New South African Acanthodrilinae Earthworm Species, with New Data for Some Earlier Known Members of the Genus Parachilota(Oligochaeta: Acanthodrilidae)

Four acanthodriline species of the genus Parachilota , namely abebaios sp. n., nkandu sp. n., uysae sp. n., and timothyi sp. n., are described and illustrated. Additional information is given on the characters, geographical range and habitat of the earlier known six Parachilota species: editha , hottentotianus , karkloofi , minimus , wahlbergi , and warreni . The shape and number of spermathecal diverticula were considered as the diagnostic characters of studied species, and three species groups are recognized: unilobate, bilobate and multilobate. Species endemism is noted.


INTRODUCTION
This paper is a continuation of a survey of the acanthodriline species in South Africa, based on the collection in the Natal Museum, Pietermaritzburg, South Africa. During the present study it was observed that the structure of the spermathecae is a valuable specific character. Three species groups were recognized: (1) unilobate, with only one, externally not divided spermathecal diverticulum; (2) bilobate, with two variably shaped and located diverticula; and (3) multilobate, with spermathecae having more than two diverticula. For now, the recognition of these species groups is applied to the present material. A confident assignment of other Parachilota species to these, and probably other, species groups, requires a comprehensive revision of the entire genus, which is beyond the scope of the present paper.

MATERIAL AND METHODS
The study was based on the examination of earthworms collected by the author with assistance of Dr B.R. Stuckenberg and Mr T. Liversage, and on material donated by various collectors. Four new species and seven earlier known South African acanthodriline species kept at the Natal Museum were compared with the material described or revised by Pickford (1927Pickford ( , 1937 housed at the South African Museum in Cape Town and kindly loaned to the author. Although some studied specimens were preserved differently, most were narcotised in 45 % alcohol, then fixed in 4 % formalin solution and thereafter transferred to 75 % alcohol. For the internal anatomy, dorsal longitudinal dissections were performed. Data were taken from the original labels. Coordinates are given in brackets for all place names as recorded on the original labels. Data not found on the original labels are given in square brackets. Measurements of the setal formulae are expressed in intersetal ratio. The photographs were taken with a Wild Heerbrugg M8 stereomicroscope. The methods and terminology used follow earlier publications (Plisko 2004(Plisko , 2007. The shape and number of spermathecal diverticula considered as valuable specific characters are employed to separate species in the unilobate, bilobate and multilobate species groups. Types of the new species are deposited in the Natal Museum, Pietermaritzburg.
The following abbreviations and acronyms are used: BMNH -Natural History Museum, London, UK; NMSA -Natal Museum, Pietermaritzburg, South Africa; SAMC -South African Museum, Cape Town, South Africa, with A followed by a number of the SAMC Oligochaeta Collection; ZMUH -Zoological Museum and Institute, University of Hamburg, Germany; BRS -B.R. Stuckenberg; JDP -J.D. Plisko; KZN -KwaZulu-Natal province of South Africa; cl -clitellate; juv -juvenile.

