Revision of the Gila Robusta (Teleostei, Cyprinidae) Species Complex: Morphological Examination and Molecular Phylogenetics Reveal a Single Species Joshua M

Home , Gila (fish), Richardsonius

Revision of the Gila robusta (Teleostei, Cyprinidae) species complex: morphological examination and molecular phylogenetics reveal a single species Joshua M. Copus1, Zac Foresman1, W.L. Montgomery2, Brian W. Bowen1, and Robert J. Toonen1

1Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744, USA 2Department of Biology, Northern Arizona University, Flagstaff, AZ 86011, USA

* Corresponding author: [email protected]

Report to the Joint ASIH-AFS Committee on Names of Fishes

Introduction Historical systematics and taxonomy There has been considerable confusion about the systematics and taxonomy of the Gila robusta complex within the Lower Colorado River Basin, largely due to a complex array of phenotypes (Demarias et al. 1992). Recent treatments of this complex recognize three species: Gila robusta Baird and Girard 1853a, G. intermedia (Girard 1856) and G. nigra Cope 1875. Here we begin with an outline of the taxonomic history of this group in an effort to clarify and provide a foundation for subsequent morphological and molecular examinations. This is not meant to be a comprehensive review of the entire genus, but instead focuses on taxonomic treatments of the G. robusta group. Many species within the genus Gila Baird and Girard 1853a have been described multiple times under different names and the G. robusta complex is no exception. We identify fifteen specific names and seven generic names applied to these species. Subsequent to our review of taxonomic history, we provide evidence that these three species are not monophyletic, and that morphological and genetic divergences among these taxonomic entities are insufficient to warrant recognition at the species level.

Gila robusta Baird and Girard 1853a Gila robusta Baird and Girard 1853a. Zuni River, New Mexico. Syntypes: USNM 246 (USNM 47983; 3, plus 1 pharyngeal arch as #2798) Gila gracilis Baird and Girard 1853a (Evermann and Rutter 1895;Jordan and Evermann 1896; Gilbert and Scofield 1898; Gilbert 1998) Gila grahamii Baird and Girard 1853b (Cope 1871; Evermann and Rutter 1895; Jordan and Evermann 1896; Fowler 1924; La Rivers 1994 [as subspecies: Rinne 1976; Lee 1980; Gilbert 1998]) Gila gibbosa Baird and Girard 1854 (Jordan and Gilbert 1883; Evermann and Rutter 1895; Jordan and Evermann 1896; Gilbert and Scofield 1898; Gilbert 1998, Snyder 1915; Fowler 1924) Tigoma gibbosa (Baird and Girard 1854) (Girard 1856; Jordan and Gilbert 1883; Evermann and Rutter 1895; Jordan and Evermann 1896; Gilbert and Scofield 1898; Gilbert and Scofield 1898 Richardsonius gibbosus (Baird and Girard 1854) (Snyder 1915) Tigoma intermedia Girard 1856 (Evermann and Rutter 1895; Jordan and Evermann 1896; Gilbert and Scofield 1898; Fowler 1924; Gilbert 1998; Snyder 1915 [as subspecies: Miller 1945, 1946; La Rivers 1994; Uyeno and Miller 1965; Barber and Minckley 1966] treated as a full species within Gila by Rinne 1969 and most subsequent authors. Squalius intermedius (Girard 1856) (Jordan and Gilbert 1883; Jordan and Evermann 1896; Gilbert and Scofield 1898) Leuciscus intermedius (Girard 1856) (Evermann and Rutter 1895; Gilbert and Scofield 1898; Jordan and Evermann 1896; Fowler 1924) Ptychocheilus vorax Girard 1856 (Evermann and Rutter 1895;Jordan and Evermann 1896; La Rivers 1994) Gila affinis Abbott 1860 (Jordan and Evermann 1896; Fowler 1924) Leuciscus zunnensis Günther 1868 (Jordan and Evermann 1896; Gilbert and Scofield 1898; La Rivers 1994; Gilbert 1998) Leuciscus robustus (Baird and Girard 1853a (Günther 1868) (Jordan and Evermann 1896; La Rivers 1994) Leuciscus grahami Günther 1868 (Jordan and Evermann 1896) Gila nacrea Cope 1871 (Evermann and Rutter 1895; Jordan and Evermann 1896; Gilbert 1998) Gila nigra Cope 1875 in Cope and Yarrow 1875 (Jordan and Gilbert 1883; Gilbert and Scofield 1898; Snyder 1915; Fowler 1924; Gilbert 1998) Leuciseus niger (Cope 1875) (Evermann and Rutter 1895; Jordan and Evernmann 1896; Gilbert and Scofield 1898) Squalius niger (Cope 1875) (Jordan and Gilbert 1883; Jordan and Evermann 1896) Squalius nigra (Cope 1875) (Gilbert and Scofield 1898, misspelling of S. niger) Squalius lemmoni Smith 1884 (Jordan and Evermann 1896; Gilbert and Scofield 1898; Gilbert 1998)

