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 μm. 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 μm 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 μm 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) μm; (2) straight or slightly sinuous, thickened centrally, fusiform: 584 (509–653) × 19.2 (19.2) μm. Microscleres are isochiadiscorhabds with three whorls of cylindrical-conical tubercles, the apex of each tubercular projection is acanthose: 45 (38–58) × 7 (4 –7) μm (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 μm; thin 669 (585–738) × 17 μm] than Tsitsikamma (T.) amatholensis sp. nov. [thick 721 (681–758) × 19.2 (19.2) μm; thin 584 (509–653) × 19.2 (19.2) μm] while T. (T.) favus [thick 621 (537–700) × 14 (14) μm; thin 530 (480–566) × 9.6 (9.6) μm] and T. (T.) nguni [thick 555 (428–672) × 14 (10–19) μm; thin 561 (449–832) × 10 (3–14) μm] 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 μm], but smaller in average than those in T. (T.) favus [48 (41–60) × 9 (7.2–9.6) μm] 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.