Cytotaxonomic Survey of Black Fly Immatures within Ohio’s Erie Drift Plain J.A. Bryant, B.R. Dunning, M.R. Gwinn, N.J. Kohler, M.A. McCain, T.A. Moore, A.A. Schroer, K.A. Stevenson, M.J. Mendel Introduction Results Black flies are insects of the order Diptera and family Simuliidae that are largely associated with In this study, a total of 86 sites were sampled within 21 counties in Ohio between March 10 and April running water, as their immature stages require such conditions. These insects are of particular 27. Since this is the timeframe within which the species diversity of black fly larvae is highest, a interest because of their potential as model organisms in ecological studies of running waters. They maximum number of species were collected. Individuals were identified from 22 species and four are often used in ecological assessments because they are widely distributed across many regions, distinct genera (Prosimulium, Simulium, Stegopterna, and Cnephia) (Table 1). Three species with nearly 2,000 described species worldwide. Although black flies are some of the most (Prosimulium fuscum, P. magnum, Simulium claricentrum) had not been found in Ohio prior to this taxonomically well-known aquatic insects, it appears that the current estimate of species is low. This sampling, and an additional four species (S. decorum, S. jenningsi, S. pilosum, and S. verecundum) is largely due to the prevalence of cryptic (sibling) species which are morphologically indistinguishable were each only identified at one site (1.2% of sites). This identification of new species is likely due to from one another. One method that has proven useful in distinguishing between cryptic species of the fact that they are rare, and difficult to differentiate from similar sibling species. By using black flies is the cytotaxonomic method involving large, polytene chromosomes found in the larval silk cytotaxonomy these rare species were able to be identified. glands. The aim of this research was to conduct a survey of black flies throughout the Erie Drift Plain All seven of these species were identified in the northeastern portion of Ohio, in either Ashtabula (P. ecoregion of Ohio using cytological and morphological characteristics to identify black fly species. fuscum and P. magnum), Geauga (S. decorum, S. verecundum), Cuyahoga (S. pilosum) or Lake (S. Materials and Methods claricentrum, S. jenningsi) County. Several species collected were widespread, with three species (P. arvum, P. albionense, S. venustum) identified at more than 25% of sites and four others (S. There are six ecoregions in Ohio (Fig. 9). Ecoregions are delineated by differences in soil taxonomy, tribulatum, Stegopterna mutata, P. mixtum, S. perissum) identified at more than 15% of sites. Two potential natural vegetation, land use, and land-surface form. Within an ecoregion, these species (P. albionense and S. venustum) were identified at more than 75% of counties sampled, and characteristics are similar. If differences occur, they will not contrast to the extent that the whole were evenly distributed throughout the ecoregion. The eleven remaining species were identified in picture of the ecoregion will be fragmented. Therefore, repeating characteristic patterns within the Figure 6. Estimate of black fly species richness in the Erie Drift Plain of Ohio less than 15% of counties, all of which were only found at four or fewer sites. This study also collected ecoregion will keep it homogeneous. Streams of the Erie Drift Plain ecoregion were sampled in this This graph shows a rarefaction estimate for black fly species richness. “S(est)” in the above graph legend stands for “estimate of species richness”. The solid blue line is based on rarefaction of data from 86 sampled state records for six species, including those previously not identified in Ohio. study, which includes the upper northeastern portion of Ohio and extends in the southeasterly sites, and the solid red and orange lines indicate the upper and lower bounds of the 95% confidence interval of direction to the middle of the state. that rarefaction. The blue dashed line is an extrapolation of the species richness from the 86 sample sites Black flies have particularly large polytene chromosomes (Fig. 8) that can be easily located in their based on an additional 20 randomly generated sites. The red and orange dashed lines represent the 95% silk glands. These chromosomes may be used to identify cryptic species, while previously they were The Erie Drift Plain is characterized by level plains with minimal local relief, and it extends from confidence interval of the extrapolated data. often conflated with other species. The chromosomes’ large size is the result of cell enlargement, and eastern Ohio to southwestern New York, passing through northwestern Pennsylvania. The region Species Percent of counties Number of sites Percent of sites not cell proliferation. These polytene chromosomes also have distinct banding patterns and consists of