Gila robusta Baird and Girard 1853a is the type species for the genus Gila Baird and Girard 1853a. The type series of the species are cataloged as USNM 246, but a note included with the type specimens states, “These, the types of Gila robusta B.+G., are cat. No. 246. They were reentered by error as 47983 and attributed to nos. 276+273, which are cods! Nos. 276+277 were attributed as type nos. of this species, by error, by Jordan and Evermann 1896:227. R.R. Miller III: 1945” The original description reported the collection locality for the syntypes as the Zuni River, New Mexico, but Smith et al. (1979) suggest that this locality is an error, based on the argument that the Zuni River was unsuitable habitat for G. robusta during the time at which the type specimens were collected. Smith et al. (1979) assumed there was a clerical error in recording the collection site of the syntypes, and suggested that the specimens were actually from the previous collection site of the Sitgreaves expedition, the Little Colorado River, below Grand Falls, Coconino County, Arizona. Sublette et al. (1990) dispute the assertions of Smith et al. (1979), and contend that the syntypes were collected on the Zuni River in 1851, and additional specimens subsequently collected twice in 1873 by different collectors, and once in 1879. It is highly unlikely that multiple clerical errors on different expeditions would have occurred at this locality with this species. Sublette et al. (1990) suggest that the Zuni River represented marginal habitat that may have received recruits from the Rio Pescado and that they have since been extirpated from the Zuni River. The argument that the syntypes were collected on the Little Colorado River rather than the Zuni is dubious and we agree with Sublette et al. (1990) that the originally reported collection location for the syntypes of this species is more plausible. Until unambiguous evidence is presented to the contrary, we maintain the type locality of the Zuni River for this species. Girard (1856) described Tigoma intermedia from specimens collected on the Rio San Pedro AZ, originally placed this species in the genus Tigoma Girard 1856, and noted that it was morphologically intermediate between T. pulchella (Baird and Girard 1854) and T. purpurea Girard 1856 (both of which are now regarded as valid species within the genus Gila). Evermann and Rutter (1895) placed T. intermedia within the genus Leuciscus Cuvier 1816. Jordan and Evermann (1896) synonymized Tigoma and Richardsonius Girard 1856 with Leuciscus, and suggested that L. intermedius and L. niger (Cope 1875) (now Gila nigra Cope 1875) may be conspecific. Fowler (1924) retains this genus placement for L. intermedius. Snyder (1914), regarded T. intermedia and G. nigra as synonyms of Gila gibbosa Baird and Girard 1854 and placed them within the genus Richardsonius. However, gibbosus Baird and Girard 1854 was unavailable due to the homonymy of gibbosus created by Jordan and Gilbert 1883 in Squalius Bonaparte 1837 and then by Jordan and Evermann (1896) within Leuciscus Cuvier 1816 (see below for further explanation). Miller (1945) treated intermedia as a subspecies of G. robusta (Baird and Girard 1853a), which was followed by subsequent authors (Miller 1946; Uyeno 1961; Miller 1961; LaRivers 1962; Sigler and Miller 1963; Miller and Lowe 1964; Uyeno and Miller 1965; Barber and Minckley 1966; Miller and Lowe 1967; Cole 1968; Minckley and Alger 1968; Minckley 1969; Lee 1980; Robins et al. 1980). Rinne (1969) recognized three distinct species of Chub within the Lower Colorado River Basin, including G. elegans Baird and Girard 1853a, G. robusta, and a third species for populations principally distributed in central and southern Arizona, to which he applied the name G. intermedia. Within the synonymy of G. intermedia, he also included G. gibbossus, G. nigra, and G. lemmoni, noting that the former was unavailable due to homonymy, thereby asserting G. intermedia as the next available name. Rinne (1969) does not appear to have examined any of the type series of G. intermedia, but states “in all respects, they [populations he labels as G. intermedia] correspond to the original description of Tigoma (=Gila) intermedia Girard 1856”. The original description of G. intermedia consisted of the following text: “Intermediate between T. pulchella and T. purpurea, more closely related however to the former than to the latter. The fins are much less developed, the inferior fins especially are quite small.” This description is not sufficient to confidently assign the type specimens of G. intermedia to Rinne’s third species and there is no evidence that Rinne examined either pulchella or purpurea. Assignment of G. intermedia and of Rinne’s third species in this context is further complicated by the