Stygoregions – a promising approach to

a bioregional classification of
SUBJECT AREAS:
SUSTAINABILITY groundwater systems
ZOOLOGY
Heide Stein1, Christian Griebler2, Sven Berkhoff1, Dirk Matzke3, Andreas Fuchs4 & Hans Jürgen Hahn1
BIODIVERSITY
METHODS
1
University of Koblenz-Landau, Campus Landau, Institute for Environmental Sciences, Fortstr. 7, 76829 Landau, Germany,
2
Helmholtz Zentrum München, Institute of Groundwater Ecology, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany,
3
Received Institute for Groundwater Ecology, Regional Office North, Unter den Eichen 24, 39517 Burgstall, Germany, 4Institute for
6 March 2012 Groundwater Ecology, Marktplatz 5, 3970 Weitra, Austria.

Accepted
29 August 2012 Linked to diverse biological processes, groundwater ecosystems deliver essential services to mankind, the
most important of which is the provision of drinking water. In contrast to surface waters, ecological aspects
Published of groundwater systems are ignored by the current European Union and national legislation. Groundwater
19 September 2012 management and protection measures refer exclusively to its good physicochemical and quantitative status.
Current initiatives in developing ecologically sound integrative assessment schemes by taking groundwater
fauna into account depend on the initial classification of subsurface bioregions. In a large scale survey, the
regional and biogeographical distribution patterns of groundwater dwelling invertebrates were examined
Correspondence and
for many parts of Germany. Following an exploratory approach, our results underline that the distribution
requests for materials patterns of invertebrates in groundwater are not in accordance with any existing bioregional classification
should be addressed to system established for surface habitats. In consequence, we propose to develope a new classification scheme
H.J.H. (hjhahn@uni- for groundwater ecosystems based on stygoregions.
landau.de)

G
roundwater systems are diversely populated habitats. The world wide number of described obligate
groundwater invertebrates, the so-called stygofauna, sums up to 7000 species1. The true species richness
though, is believed to exceed that number by far taking into account that groundwater systems have not
been studied to a great extent and a multitude of species await detection and description2–5. The groundwater
fauna is mainly composed of small crustaceans, oligochaetes, nematodes, acari, and molluscs, less than 1 mm to
several centimeters in body size4 (Fig. 1 a–d).
The goods and services provided by groundwater ecosystems (i.e ecosystem services) which include the
purification of water by the breakdown of organic matter, nutrients and contaminants, are fundamental to both
the integrity of the ecosystems and the sustaining of human life6–10. Intimately connected to microbiological
remineralization processes, invertebrates are believed to play an important role in the purification of subterranean
water and thus, the maintainance of good water quality11,12. Feeding, burrowing and bioturbating activities of the
fauna in highly active transition zones (e.g. hyporheic zone) contribute to maintain the hydraulic connectivity
between surface systems and aquifers11, a service groundwater dependant ecosystems (e.g. soils, wetlands, rivers)
strongly rely on11. Ecosystem services such as those mentioned above are only provided sustainably by ground-
water communities with a structural and functional integrity13,14.
Recently, the need for an ecologically sound management of groundwater resources has become increasingly
recognised by national and international political authorities15–19. The European Union Groundwater Directive20
(EU-GWD), released in 2006, emphasize the importance of protective measures for groundwater ecosystems
proposing the conductance of more ecological research in order to provide better criteria for ensuring ground-
water ecosystem quality in the future13. This triggered new scientific research on identifying and evaluating
biological criteria related to groundwater ecosystem health and water quality, and vice versa13,14,19,21.
Organisms integrate impacts over space (their habitat) and time, clearly extending the information obtained
from abiotic monitoring21. Indeed groundwater invertebrates have been shown to be sensitive sentinels indicating
the influence from surface water infiltration, changes of hydrological conditions, the impact of organic contami-
nants and nutrients, and were shown valuable biomonitors of heavy metal pollution as well as heat discharge 14 and
citations therein
. Only recently, first integrative approaches for the assessment of groundwater ecosystem health, which
define criteria, reference conditions and thresholds for individual physicochemical, microbiological and faunistic

