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Cyclacanthia rethahofmeyri Samaai, Kelly, Payne and Ngwakum sp. nov. (Fig. 15B, Tables 6, 7) Material examined. Holotype SAMC-A090895, Amathole region Stn 3872, Eastern Cape, 32.950�� S, 28.066�� E, 40.5 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 31 Aug 2016. Paratypes. SAMC-A090896, Amathole region Stn 3737, Eastern Cape, 32.751�� S, 28,415�� E, 31 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 23 Feb 2016; SAMC-A090897, SAMC-A090898, SAMC-A090899, Amathole region Stn 3832, Eastern Cape, 32.759�� S, 28.410�� E, 45 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 27 Aug 2016. Type locality. Amathole region, off East London, Eastern Cape. Distribution. Amathole region, off East London, 40.5 m. Description. Shape varies from either flattened low lying hemispherical (flattened), ranging from 24 mm long by 24 mm wide by 6 mm thick to hemispherical sponges, 13 mm long by 34 mm wide by 27 mm thick (Fig. 15D). Surface undulating but smooth, with volcano-shaped oscules, 5 mm high by 3 mm wide at base, 1 mm at apex being closely packed (5mm apart), and a few nodular truncate areolate porefields, 3 mm high by 3 mm wide, with no poral membrane covering the opening. Texture firm, rubbery and tough. Medium to barely compressible, difficult to tear or break. Colour in life either black, dark green/black or dark brown; in preservative dark green (Fig. 15D). Green exudate visible, no smell. Skeleton. The choanosomal skeleton forms an irregular polygonal reticulation formed by wispy tracts of smooth styles (Fig. 15H). The tracts range in width from 85 ���150 ��m in thickness, and form meshes that are 195 ��m wide. Within the inner choanosome, tracts diverge towards the surface and are 172���205 ��m wide. Interstitial spicules are abundant. The ectosome has a palisade of densely packed interlocking anisodiscorhabds, which are absent from the surface structures. The paratangential layer beneath the palisade is approximately 320 ��m deep, and this extends into the oscular fistules. Spicules. Megascleres are anisostyles in one size category: Large, thick, smooth, straight, occasionally sinuous, centrally thickened, narrow proximal region, fusiform: 696 (480���796) �� 18 ��m. Microscleres are isospinodiscorhabds I (Fig. 15E, F) with 50% of microscleres either having three or two whorls. In some spicules the median whorl is absent or rudimentary. The manubrium is identical to the apical whorl. The median whorl is equidistant from both apical whorl and manubrium, but smaller and shorter: 49 (38���60) ��m. Large isospinodiscorhabds II (Fig. 15G) with two whorls, 75 (64���88) �� 15 (15) ��m. Substratum, depth range and ecology. Occurs off East London, Amathole region at a depth range of 30���42 m. DNA sequence data. We sequenced partial COI of collected material from different localities; GenBank accession numbers: COI MK153269 ��� MK153271; MK016476. Etymology. The species name rethahofmeyri is given in memory of Emeritus Professor Dr Retha Hofmeyr who joined the Zoology Department at the University of Western Cape in 1974 and served as Head of Department from 1991 to 1992 before she passed away in 2020. Retha will always be cherished and remembered for her kindness and encouragement of her students and staff. Remarks. Cyclacanthia rethahofmeyri sp. nov. have acanthose isospinodiscorhabds as microscleres (Fig. 14) and conforms to the general skeletal characteristics of the genus. The examination of this material revealed the presence of a second category of large isospinodiscorhabds (Fig. 15G, a spicule not present in any other Cyclacanthia species described to date (Samaai et al. 2004). The definition of Cyclacanthia has been expanded to include species with a 2 nd category of isospinorhabds. The COI sequences of the Cyclacanthia specimens was clearly separated from Tsitsikamma and formed one, strongly supported phylogenetic cluster characterised by a single distinct haplotype (Fig. 6). Table 7 highlights the morphological characters of the different Cyclacanthia species and figure 14 shows the different types of isospinodiscorhabds. The family Latrunculiidae is monophyletic as indicated by the COI phylogeny (Fig. 6)., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 435-437, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Samaai, T., Govender, V. & Kelly, M. (2004) Cyclacanthia n. g. (Demospongiae: Poecilosclerida: Latrunculiidae incertae sedis), a new genus of marine sponges from South African waters, and description of two new species. Zootaxa, 725 (1), 1 - 18. https: // doi. org / 10.11646 / zootaxa. 725.1.1"]}

