Research Article |
Corresponding author: Vladimir Pešić ( vladopesic@gmail.com ) Academic editor: Farid Faraji
© 2023 Vladimir Pešić, Milica Jovanović, Amália Espiridião Oliveira, Ana Pedro, Marvin Freira, Maria Manuela Morais.
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.
Citation:
Pešić V, Jovanović M, Espiridião 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
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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.
New records, new species, systematics, taxonomy
The water mites of Portugal are still insufficiently known. Water mites of mainland Portugal and its archipelagos (Madeira and Azores) were studied by
In recent years, the use of the mitochondrial cytochrome c oxidase subunit I (COI) 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 (
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.,
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.
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 molecular analyses (see below). Water-mite specimens used for the molecular study are listed in Table
Details on barcoded specimens, including data and coordinates of sampling sites, the barcode index number (N 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 = species assigned to nearest neighbor BIN. BOLD data presented here was last accessed on 10 January 2023.
Species | Locality | Coordinates | Voucher Code | BOLD/GenBank Acc Nos | BIN BOLD | DNN (%) | NN BIN BOLD: | NN taxonomy |
---|---|---|---|---|---|---|---|---|
Lebertiidae | ||||||||
Lebertia pusilla | Santarém, Caniceira | 39.4110°N, 8.2615°E | CCDB_39397_B06 | HYDAS018-22/OQ211647 | – | – | – | – |
CCDB_39397_C03 | HYDAS027-22/OQ211648 | |||||||
Torrenticolidae | ||||||||
Torrenticola hispanica | Santarém, Caniceira | 39.4110°N, 8.2615°E | CCDB_39397_B10 | HYDAS022-22/OQ211664 | N BOLD:AES2742 | 14.02 | BOLD:AEW2607 | Torrenticola sp. |
Monatractides stadleri | Beja, Corgo da Ponte Quebrada | 37.6961°N, 8.7122°E | CCDB_39397_B05 | HYDAS017-22/OQ211649 | BOLD:AEU1504 | 8.98 | BOLD:AED3802 | Monatractides stadleri |
Oxidae | ||||||||
Oxus angustipositus | Porto, Silveirinhos | 41.1727°N, 8.5007°E | CCDB_39397_A06 | HYDAS006-22/OQ211652 | N BOLD:AET9442 | 5.59 | BOLD:AED9576 | Oxus angustipositus |
CCDB_39397_A08 | HYDAS008-22/OQ211651 | |||||||
CCDB_39397_A07 | HYDAS007-22/OQ211650 | |||||||
Hygrobatidae | ||||||||
Atractides marizae sp. nov. | Santarém, Caniceira | 39.4110°N, 8.2615°E | CCDB_39397_B12 | HYDAS024-22/OQ211637 | N BOLD:AER7878 | 12.98 | BOLD:AEN9154 | Atractides giustinii |
CCDB_39397_C04 | HYDAS028-22/OQ211643 | |||||||
CCDB_39397_C05 | HYDAS029-22/OQ211642 | |||||||
CCDB_39397_C02 | HYDAS026-22/OQ211640 | |||||||
Atractides allgaier | Beja, Corgo da Ponte Quebrada | 37.6886°N, 8.7043°E | CCDB_39397_B02 | HYDAS014-22/OQ211639 | N BOLD:AEU1287 | 14.58 | BOLD:ACS0163 | Atractides distans |
CCDB_39397_A09 | HYDAS009-22/OQ211641 | |||||||
Atractides cultellatus | Santarém, Caniceira | 39.4110°N, 8.2615°E | CCDB_39397_B11 | HYDAS023-22/OQ211638 | N BOLD:AEU1503 | 16.01 | BOLD:ADG8744 | Atractides rivalis |
Pionidae | ||||||||
Piona nodata | Herdade do Pinheiro | 38.4953°N, 8.7097°E | CCDB_39397_C06 | HYDAS030-22/OQ211655 | N BOLD:AET0101 | 10.43 | BOLD:ACR9882 | Piona nodata |
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
All measurements are given in μm. The photographs of selected structures were made using the camera of a Samsung Galaxy smartphone. The following abbreviations are used:
Ac-1 = first acetabulum;
Cx-I = first coxae;
Dgl-4 = dorsoglandularia 4;
dL = dorsal length;
H = height;
I-L-4-6 = fourth-sixth segments of first leg;
L = length;
lL = lateral length;
mL = medial length;
P-1-P-5 = palp segment 1-5;
S-1 = proximal large ventral seta at I-L-5;
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 (
DNA barcode sequences were aligned using MUSCLE alignment (
Data related to each BIN, including the minimum p-distance to the nearest neighboring BIN, was estimated through BOLD. Intra- and interspecific genetic distances were calculated based on the p-distance model using MEGA X (
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 genera (Table
Holotype
♂ (sequenced, CCDB_39397_C02, Table
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.
