Sie sind auf Seite 1von 20

See

discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/227906862

The response of Chironomidae to sediment


pollution and other environmental
characteristics in urban...

Article in Freshwater Biology December 2007


DOI: 10.1111/j.1365-2427.2007.01840.x

CITATIONS READS

55 157

4 authors, including:

Melissa E Carew Vincent Pettigrove


University of Melbourne University of Melbourne
37 PUBLICATIONS 707 CITATIONS 82 PUBLICATIONS 947 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Lower Goulburn River Long-Term Intervention Monitoring Project View project

All content following this page was uploaded by Melissa E Carew on 05 November 2014.

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document
and are linked to publications on ResearchGate, letting you access and read them immediately.
Freshwater Biology (2007) doi:10.1111/j.1365-2427.2007.01840.x

APPLIED ISSUES

The response of Chironomidae to sediment pollution and


other environmental characteristics in urban wetlands
M E L I S S A E . C A R E W * , V I N C E N T P E T T I G R O V E , R E N E E L . C O X * A N D A R Y A . H O F F M A N N *
*Centre for Environmental Stress and Adaptation Research, Departments of Zoology and Genetics, The University of Melbourne,
Vic., Australia

Research and Technology, Melbourne Water Corporation, Melbourne, Vic., Australia

SUMMARY
1. We investigated the distribution of chironomid taxa in urban wetlands in the greater
Melbourne area, Australia, to test if their distribution was influenced by sediment
pollution and other environmental variables.
2. For identification of the Chironomidae, DNA markers generated via polymerase chain
reactionrestriction fragment length polymorphism of cytochrome c oxidase sub unit I
(COI) were validated against morphology and reference specimens for more than 5000
chironomids representing over 80 species. DNA-based identification generally concurred
with morphological separation, but also indicated the existence of cryptic diversity in some
genera.
3. Non-metric multidimensional scaling (NMS) and canonical correspondence analysis
(CCA) showed chironomid assemblages were structured among wetlands and could be
linked to several habitat characteristics. However, Chironomidae assemblages were only
weakly linked to sediment pollution.
4. Logistic regressions identified potential bioindicators of sediment pollution. Riethia
stictoptera, Tanytarsus inextentus, Coelopynia and Chironomus februarius were negatively
associated and Chironomus duplex was positively associated with sediment pollution.
Thresholds for the pollution sensitivities of specific species were mostly similar to those
established with previous microcosm tests.
5. Several other environmental factors influenced the distribution of specific chironomid
taxa. Salinity, substratum type and submerged and riparian vegetation were particularly
important.
6. We conclude that specific chironomid taxa rather than assemblages have potential as
bioindicators of sediment pollution provided their ecological preferences are considered
and their pollution sensitivities are characterized using multiple methods. The integration
of DNA-based techniques should facilitate accurate and rapid identification of bioindi-
cators species.

Keywords: Chironomidae, bioindicators, urban aquatic ecosystems, DNA identification, polymerase


chain reactionrestriction fragment length polymorphism

Introduction

Correspondence: Melissa Carew, Departments of Genetics and Urbanization can lead to numerous detrimental chan-
Zoology, Level 2, Bio21 Institute, The University of Melbourne, ges in aquatic ecosystems, including deterioration of
3010 Vic., Australia. E-mail: mecarew@unimelb.edu.au aquatic and riparian habitats, invasion by exotic
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd 1
2 M. E. Carew et al.
species, changes to hydrology, and increased water many environmental parameters, including sediment
temperatures, turbidity and contamination by pollut- pollution. Multiple evaluations are important for
ants (Ellis & Hvitved-Jacobsen, 1996; Marsalek, 1998; ensuring potential bioindicator species are respond-
Paul & Meyer, 2001). Sediment pollution is likely to ing in a predictable and specific manner to sediment
play an important role in biological impacts associated pollution.
with urbanization (Pitt, 1995), as it is known to reduce Many larval chironomids are difficult to morpholog-
macroinvertebrate diversity and change community ically identify to species; this means that some poten-
composition (Bendell-Young et al., 2000; Pettigrove & tially good bioindicators cannot be easily identified.
Hoffmann, 2005). As a result, interim guidelines have Developing species-specific DNA markers may pro-
been developed for acceptable concentrations of pol- vide a simple means to identify morphologically
lutants in sediments (e.g. Australian and New Zealand similar chironomid species (Sharley, Pettigrove &
Environment and Conservation Council/Agriculture Parsons, 2004; Carew, Pettigrove & Hoffmann, 2005).
and resource Management Council of Australia and Once established, a DNA-based identification system
New Zealand (ANZECC/ARMCANZ), 2000; MacDon- has the added advantage of becoming automated,
ald, Ingersoll & Berger, 2000). which could also reduce the time and cost of routine
Aquatic sediments are well-known sinks, seques- identification. Although DNA-based identification has
tering many types of pollutants in urban areas (Pitt, been successfully developed for some chironomid
1995). Although chemical analyses may indicate groups (Newburn & Krane, 2002; Carew, Pettigrove &
whether sediments are toxic to aquatic life, this Hoffmann, 2003; Sharley et al., 2004; Carew et al., 2005),
assumes that all potential pollutants are analysed, it has not been used to identify the wide range species
and that detection limits are adequate to detect that are collected in field surveys or to determine
pollutants at potentially toxic levels. Furthermore, whether morphologically cryptic larvae exhibit differ-
the bioavailability of some sediment pollutants can ent environmental or pollution sensitivities.
vary with changes in pH, temperature and redox This study investigates whether chironomid assem-
potential (Ankley & Schubauer-Berigan, 1995). blage structure and the distribution of specific chir-
Catchment geology and edaphic characteristics can onomid taxa can be linked to sediment pollution and
also influence what levels of urbanization sediment other environmental variables in the greater Mel-
are likely to become toxic (Pettigrove & Hoffmann, bourne area (GMA), Australia. We develop a sampling
2003). For these reasons, the effects of pollutants are protocol to collect common chironomids from wet-
often better described by examining the response of lands, and test for associations between the chirono-
the resident biota. mid taxa and pollution or habitat features. Species
Sediment-dwelling Chironomidae are potentially identification was undertaken with the assistance of
useful because they are typically diverse and abun- polymerase chain reactionrestriction fragment length
dant in urban freshwaters, and often have direct and polymorphism (PCRRFLP) analysis of a region of the
prolonged contact with sediment (Berg, 1995; Bea- cytochrome c oxidase subunit I (COI) gene to assess, if a
van, Sadler & Pinder, 2001; Paul & Meyer, 2001; molecular approach could be useful for recognizing
Beasley & Kneale, 2002). As different chironomid chironomid species in routine monitoring.
species vary in their sensitivities to environmental
stressors, they can make good indicators of anthrop-
ogenic change (Bazzanti & Bambacigno, 1987; Cran- Methods
ston et al., 1997; Cranston, 2000a; Ruse, Herrmann &
Study area
Sublette, 2000; Mousavi, Primicerio & Amundsen,
2003). Furthermore, the relationship of many indi- Surveys of Chironomidae from wetlands in the GMA
genous chironomid taxa with sediment pollution can (within a 100 km radius of the city of Melbourne) were
be compared using both field-based microcosms and undertaken in spring 2003 and 2004 (Fig. 1). Wetlands
field surveys (Pettigrove & Hoffmann, 2005). Field- were selected due to the high densities of chironomids
based microcosms can isolated the impact of sedi- in these environments and also because wetlands
ments alone, while field surveys provide a holistic typically accumulate sediment and associated pollut-
perspective by comparing species distributions to ants (Pitt, 1995; King & Richardson, 2002). Two major
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 3

Clay

Australia
Macedon ranges

Clay Melbourne

Basalt SRP
RLM DRH
TLT

Brisbane ranges

Dandenong ranges
Clay
OWL
SWC

CLA
Melbourne
SLP city centre
HBW

N
LEW
Port Phillip Bay
Fig. 1 Sites in the greater Melbourne area
(GMA), Australia, where chironomids 0 12.5 25 50 Coastal plain
were sampled. Sites used for the initial Kilometres
chironomid and sediment surveys are
indicated by three letter codes.

