A peer-reviewed open-access journal Zookeys 1151: 205-222 (2023) doi: 10.3897/zookeys. | 15 1.100766 RESEARCH ARTICLE #ZooKey S https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research New records of water mites (Acari, Hydrachnidia) from Portugal revealed by DNA barcoding, with the description of Atractides marizae sp. nov. Vladimir Pesic', Milica Jovanovic', Amalia Espiridiao Oliveira’, Ana Pedro?, Marvin Freira?, Maria Manuela Morais?*° | Department of Biology, University of Montenegro, Cetinjski put 6.b., 81000 Podgorica, Montenegro 2 Mediterranean Institute for Agriculture, Environment and Development (MED), CHANGE — Global Change and Sustainability Institute, Institute for Advanced Studies and Research, Universidade de Evora, Pélo da Mitra, Ap. 94, 7006-554, Evora, Portugal 3 Water Laboratory, University of Evora, PI.T:E. Rua da Barba Rala No. 1, 7005-345 Evora, Portugal 4 Institute of Earth Sciences-ICT, University of Evora, Rua Romdo Ramatho 59, 7000-671, Evora, Portugal 5 Department of Biology, School of Sciences and Technologies, Pélo da Mitra Apartado 94 7002-554, Evora, Portugal Corresponding author: Vladimir Pesi¢ (vladopesic@gmail.com) Academic editor: Farid Faraji | Received 18 January 2023 | Accepted 16 February 2023 | Published 1 March2023 https://zoobank. org/6F3BA97A-4E B4-47B4-8ADD-4375417D9C78 Citation: Pesi¢ V, Jovanovic M, Espiridiao Oliveira A, Pedro A, Freira M, Morais MM (2023) New records of water mites (Acari, Hydrachnidia) from Portugal revealed by DNA barcoding, with the description of Atractides marizae sp. nov. ZooKeys 1151: 205-222. https://doi.org/10.3897/zookeys.1151.100766 Abstract This study presents the first results of DNA barcoding of water mites from Portugal. DNA barcodes were recovered from 19 water mite specimens morphologically assigned to eight species, seven of them newly reported from Portugal. Two species, Torrenticola hispanica (Lundblad, 1941) and A. cultellatus (K. Viets, 1930) were discovered more than 80 years after they were first described, and Atractides marizae sp. nov. is described as new for science. Keywords New records, new species, systematics, taxonomy Copyright Viadimir Pesi¢ et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 206 Vladimir PeSié et al. / ZooKeys 1151: 205-222 (2023) Introduction The water mites of Portugal are still insufficiently known. Water mites of mainland Portugal and its archipelagos (Madeira and Azores) were studied by Barrois (1887, 1896), Koenike (1895), Thor (1898), Viets (1918), Lundblad (1941, 1942, 1954, 1956), and Cantallo et al. (2021, 2022). The most recent check list of water mites of Portugal and its archipelagos was published by Cantallo et al. (2022), who reported 93 hydrachnid species from 34 genera and 16 families. All of these species were ex- clusively identified on the basis of morphological characters, and until now there have been no studies analyzing the genetic diversity of this important but often neglected limnofaunistic group. In recent years, the use of the mitochondrial cytochrome c oxidase subunit I (COT) gene, has proven to be a highly effective tool for delimiting and identifying water mites, in particular for recognizing species complexes with potential cryptic diversity (Martin et al. 2010; Pesié et al. 2012, 2017, 2019, 2020, 2022; Fisher et al. 2017; Pesi¢ and Smit 2020). The use of this system, known as DNA barcoding, in recent taxonomic studies has been accelerated by the formation of worldwide databases for the storage and public identification of sequences, such as GenBank and the BOLD system (DNA Barcode of Life Data System). In some regions, COI data on water mites has been intensively accumulated in recent years and has led to the compilation of national and regional DNA barcode libraries (e.g., Blattner et al. 2019; Pesi¢ et al. 2021a, b; Pesié and Smit 2022). This has enabled a better assessment of the molecular diversity of water mites in specific habitats, as well as the identification of problematic species groups, resulting in the de- scription of a number of cryptic or pseudocryptic species that would probably remain undescribed using solely classical taxonomic methods. The main aim of this study is to enrich the existing reference library with new sequences of specimens collected in Portugal and