uncertainty of the type locality for G. robusta. Nevertheless, most subsequent authors followed Rinne (1969) in treating G. intermedia as a valid species (e.g. Rinne and Minckley 1970; Stout et al 1970; Minckley 1971, 1973; Rinne 1976; Hocutt and Wiley 1986; Minckley et al 1986; Sublette et al. 1990; Robins et al. 1991; Winfield and Nelson 1991; Gilbert 1998; Espinosa Pérez et al. 1993; Gilbert 1998; Minckley & DeMarais 2000; Norris et al. 2003; Nelson et al. 2004; Scharpf 2005; Miller 2006; Minckley & Marsh 2009; Page & Burr 2011; Page & Burr 2011; Page et al. 2013). Baird and Girard 1853b described Gila grahamii (often misspelled in the literature as grahami) from specimens collected in the Rio San Pedro, Gila River basin. Günther (1868) placed it within the genus Leuciscus Cuvier 1816. Cope and Yarrow 1875 placed the species back in the genus Gila and recognized it as distinct from both G. robusta Baird and Girard 1853a and G. nigra Cope 1875. Evermann and Rutter (1895) treated G. grahamii as a synonym of G. robusta, and this was followed by subsequent early authors. This synonymy remained stable until Rinne (1969), who regarded grahamii as a subspecies of G. robusta, recognizing populations collected from the tributaries of the Verde River and the upper Gila River system as distinct from G. r. robusta in the main stem Verde and Gila Rivers. Interestingly, Rinne (1969) recognized current San Pedro populations (type locality of G. grahamii) to belong to the species he referred to as G. intermedia, even though he accepted G. r. grahamii for this "headwater" form (again, apparently without examining any of the type specimens). In the years between 1969 and 2000, there was not consistent recognition of G. r. grahamii as a valid subspecies, but no authors treated it as a valid species, or as a synonym or subspecies of any species other than G. robusta (e.g., Winfried and Nelson; Robins 1980; Holden and Minckley 1980; Mayden 1992; Lee et al. 1980; La Rivers 1994; Rinne 1976; Lee 1980; Gilbert 1998). Minckley and Demarias (2000) regarded the populations referred to by Rinne (1969) as G. r. grahamii as representing a distinct species, even though they note that it is likely of hybrid origin and paraphyletic. However, they also noted that the syntypes of G. grahamii belong to what Rinne (1969) regarded as the subspecies G. r. robusta, (citing personal communication from RR Miller and WL Minckley). Therefore they recognized G. nigra Cope 1875, as the earliest available name for the species previously referred to by Rinne (1969) as G. r. grahamii. Cope and Yarrow (1875) described G. nigra Cope 1875 from specimens collected in Ash Creek and at San Carlos, Arizona. Jordan and Gilbert 1883 placed it in the genus Squalius Bonaparte 1837, and later Jordan and Evermann (1896) placed it in the genus Leuciscus. They also regarded Gila gibbosa Baird and Girard 1854 as conspecific, but unavailable due to homonymy with L. gibbosus Storer 1845 (as well as L. gibbossus Ayers 1845). Gilbert and Scofield (1898) synonymized G. nigra with T. intermedia, which they placed in the genus Leusciscus. Snyder (1915) regarded G. niger as a synonym of G. gibbosa (within the genus Richardsonius), failing to recognize that the latter species name was not available due to homonymy. Fowler (1924) followed Gilbert and Scofield (1898) in treating G. nigra as a synonym of intermedius, within the genus Leusciscus. Subsequent treatments place nigra in synonymy with intermedia (see above). This synonymy was broadly followed until Minckley and Demarias (2000) recognized G. nigra as the earliest available name to refer to the species treated by Rinne (1969) as G. r. grahamii. Along with the considerable synonymy of Gila robusta are species that were at one time considered synonyms of G. robusta and are now considered valid. Gila elegens Baird and Girard 1853a was treated as a synonym of G. robusta by Ellis (1914) and as a subspecies by Miller (1945) and La Rivers (1994). G. elegans was treated as valid by Vanicek (1967) and subsequent authors. Gila jordani Tanner 1950 was treated as a subspecies of G. robusta by Rinne (1976), Lee (1980), La Rivers (1994), Gilbert (1998). G. jordani was treated as valid by Minckley and Marsh (2009) and subsequent authors. Clinostomus pandora Cope 1872 was treated as a synonym of G. robusta by Ellis (1914) but valid as Gila pandora (Cope 1872) by subsequent authors. Tigoma egregia Girard 1858 was treated as a synonym of G. robusta by Ellis (1914) but regarded as valid as Richardsonius Girard 1856 egregius (Girard 1858) by subsequent authors. Finally, Gila seminuda Cope and Yarrow 1875 was treated as a subspecies of G. robusta by Ellis (1914), Snyder (1915), Rinne (1976), and Lee (1980) but valid as Gila seminuda by Gilbert (1998) and subsequent authors.

