First Insight Into Groundwater Copepods of the Polish Lowland

Home , Cyclopidae, Cyclopoida, Cyclops (copepod), Cyclops strenuus, Diacyclops, Folwarki Wielkie

water

Article First Insight into Groundwater Copepods of the Polish Lowland

Maciej Karpowicz 1,* , Sabina Smolska 1, Magdalena Swisłocka´ 2 and Joanna Moroz 3

1 Department of Hydrobiology, Faculty of Biology, University of Białystok, Ciołkowskiego 1J, 15-245 Białystok, Poland; [email protected] 2 Department of Zoology and Genetics, Faculty of Biology, University of Białystok, Ciołkowskiego 1J, 15-245 Białystok, Poland; [email protected] 3 Doctoral School of Exact and Natural Sciences, University of Białystok, Ciołkowskiego 1K, 15-245 Białystok, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-662-154-646

Abstract: Our results are the first insight into groundwater copepods of the Polish Lowland. The sampling was conducted in 28 wells in north-eastern Poland, and Copepoda were present in 16 wells. We have identified six Copepoda species and one Cladocera. We have classified four species as stygophiles—Eucyclops serrulatus, Diacyclops bisetosus, Diacyclops crassicaudis, and Cyclops furcifer. These species were frequently found in studied wells of different regions of north-eastern Poland, often in high numbers, and females with egg sacs were observed. We present a detailed morphological description of these species, together with molecular characteristics based on mitochondrial DNA markers (COI gene) for E. serrulatus, D. bisetosus, and D. crassicaudis, and 12S ribosomal RNA for C. furcifer. We also present the development of abnormal structures in one specimen of D. crassicaudis, where the upper part of furcal rami was fused to form a single plate. Keywords: subterranean habitats; wells; Cyclopoida; Eucyclops serrulatus; Diacyclops bisetosus; Diacy-

Citation: Karpowicz, M.; Smolska, S.; clops crassicaudis; Cyclops furcifer; morphology; COI gene; abnormal structures Swisłocka,´ M.; Moroz, J. First Insight into Groundwater Copepods of the Polish Lowland. Water 2021, 13, 2086. https://doi.org/10.3390/w13152086 1. Introduction Subterranean habitats fulﬁll the requirements of experimental model systems to Academic Editors: Agnieszka address general questions in ecology and evolution [1]. Groundwaters are populated Pociecha, Henri J. Dumont and by a specialized fauna called stygobionts and by accidental taxa (stygoxenes) temporar- Joanna Czerwik-Marcinkowska ily imported from the surface. Stygophiles are intermediate between stygobionts and stygoxenes—they spend part of their life below ground and may even be more common Received: 11 July 2021 in groundwater than in surface waters [2]. It is estimated that about 40% of the European Accepted: 28 July 2021 Published: 30 July 2021 crustacean fauna is represented by stygobiontic species, among which dominate copepods with about 1000 species and subspecies known from continental groundwater [3]. Most of

Publisher’s Note: MDPI stays neutral them belong to the Harpacticoida (about 600 species) and Cyclopoida (about 300 species). with regard to jurisdictional claims in Moreover, almost half of the newly described copepods are from groundwater habitats [3]. published maps and institutional afﬁl- There are some regions with well-recognized groundwater copepods, and there are iations. regions for which there is no information. Generally, groundwater fauna is quite well explored in mountain regions of southern Europe (Alps, Apennines, Pyrenees, Carpathian, and Balkan Mountains), and these regions are considered biodiversity hotspots of stygobiontic Harpacticoida [4]. There is some information about groundwater copepods from the western part of the European lowlands (Netherland, Germany, Denmark), contrary to the Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. central part of the European lowlands. The groundwater of central and northern Poland is This article is an open access article quite a forgotten habitat, contrary to the mountain region of Poland. A preliminary study distributed under the terms and of groundwater copepods from wells in Carpathian ﬂysch (Poland) revealed the presence conditions of the Creative Commons of four species [5], while the study of different subterranean habitats of southern Poland by Attribution (CC BY) license (https:// Kur (2020) indicated the presence of 22 Cyclopoida species, but only one stygobiont [6]. creativecommons.org/licenses/by/ The preliminary research in the European lowlands revealed two Harpacticoida species 4.0/). new for the Polish fauna in the springs and wells of north-eastern Poland, Elaphoidella

Water 2021, 13, 2086. https://doi.org/10.3390/w13152086 https://www.mdpi.com/journal/water Water 2021, 13, 2086 2 of 19

presence of four species [5], while the study of different subterranean habitats of southern Water 2021, 13, 2086 Poland by Kur (2020) indicated the presence of 22 Cyclopoida species, but only one stygo-2 of 17 biont [6]. The preliminary research in the European lowlands revealed two Harpacticoida species new for the Polish fauna in the springs and wells of north-eastern Poland, Elaphoi- della elaphoides (Chappuis, 1923) and Bryocamptus (Rheocamptus) spinulosus (Borutzky, elaphoides (Chappuis, 1923) and Bryocamptus (Rheocamptus) spinulosus (Borutzky, 1934) [7]. 1934) [7]. One problem is that Harpacticoida is the least known group of Copepoda in One problem is that Harpacticoida is the least known group of Copepoda in freshwater habitatsfreshwater in Polandhabitats because in Poland the because last detailed the last taxonomic detailed taxonomic studies of studies Harpacticoida of Harpacticoida in Polish inlandin Polish waters inland were waters carried were out carried about out 100 about years ago100 years [7]. ago [7]. TheThe mainmain aimaim ofof thisthis researchresearch isis toto givegive newnew insightsinsights intointo groundwatergroundwater copepodscopepods (Cyclopoida,(Cyclopoida, Harpacticoida,Harpacticoida, Calanoida)Calanoida) ofof thethe PolishPolish lowlands.lowlands. We presentpresent aa detaileddetailed morphologicalmorphological descriptiondescription ofof eacheach speciesspecies togethertogether withwith molecularmolecular characteristicscharacteristics (COI(COI genegene oror 12S12S RNA).RNA). Currently,Currently, internationalinternational databasesdatabases (GenBank,(GenBank, BOLDBOLD System)System) containcontain singlesingle barcodesbarcodes forfor eacheach genusgenus ofof copepodscopepods thatthat areare foundfound inin thethe groundwater.groundwater. TheThe otherother problemproblem is that most most of of the the available available barcodes barcodes did did not not have have sufficient sufficient morphological morphological de- descriptionscription and, and, thus, thus, proper proper taxonomic taxonomic identi identification,fication, due due to to the well-knownwell-known taxonomytaxonomy crisiscrisis [[8].8]. Therefore, Therefore, presented presented in in this this article article are are detailed detailed morphological morphological descriptions descriptions of ofeach each species species together together with with genetics genetics analyzes analyzes of mtDNA of mtDNA molecular molecular markers, markers, which whichwill be willthe cornerstone be the cornerstone for future for biodiversity future biodiversity analyzes analyzes based on based eDNA on metabarcoding eDNA metabarcoding because becausethis method this needs method reference needs reference databases databases with nucleotide with nucleotide sequences sequences for accurately for accurately identified identifiedspecies [9]. species The other [9]. aim The of other this aimstudy of is this to determine study is to which determine species which found species in the found ground- in thewater groundwater of Polish lowlands of Polish could lowlands be considered could be considered as stygobiont as stygobiontor stygophiles. or stygophiles.

