Phylogenetic Relations among Percid Fishes as Inferred from Mitochondrial CytochromebDNA Sequence Data

Hypotheses of relationship among genera of Percidae have been conflicting. Based on different phylogenetic premises, the evolution of small benthic forms in Percidae has been interpreted as resulting from either convergence or common ancestry. In order to assess various phylogenetic hypotheses of Percidae we collected complete sequences (1140 bp) of mitochondrially encoded cytochromebfor 21 species of percids. Seven species representing four additional families of Perciformes were used as outgroups. Maximum parsimony and minimum evolution analyses both recovered single shortest trees, and the results of these analyses were generally congruent with one another. All analyses consistently recovered three monophyletic groups in Percidae: Etheostomatinae (Ammocrypta, Crystallaria, Etheostoma,andPercina), Percinae (PercaandGymnocephalus), and Luciopercinae (Stizostedion, Zingel,andRomanichthys). As a result of this analysis we present a revised classification of Percidae and discuss the phylogenetic evidence for the independent evolution of small benthic species within Etheostomatinae and Luciopercinae.     

A molecular phylogeny of the Percidae (Teleostei, Perciformes) based on mitochondrial DNA sequence

The family Percidae is among the most speciose families of northern hemisphere fishes with > 178 178 North American species and 14 Eurasian species. Previous phylogenetic studies have been hampered by a lack of informative characters, inadequate taxonomic sampling, and conflicting data. We estimated phylogenetic relationships among 54 percid species (9 of 10 genera and all but one subgenus of darters) and four outgroup taxa using mitochondrial DNA data from the 12S rRNA and cytochrome b genes. Four primary evolutionary lineages were consistently recovered: Etheostomatinae (Ammocrypta, Crystallaria, Etheostoma, and Percina), Perca, Luciopercinae (Romanichthys, Sander, and Zingel), and Gymnocephalus. Except Etheostoma and Zingel, all polytypic genera were monophyletic. The Etheostoma subgenus Nothonotus failed to resolve with other members of the genus resulting in a paraphyletic Etheostoma. The subfamily Percinae (Gymnocephalus and Perca) was not recovered in phylogenetic analyses with Gymnocephalus sister to Luciopercinae. Etheostomatinae and Romanichthyini were never resolved as sister groups supporting convergent evolution as the cause of small, benthic, stream-inhabiting percids in North American and Eurasian waters.     

The evolution of male nuptial colour in a sexually dimorphic group of fishes (Percidae: Etheostomatinae)

To test hypotheses explaining variation in elaborate male colouration across closely related species groups, ancestral‐state reconstructions and tests of phylogenetic signal and correlated evolution were used to examine the evolution of male body and fin colouration in a group of sexually dichromatic stream fishes known as darters (Percidae: Etheostomatinae). The presence or absence of red–orange and blue–green male colour traits were scored across six body regions in 99 darter species using a recently estimated amplified fragment length polymorphism (AFLP) phylogeny for comparative analyses. Ancestral‐state reconstructions infer the most recent common ancestor of darters to lack red–orange colour and possess blue–green colour on different body regions, suggesting variation between species is due to independent gains of red–orange and losses of blue–green. Colour traits exhibit substantial phylogenetic signal and are highly correlated across body regions. Comparative analyses were repeated using an alternative phylogenetic hypothesis based on one mitochondrial and two nuclear genes, yielding similar results to analyses based on the AFLP phylogeny. Red–orange colouration in darters appears to be derived; whereas, blue–green appears to be ancestral, which suggests that different selection mechanisms may be acting on these two colour classes in darters.     

Evolution of the premaxillary fraenum and substratum in snubnose darters and allies (Percidae: Etheostoma)

Darters (Percidae: Etheostomatinae), a species‐rich group of North American freshwater fishes, vary in the presence of a premaxillary fraenum, a strip of skin that connects the premaxillary bones to the snout, and it is hypothesized that this trait is a trophic adaptation to particular substrata. Ancestral state reconstructions and analyses of phylogenetic associations between presence of the premaxillary fraenum and preferred stream substratum were conducted in a clade of closely related darters (snubnose darters and allies) that vary in morphology and habitat preferences. The most recent common ancestor of this clade was inferred to possess a fraenum and to inhabit rocky substrata, consistent with previous hypotheses, but a significant correlation between fraenum presence and substratum type across the phylogeny was not found.     

