A new species of Agelas from the Zanzibar Archipelago, western Indian Ocean (Porifera, Demospongiae)

Abstract A new sponge species (Demospongiae: Agelasida: Agelasidae) is described from the eastern coast of Unguja Island in the Zanzibar Archipelago. Agelas sansibarica sp. n. is compared to all other Agelas species described so far. The new species differs from its congeners mainly in its three categories of verticillate spicules (acanthostyles, acanthostrongyles, and acanthoxeas) and their sizes. Acanthostrongyles, well represented in the spicular complement, are an exclusive trait of the new species widening the morphological range of the genus. Summarizing on spicular complement and spicular morphotraits of 36 species belonging to the genus Agelas: i) 32 species show only acanthostyles from Indo-Pacific (n = 14), Atlantic (n = 17), and Mediterranean (n = 1); ii) three Indo-Pacific species show acanthostyles and acanthoxeas; iii) one species Agelas sansibarica sp. n. from the western Indian Ocean is characterised by the unique trait of three categories of verticillate spicules (acanthostyles, acanthostrongyles and acanthoxeas). A key for the Indo-Pacific species is supplied together with short descriptions, illustrations, and geographic range; literature on chemical bioprospecting of the genus Agelas is also provided.


Introduction
The sponge fauna of the Zanzibar Archipelago is poorly studied and data are reported almost exclusively in very old papers (Lendenfeld 1897, Baer 1906, Jenkin 1908, Sollas 1908, Thomas 1976. In none of these papers species belonging to the genus Agelas Duchassaing & Michelotti, 1864 (Porifera: Demospongiae: Agelasida: Agelasidae) are reported. The presence of Agelas mauritiana (Carter, 1883) along the Zanzibar coasts was recently recorded (Said et al. 2010) as producer of bioactive compounds.
The widespread genus Agelas, including until now 35 valid species, was established by Duchassaing and Michelotti (1864: 76) describing the type species Agelas dispar from the Caribbean Sea. Agelas oroides is the only Mediterranean species, and is endemic. The western Atlantic (Gulf of Mexico, Caribbean, and Brazil) hosts 17 species. The majority of the latter (13) were recently revised while the remaining four species were considered dubious or suggested as synonyms (Van Soest 2002, Muricy et al. 2011, Parra-Velandia et al. 2014. The Indo-Pacific species of Agelas number 18, including the new species here described. The most widespread species is Agelas mauritiana (including its oxeata variety) recorded in the Australian western Pacific, and the Indian Ocean from the Mascarenes Archipelago (type locality), Seychelles Archipelago, Madagascar, and Mozambique Channel to the southern Red Sea and east to Sri Lanka.
The present paper aims to report the discovery of a new species of Agelas from the Zanzibar Archipelago comparing it to all species belonging to the genus. To support global sharing of information on faunistics and taxonomy of Porifera from not widely accessible data sources an updated overview on the morphology and geographic distribution of Agelas species from the Indo-Pacific area is also provided together with a brief description and exhaustive iconography, as well as a dichotomous key to Indo-Pacific species.

Materials and methods
Representative fragments of six sponge specimens from the Unguja Island were studied. Growth form, surface traits, skeletal architecture, shape and size of the spicules are considered diagnostic morphotraits. Spicule dimensions are given as maximum, minimum, and medium lengths and widths of ca. 400 spicules.
The skeletal architecture was investigated by hand-cut sections of the ectosome and choanosome. To study the three-dimensional arrangements of fibres and spicules in the skeleton, fragments ca. 10 × 10 × 2 mm were cut, cleaned with 5% sodium hypochlorite (NaClO) for 24 h in a warm temperature (35-40 °C), then washed and stirred five times in abundant double distilled water, washed and stirred twice in ethanol 95%, and finally allowed to air dry and gold-sputtered or mounted in Eukitt. The skeletal samples were than studied by light microscope (LM) and scanning electron microscope (SEM). Spicule preparations were made by dissolving a small fragment of the specimen in 65% boiling nitric acid (HNO 3 )and cleaned spicules were rinsed four times with water, once with 95% ethanol. The spicules were air-dried on slides, mounted in Eukitt, and observed by a Leitz Dialux 20 EB (LM), as well as on aluminium stubs and coated with gold for the observation with a Vega3 TESCAN type LMU (SEM).
Specimens were deposited at the Museo civico di Storia Naturale "Giacomo Doria" of Genoa, Italy (acronym MSNG). For the taxonomy of genus and species level the Systema Porifera (Hooper and Van Soest 2002) and the World Porifera Database (Van Soest et al. 2015) are considered as reference.
conulose surfaces provided with small rounded and/or key-hole shaped apertures. Colour usually orange or brownish-orange. Consistency toughly compressible, firm. No ectosomal specialization. Choanosomal skeleton isotropic or anisotropic, occasionally irregular, network of primary ascending spongin fibres and secondaries. Main fibres mostly cored by megascleres. Main and interconnecting fibres echinated by megascleres in most cases. Spicules as verticillate styles, or styles and oxeas, or styles, oxeas and strongyles. Biogeographic pattern of 36 nominal species mostly matches tropical waters, a single species occurs in the Mediterranean. The genus has not been recorded from the eastern Pacific, West Africa, and the northern Atlantic European coasts. Diagnosis. Agelas with unique spicular complement composed of three spicular categories, oxeas, styles and strongyles with spines arranged in a variable number of verticilles.

