Discovery of the freshwater sponge genus Corvospongilla Annandale (Porifera: Spongillida) in Australia with the description of a new species and phylogeographic implications

A recent discovery of freshwater sponges in an unexplored hydrographic basin in north-western Australia provided the opportunity to investigate the genus Corvospongilla Annandale (Spongillida: Spongillidae) using integrative systematics. Emendation of the genus diagnosis is provided. A comparative analysis of a Corvospongilla global dataset of morphological traits together with biogeographic patterns disclosed a new Australasian Corvospongilla species and along with molecular analyses provided the basis for a phylogenetic and phylogeographic tree for some Asian, Afrotropical and Australasian lineages.


Introduction
The Australian freshwater sponge fauna (Demospongiae: Spongillida) is poorly studied. Species richness and endemicity values are comparatively high, i.e., Australia has 26 recorded species, 23 of which are Australian endemics, while the Australasian Bioregion has 36 recorded species (Racek 1969;Stanisic 1978Stanisic -1979Manconi et al. 2016;Manconi 2019, 2021). To shed more light on Australian freshwater sponge diversity, the collection of the Western Australian Museum is currently being studied from both a morphological and molecular standpoint.
The present focus is on Corvospongilla Annandale, 1911 one of the most species rich genera (n = 19 species until now) of the order Spongillida. This genus is characterized by 'pseudobirotules as skeletal microscleres', 'three gemmular morphs with variable architecture of theca' and 'strongyles to oxeas as megascleres' all traits supporting the need for deep integrative analyses Pronzato 2002, 2019;Pronzato et al. 2017). We report on the integrative analysis of congeneric species assigned to Corvospongilla in a biogeographic context. This has allowed us to report on (1) the first record of the genus in the Australasian Region; (2) the discovery of a new species from North Western Australia on the basis of a comparative morphological analysis with the other species of the genus; (3) assessment of morphological and molecular traits to delineate a species group from Australia, Africa and Asia; (4) determination of the phylogeographic pattern of the genus Corvospongilla confirming Gondwanian tracks in its evolutionary history.

Studied museum collections
Representative samples of Australian Corvospongilla were compared to type material and specimens from historical collections and to the original descriptions of congeneric species (see Table 1). Holotype and paratypes of the new species were registered at the Western Australian Museum (WAM) and schizotypes in the FW-POR collection (Italy).

Morphological analysis
In vivo images were not available. Although the WAM ethanol preserved samples were in a poor status of preservation a set of macro-and micro-morphotraits (architecture of skeleton, traits of skeletal megascleres and microscleres,  Pronzato 2002, 2019). Representative fragments of sponges were dissected by hand for light microscopy (LM) and scanning electron microscopy (SEM, Vega3Tescan, Czech Republic). Spicules processed by dissolution of organic matter in boiling 65% nitric acid, were rinsed in water, suspended in ethanol and dropped onto slides and/or stubs Pronzato 2000, 2015). Dry body fragments, dissociated spicules, entire gemmules and their cross-sections were sputter-coated with gold and observed under SEM. Measurements of spicules of each diagnostic spicular type and gemmules, were performed by LM and by SEM. The terminology of diagnostic morphotraits follows Manconi and Pronzato (2002). Taxonomic status was checked in the World Porifera Database (Van Soest et al. 2020).

