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Sharma, Prashant Pradeep

This dissertation investigates evolutionary relationships within a diverse and ancient group of arachnids, the order Opiliones, with emphasis on relationships within the suborder Laniatores. Laniatorid harvestmen encompass over two-thirds of opilionid diversity, but this suborder has received far less than commensurate phylogenetic attention. The ancient age and narrow species distributions of Laniatores, in conjunction with their representation throughout tropical and subtropical regions of the world, herald models of unparalleled utility for study of tropical diversity and biogeography. Herein I infer the phylogeny of Laniatores using a ten-gene molecular dataset, which samples for the first time every described family of the group. Based upon morphological and molecular sequence data, I describe three new families of Laniatores--two endemic to Southeast Asia and one to the Afrotropics. Focusing on the biogeography of Australasia, I contrast the evolutionary histories of two families: Sandokanidae, which are restricted to the Indo-Malay Archipelago, and Zalmoxidae, which occurs in the Neotropics and Australasia. Using molecular phylogenetic tools, I demonstrate that sandokanid distribution is largely attributable to continental vicariance in Sundaland, whereas zalmoxid distribution is attributable to a New World origin, followed by colonization of the Indo-Pacific by transoceanic dispersal.

The internal phylogeny of the arachnid order Opiliones is investigated by including molecular data from five molecular markers for ca. 140 species totalling 43 families of Opiliones. The phylogenetic analyses consisted of a direct optimization (DO) approach using POY v. 4 and sophisticated tree search algorithms as well as a static alignment analysed under maximum likelihood. The four Opiliones suborders were well-supported clades, but subordinal relationships did not receive support in the DO analysis, with the exception of the monophyly of Palpatores (=Eupnoi + Dyspnoi). Maximum-likelihood analysis strongly supported the traditional relationship of Phalangida and Palpatores: (Cyphophthalmi ((Eupnoi + Dyspnoi) Laniatores)). Relationships within each suborder are well resolved and largely congruent between direct optimization and maximum-likelihood approaches. Age estimates for the main Opiliones lineages suggest a Carboniferous diversification of Cyphophthalmi, while its sister group, Phalangida, diversified in the Early Devonian. Diversification of all suborders predates the Triassic, and most major lineages predate the Cretaceous. The following taxonomic changes are proposed. Dyspnoi: Hesperonemastoma is transferred to Sabaconidae. Insidiatores: Sclerobunidae stat. nov. is erected as a family for Zuma acuta.

The robustness of clades to parameter variation may be a desirable quality or even a goal in phylogenetic analyses. Sensitivity analyses used to assess clade stability have invoked the incongruence length difference (ILD or (_WILD)) metric, a measure of congruence among datasets, to compare a series of most-parsimonious results from re-running analyses under different analytical conditions. It is also common practice to select a single “optimal” parameter set that minimizes (_WILD) across all parameter sets. However, the divergent molecular evolution of ribosomal genes and protein-encoding genes—specifically the bias against transversion events in coding genes of conserved function—suggests that deployment of multiple parameter sets could outperform the use of a single parameter set applied to all molecules. We explored congruence in five published datasets by including mixed parameter sets in our sensitivity analysis. In four cases, mixed parameter sets outperformed the previously reported, single optimal parameter set. Conversely, multiple parameter sets did not outperform a single optimal parameter set in a case in which actual strong topological conflict exists between data partitions. Exploration of mixed parameter sets may prove useful when combining ribosomal and protein-encoding genes, due to the relatively higher frequency of single- and double-base pair indel events in the former, and the relatively lower frequency of transversions in the latter.

