Mideopsis milankovici sp. nov. a new water mite from Montenegro based on morphological and molecular data (Acariformes, Hydrachnidia, Mideopsidae)

Water mite species of the genus Mideopsis Neuman, 1880 are common in running and standing waters in the Palaearctic. In the present study we used an integrative taxonomic approach by applying partial COI sequences (DNA-barcodes) and morphological characteristics to describe a new species,Mideopsis milankovici sp. nov. from the Mediterranean region of Montenegro. A high genetic distance (18.8-26% K2P) from three other known European congeners, M. crassipes Soar, 1904, M. orbicularis (Müller, 1776), and M. roztoczensis Biesiadka & Kowalik, 1979, support M. milankovici sp. nov. as a distinct species. FromM. persicus Pešić & Saboori, 2015, a species known from South Iran, which resembles the new species in the shape of the ejaculatory complex,M. milankovici sp. nov. differs by the morphology of dorsal shield.


Introduction
Water mites of the genus Mideopsis Neuman, 1880 are known from the Holarctic and Neotropical regions (Cook 1974;Pešić et al. 2013). Recently, the genus was revised by Pešić et al. (2013). The distribution is disjunct, with one group of species with a Holarctic distribution extending with a few species into the Oriental region, and a few species extending into Costa Rica in the New World, and another group limited to South America. According to Pešić et al. (2013) it is likely that the South American taxa which are characterized by extensive setal patches in the male genital field area could represent a distinct genus.
The present study, from a methodological point of view, is based on techniques established in the past decade (e.g., Fisher 2015, Pešić et al. 2019b and bibliography cited therein). Thus, with the exception of Xystonotus willmanni (K. Viets, 1920) (see Blattner et al. 2019) so far molecular methods have not been used in studies of Palaearctic mideopsid mites.
In this paper, Mideopsis milankovici sp. nov. is described from Montenegro. In order to gain insight into the position of the new species in relation to other Mideopsis species from the Palaearctic region, we combined morphological and molecular analyses to infer molecular similarities among the studied species.

Materials and methods
Sampling Water mites were collected by hand netting, sorted live 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 well slide with a central depression. After DNA extraction, some specimens were dissected and slide mounted in Hoyer's medium. DNA sequences prepared in the course of this study were deposited in Bold with voucher codes and accession numbers indicated in Table 1. The holotype and paratypes of the new species will be deposited in Naturalis Biodiversity Center in Leiden (RMNH).

Molecular analysis
For the methods used for COI gene amplification and sequencing see Pešić et al. (2017Pešić et al. ( , 2019a. For this study, DNA was extracted from 16 specimens of the genus Mideopsis from Montenegro and The Netherlands (Table 1). Xystonotus willmanni (K. Viets, 1920) from The Netherlands was used as an outgroup.

Phylogenetic analysis
Sequences were aligned by MUSCLE 3.8.425 algorithm as implemented in Geneious Prime 2020.1.1 (Biomatters Ltd.). The neighbor-joining (NJ) and maximum-likelihood (ML) were constructed using the MEGA X software (Kumar et al. 2018). Pairwise distance calculations between nucleotide sequences were computed using Kimura's 2-parameter (K2P) distance model (Kimura 1980) for all codon positions and transition/transversion ratio was calculated using MEGA X (Kumar et al. 2018). The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei 1993). We used ML analyses with 500 bootstrap replicates using GTR+I as the most appropriate model of sequence evolution for our data set based on the Bayesian (BIC) and corrected Akaike Information Criterion (AICc) in the ML model selection feature of MEGA X. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Results and discussion COI sequences analysis
The analysis involved 18 nucleotide sequences. The nucleotide sequences could be translated into amino acid sequences without any stop codons. The final alignment for the species delimitation using COI sequence data comprised 668 nucleotide positions (nps) for five species including Xystonotus willmanni to root the tree. In the analysis of our COI data, the neighborjoining (NJ) and maximum-likelihood (ML) result trees gave similar results. Other than minor differences in bootstrap support values, no notable differences were found. The ML result tree is presented in Figure 1. The COI sequence found in the Mideopsis specimen collected in Montenegro was recovered as a sister clade of the clade formed by Mideopsis orbicularis and M. roztoczensis (Figure 1). The genetic distance between the COI sequence of the specimen from Montenegro here assigned to M. milankovici sp. nov. and M. orbicularis was 18.8%. Only one specimen of the new species could be acquired for use in molecular analysis, so intraspecific differences in COI sequence could not be investigated.
The mean genetic distance between congeneric COI sequence groups recovered in the molecular analysis ranged from 18% between M. orbicularis and M. roztoczensis, to 29% between M. orbicularis and M. crassipes ( Table 2). The intraspecific distance of M. orbicularis was 1% whereas the other two species, i.e., M. crassipes and M. roztoczensis showed no intraspecific variation.  Diagnosis -Dorsal shield flattened, in the centre slightly elevated, V-shaped area formed by anteriorly diverging lines of fine porosity little evident. Ejaculatory complex with well sclerotized anterior keel, anterior ramus wedge-shaped. Postgenital area short (males 116-125 µm, 18% dorsal shield L; females 85 µm, 12% dorsal shield L), excretory pore closer to posterior idiosoma margin (distance 25-35 µm).
Due to similarity in shape of ejaculatory complexes (see Figure 3e), M. milankovici sp. nov. resembles M. persicus Pešić & Saboori, 2015. The latter species is known from a single male collected in a stream in Fars Province of South Iran (Pešić and Saboori 2015). Mideopsis persicus can be separated by the shape of the dorsal shield with distinct, anteriorly diverging lines of fine porosity forming a well visible V-shaped area. In the new species from  Figure  4d).
Mideopsis crassipes, a species widely distributed in the Holarctic (Gerecke et al. 2016) and M. rossicus, a species known from Russia (Tuzovskij 2002), can be separated from all above-mentioned species including M. milankovici sp. nov. in having an egg-shaped idiosoma and a ventral extension of P-4 strongly curved with anteriorly directed tips.
Habitat -Characteristics of sampling sites indicate a preference for intermittent habitats. Both streams in which M. milankovici sp. nov. was collected are located in the narrow coastal region of Montenegro, their middle and lower courses regularly dry up in summer (for an overview of the species and communities that inhabit intermittent rivers in the southern part of Montenegro see Pešić et al. 2020). The upper part of the Međurječka rijeka stream is perennial ( Figure 5) but runs dry in its lower reach.