present the taxonomic results of this collecting effort. Materials and methods Water mites were collected by hand netting, sorted live from other organisms and debris in the field, and immediately preserved in 96% ethanol for the purpose of the molecu- lar analyses (see below). Water-mite specimens used for the molecular study are listed in Table 1. After DNA extraction, the specimen vouchers were stored in 96% EtOH and morphologically examined. Some of these vouchers were dissected and slide mounted in Faure’s medium, while the rest was transferred to Koenike’s fluid and stored in the collection of the first author. DNA sequences prepared in the course of this study were deposited in BOLD and GenBank. The DNA extracts were archived in -80 °C freez- ers at the Centre for Biodiversity Genomics (CBG; https://biodiversitygenomics.net). Water mites of Portugal 207 Table |. Details on barcoded specimens, including data and coordinates of sampling sites, the barcode index number ( indicates a new BIN that contains only current sequences) and associated data obtained from BOLD. DNN = distance to nearest neighbor; NN BIN = nearest neighbor BIN; NN taxonomy = spe- cies assigned to nearest neighbor BIN. BOLD data presented here was last accessed on 10 January 2023. Species Locality Coordinates VoucherCode BOLD/GenBank BIN DNN NNBIN NN taxonomy Acc Nos BOLD (%) BOLD: Lebertidae Lebertia pusilla Santarém, 39.4110°N, CCDB_39397_B06 HYDAS018-22/ - - - - Caniceira 8.2615°E OQ211647 CCDB_39397_C03 HYDAS027-22/ 0Q211648 Torrenticolidae Torrenticola Santarém, 39.4110°N, CCDB_39397_B10 HYDAS022-22/ N AES2742 14.02 AEW2607 Torrenticola sp. hispanica Caniceira 8.2615°E OQ211664 Monatractides Beja, Corgo da 37.6961°N, CCDB_39397_B05 HYDAS017-22/ AEU1504 8.98 AED3802 Monatractides stadleri Ponte Quebrada —8.7122°E OQ211649 stadleri Oxidae Oxus Porto, Silveirinhos 41.1727°N, CCDB_39397_A06 HYDAS006-22/ “ AET9442 5.59 AED9576 Oxus angustipositus 8.5007°E OQ211652 angustipositus CCDB_39397_A08 HYDAS008-22/ 0Q211651 CCDB_39397_A07 HYDAS007-22/ OQ211650 Hygrobatidae Atractides marizae Santarém, 39.4110°N, CCDB_39397_B12 HYDAS024-22/ N AER7878 12.98 AEN9154 Atractides sp. nov. Caniceira 8.2615°E OQ211637 giustinii CCDB_39397_C04 HYDAS028-22/ OQ211643 CCDB_39397_C05 HYDAS029-22/ OQ211642 CCDB_39397_C02 HYDAS026-22/ OQ211640 Atractides allgaier Beja, Corgoda_ —37.6886°N, CCDB_39397_B02 HYDAS014-22/ “AEU1287 14.58 ACS0163 Atractides Ponte Quebrada _—8.7043°E OQ211639 distans CCDB_39397_A09 HYDAS009-22/ OQ211641 Atractides Santarém, 39.4110°N, CCDB_39397_B11 HYDAS023-22/ “AEU1503 16.01 ADG8744 — Abractides cultellatus Caniceira 8.2615°E OQ211638 rivalis Pionidae Piona nodata Herdade do 38.4953°N, CCDB_39397_C06 HYDAS030-22/ “ AETO101 10.43 ACR9882 Piona nodata Pinheiro 8.7097°E OQ211655 CCDB_39397_C07_ HYDAS031-22/ OQ211656 CCDB_39397_C08 HYDAS032-22/ OQ211657 CCDB_39397_C09 HYDAS033-22/ OQ211653 CCDB_39397_C10 HYDAS034-22/ OQ211654 Morphological nomenclature follows Gerecke et al. (2016). The genital acetabula in both sexes and the genital plate in the female were measured on both sides; there- fore, their dimensions are given as a range of values, rather than a single value. The holotype and paratypes of the new species are deposited in the Naturalis Biodiversity Center in Leiden (RMNH). 208 Vladimir PeSi¢ et al. / ZooKeys 1151: 205-222 (2023) All measurements are given in um. The photographs of selected structures were made using the camera of a Samsung Galaxy smartphone. The following abbrevia- tions are used: Ac-1 = first acetabulum; Cx-I = first coxae; Dgl-4 = dorsoglandu- laria 4; dL = dorsal length; H = height; I-L-4-6 = fourth-sixth segments of first leg; L = length; IL = lateral length; mL = medial length; P-1-P-5 = palp segment 1-5; S-1 = proximal large ventral seta at I-L-5; RMNH = Naturalis Biodiversity Center, Leiden; S-2 = distal large ventral seta at I-L-5; Vgl-1 = ventroglandularia 1; W = width. Molecular and DNA barcode analyses The molecular analysis was conducted at the Canadian Centre for DNA Barcoding (Guelph, Ontario, Canada; CCDB; http://ccdb.ca/). The specimens were sequenced for the barcode region of COI using standard invertebrate DNA extraction (Ivanova et al. 2007), amplification (Ivanova and Grainger 2007a), and sequencing (Ivanova and Grainger 