Southeastern Naturalist 373 C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 22001199 SOUTHEASTERN NATURALIST 1V8o(3l.) :1387,3 N–3o8. 03 Behavior of Chrosomus saylori (Laurel Dace) During the Breeding Season Christopher T. Cronnon1,2, Meredith Harris3, Bernard Kuhajda3, and Hope Klug1,2,4,* Abstract - Chrosomus saylori (Laurel Dace) is an endangered fish species that exclusively inhabits 2 creeks in Rhea County, TN: Bumbee Creek and Youngs Creek. To the best of our knowledge, the behaviors that Laurel Dace exhibit both during and outside of the breeding season have not been previously documented. In this study, we recorded, analyzed, and documented behaviors that are exhibited by captive Laurel Dace during the breeding season. The behaviors that the Laurel Dace exhibited were chasing, attacking, a sigmoid display, chafing, territoriality, following behind, shoaling, biting of the cloaca, and a dance. We discuss the occurrence of these behaviors in relation to reproduction and the behaviors of relatives of the Laurel Dace. Introduction Chrosomus saylori (Skelton) (Laurel Dace) is an endangered minnow that is endemic to Tennessee. The Laurel Dace is currently thought to inhabit only 2 creeks in Rhea County, TN: Bumbee Creek and Youngs Creek. Several aspects of the Laurel Dace’s diet (George et al. 2016, Skelton 2001), habitat (George et al. 2016), life history (George et al. 2016, Skelton 2001), morphology (Skelton 2001), and population genetics (George et al. 2016) have previously been described. However, little is known about the behavior of Laurel Dace. Indeed, to the best of our knowledge, no previous studies have formally documented the behavior of the Laurel Dace during or outside of the breeding season. Understanding the behavior of this species is important from a natural history perspective and to help facilitate captive breeding and re-introduction programs. We describe the Laurel Dace behaviors we observed during a portion of the natural breeding season. Specifically, we focused on documenting behaviors that were likely to be associated with reproduction. We filmed the behavior of a captive group of Laurel Dace and noted any potential reproductive behaviors, including courtship behaviors and behaviors that occurred after egg fertilization. We then developed an ethogram of observed behaviors and quantified the relative frequency of each. 1Department of Biology, Geology, and Environmental Science, University of Tennessee at Chattanooga, Chatanooga, TN 37403. 2Honors College, University of Tennessee at Chattanooga, Chattanooga, Chatanooga, TN 37403. 3Tennessee Aquarium Conservation Institute, Chatanooga, TN 37405. 4SimCenter, University of Tennessee at Chattanooga, Chattanooga, Chatanooga, TN 37403. *Corresponding author - Hope-Klug@utc.edu. Manuscript Editor: Nathan Franssen Southeastern Naturalist C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 374 Materials and Methods Fish collection On 22 November 2016, staff from the Tennessee Aquarium Conservation Institute (TNACI) collected 6 Laurel Dace juveniles from Bumbee Creek (Rhea County, TN) and held them at the facility. On 10 April 2018, another 20 adult Laurel Dace were caught and relocated to TNACI using 3.05 m x 1.83 m seines in Bumbee Creek. One fish died prior to the start of the present study. Laurel Dace were collected under Scientific Collection Permit number 1308 from the Tennessee Wildlife Resource Agency, Endangered and Threatened Wildlife Permit number TE22311A-4 from the US Fish and Wildlife Service, and IACUC Approval Number 17-01 from the Tennessee Aquarium and Tennessee Aquarium Conservation Institute Animal Health and Welfare Committee. We were unable to quantify the number of males and females obtained because the sex of individual Laurel Dace cannot be externally identified with complete confidence. Coloration can vary daily, and males and females appear relatively similar. Housing During the present study (April–June 2018), we housed 25 Laurel Dace in a single aquarium (242.27 L capacity; 76.20 cm long x 76.20 cm wide x 45.72 cm high) on a rack that was 1.83 m high. The aquarium was part of a recirculating water flow system that provided a continuous flow of filtered and well-oxygenated water. However, this setup did not create directional flow within the tank. The bottom of the aquarium was covered in a cobble–sand mixed substrate. We also fully submerged a piece of a tree limb (~63.50 cm long and ~12.70 cm wide) in the tank. We added the cobble–sand substrate and tree limb to the aquarium to provide a semi-natural environment for the Laurel Dace. Approximately 2 weeks after the fish were introduced to the aquarium, but prior to data