Evolutionary relationships of Fish Lake Valley Tui Chub Siphateles obesus ssp. (Teleostei, Cypriniformes, Leuciscidae) and a new genus of leuciscid minnows from the Alvord Basin, western United States

39 Evolutionary relationships of Fish Lake Valley Tui Chub Siphateles obesus ssp. (Teleostei, Cypriniformes, Leuciscidae) and a new genus of leuciscid minnows from the Alvord Basin, western United States Matthew A. Campbell1,2 , Serra C. Perry1, Khyana N. Yearwood1, Grace Auringer1, Nick Buckmaster3, Amanda J. Finger1 1 Genomic Variation Laboratory, Department of Animal Science, University of California, One Shields Avenue, Davis CA, 95616, USA 2 Fishes and Marine Invertebrates, University of Alaska Museum of the North, 1962 Yukon Drive, Fairbanks AK, 99775, USA 3 California Department of Fish and Wildlife, Inland Deserts Region, Heritage and Wild Trout Program, Bishop, CA 93514, USA Corresponding authors: Matthew A. Campbell ([email protected]); Amanda J. Finger ([email protected]) Copyright: © Matthew A. Campbell et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract The Tui Chubs, Siphateles spp., are found widely across the Great Basin and in some adjacent regions. Nearly all diversity of Siphateles has been consolidated under the name S. bicolor and there are numerous isolated populations of Tui Chubs of uncertain taxonomic standing and therefore unclear conservation priority. The Fish Lake Valley Tui Chub (FLVTC) has been recognized informally as S. bicolor ssp. 4 with a limited natural distribution in Fish Lake Valley in southwest Nevada. Considering that a rigorous examination of the phylogenetic relationships of the FLVTC and other Tui Chubs has not been conducted, the FLVTC is placed in a taxonomic framework by first applying a species delimitation method to S. bicolor sensu lato using mitochondrial data and then conducting phylogenetic analyses of genome-wide SNP data. Siphateles bicolor is better characterized by seven species, all with existing names, which here are considered to be valid Siphateles species. Furthermore, the separation of Alvord Basin Siphateles from other Siphateles is apparent as a deeply divergent lineage. As a result, we propose Epizon Campbell & Finger, gen. nov. to contain these fishes. The Fish Lake Valley Tui Chub is found to be the earliest-branching lineage of S. obesus in our SNP data set and are highly differentiated from other S. obesus. These findings are concordant with geologic evidence that indicates that Fish Lake Valley became connected to the broader Lahontan Basin ~2 million years ago, with gene flow possible until ~0.5 million years ago. Based on the geographic distribution and magnitude of genetic divergence, we find the recognition of FLVTC as a subspecies of S. obesus is appropriate. Key words: ASAP, Epizon, Great Basin fishes, molecular phylogenetics, phylogenetic networks, species delimitation methods Academic editor: Pedro Bragança Received: 28 February 2025 Accepted: 6 October 2025 Published: 21 November 2025 ZooBank: https://zoobank. org/5D51F97E-4F34-4E86-A2143339985E5632 Citation: Campbell MA, Perry SC, Yearwood KN, Auringer G, Buckmaster N, Finger AJ (2025) Evolutionary relationships of Fish Lake Valley Tui Chub Siphateles obesus ssp. (Teleostei, Cypriniformes, Leuciscidae) and a new genus of leuciscid minnows from the Alvord Basin, western United States. ZooKeys 1261: 39–67. https://doi.org/10.3897/ zookeys.1261.151636 ZooKeys 1261: 39–67 (2025) DOI: 10.3897/zookeys.1261.51636 40 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Introduction Encompassing a vast area of western North America, the Great Basin is generally bordered by the Columbia River to the north, the Sierra Nevada to the west, and by the Rocky Mountains to the east (Houghton 1976). The name Great Basin refers to the fact it is endorheic, and it is composed of large sub-basins, the Lake Bonneville System, the Central Basins, the Death Valley System, the Northwest Lakes Basin, and the Lake Lahontan System (Houghton 1976). The ichthyofauna of the Great Basin is taxonomically diverse, but not consistently distributed across all sub-basins and limited in species diversity. Aquatic species diversity in a biogeographic region is largely a function of historical processes that generate and preserve species diversity. For fishes in the Great Basin, two of these processes may be dispersal into and subsequent speciation within the Great Basin. Processes that generate diversity are opposed by processes that reduce aquatic species diversity, such as long periods of habitat reduction, aridity, and isolation. Isolation may also contribute to species diversity if populations are not reconnected or, if they do, reproductive isolation is in place. In the Great Basin, the Pleistocene was characterized by dramatic changes in habitat, with large pluvial lakes appearing and disappearing (Mifflin and Wheat 1979), alternating periods of dispersal and high habitat availability with periods of isolation, differentiation, and extinction. These periods of high and low water availability operated over drainage patterns that have been shaped on million-year time scales (e.g., Minckley 1986). During the history of the Great Basin, ecological and habitat preferences of fishes also influenced dispersal and population connectivity. High-gradient systems in foothills and mountains were subject to a higher frequency of inter-drainage headwater stream capture in comparison to lacustrine spillover affecting low-gradient systems. These higher gradient systems are typically occupied with mountain suckers (Pantosteus spp.), salmonids (Oncorhynchus spp. and Prosopium spp.) and sculpins (Cottus spp.). The low gradient and low elevation habitats more typically are occupied by suckers (Catostomus spp. and Chasmistes spp.), minnows (Leuciscidae), pupfishes (Cyprinodon spp.), and salmonids with this habitat preference (Smith et al. 2002). The distribution and diversity of Great Basin fish species result from these habitat associations and shared historical processes as well as chance. Nonetheless, the evolutionary history of all Great Basin fishes is reflected in their genetic structuring and genetic diversity (e.g., Finger et al. 2022; Su et al. 2022; Campbell et al. 2023). While species diversity is generated, maintained, and reduced by natural processes, its recognition is heavily influenced by scientific effort (Lundberg et al. 2000). Great Basin fishes remain incompletely documented and characterized taxonomically, and number ~55 recognized species-level taxa. Over such a large area, the Great Basin contains about 8% (55/683) of recognized fish species diversity in North America (Jenkins et al. 2015). These are from the Gasterosteidae (n = 1), Cottidae (n = 3–4), Cyprinodontidae (n = 5–7), Goodeidae (n = 4), Salmonidae (n ~ 10), Catostomidae (n ~ 13) and Leuciscidae (n ~ 16) (Smith et al. 2002; Sigler and Sigler 2014; Unmack et al. 2014; Campbell et al. 2023; Harris et al. 2025). 