Tsitsikamma (Tsitsikamma) favus Samaai & Kelly, 2002 (Figs 8, 9, Table 1, 2) Tsitsikamma favus Samaai and Kelly, 2002: 718; fig. 6 A���G. Tsitsikamma favus, Samaai et al. 2003: 11; fig. 3f; 17; fig. 6A; Parker-Nance et al. 2019: 105; fig. 1a���p; table 1. Type & locality (not examined). Holotype ��� NHMUK 1997.7.3.2, Rheeders Reef, Tsitsikamma National Park, 34.166�� S, 23.900�� E, 22 m, collected by P. Coetzee, 1995. Material examined. NIWA 110724, Table Top Reef, Algoa Bay, Port Elizabeth, 33.980�� S, 25.693�� E, 18 m, collected by Patrick L. Colin, CRRF, 4 Oct 1998, identified by Michelle Kelly, NIWA, Auckland; NIWA 110826, White sands Reef, Algoa Bay, Port Elizabeth, 33.995�� S, 25.707�� E, 14 m, collected by Patrick L. Colin, CRRF, 17 Feb 1999, identified by Michelle Kelly, NIWA; NIWA 111059, Grootbank, Plettenberg Bay, 34.007�� S, 23.496�� E, 10���14 m, collected by Patrick L. Colin, CRRF, 22 Mar 2000, identified by Michelle Kelly, NIWA, Auckland; SAMC-A091443, Grootbank, Plettenberg Bay, 34.007�� S, 23.496�� E, 10���14 m, collected by Patrick L. Colin, CRRF, 22 Mar 2000, identified by Michelle Kelly, NIWA, Auckland. SAMC-A090900, SAMC-A090902, Flat Rock, Jeffreys Bay, 34.102�� S, 24.958�� E, 13 m, collected by M. Pfaff, S. Kirkman, I. Malick and L. Snyders, 8 Aug 2016; SAMC-A090901, Grootbank, Plettenberg Bay, 34.006�� S, 23.495�� E, 40 m, collected by T. Samaai, 25 Feb 1999; SAMC-A090903, Seal Point, Cape St Francis, 34.223�� S, 25.852�� E, 15 m, collected by T. Haupt & I. Malick, 9 Aug 2016; SAMC-A090904, SAMC-A091416, SAMC-A091417, SAMC-A091418, Evans Peak, Algoa Bay, Port Elizabeth, 33.842�� S, 25.816�� E, 30 m, collected by Rhodes University, May 2010; SAMC-A090905, Bell Buoy, Sardinia Bay, 33.980�� S, 25.698�� E, Algoa Bay, Port Elizabeth, 16 m, collected by C. Evans, 25 May 2009; SAMC-A090906, Tsitsikamma National Park, 34.091�� S, 23.895�� E, 14 m, collected by T. Samaai, May 2009; SAMC-A091414, Rheeders Reef, Tsitsikamma National Park, 34.166�� S, 23.900�� E, 18 m, collected by M. Davies-Coleman, 1 Oct 1998; SAMC-A091415, Rheeders Reef, Tsitsikamma National Park, 34.166�� S, 23.900�� E, 22 m, collected by M. Davies-Coleman, 25 Feb 1994; SAMC-A091419, SAMC-A091420, SAMC-A091421, Evans Peak, Algoa Bay, Port Elizabeth, 33.842�� S, 25.816�� E, 30 m, collected by Rhodes University, May 2009; SAMC-A091422, SAMC-A091423, SAMC-A091424, Phillips Reef, (33.974�� S, 25.681�� E), Algoa Bay, Port Elizabeth, 30 m, collected by Rhodes University, 10 Aug 2010; SAMC-A091425, Suicide Reef, Sardinia Bay, 34.005�� S, 25.636�� E, Algoa Bay, Port Elizabeth, 16 m, collected by C. Evans, 25 May 2009; SAMC-A091426, off Christian Centre, Sardinia Bay, 34.053�� S, 25.648�� E, Algoa Bay, Port Elizabeth, 16 m, collected by C. Evans, 25 May 2009; SAMC-A091427, Swim Thru, Sardinia Bay, 34.049�� S, 25.648�� E, Algoa Bay, Port Elizabeth, 16 m, collected by C. Evans, 25 May 2009; SAMC-A091428, Seal Point, Cape St Francis, 34.223�� S, 25.852�� E, 15 m, collected by T. Haupt & I. Malick, 8 Aug 2016; SAMC-A091429, Table Top Reef, Algoa Bay, Port Elizabeth, 33.980�� S, 25.693�� E, 16 m, collected by Patrick L. Colin, CRRF, 4 Oct 1998, identified by Michelle Kelly, NIWA. SAMC-A091442, Grootbank, Plettenberg Bay, 34.006�� S, 23.384�� E, 16 m, collected by T. Samaai, 25 February 1999. Additional material (not examined). CASIZ 300535, Table Top Reef, Algoa Bay, Port Elizabeth, 33.980�� S, 25.693�� E, 16 m, collected by Patrick L. Colin, CRRF, 4 Oct 1998, identified by Michelle Kelly, NIWA; CASIZ 300636, White sands Reef, Algoa Bay, Port Elizabeth, 33.995�� S, 25.707�� E, 14 m, collected by Patrick L. Colin, CRRF, 17 Feb 1999, field identification by L. J. Bell, CRRF, listed in Parker-Nance et al. (2019); CASIZ 301054, Grootbank, Plettenberg Bay, 34.007�� S, 23.496�� E, 10���14 m, collected by Patrick L. Colin, CRRF, 22 Mar 2000, identified by Michelle Kelly, NIWA, Auckland; SAIAB 141112, Knoll Reef, Tsitsikamma National Park, 34.166�� S, 23.900�� E, 18 m, collected by C. Buxton & M. Davies-Coleman, 2 May 1993; SAIAB 141356, Rheeders Reef, Tsitsikamma National Park, 34.166�� S, 23.900�� E, collected by P. Coetzee, R. Palmer & B. Cart��, 18 Mar 1995. Type location. Rheeders Reef, Tsitsikamma National Park, 22 m. Distribution. Plettenberg Bay, Tsitsikamma Marine Protected Area, St. Francis Bay, St. Jeffreys Bay, Sardinia Bay and Algoa Bay. Diagnosis. Varying morphology being either massive, lobate sometimes hemispherical to thickly encrusting, 5���10 cm thick and 10���20 cm diameter, surface very rugose and crowded with large single to multichambered cylindrical lance-shaped (button shaped) oscules, 1���4 mm wide, 6 mm high and with cauliform areolate porefields, 3 mm wide, 3���5 mm high, with no membrane (Fig. 8). Sponge is hard and slightly compressible or soft and fleshy; colour in life vary being either dark brown, liver brown, turquoise or dark green (Fig. 8; Table 1), internally olive or dark green/brown; in alcohol, ectosome black or dark brown, choanosome black. The choanosome is divided into honeycomb-like chambers and convoluted layers by very thick reinforced tracts of anisostyles, forming meshes that are elliptical in shape and 5800 ��m wide (Fig. 9). The ectosome is composed of a thick, dense feltwork of tangential and paratangential anisostyles. A single layer of erect isochiadiscorhabds lines the surface of the ectosome (Fig. 9). Megascleres (Table 1, 2) are anisostyles in two size categories: (1) slightly curved and thickened centrally, 621 (537���700) �� 14 (14) ��m); (2) thinner slightly curved centrally: type species: 530 (480���566) �� 9.6 (9.6) ��m) (Table 2). Microscleres (Fig. 9; Table 1, 2) are isochiadiscorhabds with three whorls of cylindrical, conical tubercles, the apex of each tubercular projection is acanthoses; some isochiadiscorhabds (10%) malformed or having a rudimentary forth whorl (Fig. 9): 48 (41���60) �� 9 (7.2���9.6). Sponge occurs in exposed coastal bays from Plettenberg Bay to Algoa Bay at depths of 9���40 m; abundant between 15���20 m in areas of strong surge where coastal upwelling occurs. Attached to hard rocky substrata associated with other invertebrates. Specimens are often abundant and occur within a metre distance from one another. The species contains biologically active pyrroloiminoquinone alkaloids, tsitsikammamines A and B and discorhabdin derivatives +14-Bromo-3-dihydrodiscorhabdin C and 14-Bromodiscorhabdin C that may have pharmacological potential [Cytotoxicity against Human Colon Tumour cell line (HCT 116)] (Antunes et al. 2004, 2005). ...Continued on the next page DNA sequence data. We sequenced partial COI of collected material from different localities; GenBank accession numbers: COI MK153285 ��� MK153287, MK153289, MK153292 ��� MK153293, MK153288, MK153290 ��� MK153291. Remarks. When T. (T.) favus was first described, it was considered to be rare (Samaai and Kelly 2002; Samaai et al. 2006) as it was only known from reefs shallower than 20 m at Tsitsikamma MPA and Algoa Bay. We have made spicule measurements and compared the isochiadiscorhabd morphology for a variety of Tsitsikamma specimens collected from St. Francis Bay (FB), Jeffreys Bay (JB), and the deeper reefs (30���40 m) off Grootbank, Plettenberg Bay (PB) (see also Parker-Nance et al. 2019) and found them to be similar in morphology and spiculation to T. (T.) favus described by Samaai et al. (2004) from Tsitsikamma MPA (T) and Algoa Bay (AB). These locations are within a 100 nautical mile (nmi) radius (T ��� PB = approx. 20 nmi; PB ��� JB = approx. 50 nmi; JB ��� AB = approx. 40 nmi) and form part of an extensive subsurface cold-water ridge area on the south coast (Agulhas ecoregion) (see Hutchings et al. 2002). Extensive surveys conducted along the South African coast in the last couple of years, failed to spot any T. favus south of 30��� S or north of 33��� S, thus suggesting this species has a geographic range that is influenced by coastal upwelling (see Fig. 2, 3). This is in marked contrast to the southeast coast environment were the three new Tsitsikamma spp. were retrieved. While we have expanded both the geographical and depth range of this species, its range is nonetheless small, and restricted to coastal upwelling zones on the south coast of South Africa. The morphology, spicule complement, and spicule sizes of the new material all fall within the size range of the type specimen (Table 2). Examination of the numerous T. (T.) favus specimens across its geographical ranged allowed us to assess the intra-specific morphological variation of the external morphology, colour, shape of oscula and areolate porefields and the shape of the isochiadiscorhabds and structure of the conical tubercles. We found that the majority of isochiadiscorhabds possess three whorls, with a small percentage being malformed or having a rudimentary forth whorl (Fig. 9). The variation in isochiadiscorhabd morphology of T. (T.) favus was also observed by Samaai and Kelly (2002), Kalinski et al. (2019) and Parker-Nance et al. (2019). Tsitsikamma (T.) favus have a characteristic base pigmentation of deep brownish black or liver brown that is often and variously tinged with deep green to dark turquoise; the ethanol preservative is always oily-looking and deeply pigmented; while the preserved specimens are dark brown. Some species are a lighter oak brown throughout. It is interesting to note that specimens of T. (T.) favus without discorhabdins are a lighter brown than their discorhabdin-containing siblings (Samaai and Kelly 2002). Tsitsikamma (T.) favus occurs with T. (T.) scurra in areas of local coastal upwelling or high wave surge. Tsitsikamma (T.) scurra differs from T. (T.) favus in the following characteristics: 1) Tsitsikamma (T.) scurra is lime green with a brownish surface in situ, and has long, hollow, strappy oscular fistules (Fig. 10), while T. (T.) favus is turquoise to dark brown or olive brown, semispherical and with short surface extensions (Fig. 8); 2) Tsitsikamma (T.) scurra has a folded globular thick encrusting growth structure with thin sandpaper-like ectosome (Fig. 10) (Samaai et al. 2006; Parker-Nance et al. 2019) while T. (T.) favus has a globular to semi-spherical morphology with a dense, thick ectosome; 3) Tsitsikamma (T.) scurra has larger, thicker anisostyles [thick 829 (774���882) �� 24 ��m; thin 669 (585���738) �� 17 ��m] than T. (T.) favus [thick 621 (537���700) �� 14 (14) ��m; thin 530 (480���566) �� 9.6 (9.6 ��m], but the isochiadiscorhabds are smaller than those found in the type species [T. (T.) scurra: 41 (38���45) �� 8 ��m; T. (T.) favus: 48 (41���60) �� 9 (7.2���9.6) ��m] (see Table 1); 4) Isochiadiscorhabds of Tsitsikamma (T.) scurra (Fig. 10) have three whorls of conico-cylindrical tubercules project at 45�� to the shaft in pairs whereas up to five complete whorls as well as many intermediate forms are found in T. (T.) favus . The tubercles projections are either arranged in pairs or in groups of three in T. (T.) favus; 5) The distribution of the two species is disparate; Tsitsikamma (T.) scurra is recorded from Hout Bay in the southern Benguela ecoregion while T. (T.) favus is recorded from the Agulhas ecoregion; 6) Tsitsikamma (T.) scurra only contains the biologically active pyrroloiminoquinone alkaloid derivative 14-Bromodiscorhabdin C (Antunes et al. 2004), while in T. (T.) favus the biologically active pyrroloiminoquinone alkaloid tsitsikammamines A and B and discorhabdin derivatives +14-Bromo-3-dihydrodiscorhabdin C and 14-Bromodiscorhabdin C are found (Samaai and Kelly, 2002; Antunes et al. 2004, 2005). Tsitsikamma (T.) favus can also be compared to T. (T.) nguni: (1) Tsitsikamma (T.) nguni is dark slate to black coloured in situ, thickly encrusting or hemispherical, and has small short, blunt rounded knob-shape or button like oscula and small round porefields; (2) Tsitsikamma (T.) nguni are also firm and ridged, with a thick ectosome visible, like in T. (T.) favus (Parker-Nance et al. 2019); (3) Tsitsikamma (T.) nguni have on average smaller anisostyles [thick 555 (428���672) �� 14 (10���19) ��m; thin 561 (449���832) �� 10 (3���14) ��m] (see also Tables 1, 2) (Parker-Nance et al. 2019) than T. (T.) favus [thick 621 (537���700) �� 14 (14) ��m; thin 530 (480���566) �� 9.6 (9.6) ��m]; (4) the isochiadiscorhabds are similar in average size (51 (40���60) ��m) to that found in type species [T. (T.) favus: 48 (41���60)] (Table 1, 2). Tsitsikamma (T.) favus and T. (T.) nguni differ in the number of the cylindrical-conical tubercles found on the isochiadiscorhabds; three per group in T. (T.) favus and four to six in T. (T.) nguni (Parker-Nance et al. 2019) (see also Fig. 5). The other apparent difference between T. (T.) favus and T. (T.) nguni is in the surface morphology, where T. (T.) favus have short, thick cauliform areolate porefields and lance-shaped oscula, oppose to blotchedshaped porefields and small button-shaped oscula in T. (T.) nguni. No intraspecific genetic diversity was found for the COI gene sequence of specimens of T. (T.) favus in this study. The same results were also found by Parker-Nance et al. (2019) for specimens of T. (T.) favus using the 28S rRNA gene sequences., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 417-423, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T. & Kelly, M. (2002) Family Latrunculiidae Topsent, 1922. In: Hooper, J. N. A. & Van Soest, R. W. M. (Eds.), Systema Porifera. A guide to the classification of sponges. Vol. 1. Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London and Moscow, pp. 708 - 720. https: // doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 78","Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Antunes, E. M., Beukes, D. R., Kelly, M., Samaai, T., Barrows, L. R. & Marshall, K. M. (2004) Cytotoxic pyrroloiminoquinones from four new species of South African latrunculid sponges. Journal of Natural Products, 67, 1268 - 1276. https: // doi. org / 10.1021 / np 034084 b","Antunes, E. M., Copp, B. R., Davies-Coleman, M. T. & Samaai, T. (2005) Pyrroloiminoquinone and related metabolites from marine sponges. Natural Products Reports, 22, 62 - 72. https: // doi. org / 10.1039 / b 407299 p","Samaai, T., Gibbons, M. J. & Kelly, M. (2006) Revision of the genus Latrunculia du Bocage, 1869 (Porifera: Demospongiae: Latrunculiidae) with descriptions of new species from New Caledonia and the Northeastern Pacific, Zootaxa, 1127 (1), 71. https: // doi. org / 10.11646 / zootaxa. 1127.1.1","Samaai, T., Govender, V. & Kelly, M. (2004) Cyclacanthia n. g. (Demospongiae: Poecilosclerida: Latrunculiidae incertae sedis), a new genus of marine sponges from South African waters, and description of two new species. Zootaxa, 725 (1), 1 - 18. https: // doi. org / 10.11646 / zootaxa. 725.1.1","Hutchings, L., Beckley, L. E., Griffiths, M. H., Roberts, M. J., Sundby, S. & Lingen, C. van der (2002) Spawning on the edge: spawning grounds and nursery areas around the southern African coastline. Marine and Freshwater Research, 53 (2), 307 - 318. https: // doi. org / 10.1071 / MF 01147","Kalinski, J. C. J., Waterworth, S. C., Siwe Noundou, X., Jiwaji, M., Parker-Nance, S., Krause, R. W. M., McPhail, K. L. & Dor- rington, R. A. (2019) Molecular Networking Reveals Two Distinct chemotypes in Pyrroloiminoquinone-Producing Tsitsikamm a favus Sp onges. Mar. Drugs, 17, 60. https: // doi. org / 10.3390 / md 17010060"]}

