Abstract
This study presents the first results from the analysis of water mites collected in Portugal as part of the Biodiversity Genomics Europe project. 307 COI DNA barcodes clustered into 75 BINs are provided, with 38 BINs being unique and deposited for the first time in the Barcode of Life Data Systems (BOLD). 65 species have been identified, of which 36 are new to the water mite fauna of Portugal. Two species, Torrenticolasoniae Pešić, sp. nov. and T.elisabethae Pešić, sp. nov. (Torrenticolidae), are described as new to science. 47% of the water mite species currently known from Portugal now have reference barcodes in BOLD. High intraspecific distances were recorded for some species, suggesting the presence of cryptic diversity and species complexes that needs further study. Our results improve the DNA barcode reference database for Portuguese water mites, enhancing species identification accuracy and stimulating future investigation.
Key words: Cytochrome c oxidase subunit I (COI), DNA barcoding, Iberian Peninsula, integrative taxonomy, Portugal, systematics
Introduction
With nearly 7,500 species grouped into 550 genera (Smit 2020), water mites (Hydrachnidia) are the most diverse and abundant group of arachnids in freshwater habitats (Davids et al. 2007). They inhabit a wide range of aquatic habitats, including lotic, lentic, temporary, and interstitial waters (Davids et al. 2007). Despite the fact that water mites can be good indicators of ecosystem health, especially for groundwater dependent ecosystems, such as springs, the more significant involvement of this limnofaunistic group in rapid assessment programs is still limited by their often time-consuming taxonomic identification (Pešić et al. 2021a).
Knowledge of water mites in Portugal is still insufficient. The checklist published by Cantallo et al. (2022) listed 93 species from 34 genera and 16 families for Portugal and summarized all previous research on water mites in Portugal and its archipelagos (Madeira and Azores). Recently, Pešić et al. (2023b) added seven more species new for the water mite fauna of Portugal, one of which, Atractidesmarizae Pešić, 2023, was new to science.
In recent years, the use of the DNA barcode fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene has proven to be a very effective tool for the identification of water mites (e.g., Pešić et al. 2017, 2022b, 2023a; Blattner et al. 2019). A large number of regional DNA barcoding initiatives resulted in the formation of national and regional water mites DNA barcode libraries in some parts of Europe, such as, for example, Montenegro (http://dx.doi.org/10.5883/DS-MNEHYD; Pešić et al. 2021a), Corsica (http://dx.doi.org/10.5883/DS-CORHYD; Pešić and Smit 2022), Iran and Turkey (http://dx.doi.org/10.5883/DS-TRIRHYD; Pešić et al. 2023c), Norway (http://dx.doi.org/10.5883/DS-NOHYD; Gerecke et al. 2022). However, extensive sampling for DNA barcode reference library building is yet to be performed for a large part of Europe, including for countries like Portugal which have only been partially sampled (Pešić et al. 2023b).
In 2015, the Research Network in Biodiversity and Evolutionary Biology (InBIO) launched the DNA Barcoding Initiative (IBI) with the aim of forming a reference collection of DNA barcodes, focusing on Portuguese invertebrate taxa (Ferreira et al. 2020a). As a result, several datasets holding DNA barcodes of freshwater organisms have been published, namely on Megaloptera, Plecoptera and Trichoptera (Ferreira et al. 2019; Ferreira et al. 2020b; Pauperio et al. 2023). More recently the local efforts to represent countries’ biodiversity in DNA barcode reference collections have been combined with wider endeavors. The implementation of the project Biodiversity Genomics Europe (BGE, https://biodiversitygenomics.eu/) aims to address the global biodiversity crisis by providing a deeper understanding of the diversity of life on Earth through genomics. Additional goals are to develop and strengthen functioning communities of practice, and to build capacity and complementarity across activities and borders. The barcode stream of the project focuses on increasing the geographic and taxonomic representation of understudied organisms in publicly accessible databases. By contributing to the DNA barcode reference library of invertebrate taxa, the project promotes improved assessment and monitoring of macroinvertebrates, including water mites. Moreover, the DNA barcode data provides valuable input for integrative taxonomic research of challenging species groups, especially of cryptic or pseudocryptic species that likely would remain undiscovered using only morphological features.
This is the first in a series of papers related to the diversity of Portuguese water mites that will present the results and ongoing public releases of the DNA barcodes in BOLD. The paper is based on specimens collected in continental Portugal in 2023 and COI records that are publicly available in the Barcode of Life Data Systems (BOLD). As a result of this investigation, we describe two new species to science, and report 36 species of water mites from Portugal for the first time.
Material and methods
Water mites were collected with kick nets and immediately preserved in 96% ethanol for the purpose of molecular analyses. Water mite specimens used for the molecular study are listed in Table 1. After non-destructive, whole-body DNA extraction, the specimen vouchers were stored in 96% ethanol 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.
Table 1.
Details of DNA barcoded specimens, including localities and coordinates of sampling sites, sample codes, and the barcode index number codes (N indicates a new BIN that contains only sequences from this study). BOLD data presented here was last accessed on 10 May 2024.
| Taxa | Locality | Coordinates | Sample ID | Process ID | BIN |
|---|---|---|---|---|---|
| Eylaidae | |||||
| Eylaistantilla | Beja, São João dos Caldeireiros (stream) | 37.625°N, 7.810°W | BGE_00110_B03 | BSNTN490-23 | N BOLD:AFP3591 |
| BGE_00110_B04 | BSNTN491-23 | ||||
| Beja, Herdade de Alagães (stream) | 37.676°N, 7.853°W | BGE_00110_D01 | BSNTN512-23 | ||
| BGE_00110_D02 | BSNTN513-23 | ||||
| BGE_00110_D03 | BSNTN514-23 | ||||
| Beja, Herdade de Alagães (pond) | 37.673°N, 7.848°W | BGE_00110_D08 | BSNTN519-23 | ||
| Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00228_C09 | BSNTN888-23 | ||
| Beja, São Miguel do Pinheiro | 37.552°N, 7.850°W | BGE_00228_G01 | BSNTN928-23 | ||
| Beja, Herdade de Alagães | 37.676°N, 7.853°W | BGE_00228_H01 | BSNTN940-23 | ||
| Limnocharidae | |||||
| Limnocharesaquatica | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_B10 | BSNTN972-23 | N BOLD:AFV0270 |
| BGE_00108_B03 | BBIOP110-24 | ||||
| BGE_00108_B04 | BBIOP111-24 | ||||
| Guarda, Casa do Loureiro | 40.433°N, 7.701°W | BGE_00109_D05 | BBIOP041-24 | ||
| Hydryphantidae | |||||
| Hydrodromadespiciens | Guarda, Covão do Forno | 40.369°N, 7.638°W | BGE_00227_D11 | BSNTN997-23 | BOLD:ACS0426 |
| BGE_00108_A02 | BBIOP097-24 | ||||
| Protziaannularis | Faro, Parque do Barranco dos Pisões | 37.333°N, 8.567°W | BGE_00228_E09 | BSNTN912-23 | N BOLD:AFX2700 |
| Lebertiidae | |||||
| Lebertiafimbriata | Beja, São João dos Caldeireiros | 37.626°N, 7.810°W | BGE_00110_A02 | BSNTN477-23 | BOLD:AEI5359 |
| BGE_00110_B02 | BSNTN489-23 | ||||
| Lebertiasparsicapillata | Beja, Zambujeira do Mar | 37.399°N, 8.723°W | BGE_00110_F01 | BSNTN536-23 | BOLD:AFN4501 |
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F12 | BSNTN1022-23 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A10 | BSNTN865-23 | ||
| Guarda, Casa do Loureiro | 40.433°N, 7.701°W | BGE_00109_H05 | BBIOP089-24 | ||
| Lebertiavariolata | Beja, Zambujeira do Mar | 37.398°N, 8.68°W | BGE_00228_D06 | BSNTN897-23 | BOLD:ADK0996 |
| Faro, Portimão | 37.237°N, 8.546°W | BGE_00228_F08 | BSNTN923-23 | ||
| Lebertiapilosa | Beja, Zambujeira do Mar | 37.399°N, 8.723°W | BGE_00110_E11 | BSNTN534-23 | BOLD:AEJ2601 |
| BGE_00110_E12 | BSNTN535-23 | ||||
| BGE_00110_F07 | BSNTN542-23 | ||||
| BGE_00228_F01 | BSNTN916-23 | ||||
| Lebertiagibbosa | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A08 | BSNTN958-23 | BOLD:ACR9744 |
| BGE_00227_B03 | BSNTN965-23 | ||||
| Guarda, Ponte dos Frades | 40.403°N, 7.526°W | BGE_00108_D12 | BBIOP143-24 | ||
| Guarda, Central hidroelétrica de Ponte dos Jugai | 40.385°N, 7.706°W | BGE_00108_F01 | BBIOP156-24 | ||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00108_F06 | BBIOP161-24 | ||
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00108_G06 | BBIOP173-24 | ||
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00108_G11 | BBIOP178-24 | ||
| Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_F05 | BBIOP065-24 | ||
| Lebertiaalgeriensis | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F07 | BSNTN1017-23 | N BOLD:AFV0271 |
| Guardia, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A03 | BSNTN858-23 | ||
| BGE_00108_E09 | BBIOP152-24 | ||||
| Lebertiainsignis | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_D05 | BSNTN991-23 | BOLD:AEB9107 |
| Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_E11 | BBIOP059-24 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A04 | BSNTN859-23 | N BOLD:AFW6960 | |
| Lebertiaporosa aggr. sp. A | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A10 | BSNTN960-23 | BOLD:ACP5319 |
| BGE_00109_A02 | BBIOP002-24 | ||||
| Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_D01 | BSNTN987-23 | ||
| BGE_00108_B08 | BBIOP115-24 | ||||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00228_C02 | BSNTN881-23 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00108_E06 | BBIOP149-24 | ||
| BGE_00108_E08 | BBIOP151-24 | ||||
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00108_F11 | BBIOP166-24 | ||
| BGE_00108_G03 | BBIOP170-24 | ||||
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00108_G10 | BBIOP177-24 | ||
| Lebertiaporosa aggr. sp. ACS0974 | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_B07 | BSNTN969-23 | BOLD:ACS0974 |
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_E12 | BSNTN1010-23 | ||
| BGE_00227_F09 | BSNTN1019-23 | ||||
| Guarda, Poio do Leão | 40.399°N, 7.541°W | BGE_00227_H10 | BSNTN1044-23 | ||
| Guarda, Ponte dos Frades | 40.403°N, 7.526°W | BGE_00108_D10 | BBIOP141-24 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00108_E05 | BBIOP148-24 | ||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00108_F08 | BBIOP163-24 | ||
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_C01 | BBIOP025-24 | ||
| Bragança, Gasparona | 41.85°N, 7.013°W | BGE_00109_C12 | BBIOP036-24 | ||
| Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_F01 | BBIOP061-24 | ||
| Lebertiapusilla | Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_E09 | BBIOP057-24 | BOLD:AFW6961 |
| Oxidae | |||||
| Oxusangustipositus | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_B12 | BSNTN974-23 | BOLD:AET9442 |
| Guarda, Casa do Cantoneiro | 40.418°N, 7.603°W | BGE_00227_E02 | BSNTN1000-23 | ||
| BGE_00108_H04 | BBIOP183-24 | ||||
| Oxuslusitanicus | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_E09 | BSNTN1007-23 | N BOLD:AFX3224 |
| BGE_00227_E11 | BSNTN1009-23 | ||||
| Beja, Moinho de Alferes 2 | 37.503°N, 7.687°W | BGE_00228_E05 | BSNTN908-23 | ||
| BGE_00228_E06 | BSNTN909-23 | ||||
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_A07 | BBIOP007-24 | ||
| BGE_00109_A08 | BBIOP008-24 | ||||
| Vila Real, Noura stream | 41.409°N, 7.417°W | BGE_00109_D11 | BBIOP047-24 | ||
| Oxusmusculus | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00228_C10 | BSNTN889-23 | BOLD:AFC2154 |
| Oxusovalis | Beja, Zambujeira do Mar | 37.399°N, 8.723°W | BGE_00110_F08 | BSNTN543-23 | N BOLD:AFP5747 |
| BGE_00228_F02 | BSNTN917-23 | ||||
| Oxussetosus | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_E10 | BSNTN1008-23 | BOLD:ACS0808 |
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00228_C03 | BSNTN882-23 | ||
| Sperchontidae | |||||
| Sperchonalgeriensis | Faro, Portimão | 37.237°N, 8.546°W | BGE_00228_F12 | BSNTN927-23 | BOLD:AES2436 |
| Sperchonclupeifer | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F04 | BSNTN1014-23 | N BOLD:AFX0389 |
| Sperchoncompactilis | Faro, Portimão | 37.237°N, 8.546°W | BGE_00110_E03 | BSNTN526-23 | BOLD:AER7687 |
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A07 | BSNTN862-23 | ||
| Torrenticolidae | |||||
| Monatractidesmadritensis | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A12 | BSNTN962-23 | BOLD:AED3803 |
| Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_C09 | BSNTN983-23 | ||
| Guarda, Poio do Leão | 40.399°N, 7.541°W | BGE_00227_H07 | BSNTN1041-23 | ||
| BGE_00108_D02 | BBIOP133-24 | ||||
| Bragança, Gasparona | 41.850°N, 7.013°W | BGE_00109_C06 | BBIOP030-24 | ||
| Monatractidesstadleri | Faro, Parque do Barranco dos Pisões | 37.333°N, 8.567°W | BGE_00110_G03 | BSNTN550-23 | BOLD:AEU1504 |
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F01 | BSNTN1011-23 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A06 | BSNTN861-23 | ||
| BGE_00108_E01 | BBIOP144-24 | ||||
| Guarda, Central hidroelétrica de Ponte dos Jugais | 40.385°N, 7.706°W | BGE_00228_B01 | BSNTN868-23 | ||
| Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_E07 | BBIOP055-24 | ||
| Guarda, Casa do Loureiro | 40.433°N, 7.701°W | BGE_00109_G11 | BBIOP083-24 | ||
| Monatractidesstenostomus | Beja, Corte do Pinto | 37.682°N, 7.512°W | BGE_00228_G08 | BSNTN935-23 | N BOLD:AFU3082 |
| Torrenticolaelliptica | Bragança, Gasparona | 41.850°N, 7.013°W | BGE_00109_C09 | BBIOP033-24 | BOLD:AEI9183 |
| Torrenticolatenuipalpis | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F03 | BSNTN1013-23 | N BOLD:AFV2021 |
| Torrenticolasoniae sp. nov. | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A11 | BSNTN961-23 | N BOLD:AFW5337 |
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F02 | BSNTN1012-23 | ||
| Guarda, Ponte dos Frades | 40.403°N, 7.526°W | BGE_00108_D09 | BBIOP140-24 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00108_E02 | BBIOP145-24 | ||
| BGE_00108_E07 | BBIOP150-24 | ||||
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_C03 | BBIOP027-24 | ||
| Torrenticolaelisabethae sp. nov. | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_C10 | BSNTN984-23 | N BOLD:AFW5336 |
| Guarda, Poio do Leão | 40.399°N, 7.541°W | BGE_00227_H06 | BSNTN1040-23 | ||
| BGE_00108_D04 | BBIOP135-24 | ||||
| Limnesiidae | |||||