Introduction: Traditionally, genomic or transcriptomic data have been restricted to a few model or emerging model organisms, and to a handful of species of medical and/or environmental importance. Next-generation sequencing techniques have the capability of yielding massive amounts of gene sequence data for virtually any species at a modest cost. Here we provide a comparative analysis of de novo assembled transcriptomic data for ten non-model species of previously understudied animal taxa. Results: cDNA libraries of ten species belonging to five animal phyla (2 Annelida [including Sipuncula], 2 Arthropoda, 2 Mollusca, 2 Nemertea, and 2 Porifera) were sequenced in different batches with an Illumina Genome Analyzer II (read length 100 or 150 bp), rendering between ca. 25 and 52 million reads per species. Read thinning, trimming, and de novo assembly were performed under different parameters to optimize output. Between 67,423 and 207,559 contigs were obtained across the ten species, post-optimization. Of those, 9,069 to 25,681 contigs retrieved blast hits against the NCBI non-redundant database, and approximately 50% of these were assigned with Gene Ontology terms, covering all major categories, and with similar percentages in all species. Local blasts against our datasets, using selected genes from major signaling pathways and housekeeping genes, revealed high efficiency in gene recovery compared to available genomes of closely related species. Intriguingly, our transcriptomic datasets detected multiple paralogues in all phyla and in nearly all gene pathways, including housekeeping genes that are traditionally used in phylogenetic applications for their purported single-copy nature. Conclusions: We generated the first study of comparative transcriptomics across multiple animal phyla (comparing two species per phylum in most cases), established the first Illumina-based transcriptomic datasets for sponge, nemertean, and sipunculan species, and generated a tractable catalogue of annotated genes (or gene fragments) and protein families for ten newly sequenced non-model organisms, some of commercial importance (i.e., Octopus vulgaris). These comprehensive sets of genes can be readily used for phylogenetic analysis, gene expression profiling, developmental analysis, and can also be a powerful resource for gene discovery. The characterization of the transcriptomes of such a diverse array of animal species permitted the comparison of sequencing depth, functional annotation, and efficiency of genomic sampling using the same pipelines, which proved to be similar for all considered species. In addition, the datasets revealed their potential as a resource for paralogue detection, a recurrent concern in various aspects of biological inquiry, including phylogenetics, molecular evolution, development, and cellular biochemistry.

We investigated the internal phylogeny of Laniatores, the most diverse suborder of Opiliones, using sequence data from 10 molecular loci: 12S rRNA, 16S rRNA, 18S rRNA, 28S rRNA, cytochrome c oxidase subunit I (COI), cytochrome b, elongation factor-1α, histones H3 and H4, and U2 snRNA. Exemplars of all previously described families of Laniatores were included, in addition to two families – Petrobunidae, fam. nov. and Tithaeidae, fam. nov. – that we erect herein. Data analyses were based on maximum likelihood and Bayesian approaches on static alignments, and included phylogenetic tree estimation, molecular dating, and biogeographic analysis of ancestral area reconstruction. The results obtained include the monophyly of Laniatores and the infraorder Grassatores – the focus of this study – as well as support for numerous interfamilial relationships. The two new families described cluster with other South-east Asian families (Podoctidae and Epedanidae). Diversification of Laniatores is estimated at ~348 Mya, and origin of most Grassatores superfamilies occurs in a ~25 million year span of time immediately after the end-Permian mass extinction (254 Mya). Ancestral range reconstruction of the clade (Samooidea + Zalmoxoidea) suggests that the ancestral range of Samooidea comprises West Tropical Gondwana (West Africa + Neotropics), whereas that of Zalmoxoidea is exclusively Neotropical. The following additional taxonomic changes are proposed: (1) Remyus is transferred to Phalangodidae, and (2) Escadabiidae and Kimulidae are transferred to Zalmoxoidea.

A revised catalogue of the Paleotropical Zalmoxidae including images of selected available type specimens is presented. Distribution data are provided to the best of our knowledge. The genera Acrozalmoxis Roewer, 1915, Camanastus Roewer, 1949, Papuastus Roewer, 1949, Savoa Roewer, 1949, and Zalmoxomma Roewer, 1949 are newly synonymized with the type genus Zalmoxis Sørensen, 1886. The following new combinations are established: Zalmoxis australis, Zalmoxis bon- ka, Zalmoxis insularis, Zalmoxis maculosus, Zalmoxis occidentalis, and Zalmoxis ponapeus. Bogania Forster, 1955 and Bunofagea Lawrence, 1959 are removed from Zalmoxidae and transferred to Phalangodidae. Gjellerupia Roewer, 1913 and Spalicus Roewer, 1949 are considered Grassatores incertae sedis. The incidence of preoccupied taxon names is re- dressed by designation of new specific epithets as follows: Zalmoxis neoguinensis (Roewer, 1915) is renamed Zalmoxis thorelli; Zalmoxis neoguinensis (Müller, 1917) is renamed Zalmoxis muelleri; Zalmoxis neoguinensis (Roewer, 1949) is renamed Zalmoxis mutus; and Zalmoxis minima (Roewer, 1915) is restored to Gjellerupia minima. Zalmoxis pallicolor Strand, 1910 is synonymized with Zalmoxis armatipes Strand, 1910. Diagnoses of genera are provided. Species richness and familial level relationships of Zalmoxidae are discussed.