Tsitsikamma (Clavicaulis subgen. nov.) beukesi Samaai, Kelly, Payne and Ngwakum sp. nov. (Fig. 13, Table 1, 5) Material examined. Holotype SAMC-A090892, Amathole region Stn 3876, Eastern Cape, 33.285�� S, 27.909�� E, 103 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 02 Sep 2016. Type locality. Amathole, Eastern Cape Province, South Africa. Distribution. Amathole region off East London. Description. Sponge pedunculate, stalk length 30 mm, bulb area 45 mm diameter, stalk base 10 mm wide and stalk width 5 mm (Fig. 13A) with overall length of sponge 81 mm high. A very tough leathery purse surrounds a much softer choanosome that pulls away from the outer case upon collection. Surface undulating but smooth with cauliform areolate porefields, 2 mm diameter, 5 mm high. Oscules not visible in preserved specimens (Fig. 13A). Texture tough and rubbery. Barely compressible, tears so-so and breaks hard. No spicules or fibres. Colour in life is dark olive-green brown; in preservative the ectosome is brown, the choanosome is dark brown. Brown-green exudate. Skeleton. The overall architecture is purse-like, consisting of a thick ���purse��� of tangential megascleres (the ectosome) surrounding a much softer choanosome containing an irregular reticulation of wispy tracts of anisostyles (Fig. 13C). The choanosome detaches and falls away from the ectosomal purse in preservative. The choanosomal tracts range in width from 30���45 ��m thick and form meshes that are elliptical in cross section and 120 ��m wide. Microscleres are abundant throughout the choanosome and form an irregular palisade of oblique or erect microscleres, below which is a dense feltwork of tangential and paratangential styles approximately 144���173 ��m deep (Fig. 13C). Spicules (Table 1, 5; Fig. 13). Megascleres are anisostyles, in two size categories: (1) Large, straight or slightly sinuous, thickened centrally, fusiform, 740 (710���787) �� 14 (14) ��m; (2) straight or slightly sinuous, thickened centrally, fusiform: 622 (537���662) �� 14 (19) ��m (Table 1, 5). Microscleres are isochiadiscorhabds with three whorls of cylindrical, conical tubercles, medium whorl shorter, the apex of each tubercular projection is acanthose. Immature spicules have medium whorl present: 35 (28���38) �� 5 (5) ��m (Fig. 13B; Table 5). Substratum, depth range and ecology. Rocky reef and sandy plains between a depth of 50���100 m depth. DNA sequence data. No COI sequences. Etymology. This species is named after Prof. Denzil Beukes, in recognition for his contribution to the taxonomy and marine natural products chemistry of South African sponges, especially the family Latrunculiidae. Remarks. The live specimen of T. (C.) beukesi subgen. et sp. nov. is dark olive-green brown in colour with a smooth surface along with cauliform areolate porefields, unlike T. (C. subgen. nov.) pedunculata, where the surface is crowded with small, button-shaped oscules and with numerous circular fungiform areolate porefields. Tsitsikamma (C. subgen. nov.) pedunculata is also extremely resilient and leathery with a thick ectosome and salmon pink to pinkish brown in colour. The preserved specimen of T. (C.) beukesi subgen. et sp. nov. is a uniform brown colour with the choanosome being dark brown. In addition, Tsitsikamma. (C.) beukesi subgen. et sp. nov. differs from Tsitsikamma (C. subgen. nov.) pedunculata in the following characteristics: 1) Tsitsikamma (C. subgen. nov.) pedunculata differs considerably from Tsitsikamma (C.) beukesi subgen. et sp. nov. in the texture and thickness of the ectosome, internal tracts and surface structures which is smooth and undulating in Tsitsikamma (C.) beukesi subgen. et sp. nov.; 2) Tsitsikamma. (C.) beukesi subgen. et sp. nov. has larger, thicker anisostyles [thick 740 (710���787) �� 14 (14) ��m; thin 622 (537���662) �� 14 (19) ��m] than Tsitsikamma (C. subgen. nov.) pedunculata [thick 684 (591���728) �� 16 ��m; thin 536 (500���555) ��m], and the isochiadiscorhabds are larger than those found in T. (C. subgen. nov.) pedunculata [29 (27���30) �� 7 ��m]; Tsitsikamma. (C.) beukesi subgen. et sp. nov. [35 (28���38) �� 5 (5) ��m] (see Table 1); 3) Tsitsikamma. (C.) madiba subgen. et sp. nov. have isochiadiscorhabds with three whorls of long cylindrical, conical tubercles, and well-developed manubrium, as opposed to T. (C. subgen. nov.) pedunculata having two whorls of cylindrical conical spines; 5) Tsitsikamma (C.) beukesi subgen. et sp. nov. is recorded from the Amathole region, where the shelf is narrow and Agulhas current prevalent, while Tsitsikamma (C. subgen. nov.) pedunculata is recorded from Algoa Bay. Tsitsikamma (C.) beukesi subgen. et sp. nov. can also be compared to T. (C.) Madiba subgen. et sp. nov.: (1) T. (C.) madiba subgen. et sp. nov. is dark green to dark green brown coloured in situ, ovate, purse-like, not stalked, and has small, button-shaped oscules and cauliform areolate porefields; 2) T. (C.) madiba subgen. et sp. nov. has a very tough leathery purse like in T. (C. subgen. nov.) pedunculata; 3) Tsitsikamma. (C.) beukesi subgen. et sp. nov. have on average similar size range anisostyles (thick 740 (710���787) �� 14 (14) ��m; thin 622 (537���662) �� 14 (19) ��m) to T. (C.) madiba subgen. et sp. nov. (thick 737 (681���758) �� 19 (19) ��m; thin 612 (576���672) �� 19 (19) ��m), but the isochiadiscorhabds are smaller than those found in Tsitsikamma (C.) beukesi subgen. et sp. nov. [35 (28���38) �� 5 (5) ��m]; T. (C.) madiba subgen. et sp. nov. [32 (28���36) �� 5 (5) ��m] (see Table 4). 4) Tsitsikamma (C.) beukesi subgen. et sp. nov. have isochiadiscorhabds with three whorls of long cylindrical, conical tubercles, and well-developed manubrium, oppose to T. (C.) madiba subgen. et sp. nov. having two whorls of cylindrical conical spines. Like T. (C. subgen. nov.) michaeli, T. (C.) beukesi subgen. et sp. nov., is purse-like, with isochiadiscorhabds having three whorls of cylindrical, conical tubercles. Defining the differences between T. (C.) beukesi subgen. et sp. nov. and T. (C. subgen. nov.) michaeli is more challenging. Most apparent is the surface morphology. The surface of T. (C. subgen. nov.) michaeli are dominated by small short tube-shaped oscula, and large stalked cauliform porefields, with the porefields spilling over the supporting stalk (Parker-Nance et al. 2019). In contrast, T. (C.) beukesi subgen. et sp. nov. has very few oscules and cauliform porefields visible giving the surface a smooth appearance. Tsitsikamma (C. subgen. nov.) michaeli has a very tough leathery purse like appearance and the species does not have a stalk, although the basal attachment area of Tsitsikamma (C. subgen. nov.) michaeli is reinforced by a thickening of the ectosome (Parker-Nance et al. 2019). Tsitsikamma. (C.) beukesi subgen. et sp. nov. is stalked. Tsitsikamma (C.) beukesi subgen. et sp. nov. have on average smaller and thinner size range anisostyles (thick 740 (710���787) �� 14 (14) ��m; thin 622 (537���662) �� 14 (19) ��m) to T. (C. subgen. nov.) michaeli (thick 713 (537���935) �� 21 (12���30) ��m; thin 622 (439���769) �� 9 (4���13) ��m). The isochiadiscorhabds of Tsitsikamma (C.) beukesi subgen. et sp. nov. [35 (28���38) �� 5 (5) ��m] are smaller than those found in T. (C. subgen. nov.) michaeli [38 (34���44) ��m] (see Table 4). T. (C. subgen. nov.) michaeli have isochiadiscorhabds with three or four whorls and the acanthose tubercles are arranged in sets of two to four, radiating from the terminal ends (Parker-Nance et. al. 2019). Tsitsikamma (C. subgen. nov.) michaeli is often encrusted by a yellow species of Mycale (Mycale) also found on the surface of T. (C. subgen. nov.) pedunculata. Tsitsikamma (C.) beukesi subgen. et sp. nov. have isochiadiscorhabds with three whorls of long cylindrical, conical tubercles, and well-developed manubrium similar in size and structure to the apical whorl. The acanthose tubercles are well defined and arranged in sets of three on the manubrium and apical whorls and singular along the medium whorl. In summary, it is the general appearance of Tsitsikamma. (C.) beukesi subgen. et sp. nov., the shape and abundance of the porefields, and smaller size of the oscula, the colour, both in life and preserved, the shorter in average styles and isochiadiscorhabds, the slight difference in the arrangement of the acanthose tubercles, the similar appearance and size of the microsclere manubrium and apical whorl, the slightly thinner ectosome, that contribute as a whole to a species that is distinctly different in appearance from T. (C. subgen. nov.) pedunculata, T. (C.) madiba subgen. et sp. nov. and T. (C. subgen. nov.) michaeli., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 431-432, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683

