Nematodes from the Bakwena Cave in Irene, South Africa

Candice Jansen van Rensburg

Academic year: 2009-2010

Thesis submitted in partial fulfillment of the requirements for the award of the degree Master of Science in Nematology in the Faculty of Sciences, Ghent University

Promoter: Prof. Wilfrida Decraemer Co-Promoters: Dr. Wim Bert Dr. Antoinette Swart

Nematodes from the Bakwena Cave in Irene, South Africa

Candice JANSEN VAN RENSBURG 1*,2

1Nematology section, Department of Biology, Faculty of Sciences, Ghent University; K.L. Ledeganckstraat 35, 9000 Ghent, Belgium 2Dept. Zoology & Entomology, P.O. 339, University of the Free State, Bloemfontein, 9300, South Africa; [email protected]

*Corresponding e-mail address: [email protected]

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Summary

A survey forming part of the Bakwena cave project was carried out from January 2009 to February 2010 at the Bakwena Cave South Africa. A total of 27 nematode genera belonging to 23 families were collected, 19 genera are reported for the first time from cave environments. Of the six localities sampled, the underground pool of the cave showed the highest species diversity with lowest diversity associated with fresh and dry guano deposits. Four of the sampling localities were dominated by bacterial feeders the remaining two localities being comprised of fungal feeders, obligate and facultative plant feeders and omnivores. Multidimensional scaling indicated six nematode assemblages corresponding with six localities, which might reflect substrate associated patterns. Three species are also described, two being new to science. Diploscapter coronatus is characterised by having a visibly annulated cuticle; a pharyngeal corpus clearly distinguishable from the isthmus, the vulva situated about mid-body and the stoma almost twice as long as the lip region width. Panagrolaimus n. sp. is characterised by an almost straight habitus for females and J-shaped habitus for males, lips amalgamated into three pairs, lateral field with two incisures, ovary with straight reflexed part and germinal zone extending till anal level or even beyond into the tail cavity, post uterine sac shorter than body width, vulva at 69% (mean value) with protruding lips, female tail elongate conoid to conoid, male tail conoid with short mucron, spicules robust with hemispheroid manubrium, a short calamus and lamina with dorsal hump and ventral wing, gubernaculum curved ventrad. Plectus n. sp. is characterised by its small body size, labial region not set off, cephalic setae almost as long as half labial diameter, amphidial fovea at posterior end of stoma, vulva at mid-body, each genital branch about two body widths long, tail ventrally arcuate and relatively short. In addition to the morphological diagnostic features described for Plectus n. sp. a polytomous key for females of the genus Plectus is also presented. Descriptions, measurements and illustrations including SEM micrographs are provided for the three species.

Keywords: Ecology, karst system, Pretoria, morphology, new species, Diploscapter coronatus , SEM, taxonomy, key

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The study of cave systems and particularly karst systems of South Africa are still in their initial stages (Durand, 2008). Karst is a special kind of landscape that is formed by the dissolution of soluble rocks such as dolomite. Sinkholes, caves and complex underground water flow networks are found associated with a karst system (Williams, 2008). Approximately 25% of the world’s population inhabit areas above karst regions and obtain water from its aquifers (Buchanan, 2008). Karst regions are therefore very important to be protected from any kind of pollutants moreover because they appear extremely sensitive to such disturbance (Buchanan, 2006).

South Africa holds 12% of the world’s karst systems, with the Gauteng Province having one of the largest single repositories of karst in the world (Buchanan, 2008). In South Africa, Karst systems are facing immense pressures from unsustainable over-exploitation. This over- exploitation comes in the form of pollution of groundwater and surface habitats, urban development and roads, unauthorized removal of dolomites, fossils and cave formations by public and the over-use of natural resources in the areas, including lime operations and agriculture.

Cave environments have been considered by many authors (Culver et al. , 1999, 2004; Culver & Sket, 2000; Hodda et al. , 2006) as extreme habitats which are inhabited by only a few specialised species (Abolafia & Peña-Santiago, 2006a).

Cave fauna can include obligate (troglobites), edaphic (edaphobites) and aquatic (stygobites) cave dwellers while some species may be facultative (troglophiles), indifferent cave soil dwellers (trogloxenes) or even accidentals (Abolafia & Peña-Santiago, 2006a). A cave may be classified into three main zones according to light intensity: the twilight zone, middle zone and deep zone (Durand, 2008). The most diverse fauna in a cave occurs in the twilight zone near the entrance. The middle zone is in complete darkness but has variable temperature and supports several species, some of which may commute to the surface (Poulson & White, 1969). The deep zone is characterised by complete darkness and constant temperatures.

The different fauna associated with these three zones show varying degrees of adaptivity towards troglomorphism, with many cave-dwelling organisms becoming obligatory troglobites, which reproduce, feed and spend their whole life underground (Poulson & White, 1969). Other organisms such as certain bat species need caves for roosting, hibernation and

3 reproduction, but hunt outside caves. The guano deposits of these bats serve as food for decomposers such as bacteria and fungi (Durand, 2008).

The most widely studied group of organisms from cave environments are arthropods (Welbourn, 1999) with other taxa such as nematodes only being mentioned as present without specification (Abolafia & Peña-Santiago, 2006a).

Little is known on the nematofauna of cavernous ecosystems especially of those forming part of a karst system. Altogether 28 nematode genera have been reported from cave habitats worldwide (Hodda et al. , 2006). The nematode species found in this unusual type of environment seem to consist of a few endemic species on the one hand and accidental occupants that can tolerate a wide variety of environmental conditions on the other hand (Hodda et al. , 2006).

Muschiol & Traunspurger (2007) showed that bacterial mats in caves are dominated by five nematode genera: Chronogaster Cobb, 1913, Monhystrella Cobb, 1918, Panagrolaimus Fuchs, 1930, Poikilolaimus Fuchs, 1930 and Udonchus Cobb, 1913. Chronogaster troglodytes Poinar & Sarbu, 1994 can be considered as a truly cavernicolous nematode which is regularly found associated with bacterial mats relying on autochthonous primary production from thermal springs (Poinar & Sarbu, 1994).

Members of the genera Plectus Bastian, 1865 and Panagrolaimus have previously been reported from cave environments (Cayrol, 1973; Poinar & Sarbu, 1994) from the south of France, and Romania respectively. Diploscapter Cobb, 1913 on the other hand is not listed from this extreme habitat (Hodda et al. , 2006).

A checklist of free living nematodes recorded from freshwater habitats in southern Africa which catalogued about 37 families, 66 genera and 140 nematode species (Heyns, 2002), did not report on any nematodes from cave habitats. The available information of species from the families Panagrolaimidae Thorne, 1937, Diploscapteridae Micoletzky, 1922 and Plectidae Örley, 1880 in South Africa is limited with only a few records being known (Heyns, 1971;

Dassonville, 1981; Botha & Heyns, 1993; De Bruin & Heyns, 1993). Since the passing of Juan Heyns no recent taxonomic information is available on free living nematodes from South Africa.

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The current study is part of the Bakwena Cave Project initiated by the South African Karst Ecology Study Group of the University of Johannesburg and the Biosystematics division of the Plant Protection Research Institute (PPRI) of the Agricultural Research Council (ARC), Pretoria.

The objectives of the study undertaken herein were: 1) to identify all nematodes from the Bakwena cave to genera level; 2) to taxonomically describe some key species or new species from the different habitats within the cave; 3) to determine possible distribution patterns of the nematode genera and 4) to determine if the cave shows any signs of an ecological disturbance

Overall the above mentioned objectives aim to contribute to the Bakwena cave biodiversity study, as well as to increase our current knowledge on free living nematodes from South Africa.

Material and methods

STUDY AREA : BAKWENA CAVE The Bakwena Cave located south of Pretoria in the Gauteng Province, was chosen as study area. The cave appears as a sinkhole in dolomite with its entrance obscured by a thicket and a few Celtis africana Burm.f. trees along the periphery . The perimeter of the cave is about 10m in diameter and tapers down to an almost vertical shaft of about 9m in diameter and 21m deep, sloping in a north-easterly direction (Fig. 1B). At the bottom of the shaft there is a talus slope consisting of scree (about 13m along its east-west axis) forming the entrance to the main chamber which is approximately 15m wide along its north-south axis, and 21m long along its east-west axis.

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Fig. 1: Diagram showing simplified map of A) a vertical and B) a horizontal section through the Bakwena Cave in Irene South Africa (courtesy of Francois Durand, Dept. Zoology, University of Johannesburg and Elsa van Niekerk, graphical designer, ARC-PPRI, South Africa).

The depth of the main chamber i.e. between roof and scree floor varies from 1.5m to 4m. In the north-western corner of the main chamber are two fissures that serve as passageways connecting the main chamber to two smaller side chambers (Fig. 1A: b, c), in which a colony of Minopterus schreibersii Kuhl, 1817 bats roost. An easterly running fissure opens in the farthest eastern corner of the main chamber opposite the entrance (Fig. 1A: a). This fissure slopes downwards, intersects with the water table, and continues as far as can be seen, for meters under the water table. The three fissures (Fig. 1A: a, b, c) that lead from the main chamber continue for many meters in an easterly and westerly direction, which is basically the same as the direction of the slope of the shaft and the main chamber. According to Durand (2007) these fissures could be part of a more extensive fissuring pattern in the dolomites in the region.

