Genetic analysis confirms the freshwater origin of the endemic Caspian sponges (Demospongiae, Spongillida, Metschnikowiidae).

The Caspian Sea is a unique inland brackish waterbody inhabited by highly endemic fauna. This fauna consists of species of both marine and freshwater origin. Some Caspian invertebrates cannot be confidently referred to as animals of either origin. The endemic monophyletic family of sponges, Metschnikowiidae, is among them. Although these sponges are considered as fresh water in the modern literature, no researcher has seen them alive for many years, and its status is actually unconfirmed. Here, we present the first photos of Metschnikowia tuberculata Grimm, 1877 and report evidence for its freshwater origin based on analysis of ITS1 and ITS2 sequences and partial sequences of CO1 gene. According to the genetic analysis, M. tuberculata belongs to the order Spongillida. We observed specimens of diverse appearance, but their spicule complement proved to be similar, and ITS sequences were identical. Thus, we conclude that they belong to the same species. The obtained results expand our knowledge about the dispersal ability of freshwater sponges.

Symbiosis, Selection, and Novelty: Freshwater Adaptation in the Unique Sponges of Lake Baikal.

Freshwater sponges (Spongillida) are a unique lineage of demosponges that secondarily colonized lakes and rivers and are now found ubiquitously in these ecosystems. They developed specific adaptations to freshwater systems, including the ability to survive extreme thermal ranges, long-lasting dessication, anoxia, and resistance to a variety of pollutants. Although spongillids have colonized all freshwater systems, the family Lubomirskiidae is endemic to Lake Baikal and plays a range of key roles in this ecosystem. Our work compares the genomic content and microbiome of individuals of three species of the Lubomirskiidae, providing hypotheses for how molecular evolution has allowed them to adapt to their unique environments. We have sequenced deep (>92% of the metazoan "Benchmarking Universal Single-Copy Orthologs" [BUSCO] set) transcriptomes from three species of Lubomirskiidae and a draft genome resource for Lubomirskia baikalensis. We note Baikal sponges contain unicellular algal and bacterial symbionts, as well as the dinoflagellate Gyrodinium. We investigated molecular evolution, gene duplication, and novelty in freshwater sponges compared with marine lineages. Sixty one orthogroups have consilient evidence of positive selection. Transporters (e.g., zinc transporter-2), transcription factors (aristaless-related homeobox), and structural proteins (e.g. actin-3), alongside other genes, are under strong evolutionary pressure in freshwater, with duplication driving novelty across the Spongillida, but especially in the Lubomirskiidae. This addition to knowledge of freshwater sponge genetics provides a range of tools for understanding the molecular biology and, in the future, the ecology (e.g., colonization and migration patterns) of these key species.

Endemic Lake Baikal sponges from deep water. 2: Taxonomy and Bathymetric Distribution.

Unique samples of deep-water sponges of Lake Baikal were collected between 120 and 1450 m depth and their taxonomy and bathymetric distribution were studied. Based on morphological studies with scanning electron microscopy (SEM) and molecular analyses (CO1, ITS) we describe a new species, Baikalospongia abyssalis sp. nov. Spicule morphology of this new species is similar to Palaeoephydatia sp., a species previously known only from fossils in Late Pliocene (3.2-2.8 mya) sediments. Other sponge samples collected were identified as Baikalospongia intermedia intermedia, B. intermedia profundalis, B. bacillifera, B. fungiformis, B. martinsoni and Swartschewskia papyracea, all from the family Lubomirskiidae. Sponge specimens with giant spicules, identified as B. fungiformis, were found at great depths. B.i. intermedia and B. i. profundalis are dominating species at great depth. Light is a limiting factor for distribution of Lubomirskia baicalensis, possibly due to its symbiosis with photosynthetic protists. The current study extends our knowledge on the distribution boundaries of Lubomirskiidae at great depths.

Endemic Lake Baikal sponges from deep water. 1: Potential cryptic speciation and discovery of living species known only from fossils.

