2011 NORTHEASTERN NATURALIST 18(4):411–444 Characteristics of Macroinvertebrate and Fish Communities From 30 Least Disturbed Small Streams in Connecticut Christopher J. Bellucci1,*, Mary Becker1, and Mike Beauchene1 Abstract - Water quality programs in Connecticut and nationally have focused on restoring impaired waters, while modest attention has been allocated to healthy watersheds in the least disturbed condition. The objective of our study was to document the geographic location of least disturbed streams in Connecticut, describe the aquatic biota from these systems, and describe important environmental variables that may help explain the distribution of these biota. We used geographic information systems to select drainage basins by their natural attributes and by eliminating anthropogenic stressor variables in order to best approximate a least disturbed watershed condition in Connecticut. We then sampled the fish and macroinvertebrate communities, water chemistry, and associated GIS-derived watershed attributes to determine the variables that best described the sampled biota. We identified 30 least disturbed streams that had drainage areas <29 km2, whose stream order ranged from 1–4, and that contained <4% total impervious cover in the upstream watershed. Least disturbed streams were generally located in three geographic areas of the state—northwest Connecticut, northeast Connecticut, and the central Connecticut valley—and were absent from the southern coast of Connecticut and southwestern Connecticut. Cluster analysis and nonmetric multidimensional scaling of macroinvertebrate taxa in the Orders Ephemeroptera, Plecoptera, and Trichoptera showed 3 macroinvertebrate stream classes, with 12 significant indicator species (P < 0.05). Drainage area, water temperature, alkalinity, hardness, chloride, ammonia, total nitrogen (TN), and total phosphorus (TP) may explain some of the differences in taxa between macroinvertebrate stream classes. Cluster analysis and nonmetric multidimensional scaling of fish species also showed three fish stream classes, with 9 significant indicator species (P < 0.05). Drainage area, stratified drift, dam density, water temperature, total suspended solids, alkalinity, hardness, ammonia, TN, and TP may explain some of the differences in species between fish stream classes. Ninety percent of the least disturbed streams sampled contained Salvelinus fontinalis (Brook Trout), which can be considered a sentinel fish species for small, least disturbed streams in Connecticut. Introduction The history of water quality management in Connecticut dates back to the Connecticut Water Pollution Control Act (CWPCA) of 1967. Public concern over poor water quality led to the CWPCA, which gave the state authority to require more stringent wastewater treatment for municipal sewerage facilities and industrial discharges to the states waters, and is now incorporated into the General 1Connecticut Department of Environmental Protection, Bureau of Water Protection and Land Reuse, 79 Elm Street, Hartford, CT 06106. *Corresponding author - christopher. bellucci@ct.gov. 412 Northeastern Naturalist Vol. 18, No. 4 Statutes of Connecticut (Chapter 446k, Sections 22a-416 to 22a-599). Nationally, amendments to the Federal Water Pollution Control Act in 1972 and 1977 (FWPCA) resulted in the first comprehensive water pollution law for the nation. This legislation and subsequent amendments still serve as the foundation of surface water quality regulations in the United States. As a result of public concern over poor water quality and the promulgation of these state and federal laws, monitoring the chemical and biological quality of the state’s water resources became a priority issue to track progress of clean water regulations. Biological monitoring has been the foundation for assessing water quality in Connecticut’s rivers and streams since the early 1980s. The concept behind biological monitoring is to use organisms living in streams (e.g., macroinvertebrates, fish) to measure the health of the waters. Karr (1981) first introduced an index of biological integrity (IBI), a composite measure of ecological characteristics, as an index of fish population health. In 1989, the United States Environmental Protection Agency (EPA) introduced guidance that included assessment protocols for fish and macroinvertebrates that expanded the development of multimetric indices to assess stream health (Plafkin et al. 1989). Following EPA’s guidance, the Connecticut Department of Environmental Protection (CTDEP) implemented bioassessment protocols focusing on macroinvertebrates as the foundation of stream health assessment to evaluate the goals of CWPCA and the FWPCA The goal of the FWPCA is to “restore and maintain the chemical, physical, and biological integrity of the nation’s waters.” However, much of the national and state effort to monitor and assess its waters from the 1980s to late 1990s focused on the restoration of “impaired” streams that fell on the “high” portion of the stressor gradient rather than the maintenance or preservation of streams that fell on the “low” portion of stressor gradient (Fig.1). Davies and Jackson (2006) introduced the biological condition gradient (BCG) conceptual model of ecological community change in flowing waters with increased anthropogenic stressors. The BCG describes the ecological community change as a continuum, with one end representing communities exposed to low stress and natural biological condition and the other end representing high stress and degraded biological condition. Since much of the historic monitoring of stream biological communities in Connecticut has focused on impaired waters (i.e., mid–high stress on the BCG), biological communities from natural streams under low stress on the BCG continuum are not well documented. This paper identifies the location of 30 streams in the natural/low-stress portion of the BCG continuum, or least disturbed condition in Connecticut. Given Connecticut’s long history of land-use disturbance (Bell 1985), we follow the definition of Stoddard et al. (2006) that the least disturbed condition is the “best available physical, chemical, and biological habitat conditions given today’s state of the landscape.” We used geographic information system software (GIS, ESRI Arc Map Version 9.2) to select drainage basins by their natural attributes and by eliminating known or suspected anthropogenic stres2011 C.J. Bellucci, M. Becker, and M. Beauchene 413 sor variables in order to best approximate a least disturbed watershed condition. Our goal was to describe important fish species and macroinvertebrate taxa, and to use watershed attributes derived from GIS and water chemistry samples to highlight variables that best described these sampled biota. The results of this study can aid our understanding of fish and macroinvertebrate communities along the low-stress/natural portion of the BCG gradient (Fig 1) in Connecticut and lead to a better understanding of how these streams compare to streams with higher anthropogenic stress. Methods Selection of least disturbed streams We used GIS to select least disturbed streams in Connecticut by evaluating land-use characteristics, water quantity stress (diversions), habitat fragmentation (dams and reservoirs), and salmonid fry stocking records. We used a hierarchical approach to select study streams first by screening at the subregional drainagebasin scale using GIS, followed by catchment-level screening using GIS, and we then followed GIS screening with field checks to determine habitat suitability (i.e., wadeable, good mix of riffle habitat and pool habitat) and validate dam Figure 1. A schematic of the biological condition gradient based on Davies and Jackson (2006), showing focus of water quality efforts on moderately to highly stressed waters since the adoption of Connecticut Clean Water Act of 1967 and Federal Clean Water Act in 1972. 