41 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub The leuciscid minnow lineages have a long presence in the Great Basin fossil record despite the challenges associated with preservation and recovery of fossils from these fishes, and several species are still present in this system. Fossil leuciscid minnows are known from at least the middle Miocene in the Great Basin (~16 mya) (Cavender 1986). Three genera have been persistent and are found widely, these are Gila Baird & Girard, 1853, the senior synonym of Moapa Hubbs & Miller, 1948, Rhinichthys Agassiz, 1849, and Siphateles Cope, 1883. Siphateles is known to be well-represented in the fossil record in the Miocene and Pleistocene of the Lahontan Basin and Mojave River (Smith et al. 2002). The genus Siphateles, despite being widely distributed across the Great Basin, contains only three valid species (Fricke et al. 2025). Siphateles bicolor (Girard, 1856) is distributed broadly across phylogeographic regions, including the Great Basin, in the Klamath River drainage, the Sacramento River drainage (Pit River), and the Columbia River drainage. The other two species are closely-related and are found in the Alvord Basin: S. alvordensis (Hubbs & Miller, 1972) and S. boraxobius (Williams & Bond, 1980), and they may belong to a separate genus altogether (Schonhuth et al. 2012). Divergence time estimation indicates that the Alvord Basin leuciscids has been an independent lineage for ~10 million years (Rabosky et al. 2018). All other Great Basin Siphateles have been placed under S. bicolor (Girard, 1856) and within S. bicolor there are various distinctive and isolated fish populations, but they are of uncertain taxonomic standing and thus unclear conservation priority. Molecular dating places the age of the lineage of fish classified as S. bicolor at ~ 12 million years (Kumar et al. 2017; Rabosky et al. 2018). In conjunction with fossil evidence, the distribution and ages of these fishes indicates that there was ample time and space for diversification within Siphateles beyond the currently recognized three species. Other genera with distributions spanning the Great Basin of that age exhibit distinct species such as Rhinichthys (Moyle et al. 2023), Pantosteus Cope, 1875 (Unmack et al. 2014), Catostomus Lesueur, 1817, and Chasmistes Jordan, 1878 (e.g., Campbell et al. 2023). In particular, there are geographically and genetically structured populations within these genera distributed among Columbian, Sacramento/Klamath and Lahontan geographic regions that may be recognized as species. Based on the age, wide range, occurrence across phylogeographic regions known for endemism, and lower elevation habitat preference that promotes fewer drainage basin exchanges it is plausible that S. bicolor contains several species and subspecies-level taxa. Lack of recognition of these taxa is, in part, because a rigorous and comprehensive phylogenetic examination of Great Basin Siphateles has not been undertaken to test the species boundaries of these diverse populations. An understanding of the diversity within S. bicolor and the relationships between this lineage and others will inform management and conservation decisions in this arid landscape. Here we conduct this analysis of Great Basin Siphateles and focus on the Fish Lake Valley Tui Chub (FLVTC) which has been recognized informally as S. bicolor ssp. 4. It occupies a limited natural distribution in Fish Lake Valley in southwest Nevada where it is the only extant native fish. Specifically, we conduct a molecular phylogenetic study with the aim of identifying the evolutionary relationships of FLVTC, current composition of the lineage, and its taxonomic standing. 42 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Materials and methods Mitochondrial phylogeny and species delimitation We obtained representative sequences of Siphateles from the NCBI GenBank drawing from a mitochondrial cytochrome b (cytb) PopSet 28190045 (Harris 2000). Outgroup sequences were selected from 12 leuciscid minnows in the Laviniinae subfamily with Chrosomus erythrogaster (Rafinesque, 1820) used for rooting (Schonhuth et al. 2012). Additional Siphateles sequences with identifying information that could expand representation were obtained from GenBank through blastn searches against the NCBI Nucleotide database using cytb sequences from PopSet 28190045 (Altschul et al. 1997). During the collection of public sequence data, we obtained a single potential representative mtDNA sequence from the Owens Valley (AF370056.1). The Owens Valley is known to contain distinctive endemic Siphateles lineages, but also out-of-basin genetics from human mediated movements (Chen et al. 2007). The single publicly available sequence was ambiguous due to these factors and we undertook Sanger sequencing of cytb sequences from 12 individuals from the Owens Valley of known native backgrounds to represent endemic Siphateles mitochondrial lineages. We used two primer sets to amplify two fragments of the cytb gene that were combined into a longer sequence. The primer sets were previously reported by Zardoya and Doadrio (1998) and used to generate previously published data from Siphateles (e.g., Schonhuth et al. 2012). The primers used were Glu-F 5’-GAAGAACCACCGTTGTTATTCAA-3’; Cytb-R 5’-TCTTTATATGAGAARTANGGGTG-3’, Cytb-F 5’-CACGARACRGGRTCNAAYAA-3’ and Thr-R 5’-CCTCCRATCTYCGGATTACA-3’ (Zardoya and Doadrio 1998). Polymerase Chain Reactions (PCR) were carried out in a total volume of 25 µL, using 5x Colorless GoTaq® Flexi Buffer (5 uL per reaction) with a final concentration of 200 µM of dNTP, 2 mM MgCl2, 0.2 µM of each primer, 0.625 U of GoTaq® DNA polymerase, and 15 ng of DNA extract. After an initial denaturation step of 2 min at 95 °C, we ran 40 PCR cycles consisting of 1 min at 95 °C, 1 min at 45 °C, and 1 min and 45 s at 72 °C. These cycles were followed by a 7-min final extension step at 72 °C. Forward and reverse direction chromatograms were aligned with Geneious (https://www.geneious.com), and then the two primer set contigs together were mapped to a reference sequence from NCBI (Siphateles bicolor; accession: AY096010.1) with the ‘Map to Reference’ function within Geneious. A multiple sequence alignment (MSA) of cytb sequences was made with MAFFT (Katoh et al. 2002; Katoh and Toh 2008; Katoh and Standley 2013) and Maximum-Likelihood (ML) phylogeny inferred with IQ-TREE2 (Nguyen et al. 2014; Minh et al. 2020). Model selection was conducted by specifying ModelFinder Plus (-m MFP) (Kalyaanamoorthy et al. 2017) and support for nodes evaluated with 10,000 rapid bootstraps (-bb 10000) (Hoang et al. 2018). To delimit possible species within S. bicolor, mitochondrial sequences identified as S. bicolor were uploaded to the Assemble Species by Automatic Partitioning (ASAP) webserver (Puillandre et al. 2021, https://bioinfo.mnhn.fr/abi/ public/asap/asapweb.html). Simple, uncorrected distance was specified . 