North American Journal of Fisheries Management 29:1425–1437, 2009 [Article] Ó Copyright by the American Fisheries Society 2009 DOI: 10.1577/M08-253.1 Morphological Models for Identifying Largemouth Bass, Spotted Bass, and Largemouth Bass 3 Spotted Bass Hybrids JASON D. GODBOUT,D.DEREK ADAY,* AND JAMES A. RICE Department of Biology, North Carolina State University, Campus Box 7617, Raleigh, North Carolina 27695, USA MAX R. BANGS AND JOSEPH M. QUATTRO Department of Biological Sciences, School of the Environment, University of South Carolina, Columbia, South Carolina 29208, USA Abstract.—Hybridization is common among many closely related fishes, such as the largemouth bass Micropterus salmoides and spotted bass M. punctulatus. Although these species are common members of the sport fish community in midwestern and southeastern U.S. reservoirs, fairly little is known about their ecological interactions or the potential for the introduction of one species to influence the other species. To address these ecological questions and develop appropriate management strategies, reliable field and laboratory identification of each parental species and their hybrid is required. To that end, we collected juvenile (n ¼ 60) and adult (n ¼ 78) largemouth bass, spotted bass, and largemouth bass 3spotted bass hybrids from Lake Norman, North Carolina, a system with a historically strong largemouth bass fishery that recently experienced a spotted bass introduction. We recorded a suite of morphological traits on each individual and correlated those observations with DNA sequences from one mitochondrial marker and three nuclear DNA markers in an attempt to develop morphological field and laboratory methods for identifying individuals of the parental species and their hybrid. After confirming that largemouth bass and spotted bass were hybridizing in Lake Norman, we used classification tree analyses to form dichotomous keys for field and laboratory identification of parental individuals and hybrids at juvenile (50–100 mm total length) and adult (300–500 mm) life stages. These keys should provide fishery biologists and managers with a tool to identify these two species, which commonly interact and closely resemble one another. In addition, these keys should be useful in providing evidence that largemouth bass and spotted bass are hybridizing before more expensive techniques like DNA sequencing are pursued. Many freshwater fishes hybridize in nature, and some systems where they co-exist requires methods for do so commonly (Hubbs 1955; Scribner et al. 2001). reliably identifying individuals of each parental species Largemouth bass Micropterus salmoides and spotted and, if they are intermixing, their hybrid. bass M. punctulatus are ecologically similar species and Several methods exist to quantify hybridization. co-occur in many reservoirs in the midwestern and Early work examined hybridization in laboratory tanks, southeastern United States. Both naturally hybridize creating an environment to encourage spawning with other black basses (Whitmore and Hellier 1988; between different species (Hubbs 1955). Now, natu- Morizot et al. 1991; Koppelman 1994; Avise et al. rally spawned fish can be identified as hybrid or pure 1997; Pierce and Van den Avyle 1997; Barwick et al. by using a suite of cellular analyses (see review by 2006); however, based on a review of the primary Ward and Grewe 1994). Black bass species and literature, the two species have not been recorded as subspecies can be identified using allozyme markers naturally hybridizing with each other. Hybridization is (e.g., Kassler et al. 2002), which are particularly useful possible and seems likely as spawning behaviors and in systems involving only two taxonomic groups. In locations are similar in both species and spawning systems containing three or four groups (e.g., subspe- occurs at about the same time of year, although spotted cies) that may be hybridizing, confident identification bass may construct their nests in slightly deeper water of individuals becomes more difficult given the than largemouth bass (Robbins and MacCrimmon allozyme markers currently available (see Kassler et 1974; Vogele and Rainwater 1975; Sammons et al. al. 2002). Conversely, nuclear methods that can 1999). Successful management of these species in distinguish (1) Florida largemouth bass M. salmoides floridanus and northern largemouth bass M. salmoides *Corresponding author: [email protected] salmoides, (2) Alabama spotted bass M. punctulatus Received December 16, 2008; accepted April 28, 2009 henshalli and northern spotted bass M. punctulatus Published online September 3, 2009 punctulatus, and (3) largemouth bass 3 spotted bass 1425 1426 GODBOUT ET AL. hybrids are currently available and offer the