Southeastern Naturalist 451 M.S. Piteo, M.R. Kendrick, and P.M. Harris 22001177 SOUTHEASTERN NATURALIST 1V6o3l.) :1465,1 N–4o6. 33 Life History of Labidesthes vanhyningi (Atheriniformes: Atherinopsidae; Stout Silverside) in the Black Warrior River Drainage, Alabama Matthew S. Piteo1, 2,*, Michael R. Kendrick1,3, and Phillip M. Harris1 Abstract - Little is known about the life history of Labidesthes vanhyningi (Stout Silverside) in the Mobile Basin. We made monthly collections of Stout Silverside from Lake Tuscaloosa in Northport, AL, from September 2011 to September 2012. Emergence of young-of-the-year occurred in the months of September, October, January, and May. Reproductive investment, calculated using a gonadosomatic index (GSI), was observed from March to December. We also documented evidence for internal fertilization by Stout Silverside in the Mobile Basin. Dietary analysis showed cladocerans were the numerically dominant prey item. These life-history data provide information to natural resource-management agencies about Stout Silverside in the Mobile Basin, where the species is currently under threat from invading Menidia audens (Mississippi Silverside). Introduction Labidesthes vanhyningi B.A. Bean & Reid (Atheriniformes: Atherinopsidae; Stout Silverside) is a slender-bodied, surface-dwelling fish that has been documented up to 76 mm standard length (Werneke and Armbruster 2015). This species is widely distributed throughout southeastern North America. Stout Silverside occurs in river drainages from the Gulf of Mexico to river drainages in the lower Atlantic Slope (Werneke and Armbruster 2015). Cope (1865) originally described the Brook Silverside as Chirostoma sicculum. A taxonomic revision placed the species in Labidesthes and was renamed Labidesthes sicculus (Cope 1870). Bean and Reid (1930) later identified a unique form in the southeastern US, and described it as a new species, L. vanhyningi (Fiery-Finned Silverside). Several years later, Bailey et al. (1954) synonymized L. vanhyningi with L. sicculus. Hubbs and Allen (1943) recognized variation in the southeastern populations and re-elevated the Fiery-Finned Silverside to the subspecies L. s. vanhyningi. Recent work has provided evidence for the validity of L. vanhyningi as a species. Bloom et al. (2009) found 14.7% uncorrected DNA sequence divergence and a well-supported sister relationship between L. s. sicculus and L. s. vanhyningi based on a phylogenetic analysis using mitochondrial ND2. Werneke and Armbruster (2015) documented morphological differences between L. sicculus and 1Box 870344, Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487. 2Current address - Abernathy Fish Technology Center, US Fish and Wildlife Service, 1440 Abernathy Creek Road, Longview, WA 98632. 3Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC 29422. *Corresponding author - matthew_piteo@fws.gov. Manuscript Editor: Carol Johnston Southeastern Naturalist M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 452 L. vanhyningi, elevated L. vanhyningi to the species level, and suggested Golden Silverside as a common name. However, Golden Silverside was already assigned to Menidia colei (Page et al. 2013). Here Stout Silverside is proposed as a common name for L. vanhyningi, denoting that live specimens of this species are heartier, heftier, and tougher than those of L. sicculus (D. Werneke, Auburn University, Auburn, AL; pers. comm.). Although some areas of sympatry occur between populations of L. sicculus and L. vanhyningi, only L. vanhyningi has been documented from the Mobile Basin (Werneke and Armbruster 2015). Most Labidesthes populations are considered stable throughout the entire species range, but some populations have experienced a decline due to invasions by Menidia (silversides). These invasions are prevalent in highly modified river systems such as the Tennessee–Tombigbee Waterway (TTW) (TN, MS, and AL) and Lake Texoma (TX and OK), where dams and habitat modification have transformed previously lotic habitats, preferred by Labidesthes, to lentic habitats, preferred by Menidia (Herbert and Gelwick 2003, Taylor et al. 2008). In lentic habitats, Menidia has been shown to be a superior competitor for resources used by L. sicculus (McComas and Drenner 1982). Menidia audens Hay (Mississippi Silverside) has recently invaded the Mobile Basin, likely originating from the Tennessee River and invading through the TTW; it represents