1.1. A review of the southern African ascidian literature

The history of the literature and a bibliographic list for the then known global ascidian fauna from about 330 B.C. to 1882 A.D. is given by Herdman, 1882 in his report on solitary ascidians ‘Ascidiae Simplices’ collected during the H.M.S. Challenger expedition. The voyage can be divided into a series of comparatively short stages covering the globe from 1873 to 1876. The 1882 report of Herdman includes references to the preliminary report (Part 1 and 2 of Herdman (1880)) that contains the description for Clavelina enormis Herdman, 1880 collected from Simon’s Bay, South Africa. This species description, based on a single specimen, is short and the specimen’s taxonomic status remains ambiguous (Monniot, C. et al. 2001). In the 1882 report by Herdman an account is given of eighty-two species arranged in (then) twenty genera.

Herdman’s subsequent report on the colonial ascidians ‘Ascidiae Compositae’ published in 1886 contained a supplementary list on early literature. The report gives an account of one hundred and two species arranged in (then) twenty-five genera, which includes descriptions on eight species collected from the South African coast. The South African species described were viz.: Atropogaster elongata var. pallida, Leptoclinum albidum, Goodsiria placenta (junior synonym to Gynandrocarpa placenta), Goodsiria placenta var. fisca (junior synonym to Gynandrocarpa placenta), Amaroucium colelloides (junior synonym to Aplidium colelloides), Psammaplidium exiguum (junior synonym to Aplidium exiguum), Psammaplidium subviride and Leptoclinum edwardsi. Diplosomoides molle (junior synonym to Didemnum molle) from Arrou Island (north of Australia) was subsequently described from the KwaZulu-Natal coast off South Africa by Monniot et al. (2001).

Adding to our early knowledge of the southern African ascidian fauna was the work done by Sluiter (1898a) in the report ‘Ergebrisse einer Reise van Prof. Max Weber in Jahne 1894’. In this work he described thirty-two species, thirty new to science, one solitary specimen (Microcosmus coalitus) from the Namibian coast, and nine from neighbouring Mozambique. Species that had been recollected included Amaroucium flavoliniatum (junior synonym to Aplidium flavolineatum), Polyclinum isispingense and Distoma caeruleum (junior synonym to Eudistoma caeruleum) (Michaelsen 1919, Michaelsen 1934, Millar 1956b, Millar 1962a, Lafargue and Vasseur 1989, Monniot, C. et al. 2001).

Michaelsen (1904a) reported on the collections made during the Steamer Valdivia’s 1989-1899 ‘Deutschen Tiefsee-Expedition’ off the Cape of Good Hope, Plettenberg Bay, St Francis Bay and on the Agulhas Bank. This included new species such as Halocynthia vanhöffeni (junior synonym to Pyura stolonifera), Cynthiopsis valdiviae (junior synonym to Pyura herdmandi), Microcosmus albidusand Monandrocarpa tritonis (for further information see Michaelsen, 1904b). The subsequent ascidian descriptions by Dr. R Hartmeyer (1912) contributed further to the knowledge of the group occurring off the southern African coast and led to the description of twenty-two species of which thirteen were new to science. A subsequent species list of ascidians from the South African coast, given by Hartmeyer in 1913, includes new species descriptions for Trididemnum cerebriforme and Aplidium schultzei.

Work by Michaelsen (1921a) on the Western Indian Ocean, his research in 1923 on the South African ascidians (Michaelsen 1923c) and his study on ‘The Ascidians of the Cape Province of South Africa’ (1934) are valuable contributions to our current knowledge of the ascidian fauna found in southern African waters.