Molecular analyses
Total genomic DNA was extracted from tissue fragments of the adult specimens, or in case of C. lemuriensis, from the juveniles freshly hatched from gemmules in petri dishes using the NucleoSpin ® . Tissue DNA extraction Kit (Macherey-Nagel) following the manufacturer's protocol. Primarily, amplification of the entire ITS region was attempted for all specimens using the primers ITS-RA2-fwd (5′-GTC CCT GCC CTT TGT ACA CA-3′) in combination with ITS2.2rvse (5′-CCT GGT TAG TTT CTT TTC CTC CGC-3′) (Wörheide 1998 concatenated and aligned using MAFFT (Katoh and Standley 2013) prior to maximum likelihood reconstructions using PhyML (which regards gaps as missing data, Guindon et al. 2010), as implemented in Geneious 2019.2.1 (Kearse et al. 2012) under the F81 model as suggested by jModeltest2 (Darriba et al. 2012 . Encrusting, flat to massive, lobate growth form. Consistency extremely hard to fragile. Spongin scanty except for the well-developed basal spongin plate and the gemmular theca. Skeletal network irregularly alveolar to isotropic with sometimes vague ascending pauci-to multispicular tracts toward surface supporting conules and ridges if present. Skeletal megascleres strongyles to oxeas smooth, tubercled-granulated or spiny. Skeletal microscleres as pseudobirotules frequently rare, straight to slightly curve with smooth shaft of variable length and pseudorotules at tips. Pseudorotules with hooks (corvus) variably long and curve. Gemmules of various morphs, sometimes coexistent, single or grouped, in the skeletal network (free gemmules) or adhering to the basal spongin plate (sessile gemmules) with or without a variably stout spicular cage around the theca. Foramen from apical to lateral with a short porous tube. Three gemmular morphs according to the architecture of the trilayered to mono-or bi-layered theca. Gemmular theca with variably thick pneumatic layer of rounded chambers with compact laminae. Gemmuloscleres variably embedded and tangentially arranged in the theca, from elongated, spiny to smooth, stout strongyles to oxeas to strongyloxeas, straight or variably curved to boomerang-shaped, ringshaped or oval. Spicules of larvae slender, smooth to spiny oxeas.
Etymology The genus name Corvospongilla refers to the corvus as the typical diagnostic morphotrait 'pseudobirotule as skeletal microscleres with ornamentation at tips a few long, curved, smooth hooks'. The suffix corvo refers to the particular shape of these hooked tips on the basis of the Latin term corvus meaning harpoon, hook and rising from a Roman naval boarding device (mobile catwalk with an hook at the tip) used in naval battles.    Diagnosis Corvospongilla moochalabrensis is characterized by a combination of unique traits of the spicular complement as 'smooth slender oxeas as megascleres with tips ranging from abruptly pointed to fusiform and rare oxeas thin, straight, fusiform' and 'pseudobirotules as microscleres with curved shaft and pseudorotules with long hooks' and gemmular architecture as 'gemmules of a single morph, i.e., sessile with spicular cage of smooth oxeas (megascleres)', 'sessile gemmular theca trilayered with well-developed chambered pneumatic layer', 'spiny strongyles to oxeas and strongyloxeas as spiny gemmuloscleres'.
Life cycle Sponges growing on the dam were in the active vegetative phase and always bearing gemmules both in the wet season (summer; December and February) and in the arid season (winter; June) when they were collected.
Habitat Dense sponge populations occurred in shallow water of Moochalabra Dam on grids of in-take and off-take structures within the dam and were collected during dam maintenance (grid cleaning). Water in the dam, after filtration, chlorination and disinfection meets strict Australian Drinking Water Guidelines. During the wet season, the dam inflow may contain sediment, depending on levels of rainfall in the catchment, and in winter a temperature gradient may occur in the dam, which can lead to sediments being resuspended, causing seasonal turbidity (https:// www. water corpo ration. com. au, accessed 29/8/2019). Sponge associated organisms were abundant bryozoans (with statoblasts) strictly growing with sponges, together with diatoms, nematodes, water mites, and chironomid larvae.
Geographic range Currently known only from the type locality of Moochalabra Dam, in the North Kimberley Region, north Western Australia (Fig. 1).