ecoregions occur within the GMA: a low rainfall basalt samples were used for large sites (>20 000 m2 surface
region, and a more diverse region of sedimentary area) which gave a >90% chance of detecting species
materials (Marchant, Wells & Newall, 2000; Pettigrove at a frequency of 1%. Three samples were taken for
& Hoffmann, 2003). The north-west and west of the medium size sites (c. 50020 000 m2 surface area) and
GMA are dominated by basalt, and the north-east and two samples for sites below c. 500 m2 surface area.
east consist of sands or loams overlaying clays. A third
ecoregion to the south-east of the GMA contains coastal
Chironomid collection
plains and dunes (Walsh et al., 2001; Pettigrove &
Hoffmann, 2003), but <10% of sites sampled were Chironomids were sampled only from the littoral
located within this region. habitat (depth 0.30.8 m and devoid of emergent
plants) to limit confounding habitat effects associated
with differences in surface area, water depth and
Sampling
habitat complexity between wetlands (Pettigrove,
Field sampling was conducted in spring at 100 sites in 1990; Parsons & Norris, 1996). This habitat was chosen
2003 and 73 sites in 2004. In total, 121 sites were to maximize the diversity of chironomids (White &
sampled, with 53 sites replicated across both years. Irvine, 2003), while also maximizing the proportion of
Sediment samples were taken from the 121 sites. Sites sediment-dwelling chironomids sampled (as opposed
sampled in 2003 typically had low-to-moderate pol- to those living on emergent vegetation). Samples were
lution, while 20 sites collected in 2004 received runoff collected from each site with a rectangular dip net
from industrial areas and major roads, and therefore (0.5 0.3 m; mesh size 240 lm) using a 5-min kick
were more polluted. These polluted sites were inclu- method (Chessman, 1995). Chironomids were sorted
ded to increase the size of the pollution gradient and identified from each sample independently, and
sampled and the likelihood of detecting species the information from all samples from a site was
sensitive to higher pollution levels. combined prior to analysis.
Based on the results of the initial survey outlined in An initial survey of four large wetlands (SLP, CLA,
the methods (see below), at least two chironomid OWL and DRH) was conducted to determine the
samples were obtained for each wetland. This was number of chironomid samples to be taken per site
expected to result in >90% of species present at a and the time needed to sort chironomids from each
relative abundance of 5% or greater being sampled. sample (Fig. 1). Ten samples were collected from each
As a result of sites varying substantially in size, four site and washed in the laboratory to remove any
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
4 M. E. Carew et al.
suspended solids, sand or large soil particles, and eters associated with the presence/absence of chir-
preserved in 100% ethanol at 4 C. Chironomids were onomid taxa. Habitat descriptors of the entire wetland
sorted in the laboratory by picking through the entire included the type and coverage of riparian (large and
sample; this enabled the collection of a representative small trees, shrubs, sedges and grasses) and aquatic
sample of the different chironomids present. To sort (emergent, floating and submerged) vegetation, as
chironomids, a sample was placed in a sorting tray well as the benthic habitat (determined from examin-
and chironomids were picked out for 15 min which ing the substratum and dip net contents). Other
allowed all taxa in a sample to be identified. To descriptors were the amount of sediment resuspend-
determine number of samples needed to get an ed generated during sampling (plume), predominant
adequate representation of the chironomid taxa in a catchment land use (urban, agricultural, industrial or
wetland, multiple samples were collected and the natural), volume of storm water runoff (high, med-
probability of detecting species that occurred at a ium, low), if sites were connected to streams, and
relative abundance of >1%, 5% and 10% was obvious signs of pollution (e.g. sediment odours
assessed. This showed that two samples per site were and motor oils floating on the water surface or
able to detected >90% of common taxa (at >5% overlaying sediments). Water quality parameters,
relative abundance) and that four samples detected including turbidity (i.e. clear, slight, turbid and
>90% of common taxa with a relative abundance of opaque), dissolved oxygen (DO), electrical conductiv-
>1%. For wetlands with >20 000 m2 surface area four ity (EC), temperature and pH, were also measured
samples were collected, while fewer samples were (Table 1).
obtained from smaller wetlands (the number of Sediment samples collected from sites were filtered
samples per wetland area was always similar). through a 64-lm nybolt mesh net and analysed for
total heavy metals (As, Cu, Cd, Cr, Pb, Ni, Hg & Zn),
total organic carbon (TOC), total phosphorus (TP) and
Chironomid species identification
total Kjeldahl nitrogen (TKN) (Table 1). Before deter-
In most cases, chironomid larvae were identified to the mining concentrations, filtered samples were air-dried
genus level under a dissecting microscope and then and digested using a modification of the U.S. Envi-
stored in 100% ethanol at )20 C. In the few cases ronmental Protection Agency Method 200.2. To deter-
morphologically similar genera could not be easily mine total petroleum hydrocarbon (TPH) content,
distinguished, they were sorted into the same vial (e.g. whole sediment samples were air-dried and analysed
Cladopelma and Microchironomus). For species identifi- using the U.S. Environmental Protection Agency
cations, where >20 individuals per genus were present Method 8015A. The TPH content is presented as four
at a site, a subsample of 20 individuals per genus was size fractions based on carbon chain length (Table 1).
taken for morphological/DNA analysis. Larval head Repeated weekly sampling over a month at four
capsules of genera with morphological keys were sites (RLM, SRP, LEW and HBW) was undertaken to
mounted and keyed using Cranston (2000b), while the determine, if multiple measurements of sediment and
remaining bodies were used for DNA analysis follow- pore water pollutants as well as water parameters at
ing Carew et al. (2003). Species-specific markers were sites were similar (Fig. 1). Sediment samples were
developed using PCRRFLP of the mitochondrial COI taken from four locations within sites in the first week
gene, as outlined in Carew et al. (2003). PCRRFLP and at one location per site in each of the subsequent
profiles were then matched to corresponding identifi- 3 weeks. Sediments were assessed for metals (Cr, Cu,
able larval head capsule morphology. For morpholog- Pb, Ni and Zn), TOC, TP, TKN and TPH. Water
ically cryptic larvae, cytologically identified reference quality measures (DO, EC, temperature and pH) and
specimens or other identifiable life stages were linked pore water concentrations of metals were taken from
to larvae for identification. four locations within sites in the first week and also in
each of the subsequent 3 weeks. Pore water samples
were collected according to Australian and New
Environmental variables
Zealand Environment and Conservation Council/
Habitat and physicochemical data were collected at Agriculture and resource Manangement Council of
each wetland site to examine environmental param- Australia and New Zealand (ANZECC/ARMCANZ)
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 5
Table 1 Sediment and water quality parameters from survey sites in 2003 and 2004. The range and mean of each variable are given

2003 2004

Environmental variables Range Mean Range Mean

Water
pH 4.6811.3 7.57 4.6811.3 7.51
Electrical conductivity (lS cm)1) 53.223800 1369 66.117310 952
Dissolved oxygen (ppm) 2.2329.90 10.07 1.1417.78 8.73
Water temperature (C) 10.226.0 16.7
Sediment
Total arsenic (mg kg)1) 182 5.92 <188 7.74
Total cadmium (mg kg)1) <5 <10
Total chromium (mg kg)1) 573 22.72 1393 39.26
Total copper (mg kg)1) 3139 22.75 2.6679 60.06
Total lead (mg kg)1) 5402 32.38 6870 80.65
Total mercury (mg kg)1) <4 <15
Total nickel (mg kg)1) 266 16.12 0.5424 30.98
Total zinc (mg kg)1) 72540 160.24 97340 448.31
Total phosphorus (mg kg)1) 517570 657 63.512200 917
Total Kjeldahl nitrogen (mg kg)1) 36714600 2890.11 34176500 4415
Total organic carbon (%) 0.256.06 2.04 <0.516.1 2.62
Total petroleum hydrocarbons (mg/kg)1):
C6C9 fraction <5 <10
C10C14 fraction <501630 64.60 <251830 75.24
C15C28 fraction <1007260 444.81 <1009130 621.63
C28C36 fraction <1007310 446.60 <10013200 730.77

(2000) guidelines and analysed for Ca, Cu, Pb, Mg, Ni pollutants from the weekly repeat environmental
and Mn. Pore water metal concentrations were recor- sampling of four sites (RLM, SRP, LEW and HBW).
ded as they may reflect the bioavailable proportion of Principal components (PCs) were extracted using a
heavy metals in sediments and link more directly to correlation matrix, with each PC representing uncor-
metal toxicity (Ankley et al., 1994). However, we related linear combinations of the sediment pollut-
found pore water metal concentrations were highly ants. Loadings of sediment pollutants onto each PC
variable at sites between weeks and often below axis indicated their influence on the axis. Where
detection limits. This made it difficult to relate multiple sediment pollutants loaded strongly (>0.5)
chironomid assemblages to pore water metal concen- onto a PC axis, factor scores were extracted using the
trations. For these reasons, pore water measures were regression method. Factor scores were then used to
not considered further. represent correlated sediment pollutants in A N O V A s.
We also determined if sediment and water quality Site and weekly differences for pollutant concentra-
measures varied consistently between sites over a tions in sediments were examined using A N O V A s.
period longer than a month. Sediment and water Variance components were extracted with restricted
quality measures were taken from nine sites (RLM, maximum likelihood (REML) and these provide an
SRP, LEW, HBW, OWL, SPL, DRH, TLT and SWC) in indication of variation within and/or between sites,
spring 2003 and compared with samples at the same and between sampling weeks.
sites taken in autumn 2004 (Fig. 1). Variation among collections (or years) for sediment
pollutants was also summarized using PCA (as
above). Differences between sites and collections for
Data analysis
sediment pollutants, EC and pH were examined using
All analyses were undertaken in S P S S 13.0 for unbalanced A N O V A s and variance components were
Windows (SPSS Inc., Chicago, IL, U.S.A.), except extracted with REML to allocate variation to sites as
where indicated. Principal components analysis well as collections. TPHs could not be analysed by
(PCA) was used to summarize correlated sediment A N O V A s because of the high number of samples

