Abstract
Abstract -Freshwater systems on volcanic oceanic islands have very particular characteristics as a result of their geological origins, relatively small size, distances from source areas for colonizers, and distinct catchment morphology. These factors result in freshwater communities that are clearly distinct from continental systems. Chironomid spatial and temporal distribution was investigated in chironomid assemblages at 21 sites across the Azores Archipelago. Results using PERMANOVA, a permutational multivariate analysis of variance, indicated significant longitudinal differences in assemblages but none between islands or over time.
Abstract
Links between hydromorphological and physicochemical variables and the community assemblage were assessed using DISTLM, a linear model for distance-based multivariate analysis. The percentage of variation explained by hydromorphological factors (31%) was slightly higher than that explained by physicochemical (28%) factors. Descriptors of land use (forest area, scrub area, natural area of catchment) and stream slope were found to be the best environmental predictors of chironomid assemblages in Azores. Physicochemical variables such as temperature, pH, nitrite, iron and conductivity were the principal drivers of change in chironomid composition in stream locations. Headwater sites, characterized by lower temperature, acid to neutral pH values, low conductivity, nutrient and metal concentrations were dominated by Rheocricotopus atripes. Mid-section sites, located in agricultural areas were dominated by Thienemanniella clavicornis. Urbanized lower reaches were characterized by higher temperature, pH, conductivity and nutrient levels and were dominated by Cricotopus sp. These results give essential information that allows us to predict the response of different chironomid species to hydromorphological and physicochemical gradients across the archipelago's streams contributing providing the basis for the development of tools for the implementation of the Water Frame Directive.
Introduction
Freshwater systems on volcanic oceanic islands differ from their continental counterparts due to their oceanic location (isolation), comparatively small size and geological youth. At a more local scale, catchment morphology and often a more extreme torrential seasonal flow regime also shape these systems. These biogeographical filters operate over a range of spatial scales, influencing colonizing organisms and events that shape the local freshwater assemblage. Even the absence of given taxon provides evidence on mechanisms governing these processes (Bilton et al., 2001; Covich, 2006; Cowie and Brenden, 2006) . Freshwater communities occurring on these islands tend to be depauperate but highly endemic (Borges and Brown, 1999; Walter, 2004; Hughes, 2006; Covich, 2009; . Oceanic island ecosystems tend to be simpler and offer great opportunities for the study of environmental factors determining the distribution of freshwater faunal elements such as the Chironomidae, the most abundant and diverse freshwater taxa in Azores archipelago Borges et al., 2010) . Such studies provide a better understanding of the key environmental drivers of change in freshwater communities, essential for the development and implementation of regionally appropriate conservation measures and ecological monitoring programmes.
Introduction
Many lotic ecological assessment protocols are based on benthic macroinvertebrates, one of the four biological elements specified in Water Frame Directive (WFD; European and The Council of the European, 2000) for assessing ecological status. However, many metrics, developed for continental streams and rivers, cannot be directly applied to insular systems, because of the distinct nature of their biotic assemblages [see Hughes (2005) for an overview of ecological monitoring on the Macaronesian islands]. For example, the commonly used EPT (Ephemeroptera, Plecoptera, Trichoptera) metric is inappropriate for Azores due to the absence of Plecoptera and the low diversity of both Ephemeroptera (one species reported) and Tricoptera (four species reported). Over twenty well-known metrics used to assess the suitability of macroinvertebrates in defining the ecological quality of Madeiran lotic systems based on perceived environmental impacts, were found to perform badly for the island of Madeira (Hughes, 2003) . A similar situation was reported by Gonc¸alves et al. (2008) for the Azores.
Introduction
A possible alternative approach is the study of the Chironomidae (Insecta, Diptera, Nematocera) as potential bioindicators of freshwater ecological status in Azorean streams, given their ubiquity, abundance and diversity. However, most macroinvertebrate based protocols only identify chironomids to family or sub-family or even omit them [e.g. Family Biotic Index -FBI (Hilsenhoff, 1988) ; Biological Monitoring Working Party -BMWP (Armitage et al., 1983) ]. Chironomids are often excluded because of the difficult larval taxonomy that involves time consuming preparation of slides for subsequent identification and the large numbers of larvae that tend to occur in benthic samples. This is unfortunate, since chironomid diversity can match or even exceed that of other aquatic group insects combined (Heino and Paasivirta, 2008) . Methods such as the Chironomid Pupal Exuviae Technique -CPET (Coffman, 1973; Wilson and Bright, 1973; Ruse and Wilson, 1995; Wilson and Ruse, 2005) largely overcome these difficulties because field and laboratorial protocols are well established and exuvial taxonomy is relatively easy, allowing identification to genus or even species. CPET is based upon the premise that the diversity and perceived tolerance of members of the aquatic chironomid larvae reflect local environmental conditions and that the winged adults ensure effective dispersal and colonisation of other aquatic habitats.
