Molecular phylogenetic evaluation of classification and scenarios of character evolution in calcareous sponges (Porifera, Class Calcarea).

Calcareous sponges (Phylum Porifera, Class Calcarea) are known to be taxonomically difficult. Previous molecular studies have revealed many discrepancies between classically recognized taxa and the observed relationships at the order, family and genus levels; these inconsistencies question underlying hypotheses regarding the evolution of certain morphological characters. Therefore, we extended the available taxa and character set by sequencing the complete small subunit (SSU) rDNA and the almost complete large subunit (LSU) rDNA of additional key species and complemented this dataset by substantially increasing the length of available LSU sequences. Phylogenetic analyses provided new hypotheses about the relationships of Calcarea and about the evolution of certain morphological characters. We tested our phylogeny against competing phylogenetic hypotheses presented by previous classification systems. Our data reject the current order-level classification by again finding non-monophyletic Leucosolenida, Clathrinida and Murrayonida. In the subclass Calcinea, we recovered a clade that includes all species with a cortex, which is largely consistent with the previously proposed order Leucettida. Other orders that had been rejected in the current system were not found, but could not be rejected in our tests either. We found several additional families and genera polyphyletic: the families Leucascidae and Leucaltidae and the genus Leucetta in Calcinea, and in Calcaronea the family Amphoriscidae and the genus Ute. Our phylogeny also provided support for the vaguely suspected close relationship of several members of Grantiidae with giantortical diactines to members of Heteropiidae. Similarly, our analyses revealed several unexpected affinities, such as a sister group relationship between Leucettusa (Leucaltidae) and Leucettidae and between Leucascandra (Jenkinidae) and Sycon carteri (Sycettidae). According to our results, the taxonomy of Calcarea is in desperate need of a thorough revision, which cannot be achieved by considering morphology alone or relying on a taxon sampling based on the current classification below the subclass level

Molecular phylogenies confirm the presence of two cryptic Hemimycale species in the Mediterranean and reveal the polyphyly of the genera Crella and Hemimycale (Demospongiae: Poecilosclerida)

Este artículo contiene 24 páginas, 4 figuras, 3 tablas.Background: Sponges are particularly prone to hiding cryptic species as their paradigmatic plasticity often favors species phenotypic convergence as a result of adaptation to similar habitat conditions. Hemimycale is a sponge genus (Family Hymedesmiidae, Order Poecilosclerida) with four formally described species, from which only Hemimycale columella has been recorded in the Atlanto-Mediterranean basin, on shallow to 80 m deep bottoms. Contrasting biological features between shallow and deep individuals of Hemimycale columella suggested larger genetic differences than those expected between sponge populations. To assess whether shallow and deep populations indeed belong to different species, we performed a phylogenetic study of Hemimycale columella across the Mediterranean. We also included other Hemimycale and Crella species from the Red Sea, with the additional aim of clarifying the relationships of the genus Hemimycale. Methods: Hemimycale columella was sampled across the Mediterranean, and Adriatic Seas. Hemimycale arabica and Crella cyathophora were collected from the Red Sea and Pacific. From two to three specimens per species and locality were extracted, amplified for Cytochrome C Oxidase I (COI) (M1–M6 partition), 18S rRNA, and 28S (D3–D5 partition) and sequenced. Sequences were aligned using Clustal W v.1.81. Phylogenetic trees were constructed under neighbor joining (NJ), Bayesian inference (BI), and maximum likelihood (ML) criteria as implemented in Geneious software 9.01. Moreover, spicules of the target species were observed through a Scanning Electron microscope. Results: The several phylogenetic reconstructions retrieved both Crella and Hemimycale polyphyletic. Strong differences in COI sequences indicated that C. cyathophora from the Red Sea might belong in a different genus, closer to Hemimycale arabica than to the Atlanto-Mediterranean Crella spp. Molecular and external morphological differences between Hemimycale arabica and the Atlanto- Mediterranean Hemimycale also suggest that Hemimycale arabica fit in a separate genus. On the other hand, the Atlanto-Mediterranean Crellidae appeared in 18S and 28S phylogenies as a sister group of the Atlanto-Mediterranean Hemimycale. Moreover, what was known up to now as Hemimycale columella, is formed by two cryptic species with contrasting bathymetric distributions. Some small but consistent morphological differences allow species distinction. Conclusions: A new family (Hemimycalidae) including the genus Hemimycale and the two purported new genera receiving C. cyathophora and Hemimycale arabica might be proposed according to our phylogenetic results. However, the inclusion of additional Operational Taxonomic Unit (OTUs) appears convenient before taking definite taxonomical decisions. A new cryptic species (Hemimycale mediterranea sp. nov.) is described. Morphologically undifferentiated species with contrasting biological traits, as those here reported, confirm that unidentified cryptic species may confound ecological studies.The research has been funded by MARSYMBIOMICS project (Spanish MINECO, CTM2013-43287-P), BluePharmTrain (FP7 People-INT, Ref. 2013-667786), and Grup Consolidat SGR-120, to Maria J. Uriz. Leire Garate benefited from a fellowship within the Benthomics project (Spanish MICINN, CTM-2010-22218-C02-01).Peer reviewe