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
6 M. E. Carew et al.
below detection limits in one or both collections. PCA with unrotated correlation matrices and varimax
However, an indication about the consistency of rotated covariance matrices were used to select a final
measurements across collections was obtained by list of quantitative variables and determine the most
correlating values from sites that had concentrations informative means of summarizing correlated varia-
above detection limits on two sampling occasions. bles. Variables that loaded poorly on any axes ()0.5 to
Ordination and regression methods were used to 0.5) (see below) were not included in the final PCA.
examine the relationship between chironomids and All others were considered to strongly load onto axes
environmental attributes. All analyses used qualitat- (Costello & Osborne, 2005). Axes were included only
ive chironomid data and were conducted on each if eigenvalues were >1. The final list of correlated
collection separately. Treating collections separately quantitative variables was then summarized as factor
in the analysis allowed us to determine, if relation- scores for ordination and regression analyses.
ships between taxa and environmental variables Habitat information was condensed to several un-
detected in one collection were also present when a correlated variables that best described site differences
different set of sites was included. Rare taxa (occur- between wetlands, with the exception of turbidity that
ring at <5% of sites) were excluded from the analysis was weakly correlated with EC in 2003, but not 2004.
as their occurrence may be due to chance rather than Turbidity was included in the analysis of both 2003 and
ecological conditions (see McCune & Grace, 2002). 2004 data as it is known to affect the distribution of
For the environmental data, some pollutant con- macroinvertebrates (Timms & Boulton, 2001; de Meut-
centrations were below detection limits and were left- ter, Stoks & De Meester, 2005), including chironomids
censored using Helsels robust method (HeIsel, 1990). (Cranston, 2000b). Variables describing riparian veget-
We only included censored values for variables that ation (trees, shrubs and sedges) around the entire
had no more than 40% of values below detection wetland were significantly correlated (Spearmans
limits. Variables with >40% of their values below rank, P < 0.001) and were therefore summed to pro-
detection limits were excluded. This included Cd, Hg duce a single variable that indicated riparian habitat
and the TPH fractions C6C9 and C10C14 for both quality. The proportion of submerged vegetation pre-
collections and the TPH fraction C15C28 in 2003. The sent where chironomid samples were taken was also
remaining quantitative variables were checked for included as a variable, as this indicated whether
normality using the KolmogorovSmirnov test (Sokal animals were collected in macrophyte dominated
& Rohlf, 1995), and where required log (x + 1) systems and could be living on vegetation rather than
transformed to achieve normality. To remove redund- in sediments. Plume and clay substratum were inclu-
ancy in the data set, the extent to which all environ- ded as they estimate the soft sediment/silt deposited at
mental variables were correlated was examined with a site (estimated during sampling), and the dominant
Spearmans rank correlations. Qualitative variables wetland soil type (reflecting ecoregion differences)
that were significantly correlated (r2 > 0.3) with quan- respectively. These factors are known to affect the
titative variables were eliminated. These included abundance and composition of chironomids (Bazzanti,
catchment land use, storm water volumes and silt, as 2000; Ciesielka & Bailey, 2001; Walsh et al., 2001).
well as signs of pollution which were correlated with Ordination analysis was completed using P C - O R D
various sediment pollutant measures. Qualitative version 4.34 (McCune & Mefford, 1999) and regres-
habitat variables that were significantly and positively sion analysis was conducted with S P S S 13.0 for
correlated were summed. Alternatively, single habitat Windows (SPSS Inc.). Non-metric multidimensional
variables were used to summarize site differences scaling (NMS) and canonical correspondence analysis
explained by several correlated habitat variables; for (CCA) were used to examine variation in chironomid
instance basalt and clay benthic substrates were assemblages and relate this to environmental varia-
negatively correlated so clay was selected to represent bles. NMS was performed with relative Srensens
substratum type. In these instances, habitat variables distance measures and all analyses were run in the
that are known to affect chironomid distributions autopilot slow and thorough mode to avoid the issue
were favoured. Variables exhibiting significant daily of local minima. A Monte Carlo significance test based
or weekly variation, such as DO and water tempera- on 50 random runs determined the significant axes.
ture, were eliminated from the analysis. Exploratory Multiple linear regression analysis (stepwise selection;
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 7
P 0.05 to enter and leave model) between selected correlated and could be summarized as two PCs. The
environmental variables and NMS axes were calcula- metals Cr, Cu, Pb, Ni and Zn, and phosphorus loaded
ted for both linear and nonlinear responses. Squared >0.68 on to the first PC axis, while TKN and TOC loaded
terms were used to investigate unimodal responses. >0.73 onto second axis. Pollutants that were below
However, this procedure will ignore oblique relation- detection limits (As, Cd, Hg and all TPH fractions) were
ships. Only significant regression coefficients with R2 excluded from the PCA. A N O V A s indicated that no
values >0.20 are presented. CCA eigenvalues from the pollutants measured in sediment varied significantly
first three axes were compared with variance in over the 4-week period [PC 1 (metals and TP) P 0.67,
chironomid assemblages. A Monte Carlo procedure F 0.51, d.f. 3,12; PC 2 (TKN and TOC) P 0.21,
was used to test the significance of the eigenvalues F 1.77, d.f. 3,12 respectively). Therefore, chirono-
and speciesenvironment correlation. Interset correla- mids present in these wetlands were probably exposed
tions from P C - O R D were examined to see which to a relatively constant level of pollution. PC 2 (TKN
environmental variables had the greatest influence and TOC) also varied significantly between the four
on the CCA axes. sites (P 0.003, F 8.34, d.f. 3,12) reflecting
Finally, to explore the response of specific species to potentially different pollution stresses at these sites.
environmental variables, we used the logistic regres- A N O V A s for water quality parameters established EC
sion approach outlined in Peeters & Gardeniers (1998). was the most stable water measure and did not vary
Logistic regressions are commonly used to examine over 4 weeks (P 0.52, F 0.77, d.f. 3,48), while pH
relationships between species and their environment was relatively consistent (P 0.155, F 2.22, d.f.
(Ter Braak & Looman, 1986; Peeters & Gardeniers, 1998; 3,48) between sites when considered across weeks.
Mezquita et al., 2005). For each species included in the For the comparison across years, a PCA revealed all
NMS analysis, logistic regressions were first run on pollutants (Cr, Cu, Ni, Zn, TP, TKN and TOC) loaded
presence/absence data of individual species singly for >0.52 onto the first PC axis. A N O V A on PC 1 indicated
each environmental variable. Tests were undertaken to that concentrations of sediment pollutants varied
assess whether there was a significant association significantly between sites (P < 0.01, F 2.33, d.f.
between presence/absence and the variable using the 3,22) but did not vary across years (P 0.27, F 1.40,
logit function B0 + B1x, where x is the environmental d.f. 1,22). The variance between sites was high
variable. We also included a B2x2 term in the logit (65%) compared with between years (2.4%). Water
function but this term was almost never significant and measurements were variable between years. EC was
therefore excluded. We initially tested if B1 differed the only water measure that varied significantly
significantly from 0 (P < 0.01) in either collection. across sites (P < 0.001, F 35.29, d.f. 7,27) but not
Multiple stepwise logistic regression was then applied years, with 95% of the variation attributed to between
to determine the minimal set of variables for predicting site differences. For pH, there was a significant site
the occurrence of each species, using only those collection interaction (P < 0.001, F 10.47, d.f. 7,27)
variables that were significant in the single regressions. accounting for 81% of the variance, suggesting that
To assess the minimal set, one variable was left out per pH levels at sites varies between years. Correlations of
analysis and the relative contribution of individual TPH fractions across two sampling occasions revealed
variables was then determined. The variables selected that the high molecular weight TPH fractions C15C28
for each species in these analyses were compared across and C29C36 were significantly correlated between
the two collections. These were then used to determine collections (n 28, r 0.59, P 0.03 and n 30,
species associations with environmental variables and r 0.45, P 0.03, respectively). The lower TPH C10
compared to other published data. C14 fraction was not correlated (n 7, r 0.27, P
0.55), but this may have been influenced by the small
sample size for this fraction.
Results
Sampling protocols and repeated sampling Chironomid species identification and occurrence
Sediment pollutants from the repeated sampling of Eighty-five chironomid taxa were collected in 2003
four sites (RLM, SRP, LEW and HBW) were highly and 58 taxa in 2004 (Appendix 1). The Chironominae
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
8 M. E. Carew et al.
were the most abundant and easily identifiable sub-
Chironomid taxa and environmental responses
family (55 and 38 taxa, representing 69% and 65% of
chironomid abundance in 2003 and 2004, respect- Many taxa collected were rare (Appendix 1), with 58
ively). Therefore this study is biased to species from out of 85 taxa in 2003 representing only 2.6% of the
this subfamily. All Chironominae groups could be total chironomid abundance, and 19 out of 55 taxa in
easily resolved to species using DNA markers, with 2004 representing only 3.2% of chironomid abun-
the exception of Cryptochironomus which was resolved dance. We did not include rare taxa (which typically
to genus. Cryptochironomus showed high COI PCR had a low frequency of occurrence and abundance) in
RFLP diversity compared with other species (data not our analyses, as they were unlikely to be reliably
shown), even though only a single Australian species sampled in our field surveys and lacked statistical
is described (Cranston & Martin, 1996). Similarly, high power in our analyses.
COI diversity was found within Procladius and As expected, sites in 2004 were more polluted,
Coelopynia (Tanypodinae). Procladius was the most having higher average concentrations of pollutants
dominant genus in both collection years, present at c. than the 2003 sites (Table 1). The samples collected in
90% of sites and representing >25% of the total 2004 also covered a wider range of sediment pollutant
chironomid abundance. Some Procladius species can concentrations. In contrast, the water quality param-
be recognized based on larval morphology (Roback, eters in 2003 had higher mean concentrations and
1982; Cranston, 2000b). However, high COI diversity varied more than in 2004.
between larvae suggests there may be more species Environmental data were reduced to nine variables
than currently described. Coelopynia also exhibited for the 2003 collection and seven variables for the 2004
high COI diversity despite only a single Australian collection. We found that varimax rotated PCA
species, Coelopynia pruinosa Freeman, being described yielded no additional information when compared
(Cranston & Martin, 1996). Thus, these taxa were only with unrotated PCA, so unrotated PCA was used to
identified to genus. Other Tanypodinae and the summarize correlated variables. In 2003, exploratory
majority of Orthocladiinae (with the exception of PCA showed that pH loaded poorly onto the PC axes
Cricotopus and Paratrichocladius) were also identified and was not included in the final PCA (Table 2). PCA
to the genus level because of a lack of morphological revealed three components that accounted for 75% of
characters, individuals or identifiable life stages to the variation between sites. PC 1 was loaded with As,
verify the DNA restrictions profiles. Apart from Cu, Pb, Zn, TP, TKN, TOC, Ni and Cr. PC 2 was
Procladius and Coelopynia, genera from these sub- loaded with Cr, Ni and was negatively associated
families were rarely found. with TPH (C29C36 fraction), while PC 3 was primarily

