Southeastern Naturalist 43 M.F. Cashner and H.L. Bart Jr. 22001188 SOUTHEASTERN NATURALIST Vo1l7.( 117):,4 N3–o5. 41 Spawning Community and Egg Deposition for Three Southeastern Nest-associate Minnows Mollie F. Cashner1,2,* and Henry L. Bart Jr.2 Abstract - Nest association is a symbiotic reproductive strategy in North American minnows in which a species spawns in the nest substrate of a host species. Host specificity is unknown for the vast majority of nest associates, and presence of a spawning aggregation over a particular nest site has is assumed to be evidence of egg deposition. In this study, we surveyed multiple streams for spawning aggregations throughout the ranges of 3 nestassociate species—Notropis baileyi (Rough Shiner), N. rubricroceus (Saffron Shiner), and N. chlorocephalus (Greenhead Shiner). We paired direct observation of spawning behavior with molecular verification of egg deposition. We observed all spawning aggregations in association with a host nest. We identified eggs from a number of species not directly observed over a particular aggregation site, although all species were known to aggregate as nest associates. On 2 occasions, we documented Saffron Shiner males in aggregations over Semotilus atromaculatus (Creek Chub) pit–ridge nests; however, we recovered no Saffron Shiner eggs from the nests. Our findings demonstrate that field observations of nuptial aggregations alone are not sufficient to confirm spawning associati on. Introduction North American minnows (Family Cyprinidae) exhibit a diverse array of breeding strategies with various levels of parental care. In both broadcast (with either pelagic or benthic eggs) and crevice spawning, parental care is limited to the egg-deposition site, whereas substrate manipulation (nest building and pit forming), egg clustering, and egg clumping entail more parental care investment by males via construction of appropriate spawning substrate and often some level of egg-predator defense (Johnston and Page 1992; Maurakis et al. 1990, 1992; Vives 1990). The symbiotic reproductive strategy of nest association, in which a species spawns in a nest built by another species, is an interesting combination of broadcast spawning and substrate manipulation that is exhibited in multiple minnow lineages (Johnston and Page 1992). Nest associates vary in host type and specificity, but in all cases, hosts manipulate the substrate to form a structure for egg deposition. Cyprinid nest associations are mutualistic relationships between the host and nest associates (Johnston 1994a, 1994b; Johnston and Kleiner 1994; Peoples and Frimpong 2013;Walser et al. 2000). Egg predation is the strongest selective pressure on this system; associate eggs are protected when the host buries eggs and defends the nest against predators (Johnston 1994a, Maurakis et al. 1992, Vives 1990). Due to the large numbers of eggs in each nest, host species benefit from a reduction in egg predation (dumping effect; Johnston 1Biology Department, Austin Peay State University, Clarksville, TN 37044. 2Department of Ecology and Evolutionary Biology, Tulane University, New Orleans, LA 70118. *Corresponding author - cashnerm@apsu.edu. Manuscript Editor: Carol Johnston Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 44 1994b, Peoples and Frimpong 2013). Nocomis spp. (chubs) mounds are prominent features of the stream bed, and that, coupled with spawning aggregations composed of hundreds of brightly colored fishes, may attract predators. Nest associates often outnumber hosts; thus, a dumping effect on adult fishes might also play a selective role in the evolution of this reproductive strategy. Host specificity among nest associates is largely unknown. Notropis lutipinnis (Jordan & Brayton) (Yellowfin Shiner) has been studied most rigorously, and there is convincing evidence that it is an obligate nest associate of, Nocomis leptocephalus (Girard) (Bluehead