SAMPLING AND EXTRACTION Material for this study was collected by Dr. Antoinette Swart from the Nematode Biosystematics Division, Agricultural Research Council, Pretoria. Nematode samples were taken at six different localities in the cave system (Fig. 1A & B): 1) under ferns and mosses

6 against the walls at the entrance of the cave (normal light zone); 2) the floor of the main cave chamber (twilight zone); 3) dry bat guano from the main chamber (twilight zone); 4) “the roost” consisting of fresh bat guano from one of the side chambers (total darkness); 5) from the bottom of an underground pool (total darkness); and 6) the cave floor of one of the side chambers (total darkness). Sampling was carried out every month from January 2009 to February 2010 at the five localities, with two preliminary surveys in May and October 2008.

The sieving- centrifugal- flotation method (Jenkins, 1964) was used to extract nematodes from samples except for the roost material where the modified Baermann funnel technique (Kleynhans, 1997) was applied. The latter technique resulted in much cleaner samples and higher number of nematode specimens. Nematodes were heat killed and fixed using TAF (Courtney, Polley & Miller, 1955) and dehydrated to anhydrous glycerin following the slow method (Hooper, 1970). Additional fixed specimens were sent to Ghent University, Belgium for processing and mounting following the glycerol-ethanol method (Seinhorst, 1959 as modified by De Grisse, 1969). For light microscopic observations, specimens were mounted on Cobb slides (Cobb, 1917) and sealed with ‘glyceel’ (Thorne, 1935). Fixed material which was processed in Gent was mounted on Cobb slides and sealed with a wax ring (De Maeseneer & d’Herde, 1963).

MORPHOLOGICAL OBSERVATIONS AND MEASUREMENTS Measurements and drawings were made with the aid of an Olympus CX31 microscope equipped with a drawing tube; micrographs were taken with an automatic camera system mounted on an Olympus BX 51 DIC microscope (Olympus optical, Tokyo, Japan). For Scanning Electron Microscopy mounted specimens were removed carefully from slides, hydrated in distilled water, dehydrated in a graded ethanol series, critical point dried and coated with gold and observed under a JEOL 840 microscope (Green, 1967).

The terminology used for stoma and spicule morphology follows the proposals of De Ley et al. (1995) and Abolafia & Peña-Santiago (2006b) respectively. Body length and all curved structures were measured along the median line.

Abbreviations used further in the text: a- body length divided by greatest body width; ABD- anal body diameter; Amphid c.b.d .- corresponding body diameter at level of amphidial fovea; b- body length divided by distance from anterior end to junction of pharynx and intestine; Basal phar. bulb length - basal pharyngeal bulb length; Basal phar. bulb width - basal pharyngeal bulb width; c- body 7 length divided by tail length (anus or cloaca to tail terminus); c’- tail length divided by body width at anus or cloaca; Corpus/isthmus- corpus length divided by isthmus length; Egg l- egg length; Egg w- egg width; Excretory pore-ant. end - distance of secretory-excretory

pore from anterior end; G1- length of anterior genital branch (from vulva to terminal cell of ovary via flexures) expressed as percentage of body length; G2- overall posterior genital branch length expressed as percentage of body length; Isthmus– length of isthmus measured from base of corpus to beginning of basal bulb; L– tota l body length; LRD - lip region diameter; LRH- lip region height; Nerve ring- ant. end - distance of nerve ring from anterior end; Pharyngeal corpus– length of corpus; Pharynx length– distance from anterior to end of basal bulb; Phasmid-anus distance- distance of phasmid from anus ; Stoma– length of stoma; T- distance from cloacal opening to tip of testis expressed as a percentage of total body length; V- distance of vulva from anterior end expressed as a percentage of total body length ; V-A/tail- distance vulva to anus divided by tail length; Vulva-anterior end- Distance of vulva from anterior end . (a, b, c: ratio’s (de Man,1880))

SPECIES CONCEPT Specimens were described and identified based on the Morphological Species Concept (MSC) which states that species are the smallest groups that are consistently and persistently distinct and distinguishable by ordinary means (Cronquist, 1978). This species concept is still for many taxa the concept in vogue since gene flow cannot be proven and morphological distinctiveness is assumed as surrogate to lineage independence (Decraemer et al ., 2008)

NEMATODE COMMUNITY ANALYSIS Data and material received from the Agricultural Research Council (Pretoria) was identified where possible to genera level. The genera were classified according to their feeding types (Yeates et al. , 1993) and assigned c-p (coloniser-persister) values according to their r or K life strategies (Bongers, 1990, 1999). These values were then used to calculate the Maturity Index (MI) (Bongers, 1990, 1999) as measure for identification of the environmental disturbance of the cave ecosystem. Nematode genera data was also used to calculate diversity as the number of species per sample (S), the Shannon-Wiener diversity index, as well as Hill’s N ∞ (Hill, 1973). Evenness was calculated using Pielou’s J (J’=H’/log S) (Pielou, 1975). Diversity patterns were visualised by k-dominance curves (Lambshead et al. , 1983). The nematode community structure was analysed by non metric Multi-Dimensional Scaling (MDS) using the Bray-Curtis similarity measure (Kruskal, 1964) and the program Primer v. 6.

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Remark : Material for this study was not collected in replicate, this limited the possibilities for statistical of processing. For instance, it was impossible to carry out a SIMPER test which identifies which species are responsible for the similarities and dissimilarities in the ecosystem.

Results

Ecological results Since no replicates of sampling localities exist and only incomplete data of month to month collection of the nematodes was available, possible changes to the system are not statistically measurable. This gathered information can thus only be used as a basis for future more elaborate ecological study.

NEMATODE GENERA COMPOSITION AND DIVERSITY A total of 27 nematode genera belonging to 23 families were identified from the six localities within the cave (Table 1). Most genera (20) were collected from the underground pool (
) (Table 1 & 2). Fourteen genera were collected from more than one habitat type with Panagrolaimus sp. and Acrobeloides sp. being found in three of the six sample localities.

Table 1: Nematode genera collected from the six sampling localities in the Bakwena Cave, South Africa. (Refer to Fig. 1 for description of localities) with indication of their c—p value.

SAMPLING LOCALITY    


 c-p value ENOPLIDA Filipjev, 1929 Alaimidae Micoletzky, 1922 Alaimus sp. 1 *   4 TRIPLONCHIDA Cobb, 1920 Trichodoridae Cobb, 1913* Trichodorus parorientalis*  4 Prismatolaimidae Micoletzky, 1922 Prismatolaimus sp. 1 *  3 DORYLAIMIDA Pearse, 1942 Aporcelaimidae Heyns, 1965 Aporcelaimus sp. 1 *   5 Qudsianematidae Jairajpuri, 1965 Discolaimus sp. 1 *   4 MONONCHIDA Kirjanova & Krall, 1969 Mononchidae Chitwood, 1937 Mononchus sp. 1  4 Mylonchulidae Jairajpuri, 1969 Mylonchulus sp. 1   4

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Table 1 continued: Nematode genera collected from the six sampling localities in the Bakwena Cave, South Africa. (Refer to Fig. 1 for description of localities) with indication of their c—p value.

CHROMADORIDA Chitwood, 1933 Cyatholaimidae Filipjev, 1918* Achromadora sp. 1 *  3 MONHYSTERIDA Filipjev, 1929 Monhysteridae Cobb, 1918 Eumonhystera sp. 1 *  2 Monhystrella sp. 1   2 ARAEOLAIMIDA De Coninck &Schuurmans Stekhoven, 1933 Diplopeltidae Filipjev, 1918 Cylindrolaimus sp. 1  3 PLECTIDA Malakhov, 1982 Plectidae Örley, 1880 Anaplectus sp. 1  2 Chiloplectus sp. 1 *  2 Plectus sp. 1   2 TYLENCHIDA Thorne, 1949 Tylenchidae Örley, 1880 Boleodorus sp. 1 *   2 Anguinidae Nicoll, 1935 (1926) Ditylenchus sp. 1 *   2 Hoplolaimidae Filipjev, 1934 Helicotylenchus sp. 1 *  3 Pratylenchidae Thorne, 1949 (Siddiqi, 1963) Pratylenchus sp. 1 *  3 Telotylenchidae Siddiqi, 1960 Tylenchorhynchus sp. 1 *   2 APHELENCHIDA Siddiqi, 1980* Aphelenchidae (Fuchs, 1937) Aphelenchus sp. 1 *   2 Aphelenchoididae Skarbilovich, 1947 (Paramonov, 1953) Aphelenchoides sp. 1*  2 RHABDITIDA Chitwood, 1933 Cephalobidae Filipjev, 1934 Acrobeloides sp. 1    2 Acrobeles sp. 1 *   2 Panagrolaimidae Thorne, 1937 * Panagrolaimus sp. 1    1 Diplogasteridae Steiner, 1919 * Diplogasteroides sp. 1 *  1 Rhabditidae Örley, 1880 Mesorhabditis sp. 1 *   1 Diploscapteridae Micoletzky, 1922* Diploscapter sp. 1 *  1 *New cave record (compare to Hodda et al. , 2006; Muschiol & Traunspurger, 2007)