Introduction
Situated in the North Atlantic, the Azores constitute the most remote archipelago of the Palaearctic sub region of Macaronesia, which also comprises the archipelagos of the Madeira, Canary and Cape Verde. The Azorean freshwater fauna is dominated by insects, in particular the Diptera (Gonc¸alves et al., 2008; and especially the Chironomidae, or non-biting midges (Borges, 2005; ). The Azorean chironomid fauna has been studied by several workers during the last century (e.g. Frey, 1945; Stora, 1945; Freeman, 1959) . More recent records of Chironomidae were provided by Murray et al. (2004) and who gave a total of 44 dipteran to the Azores and and Raposeiro et al. (in press) who updated the list.
Introduction
Chironomid larvae inhabit almost every kind of aquatic habitat (Pinder, 1986; Ashe et al., 1987; Armitage et al., 1995a; Osborne et al., 2000) . As a consequence they have been used as biological indicators in freshwater ecosystems (e.g. Rosenberg and Resh, 1993; Wilson and Ruse, 2005; Raunio et al., 2007b; Raunio et al., 2007a; Heino and Paasivirta, 2008) . Further, studies relating environmental variables to the spatial and/or temporal patterns of chironomid distribution are reported from Australia (Hardwick et al., 1995; Dimitriadis and Cranston, 2007) , Finland (Raunio et al., 2007b; Heino and Paasivirta, 2008) , the United Kingdom (Ruse and Wilson, 1995; Ruse, 2000; Ruse and Davison, 2000) , Spain (Calle-Martı´nez and Casas, 2006; Punti et al., 2009) and Japan (Inoue et al., 2005) . The association between the environmental variables that determine chironomid community structure is complex, determined by physical, chemical and biological processes that vary both temporally and spatially in the lotic environment.
Introduction
The principal aims of this study were to characterize chironomid communities of Azorean streams, using chironomid pupal exuviae to (i) determine if there are differences among chironomid assemblages between two islands of the Azores archipelago and (ii) identify environmental and chemical factors that determine chironomid assemblage structure both temporally and spatially.
Introduction
These results will contribute to characterising chironomid distribution patterns along these particular lotic systems and provide information on factors determining the distribution of potential chironomid bioindicators across a quality gradient, ranging from reference to degraded conditions, paving the way to the development of regionally appropriate metric to describe ecological status, in accordance with the demands of the WFD (Hughes and Malmqvist, 2005; Hughes, 2006) .
Materials and Methods

Study area and sampling procedure
The Azores is an oceanic archipelago, comprising 9 volcanic islands in three geographical groups (eastern, central and western groups), located between the coordinates 37 to 40xN, 25 to 31xW (Fig. 1) . The oceanic and temperate azorean climate is characterized by low thermal amplitude, high levels of precipitation, high air humidity, and persistent wind (Cruz, 2003) , with mean annual temperatures of 14-18 xC and mean annual precipitation of 740-2400 mm. Most annual precipitation (65-70%) occurs between October and March (Bettencourt, 1979) .
Study area and sampling procedure
Azorean stream drainage networks are typically narrow and short with very steep, shallow channels often characterized by turbulent, torrential, and seasonal flow. Substrates are predominantly coarse, comprising bedrock, boulders, cobbles and sand. Background stream water chemistry is determined by (i) atmospheric input from oceanic-influenced precipitation, (ii) weathering of volcanic substrates and (iii) geothermal activity and spring water (Louvat and Allegre, 1998) .