Tsitsikamma (Clavicaulis subgen. nov.) pedunculata Samaai & Kelly, 2003 (Fig. 11, Table 1, 4) Tsitsikamma pedunculata, Samaai, Gibbons, Kelly and Davies-Coleman, 2003: 19���20, fig. 3G, 4F, 6B. Tsitsikamma pedunculata, Parker-Nance et al. 2019: 109���112, figure 2a���l; table 2. Material examined. Holotype ��� NHMUK 2003.1.10.2, Thunderbolt Reef, Cape Recife, St. Francis Bay, 34.053�� S, 25.689�� E, 40 m, collected by Patrick L. Colin, CRRF, 25 Feb 1999. Type locality. St. Francis Bay, Algoa Bay, Agulhas ecoregion, South Africa. Distribution. Algoa Bay, Agulhas ecoregion, South Africa. Diagnosis. Sponge pedunculate, body 5 cm diameter, up to 8 cm high, stalk 3���4 cm long, surface smooth and crowded with small, button-shaped oscules, 0.5���2 mm wide, 3 mm high and with numerous circular fungiform areolate porefields, 4���5 mm wide, 6 mm high, without membrane (Fig. 11A). A very tough leathery purse surrounds a much softer choanosome that pulls away from the outer case upon collection. Sponge is extremely resilient, leathery and compressible; colour in life is salmon pink to pinkish brown; between the oscules and areolate porefields the colour is dark pink (Fig. 11A); some specimens might appear as turquoise to dark purple in situ under low light conditions (see Fig. 2 in Parker-Nance et al. 2019); In alcohol, the ectosome is either brown, olive green, cream to tan colour and the choanosome is dark brown or tan. In preservative the ectosome is brown, the choanosome is dark brown (Fig. 11A). The choanosome consists of a thick ���purse��� of tangential megascleres (the ectosome) surrounding a much softer choanosome containing an irregular reticulation of wispy tracts of smaller styles; the choanosome detaches and falls away from the ectosomal purse when bought from the water (Fig. 11D). The ectosome is composed of a thick, dense feltwork of tangential and paratangential anisostyles. A single layer of erect isochiadiscorhabds lines the surface of the ectosome. Megascleres (Tables 1, 4) are styles, in two sizes: (1) slightly curved, thickened centrally 684 (591���728) �� 16 ��m; (2) thinner slightly curved centrally 536 (500���555) �� 11 ��m. Microscleres (Tables 1, 4) are isochiadiscorhabd (Fig. 11B, C) with two whorls of cylindrical-conical tubercles, median whorl absent, the apex of each tubercular projection is acanthose: 29 (27���30) �� 7 ��m. Sponge is found in an exposed coastal bay (Algoa Bay) rooted in sandy substrata, abundant between 34��� 40 m. The sponge contains biologically active pyrroloiminoquinone alkaloids, discorhabdin V and discorhabdin derivatives +14-Bromo-3-dihydrodiscorhabdin C that may have pharmacological potential [Cytotoxicity against Human Colon Tumour cell line (HCT 116)] (Antunes et al. 2004, 2005). Remarks. Tsitsikamma (C. subgen. nov.) pedunculata was described from a single specimen, the holotype, and was considered rare. Currently, the species is found to be abundant on deep reef systems between 34���40 m (Parker-Nance et al. 2019). Note that the in-situ photographs of turquoise to dark purple specimens, observed by Parker-Nance et al. (2019), are due to low light conditions during the survey period. Tsitsikamma (T.) favus, T. (T.) amatholensis sp. nov., T. (C. subgen. nov.) pedunculata and T. (C. subgen. nov.) michaeli are sympatric in Algoa Bay, but distinguishable in the field based on colouration and external morphology (see Table 1; Figs 8, 10E, 11A): T. (C. subgen. nov.) pedunculata is either salmon pink and stalked; T. (T.) favus is semispherical and dark brown; T. (C. subgen. nov.) michaeli is small olive green purse-like with short stalk-like structure (due to thickening of the ectosome) or sessile (see fig. 4 in Parker-Nance et al. 2019). Tsitsikamma (C. subgen. nov.) pedunculata also have a purse-like ectosome, being very leathery, forming a single chamber as opposed to the numerous honeycomb chambers typical of T. (T.) favus. The key taxonomic differences are the morphology and size of the isochiadiscorhabd; in T. (C. subgen. nov.) pedunculata it is much smaller, has only two cylindrical conical tubercules which is arranged in groups of three and the primary megascleres are slightly smaller and thinner in comparison to T. (C. subgen. nov.) michaeli and T. (T.) favus (see Tables 1, 2 and 4)., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 426-427, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Antunes, E. M., Beukes, D. R., Kelly, M., Samaai, T., Barrows, L. R. & Marshall, K. M. (2004) Cytotoxic pyrroloiminoquinones from four new species of South African latrunculid sponges. Journal of Natural Products, 67, 1268 - 1276. https: // doi. org / 10.1021 / np 034084 b","Antunes, E. M., Copp, B. R., Davies-Coleman, M. T. & Samaai, T. (2005) Pyrroloiminoquinone and related metabolites from marine sponges. Natural Products Reports, 22, 62 - 72. https: // doi. org / 10.1039 / b 407299 p"]}

Genus Tsitsikamma Samaai and Kelly, 2002 Type species. Tsitsikamma favus Samaai and Kelly, 2002, pg. 718, fig. 6A–G. Diagnosis. Hemispherical, pedunculate, or encrusting Latrunculiidae with a smooth surface, sometimes folded, covered with large cylindrical or volcano-shaped oscules and raised fungiform areolate porefields. Colour in life is brown, dark liver brown, green, dark turquoise or pinkish. Texture is either extraordinarily tough and leathery or soft and compressible. Megascleres are anisostyles, thickest centrally, often slightly irregular and wavy; microscleres are isochiadiscorhabds, with typically three substructures being cylindrical-conical tubercles. A choanosome permeated with rigid honeycomb-like chambers visible to the unaided eye, surrounding a much softer interior with wispy tracts, or a single purse-like (sac-like) chamber with softer interior. Microscleres are present in an irregular palisade on the surface ectosome and lining the internal tracts. Ectosomal skeleton consists of tangential layer of anisostyles. The morphology of the cylindrical-conical tubercles is diverse. Chemistry includes pyrroloquinoline alkaloids (tsitsikammamines), and the discorhabdin derivatives 14-Bromo-3-dihydrodiscorhabdin C and 14-Bromodiscorhabdin C. The genus is endemic to the Agulhas ecoregion of South Africa and restricted to cold upwelling areas on the south and southeast coasts (modified from Samaai and Kelly 2002; Samaai et al. 2003). Remarks. Parker-Nance et al. (2019) proposed two morphological groups, “ favus ” and “ pedunculata ”, for the genus Tsitsikamma, based on growth form and analysis of 28S rRNA sequences. We hereby propose the establishment of two new subgenera, Tsitsikamma (Tsitsikamma) subgen. nov. Samaai & Kelly, 2002, and T. (Clavicaulis) subgen. nov., for the type species T. favus and T. pedunculata, respectively, based on the two very different morphologies displayed by these two species. Species in the nominotypical subgenus Tsitsikamma, containing the type species T. favus, have a thick encrusting to hemispherical growth form, a rigid honeycombed internal structure composed of dense spicule tracts supporting the choanosome, and large, thick microscleres with three (or four) whorls. Species in the new subgenus Clavicaulis, with T. pedunculata as type, have a purse or sac-like morphology, with a short thick stalk, a choanosome made up of a single chamber and with small, stout microscleres with two or three whorls. A multivariate analysis of spicule measurements (anisostyle length, discorhabd length, shaft and whorl length), was used to analyse the relative contribution of each morphometric variable in the distinction of the proposed new species and subgenera. Despite the obvious species-level differences in gross morphology, the use of morphometric variation, as an estimator of species distinction, was not able to separate the proposed new species, but separated T. favus , T. pedunculata and T. scurra Samaai, Gibbons, Kelly & Davies-Coleman, 2003 from each other (Fig. 4). Similarly, DNA barcoding of the mitochondrial COI and the nuclear ITS of Tsitsikamma specimens, failed to clearly differentiate between specimens, despite clear morphological differences (Figs 5, 6, 7), a finding also obtained by Parker-Nance et al. (2019) based on COI and 28S rRNA gene sequences. The Tsitsikamma specimens all had the same COI haplotype, which clustered among previously published sequences of Tsitsikamma (Fig. 6). All specimens were grouped together into one cluster irrespective of whether they were stalked, oval, semispherical, rigid/soft, sac-/purse- or honeycomb-like or possessed two or three whorls of isochiadiscorhabd microscleres. No intra-specimen variation was found for the COI partition for the Tsitsikamma specimens, with the notable exception of one specimen, Tsitsikamma SAMC-A091440. This is rather surprising because COI sequences are considered variable and quite informative for population genetics and phylogeographic studies of demosponges (LópezLegentil et al. 2009; Reveillaud et al. 2011; Carella et al. 2016). The high genetic similarity raises the question of how far these sponges are phylogenetically separated. The genetic results could also imply that the Tsitsikamma sponges included here represent one single species. Notwithstanding the above, strictly identical COI sequences have been reported for other sponges, e.g. Antarctic species of Rossella Carter, 1872, Cinachyra Sollas, 1886, Antarctotetilla Carella, Agell, Cárdenas & Uriz, 2016 and Siberian freshwater sponges. These studies showed that the mitochondrial COI M1-M6 partition failed to separate species within these cold-water genera, despite clear morphological differences (Schr̂der et al. 2003; Carella et al. 2016; Vargas et al. 2017). Vargas et al. (2015) also observed instances where DNA barcoding (COI) could not correctly assign a specimen to its species because multiple species share identical or near identical barcodes (e.g. Latrunculia, Artemisina Vosmaer, 1885, Acanthorhabdus Burton, 1929, Iophon Gray, 1867). In this study, as in the case for Latrunculia, Rossella and Iophon, Tsitsikamma is a cold-water genus and share identical barcode sequences, despite clear morphological differences (habit, isochiadiscorhabd morphology (Fig. 5), etc.). The ITS sequences for Tsitsikamma (Fig. 7) were also uninformative and did not discriminate between the proposed morphological species (see Table 1; Fig. 5). This was expected because of the slow evolving nature of this partition. The ITS tree recovered was different to the COI tree. The findings highlight the limitations of the morphometric measurements and gene sequences for distinguishing between specimens and the importance of rigorous morphological examinations of specimens within the family Latrunculiidae. The results may also indicate contrast-ing evolutionary rates between sponges in the family Latrunculiidae as found in other Demospongiae groups (Heim et al. 2006). As for Antarctic Tetillidae Sollas, 1886 (Carella et al. 2016), our results suggest either a particularly slow genetic evolutionary rate of the COI and ITS markers or a recent radiation with phenotypic characters evolving faster than the genetic markers studied. Until more data become available (e.g., further studies on cold-water Demospongiae families/genera vs. temperate/tropical families/genera) we prefer to maintain the proposed new species as separate due to the clear differences in morphology and isochiadiscorhabd structure, habit and distribution. The COI partition was informative enough to confirm the monophyly of family Latrunculiidae and the relationships between the various genera in the family (Fig. 6). Our results concur with Parker-Nance et al. (2019) in supporting the monophyly of Latrunculiidae. Based on morphological comparisons (habit, isochiadiscorhabd morphology, etc.) we describe three new species of Tsitsikamma within the two new subgenera: T. (T.) amatholensis sp. nov.; T. (C.) madiba subgen. et sp. nov.; T. (C.) beukesi subgen. et sp. nov., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 414-417, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T. & Kelly, M. (2002) Family Latrunculiidae Topsent, 1922. In: Hooper, J. N. A. & Van Soest, R. W. M. (Eds.), Systema Porifera. A guide to the classification of sponges. Vol. 1. Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London and Moscow, pp. 708 - 720. https: // doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 78","Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Reveillaud, J., Van Soest, R., Derycke, S., Picton, B., Rigaux, A. & Vanreusel, A. (2011) Phylogenetic relationships among NE Atlantic Plocamionida Topsent (1927) (Porifera, Poecilosclerida): under-estimated diversity in reef ecosystems. PLOS, One, 6 (2), e 16533. [PMID: 21347368] https: // doi. org / 10.1371 / journal. pone. 0016533","Carella, M., Agell, G., Cardenas, P. & Uriz, M. J. (2016) Phylogenetic reassessment of Antarctic Tetillidae (Demospongiae, Tetractinellida) reveals new genera and genetic similarity among Morphologically Distinct Species. PLOS ONE, 11 (8), e 0160718. https: // doi. org / 10.1371 / journal. pone. 0167011","Carter, H. J. (1872) On two new sponges from the Antarctic Sea, and on a new species of Tethya from Shetland; together with observations on the reproduction of sponges commencing from zygosis of the sponge animal. Annals and Magazine of Natural History, Series 4, 9 (54), 409 - 435, pls. XX-XXII. https: // doi. org / 10.1080 / 00222937208696612","Sollas, W. J. (1886) Preliminary account of the Tetractinellid sponges Dredged by H. M. S. ' Challenger' 1872 - 76. Part I. The Choristida. Scientific Proceedings of the Royal Dublin Society, New Series, 5, 177 - 199.","Vargas, S., Kelly, M., Schnabel, K., Mills, S., Bowden, D. & W ˆ rheide, G. (2015) Diversity in a Cold Hot-Spot: DNA-Barcoding Reveals Patterns of Evolution among Antarctic Demosponges (Class Demospongiae, Phylum Porifera). PLoS ONE, 10 (6), e 0127573. https: // doi. org / 10.1371 / journal. pone. 0133096","Vosmaer, G. C. J. (1885) The Sponges of the ' Willem Barents' Expedition 1880 and 1881. Bijdragen tot de Dierkunde, 12 (3), 1 - 47, pls. I-V. https: // doi. org / 10.1163 / 26660644 - 01201001","Burton, M. (1929). Porifera. Part II. Antarctic sponges. British Antarctic (' Terra Nova') Expedition, 1910. Natural History Report, London, Zoology, 6 (4), 393 - 458, pls. I-V.","Heim, I., Nickel, M. & Brummer, F. (2006) Cytochrome oxidase subunit I-Opportunities and limits for molecular species discrimination. In: Custodio, M. R., Lobo-Hajdu, G., Hajdu, E. & Muricy, G. (Eds.), Biodiversity, innovation, sustainability: book of 66 abstracts / 7 th International Sponge Symposium. Armac \" o de Buzios, Rio de Janeiro, pp. 286."]}

