Complete mitochondrial genome of Hygrobates turcicus Pešić, Esen & Dabert, 2017 (Acari, Hydrachnidia, Hygrobatoidea)

The aim of the study was sequencing of the mitogenome of Hygrobates turcicus Pešić, Esen & Dabert, 2017 to expand knowledge of the polymorphism and cryptic or pseudocryptic diversity within Hydrachnidia. The samples originated from Bulgaria, Vidima River near Debnewo, 42°56′41.4′′N, 24°48′44.6′′E, depth 0.4 m, stones on the bottom, water flow 0.71 m/s, temperature 10 °C, pH 8.53, oxygen 110%, conductivity 279 µS/cm, hardness 121 CaO mg/l; 11 males, 27 females, 2 deutonymphs 12.x.2019 leg. Zawal, Michoński & Bańkowska; one male and one female dissected and slides mounted. The study was carried out using the following methods: DNA extraction, sequencing, assembly and annotation, comparison with other populations of H. turcicus, and multigene phylogeny. As a result of the study, it was determined that the mitogenome is 15,006 bp long and encodes for 13 proteins, 2 rRNAs, and 22 tRNAs. The genome is colinear with those of H. longiporus and H. taniguchii, the difference in size originating from a non-coding region located between protein-coding genes ND4L and ND3. Five genes have alternative start-codon, and four display premature termination. The multigene phylogeny obtained using all mitochondrial protein-coding genes unambiguously associates H. turcicus with the cluster formed by H. longiporus and H. taniguchii.