Regarding the distribution of species richness (S) between the different sampling localities within the cave, sample locality
(underground pool) and sample locality  (cave entrance)

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are the most species rich, while sample localities  (dry bat guano from main cave chamber),  (fresh bat guano from side chamber ) and  (cave floor of side chamber) have the lowest number of species (Table 2). The Shannon-Wiener index (H’) shows that diversity is highest at sample locality
(underground pool) while the lowest diversity is seen at sampling localities  (dry bat guano from main cave chamber),  (fresh guano from side chamber) and  (cave floor of side chamber) (Table 2). Thus community complexity is highest at the underground pool (
). The N ∞ is highest for sample locality  (floor of main cave chamber) compared to sample locality
(underground pool); this is in contrast to the trend that is seen in the Shannon-Wiener diversity index. However, low values are still seen at  (dry guano from main cave chamber),  (fresh guano from side chamber) and  (cave floor of side chamber) (Table 2). Low evenness (J’=0.47) was observed at sample localities  (wet guano from side chamber) and  (cave floor of side chamber) which could be attributed to dominance of two species at each locality namely: Diplogasteroides sp. and Panagrolaimus sp. at sample locality  (fresh guano from side chamber) and Eumonhystera sp. and Chiloplectus sp. at sample locality  (cave floor of side chamber).

Table 2: Comparison of nematode species richness (S), Evenness (J’), Shannon-Wiener diversity (H’), Hill’s N ∞ between the different sampling localities.

Locality    




S 11 5 2 2 20 2 J’ 0.68 0.96 0.49 0.47 0.73 0.47 H’ 1.63 1.55 0.34 0.33 2.18 0.33 N∞ 2.27 3.67 1.12 1.11 2.86 1.11

TROPHIC STRUCTURE AND C-P VALUES With the exception of sample localities  (cave entrance) and  (floor of main cave chamber) the other four sampling localities were dominated by bacterial feeders (Fig. 2). Sampling locality  (floor of main cave chamber) was composed of four different feeding type’s namely obligate plant feeders, facultative plant feeders, predators and bacteriovores. While sampling locality  (cave entrance) was mainly comprised of facultative plant feeders and fungivores.

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100% O FPF 80% OPF 60% F 40% P 20% B

Relative abundanceRelative (%) 0% 1 2 3 4 5 6

Sampling localities

Fig. 2: Nematode trophic structure from the different sampling localities (B - bacterial feeders; F- fungal feeders; FPF- facultative plant feeders; O - omnivores; OPF- obligate plant feeders; P - plant feeders).

The general opportunists (c-p 1) Diplogasteroides sp. and Panagrolaimus sp. were the only two species present at sample locality  (fresh guano from side chamber) while at sample locality  (cave floor of side chamber) the enrichment opportunists (c -p 2), Eumonhystera sp. and Chiloplectus sp. were dominant. C -p values for the remaining four localities ranged from c-p 1 to c-p 4. C-p class 1 was represented by four genera, c -p class 2 was represented by 11 genera, c-p class 3 was represented by three gener a and c-p class 4 was represented by three genera (Table 1).

To investigate possible environmental disturbance of the cave ecosystem we used the Maturity Index (MI) (Bongers, 1990). The MI value varied from 1.00 to 2.67 for the six sampling localities wit h sample locality  (floor of main cave chamber) having the highest MI value and sample locality  (fresh guano from side chamber) having the lowest value (Table 3).

Table 3: The MI values for the six sampling localities within the Bakwena Cave .

Locality    


 MI 2.20 2.67 1.11 1 1.75 2

NEMATODE COMMUNITY According to MDS-analysis ( Fig. 3) all the samples are separated from each other, with only localities  (dry guano from main cave chamber) and  (fresh guano from side chamber) closest to each other.

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Fig. 3: MDS ordination on square root transformed genera abundance data with indication of the six different localities of the Bakwena Cave (Stress value: 0.0).

Diversity visualised as k-dominance curves (Fig. 4) indicates a similar pattern as presented in the diversity indices with sample localities
(underground pool),  (cave entrance) and  (floor of main cave chamber) species association being the most diverse.

120 4 100 6 80 5 60 2 40 3 1 20 0 Cumulative dominance Cumulative dominance (%) 1 10 100

Species rank

Fig. 4: K-dominance curve for nematode genera data from the Bakwena Cave.

Sample localities  (dry guano from main cave chamber),  (fresh guano from side chamber) and  (cave floor of side chamber) all show a highly elevated curve at the 1st position of species rank most likely as a result of only two species within each of the sampling localities.

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Taxonomic results

THE GENUS Diploscapter (Cobb, 1893) Cobb, 1913 The genus Diploscapter is cosmopolitan in its distribution occurring on every continent. So far, thirteen species have been assigned to the genus (Andrássy, 2005). Diploscapter has been reported from a number of unusual habitats e.g. Diploscapter lycostoma Völk, 1950 was found in the pharyngeal glands of the Argentine ant, Iridomyrmex humile Mayr, 1868 where it destroys these glands causing a pathological condition (Markin & McCoy, 1968). Diploscapter tokobaevi Lemzina & Gagarin, 1994 is known to inhabit thermal waters (33- 52˚C) in Kyrgyzstan (Lemzina & Gagarin, 1968)

Cobb in 1893 originally described D. coronatus from the rhizosphere of banana growing in Fuji as Rhabditis coronata Cobb, 1893. In 1913, Cobb erected the genus Diploscapter Cobb, 1913 to accommodate the species which he originally described and suggested that this species could be representative of a species complex (Eyualem et al ., 1998). Zimmerman (1898) described Rhabditis bicornis found associated with the roots of coffee plants, a species later synonymised with Diploscapter bicornis (Zimmerman, 1898) Goodey, 1963. Kreis (1929) reported for the first time Acrobeles armatus Kreis, 1929 from Peking, China, a species later synonymised with Diploscapter coronata (Thorne, 1937). Here we report and provide additional habitat information on Diploscapter coronatus (Cobb, 1893) Cobb, 1913 from South Africa.

Diploscapter coronatus (Cobb, 1893) Cobb, 1913 (Figs 5- 7)

MEASUREMENTS (See Table 4)

DESCRIPTION

Female Body small, largely cylindrical, maximum width at mid-body and tapering towards posterior end; appearance straight to slightly undulate upon fixation. Body cuticle with transverse striations starting at base of lip region and ending almost at tail tip; annules about 0.2-0.3µm wide at mid-body. Lateral field with four incisures or two ridges, incisures 2.1-2.2 µm apart (measured using SEM). Lateral fields beginning at posterior level of stoma and ending about

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14-16µm anterior to anus. Lip region with six lips, subdorsal and subventral lips refractive and hook-like; lateral lips membranous (laciniae), fan-shaped, bordered by 6-7 longitudinal finger-like projections (not easily observable under LM). Amphidial fovea appearing pore- like situated just behind laciniae. Stoma cylindrical, 18-28% of pharyngeal length, its posterior part (stegostom) surrounded by pharyngeal tissue. Pharynx muscular, with distinct differentiation into corpus, isthmus and basal bulb. Pharyngeal corpus well demarcated, longer than isthmus and basal bulb together. Basal bulb spheroid, with well developed sclerotized serrated valves. Cardia hemispherical, surrounded by intestinal tissue. Cells of intestinal wall granular; rectum 1.5 to 2.3 times anal body diameter. Nerve ring at level of isthmus, i.e. at 61-77% of neck length from anterior. Secretory-excretory pore at 92-97% of neck length i.e. slightly posterior to the nerve ring. Reproductive system didelphic- amphidelphic. Anterior genital branch to the right and posterior genital branch to the left of intestine. Each ovary reflexed near its tip and relatively short. Oogonia and small oocytes arranged in an irregular bunch rather than in single or multiple file. Vulva at 50-56% body length from anterior end; a transverse slit. Vagina extending inwards one fourth of body diameter. Egg, large oval shaped. No sperm or spermathecae observed. Tail conical- elongate to filiform, ending in acute tip. Phasmid pore-like situated at 13-24 % of tail length.

Male Not found.

MATERIAL EXAMINED Thirty three females extracted from sediments at the bottom of an underground pool (Fig. 1A,


) from the Bakwena Cave.

DISCUSSION Specimens from the current study closely resemble Diploscapter coronatus in having a visibly annulated cuticle; a pharyngeal corpus clearly distinguishable from the isthmus, the vulva situated about mid-body and the stoma almost twice as long as the lip region width.