2.2. Materials andand MethodsMethods TheThe sampling was was conducted conducted in in 28 28 wells wells in north-eastern in north-eastern Poland Poland in two in tworegions regions (Fig- (Figureure 1), 1which), which are are a representation a representation of of the the glaciated glaciated part part of of the the Central Central European lowlands.lowlands. WellsWells 1–91–9 werewere sampled on the the 2 2 October October 2019, 2019, while while wells wells 10–28 10–28 were were sampled sampled on on the the 15 15July July 2020. 2020. Wells Wells 1–5 1–5are located are located in the in Biebrza the Biebrza River Rivervalley valley in the inarea the ofarea Vistulian of Vistulian Glacia- Glaciation.tion. Wells Wells6–28 are 6–28 located are located in rural in rural areas areas around around Bialystok Bialystok city city (Figure (Figure 1) 1on) on the the area area of ofWarta Warta Glaciation. Glaciation. Most Most of the of the studied studied dug dug wells wells were were in good in good conditions conditions but butmainly mainly un- unusedused presently. presently. All All of the of thewells wells have have a cover a cover that thatisolates isolates them, them, and examples and examples of the of stud- the studiedied wells wells are shown are shown in Figure in Figure 2. 2.

FigureFigure 1. MapMap of of the the studied studied 28 28 wells wells in innorth-east north-easternern Poland. Poland. Numbers Numbers 1–28 1–28 are according are according to to TableTable1 .1.

Table 1. The list of studied wells with GPS locations, the total number of copepods, and the Copepoda species (Es—Eucyclops serrulatus; Db—Diacyclops bisetosus; Dc—Diacyclops crassicaudis; Eg—Eudiaptomus gracilis; Tc—Thermocyclops crassus).

Longitude Sample Total No. of Total No. of Copepoda No. Village Latitude (N) (E) Volume [l] Adult Copepodites Species

1 Cisów 53◦45007.800 23◦05025.000 100 575 2177 Es 2 Cisów 53◦44052.700 23◦05040.600 100 0 0 - 3 Wolne 53◦42037.400 23◦09018.900 100 0 8 Es, Db Water 2021, 13, 2086 3 of 17

Table 1. Cont.

Longitude Sample Total No. of Total No. of Copepoda No. Village Latitude (N) (E) Volume [l] Adult Copepodites Species 4 Wolne 53◦42043.800 23◦09015.900 100 0 4 Es 5 Wolne 53◦42037.100 23◦09029.900 100 0 3 Es 6 Pasynki 53◦01036.400 23◦16008.800 100 2 26 Db 7 Pasynki 53◦01037.200 23◦16009.800 100 0 6 Es, Db 8 Pasynki 53◦01032.000 23◦16009.000 100 13 63 Es, Db 9 Pasynki 53◦01033.200 23◦16008.400 100 14 98 Es 10 Olmonty 53◦502.2200 23◦9050.600 70 0 0 - 11 Olmonty 53◦502.4200 23◦10018.8300 50 0 0 - 12 Halickie 53◦3054.5300 23◦1400.0900 80 0 0 - 13 Nowosady 53◦1015.0300 23◦13016.3800 85 0 0 - 14 Nowosady 53◦0016.1100 23◦13021.5400 80 0 0 - 15 Zuki˙ 53◦0041.8200 23◦14054.1900 60 0 0 - 16 Zuki˙ 53◦0049.1900 23◦14058.2400 55 2 1 Dc 17 Zagruszany 53◦2013.5800 23◦17014.3300 60 12 66 Db, Es Folwarki 18 53◦2012.8000 23◦23021.4400 80 5 13 Dc Małe Folwarki 19 53◦1044.6100 23◦24022.4100 55 0 0 - Wielkie Folwarki 20 53◦1033.2700 23◦24040.3700 60 29 2 Dc Wielkie Folwarki 21 53◦106.9700 23◦25034.4200 60 68 331 Dc, Cf Tylwickie 22 Topolany 53◦109.4600 23◦3000.2200 55 34 16 Dc, Db, Cf 23 Topolany 53◦1011.4900 23◦30017.9400 50 7 4 Db, Cf 24 Karakule 53◦10021.2900 23◦15034.5400 60 0 0 - 25 Karakule 53◦10021.8900 23◦15038.4800 60 0 0 - 26 Karakule 53◦10057.6500 23◦15047.7400 60 0 0 - 27 Karakule 53◦10033.6100 23◦15042.8200 60 0 0 - 28 Białystok city 53◦06028.2600 23◦12001.0200 50 0 2 Eg, Tc

Samples were collected using a 10 L bucket and then 50–100 L of water each time (Table1) were filtered through a 50 µm plankton net and fixed with 96% alcohol. Copepods (Cyclopoida, Harpacticoida, Calanoida) were identified to species and all individuals in the samples were enumerated. Each species was dissected and the most important morphological features (thoracic legs; caudal rami, antennule, coxopodite of P4, etc.) are presented in Figures3–7. We selected few individuals from each species for the genetic analysis. Before DNA extraction, each specimen was photographed using micro imaging software for documentation of its body and distinguishing features. Photography documentation of each species together with its COI or 12S rRNA sequence was uploaded to the BOLD System. We uploaded our sequences also to GenBank. Reference materials are deposited in the Department of Hydrobiology, University of Bialystok as alcohol samples. Copepoda individuals collected from different wells were stored in sterile plastic test tubes with 70% ethanol. The genomic DNA from individuals belonging to the four different species C. furcifer, D. bisetous, D. crassicaudis, and E. serrulatus were extracted with a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Amplification of the fragment of the mitochondrial COI gene was performed for E. serrulatus (2 individuals from Zagruszany well), D. bisetosus (8 individuals from Zagruszany well), and D. crassicaudis (3 individuals from Folwarki Wielkie, 2 from Topolany, 1 from Folwarki Małe, and 1 from Zuki˙ wells) using primers dgLCO1490 and dgHCO2198 [10], while for C. furcifer (7 individuals from Folwarki Tylwickie and 1 from Topolany wells) a fragment of 12S ribosomal RNA gene was amplified using primers 12SH13845 and 12SL13337 [11]. PCRs for each primer pair were carried out in 5 µL volumes, and the reaction mixtures consisted of 2 µL of extracted Water 2021, 13, 2086 4 of 17

DNA as a template, 1.7 µL of Qiagen Multiplex PCR Master Mix (1x), 0.3 µL mix of primers, and 1 µL of Qiagen nuclease-free water. The thermocycling parameters were as follows: initial denaturation step at 95 ◦C for 15 min and 42 cycles with denaturation at 94 ◦C for 30 s, annealing at 48 ◦C (for COI gene) or at 57 ◦C (for 12S rRNA gene) for 90 s, extension at 72 ◦C for 60 s, and final elongation for 30 min at 60 ◦C. PCRs were performed in a Labcycler SensoQuest thermal cycler. Amplified PCR products were purified with EPPiC Fast (A&A Biotechnology) in an enzymatic reaction following the manufacturer’s protocol. They were then processed for cycle sequencing PCR with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Unincorporated dideoxynucleotides were eliminated from the sequencing reaction using the ExTerminator Water 2021, 13, 2086 3 of 19 Kit (A&A Biotechnology). We carried out the detection of sequencing reaction products on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems).