TAXONOMY
Subfamily Acanthodrilinae Claus, 1880 Genus Parachilota Pickford, 1937emend. Parachilota: Pickford 1937Zicsi 1998: 63;Plisko 2004: 293. Udeina Michaelsen, 1910  Diagnosis: Male reproductive organs acanthodriline. Testes and male funnels in proandric arrangement. Excretory system holoic, avesiculate. Seminal vesicles paired; in 9 and 11, or in 9, 11 and 12, variably developed, sometimes posterior pair much reduced. Two pairs of spermathecae, variably shaped, with unilobate, bilobate or multilobate diverticulum. Gizzard in 5 or 6, well developed, moderately developed, rudimentary or reduced. Last pair of lateral hearts in 12 or 13. Prostatic pores 2 pairs, not approximate towards the mid-ventral line, or situated close to the mid-ventral line, each pair on 17 and 19, or 18 and 20. Male pores 1 pair on 18 or 19. Spermathecal pores paired; in intersegmental furrows 7/8 and 8/9, or at the anterior borders of segments 8 and 9. Internal characters: Salivary glands: Do not extend backwards beyond septum 4/5. Gizzard: In 5, well developed, cylindrical, muscular. Septa: 5/6 thin but strong; 6/7-11/12 increasing in thickness, with 10/11 and 11/12 most thickened; 12/13 and following thin. Intestine: Commences abruptly in 16, with oesophagus enlarged in 13, followed by longitudinal ridged valves in 14-15. Lateral hearts: In 9-13 with last pair in 13 much enlarged. Nephridia: Holoic, avesiculate. Ovaries: Not observed. Testes and male funnels: Ventrally in 10; funnels large, free, iridescent. Vasa deferentia: Thick single ducts run from 10 to 18. Seminal vesicles: 3 pairs; in 9, 11 and 12; anterior pair small, commencing at septum 9/10 ventrolaterally; second pair commencing at 10/11, in 11, moderate; posterior pair commencing dorsolaterally at 11/12 largest, lobulated, tufted. Spermathecae (Fig. 2): Paired; in 8 and 9; ampulla oval, small, 2.5 mm long; duct elongated, 4.5 mm long, 1.4 mm wide at its ental part; unilobate, small diverticulum 1.8 mm long, attaching to duct at its basal part, then narrows its width to nearly 0.7 mm. Iridescent sperm observed in diverticulum, and in spermathecal duct at its joint with ampulla. Ectal parts of spermathecal ducts enter body wall near septa 7/8 and 8/9. Prostates: Paired, tubular, multifolded, looped. Prostatic duct commences as muscular straight tube, extending into thin, tubular, looped and slightly coiled prostatic gland. Anterior pair confined to 17; posterior pair extends backwards, conically pushing septa 19/20-22/23 into space of three segments. Ectal parts of prostatic ducts enter body wall in 17 and 19 respectively. Penial setae ( Distribution: P. abebaios is known only from the type locality in the north-eastern Drakensberg escarpment, Mpumalanga (the area earlier known as the Eastern Transvaal). The species occurs together with P. timothyi sp. n. Biological notes: The large, mature, well-developed individual was collected in summer, during the rainy season. Iridescence observed in the male funnels and diverticula confirms sexual activity. Discussion: P. abebaios is a distinct species with 3 well-developed pairs of seminal vesicles in 9, 11 and 12, the character first noted in this genus. It was observed (Pickford 1937) that in proandric Parachilota species, in which a reduction of posterior pair of testes took place, the seminal vesicles became reduced to two, having sometimes only a rudimentary posterior pair in 12 as was noted in P. stephensonianus Pickford, 1937. P. abebaios is also characterized by a distinctive shape of spermathecae with extended spermathecal duct and unilobate diverticulum. Pickford, 1937 Fig. 4 Parachilota hottentotianus: Distribution: The present record confirms the endemic occurrence of this species in the eastern part of the Hottentots Holland Mountains, extending the known range of the species slightly beyond the earlier noted site near Caledon.

Parachilota hottentotianus
Biological notes: New material was found in early spring, after a few rainy days, on the surface of a soaked pathway, together with the microchaetid Kazimierzus sirgeli (Plisko, 1996). The penial setae and spermathecae are in early development, and no sperm was observed in small diverticula, suggesting that the specimen was at the early sexual phase, although prostates were well developed, each confined to one segment (Fig. 4). Comments: New material, although being not fully mature, with only slightly marked clitellum, matches the description of this species. The unilobate spermathecae and distal end of penial setae resemble these drawn by Pickford (1937), and match characters observed in the type material.