After Michaelsen’s contribution to the taxonomy of South African ascidians two decades of inactivity was followed by the work of Dr. R.H. Millar, who in his illlustrious career (stretching from the 1940’s to 1980’s) investigated many aspects of ascidian biology (Millar 1949a, 1949b, 1951, 1952a, 1953a, 1963b, 1963c, 1971, 1974b), systematics (Millar 1950, 1952b, 1954a, 1959, 1960d, 1963d, 1966a, 1970a, 1970b, 1978) and evolution (Millar 1966b). He studied the ascidian fauna of the Scottish (Millar 1950, 1952b, 1960b, 1988d) and British coast (Millar 1970b), northern Europe including Iceland (Millar 1966c, 1967b, 1974a), Brazil (Millar 1958, 1961b, 1977), the Caribbean (Millar 1962b, Millar and Goodbody 1974), New Zealand (1982b), the Pacific (1975, 1988b, 1988c), Atlantic (Millar1955a, 1982a) and Indian Oceans (1988a), the Antarctic (Millar 1960a, Millar 1968b) as well as the deep water ascidians from Galathea (Millar 1957a, 1964a, 1969a). In addition he investigated and contributed significantly to the knowledge of the ascidian fauna of southern Africa (Millar 1954b, 1955b, 1956b, 1961a, 1962a, 1963b, 1964b), North Africa and the African Gold coast (Millar 1953b, 1956a, 1957b, 1960c, 1965, 1968c), the Vema Seamount (Millar 1968a) and Madagascar (Millar 1967a).

This preceded a second period of inactivity where again very little interest was shown in southern African ascidians and it was only in 1992 that a collection of ascidians made by Prof. C. L. Griffiths (University of Cape Town, Cape Town) and Dr. M. Schleyer (Oceanographic Research Institute, Durban) was sent to the internationally renowned ascidian taxonomists, Dr. C. Monniot and Dr. F. Monniot (at the Muséum national d’Histoire naturelle, Paris), for taxonomic identification and description. The subsequent paper, Monniot et al. (2001) describes eighty-two species of which twenty-two are new. The ascidians were collected mainly from shallow waters along the coast from Saldanha Bay in the west to Sodwana Bay in the east of the southern African coast. This publication includes a short summary of the species previously collected from the southern African coast but not recollected, as well as species of which the current taxonomic state is ambiguous.

The work by Monniot et al.(2001) concludes the literary contributions made to South Africa’s ascidian knowledge with the exception of four natural product papers that describe bioactive secondary metabolites produced by ascidians. These secondary metabolites include new antimicrobal amines (Hooper et al. 1995) and new nitrogenous constituents (Rashid et al. 2001) in Pseudodistoma species from Tsitsikamma Marine Reserve and Algoa Bay off the Eastern Cape coast. New alkaloids have also been isolated from a species of the Aplousobranchia genera, Polycitor (family Polycitoridae) from Sodwana Bay (Rudi et al. 1994) and Lissoclinum (family Didemnidae) from Aliwal Shoal, Umkomas off the KwaZulu-Natal coast (Patil et al. 1997).

Ascidians are a prominent component of the intertidal and subtidal reef fauna along the southern African coast, but despite the numerous past contributions very little is known about these animals, their species composition, distribution and diversity. Even less is known about their ecology and community structure. About fifty intertidal species have been described; about ninety-four species from subtidal reefs out to a depth of 40 m and thirty-nine species from depths of 40-150 m (Parker-Nance 2001). The above account clearly highlights the incompleteness and sporadic nature of data collection over the last century and the present meagre species list of the South African fauna is clearly not a true reflection of the richness, diversity or importance of ascidians to the structure of South African reefs. This thesis represents the first study of the taxonomy of southern African ascidian fauna by a resident South African scientist. The aim of this thesis is therefore to add to the paucity of knowledge of this unique and important group of organisms.

1.2. Biogeography

Community composition and natural processes occurring along the South African coast is better understood for the littoral zone than for subtidal habitats (Stephens 1937). Information available on intertidal communities and processes along the southern African coast suggest that different biogeographical regions exist (Stephens 1937, Ekman 1953, Briggs 1974, Branch et al. 1994).

The area between Ponta do Ouro (on the Mozambique border) to just north of East London (to the south) constitutes the subtropical component of the South African coast (Figure 1). The continental shelf fringing the coast is narrow and the warm water from the Mozambique channel flows close to shore bringing with it a number of more tropical Indo Pacific species. The northern part of KwaZulu-Natal is the only area along the South African coast that has a number of coral reefs; the rest of the coastline consists of warm temperate reefs (Figure 1).

South of East London the continental shelf increases in width pushing the warm water away from the coast resulting in cooler water temperatures. The area is characterised by a warm temperate reef fauna made up of a number of soft-bodied sessile invertebrates. The main sessile components are sponges, ascidians and bryozoans. Species diversity and composition is not known.

