Pešić, Vladimir ¹ ; Smit, Harry ² and Konopleva, Ekaterina S. ³

¹✉ University of Montenegro, Cetinjski put bb, 81000 Podgorica, Montenegro.
²Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, the Netherlands.
³N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Nikolsky Ave. 20, 163020, Arkhangelsk, Russia & Northern Arctic Federal University, Northern Dvina Emb. 17, 163002, Arkhangelsk, Russia.

2023 - Volume: 63 Issue: 1 pages: 262-274

ZooBank LSID: 33E3FBF0-FD5D-4E42-B34A-DC1AE35D0392

Original research

Keywords

Abstract

Introduction

The water mites with more than 7,500 species (Smit 2020) inhabiting all types of standing and running waters, represent a good model group for investigating diversity and distribution patterns in aquatic ecosystems. Molecular data suggest that water mites probably originated in Pangea about 235 million years ago (Dabert et al. 2016). Studies aiming to explain the evolutionary history of this group are still scarce and limited to the higher level molecular phylogeny of water mites (Dabert et al. 2016).

Water mites of the genus Hygrobates Koch, 1837 have been found in all biogeographical regions except Antarctica (Pešić et al. 2017). Hygrobates specimens with a reticulated soft integument generally have been considered as representatives of H. fluviatilis (Ström, 1768), regarded as a common and widely distributed species in the Palaearctic. However, recent integrative taxonomic studies using the DNA barcode region of the COI mitochondrial gene combined with detailed morphological studies proved that the later species represents a species complex consisting of distinct but previously overlooked (or not accepted (Viets 1930; Thor 1927)) lineages (Pešić et al. 2017, 2019b, 2020).

Currently, the H. fluviatilis-complex in Western Palaearctic encompasses ten species, i.e., Hygrobates fluviatilis, H. arenarius Smit & Pešić, 2017, both widely distributed in Central Europe (Pešić et al. 2017), H. corsicus Pešić & Smit, 2017 (Corsica, Sardinia), H. marezaensis Pešić & Dabert, 2017 (Montenegro, Albania, Croatia), H. turcicus Pešić, Esen & Dabert, 2017 (Turkey, Bulgaria), H. persicus Pešić & Asadi, 2017 (Iran, E Turkey), H. grabowskii Pešić, Saboori, Zawal & Dabert, 2019, H. ulii Pešić, Saboori, Zawal & Dabert, 2019, H. mediterraneus Pešić, 2020 (Montenegro, Greece; Pešić et al. 2021b) and H. balcanicus Pešić, 2020, the latter originally described from Bulgaria (Pešić et al. 2020) and in this study also reported from eastern Serbia. Some of these species are at least sympatric, challenging species identification but also making this complex an interesting model to study.

The existence of a large number of standardized cytochrome c oxidase subunit I (COI) sequences generated as a result of recent DNA barcoding initiatives provides the opportunity to reconstruct the diversification of the H. fluviatilis complex based on a time-calibrated phylogeny. Moreover, as a result of an integrative taxonomic approach, we discovered one new species of the H. fluviatilis-complex which will be described in this paper.

Material and methods

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 Faure's medium.

Morphological nomenclature follows Gerecke et al. (2016); for explanations concerning morphology and measurements of H. fluviatilis-complex see Figs. 1B-D in Pešić et al. (2017). The genital acetabula in both sexes and the genital plate in the female were measured on both sides, and therefore their dimensions are given as a range. The holotype and paratypes of the new species are deposited in the Naturalis Biodiversity Center in Leiden (RMNH).

All measurements are in µm. The following abbreviations are used: Ac-1 to -3 = acetabula from anterior to posterior; Cx-I to Cx-IV = coxae I to IV; dL = dorsal length; H = height; I-L-4-6 = fourth to sixth segments of first leg; L = length; Ma (mega-anum) = million years; mL = median length; n = number of specimens examined; P-1 to P-5 = palp segments 1 to 5; W = width; MRCA = the most recent common ancestor.

