Revision of Notropis stramineus (Cope, 1865), descriptions of three new species and comments on the monophyly of Miniellus Jordan, 1882 (Pisces: Cypriniformes: Leuciscidae)

699 Revision of Notropis stramineus (Cope, 1865), descriptions of three new species and comments on the monophyly of Miniellus Jordan, 1882 (Pisces: Cypriniformes: Leuciscidae) Amanda K. Pinion1, Daemin Kim2, Elizabeth P. Dolan3, David S. Portnoy3, Gary Voelker4, Kevin W. Conway4 1 Museum für Tierkunde, Senckenberg Naturhistorische Sammlungen Dresden, Dresden, Germany 2 Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637, USA 3 Marine Genomics Laboratory, Department of Life Sciences, Texas A&M University–Corpus Christi, Corpus Christi, TX 78412, USA 4 Department of Ecology and Conservation Biology and Biodiversity Research and Teaching Collections, Texas A&M University, College Station, TX 77843, USA https://zoobank.org/3FB861CA-893D-4C9A-BAC3-FDCE312A66DA Corresponding author: Amanda K. Pinion ([email protected]) Academic editor Uwe Fritz | Received 20 April 2025 | Accepted 10 November 2025 | Published 3 December 2025 Citation: Pinion AK, Kim D, Dolan EP, Portnoy DS, Voelker G, Conway KW (2025) Revision of Notropis stramineus (Cope, 1865), descriptions of three new species and comments on the monophyly of Miniellus Jordan, 1882 (Pisces: Cypriniformes: Leuciscidae). Vertebrate Zoo logy 75: 699–755. https://doi.org/ 10.3897/vz.75.e156077 Abstract Combined analyses of molecular and morphological data support a revised classification of Notropis stramineus comprising five distinct evolutionary lineages, two for which names are available, Notropis stramineus (Cope, 1865) and N. missuriensis (Cope, 1871), herein redescribed, and three for which no names are available, here formally described as new species. Four of these clades comprise a species complex, here termed the N. stramineus species complex, while these four clades, in addition to N. procne, N. topeka, N. chihuahua and the fifth clade form a monophyletic group here termed the N. stramineus species group. Notropis lucifer sp. nov., sister taxon of N. chihuahua, is distributed in the Colorado River and Rio Grande basins of Texas (USA), where it is distributed in the Devils River, and presumably also in Pinto Creek; based on distribution, populations in tributaries of the Rio Grande in Mexico are tentatively also assigned to this species. Notropis lucifer sp. nov. can be distinguished from other members of the N. stramineus species group primarily by coloration in life and pigmentation patterns which are still visible after preservation. Notropis oblitus sp. nov. is found in several Gulf Slope streams in Texas, including the upper reaches of Nueces, San Antonio, and Guadalupe River basins, though likely absent from the Colorado River basin. This species can be distinguished from other members by head shape, eye size and pigmentation patterns. Notropis multicorniculatus sp. nov. is found in the western portions of the Arkansas, Canadian and Red River basins in parts of Texas, Oklahoma, and Kansas, and likely also Arkansas and Colorado, as well as the Pecos River (New Mexico), where it is likely non-native. This species can be distinguished from other members of the species complex by comparatively larger and more abundant tubercles in males and differences in body shape. Comments are provided regarding the genus Miniellus, which has been recently suggested to comprise 21 species, including N. stramineus. Keywords Integrative taxonomy, Leuciscidae, shiners, systematics, UCEs Vertebrate Zoology 75, 2025, 699–755 | DOI 10.3897/vz.75.e156077 Copyright Amanda K. Pinion et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 700 Introduction North America is home to ~300 species across ~57 genera in the family Leuciscidae, a species-rich family of Holarctic freshwater fishes that includes the taxonomically challenging minnows and shiners (Gidmark and Simons 2014). In recent years, putative new species of leuciscids continue to be identified using molecular methods, though these are often not immediately formally described (e.g., Schönhuth et al. 2012; Kim and Conway 2014; Schönhuth et al. 2015; Ballesteros-Nova et al. 2019; Weyand and Piller 2020). Within North America, the Leuciscidae have been identified as one of several families of freshwater fishes likely to harbor significant unrecognized diversity, with the genus Notropis Rafinesque, 1818 (of the speciose subfamily Pogonichthyinae) responsible for driving this trend (April et al. 2011). Notropis was until recently the largest genus of North American minnows with ~100 spp. of generally small-bodied, superficially similar looking fishes, with even well-studied species sometimes difficult for experts to identify. Several species in the genus have large distributions across North America, though modern work employing molecular methods is increasingly revealing that many of these supposedly widespread species may in fact represent complexes of similar-looking but evolutionarily distinct species. The sand shiner Notropis stramineus (Cope, 1865) (Fig. 1) is a small-bodied minnow (reaching approximately ~60 mm in standard length) with a distribution that extends from northern Mexico to southern Canada (Fig. 2; Lee et al. 1980; Page and Burr 2011). Notropis stramineus occurs and is often abundant in a diverse array of aquatic habitats, ranging from shallow, spring-fed desert streams in the southwestern United States (U.S.) and Mexico, to wide, sandy rivers in the U.S. Midwest and northeast as well as in Canada (Hubbs and Lagler 1958; Bailey and Allum 1962; Tanyolaç 1973; Scott and Crossman 1973). Several major river systems in the north are included in this distribution, e.g., the Great Lakes and St. Lawrence River basin, and the Red River of the North (Hudson Bay drainage). In the Interior Plains, N. stramineus can be found in the Ohio, Missouri, and Mississippi River systems, and eastward in the Tennessee and Cumberland Rivers (Etnier and Starnes 1993). In the southern U.S., N. stramineus is abundant in the Red, Canadian, and Arkansas Rivers (Miller and Robison 2004). Isolated populations of N. stramineus are found within several of the independent Gulf Slope drainages of Texas, including the upper reaches of Nueces, San Antonio, Guadalupe, and Colorado River basins within the Edwards Plateau ecoregion. The species is absent from the Brazos and Lavaca, and very rarely collected in the Trinity River drainage (Hendrickson and Cohen 2022). Finally, the southernmost portion of the natural sand shiner distribution includes multiple tributaries of the Rio Grande basin, including the Devils River of Texas, and the Río San Juan and Río Salado of Mexico (Sublette et al. 1990; Miller et al. 2005). Introduced populations of N. stramineus exist in the upper Colorado River drainage of Utah and Colorado, East Clear and Chevalon creeks in Arizona, Shawangunk Kill in New York, and the Bighorn River drainage, Wyoming (Fuller et al. 1999; Nico et al. 2019). The extant population in the upper Pecos River, New Mexico is also likely introduced (Hoagstrom et al. 2025). Notropis stramineus is notably absent from regions such as the Ozarks and Ouachita highlands, and much of the Atlantic slope, excepting the St. Lawrence River drainage. The taxonomic history of Notropis stramineus sensu lato is complex and summarized in part by Suttkus (1958), Bailey and Allum (1962), Tanyolaç (1973) and Mayden and Gilbert (1989). Three subspecies were recognized by Hubbs as early as 1928 (Hubbs and Greene 1928: 377; Hubbs and Ortenburger 1929). At this time, the sand shiner was known as N. deliciosa, and according to Hubbs comprised three subspecies: N. d. deliciosa, distributed south of the Arkansas River (including Texas and Mexico populations), N. d. missuriensis, occurring in the Arkansas River and to the north in the western tributaries of the upper Mississippi River, and N. d. stramineus, occurring in the eastern tributaries of the Mississippi River. However, Suttkus (1958) demonstrated that the type series of N. deliciosa comprised a mixed lot of two species, neither representative of the sand shiner known to North American ichthyologists of the time, and placed N. deliFigure 1. Notropis stramineus, TCWC 21295.01, not measured; USA, Michigan, Washtenaw County: Huron River. Vertebrate Zoology 75, 2025, 699–755 701 ciosa in the synonymy of N. texanus. Suttkus identified Hybopsis stramineus Cope, 1865 as the next available name for the sand shiner, however this effectively left the southern subspecies of the sand shiner (N. d. deliciosa) nameless, a problem recognized by Suttkus (1958: 317): “The nominal form Moniana deliciosa Girard (now N. stramineus) was treated as a trinomial (Hubbs and Greene 1928) and the above procedure now leaves the southern population without a name. This should be treated in a comprehensive study of the subspecies.” Later, Bailey and Allum (1962), in the Fishes of South Dakota would examine squamation of Notropis stramineus across much of the midwestern portion of the distribution. Bailey and Allum (1962) did not examine specimens from southern sand shiner populations, the reason being likely a combination of their focus on a specific region of North America as well as their reference to Hubbs and Lagler (1958), who, while mentioning the existence of a southern subspecies, apparently mistakenly outlined its range as overlapping with that of N. d. missuriensis. This seems to have introduced confusion, as evident in the following line of Bailey and Allum (1962: 65): “It has been contended that the sand shiner comprises two subspecies in addition to missuriensis, both of which are said to occur in Indiana.” Bailey and Allum (1962), as well as others, found no evidence for such diversity in that region, and proposed a classification that was based on circumferential scale-row counts, dividing the sand shiners in South Dakota into an eastern subspecies, N. s. stramineus (Cope, 1865) (type locality the Detroit River in Grosse Isle, Michigan), and a Great Plains subspecies, N. s. missuriensis (Cope, 1871) (type locality a tributary of the Missouri River near St. Joseph, Buchanan County, Missouri). In a study of the fishes in the Kansas River system, Metcalf (1966) presented evidence in support of the findings of Bailey and Allum (1962) and made note of the hypothesized southern subspecies mentioned by Hubbs and Ortenburger (1929), but did not find data in support of its existence, instead finding sand shiners from the Red River with scale counts matching that of N. s. stramineus or intergrading with those of N. s. missuriensis. In a broader study, Tanyolaç (1973) examined variation across the range of N. stramineus sensu lato, including some southern populations, and expanded the distribution of the eastern subspecies N. s. stramineus to accommodate these southern populations, a valiant effort that supported a subspecies scheme that, despite persisting to the present day, has unfortunately not been often followed or recognized (some exceptions include Cross 30° N 20° N 120° W 110° W 100° W 90° W 80° W 120° W 40° N 50° N P P P T T T H Legend H P T N. stramineus N. topeka N. procne N. heterodon N. s. stramineus tissues N. s. missuriensis tissues N. procne tissues N. topeka tissues N. heterodon tissues 120° W 110° W 100° W 90° W 80° W 70° W 30° N 20° N 40° N 50° N 500 1,0000 Kilometers N Figure 2. Map of North America displaying approximate ranges of the four species belonging to the genus Miniellus sensu Mayden et al. (2006). Sample localities of the four species (and subspecies for Notropis stramineus) in the 3-gene dataset are shown as dots. The approximate native ranges are colored for each species as follows: N. stramineus sensu lato in tan, N. topeka in green, N. procne in purple, and N. heterodon in red. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 702 and Collins 1995; Gilbert 1978; Hoagstrom et al. 2007, 2011). However, Tanyolaç (1973) had closely examined meristic characters across the distribution of N. stramineus and considered their reliability to distinguish the subspecies “imperfect,” reporting intergradation. At this point in the taxonomic literature, with the southern subspecies left nameless after Suttkus’ (1958) study, Tanyolaç’s (1973) expansion of Notropis stramineus stramineus to also include all southern populations, and the lack of support of three subspecies by both Bailey and Allum (1962) and Metcalf (1966), it appears the possible existence of a southern subspecies of sand shiner was forgotten. Subsequent to Tanyolaç’s (1973) study, two unpublished dissertations that involved molecular phylogeographic analyses of Notropis stramineus populations using either mitochondrial DNA (Michels 2000) or mitochondrial DNA and microsatellite markers (Pittman 2011) contained compelling evidence for the presence of unrecognized diversity between some geographically proximate populations that did not correspond to the current subspecific scheme. However, the geographic distribution coverage of N. stramineus in both studies was limited, and as a result, in both cases the authors indicated the need for further study of the genetic diversity within N. stramineus. However, the only taxonomic work following these studies involving the sand shiner concerns the generic membership of the species. The taxonomic history of Notropis (summarized in detail by Gilbert 1978), to which the sand shiner for most of the last century has been considered to belong, is complex. Poorly defined genera, many recognized as distinct in the present day, have been variously synonymized with Notropis and treated as subgenera for much of the 20th century. There were several sporadic efforts to elevate these subgenera throughout the years (e.g., Gilbert 1964; Gidmark and Simons 2014; Stout et al. 2022), the most substantial being Mayden (1989), in which nine genera were resurrected. The genus Miniellus Jordan, 1882 is a relatively recent example. This generic name was resurrected from the synonymy of Notropis by Mayden et al. (2006) based on the results of a phylogenetic study analyzing mitochondrial cytochrome b gene (cyt b) sequences to accommodate four species that were at the time placed within Notropis: the swallowtail shiner, No tropis procne (Cope, 1865) (type species of Miniellus), the blackchin shiner, N. heterodon (Cope, 1865), the Topeka shiner, N. topeka (Gilbert, 1884), and the sand shiner, N. stramineus (Cope, 1865). This elevation of Miniellus in 2006, however, has not been embraced by the field generally and only recently recognized a handful of times (e.g., Gidmark and Simons 2014; Schönhuth et al. 2018; Tan and Armbruster 2018), however, more recent work has revisited this genus and proposed a new and expanded classification (Stout et al. 2022). Stout et al. (2022) applied exon capture to a broad but sparse representation of the “shiner” clade, a very large assemblage of North American minnows that includes Notropis (Coburn and Cavender 1992; Simons et al. 2003). As a result of this study, the authors proposed several sweeping taxonomic changes. This included the recognition and significant expansion of Miniellus to include the four species proposed by Mayden et al. (2006) as well as an additional 17 species, bringing the total included species in Miniellus to 21 (Table 1). No formal diagnosis of the genus was offered by Stout et al. (2022) and, unfortunately, two members of the four species proposed to belong to Miniellus by Mayden et al. (2006), including the type species for the genus, Notropis procne, were absent from their dataset. The two species of Miniellus sensu Mayden Table 1. Membership of the genus Miniellus (type species Notropis procne) according to different authors. Species with asterisks were not included in Stout et al. (2022) but were proposed by these authors to belong to Miniellus (see Discussion, section ‘A history of the genus Miniellus Jordan, 1882’). Species in bold in the fourth column are included in the present study. Mayden et al. (2006)1Gidmark and Simons ( 2014)2Schönhuth et al. (2018)3Stout et al. (2022)4 Notropis procne Notropis topeka Notropis heterodon Notropis stramineus Notropis procne Notropis topeka Notropis heterodon Notropis stramineus Notropis procne Notropis topeka Notropis heterodon Notropis stramineus Notropis procne* Notropis topeka* Notropis heterodon Notropis stramineus Notropis albizonatus Notropis alborus* Notropis ammophilus Notropis anogenus* Notropis boops* Notropis chihuahua Notropis greenei* Notropis longirostris Notropis mekistocholas* Notropis melanostomus Notropis nubilus Notropis ortenburgeri* Notropis perpallidus* Notropis rafinesquei* Notropis sabinae Notropis scabriceps Notropis uranoscopus* 1 Data used includes cytochrome b gene (cyt b). 2 Gidmark and Simons (2014) follow Mayden et al. (2006). 3 Data used for analysis include four genes (two mitochondrial, cyt b and COI; two nuclear, RAG1 and IRBP), concatenated. 