Phillip M. Harris

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.

Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans.

Abstract. The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulansnudC3 mutant, demonstrating evolutionary conservation of function. A C. elegansnud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.

Phylogenetic relationships of the genera of North American sunfishes and basses (Percoidei: Centrarchidae) as evidenced by the mitochondrial cytochrome b gene

Abstract The phylogenetic relationships of genera of the family Centrarchidae and its affinities to the Elassomatidae were examined using the mitochondrial DNA sequences of the cytochrome b gene. A total of 32 individuals representing 16 species from nine genera of centrarchids were sequenced. Outgroup were drawn from other perciform families including several families previously proposed to be closely related to the Centrarchidae. Phylogenetic analysis was conducted using the optimality criteria of maximum parsimony with two weighting schemes and using the maximum likelihood method. A priori and a posteriori alternative hypotheses of relationships within the Centrarchidae were investigated using the Shimodaira and Hasegawa Test. The results of all analyses support the monophyly of the Centrarchidae, although the interrelationships of centrarchid genera differed between analyses. Elassoma was never recovered as the sister taxon to the Centrarchidae; however, the sister relationship of these taxa could not be rejected. Additional data are required to unambiguously resolve relationships among centrarchid genera and determine the relationship of the Centrarchidae to other perciforms.

4 – GULF COAST RIVERS OF THE SOUTHEASTERN UNITED STATES

The river basins of the eastern Gulf Coast lie west of the Atlantic slope and east of the Mississippi River. The region has abundant water resources, including seven major rivers that arise and flow through five physiographic provinces in five states to empty into the Gulf of Mexico. These rivers vary widely in size, hydrology, geomorphology, and water chemistry, but experience a similar climate and exhibit many biological similarities. The dominant physiographic province through which the rivers flow is the coastal plain, more specifically, the sections named the eastern Gulf Coastal Plain and the Floridan. There are five freshwater ecoregions within the eastern Gulf of Mexico region, arranged by longitude. These include portions of the Mississippi Embayment and Florida ecoregions, all of the Mobile Bay and Apalachicola ecoregions, and much of the Florida Gulf ecoregion. Two of these, the Mobile Bay and the Apalachicola, follow watershed boundaries of the Mobile and Apalachicola river basins, whereas the remaining three ecoregions include multiple river basins. All rivers in this region flow from north to south into the Gulf of Mexico, and all are located between 84°W and 91°W longitude and 30°N and 35°N latitude. The eastern Gulf Coast rivers encompass a rich variety of aquatic habitats and resources. This diversity is most evident in the fishes, mollusks, crayfishes, and caddisflies.

Molecular Systematics of the Cyprinid Genus Campostoma (Actinopterygii: Cypriniformes): Disassociation between Morphological and Mitochondrial Differentiation

Abstract Campostoma are ubiquitous across North America, yet relationships among members of the genus are poorly understood. Here we present phylogenetic hypotheses based on analyses of DNA sequence data from the mitochondrial cytochrome b gene. All analyses consistently recovered nine clades of comparable topological structure. Differentiation of the recovered clades did not follow currently accepted taxonomic boundaries, and was not consistent with previously hypothesized relationships among recognized species and subspecies. Rather, the recovered clades corresponded to broad geographic divides and to areas known either to have high rates of endemism or to represent discrete biogeographic provinces, indicating that clades not corresponding to recognized taxa represent additional diversity within the group. This result provides evidence of morphological similarity among genealogically divergent lineages, and supports several disputed descriptions of putative Campostoma taxa based on subtle variation in morphology. At least nine lineages could be recognized as distinct taxa to provisionally resolve differences among prior systematic accounts of Campostoma evolutionary diversity.

Low genetic diversity and small long-term population sizes in the spring endemic watercress darter, Etheostoma nuchale

Species endemic to coldwater springs in the southeastern United States are some of the rarest and most imperiled in this region, yet little is known about their genetic composition and conservation needs. Here, microsatellite based levels of genetic diversity and estimates of effective population size (Ne) were compared between a narrow spring endemic fish, Etheostoma nuchale, and its widespread stream-dwelling relative, E. swaini. We applied several analytical methods to assess how demographic history is reflected in contemporary levels of genetic diversity for populations of E. nuchale. Phylogenetic analyses based on mitochondrial and nuclear DNA sequence data revealed a complex history among E. nuchale and E. swaini, but suggested ancient divergence and historic periods of isolation since colonization of spring habitats by E. nuchale. Populations of E. nuchale have levels of genetic diversity approximately one-half that of E. swaini, a result most likely due to founder effects and recent bottlenecks. Statistically significant Fst values (0.05−0.27) and STRUCTURE analyses implied high levels of differentiation among E. nuchale populations. Estimates of current Ne suggest relatively consistent levels across populations of E. nuchale, but one population may suffer from habitat degradation. We suggest that high levels of population structure and low levels of genetic diversity may be typical in other spring endemics inhabiting this region. Therefore, effective management planning for these unique species will require a detailed knowledge of the genetic and demographic history of each population.

