New species in the sponge genus Tsitsikamma (Poecilosclerida, Latrunculiidae) from South Africa

Abstract The genus Tsitsikamma Samaai & Kelly, 2002 is to date exclusively reported from South Africa. Three species are known from the southern coast: Tsitsikamma favus Samaai & Kelly, 2002, from the Garden Route National Park Tsitsikamma Marine Protected Area (MPA) and Algoa Bay; T. pedunculata Samaai, Gibbons, Kelly and Davies-Coleman, 2003, collected from Cape Recife in St. Francis Bay, and T. scurra Samaai, Gibbons, Kelly and Davies-Coleman, 2003, collected from a wreck site in a small bay west of Hout Bay on the west coast of South Africa. Here two new species are described: Tsitsikamma michaeli Parker-Nance, sp. nov., a small green purse-like species, collected from Algoa Bay, and Tsitsikamma nguni Parker-Nance, sp. nov., from The Garden Route National Park, Tsitsikamma MPA. Additional morphological characteristics, spicule morphology, and distribution records are provided for T. favus and T. pedunculata from Algoa Bay. The phylogenetic relationship of these five Tsitsikamma species is investigated.

Tsitsikamma are similar in their general morphology to other Latrunculiidae, with fistular oscula and areolate porefields distributed over the sponge surface. However, within Latrunculiidae, Tsitsikamma species are notably tough and leathery due to the reinforced densely spiculous nature of the ectosome, and firmness is added where species are internally reinforced with dense spiculose tracts dividing the interior into discrete chambers, visible to the unaided eye Kelly 2002, Samaai et al. 2003). The main skeletal component of both the thick tracks within the choanosome, prominent in T. favus (Samaai and Kelly 2002) and T. scurra, the reinforced stalk in T. pedunculata (Samaai et al. 2003) and the delicate compressible choanosome, are anisostyles, often polytylote Kelly 2002, Samaai et al. 2003). It is, however, the shape of the isochiadiscorhabd microscleres that is characteristic of the genus. Spines develop simultaneously on the end of a straight thin protoisochiarhabd shaft followed, when present, by median spines. The spines develop into truncate tubercles with rounded acanthose ends on a stout shaft (Samaai and Kelly 2002). A medium whorl is present in T. favus and T. scurra but lacking on the very short microscleres of T. pedunculata Kelly 2002, Samaai et al. 2003).
The genus has attracted much interest due to the production of cytotoxic pyrroloiminoquinone alkaloids including tsitsikammamines and brominated discorhabdins (Hooper et al. 1996, Beukes 2000, Antunes et al. 2004. The tsitsikammamines, were once thought to be unique to T. favus, and considered taxonomic markers differentiating this species from others in the genus and family Kelly 2002, Samaai et al. 2003). However, tsitsikammamines have since been reported from Australian Zyzzya fuliginosa (Davis et. al 2012) and Antarctic Latrunculia (Latrunculia) biformis . Recent research reporting makaluvamines for the first time in a Tsitsikamma species also discovered the existence of two distinct T. favus chemotypes, the one producing predominantly discorhabdins and tsitsikammamines while the second produces makaluvamines (Kalinski et al. 2019). The source of the bioactive properties of these sponges has been hypothesized to be microbial in origin with a close relationship between the sponges and their microbial symbionts (Walmsley et al. 2012, Matcher et al. 2017. Tsitsikamma favus is the first sponge reported to have Betaproteobacteria and Spirochetes as the dominant microbial taxon (Walmsley et al. 2012), which are conserved within the microbiomes of six species in three genera within the Latrunculiidae (Matcher et al. 2017).