| Limnesiaacuminata | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00228_C12 | BSNTN891-23 | N BOLD:AFU7587 |
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_B01 | BBIOP013-24 | ||
| BGE_00109_B03 | BBIOP015-24 | ||||
| BGE_00109_B04 | BBIOP016-24 | ||||
| Limnesiaiberica | Beja, São João dos Caldeireiros | 37.626°N, 7.810°W | BGE_00110_A03 | BSNTN478-23 | N BOLD:AFN8367 |
| BGE_00110_A04 | BSNTN479-23 | ||||
| BGE_00110_A05 | BSNTN480-23 | ||||
| BGE_00110_A06 | BSNTN481-23 | ||||
| BGE_00110_A11 | BSNTN486-23 | ||||
| BGE_00110_A12 | BSNTN487-23 | ||||
| BGE_00110_H08 | BSNTN567-23 | ||||
| BGE_00110_H09 | BSNTN568-23 | ||||
| BGE_00110_H11 | BSNTN570-23 | ||||
| Limnesiakoenikei | Guarda, Covão do Forno | 40.369°N, 7.638°W | BGE_00227_D09 | BSNTN995-23 | BOLD:ADF6559 |
| BGE_00108_A01 | BBIOP096-24 | ||||
| BGE_00108_A03 | BBIOP098-24 | ||||
| Guarda, Central hidroelétrica de Ponte dos Jugais | 40.385°N, 7.706°W | BGE_00228_B02 | BSNTN869-23 | ||
| Limnesiamaculata | Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_B05 | BSNTN967-23 | N BOLD:AFW6935 |
| Guarda, Barragem do Lagoacho | 40.385°N, 7.618°W | BGE_00108_C07 | BBIOP126-24 | ||
| Beja, Moinho de Alferes 2 | 37.503°N, 7.687°W | BGE_00228_E03 | BSNTN906-23 | ||
| Beja, Herdade de Alagães | 37.673°N, 7.848°W | BGE_00228_H04 | BSNTN943-23 | ||
| BGE_00228_H06 | BSNTN945-23 | ||||
| Limnesiawalteri | Beja, Corte do Pinto | 37.682°N, 7.512°W | BGE_00110_B11 | BSNTN498-23 | N BOLD:AFO9873 |
| BGE_00110_C02 | BSNTN501-23 | ||||
| BGE_00228_G09 | BSNTN936-23 | ||||
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_B09 | BBIOP021-24 | ||
| Hygrobatidae | |||||
| Atractidesinflatus | Beja, Zambujeira do Mar | 37.398°N, 8.680°W | BGE_00110_E05 | BSNTN528-23 | BOLD:AFI9009 |
| BGE_00228_D07 | BSNTN898-23 | BOLD:ACB4677 | |||
| Vila Real, Noura stream | 41.409°N, 7.417°W | BGE_00109_D07 | BBIOP043-24 | ||
| Atractidesmarizae | Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F11 | BSNTN1021-23 | BOLD:AER7878 |
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A08 | BSNTN863-23 | ||
| Faro, Caldas de Monchique | 37.287°N, 8.554°W | BGE_00228_D12 | BSNTN903-23 | ||
| Odeceixe, Covão da Serva | 37.374°N, 8.642°W | BGE_00228_E11 | BSNTN914-23 | ||
| Faro, Portimão | 37.237°N, 8.546°W | BGE_00228_F07 | BSNTN922-23 | ||
| Atractidesnodipalpis | Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A01 | BSNTN856-23 | N BOLD:AFV2009 |
| Atractidesrobustus | Guarda, Covão da Ametade | 40.328°N, 7.587°W | BGE_00227_G07 | BSNTN1029-23 | BOLD:AFF2463 |
| Hygrobatesbalcanicus | Faro, Portimão | 37.237°N, 8.546°W | BGE_00110_E01 | BSNTN524-23 | BOLD:AEG3198 |
| BGE_00110_E02 | BSNTN525-23 | ||||
| BGE_00228_F06 | BSNTN921-23 | ||||
| Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_D12 | BBIOP048-24 | ||
| BGE_00109_E10 | BBIOP058-24 | ||||
| Porto, Parque Molinológico e Florestal de Pias | 41.268°N, 8.256°W | BGE_00109_F09 | BBIOP069-24 | ||
| Porto, Rio Este | 41.378°N, 8.695°W | BGE_00109_G07 | BBIOP079-24 | ||
| BGE_00109_G08 | BBIOP080-24 | ||||
| BGE_00109_G09 | BBIOP081-24 | ||||
| BGE_00109_H11 | BBIOP095-24 | ||||
| Hygrobatesfluviatilis | Guarda, Casa do Cantoneiro | 40.418°N, 7.603°W | BGE_00227_E03 | BSNTN1001-23 | BOLD:ACB4846 |
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F10 | BSNTN1020-23 | ||
| Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A09 | BSNTN864-23 | ||
| BGE_00108_E03 | BBIOP146-24 | ||||
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00109_A04 | BBIOP004-24 | ||
| Hygrobateslongiporus complex | Guarda, Praia fluvial de Vila Cova a Coelheira | 40.379°N, 7.736°W | BGE_00228_A02 | BSNTN857-23 | N BOLD:AFV9997 |
| 40.379°N, 7.736°W | BGE_00108_E04 | BBIOP147-24 | |||
| Guarda, Ponte dos Frades | 40.403°N, 7.526 °W | BGE_00108_D11 | BBIOP142-24 | ||
| Gurad, Covão da ponte | 40.443°N, 7.514°W | BGE_00108_G04 | BBIOP171-24 | ||
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00109_A03 | BBIOP003-24 | ||
| Guarda, Casa do Loureiro | 40.433°N, 7.701°W | BGE_00109_H07 | BBIOP091-24 | ||
| BGE_00109_H08 | BBIOP092-24 | ||||
| Bragança, Torre de Dona Chama | 41.665°N, 7.146°W | BGE_00109_B05 | BBIOP017-24 | N BOLD:AFW1423 | |
| Vila Real, Noura stream | 41.409°N, 7.417°W | BGE_00109_D09 | BBIOP045-24 | ||
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A09 | BSNTN959-23 | N BOLD:AFV9998 | |
| BGE_00227_B01 | BSNTN963-23 | ||||
| BGE_00227_B02 | BSNTN964-23 | ||||
| BGE_00109_A01 | BBIOP001-24 | ||||
| Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_D03 | BSNTN989-23 | ||
| BGE_00227_D04 | BSNTN990-23 | ||||
| BGE_00108_B07 | BBIOP114-24 | ||||
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00227_F06 | BSNTN1016-23 | ||
| BGE_00227_F08 | BSNTN1018-23 | ||||
| Guarda, Poio do Leão | 40.399°N, 7.541°W | BGE_00108_D03 | BBIOP134-24 | ||
| Gurada, Central hidroelétrica de Ponte dos Jugais | 40.385°N, 7.706°W | BGE_00108_F02 | BBIOP157-24 | ||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00108_F10 | BBIOP165-24 | ||
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00108_F12 | BBIOP167-24 | ||
| BGE_00108_G05 | BBIOP172-24 | ||||
| BGE_00108_G07 | BBIOP174-24 | ||||
| BGE_00108_G08 | BBIOP175-24 | ||||
| BGE_00108_G09 | BBIOP176-24 | ||||
| Unionicolidae | |||||
| Neumaniaelliptica | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_D02 | BSNTN988-23 | N BOLD:AFU2122 |
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00228_B12 | BSNTN879-23 | ||
| Neumaniaimitata | Porto, Parque Molinológico e Florestal de Pias | 41.268°N, 8.256°W | BGE_00109_G02 | BBIOP074-24 | N BOLD:AFV0268 |
| Neumanialimosa | Beja, Herdade de Alagães | 37.673°N, 7.848°W | BGE_00110_D06 | BSNTN517-23 | BOLD:ACS0551 |
| BGE_00110_D07 | BSNTN518-23 | ||||
| BGE_00110_D09 | BSNTN520-23 | ||||
| BGE_00228_H05 | BSNTN944-23 | ||||
| Guarda, Lagoa | 40.350°N, 7.549°W | BGE_00227_G01 | BSNTN1023-23 | ||
| BGE_00227_G02 | BSNTN1024-23 | ||||
| BGE_00108_B01 | BBIOP108-24 | ||||
| BGE_00108_B02 | BBIOP109-24 | ||||
| Guarda, Barragem do Lagoacho | 40.385°N, 7.618°W | BGE_00227_H04 | BSNTN1038-23 | ||
| Neumaniauncinata | Faro, Caldas de Monchique | 37.287°N, 8.554°W | BGE_00228_E01 | BSNTN904-23 | N BOLD:AFV0253 |
| Porto, Rio Este | 41.378°N, 8.695°W | BGE_00109_G04 | BBIOP076-24 | N BOLD:AFV0269 | |
| Neumaniapapillosa | Beja, Corte do Pinto | 37.682°N, 7.512°W | BGE_00110_C01 | BSNTN500-23 | N BOLD:AFO2116 |
| BGE_00228_G10 | BSNTN937-23 | ||||
| BGE_00228_G11 | BSNTN938-23 | ||||
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00227_A05 | BSNTN955-23 | ||
| Unionicolaminor | Beja, São João dos Caldeireiros | 37.626°N, 7.810°W | BGE_00110_H06 | BSNTN565-23 | N BOLD:AFO2171 |
| Beja, Moinho de Alferes 2 | 37.503°N, 7.687°W | BGE_00228_E02 | BSNTN905-23 | ||
| Pionidae | |||||
| Forelialongipalpis | Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00228_C01 | BSNTN880-23 | N BOLD:AFV3893 |
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00228_C05 | BSNTN884-23 | ||
| Guarda, Barragem do Vale do Rossim | 40.4°N, 7.589°W | BGE_00108_C02 | BBIOP121-24 | ||
| BGE_00108_C03 | BBIOP122-24 | ||||
| BGE_00227_H02 | BSNTN1036-23 | ||||
| BGE_00108_C04 | BBIOP123-24 | ||||
| Foreliavariegator | Guarda, Central hidroelétrica de Ponte dos Jugais | 40.385°N, 7.706°W | BGE_00228_B06 | BSNTN873-23 | N BOLD:AFU5459 |
| Beja, São João dos Caldeireiros | 37.626°N, 7.81°W | BGE_00228_H09 | BSNTN948-23 | ||
| Porto, Parque Molinológico e Florestal de Pias | 41.268°N, 8.256°W | BGE_00109_F12 | BBIOP072-24 | ||
| BGE_00109_G01 | BBIOP073-24 | ||||
| Porto, Rio Este | 41.378°N, 8.695°W | BGE_00109_G05 | BBIOP077-24 | ||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00108_F05 | BBIOP160-24 | ||
| Hydrochoreuteskrameri | Beja, Herdade de Alagães (pond) | 37.673°N, 7.848°W | BGE_00110_D11 | BSNTN522-23 | BOLD:ACR9737 |
| Guarda, Covão do Forno | 40.369°N, 7.638°W | BGE_00227_D12 | BSNTN998-23 | ||
| Tiphystorris | Beja, Zambujeira do Mar | 37.399°N, 8.723°W | BGE_00110_F02 | BSNTN537-23 | BOLD:ACR9977 |
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.64°W | BGE_00227_A06 | BSNTN956-23 | ||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00228_B10 | BSNTN877-23 | ||
| Nautarachnacrassa | Guarda, Casa do Cantoneiro | 40.418°N, 7.603°W | BGE_00227_E01 | BSNTN999-23 | N BOLD:AFV0462 |
| Pionacarnea | Guarda, Lagoa | 40.350°N, 7.549°W | BGE_00108_A10 | BBIOP105-24 | BOLD:ACS0622 |
| Beja, São Sebastião dos Carros | 37.598°N, 7.754°W | BGE_00110_G05 | BSNTN552-23 | BOLD:ACM0527 | |
| BGE_00110_G06 | BSNTN553-23 | ||||
| BGE_00110_G07 | BSNTN554-23 | ||||
| BGE_00110_G08 | BSNTN555-23 | ||||
| BGE_00110_G12 | BSNTN559-23 | ||||
| BGE_00228_D08 | BSNTN899-23 | ||||
| BGE_00228_D10 | BSNTN901-23 | ||||
| Beja, Herdade de Alagães | 37.678°N, 7.848°W | BGE_00228_E08 | BSNTN911-23 | ||
| Vila Real, Noura stream | 41.409°N, 7.417°W | BGE_00109_D10 | BBIOP046-24 | ||
| Guarda, Lagoa | 40.350°N, 7.549°W | BGE_00108_A10 | BBIOP105-24 | BOLD:ACS0622 | |
| BGE_00108_A12 | BBIOP107-24 | ||||
| Pionavariabilis | Beja, São Sebastião dos Carros | 37.598°N, 7.754°W | BGE_00110_H01 | BSNTN560-23 | BOLD:AAU0701 |
| Pionopsislutescens | Porto, Parque Torre de Vilar | 41.287°N, 8.210°W | BGE_00109_F06 | BBIOP066-24 | N BOLD:AFV3897 |
| Porto, Parque Molinológico e Florestal de Pias | 41.268°N, 8.256°W | BGE_00109_F11 | BBIOP071-24 | ||
| Guarda, Cise | 40.419°N, 7.709°W | BGE_00109_A05 | BBIOP005-24 | ||
| BGE_00109_A06 | BBIOP006-24 | ||||
| BGE_00227_B08 | BSNTN970-23 | ||||
| Aturidae | |||||
| Aturusscaber | Porto, Moinho da Tapada | 41.263°N, 8.307°W | BGE_00109_E03 | BBIOP051-24 | BOLD:ACQ9097 |
| Porto, Parque Molinológico e Florestal de Pias | 41.268°N, 8.256°W | BGE_00109_G03 | BBIOP075-24 | ||
| Mideopsidae | |||||
| Mideopsisroztoczensis | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00110_B10 | BSNTN497-23 | N BOLD:AFP5421 |
| Beja, Pulo do Lobo | 37.805°N, 7.633°W | BGE_00110_C08 | BSNTN507-23 | ||
| BGE_00110_C09 | BSNTN508-23 | ||||
| BGE_00110_C10 | BSNTN509-23 | ||||
| BGE_00110_C11 | BSNTN510-23 | ||||
| 37.805°N, 7.633°W | BGE_00110_C12 | BSNTN511-23 | |||
| BGE_00228_G12 | BSNTN939-23 | ||||
| Vila Real, Noura stream | 41.409°N, 7.417°W | BGE_00109_D08 | BBIOP044-24 | ||
| Beja, Zambujeira do Mar | 37.398°N, 8.680°W | BGE_00228_D03 | BSNTN894-23 | ||
| Bragança, Gasparona | 41.850°N, 7.013°W | BGE_00109_C07 | BBIOP031-24 | N BOLD:AFU6108 | |
| BGE_00109_C10 | BBIOP034-24 | N BOLD:AFW3785 | |||
| Guarda, Ponte dos Frades | 40.403°N, 7.526°W | BGE_00108_D06 | BBIOP137-24 | ||
| Guarda, Casa do Loureiro | 40.433°N, 7.701°W | BGE_00109_D03 | BBIOP039-24 | N BOLD:AFU6108 | |
| BGE_00109_D04 | BBIOP040-24 | ||||
| BGE_00109_G10 | BBIOP082-24 | ||||
| BGE_00109_G12 | BBIOP084-24 | N BOLD:AFW3785 | |||
| BGE_00109_H02 | BBIOP086-24 | N BOLD:AFU6108 | |||
| Guarda, Covão da Ametade | 40.328°N, 7.587°W | BGE_00108_A06 | BBIOP101-24 | ||
| BGE_00227_G05 | BSNTN1027-23 | N BOLD:AFU6108 | |||
| BGE_00227_G09 | BSNTN1031-23 | ||||
| Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00108_B09 | BBIOP116-24 | ||
| BGE_00227_C04 | BSNTN978-23 | N BOLD:AEV2909 | |||
| BGE_00227_C06 | BSNTN980-23 | ||||
| BGE_00227_C08 | BSNTN982-23 | N BOLD:AFU6108 | |||
| Guarda, Casais de Folgosinho | 40.454°N, 7.493°W | BGE_00108_G12 | BBIOP179-24 | ||
| BGE_00227_F05 | BSNTN1015-23 | ||||
| Guarda, Poio do Leão | 40.399°N, 7.541°W | BGE_00108_C12 | BBIOP131-24 | ||
| BGE_00227_H08 | BSNTN1042-23 | ||||
| Guarda, Central hidroelétrica de Ponte dos Jugais | 40.385°N, 7.706°W | BGE_00228_B03 | BSNTN870-23 | ||
| BGE_00228_B08 | BSNTN875-23 | ||||
| BGE_00228_B09 | BSNTN876-23 | ||||
| BGE_00108_E10 | BBIOP153-24 | ||||
| BGE_00108_E11 | BBIOP154-24 | ||||
| Guarda, Nossa Senhora do Desterro | 40.395°N, 7.694°W | BGE_00108_F04 | BBIOP159-24 | ||
| BGE_00108_F09 | BBIOP164-24 | ||||
| Guarda, Covão da ponte | 40.443°N, 7.514°W | BGE_00108_G01 | BBIOP168-24 | ||
| Guarda, Casa do Cantoneiro | 40.418°N, 7.603°W | BGE_00108_H02 | BBIOP181-24 | ||
| BGE_00227_E04 | BSNTN1002-23 | N BOLD:AFV6334 | |||
| BGE_00227_E05 | BSNTN1003-23 | N BOLD:AFU6108 | |||
| Guarda, Praia Fluvial de Sabugueiro | 40.401°N, 7.640°W | BGE_00108_H09 | BBIOP188-24 | ||
| BGE_00227_A07 | BSNTN957-23 | ||||
| Momoniidae | |||||
| Momoniafalcipalpis | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00108_B10 | BBIOP117-24 | N BOLD:AFX3396 |
| Arrenuridae | |||||
| Arrenurusalbator | Guarda, Barragem do Vale do Rossim | 40.400°N, 7.589°W | BGE_00227_G11 | BSNTN1033-23 | BOLD:ACR9639 |
| BGE_00227_G12 | BSNTN1034-23 | ||||
| BGE_00227_H01 | BSNTN1035-23 | ||||
| BGE_00108_B11 | BBIOP118-24 | ||||
| Guarda, Barragem do Lagoacho | 40.400°N, 7.589°W | BGE_00108_C08 | BBIOP127-24 | ||
| Arrenurusszalayi | Beja, Moinho de Alferes | 37.502°N, 7.690°W | BGE_00110_B05 | BSNTN492-23 | BOLD:ACS0403 |
| BGE_00110_B06 | BSNTN493-23 | ||||
| BGE_00228_C06 | BSNTN885-23 | ||||
| BGE_00228_C07 | BSNTN886-23 | ||||
| BGE_00228_C08 | BSNTN887-23 | ||||
| Arrenurusleuckarti | Guarda, Poço do Inferno | 40.373°N, 7.516°W | BGE_00227_B11 | BSNTN973-23 | BOLD:ACR9670 |
| Guarda, Casa do Cantoneiro | 40.418°N, 7.603°W | BGE_00108_H03 | BBIOP182-24 | ||
| Arrenurusneumani | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00110_B07 | BSNTN494-23 | N BOLD:AFP6143 |
| BGE_00110_B08 | BSNTN495-23 | ||||
| BGE_00228_C11 | BSNTN890-23 | ||||
| BGE_00228_H10 | BSNTN949-23 | ||||
| Arrenurustricuspidator | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00228_D01 | BSNTN892-23 | N BOLD:AFU3639 |
| Arrenurusglobator | Beja, Moinho de Alferes 1 | 37.502°N, 7.690°W | BGE_00110_B09 | BSNTN496-23 | N BOLD:AFO3503 |
| Beja, São Sebastião dos Carros | 37.598°N, 7.754°W | BGE_00110_H02 | BSNTN561-23 | ||
| BGE_00110_H03 | BSNTN562-23 | ||||
| BGE_00110_H04 | BSNTN563-23 | ||||
| Beja, São Miguel do Pinheiro | 37.552°N, 7.850°W | BGE_00228_G02 | BSNTN929-23 | ||
| BGE_00228_G03 | BSNTN930-23 | ||||
| BGE_00228_G04 | BSNTN931-23 | ||||
| Arrenuruszachariae | Bragança, Gasparona | 41.850°N, 7.013°W | BGE_00109_C08 | BBIOP032-24 | N BOLD:AFU0319 |
Morphological nomenclature follows Gerecke et al. (2016). The distribution data are from Cantallo et al. (2022) unless stated otherwise. The dorsal platelets of Torrenticola spp. were measured on both sides, therefore their dimensions were given as a range of values, rather than a single number. The holotype and paratypes of the new species are deposited in the Naturalis Biodiversity Center in Leiden (RMNH).
