Creating a Molecular Database for Identifying Two ...

MarSci: The Undergraduate Journal for the Marine and Aquatic Sciences Fall 2008

Creating a Molecular Database for Identifying Two Subspecies of Largemouth Bass (Micropterus salmoides) Max R. Bangs1*, Joseph M. Quattro2, Kenneth J. Oswald2, Jean Leitner3 1

University of South Carolina, Dept. of Biological Sciences, Columbia, SC, 29225 University of South Carolina, Marine Science Program, Columbia, SC, 29225 3 South Carolina Department of Natural Resources 2

Two subspecies of largemouth bass, the northern largemouth bass (Micropterus salmoides salmoides) and the Florida bass (Micropterus salmoides floridanus) occur commonly throughout the southeastern United States. Prior to 1949 the Florida bass was known only from peninsular Florida; unfortunately, due to greater maximum size and longevity the Florida subspecies has been introduced into lakes and other impoundments throughout the east coast and southern United States. This expansion of Florida bass into the habitat of northern largemouth bass could result in hybridization of the two subspecies and thus may affect the fitness of the northern largemouth bass populations in these areas. We characterized genetic diversity at one mitochondrial locus (ND2) and four single copy nuclear loci (ITS2, Actin, S7, and Calmodulin). The resulting DNA sequences were used to create a genetic database useful for differentiating these two subspecies. Keywords: Micropterus salmoides salmoides, Micropterus salmoides floridanus, habitat expansion, subspecies, hybridization

Introduction There are currently two subspecies of largemouth bass: the northern largemouth bass (Micropterus salmoides salmoides) and the Florida bass (Micropterus salmoides floridanus). The Florida bass was first described based on a diverse suite of meristic and morphometric characteristics including scale count, body length, and distinctive coloration (Bailey and Hubbs, 1949). It is believed that these subspecies share a recent common ancestor and were isolated due to sea level fluctuation near the Florida peninsula (Near et al., 2003). Bailey and Hubbs also described the natural range of the two subspecies. The Florida bass was described as living in the Florida peninsula south and east of the Suwannee River drainage, whereas the northern largemouth bass was described as living north and west of that range, with a small intergrade zone between the two (Bailey and Hubbs, 1949).

In 1983, Philipp et al. expanded this intergrade zone to include the east coast as far north as Maryland as well as large parts of Alabama and Mississippi (Philipp et al., 1983). They believed that this expansion was due to the stocking of Florida bass throughout the southeastern United States (Philipp et al., 1983). In fact, this intergrade zone has probably expanded even more because the largemouth bass is one of the most widely introduced species of freshwater fishes (Jackson, 2002). Today, as a result of stocking, the range of largemouth bass has greatly expanded to include the United States, warm regions of Canada, most of Mexico and Central America, large areas of Europe, Southern Africa, South America and even parts of Asia and the Caribbean (Jackson, 2002). This expansive stocking, conducted mostly by management agencies, is done to facilitate angling by enhancing existing populations and stocking in locations where

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these fish do not occur naturally (Jackson, 2002). In most of these stocking programs, Florida bass are used because their maximum size surpasses that of the northern largemouth bass, and they also have a greater longevity (Fries, 2002). The stocking of Florida bass may have an effect on the United States largemouth bass population, especially along the east coast where northern largemouth bass already reside and Florida bass are being successfully introduced. Stocking could result in hybridization of the two subspecies. This change may have multiple effects on the largemouth bass populations including a hybridization of the northern largemouth bass out of existence, or reduced fitness of the population due to the presence of hybrids (Philipp et al., 2002). Unfortunately, hybrids are hard to identify based solely on taxonomic features. Genetic markers have been successfully employed to identify hybrids in a wide range of species, including largemouth bass (Near et al., 2005). Our objective is to create a database of genetic markers, which will help identify hybrids and distinguish northern largemouth bass (M. s. salmoides) from Florida bass (M. s. floridanus) for future studies.

Materials and Methods Thirty samples of pure northern largemouth bass (M. s. salmoides) were collected from Wisconsin and Minnesota (15 from each state) using single-line sampling and/or electrofishing. Thirty samples of pure Florida bass (M. s. floridanus) were collected from Florida using similar methods. Fin clips were taken and stored in 95% ethanol for later laboratory work. Once in the lab, total genomic DNA was extracted from fin clips using the QIAGEN Dneasy Tissue Kit (QIAGEN Corporation, Maryland, USA) following the manufacturer’s protocol. Presence of DNA was confirmed using agarose gel electrophoresis. The extractions were then used as template in polymerase chain reaction (PCR) to amplify one mitochondrial

(mtDNA) locus and four single copy nuclear (scnDNA) loci. The mtDNA locus was NADH dehydrogenase subunit 2 (ND2; primers from Breden et al., 1999) while the scnDNA loci included the second intron of the intertransgenic spacer locus (ITS2; primers from Presa et al., 2002), , the first intron of the beta-actin gene (Actin; primers from McDowell and Graves, 2002), the first intron of the ribosomal S7 gene (S7; primers from Chow and Hazama, 1998), and the forth intron of the calmodulin gene (Calmodulin; primers from Near, 2005). PCR amplifications were performed as in Oswald (2007) except for Calmodulin which used a 52 degrees Celsius annealing temperature. PCR amplifications were confirmed using agarose gel electrophoresis. Successful PCR amplifications were sequenced as in Oswald (2007). DNA sequences were edited and assembled using Sequencher (version 4.1; Genecodes Corporation, Michigan, USA). The sequences were then exported and aligned using BioEdit (version 7.0; Hall, 1999). Aligned files were then analyzed and organized using Excel and MacClade (version 3.0; Maddison, 1992).

