Phylogenetic relationships and classification of the Holarctic family Leuciscidae (Cypriniformes: Cyprinoidei).

The phylogenetic relationships and classification of the freshwater fish order Cypriniformes, like many other species-rich groups of vertebrates, has evolved over time with some consistency and inconsistencies of relationships across various studies. Within Cypriniformes, the Holarctic family Leuciscidae is one of the most widely distributed and highly diverse monophyletic groups of cyprinoids. Despite several studies conducted on this group, alternative hypotheses exist as to the composition and relationships within Leuciscidae. Here we assess the extent, composition, phylogenetic relationships, and taxonomy of this highly diverse group of fishes, using multiple mitochondrial and nuclear loci and a comprehensive and dense taxonomic sampling. Analyses of 418 specimens (410 species) resolve a well-supported Leuciscidae including 362 specimens (358 taxa) in six well-supported subfamilies/major clades: Pseudaspininae/Far East Asian clade (FEA); Laviniinae/North American Western clade (WC); Plagopterinae/North American Creek Chub-Plagopterin clade (CC-P); Leuciscinae/Eurasian Old World clade (OW) (minus Phoxinus) plus North American Notemigonus; Phoxininae/Eurasian Phoxinus clade (PHX); and Pogonichthyinae/North American clade (NA) including all remaining leuciscids. Within Leuciscidae, neither the traditional phoxinins (Phoxinus, FEA, Nearctic genera) nor all Nearctic genera (minus Notemigonus) are resolved as monophyletic; whereas the WC and CC-P form two independent lineages from remaining North American cyprinoids. A close relationship exists between Eurasian Phoxinus, NA, and OW clades, while FEA is the sister group to all remaining Leuciscidae. Major lineages resolved within these six subfamilies are mostly congruent with some previous studies. Our results suggests a complex evolutionary history of this diverse and widespread group of fishes.

Phylogeography of the widespread creek chub Semotilus atromaculatus (Cypriniformes: Leuciscidae).

The extent and nature of genetic differentiation in Semotilus atromaculatus, one of the most abundant and widespread leuciscids in North America, were evaluated based on mitochondrial (mt) and nuclear DNA sequence variation. Phylogenetic relationships were first inferred based on a fragment of the cytochrome b (cytb) region and the nuclear intron s7 gene for S. atromaculatus and all other congeners as well as representative species from all other genera in the creek chub-plagopterin clade. The phylogeography of major haplogroups of S. atromaculatus was also assessed according to variation in a fragment of the mitochondrial cytb region from 567 individuals across its range. All analyses identified S. thoreauianus, S. lumbee and S. corporalis as reciprocally monophyletic groups. Analyses of nuclear sequence variation resolved S. atromaculatus as a single clade, where S. thoreauianus and S. lumbee were recovered as the sister group to S. atromaculatus, and S. corporalis was resolved as sister to all other species in the genus. Analyses of mtDNA sequence variation recovered S. atromaculatus as three well supported and differentiated monophyletic groups, with a widespread genetically homogeneous lineage extending across most of the current range of the species; a more geographically restricted and geographically structured lineage in the southern Appalachians, sister group to S. lumbee; and a geographically restricted lineage was identified from two Gulf Slope basins. Evidence of complex mito-nuclear discordance and phylogeographic differentiation within S. atromaculatus illustrates that further analysis of widespread species is warranted to understand North American freshwater fish diversity and distributions.

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

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

Phylogeny, diversity, and species delimitation of the North American Round-Nosed Minnows (Teleostei: Dionda), as inferred from mitochondrial and nuclear DNA sequences.