Molecular Phylogeny of the Astrophorida (Porifera, Demospongiaep) Reveals an Unexpected High Level of Spicule Homoplasy

Background: The Astrophorida (Porifera, Demospongiaep) is geographically and bathymetrically widely distributed. Systema Porifera currently includes five families in this order: Ancorinidae, Calthropellidae, Geodiidae, Pachastrellidae and Thrombidae. To date, molecular phylogenetic studies including Astrophorida species are scarce and offer limited sampling. Phylogenetic relationships within this order are therefore for the most part unknown and hypotheses based on morphology largely untested. Astrophorida taxa have very diverse spicule sets that make them a model of choice to investigate spicule evolution. Methodology/Principal Findings: With a sampling of 153 specimens (9 families, 29 genera, 89 species) covering the deepand shallow-waters worldwide, this work presents the first comprehensive molecular phylogeny of the Astrophorida, using a cytochrome c oxidase subunit I (COI) gene partial sequence and the 59 end terminal part of the 28S rDNA gene (C1-D2 domains). The resulting tree suggested that i) the Astrophorida included some lithistid families and some Alectonidae species, ii) the sub-orders Euastrophorida and Streptosclerophorida were both polyphyletic, iii) the Geodiidae, the Ancorinidae and the Pachastrellidae were not monophyletic, iv) the Calthropellidae was part of the Geodiidae clade (Calthropella at least), and finally that v) many genera were polyphyletic (Ecionemia, Erylus, Poecillastra, Penares, Rhabdastrella, Stelletta and Vulcanella). Conclusion: The Astrophorida is a larger order than previously considered, comprising ca. 820 species. Based on these results, we propose new classifications for the Astrophorida using both the classical rank-based nomenclature (i.e., Linnaean classification) and the phylogenetic nomenclature following the PhyloCode, independent of taxonomic rank. A key to the Astrophorida families, sub-families and genera incertae sedis is also included. Incongruences between our molecular tree and the current classification can be explained by the banality of convergent evolution and secondary loss in spicule evolution. These processes have taken place many times, in all the major clades, for megascleres and microscleres

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

Coevolution of Sponges and Microbial Symbionts Across Ocean Basins

Sponges (phylum Porifera) can comprise a large portion of benthic communities in freshwater and marine environments. Because sponges possess simple body plans and can have substantial phenotypic plasticity within species, reconstructing phylogenies of Porifera based upon morphologies alone has proven difficult. With the advent of nucleic acid sequencing technologies, interest in sponge phylogenetics has significantly increased among researchers. Additionally, sponges host a wide range of microbial symbionts, including the cyanobacterium Synechococcus spongiarum. This study examined the evolution of both sets of organisms in the symbiotic relationship between Aplysina sponges and Synechococcus cyanobacteria. In Chapter 3, gene sequences coding for the large subunit (28S) of nuclear ribosomal RNA (rRNA), as well as the internal transcribed spacer region, were used to reconstruct a phylogeny of Aplysina to determine the evolutionary relationships of sponges among three geographic locations. Aplysina species from the Eastern Pacific Ocean and the Caribbean Sea were more closely related to each other than they were to species from the Mediterranean Sea. In Chapter 4, cyanobacterial 16S rRNA gene sequences were used to reconstruct a phylogeny of Synechococcus to test the hypothesis that each sponge species hosts a unique clade of Synechococcus. By reconstructing the cyanobacterial phylogeny, new hosts were found for existing clades. iv Multiple statistical analyses indicated that symbiont phylogeny was significantly associated with geographic location and host species identity. Additionally, a ParaFit analysis was used to test the hypothesis of coevolution among hosts and symbionts. This analysis showed that several, but not all, host-symbiont parings displayed significant evidence of coevolution. This study implemented new statistical tests to improve previously established methods. These updated methods demonstrate the recent rapid progression of new statistical tools and methods, and with further development of new analytical approaches, these results can be further refined