Materials and Methods Materials examined: Due to the inaccuracy of the taxonomic key (Minckley and Demarias 2000) in assigning individuals correctly to Figure 1: Map of collecting locations of specimens of G. robusta (black), G. nigra (blue), and G. intermedia (red) species, the current practice of within the Lower Colorado River Basin. species identification for managers and researchers working with G. robusta, G. intermedia, and G. nigra within the Lower Colorado River Basin is based on stream drainage of collected specimens. The populations of fish within these localities were assigned species names by Rinne (1969, 1976; and later revised my Minckley and Demarias 2000) who examining large numbers of individuals, was able to see mean differences in populations based on morphology. Therefore ID's used in this study are based on collecting location, conforming to common practice and because there is no other known method of identification. Five specimens of each species of the G. robusta complex (G. robusta, G. intermedia, and G. nigra) and 1 each of G. elegans and G. cypha as out-groups were analyzed from streams throughout Arizona. One individual per location across the range of each species was analyzed in an attempt to capture as much within species variation as possible (Figure 1). Tissue samples were collected from each and stored in both Salt- Saturated DMSO (20% dimethyl sulfoxide, 0.25M EDTA, pH 8.0, saturated with NaCl; Seutin et al. 1991) and RNA Later (ThermoFisher Scientific, Waltham, MA) for RAD genomic sequencing using the ezRAD protocol (Toonen et al. 2013). Specimens were frozen prior to morphological analyses. Type material for each of the currently recognized species of the G. robusta species complex as well as the type for the synonym G. grahamii were obtained from the Smithsonian National Museum of Natural History (G. robusta (USNM 246), G. nigra (USNM 16972), G. intermedia (USNM 232), and G. grahamii (NMNH 253). Morphological Analysis: Fresh specimens were thawed and radiographed. Meristic and morphometric analysis largely follow Hubbs and Lager (1958). Standard length is the horizontal straight-line distance from the tip of the snout to the base of the caudal fin at the end of the hypural plate. Body depth is the maximum depth just posterior to the gill opening. Head length is the median anterior point to the greatest extent of fleshy opercle. Head width is the maximum width of the head. Head depth is the maximum depth of the head. Snout length is the fleshy edge of the orbit to the front of the upper lip in the median plane. Mandible length is the tip of the lower jaw to the posterior most end of the articular. Orbit diameter is the greatest fleshy diameter. Interorbital width is the least fleshy width. Upper-jaw length is the front of the upper lip to the posterior end of the maxilla. Caudal peduncle depth is the least depth. Caudal peduncle length is the horizontal distance between the verticals at the rear base of the anal fin and the caudal-fin base. Predorsal length is measured from the tip of the snout to a vertical at the base of the anterior most fin ray. Preanal length is the tip of the snout to the anus. Pectoral insertion to pelvic insertion is a straight-line distance from posterior-most point where the pectoral fin connects with the body to the posterior most point where the pelvic fin connects with the body. Anal to caudal length is a straight-line measurement from the anus to the base of the origin of the caudal fin. Origin of anal fin to hyperal plate is a straight-line distance from the anterior most point on the anal fin to the posterior most point of the hyperal plate. Prepelvic length is measured from the tip of the snout to the base of the origin of the pelvic fin. Caudal fin length is from the hypural plate to the tip of the upper lobe of the caudal fin. Caudal concavity is the horizontal distance between the verticals at the tip of the longest and shortest rays. Fin length is measured from the base of the fin to the tip of the longest ray. Lateral line scale counts include all normal, pored scales. Scales above the lateral line is the number of scales from the lateral line to the anterior base of the dorsal fin. Scales below the lateral line are the number of scales from the lateral line to the anterior base of the anal fin. Neither of the previous two counts include the lateral line scale. Gill rakers are counted on the first gill arch. Counts of the rakers of the upper limb are given first followed by the lower limb. Gill raker at the angle is included in the lower limb count. Vertebrae counts include the hypural. Linear regression was performed on each of the morphometric characters to test for allometric growth and confirm that each of these characters scale linearly with size. Variables were then standardized by length measures for comparison. Values are presented as a percentage of standard length, head length, or body depth. Genomic Data Production: Genomic DNA was extracted from tissue using the Omega E.Z.N.A Tissue DNA Kit (Omega Biotek, Norcross, GA) following the manufacturer's protocol. HPLC grade H2O was substituted for the elution buffer. DNA aliquots were viewed on a 1% agarose gel to assess quantity and quality. For extractions that did not yield ample high molecular weight DNA (> 1μg), multiple extractions were completed and samples were pooled and concentrated using an Eppendorph Vacfuge plus (Eppendorph, Hauppauge, NY). Extractions were quantified with AccuBlue (Biotium, Inc, Hayward, CA) high sensitivity dsDNA quantification kit and measured on a SpectraMaz M2 microplate reader (Molecular Devices, Sunnyvale, CA). All extractions were subsequently stored at -200C until used for library preparation as outlined below. Genomic libraries were generated following the ezRAD protocol (Toonen et al. 2013; Knapp et al. 2016) Tissue samples were cleaned with Agencourt AMPure XP (Beckman Coulter, Indianapolis, IN) beads following manufactures protocols. DNA digestions were completed using the enzyme DpnII to cleave at all GATC cut sites using 25μl of mastermix (5μl Buffer, 19μl HPLC grade H2O, 1 μl DpnII) to 1μg of template DNA in 25 μl. Samples were incubated for 3 hours at 37OC followed by 20 minutes at 65OC to denature the enzyme. Following digestion, samples underwent a second bead clean with AMPure XP beads. Library preparation for Ilumina sequencing was completed with use of the KAPA HyperPrep kit (Kapa Biosystems, Wilmington, MA) following manufacturers protocols. All libraries were size selected for 300- 500 base pair (bp) fragments and passed through quality control steps (bioanalyzer and qPCR). Illumina Paired-end fragments were sequenced at the Hawaii Institute of Marine Biology Genetics Core Facility using the Ilumina MiSeq genomic analyzer (Ilumina, San Diego, CA). Mitochondrial genome mapping: Raw Illumina reads were paired and trimmed on both the 5' and 3' ends in Geneious v.6.1.8 (Biomatters, Newark NJ). Paired reads were then mapped to the mitochondrial genome of G. robusta (Genbank DQ536424.1) using Geneious. Consensus sequences were extracted from reads that mapped successfully to the reference mitochondrial genome. With this method, we were able to successfully construct >95% of complete mitochondrial genomes for all specimens. Nuclear DNA SNP analysis: Raw Illumina reads were paired, validated, and merged using PEAR v.0.9.6 with default settings (Zhang et al. 2014). Merged and unmerged reads were concatenated into a single file for each library. Libraries were then trimmed to 100 bp and clustered using AftrRAD v.5.0 (Sovic et al. 2015). The quality filtering step was completed with the following protocols: minQial: 20; minDepth: 3; minIden: 0%; numIndels: 3; minParlog: 5; Phred: 33; dplexedData: 1; stringLength: 15; MaxH: 90%. Genotypes were then scored using the following protocol: MinReads: 3; pvalLow: 0.0001; pvalHigh: 0.00001; pvalThresh: 100. Finally, data was filtered using the following settings: pctScored: 100%; maxSNP: 0; MinReads: 3. This process resulted in 2633 polymorphic loci with 100% coverage across all samples. Phylogenetic analysis: RAxML v.8 (Stamatakis 2014) was used for rapid bootstrap best- scoring maximum likelihood phylogenetic analyses using the GTR model of evolution including optimization of substitution rates, the Table 1: Proportional measurements of the type material of each of the GAMMA model of currently recognized species within the G. robusta species complex, G. robusta, G. nigra, and G. intermedia, as well as G. grahamii. Values rate heterogeneity, presented as a percentages of standard length1, head length2, or body depth4. random seed of 12345, and 500 bootstrap replicates. Phylogenetic trees were constructed and visualized using FigTree Table 2: Meristic data of the type material of each of the currently v.1.4.2 recognized species within the G. robusta species complex, G. robusta, G. nigra and G. intermedia, as well as G. grahamii. (http://tree.bio.ed.ac.uk/softwar e/figtree/). Uncorrected pairwise divergence times were estimated using Mega v.7.0 (Kumar et al 2016).