Chub; Clayton 2000; McAuliffe and Bennett 1981; Wallin 1989, 1992). Field observations of, N. chlorocephalus (Cope), (Greenhead Shiner), N. rubricroceus (Cope) (Saffron Shiner), and, Notropis baileyi Suttkus & Raney (Rough Shiner) have identified several host taxa: Nocomis micropogon (Cope) (River Chub), Nocomis leptocephalus (Girard) (Bluehead Chub), Semotilus atromaculatus (Mitchill) (Creek Chub), and various Campostoma spp. (stonerollers) (Cochran and Lyons 2001, Johnston 1991, Johnston and Kleiner 1994, Outten 1961). At least 27 minnow species are known to use Nocomis nests as spawning substrate (Johnston and Page 1992). Although participants in nest-association spawning aggregations typically display nuptial coloration and reproductive behaviors, empirical evidence of egg deposition by all species involved is generally lacking. The community structure of spawning aggregations has primarily been reported via in-stream observations of adults at or near a nest site (e.g., Cochran and Lyons 2001, Johnston and Kleiner 1994, Outten 1961). Two studies have documented egg deposition in a chub nest with subsequent rearing of collected eggs to larval stage (Cooper 1980, Peoples et al. 2017), a relatively time-consuming process. Recent model-based approaches to investigate evolutionary and community-ecology components of nest association depend on published reports of spawning aggregations to identify host specificity (e.g., Pendelton et al. 2012, Peoples and Frimpong 2013); however, confirmation of egg deposition is absent from much of the published literature. Determining whether species seen in aggregations over a particular site are actually depositing eggs is crucial to the understanding of nest association and host specialization. Cyprinid eggs have few distinguishing morphological characters, and egg size is similar among species with shared reproductive behaviors (Coburn 1986). In order to identify cyprinid eggs to species, Cashner and Bart (2010) identified a reliable molecular method using restriction-fragment length polymorphisms (RFLP) of the maternally inherited mitochondrial coded ND2 gene double-digested with restriction enzymes HinfI and HhaI. This method can be employed to distinguish closely related species in a diverse community (Cashner and Bart 2010). To date, no single study has attempted to survey multiple streams within the range of multiple nest-associate species in order to assess egg-deposition success. The objective for this study was to assess whether observations of putative spawning aggregations are accurate measures of reproductive activity for 3 nest-associate species (Rough Shiner, Saffron Shiner, and Greenhead Shiner). Southeastern Naturalist 45 M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 Field-site Description We sampled 4–6 streams for each focal species (Fig. 1, Appendix 1). All streams were 2nd- or 3rd-order with diverse, immediately adjacent land uses. Rough Shiner sites were in Mississippi (Pascagoula River System) and Alabama (Tennessee River System) and had substrates dominated by sand with small gravel deposits. Sites sampled for Saffron Shiner were all within the French Broad River System in western North Carolina and had substrates primarily composed of cobble and Figure 1. Sample localities and known ranges of Rough Shiner (squares and intermediate gray), Saffron Shiner (circles and dark gray), and Greenhead Shiner (triangles and light gray). Numerals designate egg-identification localities. Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 46 slab. Greenhead Shiner sites ranged throughout the Catawba River System in North Carolina. Sites in the lower Catawba River System contained more sand substrate and were surrounded by agricultural land. We observed Castor canadensis Kuhl (North American Beaver) activity in 1 or 2 streams for each focal species. Materials and Methods Stream surveys We surveyed bank and in-stream transects of 0.3–0.5 km in each stream from April to June over 6 years. We visited streams at least 2 times, and surveyed most streams multiple times throughout the season. We identified spawning aggregations (multiple nuptial individuals aggregated in a small area) during walking transects; underwater observations were made when water clarity permitted, otherwise observations were conducted from the bank using binoculars with polarized lenses. We recorded the species engaged in spawning behavior (nuptial coloration, territoryholding, spawning), water temperature, and substrate type at each putative spawning aggregation. Our observations periods were 30–60 min, and we made video recordings at most sites. Egg identification We targeted a subset of spawning sites for egg identification, and selection of sites was opportunistic and dependent on availability of supplies. We collected eggs from the substrate by placing an aquarium net downstream of an active area and manually agitating the substrate. As the eggs floated downstream, they were captured in the net. After collection from the net, we transferred the eggs to 95% ethanol (ETOH). We changed the ETOH at least 2 times within the first 24 h to optimize preservation. We created restriction-fragment banding-pattern libraries for each community based on adult (known) specimens collected or observed in the immediate area (Table 1) following protocols outlined in Cashner and Bart (2010). When necessary, we employed a grid and a random-numbers table to select a subsample of 200 eggs (1000+ eggs could be collected from a single nest), and egg extraction and amplification followed the protocol of Cashner and Bart (2010); however, due to variation in amplification success for some species within the Saffron Shiner (i.e., Creek Chub) and Greenhead Shiner (i.e., Rosyside Dace) communities, we used alternate amplification primers (ND2B-L and ND2E-H from Broughton and Gold 2000). We created RFLP libraries for Rough Shiner (ASN/GLN ND2 amplicons), Saffron Shiner (BL/EH ND2 amplicons), and Greenhead Shiner (2 libraries: 1 from each primer pair ND2 amplicon set). We included 2–20 individuals of each species to design the reference library for each community (Table 1). We identified eggs to species by comparing their RFLP patterns to those in the reference library. We used the ND2 primer set appropriate for the source community to amplify the eggs. Subsequent PCR products were subjected to a double-digest with HinfI and HhaI, and the resulting fragments visualize via electrophoresis on a 3% NuSieve/Agarose gel with a 100-bp (NEB) DNA ladder used as a size standard. If egg RFLP banding patterns did not match any of our RFLP libraries, we Southeastern Naturalist 47 M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 direct-sequenced the eggs (see Cashner and Bart 2010 for primers and sequencing conditions) and compared the results to known ND2 sequences on GenBank (NCBI) for positive identification. Results We conducted a total of 138 surveys from early May to late June of 2005, 2006, 2007, and 2009. Water temperatures varied from 10.5 °C to 27 °C. We observed 28 spawning aggregations—9 in Rough Shiner communities, 13 in Saffron Shiner communities, and 6 in Greenhead Shiner communities. In all cases, spawning aggregations were in association with nest-building minnow species (Appendix 1). We collected eggs from 10 spawning aggregations, and PCR success varied from 5% to 100%, with over 60% success for most (7) sites. The focal nest associate species eggs were recovered from every Bluehead Chub and River Chub nest sampled, but none were recovered from the 2 Creek Chub nests sampled (Table 2). Moreover, Bluehead Chub and River Chub were not the most numerous eggs identified in any of the collections, with nest-associating species dominating egg numbers (Table 2). Egg composition and observed species composition differed for every