Niamh E. Redmond

Reconstruction of Family-Level Phylogenetic Relationships within Demospongiae (Porifera) Using Nuclear Encoded Housekeeping Genes

Background Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. Methodology/Principal Findings We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosap, Myxospongiaep, Spongillidap, Haploscleromorphap (the marine haplosclerids) and Democlaviap. We found conflicting results concerning the relationships of Keratosap and Myxospongiaep to the remaining demosponges, but our results strongly supported a clade of Haploscleromorphap+Spongillidap+Democlaviap. In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillidap) are sister to Haploscleromorphap rather than part of Democlaviap. Within Keratosap, we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiaep, Chondrosida and Verongida were monophyletic. A well-supported clade within Democlaviap, Tetractinellidap, composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlaviap. Within Tetractinellidap, we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. Conclusions/Significance These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.

Phylogeny and Systematics of Demospongiae in Light of New Small-Subunit Ribosomal DNA (18S) Sequences

The most diverse and species-rich class of the phylum Porifera is Demospongiae. In recent years, the systematics of this clade, which contains more than 7000 species, has developed rapidly in light of new studies combining molecular and morphological observations. We add more than 500 new, nearly complete 18S sequences (an increase of more than 200%) in an attempt to further enhance understanding of the phylogeny of Demospongiae. Our study specifically targets representation of type species and genera that have never been sampled for any molecular data in an effort to accelerate progress in classifying this diverse lineage. Our analyses recover four highly supported subclasses of Demospongiae: Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha. Within Keratosa, neither Dendroceratida, nor its two families, Darwinellidae and Dictyodendrillidae, are monophyletic and Dictyoceratida is divided into two lineages, one predominantly composed of Dysideidae and the second containing the remaining families (Irciniidae, Spongiidae, Thorectidae, and Verticillitidae). Within Myxospongiae, we find Chondrosida to be paraphyletic with respect to the Verongida. We amend the latter to include species of the genus Chondrosia and erect a new order Chondrillida to contain remaining taxa from Chondrosida, which we now discard. Even with increased taxon sampling of Haploscleromorpha, our analyses are consistent with previous studies; however, Haliclona species are interspersed in even more clades. Haploscleromorpha contains five highly supported clades, each more diverse than previously recognized, and current families are mostly polyphyletic. In addition, we reassign Janulum spinispiculum to Haploscleromorpha and resurrect Reniera filholi as Janulum filholi comb. nov. Within the large clade Heteroscleromorpha, we confirmed 12 recently identified clades based on alternative data, as well as a sister-group relationship between the freshwater Spongillida and the family Vetulinidae. We transfer Stylissa flabelliformis to the genus Scopalina within the family Scopalinidae, which is of uncertain position. Our analyses uncover a large, strongly supported clade containing all heteroscleromorphs other than Spongillida, Vetulinidae, and Scopalinidae. Within this clade, there is a major division separating Axinellidae, Biemnida, Tetractinellida, Bubaridae, Stelligeridae, Raspailiidae, and some species of Petromica, Topsentia, and Axinyssa from Agelasida, Polymastiidae, Placospongiidae, Clionaidae, Spirastrellidae, Tethyidae, Poecilosclerida, Halichondriidae, Suberitidae, and Trachycladus. Among numerous results: (1) Spirophorina and its family Tetillidae are paraphyletic with respect to a strongly supported Astrophorina within Tetractinellida; (2) Agelasida is the earliest diverging lineage within the second clade listed above; and (3) Merlia and Desmacella appear to be the earliest diverging lineages of Poecilosclerida.