FigureFigure 2. AnAn example example of ofthe thestudied studied wells. wells. A—Karakule;A—Karakule; B and BC—andŻuki;C— DZuki;—Zagruszany;˙ D—Zagruszany; E— EOlmonty;—Olmonty; F—Topolany.F—Topolany.

SequencingSamples were results collected of the using mtDNA a 10 COI L bucket gene and and 12S then rRNA 50–100 were Laligned of water and each revised time manually(Table 1) were using filtered BioEdit through [12]. Photography a 50 µm plankton documentation net and fixed of each with species 96% alcohol. together Cope- with itspods fragments (Cyclopoida, of the Harpacticoida, COI gene (E. serrulatus Calanoida), D. werebisetosus identified, D. crassicaudis to species) or and 12S all rRNA individu- gene sequencesals in the samples (C. furcifer were) were enumerated. uploaded toEach the sp BOLDecies System.was dissected We also and submitted the most to important GenBank obtainedmorphological in our features study sequences (thoracic legs; of the caudal mtDNA rami, COI antennule, and 12S coxopodite rRNA genes. of P4, To testetc.) theare phylogeneticpresented in relationshipsFigures 3–7. We among selected our newly few in obtaineddividuals COI from and each 12S species rRNA haplotypes for the genetic and sequencesanalysis. Before downloaded DNA extraction, from GenBank each (Tablespecimen S1), was we constructed photographed phylogenetic using micro trees imaging using asoftware maximum for likelihooddocumentation (ML) algorithmof its body in and Mega distinguishing v6.06 [13] using features. 1000 bootstrapPhotography replicates. docu- Thementation GTR+I+G of each model species of substitution together with was its selected COI or as 12S the rRNA best ﬁtting sequence model was by uploaded the AIC test to (Akaikethe BOLD Information System. We Criterion) uploaded with our the sequences jModelTest also [ 14to] GenBank. for the ML Reference trees. materials are deposited in the Department of Hydrobiology, University of Bialystok as alcohol samples. Copepoda individuals collected from different wells were stored in sterile plastic test tubes with 70% ethanol. The genomic DNA from individuals belonging to the four different species C. furcifer, D. bisetous, D. crassicaudis, and E. serrulatus were extracted with a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). Amplification of the fragment of the mitochondrial COI gene was performed for E. serrulatus (2 individuals from Zagruszany well), D. bisetosus (8 individuals from Zagruszany well), and D. crassicaudis (3 individuals from Folwarki Wielkie, 2 from Topolany, 1 from Folwarki Małe, and 1 from Żuki wells) using primers dgLCO1490 and dgHCO2198 [10], while for C. furcifer (7 individuals from Folwarki Tylwickie and 1 from Topolany wells) a fragment of 12S ribosomal RNA gene was amplified using primers 12SH13845 and 12SL13337 [11]. PCRs for each primer pair were carried out in 5 µL volumes, and the reaction mixtures consisted of 2 µL of extracted DNA as a template, 1.7 µL of Qiagen Multiplex PCR Master Mix (1x), 0.3 µL mix of primers, and 1 µL of Qiagen nuclease-free water. The thermocycling parameters were as follows: initial denaturation step at 95 °C for 15 min and 42 cycles with denaturation at 94 °C for 30 s, annealing at 48 °C (for COI gene) or at 57 °C (for 12S rRNA gene) for

Water 2021, 13, 2086 5 of 17

3. Results The groundwater copepods were found in 16 of the 28 studied wells in north-eastern Poland (Table1). The highest abundance was recorded in Cis ów village, with 27.5 individuals per liter (Table1). The average abundance of groundwater copepods was 2.5 ± 6.9 ind. L−1. We identified six Copepoda (five Cyclopoida, one Calanoida) and one Cladocera species in the groundwater of north-eastern Poland. Most studied wells were dominated by one species, nevertheless, two species were found in six wells, and three species were found in two wells (Table1). The most common species were: Eucyclops serrulatus (eight wells), Diacyclops bisetosus (seven wells), Diacyclops crassicaudis (five wells), and Cyclops furcifer (three wells). We observed abnormal morphology of the caudal rami in D. crassicaudis. Detailed descriptions of the above-mentioned species are presented below, as well as a description of abnormal structures in a single individual of D. crassicaudis. Other species were single records and represented stygoxenes, which were temporarily imported from the surface. Among stygoxenes, one individual of Eudiaptomus gracilis and Thermocyclops crassus were found in the well in Białystok city (district of Dojlidy) (Figure S1). The analyzed individual of E. gracilis was a male with a flattened body (Figure S1A); the fourth leg (P4) and fifth leg (P5) are shown in Figure S1B,C. Pictures of the Thermocyclops crassus body, furcal branches, and exopodite P4 are presented in the Figure S1D–F. Moreover, the cladoceran Chydorus sphaericus was found in the Zagruszany well (Figure S1G–I) in the number of eleven individuals. Cyclops furcifer Claus, 1857 Cyclops furcifer was found in high abundance in the Folwarki Tylwickie village, and in low numbers in two wells in the Topolany village (Table1). Females with egg sacs were also observed. The body size of the examined adult female was 1578 µm. The ends of the fourth and fifth thoracomer (Th4 and Th5) extended outwards forming ‘wings’ (Figure3A) . The furcal branches were long and slender with a proportion length to width of 6:1 (Figure3B) . The inner margin of furcal branches had hair setae. Terminal internal seta was shorter than the furcal branches (about 0.6) (Figure3B). Terminal external seta was shorter than the terminal inner seta (Figure3B). Antennule 17-segmented reaching the middle of Th2 (Figure3C). The spine formula of swimming legs was 3.4.3.3 (Figure3E–H). The last exopodal article was bearing five setae in all swimming legs (Figure3E–H). The last endopodal article of P4 was almost twice as long as wide, the inner apical spine was longer than the article (Figure3H). The coupler of P4 had two/three lines of hair-like spinules (Figure3I). The coupler of P3 was also pilose caudally, which distinguishes it from all the other Cyclops occurring in Poland, except for C. insignis, but this species had 14-segmented antennule and very thick P4 coxopodite seta. The ornamentation of P4 coxopodite is shown in Figure3J . The P5 had two segments, and the medial spine of second segment was long, reaching far beyond the end of the segment (Figure3D). At the end of the second segment of P5, there were rows of small spinules at insertion of medial and apical seta (Figure3D). The spinules were present also on the first segment of P5 at insertion of the lateral seta. The molecular analysis of a 369-bp of a fragment of the 12S ribosomal RNA gene yielded among studied individuals of C. furcifer from the Folwarki Tylwickie (N = 7) and Topolany (N = 1) wells one new haplotype (GenBank accession no. MZ502237; BOLD sample ID: UwB_4_Cf4). The maximum likelihood phylogenetic reconstructions produced a strong topology (Figure8A). The ML tree revealed that our 12S rRNA newly discovered haplotype of C. furcifer was grouped with haplotypes described for C. scutifer (MK329326 and MK329328) by Hołyńskaand Wyngaard (2019) [15]. Water 2021, 13, 2086 6 of 17 Water 2021, 13, 2086 7 of 19

Figure 3.FigureCyclops 3. Cyclops furcifer :furciferA—habitus;: A—habitus;B—furcal B—furcal branches; branches;C—cephalothorax C—cephalothorax with with antennule antennule (A1) (A1) andand antennaantenna (A2); (A2); DD——P5; E—P1; FP5;—P2; E—P1;G—P3; F—P2;H—P4; G—P3;I—coxopodite H—P4; I—coxopodite of P4; J —couplerof P4; J—coupler of P4. of P4.