site; 50% of the time we recovered eggs of more species than we observed in the spawning aggregation, and 50% of the time there were eggs of Table 1. Species used to generate RFLP libraries for 3 nest-associate communities. The number of individuals used per species is in brackets. Rough Shiner community Saffron Shiner community Greenhead Shiner community Notropis baileyi Suttkus & Notropis rubricroceus (Cope) Notropis chlorocephalus (Cope) Raney (Rough Shiner) [10] (Saffron Shiner) [10] (Greenhead Shiner) [10] Notropis longirostris (Hay) Notropis leuciodus (Cope) Notropis chiliticus (Cope) (Longnose Shiner) [20] (Tennessee Shiner) [10] (Redlip Shiner) [8] Notropis amplamala Pera & Notropis spectrunculus (Cope) Clinostomus funduloides Girard Armbruster (Longjaw (Mirror Shiner) [8] (Rosyside Dace) [9] Minnow) [10] Notropis texanus (Girard) Luxilus coccogenis (Cope) Central Stoneroller [10] (Weed Shiner) [10] (Warpaint Shiner) [10] Lythrurus roseipinnis (Hay ) Campostoma anomalum Warpaint Shiner [10] (Cherryfin Shiner) [10] (Rafinesque) (Central Stoneroller) [10] Luxilus chrysocephalus Semotilus atromaculatus Bluehead Chub [10] Rafinesque (Striped Shiner) (Mitchill) (Creek Chub) [8] [10] Cyprinella venusta Girard Nocomis micropogon (Cope) (Blacktail Shiner) [10] (River Chub) [10] Hybopsis winchelli Girard Rhinichthys cataractae (Valenciennes) (Clear Chub) [3] (Longnose Dace) [2] Nocomis leptocephalus (Girard) (Bluehead Chub) [10] Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 48 fewer species than expected from direct observations (1 site had 5% PCR success, and the only eggs recovered were of the focal nest-associate species, Table 2). Discussion We employed a combination of methods, from in-situ observations to laboratory- based egg identification, to describe nest-site use and egg deposition by several Table 2. Species observed in spawning aggregations compared to percent egg identification and percentage of total eggs identified. Site ID number is referenced i n Figure 1. Community Site ID Species observed Egg ID % (n) Rough Shiner Martin Branch (1) Rough Shiner Striped Shiner 51% (110) Rough Shiner 33% (72) Bluehead Chub 16% (35) Beaver Creek (2) Bluehead Chub Rough Shiner 53% (53) Rough Shiner Striped Shiner 27% (26) Bluehead Chub 20% (20) Saffron Shiner South Fork Mills (3) Saffron Shiner Saffron Shiner 100% (2) (26 May 2005) Tennessee Shiner Central Stoneroller Warpaint Shiner River Chub South Fork Mills Saffron Shiner Warpaint Shiner 42% (22) (10 June 2006 -1) Tennessee Shiner Saffron Shiner 42% (22) Central Stoneroller River Chub 8% (4) River Chub Central Stoneroller 6% (3) Tennessee Shiner 2% (1) South Fork Mills Saffron Shiner Saffron Shiner 93% (43) (10 June 2006 -2) Tennessee Shiner Warpaint Shiner 7% (3) Central Stoneroller River Chub South Fork Mills Saffron Shiner Creek Chub 100% (57) (23 May 2009) South Fork Mills Saffron Shiner Creek Chub 100% (40) (2 June 2009) Creek Chub Greenhead Shiner Lippard Creek (4) Greenhead Shiner Central Stoneroller 52% (23) Rosyside Dace Greenhead Shiner 27% (12) Bluehead Chub 20% (9) Ballard Creek (5) Greenhead Shiner Greenhead Shiner 49% (23) Redlip Shiner Bluehead Chub 23% (11) Rosyside Dace Central Stoneroller 6% (13) Bluehead Chub Rosyside Dace 5% (11) Redlip Shiner 2% (4) Cox Creek (6) Greenhead Shiner Greenhead Shiner 67% (20) Rosyside Dace Bluehead Chub 33% (10) Bluehead Chub Southeastern Naturalist 49 M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 nest-associating North American minnows. We documented egg deposition for species known to spawn independent of a host (and thus considered facultative nest associates), such as Campostoma anomalum Rafinesque (Central Stoneroller) and Luxilus chrysocephalus Rafinesque (Striped Shiner), in mound nests built by chub species. All documented egg deposition by Rough Shiner, Saffron Shiner, and Greenhead Shiner occurred over mound nests of chubs, indicating