In this study examination of the morphological features of multiple specimens suggests the grouping of Tsitsikamma species into two morphological forms. The first resembles T. favus, with a thick encrusting or hemispherical growth form, large attachment area and a choanosome structurally reinforced by dense spiculose tracts (Samaai and Kelly 2002) together with T. scurra and T. nguni sp. nov. The second morphological group has a reinforced peduncle that supports a rounded body without any reinforcing tracts subdividing the delicate interior (Samaai et al. 2003) and is represented by T. pedunculata and T. michaeli sp. nov. We provide additional morphological characteristics and new information on the geographical distribution of known species, describe two new species and investigate the integrity of two morphological groups considered using 28S rRNA gene sequence analysis.

Materials and methods
Samples were collected by SCUBA or Remotely Operated Vehicle equipped with a collection arm and deployed from the coastal Research Vessel uKwabelana. Specimens were collected from Tsitsikamma Marine Protected Area and Algoa Bay within the Agulhas Ecoregion from depths of 18-40 m. All sponges were preserved in 70% ethanol or frozen at -20 °C. Photographic records were collected in situ, of freshly collected and preserved specimens, where possible. The majority of the samples, type specimens and reference material are lodged with the South African Institute for Aquatic Biodiversity (SAIAB) a National Research Foundation (NRF) National Collection facility (for further information please visit www.saiab.ac.za) and have the prefix SAIAB. Additional samples collected by the Coral Reef Research Foundation (CRRF) on behalf of the United States National Cancer Institute shallow-water collection programme, are now held at the California Academy of Sciences. Specimens belonging to collections held at South African Museum (SAM), Cape Town, the British Natural History Museum (NHMUK), London, and California Academy of Sciences Invertebrate Zoology Collection (CASIZ), San Francisco, are as such indicated by the abbreviation as prefix to the sample number. Voucher specimens of all newly collected specimens will be sent to the South African Museum. All specimens listed in this publication were collected by Shirley Parker-Nance except where otherwise indicated.
Chiadiscorhabd microsclere morphology changes as the spicules develop (Samaai and Kelly 2002) and vary intra-and interspecifically within the genus. Measurements of the spicule shaft length and width, the apical whorl and manubrium diameter and the total length of the spicules were made to quantify differences. The largest of 40 megascleres and 20 microscleres presented in 20 images taken from permanent pre-pared slides were used to define size attributes. A distinction was made between the smaller apical whorls and a larger manubrium, and both are provided for the microscleres measured.
Sponge DNA was extracted either according to the method described by Walmsley et al. (2012) or using the ZR Soil Microbe DNA MiniPrep kit (Zymo Research,Cat. No. D6001). Partial 28S rRNA gene sequence was PCR amplified as in Waterworth et al. (2017) using primer pairs (SP18cF: 5'-GACCCGTCTTGAAACACGA-3'and SP18dR: 5'-ACACACTCCTTAGCGGA-3). The PCR products were either cloned into the pGEM-T Easy vector (Promega) or sequenced directly by Sanger sequencing. Primer pairs (The LCO1490: 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and (HCO2198:5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) were used to amplify COI gene fragment as per Walmsley et al. (2012). Seventeen 28S rRNA sequences (629 bp) and two COI sequences (658 bp), supplemented by relevant sequence obtained from GenBank, were aligned using ClustalW and the phylogenetic trees constructed using the Neighbour-Joining method with 500 bootstrap replicates in MEGA X (Kumar et al. 2018 Tsitsikamma favus Samaai & Kelly, 2002 Tsitsikamma pedunculata Samaai, Gibbons, Kelly & Davies-Coleman, 2003 Tsitsikamma scurra Gibbons, Kelly & Davies-Coleman, 2003 Tsitsikamma michaeli Parker-Nance, sp. nov. Tsitsikamma nguni Parker-Nance, sp. nov. Diagnosis. Hemispherical, thick encrusting or pedunculate Latrunculiidae with a smooth, in some species generously folded, surface with cylindrical or volcano-shaped oscula and prominent areolate porefields. The ectosome is resident and leathery, the colour varies between species from pinkish to dark liver brown, dark turquoise or green in life. Megascleres are anisostyles with isochiadiscorhabd microscleres. The microscleres are present in an irregular palisade layer on the surface ectosome and line the internal tracts (from Kelly 2002, Samaai et al. 2003).