2007b) protocols. DNA barcode sequences were aligned using MUSCLE alignment (Edgar 2004). Primer nucleotide sequences were removed, and chromatograms were checked for the presence of double peaks, stop codons, and frameshifts, which could indicate the amplification of nuclear mitochondrial pseudogenes. None of the DNA sequences showed evidence of pseudogenes. Data related to each BIN, including the minimum p-distance to the nearest neigh- boring BIN, was estimated through BOLD. Intra- and interspecific genetic distances were calculated based on the p-distance model using MEGA X (Kumar et al. 2018). MEGA X 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 posi- tions included were 1°+2"'+3"'+Noncoding. Results and discussion This study represents first DNA barcodes of water mites from Portugal with a COI barcode dataset obtained from 19 specimens and morphologically assigned to six gen- era (Table 1). The two species, Torrenticola hispanica (Lundblad, 1941) and Atractides cultellatus (K. Viets, 1930), which are both endemic to the Iberian Peninsula, were up- loaded into the BOLD database; these contribute to the formation of a DNA barcode reference library for the reliable identification of water mite species in future studies. Moreover, one species is described as new for science, and seven species are reported as new for water-mite fauna of Portugal. Water mites of Portugal 209 Description of new species Family Hygrobatidae Koch, 1842 Atractides (Atractides) marizae PeSi¢, sp. nov. https://zoobank.org/97384632-7c6e-4387-9a59-d6d907670250 Figs 1, 2A—D, 3 Type material. Holotype GS (sequenced, CCDB_39397_C02, Table 1), dissected and slide mounted (RMNH), PorruGat, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022 leg. Jovanovic. Paratypes: 34, 29, same site and data as the holotype, 23, 19 sequenced (Table 1), 14° (CCDB_39397_C0) damaged (one palp and I-legs missing), 1 9 (CCDB_39397_B12) dissected and slide mounted (RMNH). Diagnosis. Characters of the nodipalpis-species group (integument finely striated, muscle insertions unsclerotized; males with anteriorly and posteriorly indented genital field, P-2 with distoventral projection and ventral margin of P-4 projecting); excretory pore smooth, acetabula relatively small, arranged in an obtuse triangle. Description. General features—Integument striated, muscle insertions unsclerotized; mediocaudal margin Cx-I strongly convex, apodemes of Cx-II in an acute angle with the median line. Excretory pore smooth; Vgl-1 not fused to Vgl-2. Palp with strong sexual dimorphism in shape of P-2 and P-4, in both sexes medial peg-like seta inserting halfway between ventral setae, seta insertions dividing ventral margin into three equal sectors. I-L- 5 proximally subrectangular, distally protruding near insertion S-1, with seta S-1 slender and bluntly pointed, S-2 shorter and pointed, proximally enlarged; I-L-6 slender, curved, basally slightly thickened from the centre to the claw furrow with parallel dorsal and ven- tral margins (Figs 2C, 3C). Male—Anterior margin of genital plate with a notch and bead structure, a fine median tip projecting in a deep indentation; caudal margin with a deep indentation extending to about 1/2 L of Ac-3, Ac rounded to subtriangular, arranged in an obtuse triangle (Fig. 1B, C); ventral margin P-2 with a strongly developed distal exten- sion, P-3 strongly concave, P-4 proximally concave, inflated near proximoventral seta. Female—Caudal apodemes of Cx-I +II strongly protruding, Cx-IV with well-developed apodemes at medial margins (Fig. 3A), P-2 nearly straight with a right-angled ventrodis- tal edge, P-3 dorsal margin slightly concave, P-4 more slender than in the male (Fig. 3B). Measurements. Male (holotype, CCDB_39397_C02; in parentheses some meas- urements of paratype, CCDB_39397_C04)—Idiosoma L 559 (538), W 458 (425); maximum diameter Dgl-4, 28. Coxal shield L 344 (303); Cx-II W 388 (334); Cx-I+ I mL 117 (122), Cx-I+II IL 244 (206). Genital field L/W 91(94)/129(117), L Ac-1-3: 25-28 (25-28), 23-27 (26-30), 30-31 (32-34). Ejaculatory complex L 94. Palp—Total L 338; dL/H, dL/H ratio: P-1, 31/30, 1.05; P-2, 73/58, 1.26; P-3, 83/45, 1.83; P-4, 111/41, 2.73; P-5, 40/14, 2.8; L ratio P-2/P-4, 0.66. Gnathosoma vL 125, chelicera total L 222. 