414 Northeastern Naturalist Vol. 18, No. 4 locations shown on our GIS. We only considered wadeable perennial streams with watersheds 2–2000 km2 for our study. We first selected subregional drainage basins, as defined in Nosal (1997), with greater than 80% natural land cover. Percent natural land cover was calculated from 2002 land-cover data produced by the University of Connecticut Center for Land-use Education and Research program and derived from 2002 LandSat satellite imagery. Percent natural land cover was an aggregate percentage of deciduous forest, coniferous forest, open water, and wetland land-cover categories. We calculated the percent natural land cover for each of the 334 subregional basins in Connecticut. Subregional basins in Connecticut range in size from 0.21–457.81 km2, although 95% are less than 101.01 km2 (median = 27.07 km2). For those subregional basins that met the >80% natural land-cover criterion, we applied additional criteria for total percent impervious cover (IC)—water diversions, dams and reservoirs, and salmonid fry stocking in catchments within those subregional basins—to obtain a list of least disturbed streams. Impervious cover has been shown to act as a surrogate measure of negative impacts to aquatic life in streams (Bellucci 2007, Morse et al. 2003, Roy et al. 2005, Stranko et al. 2008, Wang et al. 2001) and therefore is an appropriate screening tool at a broad spatial scale. Subregional basins containing <4% IC were selected for potential study. Subregional basins >4.1% IC were excluded from further analysis. IC was calculated using the Impervious Surface Analysis Tool, an ESRI Arc Map version 9.2 extension, using 2002 Connecticut Land Cover data following the guidelines in Prisloe et al. (2002). The reduction in stream flow from water diversions can reduce the available aquatic habitat and therefore negatively impact the abundance and diversity of aquatic life in streams (Bain et al. 1988, Freeman and Marcinek 2006, Konrad et al. 2008, Poff et al. 1997). The location of water diversions was evaluated using best available data from the CTDEP Inland Water Resources Division. The diversion database contained the locations of approximately 2236 diversions, and we used GIS to select catchments that did not contain diversions. All catchments that contained diversions were excluded. Dams are ubiquitous in Connecticut’s landscape, and can contribute to stream habitat fragmentation and change the natural dynamics of stream ecosystems (Braatne et al. 2008, Graf 1999, Ligon et al. 1995, Poff and Hart 2002, Stanford and Ward 1989). Because dams are so widespread and common, we could not completely eliminate their presence or we would risk having no streams left in our study population. Therefore, we attempted to eliminate large dams from our analysis and included an acceptable threshold distance downstream from smaller dams. To infer the presence of large dams, we used a combination of a CTDEP database containing Hazard Class C dams and a Connecticut Department of Public Health (CTDPH) database containing information on reservoir size. Hazard Class C dams are defined as dams that impound large volumes of water and could be hazardous if the dam were breached. Waterbodies listed as reservoirs in the CTDPH database are typically used for public water supply storage and 2011 C.J. Bellucci, M. Becker, and M. Beauchene 415 are usually not run of river. First, we screened stream segments using GIS and excluded those with Hazard Class C dams or reservoirs in upstream segments. Second, we used the CTDEP dam location database to eliminate stream reaches that were within 1.6 km of a dam and selected free-flowing sections of stream that were located greater than 1.6 km from a dam. We thought that 1.6 km was a reasonable distance to filter immediate ecological impacts from small dams for our study, while still retaining some sections of stream for our study. Fish stocking can have negative impacts on natural fish populations (Faush 1988, Kreuger and May 1991) and was therefore a consideration to identifying least disturbed streams in Connecticut. Salmo trutta L. (Brown Trout) fry and Salmo salar L. (Atlantic Salmon) fry stocking records were obtained from the CTDEP Fisheries Division, and streams stocked with these species were eliminated because it is not possible to discriminate naturally reproduced Brown Trout fry from stocked fry; most occurrences of juvenile Atlantic Salmon in Connecticut are stocked fish. We then used GIS to select stream segments that were not influenced by fry stocking of these species. We did not exclude streams that were stocked with adult salmonids because our selection criteria dictated small, remote streams which are typically not stocked with adult salmonids. In addition, we hypothesized that there would be few, if any, adult stocked streams in the potential stream choices given our other selection criteria, and that if captured, adult stocked salmonids would be easily identified in the field. Field checks were used to evaluate site accessibility, standardize sampling habitat (e.g., reaches with no riffle habitat or too deep to wade were eliminated), and verify dam locations. For watersheds that met all the above GIS screening criteria and field checks, the latitude and longitude of the sampling sites were recorded with a Garmin Model 76 GPS. We then used those coordinates and the Arc Hydro extension of GIS to delineate the watershed upstream of the sampling point. Our GIS selection criteria, followed by site visits, resulted in 30 small least disturbed streams as our study population. Biological communities and water quality Benthic macroinvertebrate samples were collected September–October 2007 using an 800-um-mesh kick net. A total of 2 m2 of riffle habitat (12 kicks composited from multiple riffles of a stream reach) was sampled at each location. Samples were preserved in 70% ethyl alcohol and brought back to the laboratory for subsampling. A 200-organism subsample was taken using a random grid design (Plafkin et al. 1989) from each sampling location. Organisms were identified to the lowest practical taxon, generally species. A macroinvertebrate multimetric index (MMI) score for each site was calculated using a 200-organism subsample at the genus level (Gerritsen and Jessup 2007). The MMI is composed of 7 metrics: Ephemeroptera (E) taxa, Plecoptera (P) taxa, Trichoptera (T) taxa, percent sensitive EPT, scraper taxa, BCG taxa biotic index, and percent dominant genus (Table 1). The MMI score is the average score of all seven metrics and ranges from 0–100, with low values representing 416 Northeastern Naturalist Vol. 18, No. 4 high stress and high values representing least stressed sites. For this paper, we followed the convention of CTDEP to aid in interpretation of the MMI scores as follows: MMI < 44 fails aquatic life goals, MMI range of 45–55 is an inconclusive assessment, and MMI > 56 passes aquatic life goals. These MMI values are typically used by CTDEP as part of the decision criteria for assessing aquatic life for Clean Water Act 305 (b) reporting and Section 303 (d) impaired water listing. We evaluated the MMI values from our study streams along the humandisturbance gradient using a scatter plot of MMI and IC. We included locations in Connecticut that were sampled outside of this study to allow comparison of MMI values from this study to MMI values from streams with higher levels of human disturbance. To accomplish this, 125 sites from wadeable streams in Connecticut with macroinvertebrate samples (Bellucci 2007) collected using the same sampling protocols as in this study were included in the scatter plot. Fish sampling was conducted from June–September 2007 during periods of low streamflow to maximize sampling efficiency. Typically, 150 m of stream were electrofished using either a backpack unit or a single tow barge electrofishing unit (Hagstrom et al. 1995). A single pass was completed at each location, and all species were measured to the nearest centimeter (total length), counted, and immediately released into the stream. A surface-water grab sample was collected from mid-channel at least once during spring, summer, and fall 2007 at each site and analyzed for total nitrogen, ammonia, total phosphorus, pH, alkalinity, hardness, and chloride. Water temperature was measured concurrent with site visits from May–September 2007 using a calibrated thermometer. Statistical analysis We calculated the percent occurrence of fish taxa from 30 least disturbed study streams and macroinvertebrate taxa from 24 least disturbed study streams. We Table 1. Description of the seven metrics used to calculate the macroinvertebrate multi-metric index (MMI). The MMI is calculated as the average of the seven metrics. For more details on metrics that compose the MMI, see Gerritsen and Jessup (2007). Trend = trend in response to increasing stress. Metric Description Trend E taxa Number of genra in the Order Ephemeroptera (E). Decrease This metric is adjusted for watershed size. P taxa Number of genera in the Order Plecoptera (P). Decrease T taxa Number of genera in the Order Trichoptera (T). Decrease % EPT Number of organisms in the Orders EPT excluding the Decrease families Hydropsychidae and Baetidae divided by the total number of organisms in the samples times 100. This metric is adjusted for watershed size. Scraper taxa Number of genera in the scraper functional feeding group Decrease % dominant genus Number of organisms in the genus with the most individuals Increase divided by total number of organisms multiplied times 100. BCG taxa Average of BCG attributes for each genera. Increase 2011 C.J. Bellucci, M. Becker, and M. Beauchene 417 also compared the percent