Molecular analysis

Molecular analysis was performed in the Canadian Centre for DNA Barcoding (Guelph, Ontario, Canada; (CCDB; http://ccdb.ca/) ) using standard invertebrate DNA extraction, COI PCR amplification and sequencing protocols (Ivanova et al. 2007; Ivanova and Grainger 2007a, b). DNA sequences prepared in the course of his study were deposited in BOLD and GenBank with accession numbers indicated in Supplementary Material. DNA of the new species was extracted from three specimens of the genus Hygrobates from Corsica. For all other species, COI sequence data were taken from Pešić et al. (2017, 2019b, 2020, 2021a,b), Pešić and Smit (2022) and Klimov et al. (2022) and downloaded from the respective sequence data archives (see Supplementary Material).

In total, we used 176 sequences representing COI haplotypes of H. arenarius (n=20), H. balcanicus (n=2), H. corsicus (n=3), H. cyrnusensis sp. nov. (n=3), H. fluviatilis (n=79), H. grabowskii (n=4), H. marezaensis (n=7), H. mediterraneus (n=5), H. persicus (n=30), H. turcicus (n=17), and H. ulii (n=6) (see Supplementary Material). Hygrobates prosiliens from the H. longipalpis-complex was used as the outgroups.

Sequence comparisons were performed using MUSCLE alignment (Edgar 2004). Intra- and interspecific genetic distances were calculated based on the Kimura 2-parameter (K2P) model (Kimura 1980), using MEGA-X (Kumar et al. 2018). The COI dataset was collapsed to 113 unique haplotypes using an online FASTA sequence toolbox (FaBox v1.61; https://birc.au.dk/ palle/php/fabox/ ; Villesen 2007). Representatives of Hygrobates longipalpis and Hygrobates prosiliens were used as outgroups. Maximum likelihood phylogenetic analysis was performed using the online server for IQ-TREE v1.6.12 (W-IQ-TREE) with GTR+G evolutionary model and ultrafast bootstrapping algorithm (UFBoot) with 5000 replicates (Nguyen et al. 2015; Hoang et al. 2017).

Additionally, the sequence data were analysed using the Assemble Species by Automatic Partitioning (ASAP), a method designed to species partitioning using a hierarchical clustering algorithm based on the pairwise distance distribution (Puillandre et al. 2021). We used the online ASAP version (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html ) with default settings and K2P distance model.

Time-calibrated reconstruction of phylogeny

A time-calibrated СOI phylogeny (3 codons of СOI) was based on 31 haplotypes of the Hygrobates. We used from two or three sequences per species according to available haplotypes. Representatives of Hydrachnoidea (Hydrachna conjecta and Hydrachna globosa), Arrenuroidea (Horreolanus orphanus and Mideopsis roztoczensis) as well as Hygrobates longipalpis and Hygrobates prosiliens were used as outgroups. The best-fit evolutionary models were calculated through the Model Finder (Kalyaanamoorthy et al. 2017) according to BIC: 1st codon of COI: HKY+G; 2nd codon of COI: SYM+I+G; 3rd codon of COI: TPM3+I. Instead of using the estimated best-fit models, we used the less complex HKY model with corresponding distributions for each partition (Bolotov et al. 2017). Calculations were performed in BEAST v2.7.3 with an optimised relaxed clock and Yule speciation process as priors (Bouckaert et al. 2019). We used external mean evolutionary rate 0.0177 ± 0.0019 calculated for insects by Papadopoulou et al. (2010) for our partitioned COI dataset with log normal distribution (M=0.0177, S=0.056). We also added one fossil calibration point for the most recent common ancestor (MRCA) of Arrenuroidea from the Miocene (Cook 1957; Dabert et al. 2016) with a gamma distribution prior: mean (lambda) = 3.11, offset = 11.5.

Two independent runs of 25,000,000 generations were processed, with sampling every 1000 generations. The resulting tree sets were combined using LogCombiner v1.10.4 with 10% burn-in (Suchard et al. 2018). The ESS values were checked using Tracer v1.7 (Rambaut et al. 2018) and each value of significant parameter was recorded as \textgreater300. A maximum clade credibility tree was computed with TreeAnnotator v1.10.4 (Suchard et al. 2018).

Results

Species delimitation using DNA-barcodes

The final alignment for species delimitation using COI sequence data comprised of 1219 nucleotide positions (nps) for 176 specimens of the H. fluviatilis-complex, and one specimen each of H. prosiliens and H. longipalpis to root the tree. The nucleotide sequences could be translated into amino acid sequences without any stop codons. Both the ML and NJ trees, based on COI sequences, were in agreement regarding the general topology. The ML tree is shown in Figure 1.