Study area and sampling procedure
Forested areas (native and non-native) dominate the higher reaches of most catchments while agricultural and urban areas occupy the more accessible lower reaches. This study was conducted from early January to late November 2008, on the islands of Sa˜o Miguel (eastern group, Fig. 1 ) and Flores (western group, Fig. 1 ). A total of 21 sites (seven in the headwater reaches, close to source; seven in mid-section reaches and seven in the lower reaches, close to the river mouth) along 10 permanent streams (eight in Sa˜o Miguel and two in Flores) were selected and sampled to ensure adequate representation along both the altitudinal and anthropogenic gradient (Tab. 1). Although permanent streams, fed by lakes or spring waters, exist in Santa Maria, Sa˜o Miguel, Sa˜o Jorge, Faial and Flores islands (DROTRH and INAG, 2001) , we selected Sa˜o Miguel and Flores because they are the only islands where more than one permanent stream over 10 km 2 in catchment area can be found. Pupal exuviae were collected seasonally (winter, spring, summer and autumn) with a 250 mm hand net over a 20 minute period along the stream margins, focusing on areas where flotsam (foam and floating material) accumulated behind obstacles such as exposed rocks and boulders (see Ferrington et al., 1991; Wilson and Ruse, 2005) in order to guarantee that a minimum of 200 exuviae were available for analysis (Wilson and Ruse, 2005) . Collected material was placed in a 250 mm sieve, washed and preserved in 96% ethanol. In the laboratory the material was sorted and identified to the lowest possible taxonomic level (species or species-group) using keys by Langton (1991) and Langton & Visser (2003) . Approximately 200 exuviae were subsampled randomly from each collection to provide an unbiased, representative sample (Ruse, 1993) .
Study area and sampling procedure
Physicochemical variables were measured at each site prior to biological sampling. Water temperature ( xC), pH and dissolved oxygen (%) were measured in situ with electronic field probes (DKK, model WQC-24). Water samples were collected and then transported to the laboratory in a cool box and analyzed using certified methods within 24 hours. Analysis determined concentra-
Study area and sampling procedure
), total and fecal coliforms (ufc/100 mL) were also determined in laboratory. Hydromorphological factors were obtained in ArcGis 9 (ESRI) using 1:25000 topographic maps (altitude, latitude, longitude, stream order, distance to mouth, distance to source and slope) for each site. Land use categories and areas (non-native forest, scrub, agriculture area, natural vegetation and urban areas) were obtained from Regional Planning Department of the Azores (Quaternaire-Portugal, 2008); a circular buffer area (200 m radius) was created around each sampling site. All described variables were obtained for each site, sample site and sampling season, with the exception of cartographic variables.
Data analyses
Several indices of species diversity (Magurran, 2004) , such as Pielou's evenness, Fisher's a and Shannon-Wiener were calculated from the biological data. Non-redundant subsets of environmental and pressure parameters were obtained using the Pearson correlation coefficients to find and compare highly correlated parameters. If two environmental or pressure variables were highly correlated (threshold value of ri 0.6), then the variable with a highest overall mean correlation coefficient was excluded from subsequent analyses (Hering et al., 2006; Feld and Hering, 2007) . A total of 7 environmental and 14 chemical variables were retained from an initial list of 13 environmental and 28 chemical variables (Tab. 2). Analyses were carried out using Primer 6.0 (Clarke and Gorley, 2006) .
Data analyses
A Principal Components Analysis (PCA) was performed to identify abiotic gradients and patterns between streams. Permutational multivariate analysis of variance -PERMANOVA (Anderson, 2001; McArdle and Anderson, 2001 ) was used to determine associations between the chemical variables and chironomid assemblages. PERMANOVA partitions the total sum of squares based on the full experimental design (given below), and calculates a distance based pseudo-F statistic for each term in the model, based on the expectations of the mean squares. P-values are obtained using a permutation procedure for each term (Anderson et al., 2008) , or via Monte Carlo random drawn from an asymptotic permutation distribution if too few permutations are available for a given test (Anderson and Robinson, 2003) .
Data analyses
We tested for two spatial and one temporal distribution patterns using PERMANOVA: (i) between islands (fixed, two levels; Flores and Sa˜o Miguel), (ii) over time (random, four levels: Winter, Spring, Summer and Autumn) and (iii) along the lotic longitudinal gradient (fixed, three levels: headwater sites; mid-section sites and lower reaches sites). Because chemical and environmental data have different measurement scales and similarity methods, we normalised data, according to Clarke and Gorley (2001) . The relative abundance of the 21 species was log(x+ 1) transformed to reduce differences in scale (Clarke and Green, 1988) . For PERMANOVA, we used 9999 permutations of the data (an appropriate distribution for a pseudo-F statistic under a true null hypothesis is obtained by using a permutation test. According to Anderson and Braak (2003) the programme permutes the units identified by the denominator term of the F-ratio).