All measurements are given in μm. The photographs of selected structures were made using a camera on Samsung Galaxy smartphone. The following abbreviations are used: Ac-1 = first acetabulum; Cx-I = first coxae; Cxgl-4 = coxoglandularia 4; dL = dorsal length; H = height; I-L-4–6 = fourth-sixth segments of first leg; L = length; mL = medial length; P-1–P-5 = palp segments 1–5; Vgl-1 = ventroglandularia 1; W = width.
Molecular and DNA barcode analyses
The molecular analysis was conducted at the University of Florence (Florence, Italy). DNA was extracted using a non-destructive protocol. Samples were digested using 95 μl of extraction buffer (100 mM Tris-HCl, 5 mM EDTA, 100 mM NaCl, 0.5% SDS, pH 8) and 5 μl of proteinase K. Dilutions (1:10) of crude digested samples were used as template for the amplification of the mitochondrial cytochrome c oxidase subunit I (COI). Amplicons were amplified and barcoded in a single-step PCR using a cocktail of two barcoded primer pairs, namely Folmer primers (LCO1490, HC02198; Folmer et al. 1994) and Lep primers (LepF1, LepR1; Hebert et al. 2004). PCR was performed using the Kapa3G Plant PCR Kit according to the manufacturer’s protocol and with the following thermal profile: initial denaturation step of 3 min at 94 °C, 35 cycles of 20 s at 95 °C, annealing for 15 s at 52 °C and extension for 30 s at 72 °C, and a final extension for 1 min at 72 °C. Amplicons were checked on a 1.2% agarose gel and pooled in a single tube. The amplicon mix was used to prepare a PacBio library with the SMRTbell prep kit 3.0 according to the manufacturer’s protocol. The library was sequenced on a 8M ZMW SMRT cell on a PacBio Sequel IIe platform.
Raw reads were demultiplexed using the Pacific Biosciences SMRT Link software. Consensus sequences were generated with the PacBio Amplicon Analysis (pbaa) tool. Primer trimming, translation and stop codon checking were performed using Geneious Prime 2024.0.1.
Consensus sequences were made available in the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2007), and the Barcode Index Numbers (BIN) were obtained, grouping DNA sequences based on the Refined Single Linkage (RESL) analysis performed in BOLD (Ratnasingham and Hebert 2013). BINs are often considered proxies for species (e.g., Hebert et al. 2016). Relevant voucher information, photos, and newly generated DNA barcodes are publicly accessible through the Dataset - DS-BGEPL01 BGE Biodiversity Genomics Europe: Portuguese water mites I https://www.boldsystems.org/index.php/MAS_Management_DataConsole?codes=DS-BGEPL01) in BOLD. Data related to each BIN, including the minimum p-distance to the nearest neighboring BIN, was estimated using BOLD tools. The dataset consists of 307 sequences generated through this study (Suppl. material 1).
Sequence alignments were performed using MUSCLE (Edgar 2004). Intra- and interspecific genetic distances were calculated based on the Kimura 2-parameter model (K2P; Kimura 1980), using MEGA X (Kumar et al. 2018). The latter software was used to calculate Neighbor-Joining (NJ) trees based on K2P distances (standard for barcoding studies) using pairwise deletion for missing data. Branch support was calculated using nonparametric bootstrap (Felsenstein 1985) with 1000 replicates and shown next to the branches. All codon positions were considered in the analyses.
Results and discussion
We generated 307 DNA barcodes from 65 water mite species. The collected water mites represent 15 families of the 16 recorded in Portugal. The most sequence-rich family was Hygrobatidae with 51 sequences (16.7% of total; 10 BINs), followed by Lebertiidae wih 47 sequences (15.4%; 11 BINs), Mideopsidae with 41 sequences (13.4%; 5 BINs), Pionidae with 35 sequences (11.5%; 10 BINs), Limnesiidae with 26 sequences (8.5%; 5 BINs), Arrenuridae with 25 sequences (8.2%; 5 BINs), and Torrenticolidae with 24 sequences (7.9%; 7 BINs). Some families were rare, such as Hydryphantidae and Momoniidae, represented by a single sequence each and corresponding single BIN.
Our findings added the first records of 34 species for Portugal: Eylaistantilla Koenike, 1897 (Eylaidae), Lebertiasparsicapillata Thor, 1905, L.variolata Gerecke, 2009, L.gibbosa Lundblad, 1926, L.algeriensis Lundblad, 1942, L.insignis Neuman, 1880, L.porosa aggr. sp. A (Lebertiidae), Oxusmusculus (Müller, 1776), O.ovalis (Müller, 1776), O.setosus (Koenike, 1898) (Oxidae), Sperchonalgeriensis Lundblad, 1942, S.compactilis Koenike, 1911 (Sperchontidae), Monatractidesmadritensis (K. Viets, 1930), Torrenticolaelliptica Maglio, 1909 (Torrenticolidae), Limnesiakoenikei Piersig, 1894 (Limnesiidae), Atractidesinflatus (Walter, 1925), A.robustus (Sokolow, 1940), Hygrobatesbalcanicus Pešić, 2020 (Hygrobatidae), Neumaniaelliptica Walter, 1925, N.imitata Koenike, 1908, N.limosa (Koch, 1836), Unionicolaminor (Soar, 1900) (Unionicolidae), Forelialongipalpis Maglio, 1924, Hydrochoreuteskrameri Piersig, 1896, Nautarachnacrassa (Koenike, 1908), Pionacarnea (Müller, 1776), P.variabilis (Koch, 1836) (Pionidae), Aturusscaber Kramer, 1875 (Aturidae), Mideopsisroztoczensis Biesiadka & Kowalik, 1979 (Mideopsidae), Momoniafalcipalpis Halbert, 1906 (Momoniidae), Arrenurusleuckarti Piersig, 1894, A.neumani Piersig, 1895, A.cf.tricuspidator (Müller, 1776) and A.cf.zachariae Koenike, 1886 (Arrenuridae). Two species of the genus Torrenticola are described as new to science. Even though sampling was focused on certain districts (i.e., Beja, Bragança, Faro, Guarda, Porto, Vila Real), we recorded specimens from 47.4% of the Portuguese water mite fauna (65 of 137 species including 36 species new to Portugal).
The resulting sequences clustered into 75 BINs, with 38 BINs (51%) being unique and deposited for the first time in BOLD. The number of BINs per species ranged from one (58 species, 89%) to five for Mideopsisroztoczensis (BOLD:AFU6108, BOLD:AFP5421, BOLD:AFW3785, BOLD:AFV6334, BOLD:AEV2909). Two BINs were detected for five species, Lebertiainsignis (BOLD:AEB9107, BOLD:AFW6960), Atractidesinflatus (BOLD:AFI9009, BOLD:ACB4677), Neumaniauncinata (BOLD:AFV0253, BOLD:AFV0269), Forelialongipalpis (BOLD:AFX2876, BOLD:AFV3893), Pionacarnea (BOLD:ACM0527, BOLD:ACS0622), and one species, Hygrobateslongiporus (BOLD:AFV9997, BOLD:AFW1423, BOLD:AFV9998), has three BINs.
Our study provided the first DNA barcodes for Protziaannularis Lundblad, 1954 (BOLD:AFX2700), Monatractidesstenostomus (K. Viets, 1930) (BOLD:AFU3082), Torrenticolatenuipalpis (Lundblad, 1956) (BOLD:AFV2021), Oxuslusitanicus Lundblad, 1954 (BOLD:AFX3224), Limnesiaacuminata Walter, 1925 (BOLD:AFU7587), L.iberica Lundblad, 1954 (BOLD:AFN8367), L.walteri Migot, 1926 (BOLD:AFO9873), Neumaniaelliptica (BOLD:AFU2122), N.papillosa (Soar, 1902) (BOLD:AFO2116), Momoniafalcipalpis (BOLD:AFX3396) and Arrenurusszalayi Lundblad, 1954 (BOLD:ACS0403).
Systematics
Family Eylaidae Leach, 1815
Genus. Eylais
Latreille, 1796
81AEBB3D-CC1F-558F-AE0A-9C8AEAF4B7EB
Note.
Only one species reported from Portugal.
. Eylais tantilla
Koenike, 1897
B5402981-8F6C-5C4F-907B-D112C0CF3E39
Material examined.
Portugal, Beja: • Mértola, São João dos Caldeireiros, stream, 37.625°N, 7.81°W, 17 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂ (sequenced), dissected and slide mounted (RMNH); • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced); • Mértola, Herdade de Alagães, dry stream site 1, 37.676°N, 7.853°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio 1♀ (sequenced).
Remarks.
As a result of the treatment during the barcoding process, all vouchered individuals except one male and two females were partly or completely destroyed. With regards to the shape of the eye bridge and gnathosoma, the specimens examined in our study matches the description of E.tantilla given by K. Viets (1930) for material from Spain. The sequenced specimens from Portugal form a unique BIN (BOLD:AFP3591), with the nearest neighboring BIN being BOLD:ACS1138, which includes three public sequences of specimens from Norway assigned to E.rimosa Piersig, 1899, and three unpublished sequences of specimens from the Netherlands, two of them assigned to E.extendens and one assigned to E.setosa Koenike, 1897. The p-distance between these two BINs was estimated at 14.83%.
Distribution.
Palaearctic. New for Portugal.
Family Limnocharidae Grube, 1859
Genus. Limnochares
Latreille, 1796
C17355CF-08A5-5A15-A1CA-970776D74A9F
Note.
Only one species reported from Portugal.
. Limnochares aquatica
(Linnaeus, 1788)
047C4043-F917-5DAC-8374-C9F384CF6303
Material examined.
Portugal, Guarda: • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Casa do Loureiro, 40.433°N, 7.701°W, 415 m a.s.l., 19 Jul. 2023 leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The examined specimens in our study, keyed to L.aquatica following Davids et al. (2007), form a unique BIN (BOLD:AFV0270). The p-distance between the latter BIN and its nearest neighbor, BOLD:ACS0438, which includes specimens of L.aquatica from the Netherlands, Norway, Montenegro, and Italy, was estimated at 11.72%, indicating the need for taxonomic revision of L.aquatica complex to identify possible undescribed cryptic species.
Distribution.
Holarctic. In Portugal previously reported from Beira Alta and Alentejo (Lundblad 1956).
Family Hydrodromidae Viets, 1936
Genus. Hydrodroma
Koch, 1837
90231F63-46BD-56ED-B1F1-1AA6390B2FDE
Note.
Only one species reported from Portugal.
. Hydrodroma despiciens
(Müller, 1776)
227F79ED-7EC9-5AF5-9DC6-97C5B4EAA692
Material examined.
Portugal, Guarda: • Seia, Covão do Forno, 40.369°N, 7.638°W, 1574 m a.s.l., 19 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1 deutonymph (sequenced).
Remarks.
The sequences obtained from the specimens from Portugal fall into BOLD:ACS0426, which, in addition to the specimens used in this study for molecular analysis, includes 17 specimens of H.despiciens from the Netherlands, Norway, and Poland, available in the BOLD database.
Distribution.
Europe. In Portugal previously reported from Alentejo (Lundblad 1956).
Family Hydryphantidae Piersig, 1896
Genus. Protzia
Piersig, 1896
9AF7AB15-3662-5FE7-8998-48C8901A4F0A
Note.
Only three species reported from Portugal.
. Protzia annularis
Lundblad, 1954
AF29D7D0-ECFB-5D48-ABC9-100778E44EEF
Material examined.
Portugal, Faro: • Monchique, Ribeira de Seixe, Parque do Barranco dos Pisões, 37.333°N, 8.567°W, 480 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♀ (sequenced).
Remarks.
The single examined female from Ribeira de Seixe matches the description of P.annularis, a species known from Portugal and Spain (Lundblad 1956). The Portuguese specimen forms a unique BIN (BOLD:AFX2700), with the nearest neighboring BIN being BOLD:AEI5748, which includes specimens of P.lata from Corsica. The p-distance between these two BINs was estimated at 12.36%.
Distribution.
Iberian Peninsula. In Portugal previously reported from Beira Alta (Lundblad 1956).
Family Lebertiidae Thor, 1900
Genus. Lebertia
Neuman, 1880
E40A51FC-954F-5F95-A03F-5F33725458DC
Note.
Nine species known from Portugal, two of them, Lebertiamadericola (Lundblad, 1942) and Lebertiamaderigena (Lundblad, 1942), are endemic for Madeira.
. Lebertia (Lebertia) fimbriata
Thor, 1899
F58907BE-66B8-5F2C-8C9B-CC564120CF63
Material examined.
Portugal, Beja: • Mértola, São João dos Caldeireiros, stream, 37.626°N, 7.81°W, 17 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀, 1♂ (sequenced).
Remarks.
The specimens from Portugal match the description of Lebertiafimbriata, a species widely distributed in the Western Palaearctic (Di Sabatino et al. 2010). The Portuguese specimens were clustered within BOLD:AEI5359, which includes specimens of L.fimbriata from Germany, Spain and North Macedonia.
Distribution.