Biological notes: The Ncandu Nat. Res., earlier known as Incandu Forest Reserve and recently placed under the protection of Ezemvelo KZN Wildlife, is situated on the border of KZN and the Orange Free State. It extends to the Drakensberg northern escarpment, south-west of Newcastle (27°42'S:29°59'E). The area where the species was abundant extends over the indigenous grassland plateau along the Ulumbi R. at 1830 m above sea level. A large part of the plateau covered by indigenous grassland extends below the river with several small pockets of indigenous bushes, and continues on the other side of the river. P. ncandu was found in grassland moist soil, on the river bank, under stones, and under mosses covering large rocks with flowing surface water. A large species population at different states of development occurred in the grassland and forested areas. Iridescence observed in male funnels and in spermathecal diverticula suggests a summer phase of sexual activity. P. ncandu was collected together with P. timothyi sp. n. and the microchaetid Proandricus bourquini Plisko, 1996. Discussion: The commencement of the intestine in segment 17 and the location of the lateral heart in 12, features observed in P. ncandu and P. erythrocephalus Pickford, 1937, might suggest species relationships. However, the shape of the spermathecal diverticulum and penial setae differ substantially in both species, and a relationship between these two species cannot be confirmed. Considering the geographical distance separating the species, it may rather be suspected that these characters have evolved independently, with parallel differentiation of the spermathecal diverticulum and penial setae. The species accredited by Pickford (1937) to the P. erythrocephalus species-group, P. traegardhi (Michaelsen, 1907), P. warreni (Michaelsen, 1913), and P. nanus Pickford, 1937, known from KZN differ from P. ncandu in the specificity of the spermathecal diverticulum and also in other features. P. bainellus, P. ruficeps and P. adolphus, described by Pickford (1937) and placed in the erythrocephalus species-group, are recorded from the Western Cape and differ in the complexity of their morphological characters.

Parachilota timothyi sp. n.
Figs 6-9 Etymology: Named after Mr Timothy Liversage, who assisted with collection of the type series. Diagnosis: Spermathecal pores in intersegmental furrows 7/8 and 8/9. Clitellum saddleshaped on 13,14-1/n17,17. Male pores in 18. Prostatic pores not approximate towards the mid-ventral line, in 17 and 19. Gizzard in 5, well developed. Commencement of intestine in 16. Last pair of lateral hearts in 13. Seminal vesicles paired, in 9 and 11. Spermatheca with unilobate diverticulum attached to basal part of spermathecal duct, extending to joint duct/ampulla and invagination of ampulla. Description: External features: Body cylindrical, firm. Colour: In life dorsally violet, ventrally brownish grey; alcohol-preserved dorsally violet, extending violet colouration laterally to c setal lines, with dark tint on preclitellar and few last posterior segments. Colour fading after extended preservation. Dimensions: Holotype 140×7 mm; clitellate paratypes 120-137×6-7 mm; juvenile 30-72 mm. Segment number: Holotype 153; clitellate paratypes 163-210; juvenile 109-220. Prostomium: Tanylobous with obvious sutures. Setae: Paired; on postclitellar segments aa:ab:bc:cd = 3.5:1.8:4:2; distance between ab decreasing on 12-17, increasing on 19-27; this characters clearly observed also on juvenile specimens. Dorsal pores: Not observed. Nephridial pores: Not observed on holotype, although on some individuals noted occasionally in postclitellar intersegmental furrows, in c setal lines. Spermathecal pores: Paired; in 7/8 and 8/9, in front of b. Female pores: Paired, in 14 between aa. Clitellum (Fig. 6) ; anterior pair small, commencing at septum 9/10 ventrolaterally, little lobulated; posterior pair commencing dorsolaterally at septum 10/11 large, brownish, much lobulated. Spermathecae (Fig. 7): Paired; in 8 and 9; ampulla smooth, 2 mm long and nearly 2 mm wide, with external indentation