Table 2 Principal component loadings for quantitative environmental variables measured in 2003 and 2004

2003 2004

PC 1 (49%) PC 2 (15%) PC 3 (11%) PC 1 (61%) PC 2 (10%) PC 3 (8%)

TPH C15C28 fraction* 0.827 )0.227 0.420


TPH C29C36 fraction 0.455 )0.543 )0.319 0.810 )0.267 0.409
Arsenic 0.635 0.394 0.765 0.217
Chromium 0.576 0.717 0.774 )0.494
Copper 0.901 )0.211 0.936 )0.237
Lead 0.862 0.916
Nickel 0.577 0.744 0.769 )0.397
Zinc 0.833 )0.314 0.886
Total organic carbon 0.787 )0.307 0.861 0.293
Total Kjeldahl nitrogen 0.696 )0.316 0.307 0.862 0.219
Total phosphorus 0.806 0.259 0.864
Electrical conductivity 0.276 0.299 0.718 0.810
pH )0.633

Loadings between )0.2 and 0.2 are not shown. Variables with a high loading (>0.5 or )0.5) are in bold text.
*Total petroleum hydrocarbon (TPH) C15C28 fraction was not included in 2003 principal component (PC).

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 9
loaded with EC. In 2004, three PCs were extracted that collection were related to PC 1 (sediment pollutants),
accounted for 79% of the variance. PC 1 loaded with which represented sediment pollution. Also, no non-
sediment pollutants, while PC 2 loaded with water linear associations, excluding oblique relationships,
parameters, pH and EC. PC 3 did not have partic- were found between NMS axes and environmental
ularly high loadings for any environmental variable. variables. In 2004, when sites representing a broader
Therefore, this axis was not included in further pollution range were sampled, NMS axis 1 was
analyses. Although for both collections PC 1 primarily related to PC 1 (sediment pollution) and NMS axis 2
loaded with sediment pollutants including nutrients to habitat features (clay substratum and riparian
and metals, a second PC axis (Ni, Cr and TPH) was vegetation). No relationship was found between
found in 2003 that was not present in 2004. The Ni, Cr NMS axis 3 and any environmental variable. Nonlin-
loading on the 2003 PC 2 axis could be attributed to ear responses detected with squared terms were only
the increased proportion of sites sampled that evident for NMS axis 1 (r2 0.375) reflecting a
occurred in basalt areas in the 2003 (c. 30% basalt) relationship with PC 1 (sediment pollution) (P
compared with 2004 (c. 10% basalt), as basalt soils in 0.013, r 0.121, SE 0.048) and PC 2 (EC and pH)
the GMA have higher background Ni and Cr levels (P 0.030, r )0.106, SE 0.048). Although some
(Pettigrove & Hoffmann, 2003). However, why Ni and significant relationships between chironomid assem-
Cr are negatively associated with TPH in this collec- blages and sediment pollution were detected using
tion is less clear. TPHs loaded strongly with other NMS, overall chironomid assemblages did not
sediment pollutants in 2004. strongly group sites with respect to sediment pol-
Non-metric multidimensional scaling analyses indi- lution.
cated chironomid taxa were structured in both sur- Canonical correspondence analysis axes in both
veys. Three-dimensional NMS solutions were used in sampling periods significantly correlated with envi-
each collection as changes in stress value with further ronmental variables, but the low eigenvalues (Table 4)
dimensions were minor; this was substantiated by and high inertia (3.28 for 2003 and 4.14 for 2004)
Monte Carlo test of significance for axes in 2003 (P indicated these associations were not strong and axes
0.0196) and 2004 (P 0.0196). The first three axes contributed weakly to the total variance. In 2003, only
accounted for 73.5% of the variance in 2003 and 70.2% the first axis was significant and was associated with
in 2004. The r2 values for each axis are given in riparian vegetation, clay substratum and to a lesser
Table 3. Chironomid taxa summarized in the NMS extent EC. These results were similar to those of the
axes were related to the selected environmental NMS, showing that habitat features had an influence
variables (Table 3). In 2003, only NMS axis 2 could on chironomid assemblages in 2003. The 2004
be related to environmental variables with an associ- collection, which sampled a wider range of pollu-
ation with habitat features (clay substratum, riparian tion concentrations, showed stronger gradients and
and submerged vegetation). No axes in the 2003 higher eigenvalues in the CCA (Table 4). Inter-set

Table 3 Stepwise linear regression of NMS axes showing regression coefficients (and SE) for environmental variables included in the
regression model

Environmental variable:
Collection Axis Axis r2 Model r2 regression coefficient (SE)

2003 1 0.211
2 0.288 0.311 Clay substratum: )0.397 (0.110)*** Riparian vegetation: Submerged vegetation:
)0.050 (0.017)** 0.080 (0.036)*
3 0.236
2004 1 0.164 0.221 PC 1 (metals, TPH, nutrients): )0.275 (0.062)***
2 0.282 0.323 Clay substratum: 0.501 (0.124)*** Riparian vegetation:
0.046.(0.020)*
3 0.26

NMS, non-metric dimensional scaling; SE, standard error; PC, principal component; TPH, total petroleum hydrocarbon.
*P < 0.05; **P < 0.01; ***P < 0.001.

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
10 M. E. Carew et al.
Table 4 CCA inter-set correlations of the first three axes with environmental variables

2003 2004

Variable Axis 1 Axis 2 Axis 3 Variable Axis 1 Axis 2 Axis 3

PC 1 (metals, 0.177 )0.299 )0.042 PC 1 (metals, )0.366 0.340 )0.079


nutrients, TOC) TPH, nutrients)
PC 2 (Ni, Cr, TPH) 0.401 0.164 0.054 PC 2 (EC, pH) )0.470 0.220 )0.301
PC 3 (EC) 0.429 0.176 )0.214 Plume )0.290 )0.087 0.275
Plume 0.031 )0.345 )0.078 Riparian vegetation 0.351 )0.069 )0.312
Riparian vegetation )0.648 )0.006 )0.201 Submerged vegetation )0.334 )0.114 )0.300
Submerged vegetation )0.571 0.110 )0.127 Substratum clay 0.651 0.202 0.119
Substrate clay 0.348 0.138 )0.304 Turbidity 0.403 0.097 )0.052
Turbidity )0.159 0.282 0.462
pH )0.007 0.059 0.043
CCA eigenvalues 0.180* 0.087 0.060 0.179* 0.118 0.103
CCA eigenvalue 0.0470.125 0.0410.086 0.0300.073 0.0580.168 0.0530.119 0.0420.091
ranges (MC test)

The strongest correlations are in bold text. CCA eigenvalues describing the relative strength of gradients and variation in species
scores in the 2003 and 2004 collections and MC tests for the significance of the first axis and estimated ranges are provided from 1000
randomized permutations for all axes. Eigenvalues that were greater than expected by chance are in bold text and underlined.
CCA, canonical correspondence analysis; MC, Monte Carlo; PC, principal component; TOC, total organic carbon; TPH, total petroleum
hydrocarbon; EC, electrical conductivity.
*P 0.001.

correlations showed that the first CCA axis was and Dicrotendipes (data not shown). The number of
associated with habitat characteristics, predominantly indicator taxa for sediment pollution identified by our
clay substratum, and to a lesser extent with PC 3 regression analysis was typically low, compared with
representing EC and pH (Table 4). The second CCA that shown from previous field-based microcosm tests
axis most strongly associated with PC 1 that indicated (Pettigrove & Hoffmann, 2005). We therefore investi-
sediment pollution, while the third CCA axis was gated the threshold pollutant levels for indicator
most strongly associated with riparian vegetation. species suggested by Pettigrove & Hoffmann (2005)
However, as in the NMS analyses, relationships were and compared this to our field samples, using Zn
relatively weak. concentrations as a surrogate for sediment pollution
Potential bioindicator species and ecological asso- (Fig. 2). In general, where the same species occurred
ciations were identified using logistic regression in both studies, we found similar thresholds for
(Table 5). For 28 species, comparisons could be made specific species (defined as the level where species
in both collections to selected environmental varia- declined in their frequency of occurrence). The only
bles. Some individual taxa appeared to have potential exceptions were Paratanytarsus grimmii Schneider,
as indicators of sediment pollution. Riethia stictoptera which was present at a few highly polluted sites.
Kieffer and Coelopynia exhibited significant negative This result was also found by Pettigrove & Hoffmann
relationships with sediment pollution in both collec- (2005) when they compared P. grimmii with Paratany-
tions (Table 5; Fig. 2). Chironomus februarius Martin tarsus species found in field surveys. Kiefferulus
and Tanytarsus inextentus showed significant negative intertinctus Skuse appeared be more tolerant of
associations with increased sediment pollution during pollution in the field compared with in microcosms
at least one collection. Chironomus duplex had a or could be affected by contaminants other than Zn.
positive association with sediment pollution in one Overall this comparison of field surveys to microcosm
collection, C. duplex showed more tolerance to pol- tests indicated that the responses of many species to
lution compared with C. februarius suggesting that sediment pollution occurred at particular threshold
congeneric species vary in their pollution associations levels. Furthermore, this result is strengthened by the
(Fig. 2). This divergent pattern among congeneric repeatability of these threshold levels in field surveys
species was also evident for Tanytarsus, Polypedilum which are confounded by habitat differences
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 11
Table 5 Estimated values for parameters B0 (intercept), B1 (regression slope) and percentage deviance reduction for species that
showed significant associations with pollutants in logistic regressions across both collections

