- Research article
- Open Access
Evolution of group I introns in Porifera: new evidence for intron mobility and implications for DNA barcoding
© The Author(s). 2017
- Received: 15 September 2016
- Accepted: 28 February 2017
- Published: 20 March 2017
Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns.
For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated.
This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested.
- homing endonuclease gene (HEG)
- group I intron
- DNA barcoding
Mobile introns are self-splicing DNA sequences that play a major role in genome evolution. Group I and group II introns are distinguished based on their splicing mechanisms and secondary structures. Apart from unique splicing mechanisms, differences between group I and group II introns were observed within the core regions of their secondary structures. Depending on these structural characteristics, group I introns have been further categorized into IA-IE classes. Group II introns constitute up to six stem-loop domains and are classified I-VI respectively (e.g., ). Group I and group II introns often contain open reading frames (ORFs) in their loop regions , which can encode for different site-specific homing endonuclease genes (HEGs). The majority of group I introns include HEGs, which have a conservative single or a double motif of the amino-acid sequence LAGLIDADG. In contrast group II introns encode in most cases a reverse transcriptase-like (RT) ORF (e.g., ). Group I and group II introns are found in all domains of life: group I introns are present in bacterial, organellar, bacteriophage and viral genomes as well as in the nuclear rDNA of eukaryotes. Group II introns have a similar distribution, but are not known from the nuclear rDNA (e.g., ). More specifically, group I and/or group II introns are found, e.g., in eukaryotic viruses , slime molds , choanoflagellates , the annelid Nephtys sp. , red algae , brown algae  and plants: green algae , liverworts [58, 66, 67] and different angiosperms [58, 66, 67]. Group II introns seem to thrive especially in plants , whereas the largest abundance of group I introns currently occurs within fungi [23, 54, 68]. As an example, the mitochondrial (mt) genome of the fungus Ophioscordyceps sinensis harbors 44 group I introns and six group II introns, accounting for 68.5% of its mt genome nucleotides. Here, 12 out of 44 group I introns and only one out of six group II introns are located in the cytochrome c oxidase subunit 1 (cox1) gene , an acknowledged insertion hotspot for mt group I introns .
Fungi and Placozoa have been proposed as possible donors for group I introns among sponges [43, 47, 65]. However, these findings await corroboration with a broader and more comprehensive taxon set. Intron/HEG phylogenetic analyses and group I/II intron secondary structures are the basis for different scenarios on the origin of introns within sponges [43, 47, 82]. The presence of independent horizontal gene transfers (HGT) for introns is supported by their haphazard distribution over phylogenetically distant sponge groups [21, 43, 82]. Vertical gene transfer (VGT) of introns is assumed among closely related taxa, but never confirmed due to the lack of comprehensive taxon sampling .
To gain new insights into the evolution of sponge introns we required an intron-rich taxonomic group. Based on earlier studies on sponge introns, the order Tetractinellida represents an obvious target. Other lines of evidence support this choice, such as unsuccessful attempts to amplify cox1 in this group with standard protocols [10, 72, 81], potentially due to introns in the relevant primer regions . Consequently, this study focuses on tetractinellid cox1 mitochondrial data to broaden our knowledge on mt intron evolution in this early-branching metazoan phylum.
The data from this “intron-hotspot taxon” presented here constitutes the most representative dataset to target specific questions pivotal to understand intron structure and distribution including activity and mobility. Importance of HGT or VGT or a combination of both will be addressed. Additionally, current hypotheses on the origin of sponge mitochondrial introns will be discussed by comparing intron data across other phyla.
Mitochondrial intron diversity and characteristics in tetractinellid sponges
We discovered more introns in the Spirophorina at positions 714, 723 and 870 (Fig. 2); no intron at position 387 was found. As an example, intron 714 sequences were generated for five more Cinachyrella sp. 2 taxa; four from the Indian Ocean (Kenya, Myanmar) and one from the southwest-Pacific (Indonesia). Cinachyrella species are previously known to have only one intron insertion at a time (either 714 or 723). However, our study reveals that both introns 714 and 723 can occur together in cox1, e.g., in Cinachyrella sp. 2 from marine lakes (RMNH POR11161) and mangroves (RMNH POR11187). Intron 723 was sequenced from 11 different Cinachyrella species, and it is particularly present in the Cinachyrella alloclada complex. In total this study contains 42 sequences of Cinachyrella alloclada (intron 723) from the western Atlantic, the Caribbean Sea and the Gulf of Mexico. We added six additional intron 723 sequences including C. cf. anomala, C. cf. providentiae, C. porosa (all Indonesia), C. sp. 3 (Red Sea), C. sp. 4 (Morocco) and C. sp. 5 (Taiwan). The resulting Cinachyrella dataset covers subtropical-tropical areas from 1 to 90 m depth.
For the first time we discovered intron 723 in the Scleritodermidae (Microscleroderma, Aciculites, Setidium and Scleritoderma). Intron 870 was found in Tetilla quirimure from Brazil and Microscleroderma herdmani from the Indian Ocean (Mauritius), and the Pacific (Philippines and Hawaii). Huchon et al.  located intron 723 in combination with intron 870 in two families (Axinellidae and Agelasidae), while our study reveals this combination in three scleritodermid genera (Microscleroderma, Aciculites, Setidium).
Comparative intron and exon phylogenies of Tetractinellida
Phylogenetic reconstructions of the cox1 exon and the intron revealed a patchy distribution of intron insertions among the Scleritodermidae and Tetillidae and different levels of congruence among intron and exon phylogenies.
Intron + LAGLIDADG Phylogeny
Interestingly, Agelas oroides and Cymbaxinella verrucosa intron sequences grouped within Spirophorina in a highly supported sister group to Cinachyrella introns. The intron + LAGLIDADG phylogenies for 870 and 714 were congruent with the exon phylogeny for all supported clades (Additional file 1).
Secondary structure analyses of introns 723 and 870
Our current study expands our knowledge on Cinachyrella intron 723  thanks to five additional structures predicted for Cinachyrella porosa (RMNH POR11225), Cinachyrella sp. 4 (PC944), Cinachyrella alloclada 2 (GW3920), Cinachyrella cf. providentiae (RMNH POR11228), and Cinachyrella cf. anomala (JX177887) (Additional file 2). The structures of introns 714 and 723 only have a single-stranded P2 region, whereas intron 870 has a double stranded P2 region (Fig. 6, ). The LAGLIDADG ORF is always located in the loop of the P8 helix (Fig. 6, Additional file 2).
Intron 723 structural differences between the species are in the P6 and P9 regions. In particular, Cinachyrella sp. 4 from Morocco has reduced helices P9.1c and P9.1d compared to all others. Cinachyrella alloclada 2 differs slightly in the P6, P6a, P6b and P6d regions to other Cinachyrella species. We generated for the first time secondary structures of scleritodermid intron 723 in Microscleroderma sp. 2 (USNM 1133739), Setidium sp. 1 (HBOI 14-XI-02-3-008) and Scleritoderma sp. 2 (HBOI 25-X-95-1-010) (Fig. 6). All three species show a high variability in loops and helices within the P9 region. Only a few differences were observed in the P6 region between the species. The main difference between Cinachyrella and Scleritodermidae intron 723 is the absence of the P6d region in the latter (Fig. 6a, b and c).
The secondary structure of intron 870 was reconstructed for Tetilla quirimure (MNRJ 17891) and Microscleroderma herdmani 3 (BMNH 1918.104.22.168) (Fig. 6d and e). Both taxa contain the known core helices and conserved structures of Q, P, S, and R. Tetilla quirimure intron structure is very similar regarding P6 and P9 regions to the one from Tetilla radiata . The intron of Microscleroderma herdmani, in turn, has a reduced P6a helix and a different P5a region compared to that found in Tetilla.