a potential threat to native L. vanhyningi populations in the Mobile Basin. Other populations of Labidesthes have survived introductions of Menidia by shifting to a more terrestrial diet (Ramirez et al. 2006); however, there is currently no evidence that the introduction of M. audens in the TTW has led to a shift in the diet of native L. vanhyningi (Strongin et al. 2011). Given the lack of evidence supporting resource partitioning between L. vanhyningi and M. audens, Strongin et al. (2011) concluded that it is unlikely that these species will be able to coexist in the TTW, and M. audens is likely to replace the native L. vanhyningi populations. Although many studies have examined life-history attributes of L. sicculus (Boesel 1938, Cahn 1927, Ewers and Boesel 1935, Fogle 1959, Hubbs 1921, Mullan et al. 1968, Nelson 1968, Pratt et al. 2002, Ramirez et al. 2006, Zimmerman 1970), few studies have evaluated life-history and diet characteristics of populations representing L. vanhyningi (Grier et al. 1990, Rasmussen 1980, Strongin et al. 2011). A population of L. vanhyningi in the Mobile Basin is threatened by invading Menidia; thus, life-history and diet data for L. vanhyningi are needed to understand the potential impacts of Menidia invasion and to better inform policy makers and stakeholders in conservation decisions and planning. The goal of this study was to examine life-history aspects of a L. vanhyningi population in the Black Warrior River Drainage and compare the findings to other studies conducted on Labidesthes. Site Description and Methods We collected Labidesthes vanhyningi from Lake Tuscaloosa (North River) 17.7 km (11 mi) north of Tuscaloosa, AL, near US Highway 43 (33°20'60''N, 87°36'30''W) from September 2011 through September 2012. Our goal was to Southeastern Naturalist 453 M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 collect 100 individuals per month for this study. We seined in open water along 2 boat ramps and surrounding areas using a standard mesh, 1.2-m x 3.1-m seine (mesh size = 3.2 mm). Shoreline habitat consisted of Justicia sp. (water willow), sand, silt, gravel, cobble, and small boulders. We made our collections in the evening after 1700 h. All L. vanhyningi samples were euthanized in a lethal dose of MS-222 (80 mg/L), preserved in 10% formalin, rinsed in water, and then transferred to 70% ethanol for laboratory examination and long-term storage in the University of Alabama Icthyological Collection (UAIC 16027–16041). We measured standard lengths (SL; Hubbs and Lagler 1947) in the laboratory and plotted them against month collected. We chose the 5 largest males and females per sampling period to examine reproductive ecology. Each individual was blot dried for 5 sec, weighed to the nearest 0.01 g, and measured to the nearest 0.01 mm SL. We dissected, blot dried for 5s, and weighed to the nearest 0.001 g gonadal tissue from each individual. The gonadosomatic index (GSI; Middaugh and Hemmer 1992) for each individual was generated by using the formula: GSI = (gonad weight / total fish weight) x 100 We plotted male and female GSI scores against month collected. To characterize ova development, we removed 1–2 ovaries from females from each breedingseason month and weighed the left and right lobes of each ovary separately to the nearest 0.001 g. We classified ova into 4 developmental classes based on gross differences in size and color observed under a dissecting microscope. For this study, we counted and measured only fertilized and mature ova from each ovary lobe. We photographed mature and fertilized ova using a dissecting microscope affixed with a digital camera. We employed ImageJ software (Schneider et al. 2012) to calculate the mean diameters of fertilized and mature ova. A student’s t-test was used to test differentiation of ovarian lobe mass and mean mature-egg number between each ovarian lobe. In order to examine feeding ecology, we chose 20 individuals (40–50 mm SL) from January, April, July, and October, thus representing all seasons of the year. We dissected the digestive tract from the esophagus to the first turn of the intestine from each individual and identified stomach contents to the lowest possible taxonomic level (e.g., order or family) under a dissecting microscope using dichotomous taxonomic identification keys (Merritt et al. 2008, Thorp and Covich 2009). We determined the proportions of total stomach contents for each taxon for