A Case of Mistaken Identity: Genetic and Morphological Evidence for the Presence of Redeye Bass in the Verde River, Arizona

Black bass (Genus Micropterus) comprises one of the most important groups of game fish in the United States and these species have been introduced outside of their native range extensively (Long et al. 2015). In many areas where they have been introduced and subsequently proliferated, they directly compete with and prey upon native species and have caused declines of native fishes, including in the Desert Southwest. The Verde River is one of few perennial streams in Arizona that still flows largely unimpeded and at least three species of black bass (Largemouth Bass Micropterus salmoides, Smallmouth Bass Micropterus dolomieu, and Spotted Bass Micropterus punctulatus) were reportedly introduced in 1942 by the Arizona Game and Fish Department (AZGFD), with Spotted Bass being considered as rare or extirpated by 1973 (Miller and Lowe 1967; Minckley 1973). These introductions have caused detrimental effects on native fishes, including three species that are listed as endangered under the U.S. Endangered Species Act (ESA; ESA 1973, as amended; Minckley 1973; Rinne 2001; Bonar et al. 2004). Large portions of the Verde River and its tributaries are now Designated Critical Habitat for the endangered Spikedace Meda fulgida, Loach Minnow Rhinichthys cobitis, and Razorback Chub Xyrauchen texanus (ESA 1973, as amended).

Two lines of evidence led us to investigate the possible presence of Redeye Bass Micropterus coosae in the Verde River system of Arizona that may have long been misidentified as Smallmouth Bass. Preliminary results from a vertebrate metabarcoding study targeting a mitochondrial DNA region, the 16S ribosomal RNA gene, from environmental DNA (eDNA) collected from water found sequences matching Redeye Bass and none matching Smallmouth Bass haplotypes at each of three sites on the Verde River (Eaton et al., in review; Figure 1). We searched citizen science databases and social media reports from the Verde River and its tributaries and found several instances of suspected Redeye Bass occurrence, including two research-quality results with photographs from Wet Beaver Creek (a tributary of the Verde River), that were identified by multiple observers as Redeye Bass (iNaturalist.org 2021a, 2021b). Four additional iNaturalist occurrences with photos have since been added, and all are shown on the map in Figure 1. Redeye Bass were introduced in several states outside of their native range in the 1900s, including California, as a game species for warm water streams that were not ideal for larger species such as Smallmouth and Largemouth Bass, but were too warm to support populations of trout (Parson 1954; Fuller 2021).

Redeye Bass was originally described based on morphological data and has since undergone genetic scrutiny that has separated it into several species, each restricted to disjunct upland river systems in the Southeastern United States (Hubbs and Bailey 1940; Baker et al. 2013; Freeman et al. 2015). Here we use the term Redeye Bass complex to refer to all members of M. coosae sensu lato. We use the common name Redeye Bass to refer to M. coosae sensu stricto, native to the Coosa River system of Alabama, Georgia, and Tennessee, following the recent circumscription of new species from the Redeye Bass complex (Baker et al. 2013; Freeman et al. 2015). Other species named from within the Redeye Bass complex are referred to with the following common names: Cahaba Bass Micropterus cahabae, Chattahoochee Bass Micropterus chattahoochae, Tallapoosa Bass Micropterus tallapoosae, and Warrior Bass Micropterus warriorensis. Of these, Chattahoochee Bass could more appropriately be considered a member of the Shoal Bass Micropterus cataractae complex, but it occurs in the eastern portion of the Redeye Bass complex range as first described by Hubbs and Bailey (1940; Freeman et al. 2015).

Our goals for this study were to collect morphological and genetic data on the black bass most abundant at a site on Verde River to test the hypothesis that Redeye Bass were introduced and have subsequently proliferated in this river system. We compared meristics including scale counts along lateral line, above lateral line, and below lateral line with those reported from Smallmouth Bass and Redeye Bass to determine which species is present. We sequenced the mitochondrial gene nicotinamide adenine dinucleotide + hydrogen (NADH) dehydrogenase subunit 2 (ND2), an informative gene for phylogeny reconstruction in black bass (Near et al. 2004; Baker et al. 2013; Freeman et al. 2015), to assess the taxonomic origin of this mitochondrial gene for the black bass sampled from the Verde River. We analyzed Single Nucleotide Polymorphism (SNP) loci developed to delineate several species of black bass to determine the taxonomic origin of the suspected Redeye Bass in the Verde River using nuclear genes inherited biparentally, a method that can also indicate whether hybridization and introgression between black bass species has occurred (Thongda et al. 2019).