210 Vladimir PeSié et al. / ZooKeys 1151: 205-222 (2023) Figure |. Azractides marizae nov. sp., 6 A,B holotype, CCDB_39397_C02 € paratype, CCDB_39397_ C04 A idiosoma in ventral view B, C genital field. Scale bars: 100 um. Legs—I-L-5 dL 195, vL 142, dL/vL ratio 1.37, maximum H 49, dL/maximum H 3.99, S-1 L 98, L/W ratio 10.5, S-2 L 78, L/W ratio 4.99, distance S-1-2, 16, dL ratio S-1/2, 1.26; I-L-6 dL 141, central H 22, dL/central H ratio 6.46; L I-L-5/6 ratio 1.38. Water mites of Portugal alg Figure 2. A-D Abractides marizae nov. sp., 6 holotype, CCDB_39397_C02 A palp in medial view B palp in lateral view C I-L-5 and -6 D excretory pore E A. ruffoi, 9 CCDB_39397_C02, Corsica; excre- tory pore. Scale bar: 100 pm. Female (CCDB_39397_B12)-—Idiosoma L 686, W 531. Coxal shield L 369; Cx- III W 466; Cx-I+II mL 122, Cx-I+II IL 263. Genital field L/W 150/167, genital plates L 122-124, pregenital sclerite 84, gonopore L 119, L Ac-1-3: 41, 39-41, 42. Palp—Total L 454; dL/H, dL/H ratio: P-1, 38/38, 1.02; P-2, 97/64, 1.51; P-3, 127/52, 2.43; P-4, 147/36, 4.09; P-5, 45/19, 2.41; L ratio P-2/P-4, 0.66. Gnatho- soma vL 158, chelicera total L 280. Legs—I-L-5 dL 277, vL 194, dL/vL ratio 1.43, maximum H 66, dL/maximum H 4,22, S-1 L 145, L/W ratio 12.8, S-2 L 114, L/W ratio 6.1, distance S-1-2, 36, dL ratio S-1/2, 1.27; I-L-6 dL 202, central H 22, dL/central H ratio 9.22; L I-L-5/6 ratio 1.37. ZA2 Vladimir PeSié et al. / ZooKeys 1151: 205-222 (2023) Figure 3. Atractides marizae nov. sp., 2° paratype, CCDB_39397_B12 A idiosoma in ventral view B palp in medial view C I-L-5 and -6. Scale bar: 100 um. Water mites of Portugal 213 Etymology. The new species is named in honor of Marisa dos Reis Nunes, known professionally as Mariza, a famous Portuguese fado singer in the appreciation of the enjoyment her music brings to the authors. Species delimitation using DNA-barcodes. ‘The final alignment for species de- limitation using COI sequence data comprised 674 nucleotide positions (nps) of the 175 Atractides specimens, morphologically assigned to 40 species listed in Suppl. mate- rial 1 and one outgroup, Mixobates processifer from Norway to root the tree. The NJ tree is presented in Fig. 4. The COI tree sequences retrieved from specimens of A. marizae sp. nov. from Portugal appeared as a sister clade of A. ruffoi Gerecke & Di Sabatino, 2013, a rhitrobiontic species endemic to Corsica (Gerecke and Di Sabatino 2013). The p-distance between the COI sequences of specimens of A. marizae sp. nov. from Portugal and one specimen of A. ruffoi from Corsica was estimated at 13.34 + 1.3%, indicating genetic separation between these two clades. The mean intraspecific diver- gence within the clade of new species from Portugal was relatively low (1.09 + 0.27). Remarks. Pesi¢ and Smit (2022), by mistake, assigned the voucher specimen (CCDB 38559A09) of Atractides ruffoi from Corsica to A. guistinii Gerecke & Di Sabatino, 2013, a species endemic to Corsica and Sardinia. Therefore, the sequence NOVMB009-21/ON002561 deposited in BOLD/GenBank belongs to A. ruffoi. Discussion. In regard to the striated integument, a characteristic “notch and bead” structure of male genital field, and the shape of the palp in the male (P-2 with distoventral projection, ventral margin of P-4 projecting), the new species resembles A. nodipalpis Thor, 1899, A. robustus (Sokolow, 1940), and A. ruffoi. Both sexes of A. nodipalpis and A. robus- tus differ by having larger acetabula in a triangular arrangement. Atractides ruffoi differs by the development of a sclerite at the excretory pore (Gerecke and Di Sabatino 2013). A characteristic “notch and bead” structure of the male genital plate is found also in A. clavipalpis (Lundblad, 1956), which in males, differ from the new Portuguese species in having the ventral margin of P-2 distally slightly protruding and not forming a projection, and a distally club-shaped P-4 (Gerecke 2003). Habitat. A rhithrobiont. Collected in a low-order stream, with shaded pool reach- es having accumulations of leaf litter (Fig. 5). Distribution. Portugal; only known from the type locality. Species new for water mite fauna of Portugal Family Lebertiidae Thor, 1900 Lebertia pusilla Koenike, 1911 Material examined. PortuGat, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanovic, 24, 49, 29 sequenced (Table 1). Remarks. The Portuguese specimens molecularly analyzed in this study match the description of L. pusilla, a species widely distributed in the Palaearctic (Di Sabatino et al. 2010). They share the presence of only one short swimming seta on I-L-5 and two 214 Vladimir PeSi¢ et al. / ZooKeys 1151: 205-222 (2023) 16 40 95 94 24 45 0.05 Figure 4. Neighbour-joining a Atractides nodipalpis Atractides ruffoi HYDAS028-22|Atractides marizae sp. nov. |Portugal HYDAS026-22|Atractides marizae sp. nov.