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100,000 km2) Central Europe encompasses at least three main ecor-

egions (Nothern Lowlands, Central Uplands, Alps)38 (Fig. 2a). How-
ever, most classification systems for Germany, including the water
framework directive (WFD)33, additionally comprise the Alpine Foo-
thills (Fig. 2b). On a regional scale, these ecoregions are refined into
major physiographic units (MPU)37 (Fig. 2b). This classification
scheme, applied to Germany, but also to Austria, considers surface
and subsurface features, including climate, geology, physical geo-
graphy, and morphology37. Examples for Germany are the Black Fo-
rest (Fig. 3, MPU No. D54) the Lower Rhine Valley (Fig. 3, MPU No.
D35) or the Pfälzerwald Mountains (Fig. 3, MPU No. D51). In order
to develop a first large scale classification for Central Europe’s
groundwater systems, we evaluated the patterns obtained from our
groundwater fauna data set for its agreement with existing classifica-
tion schemes, following different approaches and levels of statistical
analysis including selected spatial aggregation.

Figure 1 | Stygobiontic invertebrates are perfectly adapted to Results

groundwater habitats. As a convergent evolutionary response to darkness, Compared to sampling surveys in surface waters, groundwater stud-
obligate groundwater animals are translucent, blind, exhibit enhanced ies very often reveal highly complex data sets, characterized by a large
tactile sense organs and lack circadian periodicity. Their bodies are percentage of samples not containing animals or only a few species,
elongated to vermiform facilitating locomotion in habitats with limited which are often rare or endemic. Statistical analysis often require
pore spaces. As an adaption to low and patchy food supply stygobites have careful selective data pre-treatment. A promising strategy for the
slow metabolic rates, low reproduction rates and they exhibit longevity, elucidation of meaningful spatial distribution patterns is a stepwise
compared to their surface water relatives. Shown are four frequently found data aggregation starting with the highest possible resolution (level of
representatives of the crustaceans; a) Niphargus laisi, Amphipoda; individual samples) followed by pooling the data according to sam-
SCHELLENBERG, 1936 b) Antrobathynella stammeri, Bathynellacea; JAKOBI, pling sites (wells) and subsequently according to meaningful units
1954, c) Fabaeformiscandona spec., Ostracoda d) Proasellus slavus, Isopoda, such as the type of aquifer, GeoRegs, hydrogeological units (HU) or
REMY, 1948) (Pictures: K. Grabow). major physiographic units (MPU). Following this protocol, no clear
distribution patterns for groundwater fauna were obtained analysing
conditions, have been developed and indices are provided21–23. at the level of single samples and sampling sites, respectively, apply-
However, a biogeoregional classification of groundwater habitats, ing multivariate statistics (ANOSIM: R , 0.09). Pre-selective data
as a necessary and solid basis for the standardized application of aggregations on the level of aquifer types and HU did not reveal
assessment schemes is still missing. meaningful and reproducible patterns as well. In contrast, analysis
For European groundwaters various classification systems are avail- of data aggregated at the level of MPU (ANOSIM: R50.896) and
able, such as the classification into hydrogeological and geochemical GeoRegs (ANOSIM: R50.221) offered reproducible distribution
units24, defined by the type of aquifer (e.g. porous, karst), its pet- patterns, statistically most striking for MPU.
rographical or hydrochemical properties, its hydraulic permeability, Thus, aggregating invertebrate community data within major
groundwater recharge and productivity. Accordingly, for Germany 17 physiographic units (MPU) best explained the groundwater faunal
groundwater landscapes (hydrogeological units) have been refined distribution on the biogeographical scale for the study area. The
by Kunkel et al.25. Opposite to the majority of ‘abiotic’ classification subsequent testing of the distribution patterns obtained by multi-
schemes26–29, only a few zoogeographic classifications are available, dimensional scaling (MDS) analysis for agreement with existing
such as the ones suggested by Botosaneanu1 or Husmann30. How- bioregional classification schemes revealed that it neither matched
ever, these proved inapplicable in practice, due to their low biogeo- the spatial units classified by the EU-WFD35 nor the classification
graphical resolution1 and enormous complexity in habitat types30. systems mentioned above25,33,34 (Fig. 3a, b). Our findings emphasize
More recent approaches, such as the pioneering PASCALIS project31 that existing classification schemes are not adequate for the fauna in
(Protocols for the ASsessment and Conservation of Aquatic Life In the groundwater. In contrary, data evaluation hints at four major clus-
Subsurface), focussed on groundwater biodiversity delivering exciting ters, in the following termed stygoregions, characterized by faunal
diversity maps for Europe, but did not have the objective of a biogeo- communities significantly distinct (p,0.0001) in their (aggregated)
graphical classification of groundwater systems. Nevertheless, at that composition (Fig. 3 a, b, Table 1). The pairwise tests, corrected by a
time the idea of a stygoregional classification has first emerged4,32 and sequential Bonferroni test (see adjusted significance level in par-
was recently set into action by Stoch and Galassi5. Unfortunately, this enthesis) show significant differences between all four stygoregional
study was limited to the edge of the Southern Alps comprising only assemblages (Northern Lowlands - Central Uplands: p50,0013 (p5
one subterranean bioregion (stygoregion), i.e. ‘North Eastern Italy’. 0.0125); Northern Lowlands - South-Western Uplands: p50,0003
On a regional scale (100–10,000 km2) Hahn and Fuchs33 proposed (p50.0083); Northern Lowlands – Southern Uplands and Northern
so called ‘georegs’ (regional geology 5 aquifer type 1 major physio- Alps: p50,0024 (p50.025); Central Uplands – South-Western Uplands:
graphic unit: MPU) for the analysis of stygofaunal distribution patterns. p50,001; Central Uplands - Southern Uplands and Northern Alps:
Indeed, the assessment of freshwater surface systems is based on p50,0087 (p50.05); South-Western Uplands - Southern Uplands and
bioregional references, strongly influenced by the zoogeographical Northern Alps: p50,0015 (p50.016)).
classification from Illies34, with the good ecological state being the Analysis of similarities (ANOSIM) support this proposal by
key criterion35. For freshwaters in Central Europe classification sys- revealing strong differences between the faunal assemblages when
tems and assessment schemes, including biological criteria, are well tested for stygoregions (R50.896, p50.001 with d51) while being
developed, but do not take groundwater ecosystems into considera- less different when tested for the EU-WFD ecoregion classification
tion. The spatial classification systems are based on biogeographical, (R50.505, p50.002 with d51). To exclude that the introduction of
geomorphological, hydrological, physicochemical, and zoogeo- a dummy species for integration of ‘zero value samples’ into the
graphical aspects24,25,27,34,36,37. On a biogeographical scale (10,000 to analysis (see Material & Methods section) may have caused these

