Distribution, structure and functioning of subterranean fauna within Irish groundwater systems

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Resource or Project Abstract

Crustacean abundance in groundwater displayed strong correlations with hydrology related parameters, such as water table dynamics and electrical conductivity, which can be used as a proxy for hydrologic exchange with surface water. Increased faunal abundance during dry periods with falling groundwater levels appeared to be the consequence of a concentration effect. Increased abundance in zones with higher exposure to exchange with surface water was presumably caused by improved food availability from surface environments. The most promising application of groundwater fauna monitoring would therefore be in bioindication of surface water intrusion (i.e. groundwater vulnerability). Obligate groundwater crustaceans tolerated a low oxygen environment, but did not occur in environments with saturation levels of 5% or lower. The most frequently encountered animal Niphargus kochianus irlandicus also displayed a low tolerance of elevated salt concentration and is therefore unlikely to inhabit coastal margins that are exposed to saline intrusions.

Genetic investigations revealed that this endemic and therefore uniquely Irish crustacean was separated from other European species several million years ago and must have survived all Quaternary glaciations in Ireland. Based on this genetic assessment, it needs to be elevated from its current taxonomic subspecies ranking to species level. It should be renamed as Niphargus irlandicus. The species has least three genetic lineages, whose biogeographic ranges are partially separated by poorly productive aquifers. The geothermal refuge hypothesis proposes that the animals survived under the ice cover in zones where geothermal activity led to the rise of warm water through deep bedrock faults. Supporting evidence for this hypothesis are the accessibility of warm and tepid springs within each lineage?s geographic range and the species? lack of adaptation to water temperatures below 4°C. It should be tested whether the hypothesis can also be applied to other endemic groundwater animals in Ireland and whether it may even be applicable to groundwater fauna in formerly glaciated non-volcanic areas beyond Ireland. After this island wide project more intensive regional surveys are needed for further advances in the knowledge of the biodiversity of Irish subterranean aquatic fauna. Aquifers adjacent to large rivers and eskers would appear to be particularly promising targets for future surveys. A sampling effort of six sampling sites per aquifer is deemed sufficient for the representation of faunal diversity. Investigations into the impact of hydrology and hydrogeology parameters on groundwater fauna require long term monitoring of groundwater fauna. Therefore, the recommendation is to identify sets of long term monitoring sites in at least two different aquifer types (Fracture flow and intergranular flow), which should be sampled at least every two months for several years. Finally, an improved understanding of groundwater ecosystems also requires further autecological studies of groundwater organisms. For practical applications water engineers may be interested to note that annual monitoring of abundance of crustacean groundwater fauna and surface water fauna within distribution networks for water transport from groundwater sources could provide early warning signals with regard to microbial growth, surface water intrusion and related contamination risks.

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Contact Information for This Resource

Dr. Jörg Arnscheidt
University of Ulster
Environmental Research Scientist
Room G062, University of Ulster Coleraine campus, School of Environmental Sciences, University of Ulster Coleraine campus Coleraine Co. Londonderry BT52 1SA, Northern Ireland
Telephone: +44 28 70124095
e-mail: j.arnscheidt@ulster.ac.uk

Prof. James Dooley
University of Ulster
Professor of Microbiology Biomedical Sciences Research Institute
Room W0080 School of Biomedical Sciences, University of Ulster Coleraine campus, Cromore Road Coleraine, Co. Londonderry BT52 1SA, Northern Ireland
Telephone: +44 28 70124427
e-mail: jsg.dooley@ulster.ac.uk

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Author(s)Arnscheidt, J. Dooley, J.
Title Of WebsiteSecure Archive For Environmental Research Data
Publication InformationDistribution, structure and functioning of subterranean fauna within Irish groundwater systems
Name of OrganisationEnvironmental Protection Agency Ireland
Electronic Address or URL http://erc.epa.ie/safer/resource?id=f107f038-e4f7-11e2-8c2d-005056ae0019
Unique Identifierf107f038-e4f7-11e2-8c2d-005056ae0019
Date of AccessLast Updated on SAFER: 2017-02-27