Phylogenetic analyses clustered COI sequences of analysed Hygrobates specimens into 11 maximally supported clades (Figure 1), ten of them corresponded to the previously known members of the H. fluviatilis-complex. The COI sequence found in the Hygrobates specimens collected in Corsica was recovered as a sister clade to the H. fluviatilis. The genetic distance between the COI sequence of H. cyrnusensis sp. nov. and H. fluviatilis amounted to 11.8±1.4% K2P. These genetic distances were higher than the barcoding gap found by the ASAP method (4 to 10%) in the genetic distances among all species belonging to the H. fluviatilis-complex (Figure 2), which additionally supported the species-status of the new species from Corsica.

Time-calibrated reconstruction of phylogeny

Fossil-calibrated modelling (Figure 3) reveals that the MRCA of H. fluviatilis-complex likely originated in the Oligocene (mean age = 31.6 Ma, 95% highest posterior densities (HPD) = 20.0-44.2 Ma, BEAST BPP = 0.89). The diversification and speciation processes mainly started from the Oligocene-Miocene around 24.0 Ma (95% HPD = 15.2-34.2 Ma, BEAST BPP = 1.0) and lasted during the Miocene. The MRCA of turcicus-group (TG), including H. turcicus, H. ulii and H. balcanicus, originated in the Early Miocene about 19.5 Ma (95% HPD = 10.4-30.2 Ma, BEAST BPP = 1.0). Initially H. balcanicus was separated and then the MRCA of H. turcicus and H. ulii diverged about 8.0 Ma (95% HPD = 3.6-13.5 Ma, BEAST BPP = 1.0). The split of persicus-group (PG) was placed in the Early Miocene about 18.4 Ma (95% HPD = 10.2-27.1 Ma, BEAST BPP = 0.70), leading the separation of H. corsicus. Other three species, i.e. H. persicus, H. grabowskii, H. mediterraneus, diverged during the Late Miocene (8.9-6.3 Ma). The MRCA of the fluviatilis-group (FG) existed approximately 17.4 Ma (95% HPD = 9.8-26.1, BEAST BPP = 0.94) and split in two ancestor groups, one of which included the MRCA of Hygrobates fluviatilis and H. cyrnusensis sp. nov. (mean age = 6.7 Ma, 95% HPD = 2.7-11.3 Ma, BEAST BPP = 1.0) and other included the MRCA of H. marezaensis and H. arenarius (mean age = 12.0 Ma, 95% HPD = 5.2-19.5 Ma, BEAST BPP = 0.78).

Systematic descriptions

Family Hygrobatidae Koch, 1842

Genus Hygrobates Koch, 1837

Subgenus Hygrobates s.s.

Hygrobates cyrnusensis Pešić & Smit sp. nov.

ZOOBANK: 828F8D38-AA4B-4DEF-B911-FD58EA7CC04F

Figs. 4-5

Material examined — Holotype ♂ (CCDB 38559 D02), sequenced (see Table 1), dissected and slide mounted (RMNH), France, Corsica, Rivière La Figarella, 42.4873° N, 8.80532° E, 147 m asl., 15 Apr. 2015 leg. Smit. Paratypes: 1♂ (CCDB_44300_C03), 1♀ (CCDB_44300_C01), same data as holotype, sequenced (see Table 1), 1♀ dissected and slide mounted (RMNH).

Diagnosis — Membranous integument dorsally reticulate, cells without "inner lines» (Figure 5B); P-2 distoventrally protruding in a long projection, large denticles covering nearly the whole ventral margin, except a small most proximal part, ventral denticles on P-2/-3 strong; male genital field with Ac-3 slightly elongated, postero-medial margin of Cx-I+II broadly rounded in female.

Description — General features — Colour yellowish to dark brown. Membranous integument dorsally and postero-ventrally reticulate. Postero-medial margin of Cx-I+II convex; Cx-IV subtriangular, with a distinct nose-like protruding medial margin (Figures 4A, 5A). Genital field with acetabula in a triangular arrangement. P-2 ventral margin in the proximal part slightly convex, disto-ventrally protruding in a long projection, large denticles covering more than half of the proximo-ventral margin including the ventral and apical surface of the projection (Figures 5C-D); P-3 with denticles covering distal two thirds of ventral margin. Male — Anterior margin of genital field convex with a knob-shaped medial projection, posterior margin deeply indented, with a central protrusion not extending beyond posterior genital plate margin (Figure 4B). Female — P-4 more slender than in male.