each season from relative abundance. Results We collected 48–168 L. vanhyningi (9 to 66 mm SL) each month, from September 2011 to September 2012, resulting in a total of 1189 individuals measured for this study. We collected individuals in high abundance (>100 individuals) during most sampling periods. Abundance ranged from 48 specimens taken from ~15 seine hauls in September 2011 to 168 individuals from 1 seine haul in December 2011. Of the total number of individuals collected, 80% ranged from 35 mm to 50 mm SL Southeastern Naturalist M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 454 (Table 1). We collected young-of-year (YOY) individuals in 5 separate months— September 2011, October 2011, January 2012, May 2012, and September 2012. Individuals of ≥50 mm SL were collected in all months except September 2011, July 2012, and August 2012 (Fig. 1). We observed 4 stages of ovum development during dissection of ovaries (Table 2). Fertilized ova were the largest in size and were easily distinguishable by having orange/yellow-colored yolk surrounded by clear albumin. Mature ova were the next largest class observed and were orange or amber in color. Maturing ova were smaller and paler in color than mature ova. Immature ova were the smallest and were white in color. We recorded mature ova in females from September Figure 1. Standard length measurements plotted against month for all Labidesthes vanhyningi individuals collected in this study from September 2011 through September 2012. Table 1. Static life-table depicting the length distribution of all 1189 individual Labidesthes vanhyningi collected for this study from September 2011 through September 2012. Individuals were binned into size classes of 5 mm increments (15–19.9, 20–24.9, etc.). Size class (mm) Number of individuals 15 5 20 11 25 16 30 95 35 228 40 311 45 237 50 171 55 70 60 36 65 7 70 2 Southeastern Naturalist 455 M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 through December 2011 and from March through September 2012. We documented fertilized eggs in early developmental stages of cleavage (Fig. 2) during October Figure 2. Fertilized eggs of Labidesthes vanhyningi in early blastulation pictured. Yolk surrounded by clear albumin. (A) Eggs dissected from ovary. (B) Fertilized eggs pictured inside the ovarian duct. Blastoderm is circled. Southeastern Naturalist M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 456 and November 2011 and in April, May, and July 2012. Distinct formation of the blastoderm was observed in numerous eggs. Fertilized eggs (n = 23) possessed 2 filaments. In most months, GSI scores were variable for both sexes (Fig. 3). The maximum and minimum GSI scores observed for all females in this study were 10.19 (October 2011) and 0.12 (October 2011), respectively. For males, we observed a maximum of 1.62 (April 2011) and a minimum of 0.08 (November 2011). Mean GSI score was 3.16 (± 0.34 SE) for females and 0.48 (± 0.06 SE) for males. We recorded below-average GSI scores for all individuals within a single month in January 2012 and February 2012 for females and in October 2011, November 2011, January 2012, February 2012, and September 2012 for males. Our tests detected no statistical differences for comparisons of left and right ovarian-lobe mass (P > 0.59) or number of mature ova (P > 0.80). Smaller-sized individuals were under sampled; thus, we did not choose multiple size classes for the seasonal-based analysis of stomach contents (Table 1). Our analysis revealed that L. vanhyningi fed on a large assortment of prey items found at or near the surface of the water. Cladocerans were consumed in higher quantities than any other prey in all seasons and ranged from 56–98% of total diet in the winter and summer, respectively (Fig. 4). Insects in an aquatic life stage at the time of collection accounted for 13% of winter prey items (Collembola 12%; Chironomidae 1%), but these 2 prey items only accounted for 3% in the spring (Collembola 1%; Chironomidae 2%). Insects in terrestrial life stages (e.g., winged), at the time of collection accounted for 10% of winter prey items and 21% of spring prey items. Eggs of unknown origin—possibly from fish, snails, or crayfish—represented 21% of the food items consumed in the fall. The fish we sampled consumed copepods in all seasons; these invertebrates constituted 5% of the diet in the spring and 9% in the fall. Less-common food items found (labeled as “other” in