R (%
Species Collection Variable B0 B1 deviance)

Chironomus duplex 2003 PC 3 (EC) )0.050 0.839 20.01


PC 1 (metals, nutrients, TOC) )0.061 0.507 7.90
2004 PC 2 (EC, pH) )0.384 0.748 16.95
Clay 0.693 )2.079 17.42
Turbidity 0.280 )0.798 29.03
Riparian vegetation 0.528 )0.241 16.34
Chironomus februarius 2003 PC 2 (Ni, Cr, TPH) )1.035 )0.697 13.74
Turbidity )0.535 )0.415 6.62
Clay substratum )2.140 1.735 12.19
2004 PC 1 (metals, TPH, nutrients) )1.089 )1.166 22.74
Coelopynia pruinosa 2003 PC 1 (metals, nutrients, TOC) )1.339 )0.557 8.23
2004 PC 1 (metals, TPH, nutrients) )2.123 )1.898 37.24
Dicrotendipes pseudoconjunctus 2003 PC 1 (EC) )0.788 0.829 20.42
Turbidity )0.154 )0.639 7.95
Submerged vegetation )1.398 0.443 8.18
2004 PC 2 (EC, pH) 0.928 1.119 26.16
Kiefferulus martini* 2004 PC 2 (EC, pH) )1.953 )1.099 24.28
Paramerina spp. 2003 PC 3 (EC) )3.212 1.112 34.43
Riparian vegetation )3.137 )0.099 29.49
Submerged vegetation )4.505 0.821 39.69
2004 PC (EC, pH) )3.272 1.209 51.36
Submerged vegetation )7.019 1.712 72.31
Riparian vegetation )1.557 )0.399 21.23
Riethia stictoptera 2003 PC 1 (metals, nutrients, TOC)* )1.097 )0.818 15.59
2004 PC 1 (metals, TPH, nutrients) )1.736 )1.131 18.92
PC (EC, pH) )1.630 )0.844 16.25
Tanytarsus semibarbitarsus 2004 PC (EC, pH) )4.020 1.577 85.00
Tanytarsus inextentus 2003 PC 1 (metals, TOC, nutrients) )2.603 )1.103 24.79
Riparian vegetation )3.043 0.195 15.23

PC, principal component; EC, electrical conductivity; TOC, total organic carbon; TPH, total petroleum hydrocarbon.
*Only in clay areas.

Absent in 2003.

No effects in 2004.

compared with the microcosm test which isolated vegetation. Kiefferulus martini Freeman and R. stictop-
impacts of sediment pollution from habitat features tera were only found at sites with a clay substratum.
and spatial differences. Chironomus februarius also showed a preference for a
Salinity (as indicated by EC) affected the distribu- clay substratum. Chironomus duplex was associated
tion of some species (Table 5). The presence of with a non-clay substratum. Tanytarsus inextentus
Tanytarsus semibarbitarsus Glover was associated with associated sites with riparian vegetation. In contrast,
increased salinity (and pH) in the 2004 collections, species such as C. duplex and Paramerina spp. were
while the presence of Paramerina spp., Dicrotendipes negatively associated with riparian vegetation. Param-
pseudoconjunctus Epler and C. duplex Walker was erina spp. was positively associated with submerged
associated with increased salinity across both years. vegetation in both collections, while D. pseudoconjunc-
Riethia stictoptera had negative associations with tus was also associated with submerged vegetation in
increased salinity (and pH) in 2004. one collection. In all cases where negative associations
Many species also exhibited significant relation- with turbidity were observed, a relationship with EC
ships with habitat variables. The most notable were was also found, except for C. februarius which was
associations with a clay substratum and riparian only associated with increased turbidity.
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
12 M. E. Carew et al.
0.7 0.7
Kiefferelus intertinctus Kiefferulus martini
0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
<50 51100 101500 5011000 >1000 <50 51100 101500 5011000 >1000

0.7
0.7
Tanytarsus fuscithorax Tanytarsus inextentus
0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
<50 51100 101500 5011000 >1000 <50 51100 101500 5011000 >1000

0.7
0.7
Riethia stictoptera Coelopynia
0.6
0.6
0.5
0.5
0.4 0.4
0.3 0.3
0.2 0.2
0.1 0.1

0 0
<50 51100 101500 5011000 >1000 <50 51100 101500 5011000 >1000

0.7 0.7
Polypedilum vespertinus Paratanytarsus grimmii
0.6 0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
<50 51100 101500 5011000 >1000 <50 51100 101500 5011000 >1000

0.7
0.7
0.6
Chironomus duplex Chironomus 'februarius'
0.6

0.5 0.5

0.4 0.4

0.3 0.3

0.2 0.2

0.1 0.1

0 0
<50 51100 101500 5011000 10012000 >2001 <50 51100 101500 5011000 10012000 >2001

Fig. 2 Frequency of occurrence of taxa versus Zn sediment concentration range for potential bioindicator taxa based on 121 sites
combined from 2003 and 2004. Arrows show the critical thresholds indicated by Pettigrove & Hoffmann (2005) for species that were
encountered in both studies. Sample sizes are 48 (<50 mg kg)1), 24 (51100 mg kg)1), 32 (101500 mg kg)1), 6 (5011000 mg kg)1), 3
(10012000 mg kg)1) and 5 (>2001 mg kg)1). Error bars indicate 1 SD.