The material from the present study agrees well with descriptions provided by Dassonville (1981) and Eyualem et al. (1998) in having similar vulva position (53%) and a-ratio value (18-19) (Table 1). Other similarities with our population and the Skinnerspruit population (Dassonville, 1981) include (b=4.2 present study vs. 4.2; c=6.3 present study vs. 6.5; c’=6.1 present study vs. 6.3). The main differences of our population from the other two populations

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are the position of the secretory-excretory pore which lies opposite the terminal bulb ( vs. before terminal bulb) and the smaller body size (L=373µm present study vs. 420µm (Dassonville, 1981) and 427.5µm (Eyualem et al. , 1998)). Other differences from D. coronatus as described by Eyualem et al . (1998) include the larger c’ value, nerve ring closer to the anterior end, (c’=6.1 present study vs. 4.9; Nerve ring= 69 present study vs. 70) and the tail which does not have enlarged annulations.

Based on the similarities of our population to those described by Dassonville (1981) and Eyualem et al. (1998) and the observed differences lying within range of intraspecific variation, we thus consider our population to be representative of D. coronatus .

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Table 4: Morphometrics of Diploscapter coronatus Cobb, 1893 (Cobb, 1913) populations from South Africa, China, Fuji and Ethiopia. All measurements in µm and in form mean±stdev(range) for Bakwena Cave and Ethiopian population. (Other populations only mean (range) presented).

Bakwena Cave, South Africa Rhabditis coronata Cobb, 1893 Acrobeles armatus Kreis, 1929 Diploscapter coronata Thorne, Diploscapter coronatus (Cobb, 1893) 33 &&& (Cobb, 1893) 1937 Cobb, 1913 (Kreis, 1929) (Dassonville, 1981) (Eyualem et al. , 1998) L 373±29.5(319-446) 360 345 (312-405) 420 (340-470) 427±25 (395-480) a 19.0±1.8(16.0-24.0) - 19.9(17.6-22.3) 18.3(15.5-23.2) 17.7±1.2(15.8-19.8) b 4.2±0.3(3.6-5.3) - 3.97(3.73-4.33) 4.2(3.6-4.8) 4.0±0.2(3.6-4.1) c 6.3±0.4(5.5-7.4) - 6.09(5.35-6.68) 6.5(5.2-7.5) 7.8±0.7(6.3-9.2)

c’ 6.1±0.6(4.9-7.7) - - 6.3(4.6-7.3) 4.9±0.7(4.1-6.5) V% 52.5±1.4(49.8-56.2) 55 54.6(52-60) 52.7(50.6-55.7) 53.2±1.5(50-55.7) LRH 3.7±0.7(1.8-5.4) - 3.9 - - LRD 9.1±0.9(7.1-10.7) - 7.8 - 9.5±1(8-13) Stoma 20.1±1.7(15.5-22.6) 17 - - 21.8±0.9(20-23) Pharyngeal corpus 38.0±2.6(32.1-43.4) - - - - Isthmus 18.3±2.2(10.1-22.0) - - - 24±2.2(22-31) Corpus/isthmus 2.1±0.4(1.7-3.5) - - - - Basal phar. bulb 15.6±1.2(13.1-17.9) - 10.4 - 20.7±2.6(15-25) length Pharynx length 89.2±5.9(74.4-96.4) - - - 108±5.2(95-114) Nerve ring 61.8±4.7(53.6-70.2 - - - 70±3(65-77) Excretory pore 84.2±3.9(76.2-93.4) - - - 79±4.9(70-89) Body width: neck 18.7±1.7(16.7-23.8) - 18.2 - - mid-body 20.0±2.3(16.1-28.0) 5.8 20.8 - 24.3±1.8(21-28) anus 9.9±1.2(7.1-12.0) 2.8 13 - -

G1 19.2±2.49(15.1-25.6) - - - 60.7±11.3(39-94)

G2 17.1±2.3(12.9-22.3) - - - 55.9±11.1(38-72) Egg L 36.6±2.7(34-39.2) Egg W 13.7±2.5(13.7-18.4) Vulva- anterior end 196±13.1(163-225) - - - 227.5±13.4(205-255) Rectum length 14.6±1.7(8.9-17.9) - - - - Tail length 59.4±4.8(49.4-67.8) - - - 55.5±6.3(45-71) Phasmid-anus distance 12.2±1.7(8.3-15.5) - - - - V-A/tail 2.0±0.2(1.6-2.5) - - - - 17

18

19

20

THE GENUS Panagrolaimus Fuchs, 1930 Andrássy (2005) distinguished the genus Panagrolaimus from other members of the family by its distinctive mouth structure with cheilostom not or hardly sclerotized; the protruding and open vulval lips and the narrowing of the body posterior to the vulva. Representatives of the genus are distributed worldwide, being recorded even from Antarctica. Panagrolaimus is represented by no less than 72 species (Andrássy, 2005), however Abolafia & Peña-Santiago (2006b) considered only 38 species to be valid. Abolafia & Peña-Santiago (2006b) remarked that Panagrolaimus displays quite a regular morphological pattern. The main diagnostic features these authors use to distinguish between the species are the lip region diameter, lip shape, shape of the gymnorhabdia, presence or absence of a postvulval sac; phasmid position in female, tail length in male and female; and lateral field incisures. Williams (1987) used the differences in lip morphology to distinguish species from each other. He divided the different lip regions into four groups: i) six free and separated lips (P. superbus Fuchs, 1930), ii) six free lips very close together ( P. detritophagus Fuchs, 1930), iii) six lips amalgamated into three pairs ( P. rigidus ), and iv) amalgamated into three lips ( P. subelongatus ). Abolafia & Peña-Santiago (2006b) regard this work as a valuable contribution and useful as a basis for understanding some of the group’s evolutionary tendencies. However, very few SEM work has accompanied descriptions of the known species to group species by their lip region characteristics beyond doubt. Here Panagrolaimus n. sp. is reported from South Africa.

Panagrolaimus n. sp. (Figs 8-10)

MEASUREMENTS (See Table 5)

DESCRIPTION

Female Body arcuate ventrad, nearly straight when relaxed by heat. Cuticle annulated; annules 0.8- 1.0µm wide in neck region. Lateral field with two incisures and one ridge extending to level of anus, sometimes further; 1.8-2.0 µm wide at mid-body, occupying 9% of the body width. Anterior end with six lips. Stoma panagrolaimoid. Stoma length 10.0µm (mean value), cheilostom weakly sclerotized; gymnostom strongly sclerotized, stegostom slightly sclerotized. Pharyngeal corpus cylindrical, slightly broadened posteriorly 2.3±0.2 (2.0-3.0) times isthmus length. Pharyngeal corpus-isthmus junction distinctly marked. Isthmus 21

slender, narrower than pharyngeal corpus. Basal bulb ovoid 21.6±2.2 (17.3-25.6) µm long with valves. Cardia surrounded by intestinal tissue. Rectum 0.4±0.2 (0.3-0.8) times anal body diameter. Nerve ring surrounding isthmus at 60-68% of neck length. Secretory- excretory pore just behind nerve ring at 70-78% neck length at level of isthmus. Reproductive system panagrolaimid, monodelphic, prodelphic, ovary with reflexed part straight and tip of germinal zone reaching level of anus and sometimes beyond. Oviduct well developed made up of flattened disc-like cells, arranged two by two. Oviduct separated from tubular uterus by constriction. Postvulval sac short. Vulva, a transverse slit, with slightly protruding lips. Vagina occupying one third of corresponding body diameter. Tail elongate- conoid with pointed terminus. Phasmid at 41-58% tail length from anal level.

Male General morphology similar to female but body with posterior region more curved ventrad, usually “J” shaped upon fixation. Lateral field with two incisures extending to start of tail mucron; 1.8-2.0 µm wide at mid-body, occupying 10% of corresponding body width. Reproductive system monorchic. Testis reflexed anteriorly. Sperm cells large, 9.2±1.6 (7.1- 11.9). Spicules robust: manubrium hemispheroid; calamus very short; lamina with dorsal hump and ventral wing. Gubernaculum curved ventrad. Tail conoid with mucron. Two pairs of preanal papillae, five pairs of caudal genital papillae: two subdorsal, two subventral and one sublateral

MATERIAL EXAMINED Fourteen females and twenty six males from fresh wet guano in side chamber of Bakwena cave (Fig. 1B, ).

DIAGNOSIS AND RELATIONSHIPS Panagrolaimus n. sp. is characterised by body length (438-762µm in female; 429-655 µm in male), lips amalgamated into three pairs, ovary with straight reflexed part and germinal zone extending till anal level or even beyond into the tail cavity, post uterine sac shorter than body width, vulva positioned at 69% (mean value) with protruding lips, female tail elongate conoid to conoid (c= 8-14; c’=3.3-5.5). Males are characterised by spicule length (23-30µm) and shape (manubrium hemispheroid, short calamus and lamina with dorsal hump and ventral wing); tail conoid with short mucron and somewhat shorter than in female (c=10-15; c’=1.9- 3.6).