Arizona Game and Fish Department collected 15 putative Redeye Bass by angling from the Upper Verde Wildlife Management Area (34.867749°N, 112.401625°W) for morphological analyses on November 16, 2018 (Figure 1). We preserved the fish in 70% EtOH before conducting scale counts for scales along lateral line, scales above lateral line, and scales below lateral line according to Strauss and Bond (1990). We compared the data with the published ranges of these scale meristics from several studies to determine whether they were Redeye Bass or Smallmouth Bass (Turner et al. 1991; Pipas and Bulow 1998; Baker et al. 2013).

Arizona Game and Fish Department collected five additional putative Redeye Bass individuals from the Verde River. We placed fin tissue clips from these individuals into sterile microcentrifuge tubes in 70% molecular biology grade EtOH and analyzed them at the Forensic Biology and Conservation Genetics Lab at Embry-Riddle Aeronautical University in Prescott, Arizona. Prior to DNA extraction, we disinfected and sterilized workspaces and equipment using 10% commercial bleach on lab benches, or DNA Away (ThermoFisher Scientific, Waltham, MA) on metal surfaces of equipment and pipettes. We extracted DNA from fin tissue samples using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol for tissue.

We set up polymerase chain reaction (PCR) reactions in a Fisherbrand™ PCR Workstation (Fisher Scientific, Hampton, NH) with UV sterilization and using filtered pipette tips. We amplified extracted DNA samples with previously published primers targeting the mitochondrial NADH subunit 2 (ND2) gene (Kocher et al. 1995). We performed all PCR amplification reactions using Bio-Rad C1000 Touch Thermal Cyclers (Bio-Rad Laboratories, Hercules, CA). Amplification of the ND2 gene used 25-μL reaction volumes and included 1X Kapa HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA), 0.2 μM forward primer, 0.2 μM reverse primer, and 2.5 μL of template DNA. We conducted thermal cycler reactions with a heated lid and the following conditions: initial denaturation at 95°C for 3 min, followed by 5 cycles at 94°C for 30 s, annealing at 57°C for 30 s, and 72°C for 75 s; an additional 5 cycles of annealing at 56°C for 30 s and 5 cycles with annealing at 55°C for 30 s; followed by 20 cycles at 94°C for 30 s, annealing at 54°C for 30 s and 72°C for 75 s, and a final extension at 72°C for 10 min, followed by a final hold at 4°C (Baker et al. 2013). We visualized the PCR products on a 2% agarose gel to check the amplification success using a Bio-Rad Gel Doc™ EZ Gel Documentation System (Bio-Rad Laboratories). We then cleaned amplicons using the Axygen™ AxyPrep Mag™ PCR Clean-up Kit (Corning, Corning, NY) according to the manufacturer's supplemental protocol to remove primer dimers or DNA smaller than 200 basepairs, with an 8-μL dilution of the AxyPrep Mag PCR clean-up reagent.

We performed Sanger cycle sequencing of all products by Genewiz, Inc (North Plainfield, NJ) in both directions using the ND2 primers (Kocher et al. 1995). We constructed consensus sequences of ND2 by analyzing the forward and reverse sequencing trace files using Geneious Prime (2019.2.1; Biomatters, Inc, San Diego, CA). We manually inspected the forward and reverse reads for quality and for any discrepancies in base calls. We aligned the resulting five consensus sequences in Geneious Prime (2019.2.1) to determine the number of unique haplotypes present for subsequent analyses. We annotated the three unique ND2 sequences in Geneious Prime (2019.2.1) and submitted them to GenBank (NCBI 2021), under the accessions MN657205 (AZMICO1), MN657206 (AZMICO2), and MN657207 (AZMICO3).