Fig. 4) were arachnids, crustaceans, arthropods, larval fish, plant materials, and nematodes. Discussion We observed an elevated GSI score in at least 1 individual of both sexes in most sampling months. GSI scores were low for both sexes in January 2012 and February 2012, indicating a period of minimal spawning activity. Although we documented minimal reproductive investment for males in October 2011 and November 2011, Table 2. Counts, measurements, and description of ova obtained from female Labidesthes vanhyningi used in this study from September 2011 through September 2012. n = total number of ova measured, and SE = standard error. Mean number of ova per Diameter (mm) Type of ova Color n individual (SE) Min. Max. Mean (SE) Fertilized Orange/yellow yolk 85 12.1 (5.22) 1.08 1.32 1.20 (0.005) with clear albumin Mature Orange or amber 908 53.4 (4.05) 0.61 1.33 0.98 (0.005) Southeastern Naturalist 457 M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 it was observed in September 2011 and December 2011. This result indicates that spawning activity likely occurred in these months, but we observed no individuals with elevated GSI scores, possibly due to the sample size chosen for the GSI analysis (n = 5). We collected YOY in September 2011, October 2011, January 2012, May 2012, and September 2012. It is possible that YOY were present in other months, but were not captured due to the sampling protocol for this study; e.g., mesh size of net and/or under-sampling of habitat used by larvae and juveniles. Given that we observed YOY in multiple seasons, and individuals invested in reproduction for most of the year, it appears that L. vanhyningi in the Black Warrior River Drainage spawn Figure 3. Gonadosomatic index (GSI) plotted against month collected for male and female Labidesthes vanhyningi examined in this study from September 2011 through September 2012. Southeastern Naturalist M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 458 from at least March through December. This finding contrasts with Nelson (1968), who found that Tippecanoe River (Wabash/Ohio River Drainage, IN) L. sicculus spawned from about mid-June to early August. Similar to Nelson’s (1968) result, Powles and Sandeman (2008) found that L. sicculus from the Kawartha Lake district of central Ontario spawned from late-May to mid-August. The increased duration of spawning in Alabama compared with northern populations is likely due to Alabama populations experiencing optimal spawning temperatures for a longer period than northern populations, which permits earlier and longer spawning events (Conover 1992). Cahn (1927) reported that spawning activities for L. sicculus began at 18 °C and peaked at 24 °C. Applying Cahn’s (1927) observations to water temperature data collected for this study would indicate that spawning should have occurred only from March through September. The emergence of YOY in May 2012 (34 °C), September 2011 and 2012 (17 °C and 26 °C, respectively), October 2011 (19 °C), and January 2012 (temperature data not available, but estimated 13–15 °C from December 2011 and February 2012 records) for this study suggests that the Black Warrior River Drainage populations either: (1) spawn at temperatures lower than indicated by Cahn (1927); or (2) spawn at similar water temperatures, but water temperature data collected for this study may not be representative of average monthly temperatures. Few studies have characterized, counted, and measured ova from preserved L. sicculus/vanhyningi specimens. Rasmussen (1980) conducted an in-depth study Figure 4. Proportion of food items found in the stomachs of Labidesthes vanhyningi in months of October 2011, January 2012, April 2012, and July 2012, representing the seasons of autumn, winter, spring, and summer, respectively. We examined n = 20 stomachs per season were examined. Insect (aquatic) refers to insects that were in an aquatic life stage when collected and Insect (terrestrial) refers to insects that were collected in a terrestrial (e.g., winged) life stage. Southeastern Naturalist 459 M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 exploring egg morphology and development of L. sicculus eggs collected using a plankton net. Cahn (1927) and Hubbs (1921) detailed observational accounts of eggs after they had been laid. Fogle (1959) characterized mature eggs as clear and ready to be fertilized and found mean egg diameter was 0.95 mm. Mature ova found