cropland, pastureland, and woodland. One-tenth of the region provides pasture for cattle, micromorphological characteristics such as wide diameters in certain areas that are unique to each S. venustum 86% 33 38.4% cropland comprises nearly one-third of the region, and 20 percent of the region is urbanized. species. By examining the micromorphological characteristics of these large, easily accessible Interspaced throughout these regions are areas of northern hardwood forest, which largely influence P. albionense 76% 29 33.7% polytene chromosomes, black fly sibling species can be determined. the soil traits of the region. Large watersheds throughout the region cover nearly 500 square miles on S. tribulatum 52% 20 23.3% average, with most streams being perennial. The rarefaction curve (Fig. 6) demonstrates that the sample size of 86 sites has resulted in a species P. arvum 48% 25 29.0% richness estimate beyond the asymptote of the curve. This indicates that further sampling of the Erie For this research, black fly larvae and pupae were removed from various substrates in the streams S. perissum 43% 15 17.4% Drift Plain is not needed to establish species richness. Likewise, when extrapolating our data set by and placed in Carnoy’s fixative, a solution of glacial acetic acid and 95%-100% ethanol mixed in a 1:3 P. mixtum 38% 16 18.6% 20 samples, the measured black fly species richness of 22 fell within the 95% confidence interval of ratio. Species were primarily determined using cytotaxonomy, a method that uses an organism’s 18-29 species (Fig. 6). Additional extrapolation by doubling our sample size also indicated that the chromosomal morphology to distinguish between sibling species. In addition, some morphological Stegopterna mutata 33% 18 21.0% measured species richness was within the projected 95% confidence interval for species richness. characteristics were also used in species identification. EstimateS (a free software program) S. tuberosum 24% 7 8.1% This also signifies that the data is a reasonably accurate representation of the black fly species developed by Colwell (2013) was used to develop rarefaction and extrapolation curves (including P. canutum 19% 7 8.1% richness for Ohio’s Erie Drift Plain and further sampling is not required. associated confidence intervals) for black fly species richness using Chao1 and Chao2 estimators. S. hematophilum 19% 4 4.7% Stegopterna diplomutata 14% 4 4.7% S. vittatum 14% 3 3.5% S. vandalicum 9.5% 2 2.3% Cnephia dacotensis 9.5% 2 2.3% P. multidentatum 4.8% 2 2.3% S. decorum 4.8% 1 1.2% S. jenningsi 4.8% 1 1.2% S. pilosum 4.8% 1 1.2% S. verecundum 4.8% 1 1.2% Figure 1. Slate substrate riffles P. fuscum 4.8% 1 1.2% Demonstration of a riffle area - where sampling took place, with a bottom substrate of slate rock. P. magnum 4.8% 1 1.2% S. claricentrum 4.8% 1 1.2% Table 1. Results of black fly sampling “Species” lists all the species in order of frequency by county. “Number of counties” gives the number of Figure 8. Black fly chromosomes species found per county. “Percent of counties” lists the species as a percentage in all counties. “Number of Polytene chromosomes of black fly larva used in cytotaxonomic study. sites” enumerates the amount of site where that particular species was present. “Percent of sites” gives the Figure 9. Ohio Ecoregions species presence as a percentage of all sites. Ecoregions present in the state of Ohio. This study focused on the Erie Drift Plain, in green, at the upper right corner. Conclusions Multiple species, including newly found species for Ohio, were identified through collection. The new and rare species were all found in the northeastern region of Ohio, near the boundary of the Ohio portion of the Erie Drift Plain. Introducing cytotaxonomy into the methods of research allowed us to Figure 2. An Ohio stream with pools Figure 4. Map of sampling locations identify these new and rare species that had not yet been identified in Ohio. Our rarefaction curves This is an example of a stream that was sampled, The purple dots indicate a stream location in the Erie Drift Plain indicate that the sampling done in this study was sufficient to accurately identify the total black fly this picture shows pools, in contrast to riffles. where black fly sampling took place. species richness in this region, and that additional sampling is not necessary. The species found through a wider geographic range are likely more capable of living in a broader set of environmental conditions, while the novel and rare species may be specialized to unique environments. Investigations of black fly species distributions provide insight on diverse ecological patterns between species. This research could be useful in developing innovative and more precise methods of water quality evaluation. Future studies exploring the environmental characteristics of northeastern Ohio should provide greater understanding of the ecological factors driving the unique black fly biodiversity found within this region.