We then analyzed the unique haplotypes from the Verde River samples in a phylogenetic framework to determine their likely origin from within the Redeye Bass complex. We downloaded 17 ND2 sequences (Figure S1, Supplemental Material) from species in the Redeye Bass complex included in Baker et al. (2013), along with Largemouth Bass M. salmoides for outgroup comparison, from NCBI GenBank (NCBI 2021). We did not include Smallmouth Bass in the phylogenetic analysis because multiple studies have shown that they are quite distantly related to Redeye Bass and our goal was not to construct a phylogeny for the entirety of Genus Micropterus, focusing instead on fine-scale relationships within the Redeye Bass complex and closely related species (Near et al. 2004; Baker et al. 2013; Freeman et al. 2015). We aligned these sequences with the unique haplotypes from the Verde River individuals with Geneious Prime (2019.2.1) using the Geneious global alignment with free end gaps and a cost matrix of 65% similarity. We then trimmed the alignment to a 928-basepair region of the ND2 gene covered by the Baker et al. (2013) sequences and the complete ND2 Verde River sequences. We excluded three sequences from Baker et al. (2013) from the analysis because they did not fully cover this region.

We generated a phylogenetic hypothesis for the putative Redeye Bass haplotypes from the Verde River from the aligned ND2 sequences using Bayesian inference implemented in Mr. Bayes 3.2.6 (available at http://nbisweden.github.io/MrBayes/; Ronquist and Huelsenbeck 2003). The parameters for Bayesian phylogeny reconstruction included the HKY 85 substitution model with a gamma rate variation, 1,100,000 chain length with 4 heated chains and a chain tempo of 0.2. The chain temperature was 0.2, subsampling frequency was 200, and burn-in length was 100,000. Branch lengths were unconstrained with the setting GammaDir (1,0.1,1,1). We ran the Bayesian analysis twice to ensure stability of the resulting phylogeny topology and of the posterior probabilities supporting clades.

We used the DNA extracted from the same five individuals from the mitochondrial ND2 sequencing for the SNP analyses. We plated samples in triplicate and performed amplification and extension reactions (iPLEX™ Gold Assay, Agena Bioscience® Inc, San Diego, CA) using 2 μL of input DNA at concentrations ranging from 168 to 351 ng/μL. We processed samples using the MassARRAY (Agena Bioscience® Inc) system according to manufacturer's protocols. We generated the SNP genotyping calls automatically using the MassARRAY Typer 4.0 Analysis software. The SNP panels consisted of 64 loci described in Thongda et al. (2019), that were developed to use as an informed prior of reference individuals for each of six common Micropterus spp. found in the native range of Redeye Bass and among those translocated to the western United States.

We analyzed a consensus genotype for each sample to determine the species identity of the putative Redeye Bass samples from the Verde River system in Arizona using a Bayesian clustering algorithm-based program, STRUCTURE version 2.3.4 (Pritchard et al. 2000). We performed STRUCTURE analysis using the admixture model in conjunction with the USEPOPINFO and POPFLAG models to group unknown individuals (POPFLAG = 0) by comparing them with the established reference individuals for those species (POPFLAG = 1; Porras-Hurtado et al. 2013). The parameters used were a burn-in of 20,000 iterations followed by 150,000 repetitions of Markov chain Monte Carlo simulation with a migration prior set to 0.01 with correlated allele frequencies. We performed STRUCTURE runs in triplicate using the Program R package strataG (Archer et al. 2017) with the Q-values averaged across replicates. For the first comparison, we used six common black bass species known to have been translocated outside of their native range as reference individuals: Alabama Bass Micropterus henshalli (n = 70), Largemouth Bass (n = 70), Redeye Bass (n = 60), Shoal Bass (n = 70), Smallmouth Bass (n = 70), and Spotted Bass Micropterus punctulatus (n = 70). To determine the taxonomic status of the Verde River samples within the Redeye Bass complex, we used reference individuals from seven black bass species : Alabama Bass (n = 70), Spotted Bass (n = 70), Cahaba Bass (n = 29), Redeye Bass (n = 60), Tallapoosa Bass (n = 59), Warrior Bass (n = 37), and Chattahoochee Bass (n = 42). Alabama Bass and Spotted Bass are not members of the Redeye Bass complex; however, we included them as outgroups to improve the resolution of these closely related species. STRUCTURE results yielded mean individual genomic proportion values (Q-value) for each individual in relation to each reference population. We considered a Q-value of ≥0.05 to be evidence of a potential genomic contribution from a reference species (Thongda et al. 2019).