History repeated: recent and historical mitochondrial introgression between the current darter Etheostoma uniporum and rainbow darter Etheostoma caeruleum (Teleostei: Percidae)

Incongruence between recognized taxonomy and phylogenetic relationships between two species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus , Etheostoma uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout their sympatric distribution in the Ozark Highlands of the central United States. Sequences from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum . However, both species were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by those of E. caeruleum . Some E. uniporum haplotypes are shared with geographically proximate E. caeruleum , and this is consistent with recent hybridization, while other E. uniporum haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression is likely asymmetric sneaking behaviour by male E. uniporum , a mating tactic observed in related species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in a historically small female effective population. Additional evidence for darter hybridization will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern North America.     

Habitat alteration and its effects on native fishes in the upper Tennessee River system, east-central U.S.A.

The upper Tennessee River drainage, which includes portions of the States of Virginia, North Carolina, and Tennessee, supports an exceptionally diverse fish fauna. Recent reductions in abundance and geographic ranges of several freshwater fishes have promulgated the imposition of protective measures for about 115 species among the three states, with nearly half of those species occurring in the upper Tennessee River. Most protected species are darters (Percidae: Etheostomatinae) or minnows (Cyprinidae), and are typically small, benthic invertivores. Major impacts on the fish fauna have resulted from dams, introduced species, toxic spills, mining and agriculture. An important cumulative effect of these impacts is fragmentation of the watershed; nearly 40% of the riverine habitat in major tributaries is either impounded or altered by tailwater discharges. The isolation and stress imposed on tributaries of the river have caused and will continue to cause extirpations of fishes, mussels and other aquatic fauna. Numerous federal, state, and private organizations are co-operating in efforts to protect rare species and habitats, improve agricultural and coal-producing practices, and enforce regulations for industrial and municipal effluents.     

AFLP phylogeny of the snubnose darters and allies (Percidae: Etheostoma) provides resolution across multiple levels of divergence

The snubnose darters (Percidae: subgenus Ulocentra) are a group of small, brightly colored North American freshwater fish belonging to the genus Etheostoma. Phylogenetic relationships among snubnose species have been a challenge to resolve at all levels of divergence, from the monophyly of species to deeper relationships among subgenera. Here, we used amplified fragment length polymorphisms (AFLPs) to estimate phylogenetic relationships among species from three closely related subgenera: Ulocentra, Etheostoma, and Nanostoma. With nearly complete sampling of recognized species, our analysis yielded a robust tree with statistical support at all nodes. Support was strongest for shallower relationships; support for internal nodes was either comparable to or greater than that of previous studies based on mitochondrial sequence data. Most recovered relationships were consistent with earlier hypotheses based on morphology or mtDNA sequences, with the exception of Etheostoma histrio, which was recovered as sister to Ulocentra. Our analysis indicates that careful use of AFLPs can yield statistically robust estimates of evolutionary relationships across multiple levels of divergence.     

Reconciling gene trees of eastern North American minnows

Most eastern North American cyprinid fishes belong to a clade known as the "open posterior myodome" (OPM) minnows, but phylogenetic relationships within this clade have been difficult to ascertain. Previous attempts to resolve relationships among the generally benthic "chubs" and the more pelagic "shiners", that constitute the majority of OPM minnows, have led to highly discordant phylogenetic hypotheses. To further examine relationships among the OPM minnows, we utilized both a concatenated Bayesian approach and a coalescent-based species tree method to analyze data from six protein coding nuclear loci (Enc1, Ptr, Ryr3, Sh3px3, Tbr1, and Zic1), as well as the mitochondrial locus (Cytb). We focused our analyses on the chub-like genus Phenacobius, a group that has drifted topologically between other benthic chubs and the more pelagic shiners, and also included exemplar taxa from 11 other OPM lineages. Individual gene trees were highly discordant regarding relationships within Phenacobius and across the OPM clade. The concatenated Bayesian analysis and coalescent-based species tree reconstruction recovered slightly different phylogenetic topologies. Additionally, the posterior support values for clades using the coalescent-based approach were consistently lower than the concatenated analysis. However, Phenacobius was resolved as monophyletic and as the sister lineage to Erimystax regardless of the combined data approach taken. Furthermore, Phenacobius+Erimystax was recovered as more closely related to the shiners we examined than to other chubs. Relationships within Phenacobius varied depending on the combined phylogenetic method utilized. Our results highlight the importance of multi-locus, coalescent-based approaches for resolving the phylogeny of diverse clades like the eastern North American OPM minnows.     