Type species. Tsitsikamma favus Samaai & Kelly, 2002 Remarks. The diagnostic character that unites species of Tsitsikamma is the possession of isochiadiscorhabd microscleres. Isochiadiscorhabd or isochia(acantho)dis-corhabds have a short straight smooth shaft bearing an apex whorl and manubrium and when present median whorls. These whorls consist of singular or grouped conicocylindrical tubercles, radiating from the shaft, with the distal end acanthose. These differ from microscleres present in other Latrunculiidae such as the acanthose isospinodiscorhabds with stout straight shaft, with similar terminal whorls and discrete conical spines unevenly distributed along it in Cyclacanthia; microscleres with disk-like whorls of spines that are different in shape and size, such as the anisodiscorhabds found in Latrunculia; or isoconicodiscorhabds or 'sceptres' with stout straight shaft and undifferentiated terminal whorls found in Sceptrella (Samaai and Kelly 2002, Samaai et al. 2006, Kelly et al. 2016. The ontogeny of the microscleres further set the genera within this family apart, as the protorhabd projections develop simultaneously in Tsitsikamma, Cyclacanthia, and Sceptrella but not so in Latrunculia Kelly 2002, Samaai et al. 2004).
Interestingly, Tsitsikamma species occur in two very different growth forms. In two of the species, T. favus and T. scurra, the interior of the sponge is partitioned by reinforced dense spiculose tracks through the delicate choanosome. The third species, T. pedunculata, has a spicule dense stalk that supports a spherical pouch without the characteristic spicule tracts penetrating into the choanosome. The description of an additional two Tsitsikamma species, presented in this work, support this separation further as one has internal tracts and the other is purse-shaped.    Diagnosis (emended from Samaai and Kelly 2002). Large, firm, dark brown hemi-spherical to thick encrusting sponges, up to 15 cm high and 20 cm in diameter, sessile with a large area of attachment. Surface smooth and firm although undulant presenting a folded or bumpy appearance in some specimens ( Fig. 1a-c), only slightly to moderately compressible, resilient and leathery. Surface with large single to multichambered cylindrical lance-shaped oscula, and pedunculate cauliform areolate porefields, colour in life is light to dark brown or liver brown.

Tsitsikamma favus Samaai & Kelly, 2002
Skeleton. The ectosome is composed of a thick dense feltwork of anisostyles with a single layer of erect isochiadiscorhabds arranged perpendicular to the underlying megascleres (Fig.1e). The ectosome is generally thinner than the dense spiculose tracts that penetrate and divide the soft choanosome into honeycomb-like chambers (Table 1, Fig. 1d, f ).
Spicules. Megascleres. Slightly sinuous anisostyles, hastate, mucronate or blunt, occasionally tylote form the main structural components with two categories pre-sent; (i) long slightly curved and thickened centrally and (ii) shorter, thinner slightly curved centrally (Table 1, Fig. 1g-j). Short thick anisostrongyles, may also be present (Fig. 1k). Microscleres. Isochiadiscorhabd, with three whorls of conico-cylindrical tubercles terminally acanthose (Fig. 1p), line the tracts and are found abundantly throughout the choanosome (Fig. 1l). In addition to the three whorled microscleres, as described by Samaai and Kelly (2002) for the type material, are chiadiscorhabds with up to five complete whorls as well as many intermediate forms ( Fig. 1m-o). Typically, the manubrium and the apical whorl differ slightly in diameter (Table 1) and tubercles projections arranged pairwise or in groups of three respectively ( Fig. 1l-p). Isochiadiscorhabds with the terminal tubercles arranged in such a way to give a flattened appearance are also present (basal whorl in Fig. 1o). Oocytes were present in specimens (after Samaai and Kelly 2002).
