Springer 2005 Hydrobiologia (2005) 543: 119–133 DOI 10.1007/s10750-004-6950-0 Primary Research Paper Species composition and assemblage structure of chironomid larvae (Diptera: Chironomidae) attaching to the artificial substrates in a Japanese temperate basin, in relation to the longitudinal gradient Eiso Inoue*, Koichiro Kawai & Hiromichi Imabayashi Laboratory of Aquatic Ecology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima, Hiroshima 739-8528, Japan (*Author for correspondence: Tel.: +81-(0)82-424-7895, Fax: +81-(0)82-422-7059, E-mail: inoueiso@hiroshima-u.ac.jp) Received 15 March 2004; accepted in revised form 30 November 2004 Key words: Chironomidae, artificial substrate, topographic type, riparian canopy, bank protection, longitudinal distribution Abstract The relative importance of natural and anthropogenic factors, especially topographic type, riparian canopy, altitude, temperature and bank protection, on larval chironomid assemblage was investigated in a Japanese basin. To focus on the macro-scale factors, a concrete block, as an artificial substrate, was used for chironomid collection so that sampling regime may be identical among the sites. Partial CCA using sampling month as a covariable revealed that topographic type, riparian canopy coverage, water temperature and altitude were the main factors influencing species distribution. Stempellinella tamaseptima, Polypedilum tamanigrum and five Rheotanytarsus species showed positive, whereas five Cricotopus species showed negative associations with canopy coverage. Some traditional longitudinal zonations of species were still shown. Chironomus flaviplumus and Chironomus yoshimatsui were merely associated with lower reaches. Stepwise multiple regressions of the assemblage indices on the environmental variables were applied. Bank protection and depth showed negative correlations with Shannon diversity H0 . Both topographic type and depth showed negative correlations with Pielou equitability J. Topographic type (lower reach) and specific conductance showed positive, while bank protection showed a negative correlation with abundance. Species richness was not explained by any variables. As a whole, topographic type was the most directly related factor to chironomid assemblages. Introduction Horton (1945) developed stream order, a hierarchical classification system of streams, subsequently modified by Strahler (1952, 1964), that remains in wide use (Allan, 1995). That is, the smallest, permanently flowing stream is termed first order, and the union of two streams of order n creates a stream of order n þ 1. This provides a convenient system in which rivers increase in order as they increase in size (Allan, 1995). Vannote et al. (1980) used stream order as physical template and proposed the River Continuum Concept, a bold attempt to construct a single synthetic framework to describe the function of lotic ecosystems from source to mouth, and to accommodate the variation among sites that results from differences in their terrestrial setting. Particularly at headwater streams, condition of riparian vegetation is likely to impact strongly on aquatic communities and may be the single most important anthropogenic factor influencing the structure, function and water quality (Hynes, 1975; Sweeney, 1992). Recently, Roy et al. (2003) have shown that increase in urban land cover resulted in less diverse 120 Figure 1. Schematic drawings showing three configuration types of riffle (R)/pool (P) unit, longitudinal cross section. Streams flow rightward: (a) type a (topographic type Aa); (b) type b (Bb); (c) type c (Bc) (After Kani, 1944). and more tolerant stream macroinvertebrate communities. On the other hand, as a landscape scale approach, Kani (1944) has proposed a system for ecological classification of Japanese streams, topographic type, by riffle/pool distribution and configuration patterns as the following. The distribution patterns can be divided into two types, A and B. In type A, one reach from a meandering point to the next meandering point comprises two or more riffle/pool units. In type B, one reach comprises a single unit. The unit configuration has three types: a, b and c (Fig. 1). In type a, the riffle/ pool units are arranged as if in a series of stairs, and the stream is more or less torrential and forms small waterfalls. In type b, no waterfalls are found, but riffles are partially wavy and foaming. In type c, successive stream units are almost leveled, and the water surface is usually waveless. Kani (1944) found that distribution type A is always accompanied by the configuration type a, while B is combined with either b or c. Therefore, three binary combinations of distribution and configuration types are found: Aa, Bb and Bc. Even a stream that at first sight does not seem well fit to any one of the three can be classified as a transition in between Aa and Bb or Bb and Bc, and Aa–Bb is frequently found in natural streams (Fig. 2; Kani, 1944, 1981). Stream order system is the most widely used in the world for describing longitudinal position of streams (Allan, 1995). However, the topographic type system has so far been well established as one of the most useful measure of riverine landscape features and used in studies on distribution of lotic fishes and macroinvertebrates as well (Mizuno & Gose, 1972; Okino, 2002; Ohgushi, 2004). Chironomidae are the most widely distributed, frequently the most abundant insects in freshwater ecosystems (Pinder, 1986; Cranston, 1995), and the Figure 2. Schematic drawings showing distribution types of riffle (scratches)/pool (dashed circles) unit, top view. Streams flow downward: (a) type A (topographic type Aa); (b, c) transition types between types A and B (Aa–Bb); (d) type B (Bb); (e) type B (Bc). (After Kani, 1944.) 