Measurements — Male (holotype) — Idiosoma L 1390, W 1188; coxal field: L 553; Cx-III W 781; mL of Cx-I + gnathosoma L 395; distance between lateralmost ends of Cx-II apodemes, 200. Genital field L/W 228/300, ratio 0.76; L Ac 1-3: 81-83, 81-88, 75-78. Ejaculatory complex L 156.

Palp — Total L 744; dL/H, dL/H ratio: P-1, 39/56, 0.69; P-2, 184/122, 1.51; P-3, 163/109, 1.49; P-4, 278/50, 5.56; P-5, 80/22, 3.64; P-2/P-4 ratio 0.66; distance between P-4 ventral setae 88. Chelicera total L 388.

Legs — dL of I-L-2–6: 159, 205, 306, 316, 234. dL of IV-L-1–6: 191, 225, 294, 444, 459, 334.

Female (paratype) — Idiosoma L 1520, W 1310; coxal field: L 684; Cx-III W 942; mL of Cx-I + gnathosoma L 466; distance between lateralmost ends of Cx-II apodemes, 247. Genital field L/W 291/400; pregenital sclerite W 144; genital plate L 239-241; gonopore L 191; L gonopore/genital plate ratio 0.79-0.8; L Ac 1-3: 100, 100-103, 85-91. Egg maximum diameter (n=3) 172-175.

Palp — Total L 878; dL/H, dL/H ratio: P-1, 56/66, 0.86; P-2, 203/134, 1.51; P-3, 191/125, 1.53; P-4, 334/59, 5.66; P-5, 94/27, 3.48; P-2/P-4 ratio 0.61; distance between P-4 ventral setae 118. Chelicera total L 506, L basal segment 306, claw 175, L basal segment/claw ratio 1.75.

Legs — dL of I-L-1–6: 114, 178, 238, 348, 369, 284. dL of IV-L-1–6: 219, 247, 356, 525, 531, 400.

Etymology — Named after the island (known in Latin as Cyrnus or Corsica) where the new species was collected.

Discussion — Phylogenetic analysis based on COI data placed Hygrobates cyrnusensis sp. nov. as a sister species of H. fluviatilis, a species with a probably wide distribution in Europe. Morphologically, the latter species can be separated from the new species from Corsica in the shape of the palp bearing finer ventral denticles on P-2/-3 in both sexes, and by the male genital field with a comparatively more elongated Ac-3. From the latter species the female of H. cyrnusensis sp. nov. differs in a broadly rounded postero-medial margin of Cx-I. Hygrobates corsicus, another member of the fluviatilis-complex from Corsica, in addition to the red colour of sclerotized parts, differs from H. cyrnusensis sp. nov. by more rounded Ac in male, arranged in an obtuse triangle (Pešić et al. 2017).

Distribution — Corsica (France); known from type locality only.

Remarks — Pešić et al. (2017) stated that all records of H. fluviatilis from Corsica published by Angelier (1959), Giudicelli (1970) and Santucci (1965, 1971), probably refer to H. corsicus. However, in regard to the presence of two distinct lineages, all former records of H. fluviatilis from Corsica, a species whose presence on the island has not been confirmed by molecular analysis, but also the record of a juvenile individual from the Figarella river assigned by Pešić et al. (2017) to H. corsicus, require confirmation, possible by application of molecular techniques.

Discussion

Regardless of the limitation of using a single gene barcode, its usefulness in detecting previously overlooked lineages has been demonstrated for different complexes within the genus Hygrobates (see Martin et al. 2010; Pešić et al. 2017, 2019a, 2021b, 2022). Recently, a high cryptic/pseudocryptic diversity within the Hygrobates fluviatilis-complex has been identified (Pešić et al. 2017, 2019b, 2020), with eleven species know so far, one of them described in this study. As pointed out by Pešić et al. (2017, 2020) morphological identification in the H. fluviatilis-complex can be challenging due to the occurrence of intermediate specimens and often a lack of diagnosable morphological features. This study showed, that despite morphological stasis, diversification of cryptic species in the H. fluviatilis-complex have even started in early Miocene.