collection, we constructed a mound of cobble stones in the middle of the aquarium housing the Laurel Dace. The mound was intended to resemble the nest of other minnows that inhabit the creeks in which Laurel Dace are found, such as Campostoma oligolepis C.L. Hubbs & Greene (Largescale Stoneroller). In one of the few observations of nest association with Laurel Dace, nuptial Laurel Dace have been observed swimming over the nests of Largescale Stonerollers, although spawning did not occur (Skelton 2001). The mound was added because we hypothesized that a mound mimicking the nest of other minnows might stimulate spawning by the Laurel Dace, as all other closelyrelated Chrosomus (dace) species utilize the nests of mound and pit–ridge-building minnows, such as various chub species and stonerollers, for spawning (Starnes and Starnes 1981). We provided natural lighting through windows, allowing for ~13 h of light to 11 h of dark during the duration of the study. The facility was climatecontrolled, and temperature was maintained at 20.8–23.7 °C thro ughout the study. Video-recording process We videoed Laurel Dace 2–3 times per week for ~20 min per recording session (see Table 1 for full details of recordings). We made all recordings between 12:00 Southeastern Naturalist 375 C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 and 16:00 from 27 April through 14 June, which corresponds with the majority of the expected Laurel Dace breeding season (George et al. 2016). For each recording session, we used 2 GoPro Hero 5 Blacks and/or a GoPro Hero 3 mounted on tripods at a height of 190.50-cm. Specifically, we used each of the 2 GoPros to record 1 half of the tank. The combination of recordings from the GoPros allowed us to have a detailed and complete recording of the entire tank for each recording session, which subsequently allowed us to see all individuals in the tank and gather detailed behavioral data. During each recording, the observer remained out of sight so that disturbance would be reduced, and each recording began as soon as the observer was out of sight. Behavioral data collection For each recording, we documented all behaviors hypothesized to be associated with reproduction. Specifically, we focused on 13 potential behaviors (Table 2). These potential Laurel Dace behaviors were based on the behaviors of other small fish species, including Chrosomus sp. cf. saylori (Skelton) (Clinch Dace; Hatcher et al. 2017), C. tennesseensis (Starnes & Jenkins) (Tennessee Dace; Hamed et al. 2009), and C. cumberlandensis (Starnes & Starnes) (Blackside Dace; Starnes and Starnes 1981). Attacking (i.e., biting and nudging) and chasing tended to occur with the territoriality behavior; thus, we quantified both the total frequency of attacking and chasing, as well as the frequency of these behaviors when they occurred during territoriality behavior. Table 1. A summary of all of recording sessions. Recording session Device used Date Time Duration (min) 1 GoPro Hero 3 27 April 2018 15:00–15:15 15 2 GoPro Hero 3 27 April 2018 15:20–15:35 15 3 GoPro Hero 3 27 April 2018 15:40–15:51 11 4 GoPro Hero 3 30 April 2018 13:00–13:20 20 5 GoPro Hero 5 30 April 2018 13:00–13:20 20 6 GoPro Hero 5 30 April 2018 13:00–13:20 20 7 GoPro Hero 5 8 May 2018 15:00–15:20 20 8 GoPro Hero 5 8 May 2018 15:00–15:20 20 9 GoPro Hero 5 9 May 2018 13:00–13:20 20 10 GoPro Hero 5 9 May 2018 13:00–13:20 20 11 GoPro Hero 5 15 May 2018 12:15–12:35 20 12 GoPro Hero 5 15 May 2018 12:15–12:35 20 13 GoPro Hero 5 21 May 2018 12:40–13:00 20 14 GoPro Hero 5 21 May 2018 12:40–13:00 20 15 GoPro Hero 5 22 May 2018 14:40–15:00 20 16 GoPro Hero 5 22 May 2018 14:40–15:00 20 17 GoPro Hero 5 30 May 2018 15:40–16:00 20 18 GoPro Hero 5 30 May 2018 15:40–16:00 20 19 GoPro Hero 5 8 June 2018 14:20–14:40 20 20 GoPro Hero 5 8 June 2018 14:20–14:40 20 21 GoPro Hero 5 14 June 2018 15:30–15:50 20 22 GoPro Hero 5 14 June 2018 15:30–15:50 20 Southeastern Naturalist C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 376 We manually logged the behavior observed in each recording in Microsoft Word. We frequently stopped and re-watched the recordings, often in slow motion, to ensure that we documented all relevant behaviors occurring in a given video clip. After identifying all relevant behavior of all fish in each recording, we quantified the total number of times a behavior occurred during the recording period, in order to determine the relative frequency of each behavior observed over all observation periods. Table 2. Potential Laurel Dace behaviors with descriptions, the total number of observations (#), and frequencies (freq.) at which we observed each behavior. The behaviors are presented in order from most to least frequently observed. Behavior Description # Freq. Chasing One fish rapidly accelerates towards another individual. 