at link with spermathecal duct; duct 1 mm long; unilobate diverticulum commencing at basal part of spermathecal duct, extending to joint duct/ampulla locates its global ental part in ampulla's external dent. Iridescent sperm was observed in ental part of diverticulum. Ectal parts of spermathecal ducts enter body wall at 7/8 and 8/9. Prostates (Fig. 8) Distribution: Known from the outskirt of the Drakensberg Escarpment in the northwestern part of KZN, and in Mpumalanga. Biological notes: The species occurs in grassland and forest soil, on river banks, and near roads. Abundant in Ncandu Nat. Res. grassland plateau around the Ulumbi R., at ca 1830 m in moist soil, and between various plant roots on the river bank, in indigenous riverine bush, and on scattered grassland plateau rocks covered by moist moss watered by flowing water. The new species occurs together with the indigenous acanthodriline Parachilota ncandu sp. n., the microchaetids Proandricus bourquini Plisko, 1996 and Tritogenia palusicola Plisko, 1997, and with the exotic lumbricids Aporrectodea rosea (Savigny, 1826), Aporrectodea trapezoides (Dugès, 1828), Octolasion lacteum (Örley, 1881), Dendrodrilus rubidus (Savigny, 1826), and megascolecid Amynthas sp. Discussion: P. timothyi, having the last pair of lateral hearts in 13 and the intestine commencing in 16, may be related to P. wittebergensis Pickford, 1937, known from Witteberg Mountains and Mont-aux-Sources, both sites located south of the occurrence of the new species, in the Drakensberg Mountains. The species differ in the shape of the spermathecae, which are bilobate in witteberngensis, attached to anterior face of spermathecal duct, while in timothyi the unilobate diverticulum is at the base of the duct, extending to the junction with the ampulla.
Parachilota wahlbergi (Michaelsen, 1899) Fig. 10 Chilota wahlbergi: Michaelsen 18991900: 147;1912: 146. Chilota wahlbergi f. typicus: Michaelsen 1913a: 416. Chilota wahlbergi n. f. pulchior Michaelsen, 1913aPickford 1937 Biological notes and distribution: Pickford (1937), who summarized collection data published by Michaelsen (1899Michaelsen ( , 1900Michaelsen ( , 1913a and the sites of the material studied by herself, accepted a broad occurrence of this species in north-eastern South Africa. It was known that the species was collected in moist or semi-moist localities, and in the neighbourhood of waterfalls, in KwaZulu-Natal, Gauteng, Mpumalanga and Limpopo. She also observed that some of the individuals from Krugersdorp area in Gauteng discharged a phosphorescent fluid. During my extended study on acanthodrilinae in South Africa, only one specimen of wahlbergi, the presently studied one, was collected in KZN in a small drying out artificial pool, in the neighbourhood of a man-made lake where environmental changes are taking place. It is possible that this species may be on the way to extinction as a result of anthropogenic environmental changes. The studied specimen was donated alive to the Natal Museum, and no phosphorescence was observed before and during preservation. Comments: The new material matches descriptions of the type material (Michaelsen 1899(Michaelsen , 1900) extended by additional data by Michaelsen (1913a) and Pickford (1937). Pickford (1937), concluding that in this species the degree of pigmentation is not a satisfactory taxonomic feature, noted body colouration fading after a few years of preservation and accounted Michaelsen's distinction of wahlbergi f. pulchior on the base of pigmentation not valid, and synonomised it with f. typicus. The currently examined specimen was violet pigmented dorsally in life, this colour extending to the middle of the body, and kept its tint for a short time in alcohol before the colour faded. The spermathecal ampulla is oval, slightly enlarged at its ental part. The diverticulum (Fig. 10) is unilobate, tubular, almost twice as long as ampulla. The anterior pair of the prostatic glands stretches through four segments, the posterior pair extending backwards through five segments. No penial setae were observed in the studied specimen.
