Phylogenetic relationships of North American western chubs of the genus Gila (Cyprinidae, Teleostei), with emphasis on southern species

Molecular Phylogenetics and Evolution 70 (2014) 210–230

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Phylogenetic relationships of North American western chubs of the genus Gila (Cyprinidae, Teleostei), with emphasis on southern species Susana Schönhuth a,⇑, Anabel Perdices b, Lourdes Lozano-Vilano c, Francisco J. García-de-León d, Héctor Espinosa e, Richard L. Mayden a a

Department of Biology, Saint Louis University, 3507 Laclede Avenue, St. Louis, MO 63103, USA Departamento de Biodiversidad y Biología Evolutiva, Museo Nacional de Ciencias Naturales (CSIC), Madrid, Spain c Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Nuevo León, Mexico d Laboratorio de Genética para la Conservación, Centro de Investigaciones Biológicas del Noroeste, S.C. (CIBNOR), La Paz, Baja California, Mexico e Instituto de Biología, Universidad Nacional Autónoma de México, México DF, Mexico b

a r t i c l e

i n f o

Article history: Received 8 February 2013 Revised 20 August 2013 Accepted 23 September 2013 Available online 4 October 2013 Keywords: Western cyprinids Arid–semiarid regions Western Clade Gila Moapa Hybrids

a b s t r a c t Species of Gila comprise a heterogeneous and widespread group of freshwater fishes inhabiting drainage systems of western North America. The classification of species of Gila and relatives has been complicated and sometimes compromised by differences in body shapes, sizes, habitats, variable taxonomic placement by early taxonomists, and instances of hypothesized hybridization. While most attention on Gila has focused on hybridization in USA, little is actually know about their intra and intergeneric relationships. We present a molecular phylogeny using 173 specimens for all 19 recognized species of Gila, covering their entire distributions in 31 major drainages. Using one mitochondrial and three nuclear genes, specimens of Gila were analyzed with 10 other North American genera that comprise the Revised Western Clade. All analyses identified most species of Gila in a lineage that always included the monotypic genera Moapa and Acrocheilus, and we recommend the synonymy of both genera with Gila. The composition of this Gila lineage varied depending on the genes analyzed. Within the Gila lineage, similar morphotypes (forms adapted to fast currents vs. general forms) were not resolved as closest relatives. Analyses of mitochondrial DNA resolved all species of Gila from Mexico in reciprocally monophyletic clades except G. modesta. Most species of Gila in the USA were nested in 3 major clades, potentially indicating some level of historic or contemporary interspecific hybridization. Herein, we redefine the ranges for all species of Gila in Mexico. Relevant taxonomic and conservation implications stemming from the results are discussed. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Cyprinids from western North America have long presented an interesting problem as to their taxonomy and classification (Miller, 1959; Simons and Mayden, 1997; Dowling et al., 2002). Many of these genera are part of a highly distinctive group inhabiting arid environments and identified previously as the Western Clade by both Coburn and Cavender (1992) and Simons and Mayden (1998), although composition of this clade differed between studies. This Western Clade includes taxa showing high levels of endemism and several morphologically distinctive monotypic genera (Miller, 1959). Many species in the Western Clade have been assigned to the genus Gila Baird & Girard 1853.

⇑ Corresponding author. E-mail address: [email protected] (S. Schönhuth). 1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.09.021

Gila, generally including species referred to as western chubs, represents a taxonomically diverse group and currently spans most of the nonglaciated regions in western North America (Fig. 1). Gila has been proposed to be an old group, with a fossil record extending from the Miocene to the Pleistocene. The oldest known fossil assigned to Gila (Gila turneri, authority Eastman 1917) dates from the middle Miocene (approx. 12 million years ago (MA)) (Uyeno and Miller, 1963; Smith et al., 2002); several fossils of Gila sp. cf. robusta and other extinct but proposed new species of Gila date from the Pliocene to the Pleistocene (approx. 6–2 MA) (Uyeno and Miller, 1963, 1965; Smith et al., 2002). Currently, there are at least 19 described species of Gila that are mainly distributed in western drainages, with some southern species also occurring east of the Continental Divide (Fig. 1, Table 1). As with many other freshwater fishes inhabiting arid– semiarid regions in western North America, species of Gila are

Table 1 Summary of taxonomic history showing taxonomic changes and group combinations for species included in the Gila lineage (genus Acrocheilus, Moapa, Gila, and Ptychocheilus lucius), and genera previously considered Gila (Siphateles and Snyderichthys). Status of threatened species listed: (E): endangered; (T): threatened; (CS): candidate species; (R): Rare (following NOM-59ECOL-1994 SEDESOL; NOM-059-SEMARNAT, 2010; U.S. Fish and Wildlife Service, 2010; IUCN, 2013). Original descriptions and authors (1853– New species and 1945) taxonomic changes (1945–1959)

After Uyeno (1960)

New species, group combinations, and reelevations (1961–2011)

Acrocheilus alutaceus Agassiz and Pickering 1855

Gila robusta Braid & Girard 1853 Gila elegans Braid & Girard 1853 Lavinia crassicauda Braid & Girard 1854

Moapa coriacea Hubbs and Miller 1948 New species: G. cypha Miller 1946

Cheonda modesta Garman 1881 Gila conspersa Garman 1881 Tigoma orcutti Eigenmann & Eigenmann 1890 Gila minacae Meek 1902 Gila diatenia Miller 1945

Subgenus Temeculina: G. (Tigoma) orcutti G. (Tigoma) purpurea G. diatenia

Subgenus Siboma: G. (Siboma) atraria G. (Lavinia) crassicauda

Ptychocheilus lucius Girard 1856 Siphateles Cope 1883 to include Algansea bicolor Girard 1856 Squalius copei Jordan & Gilbert 1881 Genus Snyderichthys Miller 1945

Current study (status)

Gila lineage:

Gila lineage:

Acrocheilus alutaceus Moapa coriacea

Acrocheulus alutaceus Moapa coriacea (E)

G. conspersa c,d G. cypha

New species: G. eremica DeMarais 1991 G. atraria G. brevicauda Norris, Fischer G. brevicauda & Minckley 2003 G. coerulea (#) Gila robusta complex: G. conspersa

G. crassicauda (&e)

G. cypha g

G. atraria G. brevicauda* G. coerulea (#) G. conspersa* (T)

G. cypha

G. cypha (E)

G. diatenia G. modesta c,d

G. elegans G. intermedia h,i

G. diatenia G. elegans

G. diatenia* (T) G. elegans (E)

G. G. G. G.

G. nigra i G. robusta G. robusta jordani G. seminuda (G. robusta  G. elegans) j

G. G. G. G.

G. G. G. G.

nigrescens orcutti pandora c,f pulchra c,d

eremica intermedia minacae modesta

eremica* intermedia (E) minacae* (R) modesta* (E)

G. purpurea

G. nigra

G. nigra (CS)

G. robusta robusta G. robusta elegans

G. nigrescens G. orcutti

G. nigrescens* (T) G. orcutti

G. robusta intermedia

G. G. G. G.

G. G. G. G.

Siphateles is merged within Gila as subgenus (including: G. bicolor, G. mohavensis) Snyderichthys is merged within Gila as subgenus (including: G. copei)

pandora pulchra purpurea robusta

pandora pulchra* purpurea* (E) robusta (CS)

G. seminuda

G. seminuda (E) Gila sp. 1*

Ptychocheilus lucius (#) Siphateles (genus not monophyletic, member of the Revised Western Clade (RWC)) Snyderichthys m (within CC-Plagopterin Clade)

Ptychocheilus lucius (#) Siphateles (not monophyletic, member of the RWC) Snyderichthys m (not member of the RWC)

Re-elevation of genus Siphateles k Re-elevation of genus Snyderichthys l

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Tigoma nigrescens Girard 1856 Taxonomic changes a: Tigoma bicolor or Cheonda coerulea Girard Subgenus Gila: 1856 Tigoma pulchra Girard 1856 G. robusta b (including G. elegans, G. seminuda, G. intermedia) Tigoma purpurea Girard 1856 G. minacae Tigoma intermedia (=Gila gibbosa) Girard G. (Tigoma) nigrescens 1856 Siboma atraria Girard 1856 Clinostomus pandora Cope 1872 Subgenus Klamathella: Gila seminuda Cope & Yarrow 1875 G. (Cheonda) coerulea Gila nigra Cope 1875

Subgenus Gila: G. atraria G. coerulea

After Schönhuth et al. (2012b)

a

Miller (1945a). Miller (1946). c Uyeno (1960) indicated it to be valid species. d Miller (1976) removed them from synonymy of G. nigrescens. f Miller and Hubbs (1962) removed it from synonymy of G. nigrescens. &e Extinct, Miller (1961) reported last collected specimen was in 1957. g Holden and Stalnaker (1970) recognized the species. h Rinne (1976) recognized the species. i Minckley and DeMarais (2000) recognized the species. j Species of hybrid origin (DeMarais et al., 1992; Gerber et al., 2001). k Proposed by Simons and Mayden (1998). l Proposed by Simons and Mayden (1997). m Proposed within genus Lepidomeda by Johnson et al. (2004). * Native species in Mexico. # Species included in the Gila lineage (95PP) in mtDNA analyses, but not in the nDNA analyses (96PP). b

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often confined to small portions of river systems, that, together with habitat degradation and non-native fish introductions have put several of these species at risk of extinction. One species, G. crassicauda, is thought to have gone extinct in 1957 (Miller, 1961; Miller et al., 1989), and 10–12 species are currently listed as under threat (U.S. Fish and Wildlife Service, 2010; NOM-ECOL1994 SEDESOL; NOM-059-SEMARNAT, 2010). Within Gila, several studies based on morphology, allozymes, or DNA have identified hybrids between some species with morphological intergrades and/or incomplete lineage sorting, making the taxonomy of these particular species more complicated (Holden and Stalnaker, 1970; Minckley, 1973; Minckley and DeMarais, 2000; Gerber et al., 2001). While hybridization can make species differentiation difficult, molecular studies have also emphasized that hybridization has been a key factor in Gila diversification (Rosenfeld and Wilkinson, 1989; DeMarais et al., 1992; Dowling and DeMarais, 1993; Gerber et al., 2001). Regardless of interests of evolution and hybrids, there is a significant lack of phylogenetic knowledge of species now classified as Gila, especially for those that occur in Mexico (Simons and Mayden, 1998; Simons et al., 2003). Therefore, phylogenetic relationships among species of Gila are essential to understand current and/or historical hybridization among species and to distinguish this from cases of incomplete lineage sorting. Phylogenetic studies are also critical for species delimitation, to prevent inappropriate conclusions about diversity by eliminating possibilities that morphological variation could be interpreted as evidence of intergradation (Wiley, 1981; Mayden, 1999, 2002, 2013). In a recent large-scale molecular phylogenetic analysis of all western North American cyprinids (Schönhuth et al., 2012b) the

composition of the Western Clade was redefined. This Revised Western Clade (RWC) represents a new phylogenetic hypothesis placing the genus Chrosomus sister to 11 western genera, including Acrocheilus, Eremichthys, Gila, Hesperoleucus, Lavinia, Moapa, Mylopharodon, Orthodon, Ptychocheilus, Relictus and Siphateles (Schönhuth et al., 2012b; Fig. 2D). Within the RWC most of the extant species of Gila form a so-called Gila lineage that also includes two monotypic genera, Moapa and Acrocheilus, nested well within this lineage. Herein, we focus on the phylogenetic relationships of the redefined Gila lineage, paying special attention to the species diversity in Mexico. Analyses include evaluations of DNA sequences from multiple individuals of each extant species of Gila. The broader sampling serves to expand previous analyses and include representatives of most of the species from across their respective distributions. Phylogenetic analyses are derived from one mitochondrial and three nuclear genes to provide a more thorough understanding of genetic variation. Genetic variability within species is used to delineate species, and to identify cryptic lineages. Incongruences between mitochondrial and nuclear phylogenies are examined to identify possible cases of interspecific hybridization. Overall, phylogenetic inferences are used to investigate the evolutionary history of the Gila lineage and to propose a new classification of this complex group. 1.1. Phylogeny, taxonomy and systematic history of Gila Gila Baird & Girard 1853 has had a complex taxonomic and systematic history (Fig. 2, Table 1). The group includes species that are

Fig. 1. Study area indicating native species of Gila from Mexico used in this study. Bottom left – distribution of Gila and related genera in western North America. Names for major drainages are indicated in the map. BvL: Bavicora Lagoon; BsL: Bustillos Lagoon; SgL: Santiaguillo Lagoon.