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Figure 2 | a) European main ecoregions and b) a topographic map of Germany depicting the proposed stygoregions: Coloured areas show the major
physiographic units37 (MPU), which were studied. The white areas compile MPU were no data was available. The colours refer to the individual
stygoregions, which were delineated according to invertebrate distribution patterns found in groundwater. The affiliation of the Lower Rhine Valley is
under debate, indicated by the blue-white hatching. Topographic map/GIS: http://www.eea.europa.eu/legal/copyright.

patterns, the ANOSIM was re-calculated without the dummy. Now, forelands, including the Pfälzerwald Mountains. The Central
the differences between the two classification systems were even Mountain Ranges were not covered by ice shields but were
more pronounced (stygoregions: R50.475, p50.001, EU-WFD clas- strongly affected by permafrost soils and low precipitations. The
sification: R50.196, p.0.05). To further test the reproducibility and forelands mark the southern borders of the ice shields 4 0 .
statistical quality of the distribution patterns obtained, the data set Invertebrate groundwater communities are mainly characterized
was split into two sets of different sampling times and the MDS was by ubiquist species, so-called post-glacial recolonisers5,7,33, with only
recalculated. Analysis of the two individual data sets revealed almost few endemic species41 (Table 1). Invertebrates were found in around
identical results (data not shown). 65% of the wells sampled.
In the following, the four stygoregions proposed for Central 3) The South-Western Uplands are generally characterized by a
Europe are described in brief: highly diverse fauna in groundwater that have not been affected by
the periods of glaciation. The proportion of stygobites (obligate
1) The Northern Lowlands comprise groundwater systems that had groundwater species) is high and the invertebrate communities pre-
been strongly affected by pleistocene ice shields and are character- sent generally reflect in their composition the pleistocenious Danube
ized by fine porous sediments and low oxygen concentrations. As a catchment area situation with a diverse amphipod and ostracod
result, these groundwaters are naturally unpopulated or stygofauna fauna33 (Table 1). Because of major overlaps in species composition,
is scarce18,39. Only 15% of the wells investigated were populated by so far, the Lower Rhine Valley was included in this stygoregion,
invertebrates (Table 1). although the groundwater dwelling invertebrate fauna of the
2) The Central Uplands comprise the groundwater habitats of the Lower Rhine Valley exhibits a lower diversity. 76% of the wells
Central Mountain Ranges and the adjacent sub-mountainous investigated were populated by invertebrates.

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Figure 3 | Non-metrical multi-dimensional-scaling plot (MDS) depicting biogeographical distribution patterns of groundwater dwelling
invertebrates. Symbols refer to a) freshwater surface systems according to the EU-WFD35; b) stygoregions recommended for Central European
groundwater systems. The labels of the symbols refer to the official German MPU37 (not listed in detail). Plotted are Bray-Curtis similarities of faunal
means that were pooled for the major physiographic units of Germany37.

4) The Southern Uplands and Northern Alps comprise those areas by the EU-WFD (Central Uplands, Alpine foothills, Alps) are very
that were covered by the pleistocenious ice shields of the Alps and the different from the stygoregions (Fig. 3a, b). The southern assem-
Black Forest. The species composition is similar to those of the blages differed markedly from those of the surface aquatic fauna of
South-Western Uplands, but generally less diverse (Table 1). Charac- the Central Uplands and the Alps (EU-WFD 2000) (Fig. 2a, b).
teristic for this stygoregion is some species (i.e. Niphargus strouhali; Another striking difference to the surface aquatic fauna is the far
Amphipoda) which have so far only be recorded from Austria, the outreach of stygofaunal species from the Uplands into the EU-
southeast neighbour state (Schellenberg 1942b). Invertebrates were WFD-boundary areas of the Northern Lowlands (Fig. 3a, b).
found in almost 80% of the wells sampled.
Compared to bioregional classification systems for surface waters, Discussion
groundwater stygofaunal distribution patterns differ most notably in The biogeographical distribution patterns found for groundwater
the central and southern groundwater habitats: Here, the ecoregions dwelling invertebrates in Central Europe differ significantly from

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Table 1 | Species matrix of groundwater dwelling invertebrates and their distribution in different stygoregions. Species are classified as
stygobiontic and non-stygobiontic. Abbreviations: NL: Northern Lowlands, CU: Central Uplands, SWU: South-Western Uplands, SU &
NA: Southern Uplands and Northern Alps. C. 5 Crustacea
Stygoregion NL CU SWU SU & NA

