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Nest Association and Reproductive Microhabitat of the Threatened Blackside Dace, Chrosomus cumberlandensis
Hayden T. Mattingly and Tyler R. Black

Southeastern Naturalist, Volume 12, Special Issue 4 (2013): 49–63

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49 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 Nest Association and Reproductive Microhabitat of the Threatened Blackside Dace, Chrosomus cumberlandensis Hayden T. Mattingly1,* and Tyler R. Black1,2 Abstract - Chrosomus cumberlandensis (Blackside Dace) is a federally protected cyprinid fish found in small tributaries of the upper Cumberland River system in southeastern Kentucky and northeastern Tennessee. Relatively little is known about the species’ reproductive ecology and early life history. From a small number of field observations, the species is known to spawn as an associate with other cyprinid nest-building hosts, namely Campostoma anomalum (Central Stoneroller) and Semotilus atromaculatus (Creek Chub). In the present study, we first analyzed Blackside Dace co-occurrence patterns with other cyprinids to predict the relative importance of each species to Blackside Dace nestassociation behavior. We next studied Blackside Dace spawning activities in seven 200-m reaches in five Kentucky streams during May–July 2006 to document nest associations and measure microhabitat conditions at spawning and non-spawning locations. Three of the seven study reaches were impacted by active logging operations. We observed 25 Blackside Dace spawning events, and all 25 were associated with Creek Chub nests, consistent with predictions from our species co-occurrence analysis. Spawning microhabitats were located in areas with significantly greater mean wetted-channel widths, slower column and bottom velocities, lower silt levels, lower substrate embeddedness, and larger subdominant substrate particles compared to non-spawning microhabitats. Study reaches with adjacent active logging had significantly greater mean silt levels, substrate embeddedness, water temperature, and conductivity values compared to reaches with no active logging, although 4 of the 25 spawning events occurred in reaches with active logging. Our results highlight the importance of cyprinid nest-building hosts (especially Creek Chub) to Blackside Dace reproductive ecology, and they also reinforce the need to maintain the integrity of Blackside Dace streams at the whole-commu nity level. Introduction Strategies for conserving biodiversity are best developed with knowledge of important life-history and ecological characteristics of imperiled species. However, the reproductive ecology of many imperiled aquatic species remains unknown or only partially studied for a variety of reasons. An illustration of this pattern is provided by the species-rich minnow family, Cyprinidae, in which spawning modes are known for only 13 of 46 imperiled species (Johnston 1999). Johnston and Page (1992) reviewed the reproductive strategies of minnows and classified the strategies into eight categories. The most primitive and common behavior is the broadcasting of gametes with no preparation of the substrate. Another strategy with no substrate preparation is termed crevice spawning. The other six strategies involve some preparation or use of substrate to form a nest in 1Department of Biology, Box 5063, Tennessee Technological University, Cookeville, TN 38505. 2Current Address - North Carolina Wildlife Resources Commission, 1718 NC Hwy 56 W, Creedmoor, NC 27522. *Corresponding author - Ecology and Conservation of the Threatened Blackside Dace, Chrosomus cumberlandensis 2013 Southeastern Naturalist 12(Special Issue 4):49–63 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 50 Vol. 12, Special Issue 4 which eggs are deposited and fertilized. Parental care in the form of nest guarding is provided by males in some instances. One of the fascinating and complex facets of cyprinid reproduction is the nestassociation behavior displayed by certain species. Nest association occurs when one species, the host, prepares a nest that another species, the associate, uses for spawning (Johnston and Page 1992). The host species is typically another cyprinid, but some associates are known to use centrarchid nests (e.g., Fletcher 1993, Johnston and Page 1992). More than one associate species may spawn in a host’s nest. For example, Cashner and Bart (2010) found eggs of two associate species along with host eggs in the nest of Nocomis leptocephalus (Girard) (Bluehead Chub), and Johnston and Page (1992) observed as many as six associate species at one time over Nocomis nests. Nest-association behavior has been reported for at least 33 cyprinid species (Johnston and Page 1992). Some cyprinids show