|Portugal ear HYDAS024-22|Atractides marizae sp. nov.|Portugal 99 67 ~HYDAS029-22|Atractides marizae sp. nov.|Portugal Atractides cf. robustus (Turkey, Iran) Atractides robustus Atractides subasper Atractides cf. nodipalpis (Montenegro) Atractides tener Atractides cf. nodipalpis (Norway, Germany) Atractides protendens Atractides inflatus Atractides gibberipalpis Atractides latipalpis Atractides samsoni 66 Atractides adnatus Atractides separatus Atractides vaginalis 100 Atractides brendle 82 Atractides macrolaminatus 100 Atractides sp. A a | Atractides sp. B 29 Atractides walteri Atractides latipes Atractides inflatipes 100 Atractides castor Atractides stankovici Atractides cf. allgaier (Corsica) Atractides corsicus HYDAS023-22|Atractides cultellatus| Portugal Atractides cf. inflatipalpis Atractides fluviatilis Atractides ovalis Atractides distans HYDAS009-22|Atractides allgaier|Portugal 109 -HYDAS014-22|Atractides allgaier|Portugal Atractides panniculatus Atractides rivalis Atractides fissus Atractides fonticolus wae Atractides pennatus (Germany) 100 Atractides anae 99 Atractides pennatus (Montenegro, Netherlands, Germany) Mixobates processifer tree of the genus Atractides obtained from 175 nucleotide COI sequences. or three swimming setae on anterior IV-L-5. It is likely that the lineage from Portugal represents a cryptic species, with a p-distance of 9.39-9.79% to the nearest sequence (NLACA493-15) of L. pusilla from the Netherlands. Distribution. Europe. Water mites of Portugal 215 Figure 5. Photograph of locus typicus (Caniceira stream, Santarém, Portugal) of Atractides marizae sp. noy. (inset). Photographs by M. Jovanovié. Family Oxidae K. Viets, 1926 Oxus (Oxus) aff. angustipositus K. Viets, 1908 Material examined. PorruGat, Porto, Silveirinhos stream, 41.1727°N, 8.5007°W, 25.v.2022, leg. Jovanovic, hind? (sequenced; Table 1). Remarks. The Portuguese specimen molecularly analyzed in this study match- es the description of O. angustipositus. These individuals form a unique BIN (BOLD:AET9442), with the nearest neighboring BIN being BOLD:AED9576, which consists of a specimen from Lake Ohrid, North Macedonia. The p-distance between the specimens from Portugal and GenBank O. angustipositus (Montenegro; OL870273, OL870215, OL870142, OL870101) is 8.7—9.3%; this demonstrates the need for taxonomic revision of the O. angustipositus complex for identifying possibly undescribed cryptic species. Distribution. Western Palaearctic. Family Torrenticolidae Piersig, 1902 Torrenticola (Torrenticola) hispanica (Lundblad, 1941) Fig. 6 Material examined. PorruGaL, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanovic, 1 3, (sequenced; Table 1), dissected and slide mounted (RMNH). Remarks. The Portuguese specimen molecularly analyzed in this study perfectly matches the description of 7’ hispanica, a species originally described on basis of speci- mens collected from a stream near Algeciras in Spain (Lundblad 1956). Description. Male—Dorsal shield without a colour pattern, as shown in Fig. 6A; area of primary sclerotization of the dorsal plate with two dorsoglandularia; gnathosomal 216 Vladimir PeSi¢ et al. / ZooKeys 1151: 205—222 (2023) Figure 6. Selected parts of Torrenticola hispanica, 6, CCDB_39397_B10 A dorsal shield B ventral shield C palp, lateral view (P-1 lacking) D gnathosoma and chelicera in lateral view E ejaculatory com- plex. Photographs by V. Pesic¢. bay U-shaped, proximally rounded; Cxgl-4 subapical; suture line of Cx-IV evident, medially starting from posterior margin of genital field in a right angle to the main idiosoma axis; genital field subrectangular; ejaculatory complex conventional in shape (Fig. GE); excretory pore located on the line of primary sclerotization; gnathosoma ventral margin curved, rostrum strongly elongated (Fig. 6D); P-2 longer than P-4; P-2 ventral margin straight, P-2 