Species of Corvospongilla from all biogeographic realms in which the genus occurs have been considered for morphological analyses. Collections of sponges in North Western Australia were carried out during technical surveys of the Moochalabra Dam (Fig. 1). Staff of the Water Corporation of Western Australia discovered sponge growth on off-take structures at site 1(15°36′54.83″S 128°06′13.19″E) and site 2 (15°36′56.85″S 128°06′12.83″E) within the dam and collected specimens for identification (Table 1). This reservoir is fed by the Moochalabra Creek which starts at an elevation of 205 m and ends at 5.24 m when it flows into the King River (tributary of Cambridge Gulf, Timor Sea), dropping ~ 200 m over its 21.7 km length (http://bonzle.com/c/a?a=p&cmd=sp&zix=r&p=162783&st=&s=Moochalabra%20Dam&pg=1&m=0&c=1&x=128%2E12429&y=%2D15%2E4869&w=40000&mpsec=0, accessed 29/8/2019). The Dam supplies the town of Wyndham with drinking water (https://www.watercorporation.com.au/about-us/news/media-statements/media-release/swimming-prohibited-in-moochalabra-dam, accessed 29/8/2019). The Wyndham area is characterised by a hot semiarid climate (BSh) by Köppen climate classification (Geiger 1954, 1961; Peel et al. 2007).
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).
Institutional acronyms: NMHUK(BMNH), The Natural History Museum, London, United Kingdom; FW-POR, R. Manconi and R. Pronzato collection, Italy; IM, Indian Museum [including ZEV, Zoological Survey of India], Calcutta, India; MCN, Museu de Ciências Naturais, Fundação Zoobotânica, Puerto Alegre, Brazil; MRAC, Musée Royal de l’Afrique Centrale de Tervuren (KMMA), Belgium; MSNG, Museo civico di Storia Naturale ‘G. Doria’, Genova, Italy; QM, Queensland Museum, Australia; SNSB-BSPG, Bavarian State Collection for Palaeontology and Geology; USNM, National Museum of Natural History, Smithsonian Institution, Washington D.C., USA; WAM, Western Australian Museum, Perth, Western Australia; ZMB, Museum für Naturkunde, Humboldt Universität, Berlin, Germany.
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, gemmular architecture and gemmuloscleres morphology) was considered diagnostic at the family, genus and species levels (Manconi and 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 (Manconi and 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).
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 ITS2.2-rvse (5′-CCT GGT TAG TTT CTT TTC CTC CGC-3′) (Wörheide 1998). Alternatively, two minimalist ITS2 barcoding regions were amplified (“5.8S-ITS2” and “ITS2-28S” cf. Erpenbeck et al. 2019) [Primers: 5.8_Freshies_1180_9f: 5′-GCA CGT CTG TCT GAG CGT CCG-3′ / ITS2_Freshies_1174_3r: 5′-GCT TCG CAC TTS AAG GGA CGC-3′ (5.8S-ITS2) and ITS2_Freshies_1176_5f: 5′-TTG CGC GTC GGG AAC TCG AC-3′ / 28S_Freshies_1178_7r: 5′-GCT TAT TGA TAT GCT TAA ATT CAG C-3′ (ITS2-28S)]. The 25 µL PCR mix comprised 5 µL 5 × green GoTaq® PCR Buffer (Promega Corp, Madison, WI), 4 µL 25 mM MgCl2 (Promega Corp, Madison, WI), 2 µL 10 mM dNTPs, 2 µL BSA (100 µg/ml), 1 µL each primer (5 µM), 7.8 µL water, 0.2 µL GoTaq® DNA polymerase (5 μ/μL) (Promega Corp, Madison, WI) and 2 µL DNA template. The PCR regime comprised an initial denaturation phase of 94 °C for 3 min followed by 35 cycles of 30 s denaturation at 94 °C, 20 s annealing (45 °C for ITS-RA2-fwd / ITS2.2-rvse; 52 °C for 5.8_Freshies_1180_9f / ITS2_Freshies_1174_3r; 55 °C for ITS2_Freshies_1176_5f / 28S_Freshies_1178_7r). Elongation time was 60 s (for ITS-RA2-fwd / ITS2.2-rvse), respectively, 45 s (for 5.8_Freshies_1180_9f / ITS2_Freshies_1174_3r and ITS2_Freshies_1176_5f / 28S_Freshies_1178_7r) at 72 °C each followed by a final elongation at 72 °C for 5 min after the 35th cycle. PCR products were purified with a Freeze-Squeeze Method (Thuring et al. 1975) before cycle sequencing using the BigDye-Terminator Mix v3.1 (Applied Biosystems) following the manufacturer’s protocol. Both strands of the template were sequenced on an ABI 3730 automated sequencer. PCR products were cleaned with the Freeze-Squeeze methods following Thuring et al. (1975), cycle sequenced with BigDye-Terminator Mix v3.1 (Applied Biosystems) and sequenced on an ABI 3730 automated sequencer. Sequences were basecalled, trimmed, assembled and checked in CodonCode Aligner v 220.127.116.11 (www.codoncode.com). Origin of the sequences was verified with BLAST against NCBI GenBank (www.ncbi.nlm.nih.gov/genbank). Sequences of this project are deposited in the European Nucleotide Archive (ENA, www.ebi.ac.uk/ena) under study accession number PRJEB41019 and in the Sponge Barcoding Database (SBD, www.spongebarcoding.org, Wörheide and Erpenbeck 2007). Sequences were 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). Sequences of Ephydatia muelleri were chosen as outgroup as they provided comparatively short distances to the Corvospongilla ingroup. Median Joining network (Bandelt et al. 1999) reconstructions on Corvospongilla were performed with PopART (http://popart.otago.ac.nz) under an epsilon parameter of zero.