Tsitsikamma (Clavicaulis subgen. nov.) madiba Samaai, Kelly, Payne and Ngwakum sp. nov. (Fig. 12, Table 4) Material examined. Holotype SAMC-A090883, Amathole region Stn 3813, East London, Eastern Cape, 32.681° S, 28.458° E, 52–54 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 26 Aug 2016. Paratypes. SAMC-A 090882, Thunderbolt reef, Cape Recife, Port Elizabeth, 34.052° S, 25.689° E, 38–40 m, collected by Patrick L. Colin, CRRF, 30 Mar 2000; SAMC-A090884, SAMC-A090885, SAMC-A090886, SAMC-A090889, Amathole region Stn 3893, East London, Eastern Cape, 33.163° S, 27.774° E, 36 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 03 Sep 2016; SAMC-A090887, Amathole region Stn 3832, East London, Eastern Cape, 32.759° S, 28.410° E, 45 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 27 Aug 2016; SAMC-A090888, Amathole, Stn 3872, East London, Eastern Cape, 32.950° S, 28.066° E, 40.5 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 31 Aug 2016. Type locality. Amathole, Eastern Cape Province, South Africa. Distribution. Algoa Bay, Port Elizabeth and Amathole region off East London. Description. Sponge wrinkled and purse-like, not stalked, but ovate, attached to a narrow base, 20 mm wide by 45 mm length by 36.5 mm bulb diameter (Fig. 12A, B). A very tough leathery purse surrounds a much softer choanosome that pulls away from the outer case upon collection. Surface undulating but smooth, crowded with small, button-shaped oscules, 1–2 mm diameter, 2 mm high and cauliform areolate porefields, 2–3 mm diameter, 6 mm high (Fig. 12A).. In preserved specimens the oscules are not visible (Fig. 12B). Texture rubbery, leathery and tough. Moderately compressible, breaks easily. No spicules visible. Colour in life is dark green to dark green brown; in preservative, the ectosome is brown, the choanosome is dark brown (Fig. 12A). Green/brown exudate present. Skeleton. The overall architecture is purse-like, consisting of a thick ‘purse’ of tangential megascleres surrounding a much softer choanosome containing an irregular reticulation of wispy tracts of anisostyles. No honeycomb chambers present as in the Tsitsikamma subgenus. The choanosome detaches and falls away from the ectosomal purse in preservative. The choanosomal tracts range in width from 60–100 μm thick and form meshes that are elliptical in cross section and 350 μm wide. Microscleres are abundant throughout the choanosome and form an irregular palisade of oblique or erect microscleres, below which is a dense feltwork of tangential and paratangential styles approximately 560–960 μm deep (Fig. 12G, H). Spicules (Tables 1, 4; Fig. 12 C-F). Megascleres anisostyles, in two size categories: (1) Large, thick, straight or slightly sinuous, thickened centrally, hastate, 737 (681–758) × 19 (19) μm, sometime strongylote modification present; (2) straight or slightly sinuous, thickened centrally, hastate: 612 (576–672) × 19 (19) μm (Tables 1, 4). Microscleres are small isochiadiscorhabds (Fig. 12 C-F) with two whorls of cylindrical, conical tubercles, medium whorl absent, the apex of each tubercular projection is acanthose. Some isochiadiscorhabds (15%) shows signs of a small developing rudimentary spine centrally (Fig. 12F): 32 (28–36) × 5 (5) μm (Table 4). Substratum, depth range and ecology. Rocky reef with 1 m relief rocky bottom with areas of sand, 38–40 m depth. DNA sequence data. We sequenced partial COI of collected material from different localities; GenBank accession numbers: COI MK153272 – MK153275; MK016477. Etymology. Named in honour of the great South African president Nelson Rolihlahla Mandela, also known by his clan name “Madiba”, liberation struggle hero and Nobel Peace Prize laureate, who passed away on 5 December 2013 at the age of 95. Remarks. The anisodiscorhabds of Tsitsikamma (C.) madiba subgen. et sp. nov. and T. (C. subgen. nov.) pedunculata have two whorls of cylindrical conical spines, the median and subsidiary conical spines are absent. In about 20% of isochiadiscorhabds in all specimens of T. (C.) madiba subgen. et sp. nov., there is a small medium rudimentary conical-cylindrical spine protruding from the shaft (Fig. 12F). This character is absent in the isochiadiscorhabds of T. (C. subgen. nov.) pedunculata, which are stouter, shorter and have a wide manubrium. The shaft length in T. (C.) madiba subgen. et sp. nov. is longer than in T. (C. subgen. nov.) pedunculata. Tsitsikamma (C.) madiba subgen. et sp. nov. has larger, thicker anisostyles [thick 737 (681–758) × 19 (19) μm; thin 612 (576–672) × 19 (19) μm] than T. (C. subgen. nov.) pedunculata (thick 684 (591–728) × 16 μm; thin 536 (500–555) µm), but the isochiadiscorhabds are smaller than those found in T. (C. subgen. nov.) pedunculata [29 (27–30) × 7 µm]; T. (C.) Madiba subgen. et sp. nov. [32 (28–36) × 5 (5) μm] (see Table 1, 4). Tsitsikamma (C. subgen. nov.) michaeli differs from T. (C.) madiba subgen. et sp. nov. in the following characteristics: 1) Tsitsikamma (C. subgen. nov.) michaeli is a small light olive-green sponge with a short stalk and surface dominated by small short tube-shaped oscula and large stalked cauliform porefields, while T. (C.) madiba subgen. et sp. nov. is dark green, oval shaped with small button-like oscules and large cauliform porefields (see Table 1); 2) Tsitsikamma (C. subgen. nov.) michaeli have smaller isochiadiscorhabd (Table 4), and large anisostyles similar in average size to that found in T. (C. subgen. nov.) madiba sp. nov.; 3) The isochiadiscorhabds of T. (C. subgen. nov.) michaeli differ in the number of the conical-cylindrical tubercles having four or more groups, as opposed to two to three per group in T. (C.) madiba subgen. et sp. nov.; 4) the anisodiscorhabds of T. (C.) madiba subgen. et sp. nov. have two whorls of cylindrical-conical spines, the median and subsidiary conical spines are absent, as opposed to T. (C. subgen. nov.) michaeli having three to four whorls of cylindrical conical spines (Fig. 5; Table 1). Tsitsikamma (C.) madiba subgen. et sp. nov. is distinguishable from other Tsitsikamma species in the form of the sponge, being wrinkled and purse-like, no stalk, oval shaped, but attached to a narrow base, and in the microornamentation and structure of the isochiadiscorhabd. The Tsitsikamma (C.) madiba subgen. et sp. nov. is also encrusted by a grey/white encrusting didemnid ascidian (Fig. 12A). There was no intraspecific genetic diversity for the COI gene sequences for specimens of T. (C. subgen. nov.) michaeli and no interspecific genetic diversity for T. (C. subgen. nov.) michaeli and the other specimens., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 429-431, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683

Subgenus Tsitsikamma Samaai & Kelly, 2002 Type species. Tsitsikamma favus Samaai and Kelly, 2002: 718; fig. 6A–G. Diagnosis. Tsitsikamma species that are thickly encrusting to hemispherical attached to the substratum through a common base; have a rigid, thick honeycomb chamber like choanosome reinforced by very large, thick anisostyles. The isochiadiscorhabd microscleres are large, having a thick shaft and three sets of cylindrical-conical tubercles protruding from the shaft. Remarks. We herewith establish the nominotypical subgenus Tsitsikamma subgen. nov. Samaai and Kelly, 2002, for the species T. favus, T. scurra, T. nguni Parker-Nance, 2019 and T. amatholensis sp. nov., based on the possession of dense megasclere tracts that form a rigid, honeycomb-like structure, with chambers visible to the unaided eye., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on page 417, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T. & Kelly, M. (2002) Family Latrunculiidae Topsent, 1922. In: Hooper, J. N. A. & Van Soest, R. W. M. (Eds.), Systema Porifera. A guide to the classification of sponges. Vol. 1. Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London and Moscow, pp. 708 - 720. https: // doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 78"]}

