edgar 2004). Intra-and interspecific genetic distances were calculated based on the Kimura 2-parameter model (K2P; Kimura 1980), using MEGA-X (Kumar et al. 2018). MEGAX software was used to calculate Neighbour-Joining (NJ) trees based on K2P distances (standard for barcoding studies) and pairwise deletion of missing data. The support for tree branches was calculated by the nonparametric bootstrap method (Felsenstein 1985) with 1000 replicates and shown next to the branches. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair.
In order to assess the genetic differentiation of species we used the online ASAP version (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html) with default settings and the K2P distance model. The latter procedure was designated to a list of partitions of species hypotheses using genetic distances, calculated between DNA sequences and ranked by their ASAP-scores: the lower the score, the better the partition (Puillandre et al. 2021).

Species delimitation using DNA-barcodes
The final alignment for species delimitation using COI sequence data comprised 670 nucleotide positions (nps) for 59 specimens of the H. nigromaculatus-complex as well as one H. longipalpis and one H. prosiliens specimen as outgroups (Tab. 1). The nucleotide sequences could be translated into amino acid sequences without any stop codons. Neighbour-Joining (NJ) analysis clustered the COI sequences of the H. nigromaculatus-complex into five strongly supported clades (Fig. 1) The genetic distance between the COI sequence of H. calabricus sp. nov. and its closest relative, H. setosus, was estimated at 8.7±1.2% K2P. This value is more than fifteen times higher than the mean intraspecific differences in the COI sequence of H. setosus (0.52±0.13% K2P; Table 2), which additionally supported the species-status of the new clade from Italy. As only one specimen of the new species could be acquired for use in molecular analyses, intraspecific differences in COI sequence could not be investigated. Hygrobates limnocrenicus was separated from H. calabricus sp. nov. with a genetic distance of 12.2±1.5% K2P (Table 2). Table 2. Estimates of average genetic distance (K2P) (given as distance ± standard error) between clades (lower diagonal) and within each clade (diagonal) of examined species of the H. nigromaculatus-complex sequence pairs.
Diagnosis (Male) -Anterior margin of male genital field with a less pronounced medial projection, posterior margin without indentation, with a small convexly rounded projection; L of IV-L-6 proximoventral seta 38 μm.
Description: Male -Colour yellowish. Integument finely striated. Posteromedial margin of Cx-I rounded, caudo-lateral apodemes of Cx-I+II slightly developed ( Fig. 2A); Cx-IV subtriangular, with a distinct noselike protruding medial margin. Anterior margin of genital field convex, with a small medial projection, typically with irregular margin of a secondary sclerotization, posterior margin due to the secondary sclerotization without indentation, with a small centrally rounded projection, Ac in triangular arrangement (Fig. 2B). Gnathosoma anteriorly with clearly offset projections. P-2 ventral margin straight, distally forming a right angle, denticles covering distal half of ventral margin; P-3 with denticles covering distal two thirds of ventral margin; P-4 ventral setae on the same level (Figs. 2C-D).

Female -Unknown.
Etymology -Named after the (so far eclusive) occurrence of the species in Calabria (Italy).
Remarks -The phylogenetic analysis based on COI data placed Hygrobates calabricus sp. nov. as a sister clade to H. setosus, a species with a preference for running water habitats as well. The average K2P genetic distance between these two species was estimated to be 8.7±1.2% K2P indicating their genetic isolation. The results of ASAP analysis strongly supported the species status of the Hygrobates specimens collected in Calabria.
Morphologically, the male of H. calabricus sp. nov. differs from H. setosus in the posterior margin of the genital field without indentation (posterior margin indented in H. setosus) and a longer proximoventral seta on IV-L-6 (30 µm in H. setosus). From H. limnocrenicus (in parentheses) the new species differs in a narrower genital field (L/W ratio 0.8), smaller acetabula (Ac-3 >110 μm) and posterior margin of the genital field without indentation (vs. indented in H. limnocrenicus).
Distribution: Italy (so far only known from one stream in South Italy (Calabria, Fig. 3).