No. of GW-monitoring wells 40 60 376 38

No. of samples 116 223 821 81

Stygobiontic species Taxonomic group
Parastenocaris phreatica Copepoda, C. x
Parastenocaris phyllura Copepoda, C. x x
Bogidiella albertimagni Amphipoda, C. x x x
Crangonyx subterraneus Amphipoda, C. x x x
Niphargellus nolli Amphipoda, C. x x x
Niphargus fontanus Amphipoda, C. x x x
Diacyclops languidoides Copepoda, C. x x x
Graeteriella unisetigera Copepoda, C. x x x
Proasellus cavaticus Isopoda, C. x x x
Niphargus aquilex Amphipoda, C. x x
Microniphargus leruthi Amphipoda, C. x x
Parastenocaris germanica Copepoda, C. x x
Chappuisius singeri Copepoda, C. x x
Bathynella natans Syncarida, C. x x
Fabaeformiscandona breuili Ostracoda, C. x
Fabaeformiscandona latens Ostracoda, C. x
Fabaeformiscandona wegelini Ostracoda, C. x
Parastenocaris psammica Copepoda, C. x
Schellencandona belgica Ostracoda, C. x
Schellencandona insueta Ostracoda, C. x
Schellencandona triquetra Ostracoda, C. x
Niphargopsis casparyi Amphipoda, C. x
Niphargus kochianus Amphipoda, C. x
Niphargus laisi Amphipoda, C. x
Niphargus puteanus Amphipoda, C. x
Niphargus tatrensis Amphipoda, C. x
Acanthocyclops gmeineri Copepoda, C. x
Acanthocyclops kieferi Copepoda, C. x
Bryocamptus typhlops Copepoda, C. x
Chappuisius inopinus Copepoda, C. x
Echinocamptus pilosus Copepoda, C. x
Elaphoidella elaphoides Copepoda, C. x
Graeteriella laisi Copepoda, C. x
Moraria fontinalis Copepoda, C. x
Nitocrella omega Copepoda, C. x
Parapseudoleptomesochra spec. Copepoda, C. x
Parastenocaris c.f. glacialis Copepoda, C. x
Anthrobathynella stammeri Syncarida, C. x
Bathynella freiburgensis Syncarida, C. x
Parabathynella c.f. ferdii Syncarida, C. x
Pseudantrobathynella husmanni Syncarida, C. x
Proasellus coxalis Isopoda, C. x
Proasellus walteri Isopoda, C. x x
Niphargus auerbachi Amphipoda, C. x x
Niphargus bajuvaricus Amphipoda, C. x x
Schellencandona schellenbergi Ostracoda, C. x x
Niphargus inopinatus Amphipoda, C. x x
Niphargus foreli Amphipoda, C. x x
Niphargus kieferi Amphipoda, C. x x
Parastenocaris c.f. moravica Copepoda, C. x x
Cryptocandona kieferi Ostracoda, C. x x
Fabaeformiscandona bilobata Ostracoda, C. x x
Mixtacandona laisi Ostracoda, C. x x
Acanthocyclops rhenanus Copepoda, C. x x
Proasellus slavus Isopoda, C. x x
Acanthocyclps venustus Copepoda, C. x x
Acanthocyclops sensitivus Copepoda, C. x x
Parastenocaris c.f. aedis Copepoda, C. x
Bathynella chappuisi Syncarida, C. x
Nitocrella hirta tirolensis Copepoda, C. x
Niphargus strouhali Copepoda, C. x