Distribution. Plettenberg Bay, Tsitsikamma Marine Protected Area and Algoa Bay. Substrate, depth range, and ecology. Collected from rocky benthic reef, 9-33 m deep, occurring singly or in clumps of two or three, in abundance on both shallow reef systems such as Bell Buoy on the top of medium profile reef and at Evans Peak on the sides of high steep profile reef. Note that for some of the older collections the GPS position of the collection site is not available or inaccurate; for clarity Rheeders Reef is an inshore reef system within the Tsitsikamma MPA situated east of Storms River Mouth and the Knoll between -34.025730, 23.906138 and -34.032780, 23.960138 inshore and -34.044530, 23.906138 and -34.04453, 23.96013 off shore.
Remarks. Examined material compares well with the type description given by Samaai and Kelly (2002) including the shape of the oscula, distribution of the pedunculate cauliform areolate porefields, arrangement, and distribution and size of megascleres and microscleres (Table 1). The structure of the chiadiscorhabds corresponds with the type description Samaai and Kelly (2002); however, some sponges exhibited various ratios of typical microscleres with three whorls, as per the type description (Samaai and Kelly 2002), to microscleres with tubercles not arranged in or missing from or present between complete whorls (Fig. 1m-o). These variations were suggested but not discussed in the type description (see Samaai and Kelly 2002: fig. 6J, central  fig. 2B, central image) which suggests that spicules of this nature were observed in the type specimen. It is interesting to note that T. favus specimens, even some collected no more than 10 cm apart and although clearly T. favus with respect to 28S rRNA sequence analysis (as shown by sequence identity or a maximum of one nucleotide difference), differ in the firmness or compressibility of the individual sponge. Closer inspection of the spicules showed an increased occurrence of misshaped or irregular microscleres and a distinct chromatographic profile in these T. favus sponges (Kalinski et al. 2019). Live or freshly collected specimens are dark brown, olive or dark green in colour and may be heavily encrusted with soft corals, hydroids, ascidians and other encrusting sponges with the oscula and porefields protruding through the surface epibionts. As freshly collected specimens are preserved, the extract dyes the preservative (70% ethanol) a deep brown colour which intensifies as the tissue lightens; long exposure to the stained preservative darkens the tissue again. Successive preservative changes (long-term curated specimens) remove the pigment and the specimens are beige in colour. Frozen material may be dark slate green to tan externally, and the tracks are prominently tan and the choanosome dark brown.
Examination of specimens collected from Tsitsikamma in 1993 showed that one sample contained two distinct species, the one clearly T. favus the other a new species included below (SAIAB 207216: The Knoll, Tsitsikamma MPA Garden Route National Park, Eastern Cape Province, 18 m, 2 May 1993, collected by Colin Buxton).   Diagnosis (emended from Samaai et al. 2003). Characteristic dirty pink, pinkbrown pedunculate species with well-defined, ball-shaped head, up to 7 cm in diameter, on a narrow stalk, 1-3 cm wide and up to 7 cm long (Fig. 2a, b). Living sponges appear dirty pink although this is often obscured by epibionts, especially the yellow encrusting Mycale (Mycale) sponge also found growing on other members of this genus (Fig. 2a-c). Freshly collected material is a dusty pink to pink-brown to dark purple while preserved material has an olive green, cream to tan colour (Fig. 2c). Small wellspaced cone-shaped oscula 1.5-2 mm high and 1.5-3 mm in diameter are present over the upper part of the head gradually replaced by small to bigger elevated circular fungiform areolate porefields, 1-4.5 mm high and 2-7.5 mm in diameter, toward the base where the stalk is attached (Fig. 2a). In preserved specimens the oscula retain their shape but the upper border of the porefields contracts inwards giving it a button like appearance. A tough, resistant leathery ectosome surrounds a much softer choanosome. The sponge is resilient, but compressible. Salmon pink to pinkish brown between the oscula and dark pink between the areolate porefields.