these taxa are likely obligate nest associates. We did not observe active chub males at all sites, but nests were free of silt, which indicated ongoing maintenance. Chub males are skittish and difficult to observe, and we made our best observations of their behavior underwater. Our evidence supports a primary association with Bluehead Chub or River Chub and secondary associations with pit-forming species such as stonerollers. In all cases, chub eggs were not the dominant component of a nest, supporting the selfish herd or dumping effect benefit to hosts in nest associations (Johnston 1991, Peoples and Frimpong 2013, Shao 1997). We found that visual observations of spawning-aggregation composition was not sufficient to accurately describe egg deposition. In 100% of the nests sampled for egg identification, species aggregating over the site did not completely match the egg composition. Despite these discrepancies, all eggs collected were from species observed in multiple spawning aggregations (Appendix 1). We did not identify new nest-associate species in this study. Quantification and confirmation of egg deposition is made more difficult by the temporal nature of spawning aggregations. Variation in nest-associate aggregation communities compared to egg-deposition success may be the result of nests housing eggs from multiple spawning events over a series of days, or differential deposition-success at any one given spawning aggregation. There was considerable variation in PCR success across all nests sampled (5–100%); however, we were able to identify over half of the eggs sampled for the majority of nests. Inability to amplify some eggs may have been the result of eggpreservation error or of fresh spawning events which did not allow enough time for cell division to generate detectable quantities of DNA. At one site, we were unable to identify any host eggs (South Fork Mills River, 10 June 2006, site 2). This result was likely an amplification error: all eggs ~2 mm in diameter (nearly twice the diameter of other eggs and hence likely to be hosts' eggs that had been deposited earlier and thus had more time to develop) did not successfully amplify, indicating there were River Chub eggs present despite lack of molecular evidence. Nests with aggregations of individuals in peak nuptial coloration yielded eggs from focal species except in the 2 Creek Chub pit–ridge nests. Eggs collected at these sites were more uniform in size than at any other site, and all were identified as Creek Chub eggs. Woolcott and Maurakis (1988) and Cochran and Lyons (2001) both noted high-colored Saffron Shiner in association with Creek Chub pit–ridge nests; however, neither study directly observed spawning or spawning behavior. Moreover, based on these studies, Pendleton et al. (2012) identified Saffron Shiner as a weak nest-associate species. Our data suggest that Saffron Shiner aggregated over Creek Chub pits, but did not spawn at the nests we observed. In 1 active Creek Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 50 Chub pit–ridge nest observed on 2 June 2009, aggressive behavior of the resident chub seemed to prevent Saffron Shiner spawning. Saffron Shiner males in peak nuptial coloration were able to aggregate un-assaulted over the active pit; however, all non-red fish that approached the site were aggressively chased away by the resident Creek Chub. Saffron Shiner females do not develop red body coloration during spawning and were chased away as frequently as other Creek Chub individuals and non-red fish in the area. Thus, while Saffron Shiners were present at both Creek Chub nests we observed, our study demonstrates that the mere presence of Saffron Shiners over a pit–ridge nest does not indicate successful egg deposition, and it is unclear whether Saffron Shiners utilize Creek Chub nests for reproduction. Vives (1990) suggested that Nocomis biguttatus Kirtland (Hornyhead Chub) is a