The area between East London and Cape Point forms a transition zone between the warm Agulhas on the east coast and the colder Benguela current on the west coast. The west coast of South Africa has a smaller number of species but sustains highly productive food chains fuelled by up-welling events when cold nutrient rich water is brought up to the surface. This has resulted in the area sustaining a lucrative fishing industry (Lubke and de Moor 1998; Branch et al., 1994).

1.3. Collection sites off the southern African coast

1.3.1. Western Cape Province

The Western Cape Province has a coastline inclusive of the area between the Komkans on the west coast to the Bloukrans river mouth east of Knysna. Specimens were collected from South Paw in a depth of 17 to 20 m off Clifton beach between Bantry Bay in the north and Camps Bay in the south. Specimens were also collected from Vulcan Rocks situated southwest of Hout Bay, which is situated on the western side of Cape Peninsula. The reef is composed of a number of large granite boulders and pinnacles, which causes the surface to break. Collections were made from a depth of 20 to 27 m. Further collections were also made from False Bay, a large bay situated on the east side of the Cape Peninsula from Cape Point in the west to Kaap Hangklip on the east. Bakoven is situated just south of Millar’s Point between Simon’s Town in the north and Smitswinkel Bay in the south. Specimens were collected from a depth of between 17 to 19 m. The collection of specimens from Plettenberg Bay, situated on the eastern border of the Western Cape Province, were made from depths of 16 m from Groot Bank and Playground reefs situated north east of Plettenberg Bay between Plettenberg Bay and Natures Valley (Figure 1).

1.3.2. Eastern Cape Province

The Eastern Cape Province lies between the Bloukrans River mouth on the west and Port Edward north of East London to the east. Specimens were collected from Tsitsikamma Marine Reserve, St Francis Bay, Algoa Bay and East London. Collections were made from Middelbank Reef at depths of 27 to 30 m and Rheeders Reef from a depth of 5 to 30 m within the Tsitsikamma Marine Reserve at the mouth of the Storms River. Specimens were collected from Thunderbolt reef situated in the eastern side of St Francis Bay just west of the Cape Recife lighthouse from 18 to 40 m. The area is not protected and sea conditions may be unpredictable with a large sea and low visibility. Intertidal collections were made from the Table Mountain sandstone rocky shore off Cape Recife. This rocky cape divides St Francis Bay to the west from Algoa Bay in the east (Figure 1).

Algoa Bay is a large crescent-shape bay with depths within the bay up to 66 m. The subtidal reefs in the western part of the bay are protected and easily accessible. The reefs that have formed part of specimen collections include Postman’s Reef situated close inshore at 5 m, Philip’s Reef (Night Reef) from a depth of between 12 to 17 m, off shore from Shark Rock and the Old Slipway. Specimens were also collected from White Sands Reef from a depth of 12 to 30 m and Bell Buoy at a depth of 20 m. The East London coastline was sampled at Three Sisters off Danger Point north of Nahoon Point at a depth of between 14 to 19 m and off shore from Cintsa north of East London at a depth of 16 to 17 m.

1.3.3. Mozambique

Three specimens from a small collection made off the coast of Mozambique by Dr. P.S. Coetzee (the collection is currently held by the University of Port Elizabeth) are described in this study. The Mozambique specimens belong to a new species with a know distribution from Tsitsikamma Marine Reserve in the south to Mozambique in the north (see Chapter 4 for species descriptions and details on the sample localities).

1.4. Materials and Methods

Ascidian specimens must be collected and preserved with the utmost care. Collected specimens should be kept alive and be relaxed with menthol as soon as possible after collection. If not properly relaxed specimens are very difficult to examine and describe.

Once collected, specimens were submerged in fresh seawater and left to stand for about an hour without disturbance. A small menthol crystal (ca. 2 mg) was then added to each of the specimens and the collection was placed in a fridge (ca. 4°C). Samples were then left undisturbed for between 4 to 8 hours after which sufficient concentrated formaldehyde (37%) was added to give an approximately 10% formalin solution, without disturbing the samples. The sample containers were then sealed once mortality had set in. A more concentrated final formalin solution in seawater was used than the 4 % used by Monniot et al. (2001) to fix and preserve specimens. Experience has shown that due to the large size of the majority of the specimens collected a lower concentration sometimes leads to the degradation of the sample due to decay. Samples left at room temperatures (22 - 30°C) died relatively quickly and didn’t relax as well as those kept at lower temperatures.