Phylogenetic Relationships of the Marine Haplosclerida (Phylum Porifera) Employing Ribosomal (28S rRNA) and Mitochondrial (cox1, nad1) Gene Sequence Data

The systematics of the poriferan Order Haplosclerida (Class Demospongiae) has been under scrutiny for a number of years without resolution. Molecular data suggests that the order needs revision at all taxonomic levels. Here, we provide a comprehensive view of the phylogenetic relationships of the marine Haplosclerida using many species from across the order, and three gene regions. Gene trees generated using 28S rRNA, nad1 and cox1 gene data, under maximum likelihood and Bayesian approaches, are highly congruent and suggest the presence of four clades. Clade A is comprised primarily of species of Haliclona and Callyspongia, and clade B is comprised of H. simulans and H. vansoesti (Family Chalinidae), Amphimedon queenslandica (Family Niphatidae) and Tabulocalyx (Family Phloeodictyidae), Clade C is comprised primarily of members of the Families Petrosiidae and Niphatidae, while Clade D is comprised of Aka species. The polyphletic nature of the suborders, families and genera described in other studies is also found here.

Nearly complete 28S rRNA gene sequences confirm new hypotheses of sponge evolution.

The highly collaborative research sponsored by the NSF-funded Assembling the Porifera Tree of Life (PorToL) project is providing insights into some of the most difficult questions in metazoan systematics. Our understanding of phylogenetic relationships within the phylum Porifera has changed considerably with increased taxon sampling and data from additional molecular markers. PorToL researchers have falsified earlier phylogenetic hypotheses, discovered novel phylogenetic alliances, found phylogenetic homes for enigmatic taxa, and provided a more precise understanding of the evolution of skeletal features, secondary metabolites, body organization, and symbioses. Some of these exciting new discoveries are shared in the papers that form this issue of Integrative and Comparative Biology. Our analyses of over 300 nearly complete 28S ribosomal subunit gene sequences provide specific case studies that illustrate how our dataset confirms new hypotheses of sponge evolution. We recovered monophyletic clades for all 4 classes of sponges, as well as the 4 major clades of Demospongiae (Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha), but our phylogeny differs in several aspects from traditional classifications. In most major clades of sponges, families within orders appear to be paraphyletic. Although additional sampling of genes and taxa are needed to establish whether this pattern results from a lack of phylogenetic resolution or from a paraphyletic classification system, many of our results are congruent with those obtained from 18S ribosomal subunit gene sequences and complete mitochondrial genomes. These data provide further support for a revision of the traditional classification of sponges.

Molecular Phylogenies Support Homoplasy of Multiple Morphological Characters Used in the Taxonomy of Heteroscleromorpha (Porifera: Demospongiae)

Sponge classification has long been based mainly on morphocladistic analyses but is now being greatly challenged by more than 12 years of accumulated analyses of molecular data analyses. The current study used phylogenetic hypotheses based on sequence data from 18S rRNA, 28S rRNA, and the CO1 barcoding fragment, combined with morphology to justify the resurrection of the order Axinellida Lévi, 1953. Axinellida occupies a key position in different morphologically derived topologies. The abandonment of Axinellida and the establishment of Halichondrida Vosmaer, 1887 sensu lato to contain Halichondriidae Gray, 1867, Axinellidae Carter, 1875, Bubaridae Topsent, 1894, Heteroxyidae Dendy, 1905, and a new family Dictyonellidae van Soest et al., 1990 was based on the conclusion that an axially condensed skeleton evolved independently in separate lineages in preference to the less parsimonious assumption that asters (star-shaped spicules), acanthostyles (club-shaped spicules with spines), and sigmata (C-shaped spicules) each evolved more than once. Our new molecular trees are congruent and contrast with the earlier, morphologically based, trees. The results show that axially condensed skeletons, asters, acanthostyles, and sigmata are all homoplasious characters. The unrecognized homoplasious nature of these characters explains much of the incongruence between molecular-based and morphology-based phylogenies. We use the molecular trees presented here as a basis for re-interpreting the morphological characters within Heteroscleromorpha. The implications for the classification of Heteroscleromorpha are discussed and a new order Biemnida ord. nov. is erected.