and P-3 ventrodistal protrusions blunt, laterally flattened, P-4 with a well-developed ventral tubercle bearing one longer and three shorter setae (Fig. 6C). Measurements. (CCDB_39397_B10)—Idiosoma L 784, W 572; dorsal shield L 644, W 483, L/W ratio 1.33; dorsal plate L 598; shoulder plate L 203-206, W 75-81, L/W ratio 2.54-2.71; frontal plate L 142-147, W 70, L/W ratio 2.0—2.1; shoulder/ frontal plate L 1.38-1.45. Gnathosomal bay L 172, Cx-I total L 338, Cx-I mL 164, Cx-Il+UI mL 128; ratio Cx-I L/Cx-II+II] mL 2.64; Cx-I mL/Cx-H+I mL 1.28. Genital field L/W 159/134, ratio 1.19; distance genital field-excretory pore 113, geni- tal field-caudal idiosoma margin 156. Palp: total L 342, dL/H, dL/H ratio: P-1, 39/31, 255 Pelt 58 R975 Pt3.64/ 54,126 P24 1 06150,5.55. P2113, 5s biratie P-2/P-4 1.08; gnathosoma vL 337, chelicera L 400. Distribution. Spain and Portugal. Monatractides (Monatractides) stadleri (Walter, 1924) Material examined. PorruGat, Beja, Corgo da Ponte Quebrada, stream, 37.6961°N, 8.7122°W, 23.v.2022, leg. Jovanovic, 1¢ (sequenced; Table 1), gnathosoma, palps and I-legs dissected and slide mounted (dorsal and ventral shield stored in Koenike fluid). Water mites of Portugal DNF Remarks. The Portuguese specimen molecularly analyzed in this study matches the description of M. stadleri, a species widely distributed in the Mediterranean region and often very frequent in lowland, running waters (Di Sabatino et al. 2010). The sequenced specimen clusters within BOLD:AEU1504, which includes two specimens of M. stadleri from Belgium and one specimen from Spain (identified as Torrenticola sp., deposited in Taxus Medio Ambiente, Spain). The p-distance between the latter BIN and its nearest neighbour, BOLD:AED3802, which includes specimens from Montenegro and Greece, is estimated at 8.98%. This suggests the need for taxonomic revision of the M. stadleri complex to identify possible undescribed cryptic species (see PeSi¢ and Smit 2022 for a discussion). Distribution. Central, Western, and Southern Europe. Family Hygrobatidae Koch, 1842 Atractides (Atractides) cultellatus (K. Viets, 1930) Fig. 7 Material examined. PorruGat, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanovi¢, 19 (sequenced; Table 1), dissected and slide mounted (RMNH). Remarks. The single female specimen from Portugal generally matches the de- scription of A. cultellatus, which was originally described from a single female collected from Rio Manzanares, Spain (K. Viets, 1930). Atractides valencianus (K. Viets, 1930), a species originally described from Spain and later reported by Gerecke (2014) from Sardinia, resembles A. cultellatus in the presence of a lineated integument, a slenderer I-L-6, the more spaced sword setae of I-L-5, and Vgl-1 not fused to Vgl-2, but it differs in having P-2 completely devoid of thickening or rounding in females (Gerecke 2003). Measurements. Female (CCDB_39397_B11)—Idiosoma L 691, W 520. Coxal shield (Fig. 7A) L 378; Cx-III W 489; Cx-I+II mL 94, Cx-I+II IL 216. Genital field L/W 163/159, genital plates L 115-118, pregenital sclerite 78, gonopore L 131, L Ac- 1-3: 33-36, 28, 33. Egg maximum diemeter (7 = 1) 147. Palp (Fig. 7B): total L 354; dL/H, dL/H ratio: P-1, 36/33, 1.1; P-2, 77/51, 1.49; P-3, 95/39, 2.43; P-4, 108/31, 3.45; P-5, 38/13, 3.0; L ratio P-2/P-4, 0.71. Gnathosoma vL 119, chelicera total L 195. Legs: I-L-5 dL 229, vL 139, dL/vL ratio 1.65, maximum H 59, dL/maximum H 3.96, S-1 L 122, L/W ratio 11.1, S-2 L 102, L/W ratio 6.5, distance S-1-2, 38, dL ratio S-1/2, 1.2; I-L-6 dL 181, central H 19, dL/central H ratio 9.63; L I-L-5/6 ratio 1.27. Distribution. Spain and Portugal. Atractides (Atractides) allgaier Gerecke, 2003 Material examined. Portucat, Beja, Corgo da Ponte Quebrada stream, 37.6886°N, 8.7043°W, 23.v.2022, leg. Jovanovic, 29 (sequenced; see Table 1); Corgo da Ponte Quebrada stream, 37.6961°N, 8.7122°W, 23.v.2022, leg. Jovanovié 19. 