Soft sponges with tricky tree: On the phylogeny of dictyoceratid sponges

Keratose (horny) sponges constitute a very difficult group of Porifera in terms of taxonomy due to their paucity of diagnostic morphological features. (Most) keratose sponges possess no mineral skeletal elements, but an arrangement of organic (spongin) fibers, with little taxonomic or phylogenetic information. Molecular phylogenetics have targeted this evolutionary and biochemically important lineage numer-ous times, but the conservative nature of popular markers combined with ambiguous identification of the sponge material has so far prevented any robust phylogeny. In the following study, we provide a phylogenetic hypothesis of the keratose order Dictyoceratida based on nuclear markers of higher resolution potential ( ITS and 28S C-region), and particularly aim for the inclusion of type specimens as reference material. Our results are compared with previously published data of CO1 , 18S , and 28S (D3-D5) data, and indicate the paraphyly of the largest dictyoceratid family, the Thorectidae, due to a sister group relationship of its subfamily Phyllospongiinae with Family Spongiidae. Irciniidae can be recovered as monophyletic. Results on genus level and implications on phylogenetic signals of the most frequently described morphological characters are discussed.

This resulted in a fundamentally revised classification at order level (Morrow & Cárdenas, 2015). However, revisions of most intra-ordinal relationships are still due for revision. A particularly difficult order of sponges is the Dictyoceratida (Subclass Keratosa), which possess a skeleton of organic material (spongin) only and lack mineral skeletal elements (with the exception of Vaceletia, which possesses a hypercalcified secondary limestone skeleton instead of spongin fibers, see Wörheide, 2008). Therefore, these sponges were historically assigned to the "horny" sponges. The spongin skeleton renders specimens of some genera useful as bathing sponges, but at the same time limits the suite of diagnostic features for morphological classification and phylogeny. Morphologically, all dictyoceratids share the presence of this anastomosing spongin fiber skeleton that often make up a significant proportion of the body volume. Fibers develop from multiple points and are organized into primary, secondary, and sometimes tertiary fibers (Cook & Bergquist, 2002e).
So far, molecular studies targeting shallow-level relationships of Dictyoceratida provided insufficient resolution or conflicting data: The first comprehensive molecular approach based on the partial mitochondrial cytochrome c oxidase subunit 1 gene (CO1) and the D3-D5 partition of the nuclear large ribosomal subunit gene (28S) confirmed monophyly of the families Dysideidae and Irciniidae, and confirmed Dysideidae as sister to all other families as well, but failed to resolve Spongiidae and Thorectidae relationships . Likewise, Redmond et al. (2013) andThacker et al. (2013) confirmed the distinct position of Dysideidae, based on the nuclear small ribosomal subunit gene (18S) and full-length 28S, respectively, but could not robustly resolve the relationship of other dictyoceratid taxa either. Undoubtedly, the molecular markers used so far bear insufficient resolution potential to answer all dictyoceratid phylogenetic questions.
Conclusive (molecular) phylogenies must be based on well-identified species. Most dictyoceratid phylogenies, however, suffer from incomplete and ambiguous specimen identification Redmond et al., 2013;Thacker et al., 2013) due to the difficult (morphology-based) taxonomy (see also Cook, 2007).
Type specimens, particularly holotypes, are the only unambiguous reference points for taxonomic delineation, but not frequently used for sponge molecular phylogenetic studies due to difficult accessibility and bad DNA qualities (see review in Erpenbeck, Ekins, et al., 2016). The present study therefore attempts to use type material where possible, or other well-identified specimens such as Systema Porifera reference material. The results of the new dictyoceratid ITS and 28S (C-region) molecular analyses are compared with phylogenies obtained from 18S , CO1, and 28S (D3-D5)  markers in order to summarize our current knowledge and formulate a phylogenetic hypothesis for dictyoceratids.

| MATERIAL S AND ME THODS
Sponge specimens or fractions thereof, including type material, were borrowed or obtained from the Queensland Museum  Table 1), and 72°C for 1 min (extension), followed by 72°C for 5 min (final extension). For some samples, touchdown PCRs prove to be more efficient than the standard protocol: 3 min at 95°C (denaturation), 20 cycles at 95°C for 30 s (heating), 55-45°C (annealing; −0.5°C per cycle), and 72°C for 1 min (extension), followed by 20 cycles at 95°C for 30 s (heating), 50°C (annealing), and 72°C for 1 min (extension), concluded by 72°C for 5 min (final extension). PCR products were isolated cleaned up with the freeze-squeeze method (Tautz & Renz, 1983) from 1.5% agarose gels. Cycle sequencing products were generated with BigDye Terminator v3.1 followed by Sanger sequencing on an ABI 3730 in the Genomic Sequencing Unit of the LMU Munich. Forward and reverse reads were assembled and corrected with CodonCode Aligner 3.7.1 (http:// www.codon code.com) after checking for contaminants by BLAST against NCBI GenBank. Intragenomic polymorphisms (IGP) were recoded following the IUPAC ambiguity codes for nucleotides.
The assembled and checked sequences were aligned with MAFFT (Katoh & Standley, 2013)