Western Palaearctic. In Portugal previously reported from Fonte Fria in Mealhada (Buçaco mountain; Lundblad 1956).
. Lebertia (Lebertia) sparsicapillata
Thor, 1905
AD28B421-67B1-518E-8B79-C5E911DB435B
Material examined.
Portugal, Beja: • Odemira, Ribeira de Seixe, Zambujeira do Mar, river, 37.399°N, 8.723°W, 45 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♂ (sequenced). Guarda: • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Casa do Loureiro, 40.433°N, 7.701°W, 415 m a.s.l., 19 Jul. 2023 leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The specimens from Portugal match the description of L.sparsicapillata, a species widely distributed in Europe except for the most northern and eastern parts (Di Sabatino et al. 2010). The sequenced specimens cluster within BOLD:AFN4501, which includes two specimens from Germany. The p-distance between the latter BIN and its nearest neighboring BOLD:ADF6063, which include specimens of L.sparsicapillata from Germany, was estimated at 2%.
Distribution.
Europe. New for Portugal.
. Lebertia (Lebertia) variolata
Gerecke, 2009
E134BC45-EACE-52F1-809F-463022D8019C
Material examined.
Portugal, Beja: • Odemira, Ribeira de Seixe, Zambujeira do Mar, 37.398°N, 8.68°W, site 32, 75 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♀ (sequenced); • Faro, Portimão, 37.237°N, 8.546°W, 23 May 2023, leg. Ferreira & Turaccio 1♀ (sequenced).
Remarks.
The sequences obtained from two specimens from Portugal fall into the Lebertiavariolata cluster (BOLD:ADK0996). In addition to specimens used in this study, the BIN includes specimens from Montenegro, North Macedonia and Turkey morphologically assigned to L.variolata, and one private sequence of a non-identified specimen from France (GBMIN118138-1). Lebertiavariolata is a characteristic inhabitant of streams that regularly dry up in the summer (Gerecke 2009).
Distribution.
Mediterranean region. New for Portugal.
. Lebertia (Pilolebertia) gibbosa
Lundblad, 1926
D233F93E-6CDD-5BCB-A570-B274ABF3926E
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂ (sequenced); • Manteigas, Zêzere, Ponte dos Frades, 40.403°N, 7.526°W, 672 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur 1♂ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Gouveia, Rio Mondego Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Manteigas, Mondego, Covão da ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1 deutonymph (sequenced). Porto, • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 178 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1 deutonymph (sequenced).
Remarks.
This species was originally described from the island of Gotland, Sweden (Lundblad 1956), but later on synonymized with L.porosa. Recently Tyukosova et al. (2022) used material from Norway to redescribe L.gibbosa based on morphological and molecular evidence and showed that this species is widely distributed in southern Norway.
The sequenced specimens from Portugal were clustered within BOLD:ACR9744, which, in addition to the specimens from Portugal, includes specimens of L.gibbosa from the Netherlands, Norway, Poland, and Germany, available in BOLD.
Distribution.
As this species has been widely overlooked with other species of L.porosa complex, the full geographical distribution of L.gibbosa cannot be defined without additional research.
. Lebertia (Pilolebertia) algeriensis
Lundblad, 1942
5A96CDD1-A847-59AA-9B13-B83AA53699D1
Material examined.
Portugal, Guarda: • Gouveia, Rio Mondego Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced; Table 1); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀ (sequenced).
Remarks.
The Portuguese specimens molecularly analyzed in this study match the description of Lebertiaalgeriensis. Genetic data indicate that all examined specimens form a unique BIN (BOLD:AFV0271), and the closest neighboring BIN is that of L.inaequalis (BOLD:AEF5683) from North Macedonia. The p-distance between these two BINs was estimated at 4.53%.
Distribution.
Palaearctic. Gerecke (2009) mentioned that several published records of L.inaequalis (Koch, 1837) from the Mediterranean region could refer to similar L.longiseta or L.algeriensis. New for Portugal.
. Lebertia (Pilolebertia) insignis
Neuman, 1880
3D811FDC-E757-5CBA-83E6-839B9DE383BA
Material examined.
Portugal, Guarda: • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced). Porto, • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 178 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal clustered within two BINs, BOLD:AEB9107, which includes specimens of L.insignis from Norway, Montenegro, Poland and Slovakia, and the unique BOLD:AFW6960, which includes one specimen from this study collected in Rio Alva in Guarda Province. The p-distance between these two BINs was estimated at 9.79%, indicating the need for taxonomic revision of L insignis complex to identify possible undescribed cryptic species.
Distribution.
Central, Western and Northern Europe. Rare in the Mediterranean and on the Iberian Peninsula previously known only from Oviedo in Spain (Lundblad 1956). New for Portugal.
. Lebertia (Pilolebertia) porosa
aggr. sp. A
9778E366-6F01-5875-92C6-1A8D03C4BBA2
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Manteigas, Mondego, Covão da ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal clustered within BOLD:ACP5319, which includes porosa-like specimens from Serbia and Spain, and specimens from Germany and Norway provisionally assigned by Tyukosova et al. (2022) to L.porosa aggr. sp. A.
Distribution.
As this species has been widely overlooked with other species of L.porosa complex, the full geographical distribution of L.porosa aggr. sp. A. can be defined only on the basis of the records available in BOLD.
. Lebertia (Pilolebertia) porosa
aggr. sp.
856B45BC-B41B-584D-A102-BDE95944DE9A
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀ (sequenced); • Manteigas, Poio do Leão, 40.399°N, 7.541°W, 734 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Manteigas, Zêzere, Ponte dos Frades, 40.403°N, 7.526°W, 672 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur, 1♀ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1 deutonymph (sequenced). Bragança: • Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha, 1 deutonymph (sequenced); • Vinhais, Gasparona, 41.85°N, 7.013°W, 693 m a.s.l., 6 Jul. 2023, leg. Ferreira & Padilha, 1 deutonymph (sequenced). Porto • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 178 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal clustered within BOLD:ACS0974, which includes porosa-like specimens from different parts of Europe, except Fennoscandia, available in BOLD. In the phylogenetic tree, the BIN is positioned as a sister clade of L.porosa as defined by Tyukosova et al. (2022). The latter species was recently redefined by Tyukosova et al. (2022) based on the specimens from the type locality that were shown to belong to BOLD:ACQ9049. The taxonomic status of L.porosa like species of BOLD:ACS0974 needs to be clarified by resolving taxonomic status of numerous species listed as synonyms in Gerecke (2009). As emphasized by Gerecke et al. (2022), a more extensive study of L.porosa complex is needed to establish a stable taxonomy for this group.
Distribution.
As this species has been widely overlooked with other species of L.porosa complex, the geographical distribution of L.porosa like species of BOLD:ACS0974 can be defined only on the basis of the records available in BOLD.
. Lebertia (Pilolebertia) pilosa
Maglio, 1924
B4E5898B-7B28-537B-91C8-5CC94EFA070C
Material examined.
Portugal, Beja: • Odemira, Ribeira de Seixe, Zambujeira do Mar, river, 37.399°N, 8.723°W, 45 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 4♀ (sequenced), 1♀ dissected and slide mounted (RMNH).
Remarks.
The sequenced specimens from Portugal were clustered within BOLD:AEJ2601, which, in addition to material from this study, include one unidentified Lebertia specimen from Spain. The p-distance between the latter BIN and its nearest neighbor, BOLD:ACS0974, which include L.porosa like specimens from different parts of Europe, was estimated at 12.01%.
Distribution.
Europe. In Portugal previously reported from Minho River (Cantallo et al. 2021), and from São Pedro da Torre in Valença municipality (Cantallo et al. 2022).
. Lebertia (Lebertia) pusilla
Koenike, 1911
C6EF2CBA-79B8-542D-AB21-CF7D77A2B07D
Material examined.
Portugal, Porto: • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 178 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1 ♂ (sequenced).
Remarks.
The sequenced specimen from Moinho da Tapada cluster together with specimens collected by Pešić et al. (2023b) from Santarém, Portugal, and morphologically assigned to L.pusilla. These specimens form a unique BIN (BOLD:AFW6961), with a p-distance of 10.43% to the nearest sequence (NLACA493-15) of L.pusilla from the Netherlands.
Distribution.
Europe.
Family Oxidae K. Viets, 1926
Genus. Oxus
Kramer, 1877
6733003B-5B79-57E7-A0D2-825E014AA9F0
Note.
Four species known for Portugal: two of them, Oxushastata (Lundblad, 1954) and O.lusitanicus Lundblad, 1954, originally described from Portugal. Oxusoblongus Kramer, 1879, reported by Lundblad (1956) from Sintra, is a possible synonym of O.strigatus (Di Sabatino et al. 2010; Smit and Gerecke 2010).
. Oxus (Oxus) cf.angustipositus
K. Viets, 1908
09B3B135-ED7B-5F2F-9136-69371DF28061
Material examined.
Portugal, Guarda: • Manteigas, Casa do Cantoneiro, 40.418°N, 7.603°W, 1378 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂ (sequenced); • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The examined specimens cluster within BOLD:AET9442, which includes specimens from Portugal assigned by Pešić et al. (2023b) to Oxuscf.angustipositus. As emphasized by Pešić et al. (2023b) taxonomic revision of the O.angustipositus complex is required for identifying possibly undescribed cryptic species.
Distribution.
Europe. In Portugal previously reported from Porto (Pešić et al. 2023b).
. Oxus (Oxus) lusitanicus
Lundblad, 1954
028247DE-9BB5-5A78-B425-A678B817FD8E
Material examined.
Portugal, Guarda: • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀, 1 deutonymph (sequenced). Beja • Mértola, Moinho de Alferes 2, 37.503°N, 7.687°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂, 1♀ (sequenced). Bragança • Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha, 1♂, 1♀ (sequenced). Vila Real • Murça, Noura stream, 41.409°N, 7.417°W, 421 m a.s.l., 12 Jul. 2023 leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The examined specimens match the description of Oxuslusitanicus, a species originally described by Lundblad (1954) based on a single male collected in Côa River in Portugal. The specimens from Portugal used in this study for molecular analysis form a unique BIN (BOLD:AFX3224) with the nearest neighboring BIN being BOLD:ACL5934, which includes specimens of unidentified Oxus sp. from Canada, with the p-distance estimated at 14.34%.
Distribution.
Portugal previously recorded from Beira Alta (Lundblad 1956).
. Oxus (Oxus) musculus
(Müller, 1776)
3DAC928B-874B-50EF-8E9E-5A4583AFB0A5
Material examined.
Portugal, Beja • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal were clustered within BOLD:AFC2154, which includes specimens of O.musculus from Norway.
Distribution.
Palaearctic. Widespread in Europe, but here reported for the first time for Portugal.
. Oxus (Oxus) ovalis
(Müller, 1776)
225BB002-8575-507C-AC19-B8015140147B
Material examined.
Portugal, Beja • Odemira, Ribeira de Seixe, Zambujeira do Mar, 37.399°N, 8.723°W, 45 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♂, 1♀ (sequenced).
Remarks.
The sequenced specimens from Zambujeira do Mar, keyed to Oxusovalis following Di Sabatino et al. (2010), form a unique BIN (BOLD:AFP5747).
Distribution.
Widespread in Europe, but here reported for the first time for Portugal.
. Oxus (Gnaphiscus) setosus
(Koenike, 1898)
0129B0C6-B522-59F2-84F9-73731795966A
Material examined.
Portugal, Guarda: • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Manteigas, Mondego, Covão da ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal clustered within BOLD:ACS0808, which includes specimens of O.setosus from the Netherlands.
Distribution.
Palaearctic. Widespread in Europe, but here reported for the first time for Portugal.
Family Sperchontidae Thor, 1900
Genus. Sperchon
Kramer, 1877
CFCA4159-25F1-522C-BBCE-D5C3BF34DFD4
Note.
Five species known from Portugal.
. Sperchon (Hispidosperchon) algeriensis
Lundblad, 1942
F39CFB6C-5E57-546C-A116-6BBE121B7D0C
Material examined.
Portugal, Faro • Portimão, 37.237°N, 8.546°W, 23 May 2023, leg. Ferreira & Turaccio 1♀ (sequenced).
Remarks.
The single female from Portugal clusters within BOLD:AES2436, which includes one specimen of S.algeriensis recently collected from eastern Spain (López-Peña et al. 2022). The species was described from northern Africa (Lundblad 1942) and subsequently recorded from many sites in the central and western Mediterranean area (Di Sabatino et al. 2010). The species is considered as a characteristic species of warm Mediterranean streams that regularly dry up in the summer (Gerecke 1991). The hydrography of the sampling site where S.algeriensis was found in our study is characterized by summer drought.
The high genetic distance of 15.4% between Iberian populations of S.algeriensis and a specimen from Iran, attributed to S.algeriensis, suggests that the latter belongs to a further distinct species (Pešić et al. 2022a). It is likely that the latter species represents S.beneckei Bader & Sepasgosarian, 1982, a species proposed to be a synonym of S.algeriensis by Asadi et al. (2010). Therefore, the known populations of S.algeriensis from Eastern Mediterranean should be checked using molecular methods to see if they can be assigned to S.beneckei.
Distribution.
North Africa, west Mediterranean. New record for Portugal.
. Sperchon (Hispidosperchon) clupeifer
Piersig, 1896
39AE44BF-5138-52CA-B7E2-0A5A5F56F907
Material examined.
Portugal, Guarda • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced).
Remarks.
The single male from Portugal used in this study for molecular analysis matches the description of S.clupeifer. This specimen forms a unique BIN (BOLD:AFX0389). The p-distance to its nearest neighboring BIN (BOLD:ACS1100), which includes specimens of S.clupeifer from the Netherlands, Germany, Norway, Macedonia, Montenegro, Russia, Serbia, Austria, and Italy, was estimated at 7.06%.
Distribution.
Western Palaearctic. In Portugal previously reported from Fonte Fria in Mealhada (Buçaco Mountain; Lundblad 1956).
. Sperchon (Hispidosperchon) compactilis
Koenike, 1911
B6108B6F-7829-5ADD-9B27-0CC0821CEB16
Material examined.
Portugal, Beja • Faro, Portimão, 37.237°N, 8.546°W, 23 May 2023, leg. Ferreira & Turaccio 1♀ (sequenced). Guarda • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The sequences obtained from two Portuguese specimens, keyed to S.compactilis following Di Sabatino et al. (2010), clustered in BOLD:AER7687, which, in addition to specimens used in this study, includes two specimens from Austria and Spain.
Distribution.
Western Palaearctic. New record for Portugal.
Family Torrenticolidae Piersig, 1902
Genus. Monatractides
K. Viets, 1926
86F8A74D-2CE7-5018-B283-6B33D933252F
Note.
So far, two species of the genus are known from Portugal.
. Monatractides (Monatractides) madritensis
(K. Viets, 1930)
4E4B1F51-4C16-5CE2-9080-A88FF173CDB6
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Manteigas, Poio do Leão, 40.399°N, 7.541°W, 734 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced). Bragança • Vinhais, Gasparona, 41.85°N, 7.013°W, 693 m a.s.l., 6 Jul. 2023, leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The Portuguese specimens match the description of M.madritensis. The specimens clustered within BOLD:AED3803, which includes specimens of M.madritensis from Montenegro, Serbia and Italy, available in BOLD.
Distribution.
Europe. New record for Portugal.
. Monatractides (Monatractides) stadleri
(Walter, 1924)
AE6EB570-604A-5824-A015-7501B7896400
Material examined.
Portugal, Faro • Monchique, Ribeira de Seixe, Parque do Barranco dos Pisões, stream, 37.333°N, 8.567°W, 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♀ (sequenced). Guarda: • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Casa do Loureiro, 40.433°N, 7.701°W, 415 m a.s.l., 19 July 2023 leg. Ferreira & Padilha, 1♀ (juv.) (sequenced).
Remarks.
The females used in this study for molecular analysis were clustered within BOLD:AEU1504, which includes two specimens of M.stadleri from Belgium, one specimen from Spain (identified as Torrenticola sp., deposited in Taxus Medio Ambiente, Spain), and one specimen recently collected from the stream in Beja Province and assigned to M.stadleri by Pešić et al. (2023b).
Distribution.
Europe. In Portugal previously reported from Corgo da Ponte Quebrada, Beja (Pešić et al. 2023b).
. Monatractides (Monatractides)
s tenostomus (K. Viets, 1930)
ACE14194-0BC6-56A6-A93B-AFA25E3059B0
Material examined.