Large expansion segments in 18S rDNA support a new sponge clade (Class Demospongiae, Order Haplosclerida)

Newly emerging molecular phylogenetic hypotheses involving the sponge Order Haplosclerida (Class Demospongiae) are far removed from traditional views on their classification using morphology. In the new grouping of marine haplosclerid taxa by molecular data all members of one highly supported clade were found to have three large indels in the 18S rRNA gene. These indels were not found in this gene in other marine haplosclerids or in any other demosponges analysed. These indels were found in the variable V4 and V7 region of the gene, had high GC contents and formed stable double stranded helices in the 18S rRNA secondary structure. These indels are very important synapomorphies, provide high support for an alternative taxonomic scheme and could help resolve the phylogeny of this order in conjunction with other phylogenetically informative characters.

Ribosomal internal transcribed spacer regions are not suitable for intra- or inter-specific phylogeny reconstruction in haplosclerid sponges (Porifera: Demospongiae)

Sequences of the ribosomal internal transcribed spacer regions 1 and 2 (ITS-1 and ITS-2) were employed to investigate relationships between putatively very closely related species of marine haplosclerids and to investigate the species status of Haliclona cinerea . Results indicate that intra-genomic and intra-specific levels of diversity are equivalent, and sequences from multiple clones from a number of individuals of a single species could not be separated on phylogenetic trees. As a result, the ITS regions are not suitable markers for population level studies in marine haplosclerids. Sequences of these regions were highly species specific, and large differences were found between species. ITS sequences from three Callyspongia and three Haliclona species could not be aligned successfully and therefore this locus could not be used to investigate relationships between these putative close relatives. However, ITS sequences retrieved from one H. cinerea were very different from sequences generated from other H. cinerea individuals indicating that this species comprises more than one taxon.

Mycalina: Another Crack in the Poecilosclerida Framework

This is the first phylogenetic analysis integrating both morphological and molecular data of the sponge suborder Mycalina (Poecilosclerida), which was erected in 1994. A cladistic analysis of morphology supported the monophyly of Cladorhizidae (including Euchelipluma), Guitarridae (excluding Euchelipluma), Isodictyidae, Latrunculiidae, and Podospongiidae but rejected monophyly for Desmacellidae, Esperiopsidae, Hamacanthidae, and Mycalidae. Analyses of partial 16S and partial 28S rRNA datasets combined, as well as that of a complete 18S rDNA dataset, suggest that Mycalina is not monophyletic; Biemnidae is only distantly related to other poecilosclerids; Merlia and Desmacella branch near the base of a diverse Poecilosclerida clade; Mycalidae is monophyletic (excluding Mycale [Anomomycale] titubans in 18S); and Esperiopsidae and Isodictyidae form a clade. Analyses of the two molecular datasets differed on the monophyly of Podospongiidae and about the relationship of Podospongiidae to Isodictyidae + Esperiopsidae.

Vansoestia caribensis gen. nov., sp nov.: first report of the family Ianthellidae (Verongida, Demospongiae) in the Caribbean

A thin fiber-less sponge from Caribbean reefs (Bocas del Toro, Panama) with close genetic affinities (based on 18S and 28S nuclear ribosomal RNA gene sequences) to large fan-shaped fiber-bearing sponges (Ianthella and Anomoianthella) from the Indo-Pacific Ocean is here presented. We describe its overall external morphology, histological features, and ultrastructure. Its genetic distance from the only previously known fiber-less verongid genus, Hexadella, prompted the need to erect a new genus to classify this species. This novel species constitutes the first record for a member of the family Ianthellidae in the Caribbean. The characterization of the family Ianthellidae (sensu Cook and Bergquist, 2000) is here modified by: i) highlighting the cavernous nature of the choanosome, with many lacunae and channels reported for all genera included in the family; ii) extending the family distribution to the Caribbean; and iii) adding a fourth genus to the group of verongids with eurypylous chambers. The possession of a cellularized cortex (10-300 µm in thickness) is here proposed as a potential synapomorphic character of the Ianthella-Anomoianthella-Vansoestia clade. The main issues regarding the suprageneric classification of verongids are discussed.