43 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Individual and genomic sampling Access to Siphateles tissues was possible through recently collected specimens or from tissue archives. Material from across the Great Basin range of the genus (Fig. 2, Table 1), representing known divergent lineages of Siphateles with emphasis on potential near relatives of Fish Lake Valley Tui Chub was targeted (Fig. 1, Harris 2000). Fin clips were either dried and stored in coin envelopes or in 95% ethanol prior to extraction of DNA. Sampling included representatives of S. alvordensis (n = 13, one location) and a variety of potential species-level lineages currently classified as subspecies under S. bicolor: S. mohavensis Snyder, 1918 (n = 74, two locations); S. isolatus (Hubbs & Miller, 1972) (n = 25, one location); S. newarkensis (Hubbs & Miller, 1972) (n = 25, two locations); S. bicolor (n = 74, six locations); S. thalassinus (Cope, 1883) (n = 43, three locations); S. snyderi (Miller, 1973), including ‘toikona’ lineage fish (Chen et al. 2007) (n = 66, four locations and n = 50, two locations respectively). Siphateles obesus (Girard, 1856) were considered potential near relatives of FLVTC and nine locations were sequenced (n = 223). One location of S. obesus is located in Fish Lake Valley (McNett Ranch) and was considered representative of the FLVTC lineage. Sequences for rooting phylogenetic analyses were obtained from Gila orcuttii (Eigenmann & Eigenmann, 1890) and Pogonichthys macrolepidotus (Ayres, 1854). Determination of Siphateles lineages was in part informed by mitochondrial phylogenetic results (Suppl. material 1: fig S1), inclusion in previous studies, and by geographic locations. Siphateles alvordensis samples are from the Alvord Lake basin, and not from Borax Lake and can represent S. alvordensis. The location, Thousand Creek Gorge was investigated previously in genetic study of numerous Alvord Lake basin Siphateles populations by Smith et al. (2019) and is representative of S. alvordensis in that study. Siphateles mohavensis is extinct from its type locality, was translocated for conservation, and we sampled the descendants of this management action (Chen et al. 2013). Siphateles isolatus is also extinct from its type locality, but was sampled from nearby locations in Independence Valley. Siphateles newarkensis is endemic to springs in the Newark Valley with a restricted distribution, described from a spring on the western side of the valley. The NV12 sampling location is located ~5.5 km from the likely type locality as indicated by Hubbs and Miller (1972) as springs on an alluvial fan near Diamond Peak, on the west side of Newark Valley (Harris 2000). Siphateles bicolor is described from Upper Klamath Lake, with phylogenetic affinities to the material examined in this study indicated as S. bicolor, with Rock Creek in Catlow Valley sequenced for both mitochondrial and nuclear data types (Suppl. material 1: fig S1). Siphateles thalassinus is described from Goose Lake, in Oregon and we did not obtain material from that location for this study. However, we obtained material from close geographically proximity from the Warner Valley and the Pit River (Goose Lake may be considered part of the Pit River drainage). There are mtDNA lineages similar to Goose Lake samples present in Cowhead Slough (Suppl. material 1: fig S1) as well. Samples of S. thalassinus from Twenty Mile Slough and Big Sage Reservoir were 44 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub previously analyzed by Chen et al. (2009) with associated museum specimens OS 17847 and OS 17853 and are representative of S. thalassinus in that study. Siphateles snyderi and S. snyderi ‘toikona’ tissues were obtained largely from refuge populations maintained for this taxon, and may be considered representative of this lineage. The Mule Spring sampling location has been previously investigated as a refuge population of the ‘toikona’ genetic lineage, founded by translocation from Cabin Bar Ranch in 1990 (Chen et al. 2007). Siphateles obesus was described from the Humboldt River, and we were able to sample from this drainage basin for this study, and the mitochondrial phylogenetic analysis and geographic distribution are concordant with recognizing the sampling as being representative of S. obesus. Figure 1. Maximum Likelihood phylogeny of mitochondrial (mt) cytochrome b (cytb) data featuring ‘Siphateles bicolor’ labeled sequences with results of species delimitation indicated for six clusters. Nodal support is indicated by gray circles for bootstrap support (BS) > 90%, white circles for 90% > BS > 75% and not indicated for BS < 75%. Chrosomus erythrogaster Ptychocheilus grandis Epizon boraxobius Epizon boraxobius Epizon alvordensis Epizon alvordensis Epizon alvordensis Epizon boraxobius Epizon alvordensis Epizon boraxobius Ptychocheilus lucius Gila orcuttii Acrocheilus alutaceus Gila robusta Gila coerulea Relictus solitarius Hesperoleucus symmetricus Eremichthys acros Mylopharodon conocephalus Lavinia exilicauda S. obesus mt lineage S. bicolor mt lineage S. thalassinus mt lineage S. newarkensis mt lineage S. isolatus mt lineage S. mohavensis mt lineage S. snyderi samples 45 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Table 1. Taxonomic and geographic sampling. The larger geographic area (Vicinity) is indicated along with each sampling location. The sample size (n) used in analyses with the median read count after filtering is given as well as the total number of individuals sequenced (Total n). For the year collected, if collected in different years, the number retained from each year for analyses is given. The latitude and longitude and source of samples are also provided with any additional pertinent information. The taxonomic sampling is from Pogonichthys macrolepidotus (Ayres, 1854), Gila