occurrence of macroinvertebrates and fish taxa from this study to other streams in Connecticut that were subjected to greater human disturbance. To accomplish this, we established 3 bins using IC as a measure of human disturbance. Bin 1 consisted of the streams for this study with IC < 4%, bin 2 included mid-level stress sites with IC = 4.1–11.9%, and bin 3 contained high-level stress sites with IC > 12%. IC was calculated as described above. We then queried the CTDEP ambient monitoring database for wadeable stream sites where fish and macroinvertebrate taxa were collected using the same methodology used in this study, and we calculated the percent occurrence of taxa for each bin. We only report taxa that were found in this study since our goal was to compare the taxa from least disturbed smaller streams in Connecticut (i.e., taxa that occurred exclusively in bins 2 and 3 were not included in this analysis). Cluster analysis (CA) was used to explore taxa similarities between least disturbed streams separately for fish species and macroinvertebrate taxa. For macroinvertebrate stream classes, we evaluated taxa from the orders Ephemeroptera or E taxa (mayflies), Plecoptera or P taxa (stoneflies) and Trichoptera or T taxa (caddisflies). EPT were selected because these orders are known to be a dominate component of community richness in least disturbed conditions and as such would provide the most instructive information. For fish, we initially evaluated all species to determine stream fish classes. For both EPT taxa and fish species, taxa proportional abundances were arcsine square-root transformed to improve normality. The Sorensen distance measure with the flexible beta linkage method (beta = -0.25) was used in all CA. Species that occurred in less than 5% of the samples (McCune and Grace 2002) were removed from the analysis for both the EPT and fish analysis. For fish, in addition to eliminating rare species, stocked salmonids and Cyprinidae <3 cm were also eliminated from the data matrix. The 44 EPT taxa by 24 site matrix for EPT and 17 fish species by 30 site matrix were used to produce dendrograms using PC ORD Version 5 (MjM Software Design, Gleneden Beach, OR). Indicator species analysis (Dufrene and Legendre 1997) was used as an objective criterion to prune the dendrograms. The P-values from the Monte Carlo tests (1000 permutations) were averaged for all species after pruning the cluster dendrogram into 2, 3, 4, 5, 6, and 7 clusters, and the lowest average P-values determined the appropriate number of clusters (McCune and Grace 2002). We also used Wishart’s (1969) objective function and percent- information-remaining statistic to interpret the site dissimilarity. The percent-information-remaining statistic indicates the relative distance between sites as defined by the location of the dendrogram branches. Sites that span a short distance of percent information remaining have more homogeneous taxa than sites that span a greater distance. Cluster analysis results were displayed as a dendrogram that graphically displays the relationship of sites to each other based on the proportions of taxa present at each site. Sites that span a short distance of the dendrogram (i.e., percent-information-remaining statistic) have more homogeneous taxa than sites that span a greater distance. 418 Northeastern Naturalist Vol. 18, No. 4 Ordination plots using nonmetric multidimensional scaling (NMS) were used as another graphical interpretation of taxa similarities between small, least disturbed streams. We followed recommendations in McCune and Grace (2002) to seek solutions with low stress and select the appropriate number of dimensions. We used the Sorensen distance measure and ran 250 iterations with real data, and then performed a Monte Carlo simulation with random data over 250 iterations to compare the solutions with real data to solutions that might be obtained by chance. We used these results, combined with a scree plot, to determine the solution with lowest stress in relation to dimensionality, then reran the NMS to obtain the final ordination plots for macroinvertebrate stream classes and fish stream classes. After determining the macroinvertebrate and fish site classes using CA and NMS, indicator species analysis (Dufrene and Legendre 1997) was used to highlight taxa that were indicative of each of the macroinvertebrate and fish stream classes. Indicator species analysis combines a measure of taxa relative abundance and relative frequency of taxa into an indicator value score ranging from 0% (no indication) to 100% (perfect indication). A taxon with perfect indication of 100% would mean that it occurs at all sites in a group and is exclusive to that group (i.e., does not occur in other groups). We noted species that had indicator values greater than expected by chance using a 1000 permutation Monte Carlo test (P < 0.05). We used watershed attributes and water chemistry parameters collected during the study to describe variables that may influence the fish and macroinvertebrate stream classes as determined by the CA. For each catchment, we calculated MMI, drainage area (km2), percent stratified drift, road density (number per km2), and dam density (number per km2) using GIS. For each variable, differences in the data distribution among fish sites class and macroinvertebrate sites class were determined using the Kruskal Wallace test (P < 0.05). Results Description of 30 least disturbed streams in Connecticut The 30 least disturbed streams had drainage areas <29 km2 and Strahler stream order that ranged from 1–4; all contained <3.5% IC in the upstream watershed, and contained a high percentage of forested land use (Table 2). In general, the 30 least disturbed streams were located in three geographic groups: northwest Connecticut, northeast Connecticut, and the central Connecticut River valley (Fig. 2). Pendleton Hill Brook (SID 1748) was the only least disturbed stream that was located outside of these three groups. Four least disturbed streams were located in the town of East Haddam. Ashford, Canaan, and Lyme each contained three least disturbed streams and Barkhamsted, East Hampton, and Torrington each contained two least disturbed streams. Eleven towns contained one least disturbed stream. Least disturbed streams were absent from southwestern Connecticut and along the southern coast because the combination of urbanization, dams, diversions, and stocking practices excluded these streams. 2011 C.J. Bellucci, M. Becker, and M. Beauchene 419 Table 2. Location, drainage area, stream order, percent impervious cover, percent coniferous forest, and percent deciduous forest of thirty least disturbed streams in Connecticut, listed by station identification number (SID). SID’s correspond with Figure 2. Drainage Stream % impervious % coniferous % deciduous SID Stream Town Latitude Longitude area (km2) order cover forest forest 766 Stickney Hill Brook Union 41.9833 -72.2179 6 3 2.06 58.95 27.65 1236 Beaver Brook Lyme 41.4100 -72.3289 21 4 2.43 1.46 78.85 1239 Burhams Brook East Haddam 41.4603 -72.3343 3 1 2.19 7.90 81.09 1435 Cedar Pond Brook Lyme 41.4119 -72.3128 21 3 2.66 1.50 78.17 1748 Pendleton Hill Brook Stonington 41.4748 -71.8342 10 2 2.51 2.70 76.72 1941 Bebbinton Brook Ashford 41.8447 -72.1593 6 3 3.24 2.17 55.46 1981 Carse Brook Sharon 41.8552 -73.3755 14 3 2.43 0.66 84.99 2291 Branch Brook Eastford 41.9108 -72.1245 13 3 1.97 65.78 18.88 2293 Knowlton Brook Ashford 41.8492 -72.1783 18 4 2.89 1.24 73.13 2294 Gardner Brook Ashford 41.8643 -72.1598 4 2 3.37 1.42 67.21 2295 Mott Hill Brook Glastonbury 41.6615 -72.5365 7 2 2.17 1.97 83.75 2296 Beaver Meadow Brook Haddam 41.4553 -72.5288 4 2 2.97 27.42 60.03 2297 Hemlock Valley Brook East Haddam 41.4283 -72.4226 7 3 3.00 5.53 65.14 2298 Hungerford Brook Lyme 41.4255 -72.4094 7 3 3.41 1.35 69.16 2299 Rugg Brook Winchester 41.9328 -73.1214 5 2 1.93 59.95 23.08 2301 Kettle Brook Barkhamsted 41.9324 -72.9442 4 3 1.77 63.53 31.37 2302 Roaring Brook Barkhamsted 41.9454 -72.9475 4 2 1.53 77.61 15.20 2303 Powder Brook Harwinton 41.7541 -73.0170 3 2 2.23 1.00 62.97 2304 Day Pond Brook Colchester 41.5623 -72.4338 3 2 3.17 6.57 72.88 2305 Elbow Brook East Hampton 41.5211 -72.4869 2 2 2.67 0.00 87.66 2306 Flat Brook Central East Hampton 41.5544 -72.4523 6 2 3.09 1.10 81.88 2307 Early Brook East Haddam 41.4978 -72.3435 6 2 3.17 0.69 81.49 2308 Muddy Brook East Haddam 41.4756 -72.3420 3 2 2.91 7.26 79.04 2309 Flat Brook North Canaan 41.9459 -73.3200 7 2 2.45 15.24 68.84 2310 Whiting Brook Canaan 41.9730 -73.3178 2 2 1.21 48.89 47.37 2311 Hall Meadow Brook Torrington 41.8861 -73.1689 27 3 2.13 35.67 50.13 2312 Jakes Brook Torrington 41.8646 -73.1679 4 3 2.08 23.09 63.94 2331 Stonehouse Brook Chaplin 41.7812 -72.1509 14 4 2.66 0.29 77.98 2334 Chatfield Hollow Brook Madison 41.3314 -72.5950 29 4 3.20 0.52 75.86 2342 Brown Brook Canaan 41.9267 -73.2799 14 3 1.22 50.63 39.66 420 Northeastern Naturalist Vol. 18, No. 4 Figure 2. Location of the 30 least disturbed streams in Connecticut. Station identification number (SID) correspond to sites listed in Table 1. Figure 3. Scatter plot of macroinvertebrate multimetric index (MMI) and percent total impervious cover (IC) upstream of the sampling site. Solid triangles are the 24 least disturbed study streams, and the open circles are other site locations in Connecticut with samples collected in the same manner as used in this study (Bellucci 2007). 