Eucyclops serrulatus s. stricto (Fischer, 1851) Eucyclops serrulatus was found in eight wells in villages of Cisów, Wolne, Pasynki, and Zagruszany (Table1). The high abundance of this species was found in well no. 1 in the Cisów village (27.5 ind. L−1) and Pasynki village (Table1). The adult specimens accounted for about 15–25% of all copepods (Table1). The body size of the analyzed adult female was

1240 µm. The furcal branches in examined female were slender, six times as long as wide (Figure4B), but we also observed adult females with shorter furcal branches. The outer Water 2021, 13, 2086 7 of 17

margins of the furcal branches serrulated (Figure4B). The ratio of the innermost caudal seta to the outermost caudal seta was 1.4 (Figure4B). Antennule (A1) were 12-segmented (Figure4C), long, and reaching almost the genital segment (Figure4A), which is quite unusual for this species. The last three segments of A1 had a smooth hyaline membrane (Figure4C). P1–4 had 3-segmented exopod and endopod (Figure4E–G,I). The first and second segments of exopodites of the swimming legs had one seta each, while the third segment had five setae. The spine formula was 3.4.4.3., respectively (Figure4E–G,I). The last endopodal segment of P4 had two long apical spines, but we found a different arrangement of these spines (Figure4J,L). The ornamentation of P4 coxopodite is shown in Figure4K. The coxal spine of P4 had hair-like setules, but on the outer margin, there was a gap in the setulation (Figure4M,N). P5 was one segmented, with a large knife-like inner spine and two setae of a similar length to the spine (Figure4H). The analysis of an mtDNA COI gene fragment of 631-bp long yielded among two individuals from the Zagruszany well two new haplotypes of E. serrulatus: haplotype H1 (GenBank accession no. MZ503631; BOLD sample ID: UwB_7_Es7) and haplotype H2 (GenBank accession no. MZ503632; BOLD sample ID: UwB_8_Es8), as defined by one polymorphic site being transition. The maximum likelihood phylogenetic reconstructions produced a strong topology (Figure8B). The ML tree revealed that our two mtDNA COI haplotypes was grouped with haplotypes belonging to the E. serrulatus described for Ukraine, Russia (GenBank accession no. KC627312 and KC627313; unpublished). Diacyclops bisetosus (Rehberg, 1880) Diacyclops bisetosus was found in low numbers in seven wells in the villages of Wolne, Pasynki, Zagruszany, Topolany (Table1). The body size of the analyzed adult female was 1074 µm. Antennule were 17-segmented (Figure5B), relatively short reaching the end of the cephalothorax (Figure5A). Furcal branches were four times as long as wide in the female measured (Figure5D), however, we also observed longer furcal branches with a proportion of 7:1 in this species. The innermost and outermost caudal setae were subequal. The swimming legs were three-segmented with a spine formula of 2.3.3.3 (Figure5E–H). The last exopodal article was bearing four setae in all swimming legs (Figure5E–H). One of the distinguishing features was the length proportion of the apical spines on the third endopodal segment of P4, where the inner spine was longer than the outer one (Figure5H). Coxopodite of P4 had ornamentation (Figure5I). P5 consists of two segments, segment two had a long and slim inner spine (Figure5C). The analysis of an mtDNA COI gene fragment of 578-bp long yielded among eight individuals from the Zagruszany well two new haplotypes of D. bisetosus: haplotype H1 (GenBank accession no. MZ503633; BOLD sample ID: UwB_5_Db5) and haplotype H2 (GenBank accession no. MZ503634; BOLD sample ID: UwB_6_Db6), as defined by one polymorphic site being in transition. Haplotype H1 was detected in 5/8 (62.5%) of the studied individuals, while haplotype H2 was in 3/8 (37.5%). The maximum likelihood phylogenetic reconstructions produced a strong topology (Figure8C). The ML tree revealed that our two mtDNA COI haplotypes of D. bisetosus created a separated branch among different species of Diacyclops genus, which also grouped haplotype of D. bicuspidatus (GenBank accession no. KR048968) described by Baek et al. (2016) [16] and also our newly described from Poland haplotypes of D. crassicaudis (GenBank accession no. MZ503635–MZ503637). Diacyclops crassicaudis (Sars G.O., 1863) Diacyclops crassicaudis was found in four wells in the villages of Zuki,˙ Folwarki Wielkie, Folwarki Tylwickie, Topolany (Table1). The species occurred in high abundance in well no. 21 in Folwarki Tylwickie (6.6 ind. L−1), where the adult specimens accounted for about 20%. In Folwarki Wielkie and Topolany, adult specimens dominated over copepodites (Table1), and females with egg sacs were present. The body length was 1100 µm in the adult female measured, and the two egg sacs had nine eggs each (Figure6A). Thoracic segments 2, 3, and 4 were rounded and bulging (Figure6A). Antennule were 12-segmented Water 2021, 13, 2086 8 of 17

(Figure6B), relatively short and reaching the end of the cephalothorax (Figure6A). The caudal rami were ﬁve times as long as wide, with a wide gap between them, especially at the posterior end of the rami (Figure6A). The spine formula of swimming legs was 2.3.3.3 Water 2021, 13, 2086 8 of 19 (Figure6C–F). P5 consisted of two segments, with quite a large and massive inner spine (Figure6G).

Figure 4. FigureEucyclops 4. Eucyclops serrulatus serrulatus: A—body;: A—body;B—furcal B—furcal branches; branches; C—antennule; D—hyalineD—hyaline membrane membrane on the on10th the and 10th 11th and 11th segmentssegments of the antennule; of the antennule;E—P1; EF—P1;—P2; F—P2;G—P3; G—P3;H—P5; H—P5;I—P4; I—P4;J ,JL, L—last—last segment of of P4 P4 endopodite; endopodite; K—P4K—P4 coxopodite; coxopodite; M, N—coxalM spine, N—coxal of P4. spine of P4.

Water 2021, 13, 2086 9 of 17

Water 2021, 13, 2086 11 of 19

FigureFigure 5. Diacyclops 5. Diacyclops bisetosus bisetosus: :A A—body;—body; BB—antennule;—antennule; C—P5;C—P5; D—furcalD—furcal branches; branches; E—P1;E —P1;F—P2;F G—P2;—P3; GH—P3;—P4; IH—cox-—P4; I—coxopoditeopodite of of P4; P4; J—J—mouthparts.mouthparts.