Data analyses
SIMPER analysis was used to identify species' relative contribution to the groups resulting from PERMANOVA analyses. Bray-Curtis dissimilarities were calculated for all pairs of observations for the analyses below.
Data analyses
Links between the non-redundant set of hydromorphological and physicochemical variables (see Tab. 2) and the community assemblage were assessed using distancebased multivariate analysis for a linear model -DISTLM (Legendre and Anderson, 1999; McArdle and Anderson, 2001 ). DISTLM analyses and models the relationship between the multivariate data cloud, for one or more predictor variables (Anderson et al., 2008) . DISTLM allows predictor variables to be fitted individually or together in user specified sets. The DistLM routine was based on the AIC model selection criterion (Burnham and Anderson, 2004 ) using a Step-wise selection procedure. The AIC model (Akaike, 1974; Akaike, 1973) was used to select the model that explained more variation, and therefore the 'best' model for that particular data set. Primer 6.0 and PERMANOVA + for PRIMER software were used (Clarke and Gorley, 2006; Anderson et al., 2008) . For visual interpretation of the resulting model in multi-dimensional space, we used distance based redundancy analysis (dbRDA) to investigate the relationship between location, environmental and chemical variables (Anderson et al., 2008) . Each vector begins at the centre of the circle (the origin) and ends in the coordinates (x and y). The length and direction of each vector indicates the strength and sign of the relationship between the given variable and the dbRDA axes. The overlaying vector illustrates the relationship between the eight variables identified by the DistLM procedure.
Results

Physicochemical characterization
PERMANOVA results (Tab. 3) showed significant temporal (Time -F 3,57 = 2.31, P < 0.002) and spatial variation along longitudinal gradient (Location -F 2,57 = 5.49, P< 0.007), but not between islands (P > 0.1). The PCA ordination also reflected this pattern (Fig. 2) . The first two PCA axes explained 39% of the variation and cumulatively 49% with the inclusion of the third axis. Temperature, conductivity, ammonium, nitrite, nitrate and phosphate compounds increased along the longitudinal gradient (Table 2 ) while dissolved oxygen, biological and chemical oxygen demand decreased. At most sites, dissolved oxygen values and pH were generally high during spring but decreased from summer and fall as a result of baseflow conditions.
Azorean chironomids communities
A total of 24 taxa were identified from 3 subfamilies and 5 tribes. The Orthocladiinae (15 taxa) showed the greatest richness, followed by Chironominae (6 taxa) and Tanypodinae (3 taxa). The Orthocladiinae was the dominant subfamily with a mean composition of 96%. Orthocladiinae exuviae comprised between 27% and 100% of the total exuvias found.
Azorean chironomids communities
Distinct spatial chironomid distribution patterns were apparent. Cricotopus sylvestris, Polypedilum nubeculosum and Pseudorthocladius curtistylus were exclusive to Sa˜o Miguel Island, while Psectrocladius limbatellus, Pseudosmittia sp1 and Telmatopelopia nemorum were only collected from streams on Flores. T. nemorum and Zavrelimyia nubila occurred only at headwater sites, while Chironomus riparius, C. cingulatus, C. sylvestris, C. ornatus and P. nubeculosum were consistently absent from such sites (Appendix I). The most dominant species were Thienemanniella clavicornis and Orthocladius. fuscimanus, with mean composition per site of 39% and 17%, respectively. These species were detected at least once for all sampling sites. Cardiocladius freyii was present at all sampling sites, with mean composition of 10% per site, with the exception of RFT1. Thienemanniella clavicornis, Orthocladius (Eudactylocladius) fuscimanus, C. freyii, Parametriocnemus stylatus and R. atripes occurred at more than 60% of the sampling sites. Species diversity indices varied considerably among sites (Tab. 4). The highest number of taxa was at RPV4 (n = 13) during the winter while the lowest number of taxa was at RGU2 and RFT0 in autumn, and RTX2 and RG1 in winter (n = 2).