The LAGLIDADG protein phylogeny
LAGLIDADG (intron 870) of Plakina/Tetilla and the Scleritodermidae are closely related to Hexacorallia (Zoantharia) LAGLIDADG sequences, but with considerable genetic distance. The genealogical affinities of intron 714 appear unresolved.
Characterisation and mobility mechanisms of group I intron
The cox1 gene in demosponges has the lowest substitution rate of all mt protein coding genes . In fact, the mitochondrial genomes of the sponge classes Homoscleromorpha and Demospongiae possess features shared with non-metazoan opisthokonts rather than Bilateria such as the presence of intergenic regions, genes of foreign origin, a low substitution rate, selfish elements and introns . Mt introns are also found in plants , fungi , Placozoa , and Hexacorallia [13, 37, 74] all known to have slow rates of evolution. It is assumed that this lower substitution rate slows down the elimination of ribozyme activities within group I introns, therefore HEGs would degenerate slower in most fungi , anthozoans [27, 31] and placozoans . Intron mobility is particularly dependant on secondary structure and therefore mutation pressure, so sponge introns survive in the most conserved mt gene (cox1), and the most conserved regions of this gene (Fig. 8), where their HEG is most likely to degenerate slowly. On the other hand, hexactinellid and particularly calcareous sponges possess an accelerated substitution rate [51, 52] and no known mt introns to this date. This correlation between higher mutation rate and absence of mt intron is shared by Cubozoa , Ceriantharia ; Ctenophora [48, 59], Hydrozoa and Scyphozoa . One exception to this pattern is the apparent lack of introns in Octocorallia, despite their lower substitution rates compared to the intron-bearing Hexacorallia [37, 74]. This might be due to the presence of a unique MutS gene, which encodes a DNA mismatch repair machinery , which prevents intron insertions. The mt DNA mismatch repair machinery in sponges remains unknown.
Sponge group I introns consist of complex catalytic ribozymes (RNAs) that fold into a conserved three-dimensional core structure of ten helices. Within this structure, the sponge HEGs are found to be always located within the loop region of the catalytic domain (helix P8, Fig. 6) as in Hexacorallia . HEGs and their intron partners are thought to move either independently from each other  or as a single unit . Whether those HEGs are actively expressed or not often depends on their functionality. The functional expression of HEG group I introns and the resulting gains and/or losses are considered as a cyclical process of different stages . Emblem et al.  applied this into an evolutionary model for a group I intron in sea anemones and reported five stages: 1) Intron with HEG expressed and fused in frame with the upstream host gene exon; 2) Intron with expressed free-standing HEG; 3) Intron with shortened/degenerated HEG; 4) Intron without a conserved HEG and 5) Exon cox1 without intron. Until now, only a few insights into this evolutionary model were given for sponges. Different stages are observed for intron 723 and intron 870 in different sponge species . However, no detailed information has been provided yet on the potential start and stop codons, which are crucial diagnostic features for their categorisation. The potential start and stop codons, observed in all group I introns (Fig. 2), in addition to the predicted secondary structures (Fig. 6, Additional file 2), provide insights into the respective evolutionary stages of all sponge group I introns. In detail, we classified intron 387 of Stupenda singularis in stage 1. Intron 714 of all Cinachyrella sp. 2 appear in stage 1, and in stage 4 for Plakinastrella sp. due to several start and stop codons and no HEG. Intron 723 is found to be in stage 1 among all Cinachyrella species except C. sp. 2 (see below), which is in concordance with the already published data . Intron 723 in Microscleroderma sp. 1 & 2 and Scleritoderma sp. 1 & 2 are also found to be in stage 1. A study comparing the length of DNA and RNA in combination with RT-PCR on a Cinachyrella intron 723 from Taiwan (probably Cinachyrella sp. 5) suggests that it can self-splice in vivo or in vitro . It confirms that this particular stage 1 intron 723 is active. We observe intron 723 also in stages 3 and 4 in Aplysinella rhax, Microscleroderma sp. 3, Cinachyrella sp. 2, Setidium sp.1 (degenerated HEG) and Aciculites sp. 1 (short sequence and no HEG) respectively, which rebuts the suggested recent infection of intron 723 in sponges . Intron 870 was at stage 1 for Tetilla radiata, Plakina crypta and Plakina trilopha  and now shown for Microscleroderma herdmani 1–3, Tetilla quirimure and Setidium sp. 1. Interestingly, both stage 3–4 intron 870 (A. polypoides, A. oroides) previously described  co-occur with stage 1 intron 723. Also, the only stage 4 intron 714 (Plakinastrella sp.) co-occurs with a stage 1 intron 723. Similarly, all of the stage 3–4 intron 723 (Microscleroderma sp. 3, Cinachyrella sp. 2, Aciculites sp. 1, Setidium sp. 1) co-occur with stage 1 introns (either 714 or 870). Overall, two stage 1 introns never co-occur, one of the two is always degenerating. We can therefore hypothesize that the presence of two group I introns is unstable or that maybe the degeneration of one somehow enables the insertion of a different intron. More double-intron-bearing cox1 sequences are needed to study this further. Moreover, we noted that although Scleractinia (Hexacorallia) possesses intron 723 or 888, no evidence of double-intron cox1 sequences in this group is given, which applies for Cnidaria in general. Since double-intron cox1 sequences are also absent in Placozoa, sponges (e.g., demosponges and homoscleromorphs) are to date the only metazoans with double-intron cox1 sequences.
HGT versus VGT of group I introns
The sporadic detections and patchy distributions of group I introns not only among sponges, but also among other Metazoa in e.g., scleractinian corals [27, 31], plants  and fungi  are the main arguments for HGT.
Similarities in intron secondary structures of distantly related sponges are further evidence for HGT . Hence, independent insertion events in Tetillidae, Axinellidae and Agelasida were proposed for intron 723 . This is confirmed by secondary structure differences we observed in closely related families (Tetillidae and Scleritodermidae) (Fig. 6, Additional file 2). Additional loops (P9.1e,f), reduced stems (e.g., P9.1d) and the absence of the P6d region in Sceritodermidae (Fig. 6) result in a higher structure similarity to e.g., Axinella polypoides , rather than to other Tetillidae structures (; Additional file 2, ), which confirms independent insertions of intron 723 in Scleritodermidae and Tetillidae. No major structural differences of intron 723 in the P9 and P6 regions were observed within different species of Cinachyrella (Additional file 2) except for Cinachyrella sp. 4 from Morocco, which showed reduced P9.1c and P9.1d helices. A few minor differences were also noted between Cinachyrella and L. levantinensis structures (Fig. 2 in ): the latter had an additional loop in P5a, a reduced P9.1d and a loop at the end of P6d. Interestingly, the latter two features are also observed in C. sp. 4 (Additional file 2), which could be explained by their common origin resulting from a HGT (Fig. 9). The relative similarity of intron 723 between Cinachyrella and Levantiniella is a strong argument in favor of a single insertion event in this clade, which therefore implies at least two losses of intron 723 to account for the two major Cinachyrella/Paratetilla/Amphitethya clades without any intron (Fig. 9). These would be the first reported cases of mt intron secondary loss in sponges.