Molecular Techniques Reveal Wide Phyletic Diversity of Heterotrophic Microbes Associated with Discodermia spp. (Porifera: Demospongiae)

Sponges are well known to harbor large numbers of heterotrophic microbes within their mesohyl. Studies to determine the diversity of these associated microbes have been attempted for only a few shallow water species. We cultured various microorganisms from several species of Discodermia collected from deep water using the \u27Johnson-Sea-Link\u27 manned submersibles, and characterised them by standard microbiological identification methods. Characterisation of a small proportion (ca. 10%) of the total and potential eubacterial isolate collection with molecular systematics techniques revealed a wide diversity of microbes. Phylogenetic analyses of 32 small subunit (SSU) 16S-like rRNA gene sequences from different micorbes indicated high levels of taxonomic diversity assoiated with this genus of sponge. For example, bacteria from at least five cubacterial subdivisions - gamma, alpha, beta, Cytophaga and Gram positive - were isolated from the mesohyl of Discodermia. Several strains were unidentifiable from current sequence databases. No overlap was found between sequences of 24 isolates and 8 sequences obtained by PCR and cloning directly from sponge samples. The abundance and diversity of microbes associated with sponges such as Discodermia suggest that they may play important roles in marine microbial ecology, dispersal and evolution

Transcriptomic analysis of differential host gene expression upon uptake of symbionts: a case study with Symbiodinium and the major bioeroding sponge Cliona varians

Background: We have a limited understanding of genomic interactions that occur among partners for many symbioses. One of the most important symbioses in tropical reef habitats involves Symbiodinium. Most work examining Symbiodinium-host interactions involves cnidarian partners. To fully and broadly understand the conditions that permit Symbiodinium to procure intracellular residency, we must explore hosts from different taxa to help uncover universal cellular and genetic strategies for invading and persisting in host cells. Here, we present data from gene expression analyses involving the bioeroding sponge Cliona varians that harbors Clade G Symbiodinium. Results: Patterns of differential gene expression from distinct symbiont states (“normal”, “reinfected”, and “aposymbiotic”) of the sponge host are presented based on two comparative approaches (transcriptome sequencing and suppressive subtractive hybridization (SSH)). Transcriptomic profiles were different when reinfected tissue was compared to normal and aposymbiotic tissue. We characterized a set of 40 genes drawn from a pool of differentially expressed genes in “reinfected” tissue compared to “aposymbiotic” tissue via SSH. As proof of concept, we determined whether some of the differentially expressed genes identified above could be monitored in sponges grown under ecologically realistic field conditions. We allowed aposymbiotic sponge tissue to become re-populated by natural pools of Symbiodinium in shallow water flats in the Florida Keys, and we analyzed gene expression profiles for two genes found to be increased in expression in “reinfected” tissue in both the transcriptome and via SSH. These experiments highlighted the experimental tractability of C. varians to explore with precision the genetic events that occur upon establishment of the symbiosis. We briefly discuss lab- and field-based experimental approaches that promise to offer insights into the co-opted genetic networks that may modulate uptake and regulation of Symbiondinium populations in hospite. Conclusions: This work provides a sponge transcriptome, and a database of putative genes and genetic pathways that may be involved in Symbiodinium interactions. The relative patterns of gene expression observed in these experiments will need to be evaluated on a gene-by-gene basis in controlled and natural re-infection experiments. We argue that sponges offer particularly useful characteristics for discerning essential dimensions of the Symbiodinium niche. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-376) contains supplementary material, which is available to authorized users