Accurate delimitation of species is a critical first step in protecting biodiversity. Detection of distinct species is especially important for groups of organisms that inhabit sensitive environments subject to recent degradation, such as creeks, springs, and rivers in arid or semi-desert regions. The genus Dionda currently includes six recognized and described species of minnows that live in clear springs and spring-fed creeks of Texas, New Mexico (USA), and northern Mexico, but the boundaries, delimitation, and characterization of species in this genus have not been examined rigorously. The habitats of some of the species in this genus are rapidly deteriorating, and many local populations of Dionda have been extirpated. Considering the increasing concerns over degradation of their habitat, and pending a more detailed morphological revision of the genus, we undertook a molecular survey based on four DNA regions to examine variation over the range of the genus, test species boundaries, and infer phylogenetic relationships within Dionda. Based on analyses of two mitochondrial (cytb and D-loop) and two nuclear (Rag1 and S7) DNA regions from specimens collected throughout the range of Dionda, we identified 12 distinct species in the genus. Formerly synonymized names are available for two of these species, and four other species remain undescribed. We also redefine the known range of six species. The limited distribution of several of the species, coupled with widespread habitat degradation, suggests that many of the species in this genus should be targets for conservation and recovery efforts.

Phylogenomics and classification of Notropis and related shiners (Cypriniformes: Leuciscidae) and the utility of exon capture on lower taxonomic groups.

North American minnows of the Shiner Clade, within the family Leuciscidae, represent one of the most taxonomically complex clades of the order Cypriniformes due to the large number of taxa coupled with conserved morphologies. Species within this clade were moved between genera and subgenera until the community decided to lump many of the unclassified taxa with similar morphologies into one genus, Notropis, which has held up to 325 species. Despite phylogentic studies that began to re-elevate some genera merged into Notropis, such as Cyprinella, Luxilus, Lythrurus, and Pteronotropis, the large genus Notropis remained as a taxonomic repository for many shiners of uncertain placement. Recent molecular advances in sequencing technologies have provided the opportunity to re-examine the Shiner Clade using phylogenomic markers. Using a fish probe kit, we sequenced 90 specimens in 87 species representing 16 genera included in the Shiner Clade, with a resulting dataset of 1,004 loci and 286,455 base pairs. Despite the large dataset, only 32,349 bp (11.29%) were phylogenetically informative. In our maximum likelihood tree, 78% of nodes are 100% bootstrap supported demonstrating the utility of the phylogenomic markers at lower taxonomic levels. Unsurprisingly, species within Notropis as well as Hudsonius, Luxilus, and Alburnops are not resolved as monophyletic groups. Cyprinella is monophyletic if Cyprinella callistia is excluded, and Pteronotropis is monophyletic if it includes Hudsonius cummingsae. Taxonomic changes we propose are: restriction of species included in Alburnops and Notropis, elevation of the subgenus Hydrophlox, expansion of species included in Miniellus, movement of Hudsonius cummingsae to Pteronotropis, and resurrection of the genera Coccotis and Paranotropis. We additionally had two specimens of three species, Notropis atherinoides, Ericymba amplamala, and Pimephales vigilax and found signficant differences between the localities (1,086, 1,424, and 845 nucleotides respectively).

Phylogenetic relationships in the genus Cyprinella (Actinopterygii: Cyprinidae) based on mitochondrial and nuclear gene sequences.