orcuttii (Eigenmann & Eigenmann, 1890), Epizon alvordensis (Hubbs and Miller, 1972) and Siphateles Cope, 1883. Siphateles is abbreviated S. in the taxon column with S. thalassinus (Cope, 1883), S. bicolor (Girard, 1856), S. newarkensis (Hubbs & Miller, 1972), S. isolatus (Hubbs & Miller, 1972), S. snyderi (Miller, 1973), S. mohavensis Snyder, 1918, and S. obesus (Girard, 1856). Taxon Vicinity Sampling location Median read count nTotal n Year collected Latitude, Longitude Source Pogonichthys macrolepidotus Unknown Unknown 2461487 912 CDFW Archives Gila orcuttii Mojave River Drainage Desert Discovery Pond 2053059 2 2 2012 Epizon alvordensis Sheldon Wildlife Refuge Thousand Creek Gorge 7589069 10 13 2023 41.8873, -118.9520 S. thalassinus Pit River Big Sage Reservoir 4944824 12 12 2005 41.5957, -120.6405 S. thalassinus Valley Falls Honey Creek 2065216 815 2011 42.4262, -120.1009 S. thalassinus Warner Spring Twenty Mile Slough 1439984 316 2004 42.1291, -119.8177 S. thalassinus Modoc County Cowhead Slough 2964294 12 20 2023 41.9193, -120.0322 S. bicolor Catlow Valley Rock Creek 1136559 3 6 2011 42.6854, -119.1888 S. bicolor Harney County Kueny Canyon 3307963 11 12 2005 42.6818, -118.9958 S. bicolor Sheldon Wildlife Refuge Andy’s Place 2348208 15 16 n = 4, 2022 n = 11, 2023 41.7916, -119.3846 S. bicolor Sheldon Wildlife Refuge Bitner Ranch 3167076 16 16 n = 4, 2022 n = 12, 2023 41.7368, -119.4686 S. bicolor Sheldon Wildlife Refuge Horse Canyon 7898712 4 4 2022 41.7863, -119.3244 S. newarkensis Newark Valley NN4 3110134 20 25 2022 39.7227, -115.6919 S. newarkensis Newark Valley NV12 5185390 25 25 2022 39.6498, -115.7700 S. isolatus Independence Valley Warm Springs 3322395 14 25 2022 40.9545, -114.7490 S. snyderi Mammoth Mountain Sotcher Lake 1571551 11 23 n = 1, 1998 n = 10, 2022 37.6286, -119.0737 S. snyderi ‘toikona’ Owens River Cottonwood Pond (White MT Research Center) 1787193 21 25 2010 37.3606, -118.3276 S. snyderi ‘toikona’ Owens River Mule Spring 1410489 10 25 2010 37.1061, -118.3276 S. snyderi Owens River NE Pond (White Mountain Research Center) 2154310 22 25 2017 37.3606, -118.3293 S. snyderi Owens River SE Pond (White Mountain Research Center) 2307926 210 2022 37.3604, -118.3294 S. snyderi Owens River SW Pond (White Mountain Research Center) 2532879 2 8 2022 37.3604, -118.3296 S. mohavensis Mojave River Camp Cady 2230368 16 19 n = 6, 1997 n = 10, 2005 34.9451, -116.5993 S. mohavensis Mojave River Drainage Tui Slough 1662314 35 55 2011 34.3483, -117.2411 S. obesus Carson River, Carson Desert Little Soda Lake 2696470 22 25 2006 39.5147, -118.8833 S. obesus Fish Lake Valley Lida Pond 2590847 928 2022 37.4571, -117.4959 S. obesus Fish Lake Valley McNett Ranch 2008366 625 2021 37.8443, -118.0086 S. obesus Hot Creek Valley Twin Springs Slough 3127174 38 49 n = 16, 2011 n = 22, 2022 38.1969, -116.1656 S. obesus Humboldt River System Upper Humboldt River 2920540 25 25 2022 41.1564, -115.0271 S. obesus Lassen County Eagle Lake 6658527 20 20 2023 40.6015, -120.7589 S. obesus Little Fish Lake Valley Little Fish Lake 1754730 612 2011 38.6181, -116.4710 S. obesus Railroad Valley Flowing Wells 1647665 15 23 n = 14, 2011 n = 1, 2022 38.4081, -115.7003 S. obesus Walker Lake Rose Creek Reservoir 3571471 14 16 2023 38.5899, -118.6329 46 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Genomic DNA was extracted from the chub fin clips using the QIAGEN DNeasy Blood and Tissue Kit according to the manufacturer’s protocol. Because the fin clips used in this project could be of substantial age (25+ years, Table 1), we adjusted the elution step to increase DNA yields by incubating molecular grade water at 56 °C for 10 min. DNA was eluted in 25 μL of warmed molecular grade water then centrifuged for 4 min at 5788xg. This step was repeated in order to produce a total of 50 μL of final DNA product. Library preparation followed the BestRAD protocol (Ali et al. 2016) with the SbfI restriction enzyme and individual barcodes ligated to sequences. Libraries were pooled and sequenced across two lanes on an NovaSeqX with paired-end Figure 2. Sampling map of Siphateles spp. sequenced in this study with RADseq included in analyses (n = 427). The outgroup taxa of Arroyo Chub (Gila orcuttii, n = 2) and Sacramento Splittail (Pogonichthys macrolepidotus, n = 9) are not shown. Specific collection localities for S. obesus are labeled. The inset indicates the extent of the main map within North America. Eagle Lake Flowing Wells Lida Pond Little Fish Lake Little Soda Lake McNett Ranch Rose Creek Reservoir Twin Springs Slough Upper Humboldt River 35.0 37.5 40.0 42.5 −122 −120 −118 −116 Longitude Latitude Epizon alvordensis S. thalassinus S. newarkensis S. isolatus S. mohavensis S. obesus Sample Size 10 20 30 S. snyderi 400 km N S. bicolor S. bicolor 47 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub 150 bp 25B sequencing chemistry. Sequence data were demultiplexed to the plate level then combined from different lanes for individual demultiplexing. Paired reads from individuals were aligned to the G. orcuttii reference genome (GCA_026230005.1) with the Burrows-Wheeler aligner and the MEM algorithm (bwa mem) (Li and Durbin 2010). Alignments were sorted, filtered for proper pairing, PCR duplicates removed, and number of aligned reads calculated with SAMtools (Li et al. 2009). Individuals with more than 1 million filtered and aligned reads were retained for analyses. Genome-wide phylogenetic analyses A set of SNP genotypes was generated by first calling SNPs with Analysis of Next Generation Sequence Data (ANGSD) (Korneliussen et al. 2014). Quality control of SNPs was enforced by requiring SNPs to be present in 95% of individuals, minimum mapping and base quality values of 20 (-minMapQ 20, -minQ 20), a significance value of 1.0 x 10-6 (-SNP_pval 1e-6), a posterior probability value of 0.95 (-postCutoff 0.95) and a minimum Minor Allele Frequency of 0.01 (-minMaf 0.01). A SAMtools genotype calling model was used and a PLINK-formatted file generated. Subsequently, the PLINK-formatted file was converted to a VCF-formatted file with PLINK (Purcell et al. 2007) and the VCF-formatted file was filtered to a MAF of 0.05 and ‘pruned’ with BCFtools to obtain unlinked SNPs (Danecek et al. 2021). The specific options supplied for pruning with BCFtools were +prune -m 0.20 -w 10000. A Multiple Sequence Alignment (MSA) of SNPs in PHYLIP format was made with the vcf2phylip.py script (https://github.com/ edgardomortiz/vcf2phylip/blob/master/vcf2phylip.py) from the pruned VCF file. Subsequent conversion of the PHYLIP-formatted file to NEXUS