52 35.62% A particular form of chasing is corralling, which occurs when individuals chase another individual back towards the group. Corralling tends to co-occur with shoaling (described below). Attacking One individual swims up to another individual and bites or 43 29.45% nudges the chest, head, or caudal fin of the other individual. Sigmoid display The body of one fish bends into a “C” or “S” shape while 30 20.55% swimming towards or near another fish. The body of the fish often flops while in the “C” or “S” shape. Chafing One individual rubs any part of its body against another 5 3.42% individual. Territorial behavior One individual is showing signs of aggression while staying 4 2.74% close to a specific area or spot. This aggression can be demonstrated through behaviors such as attacking and chasing any other fish that come too close to the specific area or spot. Following One individual or multiple individuals are following another 4 2.74% individual, either from behind or from the side. Shoaling Occurs when a group of individuals are gathered together for 3 2.05% social reasons, including in order to reproduce. Biting of the cloaca One individual bites the cloacal region of another individual; 3 2.05% this is not an aggressive bite as would be seen in attacking. Dance One individual swims close to another individual. The first 2 1.37% individual then positions itself upright and rapidly “flaps” its pectoral fins. Cloacal contact One individual briefly touches another individual’s cloaca 0 0.00% with its cloaca Cloacal swing One individual brings their cloaca forward from a resting 0 0.00% position without directing it towards another individual’s cloaca. Cloacal thrust One individual swings their cloaca toward another 0 0.00% individual’s cloaca without making contact Nest takeover An individual uses another species’ nest for spawning 0 0.00% Southeastern Naturalist 377 C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 Results Observed behaviors In the 421 minutes of recording across 22 recording episodes, we observed a total of 146 events representing 9 relevant behaviors (Table 2). Below, we provide a description of the behaviors observed, in order of highest observed frequency to lowest frequency. Chasing. The behavior that occurred most frequently was chasing (52 observations) which accounted for 35.62% of the behaviors observed. Chasing was also potentially underestimated in our study, as some chasing might have occurred behind the tree branch while individuals were shoaling and out of view. Regardless, chasing appears to be frequent among breeding-season Laurel Dace. We often observed chasing when 1 territorial individual appeared to chase another individual away from a specific spot. This territorial chasing was sometimes associated with the mound. Numerous episodes of chasing also occurred when individuals were gathered under the tree limb. When 1 or more individuals swam away from the tree limb, multiple individuals chased that individual back towards the group under the tree limb. The Laurel Dace exhibiting this type of chasing behavior appeared to be corralling other individuals back to the tree limb. We observed this corralling 3 times (all of which were associated with the shoaling behavior) and it accounted for 5.77% of the total chasing behavior. While individuals were gathered under the tree limb, multiple individuals also frequently chased each other around the tree limb and back toward the group. In other instances, chasing occurred when all individuals were swimming. This chasing typically lasted ~5 sec, and then the individuals dispersed. The short duration for the chasing behavior could be due to the size of the aquarium. Longer chasing durations might be expected in a l arger aquarium. Attacking. We made 43 observations of attacking behavior, which accounted for 29.45% of the behaviors observed. The attacking behavior typically involved 1 territorial individual biting or nudging the body or caudal fin of another individual. In a few cases, 1 individual attacked another individual in the absence of territoriality. The most extreme case of this attacking behavior occurred in 1 recording during which 2 individuals attacked each other approximately every 15 sec over a 2-min period. Sigmoid display. A relatively short variation of a sigmoid display accounted for 20.55% of the behaviors observed (30 observations). In this behavior, an individual flexed into a strong “S” position and rapidly flopped its body once, either at another individual, on the substrate close to another