For intron 870 no structure differences were observed between Tetilla quirimure (MNRJ 17891) (Fig. 6) and Tetilla radiata (HM032742) . Remarkably, Tetilla japonica (JX177901), which is sister to Tetilla radiata (with a strongly supported node) does not posses intron 870. We therefore assume that Tetilla japonica secondarily lost intron 870, which would represent another case of mt intron loss in sponges. The structure of intron 870 in Microscleroderma herdmani 3 displays a reduced P6a and an additional P5d region (Fig. 6d) compared to Tetilla radiata / quirimure, which suggests an independent insertion of intron 870 as the most plausible explanation. This is further corroborated by the distant phylogenetic relationship between Tetilla and Scleritodermidae (Fig. 4) and the LAGLIDADG phylogeny (Fig. 7).
Until now VGT was only assumed within sponges , but awaited proof with a wider sampling. For the first time our study on 63 Cinachyrella sequences provides conclusive evidence that introns were vertically transmitted due to 1) mostly congruent cox1 versus intron phylogenies and 2) similarity of secondary structures among closely related species. Introns 714, 723 and 870 have all undergone VGT, but this is especially apparent for intron 723 for which we have the largest sampling (Figs. 5 and 9). VGT for group I introns are also known e.g., from hexacorals (nad5-717 intron, ), but is often difficult to ascertain due to the patchy distribution of introns. To conclude, our results demonstrate that introns 714, 723 and 870 undergo VGT, HGT and secondary loss events, and that both VGT and HGT can occur within one genus (e.g., Cinachyrella) (Fig. 9).
Origin of group I introns
The origins of group I introns has been debated for many eukaryotic organisms (e.g., ) including sponges . Fungi are proposed as the primary donor of mt group I introns not only in plants (e.g., ), but also in cnidarians  and sponges [47, 65, 82]. Placozoa have also been suggested as possible donors in sponges, but only for intron 387 in one species . Sponge-fungal associations, pivotal for such HGT, are well-known for sponges (e.g., [40, 71]). However, only little is known about the specific fungal lineages associated with intron-bearing sponge taxa. For Cinachyrella, however, deep sequencing analysis recently identified Ascomycota as a dominant fungal phylum (Cinachyrella cf. australiensis and Cinachyrella sp. from the China Sea ). This is corroborated by data for Cinachyrella alloclada from the Caribbean that showed that the cosmopolitan Phoma sp. (Ascomycota) is the dominant sponge-associated fungus, while nine more ascomycete species were found . Indeed, in the reconstructed LAGLIDADG protein phylogeny (Fig. 7) all intron 723 sequences of Cinachyrella and other sponge taxa display a close relationship to ascomycete intron sequences, but also Viridiplantae (Chlorophyta and Streptophyta). The latter is not surprising, as intron of chlorophytes and Viridiplantae similarly have their origin from Ascomycota [53, 87]. The huge assemblage of group I introns described in fungi increases the chance of a HGT event. When metazoans and plants host one or two group I introns in their cox1, fungus like Ophiocordyceps sinensis contains 21 group I introns within its cox1 region alone .
The non-bilaterian LAGLIDADG-protein sequence dataset (Fig. 7) identifies Scleractinia (stony corals) and Zoantharia (zoanthids) sequences as the only sequences respectively homologous to the sponge introns 723 and 870. Although this is not very well supported, our results show a sister relationship between LAGLIDADG in Scleritodermidae and Zoantharia, which suggests they may have contaminated each other (the direction of the HGT is unclear at this point); alternatively they were contaminated by the same donor, un-sequenced as of today. Because the Scleractinia intron 723 LAGLIDADG sequences are nested within the sponge sequences (Fig. 7), Fukami et al.  suggested two alternative scenarios: 1) Scleractinia and sponges have a similar fungi donor which independently transferred intron 723 in each group or 2) HGT events from each other (sponges to coral, or vice versa). However, although intron 723 has a patchy distribution in the Scleractinia , the Scleractinia LAGLIDADG sequences form a well supported clade (Fig. 7), which suggests that the origin and therefore the donor must have been the same. Different sponges can be excluded as donors, because otherwise the Scleractinia sequences would be partly mixed with the sponge sequences. Also, the possibility of one single sponge donor is unlikely, because there is no sponge species living in close contact with all these Scleractinia species from the Indo-Pacific and the Atlantic. Thus, we are in favor of a donor, most probably a fungus, which transferred intron 723 to different Scleractinia, while similar donors transferred intron 723 to different sponges. These donors can probably also act as vectors, thereby enabling HGT between Cinachyrella species, as shown above. Intron 387 of Stupenda singularis is the only sponge LAGLIDADG sequence apparently unrelated to any coral LAGLIDADG in the data set, but is closely related to LAGLIDADG found in fungi and Marchantiophyta (liverworts). Liverworts are thought to have received their introns from fungi , and the close relationship of the Stupenda singularis LAGLIDADG 387 likewise suggests a fungal origin (see also ).
Unfortunately, the origin of sponge intron 714 remains unresolved, since no supported relationship to any of the included taxonomic groups is given. Therefore, we cannot exclude the possibility that group I introns in sponges may originate from an undiscovered and/or un-sequenced sponge-associated symbionts, e.g., fungi, Archaea, Bacteria or dinoflagellates, since all are known to posses group I introns. In particular sponge bacterial symbionts, which can contribute to over 50% of the sponge biomass [39, 69], may play an essential role as potential intron donors. However, according to our results (Fig. 7) it is unlikely that bacteria, archaea or dinoflagellates are donors, because of the absence of a homologous LAGLIDADG motif. Blastx of intron 714 specified fungi as best hits, therefore fungi remains a good donor candidate for this intron. According to the diversity of habitats of intron-bearing sponges, from shallow water (in reefs, marine lakes and mangrove) to the deep sea (Additional file 3), we can hypothesize that the putative intron donor may also be ubiquitous, and present in all these different environments.
Implications for DNA barcoding
Intron insertion in highly conserved cox1 regions decreases the possibility of intron elimination, because the removal must be specific in order to avoid any disruption of the protein function. The most widespread intron 723 is located at the most conserved site in the cox1 gene (Fig. 8), suggesting this position as an “intron hotspot”. In addition to the high number of intron 723 in Tetillidae, we discovered several more intron 714 (Cinachyrella) and intron 870 (Scleritodermidae, Tetilla), both in conserved cox1 regions. Intron presence in conserved cox1 gene regions has major consequences for other fields of science such as molecular taxonomy. In order to gain a better understanding of locations and conservation of the currently recommended barcoding primers [22, 24, 91], we plotted the 5′ site of each primer on the cox1 conservation profile line (Fig. 8). Interestingly, for the standard barcoding fragment and the I3M11 extension our analysis shows that all previously applied sponge barcoding primers are located in comparatively less conserved regions (Fig. 8). Moreover, our results indicate that group I intron 723 and group II intron 960 are in close proximity to barcoding reverse primer sites (HCO2198 and diplo-cox1) or interrupt the priming regions (intron 723), which corroborates earlier findings from Szitenberg (2010). These findings may partly explain the low (~25% mean) amplification success reported for barcoding museum samples using standard barcoding primers . We therefore recommend for future sponge barcoding studies to test the reverse COX1-R1 primer, which is more distant to the intron insertions, instead of the HCO2198 primer (Fig. 8). The COX1-R1 primer, originally designed to amplify the cox1 of Tetillidae , has been shown to successfully amplify cox1 in Poecilosclerida , Agelasida and Axinellida , Chondrosiida and Dictyoceratida , some Astrophorina (P. Cárdenas, unpublished results) and Spirophorina (this study).