Preserved ascidian specimens were then washed with fresh water to remove formalin before dissection. Organs were stained with Mayer’s hemalum stain (Humason 1979). Specimens were dehydrated with ethyl and butyl alcohols prior to mounting on microscope slides. Canada balsam was used in the mounting and slides were left to dry and clear. Specimens were then examined and line drawings were made with the aid of a camera lucida. Measurements were made with the aid of an ocular micrometer. Colour images were taken of some specimens by means of an Image Analyser camera system.

All type specimens, holotypes and paratypes, are registered in the South African Museum, Cape Town and have been allocated a SAM A registration number. Additional material is stored in the Tunicate and Marine Invertebrate Collection housed at the University of Port Elizabeth. TIC (Tunicate and Invertebrate Collection) numbers are allocated to all specimens housed in this collection.

1.5. Collection information

Early collections were made by Dr. P.S. Coetzee and are indicated together with the collection information for each specimen.

Collections by the Coral Reef Research Foundation (CRRF):

This research collaborative programme was made by a group of scientists representing the Coral Reef Research Foundation under the leadership of Dr. P. Colin and collection manager Dr. L. Bell-Colin. The CRRF collection team was also accompanied by Mr. J. Starmer and Mr. D. deMaria, others involved in this collection programme include Prof. M. Davies-Coleman, Dr. K. McPhail and Dr. C. Gray from the Chemistry Department, Rhodes University and Ms. S. Parker-Nance and Mr. A. Cloete from the Zoology Department of the University of Port Elizabeth.

Scripps Institution of Oceanography (SIO) collections:

This research collaboration was made by a group of scientists representing Dr. J. Faulkner’s laboratory at the Scripps Institute of Oceanography represented by Ms. C. Sincich, Dr. L. West, Dr. C. Ridley and Dr. D. Mustra by in association with Prof. M. Davies-Coleman and Dr. C. Gray from the Chemistry Department, Rhodes University and Ms. S. Parker-Nance and Mr. A. Cloete from the Zoology Department of the University of Port Elizabeth.

All other collections not cited were made and photographs taken by S. Parker-Nance.

1.5.1. Acronyms used in this study

CRRF - Coral Reef Research Foundation.

SAM - South African Museum, Cape Town, registration numbers allocated to holotypes and paratypes.

SIO - Scripps Institution of Oceanography.

TIC - Tunicate and Invertebrate Collection numbers for the collection of ascidians and invertebrates held by the University of Port Elizabeth.

0CDN - Collection of the Coral Reef Research Foundation housed in Koror, Palau.

1.6. A brief introduction to ascidian biology

1.6.1. General body structure

The growth form of ascidians can be either solitary (Kott 1985, Monniot, C. et al. 1991) or colonial (Kott 1990, 1992, 2001). Solitary species consist of a single individual surrounded by a tunic (Goodbody 1974), which in the case of Pyura stolonifera (Heller, 1878) is tough and protective and may be covered by numerous epibiont species (Figure 2a) (Millar 1955b, Millar 1962a, Kott 1985). Ciona intestinalis (Linnaeus, 1767) which, is common in harbours and marinas and becomes problematic because of its fouling tendencies, has a soft, almost transparent, outer test without epibionts (Kott 1990). Solitary species generally have a sac-like body with the branchial sac occupying the greater part of the individual. The gut, the gonads and the heart are situated along side of the branchial sac (Figure 3a) (Kott 1985, Monniot, C. et al. 1991).

Colonial species are characterised by a large number of zooids that are connected or embedded to varying degrees by or in a common test. In Pseudodistoma africanum Millar, 1954b and P. digitum sp. nov. the zooids are completely embedded (Figure 2b,c). In the cosmopolitan species Clavelina lepadiformis (Müller, 1776) the zooids are connected only by the basal stolons (Figure 2d) (Monniot, C. et al. 2001). The individuals reproduce sexually but colonies grow by asexual reproduction e.g. strobilation and, or budding (Berrill 1950, Kott 1992). For developmental information and aspects concerning the gametes, fertilization and embryogenesis, metamorphosis and asexual development in solitary and colonial ascidians see Berrill (1950) and Satoh (1994).