flexibility in their reproductive behavior by spawning either as an associate in the nests of hosts or independent of the host by broadcasting or by building their own nests (Johnston and Page 1992). Understanding the degree of host dependence and host specificity exhibited by a particular species is important. For example, if an imperiled broadcasting species is obligated to only spawn in nests of one or more host species, then conservation of the associate is intimately tied to conservation of the hosts, a situation paralleling that of freshwater mussels. Johnston (1999) has emphasized the importance of understanding cyprinid reproductive strategies to better inform conservation efforts. Chrosomus cumberlandensis (Starnes and Starnes) (Blackside Dace) is a threatened cyprinid species endemic to the upper Cumberland River system in Kentucky and Tennessee (Eisenhour and Strange 1998, Starnes and Starnes 1978). Blackside Dace reproductive behavior was first reported by Starnes and Starnes (1981) who confirmed the species used a broadcasting spawning mode over the nest of Campostoma anomalum (Rafinesque) (Central Stoneroller). Cicerello and Laudermilk (1996) later observed a school of nuptial Blackside Dace over the nest of Semotilus atromaculatus (Mitchill) (Creek Chub), although actual spawning was not observed. To date, no field observations of Blackside Dace spawning independent of nest-building cyprinids have been reported, although most authors have presumed that independent spawning may occur . Cicerello and Laudermilk (1996) offered three findings regarding Blackside Dace nest association that needed confirmation by additional research. First, their observations suggested that Blackside Dace spawn in Creek Chub nests, even in relatively silt-free streams. Second, they predicted that Creek Chub was a more important host than either of the two upper Cumberland River stoneroller species (Central Stoneroller and Campostoma oligolepis Hubbs and Greene [Largescale Stoneroller]) because Blackside Dace co-occurred more often with Creek Chub than stonerollers in their collections. Third, they opined that cyprinid hosts play a vital role in Blackside Dace conservation by providing spawning habitat both in streams with relatively clean substrates, as well as streams degraded by siltation. 51 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 Conservation efforts for Blackside Dace would be enhanced by a more comprehensive understanding of its reproductive behavior. Our objectives in the present study were to (1) analyze patterns of Blackside Dace co-occurrence with other cyprinid species to allow predictions of the relative importance of cyprinid hosts, (2) observe Blackside Dace spawning events in the field under varied conditions of substrate cleanliness to determine host species and possibly document independent spawning, and (3) measure microhabitat conditions at spawning and non-spawning locations to characterize the range of conditions under which Blackside Dace reproduction occurs in the field. Our approach to addressing these objectives entailed two phases. We first used two fish-collection datasets from the upper Cumberland River system to analyze Blackside Dace co-occurrence patterns with selected cyprinid species. We next conducted a field study of Blackside Dace reproductive activities at seven sites located in five streams in southeastern Kentucky. Two of the study streams had active logging operations during our field work, and substrates in these streams were subjected to elevated levels of siltation. The other three streams had no active logging operations adjacent to or upstream of our study sites, thereby providing less-silted substrate conditions for observation of spaw ning activities. Methods Species co-occurrence patterns We used two upper Cumberland River fish-collection datasets to analyze patterns of cyprinid species co-occurrence. The first dataset was provided by Laudermilk and Cicerello (1998) who reported 450 collections from Kentucky during 1982–1994, with most being made during 1993–1994. The second dataset was provided by Black et al. (2013 [this issue]) who sampled 119 sites in Kentucky and Tennessee during 2003–2006. For each set of data, collection records were selected if at least one of the following cyprinid species was present: Central Stoneroller, Largescale Stoneroller, Blackside Dace, Chrosomus erythrogaster (Rafinesque) (Southern Redbelly Dace), Luxilus chrysocephalus Rafinesque (Striped Shiner), Nocomis micropogon (Cope) (River Chub), Rhinichthys obtusus Agassiz (Western Blacknose Dace), and Creek Chub. The two stoneroller species were combined for analyses because of their ecological similarity and lack of spatial overlap (Central Stoneroller occurs above Cumberland Falls, and Largescale