Portugal, Beja • Mértola, Corte do Pinto, 37.682°N, 7.512°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂ (sequenced), dissected and slide mounted (RMNH),
Remarks.
The Portuguese specimen molecularly analyzed in this study matches the description of M.stenostomus. This individual forms a unique BIN (BOLD:AFU3082), with the nearest neighboring BIN being BOLD:ADZ9854, which includes three specimens of an unidentified Monatractides sp. from Morocco, with the p-distance estimated at 6.74%.
Distribution.
Spain, France. In Portugal previously reported from Beira Alta (Santa Comba Dão; Lundblad 1956).
Genus. Torrenticola
Piersig, 1896
832858C4-6453-5862-99D3-C212191F9CF5
Note.
So far 12 species of the genus were reported from Portugal, nine of them known from Madeira Island and four species from the mainland
. Torrenticola (Torrenticola) elliptica
Maglio, 1909
706B0B78-AAE0-54F0-A8FE-3B8B046CAC58
Material examined.
Portugal, Bragança • Vinhais, Gasparona, 41.85°N, 7.013°W, 693 m a.s.l., 6 Jul. 2023, leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The single Portuguese specimen molecularly analyzed in this study was a juvenile female that does not allow for a confident identification to the species level. Molecular data, however, revealed that the DNA barcode of the Portuguese specimen falls into BOLD:AEI9183, which includes one specimen from Montenegro, morphologically assigned by the first author to T.elliptica.
Distribution.
Palaearctic. New for Portugal.
. Torrenticola (Torrenticola) soniae
Pešić sp. nov.
C59EC9CA-BCE5-5F8B-B629-7DB467323F88
https://zoobank.org/E0A41268-B54C-4366-9886-E2954F1BF00B
Figure 1.
Torrenticolasoniae sp. nov., ♂ holotype A dorsal shield B ventral shield C palp, medial view (inset: ventrodistal projections of P-2 and P-3, enlarged 2×) D palp, lateral view E gnathosoma and chelicera F ejaculatory complex. Scale bars: 100 μm.
Figure 2.
Torrenticolasoniae sp. nov., ♀ paratype A dorsal shield B photograph of dorsal shield C ventral shield D palp, medial view (P-1 lacking). Scale bars: 100 μm.
Figure 5.
A–C Photographs of dorsal shield ATorrenticolasoniae sp. nov., ♂ holotype BT.elisabethae sp. nov., ♂ holotype CT.tenuipalpis, ♀ (BGE_00227_F03) D–E Photographs of selected sampling sites D Praia Fluvial de Sabugueiro, locus typicus of Torrenticolasoniae sp. nov. E Poço do Inferno, type locality of T.elisabethae sp. nov. Photographs by JC (5D) and SF (5E).
Type material examined.
Holotype • ♂, dissected and slide mounted (RMNH), Portugal, Guarda, Seia, Rio Alva, Praia Fluvial de Sabugueiro (Fig. 5D), river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, sequenced (BOLD ID: BSNTN961-23). Paratypes: • 2♂ (sequenced), Portugal, Guarda, Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha; • 1♂ (sequenced); Manteigas, Zêzere, Ponte dos Frades, 40.403°N, 7.526°W, 672 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur; • 1♀ (sequenced), dissected and slide mounted (RMNH), Guarda, Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha; • 1♀ (juv.; sequenced), Bragança, Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha.
Diagnosis.
Morphological: Cx-I relatively short, anteriorly broad; suture lines of Cx-IV prominent, starting at right angle from genital field; ejaculatory complex with well-developed anterior keel and proximal arms; gnathosomal rostrum short, less than width of gnathosoma; P-3 with a subrectangular, apically serrated ventrodistal projection. Molecular: this lineage represented by a unique BIN (BOLD:AFW5337) differs from T.brevirostris clade by 12.27% K2P for COI.
Description.
General features. Idiosoma roundish; dorsal shield without a color pattern as photographed in Figs 2B, 5A; area of primary sclerotization of the dorsal plate with two dorsoglandularia (Fig. 1A); frontal platelets broad, relatively short; Cx-I relatively short, anteriorly broad; gnathosomal bay U-shaped, proximally rounded; Cxgl-4 subapical; medial suture line of Cx-II+III relatively short; postgenital area extended; excretory pore and Vgl-2 away from the line of primary sclerotization, excretory pore on the level of Vgl-2; gnathosomal rostrum short, less than depth of gnathosoma (Fig. 1E); P-2 ventral margin nearly straight or slightly convex, P-2 ventrodistal protrusion bluntly pointed, apically serrated, P-3 with a subrectangular, apically serrated ventrodistal projection, P-4 with a ventral tubercle bearing one long and three shorter setae (Fig. 1C, D). Male. 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 large, subrectangular; ejaculatory complex conventional in shape, anterior keel, proximal and distal arms well developed (Fig. 1F). Female. Genital field large and pentagonal in shape, suture lines of Cx-IV extending posteriorly beyond posterior margin of genital field, laterally curved.
Measurements.
Male (holotype). Idiosoma (ventral view: Fig. 1B) L 912, W 669; dorsal shield (Fig. 1A) L 756, W 581, L/W ratio 1.3; dorsal plate L 700; shoulder plate L 220–222, W 97, L/W ratio 2.27–2.29; frontal plate L 150–156, W 78–88, L/W ratio 1.8–1.9; shoulder/frontal plate L 1.42–1.47. Gnathosomal bay L 171, Cx-I total L 359, Cx-ImL 188, Cx-II+III mL 137; ratio Cx-I L/Cx-II+III mL 2.63; Cx-ImL/Cx-II+III mL 1.37. Genital field L/W 191/163, ratio 1.17; distance genital field-excretory pore 116, genital field-caudal idiosoma margin 209. Ejaculatory complex L 291.
Gnathosoma vL 331, chelicera L 375; palp total L 398, dL/H, dL/H ratio: P-1, 39/39, 1.0; P-2, 117/73, 1.59; P-3, 84/63, 1.35; P-4, 120/42, 2.86; P-5, 38/17, 2.18; L ratio P-2/P-4, 0.98. dL of I-L-4–6: 134, 150, 136; I-L-6 H 100; dL/H I-L-6 ratio 1.36.
Female (paratype from Casais de Folgosinho, BGE_00227_F02). Idiosoma (ventral view: Fig. 2C) L 1033, W 828; dorsal shield (Fig. 2A, B) L 844, W 725, L/W ratio 1.16; dorsal plate L 781; shoulder plate L 222–235, W 100–102, L/W ratio 2.2–2.3; frontal plate L 173–175, W 97–98, L/W ratio 1.79; shoulder/frontal plate L 1.28–1.34. Gnathosomal bay L 200, Cx-I total L 384, Cx-ImL 184, Cx-II+III mL 18; ratio Cx-I L/Cx-II+III mL 21.3; Cx-ImL/Cx-II+III mL 10.2. Genital field L/W 221/198, ratio 1.12; distance genital field-excretory pore 250, genital field-caudal idiosoma margin 391.
Gnathosoma vL 350, chelicera L 409; palp total L 435, dL/H, dL/H ratio: P-1, 44/41, 1.08; P-2, 127/76, 1.67; P-3, 93/65, 1.44; P-4, 134/42, 3.18; P-5, 37/18, 2.0; L ratio P-2/P-4, 0.94.
Etymology.
The species is dedicated to Sónia Ferreira (CIBIO, Portugal) for collecting numerous specimens used in this study and her enthusiastic support in the study of Portuguese water mites.
Species delimitation using DNA barcodes.
The final alignment for species delimitation using COI sequence data comprised 669 nucleotide positions (nps) of the 130 Torrenticola specimens listed in Suppl. material 2 and one outgroup, Monatractidesmadritensis from Portugal to root the tree. The NJ tree is presented in Fig. 6. The COI tree sequences retrieved from Torrenticola specimens from Portugal, here described as T.soniae sp. nov., appeared as a sister group to the cluster of sequences belonging to T.brevirostris (Halbert, 1911), a rhitrobiontic species widely distributed in Europe. The mean genetic distance between COI sequences of these two clusters was estimated at 12.27 ± 1.42% K2P. The genetic distance was considerably higher than the estimated barcode gap found by ASAP analyses (3–5%) of all studied Torrenticola, supporting the species-status of the new taxon. The mean intraspecific K2P-divergence within the cluster of the new species was 0.63 ± 0.19%.
Figure 6.
Neighbor-Joining tree of the genus Torrenticola, obtained from 130 nucleotide COI sequences. Bootstrap values > 50% from 1000 bootstrap replicates on branches.
Discussion.
With regards to the presence of an anteriorly broad and short Cx-I, a robust and compact palp, and a deep gnathosoma with a short rostrum, the new species resembles T.brevirostris. The latter species can be separated from T.soniae sp. nov. by only slightly protruding ventrodistal projections of P-2 and particularly of P-3.
Distribution.
Portugal (this study).
. Torrenticola (Torrenticola) elisabethae
Pešić sp. nov.
DF59C6E0-AC8B-5124-9C35-2001988D5B38
https://zoobank.org/354EB35B-1F5E-4FBB-9E8B-06B956A47467
Figure 3.
Torrenticolaelisabethae sp. nov., ♂ holotype A dorsal shield B ventral shield C palp, medial view D gnathosoma and chelicera E ejaculatory complex F photograph of ejaculatory complex. Scale bars: 100 μm.
Figure 4.
Torrenticolaelisabethae sp. nov., ♀ paratype A dorsal shield B photograph of dorsal shield C ventral shield D palp, medial view. Scale bars: 100 μm.
Type material examined.
Holotype • ♂, dissected and slide mounted, Portugal, Guarda, Manteigas, Poço do Inferno (Fig. 5E), 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, sequenced (BOLD ID: BSNTN984-23). Paratypes: • 1♂, 1♀ (sequenced), Portugal, Guarda, Manteigas, Poio do Leão, 40.399°N, 7.541°W, 734 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ dissected and slide mounted (RMNH).
Diagnosis.
Morphological: Shoulder platelets fused with dorsal plate; dorsal shield with color pattern as illustrated in Figs 4B, 5B; Cxgl–4 subapical; medial suture line of Cx-II+III in male relatively long; ejaculatory complex with poorly developed anterior keel and a relatively large proximal chamber. Molecular: this lineage represent by a unique BIN (BOLD:AFW5336) differs from T.lundbladi clade by 9.8% K2P for COI.
Description.
General features. Idiosoma oval; shoulder platelets fused to dorsal plate, but suture line visible; dorsal shield with a color pattern as illustrated in Figs 4B, 5B; area of primary sclerotization of the dorsal plate with four dorsoglandularia (Fig. 3A); gnathosomal bay U-shaped, proximally rounded; Cxgl–4 subapical; excretory pore and Vgl-2 on the the line of primary sclerotization, excretory pore on the level of Vgl-2; gnathosomal ventral margin curved, rostrum elongated (Fig. 3D); P-2 ventral margin nearly straight or slightly concave, P-2 and P-3 ventrodistal protrusions bluntly pointed, P-4 with a ventral tubercle bearing one long and three shorter setae (Figs 3C, 4D). Male — Medial suture line of Cx-II+III relatively long; genital field subrectangular; ejaculatory complex with poorly developed anterior keel, proximal chamber relatively large; Fig. 3E). Female — Genital field large and pentagonal in shape.
Measurements.
Male (holotype). Idiosoma (ventral view: Fig. 3B) L 856, W 691; dorsal shield (Fig. 3A) L 731, W 619, L/W ratio 1.18; dorsal plate L 681; frontal plate L 173–183, W 64–66, L/W ratio 2.7–2.8. Gnathosomal bay L 194, Cx-I total L 383, Cx-ImL 188, Cx-II+III mL 131; ratio Cx-I L/Cx-II+III mL 2.92; Cx-ImL/Cx-II+III mL 1.43. Genital field L/W 183/150, ratio 1.22; distance genital field-excretory pore 103, genital field-caudal idiosoma margin 127. Ejaculatory complex L 275.
Gnathosoma vL 367, chelicera L 448; palp total L 390, dL/H, dL/H ratio: P-1, 44/38, 1.17; P-2, 133/64, 2.08; P-3, 79/58, 1.36; P-4, 112/38, 2.98; P-5, 22/14, 1.55; L ratio P-2/P-4, 1.19. dL of I-L-4–6: 145, 160, 131; I-L-6 H 46; dL/H I-L-6 ratio 2.85.
Female (paratype from Poio do Leão, BGE_00227_H06). Idiosoma (ventral view: Fig. 4C) L 975, W 794; dorsal shield (Fig. 4A, B) L 806, W 663, L/W ratio 1.22; dorsal plate L 766; frontal plate L 172–175, W 63–68, L/W ratio 2.6–2.75. Gnathosomal bay L 203, Cx-I total L 391, Cx-ImL 188, Cx-II+III mL 0. Genital field L/W 214/204, ratio 1.05; distance genital field-excretory pore 256, genital field-caudal idiosoma margin 347. Egg (n = 1) maximum diameter 227.
Gnathosoma vL 379, chelicera L 478; palp total L 389, dL/H, dL/H ratio: P-1, 41/36, 1.15; P-2, 130/64, 2.0; P-3, 80/59, 1.35; P-4, 116/40, 2.87; P-5, 22/14, 1.55; L ratio P-2/P-4, 1.13.
Etymology.
The new species is dedicated to Elisabeth Stur (NTNU University Museum Trondheim, Norway), who facilitated a number of barcoding projects on water mites in Europe.
Species delimitation using DNA barcodes.
The sequences retrieved from Torrenticola specimens from Portugal, here described as T.elisabethae sp. nov., appeared as a sister group to the cluster containing sequences of T.lundbladi (K. Viets, 1930), a rhitrobiontic species known from Spain (Lundblad 1956; Pešić et al. 2012). The mean K2P genetic distance between COI sequences of T.elisabethae sp. nov. and T.lundbladi was estimated at 9.8 ± 1.25%. The genetic distance was also here higher than the barcode gap found for Torrenticola in the ASAP analysis, supporting the species-status of the new taxon. The mean intraspecific divergence within the cluster of barcodes belonging to T.elisabethae was relatively low (0.2 ± 0.14% K2P).
Discussion.
The new species is most similar to Torrenticolalundbladi K. Viets, 1930, a species originally described from central Spain (K. Viets 1930). Both species have dorsal shield with the shoulder platelets partially fused with the dorsal plate, a similar color pattern of the dorsal shield, a Cxgl-4 situated subapically and a relatively long median suture line of Cx-II-III in male. Torrenticolalundbladi differs by the characteristic shape of the ejaculatory complex (proximal and distal arms short, proximal chamber large, proximal horns reduced, see Lundblad 1956: fig. 83E).
Distribution.
Portugal (this study).
. Torrenticola (Torrenticola) tenuipalpis
Lundblad, 1956
C5BED193-FAAB-5CA5-B8D6-547CB9B57832
Material examined.
Portugal, Guarda • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced), dissected and slide mounted (RMNH).
Remarks.
The Portuguese specimen matches the description of Torrenticolatenuipalpis, a species originally described by Lundblad (1956) as a “variatio” of T.amplexa based on a single female collected in a stream in Santa Comba Dão, Portugal. Recently, Cantallo et al. (2022) ranked the latter taxon as a distinct species. The sequenced specimen from Portugal forms a unique BIN (BOLD:AFV2021) with the nearest neighboring BIN (p-distance 12.2%) being BOLD:AFF4076, which consists of a single specimen of T.ramini from Iran.
Distribution.
Portugal; known from Beira Alta (Lundblad 1956).
Family Limnesiidae Thor, 1900
Genus. Limnesia
Koch, 1836
7A358908-2576-5B84-BDEC-E2CF540862F2
Note.
In Portugal, represented by eight species, seven of them known from the mainland and one (L.atlantica Lundblad, 1941) known only from Madeira.
. Limnesia (Limnesia) acuminata
Walter, 1925
25443953-57F8-554D-B13E-55040C159661
Material examined.
Portugal, Beja • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂ (sequenced). Bragança: • Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha, 2♂, 1♀ (sequenced).
Remarks.
The Portuguese specimens match the description of L.acuminata. Genetic data indicate that specimens from Portugal form a unique cluster (BOLD:AFU7587).
Distribution.
Western Mediterranean (Iberian Peninsula, southern France, Sardinia, Sicily, north Africa). In Portugal previously reported from Beira Alta, Alentejo, and Estremadura (Lundblad 1956).