The number of lateral-line scales for the 15 Verde River individuals averaged 68 ± 1.6 (SD), with a range of 65–70 (Table 1; Table S1, Supplemental Material). Counts of scales above lateral-line for the Verde River individuals averaged 8.2 ± 0.41 with a range of 8–9 (Table 1; Table S1). The bass from the Verde River averaged 12.93 ± 0.88 scales below lateral line with a range of 12–15 (Table 1; Table S1). For all three meristic traits, scale counts for the Verde River samples were outside of the ranges reported for Smallmouth Bass and within the ranges reported for Redeye Bass (Turner et al. 1991; Pipas and Bulow 1998; Baker et al. 2013). The ranges of scale counts for the three meristic traits in hybrid individuals at a site in Tennessee where Redeye Bass were introduced encompassed the range of variation reported for both Redeye Bass and Smallmouth Bass (Turner et al. 1991). All of the specimens used for the meristic and genetic analyses exhibited Redeye Bass coloration, specifically white upper and lower margins of the caudal fin that are not found in Smallmouth Bass. We did not photograph any of the specimens used for the meristic or genetic analyses but include a representative photograph of a Redeye Bass caught by angling at the sample site in October 2019 (Figure S2, Supplemental Material).

Phylogenetic analysis of the partial ND2 gene supported the inclusion of the three unique haplotypes found from the Verde River in a well-supported clade (posterior probability 0.95) including Redeye Bass from the Coosa River system of Alabama, Georgia, and Tennessee, and Cahaba Bass from the Cahaba River system of Alabama (Figure 2, and Figure S1 for GenBank Accession numbers). Two of the sequences (AZMICO1 and AZMICO2) belong to their own clade supported at 1.00 posterior probability embedded within the larger Redeye Bass and Cahaba Bass clade. The AZMICO3 haplotype was identical to GenBank Accession JX502867 from a Redeye Bass collected from Weoka Creek, Coosa River system, Alabama (Baker et al. 2013; NCBI 2021).

The results of the SNP analysis of the five individuals collected from the Verde River, when compared with other species of black bass but excluding members of the Redeye Bass complex, showed that all individuals were assigned to Redeye Bass with Q-values of > 0.90 and no other species exceeding a Q-value of 0.05 (Figure 3; Table 2; Table S2, Supplemental Material). The results of the comparison of the Verde River samples' SNP genotypes with members of the Redeye Bass complex included significant taxonomic assignments of Redeye Bass (Q-values of > 0.05) for all samples (Figure 4; Table 3; Table S3, Supplemental Material). In this comparison, two individuals also showed potential genomic contributions of Spotted Bass (MISP-1 and MISP-5, Q-values of 0.081 and 0.096, respectively). One individual (MISP-3) showed potential genomic contributions from Cahaba Bass (Q-value of 0.087).

The meristic results support the hypothesis that the black bass we collected from the Verde River are Redeye Bass and not Smallmouth Bass. All pairwise comparisons for scales along lateral line, scales above lateral line, and scales below lateral line were outside of the ranges reported for Smallmouth Bass and within the ranges reported for Redeye Bass. However, ranges reported from hybrid individuals at a site in Tennessee where Redeye Bass were introduced overlap with both the counts reported for Smallmouth Bass and Redeye Bass. These meristic traits would not be able to discriminate between Redeye Bass and Redeye Bass × Smallmouth Bass hybrids. Meristic analysis may be useful in the field for discrimination of pure Smallmouth Bass from pure Redeye Bass in Arizona, especially for the scales above lateral line that were all 8–9 scales for the individuals sampled in this study. This would be complicated if Smallmouth Bass occur in other reaches of the Verde River and if hybrids were present.