Stoneroller occurs below the falls; Burr and Warren 1986). Three hundred and ninety-five of 450 collection records by Laudermilk and Cicerello (1998) and 119 of 119 records by Black et al. (2013 [this issue]) noted the occurrence of at least one of these cyprinid species. All of the cyprinids listed above could potentially have strong ecological interactions with Blackside Dace during reproductive activities. Blackside Dace, Southern Redbelly Dace, and Western Blacknose Dace are broadcasting spawners that do not prepare their own nest during spawning. However, all three species at least occasionally interact with nest-building cyprinids by spawning in prepared nests of the hosts. Southern Redbelly Dace are known to H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 52 Vol. 12, Special Issue 4 use Campostoma, Luxilus, and Nocomis nests and Western Blacknose Dace are known to use Nocomis nests (Etnier and Starnes 1993, Johnston and Page 1992). However, Blackside Dace spawning activities have only been observed over Central Stoneroller and Creek Chub nests, and it remains unknown whether it can spawn independent of a host species (Cicerello and Laudermilk 1996, Starnes and Starnes 1981). Central Stoneroller, Largescale Stoneroller, Striped Shiner, River Chub, and Creek Chub are nest-building species that could potentially serve as hosts to the three aforementioned broadcasting dace species. The stonerollers and Striped Shiner are pit-building species that provide no parental care subsequent to egg deposition, Creek Chub is a pit-ridge-building species in which males cover eggs with gravel substrate after egg deposition, and River Chub is a mound-building species in which males also cover eggs with gravel (Boschung and Mayden 2004, Etnier and Starnes 1993, Johnston 1999). Creek Chub and River Chub males therefore provide rudimentary parental care with their nest-maintenance and eggcovering activities. We used two metrics, constancy and fidelity, to express co-occurrence patterns of the selected cyprinid species, considering each dataset separately. For any given species, constancy was the number of occurrences with Blackside Dace as a percentage of total Blackside Dace occurrences. Fidelity was the number of occurrences with Blackside Dace as a percentage of total occurrences of the given species. Pflieger (1978) originally defined constancy, dominance, and fidelity in his study of fish species co-occurrence patterns with Etheostoma nianguae Gilbert and Meek (Niangua Darter) in Missouri, and Wagner et al. (2010) recently used these metrics in their distributional analysis of Orconectes williamsi Fitzpatrick (Williams’ Crayfish) in Arkansas. Although other co-occurrence metrics are available in the literature (e.g., Peres-Neto 2004), constancy and fidelity were ideally suited for our study to identify patterns that could indicate strong species interactions during Blackside Dace reproductive activities. Different ecological and evolutionary processes certainly could influence the observed co-occurrence patterns, but we interpreted high constancy of a species with Blackside Dace as a necessary prerequisite for a strong nest-association relation ship. Field study of Blackside Dace spawning activities Study sites. We selected seven 200-m stream reaches in which to observe Blackside Dace spawning activities. The reaches were located in 5 different streams occupied by Blackside Dace in Knox, McCreary, and Pulaski counties in southeastern Kentucky (Fig. 1, Table 1). We identified reaches with a variety of Blackside Dace background densities, and with presumably different levels of siltation on the substrate. Black et al. (2013 [this issue]) determined density of Blackside Dace at 6 of the 7 reaches in 2003 and 2005, and densities ranged from 0.0–49.8 dace per 100 m2 (Table 1). We selected active logging as a land-use disturbance that was likely to introduce fine sediments to the stream, and thereby create conditions that might discourage independent spawning by Blackside 53 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 Dace, and encourage reliance on a nest-building host species to provide clean substrate for spawning. In 3 reaches located in 2 streams, we observed active logging operations adjacent to or upstream from the study reaches (Table 1). We Figure 1. Map of the study area in southeastern Kentucky. Triangles indicate 200-m study reaches with active logging and circles indicate study reaches without active logging. See Table 1 and text for additional details about the study reaches. Table 1. Characteristics of 200-m stream reaches in southeastern Kentucky where Chrosomus cumberlandensis (Blackside Dace) spawning activities were monitored weekly from 4 May–7 July 2006. Stream reach numbers refer to relative downstream (1) and upstream (4) locations within streams, and are consistent with Black et al. (2013 [this issue]). Blackside Dace background densities were determined for 6 of the sites in 2003 or 2005 by Blac k et al. (2013 [this issue]). Blackside Dace Blackside Dace background density spawning surveys in 2006 Spawning Dace per Year Sampling events Active Stream reach County 100 m2 measured dates observed logging? Big Lick Branch 3 Pulaski 49.8 2003 4 May–29 June 14 No Big Lick Branch 4 Pulaski - - 5 May–30 June 6 No Grubb Branch 1 Knox 23.2 2005 12 May–5 July 0 No Rock Creek 4 McCreary 44.4 2003 4 May–6 July 1 No Roaring Fork 1 Knox 10.2 2005 13 May–7 July 2 Yes Roaring Fork 2 Knox 0.0 2005 10 May–7 July 0 Yes Right Branch Moore Creek 1 Knox 9.3 2005 13 May–6 July 2 Yes H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 54 Vol. 12, Special Issue 4 did not measure the spatial extent of the logging activities, nor did we otherwise attempt to quantify the amount of logging disturbance. In contrast, in the remaining four reaches (Table 1), we observed no active or recent logging operations adjacent to or upstream from the reach. Spawning activities. A two-person crew performed weekly surveys from 4 May–7 July 2006 to observe Blackside Dace spawning activities. Each person walked slowly upstream through the reach, on or near opposite stream banks when possible. Spawning activities were observed from the nearest bank or the bank with greatest visibility; binoculars were used if greater resolution was warranted. If a school of nuptial individuals was spotted, we observed its behavior and/or spawning activities for 20 min. We used three categories to classify Blackside Dace reproductive activities: (1) schooling nuptial individuals, (2) males corralling females to spawning area, and (3) males pressing female to substrate and vibrating in unison (clasping). We defined a “spawning event” as either category (2) or (3), because it was unclear when gametes were actually released, and because both corralling and clasping indicated that appropriate spawning habitat had been selected. If a spawning event was observed, we marked the location with survey flagging to allow later measurements of microhabitat conditions at the spawning site. When spawning events occurred over a host nest, we visually identified the host-species, if present, and assessed its nest. Our criteria for comparing nests of taxa currently known or suspected to host Blackside Dace spawning were as follows: the stoneroller species excavate shallow pits with dislodged pebbles lining the periphery of the nest, and Creek Chubs excavate a nest that contains a pit and ridge (Boschung and Mayden 2004). Survey observations were continued until the 200-m marker was reached, after which surveyors returned to the lower margin of the reach to begin assessing habitat variables. Spawning habitat measurements. If Blackside Dace spawning events were observed during a survey, we returned to the flagging markers and measured habitat conditions at the spawning site. The center of the spawning site itself was evaluated as a microhabitat with a 10-cm radius, hereafter termed a spawning microhabitat. We measured 13 habitat variables at each spawning microhabitat, including wetted-channel width, canopy cover, silt depth, embeddedness, substrate composition, temperature (oC), dissolved oxygen (mg/L), conductivity (μS), water depth (cm), water velocity (cm/s) at the substrate and in the water column, and turbidity (NTU). Wetted-channel width was measured (nearest 0.1 m) by stretching a meter tape perpendicular to stream-flow across the spawning microhabitat. Canopy cover was visually categorized considering the entire wetted-channel width crossing the spawning microhabitat (0 = no canopy cover, 1 = 1–25% cover, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100%). Visual estimates of silt depth, substrate embeddedness, and substrate composition were made across the entire area of each 10-cm-radius microhabitat. Visual estimates were checked with a ruler at the beginning of the field season. Silt depth was the most commonly occurring depth of silt covering the protruding portion 55 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 of the substrate and was scored with a silt index (0 = 0 mm silt, 1 = 0.1–1.0 mm silt, 2 = 1.1–2.0 mm, 3 = 2.1–3.0 mm, and 4 = >3 mm) similar to that used by Mattingly and Galat (2002). Substrate embeddedness was categorized as the percentage of gravel, pebble, cobble, and boulder particle surfaces covered by fine sediment (fine sediment includes material <2 mm in diameter [sand, silt, and clay]) as follows: 0 = “negligible” = <5% coverage with fines, 1 = “low” = 5–25%, 2 = “moderate” = 25–50%, 3 = “high” = 50–75%, and 4 = “very high” = >75% (Bain 1999, Platts et al. 