Results and Discussion Morphological analysis Type material: Examination of the type series' of G. robusta (n=2), G. intermedia (n=4), G. nigra (n=3), and G. grahamii (n=1) reveal differences between each of the type series within this complex. Interestingly, similar levels of Table 3: Range of morphological variation within individuals of G. robusta, G. nigra, and G. intermedia as currently recognized. Values difference between G. grahamii presented as a percentages of standard length1, head length2, or body depth4. (now considered a synonym of G. robusta; see above) and each of the other species was also evident. (Table 1, Table 2). However, the taxonomic key (Minckley and Demarias 2000) developed from the data used to assign the names G. intermedia and

G. nigra to the populations Table 4: Range of morphological variation within fresh material of G. robusta, G. nigra, and G. intermedia as currently recognized. that Rinne (1969, 1976) and Minckley and Demarias (2000) perceive as distinct species fails to correctly assign all four type specimens of G. intermedia in the characters of lateral line scales and head length to caudal peduncle depth and one type specimen of G. nigra in the character of caudal length to depth. In this regard, the International Code of Zoological Nomenclature is quite clear: "The application of each species-group name is determined by reference to the name-bearing type [Arts. 61, 71-75] of the nominal taxon denoted by the combination in which the species-group name was established (ICZN 1999, Article 45.3) In this case the name bearing types do not align with the forms to which the names have been applied. In consequence, these names must not be applied to the populations with which they are currently in use. Fresh material: Analysis of fresh material across the range of each species reveals much greater morphological variation within any given species than among the nominal species groups (Table 3, Table

4). There is no single diagnostic

Figure 2: Principle Coordinates Analysis of 28 morphological character that can be used for measurements of the type series' (circles) and collected specimens (diamonds) of the G. robusta complex, G. robusta (black), G. nigra species identification, and with (blue) G. intermedia (red), and G. grahamii (yellow). considerable overlap among species in every morphological character there is also no suite of characters that can distinguish species unambiguously. Comparisons of fresh specimens to type material: A principal components analysis of the 28 morphological variables taken on the type material as well as the fresh sample failed to resolve the species as currently recognized (Figure 2). In fact, type specimens as well

Table 5: Proportion of 23 morphological characters measured that were able to be assigned to each of the type series of the G. robusta species complex. Combined values are greater than 1 due to overlap in characters between type series'. Values of morphological characters outside the range of any type series were marked as unclassified. as fresh material of the same species exhibit as much or more separation within species as they do between them. Similarly, it was impossible to assign any of the fresh specimens back to the type material. For example, the currently recognized G. robusta only fit the type material with 30% of morphological characters while it was assigned to G. nigra and G. intermedia 51% and 54% respectively (Table 5). There was also no single morphological character that correctly assigned to the type series in all specimens examined, but rather each character was assigned to multiple type series. Similar patterns are observed with both G. nigra and G. intermedia (Table 5). These data further supports the conclusion of a single species with high morphologically variation.

Molecular analysis Phylogenetic analysis: Phylogenetic analysis of the fresh material from 17 locations across the range of the G. robusta species complex for the mitochondrial genome and SNP datasets revealed high concordance between tree topologies. Only a minor discrepancy exists between the mtDNA and SNP trees where the SNP tree assigns Silver Creek (G. intermedia) to Clade 4. Therefore, we present here Figure 3: Phylogenetic tree of mtDNA genome of populations across the mtDNA tree for reference the Lower Colorado River Basin for currently recognized species of G. robusta (black), G. nigra (blue) and G. intermedia (red) and rooted (Figure 3). High bootstrap with G. elegans and G cypha as out groups. Branch values are bootstrap support for each node. values support each of the 5 clades presented here. These molecular analyses reveal that none of the currently accepted taxonomic units are monophyletic, concordant with the morphological analyses. With the exception of Clade 5, which is almost certainly the result of the close geographic proximity of the locations (Figure 1), no other clade consists of a single species as currently recognized. No other geographic pattern is evident within mtDNA and nuclear genomic trees. While clade 5 consists exclusively of G. nigra, this species also occurs in clade 4 with G. robusta. Previous studies based on allozyme, mtDNA, and microsatellite genetic markers likewise did not find diagnostic characters among these three species (DeMarais 1992; DeMarais et al. 1992; Dowling and DeMarais 1993; Dowling et al 2015). Estimating rates of molecular evolution is challenging, however for a first order approximation if we assume that the mitochondrial genomes evolve in a clock like fashion at a rate of approximately 2% per million years (Brown et al 1979; Bowen et al 2001; Reece et al 2010), then uncorrected pairwise divergence between lineages place the oldest divergence times of these species at approximately 10,000 years. These dates may indicate isolation events linked to post glaciation warming and subsequent transitions from the wetter climate of the Last Glacial Maximum to the more arid and dryer climate of the current southwest U.S. (Williams et al. 1985; Meffe and Vrijenhoek 1988). Table 6: Morphological variation in populations of each clade of the molecular phylogeny. Values presented as a Morphological analysis of percentages of standard length1, head length2, or body depth4. phylogeny: Morphological analysis of the phylogenetic tree revealed nearly 100% overlap of characters between each clade (Table 6). These results reinforce the finding of no morphological basis for taxonomic distinctions within the G. robusta species complex and prompt the conclusion of a single morphologically plastic species inhabiting the Lower Colorado River Basin. Gila intermedia and G. nigra were resurrected based on mean differences between populations inhabiting different streams. Some authors suggest that these differences are based on differences in water depth and speed (Miller 1946, but see Rinne 1976). However, G. robusta is not the only species that exhibits a gradation in characters by stream size and current, this pattern is known in many groups of fish (Hubbs 1940). For instance, the bluehead sucker, Catostomus discobolus, like G. robusta, varies according to size and flow of the water it inhabits (Sigler and Sigler 1979). Using mean differences in populations to justify species distinction ignores the extent of natural variation among conspecific populations, but also, as is the case here can lead to paraphyly within species.