Zooids of the Aplousobranchia have the gut loop below the branchial sac. The body of the zooid can be divided in two or three regions. Zooid of e.g. the family Didemnidae has no posterior abdomen with the thorax containing the branchial sac and an abdomen that contains the gut and gonads (Kott 2001). Zooids of e.g. the genus Pseudodistoma and Polyclinum have a posterior abdomen in which the gonad and heart are situated (Berrill 1950, Monniot, C. et al. 1991, Kott 1992). The posterior abdomen is continuous with the abdomen in Pseudodistoma but is constricted off the abdomen in Polyclinum species. The structure of the branchial sac, the gut and the position and structure of the gonads are all important characteristics in identifying the various genera and species (Kott 1992).

The branchial opening forms the inhalant aperture and leads to the branchial sac. Water is pulled into the branchial sac by the action of the cilia on the stigmata within the branchial sac wall. The atrial opening is the exhalant aperture through which the filtered water and faecal matter leaves the zooid. Depending on the species the atrial aperture may open directly to the surface of the colony (as is in the genus Euherdmania and Pseudodistoma), alternatively, a number of zooids may open into a chamber within the colony as found in the genera Polyclinum and Polysyncraton (Kott 1990, 2001). This chamber is connected to the exterior by a single opening. These structures are known as common cloacal systems and the zooids may be arranged in simple circular systems or the systems may be extensive and elaborate.

1.6.2 The branchial sac

Ascidians are filter feeders inhaling water through the branchial aperture into the branchial sac, which is a sac-shaped filtering device with perforated walls. These perforations, or stigmata, are lined with cilia. The synchronously beating of the cilia on the stigmata generates the current necessary for the movement of water into the branchial sac (Berrill 1950, Goodbody 1974, Monniot, C. et al.1991). The internal surface of the branchial sac is lined with a thin net-like mucus lining, which is secreted by cells in the endostyle, situated along the mid-ventral line (Figure 3a(3)). The mucus net moves over the branchial sac dorsally and posteriorly in the direction of the oesophagus. The food particles suspended in the incoming water are caught in this mucus layer or net and the filtered water leaves the branchial sac through the stigmata. From the branchial sac the filtered water moves into the peribranchial or atrial cavity. The mucus net is rolled into a strand dorsally by the dorsal languets, then the strand moves posteriorly into the oesophagus. This strand is laden with captured food particles and is pulled gradually towards the oesophagus to the stomach where the food particles are digested.

1.6.3. The gut loop (digestive tract)

The gut loop is situated along side (as in solitary ascidians Figure 3a) or beneath the branchial sac (as in colonial species Figure 3b) and divided into a number of sections (Goodbody 1974, Kott 1990, Monniot, C. et al. 1991). Food particles are digested in the stomach, which in turns leads into a mid-intestinal region and a post-stomach. The faecal matter in the rectum is deposited in the peribranchial cavity (also known as the atrial cavity) via the anus. From the peribranchial cavity the faecal matter is removed from the animal by filtered water flowing out of the branchial sac and pumped to the exterior via the atrial aperture.

1.6.4. Larvae

Ascidian larvae of the Aplousobranchia are generally the shape of tadpoles and can be divided into the larval trunk and a tail. The trunk contains adhesive papillae (usually three) at its anterior end while interiorly there is a cerebral vesicle (containing an ocellus and a unicellular otolith) as well as the developing branchial sac and gut. The larvae are non-feeding and the tail is used for mobility aiding in the dispersion of the larvae. The tail muscles are arranged on each side of the single row of around forty cells that represent the notochord (Berrill 1950). After dispersal and settlement the larvae undergoes metamorphoses and becomes a feeding and reproducing zooid. Larvae have a number of characteristics (Berrill 1950) that may be helpful with the identification and classification of an ascidian specimen. These characteristics include the position of the adhesive organs, the shape number and position of the ampullae and vesicles as well as the size of the larvae. In the colonial ascidians the larvae are incubated in the peribranchial cavity, oviduct or in an incubation pouch constricted off the zooid (Monniot, C. et al. 1991). Larvae are not always present in material collected and in a large percentage of species currently described the larval structure is unknown.