Tsitsikamma pedunculata Samaai, Gibbons, Kelly & Davies-Coleman, 2003
Skeleton. Microscleres are abundant throughout the choanosome and form an irregular palisade of oblique or erect microscleres over the dense feltwork of tangential and paratangential styles together forming the ectosome (Table 2, Fig. 2e) The resistant ectosome encapsulate soft choanosome with delicate tracts (Samaai et al. 2003) (Fig. 2d). The stalk consists of densely arranged spicules and has longitudinal cavities filled with soft choanosome tissue distributed regularly along the axis of the reinforced stalk (Fig. 2c, f ).
Spicules. Megascleres consist of two size classes of styles; (i) slightly sinuous, robust centrally thickened, acerate, conical, hastate or somewhat blunt even mucronate styles, and (ii) thin conspicuously sinuous and sometimes conspicuously centrally thickened styles (Table 2, Fig. 2g, h). Microscleres. Isochiadiscorhabds with only two whorls of cylindrical, conical tubercles acanthose on apex, arranged on the ends of a short shaft (Samaai et al. 2003). The large manubrium is easily distinguishable from the conspicuously smaller apex with terminally acanthose tubercles arranged in a pincushion-like way to form the apex whorl of the microsclere (Table 2, Fig. 2i-l).
Distribution. Algoa Bay and St. Francis Bay Substrate, depth range and ecology. Abundant on deep reef systems between 34-40 m. All specimens collected were attached to rock on the sides of medium profile reef adjacent to sandy gullies. A thin delicate light yellow Mycale (Mycale) species is commonly found growing on the globular head surface around the oscula and porefields. Remarks. The shape of the sponge, the long peduncle, round head, colour and the shape of the microscleres set this species well apart from any other species in this genus.
No intraspecific genetic diversity was found for the 28S rRNA gene sequence of specimens of T. pedunculata included in this study. An interspecific genetic diversity of 0.32-0.65 % for the 28S rRNA gene sequence was found between T. pedunculata and T. favus (Suppl. material 1: Table S1). Diagnosis (emended from Samaai et al. 2003). Sponge massive, semispherical to thick encrusting and lime green in life, compressible with a tough sandpapery ectosome. Samaai et al. (2003) noted the surface crowded with large hollow strap-like oscula with the apex slightly expanded and fungiform areolate porefields, with the overall skeleton dominated by an ectosomal envelope of tangential megascleres, extending up into the large oscular tubes (Fig. 3a, b). In the preserved specimen small pear-shaped oscula (2-5.5 mm high and 1-1.5 mm in diameter) and long narrow stalked areolate porefields (7-9 mm high, 2.5-5 mm in diameter) are distributed over the folded surface ( Fig. 3a-d).

Tsitsikamma scurra Samaai, Gibbons, Kelly & Davies-Coleman, 2003
Skeleton. The ectosome is thin with a fine sandpapery feel that seems to continue and fold within the interior of large specimens to form smaller subunits or internal chambers (Table 3, Fig. 3c-e). The choanosome is soft and may contain varying amounts of sand, shell and other foreign material (Fig. 3d).
Spicules. Megascleres consist of slightly curved styles, conspicuously thickened centrally sometimes bend basally and thinner styles, slightly curved centrally (Table 3, Fig. 3f-h). Microscleres. Isochiadiscorhabds with three whorls of conico-cylindrical tubercles, the apex of each is acanthose. The median whorl is polar and situated closer to the apex whorl than to the slightly larger manubrium (Table 3), this polarity may be less pronounced in larger microscleres (Fig. 3i). The acanthose tubercles arranged in pairs in the apex whorl and manubrium (Fig. 3i-k). Microscleres are abundant throughout the choanosome (after Samaai et al. 2003). Distribution. West of Hout Bay, a local area known as Maori Bay along the Western Cape Province coast.