BILOBATE species-group Parachilota editha (Pickford, 1927)  Biological notes and distribution: The species was described from a single specimen from the Eastern Cape, found in the graveyard area in the neighbourhood of Stutterheim [32°32'S:27°29'E], and Pickford suspected its accidental transportation from the Drakensberg foothills. Zicsi (1998) reported this species from the Karkloof Nat. Res. (29°18'S:30°13'E) in KZN. The present record from Giant's Castle supports Pickford's supposition that the species occurs in a broader area of the Drakensberg Mountains. Comments: The new material matches the species description (Pickford 1927) and its re-description (Pickford 1937). However, on the drawing made by Pickford (1937: fig.  279) a diverticulum is presented as one lobe extended over the spermathecal duct, with no middle division. In currently studied material the spermathecal diverticulum is bilobate, with a groove in the middle of the diverticulum face, a character not previously noted. The ornamentation of the penial setae indicated in the re-description (Pickford 1937) was not observed in new material. Zicsi, 1998 Fig. 11 Parachilota karkloofi: Distribution: This species is known only from the type locality in KZN. An assessment of its range must await the discovery of additional material. Comments: The present study of the type material housed at the Natal Museum (Plisko 2006) allows this species to be assigned to the bilobate species-group. The spermathecal ampulla is ovoid, duct elongated with two separated, slightly chambered lobes (Fig. 11). Distribution: The species was reported by Zicsi and Pajor (1992) from the Cathedral Peak area, including the Ndumeni Forest, Doreen Falls, and the vicinity of the hotel buildings, all in the eastern Drakensberg Mountains. Present records extend the known range to Drakensberg foothills in southern KZN. Comments: The studied material matches the description of the species (Zicsi & Pajor 1992). The species is characterized by a bilobate spermathecal diverticulum, clearly divided by a narrow furrow (Fig. 12). The ornamentation of the penial setae is similar to that shown in the type description, although it was observed only under 500× magnification.

Parachilota karkloofi
MULTILOBATE species-group Parachilota uysae sp. n. Fig. 13 Etymology: Named after Mrs C. Uys, who collected the type series. Diagnosis: Spermathecal pores in intersegmental furrows 7/8 and 8/9. Clitellum ringshaped on 13-17. Prostatic pores not approximate towards the mid-ventral line, in 17 and 19. Male pores in 18. Gizzard in 5, moderately developed. Commencement of intestine in 16. Last pair of lateral hearts in 12. Spermathecal multilobate diverticulum with three lobes attached to spermathecal duct at its basal part. Description: External features: Body cylindrical. Alcohol-preserved over three years: on preclitellar segments dorsally violet reddish, postclitellarly and ventrally yellowish grey. Biological notes and distribution: The presence of sperm in male funnels and spermathecal diverticula indicates sexual activity during the summer, extending to early autumn. This species was collected during a study of invertebrate diversity in Afrotemperate forests of the Drakensberg Mountains undertaken by C. Uys. The study area falls under the protection of Ezemvelo KZN Wildlife, although beforehand it was variably exploited by people (Pooley & Player 1995). Human impact on the environment has had many serious implications for the survival of the endemic fauna. Uys (2006) evaluated patches of indigenous forests as refuges for many indigenous invertebrates. Although only three specimens of P. uysae were found in the investigated protected area, in the close neighbourhood the acanthodriline Udeina adriani Plisko, 2004, the microchaetid Proandricus injasuti Plisko, 2002 and other species: Dichogaster sp. of Benhamiinae, megascolecid Amynthas sp., and exotic lumbricids Apporrectodea rosea (Savigny, 1826), Dendrodrilus rubidus (Savigny, 1826) were also found, confirming the value of protected indigenous areas. P. uysae is known only from its type locality in the foothills of the Drakensberg Mountains in central KZN.

Dimensions
Parachilota warreni (Michaelsen, 1913) Fig. 14 Biological notes and distribution: The species is known from distinct habitats, preferring moist or wet biotopes. It appears at distantly separated sites in the Drakensberg foothills in KZN, extending its range to the KZN midlands, and to Auckland Forest in the Eastern Cape. Although our knowledge of its distribution is still limited, it may be noted that its fragmented appearance is probably affected by anthropogenic changes to the environment.