Agelas sansibarica
Etymology. The speciphic epithet refers to the Zanzibar Archipelago.
Geographic distribution. Western Indian Ocean, but only recorded from the type locality to date.
Description. Growth form massive, thick, rounded, 6-10 cm in diameter. Colour in life purple to orange and light brown. Consistency firm and elastic. Surface rough to the touch, finely hispid, finely conulose for tips of ascending fibres supporting the dermal membrane, with regularly scattered circular and convoluted depressions (0.5 cm in diameter) very similar to those of A. cerebrum. Oscules few, small, irregularly scattered. Choanosomal skeleton as an irregularly reticulate network of spongin fibres. Primary fibres 50-110 (71.67 ±17.63) μm in diameter, strongly echinate by single, scattered spicules to groups of diverging spicules; ascending primary fibres cored by spicules also present. Secondary fibres 20-50 (35 ± 9) μm in diameter notably echinate and cored by spicules. Tertiary network not observed.
Remarks. The new species is characterized by the co-presence of three categories of spicules never recorded in other Agelas species. Acanthostrongyles are abundant, ca. 20-30 % of the total number of spicules.

Geographic range of Indo-Pacific Agelas species
Madagascar, Mozambique Channel, Seychelles and Mascarene archipelagos (Western Indian Ocean province) harbour four species, whereas Japan (Ryukyu Archipelago) and New Caledonia enumerates two species each. Only one species each is recorded from Philippines, Papua New Guinea, and Funafuti. Only one species each is harboured in the Red Sea/Gulf of Aden, Sri Lanka, Moluccas, Sunda Shelf/Java Sea (Indonesia), Hong Kong, Funafuti, and Australia ( Fig. 1).

Diagnostic morphotraits comparative analysis of Agelas Indo-Pacific species
To discriminate between all 36 Agelas species by diverging diagnostic morphotraits is notably difficult, as highlighted in the previous section. Morphotraits of the genus are extremely conservative and different species appear very similar. Focusing on the Indo-Pacific species our attempt was not as completely successful as is also the case for the Atlantic species by Parra-Velandia et al. (2014).
Atlanto-Mediterranean Agelas species (18)   Among the 17 previously known Indo-Pacific Agelas species, the spicular complement of 14 species is composed of acanthostyles in a single or two-dimensional classes (see Appendix 1, Figs 6-21 for details).
The most common and studied Indo-Pacific species, i.e. A. mauritiana, are characterized by a single spicular type acanthostyles, which are extremely variable in morphology, abundance of spines, and dimensional range (sometime more than three times in length) (see Table 1). The Atlantic A. dispar and the Mediterranean A. oroides show a similar size variability of acanthostyles. Only the Indo-Pacific A. axifera, A. mauritiana var. oxeata, and A. novaecaledoniae show two different categories of spicules, i.e. acanthostyles and acanthoxeas.
The new species A. sansibarica is characterized by the unique morphotrait of three categories of megascleres, i.e. acanthostyles, acanthoxeas, and acanthostrongyles with spines arranged in verticilles regularly scattered along the entire spicule. No other Agelas species exhibit this spicular combination. Acanthostrongyles, well identifiable and abundant, represent an exclusive diagnostic trait of the new species. The functional role of acanthostrongyles is doubled since echinanting spicules arm both the fibres surface and the core of the axial part of fibres.
Summarizing spicular complements and spicular morphotraits of 36 Agelas species: i) 32 species show only acanthostyles from Atlantic (17), Mediterranean (1), and the majority (14) of the Indo-Pacific areas; ii) three Indo-Pacific species show acanthostyles and acanthoxeas; iii) only one species A. sansibarica sp. n. from the western Indian Ocean show a spicular component composed of acanthostyles, acanthoxeas, and acanthostrongyles.