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 13
polluted sites. Most relationships of chironomid taxa
Discussion with environmental variables using ordinations were
weak, though sediment pollution had a relationship
Sampling
with chironomid assemblages in 2004 when a wider
Biological methods are now extensively integrated range of polluted sites was sampled. We concur with
into water quality monitoring programmes world- Hawkins & Norris (2000) and Nijboer, Verdonschot &
wide. However, they often rely on collecting addi- Van Der Werf (2005), who found that chironomid taxa
tional physicochemical information to assist in alone are poor for classifying sites when compared
interpretation. Typically, physicochemical monitoring with taxonomically broader macroinvertebrate com-
utilizes spot measures which can vary substantially munities. Despite this, chironomid taxa identified to
over time and consequently this may limit the ability lower taxonomic levels are commonly found to
to relate biota to physicochemical information (van provide more information when included with other
Dam, Camilleri & Finlayson, 1998). Our study indica- macroinvertebrates in bioassessment studies, partic-
ted that, in the wetlands surveyed, some physico- ularly in urban areas (Beavan et al., 2001; Riva-
chemical measures did vary over different temporal Murray et al., 2002). King & Richardson (2002) sug-
scales, but sediment pollution and EC were typically gested that in wetland assessment, chironomids are
stable within sites throughout the study. This ensured best resolved to genus or species while other macro-
that chironomids were likely to be exposed to a invertebrates are resolved to family because many
constant stress and that measures could be related to chironomid genera and species show different affinit-
species distributions. Moreover, the high correlation ies to specific types of impairment. Congeners of
of different sediment pollutants showed that chirono- Chironomus, Kiefferulus, Dicrotendipes, Cricotopus and
mids were probably exposed to multiple pollutants Tanytarsus can exhibit markedly different sensitivities
at most sites. to pollutants (Cranston, 2000a; Bailey, Norris &
Repeat sampling was important in relating chir- Reynoldson, 2001; Beavan et al., 2001; Riva-Murray
onomid taxa to sediment pollution, salinity and et al., 2002). We observed this among species from
habitat. Our study showed at least two samples common genera; for example, C. duplex was tolerant
taken from large sites and sorted for 15-min detected to sediment pollution and salinity, whereas C. feb-
>90% of the common (>5% relative abundance) ruarius was more sensitive to high sediment pollution
chironomid taxa. Sample number was more import- and salinity.
ant than subsampling time. Fewer samples per site Chironomidae may be more useful at finer biomon-
would result in common taxa potentially going itoring scales, by selecting specific indicator species of
undetected, while subsampling for 15 mins resulted sediment pollution rather than focusing on entire
in no taxa remaining undetected. Similar macro- chironomid assemblages. We found several taxa that
invertebrate sampling methods in wetlands recom- showed associations with pollution and are potential
mend one- to six-samples for bioassessment (Cheal bioindicators. Riethia stictoptera, Coelopynia and T.
et al., 1993; Chessman, Traylor & Davis, 2002). These inextentus were common in the GMA, and were not
recommendations, however, are aimed at collecting detected at sites with moderate to high pollution. For
multiple macroinvertebrate families and/or include R. stictoptera this result is consistent with Pettigrove &
multiple habitats. The more limited sampling we Hoffmann (2005) who have shown this species to be
used was aimed at a single habitat, and this may sensitive to sediment pollution. However, our results
have reduced the diversity of chironomids, favouring suggest R. stictoptera is also sensitive to salinity and
benthic species, and lessened confounding effects of prefers sites with a clay substratum and riparian
other habitat features (Pettigrove, 1990; Parsons & vegetation. Such ecological preferences are likely to
Norris, 1996). influence the use of some chironomids as bioindicator
species. Substratum has previously been linked to the
distribution of many macroinvertebrate assemblages
Chironomids as bioindicators of sediment pollution
in the GMA (Marchant et al., 2000; Walsh et al., 2001).
Chironomidae assemblages were not particularly Similarly, salinity was linked to the presence
useful for discriminating between non-polluted and of certain chironomid species. Chironomus duplex,
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
14 M. E. Carew et al.
D. pseudoconjunctus Paramerina spp. and T. semibarbi- RFLP proved reliable in distinguishing chironomid
tarsus were associated with relatively high levels of species, which was concordant with other studies of
salinity in wetlands. Tanytarsus semibarbitarsus and molecular chironomid identification (Newburn &
C. duplex are already known to be common in saline Krane, 2002; Carew et al., 2003; Sharley et al., 2004;
environments (Glover, 1973; Paterson & Walker, 1974; Carew et al., 2005). However, some taxa proved
Timms, 1983; Kokkinn, 1986). However, C. duplex has difficult to identify with the COI PCRRFLP tech-
also been known to prefer nutrient enriched environ- nique. Cryptochironomus, Procladius and Coelopynia all
ments (Timms, 1983). As we found C. duplex to be showed high COI diversity that was not consistent
relatively more common in polluted wetlands, it may with existing taxonomy. Further investigation of these
serve as an indicator of pollution in more saline genera using DNA sequencing of COI, the use of
freshwater environments. nuclear markers and the morphological analysis of
Overall the number of chironomid taxa that showed pupal and adult life stages are required to adequately
significant relationships with sediment pollution was resolve these groups. Approaches combining mor-
lower than found by Pettigrove & Hoffmann (2005) in phological and DNA analysis have proven useful for
their field-based microcosm study utilizing non-pol- determining putative species in chironomids inclu-
luted and polluted sediments. Although many of the ding Cladopelma (Carew et al., 2005) as well as other
same species were found in both studies, our field invertebrates (Hebert et al., 2004; Page, Choy &
study did not show associations with pollution for Hughes, 2005).
P. grimmii, T. fuscithorax, P. vespertinus, K. martini and While PCRRFLP offers an inexpensive and rapid
K. tinctus which were evident in Pettigrove & means of screening COI diversity in chironomids,
Hoffmann (2005). Nevertheless, the same critical there are limitations. In this study, chironomids were
thresholds at which many of these species declined identified to genus prior to DNA analysis, enabling
tended to support Pettigrove & Hoffmann (2005), and specimens to be compared with close relatives. This
the lack of significant association in our field survey limits the number of profiles on gels, making manual
may reflect a lack of power and/or the influence of scoring easier. Another issue is that as the number of
other environmental factors on the distribution of species identified with PCRRFLP analysis increases,
these species. Kiefferulus tinctus may be more tolerant the chance of generating the same profile for two
and C. februarius may be less tolerant to sediment different species increases, particularly for genera
pollution than suggested by data in Pettigrove & where the number of restriction sites is low. This was
Hoffmann (2005), although tolerance levels of C. encountered when species profiles in one genus
februarius can be confounded by adaptation (Bah- resembled those in another genus. Although this
rndorff et al., 2006). A larger number of sites might was resolved by sorting to genus prior to PCRRFLP
better resolve pollution sensitivities of animals in the analysis and subsequent validation using morphol-
field. For example, over 1500 sites were used to ogy, it demonstrates that misidentification can occur,
predict the occurrence of macroinvertebrate families if chironomids are not identified to genus prior to
for AUSRIVAS models (Simpson & Norris, 2000). PCRRFLP. Despite these limitations, molecular
Moreover, additional information is needed on other approaches are becoming increasingly popular for
factors that may affect chironomid distributions, like ecological surveys of invertebrates. Sequencing COI
species succession, competition and seasonal variation or the popular COI barcoding (Hebert et al., 2003)
(Lenat, 1983; Johnson, 1995). rather than using PCRRFLP would probably circum-
vent the need to sort to genus as all nucleotide
differences in the PCR product are screened giving
Chironomid species identification
greater power in determining species. However, more
Accurate species diagnosis is essential to using chir- cost-effective high-throughput approaches for species
onomid species as bioindicators of sediment pol- identification are emerging that can enable species to
lution. The DNA methods for species identification be identified for a fraction of the time and cost of
used here were validated using larval, pupal and sequencing, PCRRFLP or morphological methods.
adult morphology, or cytologically identified refer- For instance, single nucleotide polymorphism pri-
ence specimens. DNA methods based on COI PCR mers based on PCRRFLP restriction sites could be
 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 15
developed to detect the presence of chironomid sediments: a proposed approach. Environmental
bioindicator species, eliminating the need to sort all Management, 18, 331337.
individuals to species. The emergence of microarray Australian and New Zealand Environment and Conser-
technologies based on species-specific oligos or vation Council / Agriculture and resource Manange-
probes, like those proposed in Summerbell et al. ment Council of Australia and New Zealand
(ANZECC/ARMCANZ) (2000) Australian and New
(2005), may also become useful for the rapid detec-
Zealand Guidelines for Fresh and Marine Water Quality.
tion of chironomid bioindicators. Assessments of
Vol. 1 The Guidelines. Australia Water Association,
sites could eventually be based on the automated Artarmon, NSW.
analysis of unsorted macroinvertebrate samples, Bahrndorff S., Ward J., Pettigrove V. & Hoffmann A.A.
rapidly identifying bioindicator species. Nevertheless, (2006) A microcosm test of adaptation and species
validation against morphology, and an understanding specific responses to polluted sediments applicable to
of intra-species molecular variability and species indigenous chironomids (Diptera). Environmental
boundaries, are essential steps to their implementa- Pollution, 139, 550560.
tion (Lee, 2004). Bailey R.C., Norris R.H. & Reynoldson T.B. (2001)
Our study suggests that particular chironomid Taxonomic resolution of benthic macroinvertebrate
species rather than assemblages may provide a communities in bioassessment. Journal of North
means of assessing the impact of sediment pollution American Benthological Society, 20, 280286.
Bazzanti M. (2000) Ecological requirements of
in urban areas, provided the ecology and pollution
chironomids (Diptera: Chironomidae) on the soft
sensitivities of species are understood. The use of
bottom of the river Arrone, central Italy. Journal of
field-based microcosms in conjunction with the Freshwater Ecology, 15, 397409.
ecological data collected in field surveys will be Bazzanti M. & Bambacigno F. (1987) Chironomids as
vital to characterizing pollution responses and water quality indicators in the river Mignone central
developing bioindicator species. Although chirono- Italy. Hydrobiological Bulletin, 21, 213222.
mids can be difficult to identify, DNA identification Beasley G. & Kneale P. (2002) Reviewing the impact of
could be applied in the future to recognize key metals and PAHs on macro invertebrates in urban
indicator species which could decrease the time and watercourses. Progress in Physical Geography, 26, 236270.
cost involved in assessing the impact of pollution in Beavan L., Sadler J. & Pinder C. (2001) The invertebrate
aquatic environments. fauna of a physically modified urban river.
Hydrobiologia, 445, 97108.
Bendell-Young L.I., Bennett K.E., Crowe A., Kennedy
Acknowledgments C.J., Kermode A.R., Moore M.M., Plant A.L. & Wood
A. (2000) Ecological characteristics of wetlands
We thank Leon Barmuta, Torbjrn Ekrem, Paul receiving an industrial effluent. Ecological Applications,
Umina, Andrew Weeks and two anonymous review- 10, 310322.
ers for comments on this manuscript. We thank Berg M.B. (1995) Larval food and feeding behavior. In:
Joanne Kearns, Renae Ayres, Dave Sharley, Matt Hall The Chironomidae: The Ecology and Biology of Non-biting
and Nick Lewis for their assistance with field samp- Midges (Eds P.D. Armitage, P.S. Cranston & L.C.V.
ling and processing. This study was funded primarily Pinder), pp. 136168. Chapman and Hall, London.
by the Australian Research Council through their Carew M.E., Pettigrove V. & Hoffmann A.A. (2003)
Special Research Scheme, with additional support Identifying chironomids (Diptera: Chironomidae) for
from Melbourne Water Corporation. biological monitoring with PCR-RFLP. Bulletin of
Entomological Research, 93, 483490.
Carew M.E., Pettigrove V. & Hoffmann A.A. (2005) The
References utility of DNA markers in classical taxonomy: using
cytochrome oxidase I markers to differentiate
Ankley G.T. & Schubauer-Berigan M.K. (1995)
Australian Cladopelma (Diptera: Chironomidae)
Background and overview of current sediment
midges. Annals of the Entomological Society of America,
toxicity identification evaluation procedures. Journal
98, 587594.
of Aquatic Ecosystem Health, 4, 133145.
Cheal F., Davis J.A., Growns J.E., Bradley S.J. & Whittles
Ankley G.T., Thomas N.A., Garrison A.W. et al. (1994)
F.H. (1993) The influence of sampling method on the
Assessing potential bioavailability of metals in