AFLPs resolve cytonuclear discordance and increase resolution among barcheek darters (Percidae: Etheostoma: Catonotus)

The use of mitochondrial DNA (mtDNA) sequences in phylogenetic analysis has been the subject of increasing scrutiny. A recent phylogenetic study of barcheek darters (Percidae: Etheostoma: Catonotus) revealed cytonuclear discordance, discordance among mtDNA loci, and discordance between mtDNA and morphometric hypotheses. In particular, mtDNA analyses hypothesized a paraphyletic barcheek darter clade, and a combined mtDNA and nuclear sequence topology was not well resolved. Here, we used amplified fragment length polymorphisms to test the monophyly of barcheek darters and to resolve relationships within the group. By including multiple populations of each species, we were able to generate a highly resolved tree that supports both the monophyly of barcheek darters as well as recently elevated species within the group. Analysis of three mtDNA loci indicates that saturation of highly variable sites best explains the discordant topologies among mtDNA partitions.     

Molecular phylogeny of the gobioid fishes (Teleostei: Perciformes: Gobioidei)

The phylogeny of groups within Gobioidei is examined with molecular sequence data. Gobioidei is a speciose, morphologically diverse group of teleost fishes, most of which are small, benthic, and marine. Efforts to hypothesize relationships among the gobioid groups have been hampered by the prevalence of reductive evolution among goby species; such reduction can make identification of informative morphological characters particularly difficult. Gobies have been variously grouped into two to nine families, several with included subfamilies, but most existing taxonomies are not phylogenetic and few cladistic hypotheses of relationships among goby groups have been advanced. In this study, representatives of eight of the nine gobioid familes (Eleotridae, Odontobutidae, Xenisthmidae, Gobiidae, Kraemeriidae, Schindleriidae, Microdesmidae, and Ptereleotridae), selected to sample broadly from the range of goby diversity, were examined. Complete sequence from the mitochondrial ND1, ND2, and COI genes (3573 bp) was used in a cladistic parsimony analysis to hypothesize relationships among the gobioid groups. A single most parsimonious topology was obtained, with decay indices indicating strong support for most nodes. Major phylogenetic conclusions include that Xenisthmidae is part of Eleotridae, and Eleotridae is paraphyletic with respect to a clade composed of Gobiidae, Microdesmidae, Ptereleotridae, Kraemeriidae, and Schindleriidae. Within this five-family clade, two clades are recovered. One includes Gobionellinae, which is paraphyletic with respect to Kraemeriidae, Sicydiinae, Oxudercinae, and Amblyopinae. The other contains Gobiinae, also paraphyletic, and including Microdesmidae, Ptereleotridae, and Schindleriidae. Previous morphological evidence for goby groupings is discussed; the phylogenetic hypothesis indicates that the morphological reduction observed in many goby species has been derived several times independently.     

Molecular evidence for an independent origin of modern triserial planktonic foraminifera from benthic ancestors

Gallitellia vivans is the only Recent representative of the triserial planktonic foraminiferal family Guembelitriidae. The origin and evolution of this interesting albeit poorly known family are enigmatic. To elucidate the phylogenetic relationships between G. vivans and other planktonic foraminifera, we sequenced the small subunit ribosomal DNA (SSU rDNA) for comparison to our extensive database of planktonic and benthic species. Our analyses suggest that G. vivans represents a separate lineage of planktonic foraminifera, which branches close to the benthic rotaliids Stainforthia and Virgulinella. Both genera resemble Gallitellia in general morphological appearance, having elongate triserial tests at least in their early ontogenic stages. The divergence time of G. vivans is estimated at ca. 18 Ma (early Miocene), suggesting an origin independent from the Cretaceous and Paleogene triserial planktonic foraminifera. Our study thus indicates that modern triserial planktonic foraminifera are not related to the Cretaceous–Paleogene triserial species, and that the sporadic occurrences in the fossil record are not the result of poor preservation, but reflect multiple transitions from benthic to planktonic mode of life.     

Evolution of development type in benthic octopuses: holobenthic or pelago-benthic ancestor?