1.6.5. Systematics

The class Ascidiacea can be divided into three orders: Aplousobranchia, Stolidobranchia and Phlebobranchia (Berrill 1950, Monniot, C. et al. 1991, Kott 1992). The Aplousobranchia are represented by colonial forms and the Stolidobranchia and Phlebobranchia mainly by solitary species. The present study focuses on four Aplousobranchia families; Euherdmaniidae, Polyclinidae, Pseudodistomidae and Didemnidae (as defined by Kott 1992, 2001).

The family Euherdmaniidae Ritter, 1904 emend. Kott, 1992 is represented by a single genus Euherdmania (Ritter, 1904). This small genus has twelve known species worldwide with a tropical or warm temperate distribution. Only one species, Euherdmania divida Monniot, F., 2001 (Monniot, C. et al. 2001) an intertidal species collected from Algoa Bay, Eastern Cape Province and Isipingo, KwaZulu-Natal has thus far been described from South Africa (Chapter 2).

The family Polyclinidae Milne Edwards, 1842 emend. Kott, 1992 is represented by six genera: Aplidium Savigny, 1816; Aplidiopsis Lahille, 1890; Morchellium Giard, 1872; Polyclinum Savigny, 1816; Sidneioides Kesteven, 1909 and Synoicum Phipps, 1774. Aplidiopsis is a small genus with fifteen known species worldwide. Only one species is known to occur along the South African coast (Monniot, C. et al. 2001). New collections of A. tubiferus Monniot, F., 2001 (Monniot, C. et al. 2001) extends the previous known distribution of the species (Chapter 3). The genus Polyclinum is represented by forty species, P. isipingense Sluiter, 1898a and P. arenosum Sluiter, 1898a have been recorded from the Western Cape Province to the east coast of KwaZulu-Natal. P. neptunium Hartmeyer, 1912 has been recorded only for the Western Cape Province and P. constellatum Savigny, 1916 is only known to occur off the KwaZulu-Natal coast. In the present study, two new species are described, one collected off Tsitsikamma Marine Reserve and the second from Tsitsikamma Marine Reserve and Algoa Bay within the Eastern Cape Province, South Africa (Chapter 3).

The Pseudodistomidae Kott, 1992 is represented by the genus Pseudodistoma Michaelsen, 1924; Anadistoma, Kott, 1992 and probably the monotypic genus Citoclinum Monniot, F., et al. 1988. The genus Pseudodistoma has thirty-one known species, of which two are known from the South African coast. Current research has shown that this is a genus commonly encountered and specimens are abundant along the South African coast. Most of the species, with the exception of P. delicatum Monniot, C. et al. 2001, are large robust colonies. A number of new species have been found of which two are described in Chapter 4.

The family Didemnidae Giard, 1872 has eight genera; Atriolum Kott, 1983; Didemnum Savigny, 1816; Diplosoma MacDonald, 1859; Clitella Kott, 2001; Leptoclinides Bjerkan, 1905; Lissoclinum Verrill, 1871; Polysyncraton Nott, 1892 and Trididemnum Della Valle, 1881. Atriolum has only six known species worldwide (Kott 2001) but only one has been recorded from the southern African coast (Chapter 5). Although this species has been recorded from the Mozambique Channel, New Caledonia and the Coral Sea this is the first recorded occurrence from South African. The genus Polysyncraton is extensive with sixty-eight known species worldwide. Five species are known to occur around the South African coast (Chapter 5). One new species is described for this genus; currently the only recorded Polysyncraton species, from the Eastern Cape Province.

The following section is divided in to five chapters and two Appendices. Chapters 2 to 5 each concerns one of four families. Chapter 2 examine the family Euherdmaniidae and the genus Euherdmania, Chapter 3 examine the family Polyclinidae and the genera Aplidiopsis and Polyclinum, Chapter 4 examine the family Pseudodistomidae and the genus Pseudodistoma and Chapter 5 examine the family Didemnidae and the genera Atriolum and Polysyncraton. Each chapter is divided into a short introduction, a discussion and a literary overview of all the species known globally within the selected genus. This includes a summary of taxonomic characters relevant to the identification of the species and a short discussion on the global distribution of the species within the genus. This is followed by species descriptions for the relevant taxa. The species description is divided into three sections: a distribution, description and remarks section. Chapter 6 contains a conclusion.

Appendix A lists the species recorded for specific geographical areas world wide for the six genera discussed in Chapters 2 to 5. Appendix B gives the synonymy for all species in the selected genera focussed on in this study.