Remarks. The specimens examined compared well with the description given by Samaai et al. (2003) except that the colour in life of the type specimen was described as lime green and colour photographs of the freshly collected specimen indicate a brownish colouration (Fig. 3a). Preserved specimens are a medium to dark brown colour in ethanol (Fig. 3c, d). Tsitsikamma scurra differs from all other known Tsitsikamma species in the folded globular thick encrusting growth structure (Fig. 3d) with thin sandpaper-like ectosome (Table 3). Epifauna may be present on the sponge surface and the interior may contain a substantial amount of sand particles and shell fragments.
We obtained 28S rRNA gene sequences for only one T. scurra specimen. The interspecific diversity of the 28S rRNA gene sequence for T. scurra and other Tsitsikamma did not support clear genetic identity, with between 0.16-0.32 % at 28S for T. favus and 0.32 % for T. pedunculata. Description. Small olive-green, purse shaped sponge up to 5 cm high (2 cm stalk and 3 cm rounded head) or sessile, 5-10 cm in diameter. In some cases, the large  sponge may be loosely subdivided into sections (Fig. 4a-c). Small short tube-shaped oscula, 2.5-4 mm high and 1.8-5.5 mm wide at the base narrows to a point and may be laterally flattened in preserved material. The particularly large stalked cauliform porefields are 3-7 mm high and 3-6.5 mm wide, with the porefields spilling over the supporting stalk (Fig. 4d). The freshly collected sponge is a dark to olive green colour with light cream tipped oscula and darker brown green areolate porefields (Fig. 4d). The interior choanosome is bright green. Preserved specimens are olive to tan in colour. Skeleton. The soft delicate, bright green, interior choanosome is encapsulated by a protected firm resilient green ectosome 1000 (200-1500) µm thick (Fig. 4d, e). The attachment area or short stalk is represented by a thickening of the ectosome. No reinforced tracts are present through the interior.
Etymology. Tsitsikamma michaeli sp. nov. is named after Professor Michael T Davies-Coleman, Dean of Science, Department of Medical BioScience, University of the Western Cape in recognition of his outstanding contributions to our knowledge of the diversity of South African marine fauna and their production of bioactive secondary metabolites.
Distribution. Algoa Bay Substrate, depth range, and ecology. Tsitsikamma michaeli sp. nov. is a small species found on similar reef habitat as to T. pedunculata in Algoa Bay, sometimes in close proximity, at depths between 33-38 m. It shares the same epibiont Mycale (Mycale) species, which grow on the sponge surface between the oscula and porefields.
Remarks. The absence of reinforcing spicule-dense tracts through the interior choanosome differentiates this new species from T. favus and T. scurra. The sac-or purselike shape of the T. michaeli sp. nov. and the well-spaced oscula and porefields resemble Table 4. Comparison between morphological structures (µm) T. pedunculata and T. michaeli sp. nov. those of T. pedunculata but the species differs in colour, bright to olive-green compared to the purplish pink to brown of T. pedunculata. It does not have a stalk, although the basal attachment area of T. michaeli sp. nov. is reinforced by a thickening of the ectosome (Fig. 4d). The resistant ectosome is of similar thickness for the two species (Table 4). The category (i) megascleres are shorter and thinner in T michaeli sp. nov. while the category (ii) styles are longer and more robust than that of T. pedunculata (Table 4).
The microscleres of these two species have a similarly structured manubrium with tubercle in groups of four or more, but the tubercles are arranged in groups of three in T. pedunculata and in pairs in the apex whorl of T. michaeli sp. nov. Tsitsikamma pedunculata lack the median whorl and the spicule is shorter (Table 4), while T. michaeli sp. nov. may have up to two whorls between the apical whorls and manubrium. There was no intraspecific genetic diversity for the 28S rRNA gene region for T. michaeli and no interspecific genetic diversity for T. michaeli and T. pedunculata was observed in this work (Suppl. material 1: Table S1). There was, however, interspecific genetic diversity of between 0.48-0.65 % between T. michaeli and T. favus and 0.32 % between T. michaeli and T. scurra. Description. Large thick encrusting or sessile hemispherical or convex cushions, dark slate-coloured when alive but very dark brown to black in preservative. The sponge is very firm and rigid, 3-6 cm high and 3-10 cm in diameter (Fig. 5a-d). The upper third to half of the sponge surface is dominated by small short, blunt rounded knob-shaped or button-like oscula, 2-5 mm high and 2.5-5 mm wide at the base. The surface surrounding the upper osculate area, the shoulder and upper side of the sponge, has well-spaced small round slightly elevated or sessile porefields. These gradually merge to form larger round porefields that join to form irregular or blotch-shaped structures along the base of the sponge. In general, porefields are 1-4 mm high and 3-14 mm in diameter (Fig. 5a-c).