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Table 1 | Continue

Stygoregion NL CU SWU SU & NA

No. of GW-monitoring wells 40 60 376 38

No. of samples 116 223 821 81

Non-stygobiontic species
Pristina proboscidea Oligochaeta x
Tubifex tubifex Oligochaeta x
Diacyclops crassicaudis Copepoda, C. x x x x
Dorydrilus michaelseni Oligochaeta x x x x
Marionina riparia Oligochaeta x x x
Aelosoma hyalina Oligochaeta x x
Cernovsvitoviella atrata Oligochaeta x x x
Bryocamptus minutus Copepoda, C. x
Paracyclops poppei Copepoda, C. x
Potamothrix ssp. Oligochaeta x
Troglochaetus beranecki Polychaeta x x x
Paracyclops fimbriatus Copepoda, C. x x x
Diacyclops bisetosus Copepoda, C. x x
Diacyclops languidus Copepoda, C. x x
Mesenchytraeus armatus Oligochaeta x x
Bryochamtus echinatus Copepoda, C. x x
Parastenocaris brevipes Copepoda, C. x x
Aelosoma niveum Oligochaeta x x
Haplotaxis gordioides Oligochaeta x x
Tubifex ignotus Oligochaeta x x
Tubifex species A Oligochaeta x x
Tubificidae species B Oligochaeta x x
Bythiospeum ssp. Gastropoda x x
Acanthocyclops robustus Copepoda, C. x
Acanthocyclops vernalis Copepoda, C. x
Cyclops strenuus Copepoda, C. x
Cyclops vicinus Copepoda, C. x
Cypria ophtalmica Ostracoda, C. x
Diacyclops bicuspidatus Copepoda, C. x
Eucyclops serrulatus Copepoda, C. x
Eudiaptomus gracilis Copepoda, C. x
Macrocyclops albidus Copepoda, C. x
Megacyclops viridis Copepoda, C. x
Moraria brevipes Copepoda, C. x
Moraria pectinata Copepoda, C. x
Nitocra hibernica Copepoda, C. x
Thermocyclops crassus Copepoda, C. x
Tropocyclops prasinus Copepoda, C. x
Candona weltneri Ostracoda, C. x
Ancylus fluviatilis Gastropoda x
Aelosoma hemprichi Oligochaeta x
Aelosoma psammophylum Oligochaeta x
Aelosoma quaternarium Oligochaeta x
Amphichaeta leydigi Oligochaeta x
Buchholzia appendiculata Oligochaeta x
Eiseniella tetraedra Oligochaeta x
Fridericia perrieri Oligochaeta x
Marionina argentea Oligochaeta x
Nais c.f. variabilis Oligochaeta x
Phyllognathopus viguieri Oligochaeta x
Potamothrix hammoniensis Oligochaeta x
Pristinella bilobata Oligochaeta x
Psammoryctides albicola Oligochaeta x
Pseudocandona albicans Oligochaeta x
Rhyacodrilus falciformis Oligochaeta x
Uncinais uncinata Oligochaeta x
Paracamptus schmeili Copepoda, C. x
Cryptocandona vavrai Ostracoda, C. x
Spirosperma velutinus Oligochaeta x
Vejdovskiella comata Oligochaeta x

existing surface classification systems such as the aquatic bioregions goregions. In Central Europe, the distribution of groundwater fauna
by Illies34 and the European ecoregions35, implying the need of an on the large scale is mainly a result of quarternary glaciations, which
independent classification system of groundwater habitats, i.e. sty- have severely affected species richness and composition5,42. Our data