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
16 M. E. Carew et al.
classification of wetland macroinvertebrate com- Hebert P.D.N., Cywinska A., Ball S.L. & de Waard J.R.
munities. Hydrobiologia, 257, 4756. (2003) Biological identification through DNA barcodes.
Chessman B.C. (1995) Rapid assessment of rivers using Proceedings of the Royal Society of London B Biological
macroinvertebrates: a procedure based on habitat- Sciences, 270, 313321.
specific sampling, family level identification and a Hebert P.D.N., Penton E.H., Burns J.M., Janzen D.H. &
biotic index. Australian Journal of Ecology, 20, 122129. Hallwachs W. (2004) Ten species in one: DNA
Chessman B.C., Traylor K.M. & Davis J.A. (2002) barcoding reveals cryptic species in the neotropical
Family- and species-level biotic indices for skipper butterfly Astraptes fulgerator. Proceedings of the
macroinvertebrates of wetlands on the Swan Coastal National Academy of Sciences of the United States of
Plain, western Australia. Marine and Freshwater America, 101, 1481214817.
Research, 53, 919930. HeIsel D.R. (1990) Less than obvious: statistical treatment
Ciesielka I.K. & Bailey R.C. (2001) Scale-specific effects of of data below the detection limit. Environmental Science
sediment burial on benthic macroinvertebrate and Technology, 24, 17671774.
communities. Journal of Freshwater Ecology, 16, 7381. Johnson R.K. (1995) The indicator concept in freshwater
Costello A.B. & Osborne J.W. (2005) Best practices in biomonitoring. In: Chironomids: From Genes to
exploratory factor analysis: four recommendations for Ecosystems (Ed. P.S. Cranston), pp. 1127. CSIRO,
getting the most from your analysis. Practical Melbourne, Vic.
Assessment Research and Evaluation, 10, 19. King R.S. & Richardson C.J. (2002) Evaluating sub-
Cranston P.S. (2000a) Monsoonal tropical Tanytarsus van sampling approaches and macroinvertebrate
der wulp (Diptera: Chironomidae) reviewed: new taxonomic resolution for wetland bioassessment.
species, life histories and significance as aquatic Journal of North American Benthological Society, 21,
environmental indicators. Australian Journal of 150171.
Entomology, 39, 138159. Kokkinn M. (1986) Identification of two Australian salt-
Cranston P.S. (2000b) Electronic Identification Guide to the lake chironomid species form sub-fossil larval head
Australian Chironomidae. http://entomology.ucdavis. capsules. Palaeogeography, Palaeoclimatology,
edu/chiropage/index.html. (Last accessed on 27-June- Palaeoecology, 54, 317328.
06) Lee M.S.Y. (2004) The molecularisation of taxonomy.
Cranston P.S. & Martin J. (1996) Australasian/Oceanian Invertebrate Systematics, 18, 16.
Diptera Catalogue Web Version. http://hbs.bishop Lenat D.R. (1983) Chironomid taxa richness: natural
museum.org/aocat/chiro.html. (Last accessed on 27- variation and use in pollution assessment. Freshwater
June-06) Invertebrate Biology, 2, 192198.
Cranston P.S., Cooper P.D., Hardwick R.A., Humphrey MacDonald D.D., Ingersoll C.G. & Berger T.A. (2000)
C.L. & Dostine P.L. (1997) Tropical acid streams: the Development and evaluation of consensus-based
chironomid (Diptera) response in northern Australia. sediment quality guidelines for freshwater
Freshwater Biology, 37, 473483. ecosystems. Archives of Environmental Contamination
van Dam R.A., Camilleri C. & Finlayson C.M. (1998) The and Toxicology, 39, 2031.
potential of rapid assessment techniques as early Marchant R., Wells F. & Newall P. (2000) Assessment of
warning indicators of wetland degradation: a review. an ecoregion approach for classifying macro-
Environmental Toxicology and Water Quality, 13, invertebrate assemblages from streams in Victoria,
297312. Australia. Journal of the North American Benthological
Ellis J.B. & Hvitved-Jacobsen T. (1996) Urban drainage Society, 19, 497500.
impacts on receiving waters. Journal of Hydraulic Marsalek J. (1998) Challenges in urban drainage. In:
Research, 34, 711783. Hydroinformatic Tools for Planning, Design, Operation and
Glover B. (1973) The Tanytarsini (Diptera: Chirono- Rehabilitation of Sewer Systems (Eds J. Marsalek, C.
midae) of Australia. Australian Journal of Zoology, Maksimovic, E. Zeman & R. Price), pp. 123. NATO
Supplementary Series, 23, 403478. ASI Series 2. Kluwer Academic Publishers, Dordrecht/
Hawkins C.P. & Norris R.H. (2000) Effects of taxonomic Boston/London.
resolution and use of subsets of the fauna on the McCune B. & Grace J.B. (2002) Analysis of Ecological
performance of RIVPACS-type models. In: Assessing Communities. with a Contribution from Dean L. Urban.
the Biological Quality of Fresh Waters RIVPACS and Other MjM Software Design, Gleneden Beach, OR.
Techniques (Eds J.F. Wright, D.W. Sutcliffe & M.T. McCune B. & Mefford M.J. (1999) PC-ORD for Windows.
Furse), pp. 217228. Freshwater Biological Association, Multivariate Analysis of Ecological Data. MjM Software,
Ambleside, Cumbria. Gleneden Beach, OR.

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 17
de Meutter F.V., Stoks R. & De Meester L. (2005) The macroinvertebrates. Environmental Toxicology and
effect of turbidity state and microhabitat on Chemistry, 24, 170180.
macroinvertebrate assemblages: a pilot study of six Pitt R.E. (1995) Biological effects of urban runoff
shallow lakes. Hydrobiologia, 542, 379390. discharge. In: Stormwater Runoff and Receiving Systems:
Mezquita F., Roca J.R., Reed J.M. & Wansard G. (2005) Impact, Monitoring and Assessment (Ed. E.E. Herrick),
Quantifying species-environment relationships in non- pp. 127162. CRC, Boca Raton, FL.
marine Ostracoda for ecological and palaeoecological Riva-Murray K., Bode R.W., Phillips P.J. & Wall G.L.
studies: examples using Iberian data. Palaeogeography (2002) Impact source determination with
Palaeoclimatology Palaeoecology, 225, 93117. biomonitoring data in New York State: concordance
Mousavi S.K., Primicerio R. & Amundsen P.-A. (2003) with environmental data. Northeastern Naturalist, 9,
Diversity and structure of Chironomidae (Diptera) 127162.
communities along a gradient of heavy metal Roback S. (1982) The immature stages of some Australian
contamination in a subarctic watercourse. Science of Tanypodinae (Diptera), with some notes on adults.
the Total Environment, 307, 93110. Journal of the Australian Entomological Society, 21,
Newburn E. & Krane D. (2002) Molecular identification 147160.
of chironomid species. In: Chemicals in the Environment: Ruse L.P., Herrmann S.J. & Sublette J.E. (2000)
Fate, Impacts and Remediation (Eds R.L. Lipnick, R.P. Chironomidae (Diptera) species distribution related
Mason, M.L. Phillips & C.U. Jr Pittman), pp. 363383. to environmental characteristics of the metal-polluted
ACS Symposium series. Oxford University Press, New Arkansas River, Colorado. Western North American
York. Naturalist, 60, 3456.
Nijboer R.C., Verdonschot P.F.M. & Van Der Werf D.C. Sharley D.J., Pettigrove V. & Parsons Y.M. (2004)
(2005) The use of indicator taxa as representatives of Molecular identification of Chironomus spp. (Diptera)
communities in bioassessment. Freshwater Biology, 50, for biomonitoring of aquatic ecosystems. Australian
14271440. Journal of Entomology, 43, 359365.
Page T.J., Choy S.C. & Hughes J.M. (2005) The taxonomic Simpson J.C. & Norris R.H. (2000) Biological assessment
feedback loop: symbiosis of morphology and of river quality: development of AUSRIVAS models
molecules. Biology Letters, 1, 139142. and outputs. In: Assessing the Biological Quality of Fresh
Parsons M. & Norris R.H. (1996) The effect of habitat- Waters RIVPACS and Other Techniques (Eds J.F. Wright,
specific sampling on biological assessment of water D.W. Sutcliffe & M.T. Furse), pp. 125142. Freshwater
quality using a predictive model. Freshwater Biology, 36, Biological Association, The Ferry House: Far Sawrey,
419434. Ambleside, Cumbria.
Paterson C.G. & Walker K.F. (1974) Recent history of Sokal R.R. & Rohlf F.J. (1995) Biometry: The Principles and
Tanytarsus barbitarsus Freeman (Diptera: Practice of Statistics in Biological Research. W.J. Freeman
Chironomidae) in the sediments of a shallow, saline and Co, New York.
lake. Australian Journal of Marine and Freshwater Summerbell R.C., Levesque C.A., Seifert K.A., Bovers M.,
Research, 25, 315325. Fell J.W., Diaz M.R., Boekhout T., de Hoog G.S.,
Paul M.J. & Meyer J.L. (2001) Streams in the urban Stalpers J. & Crous P.W. (2005) Microcoding: the second
landscape. Annual Review of Ecological Systematics, 32, step in DNA barcoding. Philosophical Transactions of
333365. the Royal Society B Biological Sciences, 360, 18971903.
Peeters E. & Gardeniers J. (1998) Logistic regression as a Ter Braak C.J.F. & Looman C.W.N. (1986) Weighted
tool for defining habitat requirements of two common averaging, logistic regression and the Gaussian
gammarid. Freshwater Biology, 39, 605615. response model. Plant Ecology, 65, 311.
Pettigrove V. (1990) Importance of site selection in Timms B.V. (1983) A study of benthic communities in
monitoring the macroinvertebrate communities of the some shallow saline lakes of western Victoria,
Yarra River, Victoria. Environmental Monitoring and Australia. Hydrobiologia, 105, 165177.
Assessment, 14, 297313. Timms B.V. & Boulton A.J. (2001) Typology of arid-zone
Pettigrove V. & Hoffmann A. (2003) Impact of floodplain wetlands of the Paroo River (inland
urbanisation on heavy metal contamination in urban Australia) and the influence of water regime,
stream sediments: influence of catchment geology. turbidity, and salinity on their aquatic invertebrate
Australasian Journal of Ecotoxicology, 9, 119128. assemblages. Archiv Fur Hydrobiologie, 153, 127.
Pettigrove V. & Hoffmann A. (2005) A field-based Walsh C.J., Sharpe A.K., Breen P.F. & Sonneman J.A.
microcosm method to assess the effect of (2001) Effects of urbanization on streams of the
polluted urban stream sediments on aquatic Melbourne region, Victoria, Australia. I. Benthic