This study provides novel insights into the taxonomic distribution, diversity and mobility of mitochondrial group I introns in sponges, and validates the subclass Spirophorina (Tetractinellida), as an intron hotspot in sponges, notably by increasing the number of Tetillidae introns known by a factor of 5. We wonder whether this could be linked to a lower mt mutation rate in the Spirophorina with respect to other sponges, as suggested for some intron hotspot fungi groups . We show that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates a potentially active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. Consequently, such a high level of HGT in combination with the relative low variation in case of VGT (e.g., intron 723, Fig. 9), rejects any alternative use of mt introns as phylogeographic markers. Since the majority of sponge introns encode a HEG in frame with the 5′ exon, activity of those introns is assumed. We further demonstrate that introns are not restricted to shallow water sponge species, but also occur in species from deeper (~500 m) habitats and extreme environments (mangroves and marine lakes). Conservation profile analysis reveals that all group I and possibly also group II intron insertions in sponges are located within the most conserved regions of their host protein, which may partly explain why they persist in their host genes. At the same time, we show that the currently used sponge barcoding primers are usually located in less conserved regions compared to the introns, but can also overlay intron insertion sites. Therefore, we recommend applying different primers (in particular reverse primers) when standard barcoding primers fail to amplify the cox1 gene. Finally, our study enhances the support for a fungal origin for the majority of introns in sponges.
Sampling and identification of specimens
Cinachyrella samples were collected in Florida (U.S.A.) by snorkeling in the seagrass meadows adjacent to the Mote Marine Laboratory/Tropical Research Laboratory (Summerland Key, Florida U.S.A.) and by scuba-diving on the Broward County reef located off Fort Lauderdale (26° 10.498, −80° 05.632). More Cinachyrella spp. were collected in Indonesia by diving on reefs and snorkeling in mangroves and marine lakes in West Papua and East Kalimantan, Indonesia. The remaining material was obtained through collaborators or sampled in several museum collections (Additional file 3). Because of ambiguous sequences or missing data, some Cinachyrella specimens from Szitenberg et al.  were successfully re-sequenced (JX177885, JX177886, JX177887 and JX177913). Taxonomic identification to genus and species level was performed by the authors and follows the findings of Carella et al.  on Tetillidae. The species Craniella quirimure from Brazil was re-assigned to the genus Tetilla based on the absence of a clear double-layered cortex. In some cases identification of species was adopted from collections and earlier publications. Numbers were added for lineages of species that could not be recovered as monophyletic and await revision (e.g., C. alloclada 1–3). A detailed list of species origin including collector, voucher numbers and accession numbers, location and depth are provided in Additional file 3. A Cinachyrella cox1 sequence (including a group I intron) from Taiwan was manually copied from Hsiao . This species was first identified as Cinachyrella australiensis and is identified as Cinachyrella sp. 5 based on our cox1 CDS phylogeny. One complete cox1 sequence of Microscleroderma sp. (USNM 1133739) with an intron was kindly provided by D. V. Lavrov (Department of Ecology, Evolution, and Organismal Biology, Iowa State University, USA). The higher level demosponge classification follows Morrow & Cárdenas .
Genomic DNA was isolated from the choanosome of the sponge tissue by using the NucleoSpin (Machery-Nagel) or the DNeasy (Qiagen) Blood and Tissue Kit according to the manufacturer’s protocol. An additional centrifugation step was added before transferring the lysate to the Spin Column in order to avoid any clogging of the membrane, caused by sponge spicules. Quantification of the isolated genomic DNA was performed using a NanoDrop 1000 Spectrophotometer (Thermo Scientific).
Amplification of the partial cox1 was performed by using different primers and PCR conditions. Detailed information of primers used for each sample is provided in Additional file 4. For most Tetillidae the cox1 fragment was amplified using the primers LCO1490  and COX1-R1  and for most Scleritodermidae we used the primers diplo-cox1-f1 and diplo-cox1-r1 . For both primer pairs the PCR settings were: 94 °C, 5 min; (94 °C, 1 min; 50–52 °C, 1:30 min; 72 °C, 1:30 min) × 40 cycles; 72 °C, 10 min. Amplified fragments were visually checked for introns by length on a 1.5% agarose gel. For the majority of the Cinachyrella samples with introns, we observed an additional non-specific band at position ~600 bp of bacteria and fungi cox1 fragments. Separation of double bands and PCR clean-up was performed using a modified freeze-squeeze method  in which 20 μl of the PCR product were cut from the gel and stored at −80 °C for one hour, followed by a 40 min centrifugation step at 14,000 rpm. The supernatant (6 μl) was used for cycle sequencing with different and multiple sequencing primers (Additional file 4) together with BigDye Terminator v3.1 (Applied Biosystems, Forster City, CA, USA) chemicals and sequenced by an ABI 3730 Genetic Analyzer at the Sequencing Service of the Department of Biology (LMU München), or by Macrogen (South Korea).
Positions and secondary structures of group I introns within Tetractinellida
Insertion sites for each intron were ascertained in an alignment including other intron-bearing sequences [21, 43, 82]. Intron specific positions were defined according to the cox1 sequence of the sponge Amphimedon queenslandica following Szitenberg et al. . Blast hits and sequence similarity to already published group I intron insertions were used to distinguish between different insertion sites and group I and group II introns. An overview of the different group I (intron 387, 714, 723, 870) insertion sites as well as group II (intron 966 & 1141) insertion sites is given in Fig. 2. Identification of the HEG for each ORF was conducted by blastp against NCBI Genbank . The class of group I introns (IA, IB, IC, ID or IE) was obtained using the RNAweasel Website http://megasun.bch.umontreal.ca/RNAweasel/ . Initiation and stop codons of the HEG ORFs were located using the ORF finder as implemented in Geneious v.8.1.8 (www.geneious.com) with the following settings: translation Table 4 (Mold and Protozoan mitochondrial) with start codons ATG, GTG, TTG and ATT , minimum size 100 bp, including interior ORFs. Although considered as potential start codon in sponge group I introns [47, 65], there is actually no evidence that TTA is used in sponges as start codon; it has only been found so far in Trypanosoma . We therefore excluded the TTA start codon in our searches and also revisited the ORFs of previously reported sponge group I introns.
In order to predict the secondary structures of group I introns, we manually converted the given secondary core structures into a dot-bracket notation including pseudoknot informations in square brackets. As secondary structure references, we used Cinachyrella alloclada (HM032738) for intron 723 and Tetilla radiata (HM032742) for intron 870 . In order to ensure the right structure annotation for short variable (mainly P6 and P9) domains, we used Mfold http://unafold.rna.albany.edu/  under the general settings, presupposing the exclusion of additional pseudoknots, which cannot be predicted by this program. Those Mfold structures were then manually converted into a dot-bracket notation and implemented to the already established core structure sequence. SeaView v4  was used to align the sequences to their structure annotation. The LAGLIDADG regions were removed from the sequences for further analysis. The rest of the intron sequence together with its structure information, was converted to a ct-format using the Perl-script (2ct.zip) of Voigt et al.  (available at http://www.palaeontologie.geo.lmu.de/molpal/RRNA/index.htm). All secondary structures were visualized in RNAViz 2.0.3 http://rnaviz.sourceforge.net/ . Helix names follow Szitenberg et al. .
Tetractinellida phylogenies predicted by cox1 CDS
Sequence alignments and outgroup choice
Newly generated sequences as well as additional GenBank sequences were manually aligned to the datasets from Szitenberg et al. [81, 82]. Aligned sequences were subsequently controlled for discrepancies and corrected by eye. Two Astrophorina species (Geodia barretti and Alectona millari) were used as outgroups in Tetillidae phylogenetic analyses. Astrophorina has been established as the sister clade of Spirophorina in previous studies [6, 57]. For the analysis of the tetractinellid phylogeny we chose Halichondria melanodocia and Axinella corrugata, which were already successfully used as outgroups in previous studies on the molecular phylogeny of the Tetractinellida (e.g., ).