So far the historical framework of cryptic diversity of particular water mite complex has not been studied. Pešić et al. (2020) showed that species of the H. fluviatilis-complex encompasses three monophyletic clades, tentatively named as H. fluviatilis, H. turcicus and H. persicus groups. The fluviatilis-group (FG) encompasses four species, i.e., H. fluviatilis, H. marezaensis, H. arenarius and H. cyrnusensis sp. nov. The persicus-group (PG) consists of four species, i.e., H. persicus, H. grabowskii, H. mediterraneus and H. corsicus, while the turcicus-group (TG) includes H. turcicus, H. ulii and H. balcanicus. Previous studies (Pešić et al. 2017, 2020) identified Hygrobates turcicus as the earliest branching lineage within the H. fluviatilis-complex.

The initial divergence of the H. fluviatilis-complex produced two groups of lineages (TG and PG+FG) composed of a set of lineages that have started speciation processes from the Oligocene-Miocene (ca. 24 Ma), much before the beginning of Pleistocene glaciations, which is often considered as a main driver of diversification of the European biota (Hewitt 1996).

Divergence within all three clades started in the early Miocene (ca. 19-17 Ma), and continued to individual species during all Miocene. In a few cases we could associate particular paleogeological events with the present-day distribution and the presumed age of formation of some phylogenetic lineages. For example, Pešić et al. (2020) speculated that present-day distribution of H. fluviatilis and H. balcanicus in the Balkans, can be to associated with emergence of the Miocene Dinaride lake System (ca. 18.2–14.8 Ma; De Leeuw et al. 2012) which acted as a corridor between the Central and Eastern Paratethys Sea (Neubauer et al. 2015) and could have been an effective barrier for the distribution of these species.

Distribution range of H. fluviatilis in the Balkans does not reach the southern border of the Dinaric Karst in Serbia, with the easternmost records of this species known from western Serbia (Pešić et al. 2020, 2021b). This species has never been found in eastern Serbia, which belongs to the old massif of Rhodopes and which is inhabited by H. balcanicus and H. turcicus. The estimation of the appearance time frames of the most common ancestors of H. balcanicus and H. fluviatilis, respectively, in the early Miocene, support the former hypothesis of Pešić et al. (2020) suggesting that geological events that occurred in early Miocene connected with a splitting the Anatolian-Balkan landmass and a rapid regression process in the eastern Paratethys (Görür et al. 2020) are possibly responsible for the present day distribution of these species in the Balkans.

According to our calibrated molecular clock data, splitting between the other two species of the TG clade, H. turcicus (inhabiting the Anatolian landmass), and H. ulii (known from northern Iran), which occurred about 8 Ma, probably was caused by the uplift of the mountains Elburz (12-10 Ma) in northern Iran and the continuous uplift of the Zagros Mountains from the late Miocene onwards (Ballato et al. 2010). Further secondary expansion of H. turcicus likely shaped present-day distribution of the latter species.

Corsica represents a peculiar situation, as it is inhabited by two divergent lineages (H. corsicus and H. cyrnusensis sp. nov.), each of different age. This suggests multiple colonization of this island at different stage of its geological history. In the following, we tried to link the time frames of the appearance of the two above-mentioned species with certain events in the turbulent geological history of Corsica.

The first such event refers to the separation of the CSb microplate (that includes Corsica, Sardinia, part of Calabria, the Balearic Islands and both, Grande and Petite Kabylies) from the Iberian Peninsula and which is dated to about 29 Ma (Alvarez 1972). This process was followed by the clockwise rotation of the Corsican-Sardinian microplate from Eurasia (Alvarez 1972), during which the microplate, through a land bridge, likely remained in contact with the southern edge of Palaeo-Europe (Meulenkamp and Sissingh 2003).

The presumed time frame of separation of H. corsicus from continental ancestors that colonized the Corsica-Sardinia microplate in our study is calculated at about 18 Ma. This indicates that the ancestors reached the CS-microplate subsequently via the land bridge that it maintained with the continent during its rotation. The next important geological event that had an impact on the speciation process was the separation of Corsica and Sardinia, completed about 9 Ma (Alvarez 1972; Boccaletti et al. 1990), which resulted in reaching the present locations of these islands and collided with future peninsular Italy (then connected with Sicily). Finally, the next important geological event was the splitting of Corsica and Sardinia from Calabria during the opening of the Tyrrhenian Sea, occurred between 8.6 Ma and 7.6 Ma (Rosenbaum and Lister 2004), which agrees with our dating of separation of the most common ancestors of H. fluviatilis and H. cyrnusensis sp. nov. (~7 Ma).