We investigate the phylogeny, biogeography, time of origin and diversification, ancestral area reconstruction and large-scale distributional patterns of an ancient group of arachnids, the harvestman suborder Cyphophthalmi. Analysis of molecular and morphological data allow us to propose a new classification system for the group; Pettalidae constitutes the infraorder Scopulophthalmi new clade, sister group to all other families, which are divided into the infraorders Sternophthalmi new clade and Boreophthalmi new clade. Sternophthalmi includes the families Troglosironidae, Ogoveidae, and Neogoveidae; Boreophthalmi includes Stylocellidae and Sironidae, the latter family of questionable monophyly. The internal resolution of each family is discussed and traced back to its geological time origin, as well as to its original landmass, using methods for estimating divergence times and ancestral area reconstruction. The origin of Cyphophthalmi can be traced back to the Carboniferous, whereas the diversification time of most families ranges between the Carboniferous and the Jurassic, with the exception of Troglosironidae, whose current diversity originates in the Cretaceous/Tertiary. Ancestral area reconstruction is ambiguous in most cases. Sternophthalmi is traced back to an ancestral land mass that contained New Caledonia and West Africa in the Permian, whereas the ancestral landmass for Neogoveidae included the south-eastern USA and West Africa, dating back to the Triassic. For Pettalidae, most results include South Africa, or a combination of South Africa with the Australian plate of New Zealand or Sri Lanka, as the most likely ancestral landmass, back in the Jurassic. Stylocellidae is reconstructed to the Thai-Malay Penisula during the Jurassic. Combination of the molecular and morphological data results in a hypothesis for all the cyphophthalmid genera, although the limited data available for some taxa represented only in the morphological partition negatively affects the phylogenetic reconstruction by decreasing nodal support in most clades. However, it resolves the position of many monotypic genera not available for molecular analysis, such as Iberosiro, Odontosiro, Speleosiro, Managotria or Marwe, although it does not place Shearogovea or Ankaratra within any existing family. The biogeographical data show a strong correlation between relatedness and formerly adjacent landmasses, and oceanic dispersal does not need to be postulated to explain disjunct distributions, especially when considering the time of divergence. The data also allow testing of the hypotheses of the supposed total submersion of New Zealand and New Caledonia, clearly falsifying submersion of the former, although the data cannot reject the latter.

Among Opiliones, Afrotropical lineages constitute some of the least studied groups in comparison with those endemic to other biogeographic provinces. Based upon morphological evidence, we erect Pyramidopidae, fam. nov. to distinguish a group of Laniatores from the family Phalangodidae. We review evidence from recent molecular phylogenetic studies that corroborate the independence of Pyramidopidae, fam. nov. from previously described families and support its sister relationship to another largely Afrotropical group, the family Assamiidae. The monotypic genus Maiorerus Rambla, 1993 is transferred to Pyramidopidae, fam. nov. The new family comprises 12 genera geographically restricted to Africa and the adjacent Canary Islands. Interfamilial relationships of the derived Laniatores are discussed in the context of gross and genitalic morphology.

Mutations in Deleted in Azoospermia (DAZ), a Y chromosome gene, are an important cause of human male infertility. DAZ is found exclusively in primates, limiting functional studies of this gene to its homologs: boule, required for meiotic progression of germ cells in invertebrate model systems, and Daz-like (Dazl), required for early germ cell maintenance in vertebrates. Dazl is believed to have acquired its premeiotic role in a vertebrate ancestor following the duplication and functional divergence of the single-copy gene boule. However, multiple homologs of boule have been identified in some invertebrates, raising the possibility that some of these genes may play other roles, including a premeiotic function. Here we identify two boule paralogs in the freshwater planarian Schmidtea mediterranea. Smed-boule1 is necessary for meiotic progression of male germ cells, similar to the known function of boule in invertebrates. By contrast, Smed-boule2 is required for the maintenance of early male germ cells, similar to vertebrate Dazl. To examine if Boule2 may be functionally similar to vertebrate Dazl, we identify and functionally characterize planarian homologs of human DAZL/DAZ-interacting partners and DAZ family mRNA targets. Finally, our phylogenetic analyses indicate that premeiotic functions of planarian boule2 and vertebrate Dazl evolved independently. Our study uncovers a premeiotic role for an invertebrate boule homolog and offers a tractable invertebrate model system for studying the premeiotic functions of the DAZ protein family.