Spatial and temporal distribution patterns
PERMANOVA results (Tab. 5) showed that chironomid assemblages differed significantly along the longitudinal gradient (Location -F 2,56 = 12.42, P = 0.004), but not between islands (P > 0.05 on all factors and interactions) or over time (P > 0.05 on all factors and interactions). SIMPER results (Tab. 6) of longitudinal location indicated lower levels of dissimilarity between sites situated on mid-section reaches and headwater sites (42%) and between headwater sites and mid-section sites (59%), than that observed between headwater and lower reaches sites (64%). R. atripes, T. clavicornis, Cardiocladius freyii and O. fuscimanus were identified as the principal species contributing to the observed dissimilarity (> 50%) between the headwater sites and mid-section sites and between headwater sites and sites along the lower reaches. SIMPER results revealed longitudinal changes in percent abundance; in R. atripes (2.6% -headwater sites; 1.4% -mid-section sites; 0.6% -lower reaches sites), Cardiocladius freyii (0.7% -headwater sites; 2.0% -midsection sites; 2.1% -lower reaches sites), O. fuscimanus (1.8% -headwater sites; 2.3% -mid-section sites; 2.6% -lower reaches sites) and P. stylatus (0.3% -headwater sites; 1.5% -mid-section sites; 1.7% -lower reaches sites).
Hydromorphological and physicochemical determinants of chironomids assemblages
The most important hydromorphological variables explaining differences in stream location were altitude (P < 0.001), natural vegetation area (P = 0.002) and agricultural land use (P = 0.003), followed by urban area (P = 0.004), forest production (P = 0.005), slope (P = 0.013) and scrub vegetation areas (P> 0.05) identified in DistLM procedure (Tab. 7).
Hydromorphological and physicochemical determinants of chironomids assemblages
The
Hydromorphological and physicochemical determinants of chironomids assemblages
Step-wise procedure selected altitude, production forest area, scrub area, natural vegetation area, slope and agriculture area as variables that determined chironomid composition in stream location (AIC = 561.43; R 2 = 0.34; number of variables = 6).
Hydromorphological and physicochemical determinants of chironomids assemblages
The dbRDA ordination (Fig. 3 ) illustrated how sites were clearly separated by their location along the longitudinal gradient and the principal contributing environmental variables. The first two dbRDA axes explained 89% of the relationship between the chironomid assemblage and the measured hydromorphological variables, and 31% of the total variability in the community data.
Hydromorphological and physicochemical determinants of chironomids assemblages
The first dbRDA axis was strongly related to altitude while the second axis was related to land use (production forest, agriculture and natural vegetation area).
Hydromorphological and physicochemical determinants of chironomids assemblages
The most important chemical variables identified in DistLM routine were water temperature (P = 0.001), Lim. 47 (2011) 325-338 pH (P = 0.001), conductivity (P = 0.001) and nitrate (P = 0.001) as the principal determinants of chironomid composition along the longitudinal gradient. Nitrite (P = 0.006) and total iron (P = 0.01) were also important parameters (Tab. 8). The
Hydromorphological and physicochemical determinants of chironomids assemblages
Step-wise procedure selected temperature, pH, nitrite, iron and conductivity as the strongest parameters determining chironomid composition in relation to stream location (AIC = 562.16; R 2 = 0.32; nx of variables = 5) (Tab. 8).
Hydromorphological and physicochemical determinants of chironomids assemblages
The dbRDA for chemical variables (Fig. 4 ) also showed a clear longitudinal separation of stream sampling sites and the principal contributing chemical variables (pH, nitrite, iron and temperature). The first two dbRDA axes captured 87% of the variability in the fitted model, and 28% of the total variation in the location. The vector overlays showed the relationship between the five variables found by the DistLM procedure. The first dbRDA axis was negative in relation to pH and nitrite and positive in relation to iron. The second axis showed a strong positive relationship with temperature.