WaterWater 20212021,, 1313,, 20862086 1210 of of 1719

Figure 6. Diacyclops crassicaudis:: A—body; B—antennule and antenna; C—P1; D—P2; E—P3; F—P4; G—P5.—P5.

Water 2021, 13, 2086 11 of 17 Water 2021, 13, 2086 13 of 19

FigureFigure 7. 7. DiacyclopsDiacyclops crassicaudis crassicaudis withwith aberrant aberrant development development of of caudal caudal rami: rami: AA—body;—body; BB—aberrant—aberrant development development of of furcal furcal branches;branches; CC—P1;—P1; DD—P2;—P2; EE—P3;—P3; FF—P4;—P4; GG—P5;—P5; HH—antennule.—antennule.

The analysis of a 525-bp mtDNA COI gene fragment ampliﬁed from studied individuals from four different wells (three from Folwarki Wielkie, two from Topolany, one

Water 2021, 13, 2086 12 of 17

from Folwarki Małe and Zuki)˙ yielded three new haplotypes of D. crassicaudis: haplotype H1 (GenBank accession no. MZ503635; BOLD sample ID: UwB_1_Dc1), haplotype H2 (GenBank accession no. MZ503636; BOLD sample ID: UwB_2_Dc2), and haplotype H3 (GenBank accession no. MZ503637; BOLD sample ID: UwB_3_Dc3). All individuals from the Folwarki Wielkie well possessed haplotype H1 (3/7, 42.9%), which differed from haplotypes H2 and H3 by 12 polymorphic sites (11 transitions, 1 transversion). Haplotype H2 was found in individulas from Folwarki Małe i Zuki˙ (2/7, 28.6%), and differed from haplotype H3 discovered in individuals from Topolany (2/7, 28.6%) by six transitions. The maximum likelihood phylogenetic reconstructions produced a strong topology (Figure8C). The ML tree revealed that our three COI haplotypes of D. crassicaudis created a separated branch among different species of Diacyclops genus which also grouped haplotype of D. bicuspidatus (GenBank accession no. KR048968) described by Baek et al. (2016) [16] and Water 2021, 13, 2086 also our newly described from Poland haplotypes of D. bisetosus (GenBank accession14 of no. 19

MZ503633 and MZ503634).

Figure 8.8. MaximumMaximum likelihoodlikelihood topologytopology computedcomputed withwith thethe GTR+I+GGTR+I+G model of substitution evolution, representing thethe phylogeneticphylogenetic relationships among the sequencessequences of the 12S rRNA gene found in Cyclops furcifer ((A),), andand thethe COICOI genegene found in Eucyclops serrulatus (B), Diacyclops bisetosus and Diacyclops crassicaudis (C). Numbers listed at the nodes represent found in Eucyclops serrulatus (B), Diacyclops bisetosus and Diacyclops crassicaudis (C). Numbers listed at the nodes represent the percent support for the node from 1000 bootstrap replicates. The haplotypes found in this study are marked in blue the percent support for the node from 1000 bootstrap replicates. The haplotypes found in this study are marked in blue and bolded. and bolded. 4. DiscussionWe noticed developmental abnormality in the caudal rami morphology in one speci- menOur of D. study crassicaudis presents(Figure the first7) in data well on no. the 18 gr inoundwater the Folwarki copepods Małe village. of the Polish The rami lowland. were reducedThe middle in lengthEuropean and lowlands the parts were anterior an importan to the insertiont center ofof thepost-glacial lateral caudal dispersion setae of were the fusedfreshwater (Figure zooplankton7B). The outermost [17]. Furthermore, terminal caudal Quaternary seta was glaciations abnormally may long have (Figure played7A), an important role in the extinction of the surface species and increasing colonization and speciation in groundwater [3]. Currently, some phylogeographic studies have been published on the groundwater copepods in South and Eastern Europe [4], but none of them included the glaciated part of Central Europe. Generally, the groundwater fauna of the mountain region of Europe is much better known than in European lowlands, which seems to be a white spot on the map. The results of our study indicated the presence of six Copopoda species and one Cla- docera in the groundwater of north-eastern Poland. We classified three of these species as stygoxenes, and four as stygophiles. Among stygoxenes were Chydorus sphaericus, Eudiap- tomus gracilis, and Thermocyclops crassus. These species were present as a single record or in low numbers in groundwater, but they are very common in different types of surface water bodies in this region [18,19], north-western Poland [20], and Europe [21,22]. There- fore, these species seem to be accidental taxa temporarily imported from the surface. How- ever, E. gracilis has a flattened body which may indicate adaptation for an interstitial en-

Water 2021, 13, 2086 13 of 17

and other setae showed a slightly different size (Figure7B). Whereas other parts of the body were properly developed (Figure7A,G), and the legs had a spine and setae formula typical of the species (Figure7B–F).

4. Discussion Our study presents the first data on the groundwater copepods of the Polish lowland. The middle European lowlands were an important center of post-glacial dispersion of the freshwater zooplankton [17]. Furthermore, Quaternary glaciations may have played an important role in the extinction of the surface species and increasing colonization and speciation in groundwater [3]. Currently, some phylogeographic studies have been published on the groundwater copepods in South and Eastern Europe [4], but none of them included the glaciated part of Central Europe. Generally, the groundwater fauna of the mountain region of Europe is much better known than in European lowlands, which seems to be a white spot on the map. The results of our study indicated the presence of six Copopoda species and one Cladocera in the groundwater of north-eastern Poland. We classified three of these species as stygoxenes, and four as stygophiles. Among stygoxenes were Chydorus sphaericus, Eudiaptomus gracilis, and Thermocyclops crassus. These species were present as a single record or in low numbers in groundwater, but they are very common in different types of surface water bodies in this region [18,19], north-western Poland [20], and Europe [21,22]. Therefore, these species seem to be accidental taxa temporarily imported from the surface. However, E. gracilis has a flattened body which may indicate adaptation for an interstitial environment where the major constraint is the reduced living space [3,23]. We have classified four species as stygophiles—Eucyclops serrulatus, Diacyclops bisetosus, Diacyclops crassicaudis, and Cyclops furcifer. These species show an ecological preference for the groundwater habitats, and a higher frequency and occurrence in groundwater than in surface habitat. However, sometimes it is difficult to define the real ecology of the copepods found in groundwater [3]. Eucyclops serrulatus was found in eight wells from different regions of north-eastern Poland, often with high abundance to even 27.5 ind. L−1. This species is also a very common littoral copepod in Europe but are usually found in low numbers [21,22,24]. E. serrulatus is primarily benthic, swimming near the sediment layers of lakes, river, streams, and springs. It is commonly found in epikarst, alluvial aquifers, and hyporheic zones where it can live from a few centimeters below the streambed up to 1.5 m deep [25–27], as a member of the permanent hyporheos [28]. E. serrulatus is frequently the most abundant epigean cyclopoid in meiofaunal communities [21,29,30]. E. serrulatus is a morphological and ecologically variable species and was recently redescribed [31] and further serrulatus- group was redefined [32]. This group includes 17 species and subspecies, which differ from each other in the presence of microcharacters of the antennary basipodite and the P4 coxopodite [33]. Our species was E. serrulatus s. stricto, with three distalmost segments of antennule with a narrow hyaline membrane of regular shape (Figure4D), and a very characteristic spine of coxopodite of P4 with a gap among the strong teeth-like setules on the outer side (Figure4N). Furthermore, we have found a difference between populations in the arrangement of spines of the P4 endopodite (Figure4J,L). Recently, 36 European populations of E. serrulatus were deeply studied molecularly, which confirms its species complex with eight divergent lineages [34], awaiting the formal description. Further study revealed genetic and morphological heterogeneity within E. serrulatus in Europe and Asia [35]. Our molecular analysis of an mtDNA COI gene fragment discovered two new haplotypes of E. serrulatus which grouped with haplotypes described from Ukraine and Russia in the strongly supported phylogenetics branch on the ML tree. The genus Diacyclops, comprising a large number of stygobiontic species [36,37], was represented by two species in our samples; both were classified as stygophiles. Diacyclops bisetosus was found in seven wells in different regions, whereas in surface water bodies in Poland it is found occasionally in low numbers [19,20]. However, D. bisetosus is a Water 2021, 13, 2086 14 of 17