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Arnscheidt, J. Dooley, J.   "Distribution, structure and functioning of subterranean fauna within Irish groundwater systems". Associated datasets and digitial information objects connected to this resource are available at: Secure Archive For Environmental Research Data (SAFER) managed by Environmental Protection Agency Ireland http://erc.epa.ie/safer/resource?id=f107f038-e4f7-11e2-8c2d-005056ae0019 (Last Accessed: 2017-02-27)


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SAFER-Data Display URL http://erc.epa.ie/safer/iso19115/display?isoID=3011
Resource KeywordsSubterranean aquatic crustacea, survey, biogeography, Niphargus, phylogeography, phylogenetics, autecology, temperature tolerance, salinity tolerance, groundwater, hydrology, chemistry
EPA/ERTDI/STRIVE Project Code2007-WQ-MS-1-S1
EPA/ERTDI/STRIVE Project ThemeWater Quality
Resource Availability: Any User Can Download Files From This Resource
Limitations on the use of this ResourceAny attached datasets, data files, or information objects can be downloaded for further use in scientific applications under the condition that the source is properly quoted and cited in published papers, journals, websites, presentations, books, etc. Before downloading, users must agree to the "Conditions of Download and Access" from SAFER-Data. These appear before download. Users of the data should also communicate with the original authors/owners of this resource if they are uncertain about any aspect of the data or information provided before further usage.

We would like to request withholding the release of submitted data and information for the period of 1.5 years (until March 2014) in order not to compromise chances of publication in scientific journals. When published, it should be acknowledged that geological data were made available through GSI and GSNI.
Number of Attached Files (Publicly and Openly Available for Download): 2
Project Start Date Tuesday 1st April 2008 (01-04-2008)
Earliest Recorded Date within any attached datasets or digital objects Tuesday 1st April 2008 (01-04-2008)
Most Recent Recorded Date within any attached datasets or digital objects Friday 30th March 2012 (30-03-2012)
Published on SAFERThursday 4th July 2013 (04-07-2013)
Date of Last EditMonday 8th July 2013 at 12:45:13 (08-07-2013)
Datasets or Files Updated On Monday 8th July 2013 at 12:45:13 (08-07-2013)

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Geographical and Spatial Information Related To This Resource

Description of Geographical Characteristics of This Project or Dataset
The project attempted to sample suitable sites with borehole, wells, springs all over Ireland. There were sampling sites in the following 23 counties: Antrim, Cavan, Clare, Cork , Derry, Donegal, Down, Fermanagh, Galway, Kerry, Kildare, Kilkenny, Limavady, Limerick, Louth, Mayo, Monaghan, Offaly, Roscommon, Tipperary, Tyrone, Waterford and Wexford.

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Supplementary Information About This Resource

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Lineage information about this project or dataset
For the survey of Irish groundwater fauna there had been a pilot project in 2006, which was financed by the University of Ulster. This pilot tested the survey method and rationale that boreholes and wells would be suitable sampling sites. Its results were communicated to the EPA. The European Groundwater Directive (2006/118/EC) identified a knowledge gap with regard to groundwater ecology and encouraged member states support research in this area. The EPA call was a response to the pilot survey and the EU Groundwater Directive.
Supplementary Information
Key Outputs: Genetic analysis of the N. kochianus specimens from England was funded by the Systematics Research Fund (Linnean society) awarded to C. McInerney and J. Arnscheidt and a grant from Roehampton University

Funding outside EPA:
Genetic analysis of the N. kochianus specimens from England was funded by the Systematics Research Fund (Linnean society) awarded to C. McInerney and J. Arnscheidt and a grant from Roehampton University.

Beyond the project team collaborations were established with Royal Belgian Institute of Natural Sciences, Hull University and Roehampton University.

