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 3 spotted 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 do so commonly (Hubbs 1955; Scribner et al. 2001). Largemouth bass Micropterus salmoides and spotted bass M. punctulatus are ecologically similar species and co-occur in many reservoirs in the midwestern and southeastern United States. Both naturally hybridize with other black basses (Whitmore and Hellier 1988; Morizot et al. 1991; Koppelman 1994; Avise et al. 1997; Pierce and Van den Avyle 1997; Barwick et al. 2006); however, based on a review of the primary literature, the two species have not been recorded as naturally hybridizing with each other. Hybridization is possible and seems likely as spawning behaviors and locations are similar in both species and spawning occurs at about the same time of year, although spotted bass may construct their nests in slightly deeper water than largemouth bass (Robbins and MacCrimmon 1974; Vogele and Rainwater 1975; Sammons et al. 1999). Successful management of these species in systems where they co-exist requires methods for reliably identifying individuals of each parental species and, if they are intermixing, their hybrid. Several methods exist to quantify hybridization. Early work examined hybridization in laboratory tanks, creating an environment to encourage spawning between different species (Hubbs 1955). Now, natu- rally spawned fish can be identified as hybrid or pure by using a suite of cellular analyses (see review by Ward and Grewe 1994). Black bass species and subspecies can be identified using allozyme markers (e.g., Kassler et al. 2002), which are particularly useful in systems involving only two taxonomic groups. In systems containing three or four groups (e.g., subspe- cies) that may be hybridizing, confident identification of individuals becomes more difficult given the allozyme markers currently available (see Kassler et al. 2002). Conversely, nuclear methods that can distinguish (1) Florida largemouth bass M. salmoides floridanus and northern largemouth bass M. salmoides salmoides, (2) Alabama spotted bass M. punctulatus henshalli and northern spotted bass M. punctulatus punctulatus, and (3) largemouth bass 3 spotted bass *Corresponding author: derek_aday@ncsu.edu Received December 16, 2008; accepted April 28, 2009 Published online September 3, 2009 1425 North American Journal of Fisheries Management 29:1425–1437, 2009 Ó Copyright by the American Fisheries Society 2009 DOI: 10.1577/M08-253.1 [Article]