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Following the key of Abolafia & Peña-Santiago (2006b) the species from the present study should be identified as P. concolor Massey, 1964. However when looking to the original description by Massey (1964) P. concolor has a longer body (L= 860-900 µm in female and 800 µm in male vs. 570 µm in female and 540 µm in male, mean values in new species) and a shorter tail (c= 17 vs. 10 (mean) in female new species and c= 16 vs. 12 (mean) in male new species). Further, females of the two species also differ in vulva position (V= 62% in P. concolor vs. 69% in new species) and tip of ovary (germinal zone) ending two anal body widths anterior to anus vs. tip ovary extending to anus in the new species.

Panagrolaimus n. sp. also resembles P. judithae Massey, 1964, P. orientalis Korenchenko, 1986, P. spondyli Körner, 1954, P. subelongatus (Cobb, 1914) Thorne, 1937 in that all above-mentioned species don’t have an offset head, the secretory-excretory pore is situated just before the terminal bulb of the pharynx. The most prominent feature separating these species from Panagrolaimus n. sp. is the position of the vulva ( P. judithae V%=58%; P. orientalis V%=55%; P. spondyli V%=60%; P. subelongatus V%=61%). Additionally the new species can be distinguished from P. judithae in having a shorter body length (572µm vs. 775µm), the corpus is longer than the isthmus and basal bulb combined ( vs. equal to the isthmus and basal bulb combined), a post uterine sac is present ( vs. absent). It differs from P. orientalis in having a larger corpus/isthmus ratio (2.3 vs. 2.1), number of lateral line incisures (2 vs. 4); from P. spondyli in having the rectum shorter than one anal body diameter ( vs . longer), the post uterine branch smaller than one body width ( vs. twice as long as one body width), the phasmid is in the posterior half of the tail ( vs. 1st third of tail length) and in males the spicule is larger (23-30µm vs. 20-23 µm). It differs from P. subelongatus in having six lips arranged in three pairs ( vs. lips well separated), the secretory-excretory pore towards the middle of the isthmus ( vs. towards anterior end of isthmus), and the vulva anus distance is twice the tail length ( vs. 6-7 tail lengths).

Panagrolaimus n. sp. has a duplex arrangement of six lips i.e. lips arranged into three pairs, an arrangement it has in common with P. rigidus (Schneider, 1866) Thorne, 1937 (Williams, 1987). Panagrolaimus n. sp. differs from P. rigidus by a smaller body length (572µm vs. 990µm); two lateral lines vs. three in P. rigidus ; pharyngeal corpus more than twice the length of the isthmus vs. one and half times longer; rectum shorter than one anal body diameter vs. one anal body diameter and the vulva –anus distance equals twice the tail length vs. 5-6 tail lengths. Apart from a similar lip arrangement, the two species also possess a post uterine sac shorter than corresponding body width. 23

Table 5: Morphometrics of Panagrolaimus n. sp . collected from the Bakwena Cave, South Africa. All measurements in µm and in the form of mean±stdev(range).

Bakwena Cave, South Africa 14 &&& 26 %%%

L 572.3±85.0(438.3-762.5) 540±49.0(429.-655.) a 25.0±2.2(22.0-30.0) 26.0±2.3(22.0-33.0) b 4.3±0.4(3.8-5.3) 4.1±0.3(3.7-4.8) c 10.0±1.5(8.1-14.0) 12.0±1.0(10.0-15.0) c’ 4.4±0.6(3.3-5.5) 2.8±0.4(1.9-3.6) V 69.0±2.3(64.0-73.0) - T - 48.3±4.9(40.4-59.2) LRD 6.4±0.9(4.8-7.1) 6.4±0.6(5.4-7.1) Stoma 9.3±1.2(7.1-11.3) 9.3±1.2(7.1-11.3) Pharyngeal corpus 78.2±6.9(61.9-86.3) 74.3±6.5(55.3-83.3) Isthmus 33.9±4.5(25.6-39.9) 35.1±2.8(29.2-40.5) Pharynx length 134.2±11.7(105.9-148.2) 130.7±8.9(102.9-145.2) Nerve ring 88.4±7.9 (69.6-96.4) 83.7±5.3(71.4-91.0) Excretory pore 100.2±9.6(77.9-113.1) 94.6±7.9(74.4-108.3) Body width: neck base 21.6±3.3(18.5-29.2) 22.1±3.0(18.4-26.2) mid-body 23.3±3.7(17.3-30.8) 20.8±2.4(16.5-26.7) anus 13.1±2.2(8.3-17.3) 16.3±2.1(12.5-20.2) Tail length 57.2±9.4(41.7-58.2) 44.4±4.7(35.7-53.0) V-A/tail 2.0±0.3(1.5-2.5) - Spicule - 25.7±2.2(22.6-30.3) Gubernaculum - 10.4±1.7(7.1-14.3)

24

25

26

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THE GENUS Plectus Bastian, 1865 According to Andrássy (2005) the genus Plectus can be regarded as a well defined natural group comprised of some of the most common and important soil and aquatic species. Boström (1995) remarked that the taxonomy of this genus is quite complex with many species having been described over the years. From the period 1865 to present, no less than 125 species and subspecies have been described within Plectus (Andrássy, 2005). Maggenti (1961) listed 16 species, Andrássy (1985) listed 34 species, and Zell (1993) listed 53 valid species in revisions of this genus. Today, an estimated 75-80 species are accepted, depending on the evaluation of certain species by the authors (Andrássy, 2005). According to Boström (1995) many species are morphologically similar and in agreement with Kito et al. (1991) regarded the posterior region in males (spicules, gubernaculum and number of genital supplements) important for species diagnosis and identification. However, in many species males are rare which makes identification difficult. In order to objectively identify and discuss the relationship of the apparently new species from the present study, an attempt was made to provide a polytomous key (Table 6) with data available from literature.

A polytomous key for females of the genus Plectus is provided below and was compiled from the species listed in Andrássy (2005). When mean values were not given in the original description, it was calculated for current study.

Characters used in the polytomous key The code: A. Body length G. Lip region diameter 1= < 350 µm 1= < 5 µm 2= 350-450 µm 2= 5.0-7.1 µm 3= 451-650µm 3=7.2-9.2 µm 4= 651-851µm 4=9.3-11.3 µm 5= 852-1000µm 5=11.4-13.4 µm 6= >1000µm 6=13.5-15.5 µm 7=>15.5 µm

B. Epidermal glands H. Lip region 1= Present in large numbers 1= offset depression 2= Present in low numbers 2= offset constriction 3= Absent 3= not offset (continuous with body contour)

C. Tail length I. Tail curvature 1= < 50 µm 1= almost straight 2= 50-75 µm 2= ventrally arcuate 3= 76-101 µm 3= rectangularly curved 4= 102-127 µm 4= other 5= 128-153 µm 6= 154-179 µm 7= >179 µm 28

D. Position of fovea vs. stoma J. Length of stoma vs. lip region diam. 1= anterior of mid-stoma 1= <1.7 2= mid-stoma 2= 1.7-2.0 3= behind mid-stoma 3= 2.1-2.5 4= at posterior end of stoma 4= 2.6-3.0 5= 3.1-3.5 6= >3.5

E. Spinneret K. Amphidial fovea diameter 1= present 1=<2.5 2= absent 2= 2.5-3.5 3= ventral thorn 3= 3.6-4.6 4= beak shaped 4= >4.6

F. Rectum L. c' ratio 1= ≤one anal body diameter 1=>2.5 2= 1.1-1.4ABD 2=2.5-4.0 3= 1.5-2 ABD 3=4.1-5.6 4=2.1-2.6 ABD 4=5.7-7.2 5= 2.7-3.2 ABD 5=7.3-8.8 6= > 3.2ABD 6=>8.8

In the polytomous key, the “prime characters” (italic codes in key) are placed first (left), to allow easy differentiation into smaller groups and subgroups using the excel sorting tool for example (Microsoft office). No phylogenetic relationships are inferred by these groupings; division into groups is only used since it aids in identification by narrowing down the number of species to a restricted number of phenotypically similar species.

The prime characters that were chosen are D (position of the amphidial fovea vs. stoma) and H (lip region demarcation). Sorting with the ‘prime characters’ resulted in 15 groups. The three codes for measurements of a feature in the table e.g. 326 represent the average (3), minimum (2) and maximum (6) values.

Further differentiation can be obtained by use of character I in addition with one or more of the remaining characters: Group 1: Five species are present in this group, further sorting on features B,F; Group 2: Only has one species; Group 3: Is represented by three species, further sorting on C; Group 4: Has five species ; Group 5: Sixteen species can be found within this group, further sorting on A,B; Group 6 : Has only two species; Group 7 : Is represented by three species, further differentiation by features G,J; Group 8: This group has thirteen species, further differentiation based on C,G; Group 9: Is represented by six species, further differentiation based on B,E; Group 10 : Three species can be found in this group, further differentiated using features B,C ; Group 11: Seven species can be found in this group,

29

differentiation based on features A,G; Group 12: Is represented by three species, differentiated based on J,C; Group 13: Only has one species; Group 14: Five species are found in this group, differentiated based on C,F; Group 15: This group is represented by four species and further differentiated based on features A,C,L.

For details on species authorities see species list in Andrássy (2005). Plectus galeopsidis De Ley & Coomans, 1994 is also included in the list of Andrássy (2005) however this species seems to be nom. nud. since no proper description exists for this species.

