Southeastern Naturalist 735 S.L. White and D.J. Orth 22001144 SOUTHEASTERN NATURALIST 1V3o(4l.) :1733,5 N–7o4. 34 Reproductive Biology of Clinch Dace, Chrosomus sp. cf. saylori Shannon L. White1,2,* and Donald J. Orth1 Abstract - Chrosomus sp. cf. saylori (Clinch Dace) is an undescribed species that is recognized at the state and federal level as a species in need of conservation. The reproductive biology of Clinch Dace is unknown. Here we use in situ breeding observations to infer the timing and mode of reproduction and laboratory analyses to quantify primary and secondary sex characteristics. We conclude that Clinch Dace spawn from May to July using a nest association. Clinch Dace reach reproductive maturity at 2 years, and have a lower number of mature eggs per female and gonadosomatic index in comparison to other Chrosomus species. There was a 3:1 female-biased sex ratio, and pectoral fin length was the only sexually dimorphic external trait. Low reproductive potential coupled with small population sizes and a fragmented distribution places Clinch Dace at a high risk of extirpation. Introduction Chrosomus sp. cf. saylori (Clinch Dace) is a presently undescribed species with known populations patchily distributed across 8 tributaries to the upper Clinch River in Virginia. Due to small population sizes, fragmented distribution, and threats to habitat, Clinch Dace is currently listed as a federal species of concern and as Tier II—very high conservation need—in Virginia’s Wildlife Action Plan (Roble 2006, Virginia Department of Game and Inland Fisheries 2005). Typified by vibrant breeding dress and nest associations, the spawning behavior of Chrosomus minnows is among the most well documented of all species in the family Cyprinidae (Johnston 1999, Johnston and Page 1992). Although the plasticity of nest association is debatable (Pendleton et al. 2012, Smith 1908, Starnes and Starnes 1981), previous studies of Chrosomus concluded that fishes reproduce via broadcast spawning between May and July and that there is a distinct sexual dimorphism with males achieving more vibrant coloration than females and having longer, more-rounded pectoral fins (Settles and Hoyt 1978, Starnes and Jenkins 1988, Starnes and Starnes 1978). Males also develop pearl organs along the entire dorsal and lateral axes of the body (Skelton 2001, Smith 1908). It is likely that Clinch Dace reproductive behavior is comparable to that of other Chrosomus species; however, it is necessary to understand the exact timing and mode of spawning in order to develop a management plan that maximizes protections of critical spawning habitat and minimizes disruptions to watershed activities. Further, while Chrosomus reproductive behavior is well documented, there are only three species in the subgenus Chrosomus for which there are detailed accounts of sex characteristics—C. erythrogaster Rafinesque (Southern Redbelly Dace; Settles 1Virginia Tech Department of Fish and Wildlife Conservation, 100 Cheatham Hall, Blacksburg, VA 24061. 2Current address - The Pennsylvania State University 413 Forest Resources Building, University Park, PA 16802.*Corresponding author - swhite8@vt.edu. Manuscript Editor: Jennifer Rehage Southeastern Naturalist S.L. White and D.J. Orth 2014 Vol. 13, No. 4 736 and Hoyt 1978), C. eos Cope (Northern Redbelly Dace; Das and Nelson 1990), and C. tennessensis (Starnes & Jenkins) (Tennessee Dace; Hamed et al. 2008). Thus, the degree of interspecific variation in sexual morphology of Chrosomus remains uncertain. With half of Chrosomus species listed on state and/or federal endangered species acts, uncertainty in reproductive potential could result in ineffective management (Winemiller 2005). The objective of this study was to describe the reproductive biology of Clinch Dace. We observed in situ spawning behavior to determine timing and mode of reproduction. In addition, we describe primary and secondary sex characteristics to make inferences about fecundity and the degree of sexual dimorphism. We compare these results to those of similar studies to determine the extirpation risk of Clinch Dace relative to other Chrosomus species. Field-Site Description We collected fish used in this study over the entire range of Clinch Dace including 13 streams in the Clinch River watershed in northern Russell and Tazewell counties, VA (see White and Orth [2014] for a detailed description of Clinch Dace distribution and habitat). Streams ranged in size from 1st to 3rd-order and had an average depth of 11.58 cm and width of 2.16 m. Silt and small gravel were the predominant substrate, with occasional areas of bedrock. Due to low population sizes and inaccessibility at the majority of sites, we observed spawning behavior at just 2 streams, Big Lick Creek (37°5'16"N, 81°53' 40"W) and Mudlick Creek (37°8'52"N, 81°51'22"W). Methods Field observations We monitored pre-spawning behavior using streambank observations with binoculars at Big Lick and Mudlick creeks for 4 hours on 1 April and 5 May 2012. We chose these locations for study due to their accessibility and large populations of Clinch Dace discovered in 2011 (White and Orth 2014). Across 300 m of stream, we observed areas of typical spawning habitat including shallow pools and runs (Skelton 2001, Smith 1908). We observed each habitat unit for 10 minutes, or until we saw a Clinch Dace. During pre-spawning observations, Clinch Dace occupied only 1 pool in Big Lick Creek and 1 pool in Mudlick Creek. At both sites, adjacent habitat included an upstream run and another pool within 5 m downstream; Clinch Dace occupied the upstream run and the downstream pool during pre-spawning observations. All habitat units at both sites were dominated by silt and sand substrates. From 19 May to 30 June 2012, we monitored these 3 habitat units (upstream run, occupied pool, and downstream pool) at each site to observe spawning and post-spawning behavior. We observed Big Lick Creek every day for at least 2 hours. Our spawning observations at Mudlick Creek were limited to 1 hour per week due to inaccessibility. We measured stream temperature at both sites with continuous data loggers that recorded temperature every hour. Southeastern Naturalist 737 S.L. White and D.J. Orth 2014 Vol. 13, No. 4 Sexual morphology We quantified primary and secondary sex characteristics for 63 Clinch Dace (length = 29–65 mm). This sample included fish from all age classes of Clinch Dace (White and Orth 2013). We captured fish during October 2009–July 2012 using backpack electrofishing and seining. Fish were preserved in formalin for 2 weeks before being transferred to 70% ethyl alcohol. We described external morphology—color, fin size, and presence of tubercles—before preservation. Because pectoral fin length has consistently been shown to be dimorphic between sexes in Chrosomus (Settles and Hoyt 1978, Starnes and Jenkins 1988, Starnes and Starnes 1978), we compared length of the pectoral fin between males and females using an analysis of covariance (ANCOVA) with standard length as the covariate. For all fish, we measured gonad weight to the nearest 0.001 g and calculated a gonadosomatic index (GSI) by dividing gonad weight by total weight and multiplying by 100. We counted the number of mature eggs for all females. We defined mature eggs as those eggs that were opaque, yellow, and approximately 1 mm in diameter (Hamed et al. 2008, Settles and Hoyt 1978), whereas we identified eggs as immature if they were translucent and significantly smaller. We aged all fish using otoliths in the manner described by Mills (1987). Results Field observations We observed faint spawning coloration in both Big Lick and Mudlick creeks on 1 April 2012, and full coloration was achieved by 5 May 2012, including neonyellow fins, bright red abdomens, and 2 uninterrupted black lateral stripes running the entire length of the body (see White and Orth 2013). In Big Lick Creek, we observed 3 Campostoma anomalum (Rafinesque) (Central Stoneroller) making pits in the upstream run on 21 May 2012. At this time, water depth in the run was 10 cm. During pit construction, 2 Semotilus atromaculatus (Mitchill) (Creek Chub), 5 Rhinichthys atratulus (Hermann) (Western Blacknose Dace), and 3 Clinch Dace periodically swam from the occupied pool to the run, swam around and inside Central Stoneroller pits for 5–10 seconds, and then swam downstream to the occupied pool. This behavior was repeated every 5–10 minutes, and persisted for the entire observation period. We did not observe Clinch Dace in the downstream pool during this time. Average stream temperature on this day was 15.7 °C. We observed spawning in Bick Lick Creek on 23 May 2012. Average water temperature for this day was 15.4 °C. When spawning, 5–7 Clinch Dace swam from the occupied pool into a Central Stoneroller pit in the upstream run, paused briefly for several seconds, and then vibrated rapidly over the depression. The entire event lasted less than 30 seconds, and was repeated every 5–10 minutes for ~1 hour. Approximately 5 Central Stonerollers, 3 Creek Chubs, and 10 Western Blacknose Dace were intermittently present in the pits and we often saw them burrowing and tunneling into the pit after Clinch Dace spawned, possibly feeding on eggs. Southeastern Naturalist S.L. White and D.J. Orth 2014 Vol. 13, No. 4 738 Between each spawning event, Clinch Dace swam downstream to the occupied pool and remained underneath an undercut bank before returning back to the upstream run to spawn. After the spawning event on 23 May 2012, Clinch