2011 C.J. Bellucci, M. Becker, and M. Beauchene 421 Biological communities from least disturbed streams Macroinvertebrate communities were sampled from 24 of the 30 least disturbed streams. Six streams—Stickney Hill Brook (SID 766), Bebbington Brook (SID 1941), Branch Brook (SID 2291), Roaring Brook (SID 2302), Powder Brook (SID 2303, and Whiting Brook (2310)—were not sampled due to inadequate stream flow during the fall benthic sampling index period (September 15–November 30). Macroinvertebrate MMI scores ranged from 50–91 (average = 72, s.d. = 9.50), indicating the majority of the least disturbed streams passed aquatic life goals (Table 3). The one exception was an MMI value of 50 for Hall Meadow Brook (SID 2311), which was an inconclusive assessment. When compared to other streams in Connecticut along the human-disturbance gradient, the MMI scores from this study were consistent with our understanding of the BCG conceptual model (Fig. 3). That is, the majority of least disturbed streams had MMI values that scored towards the natural (least stressed) portion of the MMI scale and, therefore, the BCG scale as well. A total of one hundred forty six macroinvertebrate taxa were identified from the 24 least disturbed streams (Appendix 1). Several macroinvertebrate taxa Table 3. Macroinvertebrate multimetric index (MMI) and metrics: Ephemeroptera (E) taxa, Plecoptera (P) taxa, Trichoptera taxa, percent sensitive EPT (scoring adjusted for watershed size), scraper taxa, biological condition gradient (BCG) taxa biotic index, and percent dominant genus for 24 least disturbed streams by station identification number (SID). BCG taxa % % sensitive Scraper biotic dominant SID Sample date MMI E taxa P taxa T taxa EPT taxa index genus 1236 9/24/2007 70 100 33 38 100 64 68 87 1239 9/25/2007 85 100 83 62 100 64 89 98 1435 9/25/2007 67 38 17 85 68 82 83 96 1748 9/25/2007 73 42 50 85 82 55 100 95 1981 9/19/2007 66 53 50 38 73 64 100 80 2293 9/28/2007 63 74 17 31 88 73 93 66 2294 9/28/2007 81 100 50 54 100 73 94 100 2295 9/19/2007 61 57 33 62 51 45 83 92 2296 9/19/2007 66 71 33 54 83 45 81 92 2297 9/18/2007 76 90 50 77 74 64 81 97 2298 9/18/2007 65 47 33 69 57 64 96 90 2299 9/21/2007 70 100 33 54 70 82 75 76 2301 9/21/2007 82 100 67 77 100 55 88 89 2304 9/19/2007 80 100 50 77 100 64 70 100 2305 9/19/2007 82 100 33 85 100 64 100 92 2306 9/19/2007 69 49 33 62 84 73 84 97 2307 9/25/2007 73 90 17 77 74 64 87 100 2308 9/25/2007 73 100 33 69 100 55 66 89 2309 9/21/2007 91 89 100 77 96 91 98 84 2311 9/24/2007 50 34 17 46 35 73 57 87 2312 9/24/2007 79 76 67 69 84 73 85 97 2331 9/21/2007 58 39 33 38 70 55 84 89 2334 10/2/2007 65 57 33 69 90 45 65 97 2342 10/9/2007 76 76 67 54 83 73 87 96 422 Northeastern Naturalist Vol. 18, No. 4 documented from the 24 study streams did not occur in other mid-level (4.1–11% IC) or high-level (>12% IC) streams in the CTDEP database. For example, Adicrophleps hitchcocki Flint occurred at 16.67% of the 24 least disturbed streams sampled for macroinvertebrates, but did not occur in streams with higher levels of human disturbance. The percent occurrence of several taxa decreased with increasing human disturbance. For example, Promoresia tardella Fall, Stenelmis, Psephenus herricki DeKay, Hexatoma, Tiplua, Maccaffertium Bednarik, Nigronia serricornis Say, Acroneuria abnormis Newman, Diplectrona, and Dolophilodes all occurred in at least 75% of the of the 24 least disturbed streams sampled for macroinvertebrates, but the percent occurrence declined in streams with higher levels of human disturbance. Other taxa such as Tallaperla, Psilotreta, Ceratopsyche ventura (Ross), Rhyacophila minora Banks, Nanocladius, and Leuctra occurred in fewer than 75% of least disturbed streams, but also declined with higher levels of human disturbance. Some macroinvertebrate taxa showed a positive response to higher levels of human disturbance. For example, Antocha occurred at 4.17% of the least disturbed streams, but the percent occurrence increased to 37% and 43% as IC increased in watersheds in Connecticut. Some taxa appear to be neutral to human disturbance in that the percent occurrence is minimally affected by human disturbance. For example, the Elmid beetle Macronychus glabratus Say occurred at approximately 8% of sites across the gradient of IC. The 146 macroinvertebrate taxa contained 68 EPT taxa, but 24 taxa occurred at <5% of sites and were therefore excluded from the CA to determine macroinvertebrate classes. The indicator species analysis runs of 2–7 clusters of the 44 EPT taxa by 24 site data matrix showed that three clusters had the lowest average P value (P = 0.29098). NMS ordination plots that resulted from a 3-dimensional best fit solution (final stress = 11.30, final instability < 0.00001, 108 iterations) also supported grouping the sites into 3 classes based on the similarities in EPT taxa (Fig. 4). Therefore, three macroinvertebrate stream classes were used in subsequent analysis. Class 1 macroinvertebrate streams contained 8 streams, class 2 macroinvertebrate streams had 9 streams, and class 3 macroinvertebrate streams had 7 streams (Fig. 4). Beaver Brook (SID 1236) and Chatfield Hollow Brook (SID 2334) had the most similar EPT taxa in macroinvertebrate stream class 1. EPT taxa lists from Day Pond Brook (SID 2304) and Muddy Brook (SID 2308) were the most similar for macroinvertebrate class 2 streams. The sites with the most similar EPT taxa in macroinvertebrate stream class 3 were Beaver Meadow Brook (SID 2296) and Early Brook (SID 2307). There were 12 significant indicator taxa among the three macroinvertebrate stream classes (Table 4). Isonychia, a collector-gatherer mayfly, was the taxa most indicative of macroinvertebrate stream class 1. Isonychia had a highly significant (P = 0.0001) indicator value of 96.9%, showing that it occurred almost exclusively in macroinvertebrate class 1 sites and occurred at all class 1 sites. 2011 C.J. Bellucci, M. Becker, and M. Beauchene 423 A collector-filtering caddisfly, Diplectrona, was the taxon most indicative of macroinvertebrate class 2 sites, with an 81.1% indicator value (P = 0.0002). It is worth noting that the collection of Diplectrona from the least disturbed streams in this study represents 30% of its known occurrence in the CTDEP database. Figure 4. Dendrogram and ordination plot using nonmetric multidimensional scaling forming three macroinvertebrate stream classes (class 1 = triangles, class 2 = circles, class 3 = squares) using EPT taxa from 24 least disturbed streams. Refer to Table 1 for more information. 424 Northeastern Naturalist Vol. 18, No. 4 Acroneuria abnormis, a predatory Perlid stonefly, was the taxon most indicative of macroinvertebrate class 3, with a 54.2% indicator value (P = 0.0295). A total of 27 fish species were collected from the 30 least disturbed watersheds (Appendix 2). Natural populations of Salvelinus fontinalis Mitchill (Brook Trout) and Rhinichthys atratulus Hermann (Blacknose Dace) were the most common fish species collected from the thirty least disturbed watersheds. Ninety percent of the least disturbed streams (27/30) sampled in this study contained Brook Trout, and the percent occurrence decreased to 28% in the 4.1–11.9% IC watersheds to 17% at >12% IC watersheds. Brook Trout densities ranged from 29 to 4902 individuals per ha (mean = 630, s.d. = 1003.53) from least disturbed watersheds, but were absent from Beaver Brook (SID 1236), Carse Brook (SID 1981), and Chatfield Hollow Brook (SID 2334). Blacknose Dace occurred at 87% of the least disturbed streams sites, but their occurrence at higher levels of human disturbance remained relatively constant (Appendix 2). Two other fish species were notable since they occurred exclusively in least disturbed streams. Lota lota L. (Burbot), an endangered species in Connecticut (State of Connecticut 2004), was collected from one least disturbed stream. A species listed as endangered is any native species documented by biological research and inventory to be in danger of extirpation throughout all or a significant portion of the state, and to have no more than five occurrences in the state. Cottus cognatus Richardson (Slimy Sculpin) was collected from Mott Hill Brook (SID 2295) and is known to exist only in