Subgenus Clavicaulis Samaai, Kelly, Payne and Ngwakum subgen. nov. Type species. Tsitsikamma pedunculata Samaai, Gibbons, Kelly and Davies-Coleman, 2003: 19; 19–20, fig. 3G, 4F, 6B. Diagnosis. Tsitsikamma species that are either stalked wrinkled/smooth and non-stalked wrinkled/smooth attached by a narrow base, having a single purse-like chamber with a much softer interior of wispy tracts. The isochiadiscorhabd microscleres have either two or three cylindrical-conical tubercles projecting from the shaft, though sometimes the third whorl is rudimentary. Etymology. club-shaped (clavi - combining form NL, fr. L, fr. clava = club), Stalk (caul- or cauli- or caulocombining form (cauli - fr. L caulis = stalk) Remarks. We herewith establish the new subgenus Clavicaulis subgen. nov. for the species Tsitsikamma pedunculata, T. michaeli Parker-Nance, 2019, T. madib a sp. nov. and T. beukesi sp. nov., based on the stalked, sack or purse-shaped body morphology of the sponges, which, in effect, forms a single, reinforced chamber, unlike subgenus Tsitsikamma which is characterised by the possession of a rigid, honeycomb-like structure, with chambers visible to the unaided eye., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on page 426, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1"]}

Tsitsikamma (Clavicaulis subgen. nov.) michaeli Parker-Nance, 2019 (Fig. 11, Table 1, 4) Type & locality (not examined). Holotype — SAIAB 207202 Evans Peak, Algoa Bay, Eastern Cape Province, - 33.84548, 25.81663, 30–34 m depth, 12 November 2015. Material examined. SAMC-A090890, SAMC-A091441, SAMC-A090891, Evans Peak, 33.842° S, 25.816° E, Algoa Bay, Port Elizabeth, 30 m, collected by Rhodes University, May 2010; SAMC-A090893, Amathole region Stn 3813, Eastern Cape, 32.681° S, 28.458° E, 52–55 m, RV Ellen Khuzwayo, dredge, 26 Aug 2016. Distribution. Algoa Bay, Port Elizabeth and Amathole region off East London, Eastern Cape, South Africa. Diagnosis. Small olive-green, purse shaped sponge up to 5 cm high (2 cm stalk and 3 cm rounded head), surface crowded with small short tube-shaped oscula and numerous large stalked cauliflower porefields (Fig. 11E; see also fig. 4 in Parker-Nance et al. 2019). Sponge is fleshy and compressible in life, dark to olive green in life, olive to tan in preservative (Fig. 11E). The sponges were collected from a moderately rugged rocky bottom with patches of sand between rocks, from Evans Peak, Algoa Bay, South Africa, at 33– 38 m. Megascleres (Tables 1, 4) are hastate or blunt styles, centrally thickened; (1) 713 (537–935) × 21 (12–30) μm and (2) long slender styles 622 (439–769) × 9 (4–13) μm, with occasional short thick anisostrongyles. Microscleres (Table 1, 4; Fig. 11F) are acanthose isospinodiscorhabds with three or four whorls, 38 (34–44) μm. The terminal whorls consist of a clearly larger manubrium and apical whorls. The acanthose tubercles are arranged in sets of two to four, radiating from the terminal ends. Skeletal architecture has no reinforced tracts. The ectosome is thick and encapsulates the choanosome forming a protected layer (Fig. 11G) (modified from Parker-Nance et al. 2019). The species occupies the same reef habitat as that of T. (C. subgen. nov.) pedunculata, at depths between 33– 38 m. The sponge is often encrusted by an unidentified species of Mycale (Mycale). DNA sequence data. We sequenced partial COI of collected material from different localities; GenBank accession numbers: COI MK016478 – MK016479. Remarks. This species was described by Parker-Nance et al. (2019). For further details on the species and for comparative purposes with other Tsitsikamma species see Parker-Nance et al. (2019). There was no intraspecific genetic diversity for the COI gene sequences for specimens of T. (C. subgen. nov.) michaeli and no interspecific genetic diversity for T. (C. subgen. nov.) michaeli, T. (T.) favus and the other specimens. Parker-Nance et al. (2019) also found no intraspecific genetic diversity for the 28S rRNA gene region for T. (C. subgen. nov.) michaeli and no interspecific genetic diversity for T. (C. subgen. nov.) michaeli and T. (C. subgen. nov.) pedunculata was observed in this work. They did however find interspecific genetic diversity of between 0.48–0.65 % between T. (C. subgen. nov.) michaeli and T. (T.) favus and 0.32 % between T. (C. subgen. nov.) michaeli and T. (T.) scurra. The latter finding further strengthen the subgenera divisions., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 428-429, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683

Tsitsikamma (Tsitsikamma) amatholensis Samaai, Kelly, Payne and Ngwakum sp. nov. (Fig. 10, Tables 1, 3) Material examined. Holotype. SAMC-A090878, Amathole region Stn 3872, Eastern Cape, 32.950�� S, 28.066�� E, 40.5 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 31 Aug 2016. Paratypes. SAMC-A090877, Amathole region Stn 3813, Eastern Cape, 32.681�� S, 28.458�� E, 52���55 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 26 Aug 2016; SAMC-A090879, Amathole region Stn 3872, Eastern Cape, 32.950�� S, 28.066�� E, 40.5 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 31 August 2016; SAMC-A090880, Amathole region Stn 3807, Eastern Cape, 33.134�� S, 27.768�� E, 33 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 02 Mar 2016; SAMC-A 090881, Port Alfred, Eastern Cape, 32.933�� S, 28.080�� E, 40.5 m, dredge sampling, RV Ellen Khuzwayo, collected by R. Payne, dredge, 31 Aug 2016. Other material examined. SAMC-A091436, Evans Peak, Algoa Bay, Port Elizabeth, 33.842�� S, 25.816�� E, 30 m, collected by Rhodes University, May 2010; SAMC-A091430, SAMC-A091431, SAMC-A091432, SAMC-A 091433, SAMC-A091434, SAMC-A091435, Evans Peak, Algoa Bay, Port Elizabeth, 33.842�� S, 25.816�� E, 30 m, collected by Rhodes University, May 2010; SAMC-A091437, Amathole region Stn 3737, Eastern Cape, 32.751�� S, 28.415�� E, 31 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 23 Feb 2016; SAMC-A091438, Amathole region Stn 3807, Eastern Cape, 33.134�� S, 27.768�� E, 33 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 02 Mar 2016; SAMC-A091439, Amathole region Stn 3831, Eastern Cape, 32.759�� S, 28.411�� E, 47 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 27 Aug 2016; SAMC-A091440, Amathole region Stn 3832, Eastern Cape, 32.759�� S, 28.410�� E, 45 m, RV Ellen Khuzwayo, collected by R. Payne, dredge, 27 Aug 2016. Type locality. Amathole, Eastern Cape Province, South Africa. Description. Thickly encrusting, sometimes hemispherical, 30 mm long �� 30 mm wide �� 20 mm thick attached by a common base to the surface (Fig. 10). Specimens vary in thickness from 3���30 mm. Surface undulating but smooth and crowded with cylindrical, long lance-shaped oscules similar to those observed in T. scurra, 5 mm wide, 10 mm high and with pedunculate cauliform areolate porefields, 1 mm wide, 2���3 mm high, with no membrane. Texture tough, firm. Medium to slightly compressible, tears not easily, difficult to break. Thick ectosome visible, 0.5 mm thick. Colour in life dark brown to dark olive green; in preservative dark brown with green exudate, sometimes cream (Fig. 10; Table 1). Skeleton. The choanosome is divided into honeycomb-like chambers, thick reinforced tracts of anisostyles, forming meshes that are elliptical in shape (Fig. 10). Within and between the chambers and convoluted layers the skeleton consists of an ill-formed, irregular reticulation of small anisostyles; these tracts range in width from 100��� 350 ��m. Microscleres are isochiadiscorhabds, and these are abundant throughout the choanosome. The ectosome is composed of a thick, dense feltwork of tangential and paratangential anisostyles approximately 100 ��m wide. This layer is present in the fistulae, with anisostyles disposed in a compact regular vertical to oblique arrangement supporting the cauliform areolate structures. A single layer (sometimes double layer) of erect isochiadiscorhabds (45 ��m wide) lines the surface of the ectosome (Fig.10). Spicules (Table 1, 3). Megascleres are anisostyles, in two size categories: (1) straight or slightly sinuous, thickened centrally, fusiform, 721 (681���758) �� 19.2 (19.2) ��m; (2) straight or slightly sinuous, thickened centrally, fusiform: 584 (509���653) �� 19.2 (19.2) ��m. Microscleres are isochiadiscorhabds with three whorls of cylindrical-conical tubercles, the apex of each tubercular projection is acanthose: 45 (38���58) �� 7 (4 ���7) ��m (Fig. 10). Distribution. Southeast Agulhas ecoregion from Port Elizabeth to East London, South Africa. Substratum, depth range and ecology. Locally common off East London, Amathole region and Port Elizabeth, Evans Peaks, at a depth range of 30 to 55 m in areas with strong current. DNA sequence data. We sequenced partial COI of collected material from different localities; GenBank accession numbers: COI MK153277 ��� MK153284. Etymology. The species name reflects the type locality, Amathole, a district in the Eastern Cape, South Africa. ���Amathole��� means ���calves��� in the Xhosa language, and refers to the forested mountain range that forms the northern boundary of the district. Remarks. Tsitsikamma (T.) amatholensis sp. nov. was first collected in 2010 but identified only as ��� Tsitsikamma sp. undescribed��� based on the external morphological characteristics and isochiadiscorhabd structure (TS unpublished data) of the specimens. Matcher et al. (2017) generated partial 28S rRNA sequences for six Tsitsikamma specimens collected from the same locations as the 2009/2010 collections and confirmed the presence of two unidentified Tsitsikamma species (see Table 1 and Fig. 3b in Matcher et al. 2017). Parker-Nance et al. (2019) described a new species from these collections (Evans Peak) as T. michaeli, an observation we confirmed after re-examining all the Walmsley/Matcher specimens (Supplementary Table S1). The other Walmsley et al. (2012) specimens were identified as T. (T.) favus by TS. The second unidentified Tsitsikamma sp. is here described as a specimen of Tsitsikamma (T.) amatholensis sp. nov. Tsitsikamma (T.) amatholensis sp. nov. differs from T. (T.) favus , T. (T.) scurra and T. (T.) nguni in the following characteristics: 1) Tsitsikamma (T.) amatholensis sp. nov. is dark brown to dark olive green in situ, and has large hollow lance-shaped oscules and numerous short stalked cauliform porefields, while T. (T.) favus is turquoise to dark brown, semispherical and with short surface extensions, T. (T.) scurra is lime green with a brownish surface in situ, and has long, hollow, strappy oscular fistules and T. (T.) nguni is dark slate-coloured with small short, blunt rounded knob-shaped or button-like oscula; 2) Tsitsikamma (T.) amatholensis sp. nov. is thickly encrusting, sometimes hemispherical with a thick ectosome, while Tsitsikamma (T.) scurra has a folded globular thick encrusting growth structure with thin sandpaper-like ectosome (Samaai et al. 2006; Parker-Nance et al. 2019), T. (T.) favus and T. (T.) nguni has a thickly encrusting, globular to semi-spherical morphology with a dense, thick ectosome; 3) Tsitsikamma (T.) scurra has larger, thicker anisostyles [thick 829 (774���882) �� 24 ��m; thin 669 (585���738) �� 17 ��m] than Tsitsikamma (T.) amatholensis sp. nov. [thick 721 (681���758) �� 19.2 (19.2) ��m; thin 584 (509���653) �� 19.2 (19.2) ��m] while T. (T.) favus [thick 621 (537���700) �� 14 (14) ��m; thin 530 (480���566) �� 9.6 (9.6) ��m] and T. (T.) nguni [thick 555 (428���672) �� 14 (10���19) ��m; thin 561 (449���832) �� 10 (3���14) ��m] are smaller than those of T. (T.) amatholensis sp. nov. and thinner on average (see Tables 1, 2, 3); 4) the isochiadiscorhabds with long cylindrical, conical tubercles are similar in size found in T. (T.) scurra [41 (38���45) �� 8 ��m], but smaller in average than those in T. (T.) favus [48 (41���60) �� 9 (7.2���9.6) ��m] and T. (T.) nguni (Tables 1, 2, 3; Fig. 5, 9, 10); 5). The number of cylindrical-conical tubercles surrounding the apical whorl and manubrium in T. (T.) amatholensis sp. nov. is six per group, oppose to three and four to six per group surrounding the manubrium only in T. (T.) favus in T. (T.) nguni, respectively (Parker-Nance et al. 2019). Apart from this, the cylindrical-conical tubercles also differ; T. (T.) favus and T. (T.) nguni possess two pairs, while T. (T.) scurra and T. (T.) amatholensis possess three pairs arranged in a triangle; 6) The distribution of the two species is disparate: Tsitsikamma (T.) scurra is recorded from Hout Bay in the southern Benguela ecoregion while T. (T.) favus , T. (T.) amatholensis sp. nov. and T. (T.) nguni are recorded from the Agulhas ecoregion. In Algoa Bay T. (T.) amatholensis sp. nov. is sympatric with T. (T.) favus, T. pedunculata and T. michaeli and in Amathole the species is sympatric with the other new species (as described below), and can be differentiated on subtle differences in colour, gross morphology and isochiadiscorhabd morphology. The gross morphology of T. pedunculata and T. michaeli are, however, quite different from those of T. (T.) favus and T. (T.) amatholensis sp. nov., forming the basis for establishment in this work of a new subgenus Tsitsikamma (Clavicaulis) subgen. nov. There was no intraspecific genetic diversity for the COI gene sequences for specimens of T. (T.) amatholensis sp. nov. and no interspecific genetic diversity for T. (T.) amatholensis sp. nov., T. (T.) favus and the other specimens., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 424-426, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Matcher, G. F., Waterworth, S. C., Walmsley, T. A., Matsatsa, T., Parker-Nance, S. & Davies-Coleman, M. T. (2017) Keeping it in the family: Coevolution of latrunculid sponges and their dominant bacterial symbionts. Microbiology open, 6, 1 - 13. https: // doi. org / 10.1002 / mbo 3.417","Walmsley, T. A., Matcher, G. F., Zhang, F., Hill, R. T., Davies-Coleman, M. T. & Dorrington, R. A. (2012) Diversity of bacterial bommunities associated with the Indian Ocean Sponge Tsitsikamma favus that contains the bioactive Pyrroloiminoquinones, Tsitsikammamine A and B. Marine Biotechnology, 14 (6), 681 - 691. https: // doi. org / 10.1007 / s 10126 - 012 - 9430 - y","Samaai, T., Gibbons, M. J. & Kelly, M. (2006) Revision of the genus Latrunculia du Bocage, 1869 (Porifera: Demospongiae: Latrunculiidae) with descriptions of new species from New Caledonia and the Northeastern Pacific, Zootaxa, 1127 (1), 71. https: // doi. org / 10.11646 / zootaxa. 1127.1.1"]}