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Fig. 2. Summary of different evolutionary hypotheses of species of Gila based on morphological data: (A) Miller (1945a) indicating the 4 proposed subgenera with species analyzed in each group underlined in Uyeno’s tree; (B) Phylogenetic relationships proposed by Uyeno (1960); (C) Phylogenetic relationships proposed by Coburn and Cavender (1992). Molecular analyses based on DNA sequences: (D) Phylogenetic relationships for all 22 western genera based on 4 genes focused on species and genera included in the Revised Western Clade and showing the Gila lineage (Schönhuth et al., 2012b); (E) Phylogenetic analyses based on mtDNA (present study); (F) Phylogenetic analyses based on nDNA for all species included in the Revised Western Clade (present study). Contour drawings for some morphologically distinctive species illustrate limited congruence in patterns of morphology relative to the recovered phylogenies for the Gila lineage. In red, species of Gila and species previously allocated within Gila.

in many ways similar morphologically but are variable in body shapes (e.g. from elongated thin-bodies with long caudal peduncles, large fins and relatively high numbers of scales and fin rays, to short stubby and thick-bodies with short caudal peduncles, short fins and reduced meristics), sizes (from 8.5 cm to 62 cm), and habitats (small creeks to lakes and big rivers). Early morphological studies of North American cyprinids, proposed different classifications for species currently in Gila (Miller, 1945a; Bailey, 1956; Uyeno, 1960) (Table 1). Miller (1945a) allocated 10 species of Cheonda, Gila, Siboma and Tigoma to Gila (Table 1). In that study, he recognized the subgenera Gila Miller 1945 [including G. robusta, G. elegans, G. minacae, G. nigrescens], Siboma Girard 1856 [including G. atraria and the extinct G. crassicauda], Temeculina Cockerell 1909 [including G. orcutti, G. purpurea, G. diatenia], and erected the subgenus Klamathella for G. bicolor (Cheonda coerulea) (Miller, 1945a) (Fig. 2). Later, Bailey (1956) merged Clinostomus and Richardsonius into Gila because of the ‘‘lack of distinctive generic criteria’’. Uyeno (1960) proposed a phylogeny of American cyprinids allied to Gila and removed Richardsonius (including Clinostomus) from synonymy with Gila, and hypothesized Richardsonius was the closest relative to Gila (Fig. 2). This author also concluded that some morphological characters previously considered very important phylogenetically, such

as the pharyngeal tooth formula, were highly variable characters within the Gila group and even within species of Gila. It should be noted that Uyeno (1960) did include the genera Siphateles and Snyderichthys within Gila. Derived from this reorganization, a new arrangement of scientific names for ‘‘bicolor’’ (used in Gila and in Siphateles) was required. Siphateles bicolor became Gila bicolor (described as Algansea bicolor Girard 1856) and Gila (Tigoma) bicolor was changed to G. coerulea, the next available name for this species (Bailey and Uyeno, 1964). Furthermore, four species synonymized with G. nigrescens (Girard 1856) were resurrected by different authors as Gila pandora (Cope 1872), G. pulchra (Girard 1856), G. conspersa Garman 1881, and G. modesta (Garman 1881) (Uyeno, 1960; Miller and Hubbs, 1962; Miller, 1976). Later studies, based on either morphology or molecular data, were not able to corroborate relationships of Clinostomus and Richardsonius as closely related to Gila, nor even within the Western Clade (Coburn and Cavender, 1992; Fig. 2C; Simons and Mayden, 1997, 1998). In these studies the phylogenetic relationships of Gila (containing Gila, Siphateles and Snyderichthys) indicated that the genus was paraphyletic. In an effort to reconcile this, Simons and Mayden (1997, 1998) elevated Snyderichthys and Siphateles as separate genera. Recent molecular phylogenetic analyses for all 22 western genera resolved a more reduced Western Clade (Re-

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vised Western Clade (RWC)) including a group termed the ‘‘Gila lineage’’. This lineage included most of the species of Gila, Moapa and Acrocheilus (Schönhuth et al., 2012b; Fig. 2D). However, in that study sampling within the morphologically heterogeneous genus Gila was focused on resolving relationships at higher taxonomic levels (see Table 1 for taxonomic background for genus Gila). Despite limited understanding of phylogenetic relationships within Gila, taxonomic proposals proliferated. The subgenus Temeculina has been recognized by some authors (Miller, 1945a; Barbour and Miller, 1978); however, this recognition renders the subgenus Gila (G. robusta, G. elegans, G. minacae, G. nigrescens) paraphyletic following analyses by Uyeno (1960) (Fig. 2). Gila pulchra (Girard 1856), initially considered a synonym of G. nigrescens (hence within subgenus Gila cf. Miller, 1945a), was later recognized as a valid species (Miller, 1976), and later moved to the subgenus Temeculina (Barbour and Miller, 1978). Barbour and Miller’s study (1978) also suggested including G. crassicauda in Temeculina and hypothesized a close relationship between these species of Gila and the genus Algansea (consistent with later studies by Coburn and Cavender, 1992). Evaluation of species of Gila in Mexico reveals a much greater lack of current-day understanding of the diversity of the genus. Species of Gila in Mexico and the southwestern USA appear to follow a different pattern of differentiation. Whereas in the southwestern USA different species of Gila are co-distributed (with many studies reporting hybridization among them), species of Gila from Mexico are largely allopatric and replace one another in major drainages, suggesting a history of allopatric speciation in this arid region. Gila robusta Baird & Girard 1853, a species that inhabits the Colorado River System (USA), was designated the type species of the genus by Jordan and Gilbert (1877). Traditionally G. robusta has been hypothesized or argued to encompasses multiple subspecies (Gila r. seminuda, G. r. elegans, Gila r. grahami) now recognized as species (G. seminuda, G. elegans and G. nigra, respectively). Currently, G. robusta is also used to designate a species group (G. cypha, G. elegans, G. intermedia, G. nigra, G. robusta jordani, G. robusta robusta, G. seminuda) that occurs in the Colorado River System (Rinne, 1976; Rosenfeld and Wilkinson, 1989; Gerber et al., 2001). Considerable research has been conducted on this G. robusta species group as an evolutionary complex endemic to different regions and habitats of the Colorado River System (Gerber et al., 2001). Some studies, those based on mtDNA and allozyme data, failed to identify any diagnostic characters for the allopatrically distributed species in the lower Colorado River, including G. intermedia, G. nigra and G. robusta robusta (DeMarais et al., 1992; Dowling and DeMarais, 1993; Minckley and DeMarais, 2000). Interestingly, other studies have hypothesized a hybrid origin for the taxa G. robusta jordani and G. seminuda (allopatric taxa in the Pluvial White and Virgin rivers, respectively) (DeMarais et al., 1992; Dowling and DeMarais, 1993). The origin and taxonomic status of G. nigra (morphologically intermediate between Gila robusta robusta and G. intermedia) remains under debate (Minckley and DeMarais, 2000; Gerber et al., 2001). Contrary to G. seminuda, G. r. jordani is not recognized as a valid species (Nelson et al., 2004). Remaining species of this complex, G. cypha, G. elegans, and G. r. robusta are sympatrically distributed and in some instances hybridization has been reported among these three taxa (DeMarais et al., 1992; Dowling and DeMarais, 1993; Gerber et al., 2001). Although species of the Gila robusta complex have been extensively studied, controversy remains as to the composition of Gila, species origins, and their relationships. 2. Material and methods 2.1. Taxon sampling Sequences from 173 specimens, representing all 19 extant and recognized species of Gila, were analyzed together with all species

from 10 other North American genera that together comprise the RWC sensu Schönhuth et al. (2012b). These include the monotypic genera Acrocheilus, Eremichthys, Hesperoleucus, Lavinia, Moapa, Mylopharodon, Orthodon and Relictus, and the genera Ptychocheilus (4 species) and Siphateles (3 species). New sequences were obtained from 127 specimens of Gila; specimens were collected at 125 different localities, covering the geographic distribution of all putative species. Taxon sampling was particularly dense for Gila in Mexico as these populations have received essentially no attention, thus necessitating more broad-based sampling of representatives from as much of the entire range of each species as possible. All analyses also included Snyderichthys (S. copei), a species traditionally included within Gila (Uyeno, 1960; Coburn and Cavender, 1992), but recently found to be more closely related to members of the Creek Chub-Plagopterin Clade, a group distantly related to the RWC (Schönhuth et al., 2012b). Within Gila, morphological variation in body, head and fin shape have been commonly noted to in the literature; these are herein referred to as ‘‘morphotypes’’. Using our current understanding of the phylogenetic relationships of North American Cyprinidae (subfamily Leuciscinae) (Mayden et al., unpubl.), Platygobio gracilis, Agosia chrysogaster and Algansea lacustris were identified as most relevant outgroups for this investigation. These three genera were placed within the Open Posterior Miodome (OPM) clade (Mayden 1989), which was resolved as the sister group to the RWC in the most recent molecular phylogeny of all western cyprinid genera (Schönhuth et al., 2012b; Fig. 2D). Our sampling strategy includes specimens from 31 different independent major drainages, mainly from southwestern North America, including drainages to the Pacific or the Gulf of California (hereafter Pacific drainages), endorheic basins, and drainages of the Gulf of Mexico (hereafter Atlantic drainages) (Fig. 1). A list of specimens examined is provided in the Appendix. Voucher materials are deposited in ichthyological collections at Saint Louis University, St. Louis, Missouri, USA (SLUM); University of Alabama, Tuscaloosa, Alabama, USA (UAIC); Universidad de Nuevo León, Nuevo León, Mexico (UANL); Instituto de Biología, Universidad Nacional Autónoma de Mexico, Mexico D.F. (IBUNAM); Museum of Southwestern Biology, Alburquerque, New Mexico, USA (MSB); and Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN). 2.2. Gene sampling and molecular methods One mitochondrial (mtDNA) and three nuclear (nDNA) genes were selected for sequencing in this study. These genes have proven very useful in previous studies at this level of diversification (Chen et al., 2007; Schönhuth et al., 2008, 2012a): the complete mitochondrial cytochrome b (cytb, 1140 bp); rhodopsin (Rhod, about 850 bp); S7 ribosomal protein gene (S7, including the first intron and about 900 bp without indels); and the recombination activating gene 1 (Rag1, exon 3, 1520 bp). DNA was extracted using DNeasy Tissue extraction Kit (Qiagen, Valencia, CA, USA), and ChargeSwitch gDNA Microtissue Kit (Invitrogen, Inc.). Nuclear and mitochondrial sequences were obtained from the same individuals. Amplification and primers for cytb are detailed in Schönhuth and Doadrio (2003); for S7 in Chow and Hazama (1998); for Rhod in Chen et al. (2003); and for Rag1 in Lopez et al. (2004). When necessary, nested PCR was performed for the S7 region with two internal primers identified in Schönhuth et al. (2012b). All PCR reactions were carried out using a Peltier Thermal Cycler-200 (MJ Research, Waltham, MA, USA) and GeneAmp 2700 and 9700 Thermal Cyclers (Applied Biosystems, Madrid, Spain). When more than one product resulted from PCR amplification of S7 region, the target product was gel-extracted and purified using a DNA Gel Extraction kit (Qiagen, Valencia, CA, USA). Primers for direct sequencing of the purified PCR were the same as those used for the PCR amplification. PCR products were sequenced at the High-Throughput

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Fig. 3. Phylogenetic relationships of all specimens analyzed for the RWC using sequence variation for mitochondrial cytochrome b gene; Best RAxML tree using GTR + I + G model. Numbers on the branches are ML (BS > 75%) and Bayesian posterior probabilities (PP > 90). Asterisks indicate nodes where all values were 100. Number of identical sequences from the same river is shown in parentheses. Diamond shapes on branches indicate those clades with nested species. Underlined species names in red are those with all or most of their distributions in Mexico. Numbers after species name indicate geographic distribution as in Fig. 1.