cold, high water-quality habitat (Edwards and Cunjak 2007). Nine of 27 fish species that were collected in this study occurred in <5% of the samples and so were excluded from the CA to determine fish stream class. Despite our efforts to eliminate fry-stocked Salmo salar L. (Atlantic Salmon) from our pool of study sites, we collected Atlantic Salmon fry from Table 4. Twelve macroinvertebrate taxa indicative of each least disturbed macroinvertebrate stream class as identified using indicator species analysis. Macroinvertebrate stream classes were determined using cluster analysis. P values < 0.05 were considered statistically significant. Relative Relative Indicator Functional abundance frequency value Class Taxa feeding group (%) (%) (%) P value 1 Isonychia Collector-gatherer 97 100 96.9 0.0001 1 Paragnetina media Predator 89 63 55.7 0.0094 1 Maccaffertium modestum group Scraper 64 75 47.7 0.0482 1 Chimarra aterrima Collector-filterer 74 63 46.4 0.0356 2 Diplectrona Collector-filterer 81 100 81.1 0.0002 2 Ceratopsyche ventura Collector-filterer 97 78 75.2 0.0007 2 Tallperla Shredder 69 89 61.5 0.0059 2 Rhyacophila minora Predator 69 78 53.6 0.0211 3 Acroneuria abnormis Predator 54 100 54.2 0.0295 3 Brachycentrus appalachia Collector-filterer 100 43 42.9 0.0193 3 Rhyacophila fuscula Predator 84 43 35.9 0.0462 3 Oecetis persimilis Predator 89 43 37.9 0.0462 2011 C.J. Bellucci, M. Becker, and M. Beauchene 425 two streams in the Salmon River Basin—Day Pond Brook (SID 2304) and Flat Brook (SID 2306)—and Burnhams Brook (SID 1239) in the Eightmile River Basin. These incidental collections of Atlantic Salmon fry and any stocked adult salmonids (Oncoryhchus mykiss Walbaum [Rainbow Trout] and Salmo trutta L. [Brown Trout]) were eliminated from the analysis prior to grouping fish stream classes. The indicator species analysis runs of 2–7 clusters of the 17 fish species by 30 site data matrix showed that three clusters had the lowest average P value (P = 0.18059). NMS ordination plots that resulted from a 3-dimensional best fit solution (final stress = 13.31, final instability < 0.00001, 139 iterations) also supported grouping the sites into 3 classes based on the similarities in fish species (Fig 5). Therefore, similar to the macroinvertebrate stream class analysis, three fish stream classes were used in subsequent analyses. Fish class 1 contained 12 streams, fish class 2 had 7 streams, and fish class 3 had 11 streams (Fig. 5). There were nine significant indicator species among the three fish stream classes (Table 5). Brook Trout and Notemigonus crysoleucas Mitchill (Golden Shiner) were two fish species indicative of fish class 1 streams. Brook Trout, a fluvial specialist species, occurred in all fish class 1 streams (indicator value of 53.7%, P = 0.0026), but was also common in fish class 2 and fish class 3 streams. Golden Shiner, a macrohabitat generalist species, occurred exclusively in three fish class 1 streams and had an indicator value of 41.7% (P = 0.0145). In general, fish class 1 sites had fewer species per site than the Table 5. Nine fish species indicative of each least disturbed fish stream class as identified using indicator species analysis. Fish stream classes were determined using cluster analysis. P values < 0.05 were considered statistically significant. Relative Relative Indicator abundance frequency value Class Species Habitat use (%) (%) (%) P value 1 Salvelinus fontinalis Fluvial specialist 54 100 53.7 0.0026 (Brook Trout) 1 Notemigonus crysoleucas Macrohabitat generalist 100 42 41.7 0.0145 (Golden Shiner) 2 Esox niger Macrohabitat generalist 100 71 71.4 0.0005 (Chain Pickerel) 2 Etheostoma olmstedi Fluvial specialist 100 71 71.4 0.0002 (Tessellated Darter) 2 Semotilus corporalis Fluvial specialist 92 71 65.5 0.0005 (Fallfish) 2 Lepomis macrochirus Macrohabitat generalist 86 87 49.3 0.0105 (Bluegill) 2 Micropterus salmoides Macrohabitat generalist 80 57 45.9 0.0100 (Largemouth Bass) 2 Luxilus cornutus Fluvial dependent 69 57 39.6 0.0316 (Common Shiner) 3 Semotilus atromaculatus Macrohabitat generalist 100 36 36.4 0.0372 (Creek Chub) 426 Northeastern Naturalist Vol. 18, No. 4 2011 C.J. Bellucci, M. Becker, and M. Beauchene 427 other two classes. Five sites in fish class 1 were most similar: Kettle Brook (SID 2301), Whiting Brook (SID 2310), Elbow Brook (SID 2305), Early Brook (SID 2307), and Jakes Brook (SID 2312) all contained Brook Trout and Blacknose Dace. Fish class 2 streams had the highest species richness of the three fish classes and a mix of habitat-use requirements. Six species were significant indicators (P < 0.05) of fish class 2 streams. Esox niger Lesueur (Chain Pickerel) and Etheostoma olmstedi Storer (Tessellated Darter) both had indicator values of 71% and occurred exclusively in fish class 2 streams. Other indicator species of fish class 2 streams were Semotilus corporalis Mitchill (Fallfish), Lepomis macrochirus Rafinesque (Bluegill), Micropterus salmoides Lacepède (Largemouth Bass), and Luxilus cornutus Mitchill (Common Shiner). The sites with the most similar fish species in class 3 were Day Pond Brook (SID 2304) and Brown Brook (SID 2342). Species richness from fish class 3 sites generally fell between class 1 and class 2. The only significant indicator species was Semotilus atromaculatus Mitchill (Creek Chub), a macrohabitat generalist, which had an indicator species value of 36.4% (P = 0.0372). Neither macroinvertebrate stream classes nor fish stream classes were grouped in any noticeable geographic pattern (Fig. 6), suggesting that variables other than geographic location were more important in describing the distribution of macroinvertebrates within least disturbed watersheds. Drainage area, water temperature, alkalinity, hardness, chloride, ammonia, total nitrogen (TN), and total phosphorus (TP) were all significant variables (P < 0.05) between macroinvertebrate Figure 5 (opposite page). Dendrogram and ordination plot using nonmetric multidimensional scaling forming three fish macroinvertebrate stream classes (class 1 = triangles, class 2 = circles, class 3 = squares) using fish species from 30 least disturbed streams. Refer to Table 1 for more information. Table 6. Median site characteristics for least disturbed macroinvertebrate site classes. The Kruskal- Wallis test was used to compare site characteristics between classes and those that showed significantly differences (P < 0.05) are noted with an asterisk. Site characteristic Class 1 Class 2 Class 3 P value Drainage area (km2) 16.07 3.84 6.31 0.004* Stratified drift (%) 4.47 2.01 5.79 0.195 Road density (number per km2) 8.40 7.43 8.62 0.500 Dam density (number per km2) 1.64 0.93 0.95 0.147 Water temperature (ºC) 18.13 16.08 16.96 0.001* Total suspended solids (mg/l) 3.0 2.0 2.0 0.339 Alkalinity (mg/l) 18.5 9.0 9.0 <0.001* Hardness (mg/l) 25.0 11.0 14.0 <0.001* Chloride (mg/l) 6.28 5.48 10.70 0.001* Ammonia (mg/l) 0.011 0.008 0.015 <0.001* Total nitrogen (mg/l) 0.328 0.264 0.357 0.044* Total phosphorus (mg/l) 0.016 0.008 0.011 <0.001* Macroinvertebrate MMI 65.50 80.00 69.00 0.011* 428 Northeastern Naturalist Vol. 18, No. 4 Figure 6. Map of macroinvertebrate stream classes (A) and fish stream classes (B) (class 1 = triangles, class 2 = circles, class 3 = squares) defined using cluster analysis. Refer to Table 1 for more information. 2011 C.J. Bellucci, M. Becker, and M. Beauchene 429 stream classes (Table 6). Macroinvertebrate stream class 1 sites were, in general, larger drainage basins with warmer water temperatures and higher alkalinity and hardness. Macroinvertebrate stream class 2 sites were the smallest and had the least amount of stratified drift, but with similar water temperatures to class 3. Macroinvertebrate stream class 3 sites were intermediate in drainage area and had the highest chloride concentrations. NMS ordination plots also showed a drainage area and temperature gradient for macroinvertebrate stream classes along axis 1 (Fig. 4). Axis 1 (r2 = 0.404) and axis 2 (r2 = 0.388) accounted for approximately about 80% of the variation present in the matrix based on of the Sorensen dissimilarities between all least disturbed sites. Drainage area, stratified drift, dam density, water temperature, total suspended solids, alkalinity, hardness, ammonia, TN, and TP were all significant variables (P < 0.05) between fish stream classes (Table 7). Fish stream class 2 sites had larger drainage areas and warmer water temperatures than fish stream class 1 or 3. Fish stream class 1 sites were, in general, smaller drainage basins with low percentages of stratified drift, slightly cooler water temperatures, and the lowest alkalinity and hardness concentrations of the three fish classes. NMS ordination plots also showed a drainage area and temperature gradient for fish stream classes along axis 2 (Fig 5). Axis 1 (r2 = 0.312) and