. Limnesia (Limnesia) iberica
Lundblad, 1954
1C80227E-5DD4-57E9-A2A4-54F4098F6862
Material examined.
Portugal, Beja • Mértola, São João dos Caldeireiros, stream, 37.626°N, 7.81°W, 17 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 3♂, 6♀ (sequenced).
Remarks.
The specimens from Portugal morphologically matches description of Limnesiaiberica, a species originally described by Lundblad (1956) from a stream in Beira Alta (Santa Comba Dão, Portugal). The Portuguese specimens form a distinct BIN (BOLD:AFN8367) with the closest BIN being BOLD:ACA9272, which includes specimens from Canada, United States, and Greece assigned to L.undulata, with the p-distance estimated at 14.29%.
Distribution.
Portugal (Lundblad 1954, 1956; this study).
. Limnesia (Limnesia) koenikei
Piersig, 1894
065C2D53-D9CB-598C-991F-7F8630ECA192
Material examined.
Portugal, Guarda: • Seia, Covão do Forno, 40.369°N, 7.638°W, 1574 m a.s.l., 19 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 2♀ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced).
Remarks.
The sequences obtained from the specimens from Portugal cluster within BOLD:ADF6559, which includes one specimen from the Netherlands assigned to L.koenikei. The p-distance from the latter BIN and its nearest neighbor BOLD:ACS0816, which includes specimens of L.koenikei from Norway and the Netherlands, was estimated at 2.09%.
Distribution.
Holarctic; widely distributed in Europe but here reported for the first time for Portugal.
. Limnesia (Limnesia) maculata
(Müller, 1776)
FC4C88D1-7F6A-5B00-9176-3A0A6398E9A7
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced). Beja • Mértola, Moinho de Alferes 2, 37.503°N, 7.687°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂ (juv.) (sequenced); • Mértola, Herdade de Alagães, pond, 37.673°N, 7.848°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂, 1♀ (sequenced).
Remarks.
The Portuguese specimens molecularly analyzed in this study match the description of L.maculata. These individuals form a unique BIN (BOLD:AFW6935), with the nearest neighboring BIN being BOLD:ACS0248, which includes specimens of L.maculata from Norway, the Netherlands and France. The p-distance between these two clusters was estimated at 4.3%.
Distribution.
Holarctic. Widespread in Europe. In Portugal previously reported from Alentejo (Ribeira de Odivelas; Lundblad 1956).
. Limnesia (Limnesia) walteri
Migot, 1926
95B31EFA-E9A6-5A47-8A07-7173155E6B10
Material examined.
Portugal, Beja • Mértola, Corte do Pinto, 37.682°N, 7.512°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 3♀ (sequenced). Bragança • Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha, 1 ♀ (sequenced).
Remarks.
The sequenced specimens from Portugal form a unique BIN (BOLD:AFO9873) with the nearest neighboring BIN being BOLD:ADZ9059 (p-distance 1.6%), which includes four unidentified Limnesia specimens from Morocco.
Distribution.
North Africa, including the Sahara desert, Russia, southwestern Europe from Portugal (Lundblad 1956; Valdecasas 1988) to Greece (Gerecke et al. 2016). In Portugal previously reported from Beira Alta (Lundblad 1956).
Family Hygrobatidae Koch, 1842
Genus. Atractides
Koch, 1837
1590E703-3669-5EFF-B277-2D95E27DEB52
Note.
Nine species known from Portugal, five of them endemic to Madeira, and A.marizae Pešić, 2023 endemic to mainland Portugal
. Atractides (Atractides) inflatus
(Walter, 1925)
A36D900C-8524-5170-B6C2-14E394101AE7
Material examined.
Portugal, Beja • Odemira, Ribeira de Seixe, Herdade do Vale de Águia, river, 37.398°N, 8.68°W, 75 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 2♀ (sequenced). Vila Real • Murça, Noura stream, 41.409°N, 7.417°W, 421 m a.s.l., 12 Jul. 2023 leg. Ferreira & Padilha, 1♀ (sequenced).
Remarks.
The specimens from Portugal used in this study match the description of A.inflatus, a species widely distributed in the Mediterranean region, often very frequent in intermittent streams (Pešić et al. 2023c). The Portuguese specimens were clustered within two BINs: BOLD:AFI9009, which includes two specimens of A.inflatus from Italy, and BOLD:ACB4677, which includes specimens of A.inflatus from Iran, Morocco, Montenegro, Turkey, Greece, France, and Italy. The p-distance between these two BINs was estimated at 6.06%.
Distribution.
Mediterranean, Iran. New for Portugal.
. Atractides (Atractides) marizae
Pešić, 2023
A3DBEFBD-1A5B-5A6D-ADC3-C376AEE9DC71
Material examined.
Portugal, Guarda: • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced). Faro: • Monchique, Caldas de Monchique, 37.287°N, 8.554°W, 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♀ (sequenced); • Aljezur, Ribeira de Seixe, Odeceixe, Covão da Serva, 37.374°N, 8.642°W, 100 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco 1♀ (sequenced); • Portimão, 37.237°N, 8.546°W, 23 May 2023, leg. Ferreira & Turaccio 1♂ (sequenced).
Remarks.
The specimens from Portugal clustered within BOLD:AER7878, which includes specimens of Atractidesmarizae Pešić, 2023, a species recently described by Pešić et al. (2023b) from Santarém, Portugal. Until now, this rhitrobiontic species was known only from the type locality (Caniceira stream), and the new findings presented in this study demonstrate that A.marizae is more widely distributed in Portugal.
Distribution.
Portugal.
. Atractides (Atractides) nodipalpis
(Thor, 1899)
D34310F3-0C4F-5B23-B16B-9C7255EDD942
Material examined.
Portugal, Guarda • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The female, which keyed to A.nodipalpis following Gerecke et al. (2016), forms a unique BIN (BOLD:AFV2009). The BIN is placed as sister to BOLD:ACR0209, which includes > 200 specimens of A.nodipalpis, available in the BOLD database. The p-distance between these two BINs was estimated at 4.61%.
For a long time, A.nodipalpis has been considered the most common species of the genus in Europe. However, in the last years, genetic data revealed that the latter species consists of several distinct lineages, some of them present in the same areas (Gerecke et al. 2022; Pešić et al. 2023d). For example, Gerecke et al. (2022) mentioned that Norwegian specimens keyed as A.nodipalpis belong to two different lineages, both widely distributed in Norway. The taxonomic status of most of these lineages is still unclear as a number of species have been proposed as synonyms of A.nodipalpis in the past. Nevertheless, Gerecke and collaborators (Gerecke pers. comm. 2022) recently clarified the correct BIN assignment of the true A.nodipalpis lineage. They found that specimens of A.nodipalpis collected near its type locality in Norway belong to the BOLD:ACR0209 cluster. After that, Pešić et al. (2023d) examined specimens from the Netherlands belonging to BOLD:ACR0209 and found that A.nodipalpis can be defined primarily by the shape of male genital plate which has a distinct anteromedial peg-like fissure.
Distribution.
Based on the available records in BOLD, A.nodipalpis has a wide distribution, from SE Europe over the Fennoscandia up to Greenland. In Portugal previously reported from Beira Alta (Santa Comba Dão; Lundblad 1956).
. Atractides (Atractides) robustus
(Sokolow, 1940)
35AC45CC-0750-52D2-8B57-8CD0F54EDC48
Material examined.
Portugal, Guarda • Manteigas, Zêzere, Covão da Ametade, 40.328°N, 7.587°W, 1431 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The Portuguese specimen matches the description of A.robustus. The specimen clusters within BOLD:AFF2463, which includes specimens from Italy, Albania and Poland. The p-distance from the closest neighboring BIN being BOLD:ADZ9348, which consists of specimens of A.robustus from Germany, Austria, Montenegro, Romania, Italy, Bosnia and Herzegovina, Albania, and Greece, was estimated at 3.15%.
Recently, Pešić et al. (2023d) showed that A.robustus, a species originally described from the Caucasus (the affluents of the Kuban River), consists of two distinct lineages, one which includes populations from eastern Turkey and northern Iran, and which is likely conspecific with A.robustus, and the second lineage, which includes A.robustus like specimens from central and southern Europe. The latter lineage, to which BOLD:ADZ9348 and BOLD:AFF2463 belong, possible represents a cryptic species new to science. The final decision on the taxonomic status of A.robustus lineages has been postponed until material of the latter species from the Caucasus is available (Pešić et al. 2023d).
Distribution.
Europe. New for Portugal.
Genus. Hygrobates
Koch, 1837
A67D6A75-8E04-51ED-B7FC-B549106BDDAF
Note.
Five species known from mainland part of Portugal.
. Hygrobates balcanicus
Pešić, 2020
F91BA968-76FD-5138-81C7-F8D6EA33343B
Material examined.
Portugal, Faro • Portimão, stream, 37.237°N, 8.546°W, 23 May 2023, leg. Ferreira & Turaccio, 2♂, 1♀ (sequenced). Porto • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 176 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1♂ (sequenced); • Lousada, Parque Molinológico e Florestal de Pias, 41.268°N, 8.256°W, 170 m a.s.l., 1 Sep. 2023, leg. Ferreira, Sousa, Cruz-Oliveira & Girão, 1♂ (sequenced); • Vila do Conde, Rio Este, 41.378°N, 8.695°W, 15 m a.s.l., 7 Sep. 2023, leg. Ferreira, Cruz-Oliveira & Girão, 3♂, 1 deutonymph (sequenced).
Remarks.
The specimens from Portugal morphologically match the description of Hygrobatesbalcanicus. This species was originally described by Pešić et al. (2020) from Bulgaria, and later on reported from eastern Serbia (Pešić et al. 2023a). The sequenced specimens from Portugal cluster within BOLD:AEG3198, which, in addition to the specimens used in this study, includes specimens from Bulgaria, Serbia and Italy morphologically assigned to H.balcanicus.
Distribution.
Balkans, Italy. New for Portugal.
. Hygrobates fluviatilis
(Ström, 1768)
AA965F92-BFAD-5593-BA59-A12C335FC6B6
Material examined.
Portugal, Guarda: • Manteigas, Casa do Cantoneiro, 40.418°N, 7.603°W, 1378 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂ (sequenced); • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced).
Remarks.
Genetic data indicate that Portuguese specimens belong to BOLD:ACB4846, which includes more than 300 specimens of H.fluviatilis, available in BOLD. The latter species was recently revised using molecular and morphological data (Pešić et al. 2017).
Distribution.
Central, western, and southern Europe. In Portugal previously reported from Santa Comba Dão (Beira Alta; Lundblad 1956).
. Hygrobates longiporus
Thor, 1898 complex
B9105C9F-1D02-5007-A1F4-C88F5C49A642
Figure 7.
Neighbor-Joining tree of the Hygrobateslongiporus complex obtained from 53 nucleotide COI sequences; 23 sequences were taken from Pešić et al. (2022a, 2023f), 26 sequences from Portugal are newly generated in this study, and four private sequences (HYDIR005-23, HYDIR006-23, HYDIR019-23, HYDIR020-23) from Turkey were directly taken from the BOLD. Hygrobatesbalcanicus from Portugal was used as outgroup. BINs are based on the barcode analysis from 16 May 2024. Country codes: FR = France, GR = Greece, IR = Iran, NO = Norway, PT = Portugal, RS = Serbia, TR = Turkey. Bootstrap values > 50% from 1000 bootstrap replicates on branches.
Material examined.
Portugal, Guarda: • Seia, Rio Alva, Praia fluvial de Vila Cova a Coelheira, river, 40.379°N, 7.736°W, 312 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀, 1 deutonymph (sequenced); • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂, 1♀, 1 deutonymph (sequenced); • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀ (sequenced); • Manteigas, Mondego, Covão da ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 3♀, 2 deutonymph (sequenced); • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀ (sequenced); • Manteigas, Poio do Leão, 40.399°N, 7.541°W, 734 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Manteigas, Zêzere, Ponte dos Frades, 40.403°N, 7.526°W, 672 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur 1♂ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced). Bragança: • Mirandela, Torre de Dona Chama, 41.665°N, 7.146°W, 256 m a.s.l., 13 Jul. 2023, leg. Ferreira & Padilha, 1 ♀ (sequenced). Vila Real • Murça, Noura stream, 41.409°N, 7.417°W, 421 m a.s.l., 12 Jul. 2023 leg. Ferreira & Padilha, 1♂ (sequenced).
Remarks.
In this study, specimens keying to H.longiporus in Gerecke et al. (2016) have DNA barcodes that cluster in three unique BINs (BOLD:AFV9997, BOLD:AFW1423, BOLD:AFV9998) (Fig. 7). The p-distance to the nearest neigbour ranged between 11.38–13.62%, far exceeding the species thresholds (6.08% K2P distance) obtained in the study on the H.longiporus complex by Pešić et al. (2022a).
The identity of H.longiporus was recently questioned by Pešić et al. (2021b, 2022a) who found that DNA barcodes of the specimens assigned to the latter species in Europe and Turkey cluster within four distinct genetic lineages. The first cluster (BOLD:AEB8359) comprises a large number of longiporus-like specimens from different parts of Europe, including Norway, from where the species was originally described (Thor 1898). The second cluster (BOLD:AEM7299) is restricted to SE Europe and E Turkey, while the third cluster (BOLD:AEO0825) is known only from Corsica. Most likely the latter two clusters represent a cryptic species new to science. The fourth clade, known from northern Iran and eastern Turkey, recently was described by Pešić et al. (2022a) as H.thori Pešić & Smit, 2022.
As emphasized by Pešić et al. (2022a), a larger-scale study of H.longiporus complex is needed to establish a stable taxonomy for this group. The true identity of H.longiporus should be morphologically redefined with material from Norway. Morphological analysis revealed that in regard to the shape of palp and genital field, Portuguese specimens match the description of H.falcilaminatus, a species originally described from Azrou, Morocco on the basis of a single female (Walter 1926) and later reported from a wide area in the western Mediterranean (Spain and France). The species was synonymized with H.longiporus by Lundblad (1956) (see also Gerecke 1991 for a discussion about this species). Further research, including a redescription of H.falcilaminatus, is needed to clarify taxonomic status of the Portuguese lineages and relationship with other members of H.longiporus complex.
Family Unionicolidae Oudemans, 1909
Genus. Neumania
Lebert, 1879
4F77AE79-785B-596D-B69A-9FF73B7C3871
Note.
Three species so far reported from Portugal.
. Neumania (Neumania) elliptica
Walter, 1925
CA9CC06C-8ADC-5B23-8014-52CE48905F6B
Material examined.
Portugal, Guarda: • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The specimens from Portugal morphologically match the description of N.elliptica. This species was originally described from Algeria (Walter 1925) and later on reported by Pešić et al. (2007) from Corsica (France) and Italy (Sardinia and Sicily). Neumaniaelliptica is a characteristic colonizer of pools in summer-warm lowland streams with silty substrate (Pešić et al. 2007).
The sequenced specimens from Portugal form a unique BIN (BOLD:AFU2122), with the nearest neighboring BIN being BOLD:ACR9513 (p-distance 19.16%), which includes specimens of N.deltoides from the Netherlands, Macedonia and Turkey, available in BOLD.
Distribution.
SW-Mediterranean. New for Portugal.
. Neumania (Neumania) imitata
Koenike, 1908
BBB5777D-24A1-510A-BB9B-290120C20250
Material examined.
Portugal, Porto • Lousada, Parque Molinológico e Florestal de Pias, 41.268°N, 8.256°W, 170 m a.s.l., 1 Sep. 2023, leg. Ferreira, Sousa, Cruz-Oliveira & Girão, 1♂ (sequenced).
Remarks.
The examined male in our study keyed to Neumaniaimitata following Gerecke et al. (2016) and forms a unique BIN (BOLD:AFV0268). The p-distance between this BIN and its nearest neighbour, BOLD:ADF7924, which includes specimens of N.imitata from the Netherlands, was estimated at 8.65%, indicating the need for taxonomic revision of this species.
Distribution.
Europe; rare, reported from France, Italy, Germany, the Netherlands, Poland, and Montenegro. New for Portugal.
. Neumania (Neumania) limosa
(Koch, 1836)
17D772B1-C314-5A0B-AE53-D0EFA7EF428B
Material examined.