Octopuses of the family Octopodidae are singular among cephalopods in their reproductive behavior, showing two major reproductive strategies: the first is the production of few and large eggs resulting in well-developed benthic hatchlings (holobenthic life history); the second strategy is the production of numerous small eggs resulting in free-swimming planktonic hatchlings (pelago-benthic life history). Here, we utilize a Bayesian-based phylogenetic comparative method using a robust molecular phylogeny of 59 octopus species to reconstruct the ancestral states of development type in benthic octopuses, through the estimation of the most recent common ancestors and the rate of gain and loss in complexity (i.e., planktonic larvae) during the evolution. We found a high probability that a free-swimming hatchling was the ancestral state in benthic octopuses, and a similar rate of gain and loss of planktonic larvae through evolution. These results suggest that in benthic octopuses the holobenthic strategy has evolved from an ancestral pelago-benthic life history. During evolution, the paralarval stage was reduced to well-developed benthic hatchlings, which supports a “larva-first” hypothesis. We propose that the origin of the holobenthic life history in benthic octopuses is associated with colonization of cold and deep sea waters.     

Molecular systematics reveals the origins of subsociality in tortoise beetles (Coleoptera,Chrysomelidae, Cassidinae)

Subsocial behaviour is known to occur in at least 19 insect orders and 17 families of Coleoptera. Within the leaf beetle family, Chrysomelidae, extended maternal care is reported in only 2 of 15 subfamilies: Cassidinae and Chrysomelinae. Although the emergence of subsociality in insects has received much attention, extensive analyses on the evolution of this behaviour based on phylogenetic approaches are missing. Subsociality is recorded in 33 species of tortoise beetles belonging to the tribes Mesomphaliini and Eugenysini. A molecular phylogenetic reconstruction of these tribes and the remaining five Neotropical tribes of cassidine tortoise beetles was used to investigate the evolution of maternal care and to elucidate the phylogenetic relationships among Neotropical cassidine tribes. A phylogeny was constructed using 90 species and three loci from both mitochondrial and nuclear genes (COI, CAD and 28S). Bayesian inference and maximum likelihood analyses based on a concatenated dataset recovered two independent origins, with no evidence of reversal to solitary behaviour. One origin comprises three Mesomphaliini genera tightly associated with Convolvulaceae, and the other consists of the genus Eugenysa Chevrolat (Eugenysini), a small clade embedded within a group feeding exclusively on Asteraceae. A previous hypothesis suggesting dual origins on different host plants was confirmed, whereas other hypotheses based on a phylogenetic reconstruction of Cassidinae could not be sustained. Our analysis also revealed that the tribe Mesomphaliini is a monophyletic taxon if Eugenysini is included, and for this reason, we re-establish synonymy of both tribes. We also provide nine new records of subsociality for tortoise beetles species.     

A combined morphometric and phylogenetic analysis of an ecomorphological trend: pelagization in Antarctic fishes (Perciformes: Nototheniidae)

The Antarctic fish family Nototheniidae (Perciformes) presumably originated from a benthic ancestor, and several lineages have evolved to live or at least feed in the water column, a trend called pelagization. Here, we use information on phylogeny, allometric growth, and diet composition for an integrated analysis of morphological and ecological diversification in this group, mainly focusing on the subfamilies Trematominae and Pleuragramminae. A phylogenetic analysis of data published in earlier systematic studies produced eight equally parsimonious trees, all indicating that several previously recognized taxa are paraphyletic. These phylogenetic trees all suggest multiple origins of pelagic life styles. Multivariate morphometric analyses including nine species showed that juveniles and adults grow according to a common pattern of ontogenetic allometry. The morphometric differences among species are mosdy the result of lateral transpositions of the growth trajectories, indicating that embryonic and larval development is more important as a determinant of morphological variation than allometric growth as juveniles and adults. We studied patterns of interspecific variation with principal components and the covariation between morphometric variables and food composition with a partial least-squares analysis. Both analyses revealed a gradient from benthic to pelagic foragers. Measurements of structures involved in swimming have a prominent role in these analyses, suggesting adaptive evolution of these traits. Tracing morphometric traits on the phylogenetic trees revealed a considerable amount of evolutionary plasticity, showing that species related phylogenetically need not be morphologically similar, but can diverge considerably, perhaps as a response to natural selection and adaptation to different habitats and foraging modes. In accordance, a test of phylogenetically independent contrasts showed that bursts of increased morphological change accompanied habitat shifts.     