axis 2 (r2 = 0.248) accounted for approximately about 56% of the variation present in the matrix based on of the Sorensen dissimilarities between all least disturbed sites. Discussion This study is the first that we know of that identifies least disturbed streams in Connecticut. Identifying these 30 least disturbed streams and documenting the fish and macroinvertebrate communities, along with observations on some of the variables that influence their distribution, provides a necessary step to describing the biology of stream organisms under least disturbed conditions in Connecticut. Table 7. Median site characteristics for least disturbed fish site classes. The Kruskal-Wallis test was used to compare site characteristics between classes and those that showed significantly differences (P < 0.05 ) are noted with an asterisk. Site characteristic Class 1 Class 2 Class 3 P value Drainage area (km2) 4.22 20.69 6.31 <0.001* Stratified drift (%) 1.165 9.64 3.72 0.014* Road density (number per km2) 7.39 7.84 9.16 0.140 Dam density (number per km2) 0.00 1.94 1.06 0.003* Water temperature (ºC) 16.32 18.08 16.47 0.003* Total suspended solids (mg/l) 2.0 2.0 3.0 0.595 Alkalinity (mg/l) 8.50 13.00 14.81 <0.001* Hardness (mg/l) 12.00 16.00 21.00 <0.001* Chloride (mg/l) 5.50 8.60 8.03 0.05 Ammonia (mg/l) 0.008 0.017 0.010 <0.001* Total nitrogen (mg/l) 0.294 0.417 0.304 <0.001* Total phosphorus (mg/l) 0.008 0.020 0.010 <0.001* 430 Northeastern Naturalist Vol. 18, No. 4 These 30 least disturbed streams were generally located in three groups in the northeast, northwest, and central Connecticut River valley. The least disturbed streams and their watersheds described in this study represent a subset of the “best of what’s left” in Connecticut and are distributed in this pattern due to past land-use practices and human activities. Human activity—including town settlement, farming, forestry, canals, railroads, highways, mining, gristmills, factory mills, and urbanization—all have influenced Connecticut’s landscape (Bell 1985). The areas that we have identified as least disturbed have not been subjected to land uses, such as urbanization, that can have potential long-term effects on biological communities (Foster 1992, Foster et al. 2003, Harding et al. 1998, Maloney et al. 2008, Wenger et al. 2008). In addition, because these streams represent least disturbed conditions in Connecticut, those without existing land-protection practices could be targeted for protection and potential land acquisition. To this end, we did a cursory GIS analysis based on the best available data on a statewide scale. We calculated the percent of protected land (open space, preserved municipal land, state forests, state parks, and wildlife management areas) in the upstream drainage basin for the 30 least disturbed watersheds. We found that several of these watersheds may have opportunities for future preservation because they showed very low percentages of protected land at the scale we analyzed. It should be noted that a finer scale GIS analysis, which includes attributes such as town land records and data on local conservation and development areas, may reveal other opportunities for preservation and vulnerabilities in these watersheds, and we believe that it would be beneficial to assemble such a GIS layer to include in future analyses. Our approach to identifying least disturbed streams by eliminating known anthropogenic stressors may be valuable for other programs that seek to identify least disturbed conditions. Our attempts to reduce or eliminate anthropogenic impacts, by selecting study streams using a GIS screening followed by site checks, incorporated many potential factors that impact biological integrity of streams, including measures of land use (% IC), stream flow and geomorphology (dams, diversions), habitat fragmentation (dams), and fish stocking (Bellucci 2007, Fausch 1988, Graf 1999, Poff et al. 1997, Wang et al. 2001). We recognize that in some cases, this approach could be viewed as restrictive (e.g., location of diversion is such that it does not impact the stream) or in other cases, there could be factors that are not captured by broad scale GIS (e.g., spills, natural disturbance) and therefore may not represent actual stream conditions. Regardless, all but one of the streams in our study passed aquatic life goals (i.e., MMI > 55) using the macroinvertebrate MMI (Table 2) and, in general, plotted within the expected range of high MMI values given the level of disturbance (Fig. 3). In Hall Meadow Brook (SID 2311), the only least disturbed stream that had an inconclusive MMI score (50), the macroinvertebrate community could have been impacted by the low stream flow during the year prior to the macroin2011 C.J. Bellucci, M. Becker, and M. Beauchene 431 vertebrate index period. This period of low flow is a natural stressor that could not have been identified using GIS. Subsequent visits to this site on Hall Meadow Brook, using the same sampling methodology used in this study under average stream flow conditions, resulted in higher MMI scores (e.g., MMI of 74 collected on 4 November 2008). It is unclear why MMI scores from Hall Meadow Brook could have been more impacted by low stream flow than the other least disturbed watersheds during our study period. Our hypothesis is that low stream flow, coupled with presence of a 0.40-km2 wetland complex upstream of SID 2311, could have combined negative effects that resulted in lower macroinvertebrate abundance and diversity in the 2007 sample. SID 2311 was grouped by the CA into macroinvertebrate stream class 1 (Fig. 4), which consisted of larger drainage basins with warmer water temperatures (Table 6). Re-sampling the macroinvertebrates from least disturbed streams during additional years under varying flow conditions and adding a sampling site upstream of the wetland complex may help resolve this question. Our data show that although most of the least disturbed streams in our study have MMI scores that meet aquatic life goals for Connecticut, there can be differences in the macroinvertebrate taxa and potential influencing abiotic factors that are worth noting. The three macroinvertebrate stream classes each had distinct indicator taxa; our data suggest that drainage area, water temperature, alkalinity, hardness, chloride, ammonia, TN, and TP may potentially be important variables that influence macroinvertebrate taxa distribution in least disturbed watersheds. Our analyses also show that drainage area, stratified drift, dam density, water temperature, total suspended solids, alkalinity, hardness, ammonia, TN, and TP may potentially be important variables that influence fish species distribution in Connecticut’s least disturbed watersheds. These relationships do not show cause and effect relationships, but may help to identify parameters that could be important for monitoring least disturbed watersheds, and are worthy to consider for future monitoring efforts. Further data collection would help to confirm the importance of these variables on macroinvertebrate and fish communities and their influence in forming distinct community groups, such as the biological classes identified by the CA and indicator species analysis in this study. Our results may also reflect our incomplete knowledge of how certain factors affect fish and macroinvertebrate species. On the one hand, variables such as drainage area consistently show a strong influence on macroinvertebrate and fish assemblages (Gerritsen and Jessup 2007, Kanno and Vokoun 2008, Vannote et al. 1980). On the other hand, our knowledge on the influence of dams on macroinvertebrate and fish assemblages is incomplete. For example, although we excluded watersheds with large dams and sampled at least 1.6 km downstream of small dams, macrohabitat generalist fish species were unexpectedly found to be indicator species in all three fish stream classes (Table 5). Brook Trout and Golden Shiner were two fish species indicative of fish class 1 432 Northeastern Naturalist Vol. 18, No. 4 streams. It was unexpected to have a species such as Golden Shiner, a macrohabitat specialist typically associated with ponds, as an indicator of least disturbed streams. This finding may reflect the unavoidable influence that mill dams have on aquatic biota in Connecticut. An interesting follow-up study would be to evaluate the changes in macroinvertebrate and fish species composition with increasing distance from a dam. Our analysis of the percent occurrence of taxa from this study to higher levels of human disturbance indicate that least disturbed streams may offer important habitat for some aquatic species in Connecticut. Some macroinvertebrate taxa may occur exclusively in small, least disturbed streams in Connecticut (e.g., Adicrophleps hitchcocki), while others may be impacted by low