Portugal, Beja • Mértola, Herdade de Alagães, pond, 37.673°N, 7.848°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 4♀ (sequenced); Guarda: • Manteigas, Serra de Baixo, Lagoa, 40.35°N, 7.549°W, 1431 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 3♀ (sequenced); • Gouveia, Ribeira do Covão do Urso, Barragem do Lagoacho, 40.385°N, 7.618°W, 1438 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (juv.) (sequenced).
Remarks.
The specimens from Portugal cluster within BOLD:ACS0551, which includes specimens of N.limosa from the Netherlands. The p-distance between this BIN and its nearest neighbor BOLD:AEF5902, which includes specimens from Montenegro assigned to N.limosa, was estimated at 3.21%.
Distribution.
Palaearctic. New for Portugal.
. Neumania (Neumania) uncinata
Walter, 1927
ACDFCD3B-C2E8-57B2-AF20-57A057B48BE3
Material examined.
Portugal, Faro • Monchique, Caldas de Monchique, 37.287°N, 8.554°W, 23 May 2023, leg. Ferreira & Turaccio, 1♀ (sequenced). Porto • Vila do Conde, Rio Este, 41.378°N, 8.695°W, 15 m a.s.l., 7 Sep. 2023, leg. Ferreira, Cruz-Oliveira & Girão, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal keyed to N.uncinata following Gerecke et al. (2016), and cluster within two unique BINs (BOLD:AFV0253, BOLD:AFV0269). The p-distance between Portuguese BINs and their closest neighbor, BOLD:AER9267, which includes specimens of N.uncinata from Sardinia (see Pešić and Goldschmidt 2023), was estimated at 8.78% and 13.14%, respectively, indicating the need for taxonomic revision of N.uncinata complex from a wider geographical area.
Distribution.
Western Palaearctic. In Portugal known from Estremadura (Lundblad 1956). Neumaniaatlantida Lundblad, 1962, originally described from Madeira by Lundblad (1962), was synonymized by Pešić et al. (2007) with N.uncinata.
. Neumania (Soarella) papillosa
(Soar, 1902)
97B76798-4A9A-5F97-B073-4BC50EC68AE2
Material examined.
Portugal, Beja • Mértola, Corte do Pinto, 37.682°N, 7.512°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂, 2♀ (sequenced). Guarda • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The Portuguese specimens match the description of N.papillosa, forming a unique BIN (BOLD:AFO2116), with the nearest neighboring BIN being BOLD:ADS6560, which consists of 31 specimens of and unidentified Neumania sp. from South Africa. The p-distance between these two BINs was estimated at 14.26%.
Distribution.
Europe. In Portugal previously reported from Côa River near Santa Comba Dão (Beira Alta; Lundblad 1956).
Genus. Unionicola
Haldeman, 1842
52C3AEF8-0A93-5F80-B243-56262A4880B4
Note.
Three species so far reported from Portugal.
. Unionicola (Hexatax) minor
(Soar, 1900)
DEA57F1D-1E51-56DA-9830-020EBA476700
Material examined.
Portugal, Beja: • Mértola, São João dos Caldeireiros, stream, 37.626°N, 7.81°W, 17 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (one palp dissected and slide mounted, RMNH); • Mértola, Moinho de Alferes 2, 37.503°N, 7.687°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced).
Remarks.
Specimens keying to U.minor in Gerecke et al. (2016) have DNA barcodes that cluster in unique BIN (BOLD:AFO2171).
Distribution.
Widespread in Europe, here reported for the first time for Portugal.
Family Pionidae Thor, 1900
Subfamily Foreliinae Thor, 1923
Genus. Forelia
Haller, 1882
D2DCC453-D626-58AA-8A82-0C8A788AA6BA
Note.
Only one species reported from Portugal.
. Forelia longipalpis
Maglio, 1924
CD97C38F-6914-550C-82C5-D6EA3DE0F38E
Material examined.
Portugal, Guarda: • Gouveia, Ribeira da Fervença, Barragem do Vale do Rossim, 40.4°N, 7.589°W, 1418 m a.s.l., 22 Aug. 2023 leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur, 3♀, 1 deutonymph (sequenced); • Seia, Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Manteigas, Mondego, Covão da Ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
All barcoded specimens in our study were females. The shape of the genital field of specimens from Portugal morphologically match the description of F.longipalpis following Gerecke et al. (2016). The sequenced specimens cluster into two unique BINs (BOLD:AFX2876, BOLD:AFV3893), indicating the need for further taxonomic revision of this species. However, this revision should be postponed until males are available.
Distribution.
Widespread in Europe; new record for Portugal.
. Forelia variegator
(Koch, 1837)
0883E172-763A-5EC5-88D1-1EB976342552
Material examined.
Portugal, Beja • Mértola, São João dos Caldeireiros, stream, 37.625°N, 7.81°W, 17 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced). Porto: • Lousada, Parque Molinológico e Florestal de Pias, 41.268°N, 8.256°W, 170 m a.s.l., 1 Sep. 2023, leg. Ferreira, Sousa, Cruz-Oliveira & Girão, 2♀ (sequenced); • Vila do Conde, Rio Este, 41.378°N, 8.695°W, 15 m a.s.l., 7 Sep. 2023, leg. Ferreira, Sousa, Cruz-Oliveira & Girão, 1♂ (sequenced). Guarda • Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
Regarding the shape of the genital field, the specimens from Portugal morphologically match the description of F.variegator following Gerecke et al. (2016). The sequenced specimens from Portugal form a unique BIN (BOLD:AFU5459), with the nearest neighboring BIN being BOLD:ACS0537, which includes specimens of F.variegator from the Netherlands, Norway, North Macedonia, and Russia. The p-distance between these two BINs is estimated at 12.82%.
Distribution.
Palaearctic. In Portugal previously reported from Beira Alta and Estremadura (Lundblad 1956).
Subfamily Hydrochoreutinae K. Viets, 1942
Genus. Hydrochoreutes
Koch, 1837
1D9E4B1C-B0CA-5882-AE2E-B42CD139218D
Note.
New genus for Portugal.
. Hydrochoreutes krameri
Piersig, 1896
31F87280-A6FF-560F-8090-90BB466D1701
Material examined.
Portugal, Beja • Mértola, Herdade de Alagães, pond, 37.673°N, 7.848°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced). Guarda: • Seia, Covão do Forno, 40.369°N, 7.638°W, 1574 m a.s.l., 19 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced).
Remarks.
The sequenced specimens from Portugal cluster within BOLD:ACR9737. In addition to the specimen from Portugal, the BIN includes specimens of Hydrochoreuteskrameri from Norway, the Netherlands, and North Macedonia, available in BOLD. The p-distance between the latter BIN and its nearest neighbor, BOLD:ADZ1025, which includes specimens of H.ungulates, is estimated at 15.22%.
Distribution.
Palaearctic. New for Portugal.
Subfamily Pioninae Thor, 1900
Genus. Nautarachna
Moniez, 1888
78233157-F2CD-571C-9DC1-67F500145A04
Note.
New genus for Portugal.
. Nautarachna crassa
(Koenike, 1908)
10F174D9-ACA6-5FB5-9FE0-44592D1E79D4
Material examined.
Portugal, Guarda • Manteigas, Casa do Cantoneiro, 40.418°N, 7.603°W, 1378 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The sequenced female of N.crassa from Portugal forms a distinct BIN (BOLD:AFV0462). The p-distance between the specimen from Portugal and the barcode of a N.crassa specimen (MMHYD270-20) collected in Norway, is estimated at 16.7%, indicating the need for a taxonomic revision of N.crassa complex to identify possible undescribed cryptic species.
Distribution.
Europe; widespread but here reported for the first time for the water mite fauna of Portugal.
Genus. Piona
Koch, 1842
43A109DA-B301-5812-9975-CCD3C6D43846
Note.
So far, two species of the genus have been reported from Portugal (Pešić et al. 2023b).
. Piona carnea
(Müller, 1776)
6605F964-646F-5177-84A9-31743A5D54A1
Material examined.
Portugal, Beja: • Mértola, São Sebastião dos Carros, 37.598°N, 7.754°W, 21 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 2♂, 2♀, 3 deutonymphs (sequenced); • Mértola, Herdade de Alagães, dry stream site 2, 37.678°N, 7.848°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1 deutonymph (sequenced). Vila Real • Murça, Noura stream, 41.409°N, 7.417°W, 421 m a.s.l., 12 Jul. 2023 leg. Ferreira S, Padilha 1 deutonymph (sequenced). Guarda: • Manteigas, Serra de Baixo, Lagoa, 40.35°N, 7.549°W, 1431 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2 deutonymphs (sequenced).
Remarks.
The specimens from Portugal morphologically match the description of Pionacarnea. These specimens cluster within two BINs: BOLD:ACM0527, which includes three unidentified specimens from Canada and three specimens from the Netherlands assigned to P.carnea, and BOLD:ACS0622, which includes specimens of P.carnea from Norway, Finland, the Netherlands, and Germany.
Distribution.
Holarctic. New record for Portugal.
. Piona variabilis
(Koch, 1836)
7D798821-4174-51F6-A452-729A8E09D4FC
Material examined.
Portugal, Beja • Mértola, São Sebastião dos Carros, 37.598°N, 7.754°W, 21 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1 deutonymph (sequenced; Table 1).
Remarks.
The single deutonymph from Portugal cluster within BOLD:AAU0701, which includes 11 specimens from Sweden, Norway, and the Netherlands assigned to P.variabilis, available in the BOLD database.
Distribution.
Europe. New record for Portugal.
Subfamily Tiphyinae Oudemans, 1941
Genus. Pionopsis
Piersig, 1894
41A51D00-DE73-56E9-A22E-F8C7D559B4EE
Note.
One species of the genus reported from Portugal
. Pionopsis lutescens
(Hermann, 1804)
C741DEC2-727E-5121-9A7A-86C7256AB5FB
Material examined.
Portugal, Porto • Lousada, Parque Torre de Vilar, 41.287°N, 8.21°W, 274 m a.s.l., 1 Sep. 2023, leg. Ferreira, Sousa & Girão 1♀ (sequenced). Guarda • Seia, Cise, 40.419°N, 7.709°W, fountain, 505 m a.s.l., 25 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀, 1 deutonymph (sequenced).
Remarks.
The examined male keyed to Pionopsislutescens following Gerecke et al. (2016). The Portuguese specimens form a unique BIN (BOLD:AFV3897). The p-distance between the latter BIN and its nearest neighbor, BOLD:AET1848, which includes specimens of P.lutescens from Montenegro, was estimated at 12.34%, indicating the need for taxonomic revision of P.lutescens complex to identify possible undescribed cryptic species.
Distribution.
Holarctic. In Portugal previously reported from Sintra-Monserrate Park and Palace, Estremadura (Lundblad 1956).
Genus. Tiphys
Koch, 1836
0D1DD487-40E1-549C-9BA0-6F54749968CA
Note.
Only one species reported from Portugal.
. Tiphys torris
(Müller, 1776)
1FDFC7D0-90C8-529E-9159-1AACA93BAE4F
Material examined.
Portugal, Beja • Odemira, Ribeira de Seixe, Zambujeira do Mar, 37.399°N, 8.723°W, 45 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♀ (sequenced). Guarda • Seia, Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal form a unique BIN (BOLD:AFP3352) with the nearest neighboring BIN being BOLD:ACR9977 (p-distance 1.92%), which includes one specimen from the Netherlands assigned to T.torris and three specimens from Norway assigned to T.lapponicus.
Distribution.
Europe. In Portugal previously reported from Estremadura (Lundblad 1956).
Family Aturidae Thor, 1900
Genus. Aturus
Kramer, 1875
FCD4CA81-6810-5756-8C1B-9E097CE4A4D2
Note.
New genus record for Portugal.
. Aturus scaber
Kramer, 1875
5029C01E-1A0D-547C-875F-4E48828E6153
Material examined.
Portugal, Porto: • Lousada, Moinho da Tapada, 41.263°N, 8.307°W, 176 m a.s.l., 1 Sep. 2023, Ferreira, Sousa, Cruz-Oliveira & Girão, 1♂ (sequenced).
Remarks.
The specimen from Portugal clusters within BOLD:ACQ9097, which includes > 80 specimens of A.scaber from Norway and Germany in BOLD.
Distribution.
Western Palaearctic. New record for Portugal.
Family Mideopsidae Koenike, 1910
Genus. Mideopsis
Koenike, 1910
DB69CC2A-BCEB-58C3-9665-0BAC57053EBF
Note.
Family and genus both new for Portugal.
. Mideopsis roztoczensis
Biesiadka & Kowalik, 1979
AE5CB231-F98D-5E26-8E1A-E4FDB632EDC6
Figure 8.
Neighbor-Joining tree of the genus Mideopsis obtained from 112 nucleotide COI sequences; 71 sequences were taken from Pešić et al. (2023a) and 41 sequences from Portugal are newly generated in this study. Mundamellagermanica from Montenegro was used as outgroup. BINs are based on the barcode analysis from 16 May 2024. Country codes: BA = Bosnia and Herzegovina, BE = Belgium, CY = Cyprus, DE = Germany, FR = France, ME = Montenegro, PT = Portugal, RS = Serbia, RU = Russia, TR = Turkey. Bootstrap values > 50% from 1000 bootstrap replicates on branches.
Material examined.
Portugal, Beja: • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced); Mértola, Pulo do Lobo, 37.805°N, 7.633°W, 18 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 5♀, 1 deutonymph (sequenced); • Odemira, Ribeira de Seixe, Zambujeira do Mar, 37.398°N, 8.68°W, 75 m a.s.l., 23 May 2023, leg. Ekrem & Benitez-Bosco, 1♂ (sequenced). Bragança • Vinhais, Gasparona, 41.85°N, 7.013°W, 683 m a.s.l., 6 Jul. 2023, leg. Ferreira & Padilha, 1♀, 1 deutonymph (sequenced). Guarda: • Seia, Casa do Loureiro, 40.433°N, 7.701°W, 415 m a.s.l., 19 Jul. 2023 leg. Ferreira & Padilha, 1♂, 3♀, 1 deutonymph (sequenced); • Manteigas, Zêzere, Covão da Ametade, 40.328°N, 7.587°W, 1431 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀, 1 deutonymph (sequenced); • Manteigas, Zêzere, Ponte dos Frades, 40.403°N, 7.526°W, 672 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur 1♀ (sequenced); • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂, 1♀ (juv.), 1 deutonymph, 1♂ dissected and slide mounted (RMNH); • Gouveia, Rio Mondego, Casais de Folgosinho, 40.454°N, 7.493°W, 976 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 1♀ (sequenced); • Manteigas, Poio do Leão, 40.399°N, 7.541°W, 734 m a.s.l., 22 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂ (sequenced); • Seia, Rio Alva, Central hidroelétrica de Ponte dos Jugais, river, 40.385°N, 7.706°W, 555 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂, 2♀, 2 deutonymph (sequenced), 1♂ dissected and slide mounted (RMNH); • Rio Alva, Nossa Senhora do Desterro, river, 40.395°N, 7.694°W, 791 m a.s.l., 23 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Manteigas, Mondego, Covão da ponte, 40.443°N, 7.514°W, 999 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); • Seia, Rio Alva, Praia Fluvial de Sabugueiro, river, 40.401°N, 7.64°W, 1021 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♀ (sequenced); • Manteigas, Casa do Cantoneiro, 40.418°N, 7.603°W, 1378 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 2♂, 1♀ (sequenced). Vila Real • Murça, Noura stream, 41.409°N, 7.417°W, 421 m a.s.l., 12 Jul. 2023 leg. Ferreira S, Padilha 1♀ (sequenced).
Remarks.
The specimens examined in our study match the description of Mideopsisroztoczensis, when following Biesiadka and Kowalik (1979). Mideopsisroztoczensis is characterized by a more elevated dorsal shield with distinctly visible anteriorly diverging lines of particularly faint fine porosity, and by the the shape of the male ejaculatory complex with the wedge-shaped anterior ramus being wider and with a characteristic arrow-shaped delimited area. Recently, Pešić et al. (2023e) showed that M.roztoczensis is a genetically variable species, comprising four BINs (BOLD:ACI1492, BOLD:AEN6785, BOLD:AEA2936, BOLD:AEO2944) widely distributed in Europe. In this study, we detected an additional five unique BINs (BOLD:AFU6108, BOLD:AFP5421, BOLD:AFW3785, BOLD:AFV6334, BOLD:AEV2909) within the Portuguese M.roztoczensis-like specimens (Fig. 8), all of them unique, and some of them present at the same sites (e.g., BOLD:AFU6108 and BOLD:AFW3785 in Casa do Loureiro, BOLD:AFU6108 and BOLD:AFV6334 in Casa do Cantoneiro).