121 most species-rich group in lotic macroinvertebrate communities (Coffman, 1995). Chironomid species diversity and assemblage structure have been considered to be one of the most useful indicators of various environmental factors, e.g. water quality (Wilson & Bright, 1973; Sæther, 1979; Wilson, 1987; Kawai et al., 1989), catchment urbanization (Roy et al., 2003) and past environmental change (Walker et al., 1991; Walker, 1995). Kawai & Takahashi (1986) investigated chironomid fauna of the Ohta River basin (Hiroshima, Japan) and described longitudinal distribution of 96 species. In our previous study, it has been shown that intrageneric longitudinal zonations of the four genera, i.e. Cricotopus, Polypedilum, Rheotanytarsus and Tanytarsus, were attributable to altitude, specific conductance, topographic type and riverbed (riffle/pool) type (Kawai et al., 1998, 1999). However, the relative importance of the factors that compose river continuum on lotic chironomid distribution remained uncertain. The aim of the present study was to evaluate influences of the macro-scale variables on larval chironomid communities. Since substrate type is an important micro-scale factor for macroinvertebrate community (e.g. Hynes, 1970; Minshall, 1984; Ruse, 1994; Allan, 1995; Lindegaard & Brodersen, 1995), the condition was equalized by the use of an artificial substrate for larval sampling. A direct ordination technique and stepwise multiple regressions were used to determine which variables explain the variations in abundance, species richness, diversity, equitability and composition of the assemblages among sites. Finally, usefulness of the artificial substrate and function of river continuum on chironomids were discussed. Materials and methods Figure 3. Geographical position of the Ohta River basin and locations of the 23 sampling sites (solid circles). upper and middle reaches are often used as residential and/or agricultural land, but natural deciduous forests are relatively rich, especially in the north and west regions. The Hosomidani riparian forest, locating around the origin of the basin, is one of the most nature-rich districts in Japan although now it has been becoming a social concern as to conservation, due to plans of constructing a large scale traffic road. Conifer plantations which are mostly composed of Japanese cedar (Cryptomeria japonica D. Don) occupy many other forests. Contrariwise, areas surrounding the downstream and the river mouth, where the river runs through central Hiroshima City, are highly urbanized, and many buildings and pavements cover the land entirely. In general, water pollution has now been less severe except downstream and river mouth, but physical habitat modification and canopy removal which are mainly caused by watercourse modification is still the major disruption along the basin. Sampling sites Sampling methods A total of 23 sampling sites in the Ohta River, Hiroshima Prefecture, Japan (Fig. 3), were selected, covering its entire basin with various environmental conditions (Table 1). The main stream originates in Mt. Kanmuri (1339 m a.s.l.) of the western Chugoku Mountains and has 110 km of stretched main flow length and 1690 km2 of catchment area. Catchments of the For quantitative sampling, a concrete construction block (Fig. 4) was submerged in the stream, as a standardized artificial substrate, at a riffle in each site. The block is difficult to be flowed away and provides sufficient microhabitats for various species of chironomid larvae (Kawai et al., 1999). The blocks were set between 5 and 15 July 1994 122 Table 1. Sampling records and the environmental variables for the 23 sampling sites in 1994 Site Setting Sampling Altitude (m a.s.l.) a Water Surface temperature velocity (C) (ms ) )1 Depth (cm) Specific Topographic Canopy Bank conductance typea coverageb protectionc (lS cm ) )1 1 11 July 13 Oct. 560 17.8 0.25 20 50 2 2 0 2 7 July 13 Oct. 740 15.9 0.65 14 31 1 0 1 3 11 July 4 Oct. 770 15.6 0.42 20 35 2 0 2 4 5 14 July 14 July 4 Oct. 4 Oct. 700 720 13.7 16.2 0.16 0.93 50 30 41 39 1 2 2 0 0 2 6 14 July 4 Oct. 640 16.5 0.20 15 40 2 0 2 7 5 July 4 Oct. 640 15.5 0.41 23 29 1 0 1 8 11 July 15 Nov. 260 14.1 0.21 20 62 2 0 1 9 11 July 18 Oct. 230 14.2 0.45 9 60 2 0 2 10 7 July 15 Nov. 200 14.1 0.39 15 65 2 0 1 11 15 July 18 Oct. 100 15.5 0.49 9 46 2 1 1 12 13 7 July 7 July 4 Oct. 4 Oct. 180 220 12.1 19.9 0.41 0.65 5 22 42 88 1 1 2 1 1 1 14 7 July 4 Oct. 180 19.6 0.49 25 108 2 0 2 15 