Members of the genus Cyprinella are abundant and broadly distributed cyprinids in central and eastern rivers of North America. Currently, 30 species are placed in this genus, including seven species restricted to Mexico and three barbeled species formerly placed in different genera (Hybopsis, Erimonax). Phylogenetic analyses were performed for all recognized species of Cyprinella plus species from 14 related genera using complete nucleotide sequences of one mitochondrial (cytochrome b) and one nuclear (Rag1) gene. Cyprinella revealed high levels of genetic divergence between species in both genes compared with species from other related genera examined. Analyses of mitochondrial sequences from 98 specimens of Cyprinella revealed the gene trees of some species as non-monophyletic (i.e., C. venusta, C. lepida, C. nivea, C. lutrensis, C. whipplei). Mitochondrial analyses recovered nine well-supported species groups within a main clade inclusive of most species of Cyprinella, although resolution regarding the monophyly of the genus or its relationships with other genera was poorly resolved, except for C. callistia and C. monacha, two species that are apparently not part of the genus. Analyses of nuclear gene sequences from selected distinctive mitochondrial clades also recovered these nine clades and resulted in shorter branches but greater resolution at more basal nodes. Nuclear data also rejected the inclusion of C. monacha in Cyprinella and indicated that Cyprinella is not monophyletic if Tampichthys and Codoma are recognized as valid genera. A major Clade A (formed by Cyprinella, Tampichthys, Codoma) is more closely related to Pimephales, Opsopoeodus, Hybognathus and some Notropis than to Luxilus and Lythrurus as previously hypothesized. Although mitochondrial data are highly informative phylogenetically, our research revealed fundamental conflicts among mitochondrial and nuclear data for several species of Cyprinella. Interestingly, nuclear trees recovered some species resolved as polyphyletic with mitochondrial variation as monophyletic. While we remain highly supportive of phylogenetic studies using mitochondrial genes and genomes, this study does highlight the utility of nuclear data by identifying issues of possible hybridization or retained ancestral polymorphisms and lineage sorting that can yield erroneous hypothesis in studies using only mit-DNA analyses, while shedding invaluable light on the phylogeny of this controversial group of shiners.

Phylogeny and genetic variation within the widely distributed Bluntnose Minnow, Pimephales notatus (Cyprinidae), in North America.

This study represents a phylogenetic analysis of Pimephales notatus, a widely distributed North American cyprinid fish species, using one mitochondrial (cytb) and two nuclear (S7 and Rag1) genes. Despite the broad geographic distribution of this species that includes independent basins, results suggest a largely drainage-independent genetic structure. Results reveal a well-supported lineage in the Ozark Highlands, which is highly divergent from the remaining populations and may represent a long-term isolated lineage that should be considered as a separate conservation and management unit. All other remaining populations comprised a widely distributed lineage, covering most of the distribution for the species (from the Red River and tributaries of the Great Lakes in the north to the lower Mississippi and Mobile basin in the south, and from the Missouri River in the west to the Chesapeake and Albemarle Sound basins in the east). Phylogenetic, genetic diversity, and biogeographical data suggest that this wide-ranging lineage could have experienced a recent expansion to northern areas where populations exhibit little genetic variation. Results from molecular analyses reveal a distinctive lineage in the Ozarks and suggest that there is need for morphological analyses within P. notatus to determine its taxonomic status.

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Molecular evolution of southern North American Cyprinidae (Actinopterygii), with the description of the new genus Tampichthys from central Mexico.

Most of the recognized species of the genus Dionda inhabit drainages of the Gulf of Mexico from central Mexico to central Texas, USA, and have been considered a monophyletic group based on morphological, osteological, and allozyme investigations. Phylogenetic relationships of 15 species of Dionda and 34 species from closely related genera were inferred from one mitochondrial (cytb) and three nuclear gene sequences (S7, Rhodopsin, Rag1) totaling 4487 nucleotides. Separate analyses of all four genes yield congruent phylogenies; however the 15 putative species of Dionda evaluated were never recovered as a monophyletic group when species from nine related genera were included in the analyses. Among the ingroup taxa, one well-supported and highly divergent clade is consistently recognized and consists of six recognized and three undescribed northern species currently recognized in the genus Dionda. These nine species inhabit present or past tributaries of the Rio Grande basin of northern Mexico and southern USA, and were recovered as a basal clade in all analyses. Another large, also strongly supported clade, consisting of seven genera, include five southern recognized species currently in the genus Dionda, forming the sister group to the Codoma clade. These five species comprise the "Southern Dionda clade" and inhabit headwaters of the Pánuco-Tamesí drainage and some adjacent coastal rivers in the Tampico Embayment. The consistent and repeated identification of eight different clades recovered in most of the separate gene analyses strongly supports a division of the non-natural genus Dionda. A new genus, Tampichthys, is proposed for the clade of species endemic to east-central Mexico and formerly in Dionda. Tampichthys and the putative monotypic genus Codoma are more related to Mexican species of the genera Cyprinella and Notropis than to other species referred to Dionda sensu stricto.