The mitochondrial ND2 sequence analysis supports the hypothesis that the five fish collected for genetic analysis belong to the Redeye Bass complex. The Bayesian phylogenetic analysis of ND2 sequences places the Verde River individuals within a well-supported clade that includes Redeye Bass and Cahaba Bass. Within the Verde River system, analysis of the ND2 mitochondrial gene may be diagnostic for Redeye Bass individuals given the results of the SNP analysis discussed below. However, if hybridization has occurred between Redeye Bass and other species of black bass elsewhere in the Verde River system, Redeye Bass mitochondrial haplotypes may be found in hybrid individuals or in those that have backcrossed and have a mixture of nuclear genes from Redeye Bass and other black bass.

The analysis of SNP nuclear DNA markers from the Verde River support a hypothesis that these fish are Redeye Bass with no evidence of hybridization or introgression of Smallmouth Bass genes. When compared with other black bass species (excluding members of the Redeye Bass complex), all individuals from the Verde River were assigned to Redeye Bass with Q-values exceeding 0.90, with no other species exceeding 0.05. This result supports a hypothesis that the Redeye Bass collected from the Verde River have not experienced significant hybridization and introgression of nuclear genes from black bass outside of the Redeye Bass complex. When compared with other members of the Redeye Bass complex and outgroups including Alabama Bass and Spotted Bass, two of the five individuals were confidently assigned to Redeye Bass exclusively with Q-values exceeding 0.90. One individual (MISP_3) had a Q-value of 0.087 corresponding to Cahaba Bass, suggesting the possibility of shared alleles among the closely related Redeye Bass and Cahaba Bass that are not captured in the reference data set. An alternative explanation could be that individuals from the Cahaba River and Coosa River systems had the opportunity for gene flow through the process of their capture, rearing, transport, and introduction to the Verde River system. For two of the individuals (MISP_1 and MISP_5), Spotted Bass exceeded a Q-value of 0.05 (Q-values of 0.081 and 0.096, respectively) in the Redeye Bass complex SNP STRUCTURE analysis, but not in the broader black bass STRUCTURE results above (Q-value of 0.045 for individual MISP_5). Spotted Bass were reportedly introduced in Arizona in 1942, but have not been recognized as present in the state since 1967 (Miller and Lowe 1967). It is possible that this is evidence of past hybridization and introgression of Spotted Bass genes that occurred in the Verde River prior to their extirpation in Arizona. It is also possible that the founding population of Redeye Bass introduced in the Verde River arrived already with a low level of introgressed Spotted Bass genes because they co-occur in their native range. A third possibility is that this signal represents noise in the reference data set, which has known limitations of geographic sampling extent and taxonomic resolution (Thongda et al. 2019). The Q-values that are reported from the STRUCTURE analysis do not equate directly to the probability of correct assignment to a species with a small genomic contribution, and a power analysis to determine that relationship has not been conducted for this reference data set. Overall, these results strongly support a genomic composition for the Verde River samples of primarily Redeye Bass, with possible minor contributions of Spotted Bass and Cahaba Bass.

Previous analyses of behavior, including habitat preference and diet of supposed Smallmouth Bass from the Verde River and its tributaries, provide some hints that further support a long-standing misidentification of Redeye Bass as Smallmouth Bass in Arizona's Verde River. An analysis of habitat preference of fish identified as Smallmouth Bass from Wet Beaver Creek, a tributary of the Verde River, found an unexpected pattern of preference for shallower habitat that differed from those reported elsewhere for true Smallmouth Bass (Barret and Maughan 1994). Dietary preferences of purported Smallmouth Bass from the same sample site as this study (Upper Verde River Wildlife Area) showed a lower tendency toward piscivory (< 5% of diet by weight) and instead favored invertebrate prey, consistent with the known dietary preferences of Redeye Bass in their native range when compared with the largely piscivorous Smallmouth Bass (Parsons 1954; Bonar et al. 2004). Nonetheless, gut contents of Smallmouth Bass from the Verde River, presumed here to be instead Redeye Bass, included several species of native fishes (Bonar et al. 2004). The implications of this misidentification, possibly as far back as their introduction, include that a large body of literature on the interactions of native and nonnative fish in the Verde River needs be revisited with a strong possibility that Redeye Bass were misidentified. Redeye Bass were first described in 1940, only 2 y before the reported introduction of Spotted Bass by AZGFD in 1942 (Hubbs and Bailey 1940; Miller and Lowe 1967). It is possible that the correct taxonomic name of the introduced Redeye Bass in the Verde River was caused by a delay in the dissemination of this major taxonomic revision and has persisted until recently.