5 July 21 Nov. 50 14.2 0.57 35 61 2 0 2 16 15 July 4 Oct. 160 18.4 0.46 20 71 1 1 1 17 7 July 15 Nov. 100 13.3 0.36 7 77 1 0 2 18 15 July 15 Nov. 290 13.0 0.13 16 55 1 2 1 19 15 July 21 Nov. 360 10.8 0.45 24 74 1 2 1 20 21 15 July 15 July 21 Nov. 21 Nov. 180 250 12.1 11.0 0.53 0.27 24 8 98 36 1 1 0 2 1 0 22 5 July 21 Nov. 15 15.0 0.23 20 80 3 0 2 23 5 July 21 Nov. 10 14.5 0.53 6 71 3 0 2 1: Aa (headwater type); 2: Aa–Bb (upper-middle transitional reach type); 3: Bb (middle reach type), based on Kani (1944). Coverage above the channel, 0: = 0%; 1: <50%; 2: >50%, based on Fujitani (2002). c Degree of protected bank in 50 m, 0: = 0%; 1: <50%; 2: >50%. b 123 Figure 4. Concrete construction block, used as the standardized artificial substrate. The blocks were placed at riffles so that the longer axis was parallel to the water flow. (Table 1) and kept submerged on the riverbed for at least 2 months. The collection was replicated between 4 Oct. and 21 Nov. 1994 (Table 1). All materials attached to and caught in the block, e.g. moss, mud and litter which would be food for larvae, were collected and transported to our laboratory with gentle cooling. The samples containing larvae were put into a container (ø15 · H9 cm). The container was filled with dechlorinated water to a level of about 6 cm, covered with nylon net and aerated at room temperature (10–20 C). All emerging imagines were collected every other day for at least a month and for 2 months at the longest, until the emergence ceased. Identification to species Only male imagines were used for identification because of the difficulty in accurate identification of females and immature stages (Inoue et al., 2004). Male imagines were preserved as dried specimens and mounted on microscopic slides with gum chloral under a binocular dissecting microscope, following the method described by Sasa et al. (1980). They were identified to species using mainly the taxonomical keys provided by Pinder (1978), Wiederholm (1989) and Sasa & Kikuchi (1995). Environmental assessments At the same time of larval collection, the following environmental conditions were measured at each site: water temperature, depth, specific conductance, surface velocity, topographic type, canopy coverage and degree of bank protection (Table 1). Altitude was measured by reference to a 1:50 000 scale topographic map. Specific conductance was measured using a salinometer (YSI model 33, Yellow Springs Instrument Co., Inc. Ohio, USA), as an indicator of water quality due to its high sensitivity to a low level of trophic enrichment (Sasa et al., 1980). Surface velocity above the block was measured with a fishing float as a sign. The topographic type of the stream was classified on the basis of a criterion (Figs. 1 and 2) provided by Kani (1944), as mentioned above. In Japanese streams, types Aa, Bb and Bc are usually observed in the mountainous headwaters, middle and lower reaches, respectively, and type Aa–Bb is usually observed as a transition between headwaters and middle reaches. Canopy coverage and degree of bank protection were measured by visual observation, both as indicators of vegetation richness and anthropogenic modification of riparian terrain (Yates & Noel, 1988; Fujitani, 2002). In general, Japanese streams are extremely short and steep compared to continental ones (Okino, 2002). Combined with heavy rains, which are brought by stationary front in early summer and typhoons from late summer to autumn, many basins have often suffered from flooding from way back, and therefore bank protections were inevitably executed for flood control (Okino, 2002). Two types of bank protection were observed, crude and primitive one composed of irregular natural rocks, and massive one in which banks were covered firmly with concrete. The former was frequently found in upper reaches and sometimes difficult to be distinguished from natural rocky banks. Therefore, only the latter ones were regarded as protected banks in this study. Data analysis Preliminary to the analysis, chironomid assemblage data that contains less than 10 males were excluded. In the beginning, a Detrended Correspondence Analysis (DCA; Hill, 1979) was used to determine gradient length for the data set, which indicated that unimodal rather than linear techniques were more appropriate (Ter Braak & Prentice, 1988). Therefore, relationships between the assemblage composition and the environmental variables were assessed by Canonical Correspondence Analysis (CCA; Ter Braak, 1986), using 124 the software CANOCO (version 4.5) for Windows (Ter Braak & Šmilauer, 2002). CCA is a widely used direct ordination technique that assumes a unimodal model for the relationships between the responses of each species to the ordination (environmental) axes (Johnson et al., 1993). A partial CCA was then applied where the collecting month was used as a covariable to eliminate temporal effects on chironomid distribution pattern. The forward selection option provided by CANOCO was applied to the rest of the variables. Variables with p < 0.05 (Monte-Carlo permutation test, 199 runs) were selected for the analysis. Scaling