Dace remained in the occupied pool and no longer visited the spawning site in the upstream run. During 25 May–30 June 2012, we observed Clinch Dace in locations increasingly further away from the spawning site, including the downstream pool and more upstream sections of the run. Breeding colors were noticeably muted by 28 May 2012. After Clinch Dace had spawned, we observed no fish at the spawning location until 2 June 2012 when 4 Central Stonerollers returned to the original spawning site and began to construct pits for the next 2 days. We observed no other species at the spawning site, and Central Stonerollers vacated pits after construction. On 19 May 2012, we observed 5 Clinch Dace in close proximity to a gravel mound in the upstream run in Mudlick Creek. Although we did not observe mound construction, previous community sampling of Mudlick Creek by White and Orth (2014) indicates Creek chub is the only species present with moundbuilding behavior. We also observed ~10 Western Blacknose Dace, 3 Central Stonerollers, and 2 Catostomus commersoni Lacépède (White Sucker) near the mound. Similar to our observations in Big Lick Creek, Clinch Dace made intermittent movements from the run to the occupied pool, hid in an undercut bank, and swam back upstream to the mound every 5–10 minutes. Stream inaccessability prevented us from making further observations of Mudlick Creek until 30 May 2012, at which point Clinch Dace had likely already spawned, as evidenced by the fishes’ fading colors and their movement to the previously unoccupied downstream pool. At this point, no fish were active around the mound. Average water temperature between 19 and 30 May 2012 was 15.6 °C. Starting 9 June, no fish were observed near the mound. Sexual morphology The average number of mature ova per female was 267.30 (SE = 24.2, min = 153, max = 442, n = 12). Only age-2 females had mature ova. Age-2 female GSI peaked in early July at 7.15 before sharply declining to around 4.00. Age-2 female GSI was significantly higher than maximum age-1 and young-of-year (YOY) female GSI, which were 1.99 and 0.25, respectively (Fig. 1). Maximum GSI for age-2 males was 2.00 which was reached in April. GSI for age-2 males rapidly declined to approximately 1.00 by the middle of May, and remained low through July. Maximum GSI for age-1 males was 1.10 in July but, on average, was below 0.75 for the entire collection period. GSI for YOY males was 0.32. There was a 3:1 female-to-male sex ratio in our sample. Vibrancy of coloration was a poor predictor of sex because the most vibrant colors were often displayed by females. Only age-2 individuals displayed breeding dress. Age-2 males had pearl organs across the dorsal and lateral axes of the body. Pectoral-fin length was strongly correlated to standard length in males (r2 = 0.72, P < 0.001, n = 16) and Southeastern Naturalist 739 S.L. White and D.J. Orth 2014 Vol. 13, No. 4 females (r2 = 0.81, P < 0.001, n = 47; Fig. 2). Males had a significantly larger pectoral fin than females (by ANCOVA, P = 0.02; Fig. 2). Figure 1. Average gonadosomatic index for age-2 (circle), age-1 (diamond), and young-ofyear (square) female (A) and male (B) Clinch Dace captured from October 2009–July 2012. Southeastern Naturalist S.L. White and D.J. Orth 2014 Vol. 13, No. 4 740 Discussion This study provides the first description of Clinch Dace reproductive biology. From in situ spawning observations, mature egg counts, and the GSI of males and females, we conclude that Clinch Dace spawning lasts from at least May–July and only age-2 individuals are sexually mature. Pectoral fin length was sexually dimorphic; however, breeding coloration was homogenous across sexes. In addition, we report a 3:1 female-biased sex ratio, a finding that is atypical for Chrosomus. Spawning timing, mode, and habitat requirements documented here for Clinch Dace are consistent with reports for other Chrosomus (Hamed et al. 2008, Settles and Hoyt 1978, Smith 1908). However, Clinch Dace fecundity was significantly lower than that of any congener. The average number of mature eggs per female for Clinch Dace was less than half that of Southern Redbelly Dace (Settles and Hoyt 1978) and Tennessee Dace (Hamed et al. 2008), and maximum GSI for Clinch Dace females was 40% lower than Southern Redbelly Dace (Settles and Hoyt 1978) and 60% lower than Northern Redbelly Dace (Das and Nelson 1990). Fitness potential of Clinch Dace is further reduced by a delayed onset of sexual maturity in comparison to other Chrosomus (Settles and Hoyt 1978). Because Clinch Dace life expectancy is 2 years (White and Orth 2013) and sexual maturation is not reached until age 2, the maximum number of spawning events per individual is 1. Figure 2. Regression of pectoral fin length on standard