Further work is needed to determine the extent of the Redeye Bass presence in Arizona, whether Smallmouth Bass are also present in the Verde River system, and if hybridization of Redeye Bass and other black basses is occurring. The AZGFD conducts surveys of fish by electrofishing on the Verde River on a triennial basis. In 2019, they surveyed the stretch of Verde River from its confluence with Granite Creek downstream to Sycamore Creek with knowledge that Redeye Bass were suspected to occur (AZGFD 2019a, 2019b, 2019c). They found that Redeye Bass was the most abundant nonnative fish in this stretch of the Verde River and at many sites was the most abundant fish period. They encountered no individuals that they identified as Smallmouth Bass and 1,275 individuals that they identified as Redeye Bass. Downstream from Sycamore Creek there are suspected occurrences submitted to iNaturalist on the main stem of the Verde River near Cottonwood, Arizona and on Wet Beaver Creek (Figure 1). All of these iNaturalist suspected occurrences include a color photograph that show fish with Redeye Bass characteristics including the white upper and lower margins of the caudal fin. The results of the eDNA metabarcoding study also detected Redeye Bass mitochondrial haplotypes at a site near Camp Verde, Arizona, downstream of the convergence of the Verde River and Wet Beaver Creek. Overall, the extent of the Verde River with evidence of Redeye Bass presence is approximately 120 river kilometers. The AZGFD is conducting electrofishing surveys downstream of Camp Verde in 2021 and will release results in Winter 2021–2022. It is possible that Redeye Bass have also spread to other major tributaries of the Verde River, such as Oak Creek, West Clear Creek, Fossil Creek, and the East Verde River. It is also possible that Redeye Bass have spread to the Salt River system; however, reservoirs have existed on the lower Verde River since at least 1939 and it is unclear what their impact may be on the ability of Redeye Bass to migrate downstream to the Salt River. Unassisted upstream migration of Redeye Bass in the Salt River is prevented by a series of Salt River Project dams, that were constructed from 1911–1936.

The presence of Redeye Bass in the Verde River and its tributaries has implications for the conservation and management of native fishes. Monitoring of native and non-native species is routinely conducted by electrofishing surveys and by molecular techniques that detect species through analysis of eDNA. Recently, a quantitative PCR (qPCR) method has been developed to detect Smallmouth Bass from eDNA; however, this technique was designed explicitly to detect Smallmouth Bass and to not detect Redeye Bass (Franklin et al. 2018). In this part of Arizona, eDNA methods using qPCR for detecting an important nonnative fish in locations where they should not be would likely fail if they are targeting Smallmouth Bass and not Redeye Bass. Additionally, the behavior of Redeye Bass, a primarily upland and small stream fish in its native range, is likely very different from other black bass present in Arizona waterways (Parsons 1954). A migration study of Redeye Bass in Alabama indicated that they were routinely able to scale a 6-ft (1.83-m) vertical rock dam on a small stream presumably during flash flood events, which may have implications for the success of fish barriers constructed to exclude them from migrating upstream (Parsons 1954). In summary, these results indicate that a previously unrecognized introduction and subsequent proliferation of Redeye Bass has occurred in the Verde River system of Arizona. Further work is needed to determine the extent of their spread in Arizona and if hybridization is ongoing with other species of black bass in different reaches of the Verde River.

Please note: The Journal of Fish and Wildlife Management is not responsible for the content or functionality of any supplemental material. Queries should be directed to the corresponding author for the article.