Resolving tylenchid evolutionary relationships through multiple gene analysis derived from EST data

Sequence-based phylogenetic analyses typically are based on a small number of character sets and report gene trees which may not reflect the true species tree. We employed an EST mining strategy to suppress such incongruencies, and recovered the most robust phylogeny for five species of plant-parasitic nematode (Meloidogyne arenaria, M. chitwoodi, M. hapla, M. incognita, and M. javanica), three closely related tylenchid taxa (Heterodera glycines, Globodera pallida, and G. rostochiensis) and a distant taxon, Caenorhabditis elegans. Our multiple-gene approach is based on sampling more than 80,000 publicly available tylenchid EST sequences to identify phylum-wide orthologues. Bayesian inference, minimum evolution, maximum likelihood and protein distance methods were employed for phylogenetic reconstruction and hypothesis tests were constructed to elucidate differential selective pressures across the phylogeny for each gene. Our results place M. incognita and M. javanica as sister taxa, with M. arenaria as the next closely related nematode. Significant differences in selective pressure were revealed for some genes under some hypotheses, though all but one gene are exclusively under purifying selection, indicating conservation across the orthologous groups. This EST-based multi-gene analysis is a first step towards accomplishing genome-wide coverage for tylenchid evolutionary analyses.     

Phylogenomics of eukaryotes: impact of missing data on large alignments

Resolving the relationships between Metazoa and other eukaryotic groups as well as between metazoan phyla is central to the understanding of the origin and evolution of animals. The current view is based on limited data sets, either a single gene with many species (e.g., ribosomal RNA) or many genes but with only a few species. Because a reliable phylogenetic inference simultaneously requires numerous genes and numerous species, we assembled a very large data set containing 129 orthologous proteins ( approximately 30,000 aligned amino acid positions) for 36 eukaryotic species. Included in the alignments are data from the choanoflagellate Monosiga ovata, obtained through the sequencing of about 1,000 cDNAs. We provide conclusive support for choanoflagellates as the closest relative of animals and for fungi as the second closest. The monophyly of Plantae and chromalveolates was recovered but without strong statistical support. Within animals, in contrast to the monophyly of Coelomata observed in several recent large-scale analyses, we recovered a paraphyletic Coelamata, with nematodes and platyhelminths nested within. To include a diverse sample of organisms, data from EST projects were used for several species, resulting in a large amount of missing data in our alignment (about 25%). By using different approaches, we verify that the inferred phylogeny is not sensitive to these missing data. Therefore, this large data set provides a reliable phylogenetic framework for studying eukaryotic and animal evolution and will be easily extendable when large amounts of sequence information become available from a broader taxonomic range.     

Molecular evidence for an independent origin of modern triserial planktonic foraminifera from benthic ancestors

Gallitellia vivans is the only Recent representative of the triserial planktonic foraminiferal family Guembelitriidae. The origin and evolution of this interesting albeit poorly known family are enigmatic. To elucidate the phylogenetic relationships between G. vivans and other planktonic foraminifera, we sequenced the small subunit ribosomal DNA (SSU rDNA) for comparison to our extensive database of planktonic and benthic species. Our analyses suggest that G. vivans represents a separate lineage of planktonic foraminifera, which branches close to the benthic rotaliids Stainforthia and Virgulinella. Both genera resemble Gallitellia in general morphological appearance, having elongate triserial tests at least in their early ontogenic stages. The divergence time of G. vivans is estimated at ca. 18 Ma (early Miocene), suggesting an origin independent from the Cretaceous and Paleogene triserial planktonic foraminifera. Our study thus indicates that modern triserial planktonic foraminifera are not related to the Cretaceous–Paleogene triserial species, and that the sporadic occurrences in the fossil record are not the result of poor preservation, but reflect multiple transitions from benthic to planktonic mode of life.     