keystone species, and the evidence presented herein supports extending that description to other members of the genus (Pendleton et al. 2012). Bluehead Chub and River Chub mounds were present in nearly all observed spawning aggregations. In some cases, though the chubs may no longer have been active at a particular site, their mounds provided suitable substrate for pit–building stonerollers and shiners. Chub mounds can be large and significant features of a stream bed, and serve as egg-deposition sites for multiple species within a community (Lachner 1952). In the only 2 published studies on the subject, Johnston (1991) and Johnston and Kleiner (1994) observed spawning aggregations of Greenhead Shiners and Rough Shiners, and recorded spawning events in Bluehead Chub mound nests. Outten (1961) observed multiple spawning aggregations of Saffron Shiner over River Chub mound nests, while Woolcott and Maurakis (1988), Cochran and Lyons (2001), and Jenkins and Burkhead (1993) observed individuals in high color aggregating over Creek Chub pit–ridge nests and over apparently non-host substrates. During the course of this study, we expanded the number of streams surveyed and the number of spawning aggregations observed for these 3 species. In combination with these data, egg identification revealed that aggregations alone are not necessarily indicators of egg deposition at a putative spawning site. Quantifying variation among eggs deposited in nests within a community may lend insight into timing of spawning by various species and further our understanding of host specialization among nest associates. Acknowledgments We thank Stefan Woltmann, Rebecca Blanton, John Johansen, Veronica DelBianco, Jamie Orth, Anna Harvey, and Malorie Hayes for all of their help and support in the field. Laboratory-based egg identification was conducted in David Hurley’s and Kyle Piller’s labs, and E. Pierce Smith helped with initial RFLP protocol development. Funding was provided by the American Museum of Natural History Theodore Roosevelt Memorial Fund, American Society of Ichthyologists and Herpetologists Raney Fund, Graduate Women in Science Eloise Gerry Fellowship, Highlands Biological Station Grant-in-Aid of Research, and National Science Foundation Doctoral Dissertation Improvement Grant. Surveys were conducted under the Tulane University Institutional Animal Care and Use Committee (IACUC) protocol 0277-UT-C, and egg collection was conducted under the Tulane University IACUC protocol 0327-UT-C. Southeastern Naturalist 51 M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 Literature Cited Broughton, R.E., and J.R. Gold. 2000. Phylogenetic relationships in the North American cyprinid genus Cyprinella (Actinopterygii: Cyprinidae) based on sequences of the mitochondrial ND2 and ND4L genes. Copeia 2000:1–10. Cashner, M.F., and H.L. Bart Jr. 2010. Reproductive ecology of nest associates: Use of RFLPs to identify cyprinid eggs. Copeia 2010:554–557. Clayton, J.M. 2000. Life-history aspects of 3 minnow species of the subgenus Hydrophlox (Pisces: Cyprinidae), Notropis chiliticus, N. chlorocephalus, and N. lutipinnis. Ph.D. Dissertation. George Mason University, Fairfax, VA. 183 pp. Coburn, M.M. 1986. Egg-diameter variation in eastern North American minnows (Pisces: Cyprinidae): Correlation with vertebral number, habitat, and spawning behavior. Ohio Journal of Science 86:110–120. Cochran, P.A., and J. Lyons. 2001. The Saffron Shiner (Notropis rubricroceus) as a nest associate of the Creek Chub (Semotilus atromaculatus). Journal of the Tennessee Academy of Science 76:61–62. Cooper, J.E. 1980. Egg, larval, and juvenile development of Longnose Dace, Rhinichthys cataractae, and River Chub, Nocomis micropogon, with notes on their hybridization. Copeia 1980:469–487. Jenkins, R.E., and N.M. Burkhead. 