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Fig. 4. Phylogenetic relationships of selected specimens of the RWC based on the combined three nuclear regions; Best RAxML tree using GTR + I + G model. Numbers on the branches are ML (BS > 75%) and Bayesian posterior probabilities (PP > 85). Asterisks indicate nodes where all values were 100%. Underlined names are those species not recovered as monophyletic based on analyses of nuclear gene variability. Numbers after species name indicate geographic distribution as in Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Phylogenetic relationships of selected specimens of the RWC according to sequence variation for the combined mtDNA and nDNA gene regions; Best RAxML tree using GTR + I + G model. Numbers on the branches are ML (BS > 75%) and Bayesian posterior probabilities (PP > 90). Asterisks indicate nodes where all values were 100. Numbers after species name indicate geographic distribution as in Fig. 1.

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Fig. 6. Bar-graph showing frequency of pairwise differences (cytb uncorrected p-values genetic distances ranging from 1% to 19%) for all taxa analyzed. Black circles, double lines, and bold lines indicate average, range, and standard deviation values, respectively, for pairwise differences between all taxa analyzed (T), and for the genus Gila with respect to four other genera: (G–G) Gila–Gila; (G–Ac) Gila–Acrocheilus; (G–Mo) Gila–Moapa; (G–Sb) Gila–Siphateles; (G–Sn) Gila–Snyderichthys.

Genomics Unit in University of Washington (WA, USA), and Macrogen Inc. (Korea). Sequences specifically obtained for this study have been deposited in GenBank (accession numbers KF514144– KF514375). 2.3. Phylogenetic analyses Mitochondrial DNA analyses included 201 specimens from the RWC; 173 were representatives for all 19 extant and recognized species of Gila. A reduced subset of 105 taxa was selected for the analyses using nuclear genes. No saturation of any of the genes was detected, and no ambiguous alignments or gaps were found in cytb, Rhod or Rag1; therefore all codon positions were included in the analyses. Sequences were aligned using Clustal X ver1.85 (Thompson et al., 1997) and corrected to minimize substitutional changes. Multiple indels were detected in S7 region ranging from 1 to 38 bp and were treated as missing data. Genetic divergences provided herein are based only on cytb uncorrected pairwise distances. Phylogenetic trees were estimated separately for each gene data set (cytb, S7, Rhod, and Rag1), the combined nuclear dataset (3 genes), as well as for all regions combined (4 genes), using Maximum Likelihood (ML) and Bayesian Inference (BI). Phylogenetic trees using Maximum Likelihood (ML) were estimated as implemented in RAxML (Randomized Axelerated Maximum Likelihood, version 7.0.4) (Stamatakis, 2006). Search for the optimal ML trees and bootstrap support were performed on a high-performance iDiscover cluster computing facility (32 nodes) located at Saint Louis University. We used the GTR + I + G model with the mixed model of nucleotide substitution (4 discrete rate categories) for ML searches. Inferences included partitions by codon position for protein coding genes (all except S7, that was treated as a single partition). The ML tree search was conducted by performing 100 independent runs using default setting for random trees (-d option) as a starting tree for each run. The maximum likelihood tree was identified from among optimal trees obtained per run by comparisons of likelihood scores under the GTR + I + G model. Robust-

ness of the inferred trees was evaluated using bootstrap analysis on 1000 pseudoreplications using RAxML 7.0.4 (Felsenstein, 1985; Stamatakis et al., 2008). Resulting trees were imported into PAUP*4.0.b10 (Swofford, 2001) to obtain the consensus tree. BI analyses were conducted for each gene data set using Mr. Bayes v3.1.2 (Huelsenbeck and Ronquist, 2001). The Akaike information criterion (AIC) implemented in MODELTEST v3.4 (Posada and Crandall, 1998) was used to identify the optimal molecular evolutionary model for each partition on each sequence data set. For BI, 5,000,000 cycles were implemented for four simultaneous Monte Carlo Markov chains; sampling the Markov chain at intervals of 100 generations. Log-likelihood stability was attained after 100,000 generations; the first 1000 trees were discarded as ‘‘burn-in’’ in each analysis. The remaining trees were used to compute a 50% majority rule consensus tree in PAUP*. Support for BI tree nodes was determined based on values of Bayesian posterior probabilities. For brevity we only show those phylogenies recovered by RAxML analyses for the mitochondrial data set (cytb, 1140 bp), the nuclear data set (3 genes, 3333 bp), and all concatenated gene sequences data sets (4 genes, 4473 bp). Trees are depicted with ML bootstrap support (BS) followed by posterior probability (PP) values on well-supported [BS > 75%/PP > 90] nodes (Figs. 3–5). 3. Results All phylogenetic analyses including all species of the RWC (either by DNA region or method) resolved a lineage inclusive of most of the 19 species of Gila (including the type species) and two monotypic western genera, Moapa and Acrocheilus (Figs. 3– 5). Following our previous phylogenetic study, we refer to this morphologically heterogeneous group as the Gila lineage (Schönhuth et al., 2012b). This lineage supported by cytb analyses (BI 95PP) included all 19 described and extant species currently included in Gila (G. atraria, G. brevicauda, G. coerulea, G. conspersa, G. cypha, G. diatenia, G. elegans, G. eremica, G. intermedia, G. minacae, G. modesta, G. nigra, G. nigrescens, G. orcutti, G. pandora, G. pulchra,

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Table 2 Comparisons of support for species of Gila based on four different DNA regions analyzed. Number of base pairs (bp) in the alignment, followed by percent variable sites (VS), percent parsimony-informative sites (PS), and range of divergence are indicated for each DNA region. Species-groups well-supported by ML and/or BI based on each data set are indicated by ‘‘+’’; species-groups not supported are indicated by a ‘‘ ’’. Range of percentage of genetic divergence (uncorrected p-distances values for cytb sequences) is indicated for each group. Species

mtDNA (204 taxa) cytb

nDNA (105 taxa) Divergences S7

(1140 bp) VS: 41.4, PS: 37.5 0.2–19.4% G. atraria (Girard 1856) G. brevicauda Norris, Fischer & Minckley 2003 G. coerulea (Girard 1856) G. conspersa Garman 1881 G. cypha Miller 1946 G. diatenia Miller 1945 G. intermedia (Girard 1856) G. nigra Cope 1875 G. robusta Braid & Girard 1853 G. elegans Braid & Girard 1853 G. seminuda Cope & Yarrow 1875 G. eremica DeMarais 1991 G. G. G. G.

purpurea (Girard 1856) pulchra (Girard 1856) minacae Meek 1902 modesta (Garman 1881)

G. pandora (Cope 1872) G. nigrescens (Girard 1856) G. orcutti (Eigenmann & Eigenmann 1890) Gila sp. 1

+

(972 bp) VS: 42.1, PS: 30.1 0.2–14.8%

Rhod (843 bp) VS: 12.2, PS: 8.8 0.1–5.8%

0.0–0.70% 0.0–0.43%

Rag1

3 Nuclears combined (1518 bp) (3333 bp) VS: 12; PS: 7.7 VS: 20.6; PS: 13 0.1–3.4% 0.1–6.6%

+

mtDNA plus nDNA (105 taxa) 4 Regions combined (4473 bp)

+

+ +

+ + +(plus morphotypes G. robusta) + +G. intermedia–G. nigra-G. robusta

0.26–0.70% + + 0.0–1.57% + 0.0–0.52% +(plus morphotypes +(plus morphotypes G. robusta) G. robusta) 0.0–0.26% + + + 0.0–0.70%

+ + +(plus morphotypes G. robusta) +

+ + +(plus morphotypes G. robusta) + +G. intermedia–G. nigra–G. robusta

+G. elegans–G. seminuda

0.0–0.96%

+

+

+G. elegans–G. seminuda

+

0.0–1.05%

+

+

+ + + +G. modesta–G. pandora

0.17% 0.0–0.96% 0.0–1.92% 0.0–0.97%

+

+

+

+

+ +

0.0–0.78% 1.05%

+

0.0–0.70%

+

G. purpurea, G. robusta, G. seminuda), Acrocheilus, Moapa and Ptychocheilus lucius. This Gila lineage was also supported in nDNA analyses by BI (96PP), and included Acrocheilus, Moapa, and all species of Gila except G. coerulea. These results were congruent with previous analyses with more limited sampling within Gila (Schönhuth et al., 2012b) (Figs. 3 and 4). In all analyses the Gila lineage included Moapa and Acrocheilus nested with most of the species of Gila. However, the circumscription of the Gila lineage seems arbitrary regarding the phylogenetic position of two taxa: Gila coerulea and Ptychocheilus lucius. This lineage contains 23 or 21 species (mtDNA or nDNA, respectively) with a wide variation in body shapes and sizes, including typical generalized leuciscine forms (i.e. Gila atraria) to more specialized forms with elongated and fusiform body (i.e. G. elegans and G. cypha), as well as those with specialized trophic morphological features (i.e. Acrocheilus) (Fig. 2E–F). The remaining 13 species from eight genera also part of the well-supported RWC were never resolved within this Gila lineage (Figs. 2–5). In no analyses were species of Siphateles or Ptychocheilus resolved in respective monophyletic groups with congeners. While species of Siphateles and Snyderichthys were previously thought to be related to Gila and formed nested groupings within the genus they were never recovered within the Gila lineage herein (Figs. 3–5). Snyderichys copei was resolved as a highly distinctive species and, as in our prior molecular study, was never resolved as part of the RWC (Schönhuth et al. 2012b). Pairwise divergences among cytb sequences within the Gila lineage ranged from 0.2% to 9.6%. Interestingly, and consistent with

G. eremica–G. + purpurea + + + + +G. modesta–G. pandora – +

+

+ + + + +G. modesta–G. pandora

+

+ +

+

+

species relationships resolved herein, some distances between species of Gila and Moapa or between species of Gila and Acrocheilus were lower than those between currently recognized species of Gila. Divergences between species of Gila and different monotypic genera analyzed ranged from 4.7% (between Moapa-Gila nigra) and 4.8% (between Acrocheilus-Gila atraria) to 13.1% (between Orthodon-Gila sp. 1); divergences rose to 18.7% when Snyderichthys was included in these comparisons (divergences between species of Gila relative to Moapa, Acrocheilus, Siphateles and Snyderichthys are shown in Fig. 6). Analyses of cytb within the Gila lineage resulted in greater resolution of sister group relationships than seen in analyses using the more conserved nDNA genes, particularly on terminal nodes. All gene topologies recovered short internodes among 17 major and well-supported mitochondrial sub-clades; relationships among some of these where poorly supported or have been collapsed, particularly in analyses of nDNA genes (Figs. 3 and 4; Table 2). In some instances, the 17 well-supported sub-clades based primarily on mitochondrial sequences, did not exactly correspond to current species designations. All species of Gila from Mexico were recovered in reciprocally monophyletic lineages (>80% support) (G. brevicauda G. conspersa, G. diatenia, G. eremica, G. minacae, G. nigrescens, Gila pulchra, and Gila sp. 1), except for G. modesta where specimens of this species were always resolved nested within samples of G. pandora. Most species of Gila from Mexico (exception being G. diatenia, G. eremica, G. minacae) were resolved as a monophyletic group (75%BS, 100PP) that together encompassed a wide