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underline the general latitudinal gradient of species richness, declin- up to 100,000 km2. However, Stoch & Galassi5 describe a much
ing from south to north, which was already observed in earlier studies smaller stygoregion for north-east Italy.
and which is much less pronounced with organisms from surface Although it is a drawback that there are many areas in Germany,
freshwaters4,5,43. Cumulative species curves (data not shown) indicate Europe, and worldwide not yet sampled, we see this as a dynamic
that continued sampling will definitely expand todays groundwater development. Individual stygoregions may still be expanded or
species richness in Central Europe, which is true for most regions diminished in size in the future and new stygoregions might be
worldwide33,44–46. However, the distribution patterns obtained from defined. This was true also for the various classification systems for
the data collected in our study are robust, and the spatial distribution surface ecosystems. Attemps should be made in the future to har-
of individual groups, such as niphargids, isopods, and copepods monize and merge data sets obtained by different sampling strat-
match well with earlier studies47–51. egies, e.g. the European groundwater biodiversity data set produced
Four stygoregions are now proposed, the Northern Lowlands, the in PASCALIS, for the review and extention of the stygoregion
Central Uplands, the South-Western Uplands, and the Southern approach to the European scale. With our data set from Central
Uplands and Northern Alps. The groundwater faunal impoverish- Europe the delineation of stygoregions based on aggregation of com-
ment, which is considered typical of the Northern Lowlands (inver- munity data at the level of MPUs proved to be an appropriate ap-
tebrates were absent from almost 85% of the wells), is a consequence proach. Nevertheless, in other parts of the world other spatial sub-
of species extinction during glaciation and inhibited post-glacial units such as the type of aquifer, GeoRegs, HU or others might be
recolonization33. It left a glacially shaped subsurface, mainly com- more appropriate.
posed of sands and fine materials with limited pore space. Moreover, In conclusion, distribution patterns of fauna in groundwaters of
due to considerable amounts of organic matter and slow ground- Central Europe on a regional and continental scale proved to be
water flow, oxygen is extremely low or absent25. In comparision, the significantly different from any classification scheme related to
stygoregion of the Central Uplands was less affected by the periods of hydrogeology, geochemistry, and surface fauna. If we intend to assess
glaciation, and as such had served as refugial areas for some species - the ecological status of groundwater systems, as routine for surface
although many aquifers are assumed of have been dried out or have aquatic systems, a reliable classification along with the definition of
been affected by permafrost52. After the end of the ice age, the sur- reference conditions is required. We propose the refinement of
viving groundwater species complemented by many ubiquitous spe- Europe’s water saturated subsurface into stygoregions, which are
cies coming from the south recolonized the Central Uplands as well bioregions that comprise both surface and subsurface features. Our
as the groundwater habitats of the formerly glacified sub-mountain- investigations led to the deliniation of four different stygoregions in
ous forelands, where environmental conditions were now appropri- Germany and Central Europe, to our opinion an indispensable initial
ate (e.g. pore space and sufficient amounts of oxygen)33,42. The low step towards an ecological assessment scheme of groundwater eco-
endemism in this stygoregion is a consequence of this post-glacial systems and a really sustainable groundwater management18,51. In the
recolonisation. near future we have to investigate the yet unexplored reaches to fill
The highly diverse groundwater fauna characteristic for the sty- the groundwater fauna maps. Moreover, first attempts are already
goregion South-Western Uplands is predominately composed of made to evaluate the synchronization of fauna data with those of
ancient stygobionts, which have not been affected by glaciation. As microbial communities in groundwaters23. An integrative and eco-
such, it is likely that this region provided refuges for stygobionts in logically sound groundwater ecosystems assessment scheme will
the deep karst or in the alluvium of large streams33. The affiliation of have to take faunal, and microbiological as well as physicochemical