1983). Dominant and subdominant substrate compositions were scored using a modification of the Wentworth scale: 0 = fines <0.059 mm, 1 = sand 0.06–1.0 mm, 2 = gravel 2–15 mm, 3 = pebble 16–63 mm, 4 = cobble 64–256 mm, 5 = boulder >256 mm, and 6 = bedrock (Bain 1999). Water depth was measured with a top-setting wading rod. Water velocity in the water column above the microhabitat was measured with a Marsh-McBirney Flo-Mate 2000 portable flowmeter, with the probe located at six-tenths depth as measured down from the water surface or four-tenths depth as measured up from the streambed (Gordon et al. 1992). Water velocity was also measured at the streambed with the probe at its lowest possible setting (i.e., bottom velocity). Temperature, dissolved oxygen, and conductivity were measured with a Yellow Springs Instrument (YSI) Model 85 meter, and turbidity was measured with an HF Scientific MicroTPI. All microhabitat parameters were measured once at each spawning microhabitat except turbidity, water depth, and water velocities. Turbidity was measured above the center of the spawning microhabitat when one such site occurred, but when multiple sites occurred in close proximity (<1 m apart), only one sample was taken between spawning areas. Water depths and velocities were determined by averaging four values immediately surrounding each spawning microhabitat (i.e., upstream, downstream, right, and left) to avoid damaging eggs or embryos that might have been present in the center of the microhabitat. Non-spawning habitat measurements. Non-spawning microhabitats were established by delineating transect points as follows. Like the spawning microhabitats, each non-spawning microhabitat was considered to be a circle on the streambed with a 10-cm radius. We partitioned the 200-m study reaches using transects perpendicular to stream-flow at 10-m intervals, for a total of 21 transects per reach. We sampled 5 or 6 of the 21 transects each week. Specifically, we sampled transects 1, 5, 9, 13, 17, and 21 on the first week; transects 2, 6, 10, 14, and 18 on the second week; transects 3, 7, 11, 15, and 19 on the third week; and transects 4, 8, 12, 16, and 20 on the fourth week. On the fifth week we resampled transects 1, 5, 9, 13, 17, and 21, but sampling took place approximately 1.0 m upstream from where we sampled the first week. Throughout the 9-week study period, we continued this pattern of systematically rotating through transects on a 4-week interval, moving upstream 1 m each time a given transect was revisited. The number of microhabitat points sampled per transect was determined by the wetted-channel width, with one point sampled for every meter of width. H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 56 Vol. 12, Special Issue 4 Within each meter, the precise location of a transect point was determined by drawing a number between 1 and 9. Each number represented a possible point at 10-cm increments (e.g., the number 3 represented a transect poi nt at 30 cm). All non-spawning microhabitat measurements were taken at the transect-point as described above for the spawning microhabitats, with the following exceptions. Instead of averaging four values for water depth and velocity readings, we simply measured these variables once in the center of the microhabitat. Also, turbidity values were taken at the upper and lower periphery of each reach during surveys and then averaged to obtain one turbidity value for the reach. Each non-spawning microhabitat within the reach was then assigned the same turbidity value for that sampling date. Statistical analyses. For each habitat variable, we calculated a mean value separately for spawning and non-spawning microhabitats in each of the seven study reaches. Next we used a series of nonparametric, Kruskal-Wallis twosample tests to address the following questions: (1) did conditions differ between spawning (n = 5 reaches) and non-spawning (n = 7 reaches) microhabitat mean values for any habitat variable; and (2) did non-spawning microhabitat mean values differ between reaches with (n = 3) and without (n = 4) active logging for any habitat variable? Tests were conducted using the NPAR1WAY procedure in SAS Version 9.2 ( Statistical significance was evaluated with α = 0.05. Results Species co-occurrence patterns Blackside Dace were observed in 95 collections during 1982–1994 and 78 collections during 2003–2006. Species constancy with Blackside Dace was highest for Creek Chub, exceeding 90% in both datasets, and easily surpassing constancy values for other species (Table 2). The only other species with constancy >50% were the stonerollers at 56.4% in the 2003–2006 collections. Most other species had constancy values of 20–35%, although River Chub never occurred with Blackside Dace. Fidelity values with Blackside