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geographic distribution. This group including seven species (cytb: 3.68–5.56%) is identified as the Chihuahuan Desert Group. Species of Gila native to river systems in the USA did not form a monophyletic group, and most of them (except G. purpurea, G. orcutti, G. coerulea) were not resolved as reciprocally monophyletic groups. Gila purpurea, G. orcutti and G. coerulea are resolved in reciprocally monophyletic lineages (100%BS, 100PP), and the latter two species were highly distinctive from other species of Gila (cytb genetic distances 6.5–9.2% and 4.9–8.8%, respectively) (Fig. 3). Despite consistent morphological differences between some recognized species of Gila in the USA, cytb analyses could not resolve nine of these recognized species as being reciprocally monophyletic. These nine species were nested within four well-supported clades (>92% support) within the Gila lineage: (1) G. modesta–G. pandora group; (2) G. cypha–G. atraria group (nesting morphotypes of 3 species: G. atraria, G. cypha, G. robusta); (3) G. robusta–G. intermedia group (nesting 3 species: G. intermedia, G. nigra, G. robusta); and (4) G. elegans–G. seminuda group. In analyses of cytb, Moapa, Acrocheilus and Ptychocheilus lucius were also resolved as well differentiated species within this Gila lineage. Nuclear gene analyses offered high support for more basal nodes within the RWC, but were, in general, less informative phylogenetically for the Gila lineage in some regions of the tree as compared to mtDNA genetic variability (Fig. 4, Table 2). The Gila lineage was only well-supported in BI analyses (96PP), and this lineage included Moapa, Acrocheilus, and all 19 currently recognized species of Gila except G. coerulea. Within this Gila lineage, nDNA analyses recovered three major sub-clades and Acrocheilus as an independent lineage. The first sub-clade was a well-supported and differentiated basal group in all analyses (100%BS, 100PP). This group included three species of Gila in reciprocally monophyletic groups, with Gila purpurea sister to G. eremica and both species sister group to G. minacae. A second sub-clade resolved three species as reciprocally monophyletic in all analyses (>75 support). Here, Gila orcutti was resolved as sister to Moapa (83%BS, 100PP), and this group was sister to G. atraria (94PP). A third sub-clade represents a large well-supported group (93%BS, 100PP) that included 14 species (Gila brevicauda, G. conspersa, G. cypha, G. diatenia, G elegans, G. intermedia, G. modesta, G. nigra, G. nigrescens, G. pandora, G. pulchra, G. robusta, G. seminuda, Gila sp. 1). In these analyses, however, the Chihuahan Desert Group received no support. Contrary to results from analyses of mitochondrial gene, Gila coerulea and Ptychocheilus lucius were never recovered within this Gila lineage. Both mtDNA and nDNA gene trees, however, always recovered haplotypes of G. robusta as a paraphyletic grouping, being resolved within two distinctive lineages. All analyses (nDNA, mtDNA) resolved some specimens, identified as G. robusta, nested within G. nigra and G. intermedia, and some specimens within a G. cypha group. Other molecular incongruences also occurred for specimens of G. seminuda; mtDNA haplotypes of G. seminuda were nested within G. elegans in a well supported clade, whereas nDNA haplotypes of the same specimens resulted in some haplotypes of G. seminuda being more closely related to those of the G. robusta-intermedia group than to remaining G. seminuda or to G. elegans. Phylogenetic relationships for the Gila lineage using the four concatenated regions resulted in greater resolution than observed in instances using only nuclear genes. As in analyses of nDNA genes, Gila coerulea and Ptychocheilus lucius were not resolved within the Gila lineage, a clade that in this analysis had lower support (86PP) (Fig. 5). The Gila lineage contained the same 17 major mitochondrial lineages, including 18 species of Gila, Acrocheilus and Moapa. Some of the inter-relationships between these 17 major clades were not well-supported. A well-supported group did resolve G. minacae as the sister species to a clade formed by G. eremica and G. purpurea, as observed previously in analyses using

only nuclear genes. The Chihuahuan Desert Group, including the same six major lineages, was well-supported as in those phylogenies based on mtDNA gene variation. Haplotype variation for Gila atraria recovered specimens of this species in a monophyletic group, as also observed in analyses of nuclear genes. Gila atraria was resolved as the sister species to the G. cypha–G. robusta lineage, like analyses of mitochondrial genes.

4. Discussion 4.1. Circumscription of the Gila lineage Fossils of Eremichthys, Gila, Mylopharodon, Ptychocheilus, and Siphateles date from Middle Miocene (approx. 12 MA) and indicate an early presence of the RWC genera in aquatic systems of western North America (Uyeno and Miller, 1963; Smith et al., 2002). Interestingly, our phylogenetic hypothesis shows short internodes with poor support connecting terminal lineages for the RWC and also within the Gila lineage. One explanation for this phylogenetic pattern is a rapid radiation within the Gila lineage resulting short periods of divergences or speciation events, consistent with extensive transformations in western drainages since the Miocene that included episodic orogenies, periods of progressive aridity, and most recently the Pleistocene pluvial cycles (Miller, 1945b; Miller and Smith, 1986; Minckley et al., 1986; Smith et al., 2002). This pattern is compatible with predictions of Model III of allopatric speciation (model summarized in Wiley 1981). The age of the Gila group, combined with the isolation of lineages during periods of aridity and drainage transformation, may provide some explanation regarding conflicting phylogenetic relationships for the RWC. However, a strongly contending hypothesis for differences across previous and present phylogenetic hypotheses involves the dramatic impact of taxon and character sampling in phylogeny reconstruction (Hillis, 1998; Zwickl and Hillis 2002; Hillis et al., 2003; Mayden et al., 2008). In our phylogenetic analyses three genera were recovered as paraphyletic: Gila, Ptychocheilus, and Siphateles, congruent with our previous analyses (Schönhuth et al., 2012b). Our results confirm Siphateles and Snyderichthys as independent groups from Gila (Simons and Mayden, 1998; Simons et al., 2003), and indicate that Siphateles and Ptychocheilus might not represent natural groups. Despite differences in mtDNA vs. nDNA phylogenies, all analyses identified the Gila lineage including 18 of the 19 extant species inclusive of Gila, Moapa and Acrocheilus. The species Gila coerulea and Ptychocheilus lucius were resolved within (mtDNA) or outside (nDNA) the Gila lineage (Figs. 3–5), consistent with our prior analyses (Schönhuth et al., 2012b), and suggesting that more character and taxon sampling are warranted to more accurately infer their phylogenetic relationships. Contrary to prior phylogenetic analyses using morphological characters, Moapa was not found to be closely related to Agosia (Hubbs and Miller, 1948; Coburn and Cavender, 1992), which is likely more closely related to the Shiner Clade (as observed in Simons et al., 2003). Moapa is always nested within the Gila lineage and in analyses of nuclear gene variation was consistently recovered as a sister lineage to G. orcutti. Similarly, Acrocheilus is always recovered within the Gila lineage, but with unresolved sister-group relationships. Our results corroborate the closest relationships of Moapa and Acrocheilus to species of Gila within the Western Clade than to any species from other North American cyprinid genera (Smith et al., 2002; Simons et al., 2003; Estabrook et al., 2007; Schönhuth et al., 2012b). The recognition of Moapa and Acrocheilus renders Gila paraphyletic and misrepresents the evolutionary history and the conservation and management of the group. Thus, we recommend and propose the synonymy of both Moapa and Acrocheilus with Gila.

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Our results did not reveal close phylogenetic relationships among species of Gila with similar morphotypes (e.g. rheophilic morphotypes as in G. elegans, G. cypha, G. robusta, G. minacae). In some cases, we found these highly adapted species more closely related to species with a ‘‘typical’’ generalized leuciscin body form (Fig. 2E–F). Miller’s (1945a) subdivision of Gila into four subgenera (Temeculina, Gila, Siboma, Klamatella) is not supported in our analyses. Two of Miller’s subgenera are paraphyletic, suggesting that the diagnostic morphological characters used for these subdivisions within Gila do not represent synapomorphic features (as shown and hypothesized by Uyeno, 1960). The subgenus Temeculina, includes four morphologically similar species (G. diatenia, G. eremica, G. orcutti, G. purpurea; Miller, 1945a; Uyeno, 1960; DeMarais, 1991) or as a group also inclusive of G. pulchra (as suggested by Barbour and Miller, 1978, and Minckley et al., 1986), but is not supported in any analyses. These species previously included in Temeculina were always resolved as reciprocally monophyletic. However, none of these species were resolved as closely related to Algansea, as previously hypothesized (Barbour and Miller, 1978; Coburn and Cavender, 1992). The subgenus Gila (including G. robusta, G. elegans, G. minacae, G. nigrescens) was also resolved as a paraphyletic grouping, as also inferred by Uyeno (1960). The subgenus Siboma (currently including only G. atraria) was supported as an independent linage in analyses of variation of nDNA genes and in analyses using all four genes. In all phylogenetic reconstructions the subgenus Klamathella (G. coerulea) was resolved as a highly divergent lineage, consistent with prior hypotheses (Miller, 1945a; Uyeno, 1960; Smith et al., 2002; Estabrook et al., 2007). Our results suggest that morphotypes and ecotypes observed in different species of Gila have most likely evolved multiple times. 4.2. Systematics and distribution of southern species of the Gila lineage Phylogenetic evidence using mtDNA gene variation and four concatenated regions supported a group herein referred to as the Chihuahuan Desert Group. This lineage includes all native species occurring in the Chihuahuan Dessert and in six major lineages that do not correspond exactly to the six described species: (i) G. pulchra (area 5), (ii) G. conspersa (area 7), (iii) G. nigrescens (area 6), (iv) G. brevicauda (area 4), (v) Gila sp. 1 (area 8), and the sixth lineage with specimens of G. modesta (area 9) nested with G. pandora (area 10) (Figs. 1, 3 and 5). Previous morphological studies also provided characters uniting these six species in a clade (Uyeno, 1960). Despite this group not being resolved in nDNA analyses, grouping them with species of the G. robusta complex, it is our working hypothesis that these Chihuahuan species all shared a unique common ancestor based on morphological similarities, geographical distribution, and mtDNA support for this group. All recognized species within the Chihuahuan Desert Group share a generally similar body shape, and all except G. brevicauda (recently described by Norris et al., 2003) were, for a long time, considered synonyms of G. nigrescens (Bailey et al., 1960, 1970), distributed southward and north of the current range of G. nigrescens. The Chihuahuan Desert Group is distributed along independent drainages that were once part of a hypothesized ancestral Rio Grande System (Smith and Miller, 1986) including endorheic basins and extending south to the Rio Tunal, a drainage now part of the Rio Mezquital (Pacific drainage) (Fig. 1). These species have interesting distributional patterns, wherein each of these six lineages includes specimens from independent drainages sometimes on both sides of the North American Continental Divide. Our molecular data always supported a Gila pandora–G. modesta lineage differentiated from G. nigrescens. Previously, G. modesta and G. pandora were resurrected from G. nigrescens (Miller and Hubbs, 1962; Miller, 1976) and both species are currently recog-

