Southeastern Naturalist P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 596 2015 SOUTHEASTERN NATURALIST 14(3):596–601 First Molecular Verification of a Marine-collected Specimen of Alosa alabamae (Teleostei: Clupeidae) Paul F. Mickle1,*, Jim S. Franks2, Brian R. Kreiser4, Gary J. Gray2, Jeremy M. Higgs2, and Jeanne-Marie Havrylkoff 4 Abstract - Alosa alabamae (Alabama Shad) is an imperiled anadromous species that reproduces in northern Gulf of Mexico drainages. To date, there have only been 4 vouchered specimens collected from marine waters, but none have been verified with molecular techniques. On 28 March 2013, we collected a single adult female in proximity to a barrier island (Petit Bois) off the coast of Mississippi. Microsatellite DNA analysis corroborated the identification of this individual and suggested that the specimen was most genetically similar to the group from the Pascagoula River drainage rather than other portions of the range. Thus far, research has been focused on the species’ freshwater life history, and it is crucial that more effort be directed toward documenting and understanding the full life history of t his threatened fish. Introduction Alosa alabamae Jordan and Evermann (Alabama Shad) is an anadromous fish that has a federal status of special concern, is listed as endangered by some states, and has been extirpated from many drainages throughout its range (Meadows et al. 2006, NOAA 2015). Adults migrate from marine habitats into natal rivers to spawn during spring months (February–May) before returning to the Gulf of Mexico (GOM; Mettee and O’Neil 2003). The species’ distribution ranges from Missouri to Florida, but no published information is available about its marine distribution in the GOM. Only 4 Alabama Shad (Fishnet catalog numbers: 293755.5174309, 28671, 20627[2]) have ever been recorded in collections within the marine environment outside the boundaries of an estuary (Alabama and Florida; Fishnet2 2015). Past research of Alabama Shad was limited to riverine habitats and focused on aspects of juvenile and adult life history such as habitat use, diet, and spawning (Ely et al. 2008; Mettee and O’Neil 2003; Mickle et al. 2010, 2013). There is clearly a need for studies focused on the adult life stage in marine and estuarine environments. In this paper, we report the first molecularly verified record of an adult Alabama Shad from marine waters, thereby providing a glimpse into one important aspect of this species’ life history. 1The Mississippi Department of Marine Resources, 1141 Bayview Avenue, Biloxi, MS 39530. 2The University of Southern Mississippi, Center for Fisheries Research and Development, Ocean Springs, MS 39564. 3Department of Coastal Science, Gulf Coast Research Laboratory, 703 East Beach Drive Ocean Springs, MS 39564. 4The University of Southern Mississippi, Department of Biological Sciences, 118 College Drive # 5018, Hattiesburg, MS 39406-5018. *Corresponding author - paul.mickle@dmr.ms.gov. Manuscript Editor: Hayden T. Mattingly Southeastern Naturalist 597 P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 Materials and Methods We captured a single adult Alabama Shad on 28 March 2013 within the statedesignated marine waters of Mississippi (Fig. 1). We collected the specimen just north of Petit Bois Island and 4.8 km east of Horn Island Pass, which leads to the open GOM (Table 1). We obtained the fish during a fishery independent monitoring project (Mississippi Coast Sport Fish Study, conducted by The University of Southern Mississippi - Gulf Coast Research Laboratory) in a 11.43-cm mesh panel of a 182.88-m experimental multi-paneled gill net that was soaked for 136 min. We measured (total length [TL]), weighed (total weight [TW]), sampled (fin tissue) for genetic analysis, and deposited the specimen in the Mississippi Museum of Natural Science, Jackson, MS, Lot #61556. Some clupeid species are morphologically and meristically similar, thus, we conducted a microsatellite-DNA analysis to confirm species identification and assign the individual to a natal drainage. The molecular analysis was based on a reference collection of 473 Alabama Shad from 6 drainages—the Gasconade, Little Missouri, Ouachita, Pascagoula, Choctawhatchee, and Apalachicola rivers. This reference collection is the basis of an unpublished study of range-wide population structure (Bowen 2005). Genotyping the adult individual in our study was performed in the same laboratory and using the same methods as Bowen (2005). Briefly, the following methods were used. We genotyped individuals using the polymerase chain reaction (PCR) to amplify 16 microsatellite loci (Аѕа-2, Аѕа-4, Аѕа-9, Аѕа-16 [Waters et al. 2000]; Аа14, Аа16, Аа20 [Faria et al. 2004]; and