| RE SULTS AND D ISCUSS I ON
For a total of 236 dictyoceratid specimens, new sequences were generated (see Appendix 1). As not all fragments for every specimen were amplifiable and/or available from NCBI GenBank, the data sets   Wörheide (1998). c Chombard, Boury-Esnault, and Tillier (1998). possess laminated fibers, usually invisible with light microscopy rendering fiber lamination an unsuitable phylogenetic character.
Subfamily Phyllospongiinae, however, can be recovered, although with a taxon composition emended back to Keller's (1889) core taxa Carteriospongia and Phyllospongia, plus Strepsichordaia. F I G U R E 1 Phylogenetic hypothesis combined from the current ITS and 28S (C-region) data yielded in this study, combined with results from 28S (D3-D5), and CO1 reconstructions as calculated in this study and previously published 18S data. The 18S support is selected from Fig. 2 of Redmond et al. (2013), with occasional conflicting data (e.g., due to unverified identifications) disregarded. Thorectid taxa are shaded, of which Thorectinae are highlighted in light gray and Phyllospongiinae in dark gray. Asterisks indicate the presence of type sequences in the taxon (see text for further remarks). Shaded boxes at branches indicate the bootstrap probability (BP) for the different fragments. On the right of the taxon names are presence (+) and absence (-) of morphological features displayed (o indicates both absence and presence occurring between genera within) (cf. Cook & Bergquist, 2002a, 2002b, 2002c, 2002d, with examples given by the insert pictures on the right. Inserts are a) armor of Thorectandra excavatus (QM G303331); b) cored primary (and uncored secondary) fibers of Petrosaspongia nigra (QM G315543); c) cored secondary (and primary) fibers of Hyrtios erectus G301248; d) tertiary fibers (connecting   (Hooper & Wiedenmayer, 1994), but its reference material analyzed for the Systema Porifera was sequenced (SDCC/RF016, see Cook & Bergquist, 2002d).
Although histologically regarded as similar (Cook & Bergquist, 2002d), Thorectandra is phylogenetically distant to Thorecta (see below), prompting a re-evaluation of histological characters for keratose sponge systematics. Instead, Thorectandra is recovered close to the monotypic genus Fascaplysinopsis. Bergquist (1980) remarks Fascaplysinopsis recalling Thorectandra species in the "pronounced gelatinous appearance of the matrix, the yellow internal pigmentation and the coarse nature of the fibres" besides similarities in secondary metabolites. Unfortunately, DNA extraction from the holotype of Fascaplysinopsis reticulata Bergquist (Aplysinopsis reticulata Hentschel SMF904) was yet unsuccessful, but we managed to include the reference sample SDCC/RF017 from Systema Porifera (see Cook & Bergquist, 2002d Redmond et al., 2013). Cook and Bergquist (2002d), remark that Cacospongia species other than C. mollior and C. serta (Lendenfeld) require revision.
A partial ITS sequence of the C. serta holotype BMNH 1886.8.27.166, so far the only specimen of this species known (Cook & Bergquist, 2000), falls outside this clade, but verification from a longer sequence is required. In the past, C. mycofijiensis classification underwent numerous changes in its relatively young taxonomic history, triggered by overlapping morphological characteristics to other genera (see review in Sanders & Van Soest, 1996). An assignment of C. mycofijiensis to Petrosaspongia (suggested in Bergquist et al., 1999) can be rejected following our data, but assignment to Cacospongia (Sanders & Van Soest, 1996) or Scalarispongia (objected in Manconi, Cadeddu, Ledda, & Pronzato, 2013) requires thorough revision of the three genera.
Both Scalarispongia and Semitaspongia have been erected by Cook and Bergquist (2000) to accommodate members of the "'Cacospongia' group" which is supported by the present data.
A further major clade unites Luffariella, Thorecta, Fenestraspongia, Taonura, and Fasciospongia. Thorecta Lendenfeld is in our data set represented by T. reticulata Cook & Bergquist [reference specimen SDCC/NZ097 in Cook and Bergquist (1996) (Bergquist, 1965). Dactylospongia was subsequently assigned to Thorectidae based on its stratified fiber structure and due to morphological and pigment biochemical similarity to Smenospongia (Cook & Bergquist, 2002d). Both, distinction from Luffariella and similarity to Smenospongia, can be confirmed by our molecular data. A transfer of D. metachromia to the genus Petrosaspongia as suggested by Kwak, Schmitz, and Kelly (2000) based on terpenic compounds is in strong conflict with our molecular findings (see Uriz and Cebrian (2006) for a discussion).
Irciniidae share the apomorphic fine collagenous filaments in the mesohyl (Cook & Bergquist, 2002b). While molecular studies unequivocally supported irciniid monophyly of its largest genus Ircinia, this remains uncertain in respect to Sarcotragus ; see also Pöppe, Sutcliffe, Hooper, Wörheide, & Erpenbeck, 2010). Cook and Bergquist (2002b) regard the status of Sarcotragus, which differs from Ircinia only by the extent of fiber fasciculation and coring, as uncertain, likewise the distinction of Bergquistia, from which so far no molecular marker has been published, to Sarcotragus is uncertain (Cook, 2007). Distinction between Psammocinia and Ircinia, however, has molecularly been shown (Pöppe et al., 2010). Irciniidae frequently resemble species of Coscinoderma in shape, texture, and surface (Sim & Kim, 2014 et al., 2018). In contrast, a C. lanuga Laubenfels specimen, a species described as poorly known, but valid (Bergquist, 1980;Voultsiadou Koukoura et al., 1991), falls into the Spongiidae resulting in a paraphyletic genus Coscinoderma. Clearly, examination of the type species C. pesleonis (Lamarck, 1813) is required to resolve the classification of this genus.
For the monospecific genus Collospongia, the holotype C. auris Bergquist, Cambie & Kernan (AM Z5035) has been analyzed (Galitz et al., 2018). Cook and Bergquist (2002c) remarked on morphological similarities with the Phyllospongiinae, but with different secondary metabolite composition and a unique skeletal structure, which allegedly makes classification into any of the thorectid subclasses difficult. We recover Collospongia among the first branching thorectid genera and clearly distant from Phyllospongiinae (see also Galitz et al., 2018).
Genus Vaceletia is the only lineage among the dictyoceratids with a mineral (although secondary hypercalcified aragonitic) skeleton. It is regarded as the only extant representative of the fossil family Verticillitidae on the basis of its sphinctozoan bauplan (see Vacelet, 2002). The lack of clear synapomorphies shared with any other extant sponge lineage hampered the (morphological) classification of Vaceletia (Vacelet, 2002) until molecular data unequivocally revealed the dictyoceratid origin (Wörheide, 2008), followed by the placement of Verticillitidae as fifth family of Dictyoceratida (Morrow & Cárdenas, 2015). Molecular data recover an early branching of Vaceletia from the remaining thorectid + spongiid + irciniid taxa, probably as sister group.