Cyclacanthia bellae (Samaai & Kelly, 2003) (Fig. 15A, Tables 6, 7) Latrunculia bellae Samaai & Kelly, 2003: 14–15, fig. 3C, 4D, 5D. Cyclacanthia bellae, Samaai, Govender and Kelly (2004): 5 –6, figs. 2F, 3A, E–F, 4A, Table 1. Type & locality (not examined). Holotype—NHMUK 2003.1.10.1, Algoa Bay, Ryi Banks, 8 nm east of Port Elizabeth, South Africa, 33.992° S, 25.876° E, 22 m, collected by Patrick L. Colin, CRRF, 12 Oct 1998. Diagnosis. Thinly encrusting sponge (Fig. 15A), 5 mm thick in life, surface crowded with very small conical oscules and numerous thin-lipped crater-like areolate porefields. Compressible in life, slightly felty to the touch, emerald green in life, dark chocolate brown internally and in preservative. The sponges were collected from a moderately rugged rocky bottom with patches of sand between rocks, on Ryi Banks, Algoa Bay, south-eastern South Africa, at 10– 22 m. Spicules. Megascleres are styles. Smooth, hastate, centrally thickened straight or slightly sinuous styles, 364 (319–400) long by μm wide. Microscleres (Figs. 14, 15) are acanthose isospinodiscorhabds. The median whorl is composed of four groups of discrete spines distributed evenly around the shaft, the spines of the manubrium and apical whorl are slanted obliquely from the median whorl and the spines are orientated at different angles within each whorl. A single spike protrudes from the apex and base of the spicule, all spines are markedly acanthose, 46 (44–51) μm long. Skeleton (Fig. 15C) is made up of large dense swathes of megascleres, 230–250 μm wide, emerge from the base of the sponge towards the upper choanosome, where they diverge to form loose brushes and a wispy polygonal reticulation of tracts c. 60–180 μm wide, forming a mesh c. 230 μm wide. Interstitial megascleres and microscleres are abundant. The ectosome of tangentially arranged styles is c. 320 μm thick and is aligned by an irregular palisade of densely packed isospinodiscorhabds. The species occurs on moderate rugged rocky bottoms with patches of sand between rocks between a depth of 10– 22 m. The sponge contains novel biologically active pyrroloquilonine alkaloids, discorhabdin M and G, Makaluvic acid A and discorhabdin derivatives that may have pharmacological potential [Cytotoxicity against Human Colon Tumour cell line (HCT 116)] (Antunes et al., 2005) (modified from Samaai et al. 2004). Distribution. Algoa Bay, Eastern Cape coast, Agulhas ecoregion, South Africa Remarks. Cyclacanthia are similar in their general morphology to other Latrunculiidae, with fistular oscula and areolate porefields distributed over the sponge surface. However, within Latrunculiidae, Cyclacanthia species are notably thinly encrusting with a soft, compressible texture and have acanthose isospinodiscorhabds as microscleres (Fig. 14) (Samaai et al. 2004). Spines develop simultaneously on the end of a straight thin protoisochiarhabd shaft followed by median spines (see fig. 2D in Samaai et al. 2004). The spines develop directly from the shaft and do not transform into circular plates with serrated margins as in Latrunculia or into truncate tubercles with rounded acanthose ends as in Tsitsikamma (Samaai and Kelly 2002; Samaai et al. 2004; Parker-Nance et al. 2019). Additional diagnostic characters include the presence of broad swathes of megascleres that diverge from the base of the sponge towards the upper choanosome, where they form loose brushes and the typical wispy reticulation of most Latrunculiidae. Cyclacanthia is endemic to South Africa and is a predominantly shallow water genus occurring in warm temperate to subtropical waters, and is usually associated with rocky reef substrata, especially in areas that has cold upwelling cells and extreme current flow. The genus as presently recognised is cohesive (Samaai et al. 2004) and contains three well-defined species. The discovery of the new species from the offshore Amathole region extends the distribution of this genus further south in the Agulhas ecoregion., Published as part of Samaai, Toufiek, Kelly, Michelle, Ngwakum, Benedicta, Payne, Robyn, Teske, Peter R., Janson, Liesl, Kerwath, Sven, Parker, Denham & Gibbons, Mark J., 2020, New Latrunculiidae (Demospongiae, Poecilosclerida) from the Agulhas ecoregion of temperate southern Africa, pp. 409-442 in Zootaxa 4896 (3) on pages 433-434, DOI: 10.11646/zootaxa.4896.3.4, http://zenodo.org/record/4390683, {"references":["Samaai, T., Gibbons, M. J., Kelly, M. J. & Davies-Coleman, M. (2003) South African Latrunculiidae (Porifera: Demospongiae: Poecilosclerid): descriptions of new species of Latrunculia du Bocage, Strongylodesma Levi, and Tsitsikamma Samaai & Kelly. Zootaxa, 371 (1), 1 - 26. https: // doi. org / 10.11646 / zootaxa. 371.1.1","Samaai, T., Govender, V. & Kelly, M. (2004) Cyclacanthia n. g. (Demospongiae: Poecilosclerida: Latrunculiidae incertae sedis), a new genus of marine sponges from South African waters, and description of two new species. Zootaxa, 725 (1), 1 - 18. https: // doi. org / 10.11646 / zootaxa. 725.1.1","Antunes, E. M., Copp, B. R., Davies-Coleman, M. T. & Samaai, T. (2005) Pyrroloiminoquinone and related metabolites from marine sponges. Natural Products Reports, 22, 62 - 72. https: // doi. org / 10.1039 / b 407299 p","Samaai, T. & Kelly, M. (2002) Family Latrunculiidae Topsent, 1922. In: Hooper, J. N. A. & Van Soest, R. W. M. (Eds.), Systema Porifera. A guide to the classification of sponges. Vol. 1. Kluwer Academic / Plenum Publishers, New York, Boston, Dordrecht, London and Moscow, pp. 708 - 720. https: // doi. org / 10.1007 / 978 - 1 - 4615 - 0747 - 5 _ 78"]}

Supplementary methods, figures and tables

Rius, Marc, Teske, Peter R. (2013): Cryptic diversity in coastal Australasia: a morphological and mitonuclear genetic analysis of habitat-forming sibling species. Zoological Journal of the Linnean Society 168 (3): 597-611, DOI: 10.1111/zoj.12036, URL: http://dx.doi.org/10.1111/zoj.12036