Discussion
The number of chironomid taxa recorded in Azorean streams was low (24) compared to typologically similar continental rivers (e.g. mountain rivers) and other macaronesian streams. For example, 71 taxa were recorded at Aude River (Gendron and Laville, 1995) in France, 107 taxa at Pang River (Ruse and Wilson, 1995) , in United Kingdom, 54 taxa in Tenerife Island (Armitage et al., 1995b) and 46 taxa on the island of Madeira (Hughes, 2003) . Similar to findings from Madeira (Hughes, 2003) and Tenerife (Armitage et al., 1994) , the Orthocladiinae and Chironominae were the most frequently observed subfamilies in Azorean streams (Murray et al., 2004; . Similar to Tenerife (Armitage et al., 1994) , Thienemaniella clavicornis is the most dominant species in Azorean streams, but appears to be far rarer on Madeira (Hughes, 2003) . Bilton et al. (2001) described active and passive dispersal mechanisms for success in the colonization of freshwater systems. Actively flying chironomids adults can disperse over large areas (Massaferro and Brooks, 2002) , allowing the colonization of numerous freshwater ecosystems on the same island or even in neighbouring islands, which partly explains the high levels of similarity between the two islands in this study. In a setting such as the Azorean archipelago, active dispersal methods are important for the "stepping stone" type freshwater habitat colonization between islands (Whittaker and FernandezPalacios, 2007) .
Spatial variability: hydromorphological and physicochemical factors
The present study provides information about the physicochemical and hydromorphological variables determining chironomid assemblage distribution patterns in Azorean streams. An understanding of both local and large-scale environmental factors is vital in order to assess the importance of their role in structuring chironomid communities (Punti et al., 2007; Calle-Martı´nez and Casas, 2006; Punti et al., 2009) . Differences in chironomid assemblage structure between islands were not apparent (P = 0.068) but were significantly different along the lotic longitudinal gradient (P = 0.004). Physicochemical and hydromorphological variables explained 58.5% of the variance in chironomid assemblage composition. These values are similar to those determined in several previous studies on benthic diatoms (Soininen et al., 2004) , macroinvertebrates (Mykra¨et al., 2007; Heino and Paasivı`rta, 2008) and fish (Magalhaes et al., 2002) . The percentage of variance in chironomids assemblages explained only by hydromorphological variables in our study was considerable (30.8%) and similar to the percentage of variation explained by physicochemical variables (27.7%). The relatively high percentage of unexplained variation (41.5%) in our study is typical of noisy data sets with many 0 values (Borcard et al., 1992) , and could be result of abiotic and biotic factors not considered in this study, such as species' interactions (Kohler, 1992) , food availability (Peeters et al., 2004) , reproduction, competition and predation (MacNeil et al., 1999) .
Spatial variability: hydromorphological and physicochemical factors
The similarity of the faunal assemblages between islands appears to be related to the physical and chemical similarities of the islands' streams coupled with the chironomid dispersal abilities. PERMANOVA results did not detect significant spatial differences in physicochemical composition between islands (P > 0.1), but did detect significant differences along the lotic longitudinal gradient (P < 0.007). This emphasizes the importance of local, lower scale physicochemical and hydromorphological in determining the longitudinal distribution patterns of the chironomid community following colonization.
Spatial variability: hydromorphological and physicochemical factors
Habitat complexity also influences the composition of the freshwater assemblages (Heino, 2000; Tolonen et al., 2001; White and Irvine, 2003) . Coffman (1995 Coffman ( , 1989 proposed ecological heterogeneity as one of the main factors influencing chironomid species richness. To date, no detailed data on habitat complexity in Azorean streams is available. However, field observations indicate that key drivers such as substrate and flow are very similar between islands. Several authors (Malmqvist, 2002; Hughes and Malmqvist, 2005) have reported low spatial heterogeneity and low habitat diversity in Macaronesian systems (almost all streams are narrow, steep, with coarse substrate and typical step-pool flow patterns). The similar chemical composition and low spatial heterogeneity appears to be reflected in the expected high similarity of chironomids assemblages found in both islands.
Spatial variability: hydromorphological and physicochemical factors
T. clavicornis, O. fuscimanus and R. atripes dominated sampling sites on both Sa˜o Miguel and Flores islands. These species are known to posses a high dispersion ability and are well represented in the Palaearctic (Saether and Spies, 2009) and Macaronesian regions (Oromı´and Ba´ez, 2001; Hughes, 2008) .
Spatial variability: hydromorphological and physicochemical factors
The islands under study have suffered dramatic changes in land use following human colonization in the fifteenth century when the native vegetation at low and middle altitudes was cleared or became gradually extinct. In the 20th century, an increase in land dedicated to pasture has further depleted small fragments of native vegetation at mid range altitudes (Silva and Smith, 2004) . Nowadays, indigenous forested areas are limited to mid to high altitude (approximately > 600 m). However midrange reaches are considerably altered by human intervention including agriculture, canalization, flow diversion and organic discharge and almost all lower reaches are affected by urbanization. This study has clearly shown that changes in land use exerted a significant effect on longitudinal changes in chironomid assemblage composition along the stream represented by the secondary axes of the dbRDA.