Water mites (Hydrachnidia) are very diverse and species rich group of macroinvertebrates. They occupy almost all freshwater environments. An updated version of Limnofauna Europaea (www. water mite. org) shows the improvement of the knowledge on European water mite biodiversity. From the 1062 species listed in 1978 year, 28% have been synonymized or excluded because of their uncertain status (species incertae), while at the same time over 200 species were added 1 . There is still a clear gap in alpha-taxonomy and knowledge upon phenotypic polymorphism and cryptic diversity within Hydrachnidia. Recent publications 2-10 indicate the presence of many unrecognized species, especially in the southern part of Europe, which can be distinguished by molecular methods.
The publication of the three parts of an identification key [11][12][13] initiated a new trend in researchers on European water mites, facilitating or enabling ecological and biological research. The release of these keys was preceded by numerous revisions of individual genera [14][15][16] based on morphological data. However, the development of molecular barcoding 17 quickly suggested that alpha-taxonomy is still weakly recognised and there are many species-complex containing cryptic or pseudo-cryptic species. An important work on integrative taxonomy and phylogeny of water mites was published by Dabert et al. 18 . Their analyses, based on nuclear ribosomal genes such as 18S, 28S and fragments of the mitochondrial gene of the cytochrome c oxidase subunit 1 (cox1), provided evidence about the relationships within the group. Recent taxonomical studies 2-8 indicated a lack of knowledge about phenotypic polymorphism and cryptic or pseudo-cryptic diversity within Hydrachnidia. Therefore, more genetic data are needed. One of the ways to achieve this is to sequence complete mitochondrial genomes. There is an important, rapidly growing literature dedicated to the sequencing of complete mitogenomes, but despite this, up to now only 12 mitogenomes of Hydrachnidia have been made available and published [19][20][21][22][23] .
To participate in this effort, we have undertaken the sequencing of the complete mitogenome of Hygrobates turcicus Pešić, Esen & Dabert, 2017, a species belonging to genera extensively studied by DNA-barcoding [2][3][4][5][6]9,10 .   5,6 . It is worth noting that based on cox1 comparisons (Table 3), the specimen sequenced in the current study didn't exhibit a higher conservation with the other specimen from Bulgaria (GenBank: MN520308), more precisely from the Strymon river, than with 9 others specimens originating from Turkey. This might prove to be a limitation for accurate biogeographical studies that need to be considered when using single gene barcoding. Molecular barcoding has proven useful to unveil the genetic diversity between closely related species, if not cryptic or semi-cryptic species of water mites, especially when obtained through the sequencing of the cox1 gene. It is usually sufficient to perform molecular phylogenies within these species. For more distantly related species, e.g. belonging to different families, more conserved nuclear genes such as the small subunit of the ribosomal RNA   10,18,24 . What we would like to emphasize in our work is that complete mitogenomes can prove to be also useful. The amount of data retrieved from the concatenation of all protein-coding genes led to the obtention of a phylogenetic tree with optimal support at the nodes. The results obtained in this study combine geographically distant but taxonomically related species (H. longiporus, H. taniguchii and H. turcicus) into one clade, thereby establishing a sister group for the clade comprising the genus Unionicola, which belongs to the same superfamily (Hygrobatoidea) and contrasting the rest of the water mite species (Sperchon clupeifer and Mideospis roztoczensis). At the same time, all species of water mites constitute one group, a sister group of species belonging to Trombidia (genus Leptotrombium). This illustrates the great usefulness of complete mitogenomes for the recognition of relationship between geographically and taxonomically distant taxa. Based on our results and subsequent comparisons with the works of other authors 22 , we could also notice some differences among the genus Hygrobates for what concerns the start and stop codons of their mitochondrial genes. In the near future, it would be interesting to sequence mitogenomes of other species closely related to H. turcicus, to see how much these features are conserved.
Finally, studies such as ours will be helpful in the near future for members of the community who work on biomonitoring based on metabarcoding or environmental DNA. We might cite the recent article from Blattner et al. 25 , which includes Hygrobates norvegicus among other bioindicator species. This study was based on the www.nature.com/scientificreports/ amplification of several mitochondrial genes. Sequencing complete mitogenomes of duly identified specimen of water mites will help documenting the databases for later uses in similar studies. DNA extraction, sequencing, assembly and annotation. Water mites were collected by hand netting. Specimen were sorted out, initially identified and preserved in 96% ethanol, which is a method generally used in genetic research material 5 . Up to 50 specimens identified as H. turcicus were pooled together, and their DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) as described previously 23 . Exoskeletons were retrieved after DNA extraction and mounted in Hoyer's medium. Sequencing was performed at the Beijing Genomics Institute in Shenzhen, China, on a DNBSEQ platform in accordance with the company's procedure. A total of ca. 40 million clean 150 bp paired-end reads were obtained and assembled using SPAdes 3.14.0 26 using a k-mer of 125. The contig corresponding to the mitogenome was extracted, and the Consed 27 package was used to verify its extremities. Annotations were done with the help of MITOS 28 and manually curated. Multigene phylogeny. We aligned the 13 complete mitochondrial genomes with MAFFT version 7.510 29 ,

Materials and Methods
using Riccardoella tokyoensis and Riccardoella reaumuri as outgroup terminals. We conducted maximum likelihood (ML) analyses using RAxML-NG 30 under three different strategies. (1) One of the IR regions was removed from all mitochondrial genomes to reduce overrepresentation of duplicated sequences before we ran RAxML-NG on the unpartitioned alignment under GTR + I + G substitution model as a single partition; (2) The same data was partitioned by gene, exon, intron and intergenic spacer regions, allowing separate base frequencies, α-shape parameters, and evolutionary rates to be estimated for each; (3) we inferred the best-fitting partitioning strategy with PartitionFinder2 31 for the alignment. The best fitting nucleotide substitution models were inferred with jModelTest2 32 . Phylogenetic trees were visualized and edited with FigTree 1.4.4 33 . Support for the ML tree branches was calculated using the nonparametric bootstrap method with 1000 replicates.

Data availability
The complete mitogenome sequence of Hygrobates turcicus Pešić, Esen & Dabert, 2017 has been submitted to GenBank with the accession number OM336267. Data are available on Zenodo as the full sequence of the mitogenome in fasta format and annotations in tbl format with the following link: https:// doi. org/ 10. 5281/ zenodo. 69404 57.