the Lower Rhine Valley groundwater communities to the ones of the criteria into account.
South-Western Uplands is not fully clear yet (Fig. 2b, 3b). All clas-
sification systems, biological as well as geographical ones, consider Methods
Study regions. The faunal data presented originate from sampling surveys conducted
the Lower Rhine Valley as part of the Northern Lowlands. From a in a wide variety of aquifers across Germany (Fig. 2b), including unconsolidated
groundwater faunistic point of view, however, the Lower Rhine Val- porous aquifers, karst and fractured aquifers. A total of 515 groundwater monitoring
ley is very different from the impoverished Northern Lowlands. Our wells were repeatedly examined (2–5 times) between the years 2002 and 2009. Most
data indicate, that fauna is similar to the South-Western Uplands, samples were obtained from near-surface groundwater in approximately 10–80 m
depth. The major selection criteria for the choice of sampling areas and wells were the
implying that the groundwaters of the Lower Rhine Valley are con- geographic representativity and comparability of faunal data sets. While Hahn &
nected via the middle Rhine valley to the Pleistocenious catchments Fuchs33, frequently cited here, focused on the state of Baden-Wuerttemberg, this
of the rivers Danube and Main, which harbour a distinct fauna33. current paper encompasses samples from all over Germany.
The status of the stygoregion Southern Uplands and Northern Alps
Fauna sampling and taxonomic identification. We extracted the groundwater
is still under discussion. Although its fauna mirrors in general the dwelling invertebrates from the bottom of monitoring wells by using a phreatic net
South-Western Uplands fauna, species richness is lower and some sampler (75 mm mesh size), according to Hahn and Fuchs33. All faunal samples were
species, such as Niphargus strouhali, are unique to this region and immediately stored in a refrigerator box. Following live observations, samples were
have so far been known only from groundwater habitats located fixed with 4% formaldehyde before further processing. The taxonomic identification
was based on morphological characteristics. Crustaceans, oligochaetes, polychaetes
further south and east, i.e. from Austria47. We suppose, that recol- and gastropods were major target groups and determined to species level. Specimens
onization of the Southern Uplands and Northern Alps took place both of other taxonomic groups were excluded from further analyses. Ecological
from the South-Western Uplands and from the east via the ‘intersti- characterizations of the species follow in general Schellenberg47, Einsle48, Janetzky
tial highways’ of the Danube and its southern tributaries. These et al.49, Meisch50, and Schminke51, but were simplified to a stygobiontic vs. non-
stygobiontic classification according to Hahn22, Deharveng et al.45, and Datry et al.54.
results corroborate to the earlier findings by Hahn & Fuchs33, that
indicate a high subsurface connectivity in that area. Data analyses. Dealing with groundwater faunistic data means to analyze an
Without doubt, the fauna data available for groundwaters in extremely heterogeneous data sets with high proportions of faunistical zero values,
Germany are far from being complete. However, the study areas low species numbers per site, many rare species and strongly varying abundances.
Thus, for the biogeographical analyses pre-selective aggregation of the data was
selected are representative of the main Central European landscapes. inevidable. The problem of aggregation though, is the loss of information and possible
The delineation of stygoregions which has been proposed already pre-determination due to the choice of aggregation units. To minimize these
earlier4,5,32 stands for a common step forward in coming up with an problems, the aggregation units should be as small as possible and aggregation should
independent classification scheme for groundwater fauna and habi- be proceeded stepwise. We first analysed our data prior to aggregation. Then we the
data were aggregated on the level of sampling sites (wells), type of aquifer, geological
tats53. In dependence to the specific environmental condition of an region (GeoReg), hydrogeological unit (HU), major physiographic unit (MPU). The
area, the dimension of the stygoregions may vary considerably. quality of the aggregates was tested with respect to a potential bioregional classifi-
According to our data from Central Europe, stygoregions encompass cation for groundwater. Using this iterative approach, best results for our data were