Key to the Indo-Pacific Agelas species
The present key is an attempt to discriminate between the Indo-Pacific species, but the scenario appears very intricate mainly because morphotraits from many descriptions and illustrations are overlapping (see Table 1). A similar attempt, on the Atlantic species, was performed by Parra-Velandia et al. (2014) emphasizing that: "Caribbean Agelas taxonomy rests heavily on the external morphology"; as a consequence their key is essentially based on growth form and colour. Since this is the situation, our key is not simply dichotomous and allows the disctintion of only 13 of the 17 Indo-Pacific species (see Appendix 1). The remaining four species have acanthostyles with length ranges which are widely overlapping (from less than 150 to more than 250 μm). Three
Smooth styles (320 × 24 μm) less frequently present (Dendy 1921). Habitat. Not reported. Geographic distribution. Indian Ocean. Recorded from south India, Sri Lanka, Indonesia, and Seychelles. Remarks. Recently recorded from Indonesia (de Voogd et al. 2008). The latter authors report that acanthostyles in the Manaar Gulf (type locality) specimens have a maximum dimension of 240 × 20 μm.
Habitat. Deep water, 245-275 m of depth. Geographic distribution. Western Pacific Ocean. Recorded only from the type locality in New Caledonia. Remarks. Also small oxea-like spiny spicules reported in original illustrations but not in descriptions. de Voogd et al. (2008) reported that verticillation is absent in smaller acanthostyles.

Fig. 20
Description (modified from Pulitzer-Finali 1982). Growth form encrusting (5 mm in thickness) to massive, roundish (6 × 3 cm in diameter). Cavernous (clathrous) structure, Figure 19. Agelas novaecaledoniae. a type specimen b spicular complement with two spicular types (modified from Lévi and Lévi 1983). with narrow channels through the entire sponge. Consistency tough and resilient. Colour dull orange, dull yellowish-brown. Ectosomal skeleton not reported. Choanosomal skeleton reticulate with irregular meshes of pale spongin fibres (38-80 μm in diameter) abundantly echinate by acanthostyles. Megascleres of a single category. Acanthostyles very stout, verticillate (170-250 × 14-30 μm spine included) with 11-12 whorls of short, blunt spines. Habitat. Not reported. Geographic distribution. Pacific Ocean. Recorded from Hong Kong, Southern China. Remarks. Known only from the type locality. At present for this species only three slides are available after damaging of type materials during the recent Genova flood in 2014 (October).

Fig. 21
Agelas semiglaber Pulitzer-Finali, 1996 Description (modified from Pulitzer-Finali 1996). Growth form encrusting, very small. Skeleton architecture not reported. Megascleres of single category and two size classes. Acanthostyles type I verticillate (230-375 × 11-16 μm), spiny only towards the tips. Acanthostyles type II (75-100 × 3.5 μm) verticillate, entirely spiny. Habitat. Shallow water. Geographic distribution. Pacific Ocean. Recorded from Bismarck Sea, known only from Papua New Guinea (type locality). Remarks. The specific epithet ending with -er is masculine despite the gender Agelas is feminine, as a consequence the ending must be changed into -ra. Because of the overlap in spicule dimension de Voogd et al. (2008) report that is not possible to distinguish different size categories.

Agelas spp.
Several findings from the Seychelles Archipelago (Thomas 1973) and Kenya (Pulitzer-Finali 1993). The revisitation of the 11 Agelas specimens slides of the Pulitzer-Finali collection from East Africa highlighted the presence of only styles in the spicular complement with notable variability of thickness, length, and spinosity. This confirms his opinion: 'It would be inappropriate at the moment to try to identify some of them with established species and to propose new species.' Also in this case the original material is not available after damaging of types during the recent Genova flood in 2014 (October). Figure 21. Agelas semiglabra. Spicular complement with two dimensional categories of acanthostyles; long acanthostyles spiny only at the tips (modified from Pulitzer-Finali 1996).