by Fogle (1959) are similar in description to fertilized eggs found in this study, but have the same diameter as mature ova described in this study. Nelson (1968) found eggs 0.15–0.25 mm diameter (no color noted) from females collected in April and both orangish (0.8–1.2 mm diameter) and immature whitish ova (0.2–0.6 mm diameter) collected in June. Orangish ova found by Nelson (1968) match in size and color to mature ova found in this study. Other ova found by Nelson (1968) appear to be similar to immature ova found in this study. A future study to investigate ova development would advance the understanding of earlier stages labeled as “maturing” and “immature” in this project. Studies conducted by Hubbs (1921), Cahn (1927), and Nelson (1968) revealed that eggs possessed 1 filament, whereas our findings and those of Fogle (1959) and Rasmussen (1980) reported eggs with 2 filaments. While Rasmussen’s (1980) study examined individuals from the known range of L. vanhyningi, no discoveries to date have found the distribution of L. vanhyningi extending to Fogle’s (1959) study area of Lake Fort Smith, AR (D. Werneke, pers. comm.). Fogle’s (1959) findings suggest the possibility that L. vanhyningi was examined, which would suggest a possible increase in the known distribution of L. vanhyningi; thus, both lineages of Labidesthes occur or may have occurred in Lake Fort Smith, AR (D. Werneke, pers. comm.). More field collections and examination of museum specimens will lead to a better understanding of present and historical distributions of lineages within this genus. In this study, we made no attempt to characterize every stage of egg development in specimens examined. Instead, we characterized ova classes by distinctive gross differences in size and color. Four developmental stages of ova were easily distinguishable using a dissecting microscope. Examination with a dissecting microscope serves as a rough method to determine the most important stages of ova development, including the possibility of stages characteristic of internal fertilization (i.e., stages undergoing cleavage). We labeled the first class of ova described in this study as “fertilized”. Although no eggs presumed to be fertilized contained eyed embryos, as found in a Florida population of L. vanhyningi by Grier et al. (1990), we observed eggs in early developmental stages containing what appeared to be the beginning stages of blastulation with clear signs of developed blastoderm. The lack of eggs containing eyed embryos in this study may be due in part to the 30-d span between collecting dates or due to females possibly depositing eggs in varying stages of development. The potential for individuals containing eyed embryos could be optimized if sampling periods were conducted on a more frequent basis and a greater number of females were examined. Further evidence of internal fertilization of L. vanhyningi in the Black Warrior River Drainage would be the presence of the observed sexual dimorphic differences described by Grier et al. (1990) in which a genital palp, an intermittent sex organ for sperm transfer, was found on males and absent on females. Werneke and Armbruster (2015) found Southeastern Naturalist M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 460 presence of a genital papilla on males of both species, suggesting that L. sicculus internally fertilizes as well. It is possible that Hubbs (1921) observed copulation in L. sicculus and described the event as follows: “Only once was the actual spawning act observed, but at such close range and under such conditions of illumination that the details of movement could be closely followed: a pair upon coming to comparative rest near the surface, glided downward, counter-current, toward the bottom, moving side by side, frequently bringing their ventral surfaces into contact by a lateral turning of the body; before reaching the bottom the pair separated, each fish rapidly swimming away, although apparently not disturbed by the observer.” Internal fertilization has been discovered in other silversides and appears to be a common phenomenon within Atherinomorpha (Grier and Parenti 1994). Labidesthes sicculus appears to be an opportunistic feeder, ingesting many prey items occurring at or near the water surface. Numerous studies have examined dietary components of L. sicculus (Boesel 1938, Cahn 1927, Ewers and Boesel 1935, Mullan et al. 1968, Pratt et al. 2002, Ramirez et