The final cox1 alignment (excluding intron(s)) of the Tetillidae phylogeny comprised 133 sequences (including the two outgroups), of which 76 were newly generated from this study. The alignment was 1177 bp long, of which 829 bp were constant, 62 bp were parsimony uninformative and 286 bp were parsimony informative. The final cox1 alignment of the Tetractinellida phylogeny constituted 82 sequences (including the two outgroups) of which 33 were newly generated from this study. In total the alignment comprised 1118 bp, of which 642 bp were invariant, 77 bp parsimony uninformative and 399 bp were parsimony informative.
Phylogenetic tree reconstructions for both analyses were performed on a parallel version of MrBayes v3.2.4  and RAxML v8.0.26  on a Linux cluster. Bayesian analyses were conducted under the most generalized GTR + G + I evolutionary model, as resulted from jModelTest v.2.1.7 . Analyses were run in two concurrent runs of four Metropolis-coupled Markov-chains (MCMC) for 100,000,000 generations and stopped when the average standard deviation of split frequencies reached below 0.01. The first 25% (burn-in) of the sampled trees were removed for further analysis. For both datasets, Maximum Likelihood (ML) and bootstrap analyses (1,000 replicates) under the GTR + G model as resulted from jModelTest v.2.1.7  were performed. Tree topologies from Bayesian and ML analyses were compared and visualized using Figtree v1.4.2 http://tree.bio.ed.ac.uk/software/figtree/.
Phylogenetic inference based on intron + LAGLIDADG sequences
In order to test for vertical transmission of group I introns (including both LAGLIDADG and the non-coding regions) in the genus Cinachyrella, we conducted phylogenetic analyses on separate datasets respectively including all sponge introns 723, 714 and 870. For the analysis of intron 723, we included 74 sequences of which 63 belong to the genus Cinachyrella. One taxon Aciculites sp. 1 (HBOI 26-IX-11-2-002), was excluded from this analysis, as no putative HEG were detected in the intron. The final intron 723 alignment was 1167 bp long, of which 488 bp were constant, 307 bp parsimony uninformative and 372 bp parsimony informative. The final intron 714 dataset included 13 sequences and was 946 bp long, of which 891 bp were constant, 49 bp were parsimony uninformative and 6 bp were parsimony informative. As an outgroup for both analysis we used the introns of Plakinastrella sp. (NC 010217), a species that belongs to a different sponge class (Fig. 1). The final alignment of intron 870 contained 12 taxa and was 974 bp long, of which 615 pb were constant, 46 bp were parsimony uninformative and 313 bp parsimony informative. Plakina trilopha (HQ269356) and P. crypta (HQ269352) which belong to a different sponge class (Fig. 1) were used as outgroups. Phylogenetic tree reconstructions were performed as described above for the cox1 exon phylogeny.
In order to test whether the incongruencies between the exon and the intron/HE phylogeny were significant, we performed a series of Shimodaira-Hasegawa (SH) tests  as implemented in RAxML  on the exon tree against ML topologies constrained towards the intron tree topology. Constraints were inferred with Mesquite v.3.10 .
Phylogenetic reconstructions based on LAGLIDADG protein sequences of group I introns
In order to investigate the evolutionary origins of the putative LAGLIDADG encoding introns (387, 714, 723 and 870) in sponges the newly generated sequences were added to the LAGLIDADG dataset by Huchon et al. . Additionally, we included 12 fungal and two Marchantiophyta LAGLIDADG sequences resulting from Blastp hits of the sponge LAGLIDADG for intron 387 (Table 2, ). Subsequently, MAFFT v.7  under the L-INS-I algorithm was used to generate the protein alignment. The resulting alignment contains sequences of fungi, plants, cnidarians and sponges. Here, 291 amino-acids (aa) out of 1278 aa were parsimony-uninformative variable characters, 729 aa were constant and 708 parsimony-informative. As a result, we manually corrected the LAGLIDADG alignment. Parts with more than approximately 50% of missing data were removed manually using the custom site set selection tool in SeaView. The final alignment was 317 amino-acids long, of which one character was constant and two variable characters were parsimony-uninformative. The rest of the 314 characters were phylogenetically informative. The maximum likelihood (ML) analysis was performed using RAxML v8.0.26  on a Linux cluster with 1,000 bootstrap repeats. Using ProtTest 3.4  the best evolutionary model was found to be VT + I + Gamma + F. However, for the RAxML analysis we excluded the invariant parameter (I) from the model, as it is not recommended to use both gamma (G) and invariant (I) parameters among site-rate variations according to the RAxML manual. No root for the tree was specified, as it was not needed for our purpose.
Compilation of the conservation profile
A conservation profile was calculated from a cox1 protein alignment dataset compiled from the demosponge sequences from , complemented by Homoscleromorpha sequences from . The final protein alignment consisted of 58 sequences and 556 characters. The conservation profile was made following Swithers et al.  using the same perl script (made available in the supplementary material of Swithers et al. ) but with a slightly modification to allow ‘X’ characters in the alignment and calculation. The 5′ position of the common barcoding markers as well as all sponge intron insertion positions were plotted on the profile line.
We greatly acknowledge Dennis V. Lavrov (Iowa State University, USA) for sharing the Microscleroderma sp. 2 cox1 intron sequence and Oliver Voigt (Dept. of Earth- & Environmental Sciences, LMU Munich, Germany) for providing scripts and support for secondary structure analysis. We greatly thank Eduardo Hajdu and Cristina Castello-Branco (Universidade Federal Do Rio De Janeiro, Brasil), Carsten Lüter (Museum für Naturkunde Berlin, Germany), Helmut Lehnert (Zoologische Staatssammlung München, Germany), Sigal Shefer and Yaniv Aluma (Tel Aviv University), Sadie Mills and Kareen Schnabel (National Institute of Water and Atmospheric Research, Wellington, New Zealand), Sara Griffiths (University of Manchester, UK), Cécile Debitus (IRD, Institut de Recherche pour le Développement, Marseille, France)France), Christopher J. Freeman (Smithsonian Marine Station, Fort Pierce, USA), Belkassem El Amraoui (University Ibn Zohr, Taroudant, Morocco) , Nadia Santodomingo (Natural History Museum, London), Nicole J. De Voogd (Naturalis Biodiversity Center), Yosephine Tuti (Indonesian National Institute of Sciences) and the governments of Ecuador, Commonwealth of the Bahamas, Panama, and Martinique (France) for sampling, sharing material and help in the collections. We thank Kyle Roebuck, Nidhi Vijayan and Marissa Wickes with help in sample preparation and shipping from Florida. We thank the Systematic Biology lab (Dept. of Organismal Biology, Uppsala University, Sweden) and Gabrielle Büttner and Simone Schätzle (Dept. of Earth- & Environmental Sciences, LMU Munich, Germany) for sequencing assistance. Finally, we want to thank the three anonymous reviewers for their helpful comments and suggestions.
This work was funded by the German Science Foundation (DFG) (grant number DFG ER 611/3-1, DFG Wo896/15-1); LMUMentoring Program; HELGE AX:Son JOHNSON STIFTELSE (Sweden), Inez Johanssons HT2012 (Uppsala University, Sweden) and Netherlands Organisation for Scientific Research (NWO) Veni#863.14.020.