Regardless of the agreement in the reconstruction of paleogeological events that are possible responsible for the hypothetical radiation events within the H. fluvuatilis-complex, our study provides a robust phylogenetic framework for future research, therefore the obtained results of this study and their interpretation still warrant caution. A more accurate estimation of divergence time frames by including additional (multiple) markers, and a careful calibration and application of molecular substitution rates, which is beyond the goals of this study, is needed, gaining a much clearer insight into the evolutionary history of H. fluviatilis-complex.

Acknowledgements

We thank the two anonymous reviewers, whose constructive comments greatly improved this work.

acarologia_4589_Supplementary_material.doc

References

1. Alvarez W. 1972. Rotation of the Corsica-Sardinia microplate. Nature, 235: 103-105. https://doi.org/10.1038/physci235103a0
2. Angelier E. 1959. Acariens (Hydrachnellae et Porohalacaridae) des eaux superficielles. In: Angelier E. et collaborateurs, Hydrobiologie de la Corse. Vie et Milieu, Suppl., 8: 64-148.
3. Ballato P., Mulch A., Landgraf A., Strecker M.R., Dalconi M.C., Friedrich A., Tabatabaei S.H. 2010. Middle to late Miocene Middle Eastern climate from stable oxygen and carbon isotope data, southern Alborz mountains, N Iran. Earth Planet. Sci. Lett., 300: 125-138. https://doi.org/10.1016/j.epsl.2010.09.043
4. Boccaletti M., Ciaranfi N., Cosentino D., Deiana G., Gelati R., Lentini F., Massari F., Moratti G., Pescatore T., Lucchi F.R., Tortorici L. 1990. Palinspatic restoration and palaeogeographic reconstruction of the peri-Tyrrhenian area during the Neogene. Palaeogeogr. Palaeoclimatol. Palaeoecol., 77: 41-50. https://doi.org/10.1016/0031-0182(90)90097-Q
5. Bolotov I.N, Kondakov A.V, Vikhrev I.V., Aksenova O.V., Bespalaya Y.V., Gofarov M.Y., Kolosova Y.S., Konopleva E., Spitsyn V., Tanmuangpak K., Tumpeesuwan S. 2017. Ancient river inference explains exceptional oriental freshwater mussel radiations. Sci. Rep., 7(2135): 1-14. https://doi.org/10.1038/s41598-017-02312-z
6. Bouckaert R., Vaughan T.G., Barido-Sottani J., Duchêne S., Fourment M., Gavryushkina A., et al. 2019. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biolo., 15(4): e1006650. https://doi.org/10.1371/journal.pcbi.1006650
7. Cook D.R. 1957. Order Acarina. Suborder Hydracarina. Genus Protoarrenurus Cook, n. gen. In: Palmer A.R. (ed), Miocene Arthropods from the Mojave Desert, California. Professional Papers, Geological Survey No. 294: 248-249.
8. Dabert J., Proctor H., Dabert M. 2016. Higher-level molecular phylogeny of the water mites (Acariformes: Prostigmata: Parasitengonina: Hydrachnidiae). Mol. Phylogenet. Evol., 101: 75-90. https://doi.org/10.1016/j.ympev.2016.05.004
9. De Leeuw A., Mandic O., Krijgsman W., Kuiper K., Hrvatović H. 2012. Paleomagnetic and geochronologic constraints on the geodynamic evolution of the Central Dinarides. Tectonophysics, 530-531: 286-298. https://doi.org/10.1016/j.tecto.2012.01.004
10. Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high 679 throughput. Nucleic Acids Res., 32(5): 1792-1797. https://doi.org/10.1093/nar/gkh340
11. Gerecke R., Gledhill T., Pešić V., Smit H. 2016. Chelicerata: Acari III. In: Gerecke R. (ed.) Süßwasserfauna von Mitteleuropa, Bd. 7/2-3. Springer-Verlag Berlin, Heidelberg, pp. 1-429. https://doi.org/10.1007/978-3-8274-2689-5
12. Giudicelli J. 1970. Les biocénoses zonales d'un réseau hydrographique. Ann. Fac. Sci. Marseille, 43B: 107-125.