Starting with chapter 2 of the report we are preparing the of articles to scientific journals based on each of the chapters 1-4.
Arnscheidt, J. and Eriksson, K. (2011). The last frontier in Freshwater Biodiversity Research: Groundwater. Proceedings Asian Trans-disciplinary Karst Conference 2011, Yogyakarta Indonesia, pp. 267-274
Arnscheidt, J. (2011). Groundwater survey reveals new records and species. Biodiversity Science. 4 [online: http://www.biodiversityscience.com/2011/11/01/groundwater-survey-records-species]
Arnscheidt, J. (2010). Amazing animals in Irish groundwater. Groundwater Newsletter. 48, pp. 2-6

Special thanks to

- Phil Jordan for friendship, mentoring, help and support in initiating this project
- James Dooley for friendship and curiosity, initiative and unwavering support in all crustacean DNA matters
- Paul Wood for friendship, valuable advice, proofreading, interesting fieldwork and an introduction to the world of cavers
- Hans Jürgen Hahn and Andreas Fuchs for friendship, technology transfer and invaluable training in identification of subterranean crustaceans
- Karin Eriksson for her enthusiasm in fieldwork and patience on the microscope.
- Tara Higgins and Thomas Kieran McCarthy for their quick and efficient start on the autecology and establishing the ?Niphargus infrared CCTV?.
- Caitriona McInerney for tackling the Numts and actively engaging in collaborations
- Matthew Craig, Guiseppe Messana, Michael Usher and Alice Wemaere for advice and support during the project
- Members of the hydrometric teams who accompanied us on field work for their invaluable help John Agnew, Michael Browne, Patrick Durkin, Martin Kerr, Brendan Magennis, Margaret Maher, Hugh McGinley, Joe Reilly, Jim Ryan and Michael Stapleton.
- NIEA / GSNI staff for help with fieldwork or site identification: Silke Hartmann, Peter McConvey, Gary Mills, Gary Scott, Paul Wilson, Melanie Wrigley,
- Waterford County council staff Patrick McCarthy and colleagues for access to sites and help with fieldwork
- Nigel Macauley and Hugo McGrogan for building the temperature gradient chamber and Barry o?Hagan, Bernie Doherty and Ian Anderson for technical assistance.
- Lee Knight for the kind donation of Niphargus specimens for access to the biorecorder data.
- Mark Holmes for help with identifying challenging copepods
- Matt Zeale and Deb Triant for helpful advice on pseudogene identification
- Severine Mathjiis and Frank Fiers (Royal Belgian Institute of Natural Sciences) for the kind donation of DNA sequence data for Belgian N. kochianus specimens-
- Anne Robertson (Roehampton University) and Lou Maurice (British Geological Survey) for donating samples of N. kochianus specimens from England.
- Bernd Hänfling for permission to use DNA sequence data and assistance with data analysis and to J.M. Pujolar for helpful discussions.
- MSc students Balaji Balarahman, Govindraj Chavan and Nikhil Nikunj are thanked for their valuable work on testing PCR methodologies on Niphargus and Gammarus species.

GIS Modelling Information

Laboratory based investigations were made for the autecology research on Niphargus k. irlandicus (chapter 3 of the report) and phylogenetics (chapter 4 of the report). Autecology: Temperature tolerance
Experiments were conducted in a darkroom, whose sole light source was a darkroom light. Animals were tested in a purpose built temperature gradient chamber. An acrylic glass chamber was equipped with a Peltier element at one end and two heating plates at the other end. The floor was clad with black water resistant sandpaper. The chamber was then positioned at a slope with an angle of 13.5 degrees, so that the Peltier element was at the lower and the heating plates were at the upper end. Seven thermistors were inserted. Feedback loops from the thermistors closest to either the Peltier element or the heating plates controlled cooling and heating with the aid of a Campbell CR 10X datalogger, in order to achieve a stable temperature gradient. Convection currents in the chamber were studied in separate experiments with dye by photodocumentation.Temperature profiles were documented with a mobile temperature probe on a long and thin flexible cable. In the evening prior to the start of exposure experiments the chamber was filled with tap water, which had been aerated throughout the day. Test organisms were Niphargus k. irlandicus specimens, which had been fed and kept at 10°C in the laboratory for several months. At the end of each experiment oxygen saturation was recorded with A HACH HQ10 LOD probe after careful mixing of water in the chamber to break the thermal gradient.