Availability of data and material
New sequences from this study are stored at the European Nucleotide Archive (ENA) under the accession numbers LT628277-LT628366. All alignments generated during the current study are freely available at OpenDataLMU (doi:10.5282/ubm/data.98).
AS and PC conceived and designed the study. AS carried out PCR, sequencing, phylogenetic analyses and predicted the secondary structures. AS, LEB, MK, PC, and SAP identified the specimens. All authors contributed with samples and reagents. AS drafted the manuscript and figures. PC, JVL, SAP, GW and DE assisted in revising the MS. All authors approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study did not include protected or endangered species and requires no ethical approval. Collection permits in Indonesia were provided by the Indonesian Institute of Sciences (LIPI) and the Indonesian State Ministry of Research and Technology (RISTEK) to LEB: 0094/frp/sm/v/2009, 1810/FRP/SM/VIII/2008, 098/SIP/FRP/SM/V/2011). Collecting permits in Florida were provided by the Florida Fish and Wildlife Conservation Commission by a valid fishing license.
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- Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.View ArticlePubMedPubMed CentralGoogle Scholar
- Belfort M, Perlman PS. Mechanisms of intron mobility. J Biol Chem. 1995;270:30237–40.View ArticlePubMedGoogle Scholar
- Belinky F, Szitenberg A, Goldfarb I, Feldstein T, Wörheide G, Ilan M, Huchon D. ALG11 - A new variable DNA marker for sponge phylogeny: Comparison of phylogenetic performances with the 18S rDNA and the COI gene. Mol Phylogenet Evol. 2012;63:702–13.View ArticlePubMedGoogle Scholar
- Bilewitch JP, Degnan SM. A unique horizontal gene transfer event has provided the octocoral mitochondrial genome with an active mismatch repair gene that has potential for an unusual self-contained function. BMC Evol Biol. 2011;11:228.View ArticlePubMedPubMed CentralGoogle Scholar
- Bolaños J, Fernando De León L, Ochoa E, Darias J, Raja HA, Shearer CA, Miller AN, Vanderheyden P, Porras-Alfaro A, Caballero-George C. Phylogenetic diversity of sponge-associated fungi from the Caribbean and the Pacific of Panama and their in vitro effect on angiotensin and endothelin receptors. Mar Biotechnol. 2015;17:533–46.View ArticlePubMedGoogle Scholar
- Borchiellini C, Chombard C, Manuel M, Alivon E, Vacelet J, Boury-Esnault N. Molecular phylogeny of Demospongiae: implications for classification and scenarios of character evolution. Mol Phylogenet Evol. 2004;32:823–37.View ArticlePubMedGoogle Scholar
- Burger G, Forget L, Zhu Y, Gray MW, Lang BF. Unique mitochondrial genome architecture in unicellular relatives of animals. Proc Natl Acad Sci U S A. 2003;100:892–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Burger G, Saint-Louis D, Gray MW, Lang BF. Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae. Plant Cell. 1999;11:1675–94.View ArticlePubMedPubMed CentralGoogle Scholar
- Burger G, Yan Y, Javadi P, Lang BF. Group I-intron trans-splicing and mRNA editing in the mitochondria of placozoan animals. Trends Genet. 2009;25:381–6.View ArticlePubMedGoogle Scholar
- Cárdenas P, Menegola C, Rapp HT, Cristina DM. Morphological description and DNA barcodes of shallow-water Tetractinellida (Porifera: Demospongiae) from Bocas del Toro, Panama, with description of a new species. Zootaxa. 2009;2276:1–39.Google Scholar
- Carella M, Agell G, Cárdenas P, Uriz MJ. Phylogenetic reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) reveals new genera and genetic similarity among morphologically distinct species. PLoS One. 2016;11(8):e0160718.View ArticlePubMedPubMed CentralGoogle Scholar
- Chan H-M. Self-splicing of Group I Intron of the Mitochondrial Genome of the Sponge, Cinachyrella australiensis. Kahosiung: Master Thesis: National Sun Yat-sen University; 2009. http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0819109-130742 Accessed 20 Mar 2016.Google Scholar
- Chen I-P, Tang C-Y, Chiou C-Y, Hsu J-H, Wei NV, Wallace CC, Muir P, Wu H, Chen CA. Comparative analyses of coding and noncoding DNA regions indicate that Acropora (Anthozoa: Scleractina) possesses a similar evolutionary tempo of nuclear vs. mitochondrial genomes as in plants. Mar Biotechnol. 2009;11:141–52.View ArticlePubMedGoogle Scholar
- Cho Y, Qiu YL, Kuhlman P, Palmer JD. Explosive invasion of plant mitochondria by a group I intron. Proc Natl Acad Sci U S A. 1998;95:14244–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics. 2011;27:1164–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9:772.View ArticlePubMedPubMed CentralGoogle Scholar
- Dellaporta SL, Xu A, Sagasser S, Jakob W, Moreno MA, Buss LW, Schierwater B. Mitochondrial genome of Trichoplax adhaerens supports Placozoa as the basal lower metazoan phylum. Proc Natl Acad Sci U S A. 2006;103:8751–6.View ArticlePubMedPubMed CentralGoogle Scholar
- De Rijk P, Wuyts J, De Wachter R. RnaViz 2: an improved representation of RNA secondary structure. Bioinformatics. 2003;19:299–300.View ArticlePubMedGoogle Scholar
- Emblem Å, Karlsen BO, Evertsen J, Johansen SD. Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron. Mol Phylogenet Evol. 2011;61:495–503.View ArticlePubMedGoogle Scholar
- Emblem Å, Okkenhaug S, Weiss ES, Denver DR, Karlsen BO, Moum T, Johansen SD. Sea anemones possess dynamic mitogenome structures. Mol Phylogenet Evol. 2014;75:184–93.View ArticlePubMedGoogle Scholar
- Erpenbeck D, Aryasari R, Hooper JNA, Wörheide G. A Mitochondrial Intron in a Verongid Sponge. J Mol Evol. 2014;80:13–7.View ArticlePubMedGoogle Scholar
- Erpenbeck D, Hooper JNA, Wörheide G. CO1 phylogenies in diploblasts and the “Barcoding of Life”- are we sequencing a suboptimal partition? Mol Ecol Notes. 2006;6:550–3.View ArticleGoogle Scholar
- Férandon C, Moukha S, Callac P, Benedetto J-P, Castroviejo M, Barroso G. The Agaricus bisporus cox1 Gene: The Longest Mitochondrial Gene and the Largest Reservoir of Mitochondrial Group I Introns. PLoS One. 2010;5:e14048.View ArticlePubMedPubMed CentralGoogle Scholar
- Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294–9.PubMedGoogle Scholar
- Fontaine J-M, Rousvoal S, Leblanc C, Kloareg B, Loiseau-de GS. The Mitochondrial LSU rDNA of the Brown Alga Pylaiella littoralis Reveals α- Proteobacterial Features and is Split by Four Group IIB Introns with an Atypical Phylogeny. J Mol Biol. 1995;251:378–89.View ArticlePubMedGoogle Scholar
- Fox TD. Natural variation in the genetic code. Annu Rev Genet. 1987;21:67–91.View ArticlePubMedGoogle Scholar