4 Exon-capture was used to collect 1004 loci. Vertebrate Zoology 75, 2025, 699–755 703 et al. (2006) that were however present in Stout et al.’s (2022) dataset included one specimen each of the blackchin shiner N. heterodon, and the sand shiner N. stramineus, the most widely distributed and abundant member of the putative genus by a sizeable margin (Fig. 2). To provide an overview of the phylogeographic structure of populations of Notropis stramineus and its relatives (members of the putative genus Miniellus), three loci were collected and analyzed in a phylogenetic framework (one mitochondrial, cyt b, and two nuclear, one coding, recombination activating gene 1, RAG1, and one non-coding, S7 ribosomal protein gene intron 1). However, many studies examining subgroupings of Notropis sensu lato have found gene-tree discordance in datasets using only a few loci (usually a combination of mitochondrial and nuclear loci; e.g., Cashner et al. 2011; Hollingsworth and Hulsey 2011), and preliminary analyses of the present study indicated the same. Because this study involves a broad set of taxa, spanning shallow to deep divergences, ultra-conserved elements (UCE) were collected for a subset of Notropis stramineus samples, likely close relatives, and several other members of Notropis. The resulting datasets were analyzed to construct phylogenetic hypotheses regarding the interand intrarelationships of Notropis stramineus, including: (1) the closest relatives of N. stramineus, (2) the composition of Miniellus, and whether the genus Miniellus represents a monophyletic group as proposed by Mayden et al. (2006) or Stout et al. (2022); (3) the phylogeographic relationships of populations within N. stramineus; and (4) whether the current subspecific designations (N. s. stramineus and N. s. missuriensis) accurately reflect evolutionary history and geographic distributions of populations or lineages. Several morphological datasets are herein utilized to uncover diagnostic morphological characters useful for the discrimination of distinct lineages. These datasets include internal and external meristics, pigmentation, osteology, secondary sexual characteristics, as well as traditional and geometric morphometrics. We find that lineages uncovered as a result of these analyses can be distinguished through a combination of morphological characteristics and are thus regarded as distinct species; therefore, using these morphological datasets, descriptions are provided for three new species, and Notropis stramineus and N. missuriensis, the latter elevated from subspecific rank, are redescribed. Materials and Methods Terminology, taxon sampling, and nomenclature Analyses of the molecular datasets revealed several distinct clades of Notropis stramineus, detailed in results. Consequently, several terms are here introduced and used from this point onward. These include Notropis stramineus sensu lato, for all individuals currently considered to belong to the species N. stramineus, N. stramineus sensu stricto, for a single clade within N. stramineus sensu lato to which the nominal taxon belongs, the N. stramineus species group, which includes N. stramineus sensu lato, N. topeka, N. procne, and N. chihuahua, and lastly the N. stramineus species complex, which includes four clades of N. stramineus sensu lato Tissue samples of Notropis stramineus sensu lato (Fig. 2) were obtained from U.S. and Canadian museum collections and through fieldwork conducted between the years 2010 and 2020. Specimens were collected using sampling protocols approved by the Texas A&M University Institutional Animal Care and Use Committee (TAMU IACUC # 2017–0047, 2020–0033). Euthanized individuals were fixed in buffered formalin or preserved in 95% ETOH and deposited at the Biodiversity Research and Teaching Collections (TCWC) at Texas A&M University as voucher specimens. Tissues were obtained either from fin clips or a muscle subsample was taken from the right side of the body. Finally, in order to represent the genus Miniellus as proposed by Stout et al. (2022), sequences from additional species not sampled or borrowed from museum collections were obtained from GenBank. Ingroup samples for the 3-loci dataset included 160 individuals across the distribution of N. stramineus sensu lato (including the type localities of N. s. stramineus and N. s. missuriensis), three N. topeka, three N. procne, and one N. heterodon (Fig. 2; Table S1). Outgroup samples represent 65 species of Notropis sensu lato and members of other closely related genera (of which there are 1–3 samples per species for most, and 20 for N. chihuahua; Table S1). UCE data were collected for three to five members of each Notropis stramineus clade (including N. chihuahua) identified through preliminary analyses of Sanger-sequenced loci, and three species purported to make up the genus Miniellus sensu Mayden et al. (2006) including N. procne (type species of the genus), N. topeka, and N. heterodon. Outgroup taxa were selected to be representative of Notropis sensu lato and included N. bifenatus, N. braytoni, N. buchanani, N. chalybaeus, N. longirostris, N. mekistocholas, N. nubilus, N. sabinae, N. boops, and N. volucellus. Agosia chrysogaster, a species identified recently as the possible sister taxon to the Shiner clade (Mayden et al. 2006; Gidmark and Simons 2014; Schönhuth 2018), was also included as the most distantly related outgroup taxon. All sequences generated or obtained from GenBank are listed in Table S1 (Sanger data) and Table S2 (UCE data). Demultiplexed sequence reads have been deposited on NCBI and can be accessed through NCBI BioProject ID PRJNA1363575. Institutional abbreviations of natural history collection names throughout the manuscript, figures and tables follow Sabaj (2020). Samples for morphological study were obtained either from museum collections or were collected from the wild. Euthanized individuals were fixed in 10% buffered formalin and deposited at the Biodiversity Research and Teaching Collections (TCWC) at Texas A&M University. The distribution of samples of Notropis stramineus sensu Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 704 lato examined for morphological study by clade is shown in Figure S1. Specimens for which traditional measurements, scale counts, and meristic counts and characters were collected are listed following each species account. Male and female specimens in good condition were prioritized and include those collected near the type locality of previously described species. When possible, specimens from the localities from which genetic samples were available were analyzed for inclusion in morphological datasets. Specimens were examined and imaged using a Zeiss SteREO Discovery V20 stereomicroscope equipped with a Zeiss Axiocam MRc5 digital camera. Although it is the case that Notropis, as recognized in earlier studies (e.g., Page and Burr 2011; Page et al. 2013), is a polyphyletic assemblage that is in urgent need of systematic study and revision (see Mayden et al. 2006), in this study the recent nomenclatural changes suggested by Stout et al. (2022) are not fully embraced in which 91 of 92 species of North American leucisids previously included in Notropis (e.g., Page and Burr 2011; Page et al. 2013) are divided among multiple genera, including Alburnops, Hydrophlox, Miniellus, Notropis sensu stricto (following Stout et al. 2022), ‘Notropis’, and Paranotropis, based on a phylogenomic study of 87 species and in reference to the results of prior studies. Although these nomenclatural changes have been quickly embraced by others (e.g., Page et al. 2023), it is possible that many of these changes may be premature, especially those that relate to Miniellus in particular, for reasons outlined in detail in this study. In an attempt to facilitate navigation of this study by non-experts (that may adopt the recently published ASIH-AFS official list of common and scientific names of fishes from the United States, Canada, and Mexico; Page et al. 2023), the generic membership scheme of Stout et al. (2022) is adopted only in a subset of the figures (Figs 5, 6, S2) and Table S1, but we continue to use the generic name Notropis as applied in earlier studies (e.g., Page and Burr 2011; Page et al. 2013) in the main body of the text. For example, Notropis (Miniellus) stramineus is used in the aforementioned figures and table (following Page et al. 2023) but we use Notropis stramineus to refer to the same taxon in the text. Individual Sanger-sequenced loci dataset DNA extraction. Genomic DNA was extracted from muscle or fin-clips using a DNeasy Tissue Extraction Kit (Qiagen, Inc., CA, USA) following the manufacturer’s protocols. Three loci, one mitochondrial (cyt b) and two nuclear (S7, RAG1) were targeted as these have been demonstrated previously to be informative for inferring relationships among groups of North American leucisids (e.g., see Schönhuth et al. 2011, 2012; Conway and Kim 2016). All polymerase chain reaction (PCR) amplifications were conducted in 25 μl reactions containing 12.5 μl EmeraldAmp GT PCR Master Mix (Takara, Japan), 10.7 μl nuclease-free water, 1 μl (300 ng) of template DNA, and 0.4 μl of each primer (forward and reverse) under the following PCR conditions: cyt b was amplified using the primer pair LA-Danio and HA-Danio (Mayden et al. 2007); cycling included an initial denaturation at 94°C for 4 min, then 35 cycles at 94°C for 30 s each, 50°C for 50 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. S7 was amplified using the primer pair S7RPEX1F and S7RPEX2R (Chow and Hazama 1998); cycling included an initial denaturation at 94°C for 4 min, then 35 cycles at 94°C for 30 s each, 61°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. RAG1 was amplified using the primer pair R12533F and R14078R (Lopez et al. 2004); cycling an included initial denaturation at 94°C for 4 min, then 35 cycles at 94°C for 30 s each, 53°C for 60 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. Amplified PCR product was cleaned using ExoSAP-IT and sequenced by either the Genomics Core Lab at Texas A&M University – Corpus Christi or Psomagen (MD, USA). Sequences have been deposited in GenBank under the accession numbers provided in Table S1. Forward and reverse sequences were assembled into contigs either manually using FinchTV (Geospiza Inc.) or in Geneious Prime 11.1.5 (www.geneious.com). Base calls in all sequences were visually checked for accuracy, and uncorrected pairwise distances between select sequences were calculated using MEGA version 11.0.13 (Tamura et al. 2021). Sequence alignment and phylogenetic and haplotype network analyses. For each method of phylogenetic inference, individual gene datasets as well as a 3-loci concatenated matrix (cyt b, S7, and RAG1) were analyzed. Sequence alignment was performed with MAFFT vs. 7 (Katoh and Standley 2013) either on the webserver (https://mafft.cbrc.jp/alignment/server/) or on the CIPRES Science Gateway portal (Miller et al. 2010). PartitionFinder2 (Lanfear et al. 2017) was used to determine models of nucleotide substitution for each codon position of the two coding genes by selecting the model with the lowest AIC score. Two MCMC runs of 50 million generations were performed in MrBayes 3 (Ronquist et al. 2012) on the CIPRES Science Gateway portal under an uninformative prior with parameters allowed to vary. Tracer 1.7 (Rambaut et al. 2018) was used to visualize the posterior probability distribution and confirm convergence between runs. Maximum likelihood (ML) was implemented using RaXML version 8 (Stamatakis 2016) on the CIPRES portal under a GTR+ GAMMA model with support evaluated using 1000 bootstrap replications. Trees were rooted with Agosia chrysogaster, a species belonging to a monotypic genus previously recognized with morphological methods as a member of the western clade of North American minnows (Coburn and Cavender 1992) or alternatively as a member of the Shiner clade, as one of three possible sister taxa of the remaining members of this clade (Mayden 1992; Gidmark and Simons 2014; Schönhuth 2018). Trees were visualized using FigTree vs. 1.4.4 (http://tree. bio.ed.ac.uk/software/figtree). Median-joining haplotype networks (Bandelt et al. 1999) were constructed using PopART version 1.7 (Leigh and Bryant 2015). Vertebrate Zoology 75, 2025, 699–755 705 Ultra-conserved elements (UCE) dataset DNA extraction and library preparation. Genomic DNA was extracted from fin clips or muscle tissue using a DNeasy Tissue Extraction Kit following the manufacturer’s protocol, except in the final elution step, in which DNA was eluted in 75µl elution buffer for higher DNA concentration. Each extracted sample was checked for adequate DNA quality and concentration by running 2µl of extracted DNA on an agarose gel with a 100 bp ladder to select samples with high-weight bands (>500 bp). Library preparation was performed at the Marine Genomics Lab at Texas A&M University – Corpus Christi, Corpus Christi, TX. DNA was standardized to a concentration of 20 ng/µl in 100 µl 1X Tris-EDTA (TE) buffer, then randomly sheared to approximately 800 bp (Sonolab 7.2 program: 800 bp DF 2 time = 80 s) using an M220 Focused-ultrasonicator (Covaris, MA, USA). Because the length of the core regions of UCEs are approximately 150 bp, and fragments lacking the core region would not be captured by the UCE probe set, fragments that are approximately this length (150 bp) would not be useful as they would either not be captured or consist only of an uninformative core region with little informative flanking region on either end. Therefore, to remove short fragments (< 400 bp), sonicated samples were purified with MagBind total bind NGS (Omega BioTek, GA, USA) at 0.6x concentration. DNA concentration was calculated by quantifying samples using Accublue High-Sensitivity (HS) dsDNA quantification kit (Biotium). Subsamples of the sonicated products were then electrophoresed on a 2% agarose gel to visualize band-size distribution. To repair DNA ends from the random shearing, oligonucleotide adapters were blunt-end ligated. End prep included 60 µl reactions consisting of 50 µl sonicated DNA, 3µl end-prep enzyme and 7 µl end-prep buffer. Samples were then ligated to adaptor sequences using the NEB Ultra II DNA library prep Kit for Illumina (New England Biolabs, MA, USA), with the following conditions: 93.5µl reaction included 56.5 µl Blunt End prepped DNA, 30µl Ligation MM, 1µl Ligation enhancer, 6µl Oligo adaptor incubated at room temperature for 30 minutes. Ligated samples were again purified with MagBind total bind NGS, this time at a 1x concentration (= 93.5 µl). Samples were dual indexed with Illumina compatible PCR primers using a bead in protocol with Adapterama iTru5 and iTru7 indexed PCR primers (Glenn et al. 2016); 50 µl reactions comprised 25µl MasterMix, 5 µl iTru5 and iTru7 primer, and 15 µl ligated DNA. PCR conditions included: an initial denaturation step of 98°C for 45 s, followed by 19 cycles of 98°C for 15 s, 60°C for 30 s, 72°C for 1 min 15 s, followed by a final extension of 72°C for 5 min. Following this PCR indexing step, samples were again cleaned, this time using polyethylene glycol (PEG) at 0.9x concentration (50 µl sample * 0.9 = 45 µl PEG/sample) with the following protocol: 45 µl PEG was added to each sample and incubated for five minutes, then placed onto a magnet; supernatant was removed, followed by two washes of EtOH. Samples were eluted in 26 µl water. Cleaned and indexed samples were then quantified using a Qubit 2.0 fluorometer. Following quantification, samples were enriched for targeted UCE loci following the standard protocol with the Actinopterygian UCE baitset (myBaits UCE Actinopterygians 0.5Kv1 by Arbor Biosciences, MI, USA; Faircloth et al. 2013) that uses 2001 baits to target 500 UCE loci. Samples were standardized to 250 ng prior to pooling and eight samples were pooled per hybridization reaction. Because each of these samples were different volumes during standardization (some needing as much as 10 µl), samples were dried using a vacufuge and then re-eluted in 7 µl per the manufacturers protocol. UCE hybridization was performed per the manufacturers protocol with a hybridization temperature (TH) of 65°C (Arbor Biosciences). This temperature was chosen based on the assumption that approximately 10% of data or less will be missed, and the relatively close relatedness of the study taxa. Hybridized samples were then cleaned using Streptavidin beads (Dynabeads) following standard protocol and eluted in 30 µL of water. Clean hybridized samples were further PCR amplified with 16 cycles; 50 µl reactions each contained 1X Phusion High Fidelity buffer, 1.5 mM MgCl2, 0.25 mM of dNTP, 25 pmol of each P5 and P7 primers, 2.0 U/µl Phusion Taq, and 15 µl DNA. Cycling included: an initial denaturation step at 95°C for 5 min, 98°C for 30 s, and 16 cycles of 98°C for 15 s, 65°C for 30 s, and 72°C for 1 min, 30 s with a final extension step at 72°C for 5 min. To determine whether UCEs had been successfully enriched (at least a 50× enrichment of product compared to pre-enriched samples; Faircloth et al. 2013), quantitative PCR (qPCR) was performed using GoTaq qPCR master mix (Promega, WI, USA) and a StepOnePlus Real-Time PCR system (Applied Biosystems, inc., MA, USA). Quantitative PCR reactions of 25 µl included: water, GoTaq qPCR Master Mix, 25 pmol qPCR primer, and 2 µl standardized DNA (1 ng/µl). Quantitative PCR conditions included: an initial denaturation step at 95°C for 5 m, followed by 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 30 s, and a final cooling step at 40°C for 10 s. Forty-eight successfully