Autecology: Salinity tolerance
Video observation
Infra-red video recording was used for animal observations. Animals were videoed in darkness using a digital camcorder in night vision mode connected to a PC for live video capture. An infrared LED light source was provided.
Live video was captured on the PC using Ulead Video Studio software. Motion tracking software (MaxTraq, Innovation Systems, Columbiaville, MI) was used to measure the direction and velocity of the animals? movements . The custom-arranged video setup represented the best compromise between providing the animals with sufficient space to move around while allowing for the production of sharp images at the same time.
At the end of the 14 day acute transfer salinity bioassay with the Niphargus k. irlandicus specimens from County Galway, locomotory activity (swimming, crawling) in the remaining live animals was measured. Animals were videoed first in darkness for a period of 5 minutes and subsequently in exposure to ambient room light for 3 minutes. Recordings were analysed using MaxTraq motion analysis software (Innovation Systems, Columbiaville, MI) in order to quantify, whether salinity had non-lethal detrimental effects on the animals, in terms of their swimming/crawling movements.

After collection from the sampling site adult specimens of Niphargus from Bunatober spring, Co. Galway, were kept in aerated spring water in darkness for 48 h. For the salinity bioassays, synthetic seawater (35 psu) was diluted with filtered water collected from Bunatober spring (0.35 psu), to the following final concentrations: 0.35 (control), 1, 2, 3, and 4 psu. For the experiments, 12-14 adult animals were transferred from the lab culture using a wide-mouthed pipette to duplicate 1-L bottles containing the salt solutions (resulting in 10 bottles containing 130 animals in total) Bottles were incubated in darkness at 11°C for a period of 2 weeks. Constant gentle aeration was provided via an air stone positioned close to the top of the bottles. Bottles were checked for mortalities at 1.5, 3, 6 and 12 h intervals for the first 24 h and at 12 h intervals thereafter, and dead animals removed using a wide-mouthed pipette and their body length measured under a dissecting microscope.
As several attempts did not succeed in gathering a sufficient number of specimens from Galway, a ?replication? bioassay was performed with specimens from sites at Japanese Garden and at Milltown, County Kildare, which had been kept in aerated tap water for several weeks prior to testing. Animals were fed weekly with aquarium fish food and crab meat before experiments. As one of the aims was to determine Lc50 and Lc100 values (concentrations leading to 50% and 100% mortality respectively), the range of tested concentrations was extended for this bioassay. Due to the restricted quantity of test organisms, concentrations 1 and 2 psu were not replicated. 160 animals were used in the second bioassay with solutions of 0.35 psu (control), 3,4,5,6,7,8,9 psu. Salinity of all individual salinity treatments within the experiments was checked using a HACH sension 156 with conductivity cell.

Autecology: Feeding behaviour
Feeding trials were performed in batch; five to ten animals were kept in 500ml water with aeration at 10°C). Trials were conducted in the dark. Observations were made on an Olympus SZX 16 with the photo/video camera DP72. Tested animals came from Japanese Garden and Milltown sites in Co. Kildare. Bait was dyed with Rose Bengal.

Phylogenetics: Sample preservation and genomic DNA isolation
Samples collected were preserved in 70% ethanol. Samples were sorted under the microscope and the N. k. irlandicus specimens were separated out and stored at -20oC until further processing for genomic DNA isolation. For comparative purposes, samples of the taxonomically closely related sub-species N. k. kochianus from England and N. k. kochianus and N. k. dimorphopus from Belgium were also included in the study. Genomic DNA was isolated from the whole organism using the Nucleon® PhytoPure® kit (Genprobe) with slight modifications. Preliminary studies indicated that contaminant DNA (from gut contents etc.) did not present a significant problem with a minimal risk of its amplification during PCR (2 %). Resulting DNA was re-suspended in 20 µl sterile water and stored at ?20oC. DNA concentrations were quantified using a NanoDrop ND-1000 spectrophotometer and samples were diluted when necessary to a concentration of ca. 50ng/µl.