Summary • Type material does not align with fresh specimens as currently recognized. • The morphological characters proposed to resurrect G. nigra and G. intermedia within the G. robusta complex do not accurately distinguish between type series. • High levels of morphological variation exist within putative species, but there is insufficient variation between species to justify three taxonomic units. • Based on the fresh specimens examined, no single individual could be unambiguously assigned to a type series, but instead had morphological characters that matched all three type series. • Phylogenetic analyses of both mtDNA and nuclear genomes reveal extremely shallow coalescence of putative species, and no monophyly for currently recognized taxonomic units. • No morphological divergence was observed between molecular clades, eliminating the possibility that taxonomic units exist but are misaligned. • The G. robusta complex should be recognized as a single species.

Acknowledgements We would like to thank Richard Pyle for the help he provided in the taxonomy and nomenclature as well as his knowledge of the Code. We also thank Cassie Ka'apu-Lyons, Matthew O'neill, Clay Crowder, Julie Carter, and Ingrid Knapp the help they provided during this study.

Literature Cited Abbott, C. C. 1860. Description of four new species of North American Cyprinidae. Proceedings of the Academy of Natural Sciences of Philadelphia v. 12: 473-474. Baird, S. F. and C. F. Girard 1853a. Descriptions of some new fishes from the River Zuni. Proceedings of the Academy of Natural Sciences of Philadelphia v. 6: 368-369. Baird, S. F. and C. F. Girard 1853b. Descriptions of new species of fishes collected by Mr. John H. Clark, on the U. S. and Mexican Boundary Survey, under Lt. Col. Jas. D. Graham. Proceedings of the Academy of Natural Sciences of Philadelphia v. 6: 387-390. Baird, S. F. and C. F. Girard 1854. Descriptions of new species of fishes collected in Texas, New Mexico and Sonora, by Mr. John H. Clark, on the U. S. and Mexican Boundary Survey, and in Texas by Capt. Stewart Van Vliet, U. S. A. Proceedings of the Academy of Natural Sciences of Philadelphia v. 7: 24-29. Barber, W.E. and W.L. Minckley. 1966. Fishes of Aravaipa Creek, Graham and Pinal counties, Arizona. Southwestern Naturalist, 11:313-324. Baxter, G. T. and M. D. Stone 1995. Fishes of Wyoming. Wyoming Game and Fish Department. 1-290. Bonaparte, C. L. 1837. Iconografia della fauna italica per le quattro classi degli animali vertebrati. Tomo III. Pesci. Roma. Fasc. 19-21, puntata 94-103, 105-109, 5 pls. Bowen, B.W., A.L. Bass, L.A. Rocha, W.S. Grant, and D.R. Robertson. 2001. Phylogeography of the trumpetfish (Aulostomus spp.): ring species complex on a global scale. Evolution, 55:1029-1039. Brown, W.M., George, M. and Wilson, A.C., 1979. Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences, 76(4), pp.1967-1971. Cole, G.A. 1968. Desert Limnology. pp. 423-485 in G.W. Brown, Jr. (ed.), Desert Biology. Vol. 1, Academic Press Inc., New York. Cope, E. D. 1871. Recent reptiles and fishes. Report on the reptiles and fishes obtained by the naturalists of the expedition. U.S. Geological Survey of Wyoming & Contiguous Territories. Part 4 (art. 8): 432-442 Cope, E. D. 1872. Report on the recent reptiles and fishes of the survey, collected by Campbell Carrington and C. M. Dawes. U. S. Geological Survey Territories Annual Report v. 5 (pt. 4): 467-476. Cope, E. D. and H. C. Yarrow 1875. Report upon the collections of fishes made in portions of Nevada, Utah, California, Colorado, New Mexico, and Arizona, during the years 1871, 1872, 1873, and 1874. Engineer Department, United States Army In: Report upon the Geographical and Geological Explorations and Surveys west of the one hundredth Meridian v. 5 (Zoology) Chapter 6: 635-703. DeMarais, B. D., Dowling, T. E., Douglas, M. E., Minckley, W. L., & Marsh, P. C. 1992. Origin of Gila seminuda (Teleostei: Cyprinidae) through introgressive hybridization: implications for evolution and conservation. Proceedings of the National Academy of Sciences, 89(7), 2747-2751. Douglas, M. E. , M. R. Douglas, J. M. Lynch and D. M. McElroy 2001. Use of geometric morphometrics to differentiate Gila (Cyprinidae) within the upper Colorado River basin. Copeia 2001 (no. 2): 389- 400. Eaton, D. A. (2014). PyRAD: assembly of de novo RADseq loci for phylogenetic analyses. Bioinformatics, btu121. Ellis, Max M . 1914. Fishes of Colorado. University of Colorado Studies 11(1):1-135. Espinosa Pérez, H. , M. T. Gaspar Dillanes and P. Fuentes Mata 1993. Listados faunísticos de México. III. Los peces dulceacuícolas Mexicanos. Univ. Nacional Autónoma de México. 