Class Demospongiae Sollas, 1888
Order Spongillida Manconi and Pronzato 2002
Family Spongillidae Gray, 1867
Genus Corvospongilla Annandale 1911
[Type species Corvospongilla loricata (Weltner 1895)]
Diagnosis (revised after Manconi and Pronzato 2019; emended parts in italics). 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 multi-spicular 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, ring-shaped 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.
Corvospongilla moochalabrensis sp. n. Manconi and Erpenbeck
Figures 1, 2, 3, 4, 5, 6, 7, 8, Table 1.
Examined material Holotype: WAM Z29235/FW-POR853 schizotype, type locality Moochalabra Dam off-take site 2 (15°36′56.85″S 128°06′12.83″E), Moochalabra Creek, King River Hydrographic Basin, North Kimberley Region, east North Western Australia, coll. L. Zappia & I. Read, 22 February 2012, 75% Ethanol.
Paratypes WAM Z29234/FW-POR854 schizotype, Moochalabra Dam off-take site 1 (15°36′54.83″S 128°06′13.19″E), ibid., coll. L. Zappia & I. Read, 22 February 2012, 75% Ethanol; WAM Z29245/FW-POR 860, ibid., coll. I. Read, 6 December 2012, 75% Ethanol; WAM Z29247/FW-POR 861, ibid., 100% Ethanol; WAM Z29246/FW-POR 862, ibid., 75% Ethanol; WAM Z98322/FW-POR863, Moochalabra Dam (15°37′15.66″S 128°06′06.09″E), coll. Dalcon Environmental,18 June 2003, 75% Ethanol.
Comparative materials Corvospongilla becki Poirrier 1978 USNM topotypes/FW-POR 919, 920, 921 schizotypes, dry, Duck Lake, 25 Sept. 1975, leg. det. Poirrier, Louisiana, USA; Corvospongilla burmanica subsp. bombayensis Kirkpatrick, 1908 BMNH 18.104.22.168-3 box 6 type/FW-POR 420 schizotype, dry, Pimpli, Vashisthi Valley, Ranagiri District, India; C. burmanica (?) BMNH 22.214.171.124/FW-POR 636, River Kuano, Uttar Pradesh, India; Corvospongilla caunteri Annandale 1911 BMNH 126.96.36.199 ex-ZEV 4776/7 paratype/FW-POR 637 schizotype, Hazratgunj, Lucknow, Uttar Pradesh, India; Corvospongilla lapidosa (Annandale 1908) BMNH 08.2.11.1 paratype/FW-POR 638 schizotype, River Godavery Nasik, Maharashtra, India; C. lapidosa BMNH/FW-POR 149, River Kuano, Uttar Pradesh, India; Corvospongilla lemuriensis Manconi and Pronzato 2019 MSNG 60893a holotype/FW-POR 807 schizoholotype, FW-POR 804 topotype, Farihy Amboromalandi Reservoir, Madagascar; Corvospongilla loricata (Weltner 1895) ZMB 2093 SE325-SE37–41 type/fragment FW-POR 511, locality unknown, Africa; Corvospongilla mesopotamica Manconi and Pronzato 2004 MSNG 51766 holotype/ FW-POR 574 schizotype, River Diyala, Kurdistan, Iraq; Corvospongilla siamensis Manconi and Ruengsawang, 2012 MSNG 56533 holotype/FW-POR 733, MSNG 56533a paratype, Pong River, Lower Mekong Basin, Thailand; Corvospongilla thysi (Brien, 1968) MRAC 1311 type/FW-POR 472 schizotype, Lake Barombi-ma-Mbu, Cameroon, W-Africa; Corvospongilla ultima (Annandale 1910) BMNH 188.8.131.52 ex-ZEV 4906/7 fragment/FW-POR 639,Tanjore, Irinchinopoli District, India; C. ultima var. spinosa Annandale, 1912 BMNH 184.108.40.206 ex-ZEV 5106/7/FW-POR 640, Taloshi, Koyna Valley, Satara District, Maharashtra, India; Corvospongilla volkmeri de Rosa Barbosa, 1988 BMNH 220.127.116.11 (ex-MCN 86) schizoparatype /FW-POR 642, Lagoa Redonda, Sousa, Paraíba State, Brazil; Corvospongilla zambesiana (Kirkpatrick, 1906) BMNH 1906.2.28.2, 13IIIC/FW-POR 623 R. Zambezi, Zimbabwe; Corvospongilla sp. 1 SNSB-BSPG.GW 2354/2358-FW-POR 950/951/954, Lake Massoko/Lake Kingiri/Lake Itamba, 11 Nov. 2011, coll. M. Genner, Lake Itamba, Tanzania, Africa.