13. Görür N., Çağatay N., Sakinç M., Akkök R., Tchapalyga A., Natalin B. 2000. Neogene Paratethyan succession in Turkey and its implications for the palaeogeography of the eastern Paratethys. Geol. Soc. Spec. Publ., 173: 251-269. https://doi.org/10.1144/GSL.SP.2000.173.01.13
14. Hewitt G.M. 1996. Some genetic consequences of ice ages, and their role in divergence and speciation. Biol. J. Linn. Soc., 58: 247-276. https://doi.org/10.1006/bijl.1996.0035
15. Hoang L., Wu F-Y., Clift P.D., Wysocka A., Swierczewska A. 2009. Evaluating the evolution of the Red River system based on in situ U-Pb dating and Hf isotope analysis of zircons. Geochem. Geophy. Geosy., 10(11): 1-20. https://doi.org/10.1029/2009GC002819
16. Ivanova N.V., de Waard J.R., Hebert P.D.N. 2007. CCDB protocols, glass fiber plate DNA extraction. http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_DNA_Extraction.pdf
17. Ivanova N.V., Grainger C.M. 2007a. CCDB protocols, COI amplification. Available at: http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_Amplification.pdf
18. Ivanova N.V., Grainger C.M. 2007b. CCDB protocols, sequencing. Available at: http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_Sequencing.pdf
19. Kalyaanamoorthy S., Minh B.Q., Wong T.K.F., von Haeseler A., Jermiin L.S. 2017. Model finder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587-589. https://doi.org/10.1038/nmeth.4285
20. Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol., 16: 111-120. https://doi.org/10.1007/BF01731581
21. Klimov P.B., Stolbov V.A, Kazakov, D.V, Filimonova M.O, Sheykin S.D. 2022. A DNA barcoding and photo-documentation resource of water mites (Acariformes, Hydrachnidia) of Siberia: Accurate species identification for global climate change monitoring programs. Syst. Appl. Acarol., 27(12): 2493-2567. https://doi.org/10.11158/saa.27.12.8
22. Kumar S., Stecher G., Li M., Knyaz C., Tamura K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol. Biol. Evol., 35: 1547-1549. https://doi.org/10.1093/molbev/msy096
23. Martin P., Dabert M., Dabert, J. 2010. Molecular evidence for species separation in the water mite Hygrobates nigromaculatus Lebert, 1879 (Acari, Hydrachnidia): evolutionary consequences of the loss of larval parasitism. Aquat. Sci., 72: 347-360. https://doi.org/10.1007/s00027-010-0135-x
24. Meulenkamp J.E., Sissingh W. 2003. Tertiary palaeogeography and tectonostratigraphic evolution of the Northern and Southern Peri-Tethys platforms and the intermediate domains of the African-Eurasian convergent plate boundary zone. Palaeogeogr. Palaeoclimatol. Palaeoecol., 196: 209-228. https://doi.org/10.1016/S0031-0182(03)00319-5
25. Neubauer T.A., Harzhauser M., Kroh A., Georgopoulou E., Mandic O. 2015. A gastropod-based biogeographic scheme for the European Neogene freshwater systems. Earth Sci. Rev., 143: 98-116. https://doi.org/10.1016/j.earscirev.2015.01.010
26. Nguyen L.T., Schmidt H.A., von Haeseler A., Minh B.Q. 2015. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. Evol., 32: 268-274. https://doi.org/10.1093/molbev/msu300
27. Papadopoulou A., Anastasiou I., Vogler A.P. 2010. Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration. Mol. Biol. Evol. 27(7): 1659-72. https://doi.org/10.1093/molbev/msq051
28. Pešić V., Smit H. 2022. Water mites of Corsica: DNA barcode and morphological evidences. Int. J. Acarol., 48: 418-428. https://doi.org/10.1080/01647954.2022.2086619
29. Pešić V., Asadi M., Cimpean M., Dabert M., Esen Y., Gerecke R., Martin P., Savić A., Smit H., Stur E. 2017. Six species in one: Evidence of cryptic speciation in the Hygrobates fluviatilis complex (Acariformes, Hydrachnidia, Hygrobatidae). Syst. Appl. Acarol., 22: 1327-1377. https://doi.org/10.11158/saa.22.9.4