Tsitsikamma nguni
Skeleton. The ectosome is 780 (430-1560) µm thick guarded externally by a prominent palisade of microscleres arranged perpendicularly to the prominent inner style layer (Fig. 5e). The softer choanosome is divided into small uneven circular to oval shaped chambers 6640  µm in diameter by reinforcing tracts 1410 (530-3200) µm thick (Fig. 5c). Sand particles and shell fragments may be present in the sponge choanosome.
Etymology. The Nguni cattle breed is unique to southern Africa with characteristic dappled colour and blotchy patterns on the hide, reminiscent of the elaborate blotch-shaped areolate porefields typical of the larger T. nguni sp. nov. specimens.
Distribution. Tsitsikamma Marine Protected Area, Garden Route National Park, Eastern Cape Province.
Substrate, depth range, and ecology. The species is common in the shallow coastal zone within the Tsitsikamma Marine Protected Area on low profile reefs at a depth of 18-21 m.
Remarks. Live specimens of T. nguni sp. nov. appear a dark slate or very dark grey, almost black in colour. Freshly collected specimens consist of the dark olive-brown to black exterior with dark brown surface structures (Fig. 5a, b). The interior tracts are light olive, cartilaginous with softer withdrawn olive-brown choanosome, which may contain sand and shell fragments (Fig. 5c). Preserved specimens are a uniform dark brown colour staining the preservative (70% ethanol) a deep rich brown to almost black colour (Fig. 5d).
Tsitsikamma favus and T. nguni sp. nov. differ considerably from T. scurra in the texture and thickness of the ectosome, internal tracts and surface structures (Table 5)   Table 5. Dimensions (mm) of surface and skeletal structures (data for ectosome and tracts given as thickness, internal honeycomb-shape chamber as mean diameter and ranges, oscula and porefields as mean height and diameter with ranges). as well as the dimensions of the spicules (Table 6). Defining the differences between T. favus and T nguni sp. nov. is more challenging. Most apparent is the surface morphology. The basal part and sides of T. favus sponges are dominated by stalked cauliform porefields, densely crowded and gradually giving way to prominent lance-shaped oscula with a large basal diameter distributed over the upper surface of the sponge, giving the sponge surface an uneven, messy appearance (Samaai and Kelly 2002) (Fig. 1a).
In contrast, the lower basal parts and sides of T. nguni sp. nov. is dominated by flat to slightly raised elaborate blotch-shaped porefields which become smaller, more circular in shape and more isolated towards the upper part of the sponge where they are replaced by well-spaced, small button-shaped (in life, see Fig. 5a) or small and pointed (preserved, Fig. 5d) oscula over the upper part of the sponge. Both species have similar partitioning of the choanosome, although T. nguni sp. nov. is notably firmer, has larger more regular chambers with generally thicker spicule tracks and a slightly thicker ectosome (Table 5). The megasclere and microsclere shape and dimension are very similar (Table 6) although the species differ in the number of acanthose conico-cylindrical tubercles grouped together to make up the manubrium, three per group in T. favus (Fig. 1l) and four to six in the new species (Fig. 5j, k). The general appearance of T. nguni sp. nov., shape of the porefields, and smaller size of the oscula, the colour, both in life and preserved, the slightly shorter styles (Table 6), slight difference in the arrangement of the acanthose tubercles of the microsclere manubrium, the slightly thicker ectosome, the more robust interior spiculedense tracts, and larger chambers (Table 6) all contribute to a species that is distinctly different in appearance from T. favus. In freshly collected specimens fixed in 70% ethanol, the preservative extracts some secondary metabolites and pigment from the specimen. Tsitsikamma nguni sp. nov. colours the fixative intense dark solid brown almost Table 6. Spicule dimensions (µm) of T. scurra (n = 2), T. favus (n = 14), and T. nguni sp. nov. (n = 4) for material examined. Data in table given as mean total length (range) × shaft width (range). black colour, this is very different from the lighter brown semi-translucent colouration given to the fixative by T. favus.