- Fukami H, Chen CA, Chiou CY, Knowlton N. Novel group I introns encoding a putative homing endonuclease in the mitochondrial cox1 gene of Scleractinian corals. J Mol Evol. 2007;64:591–600.View ArticlePubMedGoogle Scholar
- Galtier N. The intriguing evolutionary dynamics of plant mitochondrial DNA. BMC Biol. 2011;9:1–3.View ArticleGoogle Scholar
- Gazave E, Lapébie P, Renard E, Vacelet J, Rocher C, Ereskovsky AV, Lavrov DV, Borchiellini C. Molecular Phylogeny Restores the Supra-Generic Subdivision of Homoscleromorph Sponges (Porifera, Homoscleromorpha). PLoS One. 2010;5:e14290.View ArticlePubMedPubMed CentralGoogle Scholar
- Goddard MR, Burt A. Recurrent invasion and extinction of a selfish gene. Proc Natl Acad Sci U S A. 1999;96:13880–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Goddard MR, Leigh J, Roger AJ, Pemberton AJ. Invasion and persistence of a selfish gene in the Cnidaria. PLoS One. 2006;1:e3.View ArticlePubMedPubMed CentralGoogle Scholar
- Gouy M, Guindon S, Gascuel O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 2010;27:221–4.View ArticlePubMedGoogle Scholar
- Hafez M, Hausner G, Bonen L. Homing endonucleases: DNA scissors on a mission. Genome. 2012;55:553–69.View ArticlePubMedGoogle Scholar
- Haugen P, Bhattacharya D. The spread of LAGLIDADG homing endonuclease genes in rDNA. Nucleic Acids Res. 2004;32:2049–57.View ArticlePubMedPubMed CentralGoogle Scholar
- Haugen P, Simon DM, Bhattacharya D. The natural history of group I introns. Trends Genet. 2005;21:111–9.View ArticlePubMedGoogle Scholar
- Hausner G. Introns, Mobile Elements, and Plasmids. In: Organelle Genetics. Heidelberg: Springer Berlin Heidelberg; 2012. p. 329–57.Google Scholar
- Hellberg ME. No variation and low synonymous substitution rates in coral mtDNA despite high nuclear variation. BMC Evol Biol. 2006;6:24.View ArticlePubMedPubMed CentralGoogle Scholar
- He L, Liu F, Karuppiah V, Ren Y, Li Z. Comparisons of the fungal and protistan communities among different marine sponge holobionts by pyrosequencing. Microb Ecol. 2014;67:951–61.View ArticlePubMedGoogle Scholar
- Hentschel U, Piel J, Degnan SM, Taylor MW. Genomic insights into the marine sponge microbiome. Nat Rev Microbiol. 2012;10:641–54.View ArticlePubMedGoogle Scholar
- Höller U, Wright AD, Matthee GF, Konig GM, Draeger S, Aust H-J, Schulz B. Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol Res. 2000;104:1354–65.View ArticleGoogle Scholar
- Hsiao S-T. Genomic Analyses of the Complete Mitochondrial DNA Sequence of the Sponge, Cinachyrella australiensis (Demospongiae: Spirophorida). Kahosiung: Master Thesis: National Sun Yat-sen University; 2005. http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0707105-161158. Accessed 20 Mar 2016.
- Huang D, Meier R, Todd PA, Chou LM. Slow mitochondrial COI sequence evolution at the base of the metazoan tree and its implications for DNA barcoding. J Mol Evol. 2008;66:167–74.View ArticlePubMedGoogle Scholar
- Huchon D, Szitenberg A, Shefer S, Ilan M, Feldstein T. Mitochondrial group I and group II introns in the sponge orders Agelasida and Axinellida. BMC Evol Biol. 2015;15:278.View ArticlePubMedPubMed CentralGoogle Scholar
- Jalalzadeh B, Saré IC, Férandon C, Callac P, Farsi M, Savoie J-M, Barroso G. The intraspecific variability of mitochondrial genes of Agaricus bisporus reveals an extensive group I intron mobility combined with low nucleotide substitution rates. Curr Genet. 2015;61:87–102.View ArticlePubMedGoogle Scholar
- Johansen S, Johansen T, Haugli F. Structure and evolution of myxomycete nuclear group I introns: a model for horizontal transfer by intron homing. Curr Genet. 1992;22:297–304.View ArticlePubMedGoogle Scholar
- Katoh K, Standley DM. MAFFT Multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol. 2013;30:772–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Kelly M, Cárdenas P. An unprecedented new genus and family of Tetractinellida (Porifera, Demospongiae) from New Zealand’s Colville Ridge, with a new type of mitochondrial group I intron. Zool J Linn Soc. 2016;177:335–52.View ArticleGoogle Scholar
- Kohn AB, Citarella MR, Kocot KM, Bobkova YV, Halanych KM, Moroz LL. Rapid evolution of the compact and unusual mitochondrial genome in the ctenophore, Pleurobrachia bachei. Mol Phylogenet Evol. 2012;63:203–7.View ArticlePubMedGoogle Scholar
- Lang BF, Laforest MJ, Burger G. Mitochondrial introns: a critical view. Trends Genet. 2007;23:119–25.View ArticlePubMedGoogle Scholar
- Lavrov DV. Mitochondrial Genomes of Two Demosponges Provide Insights into An Early Stage of Animal Evolution. Mol Biol Evol. 2005;22:1231–9.View ArticlePubMedGoogle Scholar
- Lavrov DV, Adamski M, Chevaldonné P, Adamska M. Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges. Curr Biol. 2016;26:86–92.View ArticlePubMedGoogle Scholar
- Lavrov D, Wang X, Kelly M. Reconstructing ordinal relationships in the Demospongiae using mitochondrial genomic data. Mol Phylogenet Evol. 2008;49:111–24.View ArticlePubMedGoogle Scholar
- Lindstrom SC, Pistolic J. Detection of a group I (IE) fungal intron in the green algal genus Urospora (Ulvophyceae). J Phycol. 2005;41:359–65.View ArticleGoogle Scholar
- Li Y, Hu XD, Yang R-H, Hsiang T, Wang K, Liang D-Q, Liang F, Cao D-M, Zhou F, Wen G, et al. Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis. Sci Rep. 2015;5:13892.View ArticlePubMedPubMed CentralGoogle Scholar
- Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary analyses. 2016. Version 3.10 http://mesquiteproject.org.Google Scholar
- Morrow C, Cárdenas P. Proposal for a revised classification of the Demospongiae (Porifera). Front. Zool. 2015;12:1–27.Google Scholar
- Nichols S. An evaluation of support for order-level monophyly and interrelationships within the class Demospongiae using partial data from the large subunit rDNA and cytochrome oxidase subunit I. Mol Phylogenet Evol. 2005;34:81–96.View ArticlePubMedGoogle Scholar
- Ohta E, Oda K, Yamato K, Nakamura Y, Takemura M, Nozato N, Akashi K, Ohyama K, Michel F. Group I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts. Nucleic Acids Res. 1993;21:1297–305.View ArticlePubMedPubMed CentralGoogle Scholar
- Osigus HJ, Eitel M, Bernt M, Donath A, Schierwater B. Mitogenomics at the base of Metazoa. Mol Phylogenet Evol. 2013;69:339–51.View ArticlePubMedGoogle Scholar
- Palmer JD, Adams KL, Cho Y, Parkinson CL, Qiu YL, Song K. Dynamic evolution of plant mitochondrial genomes: mobile genes and introns and highly variable mutation rates. Proc Natl Acad Sci U S A. 2000;97:6960–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Redmond NE, Morrow CC, Thacker RW, Diaz MC, Boury-Esnault N, Cárdenas P, Hajdu E, Lobo-Hajdu G, Picton BE, Pomponi SA, et al. Phylogeny and Systematics of Demospongiae in Light of New Small-Subunit Ribosomal DNA (18S) Sequences. Integr Comp Biol. 2013;53:388–415.View ArticlePubMedGoogle Scholar