Phylogenetics: DNA amplification and sequencing
Genetic variation of N. k. irlandicus was assessed at two mitochondrial genes, COI and 16s rRNA and a nuclear gene, 28s rRNA. Polymerase chain reactions (PCR) reactions were undertaken in 30 µl reaction volumes containing ca. 12.5 ng DNA, 200 µM dNTPs, 2.5 mM MgCl2, 1x PCR reaction buffer (Invitrogen), 10pM of each primer and 1.5 U Taq DNA polymerase (Invitrogen). Thermal cycling conditions consisted of an initial denaturation (5 min at 94°C) followed by 40 cycles with 1 min at 94°C, 1 min at the optimal annealing temperature and 2 min 30s at 72°C, and a final extension step of 10 min at 72°C (Table 4.2). PCR primers implemented in the multi-gene analysis are listed in Table 4.2. Additional PCR primers were designed for COI and 28s rRNA genes from N. k. irlandicus DNA sequences using PrimerSelectTM (Lasergene 4.0, DNAStar Inc.). Following amplification, an aliquot of the PCR product (5 μl) was visualised on a 1.5% agarose gel containing ethidium bromide and sized against a 100bp DNA size ladder (Invitrogen). For successful PCRs, the remaining PCR product volume (25 μl) was purified using a Wizard® SV Gel and PCR Clean-Up system (Promega). For samples that failed to amplify, an alternative PCR primer set was implemented in a repeat PCR experiment. Purified PCR products were bi-directionally sequenced (Macrogen Inc., Republic of Korea) using non-standard conditions. Both DNA template amount (5 μl) and cycling parameters (annealing time, cycle number) were increased to try to maximise sequencing reaction success. For the 16s and 28s rRNA genes, external PCR primers used in the PCR reaction were utilised for sequencing. For the COI gene, internal sequencing primers from within conserved regions of this hyper-variable gene were utilised for sequencing.

Phylogenetics: DNA sequence analysis and bioinformatic identification of Numts / Pseudogenes
Chromatograms for the forward and reverse DNA sequences were assembled and corrected in CodonCode Aligner (LI-COR Inc.). Poor quality sequence data and chromatograms containing double or triple nucleotide peaks were excluded. Sequences were compared in a BLASTn analysis against GenBank nucleotide collection to confirm gene and DNA origin. Relevant DNA sequence data for the 16s rRNA, 28s rRNA and COI genes for Niphargus was collated from GenBank. Multiple sequence alignments of Niphargus DNA sequences for each gene was carried out using ClustalW in MEGA 5. A bioinformatic strategy was then implemented for the detection and removal of numts. Aligned mitochondrial DNA sequences were translated to amino acid sequences utilising the invertebrate mitochondrial genetic code in MEGA 5. The Niphargus sequences were visually inspected for stop codons, frame-shift mutations and large insertions/deletions (ca. 20bp). Mitochondrial DNA sequences with evidence of coding errors typical of numts were subsequently removed from the dataset. Final DNA sequence alignments for the 28s rRNA (n = 149; length 843bp), COI (n = 147; length 881bp) and the 16s rRNA (n = 165; length 958bp) genes were analysed.

Phylogenetics:Phylogenetic, molecular evolutionary and genetic diversity analyses
The software DnaSP v5 was used to estimate descriptive statistics of genetic diversity: Number of haplotypes (H), haplotype diversity (h) and nucleotide diversity (π). For locations where multiple samples were of the same haplotype, a consensus sequence for each haplotype present was used in further analyses. Evolutionary relationships amongst Niphargus taxa were investigated using the Neighbor-Joining (N-J) method in MEGA 5. N-J phylogenetic trees were constructed using the Maximum Composite Likelihood method and the option ?pairwise deletion? (see MEGA 2012, MEGA 5.0 online manual). Branch support for the nodes was estimated by performing 500 bootstrap replicates. Pairwise comparisons of genetic distances (da), calculated based on the number of net nucleotide substitutions per site between populations were estimated in DnaSP v5. Based on a molecular clock approach and node depths, time of genetic divergence was calculated using the equation T = da/2*µ where µ is the rate in number of substitutions per site per year.We also applied different rates of change dependent on the gene under investigation, in order to improve calibration accuracy. Minimum and maximum T were estimated based on an interval of divergence rate assumed to be 1.40?2.60% of substitutions per site per million years for the COI 0.53?2.20% for the 16s rRNA and the global clock rate of 1.25% for the 28s rRNA gene. We evaluated whether lineages could be defined as cryptic species by applying the threshold of 0.16 subst./site in the COI gene to delimit new or uncertain species.
Links To Other Related Resources
  http://www.science.ulster.ac.uk/gei/ (Opens in a new window)
  http://www.ncbi.nlm.nih.gov/genbank/ (Opens in a new window)

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