opportu- ple, bluegills Lepomis macrochirus are known to vary nity to reliably sort species and hybrids (J.M.Q., morphologically based on habitat preference (Ehlinger unpublished data). and Wilson 1988). Nevertheless, the observation of Quantifying morphological characteristics in the intermediate and mixed phenotypic traits, coupled with field and correlating those measurements with genetic numerous prior observations of hybridization in black data may allow the development of field methods to basses, suggested the possibility that largemouth bass differentiate hybrids from pure individuals. However, and spotted bass could hybridize and were doing so in past studies examining hybridization and attempting to Lake Norman. This information, in combination with a correlate morphological field data with genetic data decline in estimated largemouth bass numbers and sometimes have produced equivocal results, often as a biomass at early life stages (Abney et al. 2007), caused result of hybrids backcrossing with pure individuals concern for North Carolina Wildlife Resources Com- (Avise et al. 1997). Results of studies attempting to use mission (NCWRC) biologists managing the lake. phenotypic characteristics to categorize pure and However, with no genetic data available and no hybrid individuals range from general failure (e.g., reliable published methods for identification of each Smith et al. 1995; Avise et al. 1997) to identification species and their hybrid in the field, questions about the with reasonable confidence (e.g., Weigel et al. 2002; extent of hybridization and its effect on the Lake Baumsteiger et al. 2005). Even with the potential for Norman fishery remained unanswered. failure, the cost savings generated by reliably differ- The objectives of this study were (1) to determine entiating hybrids from individuals of parental species whether largemouth bass and spotted bass are hybrid- in the field may make the initial investment in DNA izing in Lake Norman, and (2) if so, to develop reliable analyses worthwhile. field and laboratory methods to identify each parental Understanding hybridization also has important species and their hybrid at both juvenile and adult life ecological implications. Introgression occurs when stages. To determine whether spotted bass and hybrid individuals backcross with individuals from at largemouth bass were hybridizing and to validate field least one parental population (Rhymer and Simberloff identifications, genetic analyses were correlated with 1996). When introgression is occurring, hybrids detailed morphological observations. Classification and become particularly difficult to identify. Avise et al. regression tree analyses were used to provide straight- (1997) provided a good example of introgression forward methods that can be used in field or laboratory between two black bass species in a study that settings to identify each group by simply observing documented a drastic decrease in a population of reliable morphological traits. smallmouth bass M. dolomieu 10–15 years after spotted bass were introduced, with eventual replacement by Methods spotted bass. Using restriction fragment length poly- Study site.—Lake Norman is a 12,634-ha, mesotro- morphism digests from mitochondrial DNA (mtDNA) phic reservoir on the Catawba River in west-central and allozyme data, the authors of that study found that North Carolina. A nuclear power station and a coal- over 99% of individuals in the sample population were fired steam plant influence nearby water temperatures either spotted bass or spotted bass 3 smallmouth bass by discharging hot water, and hydroelectric power is hybrids of the F1 or later generations. More generally, generated at the dam. Much of the lake’s shoreline is Huxel (1999) investigated introgression through mod- developed and lined with riprap and has numerous eling and estimated that native species may be piers and docks. effectively replaced by nonnative species in fewer than Field.—Boat electrofishing (Smith-Root Model 7.5 five generations. The observation of hybridizing Generator-Powered Pulsator) was used to collect two species, however, does not explain why species are so size-classes of black bass (50–100 and 300–500 mm quickly replaced. Avise et al. (1997) proposed behav- total length [TL]) for genetic analyses during spring and ioral, environmental, and ecological changes as addi- summer 2007 and spring 2008. These size-classes were tional causes of the shift in dominant species. chosen in an effort to develop identification rubrics for Duke Energy biologists working on Lake Norman, both juveniles and adults, as managers often are faced North Carolina, first collected spotted bass unexpect- with the need to reliably identify species at all life stages. edly in 2000 (D.