Table 6: Polytomous key for the genus Plectus Bastian, 1865 based on type populations.

Group 1 D H I A B C E F G J K L P. australis 11 2 11 666 1 435 1 111 657 222 223 222 P. glandulatus 11 2 22 656 1 325 1 111 647 333 - 213 P. naticochensis 11 2 22 666 3 333 1 111 222 500 - 111 P. spicacaudatus 11 2 22 666 3 445 2 323 767 222 112 223 P. tolerans 11 2 22 666 2 444 1 222 555 323 333 222 Group 2 D H I A B C E F G J K L P. brzeskii 11 3 11 333 3 111 1 666 444 323 434 223 Group 3 D H I A B C E F G J K L P. patagonicus 22 1 21 545 3 334 1 212 445 324 212 323 P. americanus 22 1 22 666 3 656 1 212 545 223 434 555 P. niaensis 22 1 22 656 3 444 1 222 656 111 223 333 Group 4 D H I A B C E F G J K L P. capensis 22 2 22 445 3 334 1 323 545 222 222 333 P. minor 22 2 22 213 3 - 1 - 122 311 111 112 P. elongatus 22 2 33 445 2 657 1 222 333 323 223 666 P. parietinus 22 2 42 666 2 444 1 112 656 212 223 223 P. velox 22 2 44 656 1 324 1 112 657 416 223 222 Group 5 D H I A B C E F G J K L P. paraguayensis 22 3 11 666 3 - 1 111 - 111 - 222 P. karachiensis 22 3 12 444 3 445 1 111 323 222 323 445 P. araucanorum 22 3 22 334 3 333 1 111 334 112 - 445 P. belgicae 22 3 22 444 3 333 1 111 445 222 333 323 P. cryptoptychus 22 3 22 333 3 223 1 111 334 334 222 323 P. decens 22 3 22 435 1 324 3 545 434 222 333 545 P. fragilis 22 3 22 323 3 323 1 425 222 323 212 556 P. hyperboreus 22 3 22 666 3 445 1 112 667 112 323 333 P. meridianus 22 3 22 445 3 334 1 223 445 111 222 434 P. minimus 22 3 22 111 3 111 1 223 112 222 111 334 P. refusus 22 3 22 222 3 222 1 111 223 434 - 334 P. varians 22 3 22 434 3 435 1 113 334 323 223 445

30

Table 6 continued: Polytomous key for the genus Plectus Bastian, 1865 based on type populations.

Group 5 continued D H I A B C E F G J K L P. longicaudatus 22 3 44 333 2 324 1 325 333 213 212 556 P. palustris 22 3 44 666 3 777 1 212 545 323 223 545 P. paratenius 22 3 44 545 3 334 1 212 444 212 223 333 P. turricaudatus 22 3 44 434 3 333 1 536 434 112 213 546 Group 6 D H I A B C E F G J K L P. globocephalus 23 1 11 334 3 234 1 111 434 222 222 222 P. acuminatus 23 1 22 555 2 333 1 212 555 222 223 323 P. raabei 23 1 44 666 3 333 1 111 666 111 - 111 Group 7 D H I A B C E F G J K L P. annulatus 23 2 22 656 3 333 1 111 454 333 222 223 P. infundibulifer 23 2 22 666 1 435 1 112 767 111 223 223 P. intermedius 23 2 22 445 3 434 1 213 445 222 223 334 Group 8 D H I A B C E F G J K L P. indicus 23 3 11 656 3 435 1 112 546 222 223 434 P. sambesii 23 3 11 333 3 223 1 212 323 112 212 333 P. antarcticus 23 3 22 656 3 445 2 111 667 212 222 222 P. costatus 23 3 22 333 3 323 1 435 223 334 223 556 P. cylindricus 23 3 22 545 3 111 1 111 - - - 333 P. frigophilus 23 3 22 666 3 556 1 222 777 222 - 222 P. kyotoensis 23 3 22 656 3 556 1 222 434 223 444 545 P. montanus 23 3 22 556 3 434 1 212 434 222 222 334 P. tenuis 23 3 22 656 3 334 1 223 333 444 223 434 P. tropicus 23 3 22 222 3 111 1 213 212 324 112 333 P. rhizophilus 23 3 44 435 3 334 1 223 323 323 223 445 P. similis 23 3 44 333 3 212 1 213 333 323 212 323 P. zelli 23 3 44 334 3 223 1 111 323 536 222 324 Group 9 D H I A B C E F G J K L P. amorphotelus 33 1 11 333 3 212 2 213 434 222 212 223 P. araiensis 33 1 22 600 3 333 4 111 666 111 222 222 P. cirratus 33 1 22 666 2 434 1 212 556 222 222 223 P. communis 33 1 22 334 3 223 1 213 434 222 212 324 P. lamproptychus 33 1 22 445 1 323 4 212 545 646 111 223 P. pusteri 33 1 22 656 2 334 1 112 445 222 223 324 Group 10 D H I A B C E F G J K L P. paracuminatus 33 2 22 556 3 334 1 213 545 222 223 323 P. pseudelongatus 33 2 22 445 3 546 1 213 334 222 223 546 P. rotundilabiatus 33 2 22 656 1 435 1 212 657 112 224 223 Group 11 D H I A B C E F G J K L P. parvus 33 3 11 323 3 212 1 212 222 323 212 323 P. elegans 33 3 22 666 3 556 1 112 444 333 223 333 P. exinocaudatus 33 3 22 222 3 212 1 223 112 444 112 666

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Table 6 continued: Polytomous key for the genus Plectus Bastian, 1865 based on type populations.

Group 11 continued D H I A B C E F G J K L P. geophilus 33 3 22 213 3 111 1 123 112 334 111 333 P. makrodemas 33 3 22 545 3 324 1 212 546 222 223 323 P. minutus 33 3 22 334 3 333 1 222 323 222 121 546 P. pulcher 33 3 22 334 3 333 1 313 434 213 212 555 Group 12 D H I A B C E F G J K L P. murrayi 44 1 22 434 3 324 1 212 434 223 214 334 P. subtilis 44 1 22 222 3 444 1 111 222 111 222 555 P. magadani 44 1 44 334 3 223 1 213 434 222 213 333 Group 13 D H I A B C E F G J K L P. galapagensis 44 2 22 223 3 222 1 213 112 546 212 556 Group 14 D H I A B C E F G J K L P. chengmohliangi 44 3 22 222 3 222 1 111 222 122 - 233 P. cladinosus 44 3 22 434 3 333 1 666 333 111 222 666 P. inquirendus 44 3 22 444 3 434 1 212 233 334 334 666 P. pusillus 44 3 22 212 3 112 1 112 223 223 212 222 Plectus n. sp. 44 3 22 212 3 212 1 111 212 323 113 334 Group 15 D H I A B C E F G J K L P. insolens 44 3 44 545 3 445 1 111 334 444 222 445 P. opisthocirculus 44 3 44 334 2 223 1 212 222 434 212 333 P. sabinae 44 3 44 333 3 647 1 333 112 444 112 666 P.aquatilis 44 3 44 656 2 445 1 212 556 323 324 334

Plectus n. sp. (Figs 11-13)

MEASUREMENTS (See Table 7)

DESCRIPTION

Female Body small, largely cylindrical, tapering towards both extremities but more pronounced posteriorly; ventrally arcuate upon heat fixation. Cuticle with fine transverse striations, more prominent in lip and tail region; annules 0.4-0.5µm wide at mid-body. Lateral field with two ridges (alae) extending from about 31 st annule from anterior end to level of anus; 1.6µm wide at mid-body, occupying 10-12% of the corresponding body width. Head region narrow, continuous with body. Lip region twice as wide as high. Labial sensilla not visible under light microscope. Four cephalic setae, nearly as long as half lip region diameter, inserted on anterior most body annule i.e. just posterior to lip base. Amphidial fovea spiral, three annules 32

long, situated at posterior end of stoma. Stoma 2.0-2.5 times longer than lip region diameter. Pharynx about 100µm long, largely cylindrical, basal bulb ovate. Cardia with long process almost as long as basal pharyngeal bulb; intestine without granules; rectum, one anal body width long. Nerve ring at 46-55% of pharyngeal length. Secretory-excretory pore just posterior to nerve ring, at 61-77% of pharyngeal length from anterior. Reproductive system didelphic, amphidephic with reflexed ovaries. Both genital branches equally developed; entire reproductive tract 3.5-4.5 times mid-body diameter. Vulva a transverse slit, situated at 45-60% of body length from anterior end, vulval lips not protruding. No sperm nor spermathecae observed. Tail, five to six anal body diameters long, cylindrical and ventrally curved in posterior half with three caudal setae on each side. Spinneret present. Three caudal glands present.

Male Not found.

MATERIAL EXAMINED Females (n=15) extracted from sediments at the bottom of an underground pool (Fig. 1A,


) from Bakwena Cave. Specimens were also found under ferns and mosses on the walls of the cave entrance (Fig. 1A, ), however these specimens were not in good condition and thus not included in present study.