format was done with functions of the ape package in R (Paradis 2010). A concatenated Maximum-Likelihood (ML) phylogenetic tree was generated at the individual level with IQ-TREE2 (Minh et al. 2020). A General Time Reversible (GTR) model of nucleotide evolution with ascertainment bias correction (+ASC) was specified. Support for nodes was evaluated with the ultrafast bootstrapping algorithm (-bb 1000 -nm 2000 -bcor 0.9) (Hoang et al. 2018). A species-tree that incorporates the multispecies coalescent (explicitly modeling incomplete lineage sorting) was constructed with SVDQuartets in PAUP* (Swofford 2003; Chifman and Kubatko 2014, 2015). Individuals were pooled at the collection location level for this analysis, with 10 million random quartets evaluated. Nodal support was evaluated with 1,000 bootstrap replicates. A third analysis, an implicit phylogenetic network was undertaken to visualize possible discordance more fully as a result of hybridization and incomplete lineage sorting with SplitsTree (Huson and Bryant 2005). Uncorrected distances were used with the Neighbor-Net algorithm. Results Mitochondrial phylogeny and species delimitation We analyzed an alignment of 127 sequences with sequence accessions and metadata provided in Suppl. material 2. Mitochondrial sequences are from 12 leuciscid minnow species not classified in Siphateles (n = 12 sequences) and 115 of the sequences are indicated to be from different Siphateles 54 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub 1980). Average number of dorsal-fin rays of 7, anal rays 7, pelvic rays 8, and caudal-fin rays 19. Epizon alvordensis commonly has 13 pectoral-fin rays and E. boraxobius 14 (Williams and Bond 1980). Epizon is endemic to the Alvord Basin of Eastern Oregon of the Northwest Lakes subregion, of the Great Basin (Houghton 1976). The Alvord Basin is a north-south oriented valley bounded by Steens Mountain to the west and the Owyhee Plateau to the east, with formation of the valley during the Miocene (Orr and Orr 2012). It appears that the ancestral lineage of Epizon was isolated during this time. During the Pleistocene, the pluvial Lake Alvord was ~19 km wide and 113 km long during maximal extent (Reheis 1999). Subsequent drying isolated fishes in limited suitable habitats in a cyclical fashion, resulting in E. alvordensis being more broadly distributed within the basin of former Lake Alvord and E. boraxobius restricted to the thermal spring-fed Borax Lake and proximate habitats. Within Borax Lake, fish live typically for a year and are of a smaller size (33–45 mm SL) whereas in the diverse habitats elsewhere in Alvord Basin E. alvordensis fish live longer and grow larger, 4–5 years and ≤ 120 mm SL (Williams and Bond 1983; Sigler and Sigler 2014). A sample of 50 fish from Gridley Springs were reported to be 27–91 mm SL with a peak at 30-38 mm SL and fish distributed into larger sizes ≤ 91 mm SL (Williams and Bond 1983). As such, E. boraxobius had been noted prior to formal taxonomic description as a dwarf form of E. alvordensis. Borax Lake is fed by springs with 35–40 °C outflows resulting in a lake of typically 29–32 °C temperatures, with E. boraxobius avoiding temperatures above 34 °C and a loss of equilibrium at 34.5 °C (Williams and Bond 1983). Epizon alvordensis occupies a variety of habitats in springs, creeks, ponds, and reservoirs, in seven of eight historically occupied drainages (Scheerer et al. 2015). This taxon is found in cold, cool, and warm waters, but not above 31.1 °C (Williams and Bond 1983). Differentiation of the two species of Epizon may be driven by adaptation to the unique characteristics of the thermal-spring discharge with possible sympatric speciation of E. alvordensis and E. boraxobius (e.g., Smith et al. 2019). In addition to the characters of Williams and Bond (1980), nuclear genetic data separates the two species (Smith et al. 2019). Remple Position Epizon gen. nov. Siphateles Transition or Transversion 876 A C Tv 885 G A Ti 897 T A or G Tv 901 T C Ti 909 C G Tv 912 G A Ti 918 T C Ti 933 A G Ti 957 C T Ti 999 A T Tv 1002 T C Ti 1017 G A Ti 1023 A G Ti 1026 T C Ti 1059 A C Tv 1068 T C Ti 1122 C T Ti 55 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub (2013) reports that larger eye diameter, longer snout length and longer head length differentiating E. alvordensis and E. boraxobius are apparent in early life stages and can be used to differentiate larvae. Nuptial tubercles are present in males with sexual dimorphism reported in E. boraxobius based on relative length of fins, which are all longer in males than females (Williams and Bond 1980). This characteristic is also apparent in E. alvordensis (Sigler and Sigler 2014). Given the constant temperatures of Borax Lake, year-round spawning occurs with E. boraxobius. Epizon alvordensis in thermally fluctuating habitats, spawns only once a year (Williams and Bond 1983). Epizon are opportunistic omnivores with a diet closely related to the production of their habitats and variable with seasons (e.g., Williams and Williams 1980). Top dietary items are microcrustaceans, chironomids and diatoms (Williams and Bond 1983). Etymology. Romanized Greek version of επιζών, meaning survivor in reference to the persistence of this genus in the diverse and challenging desert habitats it has found itself. Gender masculine. Discussion Composition of Siphateles Analyses presented here (Figs 1, 3–5) and by others (Schonhuth et al. 2012, 2014, 2018; Rabosky et al. 2018;) indicate that the Alvord Basin leuciscids are substantially diverged from other members of Siphateles. Upon description, the affinities of Gila alvordensis Hubbs & Miller, 1972 were to the then subgenus Siphateles. The recognition of Siphateles as a genus by Simons and Mayden (1998) did not include representation of the Alvord Basin leuciscids in their molecular data set; however, Alvord Basin leuciscids were included in the elevation of Siphateles at that point. Subsequent molecular phylogenetic analyses of Alvord Basin leuciscids lack clear resolution of their placement, neither being a sister lineage of Siphateles nor being more-closely related to other fishes convincingly indicated (e.g., Schonhuth et al. 2012, 2014, 2018). We advocate that the Alvord Basin leuciscids should be placed in a separate genus, Epizon gen. nov., described in the results section of this manuscript. We choose to recognize two valid species of Epizon although mitochondrial barcode data does not resolve these two species phylogenetically (Fig. 1). Previous investigation of E. alvordensis and E. boraxobius with