focused on inter-species distances and no transformation of data was made except that downweighting of rare species option was set on. The significances of the first ordination axis and of the sum of all the canonical axes were evaluated with the Monte-Carlo permutation test (199 runs). Two community indices, Shannon diversity H0 (Shannon & Weaver, 1949) and Pielou evenness component diversity (equitability) J (Pielou, 1969) of the chironomid assemblage were calculated. Then stepwise multiple regressions (forward method) of these indices, species richness and total abundance on the environmental variables were performed using the software StatView (version 4.5) for Mac PowerPC (Abacus Concepts, Inc. California, USA). F-to-enter and F-to-remove were 4.000 and 3.996, respectively. All tests are two-tailed with 5% a level. Results Table 2. A list of species and abundance collected by rearing of larval samples Species Subfamily Tanypodinae Conchapelopia pallidula (Meigen) Paramerina divisa (Walker) Rheopelopia eximia (Edwards) Rheopelopia maculipennis (Zetterstedt) Table 2 shows abundance of male imagines collected for each species. A total of 673 males belonging to 69 species were recorded. The subfamilies Tanypodinae and Orthocladiinae, and tribes Chironomini and Tanytarsini of the subfamily Chironominae composed 7.6, 19.0, 33.4 and 40.0% of total males, respectively. The most abundant species was Chironomus yoshimatsui, followed by Tanytarsus tamaundecimus and Cladotanytarsus vanderwulpi. No species of the subfamily Diamesinae were collected, and species of Tanypodinae and Orthocladiinae were relatively rare, with the exception of the following 3 5 6 6 26 Rheopelopia ornata (Meigen) 7 Trissopelopia oyabetrispinosa Sasa, 1 Kawai et Ueno Subfamily Orthocladiinae Brillia japonica Tokunaga Corynoneura celtica Edwards Corynoneura lobata Edwards Cricotopus (Cricotopus) bicinctus (Meigen) Cricotopus (Cricotopus) bimaculatus 2 7 9 20 2 Tokunaga Cricotopus (Cricotopus) metatibialis Tokunaga 3 Cricotopus (Cricotopus) tokunagai Hirvenoja 3 Cricotopus (Cricotopus) tremulus (Linnaeus) Epoicocladius chuzeundecimus (Sasa) 2 1 Eukiefferiella sp. 1 Nanocladius quadrivittatus Niitsuma 2 Nanocladius seoulensis (Ree et Kim) 3 Nanocladius tamabicolor Sasa 3 Neobrillia longistyla Kawai 3 Orthocladius (Orthocladius) makabensis Sasa 2 Orthocladius (Orthocladius) tamarutilus Sasa Parametriocnemus stylatus (Kieffer) 3 8 Paratrichocladius rufiventris (Meigen) 2 Psectrocladius aquatronus Sasa 1 Psectrocladius yunoquartus Sasa Rheocricotopus chalybeatus (Edwards) Chironomid data Abundance 1 40 Rheocricotopus tamabrevis (Sasa) 1 Synorthocladius tamaparvulus Sasa 1 Thienemanniella nipponica Tokunaga Thienemanniella vittata (Edwards) 1 2 Tvetenia tamaflava (Sasa) 5 Subfamily Chironominae Tribe Chironomini Chironomus flaviplumus Tokunaga Chironomus yoshimatsui Martin et Sublette Microtendipes britteni (Edwards) 7 100 5 Microtendipes pedellus (De Geer) Microtendipes truncatus Kawai et Sasa 3 14 Polypedilum (Polypedilum) akisplendens 2 Kawai, Inoue et Imabayashi Polypedilum (Polypedilum) asakawaense Sasa 1 125 Table 2. Continued. Species Polypedilum (Polypedilum) fuscovittatum Abundance 3 Kawai, Inoue et Imabayashi Polypedilum (Polypedilum) pedestre (Meigen) 4 Polypedilum (Polypedilum) takaoense Sasa Polypedilum (Polypedilum) tamaharaki Sasa 4 20 Polypedilum (Polypedilum) tamahosohige Sasa 5 Polypedilum (Polypedilum) tamanigrum Sasa 7 Polypedilum (Polypedilum) tsukubaense Sasa 5 Polypedilum (Tripodura) unifascium (Tokunaga) 28 Polypedilum (Tripodura) sp. cf. asoprimum 1 Polypedilum (Uresipedilum) aviceps Townes 2 Polypedilum (Uresipedilum) convictum (Walker) 1 Polypedilum (Uresipedilum) cultellatum 1 Goetghebuer Polypedilum (Uresipedilum) hiroshimaense 2 Five species of Rheotanytarsus were recorded and all were relatively rare. Species richness, male abundance, Shannon diversity and equitability for each sampling site are shown in Table 3. The abundance was the highest at the site 22 followed by 23, both had the topographic type Bb and were located at the lowest reach (Table 1, Fig. 3). Contrariwise, only low abundances (n < 10) were recorded at the sites 2–4, 8, 10, 14, 19, which were usually located at the upper reaches. Thus, assemblage data of these sites were excluded for analyses hereafter. Analysis of the chironomid assemblage composition: CCA Four variables were selected by CANOCO: topographic type, canopy coverage, water temperature Kawai et Sasa Polypedilum (Uresipedilum) paraviceps Niitsuma 2 Polypedilum (Uresipedilum) surugense Niitsuma 7 Polypedilum (Uresipedilum) tamasemusi Sasa 1 Tribe Tanytarsini Cladotanytarsus vanderwulpi (Edwards) 65 Rheotanytarsus fluminis Kawai et Sasa 2 Rheotanytarsus rivulophilus Kawai et Sasa 5 Rheotanytarsus tamaquintus Sasa 6 Rheotanytarsus tamasecundus Sasa 2 Site Species Rheotanytarsus tamatertius Sasa 4 Stempellinella tamaseptima (Sasa) 9 Tanytarsus takahashii Kawai et Sasa Tanytarsus tamaduodecimus Sasa 3 16 Tanytarsus tamagotoi Sasa 17 Tanytarsus tamakutibasi Sasa Tanytarsus tamaoctavus Sasa 2 6 Tanytarsus tamaundecimus Sasa 80 Virgatanytarsus arduennensis (Goetghebuer) 52 Total Table 3. Species richness, total abundance and community indices for chironomid assemblages at the 23 sampling sites 673 richness Shannon Pielou abundance diversity H0 equitability (bit) J 1 17 86 3.34 0.82 2* 3* 5 4 8 4 – – – – 4* 3 3 – – 5 5 23 1.47 0.63 6 8 17 2.44 0.81 7 8 17 2.13 0.71 8* 6 8 – – 9 9 14 2.70 0.85 10* 3 11 8 5 15 – 2.74 – 0.91 12 8 12 2.62 0.87 13 7 10 2.72 0.97 8 – – 14* 4 species: Rheopelopia maculipennis (8 sites, 26 males), Cricotopus bicinctus (2 sites, 20 males) and Rheocricotopus chalybeatus (3 sites, 40 males). As to Chironominae, no less than 18 species of the genus Polypedilum were recorded whereas no more than 3 species of one of its affined genera, Microtendipes, were recorded. A total of 14 species of Tanytarsini were recorded and T. tamaundecimus was the most dominant species, in turn C. vanderwulpi and Virgatanytarsus arduennensis. Total 15 6 15 1.87 0.72 16 16 55 3.40 0.85 17 8 11 2.85 0.95 18 11 19* 1 24 1 3.13 – 0.90 – 20 18 77 3.45 0.83 21 12 23 3.29 0.92 22 10 121 1.24 0.37 23 21 116 3.45 0.79 *Data excluded from analyses. 126 Table 4. Result of forward selection of environmental variables used for partial CCA Variable F p Topographic type 1.911 0.010** Canopy coverage Water temperature 1.643 1.566 0.015* 0.030* Altitude 1.495 0.035* Bank protection 1.414 0.065 Surface velocity 1.366 0.140 Depth 1.189 0.245 Specific conductance 1.100 0.320 *p < 0.05, **p = 0.01 (Monte-Carlo permutation test). and altitude, in order of the significance (Table 4). The Monte-Carlo permutation test showed that the analysis was significant (p ¼ 0.005) for both the first axis and the sum of all the canonical axes. Therefore, the first two axes were used for the interpretation of the results. The first two axes explained 28.0 and 71.0% of the variance in the species data and species–environment relationship, respectively (Table 5). Topographic type was the environmental factor most strongly related to chironomid distribution, followed by canopy coverage, water temperature and altitude (Table 4, Fig. 5). The first canonical axis was interpreted as representing longitudinal gradient of the basin, since it showed a positive association with altitude and was negatively associated with topographic type and with water temperature. Species associated with the sites 22 and 23, e.g. C. bicinctus, R. chalybeatus, Chironomus flaviplumus and C. yoshimatsui were the more abundant species at the Bb reaches and at higher temperature. Cricotopus tokunagai, Psectrocladius yunoquartus, Microtendipes britteni and Polypedilum aviceps were abundant at the high-altitude sites. Canopy coverage showed highly a negative association with the canonical axis II. Therefore, the axis was interpreted as representing the degree of degradation of riparian vegetation and/or anthropogenic disturbances. Corynoneura celtica, Stempellinella tamaseptima and Polypedilum tamanigrum, for which coordinates were located on the lower part along the axis II, were regarded as the abundant species at the sites with welldeveloped riparian canopy. All the species of Rheotanytarsus were positively associated with canopy coverage, whereas all the Cricotopus species showed negative responses in some degree. Species, for which coordinates were located on the center part of the graph, e.g. Polypedilum unifascium, C. vanderwulpi and T. tamaundecimus, occurred in wider range of the environmental conditions. Analyses of the chironomid assemblage indices, species richness and abundance: stepwise multiple regression Overall, which couples of variables were the most strongly associated with chironomid community indices? The results of the stepwise multiple regressions are shown in Table 6. With respect to Shannon diversity H0 , three variables were selected as predictor and the regression model was significant (adj. R2 ¼ 0.507, p < 0.01). Bank protection was the best explanatory variable and showed a significant negative correlation with H0 (std. R ¼ )0.581, p < 0.01). Depth also showed a significant negative correlation (std. Table 5. Eigenvalues for partial CCA CCA axes I II III IV Eigenvalue 0.624 0.539 0.250 0.225 Species–environment correlations 0.979 0.931 0.918 0.939 15.0 38.1 28.0 71.0 34.0 86.2 Cumulative % variance Of species data Of species-environment relation Total inertia = 4.586. 39.4 100.0 127 Figure 5. Partial CCA triplot showing the ordination of environmental variables (arrows), chironomid assemblages (open circles) and species (crosses) on the axes I and II. R ¼ )0.451, p < 0.05) while specific conductance was positively correlated (std. R ¼ 0.389), although the correlation was not significant. As to Pielou equitability J, two variables were selected and the model was significant (adj. R2 ¼ 0.523, p < 0.005). Both topographic type (std. R ¼ )0.621, p < 0.01) and depth (std. R ¼ )0.436, p < 0.05) showed a significant negative correlation with J. No significant regression model was found to relate species richness to environmental variables (data not shown). With regard to abundance, three variables were selected and the model was significant (adj. R2 ¼ 0.631, p < 