Phylogenetic relationships and the evolution of regulatory gene sequences in the parrotfishes

Regulatory genes control the expression of other genes and are key components of developmental processes such as segmentation and embryonic construction of the skull in vertebrates. Here we examine the variability and evolution of three vertebrate regulatory genes, addressing issues of their utility for phylogenetics and comparing the rates of genetic change seen in regulatory loci to the rates seen in other genes in the parrotfishes. The parrotfishes are a diverse group of colorful fishes from coral reefs and seagrasses worldwide and have been placed phylogenetically within the family Labridae. We tested phylogenetic hypotheses among the parrotfishes, with a focus on the genera Chlorurus and Scarus, by analyzing eight gene fragments for 42 parrotfishes and eight outgroup species. We sequenced mitochondrial 12s rRNA (967 bp), 16s rRNA (577 bp), and cytochrome b (477 bp). From the nuclear genome, we sequenced part of the protein-coding genes rag2 (715 bp), tmo4c4 (485 bp), and the developmental regulatory genes otx1 (672 bp), bmp4 (488bp), and dlx2 (522 bp). Bayesian, likelihood, and parsimony analyses of the resulting 4903 bp of DNA sequence produced similar topologies that confirm the monophyly of the scarines and provide a phylogeny at the species level for portions of the genera Scarus and Chlorurus. Four major clades of Scarus were recovered, with three distributed in the Indo-Pacific and one containing Caribbean/Atlantic taxa. Molecular rates suggest a Miocene origin of the parrotfishes (22 mya) and a recent divergence of species within Scarus and Chlorurus, within the past 5 million years. Developmentally important genes made a significant contribution to phylogenetic structure, and rates of genetic evolution were high in bmp4, similar to other coding nuclear genes, but low in otx1 and the dlx2 exons. Synonymous and non-synonymous substitution patterns in developmental regulatory genes support the hypothesis of stabilizing selection during the history of these genes, with several phylogenetic regions of accelerated non-synonymous change detected in the phylogeny.     

RELATIONSHIP BETWEEN SPECIES CO-OCCURRENCE AND RATE OF MORPHOLOGICAL CHANGE IN PERCINA DARTERS (PERCIDAE: ETHEOSTOMATINAE)

When the morphological diversity of a clade of species is quantified as the among-species variance in morphology, that diversity is a joint consequence of the phylogenetic structure of the clade (i.e., temporal pattern of speciation events) and the rates of change in the morphological traits of interest. Extrinsic factors have previously been linked to variation in the rate of morphological change among clades. Here, we ask whether species co-occurrence is positively correlated with the rate of change in several ecologically relevant morphological characters using the North American freshwater fish clade Percina (Teleostei: Etheostomatinae). We constructed a time-calibrated phylogenetic tree of Percina from mtDNA sequence data, gathered data on eight morphological characters from 37 species, used a principal components analysis to identify the primary axes of morphological variation, and analyzed 16,094 collection records to estimate species co-occurrence. We then calculated standardized independent contrasts (SIC) of the morphological traits (rate of change) at each node, estimated ancestral species co-occurrence, and quantified the correlation between species co-occurrence and rate of morphological change. We find that morphology changes more quickly when co-occurrence is greater in Percina . Our results provide strong evidence that co-occurrence among close relatives is linked to the morphological diversification of this clade.     

Ultrastructure and molecular phylogeny of Stephanopogon minuta: an enigmatic microeukaryote from marine interstitial environments

Although Stephanopogon was described as a putative ciliate more than a century ago, its phylogenetic position within eukaryotes has remained unclear because of an unusual combination of morphological characteristics (e.g. a highly multiflagellated cell with discoidal mitochondrial cristae). Attempts to classify Stephanopogon have included placement with the Ciliophora, the Euglenozoa, the Heterolobosea and the Rhizaria. Most systematists have chosen, instead, to conservatively classify Stephanopogon as incertae sedis within eukaryotes. Despite the obvious utility of molecular phylogenetic data in resolving this issue, DNA sequences from Stephanopogon have yet to be published. Accordingly, we characterized the molecular phylogeny and ultrastructure of Stephanopogon minuta, a species we isolated from marine sediments in southern British Columbia, Canada. Our results showed that S. minuta shares several features with heteroloboseans, such as discoidal mitochondrial cristae, a heterolobosean-specific (17_1 helix) insertion in the small subunit ribosomal RNA gene (SSU rDNA) and the lack of canonical Golgi bodies. Molecular phylogenetic analyses of SSU rDNA demonstrated that S. minuta branches strongly within the Heterolobosea and specifically between two different tetraflagellated lineages, both named 'Percolomonas cosmopolitus.' Several ultrastructural features shared by S. minuta and P. cosmopolitus reinforced the molecular phylogenetic data and confirmed that Stephanopogon is a highly divergent multiflagellated heterolobosean that represents an outstanding example of convergent evolution with benthic eukaryovorous ciliates (Alveolata).