microsatellite and genome-wide SNP data, however, find support for a phylogenetic division of these two taxa that predates the end of Pleistocene (Smith et al. 2019). The implication is that these two lineages may have speciated sympatrically within the Alvord Basin. These lines of evidence as well as anatomical divergence and adaptation to hotsprings habitat of E. boraxobius lead us to conclude in support of its validity. Monophyly of a less inclusive Siphateles is supported across different molecular data types – mitochondrial loci, nuclear loci, and genome-wide SNP data (e.g., Schonhuth et al. 2012, 2014, 2018) and Figs 1, 3, 4 in this study. Mitochondrial, genome-wide SNP data and microsatellite data indicate that this less inclusive Siphateles may be divided into several allopatric units representing distinct clades (e.g., Figs 1–3; Harris 2000; Remple 2013). Siphateles 56 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub as defined here, contains seven species: S. isolatus, S. newarkensis, S. bicolor, S. thalassinus, S. mohavensis, S. snyderi and S. obesus. A major phylogenetic grouping present within Siphateles is of S. isolatus and S. newarkensis, found in Independence Valley and Newark Valley, Nevada respectively. These lineages are found in the Central Basins region of the Great Basin following Houghton (1976). Within these species, the subspecies S. isolatus euchila (Hubbs & Miller, 1972) from Fish Creek Valley as well as the nominal subspecies S. isolatus isolatus from Independence Valley may be reasonably recognized. Further studies specifically on this question, including more comprehensive anatomical investigation are needed to evaluate the taxonomic status of S. isolatus isolatus and S. isolatus euchila. Another major phylogenetic grouping is of S. bicolor and S. thalassinus. Overall, these lineages are found distributed in the Klamath and Pit river basins, the Northwest Lakes section of the Great Basin (Houghton 1976), and the Columbia River basin more generally (e.g., Lubinski and Scholz 2021). Siphateles bicolor contains several subspecies including the named S. bicolor columbianus (Synder, 1908) and S. bicolor eurysoma (Williams & Bond, 1981). Unlike Harris (2000), sequences from the ‘Silver Lake’ sampling location are nested within S. bicolor (Suppl. material 1: fig S1) and because of that we do not conclude it represents an undescribed distinct species as indicated by Harris (2000). We also find Siphateles thalassinus to be valid with the subspecies Gila bicolor vaccaceps Bills & Bond, 1980 placed in molecular phylogenetic analysis as a subspecies of S. thalassinus. That is, S. thalassinus vaccaceps (Bills & Bond, 1980), found in the Cow Head Basin. A third grouping of Siphateles, including S. mohavensis, S. snyderi and S. obesus is also found in our genome-wide analyses. Geographically, these lineages are found in the Death Valley System, Central Valleys, Lake Lahontan System and Northwest Lakes Great Basin sub-regions of Houghton (1976). Siphateles mohavensis are found naturally in the Mojave River basin while S. snyderi is found in the Owens Valley. As previously indicated by microsatellite analysis, the ‘toikona’ lineage is distinctive and restricted to the Owens Valley (Chen et al. 2007). With our broad geographic and lineage sampling we identify that the ‘toikona’ Tui Chub has close affinities to other Tui Chubs from the Owens Valley and may be considered a subspecies of S. snyderi, but without a formal taxonomic name yet. We represented S. obesus with numerous sampling locations and address the composition of this lineage in a separate following section. The three main lineages of this grouping, S. mohavensis, S. snyderi and S. obesus exhibit substantial mitonuclear discordance (Figs 1, 3). The mitochondrial data from S. mohavensis indicates it is the earliest branching lineage of Siphateles as defined here, however the nuclear data places S. mohavensis as clearly most closely related to S. snyderi and S. obesus. Geologic evidence indicates that the Owens Valley was the likely source of Mojave River Siphateles (Soltz and Naiman 1978) and that there were connections via the Amargosa River to the Mojave River where S. mohavensis was naturally distributed. Connections from the Owens Valley to Death Valley did occur up until the end of the Pleistocene, but how much gene flow occurred is unclear. No Siphateles are known from recent times in the Amargosa River or other intermediate systems between the Mojave River and Owens River, and if present, would be insightful. In Fig. 1 the branching order of mtDNA lineages may not reflect the true species 57 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub history and more complex scenarios are not necessary to invoke. The mitonuclear discordance observed between S. snyderi and S. obesus mtDNA and genome-wide SNP data, however, may require a more complex scenario. Previously an Owens River fish was sequenced for mitochondrial cytb (AF370056.1), and the three Northeast Pond sampling location fish we sequenced shared an identical haplotype. The four other individuals successfully sequenced from S. snyderi including toikona lineage fish, only differed by 2–4 mutational steps from each other, but were not all identical. Because these sequenced individuals come from refugial populations that are managed for conservation purposes, there were concerns about the level of potential hybridization in individuals used in translocations into the Owens Valley. However, these S. snyderi fish did not show evidence of recent hybridization in the nuclear genome (Fig. 5). While we observe deep divergences in the nuclear data-based analyses, the mitochondrial data from the Owens Valley is nested within a broadly distributed clade of S. obesus fish. There are three resultant hypotheses that can be explored. The first is that very recent introgression has occurred leading to the observed patterns. This is unlikely given that there is mitochondrial diversity across the Owens Valley fish sequenced and that all fish sequenced successfully for cytb have an obesus-like mtDNA lineage. The second hypothesis may be that there was historic gene flow between the Lahontan Basin and the prehistoric Lake Russell, represented today by Mono Lake. There is no current paleo-hydrological information supporting a connection between the Lahontan and Mono Basins after the 3.2 mya closure of the northern outlet of Lake Russell. The presence of active normal and transtensional faulting along the northern edge of the Mono Basin makes the probability of drainage capture