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nized as valid species in Mexico and USA, respectively (areas 9 and 10 Fig. 1). However, we found essentially no differentiation (cytb: 0.0–0.88%) between both nominal species (Figs. 3–5). Divergences between endangered species G. modesta and the widely distributed G. pandora were relatively small. The lack of genetic differentiation between G. modesta and G. pandora could be interpreted as different ‘morphotypes’ of the same species, incipient species, or may be due to lineage sorting. These two widely allopatric species inhabit three major tributaries of the Rio Grande drainage (Atlantic) with no other species of Gila between them. Gila pandora inhabits the upper Rio Grande and upper Pecos River basins (Texas, New Mexico and Colorado, USA), while G. modesta inhabits the upper Rio San Juan (Coahuila, Mexico). Interestingly, we found some specimens from the headwaters of the Rio San Fernando, an independent Atlantic drainage in Nuevo Leon and Tamaulipas (Mexico), nested within the G. pandora–G. modesta lineage. A more detailed study including morphological data is currently being undertaken. Another well-supported clade within the Chihuahuan Desert Group is the Gila conspersa lineage (area 7 Fig. 1). We found two mtDNA subgroups (cytb: 1.0–1.57%) within the G. conspersa lineage without either corresponding to the three major river drainages where they exist (rios Nazas, Aguanaval, Presidio). Gila conspersa inhabits two major endorheic drainages (Rio Nazas and Rio Aguanaval) in the southern part of the Chihuahuan Desert (Durango and Zacatecas, north-central Mexico) (Miller et al., 2005). We propose to extend the known range of G. conspersa to include the upper Rio Presidio (Pacific drainage) (Fig. 1), where we found specimens within the G. conspersa lineage. The Gila pulchra lineage (area 5 Fig. 1) is supported by both mtDNA and nDNA evidence (Figs. 3–5). The known range of G. pulchra has been recently restricted to the Rio Conchos (Miller et al. 2005), while other adjacent populations existing in the headwaters of the Rio Yaqui (upper Papigochic around Minaca) and the Rio Fuerte (upper Rio Verde) were referred as a ‘‘similar undescribed’’ species (Norris et al., 2003). Here, the Gila pulchra lineage includes specimens widely distributed across the Rio Conchos drainage (Atlantic; including its three main tributaries Rio Conchos, Rio Florido and Rio San Pedro) and also specimens from the headwaters of the Rio Fuerte drainage (Pacific; including its three main tributaries Rio Urique, Rio Verde and Rio Oteros), Chihuahua, northern Mexico (Fig. 1). The small genetic divergences within the G. pulchra lineage (cytb: 0.0–0.96%) between specimens occurring on both sides of the Continental Divide, may be due to the lack of anagenesis in genes examined, human introductions, shared retained primitive alleles in the group, or recent headwater connections. These phylogeographic patterns are consistent with biogeographic hypotheses suggesting drainage connections and captures as promoters of mitochondrial homogenization between populations of freshwater fishes (i.e. Campostoma ornatum, in this area of the Sierra Madre Occidental; Schönhuth et al., 2011). Smith and Miller (1986) hypothesized that specimens of Gila from Rio Papigochic (Pacific) and Laguna Bavicora (endorheic) were ‘‘not G. nigrescens of the Guzman complex but the same as that inhabiting the Rio Yaqui currently referred to as G. pulchra’’. In all mtDNA analyses herein these populations were resolved with high-support within the G. nigrescens lineage. However, in analyses of nDNA variation, these same specimens were found to be closely related to the G. pulchra and G. brevicauda lineages, albeit with low support (see discussion on Gila nigrescens below). The observed incongruence in sister group relationships for these populations in the Rio Papigochic and Laguna Bavicora could be the result of the level of variation in the genes chosen, sample sizes, the limited phylogenetic signal for distal branches, incomplete lineage sorting, or historical introgression between G. pulchra and G. nigrescens and warrants further analysis.

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The Gila nigrescens lineage is well-supported in analyses of mtDNA variation, but nuclear alleles never resolved this species as a lineage. Low mitochondrial divergences found within this lineage provide support to extend the range of G. nigrescens to include independent sub-basins of the endorheic Guzman Basin complex in Chihuahua, northern Mexico (Rio Santa Maria, Rio Casas Grandes, Rio Santa Clara), New Mexico (Mimbres River), Laguna Bustillos, Laguna Bavicora, and upper Rio Papigochic near Minaca (southern Rio Yaqui Basin, Pacific) (area 6 Fig. 1). Contrary to prior studies using morphological characters (Miller and Chernoff, 1979), mtDNA of specimens from Laguna Bustillos is indistinguishable from other populations of G. nigrescens (cytb 0.1–0.7%), while divergences between these populations and G. pulchra are 3.7– 4.3%. Although no conclusive results can be derived from nuclear genes, some evidence exists for a close relationship between the upper Rio Yaqui and Laguna Bavicora to G. pulchra and G. brevicauda than to other G. nigrescens (Fig. 4). This may be some evidence for congruence with prior studies suggesting both of the above populations being distinct from G. nigrescens and more ‘‘similar’’ to G. pulchra (Smith and Miller, 1986; Miller et al., 2005). Limited divergences for cytb observed within the G. nigrescens lineage may occur due to simply the result of retained-primitive alleles or limited divergence since these aquatic systems became isolated from a large pluvial lake system, and do not indicate any distinctive pattern of differentiation of populations from different drainages. However, while these populations can be characterized as having low to moderate levels of genetic variability across the range for the genes examined, a recent genetic analysis of populations of Gila nigrescens based on the mitochondrial genes ND2 and ND4, and microsatellites, indicated that New Mexico populations (Mimbres River) are genetically distinct from those in the Guzman Basin of Mexico as well as from populations from the Rio Papigochic, Mexico. These distinct populations were considered evolutionary significant units (ESU) (Orborne et al., 2012). The Gila brevicauda lineage (area 4 Fig. 1), another clade resolved within the Chihuahuan Desert Group, represents a well-defined group when evaluated using either mtDNA or nDNA variation. Gila brevicauda was originally described from the headwaters of the Rio Mayo drainage (Norris et al., 2003). However, recent surveys have discovered populations within the Gila brevicauda lineage (cytb: 0.0–0.43%) in headwaters of three different Pacific drainages (rios Mayo, Fuerte and Yaqui). In the present study we extend its range to include the rios Fuerte and Yaqui, two adjacent western drainages in Chihuahua south and north of the currently understood distribution of the species (Fig. 1). Mitochondrial and nuclear analyses always resolved G. brevicauda as the sister species or closely related to G. pulchra (cytb 1.92–2.80%). The last lineage resolved within the Chihuahuan Desert Group includes specimens herein referred to as the Gila sp. 1 lineage [Tunal-Mezquital]. All analyses supported these specimens as a well-differentiated lineage highly distinct from all other regional congeners (cytb: 3.8–5.8%) and supports the hypothesis of these populations representing an undescribed species (Figs. 3–5). Genetic divergences for cytb between this lineage and G. conspersa, the species geographically surrounding these populations (cytb: 4.7–5.8%), were similar to that recovered between other closely related and recognized species (i.e. G. conspersa and G. pulchra: 4.3–5.2%). Previously, these populations were morphologically differentiated from geographically close congeners and have been referred to as Gila sp. cf. pandora (Smith and Miller, 1986; Minckley et al. 1986). The Gila sp. 1 lineage is distributed in different basins in the Tunal-Mezquital system in central-north Mexico (Zacatecas and Durango) (area 8 Fig. 1). The low genetic divergences observed within Gila sp. 1 (0.0–0.7%), may indicate recent connections between these now isolated basins, a pattern also observed in the species of Dionda inhabiting this same area (Schönhuth et al.,

2012a). The close relationship between this southern Pacific species inhabiting headwaters of the Rio Mezquital with remaining species of Gila inhabiting northern endorheic and Atlantic drainages, is also consistent with previous biogeographic hypothesis for the ancestral Rio Grande (Smith and Miller, 1986). A similar pattern of congruence between geographical and genetic structure was found between species of the genus Dionda, another cyprinid group, inhabiting this area (Mayden et al., 1992; Schönhuth et al., 2012a). We resolved other three species of Gila endemic to Mexico and inhabiting the western-most Pacific drainages: G minacae, G. eremica, G. diatenia (Fig. 1). These species were always resolved as reciprocally monophyletic within the Gila lineage, but were not part of the Chihuahuan Desert Group (Figs. 3–5). The Gila minacae lineage is widespread, occurring in the rios Yaqui, Fuerte and San Lorenzo (Figs. 1 and 3–5). No specimens were collected from Sinaloa and Culiacan despite previous reports in these drainages (Miller et al., 2005). The specimen from the headwaters of Rio San Lorenzo drainage was most closely related to populations of the species from the Rio Fuerte (cytb: 0.5–0.6%) than to the Rio Yaqui (cytb: 1.8–1.9%). Gila minacae Meek 1902, was considered a synonym of G. robusta or as a subspecies of G. robusta (G. robusta minacae) based on morphological similarities (Miller, 1959, 1976; Minckley 1973; Gilbert, 1998). Gila minacae was latter resurrected by Norris et al. (2003) based on genetic differences from G. robusta (Dowling unpubl. data), and different coloration in live specimens (as noted in communication from Minckley to Norris, published by Norris et al., 2003). In this study we explicitly recognize G. minacae as one of the most highly divergent species within the Gila lineage based on molecular results. This lineage (area 3 Fig. 1) was always well-supported (100%) and not closely related to G. robusta or to any species of the G. robusta complex inhabiting the Colorado River system (Figs. 3–5). Genetic distance for cytb between G. minacae and G. robusta was 6.9–7.8% or 6.3– 9.4% in comparisons between G. minacae and any species of the G. robusta complex. No genetic structure was detected in analyses of nDNA variation, unlike that in analyses of mtDNA wherein this lineage was resolved into two well-supported sister groups (cytb 1.5–1.9%). Our results suggest that morphological similarities between these two phylogenetically distant species (G. robusta and G. minacae) may be the result of only examining museum specimens, retained primitive morphologies, or by convergence on morphological features driven by factors involved in living in similar environments. The Gila diatenia lineage and the G. eremica lineage (areas 1 and 2 respectively, Fig. 1) were always recovered in reciprocally monophyletic and well-supported groups (100%). These two species inhabit Pacific drainages of northwestern Mexico, northwest of the Rio Yaqui. Gila diatenia inhabits the upper Rio de la Concepcion drainage (Sonora, Mexico), and was resolved as a very distinctive lineage for all genes. The sister-group relationship of this species was not well-resolved in any analyses (Figs. 3–5). The Gila eremica lineage included specimens inhabiting the upper Rio Matape and the Rio Sonora drainages (Sonora, Mexico). Within the G. eremica lineage, mtDNA analyses recovered low genetic divergences among specimens from both of these drainages (cytb: 0.7–1.0%). Gila purpurea (area 11 Fig. 1), a species geographically and phylogenetically close to Gila eremica is an endangered species and is restricted to a small region in the northern Rio Yaqui (Sonora, Mexico and Arizona, USA). Gila purpurea and G. eremica were recovered as reciprocally monophyletic groups and sister species in analyses of all genes (100%), a result consistent with the hypothesis of shared-derived morphological features describing G. eremica as a different species from G. purpurea (DeMarais, 1991). Genetic divergence between these two species was 2.0–2.54% (cytb). Nuclear

S. Schönhuth et al. / Molecular Phylogenetics and Evolution 70 (2014) 210–230

DNA and four concatenated region analyses resolved G. purpurea and G. eremica as sister group to G. minacae (Figs. 3–5). Patterns of endemism per river basin and phylogenetic relationships within the Gila lineage identified 3 major areas of diversification in southern North America: Pacific drainages (areas 1, 2, 3, 11); Atlantic region including past tributaries of the ancestral Rio Grande System, including endorheic basins (areas 4, 5, 6, 7, 8, 9, 10); and the Colorado drainage (areas 12–17). The herein inferred phylogenetic relationships and distributions of the major southern lineages provide evidence that, in general, southern lineages follow a pattern of endemism per river basin, and with the exception of those species from the Colorado River basin, single species inhabit each major drainage in southern North America. Each lineage exhibited limited intraspecific genetic variability but were genetically well-differentiated from adjacent geographic lineages (Figs. 1 and 3–5). This study provides evidence, using both phylogenetic and geographic relationships, that distributional patterns of species of Gila in Mexico likely follow a evolutionary process of speciation predicted under Model I vicariance (Wiley, 1981), where species are replaced in different drainages with those of the same species or sister/closely related species. In addition to this proposed Model of allopatric speciation, other particular distributions and phylogenetic relationships among Gila are consistent with Model III speciation, a mode that involves divergences of populations from small geographic areas (Mayden, 1999, 2002). The sister group relationships observed among the widely distributed G. pulchra complex (Conchos drainage, Atlantic) and G. brevicauda (headwaters of rios Yaqui, Fuerte and Mayo, Pacific), among G. eremica (Sonora-Matape) and G. purpurea (northern Yaqui), or among G. pandora (Pecos and Rio Grande) and G. modesta (San Juan) may suggest divergences via the peripheral isolation mode of speciation. On the other hand, the effect of the continental divide in southern species of Gila is diffused by multiple new records for native species from Mexico, inhabiting geographically close headwaters of different drainages on both sides of the Continental Divide. These data may corroborate the previous hypotheses of headwater captures between drainages in this general area (Schönhuth et al., 2011, 2012a). 4.3. Hybridization within the Gila lineage Hybridization is common for freshwater fishes, and more than 30% of all hybrids are within the family Cyprinidae (Scribner et al., 2001). Interspecific hybridization has been reported among species of the genus Gila in the Colorado River system and suggested as a major mechanism for the origin of some species of Gila (DeMarais et al., 1992; Rosenfeld and Wilkinson, 1989; Dowling and DeMarais, 1993; Dowling and Secor, 1997; Minckley and DeMarais, 2000; Gerber et al., 2001). As observed in other groups of animals, mtDNA introgress more easily than nDNA, and reasons for this are unclear, but unlike nDNA, mtDNA might be free to introgress neutrally between related species (Coyne and Orr, 2004; Bachtrog et al., 2006). Incongruences between mitochondrialand nuclear-inferred gene trees have been used as evidence for identifying genetic introgression in some groups of teleosts due to this asymmetric introgression (Bossu and Near, 2009; Schönhuth and Mayden, 2010; Near et al., 2011). Our study based on mtDNA and nDNA diversity of all species of Gila and related groups provides a powerful tool to detect molecular incongruences, and examine multiple explanations rather than a priori considering them the result of hybridization. Traditionally, hybridization has been reported among different species of the Gila robusta complex in the Colorado River System, where different hybridization patterns vary geographically (DeMarais et al., 1992; Dowling and DeMarais, 1993; Minckley and