Molecular results
Full-length ITS sequences of 790-808 bp (incl. 5.8S and the flanking regions of 18S and 28S) were obtainable from C. mesopotamica, C. lemuriensis and Corvospongilla sp. 1 (Tanzania). For the remaining material only the minimalist barcodes ("5.8S-ITS2" and "ITS2-28S" cf. Erpenbeck et al. 2019) were successfully amplified. Here, the more than a century-old type material of C. burmanica, C. caunteri, C. ultima, C. ultima var. spinosa, and C. lapidosa could be amplified. These minimalist barcodes comprise lengths of 77 bp (5.8S-ITS2) and 99-101 bp (ITS2-28S), respectively. Amplification and sequencing of C. thysi, C. zambesiana and C. seckti (as C. volkmeri) was attempted, but did not lead to sequences unambiguously identifiable as Corvospongilla. From the remaining comparative specimens (Table 1) no sequences could be obtained. The concatenated data set, restricted to both minimalist barcoding regions only, comprised 15 taxa and 207 characters of ITS2. Nine character positions were variable among the Corvospongilla spp. (four transitions, four transversions, one indel, see Fig. 7, also for genetic distances). In the phylogenetic reconstructions (Fig. 7) the sequences of Corvospongilla fall in three distinct clades with an unsupported relationship to each other. Inclusion/exclusion or choice of outgroup did not affect internal relationships of the Corvospongilla ingroup. C. moochalabrensis sp. n. displays a distinct ITS2 barcode and forms a clade with C. ultima. The molecular difference between C. moochalabrensis and C. ultima is a G-C transversion. The remaining (Asian) Corvospongilla species, i.e., C. burmanica, C. caunteri, C. mesopotamica, C. lapidosa and C. siamensis share a barcode and form a supported clade, likewise C. lemuriensis and the Tanzanian Corvospongilla sp. 1 are supported and distinctive (Fig. 7). The Median Joining network reconstruction of the 177 ingroup characters resulted in a linear, unbranched arrangement of the Corvospongilla Corvospongilla moochalabrensis sp. n. from NW Australia (red) is the first Australasian record of the genus. One Miocenic fossil species (blue) is recorded from the north-western Nearctic. Emended after Manconi and Pronzato (2019) genotypes with the Australian and African/Madagascan species at either ends showing 1, respectively, 3 steps to the closest genotype from Asia (see Fig. 7).
Most species of the genus display a notably diverse morphological range and combinations of megascleres as 'spiny oxeas, smooth oxeas, smooth strongyles, spiny strongyles, or a mix of these spicule types' except for C. moochalabrensis (Penney and Racek 1968). However, the variable types of megascleres could be related to their morphofunctional roles and topographic distribution in the sponge body as (a) main skeletal spicules, (b) spicules belonging to the cage of gemmular theca, or (c) larval spicules. Moreover, in some cases details of morphotraits are unknown, e.g., gemmules are unknown in the Afrotropical C. sodenia and C. zambesiana, and gemmular cage of megascleres and larvae with their spicules are unknown for several Corvospongilla species.
In contrast the trait 'pseudobirotules as skeletal microscleres' is morphologically stable in all species, although these spicules are variable in abundance up to notably rare, and their morphometries and microtraits have a relatively narrow range. The trait 'long curved hooks of pseudobirotules (microscleres)' (Figs. 4B-D, 5A) rare in the genus is shared by the new species with three African species, i.e., C. zambesiana (Manconi and Pronzato 2009), C. micramphidiscoides (Manconi and Pronzato 2009) and C. boehmi (Penney and Racek 1968;Manconi and Pronzato 2009).
At the level of gemmular architecture the new species trait 'sessile with trilayered theca and well-developed chambered pneumatic layer' is exclusively shared with C. lemuriensis from Madagascar. The present record of 'unusual gemmular architecture as a third gemmular morph' confirms a new trait as an evolutionary novelty for the genus, i.e., 'sessile with a well-developed pneumatic layer and spicular cage'.
In support of the morphological results, molecular distinction is given to all other Corvospongilla species analysed in this study, although only by one transition to C. ultima. The minimalist barcodes resulted in a short discriminating sequence and in turn enabled the comparison with centuryold type material. The lack of molecular distinction between C. burmanica, C. caunteri, C. mesopotamica, C. lapidosa and C. siamensis with ITS2 may indicate a faster evolution of morphological traits among those Asian species than is accessible with this molecular marker (Erpenbeck et al. 2019(Erpenbeck et al. , 2020 for similar findings among other freshwater sponge species).
The genus Corvospongilla was considered absent from Australia until now Pronzato 2008, 2019). The discovery of the new species enlarges the range of extant Corvospongilla species to include the Australasian Bioregion and indicates a wider biogeographic pattern of the genus in the southern hemisphere. However, spicular remains from the stomach contents of a freshwater turtle Emydura subglobosa (Krefft, 1876) were identified as Corvospongilla caunteri (QM G317009, unpublished record) from Policeman's Crossing (13°35′11″ S 130°38′28″ E), Daly River Mission, Daly River in the Northern Territory, Welsh, M. (field coll.), Kennedy, J. (id.). New samples from the Daly River area and verification of this QM specimen will be fundamental to determining the geographic range of Corvospongilla in Australia.
The present record from Australia of the new species C. moochalabrensis increases the species number to 20 for the genus Corvospongilla (Manconi and Pronzato 2007, 2008, 2009, 2019Van Soest et al. 2020). The genus is widespread with the exception of Antarctica and the Pacific (Oceanian) (Fig. 8), and is present in Palaearctic (n = 1), Nearctic (n = 2), Neotropical (n = 1 widely spread), Oriental (n = 6), and Australasian (n = 1 present paper) bioregions, with the highest species richness in the Afrotropical Bioregion (n = 9) . Most species are extremely rare and have limited ranges i.e., endemic sensu stricto to a single hydrographic basin or a restricted area, e.g., type locality Pronzato 2004, 2019). At the global scale Corvospongilla shows a notably wide disjunct range. This biogeographic pattern better matches the vicariance versus the dispersal model and indicates a wide radiation, particularly in Afrotropical and Oriental bioregions. The morphological novelty of diverging gemmular architectures seems not to favour the species spreading widely , 2019.
The evolutionary success of Corvospongilla, in terms of radiation and geographic range, could be related to cryptobiosis and to the morphofunctional performance of asexual propagules, e.g., resistance to desiccation and hatchability for persistence and dispersal viz. 'sessile gemmules without pneuma', 'sessile gemmules with pneuma' and 'free gemmules with pneuma' Pronzato 2004, 2019;). These different functional morphs appear to fully perform their asexual reproductive and survival role in continental water with a wide range of climates and environmental conditions (Manconi and Pronzato 2016b). Unfortunately data on fossils are almost lacking .
In synthesis, the comparative study of significant representatives of Corvospongilla by molecular and morphological analysis, together with assessment of biogeographic patterns, yielded results on which to build a phylogenetic and phylogeographic tree. With the exception of C. ultima, all analysed Corvospongilla species fall into molecular clades resembling their distinct bioregions (Afrotropical, Oriental, Australasian), a pattern previously unexplored for freshwater sponges.
This new integrated assessment enlarges the geographic pattern of the genus Corvospongilla to Australia, and increases the number of species in the genus to 20. The spicular complement of strongyles and oxeas (in a variety of combinations) together with three gemmular morphs suggest that Corvospongilla is an ancient taxon. This appears to be supported by the notably disjunct biogeographic pattern of the genus, confirming it could be related to Gondwanan separation, although the enclave in the Nearctic Region suggests that the spreading of this lineage in inland waters could be more ancient.
The current data highlight the problem of Corvospongilla systematics, biogeography and phylogenetic relationships in the evolutionary history of Spongillida  and supports the need for further in depth analyses.