We revealed new deep-water species and cryptic speciation within freshwater sponges of the endemic family Lubomirskiidae (Porifera; Demospongiae; Spongillina) based on molecular and spicule morphology analyses of ITS and CO1 mtDNA. Lubomirskiidae contains a group of closely related species which are a dominant component of the benthos in Lake Baikal, the world's deepest and most ancient lake. Spicule morphology was similar between two Recent samples and species only known previously from fossils in Late Pliocene (3.2-2.8 mya) sediments. Despite the morphological similarity with the cosmopolitan family Spongillidae, molecular analysis of ITS sequences has reliably assigned these species to Lubomirskiidae. This not only indicates that species identification of freshwater fossil sponge spicules should be made with caution, but also suggests that the structure of megascleres may not be a reliable character for interpretations of paleoclimatic reconstructions for the Baikal region. Our results do not support the current classification of Lubomirskiidae into its morphologically defined genera and species, suggesting a strong discrepancy between molecular and morphological variation in Baikalian sponges. This present contribution is the first part of a study on the phylogenetic relationships of the Lake Baikal deep water sponge fauna.

Ribosomal ITS sequences allow resolution of freshwater sponge phylogeny with alignments guided by secondary structure prediction.

Freshwater sponges include six extant families which belong to the suborder Spongillina (Porifera). The taxonomy of freshwater sponges is problematic and their phylogeny and evolution are not well understood. Sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) of 11 species from the family Lubomirskiidae, 13 species from the family Spongillidae, and 1 species from the family Potamolepidae were obtained to study the phylogenetic relationships between endemic and cosmopolitan freshwater sponges and the evolution of sponges in Lake Baikal. The present study is the first one where ITS1 sequences were successfully aligned using verified secondary structure models and, in combination with ITS2, used to infer relationships between the freshwater sponges. Phylogenetic trees inferred using maximum likelihood, neighbor-joining, and parsimony methods and Bayesian inference revealed that the endemic family Lubomirskiidae was monophyletic. Our results do not support the monophyly of Spongillidae because Lubomirskiidae formed a robust clade with E. muelleri, and Trochospongilla latouchiana formed a robust clade with the outgroup Echinospongilla brichardi (Potamolepidae). Within the cosmopolitan family Spongillidae the genera Radiospongilla and Eunapius were found to be monophyletic, while Ephydatia muelleri was basal to the family Lubomirskiidae. The genetic distances between Lubomirskiidae species being much lower than those between Spongillidae species are indicative of their relatively recent radiation from a common ancestor. These results indicated that rDNA spacers sequences can be useful in the study of phylogenetic relationships of and the identification of species of freshwater sponges.

Phylogenetic analysis of freshwater sponges provide evidence for endemism and radiation in ancient lakes.

Morphologic and phylogenetic analysis of freshwater sponges endemic to lakes in Central Sulawesi, Siberia and South-East Europe is presented. We also analyzed several cosmopolitan sponge species from Eurasia and North America and included sponge sequences from public databases. In agreement with previous reports [Addis, J.S., Peterson, K.J., 2005. Phylogenetic relationships of freshwater sponges (Porifera, Spongillina) inferred from analyses of 18S rDNA, COI mtDNA, and ITS2 rDNA sequences. Zool. Scr. 34, 549-557], the metaniid sponge Corvomeyenia sp. was the most deeply branching species within a monophyletic lineage of the suborder Spongillina. Pachydictyum globosum (Malawispongiidae) and Nudospongilla vasta (Spongillidae), two morphologically quite distinct species from Sulawesi were found in a joint clade with Trochospongilla (Spongillidae) rendering Trochospongilla paraphyletic. Furthermore, Ochridaspongia sp., another Malawispongiidae, clustered far away from that clade, together with Ephydatia fluviatilis, making the latter family polyphyletic. The Lubomirskiidae endemic to Lake Baikal, Lubomirskia abietina, Baikalospongia bacillifera, B. intermedia, and Swartschewskia papyracea formed a well-supported clade that was most closely linked to the genus Ephydatia (99.9% identity over a total length of 2169 concatenated nucleotide positions). Our study indicates the frequent and independent origin of sponge species endemic to different freshwater ecosystems from a few cosmopolitan founder species. The highly specific primer sets newly developed here facilitate work on the molecular phylogeny and DNA barcoding of sponges.