223

DeMarais, 2000; Gerber et al., 2001). In these studies the differences among allozymes, morphology, and/or restriction enzymes were considered evidences for the hybrid origin of some Gila, as proposed for G. seminuda (G. robusta  G. elegans) and G. nigra (G. robusta  G. intermedia) (DeMarais et al., 1992; Rosenfeld and Wilkinson, 1989; Minckley and DeMarais, 2000). In both species, G. robusta was suggested as one parental species prone to hybridize with geographically close species. In our study the six species analyzed that occur in the Colorado River drainage, traditionally included in the Gila robusta complex, were never supported as a group (Figs. 1 and 3–5). These species did not form six reciprocally monophyletic groups, but were nested in three distinct and well-supported mitochondrial lineages: Group 1: G. cypha–G. robusta; Group 2: G. nigra–G. robusta–G. intermedia; Group 3: G. elegans–G. seminuda (Fig. 3). Analyses of nDNA supported the existence of Group 1 and a combined Groups 2 + 3 (Fig. 4). Phylogenetic analyses of mtDNA and nDNA variation never supported the monophyly of G. robusta, being resolved in two lineages (Groups 1 and 2). Resolution of G. robusta as being paraphyletic in both mtDNA and nDNA analyses could be a result of lineage sorting, shared primitive alleles in these species, or deep hybridization where two G. robusta lineages contain mtDNA and nDNA of heterospecific origin (Figs. 3–5). This pattern of phylogenetic resolution may be the result of introgression between G. cypha and G. robusta morphotypes of Group 1, as prior hypothesized by Rosenfeld and Wilkinson (1989) and Gerber et al. (2001) using morphology and mtDNA. Similarly, the close relationship of some G. robusta to G. nigra and G. intermedia in Group 2 may involve incomplete lineage sorting, hybridization (as suggested in prior studies: DeMarais et al., 1992; Dowling and DeMarais, 1993; Minckley and DeMarais, 2000) or misidentification of specimens from which tissue were supplied (Figs. 3–5). On the other hand, mtDNA variability reveals G. elegans and G. seminuda intimately related in a distinct and strongly supported Group 3, while nuclear variation revealed the haplotypes of G. seminuda as being paraphyletic. These results could suggest mitochondrial introgression and/or incomplete lineage sorting, but are also consistent with earlier studies revealing G. seminuda as a species of possible hybrid origin, possessing nuclear alleles (allozyme analyses) of both Gila robusta and G. elegans, but only mtDNA genetic variability of G. elegans (Rosenfeld and Wilkinson, 1989; DeMarais et al., 1992). MtDNA analyses strongly supported specimens of G. atraria as closely related (cytb 0.9–1.4%) to the G. cypha-robusta group, and limited phylogenetic resolution for individuals of G. atraria with respect to the cohesion of this species (cytb 0.2–0.7%). However, variation in nuclear genes did resolve G. atraria as monophyletic, it was resolved as the sister to G. orcutti-Moapa clade, well differentiated from the G. cypha-robusta group, and not included in the other two major well-supported clades within the Gila lineage. The high mtDNA genetic similarity between G. atraria and the G. cypha-robusta group, where the former species possesses a generalized morphology and the latter two species that are highly adapted forms for fast-flowing riverine habitats, can be explained by possible mtDNA transfer of G. cypha into G. atraria (as previously suggested by Smith et al., 2002), but may also be indicative of limited genetic divergence for the gene examined, or incomplete lineage sorting. MtDNA analyses placed some of the species inhabiting the Colorado River system and part of the Gila robusta complex (Groups 2 and 3) in a clade with Moapa, Gila eremica, G. purpurea and G. diatenia, all taxa from adjacent basins (Figs. 1 and 3). This clade could be indicating common ancestry. However, evidence for this group is not observed in nDNA analyses, where these four taxa are well separated from species of the Gila robusta complex (Fig. 4). These

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results may indicate ancient hybridization and mtDNA introgression, suggesting that the Colorado River basin and adjacent rivers could have been an area of mtDNA introgression in the past. Contrary to the hypothesized pattern of hybridization observed for species of Gila inhabiting the Colorado River system and some adjacent basins there are no reported cases of hybridization between species of Gila in the Chihuahuan Desert that are co-distributed in the headwaters of some drainages of the Sierra Madre Occidental (rios Yaqui and Fuerte). The limited studies of species of Gila in Mexico may account for the observed pattern for specimens of Gila inhabiting the upper Rio Yaqui and Laguna Bavicora. These specimens herein referred as G. nigrescens share very similar mtDNA haplotypes with G. nigrescens from the Guzman Basin, but nDNA variation places these specimens as closely related to G. pulchra and G. brevicauda (Figs. 3–5). Hypotheses for these particular molecular inconsistencies are discussed above under this complex. Hybridization among species of Gila in the Colorado River system is much more common than observed in southern species of the Chihuahua Desert Group. The lack of detailed studies on the Mexican species may account for these differences. However, the greater occurrence of hybrids in the Colorado River could be explained by paleogeographical data. Drainage configurations in the semi-desert areas of western North America were highly modified between the Miocene to Pleistocene (Miller, 1945b; Minckley et al., 1986; Miller and Smith, 1986). Recurrent changes in drainage configurations connecting different lineages may have contributed to the hypothesized hybridization allowing opportunities for gene exchange among these taxa. Alternatively, another possibility is highly polymorphic common ancestor and each of the species retained elements of the polymorphism that were maintained through time. Our data support geography as the most important factor to promote hybridization and cases of lateral gene transfer in Gila, independent of morphological or molecular similarity. We argue that more progress in understanding speciation and evolution within the Gila lineage will come from studies coupling biogeSpecies of Gila

Locality/drainage (field collection numbers if known)

Gila conspersa

Medina, Nazas Dr., MX. Trib. Río Aguanaval, Near Atotonilco, Zacatecas, MX. (NJL02-134)

Sampling localities for the specimens examined in this study. Collection numbers are listed for vouchers stored at institutional collections followed by tissue/DNA numbers. Dr. Drainage; Co: County; USA: United States of America; MX: Mexico. DNA region Rhod

S7

cytb

SN86 ATE17 UTGA1

SN86 ATE17 UTGA1

SN86 ATE17 UTGA1

UAIC 15346.01 (GB0650) GB0650

GB0650

GB0650

GB0650

UAIC 15284.01 (GSP251) GP251

GSP251

GP251

GSP251

Río Candamena just downstream of Basaseachic, Mayo Dr., Chihuahua, MX. (DAN06-50) Río Mayo trib. W of Basaseachic, Mayo Dr., Chihuahua, MX. (DAH2006-03-25-1) Arroyo Bravo at Cahuisori, Mayo Dr. Chihuahua, MX. (AP01.07.13.9)

Upper Klamath Lake, Klamath Co., Oregon, USA. (Population 7453)

Appendix A

SN86 ATE17 UTGA1

Gila brevicauda

Upper Klamath Lake, Klamath River, Klamath Co., Oregon, USA.

We thank Jim Brooks, John Hatch, Bernie Kuhajda, David Neely, David Propst, and Joe Tomelleri for their multi-year and steadfast assistance in multiple expeditions into Mexico sampling specimens. We are also grateful to Anabelia De Los Santos and Miguel Correa for their help with specimen collection in Mexico. We thank Tomas E. Dowling (ATE17, UTGA1, MARSA8, 10FOS125, PAN021, GPP21, AVGR2, CHGR8, LIT4, WFD4, SC178), Dennis Shiozawa (BYU 239498, BYU 88860, BYU88870, BYU 140722-23), David Ward (GE9061-2, GR9051-2), Alexandra M. Snyder (MSB 76482), and Dennis M. Stone (GE01, GE 21-22, GE 03, GE 04-05) for DNA, tissues or specimens used in this study. We thank Guillermo Orti and anonymous reviewers for comments and critical review. Our thanks are also extended to the Government of Mexico for collecting permits to Héctor Espinosa (Permisos de Pesca de Fomento DGOPA 01864/210205-0765; DGOPA 03947/250406-1606; and SEMARNAT, LCCIN FAUT-0117), Francisco J. García de León (Permiso de Pesca de Fomento DGOPA 00571/260108-0292), and Lourdes Lozano-Vilano (Permiso de Pesca de Fomento DGOPA 01430/ 060307-0479). This research was supported by the USA National Science Foundation Grants to RLM (EF-0431326, DEB 0240184, DEB-0817027, DBI-0956370, DEB-1021840) and by Spanish BBVA-Foundation supporting projects in Ecology and Conservation Biology (REF. 060501070030).

Rag1 Sevier River, Piute Co., Bonneville Basin, Utah, USA. (SN86) Sevier River, Piute Co., Bonneville Basin, Utah, USA. (ATE17) Gandy Spring, Bonneville Basin, Tooele Co., Utah, USA. (UTGA1)

Gila coerulea

Acknowledgments

Voucher (tissue or DNA)

Gila atraria

Río Basaseachic, Puente Basaseachic, Mayo Dr., Chihuahua, MX. (AP01.07.14.5) El Concheño, 8 mi NW Basaseachic, Mayo Dr., Chihuahua, MX. (CBD09-23) Río Candamena, below Basaseachic falls, Mayo Dr., Chihuahua, MX. (CBD09-25) Arroyo Banderilla at mouth of Arroyo Saucito, Yaqui Dr., Chihuahua, MX. (DAN05-18) Arroyo San Vicente at bridge, Río Oteros, Fuerte Dr., Chihuahua, MX. (CBD09-28)

ography, ecology and genetics of the species within a temporal context and phylogenetic backdrop.

MNCN 279689 (AP7139, AP71318, AP7132, AP7133) MNCN 279690 (AP7145)

AP7139,AP7132-3,AP71318

UAIC 15305.02 (GB0923)

GB0923

UAIC 15307.02 (GP0925)

GP0925

AP7145

UAIC 14969.03 (BRK514) BRK514

BRK514

BRK514

BRK514

UAIC 15310.02 (GM0928) GM0928

GM0928

GM0928

GM0928

OS 15083-1 (GC0831, SN20) OS 15082-3 (GO0833) BYU 239498 (GC498)

GC0831

GC0833

GC0833

GC0831, SN20

GC498

GC498

GC498

GC0833 GC498

(MNCN 1444) (G134)

1444 G134

1444 G134

1444 G134

1444 G134

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S. Schönhuth et al. / Molecular Phylogenetics and Evolution 70 (2014) 210–230 (continued) Species of Gila

Locality/drainage (field collection numbers if known)

Voucher (tissue or DNA)

DNA region Rag1

Gila cypha

Bajo Puentes (Molinos/Cilindro) a 100 m de Palmitos, Carretera Durango-Hidalgo del Parral, Nazas Dr., Durango, MX. Río Atotonilco at Saint Alto, Aguanaval Dr., Zacatecas, MX. Río Atotonilco at Pte. Atotonilco, Aguanaval Dr., Zacatecas, MX. Río Aguanaval at Río Grande, Zacatecas, MX. Arroyo at Saint Bajo, Aguanaval Dr., Zacatecas, MX. Arroyo at Puente Angosto 50 m. W of Entronque to Barrancas, Aguanaval Dr., Zacatecas, MX. Río Aguanaval at Rincón de la Florida, Zacatecas, MX. Río Aguanaval at Melchor Ocampo, Zacatecas, MX. Río Aguanaval at Oran, Zacatecas, MX. Río Aguanaval at Luis Moya, Zacatecas, MX. Spring at Miguel Hidalgo at Hwy 45, Aguanaval Dr., Zacatecas, MX. (SS10-17) Spring at Salitre, Río Ramos, Nazas Dr., Durango, MX. Río Aguanaval at Colonia 20 de Noviembre, Aguanaval Dr., Zacatecas, MX. Spring at Puente Cuarto I on Hwy 45, 34 km S of Rodeo, Nazas Dr., Durango, MX. (RLM10-25) Río Nazas at los Amoles, Nazas Dr., Durango, MX. Río Nazas at Abasolo, Nazas Dr., Durango, MX. Río Ramos at Santiago Papasquiaro, Río Ramos, Nazas Dr., Durango, MX. Río Tepehuanes at corrales, Río Tepehuanes, Nazas Dr., Durango, MX. Arroyo Morelos en Zape, Río Sextin, Nazas Dr., Durango, MX. Arroyo Las Escobas at San José Maria Morelos, Río Sextin, Nazas Dr., Durango, MX. Río Sextin at las Sardinas, Río Sextin, Nazas Dr., Durango, MX. Río San Juan at José María, Nazas Dr., Durango, MX. Bajo Puentes (Molinos/Cilindro), 100 m from Palmitos, road Durango-Hidalgo del Parral, Nazas Dr., Durango, MX. Presidio Lourdes Río San Diego, Pueblo Nuevo, Presidio Dr., Durango, MX. Colorado River, Coconino Co., Arizona, USA. Willow Beach National Fish Hatchery, originally from Little Colorado River, Arizona, USA.