length for males (open circles, n = 16) and females (black squares, n = 47). Length of pectoral fin was significantly larger in males compared to females (P = 0.02). Southeastern Naturalist 741 S.L. White and D.J. Orth 2014 Vol. 13, No. 4 With relatively high population sizes and fecundity, age-1 fish have been shown to contribute the most towards reproduction in other Chrosomus species (Hamed et al. 2008). As such, the semelparous life history of Clinch Dace significantly reduces the reproductive potential of the species in comparison to congeners. The presence of two life histories has been documented in Tennessee Dace, including populations comprised of some individuals that hatch and spawn in May and other individuals that hatch and spawn in June (Hamed et al. 2008). Observed spawning in May and a peak in female GSI in July suggest that two life-history patterns also exist in Clinch Dace. Unique to this study is that fish with both life histories were often parapatrically distributed within the same stream. Reproductive isolation and the patchy within-stream distribution of Clinch Dace suggests multiple subpopulations may co-occur at a single stream; an occurrence that would further decrease the effective population size of Clinch Dace. Our results showed a 3:1 female-biased sex ratio in Clinch Dace, a finding that is in contrast to previous studies of Chrosomus which suggest male-dominated populations (Hamed et al. 2008, Smith 1908, Starnes and Starnes 1981). Although a female-biased sex ratio is theoretically predicted for small, sedentary populations as a means to increase the probability of egg fertilization (Hamilton 1967, Nunney 1985, Nunney and Luck 1988), the imbalance could also be the result of inbreeding depression or differential mortality (e.g., selection for males in minnow traps used by anglers to collect bait). Irrespective of cause, a 3:1 imbalance in sex ratio is predicted to decrease effective population sizes by 25% (Hoglund 2009). If the possibility of subpopulations is disregarded, the maximum number of age-2 individuals captured at a single stream was 13, which is significantly lower than the number needed to secure short- and long-term population viability (Franklin 1980, Jamieson and Allendorf 2012). Future research is needed to determine the stability of the sex ratio, monitor population sizes, and locate additional locations of spawning activity. Chrosomus is described as a genus of extreme sexual dimorphism in breeding coloration (Settles and Hoyt 1978, Smith 1908). However, similar to Hamed et al. (2008), we find this trait is a poor predictor of sex as there was no difference in the vibrancy of coloration between males and females. The only definitive external characteristic that has been shown to be sexually dimorphic across all Chrosomus is pectoral fin shape and length (Hamed et al. 2008, Settles and Hoyt 1978), a trait that does not present until males reach sexual maturity. Thus, studies of Chrosomus using external morphology as the only predictor of sex should be viewed circumspectly. There are currently 3 species of Chrosomus listed under state and/or federal endangered species acts including C. cumberlandensis (Starnes & Starnes) (Blackside Dace), C. saylori (Skelton) (Laurel Dace), and a fourth, undescribed taxon, Tennessee Dace. Of that list, a detailed description of reproductive morphology exists only for Tennessee Dace. Comparatively, the fitness potential of Clinch Dace is significantly lower than that of Tennessee Dace, which was previously viewed as the least productive species in the genus (Hamed et al. 2008). Further, populations of Southeastern Naturalist S.L. White and D.J. Orth 2014 Vol. 13, No. 4 742 Clinch Dace are smaller, and more fragmented than any of the 3 listed Chrosomus. The combination of low fitness, narrow distribution, habitat specificity, and small population size places Clinch Dace as not only one of the most threatened Chrosomus, but one of the rarest fishes in North America (Pritt and Frimpong 2010). The absence of species-specific reproductive morphological studies for many Chrosomus could pose significant challenges to future management decisions. Here, we show that Clinch Dace fecundity is significantly lower than that of congeners, a finding that demonstrates a high degree of interspecific variation within the Chrosomus genus. Managers should exercise caution when developing conservation plans for Chrosomus and avoid extrapolating life-history parameters between species. Acknowledgments We thank Toby Coyner for Clinch Dace specimens collected in 2009 and Emmanuel Frimpong and Andy Dolloff for providing methodological advice and revisions of earlier versions of this manuscript. 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