A Mitochondrial DNA Perspective on the Molecular Systematics of the Sunfish Genus Lepomis (Actinopterygii: Centrarchidae)

Abstract Complete mitochondrial DNA cytochrome b gene sequences from 56 specimens representing all 12 species of Lepomis were used to examine phylogenetic relationships within the genus. Results supported the monophyly of Lepomis and all previously recognized subgenera, except Eupomotis, but there was no support for previously proposed relationships among subgenera. Seven species were recovered as monophyletic lineages, while five species (L. auritus, L. macrochirus, L. marginatus, L. miniatus, and L. symmetricus) were recovered as either poly- or paraphyletic or were placed as parts of unresolved polytomies with other species. Parametric bootstrapping tests rejected monophyly for only two of the five species (L. auritus and L. symmetricus). Without additional data, including increased geographic sampling and a comparable nuclear gene phylogeny, it is not possible to determine whether the failure to support monophyly for these two species reflects the presence of cryptic species or results from hybridization.

Systematics and Zoogeography of the Rock Basses (Centrarchidae: Ambloplites)

Abstract This study examines the role that historical events have played in the diversification of members of the genus Ambloplites by estimating divergence times of clades within the genus. DNA sequences of the mitochondrial cytochrome b gene were used to develop phylogenetic hypotheses for Ambloplites using maximum parsimony and Bayesian methods. The timing of diversification within and between species of Ambloplites was estimated using nonparametric rate-smoothing and penalized likelihood methods calibrated by fossil dates. Monophyly of three of the four species was supported. The ages of the most recent common ancestors of species of Ambloplites estimated in this study confirm earlier estimates that in most instances, their origins occurred prior to the Pleistocene. A comparison of estimated divergence times of lineages to sea level fluctuations indicates a correspondence to extremely high or low sea stands throughout the Neogene Period. Populations of Ambloplites in the Ozarks and Ouachita highlands previously assigned to A. ariommus appear to have either been introgressed by A. rupestris or were misidentified as A. ariommus. Ambloplites rupestris displays low genetic divergence throughout its range and is consistent with a re-colonization of previously glaciated areas following the Pleistocene.

The influence of life-history strategy on genetic differentiation and lineage divergence in darters (Percidae: Etheostomatinae).

Recent studies determined that darters with specialized breeding strategies can exhibit deep lineage divergence over fine geographic scales without apparent physical barriers to gene flow. However, the extent to which intrinsic characteristics interact with extrinsic factors to influence population divergence and lineage diversification in darters is not well understood. This study employed comparative phylogeographic and population genetic methods to investigate the influence of life history on gene flow, dispersal ability, and lineage divergence in two sympatric sister darters with differing breeding strategies. Our results revealed highly disparate phylogeographic histories, patterns of genetic structure, and dispersal abilities between the two species suggesting that life history may contribute to lineage diversification in darters, especially by limiting dispersal among large river courses. Both species also showed striking differences in demographic history, indicating that extrinsic factors differentially affected each species during the Pleistocene. Collectively, our results indicate that intrinsic and extrinsic factors have influenced levels of gene flow among populations within both species examined. However, we suggest that life‐history strategy may play a more important role in lineage diversification in darters than previously appreciated, a finding that has potentially important implications for understanding diversification of the rich North American freshwater fish fauna.

Interspecific relationships and the evolution of sexual dimorphism in pygmy sunfishes (Centrarchidae: Elassoma)

The genus Elassoma represents a small but unique component of the aquatic biodiversity hotspot in southeastern North America. We present the first phylogeny of the seven described species, corroborated by sequence data from mitochondrial and nuclear protein coding genes. This analysis reveals a Coastal Plain clade sister to the geographically isolated, and federally protected, Elassoma alabamae. The Coastal Plain clade contains the widespread E. zonatum, which is sister to a clade primarily restricted to lowland Neogene subprovinces. We analyzed morphometric data in a phylogenetic context to illustrate the evolution of sexual shape dimorphism within the genus. Sixteen univariate and three multivariate traits were tested for significant sexual dimorphism for each species, and relative transformation rates were inferred from the time tree. A simple index of interspecific sexual dimorphism revealed greater disparity among sympatric species comparisons than among allopatric comparisons. Results implicate geology as a primary factor influencing ecological diversification, and sexual selection as a mechanism reinforcing reproductive isolation in areas of secondary contact. We discuss putative roles of geological history and sexual selection in the generation and maintenance of the aquatic biodiversity gradient in southeastern North America.