Robert M. Wood

Reconstructing the phylogenetic relationships of the earth's most diverse clade of freshwater fishes—order Cypriniformes (Actinopterygii: Ostariophysi): A case study using multiple nuclear loci and the mitochondrial genome

The order Cypriniformes is the most diverse clade of freshwater fishes and is natively distributed on all continents except South America, Australia, and Antarctica. Despite the diversity of the group and the fundamental importance of these species in both ecosystems and human culture, relatively little has been known about their relationships relative to their diversity. In recent years, with an international effort investigating the systematics of the group, more information as to their genealogical relationships has emerged and species discovery and their descriptions have increased. One of the more interesting aspects of this group has been a traditional lack of understanding of the relationships of the families, subfamilies, and other formally or informally identified groups. Historical studies have largely focused on smaller groups of species or genera. Because of the diversity of this group and previously published whole mitochondrial genome evidence for relationships of major clades in the order, this clade serves as an excellent group to investigate the congruence between relationships reconstructed for major clades with whole mitogenome data and those inferred from a series of nuclear gene sequences. As descent has resulted in only one tree of life, do the phylogenetic relationships of these major clades converge on similar topologies using the large number of available characters through this suite of nuclear genes and previously published mitochondrial genomes? In this study we examine the phylogenetic relationships of major clades of Cypriniformes using previously published mitogenomes and four putative single-copy nuclear genes of the same or closely related species. Combined nuclear gene sequences yielded 3810bp, approximately 26% of the bp found in a single mitogenome; however homoplasy in the nuclear genes was measurably less than that observed in mitochondrial sequences. Relationships of taxa and major clades derived from analyses of nuclear and mitochondrial sequences were nearly identical and both received high support values. While some differences of individual gene trees did exist for species, it is predicted that these differences will be minimized with increased taxon sampling in future analyses.

Systematics of the subfamily Danioninae (Teleostei: Cypriniformes: Cyprinidae)

The members of the cyprinid subfamily Danioninae form a diverse and scientifically important group of fishes, which includes the zebrafish, Danio rerio. The diversity of this assemblage has attracted much scientific interest but its monophyly and the relationships among its members are poorly understood. The phylogenetic relationships of the Danioninae are examined herein using sequence data from mitochondrial cytochrome b, mitochondrial cytochrome c oxidase I, nuclear opsin, and nuclear recombination activating gene 1. A combined data matrix of 4117 bp for 270 taxa was compiled and analyzed. The resulting topology supports some conclusions drawn by recent studies on the group and certain portions of the traditional classification, but our results also contradict key aspects of the traditional classification. The subfamily Danioninae is not monophyletic, with putative members scattered throughout Cyprinidae. Therefore, we restrict Danioninae to the monophyletic group that includes the following genera: Amblypharyngodon, Barilius, Cabdio, Chela, Chelaethiops, Danio, Danionella, Devario (including Inlecypris), Esomus, Horadandia, Laubuca, Leptocypris, Luciosoma, Malayochela, Microdevario, Microrasbora, Nematabramis, Neobola, Opsaridium, Opsarius, Paedocypris, Pectenocypris, Raiamas, Rasbora (including Boraras and Trigonostigma), Rasboroides, Salmostoma, Securicula, and Sundadanio. This Danioninae sensu stricto is divided into three major lineages, the tribes Chedrini, Danionini, and Rasborini, where Chedrini is sister to a Danionini-Rasborini clade. Each of these tribes is monophyletic, following the restriction of Danioninae. The tribe Chedrini includes a clade of exclusively African species and contains several genera of uncertain monophyly (Opsarius, Raiamas, Salmostoma). Within the tribe Rasborini, the species-rich genus Rasbora is rendered non-monophyletic by the placement of two monophyletic genera, Boraras and Trigonostigma, hence we synonymize those two genera with Rasbora. In the tribe Danionini, the miniature genus Danionella is recovered as the sister group of Danio, with D. nigrofasciatus sister to D. rerio.

Inferring the Tree of Life of the order Cypriniformes, the earth's most diverse clade of freshwater fishes: Implications of varied taxon and character sampling