30. Pešić V., Broda Ł., Dabert M., Gerecke R., Martin, P., Smit H. 2019a. Re-established after hundred years: Definition of Hygrobates prosiliens Koenike, 1915, based on molecular and morphological evidence, and redescription of H. longipalpis (Hermann, 1804) (Acariformes, Hydrachnidia, Hygrobatidae). Syst. Appl. Acarol., 24(8): 1490-1511. https://doi.org/10.11158/saa.24.8.10
31. Pešić V., Saboori A., Zawal A., Dabert M. 2019b. Hidden but not enough: DNA barcodes reveal two new species in Hygrobates fluviatilis complex from Iran (Acariformes, Hydrachnidia, Hygrobatidae). Syst. Appl. Acarol., 24(12): 2439-2459. https://doi.org/10.11158/saa.24.12.11
32. Pešić V., Jovanović M., Manović A., Zawal A., Bańkowska A., Ljubomirova L., Karaouzas I., Dabert M. 2020. Molecular evidence for two new species of the Hygrobates fluviatilis complex from the Balkan Peninsula (Acariformes, Hydrachnidia, Hygrobatidae). Syst. Appl. Acarol., 25(9): 1702-1719. https://doi.org/10.11158/saa.25.9.15
33. Pešić V., Zawal A., Manović A., Bańkowska A., Jovanović M. 2021a. A DNA barcode library for the water mites of Montenegro. Biodiversity Data Journal 9: e78311. https://doi.org/10.3897/BDJ.9.e78311
34. Pešić V., Jovanović M., Manović A., Karaouzas I., Smit H. 2021b. New records of water mites from the Balkans revealed by DNA barcoding (Acari, Hydrachnidia). Ecol. Monten., 49: 20-34. https://doi.org/10.37828/em.2021.49.2
35. Pešić V., Esen Y., Gerecke R., Goldschmidt T., Mumladze L., Smit H., Zawal A. 2022. Evidence of cryptic speciation in the Hygrobates calliger complex (Acariformes, Hydrachnidia, Hygrobatidae) with the description of two new species. Ecol. Monten., 59: 101-122. https://doi.org/10.37828/em.2022.59.10
36. Puillandre N., Lambert A., Brouillet S., Achaz, G. 2012. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol. Ecol., 21(8), 1864-1877. https://doi.org/10.1111/j.1365-294X.2011.05239.x
37. Rambaut A., Drummond A.J., Xie D., Baele G., Suchard M.A. 2018. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Syst. Biol., 67(5): 901-904. https://doi.org/10.1093/sysbio/syy032
38. Rosenbaum G., Lister G.S. 2004. Neogene and Quaternary rollback evolution of the Tyrrhenian Sea, the Apennines, and the Sicilian Maghrebides. Tectonics, 23: 1-17. https://doi.org/10.1029/2003TC001518
39. Santucci J. 1965. Hydracariens (Hydrachnellae) des eaux superficielles du Porto (Corse). Rapp. p-v. réun. - Comm. int. explor. sci. Mer Méditerr., 18: 545-548.
40. Santucci J. 1971. Contribution à l′étude de la répartition des Hydracariens (Hydrachnellae) des eaux superficielles d'un torrent de Corse - Le Porto. Ann. Fac. Sci. Marseille, 45: 81-99.
41. Shimodaira H., Hasegawa M. 1999. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol. Biol. Evol., 16(8): 1114-1116. https://doi.org/10.1093/oxfordjournals.molbev.a026201
42. Smit H. 2020. Water mites of the world with keys to the families, subfamilies, genera and subgenera (Acari: Hydrachnidia). Monogr. Ned. Entom. Ver., 12: 1-774.
43. Suchard M.A., Lemey P., Baele G., Ayres D.L., Drummond A.J., Rambaut A. 2018. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol., 4(1): 1-5. https://doi.org/10.1093/ve/vey016
44. Thor, S. 1927. Vorläufige Revision der Gattung Hygrobates C.L. Koch, 1837, mit phylogenetischen Bemerkungen. Norsk Ent. Tidskr., 2: 118-148.
45. Viets K. 1930. Zur Kenntnis der Hydracarinen-Fauna von Spanien. Archiv Hydrobiol., 21(3): 359-446.
46. Villesen P. 2007. FaBox: An online toolbox for Fasta sequences. Mol. Ecol. Notes, 7(6): 965-968. https://doi.org/10.1111/j.1471-8286.2007.01821.x

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