Search

The performance of DNA metabarcoding approaches for characterizing biodiversity can be influenced by multiple factors. Here, we used morphological assessment of taxa in zooplankton samples to develop a large barcode database and to assess the congruence of taxonomic identification with metabarcoding under different conditions. We analysed taxonomic assignment of metabarcoded samples using two genetic markers (COI, 18S V1–2), two types of clustering into molecular operational taxonomic units (OTUs, ZOTUs), and three methods for taxonomic assignment (RDP Classifier, BLASTn to GenBank, BLASTn to a local barcode database). The local database includes 1042 COI and 1108 18S (SSU) barcode sequences, and we added new high-quality sequences to GenBank for both markers, including 109 contributions at the species level. The number of phyla detected and the number of taxa identified to phylum varied between a genetic marker and among the three methods used for taxonomic assignments. Blasting the metabarcodes to the local database generated multiple unique contributions to identify OTUs and ZOTUs. We argue that a multi-marker approach combined with taxonomic expertise to develop a curated, vouchered, local barcode database increases taxon detection with metabarcoding, and its potential as a tool for zooplankton biodiversity surveys.

We report the results of a survey of the fishes of Kāne‘ohe Bay, O‘ahu, conducted in 2017 as part of the Smithsonian Institution MarineGEO Hawaii bioassessment. We recorded 109 species in 43 families. The most speciose families were Acanthuridae (11 species), Gobiidae (11 species), Pomacentridae (10) and Chaetodontidae (9 species). Nine of the species that we collected are known or suspected to be introduced to the Hawaiian Islands. Specimens were identified, measured and photographed. All specimen vouchers were fixed in formalin and ultimately transferred to 75% ethanol for long-term storage. For nearly all species, we took multiple tissue samples from specimen vouchers prior to formalin-fixation; we preserved tissues in 95% ethanol and then stored them at −80°C. The 5′-end of the mitochondrial cytochrome oxidase I gene (mtCOI) was sequenced for 94 species to confirm their taxonomic identification. Using these barcode sequences, we also measured genetic distances between collected individuals and their conspecifics from other localities outside Hawaii to verify the hypothesis that Hawaiian populations of species broadly distributed throughout the Indo-Pacific may be genetically distinct. We present select case studies to demonstrate the potential for undiscovered endemism in the Hawaiian fish biota.

This study reinforces and extends the findings of previous molecular studies showing that there is a close relationship between species assigned to the sponge genera Halicnemia, Higginsia, Paratimea and Stelligera and that the family Heteroxyidae is polyphyletic. The present study has led to the description of one new species of Halicnemia and six new species of Paratimea, the resurrection of Halicnemia gallica and a better understanding of the characters uniting Stelligeridae. A new species of Heteroxya is also described. We demonstrate that many of the taxa assigned to Heteroxyidae are more closely related to other families, and we propose several changes to the classification of Heteroscleromorpha. Desmoxyidae is resurrected from synonymy and transferred to Poecilosclerida; Higginsia anfractuosa is transferred to Hymedesmiidae, and a new genus, Hooperia, is erected for its reception; Higginsia durissima is returned to Bubaris (Bubaridae); Higginsia fragilis is transferred to Spanioplon (Hymedesmiidae); Hemiasterella camelus is transferred to Paratimea; and Raspailia (Parasyringella) australiensis and Ceratopsion axiferum are transferred to Adreus (Hemiasterellidae).

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.

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 ofHaliclona 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 threeCallyspongiaand threeHaliclonaspecies 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 fromone H. cinereawere very different from sequences generated from otherH. cinereaindividuals indicating that this species comprises more than one taxon.

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.

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.

Synopsis Sponge classification has long been based mainly on morphocladistic analyses but is now being greatly chal- lenged 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 Levi, 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 mor- phological characters within Heteroscleromorpha. The implications for the classification of Heteroscleromorpha are discussed and a new order Biemnida ord. nov. is erected.

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.

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.

Recent molecular studies have shown that the sponge order Haploslcerida is polyphyletic as the freshwater sponges appear to be more closely related to other demosponges than they are to the marine haplosclerids. Within the marine haplosclerid clade relationships viewed via 18S and 28S rRNA gene phylogenies suggest that the suborders and many families and genera are also polyphyletic. However, both of these genes are on the same locus and do not evolve completely independently. We have analysed mitochondrial Cytochrome oxidase 1 gene fragments from 44 species of marine Haplosclerida and show conclusively that the classification of this group needs complete revision. Molecular data show a very complicated phylogeny supporting very few morphological hypotheses and little geographical pattern. However, the molecular data contain a great deal of phylogenetic signal at many taxonomic levels and support phylogenies drawn from the other genes.

In a population-based study in northern Malawi we investigated HIV-1 subtype C gag and env gene sequences associated with long-term survival. DNA samples were available from 31 individuals surviving between population surveys carried out in the 1980s and 1990s. Most survivors with paired sequences dating from the 1980s and the 1990s had a three codon deletion in the gag p17 region of the sequence retrieved from the sample collected in the 1990s that was not present in the sequence from the same individual dating from the 1980s. This deletion was also not present in any other 1980s sequences from Malawi, but was common in samples collected in Malawi in the 1990s. The deletion is equivalent to the loss of three amino acids in the D helix region of the gag protein, and may be associated with longer survival and onward transmission.

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: Keratosa p , Myxospongiae p , Spongillida p , Haploscleromorpha p (the marine haplosclerids) and Democlavia p .W e found conflicting results concerning the relationships of Keratosa p and Myxospongiae p to the remaining demosponges, but our results strongly supported a clade of Haploscleromorpha p +Spongillida p +Democlavia p . In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillida p ) are sister to Haploscleromorpha p rather than part of Democlavia p . Within Keratosa p , we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiae p , Chondrosida and Verongida were monophyletic. A wellsupported clade within Democlavia p , Tetractinellida p , 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 Democlavia p . Within Tetractinellida p , 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.