Dolores River, Montrose Co., Colorado Dr., Colorado, USA. (identified as G. robusta)

Gila elegans

Gila eremica

S7

cytb GSPH17

UANL 18751 (G038)

G038

G038

G038

G038

UANL 18749 (G039)

G039

G039

G039

G039

UANL 18783 (G050) UANL 18853 (G052) UANL 18840 (G060)

G052

G052

G052

G050 G052 G060

UANL 18867 (G063) G063 UANL 19937 (G235) UANL 18879 (G228) UANL 18886 (G231) UAIC 15384.03 (GC1017)

G063

G063

G063 G235 G228 G231 GC1017

UANL 19480 (G240) UANL 19936 (G242)

G240 G242

UAIC 15391.02 (GSP2269)

GSP2269

UANL 19240 (G166A) UANL 19245 (G169) UANL 19253 (G180)

G166A

G166A

G166A

G166A G169 G180

UANL 19255 (G181)

G181

UANL 19260 (G187)

G187

UANL 19263 (G189)

G189

UANL 19267 (G193B)

G193

G193

G193

G193

UANL 19251 (G178) IBUNAM-P15673 (GSPH15)

G178 GSPH15

UANL 21157 (GSP277, GSP278) (GC-1, GC-2) SLUM 5524-RLM8302 (GC05) SLUM 5521-RLM8306 (GE021) SLUM 5521-RLM8307 (GE022) SLUM5 523-RLM8305 (GE04) BYU 88860 (GR122011)

GSP277, GSP278

BYU 88870 (GR122012) Gila diatenia

Rhod

IBUNAM-P15673 (GSPH17)

GC2

GC2

GC2

GC1, GC2 GC05

GE021

GE021

GE021

GE021 GE022 GE04 GR122011

GR122012 GR122012 GR122012 GR122012

Arroyo Atascosa at Hwy 15, South from Nogales. Río de la Concepción, Sonora, MX. (CBD09-16) Bridge Hwy 15. Río de la Concepción, Sonora, MX. (CBD09-15)

UAIC 15299.02 (GD0916, GD0916 GSP0916) UAIC 15298.02 (GD0915) GD0915

GD0916

GD0916

GD0916, GSP0916

GD0915

GD0915

GD0915

Dexter National Fish Hatchery, New Mexico, USA.

GE01

GE01

GE01

Achii Hanyo National Fish Hatchery, Arizona, USA.

SLUM 5520-RLM8308 (GE01) SLUM 5522-RLM8303 (GE03) (GE9061, GE9062)

Río Sonora, at Arizpe, Sonora Dr., Sonora, MX. (AP01.07.10.01) Río Sonora at El Cahui (1 km La Labor), Sonora Dr., Sonora, MX. (AP01.07.09.2) Río Matape, just W San José de Pimas on Hwy 16, Sonora, MX. (CBD09-13) Arroyo San Miguel, Sonora Dr., Sonora, MX. (CBD09-14)

MNCN 279687 (AP7101, AP71021) MNCN 279686 (AP791, AP792) UAIC 15296.01 (GE9131, G09132) UAIC 15297.03 (GE00914)

GE01

GE03 GE9061 GE9062

GE9061 GE9062

GE9061 GE9062

GE9061 GE9062

AP7101

AP7101

AP7101

AP7101, AP71021

AP791

AP791

AP791

AP791-2

G09132

G09132

G09132

G09131, G09132

GE0914

GE0914

GE0914

GE0914

(continued on next page)

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S. Schönhuth et al. / Molecular Phylogenetics and Evolution 70 (2014) 210–230

(continued) Species of Gila

Locality/drainage (field collection numbers if known)

Voucher (tissue or DNA)

Gila intermedia

Spring Creek tributary to Verde River, near Cottonwood, (GSP9091, GSP9092) Gila River, Colorado Dr., Arizona, USA.

Gila minacae

Río Batopilas, 15 km upstream Batopilas, Fuerte Dr., Chihuahua, MX. (JEB05-05) Río Verde at MX Hwy 24, Fuerte Dr., Chihuahua, MX. (RLM07-07) Río Remedios at Sapioris, San Lorenzo Dr., Durango, MX. (RLM10-20) Río Verde at bridge on Hwy 24, Fuerte Dr., Chihuahua, MX. (CBD09-09) Río Bavispe at Tres Rios, Yaqui Dr., Sonora, MX. (DAN0519) Río Sahuaripa at Sahuaripa, Yaqui Dr., Sonora, MX. (AP01.07.11.5) Arroyo El Cocono at Hwy between Mesa Tres Rios and Largo, Río Negro, Yaqui Dr., Sonora, MX. (BRK07-97) Arroyo El Pedernal, Río Bavispe, Yaqui Dr., Sonora, MX. (BRK07-94) Arroyo Mesa Colorado, Río Gavilán, Yaqui Dr., Chihuahua, MX. (CBD09-20)

Gila modesta

UAIC 14983.01 (GSP14, JEB2247) UAIC 14973.03 (G0707)

DNA region Rag1

Rhod

S7

cytb

GSP9091

GSP9091

GSP9091

GSP9091

GSP9092

GSP9092

GSP9092

GSP9092

JEB2247

JEB2247

JEB2247

GSP14, JEB2247

G0707

G0707

G0707

G0707

GSP2231

GSP2231

GSP2231

UAIC 15387.01 (GSP2231) GSP2231 UAIC 15294.02 (GM0909)

GM0909

UAIC 14970.02 (DAN519) DAN519

DAN519

DAN519

MNCN 279689 (AP7115) UAIC 14278.03 (GM07201)

DAN519 AP7115

GM07201 GM07201 GM07201 GM07201

UAIC 14275.01 (GM0793)

GM0793

UAIC 15302.02 (GM0920)

GM0920

Canon del Chorro, Río San Juan, 23 km from Saltillo, Río Salado, Rio Grande Dr., Coahuila, MX. (RLM05-61) Los Chorros, near Saltillo, Río Santa Catalina, Río Salado, Rio Grande Dr., Coahuila, MX. Los Chorros, Río Salado, Rio Grande Dr., Coahuila, MX. Arroyo on Iturbide, San Fernando Dr., Nuevo Leon, MX.

UAIC 15404.01 (MEX2021) (2462)

MEX20

MEX20

MEX20

MEX20-21

2462

2462

2462

2462

UANL 16162 (G030) UANL 19274 (G200A-E)

G200A-B

G200A-B

G200A-B

G030 G200A-E

Gila nigra

Gila River, Colorado Dr., USA. Marsh Creek, Gila Co., Arizona, USA. Fossil Creek above Irving, Yavapai Co, Arizona, USA.

(SN87) (marsa8) (10fos125)

SN87 marsa8 10fos125

SN87 marsa8 10fos125

SN87GB marsa8 10fos125

SN87 marsa8 10fos125

Gila nigrescens

Mimbres River at Cooney Place, Gila National Forest, Grant Co., New Mexico, USA. Río Papigochic on Hwy 16 at hacienda San Pedro, close to Minaca, Río Yaqui Dr., Chihuahua, MX. (RLM09-06) Arroyo La Madera at Gómez Farías, Bavicora Basin, Chihuahua, MX. (SS10-02) Arroyo between Campo 7B and Campo 7C, 10 km W Alvaro Obregon, Chihuahua, Bustillos Basin, Chihuahua, MX. (SS10-04) Río Casas Grandes at Ignacio Zaragoza, Chihuahua, MX. (SS10-01) Unnamed tributary to Piedras Verdes, E of Cuesta Blanca, Casas Grandes Dr., Chihuahua, MX. (BRK07-89-03) Río Santa Clara, at Hwy in Santa Clara, Chihuahua, MX. (CBD09-05) Río Santa Maria, East Namiguipa on Hwy 15, Chihuahua, MX. (CBD09-04) Río Terreno, 3 km SE Rancho Blanco, Río Papigochic, Yaqui Dr., Chihuahua, MX. (BRK05-07) Río Santa Clara, Chihuahua, MX. Sespe River, Ventura Co., Santa Clara Dr., California, USA. Rainbow Creek, San Diego Co., California, USA.

MSB 76482 (GN821– GN824) UAIC 15291.03 (GM399, GM400) UAIC 15371.03 (GSP10021, GSP10022) UAIC 15373.01 (GN1004, GN2038)

GN821

GN821

GN821

GN821-GN824

GM400

GM400

GM400

GM399, GM400

Gila orcutti

Gila pandora

Gila pulchra

GN1004

UAIC 15370.02 (CO1001, GN1001) UAIC 14270.03 (GN0789) GN0789

GN1004

GN1004

GN1004, GN2038

CO1001, GN1001 GN0789

GN0789

UAIC 15290.03 (GN0905)

GN0789

GN0905

UAIC 15289.03 (GN0904) GN0904

GN0904

GN0904

GN0904

UAIC 15001.03 (BRK57)

BRK57

BRK57

BRK57

BRK57

(G752, G757) UAIC 11044.02 (GO-1)

GO1

GO1

GO1

G752, G757 GO1

OS 15748-2 (GO748)

Rio Chama at US Hwy 84 near Arlequin, Rio Arriba Co., SLUM 662.01 (GP662) Rio Grande Dr., New Mexico, USA. (DAN03-144) Rio Chama, Rio Grande, Rio Arriba Co., Rio Grande Dr., (PAN021) New Mexico, USA. Pecos River, Rio Grande Dr., San Miguel Co., New (GPP21) Mexico, USA. Río Rituchi, Conchos Dr., Chihuahua, MX. (DAN05-13) Trib. to Río Bucoyua, Conchos Dr., Chihuahua, MX. (DLP05-5139) Arroyo de Recachi just upstream Arroyo Hojasichi, Conchos Dr., Chihuahua, MX. (DLP05-5143) Río Rituchi, Conchos Dr., Chihuahua, MX. (RLM06-01) Arroyo Tecubichi, N of Tecubichi, Conchos Dr., Chihuahua, MX. (BRK02-66)

GSP10021 GSP10021 GSP10021 GSP10021, GSP10022

GO748

GO748

GO748

GO748

GP662

GP662

GP662

GP662

PAN021

PAN021

PAN021

PAN021

GPP21

GPP21

GPP21

GPP21

UAIC 14968.01 (BRK09) (DLP5139) UAIC 15006.03 (DLP5143) DLP5143 UAIC 14241.03 (GP0601) (G0266)

BRK09 DLP5139 DLP5143

DLP5143

DLP5143 GP0601 G0266

227

S. Schönhuth et al. / Molecular Phylogenetics and Evolution 70 (2014) 210–230 (continued) Species of Gila

Locality/drainage (field collection numbers if known)

Voucher (tissue or DNA)

DNA region Rag1

Arroyo Sapareachi, Río Verde, Fuerte Dr., Chihuahua, MX. (RLM06-02) Río Bacochi at Pueblo Hojasichi, Conchos Dr., Chihuahua, MX. (DAN06-43) Arroyo Tonachi, Conchos Dr., Chihuahua, MX. (DAN06-44) Arroyo Maguillachi, Conchos Dr., Chihuahua, MX. (DAN06-47) Rancho Viejo, Río Balleza, Conchos Dr., Durango, MX Río Conchos, Conchos Dr., Chihuahua, MX. (BRK02-68) Río Conchos at Bocoyna North of Creel on Mx Hwy 25, Chihuahua, MX. (CBD09-07) Arroyo San Antonio, Río Balleza, Conchos Dr., Chihuahua, MX. (RLM07-06) Río Oteros, Fuerte Dr., Chihuahua, MX. (DAN05-07)

Astin Spring Trans, Cochiche Co., Yaqui Dr., Arizona, USA.