individual, or on the substrate without another individual present. Fifteen of the total 30 sigmoid displays (50%) were exhibited on the substrate. Although we did not directly observe spawning, it is possible that this behavior occurred during or before spawning, given that it often involved 2 or more individuals and frequently occurred on or ne ar the substrate. Chafing. Chafing accounted for 3.42% of the behaviors observed (5 observations). However, it is possible that additional chafing occurred while individuals were shoaling; when individuals are shoaling it is difficult to observe them in the middle of the group. We typically observed chafing when individuals were Southeastern Naturalist C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 378 gathered under the tree branch. We occasionally observed chafing when individuals were swimming. Territoriality. Territoriality occurred at a frequency similar to chafing, and accounted for 2.74% of the behaviors observed (4 observations). Territoriality was always exhibited by a single individual. During territoriality, an individual appeared to guard a specific spot. This spot was sometimes associated with the mound. Unique to territoriality, other behaviors occurred simultaneously, including attacking (i.e., biting and nudging) and chasing off individuals that came in close proximity to the area appearing to be guarded. We observed these behaviors occurring with territoriality (attacking and chasing) a total of 81 times. We observed attacking behavior 43 times during all recordings (with and without territoriality), and 34 (of these 43) instances occurred during territoriality, accounting for 41.98% of the associated behaviors observed with territoriality. We observed chasing a total of 52 times during all recordings, and 47 of these instances of occurred during territoriality, accounting for 58.02% of the behaviors observed with territoriality. When observed, territoriality and its associated behaviors generally persisted for the entire recording. Following behind. Following behind accounted for 2.74% of the behaviors observed (4 observations). Typically, at least 1 individual followed a single Laurel Dace for 10–15 sec. Shoaling. Shoaling accounted for 2.05% of the behaviors observed (3 observations). In one instance, approximately 18–20 individuals gathered tightly under a small portion of the tree limb. In the other instances, ~8–12 or 15–18 individuals grouped together. Laurel Dace shoaled in what appeared to be an unorganized way. That is, in most instances, they did not all face the same direction, and they swam in either a loose or tight formation (i.e., there did not appear to be consistency in the formation of the group). The loose formation consisted of Laurel Dace swimming out and circling the group, and then returning back to the group. The tighter formation consisted of all or most of the Laurel Dace staying tightly in the group. Thus, under the conditions of this study, from a captive group of 25, it appears that shoaling groups typically involved a subset of between 8 and 20 individuals. As previously mentioned, corralling was associated with all 3 shoa ling observations. Biting of the cloaca. Biting of the cloaca accounted for 2.05% of the behaviors observed (3 observations). When we observed this behavior, 1 individual bit or nudged another individual on or around the cloaca and then either swam away or swam next to another individual and repeated the behavior with that new individual. Dancing. Dancing occurred the least frequently, and accounted for 1.37% of the behaviors observed (2 observations). When dancing occurred, 1 individual swam close to another individual, positioned itself upright, and rapidly “flapped” its pectoral fins. This behavior lasted only a few seconds, and then the individual either swam away or remained next to the other individual. We did not observe cloacal contact, cloacal swings, cloacal thrusts, or nest takeovers, nor was spawning ever directly observed. However, throughout the study period, TNACI biologists collected eggs on 5 occasions and found a total of 987 Southeastern Naturalist 379 C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 eggs and larvae. Thus, breeding did occur during the 2018 mating season under the conditions at TNACI. Discussion We have documented several behaviors that occur during the Laurel Dace breeding season. These behaviors include chasing, attacking, a sigmoid display, chafing, territoriality, following behind, shoaling, biting of the cloaca, and a dance (see also Table 2). Some of these observed behaviors are consistent with the behaviors that have been commonly observed in other dace species, while