cosmopolitan, but probably cryptic species widely distributed in Europe [22]. This species was frequently found in habitats that could be considered as an ecotone between surface water and groundwater such as semi-permanent ponds, rivers, springs, puddles, marshes, ditches, caves, and wells [22,38]. D. bisetosus was common in groundwater and caves of the Alps [39], while in the Polish mountains another similar species D. bicuspidatus was frequently reported in the groundwater [6]. Gurney (1933) found it even in a tree- hole in England [40]. D. bisetosus was also the most frequent species in the hypersaline samples from the Crimean Peninsula [41]. The international genetic databases (GenBank and BOLD System) do not have sequences for D. bisetosus; therefore, we present a detailed morphological description of this species with its COI gene sequences. Diacyclops crassicaudis was found in five wells in the rural areas, east of Białystok city, often with high abundance. Females with egg sacs were frequently reported. This species is considered as cold-stenothermic [39,42] and it was reported from Europe from temporary ponds, puddles after rains and snowmelt, and littoral or psammon habitats of small lakes [22]. D. crassicaudis was also frequently reported from groundwater in Italy, where large groundwater endemism was found and seven subspecies were described in the ‘crassicaudis’ complex [43]. The different environmental conditions in groundwater favor radiation which leads to the formation of the cryptic species [3]. The ‘crassicaudis’ species complex was also frequently reported from groundwater in Central Spain [44], Germany [45,46], Austria [39], USA [47–49], Japan [50], and Australia [51]. It also occurs in the groundwater of the mountain regions of Poland [6], while it was rarely recorded in the surface water bodies in Poland. Therefore, we consider it as a stygophiles. The international genetic databases (GenBank and BOLD System) do not have sequences for D. crassicaudis; therefore, we present a detailed morphological description of this species with its COI gene sequences. We have observed the development of abnormal structures in one specimen of D. crassicaudis, where the anterior part of furcal rami were fused together to form a single plate. Similar aberrancies were observed by Gurney (1933) in Cyclops vicinus and Eucyclops serrulatus [40]. We have also noticed differences in the caudal setae morphology, where the outermost terminal caudal seta and dorsal caudal seta were abnormal long. Both specimens described by Gurney (1933) have one large dorsal seta instead of two dorsal setae. In our case, the furcal rami were not completely fused and there are two long dorsal setae. Abnormal structures, such as supernumerary setae, or deformed setae or spine, are not very uncommon but do not seem to have any significance [40]. We have observed abnormal development of whole furcal rami with setae, which could significantly affect the functioning of these organs. The abnormalities in copepod morphology are most often related to the tumor-like anomalies [52]. Cyclops furcifer was found in three wells in rural areas east of the Białystok city. In one well it appeared in large numbers, and females with egg sacs were observed. This species is reported from Europe in small and shallow lakes, temporary waters, swamps [22,39,53], and also was found in the groundwater of the Channel Islands just off the coast of Nor- mandy, France [54]. Our phylogenetics analysis based on 12S rRNA gene sequences of different species of Cyclops genus confirmed topology obtained by Krajíˇceket al. (2016) which was based on the 2531-bp long alignment consisting of fragments of six different studied molecular markers [55]. According to this topology, C. furcifer belong together with C. scutifer to the separate phylogenetics group, named by Krajíˇceket al. (2016) Clade 2 [55]. However, Hołyńskaand Wyngaard (2019) divided kolensis-clade (C. kolensis, C. furcifer, C. kikuchii, C. vicinus, C. insignis, C. alaskaensis) from scutifer-clade (C. scutifer, C. jashnovi, C. columbianus) based on morphology and phylogenetics, along with the geographic distribution patterns and ecological traits [15]. Interestingly, we have not found any Harpacticoida, which are otherwise common in groundwater. A previous preliminary study of a few wells in this region revealed the presence of a harpacticoid Elaphoidella elaphoides (Chappuis, 1923), which was a species new to the Polish fauna [7]. Harpacticoida dominates the benthos and are rarely found Water 2021, 13, 2086 15 of 17

in groundwater plankton. They can be either benthic or, more frequently, interstitial, irrespective of grain size composition. Gravel and sand are the preferred substrate for most harpacticoid species, whereas poorly sorted or clogged sediments show a drastic decrease in species diversity [3]. While Cyclopoida can live either in the plankton of subterranean lakes and pools or occur in benthic or interstitial habitats where they prefer sediment of medium-coarse grain size [3]. Therefore, the lack of harpacticoids could be a result of the sampling method here applied, allowing the collection of mainly open-water species.