al. 2006, Zimmerman 1970). In some studies, insects and copepods constituted a major proportion of prey items consumed (Boesel 1938, Cahn 1927, Zimmerman 1970). Similar to our results from the Black Warrior River Drainage population, Ewers and Boesel (1938) found a majority of cladocerans in the stomachs of Labidesthes from Buckeye Reservoir, OH. Only 1 other dietary study, conducted in an older reservoir (Bull Shoals) and a newly constructed reservoir (Beaver) on the White River, AK, has examined seasonal-based dietary patterns in Labidesthes (Mullan et al. 1968). In that study, mostly aquatic insects and zooplankton were consumed in all seasons (except for spring) in the newly constructed reservoir. In the older reservoir, mostly terrestrial insects and zooplankton were consumed in the fall and winter, whereas mostly aquatic insects were consumed in the spring and summer. Only 1 study has examined dietary components of L. vanhyningi (Strongin et al. 2011). They found that L. vanhyningi consumed a majority of insects in allopatry with M. audens and a majority of cladocerans in sympatry with M. audens. The findings by Strongin et al. (2011) differ from the findings of this study for Labidesthes in allopatry with Menidia. In our study, cladocerans constituted a large majority of food items found in individuals from all seasons, while proportions of other food items found fluctuated among seasons. It is important to consider the consequences of ecosystem productivity on the diet of Labidesthes. Prey items found in high abundance in one system may not be present in another system. This change in composition of available prey items depends on whether the system is eutrophic or oligotrophic (Pinto-Coelho et al. 2005). In this study, we estimated diet composition from relative abundance, not biomass. Such an approach may overemphasize the importance of small, common, prey relative to less-common larger prey items for growth and energetics. It is important to consider that cladocerans are much smaller in size than insects. In terms of biomass, many cladocerans would have to be consumed to equal the biomass of a single insect. A review of published accounts indicates that Labidesthes consume the same prey items throughout its range, but different populations consume various Southeastern Naturalist 461 M.S. Piteo, M.R. Kendrick, and P.M. Harris 2017 Vol. 16, No. 3 proportions of those prey items. Another important topic to consider when examining dietary components is food quality vs. food quantity. High-quality food items, even when they constitute a small percentage of total diet, may make a greater contribution to consumer growth (Benke and Wallace 1980). A study detailing the energetic and stoichiometric basis of production and growth in Labidesthes would provide insight into the relative importance of individual prey items in this species. Some life-history characters of Black Warrior River Drainage Labidesthes differed from studies conducted on populations representing L. sicculus. The most notable difference is that L. vanhyningi have eggs with 2 filaments, whereas L. sicculus have eggs with 1 filament. Spawning also began earlier and lasted considerably longer in L. vanhyningi compared to L. sicculus populations in Indiana and Ontario. Similar to L. sicculus of Buckeye Reservoir, OH, L. vanhyningi in this study consumed mostly cladocerans. The difference in egg-filament number provides more evidence in support for the validity of L. vanhyningi as a species. Mobile Basin Labidesthes in the TTW are under threat from invading Menidia. Numerous dams have altered the natural flow regime of rivers throughout the Mobile Basin. This change has created lentic habitats favored by closely related Menidia, a superior competitor for food resources, in those environments. With 1 Mobile Basin population of Labidesthes already under threat by invading Menidia, a more comprehensive understanding of the potential for Menidia to affect L. vanhyningi in the Mobile Basin is required in modified habitats favorable t o Menidia. Acknowledgments We would like to thank Brook Fluker, Bernie Kuhajda, Alex Huryn, John Pfeiffer, Jon Benstead, Josh Jones, Mick Demi, and Mike Venarsky for providing suggestions and helpful insight. We would also like to thank Ronald Phelps and David Werneke of Auburn University for answering various questions about this project. 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