Distribution.
Europe, Turkey. New record for Portugal.
Family Momoniidae K. Viets, 1926
Genus. Momonia
Halbert, 1906
2491AE7A-DB1F-5209-B5C7-3F622E19EE35
Note.
Genus and family both new for Portugal.
. Momonia (Momonia) falcipalpis
Halbert, 1906
E406273A-205F-5980-9DD6-32B5636B0189
Material examined.
Portugal, Guarda • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The examined specimen in our study keyed to Momoniafalcipalpis and forms a unique BIN (BOLD:AFX3396).
Distribution.
Europe; rare, known from Ireland, France, Italy, and Russia. New record for Portugal.
Family Arrenuridae Thor, 1900
Genus. Arrenurus
Dugès, 1834
C59B26C4-90F9-5EA5-9D92-FEBDDB4C61A2
Note.
Seven species are reported from Portugal; one of them (Arrenurusautochthonus Lundblad, 1941) is endemic to Madeira.
. Arrenurus (Arrenurus) albator
(Müller, 1776)
3CB9C6D8-58F6-5FF1-991E-AEEF1D04534B
Material examined.
Portugal, Guarda: • Gouveia, Ribeira da Fervença, Barragem do Vale do Rossim, 40.4°N, 7.589°W, 1418 m a.s.l., 22 Aug. 2023 leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur, 2♂, 2♀ (sequenced); • Gouveia, Ribeira do Covão do Urso, Barragem do Lagoacho, 40.385°N, 7.618°W, 1438 m a.s.l., 22 Aug. 2023 leg. Ferreira, Benitez-Bosco, Padilha, Andrade & Stur, 1♀ (sequenced).
Remarks.
The examined specimens from Portugal cluster within BOLD:ACR9639, which includes one specimen of Arrenurusalbator from the Netherlands, available in the BOLD database.
Distribution.
Western Palaearctic. In Portugal previously reported from Beira Alta (Lundblad 1956).
. Arrenurus (Arrenurus) szalayi
Lundblad, 1954
B480D81F-BCFF-5B27-8428-1B2873C26B20
Material examined.
Portugal, Beja • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 2♂ incl. one juv., (both sequenced).
Remarks.
The examined males from Moinho de Alferes match the description of A.szalayi, a species originally described from Ribeira d’Odivelas in Portugal (Lundblad 1956). The sequenced specimens from Portugal clustered within one BIN (BOLD:ACS0403), which in addition to the specimens from Portugal, includes a number of specimens collected from a large geographic area, from Norway to South Africa and from the Netherlands to Kyrgyzstan, and that are morphologically assigned to A.bicuspidator Berlese, 1885, or to A.radiatus Piersig, 1894. From both species, A.szalayi, at least morphologically, can be easily separated by the characteristic shape of the male petiole (see Lundblad 1956: fig. 161A). Further research is needed to understand the factors behind this grouping and the implication of this lack of genetic differentiation between three morphological different species.
Distribution.
Portugal; previously reported from Ribeira de Odivelas, Alentejo (Lundblad 1954, 1956).
. Arrenurus (Arrenurus) leuckarti
Piersig, 1894
F04C6010-C37C-5BF2-B853-A700840F5AC8
Material examined.
Portugal, Guarda: • Manteigas, Poço do Inferno, 40.373°N, 7.516°W, 1078 m a.s.l., 21 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♂ (sequenced); Manteigas, Casa do Cantoneiro, 40.418°N, 7.603°W, 1378 m a.s.l., 24 Aug. 2023, leg. Ferreira, Benitez-Bosco & Padilha, 1♀ (sequenced).
Remarks.
The examined specimens from Portugal cluster within BOLD:ACR9670, which includes specimens of Arrenurusleuckarti from the Netherlands.
Distribution.
Western, eastern, and central Europe. New record for Portugal.
. Arrenurus (Arrenurus) neumani
Piersig, 1895
0245FD41-6B87-5ACC-88A9-13BFFD2EFF70
Material examined.
Portugal. Beja • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 3♂, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal form a unique BIN (BOLD:AFP6143) with the nearest neighboring BIN being BOLD:ACR9801, which includes specimens of A.neumani, from the Netherlands, Norway and Poland in BOLD. The p-distance between these two BINs was estimated at 5.9%.
Distribution.
Palaearctic. New record for Portugal.
. Arrenurus (Arrenurus) cf.tricuspidator
(Müller, 1776)
DDBD35F1-4726-5219-BD28-BC30A41A586A
Material examined.
Portugal, Beja • Mértola, Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1 deutonymph (sequenced).
Remarks.
One deutonymph from Portugal, used in this study for molecular analysis, forms a unique cluster BOLD:AFU3639, with a p-distance estimated at 3.21% to the closest neigbouring BIN, BOLD:ACS0825, which includes specimens of A.tricuspidator from the Netherlands, Norway, and Germany.
Distribution.
Palaearctic. New record for Portugal.
. Arrenurus (Megaluracarus) globator
(Müller, 1776)
177E68F7-7233-506E-BE1F-FD1EF162A6FB
Material examined.
Portugal: Beja: • Mértola, São Sebastião dos Carros, 37.598°N, 7.754°W, 21 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 2♂, 1♀ (sequenced); • Mértola, Ribeira de Carreiras, São Miguel do Pinheiro, 37.552°N, 7.85°W, 21 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♂, 2♀ (sequenced); • Moinho de Alferes 1, 37.502°N, 7.69°W, 19 May 2023, leg. Ferreira, Benitez-Bosco, Ekrem, Stur & Turaccio, 1♀ (sequenced).
Remarks.
The sequenced specimens from Portugal form a unique BIN (BOLD:AFO3503) with the nearest neighboring BIN being BOLD:ACS0765, which includes 34 specimens from the Netherlands, Norway, Poland and North Macedonia partially assigned to A.globator or A.tubulator. The p-distance between these two BINs was estimated at 5.45%.
Distribution.
Palaearctic. In Portugal previously reported from Alentejo (Lundblad 1956).
. Arrenurus (Megaluracarus) cf.zachariae
Koenike, 1886
B52E803F-A56C-5526-953E-5932167C291C
Material examined.
Portugal, Bragança • Vinhais, Gasparona, 41.85°N, 7.013°W, 693 m a.s.l., 6 Jul. 2023, leg. Ferreira & Padilha, 1 deutonymph (sequenced).
Remarks.
One deutonymph from Portugal forms a unique cluster BOLD:AFU0319, with a p-distance to the closest neighboring BIN, BOLD:ADF7386, which includes mostly specimens assigned to A.zachariae, estimated at 2.56%.
Distribution.
Europe. New record for Portugal.
Discussion
Our study provides the first DNA barcode reference library for Portuguese water mites. Our findings confirm the presence of 29 of the 101 previously recorded species; however, it also added 36 new species for the water mite fauna of Portugal, two of which are described as new to science. We found that 47.4% of the Portuguese water mites having sequences collected in Portugal were represented in BOLD. Intraspecific distances for some species were high indicating the incongruence between morphology and DNA barcodes, and therefore the need for further taxonomic revision of these species to identify possible undescribed cryptic diversity. The identity of some species in the absence of available males, for example Foreliavariegator and F.longipalpis, remains questionable and needs further morphological evaluation.
Our research provided 75 BINs, 38 of which were new to BOLD. Seven species in our study were represented by multiple BINs: Mideopsisroztoczensis (five BINS), Hygrobateslongiporus (three BINS), Lebertiainsignis, Atractidesinflatus, Neumaniauncinata, Forelialongipalpis, and Pionacarnea, each with two BINs. Furthermore, examining molecular diversity at a continental scale, we found more such cases. For example two BINs were detected within Limnocharesaquatica (BOLD:AFV0270, BOLD:ACS0438), a species considered to be widely distributed in the Holarctic. Three BINs were also detected within Neumaniauncinata (BOLD:AFV0253, BOLD:AFV0269, BOLD:AER9267), a species widely distributed in the Mediterranean region, and N.imitata (BOLD:AFV0268, BOLD:ADF7924, BOLD:AED4073), a species that is less common, but with a wide distribution from Sweden to Portugal. A high genetic divergence (16.7% p-distance) was found between the specimen of Nautarachnacrassa from Portugal and a specimen of this species collected in Norway. Our study, based on the available data in BOLD suggests also that Pionopsislutescens, a species common in many types of standing water in the Holarctic, includes at least four BINs in Europe. In this study we retrieved one, BOLD:AFV3897, which includes only specimens from Portugal. Two others are present in the central and northern parts of Europe, BOLD:ACS0644 in the Netherlands and Norway and BOLD:ACR9955, so far found only in the Netherlands. Finally, the fourth BIN, BOLD:AET1848, is known from Montenegro. Further research is needed to understand the taxonomic implications of these genetic divergences.
Our results show the usefulness of using BINs to detect possible cryptic species and to investigate the distribution patterns of water mite species whose presence in certain geographical areas would be difficult to confirm without molecular evidence. In this study we confirmed the presence of Hygrobatesfluviatilis in Portugal, which represent the southernmost record of this widely distributed species. We also added the first record of H.balcanicus. The latter rhitrobiontic species, so far recorded from Serbia and Bulgaria, was probably previously confused with H.fluviatilis, but new findings from Portugal indicate that this species is widespread in the Mediterranean region.
Furthermore, our results demonstrate the efficiency of using DNA barcoding to identify preadult stages, particularly deutonymphs, whose identification to the species-level is often not possible without accompanying adult stages when using morphology. In this study, two individuals of Arrenurus deutonymhs were assigned to A.tricuspidator and A.zachariae, respectively, based on matching their DNA barcodes with the BINs of these species available in BOLD. Based on morphology alone, identification of these preadult stages would be difficult, if not impossible.
In summary, this study exemplifies the high molecular diversity of Portuguese water mites as well as the need to intensify international cooperation in the generation and curation of DNA barcode reference libraries.
Supplementary Material
Acknowledgements
Biodiversity Genomics Europe is funded by Horizon Europe under the Biodiversity, Circular Economy and Environment call (REA.B.3); co-funded by the Swiss State Secretariat for Education, Research and Innovation (SERI) under contract number 22.00173; and by the UK Research and Innovation under the Department for Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme.
The authors would like to acknowledge to Estação Biológica de Mértola, to Mértola Municipality, to Lousada Municipality, to CISE and to Seia Municipality and to Faculty of Sciences – University of Porto for the logistic support during the fieldwork and to Thomas S. Ekrem, Piotr Gadawski, Roman Hodunko, José Conde, Tiago Correia, and Antonio and Luis Guilherme Sousa for the great company during fieldwork. We thank two anonymous reviewers, whose constructive comments greatly improved this work.
Citation
Pešić V, Zawal A, Ferreira S, Benitez-Bosco L, Cruz-Oliveira A, Girão D, Padilha A, Turaccio P, Rossini S, Ballini L, Staffoni G, Fratini S, Ciofi C, Iannucci A, Ekrem T, Stur E (2024) DNA barcode library of Portuguese water mites, with the descriptions of two new species (Acari, Hydrachnidia). ZooKeys 1217: 119–171. https://doi.org/10.3897/zookeys.1217.131730
Additional information
Conflict of interest
The authors have declared that no competing interests exist.
Ethical statement
No ethical statement was reported.
Funding
LBB was funded by the project TROPIBIO NORTE-01-0145-FEDER-000046, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by a PhD fellowship (UI/BD/154733/2023) attributed by the Portuguese Foundation of Science and Technology (FCT). PT benefited from an Erasmus+ Studies Program grant (2022-1-IT02_KA131-HED-000057320). SF was funded by the FCT through the program ‘Stimulus of Scientific Employment, Individual Support—3rd Edition’ (https://doi.org/10.54499/2020.03526.CEECIND/CP1601/CP1649/CT0007).
Author contributions
All authors have contributed equally.
Author ORCIDs
Vladimir Pešić https://orcid.org/0000-0002-9724-345X
Andrzej Zawal https://orcid.org/0000-0002-5838-6060
Sónia Ferreira https://orcid.org/0000-0002-6884-3966
Laura Benitez-Bosco https://orcid.org/0000-0002-8863-8353
Dinis Girão https://orcid.org/0009-0008-4842-0721
Paolo Turaccio https://orcid.org/0009-0000-8627-6718
Samantha Rossini https://orcid.org/0009-0009-6881-2527
Giorgia Staffoni https://orcid.org/0009-0003-5916-1757
Torbjørn Ekrem https://orcid.org/0000-0003-3469-9211
Data availability
All of the data that support the findings of this study are available in the main text or Supplementary Information.
Supplementary materials
BOLD TaxonID Tree
This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.
Vladimir Pešić, Andrzej Zawal, Sónia Ferreira, Laura Benitez-Bosco, Ana Cruz-Oliveira, Dinis Girão, Adriana Padilha, Paolo Turaccio, Samantha Rossini, Lorenzo Ballini, Giorgia Staffoni, Sara Fratini, Claudio Ciofi, Alessio Iannucci, Torbjørn Ekrem, Elisabeth Stur
Data type
Explanation note
Compact Neighbor-Joining tree of all analyzed water mite species based on Kimura 2-parameter distances. BINs are based on the barcode analysis from 16 May 2024. The analyses involved all 307 COI nucleotide sequences.
List of Torrenticola specimens
This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.
Vladimir Pešić, Andrzej Zawal, Sónia Ferreira, Laura Benitez-Bosco, Ana Cruz-Oliveira, Dinis Girão, Adriana Padilha, Paolo Turaccio, Samantha Rossini, Lorenzo Ballini, Giorgia Staffoni, Sara Fratini, Claudio Ciofi, Alessio Iannucci, Torbjørn Ekrem, Elisabeth Stur
Data type
doc
Explanation note
List of Torrenticola specimens used for building the Neighbor-Joining (NJ) tree (Fig. 6). Details on the specimens from Portugal are given in Table 1. GenBank numbers are indicated by *. BINs are based on the barcode analysis from 16 May 2024. Country codes (alpha-2 code): AT = Austria, BA = Bosnia and Herzegovina, CR = Croatia, DE = Germany, ES = Spain, FR = France, GR = Greece, IR = Iran, IT = Italy, NO = Norway, NL = the Netherlands, ME = Montenegro, MK = North Macedonia, PT = Portugal, RS = Serbia, TR = Turkey.
References
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
BOLD TaxonID Tree
This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.
Vladimir Pešić, Andrzej Zawal, Sónia Ferreira, Laura Benitez-Bosco, Ana Cruz-Oliveira, Dinis Girão, Adriana Padilha, Paolo Turaccio, Samantha Rossini, Lorenzo Ballini, Giorgia Staffoni, Sara Fratini, Claudio Ciofi, Alessio Iannucci, Torbjørn Ekrem, Elisabeth Stur
Data type
Explanation note
Compact Neighbor-Joining tree of all analyzed water mite species based on Kimura 2-parameter distances. BINs are based on the barcode analysis from 16 May 2024. The analyses involved all 307 COI nucleotide sequences.
List of Torrenticola specimens
This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) 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.
Vladimir Pešić, Andrzej Zawal, Sónia Ferreira, Laura Benitez-Bosco, Ana Cruz-Oliveira, Dinis Girão, Adriana Padilha, Paolo Turaccio, Samantha Rossini, Lorenzo Ballini, Giorgia Staffoni, Sara Fratini, Claudio Ciofi, Alessio Iannucci, Torbjørn Ekrem, Elisabeth Stur
Data type
doc
Explanation note
List of Torrenticola specimens used for building the Neighbor-Joining (NJ) tree (Fig. 6). Details on the specimens from Portugal are given in Table 1. GenBank numbers are indicated by *. BINs are based on the barcode analysis from 16 May 2024. Country codes (alpha-2 code): AT = Austria, BA = Bosnia and Herzegovina, CR = Croatia, DE = Germany, ES = Spain, FR = France, GR = Greece, IR = Iran, IT = Italy, NO = Norway, NL = the Netherlands, ME = Montenegro, MK = North Macedonia, PT = Portugal, RS = Serbia, TR = Turkey.
Data Availability Statement
All of the data that support the findings of this study are available in the main text or Supplementary Information.