AsaB020, AsaC249, AsaC334, AsaD021, AsaD030, AsaD055, AsaD312, AsaD392, AsaD492 [Julian and Barton 2007]) in a total volume of 12.5 μl using 1X reaction buffer (Promega Company, Madision, WI), 1.5–3.0 μM MgCl2, 200 μM dNTPs, 0.4 units of Taq polymerase (Promega Company), 0.3 μM of the M13 tailed forward primer (Boutin-Ganache et al. 2001), 0.3 μM of the reverse primer, 0.1 μM of the labeled M13 primer, 20–150 ng of template DNA, and water to the final volume. PCR cycling conditions consisted of an initial denaturing step at 94 °C for 2 min followed by 35 cycles of 30 sec at 94 °C, 1 min at 50–61 °C, and 1 min at 72 °C. A final elongation step of 10 min at 72 °C ended the cycle. We visualized microsatellite alleles on acrylamide gels using a LI-COR 4200 automated DNA sequencer (LICOR, Lincoln, NE), and scored allele sizes using GENE IMAGIR v 3.55 software (LI-COR). We conducted tests for Hardy-Weinburg equilibrium (HWE) and linkage disequilibrium (LD) using GENEPOP for the web (http://genepop.curtin.edu. au/; Raymond and Rousset 1995) and measured genetic variation by the number of alleles, observed heterozygosity (Ho), and expected heterozygosity (He) as calculated by GenAlex 6.501 (Peakall and Smouse 2006). We employed MICROCHECKER v. 2.2.3 (van Oosterhout et al. 2004) to detect the presence of null alleles and FSTAT 2.9.3 (Goudet 1995) to calculate Weir and Cockerham’s (1984) unbiased estimator of FST. Bowen (2005) suggested that there are 3 genetically discrete populations (K) represented by the data as estimated using STRUCTURE 2.3.3 (Pritchard et al. 2000). To assign our adult specimen to 1 of the genetic Southeastern Naturalist P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 598 groups defined by STRUCTURE, we performed another analysis following the recommendations for the USEPOPINFO model found in the STRUCTURE manual. We used individuals from the Apalachicola (n = 201), Pascagoula (n = 188), and Mississippi (n = 76) river basins as representatives of the 3 genetic groups where individuals had average membership coefficients (q scores) >0.95. Parameters for this analysis included 20 independent runs with a burn-in of 250,000 followed by 500,000 MCMC replications. Results The specimen was 383 mm TL, 710 g TW, and female with visually observed, well-developed ovaries (Fig. 1). The stomach was filled with what appeared to be Figure 1. Image of the collected adult female Alabama Shad (A). Image of the specimen’s well-developed ovaries (B). Southeastern Naturalist 599 P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 small invertebrates. Physiochemical (water quality) data taken at the collection site were typical (Jewel et al. 2015) for the marine zone from which the specimen was collected (Table 1). In the reference populations, none of the loci consistently deviated from HWE expectations or demonstrated LD, and we did not detect null alleles. The average number of alleles across loci at a site ranged from 3.2 to 8.1, while average Ho and He were 0.40–0.49 and 0.37–0.48, respectively. Pairwise FST values were smallest between the Choctawhatchee and Apalachicola drainages (0.006), and the largest values occurred between the Mississippi River drainages and the GOM drainages (0.092–0.119). The average q score of this specimen was 0.82 (SD = 0.06) in the Pascagoula group. Discussion This species is anadromous, and considering the collection date, it is likely the animal was preparing for a spawning run within one of the proximate rivers. The mouth of the Pascagoula River is 14.8 km north of the collection site, and this particular drainage has shown past recruitment for Alabama Shad (Mickle et al. 2010, Ross 2003). Mills (1972) and others have reported few to no diet items from adults collected in riverine habitats. As with numerous other anadromous fishes, Alabama Shad most likely feeds primarily in marine habitats, which supports the need for marine research of this species (Mettee and O’Neil 2003). Despite the existence of the FishNet collections mentioned above, it remains largely unknown what marine regions are utilized by adult Alabama Shad. The similar appearance of this species to Brevoortia patronus Goode (Gulf Menhaden) has probably contributed to misidentification and lack of reported occurrences from marine environments. With the majority of North American anadromous fishes in decline, research on these species, including Alabama Shad, must include life histories, environmental requirements, and migratory demands (Limburg and Waldman 2009). Projected increasing human populations in the northern GOM states will most likely produce additional anthropogenic pressures on the natal drainages that support anadromous fishes (US Census 2015). Considering the imperiled status of Alabama Shad, it is essential that the ecology and all