Conservation and phylogeny of a novel family of non-Hox genes of the Antp class in Demospongiae (porifera).

A survey across the most basal animal phylum, the Porifera, for the presence of homeobox-containing genes led to the isolation of 24 partial or complete homeobox sequences from 21 sponge species distributed in 15 families and 6 orders of Demospongiae. All the new sequences shared a high identity/similarity with EmH-3 (Ephydatia muelleri), a non-Hox gene from the Antp class. The Demox sequences, EmH-3, and related homeodomains formed a well-supported clade with no true affinity with any known bilaterian family, including the Tlx/Hox11 family, suggesting that the EmH-3 family of genes, comprising 31 members, represents a novel family of non-Hox genes, called the Demox family, widespread among Demospongiae. The presence of the Tlx/Hox11 specific signature in the Demox family and common regulatory elements suggested that the Demox and Tlx/Hox11 families are closely related. In the phylogenetic analyses, freshwater Haplosclerida appeared as monophyletic, and Haplosclerida and Halichondrida as polyphyletic, with a clade comprising Agelas species and Axinella corrugata. As for their expression, high levels of Demox transcripts were found in adult tissues. Our data add to the number of published poriferan homeobox sequences and provide independent confirmation of the current Demospongiae phylogenies.

Lake Baikal: a unique place to study evolution of sponges and their stress response in an environment nearly unimpaired by anthropogenic perturbation.

Lake Baikal is considered as a unique place to study evolution. In this review, we report on recent data on the evolution of endemic freshwater sponges of this ancient lake. Nucleotide sequence data support the idea that these sponges are of monophyletic origin and evolved from Spongillidae. Baikalian sponges form the dominating biomass in the benthos of the lake. Data on the expression of the biomarker heat shock protein 70, revealed that the endemic sponge species of Lake Baikal are useful as bioindicators to assess the anthropogenic impact on the lake.

Heat shock protein Hsp70 expression and DNA damage in Baikalian sponges exposed to model pollutants and wastewater from Baikalsk Pulp and Paper Plant.

Lake Baikal, a unique habitat for a great number of endemic species, is the largest freshwater reservoir in the world which is still largely unaffected by anthropogenic pollution, except for some shore regions with industrial activity. The expressions of a biomarker of exposure (heat shock protein Hsp70) and a biomarker of effect (DNA single-strand breaks) were measured for the first time in endemic Baikalian sponge species (Baikalospongia intermedia, Lubomirskia fusifera, and Lubomirskia abietina). Tissue cubes of B. intermedia and dissociated cells of L. fusifera and L. abietina reacted to temperature stress (10-16 degrees C above ambient temperature) with a time-dependent increase in expression of Hsp70. In B. intermedia, the effects of model pollutants (lead, copper, and zinc, and the organochlorines tetrachloroguaiacol, TCG, and pentachlorophenol, PCP) and of the wastewater from the final refinement and aeration reservoirs of the Baikalsk Pulp and Paper Plant (BPPP), located at the shore of the southern basin of Lake Baikal, on the expression of Hsp70 and the extent of DNA damage were investigated. It was found that lead and zinc but not copper cause a strong induction of Hsp70 in this sponge, while the frequency of DNA single-strand breaks increased after exposure to all these heavy metals tested. Induction of DNA single-strand breaks was also observed after exposure to TCG and PCP, but these compounds did not (consistently) enhance Hsp70 expression. Wastewater taken from the final water aeration pond of BPPP caused a concentration-dependent increase in Hsp70 expression in B. intermedia. However, there was no difference in the basal levels of Hsp70 between sponges collected in the shallow water at an unpolluted site near Baikalsk City and at a polluted site where the wastewaters of BPPP are discharged into the lake. There was also no clear difference in the wastewater concentration-dependent induction of Hsp70 expression between sponges collected at these sites, indicating no adaptation to continuous stress exposure.