Pyura dalbyi n. sp. Figs. 5–8 Material examined. Specimens were found subtidally under a jetty in Albany harbour, Western Australia (Table 1). Holotype: SAM-A 25988, Paratype: SAM-A 25989. Description. Individuals are squat with an extended base (Fig. 5 A). Their maximum dorsal length ranges from 35 to 95 mm, their height (measured from the base to the top of the siphonal area) from 35 to 60 mm, and their width (measured in contracted individuals) from 25 to 50 mm. The tunic is smooth (no pointed papillae on the tunic) (Figs. 5 A, B, C), with few epibionts, and not as thick and tough as that of the African representatives of the P. s t o l o n i f e r a species complex. The individuals examined were attached to one another, forming a dense aggregate. Siphonal spines are long and pointed, and their bases are slightly expanded (Figs. 6 A, B). The inner half of the siphonal lining is iridescent when observed under a dissecting microscope (Fig. 6 C). The animal has a fleshy body wall that is light orange in colour. The body size ranges in maximum length from 30 to 85 mm and in width from 20 to 45 mm. There are circular muscular bands around the siphons and the anterior longitudinal bands are present across the body wall and do not cross over each other. There are 17 to 32 ramified oral tentacles of different sizes (alternating between large and small, with more tentacles in larger individuals than in smaller ones) (Figs. 6 D, E), and the ramification complexity is of the third order branches. There are no atrial tentacles at the entrance of the exhalant siphon. The branchial stigmata are straight (Fig. 7 A) and between nine and 11 are present per mesh (i.e. between longitudinal vessels in the space between folds). There are six complete branchial folds (Fig. 7 B), with around 20 vessels per fold. The branchial formula of two individuals is: R.E. 5 (14) 4 (19) 3 (20) 3 (22) 3 (22) 3 (22) 6 D.L. 7 (22) 4 (25) 3 (25) 3 (22) 3 (20) 3 (16) 8 E.L., and R.E. 11 (20) 3 (25) 4 (26) 4 (27) 4 (30) 4 (37) 7 D.L. 9 (29) 3 (31) 3 (30) 3 (28) 4 (22) 4 (19) 10 E.L. The large sponge-like dorsal tubercle is not arranged as a double spiral cone as in P. praeputialis and P. s to l on if er a, but instead is largely spherical with two distinct bulges (Figs. 7 C, D). We found a less complex and convoluted dorsal tubercle in smaller individuals than in larger ones (Figs. 7 C, D). The dorsal lamina, which is always present, is short and contains small languets (Figs. 7 E, F). The gonads are attached to the body wall and form two rows of lobes on the right side of the body (Figs. 8 A, C). The left gonad is inside the gut loop and also forms two rows of lobes (Figs. 8 B, C). The gonoduct on the right side of the body is short (extending 3 mm away from the first gonadic block) and is located just below the gonad, whereas the one on the left side of the body is long (opening approximately 10 mm away from the first gonadic block), and it crosses below the gut and opens next to the anus (Fig. 8 D). The gut forms a sharply curved loop on the left side of the body, followed by a secondary loop that is not as sharply curved (Figs. 8 B, C). The hepatic gland is large and branched (Figs. 8 B, C) and contains 3 or 4 hepatic lobes. Endocarps are absent both around and on top of the gonads and the gut. The anal border has 6 to 10 irregular, rounded lobes (Figs. 8 D, E). Distribution. This species has been found in several localities in Victoria, on the southeast coast of Australia (Dalby 1997 a). It has also been found in Albany, Western Australia (this study). Unlike its South African and Australasian sister taxa, whose distributions extend from the subtidal to the lower intertidal, P. dalbyi has been found almost exclusively subtidally (Dalby 1997 a). Etymology. The species is named Pyura dalbyi after Dr. J. E. Dalby Jr. who reported distributional, morphometric and ecological differences between this species and P. praeputialis. Remarks. The yellow and sand-free tunic easily differentiates specimens of P. d a l b y i from the other species of the P. s t o l o n i f e r a species complex. Internally, the shape of the dorsal tubercle is one of the most conspicuous characteristics, as it is quite irregular when compared to the cone-like dorsal tubercle found in P. praeputialis and P. stolonifera. This is especially evident in smaller individuals of P. dalbyi. While the sponge-like shape of the dorsal tubercle in P. herdmani can be quite similar to that of P. d a l b y i, it lacks the two distinct bulges of the latter. The double row of gonadic blocks on the left side of the body inside the gut is a unique character of this species. All African species and P. praeputialis have just one row of gonadic blocks surrounded by the gut. The long siphonal spines of P. dalbyi are also unique, because in the other species the spines are shorter or less pointy. Pyura dalbyi lacks endocarps on top of the gonads and gut, which is uncommon in this group. None of the descriptions of Pyura species included in the extensive monograph of Australian ascidians by Kott (1985) corresponds to P. d al b yi. The presence of a picture of a large aggregate of P. dalbyi identified as P. stolonifera (see Plate VIIIa, page 420) indicates that both species were included under the same name.

Pyura dalbyi n. sp. Figs. 5���8 Material examined. Specimens were found subtidally under a jetty in Albany harbour, Western Australia (Table 1). Holotype: SAM-A 25988, Paratype: SAM-A 25989. Description. Individuals are squat with an extended base (Fig. 5 A). Their maximum dorsal length ranges from 35 to 95 mm, their height (measured from the base to the top of the siphonal area) from 35 to 60 mm, and their width (measured in contracted individuals) from 25 to 50 mm. The tunic is smooth (no pointed papillae on the tunic) (Figs. 5 A, B, C), with few epibionts, and not as thick and tough as that of the African representatives of the P. s t o l o n i f e r a species complex. The individuals examined were attached to one another, forming a dense aggregate. Siphonal spines are long and pointed, and their bases are slightly expanded (Figs. 6 A, B). The inner half of the siphonal lining is iridescent when observed under a dissecting microscope (Fig. 6 C). The animal has a fleshy body wall that is light orange in colour. The body size ranges in maximum length from 30 to 85 mm and in width from 20 to 45 mm. There are circular muscular bands around the siphons and the anterior longitudinal bands are present across the body wall and do not cross over each other. There are 17 to 32 ramified oral tentacles of different sizes (alternating between large and small, with more tentacles in larger individuals than in smaller ones) (Figs. 6 D, E), and the ramification complexity is of the third order branches. There are no atrial tentacles at the entrance of the exhalant siphon. The branchial stigmata are straight (Fig. 7 A) and between nine and 11 are present per mesh (i.e. between longitudinal vessels in the space between folds). There are six complete branchial folds (Fig. 7 B), with around 20 vessels per fold. The branchial formula of two individuals is: R.E. 5 (14) 4 (19) 3 (20) 3 (22) 3 (22) 3 (22) 6 D.L. 7 (22) 4 (25) 3 (25) 3 (22) 3 (20) 3 (16) 8 E.L., and R.E. 11 (20) 3 (25) 4 (26) 4 (27) 4 (30) 4 (37) 7 D.L. 9 (29) 3 (31) 3 (30) 3 (28) 4 (22) 4 (19) 10 E.L. The large sponge-like dorsal tubercle is not arranged as a double spiral cone as in P. praeputialis and P. s to l on if er a, but instead is largely spherical with two distinct bulges (Figs. 7 C, D). We found a less complex and convoluted dorsal tubercle in smaller individuals than in larger ones (Figs. 7 C, D). The dorsal lamina, which is always present, is short and contains small languets (Figs. 7 E, F). The gonads are attached to the body wall and form two rows of lobes on the right side of the body (Figs. 8 A, C). The left gonad is inside the gut loop and also forms two rows of lobes (Figs. 8 B, C). The gonoduct on the right side of the body is short (extending 3 mm away from the first gonadic block) and is located just below the gonad, whereas the one on the left side of the body is long (opening approximately 10 mm away from the first gonadic block), and it crosses below the gut and opens next to the anus (Fig. 8 D). The gut forms a sharply curved loop on the left side of the body, followed by a secondary loop that is not as sharply curved (Figs. 8 B, C). The hepatic gland is large and branched (Figs. 8 B, C) and contains 3 or 4 hepatic lobes. Endocarps are absent both around and on top of the gonads and the gut. The anal border has 6 to 10 irregular, rounded lobes (Figs. 8 D, E). Distribution. This species has been found in several localities in Victoria, on the southeast coast of Australia (Dalby 1997 a). It has also been found in Albany, Western Australia (this study). Unlike its South African and Australasian sister taxa, whose distributions extend from the subtidal to the lower intertidal, P. dalbyi has been found almost exclusively subtidally (Dalby 1997 a). Etymology. The species is named Pyura dalbyi after Dr. J. E. Dalby Jr. who reported distributional, morphometric and ecological differences between this species and P. praeputialis. Remarks. The yellow and sand-free tunic easily differentiates specimens of P. d a l b y i from the other species of the P. s t o l o n i f e r a species complex. Internally, the shape of the dorsal tubercle is one of the most conspicuous characteristics, as it is quite irregular when compared to the cone-like dorsal tubercle found in P. praeputialis and P. stolonifera. This is especially evident in smaller individuals of P. dalbyi. While the sponge-like shape of the dorsal tubercle in P. herdmani can be quite similar to that of P. d a l b y i, it lacks the two distinct bulges of the latter. The double row of gonadic blocks on the left side of the body inside the gut is a unique character of this species. All African species and P. praeputialis have just one row of gonadic blocks surrounded by the gut. The long siphonal spines of P. dalbyi are also unique, because in the other species the spines are shorter or less pointy. Pyura dalbyi lacks endocarps on top of the gonads and gut, which is uncommon in this group. None of the descriptions of Pyura species included in the extensive monograph of Australian ascidians by Kott (1985) corresponds to P. d al b yi. The presence of a picture of a large aggregate of P. dalbyi identified as P. stolonifera (see Plate VIIIa, page 420) indicates that both species were included under the same name., Published as part of Rius, Marc & Teske, Peter R., 2011, A revision of the Pyura stolonifera species complex (Tunicata, Ascidiacea), with a description of a new species from Australia, pp. 27-40 in Zootaxa 2754 on pages 33-35, DOI: 10.5281/zenodo.207177, {"references":["Dalby, J. E., Jr. (1997 a) Dimorphism in the ascidian Pyura stolonifera near Melbourne, Australia, and its evaluation through field transplant experiments. Marine Ecology, 18, 253 - 271.","Kott, P. (1985) The Australian Ascidiacea, Part 1. Phlebobranchia and Stolidobranchia. Memoirs of the Queensland Museum, 23, 1 - 438."]}

Key to the species of the Pyura stolonifera species complex 1. A single row of gonadic lobes forms the right gonad (African species)............................................ 2 - Two rows of gonadic lobes form the right gonad (Australasian species)........................................... 3 2. Pointed papillae on the tunic, especially around the siphons; dorsal tubercle sponge-like.................... P. herdmani - No papillae on the tunic; dorsal tubercle forms a double spiral cone.................................... P. stolonifera 3. Brown tunic, often enmeshed with sand; dorsal tubercle forms a double spiral cone...................... P. praeputialis - Tunic yellow and smooth, often with epibionts; dorsal tubercle sponge-like with two bulges............... P. dalbyi n. sp., Published as part of Rius, Marc & Teske, Peter R., 2011, A revision of the Pyura stolonifera species complex (Tunicata, Ascidiacea), with a description of a new species from Australia, pp. 27-40 in Zootaxa 2754 on page 36, DOI: 10.5281/zenodo.207177

Supplementary methods, figures and tables