Tsitsikamma nguni was found to show no genetic diversity with respect to the 28S rRNA gene sequence from T. scurra, 0.16-32 % from T. favus, and 0.32 % from T. pedunculata and T. michaeli (Suppl. material 1: Table S1).
Discussion. The species in Tsitsikamma exhibit two morphological growth forms: T. favus, T. scurra, and T. nguni sp. nov. are thick encrusting to hemispherical sponges with spicule-dense tracts that reinforce the internal choanosome while T. pedunculata and T. michaeli sp. nov. are purse-shaped species, with or without a prominent stalk. The growth form, surface architecture, colour, skeletal structure, and spicule morphology are important diagnostic characteristics Kelly 2002, Samaai et al. 2003). An identification key for the Latrunculiidae genera and species within Tsitsikamma incorporating important morphological characteristics, skeletal architecture, spicule morphology, and ontogeny has been constructed which incorporates descriptive information from Samaai and Kelly (2002), Samaai et al. (2003), Samaai et al. (2004), Samaai et al. (2009), Samaai et al. (2006, and Kelly et al. (2016) (Fig. 6). This identification key is in agreement with the relationships presented in the 28S rRNA and COI sequence based phylogenetic trees constructed for available sequences (Fig. 7, Table 7).
The phylogenetic analysis presented here of partial 28S rRNA gene sequences and COI sequences is incomplete and although lacking COI sequences for some Tsitsi- Table 7. List of species, specimen numbers, and GenBank accession numbers for 28S rRNA gene sequences and COI gene sequences used to construct phylogenetic trees.  kamma representatives, the diagnostic key constructed for morphological characteristics distinguishing members of the Latrunculiidae is not contradicted by the relatedness between taxa presented in these preliminary phylogenetic trees based on DNA sequence comparison. Both suggest that Tsitsikamma is closely related to Cyclacanthia (Fig. 7A) and Sceptrella (Fig. 7B). The separation between Tsitsikamma and Latrunculia underline the ontogenetic nature of the spicule and resulting microsclere morphology with similar terminal structures such as isochiadiscorhabds in Tsitsikamma, isospinodiscorhabds in Cyclacanthia, or isoconicodiscorhabds in Sceptrella, which are more similar in development than the anisodiscorhabds characteristic of Latrunculia [after Samaai and Kelly (2002), Samaai et al. (2003), Samaai et al. (2004), Samaai et al. (2009), Samaai et al. (2006, Kelly et al. (2016)]. The morphological similarity of species in the two morphological groups within Tsitsikamma is borne out by the similarity of their 28S rRNA gene sequences as shown (Fig. 7A) and reflected in pair wise distance analysis of the sequences. Interestingly, we observed significant intraspecific genetic diversity in T. favus but not in T. pedunculata or T. michaeli sp. nov. However, interspecific genetic diversity for the 28S rRNA gene did support the morphological species identity overall (see Suppl. material 1: Table  S1, Figs 6, 7). This study highlights the limitations of commonly used genetic markers in their current coverage for the resolution of closely related species and the importance of rigorous morphological data for taxonomic classification of the Latrunculiidae sponges. An extended phylogenetic investigation encompassing the full rRNA cistron would improve our understanding of the phylogenetic relationship of not only the higher taxa but also at species level.