- Reveillaud J, van Soest R, Derycke S, Picton B, Rigaux A, Vanreusel A. Phylogenetic relationships among NE Atlantic Plocamionida Topsent (1927) (Porifera, Poecilosclerida): Under-estimated diversity in reef ecosystems. PLoS One. 2011;6:e16533.View ArticlePubMedPubMed CentralGoogle Scholar
- Rodríguez-Trelles F, Tarrío R, Ayala FJ. Origins and evolution of spliceosomal introns. Annu Rev Genet. 2006;40:47–76.View ArticlePubMedGoogle Scholar
- Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice across a Large Model Space. Syst Biol. 2012;61:539–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Rot C, Goldfarb I, Ilan M, Huchon D. Putative cross-kingdom horizontal gene transfer in sponge (Porifera) mitochondria. BMC Evol Biol. 2006;6:71.View ArticlePubMedPubMed CentralGoogle Scholar
- Sanchez-Puerta MV, Abbona CC, Zhuo S, Tepe EJ, Bohs L, Olmstead RG, Palmer JD. Multiple recent horizontal transfers of the cox1 intron in Solanaceae and extended co-conversion of flanking exons. BMC Evol Biol. 2011;11:277.View ArticlePubMedPubMed CentralGoogle Scholar
- Sanchez-Puerta MV, Cho Y, Mower JP, Alverson AJ, Palmer JD. Frequent, phylogenetically local horizontal transfer of the cox1 group I Intron in flowering plant mitochondria. Mol Biol Evol. 2008;25:1762–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Santamaria M, Vicario S, Pappadà G, Scioscia G, Scazzocchio C, Saccone C. Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria. BMC Bioinformatics. 2009;10:1–13.View ArticleGoogle Scholar
- Santavy DL, Willenz P, Colwell RR. Phenotypic study of bacteria associated with the caribbean sclerosponge, Ceratoporella nicholsoni. Appl Environ Microbiol. 1990;56:1750–62.PubMedPubMed CentralGoogle Scholar
- Schäfer B. Genetic conservation versus variability in mitochondria: the architecture of the mitochondrial genome in the petite-negative yeast Schizosaccharomyces pombe. Curr Genet. 2003;43:311–26.View ArticlePubMedGoogle Scholar
- Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N, Perez T, Rodrigo A, Schupp PJ, Vacelet J, et al. Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J. 2012;6:564–76.View ArticlePubMedGoogle Scholar
- Schuster A, Erpenbeck D, Pisera A, Hooper J, Bryce M, Fromont J, Wörheide G. Deceptive Desmas: Molecular Phylogenetics Suggests a New Classification and Uncovers Convergent Evolution of Lithistid Demosponges. PLoS One. 2015;10:e116038.View ArticlePubMedPubMed CentralGoogle Scholar
- Sellem CH, Belcour L. Intron open reading frames as mobile elements and evolution of a group I intron. Mol Biol Evol. 1997;14:518–26.View ArticlePubMedGoogle Scholar
- Shearer TL, Van Oppen MJH, Romano SL, Wörheide G. Slow mitochondrial DNA sequence evolution in the Anthozoa (Cnidaria). Mol Ecol. 2002;11:2475–87.View ArticlePubMedGoogle Scholar
- Shimodaira H, Hasegawa M. Multiple comparisons of log-likelihoods with applications to phylogenetic inference. Mol Biol Evol. 1999;16:1114–6.View ArticleGoogle Scholar
- Signorovitch AY, Buss LW, Dellaporta SL. Comparative genomics of large mitochondria in placozoans. PLoS Genet. 2007;3:e13.View ArticlePubMedPubMed CentralGoogle Scholar
- Smith DR, Kayal E, Yanagihara AA, Collins AG, Pirro S, Keeling PJ. First complete mitochondrial genome sequence from a box jellyfish reveals a highly fragmented linear architecture and insights into telomere evolution. Genome Biol Evol. 2012;4:52–8.View ArticlePubMedGoogle Scholar
- Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–3.View ArticlePubMedPubMed CentralGoogle Scholar
- Stampar SN, Maronna MM, Kitahara MV, Reimer JD, Morandini AC. Fast-Evolving Mitochondrial DNA in Ceriantharia: A Reflection of Hexacorallia Paraphyly? PLoS One. 2014;9:e86612.View ArticlePubMedPubMed CentralGoogle Scholar
- Swithers KS, Senejani AG, Fournier GP, Gogarten JP. Conservation of intron and intein insertion sites: implications for life histories of parasitic genetic elements. BMC Evol Biol. 2009;9:303.View ArticlePubMedPubMed CentralGoogle Scholar
- Szitenberg A, Becking LE, Vargas S, Fernandez JCC, Santodomingo N, Wörheide G, Ilan M, Kelly M, Huchon D. Phylogeny of Tetillidae (Porifera, Demospongiae, Spirophorida) based on three molecular markers. Mol Phylogenet Evol. 2013;67:509–19.View ArticlePubMedGoogle Scholar
- Szitenberg A, Rot C, Ilan M, Huchon D. Diversity of sponge mitochondrial introns revealed by cox 1 sequences of Tetillidae. BMC Evol Biol. 2010;10:288.View ArticlePubMedPubMed CentralGoogle Scholar
- Tautz D, Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983;132:14–9.View ArticlePubMedGoogle Scholar
- Vallès Y, Halanych KM, Boore JL. Group II introns break new boundaries: presence in a bilaterian’s genome. PLoS One. 2008;3:e1488.View ArticlePubMedPubMed CentralGoogle Scholar
- Van Oppen MJ, Olsen JL, Stam WT. Evidence for independent acquisition of group I introns in green algae. Mol Biol Evol. 1993;10:1317–26.PubMedGoogle Scholar
- Vargas S, Schuster A, Sacher K, Büttner G, Schätzle S, Läuchli B, Hall K, Hooper JNA, Erpenbeck D, Wörheide G. Barcoding Sponges: An Overview Based on Comprehensive Sampling. PLoS One. 2012;7:e39345.View ArticlePubMedPubMed CentralGoogle Scholar
- Vaughn JC, Mason MT, Sper-Whitis GL, Kuhlman P, Palmer JD. Fungal origin by horizontal transfer of a plant mitochondrial group I intron in the chimeric cox1 gene of Peperomia. J Mol Evol. 1995;41:563–72.View ArticlePubMedGoogle Scholar
- Vicens Q, Cech TR. Atomic level architecture of group I introns revealed. Trends Biochem Sci. 2006;31:41–51.View ArticlePubMedGoogle Scholar
- Voigt O, Erpenbeck D, Wörheide G. Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera. BMC Evol Biol. 2008;8:69.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang X, Lavrov D. Seventeen new complete mtDNA sequences reveal extensive mitochondrial genome evolution within the Demospongiae. PLoS One. 2008;3:e2723.View ArticlePubMedPubMed CentralGoogle Scholar
- Wörheide G, Erpenbeck D, Menke C. The Sponge Barcoding Project: aiding in the identification and description of poriferan taxa. In: Custódio MR, Lôbo-Hajdu G, Hajdu E, Muricy G, editors. Porifera research: biodiversity, innovation and sustainability, vol. 28. Série Livros: Rio de Janeiro; 2007. p. 123–8.Google Scholar
- Yamada T, Tamura K, Aimi T, Songsri P. Self-splicing group I introns in eukaryotic viruses. Nucleic Acids Res. 1994;22:2532–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003;31:3406–15.View ArticlePubMedPubMed CentralGoogle Scholar