The phylogenetic relationships of species are fundamental to any biological investigation, including all evolutionary studies. Accurate inferences of sister group relationships provide the researcher with an historical framework within which the attributes or geographic origin of species (or supraspecific groups) evolved. Taken out of this phylogenetic context, interpretations of evolutionary processes or origins, geographic distributions, or speciation rates and mechanisms, are subject to nothing less than a biological experiment without controls. Cypriniformes is the most diverse clade of freshwater fishes with estimates of diversity of nearly 3,500 species. These fishes display an amazing array of morphological, ecological, behavioral, and geographic diversity and offer a tremendous opportunity to enhance our understanding of the biotic and abiotic factors associated with diversification and adaptation to environments. Given the nearly global distribution of these fishes, they serve as an important model group for a plethora of biological investigations, including indicator species for future cli- matic changes. The occurrence of the zebrafish, Danio rerio, in this order makes this clade a critical component in understanding and predicting the relationship between mutagenesis and phenotypic expressions in vertebrates, including humans. With the tremendous diversity in Cypriniformes, our understanding of their phylogenetic relationships has not proceeded at an acceptable rate, despite a plethora of morphological and more recent mo- lecular studies. Most studies are pre-Hennigian in origin or include relatively small numbers of taxa. Given that analyses of small numbers of taxa for molecular characters can be compromised by peculiarities of long-branch attraction and nodal-density effect, it is critical that significant progress in our understanding of the relationships of these important fishes occurs with increasing sampling of species to mitigate these potential problems. The recent Cypriniformes Tree of Life initiative is an effort to achieve this goal with morphological and molecular (mitochondrial and nuclear) data. In this early synthesis of our understanding of the phylogenetic relationships of these fishes, all types of data have contributed historically to improving our understanding, but not all analyses are complementary in taxon sampling, thus precluding direct understanding of the impact of taxon sampling on achieving accurate phylogenetic inferences. However, recent molecular studies do provide some insight and in some instances taxon sampling can be implicated as a variable that can influence sister group relationships. Other instances may also exist but without inclusion of more taxa for both mitochondrial and nuclear genes, one cannot distinguish between inferences being dictated by taxon sampling or the origins of the molecular data.

Phylogeny of the gudgeons (Teleostei: Cyprinidae: Gobioninae)

The members of the cyprinid subfamily Gobioninae, commonly called gudgeons, form one of the most well-established assemblages in the family Cyprinidae. The subfamily is a species-rich group of fishes, these fishes display diverse life histories, appearances, and behavior. The phylogenetic relationships of Gobioninae are examined using sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. This investigation produced a data matrix of 4114 bp for 162 taxa that was analyzed using parsimony, maximum likelihood, and Bayesian inference methods. The phylogenies our analyses recovered corroborate recent studies on the group. The subfamily Gobioninae is monophyletic and composed of three major lineages. We find evidence for a Hemibarbus-Squalidus group, and the tribes Gobionini and Sarcocheilichthyini, with the Hemibarbus-Squalidus group sister to a clade of Gobionini-Sarcocheilichthyini. The Hemibarbus-Squalidus group includes those two genera; the tribe Sarcocheilichthyini includes Coreius, Coreoleuciscus, Gnathopogon, Gobiocypris, Ladislavia, Paracanthobrama, Pseudorasbora, Pseudopungtungia, Pungtungia, Rhinogobio, and Sarcocheilichthys; the tribe Gobionini includes Abbottina, Biwia, Gobio, Gobiobotia, Huigobio, Microphysogobio, Platysmacheilus, Pseudogobio, Romanogobio, Saurogobio, and Xenophysogobio. The monotypic Acanthogobio is placed into the synonymy of Gobio. We tentatively assign Belligobio to the Hemibarbus-Squalidus group and Mesogobio to Gobionini; Paraleucogobio and Parasqualidus remain incertae sedis. Based on the topologies presented, the evolution of swim bladder specializations, a distinctive feature among cyprinids, has occurred more than once within the subfamily.

Molecular Systematics of the Genus Cottus (Scorpaeniformes: Cottidae)

Abstract To evaluate the monophyly of Cottus and to identify species groups within the genus we generated a data set of 1914 base pairs of mitochondrial DNA (cytochrome b and ATPase 8 and 6) for 87 individuals representing 36 of the 42 currently recognized species of the genus. Both parsimony and Bayesian analyses consistently supported non-monophyly of Cottus with respect to the Baikalian sculpins and the genus Leptocottus. Analyses also supported recognition of five clades within the genus Cottus. These are referred to herein as the Cottopsis, Uranidea, Baikalian, Cottus, and Cephalocottus clades. These clades encompass all but ten recognized Cottus species, nine whose placement was uncertain in our analyses and one not included in our analyses. Lastly, we recognize a freshwater sculpin clade comprising the most speciose freshwater component of the family Cottidae.

New primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes (Actinopterygii: Ostariophysi)

We provide 15 new primers for amplifying and sequencing the mitochondrial ND4/ND5 gene region of the Cypriniformes in an attempt to resolve relationships of this diverse group of freshwater fishes with extensive taxonomic sampling. Sequences from this region have the following desirable characteristics for phylogenetic analyses, some of which are lacking from the more commonly used cyt b and 12S/16S rRNA genes: they are (1) easy to align, (2) relatively long (ca. 3.4 kb), and (3) contain more phylogenetically informative variation at 1st and 2nd codon positions. Moreover, the ND4/ND5 gene region is easy to amplify and sequence when employing the protocol suggested herein.