0.005). Both topographic type (std. R ¼ 0.835, p < 0.001) and specific conductance (std. R ¼ 0.513, p < 0.01) showed significant positive correlations whereas bank protection showed a significant negative correlation (std. R ¼ )0.517, p < 0.05). Discussion From micro to macro-scale, many environmental factors are known to influence distribution of larval chironomid species and their local colonization (e.g. Wilson & Bright, 1973; Wilson, 1987; Kawai et al., 1998; Ruse, 1994; Lindegaard & Brodersen, 1995). In the present study, an artificial substrate was placed at a riffle and used to eliminate differences in substrate condition. In addition to topographic type, altitude, specific conductance, water temperature and depth, some riparian conditions, i.e. canopy coverage and bank protection, were shown to influence community composition. The use of the artificial substrate Chironomid assemblages, sampled by the use of the artificial substrate, may be selective and are 128 Table 6. Result of stepwise multiple regression of Shannon diversity H¢, Pielou equitability J and total abundance on environmental variables. Variables selected by forward method are shown in the table Diversity (H0 ) Equitability (J) Total abundance R2 SE df Adj. R2 Cumulative Total F p 0.606 0.490 0.507 6.144 0.009** Predictor variables Partial R SE Std. partial R F-to-remove p Bank protection )0.576 0.185 )0.581 9.707 0.009** Depth Specific conductance )0.035 0.013 0.014 0.006 )0.451 0.389 6.058 4.340 0.030* 0.059 Regression statistics R2 SE df Adj. R2 Cumulative Total F p 0.586 0.102 9.216 0.003*** Predictor variables Partial R SE Std. partial R F-to-remove p Topographic type )0.128 0.037 )0.621 12.130 0.004*** Depth )0.070 0.003 )0.436 5.966 Regression statistics R2 SE df Adj. R2 0.705 23.478 Partial R SE Regression statistics Predictor variables Topographic type Bank protection Specific conductance 0.523 0.631 Std. partial R 0.030* Cumulative Total F p 9.539 0.002*** F-to-remove p 44.906 9.845 0.835 20.805 <0.001*** )28.377 10.296 )0.517 10.106 0.017* 0.978 0.308 0.513 7.596 0.008** Fin = 4.000; Fout = 3.996 (forward method); n = 16. *p < 0.05, **p < 0.01, ***p < 0.005. unlikely to represent natural communities (Glime & Clemons, 1972). We would sample only a subset of the entire chironomid community and many species in these streams may not colonize on hard substrates used in this study. In fact, only a small numbers of males were collected at some of the sites (Table 3) and 69 species were collected in total from riffles only. Kawai & Takahashi (1986) reported a total of 96 species from the Ohta River basin, although they collected larvae at both riffles and pools. A comparison of species composition should be carried out between larval communities inhabiting in situ bottom substrates of a certain part of a riverbed and those inhabiting in an artificial substrate placed at the same part, in order to confirm the usefulness of the block as a substrate for lotic Chironomidae (Kawai et al., 1999). Nevertheless, artificial substrates are useful for comparing that subset of taxa across sites. The aim of this study was to clarify the influence of macroscale factors, rather than micro-scale ones such as substrate type. Even at an identical site, local heterogeneity in substrate condition may cause bias or distortion in assemblages that were sampled from natural substrates. On the other hand, artificial substrates can provide a degree of sampling replicability not otherwise available, especially when they are placed in comparable macrohabitats (Cairns & Pratt, 1993). In this respect, it was advantageous to apply the artificial substrate to chironomid sampling in the present study. Several micro-scale factors, e.g. depth and surface velocity, still remained not identical among the sites to some extent (Table 1), and therefore, setting methods should be improved. In addition, an interesting future study would be to use the present methods to compare leaf pack and concrete block substrates. 129 Assemblage composition and species distribution of chironomids Partial CCA showed that chironomid assemblage composition reflects topographic type, canopy coverage, water temperature and altitude (Table 4, Fig. 5). Kawai & Takahashi (1986) investigated chironomid assemblages of the Ohta River and reported that M. britteni, Brillia japonica, P. unifascium, were found only at Aa to Aa–Bb sites whereas C. yoshimatsui and C. flaviplumus occurred at only Bb to Bc sites. They also reported that R. maculipennis, P. convictum, R. chalybeatus, C. bimaculatus, M. truncatus and C. vanderwulpi occurred at all the topographic type. On the other hand, Kawai et al. (1998, 1999) showed that P. hiroshimaense, P. takaoense, P. tamaharaki, P. tamahosohige, P. tamanigrum, P. tsukubaense, R. tamasecundus and T. tamaoctavus occurred more frequently at Aa or Aa-Bb sites than Bb and Bc sites. The present study largely supports the results of Kawai & Takahashi (1986) and our previous studies (Kawai et al., 1998, 1999) in species distribution patterns along topographic type and altitude. Lindegaard & Brodersen (1995) have reviewed faunistic