William E. Browne

Broad phylogenomic sampling improves resolution of the animal tree of life.

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates

In the amphipod crustacean, Parhyale hawaiensis, the first few embryonic cleavages are total and generate a stereotypical arrangement of cells. In particular, at the eight-cell stage there are four macromeres and four micromeres, and each of these cells is uniquely identifiable. We describe our studies of the cell fate pattern of these eight blastomeres, and find that the eight clones resulting from these cells set up distinct cell lineages that differ in terms of proliferation, migration and cell fate. Remarkably, the cell fate of each blastomere is restricted to a single germ layer. The ectoderm originates from three of the macromeres, while the remaining macromere generates the visceral mesoderm. Two of the micromeres generate the somatic mesoderm, a third micromere generates the endoderm and the fourth micromere generates the germline. These findings demonstrate for the first time a total cleavage pattern in an arthropod which results in an invariant cell fate of the blastomeres, but notably, the cell lineage pattern of Parhyale reported shows no clear resemblance to those found in spiralians, nematodes or deuterostomes. Finally, the techniques we have developed for the analysis of Parhyale development suggest that this arthropod may be particularly useful for future functional analyses of crustacean development.

Neuroanatomy of sea spiders implies an appendicular origin of the protocerebral segment

Independent specialization of arthropod body segments has led to more than a century of debate on the homology of morphologically diverse segments, each defined by a lateral appendage and a ganglion of the central nervous system. The plesiomorphic composition of the arthropod head remains enigmatic because variation in segments and corresponding appendages is extreme. Within extant arthropod classes (Chelicerata, Myriapoda, Crustacea and Hexapoda—including the insects), correspondences between the appendage-bearing second (deutocerebral) and third (tritocerebral) cephalic neuromeres have been recently resolved on the basis of immunohistochemistry and Hox gene expression patterns. However, no appendage targets the first ganglion, the protocerebrum, and the corresponding segmental identity of this anterior region remains unclear. Reconstructions of stem-group arthropods indicate that the anteriormost region originally might have borne an ocular apparatus and a frontal appendage innervated by the protocerebrum. However, no study of the central nervous system in extant arthropods has been able to corroborate this idea directly, although recent analyses of cephalic gene expression patterns in insects suggest a segmental status for the protocerebral region. Here we investigate the developmental neuroanatomy of a putative basal arthropod, the pycnogonid sea spider, with immunohistochemical techniques. We show that the first pair of appendages, the chelifores, are innervated at an anterior position on the protocerebrum. This is the first true appendage shown to be innervated by the protocerebrum, and thus pycnogonid chelifores are not positionally homologous to appendages of extant arthropods but might, in fact, be homologous to the ‘great appendages’ of certain Cambrian stem-group arthropods.

De novo assembly and characterization of a maternal and developmental transcriptome for the emerging model crustacean Parhyale hawaiensis

BackgroundArthropods are the most diverse animal phylum, but their genomic resources are relatively few. While the genome of the branchiopod Daphnia pulex is now available, no other large-scale crustacean genomic resources are available for comparison. In particular, genomic resources are lacking for the most tractable laboratory model of crustacean development, the amphipod Parhyale hawaiensis. Insight into shared and divergent characters of crustacean genomes will facilitate interpretation of future developmental, biomedical, and ecological research using crustacean models.ResultsTo generate a transcriptome enriched for maternally provided and zygotically transcribed developmental genes, we created cDNA from ovaries and embryos of P. hawaiensis. Using 454 pyrosequencing, we sequenced over 1.1 billion bases of this cDNA, and assembled them de novo to create, to our knowledge, the second largest crustacean genomic resource to date. We found an unusually high proportion of C2H2 zinc finger-containing transcripts, as has also been reported for the genome of the pea aphid Acyrthosiphon pisum. Consistent with previous reports, we detected trans-spliced transcripts, but found that they did not noticeably impact transcriptome assembly. Our assembly products yielded 19,067 unique BLAST hits against nr (E-value cutoff e-10). These included over 400 predicted transcripts with significant similarity to D. pulex sequences but not to sequences of any other animal. Annotation of several hundred genes revealed P. hawaiensis homologues of genes involved in development, gametogenesis, and a majority of the members of six major conserved metazoan signaling pathways.ConclusionsThe amphipod P. hawaiensis has higher transcript complexity than known insect transcriptomes, and trans-splicing does not appear to be a major contributor to this complexity. We discuss the importance of a reliable comparative genomic framework within which to consider findings from new crustacean models such as D. pulex and P. hawaiensis, as well as the need for development of further substantial crustacean genomic resources.

Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis

The arthropod head is a complex metameric structure. In insects, orthodenticle (otd) functions as a ‘head gap gene’ and plays a significant role in patterning and development of the anterior head ectoderm, the protocerebrum, and the ventral midline. In this study, we characterize the structure and developmental deployment of two otd paralogs in the amphipod crustacean, Parhyale hawaiensis. Photd1 is initially expressed at gastrulation through germband stages in a bilaterally symmetric, restricted region of the anterior head ectoderm and also in a single column of cells along the ventral midline. Late in embryogenesis, Photd1 is expressed within the developing anterior brain and the expression along the embryonic midline has become restricted to a stereotypic group of segmentally reiterated cells. The second ortholog Photd2, however, has a unique temporal–spatial expression pattern and is not detected until after the head lobes have been organized in the developing ectoderm of the germband during late germband stages. Anteriorly, Photd2 is coincident with the Photd1 head expression domain; however, Photd2 is not detected along the ventral midline during formation of the germband and only appears in the ventral midline late in embryonic development in a restricted group of cells distinct from those expressing Photd1. The early expression of Photd1 in the anterior head ectoderm is consistent with a role as a head gap gene. The more posterior expression of Photd1 is suggestive of a role in patterning the embryonic ventral midline. Photd2 expression appears too late to play a role in early head patterning but may contribute to latter patterning in restricted regions of both the head and the ventral midline. The comparative analysis of otd reveals the divergence of gene expression and gene function associated with duplication of this important developmental gene.

MicroRNAs and essential components of the microRNA processing machinery are not encoded in the genome of the ctenophore Mnemiopsis leidyi

BackgroundMicroRNAs play a vital role in the regulation of gene expression and have been identified in every animal with a sequenced genome examined thus far, except for the placozoan Trichoplax. The genomic repertoires of metazoan microRNAs have become increasingly endorsed as phylogenetic characters and drivers of biological complexity.ResultsIn this study, we report the first investigation of microRNAs in a species from the phylum Ctenophora. We use short RNA sequencing and the assembled genome of the lobate ctenophore Mnemiopsis leidyi to show that this species appears to lack any recognizable microRNAs, as well as the nuclear proteins Drosha and Pasha, which are critical to canonical microRNA biogenesis. This finding represents the first reported case of a metazoan lacking a Drosha protein.ConclusionsRecent phylogenomic analyses suggest that Mnemiopsis may be the earliest branching metazoan lineage. If this is true, then the origins of canonical microRNA biogenesis and microRNA-mediated gene regulation may postdate the last common metazoan ancestor. Alternatively, canonical microRNA functionality may have been lost independently in the lineages leading to both Mnemiopsis and the placozoan Trichoplax, suggesting that microRNA functionality was not critical until much later in metazoan evolution.

Phylogenetic analysis of lineage relationships among hyperiid amphipods as revealed by examination of the mitochondrial gene, cytochrome oxidase I (COI)

Among metazoans, crustaceans display the greatest disparity between body plans and are second only to the insects in overall species diversity. Within the crustaceans, the Amphipoda rank as one of the most speciose extant orders. Amphipods have successfully invaded a variety of ecosystems, including the pelagic midwater environment. Despite their abundance in varied and dissimilar habitats, and the use of traditional morphological and systematic comparative analyses, phylogenetic relationships among amphipods remain uncertain. The pelagic amphipods, hyperiids, have highly divergent life histories and morphological attributes in comparison to more familiar benthic, nearshore, intertidal, and terrestrial amphipods. Some of these adaptations are likely correlated with their pelagic life history and include features such as hypertrophied olfactory and visual systems, duplications of the eyes, and an array of modifications to the appendages. Many of these morphological features may represent homoplasies, thus masking the true phylogenetic relationships among extant hyperiid amphipods. Here, we sample a wide range of amphipod taxa for the COI gene and present the first preliminary molecular phylogeny among the hyperiids.