Results ND2: Comparison between northern largemouth bass (M. s. salmoides) and Florida bass (M. s. floridanus) was done over 575 basepairs (bp). Over the 575 bp segment there were ten haplotypes (four northern and six Florida) with 32 fixed differences between the two subspecies (Table 1). Calmodulin: A 492 bp segment was obtained from the samples. Only two haplotypes were found between the samples, and the only difference between the two haplotypes was an A-G change at site 266. Both haplotypes were found in northern largemouth bass in equal frequencies. Only one haplotype

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MarSci: The Undergraduate Journal for the Marine and Aquatic Sciences (haplotype 5) was found in Florida bass (Table 2). Actin: A 513 bp segment was obtained from the samples. Like calmodulin, only two haplotypes were found between the samples, and the only difference between the two haplotypes was a G-A change at site 429. Both haplotypes were found in Florida bass (although most were haplotype 3). Only one haplotype (haplotype 3) was found in northern largemouth bass (Table 3).

ITS2: ITS2 was one of two scnDNA loci that had fix differences between the two subspecies. Over the 462 bp segment there were four haplotypes (two northern and two Florida) with three fixed differences between the two subspecies (Table 4). S7: S7 also had fixed differences between the two subspecies. Over the 622 bp segment there were four haplotypes (two northern and two Florida) with four fixed differences between the two subspecies (Table 5).

A

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Discussion The mtDNA locus ND2 and the scnDNA loci ITS2 and S7 exhibited fixed differences between the two subspecies of largemouth bass. The mtDNA locus ND2 had around ten times as many fixed differences then the scnDNA loci ITS2 and S7, because mtDNA evolves at a much faster rate than scnDNA (Brown, 1979). This may be a result of factors such as a lack of an editing function in mtDNA as well as its haploid nature in upper eukaryotes (Brown, 1979). These fixed differences can therefore be used as markers to distinguish northern largemouth bass (M.s.salmodies) and Florida bass (M.s.floridanus). These loci can be used to determine which subspecies of largemouth bass occur at different locations and can help identify hybrids between the two subspecies. Because of substantial frequency differences between alleles at the scnDNA locus calmodulin, this locus is also somewhat useful for differentiating subspecies. This database of genetic markers will be used for a variety of studies to examine habitat preference of the two subspecies (i.e. examining clines in rivers north to south to see if there is a temperature preference

or discovering if there is a preference between lakes or rivers), to identify any introductions of one subspecies into the range of the other, and to gauge the extent of hybridization between the two. These studies will then answer a multitude of ecological inquiries which include: preference in habitat, reduced fitness in the hybrid zone, and if native northern largemouth bass are being hybridized out of existence through the diffusion of genes in mixed populations. These kinds of studies are important because it was found that bass in other locations often have a reduction in fitness in hybrid zones (Philipp et al., 2002), or have become extinct in a location due to introgressive hybridization (the loss of a species in an area due to diffusion of genes resulting from interbreeding) (Whitmore, 1983). We are using this database to examine samples of largemouth bass in the lakes of the upper Savannah River to determine if Florida bass (M.s.floridanus) occur there and if they hybridizing with northern largemouth bass (M.s.salmodies). We are also using this database to confirm the existence of clinal allele frequency variation in areas where the two subspecies overlap. We expect to find a north to south cline

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MarSci: The Undergraduate Journal for the Marine and Aquatic Sciences between the two subspecies of bass with an intergrade zone which is located in the middle or found throughout. The findings of these research projects will help us better understand these fishes as well as improve management techniques.

Fries, L. T. et al. Mass Production of Polypliod Florida Largemouth Bass for Stocking Public Waters in Texas. American Fisheries Society Symposium 31. The American Fisheries Society. pp. 393-399. 2002.

Acknowledgments

McDowell J. R., J. E. Graves. Nuclear and Mitochondrial DNA Markers for Specific Identification of Istiophorid and Xiphiid Billfishes. Fish Bull. No. 100. pp. 537–544. 2002.

This research was funded by the Howard Hughes Medical Institute and a State Wildlife Grant, SCDNR. Samples of northern largemouth bass were provided by David P. Philipp. Invaluable assistance and advice was provided by Mark Roberts and other members of the Quattro lab.

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Jackson, D. A. Ecological Effects of Micropterus Introduction: The Dark Side of Black Bass. American Fisheries Society Symposium 31. The American Fisheries Society. pp. 221-232. 2002.

Near, T. J. et al. Speciation in North American Black Basses, Micropterus (Actinopterygii: Centrarchidae). Evolution. No. 57. pp. 1610-1621. 2003. Near, T.J et al. Fossil Calibrations and Molecular Divergence Time Estimates in Centrarchid Fishes (Teleostei: Centrarchidae). Evolution. No.59. pp. 1768-1782. 2005 Oswald, K. J. Phylogeography and Contemporary History of Redeye Bass (Micropterus coosae). Ph.D. Dissertation, University of South Carolina. 2007 Philipp, D. P., W. F. Childers, and G. S. Whitt. A biochemical genetic evaluation of the northern and Florida subspecies of largemouth bass. Transactions of the American Fisheries Society 112. The American Fisheries Society. pp.1-20. 1983. Philipp, D. P., et al. Mixing Stocks of Largemouth Bass Reduces Fitness Through Outbreeding Depression. American Fisheries Society Symposium 31. The American Fisheries Society. pp. 349-363. 2002. Presa, P et al. Phylogeographic Congruence Between mtDNA and rDNA ITS Markers in Brown Trout. Molecular Biology and Evolution. No. 19. pp. 21612175. 2002. Whitmore, D. H. Introgressive Hybridization of Smallmouth Bass (Micropterus dolomieui) and Guadalupe Bass (M. treculi). Copeia. No. 3. pp. 672-679. Aug, 1983.