| Implications for dictyoceratid morphological character evolution
Our Acanthodendrilla, although the extent of this character as apomorphy in dysideids has yet to be shown , particularly as secondaries in Candidaspongia are uncored (Cook & Bergquist, 2002a).
The possession of tertiary fibers is a combining character for the Phyllospongiinae, and the tertiary fiber-lacking alleged phyllospongiine Candidaspongia was revealed as dysideid (Galitz et al., 2018;Redmond et al., 2013). Tertiary fibers are further present in Luffariella and Fenestraspongia, two closely related genera. Some Spongia possess structures referred to as "pseudo-tertiary fibers" due to structural differences to those found in, for example, Luffariella (Cook & Bergquist, 2001), which leaves the possibility of tertiary fiber convergent evolution.
The arrangement of fibers into fascicles or into a regular (e.g., rectangular) skeleton does not constitute a reliable combining character either. While the closely related Thorecta and Taonura share this feature, histologically similar Thorectandra (cf. Cook & Bergquist, 2002d) are clearly distant.
In conclusion, clear-cut and unambiguous morphological apomorphies for the discrimination and classification of dictyoceratid sponges are scarce and too prone to homoplasies. The current morphology-based classification of the inter-and intrafamiliar relationships of thorectids, spongiids, Irciniidae, and Verticillitidae is incongruent to phylogenetic hypotheses of independent molecular markers and prompt for a re-classification and re-evaluation of synapomorphies based on integrative taxonomy.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information may be found online in the Supporting Information section at the end of the article. Figure S1. ITS reconstruction.   Specimens newly sequenced for this study. "HT", "NT," and "LT" following the voucher number indicate holotype, neotype, and lectotype, respectively. Accession numbers in bold indicate sequences newly obtained in the course of this study. Accession numbers of previously published sequences of the same specimen used in this study are given in regular font.