studies which cover 104 sites in 80 different watercourses and showed the succession of chironomid communities on altitude, latitude and temperature. According to their criterion, the Ohta River basin can be regarded as either of the following: lower mountain or lower montane streams for the headwaters and upper to middle reaches, and summer-warm lowland streams for the middle to lower reaches. Compared with their results in which percentages of Diamesinae and Orthocladiinae were 1–6 and 36–59%, respectively, it is remarkable that Diamesinae was not collected and Orthocladiinae was less abundant, composed 19.0% of total abundance (Table 2). These results suggest that temperature fluctuations in the rearing room might have reduced the survival of cold stenothermic taxa, especially Diamesinae (Lindegaard & Brodersen, 1995). However, imaginal community collected by light trap in a headwater of the same basin was composed of 11.6, 30.6, 33.9 and 24.0% of Tanypodinae, Orthocladiinae (excluding a terrestrial genus, Smittia (Wiederholm, 1989)), Chironomini and Tanytarsini, respectively, and Diamesinae was not collected (Kawai et al., 2002). These facts indicate that the absence or rarity of Diamesinae and relative scarcity of Orthocladiinae in the basin, and imply that larval rearing conditions could cause small bias so that its influence on our general conclusions could be substantially negligible. The vector direction of water temperature in the CCA graph was close to that of topographic type, indicating that the ordination could not clearly separate the influence of the two factors. However, the association in vector direction among topographic type, altitude and water temperature were consistent with longitudinal structure of river continuum (Vannote et al., 1980). On the other hand, the impact of canopy coverage on chironomid distribution was comparable to or greater than the three factors mentioned above (Fig. 5). It could be inferred that species distribution and community structure also reflects anthropogenic factors, such as riparian forest degradation. There was a great variety of species distribution pattern (Fig. 5). The assemblage composition for the types Aa and Aa--Bb also showed some variations. On the other hand, assemblage compositions for the type Bb sites were quite similar and some species occurred only at the sites. These results may indicate that the environments of upper reaches are more heterogeneous in microhabitats for chironomid larvae. However, possibilities that the species of the lower reaches are ecological generalists and/or are more tolerant of environmental variation cannot be discarded. S. tamaseptima, P. tamanigrum and all the species of Rheotanytarsus were positively associated with canopy coverage. Among these, larvae of Rheotanytarsus are known as collector-filterers and to construct conical catchnet for filtering food materials from the water column (Pinder & Reiss, 1983; Berg, 1995). Rheotanytarsus can not use large particles of litter (Coarse Particulate Organic Matter: CPOM) directly, but other species, especially shredders colonizing in leaf packs, should provide fine particles (Fine Particulate Organic Matter: FPOM) that can be used by collector-filterers. For instance, a North American stonefly, Pteronarcys scottii Hagen, known as a shredder (Cummins et al., 1973; Short & Maslin, 1977), was reported to fragment CPOM as much as 16% of its body weight per day (McDiffett, 130 1970). The other congeneric stonefly, P. californica Newport, was shown to inhabit resulted in significant increase in FPOM (Short & Maslin, 1977). By providing FPOM, shredders should contribute to link litter with collectors (Cummins, et al., 1989). The positive associations of Rheotanytarsus with canopy coverage suggest that riparian vegetation functions as an allochthonous food source for the species. In contrast, all the Cricotopus species were negatively associated with canopy coverage. The larvae are known as scrapers and to feed on benthic and/or epiphytic algae (Cranston et al., 1983; Berg, 1995). These results suggest that riparian vegetation functions as a light barrier for aquatic algae, resulting in effective decrease in food source for Cricotopus larvae. On the other hand, distributional patterns of all the species with respect to riparian vegetation cannot necessarily be associated with their feeding mode. Some species were even reported to exhibit considerable flexibility in larval feeding mode (Berg, 1995). Hawkins et al. (1982) showed that streams without any canopy shading had higher abundances in the collector–gatherer, filter feeder, herbivore shredder and piercer, and predator guilds of total macroinvertebrate communities and that Baetidae and Chironomidae were the most abundant taxa in each of guilds. Considering that there was no significant correlation between chironomid assemblage composition and specific conductance, riparian vegetation could merely function as a refuge of imagines for some species, rather than as allochthonous food source. Riparian canopy prevents the imagines from wind, dehydration and predators, and provides