Table S1. We sampled 15 individuals of putative Redeye Bass Micropterus coosae in 2018 for meristic analysis of scales along lateral line, scales above lateral line, and scales below lateral line. This table includes the raw count data for each individual for the three meristic measurements.

Available: https://doi.org/10.3996/JFWM-21-013.S1 (13 KB XLSX)

Table S2. We genotyped 5 individuals of putative Redeye Bass Micropterus coosae sampled from the Verde River in 2018 for 64 Single Nucleotide Polymorphism (SNP) markers and compared them with black bass species commonly introduced outside of their native range. This table includes the data input file for STRUCTURE analysis of 64 SNP loci for the samples from the Verde River and all reference individuals.

Available: https://doi.org/10.3996/JFWM-21-013.S2 (109 KB XLSX)

Table S3. We genotyped 5 individuals of putative Redeye Bass Micropterus coosae sampled from the Verde River in 2018 for 64 Single Nucleotide Polymorphism (SNP) markers and compared them with other members of the Redeye Bass complex and closely related species. This table includes the data input file for STRUCTURE analysis of 64 SNP loci for the samples from the Verde River and all reference individuals.

Available: https://doi.org/10.3996/JFWM-21-013.S3 (82 KB XLSX)

Figure S1. We sequenced the nicotinamide adenine dinucleotide + hydrogen (NADH) dehydrogenase subunit 2 (ND2) gene for five individuals of putative Redeye Bass Micropterus coosae sampled from the Verde River in 2018 and compared them with published sequences from other members of the Redeye Bass complex. We used a Bayesian phylogenetic analysis to produce a phylogenetic tree of the three unique haplotypes found in this study (indicated by a * and in bold) showing their relationship with other species of the Redeye Bass complex. Largemouth Bass Micropterus salmoides is included as an outgroup. Numbers at nodes indicate the posterior probability supporting those clades. This phylogeny has an identical topology and branch lengths to Figure 1 that shows the species names of each sample, whereas here the nodes are labeled with the GenBank accession numbers for each ND2 sequence analyzed. Branch lengths correspond to the number of DNA changes separating sequences and a scale bar indicating the length of a branch that equals 5 changes appears at the bottom of the figure.

Available: https://doi.org/10.3996/JFWM-21-013.S4 (124 KB PDF)

Figure S2. A representative photograph of a Redeye Bass caught by angling from the Upper Verde River Wildlife Area, Yavapai County, Arizona, in October 2019 showing the white upper and lower margins of the caudal fin.

Available: https://doi.org/10.3996/JFWM-21-013.S5 (5.18 MB PDF)

Reference S1.[AZGFD] Arizona Game and Fish Department Region III Fisheries Program. 2019a. Verde River Fish Survey Report: Granite Greek to Verde Ranch, June 2019. Prepared by: Gregg Cummins.

Available: https://doi.org/10.3996/JFWM-21-013.S6 (699 KB PDF)

Reference S2.[AZGFD] Arizona Game and Fish Department Region III Fisheries Program. 2019b. Verde River Fish Survey Report: Verde Ranch to Perkinsville, July 2019. Prepared by: Gregg Cummins.

Available: https://doi.org/10.3996/JFWM-21-013.S7 (828 KB PDF)

Reference S3.[AZGFD] Arizona Game and Fish Department Region III Fisheries Program. 2019c. Verde River Fish Survey Report: Perkinsville to Sycamore Creek, July–August 2019. Prepared by: Gregg Cummins.

Available: https://doi.org/10.3996/JFWM-21-013.S8 (813 KB PDF)

We thank Embry-Riddle Aeronautical University College of Arts and Sciences and Undergraduate Research Institute for funding for the ND2 sequencing and the preliminary 16S metabarcoding eDNA results that led to this study. We also thank the Arizona Game and Fish Department for providing samples for the morphological and genetic analyses. We thank Gregg Cummins and William M. Chmiel for collecting samples for the morphological analysis. We also thank iNaturalist contributers Rob Hannawacker and luj333 for sharing their observations. We are appreciative for comments and suggestions that greatly improved this manuscript from Matthew O'Neill, four anonymous reviewers, and the editors.

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