Spatial variability: hydromorphological and physicochemical factors
Many authors highlight agriculture as a major cause of degradation in stream habitats (Allan and Flecker, 1993; Allan, 2004) , and that the biological impacts of physical alterations from this activity can last for decades (Harding et al., 1998) . This type of impact contrasts with the impacts of urbanization which are mainly related to direct effects of point source pollution, hydromorphological and biological impacts (Brasher, 2003; Feld and Hering, 2007; Mykra¨et al., 2007; Munne´and Prat, 2009; Weijters et al., 2009) . The physicochemical gradient of temperature, pH and conductivity in this study illustrates the indirect, combined effect of multiple mechanisms operating over several spatial scales describing natural biotic and abiotic gradients, changes in land use an organic enrichment . Most of the retained variables e.g. increase of nitrogen and phosphate compounds, increase of coliforms also described consequences of changes of the land use along the longitudinal gradient (see Tab. 2).
Spatial variability: hydromorphological and physicochemical factors
The dbRDA ordinations revealed scale dependent environmental and chemical variables with potentially high levels of covariance. In fact, the history of colonization of the archipelago (the Azores have suffered dramatic changes in land due to colonization, remaining indigenous forested mostly in high altitude) combined with the natural spatial and temporal patterns of insular streams could obscure the ready distinction of these sources of variation. Similar situations were reported by authors for the Mediterranean systems (Dı´az et al., 2008; Hughes et al., 2009) . The increase in nutrient levels along the longitudinal gradient observed in the Azorean streams is consistent other studies identifying chironomid composition changes along the river continuum, in association with altitude and stream order (Lindegaard and Brodersen, 1995; Heino, 2005; Inoue et al., 2005; Heino and Paasivirta, 2008) .
Spatial variability: hydromorphological and physicochemical factors
Chironomid species richness, evenness and diversity tended to be lower at headwater sites (2nd-3rd order), increasing to maximum values at sites midway along the longitudinal gradient (3rd-4th order), before decreasing again at sites further downstream (5th-6th order). These findings partly agree with Coffman (1989) , who found maximum richness of lotic chironomids in 3rd order streams (e.g. RC2; RQ2) as a result of higher ecological heterogeneity. The patterns revealed by the three diversity indices are also consistent with the Connell's intermediate disturbance hypothesis, (Connell, 1978) , which describes maximal species richness at intermediate levels of disturbance (Tab. 7). Vannote et al. (1980) and Giller and Malmqvist (1998) attributed the increase in diversity from headwaters to mid section of freshwater courses to increasing temperature, improved light conditions and broader stream channels. Moore and Palmer (2005) also reported higher invertebrate biodiversity in agricultural streams which then progressively declined along a land use gradient towards urbanization. Three groups of chironomids (headwaters, mid-section and low reaches) were discernible in relation to the influence of environmental variables [identified in PERMANOVA test (F 2,54 = 3.21, P = 0.004)]. The dbRDA results clearly indicated a strong environmental framework structuring the characteristics of both the sites and taxa.
Spatial variability: hydromorphological and physicochemical factors
The right-hand side of the dbRDA ordination was occupied mainly by headwater sites in semi-pristine conditions, located in scrub or natural vegetation. These sites were characterized by low temperatures, circumneutral pH, low conductivity, nutrients and metals. R. atripes was the most abundant species at headwater sites; Z. nubila and Paramerina cingulata were restricted to these habitats, indicated by SIMPER analysis results. According to Vallenduuk and Moller Pillot (2007) , these latter two Tanypod species tend to occur in the water surface layer of soil or plants, or in pools. These features indicate more stable habitats, when compared with bedrock habitats, with low levels of disturbance (Townsend and Hildrew, 1994) . Milner and Petts (1994) proposed stream channel stability as a major environmental factor determining stream community structure. The left-hand of the dbRDA ordinations were occupied by samples from stream sites located in areas subject to high levels of human intervention. These sites were lower lying, located close to urbanized areas and displayed higher temperature, pH, conductivity and nutrient levels. C. sylvestris and P. stylatus typically occurred at lower reaches locations. Several studies have reported that Cricotopus is abundant at nutrient enriched sites (e.g. Davies and Hawkes, 1981; Wilson, 1992; Calle-Martı´nez and Casas, 2006; Maasri et al., 2008) . Increased nutrient levels at mid-section sites adjacent to agriculture and areas dedicated to grazing of livestock appeared to augment chironomid abundance. T. clavicornis and Synorthocladius semivirens were more abundant at such sites while Pseudorthocladius curtistylatus and Polypedilum nubeculosum were exclusive to these sites. T. clavicornis, Cardiocladius freyii and O. fuscimanus were the most abundant species and are tolerant to a wide range of conditions.