levels of human disturbance (e.g., Acroneuria abnormis, Diplectrona spp.). Least disturbed streams may also be important habitat for fish species such as Burbot, Slimy Sculpin, and Brook Trout. We believe that Brook Trout can be viewed as a sentinel species for small, healthy, least disturbed streams in Connecticut because they are the most important indicator fish species and are sensitive to landscape alterations (Kocovsky and Carline 2006, Stranko et al. 2008). Our study documents the occurrence of Brook Trout in 90% of the small, least disturbed streams and a decline in percent occurrence with an increase in human disturbance (Appendix 2). Similar to the use of the sentinel canary in a coal mine to warn miners of potentially lethal carbon monoxide concentrations in coal mines, monitoring shifts in age and size class of Brook Trout populations can warn natural resource managers of potential anthropogenic stress in healthy watersheds. In an investigation that included 1184 streams in Connecticut that were representative of the entire BCG range, Kanno and Vokoun (2008) found that Brook Trout were indicators of small watersheds with cool water temperatures. Similarly, Brook Trout occurred in 90% of least disturbed study watersheds in our study and were a significant indicator of fish class 1 streams. An investigation (e.g., Cormier et al. 2000, Norton et al. 2009, Yuan and Norton 2004) to determine the cause for the absence of Brook Trout from three least disturbed watersheds in our study—Beaver Brook (SID 1236), Carse Brook (SID 1981), and Chatfield Hollow Brook (SID 2334)—could provide an opportunity to learn about important stressors to these least disturbed watersheds. Monitoring water temperature, total suspended solids, alkalinity, hardness, ammonia, TN, and TP, all significant variables in our fish stream analysis, would be an important component of such an investigation. For decades, water programs were funded to support programs that focused on point-source pollution and impaired waters. This strategy has greatly improved the water quality in Connecticut and throughout the nation. However, we believe that the need for funding support to least disturbed streams is long overdue and a more holistic effort is needed to truly fulfill the requirements of the FCWA to maintain, as well as restore, the chemical, physical, and biological integrity of the nation’s waters. 2011 C.J. Bellucci, M. Becker, and M. Beauchene 433 Decades of working on impaired waters has taught us that it is labor intensive, costly, and time consuming to identify, diagnose, and fix impaired waters. While these efforts must continue, we believe that a concurrent strategy to maintain least disturbed watersheds should be employed that involves evaluating their condition and using anti-degradation policies in the FCWA to hold the line and not allow these waters to degrade. This study is an important step in achieving this goal to ensure that we are maintaining the chemical, physical, and biological integrity of the “best of what’s left” in Connecticut. Acknowledgments We thank Carol Youell and Lisa Smith at the Metropolitan District, Ken Gordon at the Bristol Water Department, and Patrick Hague at the Town of Winchester for site access to some of the study streams. Ed Machowski, Neal Hagstrom, and their seasonal crews at the DEP Fisheries Division provided assistance with fish sampling. We thank Brian Jennes, Guy Hoffman, Al Iacobucci, and Tracy Lizotte for their assistance with the macroinvertebrate samples. Mark Hoover assisted with the GIS analysis throughout the project. Paul Stacey and 2 anonymous reviewers provided critical review on earlier drafts of this manuscript. This research was funded, in part, by a 104b (3) Grant from the United States Environmental Protection Agency awarded to the Connecticut Department of Environmental Protection. Literature Cited Armstrong, D.S., T.A. Richards, and G.W. Parker. 2001. 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Biometrics 25(1):165–170. Yuan, L.L., and S.B. Norton. 2004. Assessing the relative severity of stressors at a watershed scale. Environmental Monitoring and Assessment 98:323–349. 2011 C.J. Bellucci, M. Becker, and M. Beauchene 437 Appendix 1. Percent occurrence and functional feeding group for macroinvertebrate taxa collected in this study (n = 24) compared to sites with higher levels of anthropogenic stress. The two additional categories along the human disturbance gradient were established using data collected by Connecticut DEP from wadeable streams in Connecticut 1995–2009 using similar sampling protocols used in this study. Sites were binned by percent impervious land cover (IC) as mid-level stress sites (IC 4.1-11.99%, n = 411) and high-level stress sites (IC values > 12%, n = 127). C-G = collector-gatherer, C-F = collector-filterer, SCR = scraper, SHR = shredder, and PRD = predator. % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Basommatophora Ancylidae Ferrissia 8.33 3.60 6.30 SCR Basommatophora Ancylidae Laevapex fuscus C. B. Adams 8.33 15.10 7.90 SCR Basommatophora Lymnaeidae Pseudosuccinea columella Say 4.17 0.20 0.00 C-G Basommatophora Physidae Physa 4.17 7.10 11.80 C-G Coleoptera Dryopididae Helichus 8.33 0.70 0.00 SCR Coleoptera Elmidae Macronychus glabratus Say 8.33 8.30 8.70 SHR Coleoptera Elmidae Optioservus 37.50 42.80 8.70 SCR Coleoptera Elmidae Optioservus ovalis Leconte 50.00 15.10 3.10 SCR Coleoptera Elmidae Oulimnius 54.17 10.00 3.90 SCR Coleoptera Elmidae Oulimnius latiusculus Leconte 58.33 26.50 11.00 C-G Coleoptera Elmidae Promoresia 4.17 1.90 1.60 SCR Coleoptera Elmidae Promoresia elegans Leconte 4.17 1.50 0.00 SCR Coleoptera Elmidae Promoresia tardella Fall 87.50 15.10 1.60 SCR Coleoptera Elmidae Stenelmis 83.33 29.70 39.40 SCR Coleoptera Hydrophilidae Cymbiodyta 4.17 0.00 0.00 N/A Coleoptera Psephenidae Ectopria 66.67 3.90 3.90 SCR Coleoptera Psephenidae Psephenus herricki DeKay 75.00 32.60 10.20 SCR Coleoptera Ptilodactylidae Anchytarsus bicolor Melsheimer 33.33 3.20 0.00 SHR Decapoda Cambaridae Orconectes limosus Rafinesque 4.17 0.50 1.60 C-G Diptera Athericidae Atherix 12.50 1.20 0.00 PRD Diptera Ceratopogonidae Bezzia group 25.00 1.90 0.80 PRD 438 Northeastern Naturalist Vol. 18, No. 4 % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Diptera Chironomidae Apsectrotanypus 4.17 0.00 0.00 PRD Diptera Chironomidae Brillia 4.17 4.90 8.70 SHR Diptera Chironomidae Corynoneura 25.00 1.90 2.40 C-G Diptera Chironomidae Cricotopus 8.33 7.10 14.20 SHR Diptera Chironomidae Diamesa 4.17 4.60 2.40 C-G Diptera Chironomidae Eukiefferiella 4.17 1.50 1.60 C-G Diptera Chironomidae Eukiefferiella claripennis group Lundbeck 4.17 1.50 3.90 C-G Diptera Chironomidae Eukiefferiella devonica group Edwards 4.17 3.60 0.00 C-G Diptera Chironomidae Eukiefferiella tirolensis Goetghebuer 4.17 0.70 0.80 C-G Diptera Chironomidae Limnophyes 16.67 0.20 0.80 C-G Diptera Chironomidae Lopescladius 4.17 0.50 0.00 C-G Diptera Chironomidae Micropsectra 50.00 2.40 2.40 C-G Diptera Chironomidae Micropsectra/Tanytarsus 4.17 1.20 0.00 C-G Diptera Chironomidae Microtendipes pedellus group De Geer 4.17 18.00 15.70 C-F Diptera Chironomidae Microtendipes rydalensis group Edwards 8.33 3.60 0.00 C-F Diptera Chironomidae Nanocladius 37.50 3.40 0.00 C-G Diptera Chironomidae Natarsia 4.17 0.00 0.00 PRD Diptera Chironomidae Orthocladius (Symposiocladius) lignicola Kieffer 4.17 0.00 0.00 C-G Diptera Chironomidae Parachaetocladius 41.67 3.60 0.00 C-G Diptera Chironomidae Parametriocnemus 66.67 13.40 10.20 C-G Diptera Chironomidae Paratanytarsus 20.83 0.50 0.80 C-F Diptera Chironomidae Polypedilum 12.50 5.80 8.70 SHR Diptera Chironomidae Polypedilum aviceps Townes 54.17 10.90 11.00 SHR Diptera Chironomidae Polypedilum fallax group Johannsen 8.33 1.00 1.60 SHR Diptera Chironomidae Polypedilum tritum Walker 33.33 0.50 0.00 SHR Diptera Chironomidae Rheocricotopus 8.33 2.70 5.50 C-G Diptera Chironomidae Rheotanytarsus exiguus group Johannsen 16.67 18.20 15.00 C-F 2011 C.J. Bellucci, M. Becker, and M. Beauchene 439 % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Diptera Chironomidae Rheotanytarsus pellucidus group Walker 16.67 4.60 3.10 C-F Diptera Chironomidae Stempellinella 4.17 0.20 0.00 C-G Diptera Chironomidae Stenochironomus 12.50 1.90 0.80 C-G Diptera Chironomidae Stilocladius 12.50 1.70 0.80 C-G Diptera Chironomidae Tanytarsus 20.83 4.10 10.20 C-F Diptera Chironomidae Thienemanniella 12.50 2.40 4.70 C-G Diptera Chironomidae Thienemannimyia group 37.50 11.20 18.10 PRD Diptera Chironomidae Tvetenia bavarica group Kieffer 62.50 12.70 11.00 C-G Diptera Chironomidae Tvetenia vitracies group Saether 4.17 18.50 18.90 C-G Diptera Empididae Hemerodromia 25.00 18.00 37.80 PRD Diptera Simuliidae Simulium 12.50 32.10 33.90 C-F Diptera Tabanidae Hybomitra 8.33 0.00 0.00 PRD Diptera Tipulidae Antocha 