218 Vladimir PeSi¢ et al. / ZooKeys 1151: 205-222 (2023) Figure 7. Atractides cultellatus, 9, CCDB_39397_B11 A idiosoma in ventral view B palp in lateral view C J-L-5 and -6. Scale bar: 100 um. Water mites of Portugal 219 Remarks. Populations of this species have often been confused with those of Atrac- tides distans (K. Viets, 1914); see Gerecke (2003) for a discussion. Clear morphological differences, for example the presence of a lineated integument in A. allgaier, instead of stri- ated one in A. distans, are confirmed with a large (> 14%) p-distance between these species. Distribution. Central, Western, and Southern Europe. Family Pionidae Thor, 1900 Piona aff. nodata (Miller, 1776) Material examined. PorTUGAL, Reserva Natural do Estudrio do Sado, Herdade do Pin- heiro, 38.4953°N, 8.7097°W, 10.v.2022, leg. Oliveira, EROS. (sequenced; Table 1). Remarks. The Portuguese specimens molecularly analyzed in this study match description of P nodata. Genetic data indicate that all examined specimens form a cluster (BOLD:AET0101) and belong to the same species. This BIN is solely com- posed of the Portuguese specimens; the closest neighboring BIN is that of P nodata (BOLD:ACR9882) from the Netherlands. The high p-distance (10.45%) between these two BINs indicates that the Portuguese lineage may represent a cryptic species. Distribution. Holarctic. Acknowledgements This study is part of the “DNA-Eco” scientific project, supported by a grant of the Montenegrin Ministry of Science. The mobility of the second author, which carried out the fieldwork in Portugal, was supported by the AMIGO ERASMUS project (2019-1-PT01-KA107-060632). We thank Ana Manovié (University of Podgorica) for her excellent laboratory work and the ERASMUS project. We also thank Ivana Pozojevi¢ (Zagreb) and one anonymous reviewer, whose constructive comments great- ly improved this work. References Barrois T (1887) I. Hydrachnides. In: Barrois T (Ed.) Matériaux pour Servir a Etude de la Faune des Eaux Douces des Acores. Imprimerie Le Bigot Fréres, Lille, 16 pp. Barrois T (1896) Recherches sur la faune des eaux douces des Acores. Mémoires de la So- ciété des sciences, de l’agriculture et des arts de Lille 5: 1-172. https://doi.org/10.3406/ bsef.1896.21760 Blattner L, Gerecke R, von Fumetti S (2019) Hidden biodiversity revealed by integrated mor- phology and genetic species delimitation of spring dwelling water mite species (Acari, Parasitengona: Hydrachnidia). Parasites & Vectors 12(1): e492. https://doi.org/10.1186/ s13071-019-3750-y 220 Vladimir PeSié et al. / ZooKeys 1151: 205-222 (2023) Cantallo H, Gomes N, Antunes C, Ribeiro T, Gomes MI, Costa DA (2021) Catalogue of aquatic mites from the Minho River (NW Iberian Peninsula). Environmental Smoke (Spe- cial Issue): 44-47. https://doi.org/10.32435/envsmoke/xibesymp.7 Cantallo H, Gomes N, Antunes C, Martins D, Costa DA (2022) Checklist of water mites from mainland Portugal and its archipelagos. Zootaxa 5213(4): 336-370. https://doi. org/10.11646/zootaxa.5213.4.2 Di Sabatino A, Gerecke R, Gledhill T, Smit H (2010) Acari: Hydrachnidia II. In: Gerecke R (Ed.) Chelicerata: Acari II. SifSwasserfauna von Mitteleuropa (Vol. 7, 2-2). Elsevier Spektrum Akademischer Verlag, Heidelberg, 234 pp. https://doi.org/10.1007/978-3-8274-2266-8_1 Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high 679 throughput. Nucleic Acids Research 32(5): 1792-1797. https://doi.org/10.1093/nar/ ekh340 Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39(4): 783-791. https://doi.org/10.2307/2408678 Fisher JR, Fisher DM, Skvarla MJ, Nelson WA, Dowling APG (2017) Revision of torrent mites (Parasitengona, Torrenticolidae, Torrenticola) of the United States and Canada: 90 descriptions, molecular phylogenetics, and a key to species. ZooKeys 701: 1-496. https:// doi.org/10.3897/zookeys.701.13261 Gerecke R (2003) Water mites of the genus Atractides Koch, 1837 (Acari: Parasitengona: Hygrobatidae) in the western Palaearctic region: a revision. Zoological Journal of the Linnean Society 138(2-3): 141-378. https://doi.org/10.1046/j.1096-3642.06-0.0005 1.x Gerecke R (2014) The water mites of the genus Atractides Koch, 1837 (Acari, Hydrach- nidia: Hygrobatidae) in Corsica and Sardinia. Zoosystema 36(4): 735-759. https://doi. org/10.5252/z2014n4a3 Gerecke R, Di Sabatino A (2013) The water mites (Hydrachnidia and Halacaridae) of the collection Daniele Benfatti at the Museo Civico di Storia Naturale Verona. Bollettino del Museo Civico di Storia Naturale di Verona. Botanica, Zoologia 37: 67-112. Gerecke R, Gledhill T, Pesié V, Smit H (2016) Chelicerata: Acari III. In: Gerecke R (Ed.) SiiSwasserfauna von Mitteleuropa, Bd. 7/2-3. Springer-Verlag Berlin, Heidelberg, 429 