enriched samples were then quantified, standardized, and pooled for sequencing (paired-end, 2×150 bp) on an Illumina HiSeq 4000 by Azenta, Inc. (MA, USA). Processing of sequence data. Demultiplexed data were downloaded and adapters and low-quality bases trimmed from sequence data using Trimmomatic (Bolger et al. 2014). Contigs were assembled using Trinity (Grabherr et al. 2011). Next, reads were clustered based on an 80% similarity threshold to create a consensus sequence per contig per individual (CD-hit-est; Li and Godzik 2006). A database of UCE contig matches to UCE probes was created (probe.matches.sqlite) at the beginning of the PHYLUCE pipeline using LASTZ; here contigs were screened for paralogs depending on the number of probe matches/ mismatches (Faircloth et al. 2016). Due to the broad taxon sampling of this study, it was possible that loci that could be informative for N. stramineus and closely relat- Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 706 ed species were discarded during initial filtering due to their absence in outgroup taxa. To try to account for this, an additional dataset (referred to as reduced dataset) was created that included only N. stramineus sensu lato, N. topeka, N. procne, and N. chihuahua, with N. mekistocholas, N. nubilus, and N. heterodon to root the trees. In the following outlined methods, each analysis step was performed for both the complete dataset and reduced dataset independently. Four data matrices, representing different levels of data completeness, were created for each of the two unfiltered datasets (complete and reduced, in terms of taxa included). Each set of four ranged from complete, in which no loci are missing for any taxon; i.e., all taxa share all loci (100%), as well as increasingly inclusive datasets (75%, 50%, and 25%) in which 75%, 50% and 25% of taxa are included in each locus alignment of each dataset, respectively; this is somewhat counterintuitive to these names, as the two 25% datasets will include more data than the 100% datasets (because more loci are included as the requirement for inclusions is only that 25% of taxa retained this locus in the alignment). This resulted in eight unique data matrices (Table 2). Alignment was performed using MAFFT, and taxon names were edited to remove the names of loci for use in downstream analyses in the PHYLUCE pipeline. Phylogenetic analyses of UCE data. Maximum likelihood analyses were conducted on concatenated matrices resulting from all eight datasets using RAxML vs. 8.2.12 (Stamatakis 2016), applying the GTR+Gamma substitution model. Individual gene trees for each data set were constructed using RAxML under the same nucleotide substitution model. Node support was estimated using bootstopping, an efficient method that determines whether sufficient bootstrap replicates have been completed based on a threshold of variation in branch support for a large percentage (99%) of the bootstrap replicates (a 0.03 or 3% threshold is the empirically recommended setting and was used here; Pattengale et al. 2010; Stamatakis 2016). Species trees were constructed by analyzing the best individual ML gene trees using the summary coalescent method in the program ASTRAL-III (AS; Mirarab and Warnow 2015). Simulation studies have shown that while empirical studies using concatenated datasets often result in well-supported topologies, in taxa with incomplete lineage sorting (ILS) concatenation methods perform relatively poorly and can be inconsistent (Kubatko and Degnan 2007; Roch and Steel 2015) because of gene tree species tree discordance. Therefore, while it is common to conduct an initial concatenated analysis, summary methods such as AS are considered more robust. Support values output by AS are “local posterior probabilities” (LPP) which represent the frequency a given taxon quartet is recovered in the individual UCE gene trees summarized by AS (Sayyari and Mirarab 2016), and the “best” tree maximizes the number of taxon quartets found most often in the gene trees. Internal branch lengths represent coalescent units while terminal branch lengths are not estimated (Mirarab et al. 2014). Discordance among gene trees within a dataset was estimated by calculating the normalized quartet score during species tree construction. This ranges in value from 0 to 1 and represents the proportion of quartets found in the gene trees that are present in the species tree. A normalized quartet score nearer 1 indicates very low gene-tree discordance while a low value, e.g., 0.4, represents very high levels of gene-tree discordance; i.e., in a species tree with a normalized quartet score of 0.4, 60% of quartets found in individual gene trees are not present in the estimated species tree. To visualize possible discordance in the dataset, an additional analysis was performed with the reduced, 100% dataset using BEAST2 v.2.7.5 (Bouckaert et al. 2019) under uninformative priors with a run of 10 million generations on the CIPRES Science Gateway. Clades that were consistently recovered in the other analyses in this study were constrained to be monophyletic. Convergence of the Markov chain as well as estimated sample sizes were inspected using Tracer v.1.7. DensiTree v.2.2.6 (Bouckaert and Heled 2014) was used to visualize the posterior distribution of trees resulting from this analysis. Morphological datasets Traditional morphometrics and meristic characters. Twenty measurements were collected from 330 specimens representing 50 individuals per clade of Notropis stramineus sensu lato identified in analyses of molecular data as well as ten specimens each of the other members of the N. stramineus species group. Measurements were collected from the left side to the nearest 0.1 mm using digital calipers. Measurements, collected following Hubbs and Lagler (1958), included: (1) standard length (SL); (2) body depth at origin of dorsal fin; (3) predorsal-fin distance; (4) prepelvic-fin distance; (5) pre-anal distance; Table 2. Summary statistics for the eight UCE matrices analyzed. Loci = number of loci; bp = base pairs; x  locus length = average length of loci in a given dataset; Inf. Sites = number of informative sites; Min. # Taxa/locus = the minimum number of taxa present in each alignment of a given dataset (the complete matrices contain all taxa; i.e., no taxon is missing any UCE locus). Complete taxon dataset Reduced taxon dataset Loci bp x  locus length Inf. sites Min. # Taxa/locus Loci bp x  locus length Inf. sites Min. # Taxa/locus Complete 93 116,347 1251 6784 47 125 150,103 1200 4906 36 25% Missing 416 479,353 1152 29,600 36 414 461,432 1115 15,939 27 50% Missing 436 489,518 1123 29,939 24 436 473,088 1085 16,173 18 75% Missing 449 495,616 1104 30,183 12 447 477,304 1067 16,325 9 Vertebrate Zoology 75, 2025, 699–755 707 (6) preanal-fin distance; (7) length of dorsal-fin base, (8) length of anal-fin base; (9) dorsal-caudal distance; (10) caudal-peduncle length; (11) caudal-peduncle depth; (12) head length (HL); (13) head depth through orbit; (14) head depth at occiput; (15) orbit diameter; (16) interorbit distance; (17) snout length; (18) distance from snout to occiput; (19) mouth width; and (20) length of lower jaw. Measurements 2–11 were calculated as a percentage of SL and measurements 13–20 as percentage of HL. External meristic characters were collected from 50 individuals representing each clade of N. stramineus sensu lato, as well as ten specimens each of the remaining members of the N. stramineus species group. External meristic characters follow Hubbs and Lagler (1958) except: (1) lateral line scale counts do not include those on the base of the caudal fin, which are counted separately; (2) the last dorsal-fin ray is not double-counted while the last two anal-fin rays that share a pterygiophore are both counted, and (3) scale counts (6) and (7) for which scales are counted on one side only, from dorsal midline to ventral midline. Scale counts included: (1) scales in lateral-line scale row; (2) lateral-line scales on base of caudal fin; (3) pre-dorsal scales; (4) scale rows above lateral-line scale row; (5) scale rows below lateral-line scale row (4 and 5 at dorsal-fin origin); (6) circumferential scale rows; and (7) circumpeduncular scale rows. Thirteen internal counts were taken, including: (1) total vertebrae; (2) abdominal vertebrae; (3) caudal vertebrae; (4) position of dorsal fin in relation to neural spines; (5) position of anal fin in relation to hemal spines; (6) number of ribs; (7) dorsal-fin rays; (8) anal-fin rays; (9) principal caudal-fin rays; (10) dorsal procurrent rays in caudal fin; (11) ventral procurrent rays in caudal fin; (12) pelvic-fin rays; and (13) pectoral-fin rays. Vertebral counts include the four Weberian centra and terminal compound centrum (Fink and Fink 1981). Internal meristic counts were obtained from specimens cleared and double-stained following Taylor and Van Dyke (1985). To visualize variation within the subset of measurements identified as potentially important during geometric morphometric analysis (see below) among the five clades of Notropis stramineus s. l., violin plots combined with boxplots were constructed using a combination of the R packages ggplot2 (Wickham 2011) and ggstatsplot (Patil 2021) performed in R (R Core Team 2023). These included orbit diameter, snout length, body depth, and snout to occiput. Multivariate statistics. Principal component analysis (PCA) of traditional measurements obtained from the measurement data collected (described above) was performed in base R using the function “prcomp()”. To account for the effect of size on measurements, each measurement was regressed against standard length, and the residuals were appended to the original data matrix and used in further principal component analysis. PCA plots were visualized using the package “ggbiplot”, a ggplot2 based biplot, in R. The broken-stick model was used to determine how many PCs were important to maintain for analysis. To determine whether a significant difference was present between species in the returned PCA data, a multivariate analysis of variance (MANOVA) was performed on the PC scores returned, grouped by clade. To further determine which clades differed significantly from one another, pairwise t-tests were performed for the scores obtained from principal components one through three. Geometric morphometrics. Two-dimensional geometric morphometric data were collected for use in multivariate analyses of shape data. Images were taken of the left side of 20 specimens of each member of the Notropis stramineus species group (except for N. chihuahua, for which 10 specimens were photographed) with a Canon 60D DSLR camera. Only specimens without obvious preservation artifacts (e.g., bending, desiccation, damage) were photographed and used in analyses. For photographs, specimens were placed onto a small amount of putty resting within a plastic box containing Sylguard and positioned to be parallel with the surface. A pin was placed into the underlying Sylguard and used to separate the posteriormost fin rays of the dorsal and anal fin from the body. Landmarks for the head and body include: (1) anteriormost point of rostrum; (2) posteriormost point of nare; (3) anteriormost, (4) posteriormost; (5) mid-dorsal and (6) ventral points of orbit; (7) and occiput; (8) anterior and (9) posterior insertion of dorsal fin; (10) insertion of dorsal and (11) ventral procurrent rays of the caudal fin; (12) anterior and (13) posterior insertion of anal fin; (14) anterior insertion of pelvic and (15) pectoral fins; (16) posteriormost point of opercle; (17) point of greatest curvature of preopercle; and (18) posteriormost point of the jaw (Fig. 3). Landmarks were placed using the digitizing tool available in the R package StereoMorph (Olsen and Westneat 1 2 34 5 6 7 8 910 11 13 12 14 16 15 17 18 Figure 3. Schematic representation of Notropis stramineus showing location of landmarks used in geometric morphometrics (in red). Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 714 sequence available) and thus not evaluated in the two nuclear gene trees is N. melanostomus. Haplotype network. Using a 1125 bp fragment of the cyt b gene, a total of 89 haplotypes were recovered among 164 individuals of Notropis stramineus sensu lato. Nine haplogroups were found corresponding to the six major clades recovered in the phylogenetic analyses (groups with >10 mutational steps between them were considered to be distinct haplogroups) (Fig. S4). Clades 1 and 4 were characterized by a relatively high number of haplotypes but lower number of mutations between haplotypes, while Clades 2 and 3 have both a higher number of haplotypes and a higher number of mutational steps between haplotypes. Relative to Clade 6, Clade 5 had relatively few haplotypes represented. A second analysis was performed in which the remaining members of the N. stramineus species group were included (N. procne, N. topeka, and N. chihuahua; Fig. S5). This included 189 sequences, from which 107 haplotypes were recovered. Morphological investigation Meristic characters. Scale, fin-ray and vertebral counts largely overlapped in all clades, with some differences discussed further under species accounts (scale counts, Table 5; fin ray and vertebrae counts, Table 6). Traditional morphometrics. Proportional measurements for each clade can be found in Table 7. For all measurements, ranges of values were largely overlapping for all clades of Notropis stramineus (visualized in PCA plots; Figs S6, S7), though the mean values of many of the measurements differed significantly among various clades (not shown). Loadings for PCs 1–3 are presented in Table S3. Results of MANOVA tests are presented in Table S4, along with pairwise t-tests of PC scores among clades. Hybrid violin/boxplots of select measurements are shown for visualization purposes for some of the more highly significant differences between clades, which are referenced as appropriate throughout species accounts. Differences in head measurements (% HL) were typically more drastic between clades than body measurements (% SL). For example, the orbit diameter of Clade 3 was significantly larger than the remaining clades, followed by Clade 1 and Clades 5 and 6, snout length in Clade 4 was shorter than in the other clades (Fig. S8), and distance from the snout to the occiput occupied a greater percentage of head length in Clade 3, differing significantly from all other clades except for Clade 1 (Fig. S9). Geometric morphometrics. Despite a large amount of overlap in the first few principal components, pairwise comparisons of LS means revealed statistically significant differences among many of the clades of Notropis stramineus sensu lato identified by molecular analyses, as well as between members of the N. stramineus species group (Table S5). While most pairs of clades and species were found to significantly differ in shape, several clade pairs were consistently found to not possess body or head shapes that differed significantly. For overall body shape, this includes Clade 1 and 2, Clade 1 and 4, Clade 1 and 5, Clade 2 and 4, and Clade 2 and 5. For head shape this includes only Clade 1 and 2, and Clade 1 and 4. Results of the PCA that included all members of the N. stramineus species group with vector diagrams representing extremes of principal components (minimum and maximum) are shown in Figure S10. Similar summaries for all-landmark and head-only landmark analyses including only N. stramineus sensu lato are shown in Figures 9 and S11, respectively. Pairwise analyses of those clades for Table 5. Range and mode of scale counts calculated by clade. N. stramineus sensu stricto (C1) N. multicorniculatus sp. nov. (C2) N. oblitus sp. nov. (C3) N. missuriensis (C4) range mode Holotype range mode Holotype range mode range mode Scales in lateral line 31– 39 36 34 31–38 34 34 32–36 35 31–37 35 LL scales on base of caudal fin 0–2 1 2 1–4 2 2 1–2 1 1–3 1 Predorsal scales 13–17 14 18 12–18 16 14 13–17 14 13–17 16 Above lateral line 5–7 6 6 5–8 7 6 5–7 6 5–7 6 Below lateral line 3–5 4 5 4–6 5 4 3–6 4 4–6 5 Circumferential scales 9–12 11 13 11–16 13 11 10–12 11 10–13 12 Circumpeduncular scales 6–8 7 7 7–10 9 7 7–9 7 7–9 7 N. lucifer sp. nov. (C5, 6) N. topeka N. procne N. chihuahua Holotype range mode range mode range mode range mode Scales in lateral line 33 30–38 31 34–37 36 35–37 35 33–37 35 LL scales on base of caudal fin 2 1–3 1 1–2 1 1–2 1 1–1 1 Predorsal scales 15 12–17 13 15–19 16 14–17 14 13–16 14 Above lateral line 6 5–7 6 7–8 7 6–6 6 6–6 6 Below lateral line 4 4–6 4 5–6 6 4–5 4 4–5 5 Circumferential scales 11 9–15 12 14–14 14 11–12 12 12–13 12 Circumpeduncular scales 7 6–9 7 8–10 9 7–8 7 7–8 7 Vertebrate Zoology 75, 2025, 699–755 715 which significant differences were found are shown in Figures S12 and S13. In the all-landmark dataset with all members of the N. stramineus species group (Fig. S10), principal components one to four accounted for 73% of the cumulative proportion of variance. Principal component one (33.3%) typically separated specimens along an axis that contained deeper-bodied individuals (dorsally and ventrally) with a more anteriorly-placed anal fin and a longer caudal peduncle at the positive end of the axis, and individuals that were more terete, thinner, with a more