DIAGNOSIS AND RELATIONSHIPS The species from this study can be recognized by its small body size (320-425µm), labial region continuous with body contour, cephalic setae almost as long as half labial diameter, amphidial fovea spiral and situated at posterior end of stoma, cardia as long as basal bulb and embedded in intestinal tissue, vulva at mid-body (45.1-60.6%), each genital branch about two body widths long, tail cylindrical, ventrally curved and relatively short (c’=4.5-6.1).

By applying Andrássy’s (1985) key to species one arrives at the final dichotomy between Plectus pusillus Cobb, 1893 and Plectus opisthocirculus Andrássy, 1952. Both P. pusillus and P. opisthocirculus resemble the specimens from the present study in that the lip region is not set off, the amphidial fovea’s are positioned almost at the base of the stoma and each genital branch is about 2.0 body widths long.

The South African cave specimens differ from P. pusillus and P. opisthocirculus in that the cephalic setae are almost half the lip region in length ( vs . short, hardly discernable in P. 33 pusillus and one third of lip region width in P. opisthocirculus ), the pharynx is shorter (79- 115µm vs . 100-115µm in P. pusillus and.125-135µm in P. opisthocirculus ), and vulva-anus distance to tail length is shorter (2.1-3.1 vs. 3-4 in P. pusillus and 3.2-3.5 in P. opisthocirculus ).

Using the polytomous key (Table 6), Plectus n. sp. grouped with five species, namely Plectus chengmohliangi Hoeppli & Chu, 1932, P. cladinosus Holovachov & Susulovsky, 1999, P. inquirendus Andrássy, 1958 and P. pusillus in group 14 on the basis of codes D, H and I. Hence, the species from the present is similar to the above-mentioned species in having the amphidial fovea positioned at the posterior end of the stoma, the lip region that is not set off and the tail is ventrally curved. Further similarities with P. chengmohliangi and P. pusillus include body length (code C), anal body diameter (code F) and labial region diameter (code G). The new species differs from P chengmohliangi in the length of the stoma vs. lip region diameter (2.0-2.5 vs . 1.7-2.0) (Code (J) and c ratio (6.6-7.7 vs. 5.2-5.6 (Table 7)); from P. pusillus in tail length (40-46 vs. 44.0-57.1µm) (Code C), c’ ratio (4.5-6.1 vs. 3.3-3.9) (Code M) and pharynx length (78.5-114.0 vs. 100-115 µm (Table 7)).

Furthermore Plectus n. sp. differs from P. cladinosus and P. inquirendus in body length (319- 424 vs . 660-710µm for P. cladinosus and 656-735µm for P. inquirendus ) (Code A); tail length (44.0-57.1 vs. 87.6-91 µm for P. cladinosus and 101-123 µm for P. inquirendus ) (Code C), c’ ratio (4.5-6.1 vs. for 10.4-12.0µm P. cladinosus and 9.7-9.8µm for P. inquirendus ) (Code M)

The polytomous key also shows that Plectus n. sp differs from P. opisthocirculus (with which it grouped in the dichotomous key of (Andrássy, 1985)) on the basis of Code I (tail curvature), Code A (body length) and Code B (epidermal glands). According to the polytomous key P. opisthocirculus falls into group 15. The above comparison of the Bakwena species with phenotypically similar species in our opinion shows a significant number of differences with other species to consider it a new species.

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Table 7 : Comparison and overview of Plectus species in group 13 of the polytomous key. All measurements in µm and in the form of mean±stdev(range) for South African specimens. (Other species only mean (range) is presented). Bakwena Cave, Irene 15 &&& Plectus pusillus Cobb, Plectus chengmohliangi Plectus cladinosus Holovachov & Plectus inquirendus 1893 Hoeppli & Chu, 1932 Susulovsky, 1999 Andrássy, 1958

L 360±30.2(319-424) 385 (330-440) 430 (410-450) 685 (660-710) 695.5(656-735) a 22.0±2.0(19.0-26.0) 18.5 (16-21) 20.9 (20.5-21.1) 33.9 (32.7-35.2) 32.5(30.6-34.5) b 3.9±0.2(3.6-4.3) 3.5 (3.3-3.8) 3.5 (3.1-3.6) 4.4 (4.1-4.6) 3.8 (3.6-4.0) c 7.0±0.3(6.6-7.7) 9.2 (8.4-10.0) 5.8 (5.2-5.6) 7.8 (7.7-7.9) 5.9 (5.9-6.0) c’ 5.3±0.4(4.5-6.1) 3.6 (3.3-3.9) - 11.2 (10.4-12.0) 9.7 (9.7-9.8) V 49±3.9(45.1-60.6) 51 (49-53) 51 (48-51) 46.2 (44.9-47.5) 47.6 (46.9-48.3) LRH 2.3±0.4(1.8-3.6) - - 3.5 (3.3-3.7) - LRD 5.1±0.9 (3.6-7.1) 7 (6.5-7.5) - 8.8 (8.5-9.2) - Cephalic setae length 2.4±0.6 (1.8-3.6) - - 2.5 - Amphid diameter 2.3±0.4(1.8-3.6) 2.5 (2-3) - 2.8 (2.6-3) 3.2 (2.5-4) Amphid- anterior end 9.1±1.1 (7.1-11.3) - - 14.5 (13.9-15.1) 14.2 (12.0-16.5) Amphid: c.b.d. 8.7±1.8(6.6-13.7) - - - - Stoma 13.2±1.3(11.3-15.5) - - 14.0 (13.5-14.6) 19.5(17-22) Pharynx 92.2±9.1(78.5-114.0) 107 (100-115) - 157.5 (150-165) 168 (151-186) Basal phar. bulb length 12.8±1.6(10.7-16.1) - - - - Basal phar. bulb width 10.4±1.5(8.3-14.9) - - - - Cardia 5.0±2.3(2.4-9.5) - - - - Nerve ring 48.0±4.5(40.5-59.5) - - - - Excretory pore 62.0±8.8(53.0-86.3) - - 92.5 (87-98) - Body width: neck base 15.8±2.0(13.7-21.4) - - - - mid-body 16.2±1.8(13.7-20.8) - - - - anus 9.8±0.9(8.3-12.0) - - 8.2 (7.8-8.7) -

G1 16.1±3.1(10.5-21.2) - - - -

G2 16.7±2.9(11.6-21.3) - - - - Rectum 11.2±1.1(8.3-12.5) - - 29.8(27.7-31.9) 14 (12.5-15.5) Tail length 50.9±3.3 (44.0-57.1) 50 (40-46) - 89.4 (87.6-91.2) 112 (101-123) Vulva- ant. end 175.3±16.9(150.7-213.8) - - - - V-A/tail 2.7±0.2(2.1-3.1) - - - -

35

36

37

38

Discussion

NEMATODE GENERA COMPOSITION AND DIVERSITY When comparing the nematode genera identified during this study with previous work (Joseph, 1879; Andrássy, 1959, 1973; Zullini, 1977; Abolafia & Peña-Santiago, 2006a; Hodda et al. , 2006), we see some similarities but also some differences in nematode composition. In agreement with Poinar & Sarbu (1994) we also observed that most of the genera found within the cave are also recorded from non cave environments e.g. Acrobeloides , Panagrolaimus , Plectus , Diploscapter and thus cannot be considered as typically cavernicolous. However, the authors remarked that at species level, the situation is different. Based on the genera list of Hodda et al. (2006), (28 genera belonging to 22 families) and taking into account that the type of cave and habitats described in the literature may be different from the Bakwena cave system, a total of 19 genera from this study appear to be new records for cave environments, with five families Diplogasteridae, Diploscapteridae, Trichodoridae, Aphelenchidae & Aphelenchoididae listed for the first time. Five percent (5%) of the 28 genera listed by Hodda et al. (2006) can be found within the genera list from this study.

Abolafia & Peña Santiago (2006a) reported that many of the species that end up in cave environments are accidental occupants. Such might be the case for the trichodorid species which was found only on one sampling occasion (pers. comm.) 1, from the bottom of an underground pool (
) which is 30m underground. This species was identified as Trichodorus parorientalis Decraemer & Kilian, 1992 (pers. comm.) 2 which is an endemic species to South Africa and is known from the savanna biome associated with hosts from sandy soils (Decraemer & Kilian, 1992). Although trichodorids have been found associated with soils from river banks (Lišková & Sturhan, 1999), it is unusual that this species was only found once in the year long survey. It is thus likely that Trichodorus parorientalis ended up in the underground pool as a result of flooding or from seepage from the above ground layers. Andrássy (1966) remarked that water is undoubtedly the most important factor by which nematodes can populate a cave.