appropriate swarming and oviposition sites (Downes, 1969; Sweeney, 1993; Harrison et al., 2000). Structurally complex riparian vegetation leads to a greater supply of insect recruits to all habitats (Glime & Clemons, 1972; Mauer & Brusven, 1983). In this study, larval sampling was practiced between Oct. and Nov. (Table 1). More univoltine species might be collected if it was repeated in springtime and other seasons. Abundance, species richness, diversity and equitability of chironomids Shannon diversity H0 showed a significant negative correlation with bank protection and depth while it showed a positive correlation with specific conductance although the correlation was not significant (Table 6). Bank protection may be associated with watercource modification, which in turn reduces habitat heterogeneity. In general, concrete bank protections are executed by using large construction machinery into stream and riparian terrain. For the purpose of flood control, dredging of the channel and/or even straightening of stream meandering are often accompanied (Mizuno & Gose, 1972). Moreover, the possibility that bank protection not only decreases in water movement between surface water and hyporheos, but also inhibits growth in structurally complex herbaceous vegetation along bankside cannot be discarded. In fact, Harrison & Harris (2002) showed that taxon richness and Shannon diversity H0 of benthic communities were significantly higher in ungrazed streams compared with grazed ones by cattle breeding, meaning that simplified bankside vegetation significantly decreases richness and diversity of benthic communities. Future study should be done to clarify if bank protection negatively associates with complexity of bankside vegetation. Both topographic type (lower reach) and depth showed a significant negative correlation to J (Table 6), indicating that lower reaches of the river or deeper sites tend to be dominated by a small number of species. However, no predictor variable was selected for species richness. The result suggests that richness is a more or less stochastic variable that reflects local variation in species composition, rather than environmental factors. In fact, partial CCA showed that assemblage compositions of some neighboring sites were relatively resemble each other (e.g. sites 5, 6 and 7; 11, 13 and 16; 18 and 21; 22 and 23) and vice versa. Therefore, richness itself may not be a good indicator of chironomid community structure. Nevertheless, the negative correlation between H0 and bank protection, and that the number of species positively associated with canopy coverage was larger than that of species negatively associated with canopy coverage, suggesting a substantial influence of these factors on chironomid richness. Comparisons of richness among assemblages in which the local variation in richness is minimized should be needed to clarify the influence. 131 Both topographic type and specific conductance showed significant positive correlations with abundance, whereas bank protection showed a significant negative correlation (Table 6). Kawai et al. (1989) showed a significant positive correlation between abundance and BOD. A possible explanation to this is that trophic enrichment in lower reaches may result in massive occurrence of nuisance species. In fact, C. bicinctus and C. yoshimatsui, both reported to emerge in massive swarms from polluted waters (Tabaru et al., 1987), were collected in large numbers only at the two lowest sites (Table 2, Fig. 5). Surface velocity was not a predictor variable of any analyses in the present study. This supports the results in Peckarsky & Penton (1990) and Ruse (1994) that surface current on the substrate was a poor predictor of the interstitial flows experienced by chironomid larvae. Acknowledgements We are very grateful to Mr. Shin-ichiro Nakama and Ms. Keiko Anami, formerly Tokushige, both former collaborators, for their kind help in chironomid collection. We also thank the two anonymous reviewers for their helpful comments and suggestions on the early manuscript. Figures 1 and 2 were redrawn from Kani (1944). We regret his early death in the same year in the World War II and acknowledge permission of the publisher, Kenkyusha Co., Inc. The senior author (E. I.) would like to thank Mr. Hiroshi Ashiwa of Kyoto University, Mr. Seiju Inaba and Ms. Fumie Kikkawa for their constant encouragement which inspired me to complete this work. References Conclusion As a whole, the variable most highly associated with chironomid assemblages was topographic type. Longitudinal species succession has been shown in overall benthic communities and in many taxa (Hynes, 1970; Kawai & Takahashi, 1986; Kawai et al., 1998, 1999; Fujitani, 2002). In addition, the significant correlation of the assemblages with altitude and water temperature in partial CCA and its vector direction also emphasize the longitudinal gradient. The river continuum concept predicts changes in structure and function of stream habitats along a transition from headwaters to lower reaches (Vannote et al., 1980). 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