pp. https://doi.org/10.1007/978-3-8274-2689-5 Ivanova NV, Grainger CM (2007a) CCDB protocols, COI amplification. http://ccdb.ca/site/ wp-content/uploads/2016/09/CCDB_Amplification.pdf [Accessed on: 2021-12-20] Ivanova NV, Grainger CM (2007b) CCDB protocols, sequencing. http://ccdb.ca/site/wp-con- tent/uploads/2016/09/CCDB_Sequencing.pdf [Accessed on: 2021-12-20] Ivanova NV, de Waard JR, Hebert PDN (2007) CCDB protocols, glass fiber plate DNA ex- traction. http://ccdb.ca/site/wp-content/uploads/2016/09/CCDB_DNA_ Extraction.pdf [Accessed on: 2021-12-20] Koenike F (1895) Neue Sperchon-Arten aus der Schweiz. Revue Suisse de Zoologie 3(3): 415- 427. https://doi.org/10.5962/p.318237 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi.org/10.1093/molbev/msy096 Lundblad O (1941) Neue Wassermilben aus Madeira: Vorlaufige Mitteilung. Entomologisk Tidskrift 62: 93-96. Water mites of Portugal 224 Lundblad O (1942) Die Arthropodenfauna von Madeira nach den Ergebnissen der Reise von Prof. Dr. O. Lundblad Juli-August 1935. 31. Hydrachnellae. Arkiv for Zoologi 5: 1-122. Lundblad O (1954) Neue siideuropaische Wassermilben. Zoologischer Anzeiger 153: 154-159. Lundblad O (1956) Zur Kenntnis stid- und mitteleuropadischer Hydrachnellen. Arkiv for Zo- ologi 10: 1-306. Martin P, Dabert M, Dabert J (2010) Molecular evidence for species separation in the water mite Hygrobates nigromaculatus Lebert, 1879 (Acari, Hydrachnidia): Evolutionary con- sequences of the loss of larval parasitism. Aquatic Sciences 72(3): 347-360. https://doi. org/10.1007/s00027-010-0135-x Pesi¢ V, Smit H (2020) Mideopsis milankovici sp. nov. a new water mite from Montenegro based on morphological and molecular data (Acariformes, Hydrachnidia, Mideopsidae). Acaro- logia 60(3): 566-575. https://doi.org/10.24349/acarologia/20204387 Pesi¢ V, Smit H (2022) Water mites of Corsica: DNA barcode and morphological evidences. International Journal of Acarology 48(4—5): 418-428. https://doi.org/10.1080/0164795 4.2022.2086619 Pesi¢ V, Valdecasas A, Garcia-Jimenez R (2012) Simultaneous evidence for a new species of Tor- renticola Piersig, 1896 (Acari, Hydrachnidia) from Montenegro. Zootaxa 3515(1): 38-50. https://doi.org/10.11646/zootaxa.3515.1.2 Pesi¢ V, Asadi M, Cimpean M, Dabert M, Esen Y, Gerecke R, Martin P, Savi¢ A, Smit H, Stur E (2017) Six species in one: Evidence of cryptic speciation in the Hygrobates fluviatilis complex (Acariformes, Hydrachnidia, Hygrobatidae). Systematic and Applied Acarology 22(9): 1327-1377. https://doi.org/10.11158/saa.22.9.4 PeSi¢ V, Broda £, Dabert M, Gerecke R, Martin P, Smit H (2019) Re-established after hundred years: Definition of Hygrobates prosiliens Koenike, 1915, based on molecular and morpho- logical evidence, and redescription of H. longipalpis (Hermann, 1804) (Acariformes, Hy- drachnidia, Hygrobatidae). Systematic and Applied Acarology 24(8): 1490-1511. https:// doi.org/10.11158/saa.24.8.10 Pesi¢ V, Jovanovic M, Manovi¢ A, Zawal A, Barikowska A, Broda L, Martin P, Dabert M (2020) Two new species from the Hygrobates nigromaculatus-complex (Acariformes, Hydrachnidia, Hygrobatidae), based on morphological and molecular evidence. Acarologia 60(4): 753- 768. https://doi.org/10.24349/acarologia/20204400 Pesi¢ V, Zawal A, Manovi¢é A, Batikowska A, Jovanovi¢é 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 Pesi¢ V, Jovanovié M, Manovi¢ A, Karaouzas I, Smit H (2021b) New records of water mites from the Balkans revealed by DNA barcoding (Acari, Hydrachnidia). Ecologica Montene- erina 49: 20-34. https://doi.org/10.37828/em.2021.49.2 PeSié 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, Hygro- batidae) with the description of two new species. Ecologica Montenegrina 59: 101-122. https://doi.org/10.37828/em.2022.59.10 Thor S (1898) Nye Hydrachnideformer. Fundne I Norge Sommeren. Archiv for mathematik og naturvidenskab 20: 1-10. Viets K (1918) Eine neue Limnesia-Species. Zoologischer Anzeiger 50: 111-112. 222 Vladimir PeSi¢ et al. / ZooKeys 1151: 205-222 (2023) Supplementary material | List of Atractides specimens used for building the neighbour-joining (NJ) tree (Fig. 4) Authors: Vladimir Pesi¢, Milica Jovanovic, Amalia Espiridiao Oliveira, Ana Pedro, Marvin Freira, Maria Manuela Morais Data type: word document Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1151.100766.suppl1