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obtained by aggregating at the level of MPU from which the stygoregional classifica- 19. Steube, C., Richter, S. & Griebler C. First attemps towards an integrative concept
tion derived. Since zero values (sample not containing animals) may also be a chara- for the ecological assessment of groundwater ecosystems. Hydrogeol. J. 17, 23–35
cteristic of certain areas, unpopulated wells were considered in the analysis as well. (2009).
Following the above mentioned tests, total counts of faunal populations collected in 20. European Groundwater Directive: Directive 2006/118/ECEU GWD of the
wells were averaged for each major physiographic unit (MPU) and subsequently European Parliament and of the Concil of 12 December 2006 - Official J. of the
fourth root-transformed. Data aggregation was conducted to reduce data scattering of European Comm. L372 (2006).
individual wells, which is a result of the heterogeneous faunal distribution that is 21. Korbel, K. L. & Hose, G. C. A tiered framework for assessing groundwater
naturally found in groundwater. Since the faunal data, even after fourth root-trans- ecosystem health. Hydrobiologia 661, 329–349 (2011).
formation, did not show a normal distribution, exclusively non-parametrical meth- 22. Hahn, H. J. The Groundwater-Fauna-Index: A first approach to a quantitative
ods were used for statistical analyses. Patterns in faunal community structure were ecological assessment of groundwater habitats. Limnologica 36, 119–137 (2006).
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variate analysis of variance (PERMANOVA) and analysis of similarity (ANOSIM) für Grundwasserökosysteme (Developing biological methods and criteria for
based on Bray-Curtis distance. The Bray-Curtis measure was chosen because it does groundwater ecosystems). Final report. Umweltbundesamt No FKZ 3708 23 200,
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are unpopulated22. Before generating the MDS, a dummy variable (d51) was added to overview of major groundwater composition at European scale. Eviron. Geol. 55,
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Acknowledgements How to cite this article: Stein, H. et al. Stygoregions – a promising approach to a bioregional
We thank the German Federal Environment Agency (Umweltbundesamt) and the German classification of groundwater systems. Sci. Rep. 2, 673; DOI:10.1038/srep00673 (2012).
Working Group of the Federal States on Water Issues (Bund/Länder-Arbeitsgemeinschaft

SCIENTIFIC REPORTS | 2 : 673 | DOI: 10.1038/srep00673 9