Gila robusta

Colorado River, Eagle Co., Colorado, USA. Bubbling Ponds Fish Hatchery, originally from Verde River near Cottonwood, Gila River, Colorado Dr, Yavapai Co., Arizona, USA. Aravaipa Creek, Pinal Co., Gila River, Colorado Dr., Arizona, USA. Cherry Creek, Gila Co., Gila River, Colorado Dr., Arizona, USA.

Gila seminuda

Gila sp. 1

cytb

UAIC 14988.02 (GP0643)

GP0643

UAIC 14990.03 (GSP2)

GSP2

UAIC 14993.03 (GP0647)

GP0647

(MDH3) UAIC 14967.04 (G0268) UAIC 15292.04 (CBD0907, G255) UAIC 14987.01 (G0706)

MDH3 G0268 GP0907, G255 G0706 DAN0507 DAN0507 DAN0507 DLP0540 SN69, G8724 G011 G070 G090

G070 G090

G070 G090 G122 G135 G0269

G0715

G0715

G0715 GP806 G804 G813

BYU 140722 (GP32251)

GP32251

GP32251

GP32251

GP32251

BYU 140723 GP32252)

GP32252

GP32252

GP32252

GP32252

UAIC 11624.01 (SN62) (GR9051, GR9052)

SN62 GR9051, GR9052

SN62 GR9051, GR9052

SN62 GR9051, GR9052

SN62 GR9051, GR9052

(AVGR2)

AVGR2

AVGR2

AVGR2

AVGR2

(CHGR8)

CHGR8

CHGR8

CHGR8

CHGR8

GS24701 GS24702 LIT4

GS24701 GS24702 LIT4

GS24701 GS24702 LIT4

GS24701 GS24702 LIT4

WFD4

WFD4

WFD4

WFD4

Virgin River, Washington Co., Colorado Dr., Utah, USA. BYU 56251 (GS24701) BYU 56257 (GS24702) Virgin River at Littlefield, Mohave Co., Colorado Dr., (LIT4) Arizona, USA. Washington Fields, Washington Co., Colorado Dr., Utah, (WFD4) USA. Río Frio at Veracruz, E. of Villa Unión, Endorheic Durango, MX. (RLM10-22) Río Guatimapé at Guatimapé on Hwy 23, Santiaguillo Lagoon, Durango, MX. (SS10-11) Arroyo on N side of Las Cotorras, 5 mi N of Nombre de Dios, Mezquital Dr., Durango, MX. (RLM10-21) Endorheic between Río Tunal–Río Aguanaval at Villa Unión, Durango, MX. (SS10-14) Colonia Orión, 15 km S. Sombrerete, Mezquital Dr., Zacatecas, MX. Arroyo en Progreso, 15 km N. of Canatlan, Mezquital Dr., Durango, MX. Río La Sauceda, above Francisco Villa Dam, E of Villa Unión, Endorheic, Durango, MX.

S7

GSP0602

UAIC 14995.02 DAN0507 (DAN0507) Trib. to Río Urique at Umiva, Fuerte Dr., Chihuahua, MX. UAIC 14998.02 (DLP0540) (DAN05-09) Río Santa Isabel at General Trias, Conchos Dr., UAIC 7910.01 (SN69, Chihuahua, MX. (RLM87-24) G8724) Río San Pedro at San Francisco de Borja, Conchos Dr., UANL 16072 (G011) Chihuahua, MX. Río San Pedro at Satevo, Conchos Dr., Chihuahua, MX. UANL 18914 (G070) G070 Río San Pedro at Riva Palacio, Conchos Dr., Chihuahua, UANL 18923 (G090) G090 MX. Río Conchos at Hidalgo del Parral, Conchos Dr., UANL 19011 (G122) Chihuahua, MX. Río Florido at Villa Coronado, Conchos Dr., Chihuahua, UANL 19036 (G135) MX. Lower Arroyo Tomachi–upper Arroyo Culebra, Conchos SLUM 1553.03 (G0269) Dr., Chihuahua, MX. (BRK0269) Arroyo del Molino, Conchos Dr., Chihuahua, MX. (G0715) G0715 (RLM07-03) Arroyo Rancho Viejo, Río Balleza, Conchos Dr., Durango, UAIC 14958.02 (GP806) MX. (RLM08-06) Cuevas Blancas, Río Balleza, Conchos Dr., Chihuahua, UAIC 14957.03 (GP804) MX. (RLM08-04) Arroyo el Molino, Conchos Dr., Chihuahua, MX. (RLM08- UAIC 14965.02 (GP813) 13) Gila purpurea

Rhod

UAIC 14982.01 (DSP0602)

UAIC 15389.03 (GSP2264)

GSP2264

UAIC 15379.01 GSP10111 GSP10111 GSP10111 GSP10111 (GSP10111) UAIC 15388.01 (GSP2242) GSP2242 UAIC 15382.01 (GSP10141, GSP10142) UANL 18745 (G040A, B)

GSP10141, GSP10142 G040B

G040B

G040B

G040A, B

UANL 19269 (G165)

G165

G165

G165

G165

IBUNAM-P15751 (GSPH927, GSPH930, GSPH940, GSPH941)

GSPH930

GSPH930

GSPH930

GSPH927, GSPH930, GSPH940, GSPH941

(continued on next page)

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S. Schönhuth et al. / Molecular Phylogenetics and Evolution 70 (2014) 210–230

(continued) Species of Gila

Locality/drainage (field collection numbers if known)

Voucher (tissue or DNA)

DNA region Rag1

Río Caliente, above Francisco Villa Dam, E of Villa Unión, IBUNAM-P15785 Endorheic, Durango, MX. (GSPH943, GSPH945) IBUNAM-P15769 (GSPH311, GSPH312) Río Poanas, below Francisco Villa Dam, Endorheic, (GSPH8881, 8882) Durango, MX. (WWFDGO 08-09). Species

Locality/drainage

Voucher (tissue or DNA)

S7

cytb GSPH943, GSPH945

GSPH311, GSPH311, GSPH311, GSPH311, GSPH312 GSPH312 GSPH312 GSPH312 GSPH8881, GSPH8882

DNA region Rag1

Revised Western Clade Acrocheilus Kettle River below falls at Cascade, Columbia Dr., British UAIC 11365.01 (AA1993) AA1993 alutaceus Columbia, Canada. Silvies River downstream of dam, 4.7 mi N Burns, UAIC 11650.01 (AA9743) Harney Co., Malheur Lake, Oregon, USA. Eremichthys acros

Rhod

Soldiers Meadow Spring, Lahonan Basin, Humboldt Co., UAIC 13007.01 (EA13007) EA13007 Nevada, USA. Black Rock Ranch, Desert Soldier Meadows, Lahonan (EA537) EA537 Basin, Humboldt Co., Nevada, USA.

S7

cytb

AA1993

AA1993

AA1993 AA9743

EA13007

EA13007

EA13007

EA537

EA537

EA537

Hes1

Hes1

Hes1

Hesperoleucus symmetricus

Alameda Creek, California, USA.

(Hes1)

Lavinia exilicauda

Putah Creek, Yolo Co., California, USA.

OS 015074 (SN88)

SN88

SN88

SN88

SN88

Moapa coriacea

Muddy River, Clark Co., Nevada, USA.

UAIC 13165.01 (MC6, MC9)

MC06

MC06

MC06

MC06

MC09

MC09

MC09

MC09

Mylopharodon conocephalus

Kern River, 16 km above Kernville, Tulare Co., California, UAIC 11548.02 USA. (CTOL02622) California, USA. (DAN07-43.01) (MC4301)

MC4301

MC4301

MC4301

MC4301

Orthodon microlepidotus

San Luis Reservoir, Merced Co., California, USA. (RLM96- UAIC 11546.01 (OM9659) CTOL447 59)

CTOL447

CTOL447

CTOL447

Ptychocheilus grandis

Kings River 2 mi SW of Piedra at Alta Wier, San Joaquin UAIC 11547.02 River, Fresno Co., California, USA. (PG11547) Kern River, 16 km. above Kernville, Tulare Co., UAIC 11548.01 California, USA. (RLM96-62) (CTOL02631)

PG11547

PG11547

PG11547

Ptychocheilus lucius No locality data (SN80) Dexter National Fish Hatchery Broodstock, New Mexico, (PL9041) USA. Ptychocheilus oregonensis

Ptychocheilus umpquae

Snake River at mouth of Billingsley Creek at Lower Salmon Falls boat ramp. Snake River. Gooding Co., Idaho, USA. (BRK97-57) Silvies River downstream of dam, 4.7 mi N Burns (Malheur Lake), Harney Co., Oregon, USA. (BRK97-61)

Hes1

Rhod

COTL2622 CTOL2622 CTOL2622 CToL2622

PG11547

CTOL2631 CTOL2631 CTOL2631 CTOL2631 SN80 PL9041

SN80 PL9041

SN80 PL9041

SN80 PL9041

UAIC 11633.02 (PO9739) PO9739

PO9739

PO9739

PO9739

UAIC 11650.02 (PO9761) PO9761

PO9761

PO9761

PO9761

Umpqua R. Main stem, Douglas Co., Oregon, USA (#43) OS 17899 (PU43)

PU43

PU43

PU43

PU43

Jackson Creek, South Umpqua R., Douglas Co., Oregon, OS 17887 (PU154) USA. (#154)

PU154

PU154

PU154

PU154

Relictus solitarius

Odgers Creek, Elko Co., Nevada, USA. Odgers Creek, Elko Co., Nevada, USA.

OS 15745-1 (RS745) (RS6881)

RS745 RS6881

RS745 RS6881

RS745 RS6881

RS745 RS6881

Siphateles alvordensis

Trout Creek, Albord Basin, Harney Co., Oregon, USA. (PMH9317-1)

(SA317)

SA317

SA317

SA317

SA317

Siphateles bicolor

Upper Klamath Lake, Oregon, USA. (Population 7459) Upper Klamath Lake, Klamath Co., Oregon, USA. (DAN07-90.03)

BYU 239544 (SIBI544) (SB9003)

SIBI544 SB9003

SIBI544 SB9003

SIBI544 SB9003

SIBI544 SB9003

Siphateles b. mohavensis

China Lake Naval Sta., San Bernardino Co., Mohave basin, California, USA. (BB043)

(SBM43)

SMB43

SMB43

SMB43

SBM43

Siphateles boraxobius

Borax Lake, Alvord Basin, Harney Co., Oregon, USA. (PMH9316-011)

(SB316)

SB316

SB316

SB316

SB316

Non-Western Clade Algansea lacustris Agosia chrysogaster Platygobio gracilis Snyderichthys copei

Lake Paztcuaro, Lerma Dr., Michoacán, MX. No locality data Little White River, Melette Co., South Dakota, USA. Sulfur Creek, Bear River, Uinta Co., Wyoming, USA.

(MNCN 3025, 3026) UAIC 13018.01 (SN10) UAIC 11169.03 (SN56) (SC178)

3026 SN10 GU136347 SC178

3026 SN10 SN56 SC178

3026 SN10 GU134253 SC178

3025-26 SN10 SN56 SC178

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