Conservation of arthropod midline netrin accumulation revealed with a cross‐reactive antibody provides evidence for midline cell homology

SUMMARY Although many similarities in arthropod CNS development exist, differences in axonogenesis and the formation of midline cells, which regulate axon growth, have been observed. For example, axon growth patterns in the ventral nerve cord of Artemia franciscana differ from that of Drosophila melanogaster. Despite such differences, conserved molecular marker expression at the midline of several arthropod species indicates that midline cells may be homologous in distantly related arthropods. However, data from additional species are needed to test this hypothesis. In this investigation, nerve cord formation and the putative homology of midline cells were examined in distantly related arthropods, including: long‐ and short‐germ insects (D. melanogaster, Aedes aeygypti, and Tribolium castaneum), branchiopod crustaceans (A. franciscana and Triops longicauditus), and malacostracan crustaceans (Porcellio laevis and Parhyale hawaiensis). These comparative analyses were aided by a cross‐reactive antibody generated against the Netrin (Net) protein, a midline cell marker and regulator of axonogenesis. The mechanism of nerve cord formation observed in Artemia is found in Triops, another branchiopod, but is not found in the other arthropods examined. Despite divergent mechanisms of midline cell formation and nerve cord development, Net accumulation is detected in a well‐conserved subset of midline cells in branchiopod crustaceans, malacostracan crustaceans, and insects. Notably, the Net accumulation pattern is also conserved at the midline of the amphipod P. hawaiensis, which undergoes split germ‐band development. Conserved Net accumulation patterns indicate that arthropod midline cells are homologous, and that Nets function to regulate commissure formation during CNS development of Tetraconata.

KLF/SP Transcription Factor Family Evolution: Expansion, Diversification, and Innovation in Eukaryotes

The Krüppel-like factor and specificity protein (KLF/SP) genes play key roles in critical biological processes including stem cell maintenance, cell proliferation, embryonic development, tissue differentiation, and metabolism and their dysregulation has been implicated in a number of human diseases and cancers. Although many KLF/SP genes have been characterized in a handful of bilaterian lineages, little is known about the KLF/SP gene family in nonbilaterians and virtually nothing is known outside the metazoans. Here, we analyze and discuss the origins and evolutionary history of the KLF/SP transcription factor family and associated transactivation/repression domains. We have identified and characterized the complete KLF/SP gene complement from the genomes of 48 species spanning the Eukarya. We have also examined the phylogenetic distribution of transactivation/repression domains associated with this gene family. We report that the origin of the KLF/SP gene family predates the divergence of the Metazoa. Furthermore, the expansion of the KLF/SP gene family is paralleled by diversification of transactivation domains via both acquisitions of pre-existing ancient domains as well as by the appearance of novel domains exclusive to this gene family and is strongly associated with the expansion of cell type complexity.

Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis

Hox genes play crucial roles in establishing regional identity along the anterior-posterior axis in bilaterian animals, and have been implicated in generating morphological diversity throughout evolution. Here we report the identification, expression, and initial genomic characterization of the complete set of Hox genes from the amphipod crustacean Parhyale hawaiensis. Parhyale is an emerging model system that is amenable to experimental manipulations and evolutionary comparisons among the arthropods. Our analyses indicate that the Parhyale genome contains a single copy of each canonical Hox gene with the exception of fushi tarazu, and preliminary mapping suggests that at least some of these genes are clustered together in the genome. With few exceptions, Parhyale Hox genes exhibit both temporal and spatial colinearity, and expression boundaries correlate with morphological differences between segments and their associated appendages. This work represents the most comprehensive analysis of Hox gene expression in a crustacean to date, and provides a foundation for functional studies aimed at elucidating the role of Hox genes in arthropod development and evolution.