1-98. Evermann, B. W., & Rutter, C. 1895. The fishes of the Colorado Basin. US Fish Commission Bulletin, 14(1894), 473-486. Fowler, H. W. (1924). Notes on North American cyprinoid fishes. Proceedings of the Academy of Natural Sciences of Philadelphia, 76, 389-416. Fuller, P. L. , L. G. Nico and J. D. Williams 1999. Nonindigenous fishes introduced into inland waters of the United States. American Fisheries Society, Special Publication 27 1-613. Girard, C. F. 1856. Researches upon the cyprinoid fishes inhabiting the fresh waters of the United States, west of the Mississippi Valley, from specimens in the museum of the Smithsonian Institution. Proceedings of the Academy of Natural Sciences of Philadelphia v. 8: 165-213. Girard, C. F. 1858. Fishes. In: General report upon zoology of the several Pacific railroad routes, 1857. In: Reports of explorations and surveys, to ascertain the most practicable and economical route for a railroad from the Mississippi River to the Pacific Ocean, v. 10. Beverley Tucker, Washington, D.C. [Part of Senate Ex. Doc. No. 78 (33rd Congress, 2nd Sess.). Gilbert, C. R. 1998. Type catalog of Recent and fossil North American Freshwater fishes: families Cyprinidae, Catostomidae, Ictaluridae, Centrarchidae and Elassomatidae. Florida Museum of Natural History, Special Publication No. 1. 1-284. Gilbert, C. H., & Scofield, N. B. (1898). Notes on a collection of fishes from the Colorado Basin in Arizona. Proceedings of the US National Museum, 20, 487-499. Gunther, A. K. L. G. 1868. Catalogue of the Physostomi, containing the families Heteropygii, Cyprinidae, Gonorhynchidae, Hyodontidae, Osteoglossidae, Clupeidae,…[thru]… Halosauridae, in the collection of the British Museum. Catologue of Fishes, 7, 1-512. Henkel, C. V., Dirks, R. P., Jansen, H. J., Forlenza, M., Wiegertjes, G. F., Howe, K., ... & Spaink, H. P. (2012). Comparison of the exomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio). Zebrafish, 9(2), 59-67. Hocutt, C. H., & Wiley, E. O. 1986. The zoogeography of North American freshwater fishes. Wiley- Interscience, New York. 866 pp. Hubbs, C. L. (1940). Speciation of fishes. The American Naturalist, 74(752), 198-211. Hubbs, C. L., & Lagler, K. F. (1958). Fishes of the Great Lakes region (revised). Cranbrook Inst. Sci. Bull, 26, 1-213. International Commission of Zoological Nomenclature [ICZN] (1999) International code of zoological nomenclature [the Code]. Fourth edition. The International Trust for Zoological Nomenclature, c/o Natural History Museum, London. i–xxix, + 306 pp. Jordan, D. S., & Gilbert, C. H. 1883. Synopsis of the fishes of North America (No. 16). US Government Printing Office. Jordan, D. S., & Evermann, B. W. 1898. The fishes of North and Middle America: a descriptive catalogue of the species of fish-like vertebrates found in the waters of North America, north of the Isthmus of Panama (No. 47). US Government Printing Office. Knapp, I., Pruitz, J., Bird, C., Whitney, J., Sudek, M., Foresman, Z., Toonen, R. (2016). ezRAD- an easy next- gen RAD sequencing protocol_v3.1. DOI: 10.17504/protocols.io.e9pbh5n. Kumar S., Stecher G., and Tamura K. ( 2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33:1870-1874 La Rivers, I. 1994. Fishes and fisheries of Nevada. University of Nevada Press, Reno, Nevada. 1-782. Lee, D. S. , C. R. Gilbert, C. H. Hocutt, R. E. Jenkins, D. E. McAllister and J. R., Stauffer, Jr. 1980. Atlas of North American freshwater fishes. Publication of the North Carolina Biological Survey. No. 1980- 12: 1-867. LaRivers, I. 1962. Fishes and Fisheries of Nevada. Nevada State Printing Office, Carson City, Nevada. McElroy, D. M. and M. E. Douglas 1995. Patterns of morphological variation among endangered populations of Gila robusta and Gila cypha (Teleostei: Cyprinidae) in the upper Colorado River Basin. Copeia 1995 (no. 3): 636-649. Meffe GK, Vrijenhoek RC. Conservation genetics in the management of desert fishes. Conservation Biology 1988; 2:157–169. Miller, R.R. 1946. Gila cypha, a remarkable new species of cyprinid fish from the lower Colorado River basin, Arizona. Journal Washington Academy Science, 36:206-212. Miller, R.R. 1961. Man and the changing fish fauna of the American Southwest. Paper Michigan Academy of Sciences, Arts, Letters, 46:365-404. Miller, R. R., & Lowe, C. H. 1964. Annotated checklist of the fishes of Arizona. The vertebrates of Arizona. University of Arizona Press, Tucson, Arizona, 133-151. Miller, R.R. and C.H. Lowe. 1967. Fishes of Arizona. pp. 133-151 in C.H. Lowe (ed.), The vertebrates of Arizona. University of Arizona Press, Tucson, Arizona. Miller, R. R. 2006. Freshwater fishes of México. Chicago (University of Chicago Press). 1-490. Minckley, W. L., & Alger, N. T. 1968. Fish remains from an archaeological site along the Verde River, Yavapai County, Arizona. Plateau, 40(3), 91-97. Minckley, W.L. 1969. Aquatic Biota of the Sonoita Creek Basin, Santa Cruz County, Arizona. The Nature Conservancy, Ecological Studies Leaflet, 15:1-8. Minckley, W. L. and B. D. DeMarais 2000. Taxonomy of chubs (Teleostei, Cyprinidae, genus Gila) in the American southwest with comments on conservation. Copeia 2000 (no. 1): 251-256. Minckley, W. L. and P. C. Marsh 2009. Inland fishes of the Greater Southwest. Chronicle of a Vanishing Biota. The University of Arizona Press. 