Temporal variability
Despite the distinct seasonal climatic patterns, PERMANOVA results did not detect significant temporal patterns (P > 0.05) in chironomid composition (Tab. 5) but did detect significant differences in chemical composition (Time -F 3,56 = 23814, P = 0.002). These findings indicate seasonal persistence in community patterns. SIMPER analysis results showed that T. clavicornis consistently contributed over 30% to group similarity, followed by O. fuscimanus (contribution > 18%) and Cardiocladius freyii (contribution > 8%). These species predominated in the sampled streams throughout the entire sampling period.
Temporal variability
Persistence and stability of the 3 most abundant taxa were consistently high across the entire community, suggesting a temporally persistent pool of common taxa in Azorean streams, similar to other studies in continental streams (Townsend et al., 1987; Winterbourn, 1997) . However, further research is required to understand the strategies employed by these core chironomid taxa. Seasonal persistence of the chironomid communities is consistent with the results of previous studies of macroinvertebrates communities in Mediterranean streams (Mellado, 2005; Beˆche et al., 2006) . Observed persistence in Azorean chironomid communities in this study could be the result of naturally high disturbance levels which are typical of oceanic islands (Hughes, 2005; Covich, 2006; Covich, 2009 ) and act as selective filters favouring species adapted to such conditions. Chironomid emergence patterns vary both temporally and spatially across streams and voltinism varies both interspatially and intraspatially (Wrubleski and Rosenberg, 1990) . Most chironomid lotic species are multivoltine (three or more generations per year) with peaks in emergence occurring between spring and autumn (Tokeshi et al., 1995) . The presence of several species across the four sampling seasons could be explained by the continuous emergence of species from overlapping generations or cohorts. Small sized species such T. are multivoltine and often observed to posses closely overlapping generations (Tokeshi et al., 1995) . Multivoltine species are better adapted to exploit situations of environmental change, especially when rapid fluctuations occur (Delettre, 1995) , such as rapid runoff and highly torrential flow regimes which are common after rainfall on the Azores.
Final remarks
Relatively reduced habitat diversity, low spatial heterogeneity, distance from sources of colonization and the geological youth of the Azores archipelago, limit the pool of invertebrates able to colonize and inhabit Azorean streams, a phenomenon common to other Macaronesian islands (Hughes and Malmqvist, 2005; Hughes, 2005; Hughes, 2006) .
Final remarks
Local changes in the physical environment due to changes in land use (agriculture and forestry, urbanization) alter local habitat quality, influencing community composition and biodiversity. Our results confirm that chironomids constitute a promising candidate group for ecological assessment in Azorean lotic systems, but larger data sets are required to better analyze intraannual and interannual variability and the effect of habitat scale variables. A better understanding of the ecological requirements of chironomid communities in Azorean streams and their biology is essential for developing water-quality monitoring protocols suitable for insular streams. More detailed of life cycles and emergence patterns over several seasons will provide more information on chironomid phenology. All of these findings would contribute to testing and possibly adapting the Chironomid Pupal Exuviae Technique (CPET) to Azorean surface waters.
Final remarks
The information from this study will help support current and future research of Azorean stream ecosystems and future freshwater management issues such as conservation planning, management measures, implementing biomonitoring programmes, and predicting of how human alterations will affect lotic ecosystems, allowing the development and implementation of sustainable and effective natural resource management across the archipelago.

{'paper_id': '56333049', 'title': 'Environmental factors – spatial and temporal variation of chironomid communities in oceanic island streams (Azores archipelago)', 's2_url': 'https://api.semanticscholar.org/CorpusID:56333049', 'source': 'S2ORC'}