5. Conclusions Our study is the first report on the groundwater copepods of the Polish lowland. Six copepod and one cladoceran species have been identified in the groundwater of north- eastern Poland. We have classified four species as stygophiles—Eucyclops serrulatus, Dia- cyclops bisetosus, Diacyclops crassicaudis, and Cyclops furcifer. These species are frequently found in wells in different regions of north-eastern Poland, often in high numbers, and with females with egg sacs. In the surface water bodies of Poland, these species were rarely recorded, which may indicate an ecological preference for the groundwater habitats. We have presented detailed morphological diagnoses of these species, accompanied by molecular characteristics (COI gene or 12S rRNA gene). Developmental aberrancy was observed in one specimen of D. crassicaudis, in which the anterior part of the furcal rami was fused together forming a single plate.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/w13152086/s1, Figure S1: photography of species considered as stygoxenes, Eudiaptomus gracilis, Thermocyclops crassus, and Chydorus sphaericus; Table S1: list of species and GenBank accession numbers of their gene sequences used in the phylogenetic analysis based on the maximum likelihood trees (Figure8A–C). Author Contributions: M.K.: writing—original draft preparation, conceptualization, methodology, investigation, formal analysis, validation, visualization, and supervision. S.S.: investigation, visualization, writing—review, and editing. M.S.:´ investigation, methodology, visualization, writing— review, and editing. J.M.: investigation. All authors have read and agreed to the published version of the manuscript. Funding: This article/publication has received financial support from the Polish Ministry of Science and Higher Education under subsidy for maintaining the research potential of the Faculty of Biology, University of Bialystok. Data Availability Statement: The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors. Acknowledgments: The authors are thankful to Maria Hołyńskafor consultation on identified species, valuable comments about the development of abnormal structures in copepods, and her preliminary review which significantly improve our manuscript. We are grateful to Jolanta Ejsmont- Karabin for the final language correction, and to Piotr Rode for the help in preparing Figure8. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Mammola, S.; Martinez, A. Let research on subterranean habitats resonate! Subterr. Biol. 2020, 36, 63–71. [CrossRef] 2. Brancelj, A.; Dumont, H. A review of the diversity, adaptations and groundwater colonization pathways in Cladocera and Calanoida (Crustacea), two rare and contrasting groups of stygobionts. Fundam. Appl. Limnol. 2007, 168, 3–17. [CrossRef] 3. Galassi, D.M.P. Groundwater copepods: Diversity patterns over ecological and evolutionary scales. Hydrobiologia. 2001, 453, 227–253. [CrossRef] 4. Iannella, M.; Fiasca, B.; Di Lorenzo, T.; Biondi, M.; Di Cicco, M.; Galassi, D.M.P. Spatial distribution of stygobitic crustacean harpacticoids at the boundaries of groundwater habitat types in Europe. Sci. Rep. 2020, 10, 1–10. [CrossRef][PubMed] 5. Karpowicz, M.; Pociecha, A.; Hoły´nska,M.; Dumnicka, E.; Galas, J. Groundwater copepods from Carpathian ﬂysch in south Poland. In Proceedings of the 3rd Polish Zooplankton Conference, Szczecinek, Poland, 20–22 July 2018. [CrossRef] 6. Kur, J.; Mioduchowska, M.; Kilikowska, A. Distribution of cyclopoid copepods in different subterranean habitats (southern Poland). Oceanol. Hydrobiol. Stud. 2020, 49, 255–266. [CrossRef] Water 2021, 13, 2086 16 of 17

7. Karpowicz, M. New data to the knowledge on the Harpacticoida (Crustacea, Copepoda) fauna in Poland. Fragm. Faunist. 2016, 59, 87–98. [CrossRef] 8. Agnarsson, I.; Kuntner, M. Taxonomy in a changing world: Seeking solutions for a science in crisis. Syst. Biol. 2007, 56, 531–539. [CrossRef][PubMed] 9. Bucklin, A.; Lindeque, P.K.; Rodriguez-Ezpeleta, N.; Albaina, A.; Lehtiniemi, M. Metabarcoding of marine zooplankton: Prospects, progress and pitfalls. J. Plankton Res. 2016, 38, 393–400. [CrossRef] 10. Meyer, C.P. Molecular systematics of cowries (Gastropoda: Cypraeidae) and diversification patterns in the tropics. Biol. J. Linn. Soc. Lond. 2003, 79, 401–459. [CrossRef] 11. Machida, R.J.; Miya, M.U.; Nishida, M.; Nishida, S. Complete mitochondrial DNA sequence of Tigriopus japonicus (Crustacea: Copepoda). Mar. Biotechnol. 2002, 4, 406–417. [CrossRef] 12. Hall, T.A. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. 1999, 41, 95–98. 13. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [CrossRef][PubMed] 14. Posada, D. jModelTest: Phylogenetic model averaging. Mol. Biol. Evol. 2008, 25, 1253–1256. [CrossRef][PubMed] 15. Hołyńska,M.; Wyngaard, G.A. Towards a phylogeny of Cyclops (Copepoda): (in)congruences among morphology, molecules and zoogeography. Zool Scr. 2019, 48, 376–398. [CrossRef] 16. Baek, S.Y.; Jang, K.H.; Choi, E.H.; Ryu, S.H.; Kim, S.K.; Lee, J.H.; Lim, Y.J.; Lee, J.; Jun, J.; Kwak, M.; et al. DNA barcoding of metazoan zooplankton copepods from South Korea. PLoS ONE 2016, 11, e0157307. [CrossRef] 17. Hołyńska,M. On the morphology and geographical distribution of some problematic South Palearctic Cyclops (Copepoda: Cyclopidae). J. Nat. Hist. 2008, 42, 2011–2039. [CrossRef] 18. Ejsmont-Karabin, J.; Kalinowska, K.; Karpowicz, M. Structure of ciliate, rotifer, and crustacean communities in lake systems of Northeastern Poland. In Polish River Basins and Lakes—Part II; Springer: Cham, Switzerland, 2020; Volume 87, pp. 77–101. [CrossRef] 19. Karpowicz, M.; Ejsmont-Karabin, J. Diversity and Structure of Pelagic Zooplankton (Crustacea, Rotifera) in NE Poland. Water 2021, 13, 456. [CrossRef] 20. Sługocki, Ł.; Czerniawski, R. Trophic state (TSISD) and mixing type significantly influence pelagic zooplankton biodiversity in temperate lakes (NW Poland). PeerJ. 2018, 6, e5731. [CrossRef] 21. Walseng, B.; Hessen, D.O.; Halvorsen, G.; Schartau, A.K. Major contribution from littoral crustaceans to zooplankton species richness in lakes. Limnol. Oceanogr. 2006, 51, 2600–2606. [CrossRef] 22. Bł˛edzki,L.A.; Rybak, J.I. Freshwater Crustacean Zooplankton of Europe; Springer: Cham, Switzerland, 2016; pp. 1–918. 23. Schminke, H.K. Perspectives in the study of the zoogeography of interstitial Crustacea: Bathynellacea (Syncarida) and Parasteno- carididae (Copepoda). Int. J. Speleol. 1981, 11, 83–89. [CrossRef] 24. Karpowicz, M. Biodiversity of microcrustaceans (Cladocera, Copepoda) in a lowland river ecosystem. J. Limnol. 2017, 76, 15–22. [CrossRef] 25. Galassi, D.M.P.; Stoch, F.; Fiasca, B.; Di Lorenzo, T.; Gattone, E. Groundwater biodiversity patterns in the Lessinian Massif of northern Italy. Freshw. Biol. 2009, 54, 830–847. [CrossRef] 26. Galassi, D.M.P.; Huys, R.; Reid, J.W. Diversity, ecology and evolution of groundwater copepods. Freshw. Biol. 2009, 54, 691–708. [CrossRef] 27. Galassi, D.M.P.; Lombardo, P.; Fiasca, B.; Di Cioccio, A.; Di Lorenzo, T.; Petitta, M.; Di Carlo, P. Earthquakes trigger the loss of groundwater biodiversity. Sci. Rep. 2014, 4, 6273. [CrossRef] 28. Gibert, J.; Stanford, J.A.; Dole-Olivier, M.-J.; Ward, J.V. Basic attributes of groundwater ecosystems and prospects for research. In Groundwater Ecology; Gibert, J., Danielopol, D.L., Stanford, J.A., Eds.; Academic Press, Inc.: San Diego, CA, USA, 1994; pp. 7–40. 29. Fiasca, B.; Stoch, F.; Olivier, M.-J.; Maazouzi, C.; Petitta, M.; Di Cioccio, A.; Galassi, D.M.P. The dark side of springs: What drives small-scale spatial patterns of subsurface meiofaunal assemblages? J. Limnol. 2014, 73, 71–80. [CrossRef] 30. Di Lorenzo, T.; Di Marzio, W.D.; Cifoni, M.; Fiasca, B.; Baratti, M.; Sáenz, M.E.; Galassi, D.M.P. Temperature effect on the sensitivity of the copepod Eucyclops serrulatus (Crustacea, Copepoda, Cyclopoida) to agricultural pollutants in the hyporheic zone. Curr. Zool. 2015, 61, 629–640. [CrossRef] 31. Alekseev, V.; Dumont, H.J.; Pensaert, J.; Baribwegure, D.; Vanfleteren, J.R. A redescription of Eucyclops serrulatus (Fischer, 1851) (Crustacea: Copepoda: Cyclopoida) and some related taxa, with a phylogeny of the E. serrulatus-group. Zool. Scr. 2006, 35, 123–147. [CrossRef] 32. Alekseev, V.R.; Defaye, D. Taxonomic differentiation and world geographical distribution of the Eucyclops serrulatus group (Copepoda, Cyclopidae, Eucyclopinae). In Studies on Freshwater Copepoda: A Volume in Honour of Bernard Dussart. Crustaceana Monographs; Defaye, D., von Vaupel Klein, J.C., Suárez-Morales, E., Eds.; Koninklijke Brill NV: Leiden, The Netherlands, 2011; Volume 16, pp. 41–72. [CrossRef] 33. Alekseev, V.R. Revision of the genus Eucyclops (Claus, 1893) and subfamily Eucyclopinae of the world fauna. Arthropoda Sel. 2019, 28, 490–514. [CrossRef] Water 2021, 13, 2086 17 of 17