a likely mechanism for the introduction of the S. obesus mitochondrial lineage into Lake Russell. Subsequent outflow events from Lake Russell would have led to the exchange of mitochondrial DNA lineages between the Lahontan Basin and the Owens Valley, with selection driving the introgression of mtDNA. Such events have been observed in several fish genera leading to examples of mitonuclear discordance (Moyle and Campbell 2022; Campbell et al. 2024). Finally, it is also possible that the depicted pattern cannot be fully interpreted given the few sequences generated. Our effort to generate and sequence cytb amplicons from S. snyderi was not broadly successful. We initially targeted 12 individuals but generated only seven sequences. Additional sequencing of mitochondrial data from S. snyderi and dedicated investigation of S. mohavensis, S. snyderi and S. obesus could be undertaken to clarify the source of this mitonuclear discordance. Composition of Siphateles obesus Siphateles obesus is found across the Lake Lahontan System with some locations in the Central Basins and mtDNA sequences from a region of the Northwest Lakes are placed with S. obesus though not sequenced from the nuclear genome in this study. There are three main lineages present within S. obesus in genomic sequence data examined in this study. The first division is a Fish Lake Valley lineage represented by two sampling locations (Lida Pond and McNett Ranch), with Railroad and Little Fish Lake valleys forming a second lineage, and the remainder of S. obesus sampling locations (Walker, Lahontan 58 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub drainages, etc.) forming a third lineage. The mtDNA data analyzed also contained a distinctive clade from the Summer Lake Basin in Oregon, part of pluvial Lake Chewaucan (Suppl. material 1: fig S1). This biogeographic region was not sampled in this study for genomic sequencing but may be reasoned to be another distinctive genetic lineage within S. obesus. This finding presents a disjunct distribution of S. obesus that may represent historical processes such as fault-block topography or climatic change resulting in a vicariant event (Harris 2000). Alternatively, the mtDNA may fail to accurately portray evolutionary relationships (e.g., Zink and Barrowclough 2008; Edwards and Bensch 2009). The Fish Lake Valley lineage is well-supported in phylogenetic analysis, with maximal support for monophyly in concatenated ML and species-tree analyses (Figs 3, 4). Furthermore, it is divided into two discrete units comprising the separate sampling locations (Fig. 3, Suppl. material 1: fig S2). Based on the results of the analyses presented here, it is unlikely that the Lida Pond location outside Fish Lake Valley was founded by human-mediated movement from the McNett Ranch sampling location. The Lida Pond fish appear to have unique genetic diversity within FLVTC not represented in McNett Ranch and that may now be lost from the native range. Resolution of FLVTC as the earliest-branching lineage of S. obesus is indicated by our species-tree analysis, though this result receives low (BS = 51%) support (Fig. 4). Similarly in Relict Dace Relictus solitarius Hubbs & Miller, 1972, there is clear evidence of separation of populations of this fish in Goshute, Steptoe, and Spring valleys in eastern Nevada from the western populations of this fish. Support for monophyly of eastern valleys is high, but support for branching relationships within the eastern clade are only moderate (BS = 75–78%) in a species tree analysis. Relatively rapid splitting of populations, bottlenecking and genetic drift can act to reduce the number of sites that exist to support branching patterns, producing a lower signal to noise relationship in these desert fishes (Finger et al. 2022). Geographic evidence supports a migration pathway from the Lahontan Basin into Fish Lake Valley that was disrupted ~2 million years ago by action along the Huntoon Valley fault system (Reheis et al. 2002). After this time, a pluvial lake existed until ~ 0.5 million years with periodic outflows that may have allowed gene flow (Reheis et al. 1991). The phylogenetic placement of FLVTC as closely related to Lahontan lineages with substantial divergence is concordant with this geographical information. The Railroad Valley and Little Fish Lake Valley lineage also is highly supported (e.g., Fig. 4); however, the separation of Railroad Valley sampling locations at an individual level does not occur (Suppl. material 1: fig S2). This indicates a lack of genetic structuring between Railroad Valley sampling locations. Finally, a third lineage of broadly Lahontan Basin S. obesus is present across analyses composed of sampling locations that exhibit genetic structuring (e.g., Figs 3–5). The freshwater biodiversity crisis: a perspective from the Great Basin and adjacent areas The Great Basin is a relatively accessible area in a developed country, with a limited ichthyofauna. As a result, leuciscid minnows have received substantial anatomical analysis leading to various classification schemes discussed in detail by Simons and Mayden (1998). Importantly, work by Uyeno (1961) lead 59 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub to the consolidation of numerous fishes under the genus Gila, with a reality, as interpreted through molecular phylogenetics, that is much more complex (e.g., Simons and Mayden 1998; Schonhuth et al. 2012). Indeed, the anatomical differentiation of species within Siphateles may be challenging as well, and Siphateles mohavensis, was noted by Miller (1973: 8) to be a subspecies as Gila bicolor mohavensis, accompanied by the statement “I have not been able to discover characters that will separate it specifically from all populations of Gila bicolor in the Lahontan basin”. The great amount of anatomical variation across the range of a fish species in the Great Basin, such as all Siphateles obesus in the Lahontan Basin, is likely a result of ecological plasticity and the occupation of a diversity of habitats and ecologies. Habitats in the Great Basin include hot springs, cold springs, lakes, and rivers, all with co-occurring food items and other fishes that also vary over such large geographic areas. Siphateles mohavensis is clearly differentiated from other Siphateles at mitochondrial and nuclear loci with an ancient movement into the Mojave River and extinction from other habitats that may have been occupied. Molecular phylogenetic analyses as presented here are useful to identify areas where taxonomic refinement is possible. Our work