Dace were highest for Southern Redbelly Dace in each dataset, at 43.3% for 1982–1994 and 90.3% for 2003–2006 (Table 2). Field study of Blackside Dace spawning activities Spawning activities. Creek Chubs were actively constructing nests during the first week of May, although nuptial coloration and schooling behaviors by Blackside Dace were not observed until mid-May. Twenty-five Blackside Dace spawning events (20 corralling and 5 clasping) were observed 12 May–12 June 2006, when water temperatures for such events ranged from 11.9–18.2 oC (mean ± SD = 15.3 ± 2.1 oC). Sixteen events were observed in May and nine were seen in June, with spawning activity ceasing after 12 June, and Creek Chub nest maintenance ending the following week. 57 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 All 25 Blackside Dace spawning events occurred over Creek Chub nests, and no observations of independent spawning were noted. Blackside Dace spawned in Creek Chub nests in Big Lick Branch, Rock Creek, Roaring Fork, and Right Branch Moore Creek, even in the presence of active logging disturbances (Table 1). However, most events were observed in Big Lick Branch, a stream with relatively high densities of Blackside Dace and with no active logging in its watershed. Schools of nuptial males were only observed over Creek Chub nests and ranged from 3 to ≈60 individuals. Corralling and clasping behaviors were exhibited by 2–4 males pursuing one female. In addition, most individuals in the school probed the nest after a clasping event, presumably feeding on released eggs. Differences between spawning and non-spawning microhabitats. Spawning microhabitats were located in areas with significantly greater mean wettedchannel widths, slower column and bottom velocities, lower silt levels, lower substrate embeddedness, and larger subdominant substrate particles compared to the non-spawning, transect-point microhabitats (Kruskal-Wallis χ2 ≥ 5.129; P ≤ 0.0235; Table 3). Notably, all 25 spawning microhabitats had only negligible levels of substrate embeddedness and no measurable levels of silt covering substrate particles, both presumably due to the nest-building and -maintenance activities Table 2. Patterns of species co-occurrence with Chrosomus cumberlandensis (Blackside Dace) for selected cyprinid species in the upper Cumberland River drainage. Two sets of data were evaluated: one from 1982–1994 collections in Kentucky (n = 395; Laudermilk and Cicerello 1998) and another from 2003–2006 collections in Kentucky and Tennessee (n = 119; Black et al. 2013 [this issue]) in which at least one of the selected species was collected. BSD = Blackside Dace. Constancy (C) = number of occurrences with Blackside Dace as a percentage of total Blackside Dace occurrences. Fidelity (F) = number of occurrences with Blackside Dace as a percentage of total occurrences of the species. Occurrences Species Total With BSD C (%) F (%) 1982–1994 collections Campostoma spp. (Central Stoneroller; Largescale Stoneroller) 164 28 29.5 17.1 Chrosomus cumberlandensis (Blackside Dace) 95 - - - Chrosomus erythrogaster (Southern Redbelly Dace) 60 26 27.4 43.3 Luxilus chrysocephalus (Striped Shiner) 68 3 3.2 4.4 Nocomis micropogon (River Chub) 12 0 0.0 0.0 Rhinichthys obtusus (Western Blacknose Dace) 85 20 21.1 23.5 Semotilus atromaculatus (Creek Chub) 331 87 91.6 26.3 2003–2006 collections Campostoma spp. (Central Stoneroller; Largescale Stoneroller) 65 44 56.4 67.7 Chrosomus cumberlandensis (Blackside Dace) 78 - - - Chrosomus erythrogaster (Southern Redbelly Dace) 31 28 35.9 90.3 Luxilus chrysocephalus (Striped Shiner) 26 18 23.1 69.2 Nocomis micropogon (River Chub) 0 0 0.0 0.0 Rhinichthys obtusus (Western Blacknose Dace) 40 22 28.2 55.0 Semotilus atromaculatus (Creek Chub) 116 75 96.2 64.7 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 58 Vol. 12, Special Issue 4 Table 3. Kruskal-Wallis two-sample tests to determine if reach mean values for habitat variables differed between spawning and non-spawning microhabitats in Blackside Dace streams. Values for conductivity and turbidity were rounded to nearest wh ole numbers immediately before inclusion in the table. Kruskal-Wallis Reach mean values tests Habitat variable Spawning microhabitats Non-spawning microhabitats χ2 P Wetted-channel width (m) 3.1, 3.1, 3.3, 3.6, 3.8 1.8, 1.9, 2.0, 2.1, 2.3, 2.4, 2.5 8.07 0.005 Canopy cover index 3.5, 3.5, 3.9, 4.0, 4.0 3.2, 3.3, 3.6, 3.7, 3.7, 3.8, 3.8 1.49 0.222 Silt index 0, 0, 0, 0, 0 1.0, 1.0, 1.2, 1.4, 1.6, 1.7, 2.3 8.68 0.003 Embeddedness index 0, 0, 0, 0, 0 0.6, 0.7, 0.9, 1.0, 1.6, 1.9, 2.4 8.68 0.003 Dominant substrate index 3.0, 3.0, 3.3, 3.5, 3.6 2.6, 2.9, 3.4, 3.4, 3.5, 3.5, 3.6 0.06 0.807 Subdominant substrate index 2.4, 2.5, 3.0, 4.0, 4.0 2.1, 2.2, 2.2, 2.3, 2.3, 2.4, 2.7 5.60 0.018 Temperature (°C) 14.0, 14.4, 15.0, 15.7, 17.9 15.1, 16.0, 16.5, 16.6, 16.9, 17.0, 17.1 2.38 0.123 Dissolved oxygen (mg/L) 7.9, 8.3, 8.3, 8.6, 9.1 8.1, 8.4, 8.4, 8.5, 8.5, 8.6, 8.7 0.17 0.684 Conductivity (μS) 23, 29, 47, 90, 181 23, 29, 54, 71, 74, 100, 182 0.24 0.626 Water depth (cm) 5, 8, 13, 23, 34 7, 9, 9, 9, 10, 11, 11 0.53 0.461 Bottom velocity (cm/s) 2, 2, 3, 4, 5 5, 5, 5, 5, 6, 7, 8 6.84 0.009 Column velocity (cm/s) 3, 3, 3, 5, 5 4, 5, 5, 6, 6, 6, 9 5.13 0.024 Turbidity (NTU) 1, 2, 6, 8, 11 1, 2, 7, 9, 10, 15, 21 0.66 0.416 59 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 of Creek Chub males. Mean wetted-channel widths in spawning locations were 3–4 m, consistently wider than average values for transects in non-spawning locations (Table 3). Differences between microhabitats in reaches with and without logging. Study reaches with active logging had significantly greater mean silt levels, substrate embeddedness, water temperature, and conductivity values compared to reaches with no active logging (Kruskal-Wallis χ2 = 4.50; P = 0.0339; Table 4). Despite these trends, 4 of the 25 spawning events were observed in Creek Chub nests in two reaches with active logging (Table 1). Discussion Blackside Dace spawning events were observed over a 32-day timespan (12 May–12 June 2006) during our study. Across its distributional range, however, the spring spawning season for Blackside Dace may begin earlier and extend later than these dates. As with many fishes, the exact timing of spawning is probably driven by a combination of temperature and photoperiod, and therefore spatial and temporal variation should be expected. The single Blackside Dace spawning event reported by Starnes and Starnes (1981) occurred on 17 May 1981 at a water temperature of 17.5 oC, which is within the range of dates and temperatures observed in our study. However, based on the presence of mature ova in females, Starnes and Starnes (1981) suggested that spawning most likely begins in April and extends through June. In addition, the timing of Blackside Dace spawning also appears dependent on the timing of host-species nesting, as indicated by the results of our current study. Table 4. Kruskal-Wallis two-sample tests to determine if reach mean values for non-spawning, transect-point microhabitats in Blackside Dace streams differed between reaches with and without active logging. Values for conductivity and turbidity were rounded to nearest whole numbers immediately before inclusion in the table. Reach mean values for non-spawning microhabitats Kruskal-Wallis Reaches with Reaches with tests Habitat variable active logging no active logging χ2 P Wetted-channel width (m) 1.9, 2.0, 2.4 1.8, 2.1, 2.3, 2.5 0.13 0.724 Canopy cover index 3.2, 3.7, 3.8 3.3, 3.6, 3.7, 3.8 0.13 0.724 Silt index 1.6, 1.7, 2.3 1.0, 1.0, 1.2, 1.4 4.50 0.034 Embeddedness index 1.6, 1.9, 2.4 0.6, 0.7, 0.9, 1.0 4.50 0.034 Dominant substrate index 2.9, 2.6, 3.5 3.4, 3.4, 3.5, 3.6 2.04 0.154 Subdominant substrate index 2.2, 2.2, 2.4 2.1, 2.3, 2.3, 2.7 0.13 0.724 Temperature (°C) 16.9, 17.0, 17.1 15.1, 16.0, 16.5, 16.6 4.50 0.034 Dissolved oxygen (mg/L) 8.1, 8.4, 8.5 8.4, 8.5, 8.6, 8.7 2.00 0.157 Conductivity (μS) 74, 100, 182 23, 29, 54, 71 4.50 0.034 Water depth (cm) 7, 9, 10 9, 9, 11, 11 1.24 0.266 Bottom velocity (cm/s) 5, 7, 8 5, 5, 5, 6 1.86 0.172 Column velocity (cm/s) 6, 6, 9 4, 5, 5, 6 3.43 0.064 Turbidity (NTU) 7, 15, 21 1, 2, 9, 10 2.00 0.157 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 60 Vol. 12, Special Issue 4 The relative importance of Creek Chub to Blackside Dace reproductive success seems to be much greater than that of other co-occurring cyprinid species (Fig. 2). Our constancy pattern analysis identified Creek Chub as a potentially important species, but such high constancy could have been a simple function of the cosmopolitan distribution of Creek Chub in the study area (Burr and Warren 1986, Laudermilk and Cicerello 1998). It was only after our field study that we gained greater confidence in the importance of Creek Chub to Blackside Dace reproductive ecology. All 25 observed spawning events occurred in Creek Chub nests, regardless of the presence or absence of logging disturbance at the study sites. The stream with the lowest embeddedness and silt levels in our study, Big Lick Branch, hypothetically provided the most suitable conditions under which independent spawning could occur, given the microhabitat conditions measured in that stream. However, all 20 observed spawning events in Big Lick Branch occurred in Creek Chub nests. During minnow trapping events, Detar and Mattingly (2013 [this issue]) collected a number Blackside Dace x Creek Chub hybrids (see Eisenhour and Piller 1997) in Big Lick Branch, a relatively undisturbed stream system, suggesting that rates of