34. Hamrova, E.; Krajicek, M.; Karanovic, T.; Cerny, M.; Petrusek, A. Congruent patterns of lineage diversity in two species complexes of planktonic crustaceans, Daphnia longispina (Cladocera) and Eucyclops serrulatus (Copepoda), in East European mountain lakes. Zool. J. Linn. Soc. 2012, 166, 754–767. [CrossRef] 35. Sukhikh, N.; Alekseev, V. Genetic and morphological heterogeneity within Eucyclops serrulatus (Fischer, 1851) (Crustacea: Copepoda: Cyclopidae). J. Nat. Hist. 2015, 49, 2929–2953. [CrossRef] 36. Pesce, G.L.; Galassi, D.P. New or rare species of Diacyclops Kiefer, 1927 (Copepoda, Cyclopoida) from different groundwater habitats in Italy. Hydrobiologia. 1987, 148, 103–114. [CrossRef] 37. Pipan, T. Epikarst—A Promising Habitat: Copepod Fauna, Its Diversity and Ecology: A Case Study from Slovenia (Europe); Karst Research Institute at ZRC SAZU, ZRC Publishing: Ljubljana, Slovenia, 2005; pp. 1–101. 38. Kur, J.; Wojtasik, B. Widłonogi Cyclopoida wybranych jezior zlewni górnej Raduni. In Jeziora Kaszubskiego Parku Krajobrazowego; Borowiak, D., Ed.; Uniwersytet Gdański:Gdańsk,Poland, 2007; pp. 145–163. (In Polish) 39. Gaviria, S. Checklist and distribution of the free-living copepods (Arthropoda: Crustacea) from Austria. Ann. Naturhist. Mus. Wien. 1998, 100B, 539–594. 40. Gurney, R. British freshwater Copepoda; The Ray Society: London, UK, 1933; pp. 76–78. 41. Anufriieva, E.; Hołyńska,M.; Shadrin, N. Current Invasions of Asian Cyclopid Species (Copepoda: Cyclopidae) in Crimea, with Taxonomical and Zoogeographical Remarks on the Hypersaline and Freshwater Fauna. Annal Zool. 2014, 64, 109–130. [CrossRef] 42. Lewis, J.J.; Reid, J.W. Patterns and Processes of Groundwater Invasion by Copepods in the Interior Low Plateaus of the United States. Acta Carsologica 2007, 36, 279–289. [CrossRef] 43. Pesce, G.L.; Galassi, D.M.P. Copepodi di acque sotterranee della Sicilia. Animalia 1987, 14, 193–235. 44. Iepure, S.; Rasines-Ladero, R.; Meffe, R.; Carreno, F.; Mostaza, D.; Sundberg, A.; Di Lorenzo, T.; Barroso, J.L. The role of groundwater crustaceans in disentangling aquifer type features—A case study of the Upper Tagus Basin, central Spain. Ecohydrology 2017, 10, e1876. [CrossRef] 45. Hahn, H.J.; Fuschs, A. Distribution patterns of groundwater communities across aquifer types in south-western Germany. Freshw. Biol. 2009, 54, 848–860. [CrossRef] 46. Gutjahr, S.; Schmidt, S.; Hahn, H. A proposal for a groundwater habitat classification at local scale. Subterr. Biol. 2014, 14, 25–49. [CrossRef] 47. Yeatman, H.C. Rare cyclopoid copepods from wells in North Carolina. J. Elisha Mitchell Sci. Soc. 1943, 59, 27–36. 48. Reid, J.W. Redescription of Diacyclops nearcticus (Kiefer, 1934) and description of four similar new congeners from North America, with comments on D. crassicaudis (G.O. Sars, 1863) and D. crassicaudis var. brachycercus (Kiefer, 1927) (Crustacea: Copepoda). Canad. J. Zool. 1992, 70, 1445–1469. [CrossRef] 49. Bruno, M.; Cunningham, K.; Perry, S. Copepod Communities from Surface and Ground Waters in the Everglades, South Florida. Southeast. Nat. 2003, 2, 523–546. [CrossRef] 50. Ito, T. Groundwater Copepods from South-western Japan. Hydrobiologia 1957, 11, 1. [CrossRef] 51. Pesce, G.; De Laurentiis, P. Copepods from Ground Waters of Western Australia. III. Diacyclops humphreysi n. sp., and Comments on the Diacyclops crassicaudis-Complex (Copepoda, Cyclopidae). Crustaceana 1996, 69, 524–531. 52. Jagadeesan, L.; Jyothibabu, R. Tumour-like anomaly of copepods-an evaluation of the possible causes in Indian marine waters. Environ. Monit. Assess. 2016, 188, 244. [CrossRef] 53. Einsle, U. Cyclops heberti n.sp. and Cyclops singularis n.sp., two new species within the genus Cyclops (‘strenuus-subgroup’) (Crust. Copepoda) from ephemeral ponds in southern Germany. Hydrobiologia 1996, 319, 167–177. [CrossRef] 54. Knight, L.; Brancelj, A.; Hänfling, B.; Cheney, C. The groundwater invertebrate fauna of the Channel Islands. Subterr. Biol. 2015, 15, 69–94. [CrossRef] 55. Krajíˇcek,M.; Fott, J.; Miracle, M.R.; Ventura, M.; Sommaruga, R.; Kirschner, P.; Cernˇ ý, M. The genus Cyclops (Copepoda, Cyclopoida) in Europe. Zool. Scr. 2016, 45, 671–682. [CrossRef]