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
18 M. E. Carew et al.
macroinvertebrate communities. Freshwater Biology, 46, Conservation: Marine and Freshwater Ecosystems, 13,
535551. 331351.
White J. & Irvine K. (2003) The use of littoral
mesohabitats and their macroinvertebrate assem- (Manuscript accepted 8 June 2007)
blages in the ecological assessment of lakes. Aquatic

Appendix
Taxa collected from surveys in 2003 and 2004. The number of individuals collected (n); the number of sites sampled (sites) and the
percentage occurrence at sites (frequency) are indicated. For 2003 the number of sites and percentage occurrence are equal. Species
denoted with letter-number codes are from Cranston (2000b), while those denoted as sp. then a number were found in this study

2003 2004

Taxon n Sites Taxon n Sites % occurrence

Sub family: Chironominae Sub family: Chironominae


Chironomus australis Macquat 1103 41 Chironomus australis 188 21 29
Chironomus cloacalis Archley & Martin 68 12 Chironomus cloacalis 101 12 16
Chironomus duplex Walker 2436 47 Chironomus duplex 525 30 41
Chironomus februarius Martin 166 28 Chironomus februarius 198 22 30
Chironomus oppositus Walker 1060 53 Chironomus oppositus 1162 45 62
Chironomus pseudoppositus Martin 34 4 Chironomus pseudoppositus 8 5 7
Chironomus tepperi Skuse 596 31 Chironomus tepperi 152 6 8
Cladopelma sp.1 110 16 Cladopelma sp.1 114 18 25
Cladopelma sp.2 190 14 Cladopelma sp.2 45 9 12
Cladopelma sp.3 19 1 Cladopelma sp.3 15 1 1
Cladopelma sp.4 2 1 Cladopelma sp.4 26 4 5
Cladotanytarsus australomancus Glover 448 17 Cladotanytarsus australomancus 291 13 18
Cladotanytarsus bilinearis Glover 20 2 Cladotanytarsus bilinearis 20 2 3
Cladotanytarsus sp.1 2 1 Cladotanytarsus sp. 3 2 3
Cladotanytarsus sp.2 6 1 Cryptochironomus spp. 67 13 18
Cladotanytarsus sp.7 2 1 Dicrotendipes pseudoconjunctus 294 20 27
Cryptochironomus spp. 26 9 Dicrotendipes septemmaculatus 13 5 7
Dicrotendipes sp.4 80 15 Dicrotendipes sp.4 121 13 18
Dicrotendipes sp.8 1 1 Dicrotendipes sp.6 38 1 1
Dicrotendipes sp.9 2 2 Kiefferulus cornishi 77 10 14
Dicrotendipes K4 3 2 Kiefferulus intertinctus (K. tinctus) 58 13 18
Dicrotendipes pseudoconjunctus Epler 446 32 Kiefferulus martini 76 12 16
Dicrotendipes septemmaculatus Becker 9 3 Microchironomus B1 67 13 18
Dicrotendipes spp. 3 3 Parachironomus sp.1 6 3 4
Kiefferulus cornishi 68 10 Paratanytarsus grimmii 157 19 26
Kiefferulus sp.1 16 4 Paratanytarsus kathleenae 35 2 3
Kiefferulus intertinctus Skuse (K. tinctus) 88 16 Polypedilum leei 4 2 3
Kiefferulus martini Freeman 17 11 Polypedilum nubifer 100 20 27
Kiefferulus sp. 3 2 Polypedilum S1 18 6 8
Microchironomus B1 88 10 Polypedilum spp. 2 1 1
Nilodorum sp.1 1 1 Polypedilum vespertinus 31 11 15
Parachironomus sp.1 41 9 Riethia stictoptera 86 14 19
Paratanytarsus grimmii Schneider 201 33 Tanytarsus barbitarsis 7 1 1
Paratanytarsus kathleenae Glover 21 3 Tanytarsus edwardi 1 1 1
Polypedilum leei Freeman 7 1 Tanytarsus fuscithorax 12 3 4
Polypedilum nubifer Skuse 264 25 Tanytarsus inextentus 48 14 19
Polypedilum prasiogaster Freeman 2 1 Tanytarsus semibarbitarsus 57 4 5
Polypedilum S1 36 4 Tanytarsus sp.11 15 1 1
Polypedilum vespertinus Skuse 57 13 Tanytarsus spp. 1 1 1

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x
Chironomidae as bioindicators of pollution 19
Appendix (Continued)

2003 2004

Taxon n Sites Taxon n Sites % occurrence

Riethia stictoptera Kieffer 213 19 Sub family: Orthocladiinae


Tanytarsus barbitarsis Freeman 2 2 Corynoneura sp.1 18 7 10
Tanytarsus bispinosus Freeman 5 2 Cricotopus albatarsis 1 1 1
Tanytarsus edwardi Glover 4 1 Cricotopus annuliventris 2 2 3
Tanytarsus inextentus Skuse 60 10 Cricotopus parbicinctus 11 6 8
Tanytarsus semibarbitarsus Glover 6 2 Cricotopus sp.1 11 4 5
Tanytarsus sp.2 14 5 Cricotopus sp.3 7 4 5
Tanytarsus sp.5 2 2 Cricotopus sp.5 1 1 1
Tanytarsus sp.7 2 2 Cricotopus sp.11 5 4 5
Tanytarsus. sp.11 2 13 Cricotopus/Paratrichocladius sp.17 11 2 3
Zavreliella sp.1 1 1 Cricotopus/Paratrichocladius sp.18 1 1 1
Sub family: Orthocladiinae Compterosmittia sp.1 4 3 4
Cardiocladius sp. 1 1 Orthocladiinae unknown genus 9 1 1 1
Compterosmittia sp.1 10 2 Orthocladiinae unknown genus 10 1 1 1
Corynoneura spp. 5 1 Orthocladiinae nr. Compterosmitta 17 5 7
Cricotopus annuliventris Skuse 4 1 Paratrichocladius spp. 2 2 3
Cricotopus sp.1 14 5 Sub family: Tanypodinae:
Cricotopus sp.2 1 1 Ablabesmyia spp. 4 4 5
Cricotopus sp.3 9 3 Coelopynia 163 14 19
Cricotopus sp.4 1 1 Paramerina spp. 13 5 7
Cricotopus sp.5 30 8 Procladius spp. 1947 63 86
Cricotopus/Paratrichocladius sp.6 4 2
Cricotopus sp.7 1 1
Cricotopus sp.11 5 2
Cricotopus/Paratrichocladius sp.12 1 1
Cricotopus sp.14 1 1
Cricotopus/Paratrichocladius sp.15 2 1
Cricotopus sp.16 2 1
Cricotopus parbicinctus 138 21
Cricotopus spp. 5 4
Orthocladiinae nr. Compterosmittia 16 8
Orthocladiinae unknown genus 1 1 1
Orthocladiinae unknown genus 2 2 1
Orthocladiinae unknown genus 3 1 1
Orthocladiinae unknown genus 4 1 1
Orthocladiinae unknown genus 5 1 1
Orthocladiinae unknown genus 6 2 1
Orthocladiinae unknown genus 7 2 1
Orthocladiinae unknown genus 8 1 1
Paratrichocladius sp.1 3 1
Psectrocladius sp.1 3 1
Sub family: Tanypodinae:
Ablabesmyia spp. 19 6
Coelopynia 299 22
Monopelopia sp.1 1 1
Paramerina spp. 25 6
Pentaneurini genus C 5 1
Procladius spp. 2968 90

 2007 The Authors, Journal compilation  2007 Blackwell Publishing Ltd, Freshwater Biology, doi:10.1111/j.1365-2427.2007.01840.x

View publication stats

Das könnte Ihnen auch gefallen