posteriorly-placed anal fin and shorter caudal peduncle at the negative end of the axis. Principal component two (20.5%) separated individuals with a steeper dorsal profile and a longer snout (positive end of axis) from those with a shorter snout and thinner body dorsally (negative end of the axis). In the all-landmark dataset with only Notropis stramineus sensu lato represented (Fig. 9), results closely resembled that of the previous analysis that included all members of the N. stramineus species group; however, the Euclidean distance between the LS means of Clade 4 and 5 was no longer significant (P = 0.131). Principal components one through four contained 67.5% of the cumulative proportion of variance, with principal component one and two explaining 34.2% and 12.6% of the variation, respectively. Principal component one separated individuals on the positive end of the axis from the negative end of the axis based on narrower, less steeply sloped, and shorter heads, and a dorsal profile that also slopes less steeply. Principal component two placed individuals on the positive end of the axis that have a shorter caudal peduncle, and those with the longer caudal peduncle on the negative end. Osteology. No major osteological differences were identified among clades of Notropis stramineus sensu lato. Table 6. Range and mode of meristic data (vertebral and fin-ray counts) by species. N. stramineus sensu stricto (C1) N. multicorniculatus sp. nov. (C2) N. oblitus sp. nov. (C3) N. missuriensis (C4) range mode range mode range mode range mode Total vertebrae 36–37 36/37 33–35 34 35 35 34–36 35 Abdominal vertebrae 18–19 18/19 17–18 17 18 18 18–19 18 Caudal vertebrae 17–19 18 16–17 17 17 17 15–18 16/17 Position of dorsal fin (10,11)– (12,13) 12,13 (10,11)– (11,12) (10–11) (10,11)– (11,12) (10–11)/ (11/12) (10,11)– (11,12) (11,12) Position of anal fin (18,19)– (19,20) (18,19)/ (19,20) (17,18)– (19,20) (18,19) (18,19) (18,19) (18,19), (19,20) (18,19) Number of ribs 14–15 14/15 13–14 13 14 14 14–15 14 Dorsal fin rays iii.7 iii.7 iii.4-iii.7 iii.7 iii.7 iii.7 iii.7 iii.7 Anal fin rays iii.7 iii.7 v.5-iii.7 iii.7 iii.7 iii.7 iii.7 iii.7 Caudal principal rays 10+9 10+9 10+9–11.9 10+9 10+9 10+9 (9+8)– (10+9) (10+9) D. caudal proc. rays 8–11 10 8–12 10 10 10 8–11 9 V. caudal proc. rays 8–9 9 8–9 8 9 9 7–9 8 Pelvic fin rays i.6.i i.6.i i.5.ii-i.6.i i.6.i i.6-i.6.i i.6.i i.6.i-i.7.i i.6.i Pectoral fin rays i.11.ii-i.12.ii i.11.ii i.10.iii-i.13.ii i.11.iii i.11.ii-i.12.i i.11.ii/i.12.i i.11.i-i.12.ii i.11.ii N. lucifer sp. nov. Dev. R. (C5) N. lucifer sp. nov. Col. R. (C6) N. lucifer sp. nov. (C5 & 6) range mode range mode range mode Total vertebrae 36–37 36 33–35 35 33–37 36 Abdominal vertebrae 18–20 18 16–18 18 16–20 18 Caudal vertebrae 17–18 18 16–17 17 16–18 17 Position of dorsal fin (10,11)– (12,13) (11,12) (9,10)– (11,12) (11,12) (9,10)– (12,13) (11,12) Position of anal fin (18,19)– (19,20) (18,19) (16,17)– (18,19) (18,19) (16,17)– (18,19) (18,19) Number of ribs 14–16 14 12–14 14 12–16 15 Dorsal fin rays iii.7 iii.7 iii.7-iiii.7 iii.7 iii.7-iiii.7 iii.7 Anal fin rays iii.7 iii.7 ii.7-iii.7 iii.7 ii.7-iii.7 iii.7 Caudal principal rays 10+9 10+9 9+10–10+10 10+9 9+10–10+9 10+9 D. caudal proc. rays 9–11 11 9–11 9/10 9–11 10 V. caudal proc. rays 8–9 9 8–9 8 8–9 8 Pelvic fin rays i.6.i-i.7.i i.6.i i.6.i i.6.i i.6.i-i.7.i i.6.i Pectoral fin rays i.11.ii-i.13.ii i.13.ii/i.11. ii/i.11.iii i.10.ii-i.12.ii i.11.iii i.10.ii-i.13.ii i.11.iii Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 716 Table 7. Range, mean, and standard deviation (SD) for each measurement either as a percentage of SL or HL. Mean values bolded Clade = C. N. stramineus sensu stricto (C1) N. multicorniculatus sp. nov. (C2) N. oblitus sp. nov. (C3) N. missuriensis (C4) Range Mean SD H Range Mean SD H Range Mean SD Range Mean SD Standard length (mm) 35.4–64.5 46.2 7.2 42.3 38.2–62.4 46.4 6 41.2 30.8–45.2 38.1 3.5 33.5–55.1 45.8 5.6 In percent of standard length Body depth 17.5–26.5 22.1 2 9.6 18.6–29.2 24.2 2.4 9.6 18.4–26.0 22.6 1.5 19.6–28.1 22.3 1.9 Pre-dorsal fin 48.6–55.2 51.4 1.4 21.9 47.4–53.5 51.1 1.4 21.5 49.3–54.5 51.8 1.3 47.8–55.2 51.3 1.5 Pre-pelvic fin 47.6–51.8 49.5 1.1 21.6 46.7–54.4 50.6 1.7 20.7 48.9–53.2 51.1 1.1 47.0–54.2 50.1 1.5 Pre-anus 61.3–69.6 65.6 1.8 27.7 62.1–70.1 66.4 2 26.6 64.3–69.3 66.2 1.1 63.3–71.4 66.7 1.9 Pre-anal fin 63.8–70.4 66.9 1.4 28.5 63.0–80.4 68 2.6 27.7 65.6–70.1 67.4 1 64.9–72.9 68.1 1.8 Base of dorsal fin 9.9–13.5 11.8 0.9 5.2 11.1–13.8 12.6 0.8 5 9.7–13.3 11.9 0.8 10.3–14.3 12.5 0.8 Base of anal fin 6.9–10.9 9 0.9 4.5 3.6–10.9 9.4 1.1 4.4 6.6–10.5 8.5 0.9 7.9–10.8 9.3 0.6 Dorsal-caudal distance 39.2–53.8 51.2 2.2 22.6 49.2–55.8 52.2 1.3 21.7 46.6–54.6 52.3 1.4 47.5–54.9 50.8 1.5 Caudal ped. length 18.7–28.4 24.4 1.8 10.2 21.5–29.2 23.9 1.6 9.5 21.3–27.1 24.2 1.4 17.8–26.2 23 1.9 Caudal ped. depth 7.6–11 9.7 0.7 4.5 8.5–11.8 10.3 0.6 4.3 8.93–11.1 10.2 0.5 7.9–11.1 10.1 0.6 Head length 22.5–26.6 25.1 0.8 11 24.2–28.2 26.2 1 11 23.5–28.5 26.2 1.1 24.5–29.1 26.2 1 In percent of head length Head depth through orbit 31.6–55.4 50.4 3.5 5.9 47.7–57.9 52.1 2.4 6.3 49.4–61.9 54.1 2.5 44.5–56.9 49.1 2.5 Head depth at occiput 56–70.3 63.4 3.3 7.3 59.6–82.6 67.2 3.6 7.7 61.7–73.9 66.5 2.6 58.5–71.0 63.2 2.9 Orbit diameter 24.5–36.8 30.4 2.5 2.9 19.6–30.6 25.8 2 4 28.4–37.2 32.2 1.9 23.5–31.2 27.2 1.7 Interorbit 25.9–36.8 30.1 2.9 3.8 23.8–36.5 31.6 2.6 3.3 21.7–36.6 30.6 3.4 24.3–36.3 29.1 2.6 Snout length 21.7–33.9 26.8 2.6 2.9 21.3–30.9 26.3 2.7 3.3 22.1–32.1 27.1 2.1 20.1–28.6 25 1.9 Snout to occiput 80.1–93.2 85.9 2.9 9.1 74.5–88.2 81.5 3.8 10.1 81.6–97.8 87.5 3.2 77.2–88 82.3 2.7 Mouth width 17.2–28.3 22 2.2 2.2 18.1–29.6 23.2 3 2.4 17.3–26.7 22 2.1 18.7–34.9 22.8 3.4 Lower jaw length 19.8–28.6 24.6 2 2.4 20.1–30.2 25.8 2.1 2.8 18.0–26.9 23 1.7 21.7–31.1 24.4 1.9 N. lucifer sp. nov. (C 5 and 6) N. topeka N. procne N. chihuahua H Range Mean SD Range Mean SD Range Mean SD Range Mean SD Standard length (mm) 43.2 31.6–50.6 39.6 4.5 38.7–51.8 44.0 4.6 45.4–52.1 48.6 2.5 26.4–46.8 35.0 7.2 In percent of standard length Body depth 8.8 18.2–27.0 22.1 1.9 26.6–30.1 28.7 1.0 19.7–24.5 21.6 1.6 21.8–25.2 23.4 1.2 Pre-dorsal fin 21.7 47.9–56.2 51.1 1.2 47.9–50.7 49.2 1.0 47.6–50.7 49.8 0.9 48.9–51.6 50.1 1.0 Pre-pelvic fin 20.8 46.–55.0 49.8 1.5 46.6–49.2 47.9 0.9 45.8–49.8 48.3 1.2 49.2–52.2 50.6 0.9 Pre-anus 28 61.0–71.1 64.9 1.5 60.2–63.3 61.5 0.9 47.8–65.4 62.5 5.3 62.9–68.5 65.6 1.8 Pre-anal fin 28.9 44.8–72.5 66.4 2.6 62.0–66.9 64.5 1.6 63.2–66.6 65.4 1.1 64.6–69.8 66.8 1.8 Base of dorsal fin 4.9 10.5–15.3 12.4 1.0 11.3–14.2 12.9 0.9 10.4–13.9 12.0 0.9 12.5–14.8 13.7 0.8 Base of anal fin 3.6 6.3–10.6 8.7 0.9 8.6–11.5 10.1 1.0 8.1–10.3 9.1 0.6 7.1–11.7 9.9 1.3 Dorsal-caudal distance 22.6 48.9–59.5 52.3 1.4 55.0–57.6 56.0 0.9 50.4–53.7 52.4 1.0 50.8–54.9 52.8 1.3 Vertebrate Zoology 75, 2025, 699–755 717 However, differences in the degree of ossification (i.e., presence of ossified bone vs. cartilage) of the ethmoid region, and the shape of the vomer, were apparent between individuals of Clade 5 and the remaining members of N. stramineus sensu lato (Fig. 10). Systematic Accounts Based on the information above, we consider the five recovered clades of Notropis stramineus sensu lato within the N. stramineus species group to represent distinct lineages that warrant recognition as species-level taxa, three of which are undescribed. Therefore, in the following section we provide taxonomic redescriptions for N. stramineus (Clade 1) and N. missuriensis (Clade 4), and descriptions for clades 2 (N. multicorniculatus sp. nov.), 3 (N. oblitus sp. nov.), and 5 + 6 (N. lucifer sp. nov.). Notropis stramineus (Cope, 1865) Clade 1 Figures 1, 10A, 11, 12, 13–16A Synonymy. Hybognathus stramineus Cope, 1865: 283; Detroit River, Grosse Isle, Michigan. Lectotype ANSP 4131 (designated by Fowler 1910: 274). Material examined. Lectotype ANSP 4131, 52.4 mm SL (photograph only); Wayne County: Grosse Ile, Michigan; –ROM 104002, 4, 31–49 mm SL; York RM: at Kingston Road, Glen Rouge Campground, Rouge River, 43°48′23″N 79°08′02″W; 18 September, 2017. –ROM 98646, 10, 36–54 mm SL; Lake Simcoe, 44°20′16″N 79°13′41″W; 19 June, 2007. –ROM 99745, 1, 31 mm SL; York Regional Municipality: at start of eroding cliff upstream of Dundas Street West, downstream of railway bridge, Humber River, 43°39′44″N 79°30′32″W; 5 May, 2014. –ROM 89469, 3, 39–42 mm SL; Bruce: Sauble River, 44°39′53″N 81°16′20″W; 29 May, 2007. –ROM 42057, 404, 49–65 mm SL; Huron: Maitland River (Great Lakes), 43°44′53″N 81°43′31″W; 10 June, 1982. –NYSM 70559, 45, 28–45 mm SL; Erie: Eighteenmile Creek at NYSDEC Public Fishing Access, upstream of mouth, at Old Lake Shore Drive, 42°42′44″N 78°57′59″W; 5 June 2014. –UMMZ 243085, 2, 28–37 mm SL; Wayne: South Grassy Island, Detroit River, 42°13′23″N 83°08′08″W;16 July, 1935. –UMMZ 239496, 20, 37–56 mm SL; Presque Isle: Grand Lake Creek (outlet) at Mouth in Lake Huron at Thompson Harbor Road; Lake Huron Drainage, 46°07′59″N 83°39′59″W; 30 August, 2001. –Uncat, AKP 07-16, 317, 18–58 mm SL; Wayne: Huron River at Hudson Mills Metropark, 42°22′56″N 83°54′51″W; March 2015. –NYSM 64919, 210, 31–58 mm SL; Ulster: Shawangunk Kill, Route 7a, by Route 7, 42°28′08″N 78°45′19″W; 8 July, 2009. –TCWC 17159.15, 24, 35–46 mm SL; VerCaudal ped. length 11.3 20.–30.0 24.9 1.9 26.5–28.8 27.9 0.8 23.5–26.2 24.9 0.8 20.9–25.7 23.2 1.4 Caudal ped. depth 4.3 8.6–11.8 10.2 0.7 10.3–12.8 11.6 1.0 8.5–9.7 9.1 0.3 8.3–9.8 9.2 0.5 Head length 11.2 22.9–28.6 26.1 1.2 24.6–26.8 25.7 0.7 21.5–24.4 22.6 0.8 24.6–28.2 26.2 1.0 In percent of head length Head depth through orbit 5.7 48.9–61.5 54.0 2.6 46.7–57.6 53.8 3.4 48.1–56.8 52.5 3.1 53.5–60.5 56.6 2.1 Head depth at occiput 6.8 55.3–71.9 64.9 3.3 64.4–77.6 72.4 4.3 58.6–74.7 67.0 4.4 65.7–74.7 69.6 2.5 Orbit diameter 3.4 24.3–36.1 29.7 2.1 22.3–28.1 26.3 1.9 25.7–33.0 29.3 2.1 24.1–33.3 28.7 3.0 Interorbit 2.6 22.9–36.4 29.2 3.1 36.8–43.8 39.7 2.2 31.0–36.2 33.6 1.7 32.8–34.7 33.7 0.6 Snout length 2.8 22.1–32.7 26.8 2.1 22.4–30.5 27.1 2.5 28.1–31.6 29.8 1.2 24.3–31.5 28.6 2.1 Snout to occiput 9.8 78.7–93.2 85.4 3.1 75.7–83.3 80.0 2.7 81.8–91.4 86.7 2.9 82.4–94.2 87.4 4.0 Mouth width 2.3 16–25.7 20.9 2.1 16.5–25.8 20.9 3.1 18.6–24.7 21.7 1.9 18.5–26.9 22.3 2.7 Lower jaw length 2.9 18.4 - 29.3 23.8 2.3 19.4–25.9 22.3 2.2 18.6–25.8 21.9 2.5 23.1–27.5 25.0 1.5 Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 718 milion: Vermilion River at Grape Creek Road crossing, 40°05′05″N 87°35′36″W; 10 June 2013. –JFBM 43651, 7, 45–52 mm SL; Vermilion: Middle Fork Vermilion River at Potomac Collision Rd (720E), 0.25 miles S of Potomac, 40°17′50″N 87°48′01″W; 27 July, 2004. –TCWC 17158.05, 14, 35–42 mm SL; Kendall: Aux Sable Creek at Bell Road crossing, 41°29′53″N 88°17′57″W; 9 June 2013. –ROM 43026, 4, 46–55 mm SL; Huron: Bayfield River, 43°34′09″N 81°41′51″W; 8 June, 1982. –NYSM 72734, 24, 25–50 mm SL; Tyler/Doddridge: Flint Run, 39°23′43″N 80°43′58″W; 3 June, 2015. –NYSM 69037, 18, 31–52 mm SL; Clay: Red Bird Creek, off State Route 66, near intersection with U.S. Route 421, 37°09′21″N 83°35′19″W; 3 June, 2013. –ROM 11967, 25, 42–49 mm SL; Glengarry: Lake St. Francis, 45°05′19″N 74°30′59″W; 18 June, 1938. –NYSM 71381, 26, 27–56 mm SL; Saint Lawrence: Robert Moses State Park beach, 45°00′14″N 74°51′06″W; 18 September, 2014. –NYSM 57545, 11, 35–42 mm SL; Essex: Mouth of Boquet River, to island below, Willsboro, 44°32′39″N 73°24′10″W; 3 August, 2004. –JFBM 43593, 1, 38 mm SL; Edgar: Crabapple Creek, 39°45′13″N 87°35′39″W; 28 July, 2004. Diagnosis. Notropis stramineus is distinguished from all other members of the N. stramineus species group, except N. lucifer sp. nov., by a higher number of vertebrae (36–37 vs. 33–36). Notropis stramineus is distinguished from N. lucifer sp. nov. by a diffuse and even scattering of small melanophores on the head (vs. few, large melanophore clusters), a cross-hatching pattern of melanophores on scales in rows dorsal to the lateral-line scale row (vs. scales in rows dorsal to the lateral line scale row without cross-hatching pattern of melanophores), the presence of a well-developed cleithral stripe (vs. absent or weakly developed), a higher number of lateral-line scales (31–39, modally 36, vs 30–38, modally 31), and in life a lateral bluish sheen and yellow to peachy coloration of the pectoral fin and pectoral-fin base (vs. in life, body primarily silvery, pectoral fin hyaline). Notropis stramineus is distinguished from N. topeka and N. procne by the extent of lateral pigment, existing as a series of “train track” markings along the lateral line, surrounded anteriorly and posteriorly by a sparse field of melanophores not reaching beyond two to three scale rows above and below lateral line scale row (vs. a dense, dark lateral stripe of melanophores that extends along the side of body, from snout to base of caudal fin). Notropis stramineus is further distinguished from N. topeka and N. multicorniculatus sp. nov. by the size of tubercles, with many very minute tubercles scattered across most regions of the head, the largest of these concentrated in the subopercular region (vs. fewer, large tubercles, uniform in size in all regions in which present), and further distinguished from N. topeka by tubercle distribution, with tubercles distributed across most regions of the head without clear patterns (vs. tubercles present in only a few regions, namely the rostral, lacrimal, supraorbital, internarial, interorbital, and fronto-occipital regions), and extent of nuptial coloration, with pectoral fins obtaining a yellow, peachy color in spawning males (vs. intense red and orange coloration in spawning males). Notropis stramineus can be further distinguished from N. chihuahua by the absence (vs. presence) of macromelanophores on the lateral surface of the body and head, a cross-hatching pattern on scales reaching to the lateral-line scale row and below anteriorly (vs. cross-hatching pattern restricted to three to four dorsalmost rows of scales anteriorly, and one to two dorsalmost rows of scales posteriorly). Notropis stramineus is most similar in superficial appearance to N. missuriensis and N. multicorniculatus sp. nov., particularly in pigmentation in life and in preservation, but can be distinguished from N. missuriensis and N. multicorniculatus sp. nov. most readily by aspects of tuberculation, with tubercles weakly developed in N. stramineus and inconspicuous to the eye (vs. more well-developed and obvious to the naked eye in nuptial males), and a larger eye, occupying much −0.04 −0.02 0.00 0.02 0.04 −0.075 −0.050 −0.025 0.000 0.025 0.050 Principal Component 1 (PC1): 34.18% Principal Component 2 (PC2): 13.6 % Clades Clade 1 Clade 2 Clade 3 Clade 4 Clade 5 Clade 6 Figure 9. Results of PCA using geometric morphometric characters of body taken from 20 specimens per clade of Notropis stramineus sensu lato; PC1 vs. PC2. Body shapes represented by maximum (positive) values of PCs in black, and minimum (negative) values in gray. Thin-plate spline deformation grids (top left, bottom right) show trends in shape difference along the principal component axes. Vertebrate Zoology 75, 2025, 699–755 719 of the head (orbit diameter 24.5–36.8 HL vs. 23.5–31.2 in N. missuriensis, 19.6–30.6 in N. multicorniculatus sp. nov.). Notropis stramineus is distinguished from Notropis oblitus sp. nov. by a generally greater abundance and concentration of brown and black pigmentation in rows dorsal to the lateral-line scale row in life (vs. scales in rows dorsal to lateral-line scale row with sparse scattering of dark brown pigment), the more pronounced presence of the cross-hatching pattern on the dorsal scale rows in the posterior half of the body in life and in preservation (vs. weakly developed cross-hatching pattern on the posterior half of the body in life and in preservation), as well as a prominent blue-to-purple lateral sheen in life (vs. the reduction or absence of this blue-to-purple sheen on the body side in life). Description. Body shape and general appearance as in Figures 1, 11, 17 and 12, 13A–16A. Lateral side of cranium rendered from CT scan shown in Figure 18. Hyopalatine arch, opercular bones, and infraorbital series shown in Figure 19. Morphometric and meristic data are listed in Tables 7–9. Small-bodied leuciscid fish; maximum size examined 64.5 mm SL. Body slightly compressed, elongate and slender, fusiform with gently sloping anterior profile, snout to dorsal fin insertion sloping more steeply than from dorsal fin to caudal fin. Head triangular/wedgeshaped to rounded, snout slightly rounded to sharp. Ventral profile convex anterior to pelvic-fin origin, and slightly concave from origin of pelvic fin to caudal-fin base. Body depth greatest at point slightly anterior to dorsal-fin insertion, approximately two-thirds distance between pectoral-fin base and anterior point of pelvic-fin insertion, and narrowest at caudal peduncle slightly anterior to insertion of procurrent caudal-fin rays. Eye large with anteriorly pointing pupil. Mouth gently sloped, subterminal with thin lips. Imaginary horizontal line through anteriormost point of upper jaw passes through lower third of orbit. Posteriormost point of jaw only reaches anterior margin of orbit. Nostrils slightly closer to rounded tip of snout than anterior margin of the eye. Anterior nostril small and elliptical; posterior nostril large and rounded to weakly elliptical in dorsal view. Gill membranes joined at isthmus. Pharyngeal teeth 4–4. Dorsaland anal-fin rays iii.7. Principal caudal fin rays 10+9. Dorsal procurrent caudal-fin rays 8(1), 9(1), 10(2), or 11(1). Ventral procurrent caudal-fin rays 8(2) or 9(3). Pectoral-fin rays i.11.ii (4) or i.12.ii (1). Pelvic-fin Figure 10. Cleared and stained ethmoid region of select members of Notropis stramineus species group. a, N. stramineus sensu stricto (Clade 1), TCWC 21295.01, Michigan, Huron River; b, N. multicorniculatus sp. nov. (Clade 2), TCWC 15789.07; c, N. missuriensis (Clade 4), TCWC 21299.01, Missouri, Contrary Creek, Missouri River drainage; d, N. lucifer sp. nov. (Clade 5, 6), TCWC 15689.04, 40.0 mm SL. V, vomer; P, parasphenoid; LE, lateral ethmoid. White outline demarcates border of vomer; note size and extend of cartilage in D (N. lucifer sp. nov.) relative to the other three. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 720 rays i.6.i. Dorsal fin short and rounded, slightly convex at posterior margin. Anal fin rounded with a slightly convex posterior margin. Anal-fin origin posterior to posteriormost insertion of dorsal fin. Insertion of pelvic fin slightly anterior to insertion of dorsal fin. Caudal fin forked, lobes slender, with ventralmost margin of upper lobe and dorsalmost margin of lower lobe weakly convex, approximately of equal length. Scales cycloid. Lateral line complete, perforating 31(1), 33(1), 34 (10), 35(6), 36(22), 37(5), 38(4) or 39(1) scales, plus 0(1), 1(27) or 2(22) on base of caudal fin. Scales in predorsal scale row 13(9), 14(24), 15(8), 16(5) or 17(4). Circumferential scale rows 9(4), 10(6), 11(22), or 12(18), including 5(9), 6(39) or 7(2) above the lateral line, and 3(1), 4(38), or 5(11) below. Circumpeduncular scale rows 6(1), 7(44), or 8(5). Scales absent on chest from posterior insertion of pectoral fins anteriorly. Total vertebrae 36(3) or 37(3), with either 18(3) or 19(3) abdominal vertebrae and 17(1), 18(4) or 19(1) caudal vertebrae. Insertion of first dorsal-fin pterygiophore between neural spines of vertebrae 10/12(1), 11/12(1), 12/13(4) (modally 12/13). Insertion of first anal-fin pterygiophore between hemal spines of vertebrae 18/19(4) or 19/20(2). Ribs 14(3) or 15(3). Infraorbital series comprising four or five bones (IO1– 5) (Figs 18, 19). IO1 (lacrimal) a large plate-like bone with concave posterior margin. IO2 long, tapering toward posterior half (in CT images, divided into two ossifications; Fig. 18). IO3, approximately twice as long as IO2, narrowest anteriorly, broader posteriorly. IO4 large and plate-like, approximately half the size of lacrimal. IO5, when present, consists nearly entirely of canal ossificaFigure 11. ANSP 4131, lectotype of Hybognathus stramineus, 52.4 mm SL; Michigan, Detroit River, Grosse Isle. Photo by M. Sabaj. Scale bar = 5 mm. Figure 12. Notropis stramineus sensu stricto, TCWC 21295.01, male (specimen not measured), photographed alive, in two views; Michigan, Huron River, Hudson Mills Metropark, March 2016. A lateral view; B dorso-lateral view. Vertebrate Zoology 75, 2025, 699–755 721 tion, with a small flange of dermal bone projecting anteriorly from shaft of canal. Cephalic lateral-line system comprising the following sensory canals and externally visible pores: infraorbital canal 10(2), 11(1) or 12(1); supraorbital canal 7(2), 8(1) or 10(1); preoperculo-mandibular canal 9(2), 10(1), or 12(1). with 4(3) or 5(1) in mandibular portion and 5(2), 6(1) or 7(1) in preopercular portion; otic 3(3) or 4(4); temporal 2(3) or 4(1). Canals of cephalic lateral-line system commonly exhibit asymmetry. Cephalic tubercles typically small to medium, not well developed or conspicuous, present in rostral region, sparse in interorbital region, present around outer margin of fronto-occipital region only, present in anterior part of lacrimal and supraorbital regions and throughout lateral portion of preopercular and interorbital regions. Tubercles absent from ventral regions, i.e., gular, interopercular, mandibular, branchiostegal membrane, chest, and ventral portion of preopercular regions. Males at peak of spawning activity may develop several larger tubercles in anterior part of supraorbital region. Minute cephalic tubercles present on nape, disorganized, scattered haphazardly across scales. Cephalic tubercles with wide base, recessed into a ring, with small conical tip projecting upward, small, height not extending far beyond epidermis. Pectoral-fin ray tubercles conical, small and slightly recurved, comparatively more well-developed than cephalic tubercles, and present on anteriormost pectoral-fin rays 1–8, arranged in 5–6 (sometimes more in very large males) slightly irregular rows, decreasing incrementally to one row moving distally to proximally. Tubercles on first ray typically restricted to center of ray (not developed on distal or proximal thirds of fin ray, except occasionally in very large males at peak of spawning activity), in one or two rows (sometimes as many as four in large males). Body tuberculation weakly developed, with tubercles arranged on posterior margin of scales dorsal to and including lateral line scale row, tubercles generally not developed on scales beyond point of dorsal fin insertion. Figure 13. Lateral view of heads of males of each member of Notropis stramineus species group; A Notropis stramineus sensu stricto (Clade 1), ROM 11967, 46.6 mm SL; B N. multicorniculatus sp. nov. (Clade 2), TCWC 15789.07, 43.2 mm SL; C N. oblitus sp. nov. (Clade 3), TCWC 16802.06, 41.2 mm SL; D N. missuriensis (Clade 4), KU 4186, 48.4 mm SL; E N. lucifer sp. nov., Devils River (Clade 5), TCWC 15689.04, 43.2 mm SL; F N. lucifer sp. nov., Col. drainage (Clade 6), TCWC 19721.01, 42.3 mm SL; G Notropis topeka, KU 40725, 46.1 mm SL; H Notropis procne, NCSM 12688, 51.1 mm SL; I Notropis chihuahua, UMMZ 211231, 41.8 mm SL. Scale bars = 2 mm. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 722 Coloration. In preservation, body background color pale yellow to light brown, pigment concentrated more densely on dorsal half than ventral half (Figs 11, 17). Lateral-line canals on body outlined above and below with thick speckling of melanophores, creating “train track” pattern. Train track pattern continues posteriorly to caudal-fin base, joined at approximately imaginary horizontal line through insertion of anal fin with a broader lateral stripe consisting of diffuse melanophores becoming more concentrated posteriorly. Lateral-line scale row plus one scale row above and below with band of guanine persisting in some specimens in preservation. Lateral band of guanine wider anteriorly than posteriorly, becoming constricted beginning approximately at horizontal line at midway point of pelvic fin. Scales dorsal to and including lateral-line scale row outlined in “cross-hatching” pattern, with first ca. 10 scale rows also cross-hatched ventral to lateral-line scale row, decreasing in pigmentation moving posteriorly. Males with densest concentration of melanophores on leading edge of pectoral fin and interradial membranes. Dorsal midline outlined by a thick line of melanophores, widened to a wedge at anterior insertion of dorsal fin where line terminates, to begin again at insertion of last dorsal-fin ray, proceeding posteriorly as a thin line until another widening at insertion point of dorsal procurrent caudal-fin rays. Dorsally, cephalic pigmentation is densely scattered as small fields of evenly sized melanophores in all regions (Fig. 14A). Laterally, pigmentation restricted to rostral, lacrimal, supraorbital, and fronto-occipital regions, with pigment more sparsely distributed in dorsalmost portion of opercular region (Fig. 13A). Pigment absent from all ventral regions of head, i.e., gular, interopercular, mandibular, branchiostegal membranes, chest, and ventral portion of preopercular regions (Fig. 15A). In life, dorsal-half of body straw-colored with a noticeable blue-to-purple sheen midway along body, typically along two scale rows dorsal to lateral-line scale row (Fig. 19). Peritoneum silvery. Orange tinge on pectoral fin and pectoral-fin base, fading distally; more pronounced Figure 14. Dorsal view of heads of males of each member of Notropis stramineus species group; A Notropis stramineus sensu stricto (Clade 1), ROM 11967, 46.6 mm SL; B N. multicorniculatus sp. nov. (Clade 2), TCWC 15789.07, 43.2 mm SL; C N. oblitus sp. nov. (Clade 3), TCWC 16802.06, 41.2 mm SL; D N. missuriensis (Clade 4), KU 4186, 48.4 mm SL; E N. lucifer sp. nov., Devils River (Clade 5), TCWC 15689.04, 43.2 mm SL; F N. lucifer sp. nov., Col. drainage (Clade 6), TCWC 19721.01, 42.3 mm SL; G Notropis topeka, KU 40725, 46.1 mm SL; H Notropis procne, NCSM 12688, 51.1 mm SL; I Notropis chihuahua, UMMZ 211231, 41.8 mm SL. Scale bars = 2 mm. Vertebrate Zoology 75, 2025, 699–755 723 in males. Guanine abundant laterally, including on head, where a scattering of chromatophores appear as golden flecks speckled sparsely, sometimes gathered in clusters. Dorsal midline with sporadic clusters of chromatophores that shine gold in light, the pattern of which is variable, sometimes in dashes, but also organized as short lines (Fig. 12B). Distribution. Notropis stramineus sensu stricto is widely distributed through the northeast of North America, west of the Appalachian Mountains and east of the Mississippi River mainstem, throughout the Great Lakes, Ohio, upper Illinois (though populations in the Illinois River are possibly non-native; see Discussion section “Membership and relationships of the Notropis stramineus species group – Notropis stramineus sensu stricto.”), Tennessee and Cumberland River drainages. Notropis stramineus is absent from the Atlantic Slope, except for the St. Lawrence River drainage. This distribution includes Canada (the provinces Ontario and Quebec), as well as the U.S. states Illinois, Indiana, Kentucky, Michigan, New York, Ohio, Pennsylvania, Tennessee, West Virginia and Wisconsin. Abundant in shallow, silty streams, but also abundant in deeper, slower moving water (e.g., widenings of the Huron River, MI; Fig. S14). Present, but not abundant, at the type locality (Grosse Ile, MI) as the shoreline has been heavily modified and canalized. Notropis missuriensis (Cope, 1871) Clade 4 Figures 10C, 13D–16D, 20, 21B,E,H, 22B, 23, 24 Synonymy. Hybopsis missuriensis Cope, 1871: 437; tributary to Missouri River, Buchanan County, near St. Joseph, Missouri. Lectotype ANSP 4374 (designated by Fowler 1910: 274, pl. 15, fig. 6). Figure 15. Ventral view of heads of males of each member of Notropis stramineus species group; A Notropis stramineus sensu stricto (Clade 1), ROM 11967, 46.6 mm SL; B N. multicorniculatus sp. nov. (Clade 2), TCWC 15789.07, 43.2 mm SL; C N. oblitus sp. nov. (Clade 3), TCWC 16802.06, 41.2 mm SL; D N. missuriensis (Clade 4), KU 4186, 48.4 mm SL; E N. lucifer sp. nov., Devils River (Clade 5), TCWC 15689.04, 43.2 mm SL; F N. lucifer, Col. drainage (Clade 6), TCWC 19721.01, 42.3 mm SL; G Notropis topeka, KU 40725, 46.1 mm SL; H Notropis procne, NCSM 12688, 51.1 mm SL; I Notropis chihuahua, UMMZ 211231, 41.8 mm SL. Scale bars = 2 mm. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 730 Texas State Highway 127 crossing in Concan, 29°29′44″N 99°42′42″W; 15 May, 2015, Conway, K.W., Kubicek, K.M., and Prestridge, H.L. (Fig. S16). Paratypes. TCWC 6221.03, 2, 39–44 mm SL; Kerr: Mo Ranch, 30°03′10″N 099°28′24″W; 30 April, 1985. –TCWC 6811.01, 2, 32–50 mm SL; Comal: Guadalupe Figure 23. Notropis missuriensis, KU 4186, USA, Kansas, Pottawatomie County: Missouri River, Carnahan Creek; A male, 48.4 mm SL; B female, 50.9 mm SL. Scale bars = 5 mm. Figure 24. Notropis missuriensis, TCWC uncatalogued, not measured, photographed alive, in two views; USA, Tennessee, Tipton County: Bear Creek. A lateral view; B dorsal view. Vertebrate Zoology 75, 2025, 699–755 731 River on HWY 306, 0.2 miles north of HWY 2673, 29°51′52″N 098°09′50″W; 16 July, 1983. –TCWC 174.02, 22, 28–44 mm SL; Blanco: Blanco River at Blanco, 30°05′52″N 098°24′57″W; 26 November, 1950. –JFBM 18598, 8, 21–33 mm SL; Hays: Blanco River, 6 miles W of Kyle (station 18), 30°00′17″N 097°58′07″W; 14 November, 1955. –TCWC 16802.06, 28, 31–38 mm SL; Kerr: Guadalupe River at bridge crossing on FM 1340, 30°03′08″N 099°27′04″W; 15 May 2015. –TCWC 15549.06, 11, 22–42 mm SL; Real: Kent Creek @ 336, 30°40′09″N 100°32′57″W; 15 October, 2011. –TCWC 16326.15, 40, 20–44 mm SL; Uvalde: Nueces River at FM 55, 29°37′03″N 100°00′33″W; 20 April 2013. – TCWC 16328.08, 69, 25–45 mm SL; Bandera: Sabinal River at RM337, 29°44′40″N 099°33′10″W; 21 April 2013. –TCWC 16876.1, 39, 18–39 mm SL; Uvalde: Frio River, 29°36′23″N 099°44′19″W; 18 January, 2014. Diagnosis. Notropis oblitus sp. nov. is distinguished from all other members of the N. stramineus species group, except N. stramineus, by a relatively large eye (orbit diameter ca. 32% of HL vs. 26–29% HL) in comFigure 25. Notropis multicorniculatus sp. nov. (Clade 2), USA, Texas, Hemphill County: Canadian River; A TCWC 15789.09, holotype, male, 43.2 mm SL; B TCWC 15789.07, paratype, female, 42.3 mm SL. Scale bars = 5 mm. Figure 26. Notropis multicorniculatus sp. nov. (Clade 2), male, not measured or preserved, photographed from USA, New Mexico, Guadalupe County: Pecos River. Photo by T. Kennedy. (https://www.inaturalist.org/people/tomkennedy). Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 732 bination with a low, rounded head (vs. a relatively steeply sloped head). It is further distinguished from N. stramineus by possessing fewer vertebrae (35 vs. 36–37), and from N. stramineus, N. multicorniculatus sp. nov. and N. missuriensis by a reduction in body pigmentation, with pigment on posterior half of body dorsal to lateral-line scale row reduced, cross-hatch pattern on scales sometimes absent posteriorly (vs. consistently pigmented posteriorly), as well as a lack of pigment ventral to the lateral-line scale row anteriorly, aside from cleithral streak Figure 27. Notropis oblitus sp. nov., USA, Texas: Uvalde County, Frio River; A TCWC 16882.13, holotype, male, 41.2 mm SL; B TCWC 16882.06, paratype, female, 43.1 mm SL. Scale bars = 5 mm. Figure 28. Notropis oblitus sp. nov., TCWC 20319.09, male, photographed alive, in two views; USA, Texas, Bandera County, Sabinal River. A lateral view; B dorsal view. Scale bar = 5 mm. Vertebrate Zoology 75, 2025, 699–755 733 (vs. first few scale rows with cross-hatch pattern ventral to lateral-line scale row in addition to cleithral streak). Notropis oblitus sp. nov. is further distinguished from N. lucifer sp. nov. by the presence of a typically well-developed cleithral streak (vs. cleithral streak absent or very weakly developed), the presence of pigmentation arranged in a cross-hatch pattern in all scale rows dorsal to lateral line (rarely reduced posteriorly) (vs. absent or reduced to sparse pigmentation on 2–3 scale rows dorsal to lateral-line scale row, including anteriorly), by cephalic pigmentation present in similarly-sized melanophores scattered evenly across most regions of the head (vs. restricted in distribution and consisting of a few clusters of melanophores), and by the presence of a peach-yellow pigmentation on the pectoral fins and pectoral-fin base in live males (vs. pectoral fins hyaline, and pectoral-fin base silvery or cream colored in life). Notropis oblitus sp. nov. is further distinguished from N. topeka and N. procne by the absence (vs. presence) of a dark lateral stripe along body side, from N. topeka and N. multicorniculatus sp. nov. by the presence of minute tubercles in reproductively active males (vs. large tubercles in reproductively active males), and further from N. topeka by an approximately even distribution of tubercles across the dorsal and lateral surfaces of the head (vs. densest in dorsal regions, largely absent from the lateral infraorbital, preopercular, opercular, and subopercular regions), and by a larger eye (orbit diameter 32.3% HL vs. 26.3% HL). Notropis oblitus sp. nov. is further distinguished from N. chihuahua by the presence of small, evenly distributed melanophores on all regions of the dorsal surface of the head (vs. melanophores restricted to fronto-occipital region, interorbital region, and in internarial, nasal, and rostral region present as irregularly scattered macromelanophores), as well as the presence of cross-hatching on scale rows dorsal to the lateral-line scale row anteriorly (vs. restricted to two to three scale rows ventral to dorsal midline anteriorly). Description. Body shape and general appearance as in Figures 27 and 28. Morphometric and meristic data are listed in Tables 7–9. As described for N. stramineus, except for the following. Eye very large, head low and round with rounded, blunt snout. Cephalic tuberculation weakly developed, inconspicuous and not visible without magnification, with small conical tubercles distributed sparsely across regions of head. Tubercles most densely concentrated in lacrimal, orbital, and preopercular regions, with tubercles scattered sparsely across internarial and fronto-occipital regions. Tubercles absent from rostral, opercular and subopercular regions, and in all ventral regions of head. Pectoral-fin tuberculation well developed, with four-five rows of large conical tubercles developed along pectoral-fin rays 1–7, and 1–2 rows of tubercles present on anteriormost margin of first pectoral-fin ray. Coloration. Coloration in preservative as in Notropis stramineus except for the following. Body background Figure 29. Map of southern Texas and Northern Mexico, depicting distribution of samples of Notropis oblitus sp. nov. and N. lucifer sp. nov. used in 3-gene dataset. Type localities of each new species indicated by a star. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 734 color in preservative a light yellow to whitish (Fig. 27). Melanophores below lateral-line canal sparse anteriorly, largely absent posteriorly until approximately vertical with insertion of anal fin, where melanophores ventral to lateral-line canal create weak, dusky band. Coloration in life silvery, pale, with straw or yellow-colored pigmentation restricted to uppermost part of body side, bluish sheen present but weak (Fig. 28A). Chromatophores on dorsal midline creating thick golden streak (Fig. 28B). Cleithral streak variable in intensity, often distinct, sometimes weak, in life and in preservation. Distribution. Notropis oblitus sp. nov. is distributed throughout several independent Gulf Slope streams in Texas, including the upper reaches of Nueces, San Antonio, and Guadalupe River basins (Fig. 29) in the Edwards Plateau ecoregion. It appears to be absent from the Colorado River basin. Etymology. The species name oblitus, forgotten, is the perfect participle of the Latin verb “obliterare”, to forget. The name alludes to Carl Hubbs’ recognition of the distinctiveness of southern populations of the sand shiner in the form of a third subspecies of Notropis deliciosa (Girard) 1856 (= Notropis stramineus), and the subsequent incidental loss of this recognition by other NA ichthyologists; oblitus, the perfect participle of the verb obliterare, also means “smeared” or “blurred”, a reference to the possible hybrid nature of the external morphological features with that of N. lucifer sp. nov. collected in the Colorado River basin of Texas. An adjective. Notropis lucifer sp. nov. https://zoobank.org/FB88ABF4-6B04-407D-BBDE-6E9D2F9E06CD Clades 5 and 6 Figures 10D, 13–16E,F, 30–32 Holotype. TCWC 15689.08, male, 43.2 mm SL; United States of America, Texas: Val Verde County: Devils River, Devils River Ranch, 29°40′ 34.32″N, 101°0′5.7594″W; Conway, K.W. and Prestridge, H.L., 18 April, 2012 (Fig. 30A). Paratypes. TCWC 15551.09, 10, 22–40 mm SL; Llano: Sandy Creek @SH16, 30°33′11″N 098°42′05″W; 19 September, 2018. –TCWC 922.02, 16, 38–51 mm SL; Llano: Sandy Creek, 19.0 mi SW of Llano., 30°32′04″N 098°47′52″W; 24 April, 1976. –TCWC 2069.02, 12, 23– 43mm SL; Llano: Sandy Creek at Hwy 71, 30°33′00″N 098°28′00″W; 17 September, 1977. –JFBM 18636, 4, 32–43 mm SL; Gillespie: Llano River, at Lang’s Mill (station 32), 30°28′50″N 099°07′15″W; 5 June, 1956. – TCWC 4065.01, 110, 25–37 mm SL; Gillespie: Pedernales River, Trough Springs on headwaters of Wolf Creek., 30°08′36″N 099°04′42″W; 30 October, 1971. –TNHC 47383, 34, 20–49 mm SL; Kimble: Little Devils River at CR 4301, 30°28′55″N 099°23′07″W; 9 July, 2013. – JFBM 18522, 93, 16–40 mm SL; Gillespie: Wolf Creek at Springs, 1 mile Below Texas Highway 16, 4 miles S of junction with Pedernales River, 30°10′28″N 099°00′41″W; 22 November, 1954. –TCWC 15689.04, 26, 23–43 mm SL; Val Verde: Devils River, 29°39′51″N 100°57′10″W; 19 September, 2018. –TCWC 7510.06, 106, 15–37 mm SL; Val Verde: Devils River, 29°53′08″N 100°59′36″W; 8 March, 1993. –TNHC 30952, 175, 16–49 mm SL; Val Verde: Devils River, 29°56′21″N 101°05′46″W; 27 July, 2004. –TCWC 11924.01, 2, 26–40 mm SL; Val Verde: Devils River, 29°52′23″N 100°59′35″W; October, 1997. –TNHC 30562, 559, 15–55 mm SL; Val Verde: Devils River, 29°52′23″N 100°59′35″W; 29 July, 2003. –TCWC 7509.09, 14, 23–42 mm SL; Val Verde: Devil’s River; 175 m upstream from Dolan Falls, 29°53′08″N 100°59′36″W; 17 May, 1994. –JFBM 18795, 17, 37–42 mm SL; Val Verde: Devil’s Lake, Devil’s River, 8 air miles NW of Del Rio, 29°29′40″N 100°59′19″W; 27 March, 1954. –TNHC 29785, 435, 14–49 mm SL; Val Verde: Devils River, 29°52′23″N 100°59′35″W; 30 July, 2002. –UMMZ 211231, 23, 24–47 mm SL; Brewster: Terlingua Creek, at Hwy 170, 1.3 mi W of Study Butte (JCT 170 and 118, 29°19′38″N 103°33′12″W; 14 March, 1982. Diagnosis. Notropis lucifer sp. nov. is distinguished from all members of the Notropis stramineus species group, except N. chihuahua, by the following characters: absence of cross-hatched (outlined) scales located on the 2–3 scale rows dorsal to the lateral-line canal scale row (vs. present and well developed anteriorly, and present but sometimes poorly developed posteriorly), cleithral streak absent (Devils and Colorado River drainages) or if present greatly reduced (Colorado River drainage, most obvious in males) (vs. present and well developed, most obvious in males), sparse, large melanophores in the rostral region of the head (vs. many small, diffuse melanophores), few, large melanophores laterally in the fronto-occipital region (vs. many small melanophores scattered across cranial region 8), restriction of few, large melanophores to the anterior half of the internarial region (vs. internarial region completely covered in small, diffuse melanophores), a more pronounced “wedge” of melanophores at insertion of dorsal procurrent rays, and extent of ossification of the ethmoid region reduced, with much of the ethmoid region remaining cartilaginous (vs. well-ossified), and a broad (vs. thin and tapering) vomer. Notropis lucifer sp. nov. is distinguished from N. chihuahua by smaller, fewer, and less diffuse macromelanophores generally, except in the internarial region, where melanophores are abundant (vs. sparse in N. chihuahua). Notropis lucifer sp. nov. is further distinguished from N. chihuahua by a continuous edge of medium to small melanophores outlining scales on nape and dorsal body rows (vs. scales on nape and scale rows dorsal to lateral-line scale row irregularly outlined by macromelanophores). Notropis lucifer sp. nov. is further distinguished from N. topeka and N. procne by the lack of a pronounced lateral stripe, and further distinguished from N. topeka by tubercle size and distribution, Vertebrate Zoology 75, 2025, 699–755 735 being small and distributed across most dorsal and lateral regions of the head (vs. very large and absent from the infraorbital, preopercular, opercular, and subopercular regions). Notropis lucifer sp. nov. is further distinguished from N. stramineus, N. missuriensis, N. multicorniculatus sp. nov. and N. oblitus sp. nov. by the absence (vs. presence) of a bluish sheen along the lateral body side in life, and cephalic pigmentation concentrated in the lacrimal, Figure 30. Notropis lucifer sp. nov., Texas, Val Verde County, Devils River; A TCWC 15689.08 holotype, male, 43.2 mm SL; B TCWC 15689.04, paratype, female, 38.6 mm SL. Scale bars = 5 mm. Figure 31. TCWC 19721.01, Notropis lucifer sp. nov., paratypes, Texas, Kimble County, Little Devils River; A male, 42.3 mm SL; B female, 42.2 mm SL. Scale bars = 5 mm. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 736 suborbital, dorsalmost part of opercular, fronto-occipital, and interorbital regions into clusters of melanophores (vs. cephalic pigmentation present in similarly-sized melanophores scattered evenly across most regions of the head, except ventrally), and rostral cap translucent and devoid of pigment with large clusters of melanophores on the lateral surface of the head, particularly in the lacrimal region (vs. rostral cap and lacrimal region covered in evenly distributed field of small melanophores). It is further distinguished from N. missuriensis and N. multicorniculatus sp. nov. by a larger eye (orbit diameter ca. 30% of HL vs. 25–27% of HL), and further from N. multicorniculatus sp. nov. by having physically larger pre-dorsal scales (vs. smaller scales, appearing crowded in pre-dorsal scale rows). Notropis lucifer sp. nov. is further distinguished from N. oblitus sp. nov. by the combination of an absent or weakly developed cleithral streak (vs. well-developed), and the absence of a cross-hatched pattern of melanophores in the two to three scale rows dorsal to lateral-line scale row along the length of the body (vs. presence of the cross-hatched pattern, sometimes weakly developed posteriorly). Figure 32. Notropis lucifer sp. nov. photographed at Devils River Ranch, Devils River, Texas, April 2022 (A, B, D), and at the San Saba River (C); A male, B female, C male, D, sex undetermined, demonstrating dorsal midline pigmentation. Scale bars = 5 mm. Vertebrate Zoology 75, 2025, 699–755 737 Description. Body shape and general appearance as in Figures 30–32. Morphometric and meristic data are listed in Tables 7–9. As described for N. stramineus, except for the following. Head deep (ca. 54% HL), more so in Colorado River drainage. Dorsal profile low, arch from snout to dorsal-fin origin weak. Snout rounded. Tuberculation as described for Notropis oblitus sp. nov. Ethmoid region weakly ossified; vomer broad (Fig. 10D). Coloration. Coloration in preservative (Figs 30, 31) as in Notropis oblitus sp. nov. except for the following. Melanophores below lateral-line scale row almost entirely absent anteriorly. Head pigmentation in clusters of melanophores, some forming macromelanophores, restricted to lacrimal, suborbital, dorsalmost part of opercular, fronto-occipital, and interorbital regions. Pigmentation reduced in supraorbital region and restricted to anteriormost and posteriormost half of internarial and rostral regions, respectively (Fig. 14 and 13E,F). Coloration in life silvery, pale, much of body side without yellow pigment, bluish sheen absent (Fig. 32). Chromatophores on dorsal midline creating thick golden streak, continuing onto head and covering much of dorsal surface (Fig. 32D). Scale rows in dorsal midline posterior to dorsal fin with thick margin of melanophores on posterior margin of scale. Cleithral streak absent or weakly developed and short. Pectoral fins largely hyaline; in males, pigment on pectoral fins restricted to few scattered melanophores on leading edge. Distribution. Notropis lucifer sp. nov. is distributed in the upper portions of the Colorado River basin of Texas, and in the Rio Grande basin (Fig. 29), where it is distributed in the Devils River (Fig. S17). Notropis lucifer sp. nov. is presumably also present in Pinto Creek, a tributary of the Rio Grande south of the Devils River, though recent attempts to collect the species there have failed. In Mexico, Notropis lucifer sp. nov. is presumed to be distributed in tributaries to the Rio Grande, including the Rio Salado and Rio San Juan, though genetic material is not available for these populations, making assignment of these populations to this species tentative. The report of N. stramineus sensu lato from the Río Pilón near Montemorelos by Miller et al. (2005: fig. 6.123, image of specimen from UMMZ 210715, not included in distribution map) appears to be based on a misidentification. Collections from the lower Pecos River, including Independence Creek (not evaluated herein), may also belong to this species and deserve further study. Etymology. Lucifer in Latin means light bearer or morning star. This species is so named for the comparatively bright appearance to that of the remaining members of the Notropis stramineus species group. This name also alludes to the Devils River, the type locality of this species, in reference to the connotation of Lucifer as Satan, the Devil, in Jewish and Christian literature. Additionally, the authors hope the recognition of this new species will cast a light upon the conservation risks of this geographic region, experienced by much of the arid regions of southwestern U.S. and adjacent Mexico. A noun in apposition. Remarks. Populations of Notropis lucifer sp. nov. from the Colorado River basin of Texas exhibit some morphological characteristics that are intermediate between those seen in the Devils River populations and those of individuals of N. oblitus sp. nov. (see, e.g., Figs 13–16E,F vs. 13–16C). These include a deeper body, the occasional presence of a subtle cleithral streak (most pronounced in males), and increased pigmentation posteriorly dorsal to the lateral line scale row (though typically not forming complete cross-hatching pattern). These intermediate conditions could be the result of past hybridization events between N. lucifer sp. nov. in the Colorado River basin and N. oblitus sp. nov., a scenario that likely explains the close relationship and low genetic distance in analyses of mitochondrial DNA. Discussion Throughout the course of this study, we have uncovered evidence we consider sufficient to recognize five distinct evolutionary lineages (i.e., species) currently classified as the taxon Notropis stramineus. Four of these lineages likely represent a monophyletic group and comprise the N. stramineus species complex; these include N. stramineus (Clade 1), N. multicorniculatus sp. nov. (Clade 2), N. oblitus sp. nov. (Clade 3), and N. missuriensis (Clade 4). Joining these are N. topeka (Figs 13G–16G, 34A, 33), N. procne (Figs 13H–16H, 34B, 35), N. chihuahua (Figs 13I–16I, 34C, 36) and N. lucifer sp. nov. (Clades 5 and 6) to comprise the N. stramineus species group. However, despite improving upon the breadth of sampling in prior studies, portions of the range of N. stramineus sensu lato, e.g., the western Missouri River basin, the lower Pecos River, and populations distributed in tributaries of the Rio Grande in Mexico, were not sampled in the present study and deserve attention in future work to further refine our knowledge of the geographic ranges of this species group. In addition to these findings, we demonstrate that the genus Miniellus as defined by Mayden et al. (2006) is not monophyletic, and suggest that Miniellus as composed by Stout et al. (2022) also deserves further study. A history of the genus Miniellus Jordan, 1882 The name Miniellus was first made available by Jordan (1882: 842), the type species Hybognathus procne Cope, 1865 designated later by Jordan and Evermann (1896: 254). Much like Notropis, Miniellus has had a tumultuous taxonomic history, and early on was considered by Jordan to be a subgenus of varying genera, e.g., Hudsonius (Jordan 1882: 842), Cliola (Jordan and Gilbert 1883) or as a synonym of other genera, e.g., Alburnops (Jordan Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 738 1885) and ultimately Hybopsis (Jordan et al. 1930). Later, in his type catalogue for the genus Notropis, Gilbert (1978) treated Miniellus as a synonym of Notropis, a decision that remained until two recent attempts to characterize the species comprising Miniellus and elevate it to the rank of genus. The first was Mayden et al. (2006), in which the relationships of notropin shiners and minnows (Notropis sensu lato and relatives) were inferred from analysis of the cyt b gene. Mayden et al. (2006) proposed that the species N. procne (type species), N. topeka, N. stramineus, and N. heterodon should comprise Miniellus, and recovered the topology seen in Figure 37A. However, Mayden et al. (2006) suggested that several additional species of Notropis not included in their study (viz. N. chihuahua, N. uranoscopus, N. alborus and N. albizonatus) also may need to be included in the genus based on a separate study (Warren et al. 1994). This same four-species composition of Miniellus was supported by Schönhuth et al. (2018) in which two mitochondrial (cyt b and COI) and two nuclear (RAG1 and IRBP) genes were used to construct phylogenetic hypotheses for the Leuciscidae. Schönhuth et al. (2018) included broad as well as dense taxon sampling, and despite assessing much of the family Leuciscidae, also managed to include all four original members of the genus Miniellus sensu Mayden et al. (2006). With their combined (concatenated) dataset, Schönhuth et al. (2018) recovered Miniellus as monophyletic, with the relationships of the four included species as seen in Figure 37B, a topology that differed slightly from Mayden et al. (2006). A strict consensus between the two studies would include N. stramineus, N. topeka, and N. heterodon in a trichotomy with N. procne as the sister taxon. Notably, none of the analyses in the present study recovered N. heterodon within a clade that contains N. stramineus, N. topeka, and N. procne (Figs 5, 6, S2, S3). Stout et al. (2022) also did not recover N. heterodon as closely related to N. stramineus (Fig. 37C). A possible explanation for this discrepancy is discussed in the following discussion section, “Notropis heterodon”. Recently, Stout et al. (2022) used exon capture to study the relationships of notropins, and proposed that Miniellus be expanded to comprise the aforementioned species as well as 17 more species currently placed in Notropis. This would bring the total number of species included in Miniellus to 21 (only 10 of which are represented by sequence data in the analyses of Stout et al. 2022; see Table 1). These putative members of Miniellus include (asterisks denote those species not included in the dataset of Stout et al. 2022, as in Table 1): Notropis albizonatus Warren & Burr, 1994 (palezone shiner), N. alborus Hubbs & Raney, 1947 (whitemouth shiner)*, N. ammophilus Suttkus & Boschung, 1990 (orangefin shiner), N. anogenus Forbes, 1885 (pugnose shiner)*, N. boops Gilbert, 1884 (bigeye shiner)*, N. chihuahua Woolman, 1892 (Chihuahua shiner), N. greenei Hubbs & Ortenburger, 1929 (wedgespot shiner)*, N. heterodon (blackchin shiner), N. longirostris (Hay, 1881) (longnose shiner), N. mekistocholas Snelson, 1971 (Cape Fear shiner)*, N. Figure 33. KU 40725, Notropis topeka; Kansas, Arkansas River Drainage, North Elm Creek; A male, 46.1 mm SL; B female, 41.4 mm SL. Scale bars = 5 mm. Vertebrate Zoology 75, 2025, 699–755 739 melanostomus Bortone, 1989 (blackmouth shiner), N. nubilus (Forbes, 1878) (Ozark minnow), N. ortenburgeri Hubbs, 1927 (Kiamichi shiner)*, N. perpallidus Hubbs & Black, 1940 (peppered shiner)*, N. procne (Cope, 1865) (swallowtail shiner)*, N. rafinesquei Suttkus, 1991 (Yazoo shiner)*, N. sabinae Jordan & Gilbert, 1886 (Sabine shiner), N. scabriceps (Cope, 1868) (New River shiner), N. stramineus (Cope, 1865) (sand shiner), N. topeka (Gilbert, 1884) (Topeka shiner)* and N. uranoscopus Suttkus, 1959 (skygazer shiner)*. Despite lacking over half of these species in their dataset, Stout et al. (2022) recommended their inclusion in Miniellus for a variety of reasons. These reasons vary by species, but, in general, amount to two major factors: these species have either been previously hypothesized as closely related to either one of the four originally proposed members of Miniellus (N. procne, N. topeka, N. heterodon or N. stramineus), or to species recovered as closely related to any one of these original four species in Stout et al. (2022). These previous hypotheses of relationships were often made by authors that were experts in the group (e.g., Hubbs and Raney 1947; Snelson 1971), though these were often based on overall morphological similarity (i.e., did not include formal phylogenetic methods). Stout et al. (2022) also do not test these relationships because, as mentioned above, most of the species were missing from their dataset. Notably, two of the taxa proposed to belong in Miniellus by Mayden et al. (2006) Figure 34. Live photographs of three members of the Notropis stramineus species group. A N. topeka, male; B N. procne, male; C N. chihuahua, male. Photo in (A) by L. Merry, (B) by S. Smith, and (C) by K.W. Conway. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 746 well outside of the group containing clades of N. stramineus and close relatives N. procne and N. topeka. One possible explanation for this discrepancy is that the mitochondrial DNA shared by N. lucifer sp. nov. in the Devils River and N. chihuahua has entered into the mitochondrial genome of the two taxa from a species not sampled in this study. This could have occurred through one or a combination of several possible means. In one scenario, this unsampled species, whether extinct or extant, could have hybridized with either N. chihuahua or Devils River N. lucifer sp. nov., the two later then hybridizing with each other. In a second scenario, the unsampled species hybridized with both species independently, resulting in the shared mitochondrial signal. These scenarios would represent the “indeterminate” introgressive signal of Near et al. (2011), or a “mitochondrial poltergeist” of Fritz et al. (2024). The event that caused the mitochondrial introgression either did not involve Colorado River populations of N. lucifer sp. nov., or, alternatively, populations of N. lucifer sp. nov. in the Colorado River basin were involved in the introgression event, but subsequent (more recent) hybridization events involving these populations and N. oblitus sp. nov. occurred that obscured this older signal. This would be consistent with what appears to be some signals of introgression between N. lucifer sp. nov. in the Colorado River basin and N. oblitus sp. nov. Hybridization resulting in introgression is not rare in freshwater fishes and may be a source of genetic diversity and an agent of evolutionary change rather than the evolutionary sink it is often thought to represent (Hubbs 1955; Arnold 1997; Seehausen 2004). Hybridization may be more of a general rule than an exception in many groups of North American freshwater fishes (Near et al. 2011). It has been reported from several groups, including darters (Near et al. 2011), catfishes (Egge and Simons 2006), salmonids and catostomids (Smith 1992). Recent studies of North America minnows have shown hybridization, even intergeneric hybridization, to be extremely common (Zbinden et al. 2023), and introgression resulting from hybridization has likely played a major role in shaping the mitochondrial signal seen in many groups of minnows and shiners (e.g., species of the Macrhybopsis aestivalis species complex; Echelle et al. 2018; Hoagstrom and Echelle 2022; Cyprinella in central Texas; Broughton et al. 2011). It appears that the membership of N. chihuahua in the N. stramineus species group is well-supported by molecular analyses and morphology. Consistent placement of N. chihuahua and N. lucifer sp. nov. with the remaining members of the N. stramineus species group using both nuclear loci, particularly in the slower evolving RAG1, and UCEs, as well as highly congruent morphology, points to a close relationship and a phylogenetic affinity of these taxa. A close affinity of N. chihuahua with N. stramineus has been proposed before by Burr and Mayden (1981). Though Notropis lucifer sp. nov. at present does not seem to be in immediate danger of conservation risk, its limited, disjunct distribution and the history of anthropogenic change in the region warrant keeping a close eye on the species in the near future. Notropis procne, N. alborus and N. albizonatus. Notropis procne, the swallowtail shiner, is distributed widely throughout the Atlantic Slope drainages and, importantly, is the type species of the genus Miniellus (Fig. 35). This species has long been considered to be closely related to N. stramineus sensu lato, e.g., Snelson (1971) considered Notropis procne to be “an Atlantic coast derivative” of N. stramineus. In the present study, N. procne is consistently recovered as a member of the N. stramineus species group. Snelson (1971) outlined the Notropis procne species group to include N. procne, N. alborus, N. albizonatus (at the time yet undescribed), N. heterolepis Eigenmann & Eigenmann, 1893, N. mekistocholas, N. stramineus, and N. uranoscopus. Snelson (1971) drew comparisons between N. procne and N. alborus, contrasting them with the newly described N. mekistocholas, e.g., in “… lacking herbivorous modifications, in having seven rather than eight anal rays, and in numerous other features”. Despite drawing these similarities between N. procne and N. alborus, Snelson (1971) concludes that N. alborus shares the greatest affinity with N. heterolepis, and is probably an “Atlantic coast derivative” of that species (presently, the generic placement of N. heterolepis remains unclear, with Stout et al. (2022) placing the species within the grade ‘Notropis’). Mayden (1989) tentatively placed N. alborus within the subgenus Hybopsis but cautioned that this assemblage may have been constructed based on convergent characters related to a preference for benthic habitats. Warren et al. (1994) used allozyme data to construct a hypothesis of relationships within the N. procne species group and is the only study to date to include N. alborus, N. albizonatus, N. procne, N. chihuahua and N. stramineus together in a phylogenetic study. Unfortunately, Warren et al. (1994) treat N. alborus as an outgroup a priori, a decision based on the hypothesis of Mayden (1989) that N. alborus was an outgroup of Notropis (Warren et al. 1994: 880), and thus any close relationship to N. procne may have been obscured. Schönhuth et. al. (2018) placed N. alborus as the sister taxon to a clade containing N. procne, N. heterodon (though see above discussion section “Notropis heterodon”), N. topeka and N. stramineus in a concatenated analysis. Analyses herein of the cyt b and RAG1 loci support the placement of N. alborus within the N. stramineus species group (Fig. S2A, C), likely as the sister taxon to N. procne (an S7 sequence was not available for N. alborus). Stout et al. (2022) did not include N. alborus in their analyses. All taken together, it appears as if a combination of morphological evidence, e.g., Snelson (1971), and molecular evidence (the present study, Schönhuth et al. 2018) supports the inclusion of N. alborus as a core member of the N. procne species group sensu Warren et al. (1994) and therefore also the N. stramineus species group. Snelson (1971) referred to the undescribed Notropis albizonatus as the “stramineus-like form from the Cumberland and Tennessee drainages”. Notropis albizonatus, Vertebrate Zoology 75, 2025, 699–755 747 the palezone shiner, long recognized as distinct, was eventually described as a species endemic to the Tennessee and Cumberland River drainages (Warren et al. 1994). Warren et al. (1994) also considered N. albizonatus to be a member of the Notropis procne species group, most closely related to N. ludibundus (= stramineus sensu lato). While neither the present analysis nor those of Mayden et al. (2006) or Stout et al. (2022) included samples of Notropis albizonatus, it cannot be ruled out as a member of the N. stramineus species group, as an analysis of allozymes in Warren et. al. (1994), the only study to include all of these species (see above), placed N. albizonatus as the sister taxon to a clade containing N. chihuahua and N. uranoscopus, with N. ludibundus (= stramineus sensu lato) + N. procne as the sister group to that assemblage. In other words, N. albizonatus was nested within a clade containing those species found in the present study to be closely related to N. stramineus. Mayden et al. (2006) also discuss the possibility that N. albizonatus, N. chihuahua and N. uranoscopus could belong within their Miniellus, though they did not include these species in their analysis. As recommended by Mayden et al. (2006), further study is needed regarding the position of N. albizonatus, though material is scarce due to its extremely limited distribution and federally endangered status (Warren et al. 2000). Notropis topeka. The Topeka shiner (Notropis topeka) is an endangered species distributed in the Midwest in the Mississippi and Missouri River system. This species is the most morphologically distinct member of the N. stramineus species group, with males developing a bright red or orange head and fins, and impressive tubercles distributed mainly on the top of the head and along the anteriormost portions of the fins (Figs 21C,F,I, 33, 34A). Analyses of the individual cyt b, S7 and RAG1 loci placed Notropis topeka among clades of N. stramineus sensu lato, while analyses of UCEs placed the species outside rather than within the N. stramineus species complex with strong support. This result suggests a weak or misleading phylogenetic signal due to incomplete lineage sorting, or introgression, overcome by analysis of many UCE loci. Hybridization between N. topeka and N. missuriensis or N. multicorniculatus sp. nov. would not be surprising given the sympatry of N. topeka and these two species, and the fact that N. topeka appears to have arisen within the clade comprising the N. stramineus species complex + N. procne. This could represent an example of peripatric speciation, a hypothesis that has also been proposed in the past; Schmidt and Gold (1995) uncovered a sister-group relationship of N. topeka and N. stramineus (using the mitochondrial locus cyt b) and refer to the Topeka shiner as a “regional derivative” of the “widespread N. stramineus”. Species identification in the Notropis stramineus species group. Distinguishing between members of the Notropis stramineus species group presents some challenges, due to the subtle nature of the diagnostic characters, and the fact that many of the characters relate to sexual dimorphism and are thus only useful when in the possession of adult males in breeding condition (e.g., nuptial coloration, tuberculation). Despite this, distinguishing between the species is not an impossible task, perhaps making the descriptor “cryptic” for this (and probably other similar species complexes) inappropriate. Notropis topeka, with its bright red coloration in life and extensive tuberculation (in males), deep body, large number of scale rows, small eye and relatively small head, is perhaps the most distinctive member of the N. stramineus species group (Fig. 34A), its distinctiveness approached only by N. procne with its combination of a long, slender body, terminal mouth and characteristic dark lateral stripe (Figs 34B, 35). Notropis chihuahua (Figs 34C, 36), despite an overall similar external appearance to N. stramineus sensu lato, is also readily distinguished by its distinctive pigmentation pattern comprising large macromelanophores across the body and head, more abundant than in N. lucifer sp. nov. In life, the blueish sheen along the bodyside of Notropis stramineus, N. missuriensis and N. multicorniculatus sp. nov., in combination with the scales dorsal to the lateral-line scale row comparatively densely outlined in melanophores, may be the most obvious way to distinguish these taxa from those distributed farther south in Texas and Mexico. In turn, the species in Texas and Mexico can be most readily distinguished from each other externally by the very large size of the eye in combination with peach-to yellow pectoral fin pigmentation and a cleithral streak in N. oblitus sp. nov. in comparison to a lack thereof in N. lucifer sp. nov. (cleithral stripe sometimes weakly developed, particularly in males, in Colorado River individuals). Interestingly, both N. oblitus sp. nov. and N. lucifer sp. nov. are quite pale in life, perhaps due to inhabiting somewhat similar habitats (clear, spring-fed streams flowing over calcareous bedrock) when compared to the silty, sandy streams in which the remaining members of the N. stramineus species complex are typically distributed (compare Figs S14 and S15 with Figs S16 and S17). Notropis stramineus can most readily be distinguished from N. missuriensis and N. multicorniculatus sp. nov. by a comparatively larger eye, more slender head, and elongate body. If fortunate enough to be comparing nuptial males, tuberculation in N. missuriensis and N. multicorniculatus sp. nov. is much more pronounced than in N. stramineus, a characteristic that appears to be quite consistent across the large distribution of the species. Perhaps the most difficult species to distinguish from each other are Notropis missuriensis and N. multicorniculatus sp. nov. Some evidence points to a possible sister-group relationship between the two, and perhaps a geographically proximate distribution has facilitated a higher level of geneflow in recent times than has been the case in the other lineages. However, the two can be distinguished by the size and number of predorsal scales. The higher number of scales in N. multicorniculatus sp. nov. (circumferential and predorsal) by consequence results in a predorsal region that has smaller scales and is more crowded in appearance. Further, N. multicorniculatus sp. Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 748 nov. tends to have a deeper head as well as a deeper body than N. missuriensis. Comments on the N. stramineus species complex. In the present study, unraveling species limits and relationships within what we now call the Notropis stramineus species complex proved challenging due to conflicting signals in gene trees and a conserved morphology. For example, using Astral, a value of ~0.7 was calculated for the normalized quartet scores of all species trees estimated. This means that on average, ~30% of taxon quartets found in individual gene trees were not present in the resulting species trees, suggesting gene-tree discordance is present in all of the datasets examined herein. This discordance mainly pertains to the Notropis stramineus species complex, its unclear intrarelationships further displayed by low support values (Figs 5, S3) and visualization of the posterior distribution of species trees in DensiTree (Fig. 8). This suggests that the N. stramineus species complex has been affected by several factors that make estimating phylogenetic relationships of the different clades extremely difficult. These factors most likely include incomplete lineage sorting and ancient (and potentially also recent) hybridization through secondary contact. While the most common topology suggests a sister-group relationship between Notropis stramineus sensu stricto and N. oblitus sp. nov., and N. missuriensis and N. multicorniculatus sp. nov., there is likely a great deal of shared ancestry among these groups, including possible ancient and recent hybridization and gene flow, and further study is needed to more confidently estimate the evolutionary history of this group. Notropis: the final frontier in North American ichthyology? The genus Notropis Rafinesque, 1818, as currently known (prior to changes proposed in the last couple of years, e.g., Stout et al. 2022; Page et al. 2023), is the largest genus of North American minnows, and has been alleged to harbor so-called “cryptic” diversity at a rate much higher than that of many other groups (April et al. 2011). Despite this diversity, in the present day it is extremely rare for new species of Notropis to be described, with some of the most recent examples published more than a decade ago (e.g., Lyons and Mercado-Silva 2004; Pera and Armbruster 2006; Domínguez-Domínguez et al. 2009). Difficulty surrounding the systematics of Notropis is probably due to a combination of factors, including conserved morphology, high levels of intraspecific phenotypic variation, hybridization resulting in introgression, incomplete lineage sorting resulting from relatively quick speciation events, and perhaps extinction. This is not to mention the difficulty researchers generally have with small, non-descript leuciscids: the characters necessary for the correct identification of even well-studied species often involve the careful post-preservation examination of numerous characters. Recently, a study with the main objective of generating mitogenomes for two species of Notropis, N. chlorocephalus and N. chiliticus, utilized mitogenome sequences obtained from GenBank to construct a phylogenetic hypothesis of several species of Notropis, including some of those included in Miniellus sensu Stout et al. (2022), included in the present study. The authors (Alley et al. 2025) do not adopt the classification of Stout et al. (2022) and remark on the non-monophyly of Miniellus uncovered in their study due to the sister group relationship of N. boops and N. telescopus. A quick look at GenBank and a comparison of their sequences used reveals that the identity of these two mitogenomes, including the cyt b locus, is approximately 99% identical, and likely conspecific. In fact, the origin of this sequence is a voucher specimen examined recently by two of the present authors (AKP, KWC) after concerns of specimen identification during the course of this study, and was determined to indeed be misidentified, the specimen labeled as N. telescopus actually being a specimen of N. boops (GenBank ID #AP012100). This only further emphasizes the care needed when obtaining sequences from species of Notropis and relatives, as identification to the level of species is often quite challenging and sometimes not verified by experts. Despite the difficulty in species identification and in unraveling the species limits of “cryptic” taxa within Notropis, careful work examining often overlooked morphological traits can reveal characters useful for distinguishing species that initially appeared extremely similar. A recent example is the elevation of N. megalops, a species of Notropis occurring in West Texas and Mexico, and until recently considered a junior synonym of a superficially very similar looking species, Notropis amabilis (Conway and Kim 2016). Detailed comparative work revealed several morphological characters (e.g., aspects of snout and body pigmentation, body shape and osteology) that not only make it possible to distinguish the species in the field where they are in some localities in sympatry, but also to identify specimens in natural history collections collected many years prior. A recent genomic study of Dye et al. (2024) has confirmed that N. megalops and N. amabilis represent distinct genetic groups, solidifying the taxonomic decision to recognize both as separate species. The recognition and reinstatement of N. megalops has important implications for conservation, ecological, and evolutionary studies, because prior to this nomenclatural change two distantly related species were confused under a single name. The present study follows this same path, utilizing details of pigmentation, body shape, tuberculation, osteology, and body shape to diagnose and describe distinct taxa confused under one name for over a century. Furthermore, the novel terminology of head and body regions proposed herein can facilitate the continued comparison of pigmentation and tuberculation of notropins and related minnows and shiners, as well as the continued study of the Notropis stramineus species group (and relatives, e.g., the N. procne species group; Miniellus). Future work is needed to better understand the relationships within the N. stramineus species complex, and those of related species, as well as the historical processes that shaped Vertebrate Zoology 75, 2025, 699–755 749 them. If other widespread species of Notropis (e.g., N. atherinoides; see April et al. 2011; Fields et al. 2024; N. buchanani, N. texanus, N. volucellus) are similarly comprised of multiple species, description of these has the potential to greatly increase the number of North American fishes known to exist. Conclusions We have identified unrecognized diversity within the widespread North American minnow Notropis stramineus. Using molecular and morphological evidence, we have described three distinct lineages as new species and redescribed N. stramineus and N. s. missuriensis, elevating the latter to species status. Morphological characteristics most useful in distinguishing the five species include pigmentation patterns, and extent and distribution of sexually dimorphic tubercles of males. A hypothesis of the existence of two monophyletic groups, one comprising the Notropis stramineus species group, which includes N. stramineus sensu stricto, N. missuriensis, N. multicorniculatus sp. nov., N. oblitus sp. nov., N. topeka, N. procne, N. lucifer sp. nov., and N. chihuahua, and within this, the N. stramineus species complex, comprising N. stramineus sensu stricto, N. missuriensis, N. multicorniculatus sp. nov., and N. oblitus sp. nov., is here proposed. The monophyly of both is supported by morphological and high-throughput molecular evidence (UCEs). Paraphyly of these clades in the topologies resulting from analyses of the individual Sanger-sequences loci is most likely explained by past introgressive hybridization and incomplete lineage sorting in the case of the cyt b locus and the nuclear loci, respectively. Incomplete lineage sorting in the UCE loci, where genetree discordance is clearly present, seems to have been overcome by the much larger sample of loci but is still not adequate to provide a well-supported hypothesis of the intrarelationships of the N. stramineus species complex. Future work to clarify these relationships will be necessary. A surprising result was the highly distinct lineage distributed in southern Texas (and potentially also Northern Mexico), its sister group relationship with N. chihuahua, and the highly divergent mitochondrial DNA shared between the two taxa. The origin of this captured mtDNA is unknown and deserves further study. Our study did not support the monophyly of the putative genus Miniellus sensu Mayden et al. (2006). Further, we argue that the placement of 21 species of Notropis into the genus Miniellus as proposed by Stout et al. (2022) is premature. We argue this because of the arbitrary delimitation of the genus based solely on molecular data, the sparse taxon sampling of Stout et al. (2022), including the absence of the type species of Miniellus (N. procne) in the dataset, and the extent of misidentification present in prior studies (demonstrated herein) that act as the scaffolding supporting their reclassification. Future work on the putative Miniellus should combine exhaustive taxon sampling, accurately identified voucher specimens available for reference, and careful morphological investigation that could provide diagnostic characters important for the diagnosis and description of genera described in the mid to late 19th century. The Notropis stramineus species complex, along with closely related species (viz. N. chihuahua, N. lucifer sp. nov., N. procne, and N. topeka), could in the future be a model group to study more closely and in detail present-day and ancient hybridization and introgression, as these phenomena are prevalent widely in North American leuciscids and likely have a large impact on phylogenetic inference and evolution in this taxon. Despite the long history of ichthyology in North America, information on the extent of species level diversity, basic biology, behavior, and ecology of North American minnows and shiners is still deficient. This remains the case in spite of the continued shifting of higher-level classifications using genomic tools and, more and more often, genetic data gathered from online databases where, in many cases, voucher specimen species identity is most likely not confirmed. Yet, the taxonomic impediment, combined with rapid biodiversity loss particularly in freshwaters (Jelks et al. 2008), presents a conflict when quick action in recognizing, describing and protecting species is important. Therefore, a balance between fast-moving genomic studies introducing broad taxonomic change, including species recognition, and thorough integrative taxonomic approaches ideally should be found. Acknowledgements This study represents the doctoral dissertation conducted at Texas A&M University (TAMU) by AKP under the direction of KWC. AKP thanks all members of her dissertation committee, in particular J. Light and H. Song and G. Voelker, for their helpful advice and comments which have greatly improved the contents of this manuscript. AKP also thanks R. Britz for helpful and encouraging comments and advice, and A. Summers also for advice and multiple opportunities to visit and conduct research at the Friday Harbor Laboratories. We thank A. Bentley (KU), E. Carson, A. Cohen (TNHC), E. DeArmon, M. de Lourdes Lozano Vilano, T. Turner (MSB), G. Hogue (NCSM), E. Holm (ROM), S. Huber (VIMS), R. Mooi (MM), D. Nelson, R. Singer (UMMZ), M. Sabaj (ANSP), A. Simons (JFBM) and J. Wright (NYSM) for loan of tissues, specimens, or photographs of specimens, N. Bertrand, R. Britz, G. Garrett, C. Hoagstrom, K. Kubicek, J. Light, E. Marsh-Matthews, W. Matthews, K. Mayes, T. Near, M. Osborne, H. Robison, H. Song, M. Stiassny, K. Kubicek and A. Summers for discussions and advice, C. King and K. Keith for early instruction and assistance in molecular lab techniques (to AKP), M. Pendleton and I. Freiday for instruction in SEM techniques, N. Bertrand, the Degnan family, T. Krabbenhoft, K. Keith, C. Krabbenhoft, K. Kubicek, B. Muller, S. Parker, J. Perkin, and A. Simons, for aiding fieldwork, J. Light and M. Mateos for molecular lab access, the TAMU Microscopy Imaging Center (RRID:SCR_022), P. Hollingsworth for information on Notropis alborus samples, B. Muller, L. Merry, and T. Kennedy, S. Smith and Z. Steffensmeier for photos used in figures, “Jim” of the Grosse Ile Pilot House for advice about De- Pinion AK et al.: Revision of Notropis stramineus (Cope, 1865) 750 troit River access, and the late G.R. Smith (UMMZ) for his maps, collecting advice and for introducing AKP to B. Muller, who aided in field work, shared photos and collection localities, and introduced AKP to the North American Native Fishes Association (NANFA). AKP would like to thank the Southeastern Fishes Council and C. Black for an excellent workshop in geometric morphometrics. Finally, the authors would like to thank C. Hoagstrom, B. Sidlauskas, one anonymous reviewer, and the editor (U. Fritz) for critical comments and suggestions that improved this manuscript. This research was supported financially via the TAMU Department of Wildlife and Fisheries Sciences Undergraduate Research grant program, TAMU College of Agriculture and Life Sciences Graduate Excellence Fellowship program, the Texas Chapter of the American Fisheries Society Clark Hubbs scholarship, the NANFA Conservation grant program, the AMNH Richard Gilder Graduate School Theodore Roosevelt Memorial Fund, the Texas Ecolabs Program (to AKP), and the TAMU Agrilife Research/USDA National Institute of Food and Agriculture program (HATCH TEX-09452-1 to KWC). This is publication number 1713 of the Biodiversity Research and Teaching Collections at Texas A&M University, 16 of the TAMU Aquarium Research Facility, 45 of the Marine Genomics Laboratory, and 137 of Genetic Studies in Fishes. References Adams CD, Collyer ML, Kaliontzopoulou A, Sherratt E (2020) Geomorph: Geometric morphometric analyses of 2D/3D landmark data. 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Posterior probability is shown above nodes. Asterisks denote nodes at which posterior probability is 0.95 or higher. Values < 0.75 not shown. Generic names presented in parentheses represent the generic assignment of Stout et al. (2022) and Page et al. (2023). — Figure S3. Alternative topology seen in concatenated ML analysis of the complete taxon, complete UCE dataset. Asterisks at nodes represent bootstrap (BS) values >95. — Figure S4. Median-joining haplotype network for 164 cyt b sequences of Notropis stramineus sensu lato. The size of each circle is proportional to haplotype frequency. Hatched lines represent single mutational steps, and small black circles represent hypothetical intermediate haplotypes. Broken lines with numbers in between them represent a large number of mutational steps, and the length of the branch is shortened for convenience. Clade colors match that of Figure 4. — Figure S5. Median-joining haplotype network for 189 cyt b sequences of Notropis stramineus sensu lato and closely related species (based on present study). The size of each circle is proportional to haplotype frequency. Hatched lines represent single mutational steps, and small black circles represent hypothetical intermediate haplotypes. — Figure S6. Results of PCA using traditional morphometric characters taken from 50 specimens per clade of Notropis stramineus sensu lato; PC1 vs. PC2. — Figure S7. Results of PCA using traditional morphometric characters taken from 50 specimens per clade of Notropis stramineus sensu lato; PC2 vs. PC3. — Figure S8. Hybrid violin/box plots depicting measurement data for each clade of Notropis stramineus sensu lato for selected measurements. Above, orbit diameter as a percentage of HL; below, snout length as a percentage of HL. — Figure S9. Hybrid violin/box plots depicting measurement data for each clade of Notropis stramineus sensu lato for selected measurements. Above, snout to occiput as a percentage of HL; below, body depth as a percentage of SL. — Figure S10. Results of PCA using geometric morphometric characters of body taken from 20 specimens per clade of Notropis stramineus sensu lato, and 10 specimens per closely related species N. topeka, N. procne and N. chihuahua. PC1 vs. PC2. Thin-plate spline deformation grids (top left, bottom right) show trends in shape difference along the principal component axes. — Figure S11. Results of PCA using geometric morphometric characters of head taken from 20 specimens per clade of Notropis stramineus sensu lato; PC1 vs. PC2. Head shapes represented by maximum (positive) values of PCs in black, and minimum (negative) values in gray. — Figure S12. PC1 vs PC2 for head and body shape of pairs of clades for which means differed significantly as result of pairwise comparison. Vector diagrams for PC1 are located on the bottom right of each graph, and PC2 on the top left. Body and head shapes repre- Vertebrate Zoology 75, 2025, 699–755 755 sented by maximum (positive) values of PCs in black, and minimum (negative) values in gray. Legend corresponds to graph to the left of the legend. — Figure S13. PC1 vs PC2 for head shape of pairs of clades for which means differed significantly as result of pairwise comparison that were not significantly different in body shape. Vector diagrams for PC1 are located on the bottom right of each graph, and PC2 on the top left. Head shapes represented by maximum (positive) values of PCs in black, and minimum (negative) values in gray. Legend corresponds to graph to the left of the legend. — Figure S14. USA, Michigan, Washtenaw County: Huron river, March 2016, locality of Notropis stramineus sensu stricto Photo by A.K. Pinion. — Figure S15. Canadian River, Texas, locality of Notropis multicorniculatus sp. nov. Photo by Z. Steffensmeier. — Figure S16. USA, Texas, Uvalde County: Frio River at Texas State Highway 127 crossing in Concan, type locality of Notropis oblitus sp. nov. Photo by K.W. Conway. — Figure S17. Devils River, Val Verde Co., Texas. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/vz.74.e156077.suppl1 Supplementary Material 2 Tables S1–S5 Authors: Pinion AK, Kim D, Dolan EP, Portnoy DS, Voelker G, Conway KW (2025) Data type: .zip Explanation notes: Table S1. Samples used in the 3-gene dataset. — Table S2. Samples and taxa used in UCE analyses. Notropis stramineus s.l. samples listed first, followed by outgroup taxa. — Table S3. Principal components 1–3 variable loadings, eigenvalues and cumulative proportion of variance obtained from PC analysis of morphometric data regressed on standard length comprising n=50 per clade of Notropis stramineus. — Table S4. Results from MANOVA tests of PC scores for PCs 1–3 (A) and (B) results of pairwise T-tests performed to determine which clades differed significantly from each other in the resulting PCA scores for PCs 1–3. — Table S5. Median-Pairwise distances between clade mean values of Procrustes shape coordinates for whole body dataset and head-only landmarks. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/ odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/vz.74.e156077.suppl2