4.17 37.00 43.30 C-G Diptera Tipulidae Dicranota 70.83 6.10 3.10 PRD Diptera Tipulidae Hexatoma 75.00 2.90 0.80 PRD Diptera Tipulidae Limnophila 8.33 0.50 0.00 PRD Diptera Tipulidae Limonia 4.17 0.20 0.00 PRD Diptera Tipulidae Tipula 87.50 20.20 27.60 SHR Ephemeroptera Baetidae Baetis 16.67 26.80 19.70 C-G Ephemeroptera Baetidae Baetis flavistriga McDunnough 4.17 5.40 6.30 C-G Ephemeroptera Baetidae Baetis pluto McDunnough 16.67 4.40 1.60 C-G Ephemeroptera Baetidae Baetis tricaudatus Dodds 16.67 6.10 9.40 C-G Ephemeroptera Baetidae Diphetor hageni Eaton 4.17 0.00 0.00 C-G Ephemeroptera Baetidae Heterocloeon 4.17 2.40 0.00 SCR Ephemeroptera Baetidae Procloeon 4.17 0.00 0.00 C-G Ephemeroptera Ephemerellidae Ephemerella 41.67 6.30 0.80 C-G Ephemeroptera Ephemerellidae Eurylophella funeralis McDunnough 33.33 3.90 3.10 C-G 440 Northeastern Naturalist Vol. 18, No. 4 % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Ephemeroptera Ephemerellidae Serratella deficiens Morgan 8.33 11.70 0.80 C-G Ephemeroptera Heptageniidae Epeorus 12.50 5.10 0.00 SCR Ephemeroptera Heptageniidae Maccaffertium 91.67 22.90 16.50 SCR Ephemeroptera Heptageniidae Maccaffertium modestum group Banks 54.17 31.40 26.80 SCR Ephemeroptera Heptageniidae Maccaffertium terminatum Walsh 8.33 0.20 0.00 SCR Ephemeroptera Heptageniidae Maccaffertium vicarium Walker 25.00 1.00 0.00 SCR Ephemeroptera Isonychiidae Isonychia 41.67 31.10 4.70 C-G Hoplonemertea Tetrastemmatidae Prostoma 8.33 5.10 7.90 PRD Lumbriculida Lumbriculidae 66.67 17.30 15.00 C-G Megaloptera Corydalidae Corydalus cornutus Linnaeus 4.17 10.90 4.70 PRD Megaloptera Corydalidae Nigronia serricornis Say 100.00 27.30 20.50 PRD Megaloptera Sialidae Sialis 8.33 3.20 1.60 PRD Neotaenioglossa Hydrobiidae Amnicola limosus Say 4.17 4.60 0.80 SCR Odonata Aeshnidae Boyeria vinosa Say 54.17 4.40 0.80 PRD Odonata Calopterygidae Calopterygidae 8.33 0.50 0.00 PRD Odonata Cordulegastridae Cordulegaster 4.17 0.20 0.00 PRD Odonata Cordulegastridae Cordulegaster maculate Selys 4.17 0.00 0.00 PRD Odonata Gomphidae Lanthus 20.83 0.50 0.00 PRD Odonata Gomphidae Lanthus parvulus Selys 8.33 0.70 0.00 PRD Odonata Gomphidae Lanthus vernalis Carle 4.17 0.00 0.00 PRD Odonata Gomphidae Ophiogomphus 20.83 2.70 0.00 PRD Odonata Gomphidae Stylogomphus albistylus Hagen 8.33 3.60 2.40 PRD Odonata Libellulidae 4.17 0.00 0.00 PRD Plecoptera Capniidae Paracapnia 8.33 1.20 0.00 SHR Plecoptera Chloroperlidae Sweltsa 29.17 1.50 0.80 PRD Plecoptera Leuctridae Leuctra 33.33 1.50 0.00 SHR Plecoptera Peltoperlidae Tallaperla 54.17 1.90 0.00 SHR 2011 C.J. Bellucci, M. Becker, and M. Beauchene 441 % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Plecoptera Perlidae Acroneuria 25.00 7.10 1.60 PRD Plecoptera Perlidae Acroneuria abnormis Newman 79.17 20.40 1.60 PRD Plecoptera Perlidae Agnetina capitata Pictet 4.17 0.70 0.00 PRD Plecoptera Perlidae Eccoptura xanthenes Newman 12.50 1.50 0.00 PRD Plecoptera Perlidae Paragnetina 8.33 1.20 0.00 PRD Plecoptera Perlidae Paragnetina immarginata Say 8.33 2.40 0.00 PRD Plecoptera Perlidae Paragnetina media Walker 29.17 9.70 0.80 PRD Plecoptera Perlodidae 8.33 2.20 0.80 PRD Plecoptera Pteronarcyidae Pteronarcys 16.67 0.70 0.00 SHR Plecoptera Taeniopterygidae Taeniopteryx 4.17 12.90 5.50 SHR Trichoptera Apataniidae Apatania 8.33 21.40 2.40 SCR Trichoptera Brachycentridae Adicrophleps hitchcocki Flint 16.67 0.00 0.00 SHR Trichoptera Brachycentridae Brachycentrus appalachia Flint 12.50 0.50 0.00 C-F Trichoptera Brachycentridae Micrasema 37.50 9.50 3.90 SHR Trichoptera Glossosomatidae Agapetus 4.17 0.00 0.00 SCR Trichoptera Glossosomatidae Glossosoma 62.50 38.70 22.00 SCR Trichoptera Goeridae Goera 12.50 1.50 0.00 SCR Trichoptera Helicopsychidae Helicopsyche borealis Hagen 8.33 4.10 0.00 SCR Trichoptera Hydropsychidae Ceratopsyche sparna Ross 41.67 39.90 28.30 C-F Trichoptera Hydropsychidae Ceratopsyche ventura Ross 41.67 0.20 0.00 C-F Trichoptera Hydropsychidae Cheumatopsyche 87.50 64.20 66.90 C-F Trichoptera Hydropsychidae Diplectrona 79.17 4.40 3.90 C-F Trichoptera Hydropsychidae Hydropsyche 70.83 29.40 33.10 C-F Trichoptera Hydropsychidae Hydropsyche betteni Ross 50.00 56.40 55.90 C-F Trichoptera Lepidostomatidae Lepidostoma 45.83 13.10 1.60 SHR Trichoptera Leptoceridae Mystacides sepulchralis Walker 4.17 0.20 1.60 C-G Trichoptera Leptoceridae Oecetis persimilis Banks 16.67 0.20 0.00 PRD 442 Northeastern Naturalist Vol. 18, No. 4 % occurrence % occurrence % occurrence mid-level high-level Functional this study stress stress feeding Order Family Taxon (n = 24 sites) (n = 411 sites) (n = 127 sites) group Trichoptera Limnephilidae 25.00 1.50 0.00 SHR Trichoptera Odontoceridae Psilotreta 45.83 1.90 0.00 SCR Trichoptera Philopotamidae Chimarra aterrima Hagen 29.17 39.20 22.80 C-F Trichoptera Philopotamidae Dolophilodes 91.67 18.70 12.60 C-F Trichoptera Philopotamidae 4.17 3.20 0.80 C-F Trichoptera Polycentropodidae Polycentropus 29.17 4.90 0.00 PRD Trichoptera Psychomyiidae Lype diversa Banks 8.33 0.70 0.00 SCR Trichoptera Rhyacophilidae Rhyacophila 62.50 9.00 2.40 PRD Trichoptera Rhyacophilidae Rhyacophila carolina Banks 33.33 7.10 0.80 PRD Trichoptera Rhyacophilidae Rhyacophila fuscula Walker 16.67 15.60 6.30 PRD Trichoptera Rhyacophilidae Rhyacophila manistee Ross 12.50 1.90 0.00 PRD Trichoptera Rhyacophilidae Rhyacophila minora Banks 41.67 1.20 0.00 PRD Trichoptera Uenoidae Neophylax 4.17 6.60 0.80 SCR Trombidiformes Limnocharidae Rhyncholimnochares 12.50 0.20 0.00 PRD Trombidiformes Sperchonidae Sperchon 4.17 1.70 1.60 PRD Tubificida Tubificidae Tubificidae with cap setae 4.17 0.00 0.80 C-G Veneroida Pisidiidae Pisidium 8.33 0.50 0.80 C-F 2011 C.J. Bellucci, M. Becker, and M. Beauchene 443 Appendix 2. Percent occurrence, tolerance value, and stream flow guild for fish species collected in this study (n = 30) compared to sites with higher levels of anthropogenic stress. Species collected in this study were compared to 2 additional categories along the human disturbance gradient based on data collected by Connecticut DEP from wadeable streams in Connecticut 1995–2009 using similar sampling protocols used in this study. Sites were binned by percent impervious land cover (IC) as mid-level stress sites (IC 4.1-11.99%, n = 341) and high-level stress sites (IC values > 12%, n= 109). Tolerance values and stream preferences were taken from regional references (Armstrong et al. 2001, Halliwell et al. 1999, Whitworth 1996). I = intolerant, M = intermediate, T = tolerant; FS = fluvial specialist, FD = fluvial dependent, MG = macrohabitat generalist. % occurrence % occurrence % occurrence mid-level high-level Water this study stress stress Tolerance Flow temperature Family Species (n = 30 sites) (n = 341 sites) (n = 109 sites) value guild preference Native Anguillidae Anguilla rostrata Lesueur 40.00 71.55 67.89 T FD W Yes Catostomidae Catostomus commersoni Lacepède 40.00 82.40 77.06 T FD Cool Yes Centrarchidae Lepomis auritus Linnaeus 3.33 38.12 25.69 M MG W Yes Centrarchidae Lepomis cyanellus Rafinesque 16.67 9.09 9.17 T FD W No Centrarchidae Lepomis gibbosus Linnaeus 13.33 40.76 33.03 M MG W Yes Centrarchidae Lepomis macrochirus Rafinesque 20.00 48.39 43.12 T MG W No Centrarchidae Micropterus dolomieu Lacepède 3.33 20.53 5.50 M MG Cool No Centrarchidae Micropterus salmoides Lacepède 20.00 37.24 40.37 M MG W No Cottidae Cottus cognatus Richardson 3.33 0.00 0.00 I FS Cold Yes Cyprinidae Luxilus cornutus Mitchill 26.67 30.21 18.35 M FD Cool Yes Cyprinidae Notemigonus crysoleucas Mitchill 16.67 16.13 17.43 T MG W Yes Cyprinidae Rhinichthys atratulus Hermann 86.67 82.70 81.65 T FS Cool Yes Cyprinidae Rhinichthys cataractae Valenciennes 30.00 46.92 49.54 M FS Cool Yes Cyprinidae Semotilus atromaculatus Mitchill 13.33 21.70 19.27 T MG Cool Yes Cyprinidae Semotilus corporalis Mitchill 23.33 35.78 13.76 M FS Cool Yes Esocidae Esox americanus Gmelin 6.67 14.66 11.01 M MG W Yes Esocidae Esox niger Lesueur 16.67 12.32 3.67 M MG W Yes Gadidae Lota lota Linnaeus 3.33 0.00 0.00 M FS Cold Yes 444 Northeastern Naturalist Vol. 18, No. 4 % occurrence % occurrence % occurrence mid-level high-level Water this study stress stress Tolerance Flow temperature Family Species (n = 30 sites) (n = 341 sites) (n = 109 sites) value guild preference Native Ictaluridae Ameiurus natalis Lesueur 3.33 5.57 0.92 T MG W No Ictaluridae Ameiurus nebulosus Lesueur 13.33 15.54 9.17 T MG W Yes Percidae Etheostoma fusiforme Girard 3.33 0.29 0.00 I MG W Yes Percidae Etheostoma olmstedi Storer 16.67 59.82 57.80 M FS Cool Yes Percidae Perca flavescens Mitchill 3.33 16.72 12.84 M MG Cool Yes Salmonidae Oncorhynchus mykiss Walbaum 3.33 21.99 10.09 I FS Cold No Salmonidae Salmo salar Linnaeus 10.00 6.45 1.83 I FS Cold Yes Salmonidae Salmo trutta Linnaeus 16.67 29.33 20.18 I FS Cold No Salmonidae Salvelinus fontinalis Mitchill 90.00 28.15 17.43 I FS Cold Yes