hybridization are probably related to multiple factors. Rakes et al. (1999, 2013 [this issue]) describe the re sponse of Blackside Dace in captivity to milt from other fish species. Heterospecific gamete-release cues appear to represent a strong mechanism for induction of reproductive activities by Blackside Dace. However, Rakes et al. (2013 [this issue]) recently found that Blackside Dace can spawn independently in captivity, as discovered during April—May 2013, without the presence of (or cues from) other fishes, representing the first time that independent spawning has been reported for Blackside Dace. Despite an ability to spawn independently in captivity, it remains unknown whether independent spawning is practiced in a field setting. Figure 2. Chrosomus cumberlandensis (Blackside Dace) adults in nuptial coloration swimming near a nesting male Semotilus atromaculatus (Creek Chub), photographed on 10 June 2005 by Tyler R. Black at Grubb Branch, eastern Knox County , KY. 61 H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist Vol. 12, Special Issue 4 Microhabitat conditions measured at Blackside Dace spawning sites were fairly narrow for certain variables (e.g., zero silt depth index, zero embeddedness index, 100% in Creek Chub nests, channel widths 3–4 m), and the reliance on Creek Chub nests apparently constrains Blackside Dace reproduction to microhabitats selected by Creek Chub. Blackside Dace spawning was possible in sites with generally elevated siltation and embeddedness, such as found in Roaring Fork and Right Branch Moore Creek. The nest-building and -maintenance activities of Creek Chub males apparently created isolated small microhabitat “islands” in which spawning conditions were suitable for Blackside Dace. In short, conservation of Blackside Dace now seems more intimately tied to conservation of host species, such that dace populations may reproduce in streams where conditions are somewhat degraded. Our study did not identify a threshold of siltation or embeddedness that entirely prevented Blackside Dace reproduction. Furthermore, we also did not examine lethal or sub-lethal effects of siltation on embryos or other early life history stages. Even when reproduction occurs in silted streams, we remain unsure how well embryos, larvae, and juveniles feed, grow, and survive under such conditions (e.g., Kemp et al. 2011; Sutherland 2007; Sutherland and Meyer 2007; Sutherland et al. 2008; Wood and Armitage 1997, 1999). Early life stages could be more sensitive than adults to suspended particles. In conclusion, our study represents the first quantitative description of microhabitat conditions at Blackside Dace spawning locations in multiple streams. We also documented distinct differences in microhabitat conditions for several variables between spawning locations and non-spawning transect points. Further, we documented a strong nest-association pattern with Creek Chub, confirming the earlier predictions of Cicerello and Laudermilk (1996). Stoneroller species appear to be of less importance than Creek Chub to Blackside Dace reproductive activities. The potentially obligatory relationship that Blackside Dace have with nest-building cyprinids, especially Creek Chub, reinforces the importance of maintaining the integrity of the whole stream community. Finally, we documented differences in habitat conditions between stream reaches with and without logging disturbances, and observed that Blackside Dace can spawn in both situations, most likely due to the nest-cleaning actions of male Creek Chub. However, we observed fewer Blackside Dace spawning events in sites with active logging, and it remains unknown how early life stages would fare in areas with increased silt, embeddedness, temperature, and conductivity. Acknowledgments We thank the US Fish and Wildlife Service, Tennessee Wildlife Resources Agency, The Nature Conservancy, and the Department of Biology and Center for the Management, Utilization and Protection of Water Resources at Tennessee Technological University (TTU) for financial and other support. We appreciate the cooperation of US Department of Agriculture Forest Service and several private landowners who granted access to their properties. C.J. Sutherland constructed Figure 1, J.R. Darden assisted with field work, H.T. Mattingly and T.R. Black 2013 Southeastern Naturalist 62 Vol. 12, Special Issue 4 and C. Peterson provided editorial support. Completion of the manuscript was facilitated by a TTU Faculty Non-Instructional Assignment during 2011–2012. We especially thank Y. Kanno for guidance and suggestions during the study. The manuscript was improved by comments from two anonymous reviewers and the guest editor . Literature Cited Bain, M.B. 1999. Substrate. 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