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 ventral margins (Figs
Male (holotype, CCDB_39397_C02; in parentheses some measurements of paratype, CCDB_39397_C04)–Idiosoma L 559 (538), W 458 (425); maximum diameter Dgl-4, 28. Coxal shield L 344 (303); Cx-III W 388 (334); Cx-I+II mL 117 (122), Cx-I+II lL 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.
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.
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 lL 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. Gnathosoma 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.
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.
The final alignment for species delimitation using COI sequence data comprised 674 nucleotide positions (nps) of the 175 Atractides specimens, morphologically assigned to 40 species listed in Suppl. material
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. robustus differ by having larger acetabula in a triangular arrangement. Atractides ruffoi differs by the development of a sclerite at the excretory pore (
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 (
Portugal; only known from the type locality.
Portugal, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanović, 2♂, 4♀, 2♀ sequenced (Table
The Portuguese specimens molecularly analyzed in this study match the description of L. pusilla, a species widely distributed in the Palaearctic (
Europe.
Portugal, Porto, Silveirinhos stream, 41.1727°N, 8.5007°W, 25.v.2022, leg. Jovanović, 1♂, 2♀ (sequenced; Table
The Portuguese specimen molecularly analyzed in this study matches the description of O. angustipositus. These individuals form a unique BIN (BOLD:AET9442), with the nearest neighboring BIN being OLD: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.
Western Palaearctic.
Portugal, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanović, 1♂, (sequenced; Table
The Portuguese specimen molecularly analyzed in this study perfectly matches the description of T. hispanica, a species originally described on basis of specimens collected from a stream near Algeciras in Spain (
Male–Dorsal shield without a colour pattern, as shown in Fig.
(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-II+III mL 128; ratio Cx-I L/Cx-II+III mL 2.64; Cx-I mL/Cx-II+III mL 1.28. Genital field L/W 159/134, ratio 1.19; distance genital field-excretory pore 113, genital field-caudal idiosoma margin 156. Palp: total L 342, dL/H, dL/H ratio: P-1, 39/31, 1.25; P-2, 114/58, 1.97; P-3, 64/51, 1.26; P-4, 106/30, 3.55; P-5, 19/13, 1.5; L ratio P-2/P-4 1.08; gnathosoma vL 337, chelicera L 400.
Spain and Portugal.
Portugal, Beja, Corgo da Ponte Quebrada, stream, 37.6961°N, 8.7122°W, 23.v.2022, leg. Jovanović, 1♂ (sequenced; Table
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 (
Central, Western, and Southern Europe.
Portugal, Santarém, Caniceira stream, 39.4110°N, 8.2615°W, 25.v.2022, leg. Jovanović, 1♀ (sequenced; Table
The single female specimen from Portugal generally matches the description 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
Female (CCDB_39397_B11)–Idiosoma L 691, W 520. Coxal shield (Fig.
Spain and Portugal.
Portugal, Beja, Corgo da Ponte Quebrada stream, 37.6886°N, 8.7043°W, 23.v.2022, leg. Jovanović, 2♀ (sequenced; see Table
Populations of this species have often been confused with those of Atractides distans (K. Viets, 1914); see
Central, Western, and Southern Europe.
Portugal, Reserva Natural do Estuário do Sado, Herdade do Pinheiro, 38.4953°N, 8.7097°W, 10.v.2022, leg. Oliveira, 2♂, 2♀ (sequenced; Table
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 composed 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.
Holarctic.
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 Manović (University of Podgorica) for her excellent laboratory work and the ERASMUS project. We also thank Ivana Pozojević (Zagreb) and one anonymous reviewer, whose constructive comments greatly improved this work.
List of Atractides specimens used for building the neighbour-joining (NJ) tree (Fig.
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