1993. Freshwater fishes of Virginia. American Fisheries Society, Bethesda, MD. 1079 pp. Johnston, C.E. 1991. Spawning activities of Notropis chlorocephalus, Notropis chiliticus, and Hybopsis hypsinotus, nest associates of Nocomis leptocephalus in the southeastern United States, with coments on nest association (Cypriniformes: Cyprinidae). Brimleyana 17:77–88. Johnston, C.E. 1994a. The benefit to some minnows of spawning in the nests of other species. Environmental Biology of Fishes 40:213–218. Johnston, C. E.1994b. Nest association in fishes: Evidence for mutualism. Behavioral Ecology and Sociobiology 35:379–383. Johnston, C.E., and K.J. Kleiner. 1994. Reproductive behavior of the Rainbow Shiner (Notropis chrosomus) and the Rough Shiner (Notropis baileyi), nest associates of the Bluehead Chub (Nocomis leptocephalus) (Pisces: Cyprinidae) in the Alabama River drainage. Journal of the Alabama Academy of Science 65:230–238. Johnston, C.E., and L.M. Page. 1992. The evolution of complex reproductive strategies in North American minnows. Pp. 600–621, In R.L. Mayden (Ed.). Systematics, Historical Ecology, and North American Freshwater Fishes. Stanford University Press, Stanford, CA. 969 pp. Lachner, E.A. 1952. Studies of the biology of the cyprinid fishes of the chub genus Nocomis of the northeastern United Sates. American Midland Naturalist 48:433–466. Maurakis, E.G., W.S. Woolcott, and J.T. Magee. 1990. Pebble-nests of 4 Semotilus species. Southeastern Fishes Council Proceedings 22:7–13. Maurakis, E.G., W.S. Woolcott, and M.H. Sabaj. 1992. Water currents in spawning areas of pebble nests of Nocomis leptocephalus (Pisces: Cyprinidae). Southeastern Fishes Council Proceedings 25:1–3. McAuliffe, J.R., and D.H. Bennett. 1981. Observations on the spawning habits of the Yellowfin Shiner, Notropis lutipinnis. Journal of the Elisha Mitchell Scientific Society 97:200–203. Outten, L.M. 1961. Observations on the spawning coloration and behavior of some cyprinid fishes. Journal of the Elisha Mitchell Scientific Society 77:1 18. Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 52 Pendleton, R.M., J.J. Pritt, B.K. Peoples, E.A. Frimpong. 2012. The strength of Nocomis nest association contributes to patterns of rarity and commonness among New River, Virginia cyprinids. American Midland Naturalist 168:202–217. Peoples, B.K., and E.A. Frimpong. 2013. Evidence of mutual benefits of nest association among freshwater cyprinids and impliations for conservation. Aquatic Conservation: Marine and Freshwater Ecosystems 23:911–923. Peoples, B.K., P. Cooper, E.A. Frompong, and E.M. Hallerman. 2017. DNA barcoding elucidates cyprinid reproductive interactions in a southwest Virginia stream. Transactions of the American Fisheries Society 146:84–91. Shao, B. 1997. Effects of Golden Shiner (Notemigonus crysoleucas) nest association on host Pumpkinseeds (Lepomis gibbosus): Evidence for a non-parasitic relationship. Behavioral Ecology and Sociobiology 41:399–406. Vives, S.P. 1990. Nesting ecology and behavior of Hornyhead Chub, Nocomis biguttatus, a keystone species in Allequash Creek, Wisconsin. American Midland Naturalist 124:46–56. Wallin, J.E. 1989. Bluehead Chub (Nocomis leptocephalus) nests used by Yellowfin Shiners (Notropis lutipinnis). Copeia 1989:1077–1080. Wallin, J.E. 1992. The symbiotic nest association of Yellowfin Shiners, Notropis lutipinnis, and Bluehead Chubs, Nocomis leptocephalus. Environmental Biology of Fishes 33:287–292. Walser, C.A., B. Falterman, and H.L.J. Bart. 2000. Impact of introduced Rough Shiner (Notropis baileyi) on the native fish community in the Chattahoochee River system. American Midland Naturalist 144:393–405. Woolcott, W.S., and E.G. Maurakis. 