supports the recognition of seven species previously classified under one, as valid. As species are a currency in biodiversity research, lack of recognition of species-level diversity is detrimental to successful conservation actions (Brown and Lomolino 1998). Similar studies have improved taxonomy at the species-level within Pantosteus Cope, 1875 and Rhinichthys Agassiz, 1849 in the Great Basin and adjacent regions (Unmack et al. 2014; Su et al. 2022; Moyle et al. 2023). Molecular analyses have also indicated that in western North America adjacent to and in the Great Basin Prosopium williamsoni (Girard, 1856) and Catostomus ardens Jordan & Gilbert, 1881 contain divergent and unrecognized species lineages (Miller 2003; Mock et al. 2006). Further investigations are warranted to examine these fishes for the presence or not of anatomical characteristics or other traits in support of species descriptions. At a higher level, molecular phylogenetic studies of Great Basin fishes such as this one provide additional taxonomic improvements at the genus-level. Genomic studies examining other taxa such as the Catostomidae may also be important for understanding the generic relationships of that group (e.g., Campbell et al. 2023; Harris et al. 2025). With due consideration and application of integrative approaches (Ottoni et al. 2025), the contribution of the Great Basin to North American ichthyofauna is likely to continue to increase, more accurately informing biodiversity conservation in the region. In response to the worldwide biodiversity crisis, in species rich systems conservationists advocate the conservation of ‘type locality hot spots’ where there are type localities of multiple and distinct taxonomic groups, such as plant or butterfly species, for example. For freshwater fish, such locations might be in the species-rich Amazon basin (e.g., Azevedo-Santos and Ottoni 2025). However, this conservation strategy falls short in the Great Basin, where the disconnected inland waters are often not species rich, and therefore type localities with only a single species warrant protection. Indeed, some endemic Great Basin fishes like the Relict Dace are the only naturally-distributed fishes found in their habitats even if over a rather large area. In other Great Basin fishes, the type locality may represent the only extant population such as the Wall Canyon Sucker Catostomus murivallis Harris, Markle & Campbell, 2025. 60 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub While some Great Basin fish species like Relict Dace and the Wall Canyon Sucker still exist in their type localities, many others do not, often due to the introduction of non-native fishes (Cucherousset and Olden 2011) and human alterations to habitats (Ottoni et al. 2023). For example, S. isolatus is no longer found in its type locality and S. mohavensis was extirpated from its original distribution because of non-native fish introductions (Harris 2000). Another example is the ’toikona’ Tui Chub which was first documented in 1987 from a single location and fish examined in this study descend from 24 fish translocated to artificial refuge pounds in 1989 (Chen et al. 2007). Conservation actions aimed at the type locality of a hypothetical ‘toikona’ Tui Chub taxon would not provide broad conservation benefits to freshwater fishes in the Great Basin. However, integration across aquatic taxa (e.g., including spring snails Gastropoda: Sorbeoconcha: Hydrobiidae) may be an effective conservation strategy that identifies aquatic type locality hotspots that may be emphasized within conservation frameworks in the Great Basin. Conclusions We find the fishes previously classified under Siphateles to merit taxonomic refinement. At the highest level, a deep genetic divergence between the Alvord Basin leuciscids and other fishes is present and we propose Epizon gen. nov. for the Alvord Basin leuciscids. Furthermore, what has been consolidated under S. bicolor is better characterized as seven species-level entities, all with existing names. Within S. obesus we find that Fish Lake Valley Tui Chub is phylogenetically the earliest-branching lineage and is highly genetically differentiated from other S. obesus lineages. It also occupies a limited geographic area, separate from all other Tui Chubs. Based on the level of genetic differentiation and geographic distribution of Fish Lake Valley Tui Chub, it merits recognition as a subspecies. Our search through the literature did not find an available name, therefore formal taxonomic description would be necessary to provide an official name for this subspecies. Acknowledgements We would like to recognize the sampling efforts that made this possible. Samples were provided by California Department of Fish and Wildlife Fisheries Branch Central Valley Tissue Archive and Nevada Department of Wildlife, California Department of Fish and Wildlife, Oregon Department of Fish and Wildlife, and US Fish and Wildlife Service (USFWS) personnel. Katerina Stasinopoulou assisted with the etymology and spelling of Epizon gen. nov. Pictures of the holotypes of Epizon alvordensis and Epizon boraxobius are courtesy of the University of Michigan Museum of Zoology Division of Fishes. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement Material collected in support of this study was obtained under institutional and ethical guidelines of NDOW, CDFW, ODFW, and USFWS by personnel of those agencies. 61 ZooKeys 1261: 39–67 (2025), DOI: 10.3897/zookeys.1261.51636 Matthew A. Campbell et al.: Evolutionary relationships of Fish Lake Valley Tui Chub Use of AI No use of AI was reported. Funding The authors would like to acknowledge funding for this project through the USFWS grant #F22AC00854-00. The sequencing was carried out by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center, supported by NIH Shared Instrumentation Grant 1S10OD010786-01. Author contributions Conceptualization: MAC. Data curation: KNY, SCP, MAC, GA. Formal analysis: MAC, GA. Funding acquisition: AJF, MAC. Methodology: AJF, MAC. Project administration: AJF. Resources: NB, AJF. Supervision: AJF. Visualization: MAC. Writing - original draft: AJF, MAC. Writing - review and editing: NB, SCP, KNY, AJF, GA. Author ORCIDs Matthew A. Campbell https://orcid.org/0000-0002-5826-0329 Grace Auringer https://orcid.org/0000-0002-7639-3766 Amanda J. Finger https://orcid.org/0000-0003-3850-3685 Data availability Demultiplexed NGS sequence data has been deposited to the NCBI Sequence Read Archive under BioProject PRJNA1327467. 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