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’.
Etymology The specific epithet moochalabrensis refers to the Moochalabra Dam (type locality).
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.watercorporation.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).
Description. Growth form encrusting. Colour light brown to brown in ethanol in the same specimen. Consistency hard, fragile in alcohol. Spongin scanty in the skeleton, conspicuous in the gemmular theca and basal spongin plate. Basal spongin plate notably developed around sessile gemmules. Surface slightly hispid from tips of irregularly arranged oxeas. Oscules inconspicuous. Ectosomal skeleton irregularly arranged oxeas (no special architecture). Choanosomal skeleton (on the basis of few basal fragments) as network of megascleres with multi-spicular (up to 10–15 spicules) meshes. Megascleres oxeas smooth, slender, abruptly pointed to fusiform, straight to slightly bent (167–205 × 8–14 µm, WAM Z29235/FW-POR 853; 195–233 × 9–13 µm, WAM Z98322/FW-POR 863; n = 25 spicules measured per sample). Oxeas smooth, thin, straight, fusiform (112–158 × 2–5 µm, WAM Z29235/FW-POR 853; n = 25 spicules measured per sample) also present. Microscleres pseudobirotules abundant, scattered in the skeleton and near gemmular carpets; pseudobirotules entirely smooth, with thin shaft evidently bent to straight, notably variable in length (28–56 × 1.5–2 µm, WAM Z29235/FW-POR 853; 21–37 × 1.5–2 µm, WAM Z98322/FW-POR 863; n = 25 spicules measured per sample). Pseudorotules smooth (8.7–23 µm in diameter) armed with long, acute hooks (n = 4–5). Gemmules subspherical, light brown (alcohol), exclusively sessile at the sponge basal portion, strictly adhering to the basal spongin plate in groups, sharing in part the gemmular cage of smooth oxeas. No free gemmules in the skeletal meshwork. Gemmular cage of abundant, smooth, stout oxeas (megascleres) tangentially arranged at the theca surface. Gemmular theca subspherical (~ 530 µm in diameter with cage, ~ 340 µm without cage) of a single morph, trilayered, armed by a single layer of gemmuloscleres and enclosed by the spicular cage (cage and theca easily detachable from each other). Outer layer of spongin with small concavities to bubbles (resembling pneumatic chambers). Pneumatic layer (23–26 µm in thickness) as chambered laminar spongin to form rounded small chambers and armed by scattered gemmuloscleres tangentially embedded. Inner layer with sublayers of laminar compact spongin. Foramen single, apical with a short simple collar and aperture oriented upward in gemmular carpets. Gemmuloscleres spiny from strongyles to oxeas and strongyloxeas (33–66 × 3.5–9 µm, WAM Z29235/FW-POR 853; 33–93 × 9.3 µm, WAM Z98322/FW-POR 863; n = 25 spicules per each sample) straight to slightly bent with spines and tubercles of various sizes, more dense at tips; spines simple with acute tips, to large and ornate with apical microspines.
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 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).