1988. Pit–ridge nest construction and spawning behaviors of Semotilus lumbee and Semotilus thoreauianus. Southeastern Fishes Council Proceedings 18:1–3. Southeastern Naturalist 53 M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 Appendix 1. Environmental and community data recorded for observed spawning aggregations. Arabic numerals represent multiple spawning aggregations within a single observation date and stream. Surveys which included sampleing for egg-composition analysis are indicated by an asterisk (*). Species observed engaged in pit-building activity at spawning site are indicated by a dagger (†). Focal species/ Stream Date Temp (ºC) Putative host Aggregation members Rough Shiner Martin Branch, Covington County, MS; 31.46458°N, 89.52943°W 6 May 2004 17.5 Bluehead Chub 1. Rough Shiner, Striped Shiner 2. Rough Shiner, Striped Shiner, Bluehead Chub *5 May 2006 21.5 Bluehead Chub Rough Shiner 30 April 2009 21.0 Bluehead Chub Rough Shiner, Striped Shiner, Bluehead Chub Beaver Creek, Covington County, MS; 31.47497°N, 89.40412°W 11 May 2004 20.0 Bluehead Chub Rough Shiner *6 June 2004 22.0 Bluehead Chub *1. Rough Shiner, Bluehead Chub 2. Rough Shiner, Bluehead Chub 19 June 2004 22.0 Bluehead Chub Rough Shiner, Bluehead Chub, Striped Shiner 6 May 2007 24.0 Bluehead Chub Rough Shiner, Bluehead Chub Chenault Springs, Franklin County, AL; 34.36215°N, 87.54797°W 20 May 2004 19.5 Bluehead Chub 1. Rough Shiner, †Striped Shiner, Largescale Stoneroller, Bluehead Chub 2. Rough Shiner, †Striped Shiner, Largescale Stoneroller, Bluehead Chub 21 May 2004 20.0 Bluehead Chub Same sites and species composition as 20 May 2004 Saffron Shiner Bent Creek, Buncombe County, NC; 35.50133°N, 82.59318°W 19 May 2005 16.0 River Chub Saffron Shiner, †Central Stoneroller, Warpaint Shiner, River Chub 6 June 2005 17.0 River Chub †Central Stoneroller, Saffron Shiner, Warpaint Shiner 17 May 2006 17.0 River Chub Central Stoneroller, Saffron Shiner, Tennessee Shiner, Warpaint Shiner 7 June 2006 16.0 River Chub 1. Saffron Shiner, Tennessee Shiner 2. Saffron Shiner, Warpaint Shiner, River Chub North Fork French Broad, Transylvania County, NC; 35.14363°N, 82.83918°W 24 May 2005 14.0 River Chub Saffron Shiner, River Chub, Central Stoneroller South Fork Mills River, Henderson County, NC; 35.38004°N, 82.61356°W 24 May 2005 15.5 River Chub Saffron Shiner, Warpaint Shiner, River Chub 25 May 2005 16.0 River Chub Saffron Shiner, Tennessee Shiner, Warpaint Shiner, Central Stoneroller, River Chub Southeastern Naturalist M.F. Cashner and H.L. Bart Jr. 2018 Vol. 17, No. 1 54 Focal species/ Stream Date Temp (ºC) Putative host Aggregation members *26 May 2005 15.5 River Chub Saffron Shiner, Tennessee Shiner, Central Stoneroller, Warpaint Shiner, River Chub 31 May 2005 16 River Chub Saffron Shiner, Tennessee Shiner, Central Stoneroller, Warpaint Shiner, River Chub *10 June 2006 20 River Chub *1. Saffron Shiner, Tennessee Shiner, Central Stoneroller, River Chub *2. Saffron Shiner, Tennessee Shiner, Central Stoneroller, River Chub *23 May 2009 14 Creek Chub Saffron Shiner 1 June 2009 15 Creek Chub Saffron Shiner *2 June 2009 16 Creek Chub Saffron Shiner, Creek Chub Greenhead Shiner Lippard Creek, Lincoln County, NC; 35.53616°N, 81.14895°W 5 June 2005 18.5 Bluehead Chub 1. Greenhead Shiner, Rosyside Dace, Bluehead Chub 2. Greenhead Shiner, Rosyside Dace, Bluehead Chub *1 June 2007 21.5 Bluehead Chub Greenhead Shiner, Rosyside Dace Ballard Creek, Lincoln County, NC; 35.50107°N, 81.08724°W *29 May 2005 16 Bluehead Chub 1. Greenhead Shiner, Redlip Shiner, Rosyside Dace Bluehead Chub 2. Greenhead Shiner, Redlip Shiner, Rosyside Dace Bluehead Chub *3. Greenhead Shiner, Redlip Shiner, Rosyside Dace Bluehead Chub 1 June 2007 20 Bluehead Chub Greenhead Shiner, Redlip Shiner, Bluehead Chub Mill Creek, McDowell County, NC; 35.63544°N, 82.19215°W 18 May 2005 18 Bluehead Chub Greenhead Shiner, †Central Stoneroller, Bluehead Chub, Rosyside Dace, Warpaint Shiner 16 June 2006 21 Bluehead Chub Greenhead Shiner 30 May 2007 20 Bluehead Chub Greenhead Shiner, Central Stoneroller, Bluehead Chub, Rosyside Dace, Warpaint Shiner Cox Creek, McDowell County, NC; 35.8178°N, 82.0401°W *27 May 2007 19 Bluehead Chub Greenhead Shiner, Rosyside Dace, Bluehead Chub