other behaviors appear to be unique to Laurel Dace. Behaviors commonly seen in other dace that were also exhibited by Laurel Dace include chasing (also seen in Blackside Dace, Tennessee Dace, and Clinch Dace), territorial chasing (also seen in Tennessee Dace and Clinch Dace), following (also seen in Tennessee Dace), and corralling (also seen in Blackside Dace) (T. Black, NC Wildlife Resources Commission, Creedmor, NC, pers. com.; Hamed et al. 2009; Hatcher et al. 2017; Starnes and Starnes 1981). To the best of our knowledge, the sigmoid display and the dance behavior exhibited by Laurel Dace have not previously been observed in other dace. While there have been sigmoid displays exhibited by other dace, such as the Blackside Dace, these displays differ from the one exhibited by Laurel Dace (T. Black, pers. comm.). Laurel Dace do not press another fish to the substrate or near the substrate while doing their sigmoid display, and the display is extremely rapid and brief. Additional observations are needed to determine the function of the various behaviors. Future research should also be conducted to further examine the territorial behavior of Laurel Dace. We were unable to determine if territorial individuals were protecting a given area or if they were guarding fertilized eggs deposited or buried in the substrate. If the individuals were guarding fertilized eggs, this behavior would suggest that Laurel Dace exhibit parental care. We should note that the spawning behavior of all of the mentioned sister species of Laurel Dace are associated with the nests of other minnows (Hatcher et al. 2017). However, we cannot determine if the eggs laid by Laurel Dace were associated with the mound. Future research should explore whether a mound is an essential component of Laurel Dace reproduction. Laurel Dace also exhibited cleaning behavior (i.e., biting at the substrate to remove debris). The cleaning behavior could be associated with cleaning the substrate before spawning, cleaning the substrate around fertilized eggs, or searching for food. We observed cleaning in multiple locations of the aquarium, including on and around the mound. In addition to documenting some reproductive season behavior of Laurel Dace during its breeding season, we also found that, under the conditions of our study, Laurel Dace can reproduce in a lab setting. During our study, successful reproduction occurred given that fertilized eggs were removed from the aquarium substrate and successfully hatched and reared, which indicates that individuals were in breeding condition and likely suggests that at least some of the behaviors observed were associated with reproduction. Additional research is needed to determine if similar or different behaviors are exhibited during the non-breeding season. Southeastern Naturalist C.T. Cronnon, M. Harris, B. Kuhajda, and H. Klug 2019 Vol. 18, No. 3 380 Acknowledgments The Tennessee Wildlife Resource Agency, the US Fish and Wildlife Service, and Approval Number 17-01, and the Tennessee Aquarium and Tennessee Aquarium Conservation Institute Animal Health and Welfare Committee provided our collection permits. The work presented herein served as the basis for a Departmental Honors Thesis for C.T. Cronnon at the University of Tennessee at Chattanooga. This material is based upon work supported by the National Science Foundation under Grant No. 1552721 to H. Klug. Funding for this project was also provided by the Tennessee Wildlife Resource Agency and the US Fish and Wildlife Service. We thank Warren Stiles and George Gavrielides for assistance with fieldwork and Alexandra Miles, Angela Maroti, Shannon Murphy, and Elora Hunt for assistance with Laurel Dace husbandry. We are grateful to Sarah Farnsley, at the University of Tennessee at Chattanooga, and the two referees of this manuscript for providing feedback on aspects of this work. Literature Cited George A.L., B.R. Kuhajda, and D.A. Neely. 2016. Recovery plan for the Laurel Dace (Chrosomus saylori). US Fish and Wildlife Service, Atlanta, GA. 63 pp. Hamed, M.K., F.J. Alsop III, and T.F. Laughlin. 2008. Life-history traits of the Tennessee Dace (Phoxinus tennesseensis) in northeast Tennessee. American Midland Naturalist 160(2):289–299. Hatcher, H.R., M.J. Moore, and D.J. Orth. 2017. Spawning observations of Clinch Dace: Comparison of Chrosomus spawning behavior. American Midland Naturalist 177(2):318–326. Skelton, C.E. 2001. New dace of the genus Phoxinus (Cyprinidae: Cypriniformes) from the Tennessee River drainage, Tennessee. Copeia 2001(1):118–128. Starnes, L.B., and W.C. Starnes. 1981. Biology of the Blackside Dace Phoxinus cumberlandensis. American Midland Naturalist 106(2):360–371.