Phylogenetics of Scaphirhynchus Based on Mitochondrial DNA Sequences

Abstract Species delineation and taxonomy within the sturgeon genus Scaphirhynchus is controversial. This issue is made more complex by political issues regarding the Alabama sturgeon S. suttkusi and potential hybridization between sympatric shovelnose sturgeon S. platorynchus and pallid sturgeon S. albus. We investigated phylogenetic relationships among species of Scaphirhynchus based on nucleotide sequences for two mitochondrial loci, cytochrome b and the control region (D-loop). White sturgeon Acipenser transmontanus and green sturgeon A. medirostris were used as outgroups. Phylogenetic analyses did not recover monophyletic shovelnose or pallid sturgeon; however, some populations of pallid sturgeon were resolved as sister to the Alabama sturgeon, and one specimen of shovelnose sturgeon was consistently resolved as the sister to all other ingroup taxa. The hierarchical pattern of relationships produced by analysis of mitochondrial DNA is not consistent with that produced by morphological data. It is con...

Phylogeographic analyses suggest multiple lineages of Crystallaria asprella (Percidae: Etheostominae)

The crystal darter, Crystallaria asprella, exists in geographically isolated populations that may be glacial relicts from its former, wide distribution in the Eastern U.S. An initial phylogeographic survey of C. asprella based upon the mitochondrial cytochrome b (cyt b) gene indicated that there were at least four distinct populations within the species: Ohio River basin, Upper Mississippi River, Gulf coast, and lower Mississippi River. In particular, the most divergent population was the most recently discovered, from the Elk River, WV, in the Ohio River basin, and it was postulated that this population represents an undescribed, potentially threatened species. However, differentiation observed at a single gene region is generally not considered sufficient evidence to establish taxonomic status. In the present study, nucleotide variation at the mitochondrial control region and a nuclear S7 ribosomal gene intron were compared to provide independent verification of phylogeographic results between individuals collected from the same five disjunct populations previously surveyed. Variation between populations at the control region was substantial (except between Gulf drainages) and was concordant with patterns of sequence divergence from cyt b. Only the Elk River population was resolved as monophyletic based upon nuclear S7, but significant differences based upon ΦST statistics were observed between most populations. Morphometric data were consistent with molecular data regarding the distinctiveness of the Elk River population. It is proposed that populations of C. asprella consist of at least four distinct population segments, and that the Elk River group likely constitutes a distinct species.

Phylogenetic Relationships among Selected Darter Subgenera (Teleostei: Percidae) as Inferred from Analysis of Allozymes

Twenty-six darter taxa representing 18 subgenera and three outgroup taxa were assayed electrophoretically for allozyme variation at 32 presumptive gene loci. The most-parsimonious phylogenetic tree resulted from PAUP-derived analysis enforcing a topological constraint among outgroup taxa consistent with a previously published phylogenetic hypothesis. Phylogenetic relationships depicted in this tree include a basal clade of Crystallaria asprella plus Etheostoma (Ammocrypta) beanii sister to all remaining darter taxa (genera Percina and Etheostoma). Percina is paraphyletic with respect to Etheostoma. Within Percina, R caprodes and P. vigil form a clade sister to R nigrofasciata plus all members of Etheostoma except the sole representative of subgenus Ammocrypta. Etheostoma (exclusive of subgenus Ammocrypta) consists of four clades. The basal-most clade includes Litocara as the sister taxon to Allohistium

Phylogenetic Systematics of the Darter Subgenus Nothonotus (Teleostei: Percidae)

Allozyme variation at 38 presumptive gene loci was assayed for 24 populations representing 16 species of the percid subgenus Nothonotus. One behavioral and 11 morphological characters also were evaluated. Distance-Wagner, neighborjoining, and character-based parsimony analysis were employed to generate trees from allozyme data. Nonallozyme data were analyzed in conjunction with allozyme data by using character-based parsimony. Most parsimonious solutions were chosen through FREQPARS optimization of all trees derived from distancebased analyses and of all equally most parsimonious trees resulting from character-based analyses. Results indicate that Nothonotus is monophyletic with respect to outgroup taxa analyzed. Two, near-equal most parsimonious solutions to the combined allozyme plus nonallozyme data sets were found: (E. juliae ((E. tippecanoe (E. rubrum, E. moorei)) (E. acuticeps (E. bellum ((E. camurum, E. chlorobranchium) (E. rufilineatum (E. jordani ((E. vulneratum, E. wapiti) (E. microlepum (E. aquali (E. sanguifluum, E. maculatum))))))))))); and (E. juliae ((E. tippecanoe (E. rubrum, E. moorei)) ((E. rufilineatum (E. bellum (E. camurum, E. chlorobranchium))) ((E. jordani, E. acuticeps) ((E. vulneratum, E. wapiti) (E. microlepidum (E. aquali (E. sanguifluum, E. maculatum)))))))). Both trees support existence of an upper Tennessee River/Mobile Basin connection. Hypothesized relationships in the E. maculatum species-group support a closer affinity of the Duck River to the Green and Cumberland rivers than to the Tennessee River system within the time frame relevant to speciation within this clade. Hypothesized relationships within the E. tippecanoe species-group suggest a continuous Ozarkian/Lower Mississippi River connection after the vicariant event separating the Eastern Highlands from the Interior Highlands.