life-history aspects of this species are documented for its riverine, estuarine, and marine habitats. Table 1. Physiochemical data recorded during collection of the specimen of Alabama Shad from location 30.22797ºN, 88.45806ºW. Parameter Surface Bottom Water temperature 14.9 ºC 15.1 ºC Salinity 21.8 ppt 23.4 ppt Dissolved oxygen 9.19 mg/L 8.87 mg/L Water depth 4.3 m Southeastern Naturalist P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 600 Acknowledgments The specimen was collected during a Gulf Coast Research Laboratory (GCRL) fisheries project funded by the US Fish and Wildlife Service, Sport Fish Restoration Program, and the Mississippi Department of Marine Resources. We thank The University of Southern Mississippi’s Department of Coastal Sciences and Center for Fisheries Research and Development for vessel and personnel support. Literature Cited Boutin-Ganache I., M. Raposo, M. Raymond, and M. Deschepper. 2001. M-13 tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. BioTechniques 31:24–28. Bowen, B.R. 2005. Alabama Shad phylogeography. M.Sc. Thesis. The University of Southern Mississippi, Hattiesburg, MS. Ely, P.C., S.P. Young, and J.J. Isley. 2008. Population size and relative abundance of adult Alabama Shad reaching Jim Woodruff Lock and Dam, Apalachicola River, Florida. North American Journal of Fisheries Management 28:827–831. Faria, R., B. Wallner, S. Weiss, and P. Alexandrino. 2004. Isolation and characterization of eight dinucleotide microsatellite loci from two closely related clupeid species (Alosa alosa and A. fallax). Molecular Ecology Notes 4:586–588. Fishnet2. 2015. Alabama Shad historical collection data in the Gulf of Mexico: 1957–2014. Available online at http://www.fishnet2.net. Accessed 4 January 2015. Goudet, J. 1995. FSTAT version 1.2: A computer program to calculate F-statistics. Journal of Heredity 86:485–486. Limburg, K.E., and J.R. Waldman. 2009. Dramatic declines in north Atlantic diadromous fishes. BioScience 59:955–965. Jewell, J., M.T. Hill, and M.K. Brainard. 2015. Monitoring and assessment of Mississippi’s interjurisdictional marine resources. Technical report. Mississippi Department of Marine Resources, Biloxi, MS. 12 pp. Julian, S.E., and M.L. Barton. 2007. Microsatellite DNA markers for American Shad (Alosa sapidissima) and cross-species amplification within the family Clupeidae. Molecular Ecology Notes 7:805–807. Meadows, D.W., S.B. Adams, and J.F. Schaefer. 2006. Threatened fishes of the world: Alosa alabamae (Jordan and Evermann, 1896) (Clupeidae). Environmental Biology of Fishes 82:173–174. Mettee, M.F., and P.E. O’Neil. 2003. Status of Alabama Shad and Skipjack Herring in Gulf of Mexico drainages. Pp. 157–170, In K. Limburg and J. Waldman (Eds.). Biodiversity, Status, and Conservation of the World’s Shads. American Fisheries Society, Symposium 35, Bethesda, MD. Mickle, P.F., J.F. Schaefer, S.B. Adams, and B.R. Kreiser. 2010. Habitat use of age 0 Alabama Shad in the Pascagoula River drainage, USA. Ecology Of Freshwater Fish 19:107–115. Mickle, P.F., J.F. Schaefer, D.A. Yee, and S.B. Adams. 2013. Diet of juvenile Alabama Shad (Alosa alabamae) in two northern Gulf of Mexico drainages. Southeastern Naturalist 12:233–237. Mills, J.G. 1972. Biology of Alabama Shad in northwest Florida. Florida Department of Natural Resources Technical Series 68:24–33. Southeastern Naturalist 601 P.F. Mickle, J.S. Franks, B.R. Kreiser, G.J. Gray, J.M. Higgs, and J.-M. Havrylkoff 2015 Vol. 14, No. 3 National Oceanic and Atmospheric Administration (NOAA). 2015. Species of concern. Available online at http://www.nmfs.noaa.gov/pr/species/concern. Accessed 15 April 2015. Peakall, R., and P.E. Smouse. 2006. GENALEX 6: Genetic analysis in Excel. Population genetic software for teaching and research. Molecular Ecology Notes 6:288–295. Pritchard, J.K., M. Stephens, and P. Donnelly. 2000. Inference of population structure using multilocus genotype data. Genetics 155:945–959. Raymond, M., and F. Rousett. 1995. GENEPOP (version 1.2): Population-genetics software for exact tests and ecumenicism. Journal of Heredity 86:248–249. Ross, S.T. 2003. Inland Fishes of Mississippi. University Press of Mississippi, Jackson, MS. 624 pp. US Census. 2015. Available online at http://www.census.gov/quickfacts. Accessed 15 April 2015. van Oosterhout, C.V., W.F. Hutchinson, D.P.M.Wills, and P. Shipley. 2004. MICROCHECKER: Software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology 4:535–538. Waters, J.M., J.M. Epifanio, T. Gunter, and B.L. Brown. 2000. Homing behavior facilitates subtle genetic differentiation among river populations of Alosa sapidissima: Microsatellites and mtDNA. Journal of Fish Biology 56:622–636. Weir, B.S., and C.C. Cockerham. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370.