1-426. Nelson, J. S. , E. J. Crossman, H. Espinosa Pérez, L. T. Findley, C. R. Gilbert, R. N. Lea and J. D. Williams 2004. Common and scientific names of fishes from the United States, Canada, and Mexico. Sixth Edition. American Fisheries Society, Special Publ. 29. Bethesda, Maryland. Committee Scient. Names Fishes U.S. Canada Mexico Sixth Ed.: 1-386. Norris, S. M. , J. M. Fischer and W. L. Minckley 2003. Gila brevicauda (Teleostei: Cyprinidae), a new species of fish from the Sierra Madre Occidental of México. Ichthyological Exploration of Freshwaters v. 14 (no. 1): 19-30. Page, L. M. and B. M. Burr 1991. A field guide to freshwater fishes. North America. North of Mexico. The Peterson Field Guide Series. Boston. 1991: v-xii + 1-432 Page, L. M. and B. M. Burr 2011. Peterson Field Guide to Freshwater Fishes of North America North of Mexico (Second Edition). Freshwater Fishes of North America. Page, L. M. , H. Espinosa-Pérez, L. D. Findley, C. R. Gilbert, R. N. Lea, N. E. Mandrak, R. L. Mayden and J. S. Nelson 2013. Common and scientific names of fishes from the United States, Canada, and Mexico. Seventh Edition. American Fisheries Society, Special Publication 34. 1-384. Reece, J.S., B.W. Bowen, D.G. Smith, and A.F. Larson. 2010. Molecular phylogenetics of moray eels (Murenidae) demonstrates multiple origins of a shell-crushing jaw (Gymnomuraena, Echidna) and multiple colonizations of the Atlantic Ocean. Molecular Phylogenetics and Evolution, 57:829 – 835. Robins, C. R., Bailey, R. M., & Bond, C. E. (1980). A list of common and scientific names of fishes from the United States and Canada. Robins, C. R. (1991). Common and scientific names of fishes from the United States and Canada. Special publication/American Fisheries Society (USA). Scharpf, C. 2005. Annotated checklist of North American freshwater fishes, including subspecies and undescribed forms. Part I: Petromyzontidae through Cyprinidae. American Currents v. 31 (no. 4): 1-44. Sigler, W. F. and J. W. Sigler 1996. Fishes of Utah. 1-375. Sigler, W.F. and R.R. Miller. 1963. Fishes of Utah. Utah Department of Fish and Game, Salt Lake City, Utah. Seutin, G., White, B. N., & Boag, P. T. (1991). Preservation of avian blood and tissue samples for DNA analyses. Canadian Journal of Zoology, 69(1), 82-90. Smith, G. R., Miller, R. R., & Sable, W. D. 1979. Species relationships among fishes of the genus Gila in the upper Colorado River drainage. Ann Arbor, 1001(48109), 48109. Smith, R. 1884. Description of a new species of Squalius Bonaparte 1837. Bulletin of the California Academy of Sciences No. 1: 3-4. Snyder, J.O. 1915. Notes on a collection of fishes made by Dr. Edgar A. Mearns from rivers tributary to the Gulf of California. Proceedings U.S. National Museum, 49:573-586. Sovic, M. G., Fries, A. C., & Gibbs, H. L. (2015). AftrRAD: a pipeline for accurate and efficient de novo assembly of RADseq data. Molecular ecology resources, 15(5), 1163-1171. Stamatakis, A. (2014). RAxML Version 8: A tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies". Bioinformatics Swift, C. C. , T. R. Haglund, M. Ruiz and R. N. Fisher 1993. The status and distribution of the freshwater fishes of southern California. Bulletin of the Southern California Academy of Sciences v. 92 (no. 3): 101-167. Tanner, V. M. 1950. A new species of Gila from Nevada (Cyprinidae). Great Basin Naturalist, Provo Utah v. 10 (nos 1-4): 31-36. Toonen, R. J., Puritz, J. B., Forsman, Z. H., Whitney, J. L., Fernandez-Silva, I., Andrews, K. R., & Bird, C. E. (2013). ezRAD: a simplified method for genomic genotyping in non-model organisms. PeerJ, 1, e203. Tyus, H. M. 1998. Early records of the endangered fish Gila cypha Miller from the Yampa River of Colorado with notes on its decline. Copeia 1998 (no. 1): 190-193. Uyeno, T. 1961. Osteology and phylogeny of the American cyprinid genus Gila. Ph.D. dissertation. University of Michigan, Ann Arbor, Michigan. Uyeno, T. and R.R. Miller. 1963. Summary of late Cenozoic freshwater fish records for North America. Occasional Papers Museum Zoology University Michigan, 631:1-34. Uyeno, T., & Miller, R. R. (1965). Middle Pliocene cyprinid fishes from the Bidahochi formation, Arizona. Copeia, 28-41. Vanicek, C. D. 1967. Ecological studies of native Green River fishes below Flaming Gorge dam, 1964-1966. Unpubl. Ph.D. diss.138 pp. Williams et al. 1985 Endangered aquatic ecosystems in North American deserts with a list of vanishing fishes of the region. Zhang, Y., Stupka, E., Henkel, C. V., Jansen, H. J., Spaink, H. P., & Verbeek, F. J. (2011). Identification of common carp innate immune genes with whole-genome sequencing and RNA-Seq data. J Integr Bioinform, 8(2), 169. Zhang, J., Kobert, K., Flouri, T., & Stamatakis, A. (2014). PEAR: a fast and accurate Illumina Paired-End read merger. Bioinformatics, 30(5), 614-620.