A decrease in species richness from the outermost to innermost part of the cave has been reported as common for submarine caves (Martí et al ., 2004) but this appears not to be the

1 Dr Antoinette Swart, Biosystematics Division, Plant Protection Research Institute of the Agricultural Research Council, Pretoria 2 Prof Wilfrida Decraemer, Royal Belgium Institue of Natural Sciences, Brussels 39

case for subterranean caves (Christman & Culver, 2001; Sambugar et al. , 2008). Sambugar et al. (2008) showed that habitat segregation does not show any relationship with depth from the surface but is rather influenced by local factors e.g. microhabitat structure. The pattern observed for the Bakwena cave supports that of Sambugar et al . (2008). Among the six assemblages compared from the cave entrance to the underground pool, the latter unexpectedly had the higher species richness and diversity. A possible reason for this might be because the underground pool which is 30m below ground is relatively isolated when one thinks of interferences such as visitation by animals while the cave entrance is in direct contact with the external environment and is more susceptible to interferences including human activities. Since these localities are also different, one being aquatic and the other terrestrial it would be correct in assuming that their diversity and species composition would differ.

The diversity of nematodes found within the guano deposit samples from sample locality (  dry guano from main cave chamber and  fresh guano from side chamber) is low with only three species found associated with this type of habitat. An assumption may be made that two of these nematodes species avoided competition with other nematodes for food resources by becoming specialised in living in this environment and can enter dormant state. Nicholas & Stewart (1985) reported on a free living Panagrolaimus isolate being able to survive dehydration for more than eight years by entering into an anhydrobiotic state. Previously Panagrolaimus spondyli Körner, 1954 was found living in the galleries of dark beetles while Panagrolaimus detritophagus Fuchs, 1930 was found occurring in soil rich in decaying materials (Andrassy, 2005). Hence it is no surprise that the Panagrolaimus sp. from the current study is able to survive in the guano deposits.

The reason for the higher N ∞ value observed at locality two ( ) which is the cave floor could be because this site is represented by more than one common species and thus the N ∞ value is larger than it is for locality
(underground pool) which has less common species. Low evenness (Pielou’s J) as seen at sampling localities  (fresh guano from side chamber) and  (cave floor of the side chamber) is as a result of the occurrence of only two species at these localities and thus there is a high variation in the community between species. Evenness values fall between 0 & 1, and the closer the value is to 1 the less variation in the community between species (Pielou, 1975).

40

TROPHIC STRUCTURE AND C-P VALUES The trophic structure of the Bakwena cave is driven by food resources available at each of the sampling localities. Zhou & Zhou (2008) suggested that light availability may be important for determining food resources and thus feeding composition of submarine cave nematodes. The trophic structure at the entrance of the cave  was different to the underground pool
. The latter being dominated by bacterial feeders, reflecting a possible difference in food species composition which may be related to a decreasing gradient of light level from the entrance into cave. Bacteriovore dominance can also be explained by fungi and bacteria forming the basis of the food web in the Bakwena Cave (Durand, 2008). Andrassy (1966) reported that bacteriovores constituted 80% of the Baradla cave fauna.

Making any definite comment on whether the Bakwena Cave can be regarded as a disturbed environment would be dangerous, since we have no monthly data and no reference value with which to compare the data. Our data could possibly be used later as reference material. Bongers et al . (1990) remarked that to interpret the nematofauna of a given sample a reference value is needed. Bongers & Bongers (1998) showed that the MI values can vary from 1 (in cow pats or after heavy manuring) to a value of around 4 under undisturbed or pristine conditions.

The low values for sampling localities  (dry guano form main cave chamber) and  (fresh guano from side chamber) can be explained because they are coupled to guano deposits, and bat guano being rich in organic matter can be considered in the same light as cow pats. Andrassy (1966) reported that bats play an important role in introducing food into a cave system; this food consists of bacteria and fungi off which nematodes live, so it is understandable why enrichment opportunists are associated with guano deposits.

Bongers et al. (1991) also stated that both disturbance and an increase in decomposition rate can decrease the MI value. Another way to interpret the data from this study is that an increase in organic input which serves as a food source could favour the fast reproducing species and result in a decrease in the MI value. The cave floor which had the highest MI value probably did not have as much food input than the other sampling localities and thus was not occupied by the more fast reproducing species.

NEMATODE COMMUNITY & K-DOMINANCE CURVE It would seem that the separation into six different associations depends specifically on perhaps the substrate type. Sambugar et al. (2008) showed that factors such as accumulation 41

of vegetable debris may be responsible for habitat segregation. There is of course difficulty in commenting on this ordination since no information besides the nematode data exists for the cave environment.

The k-dominance curve shows us that the diversity of species differs from locality to locality and might be related to the differences in conditions at each of the six localities.

REMARKS ON SPECIES DESCRIBED IN THIS STUDY :

Diploscapter coronatus Species belonging to the genus Diploscapter can be found in almost all soil samples particularly D. coronatus . Most species of the genus are reported from the southern hemisphere with D. coronatus being the most widespread (Andrássy, 1983). Diploscapter coronatus was only reported once before from South Africa. It was found along the banks of the Skinnerspruit River, Pretoria (Dassonville, 1981). In the list of genera of cavernicolous nematodes provided by Hodda et al. (2006) Diploscapter coronatus does not appear, and this appears to be the first record of Diploscapter coronatus from a cave environment.

Panagrolaimus n. sp. The last major revisions of the genus Panagrolaimus were made by Andrássy (1984) and Abolafia & Peña-Santiago (2006b), both providing a compendium of species of Panagrolaimus and a key to their identification. However, distinguishing these species from each other is a difficult task. Abolafia & Peña-Santiago (2006b) remarked that information for the species within this genus is very limited and the low quality of several of the original descriptions makes morphological characterisation difficult. Identifying the species from the present study was made difficult by the fact that limited information exists for this genus from South Africa. Currently, only two Panagrolaimus species have been recorded from southern Africa. Heyns (1971) described P. subelongatus from agricultural fields from the Free State and Gauteng Province, South Africa. DeBruin & Heyns (1993) described Panagrolaimus species cf australis (Cobb, 1893) Sudhaus, 1976 from the Moremi Wildlife Reserve, Botswana. The species described herein represents a new taxon & third geographical record for the genus from South Africa.

Since Panagrolaimus n. sp. was found only from guano deposits, it is proposed that the possible entry of this species into the cave is through bat guano. According to Poinar et al. (1979) members of the genus Panagrolaimus may show a phoretic association with insects

42

when their third stage resistant juveniles attach to the external surface of arthropods . Massey (1964; 1971) reported that Panagrolaimus species are often found associated with beetles and can be found in saprobic habitats (Andrássy, 2005). It is likely that the bats could feed on beetles and if Panagrolaimus n. sp. is associated with these beetles they could eventually end up in the cave; since beetle remains have been found within the guano deposits from the cave 3

Plectus n. sp. Members of the genus Plectus are bacteriovores and because of their ecological importance have received substantial taxonomic attention (Khan & Araki, 2001). The last major revision of this genus was made by Zell (1993) in which he considered only 53 species as valid. De Ley & Coomans (1994) already considered the identification of species from this genus a difficult task since many original descriptions lack the detail needed to characterise these species and type specimens are sometimes lost. In trying to identify the species from the present study a polytomous key was constructed using characters to separate the species into groups using codes. However, some difficulty was experienced as some descriptions lacked information of some of the characters used to distinguish the species from each other. It was finally possible to place Plectus n. sp. into a group (group 14) based on position of amphidial fovea in relation to stoma, lip region demarcation and tail curvature. In the checklist of free living nematodes from freshwater habitats in southern Africa (Heyns, 2002), only two species from the genus Plectus have been reported from southern Africa. Micoletzky (1916) described Plectus sambesii Micoletzky, 1916 from the Zambezi River, Zimbabwe and Botha & Heyns (1993) later reported on the presence of Plectus cirratus Bastian, 1865 from the Crocodile and Olifants River, South Africa. Thus Plectus n. sp. represents a new taxon and second geographical record for the genus in South Africa.

Future research

The nematofauna of the Bakwena cave revealed a relatively high diversity of species when comparing it to the works of Andrássy (1959) and (1973) who described 17 and 10 species respectively from the Baradla Cave. It would be important to try and describe most of these species especially those that have never been reported from South Africa before. Molecular

3 Dr Antoinette Swart 43

characterisation would also be beneficial to supplement characterisation especially for genera such as Panagrolaimus and Plectus which are difficult to identify morphologically.

Further studies should also include sediment analysis and the inclusion of abiotic data such as

pH, relative humidity (RH), temperature, and CO 2 concentrations. Analysis of water and sediment samples would also be important for eco-toxicological testing. To improve the sampling strategy that was used during this study I would: 1) collect the samples at each of the six localities in replicate (x3); 2) fix a part of the specimens in formalin for morphological observations and 3) fix the other remaining specimens in DESS (Yoder et al. , 2006) for molecular analyses.

This data would then also enable us to see which factors are responsible for distribution of the species between the sampling localities. All this additional data will allow a proper assessment to see whether anthropogenic interference by means of mining activities and urban development in anyway disturb this cave. The data could then as mentioned before be used as reference material for the Bakwena cave.

Acknowledgements The author expresses her sincere appreciation and thanks to the VLIR-UOS for financial aid, and Ghent University for use of facilities. A special thanks also to Marjolein Couvreur for her help with the SEM work and Dr Antoinette Swart (ARC-PPRI) for providing the specimens for the study.

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