Description of two new species, Aphanolaimus strilliae n. sp. (Nematoda: Aphanolaimidae) and Makatinus africanus n. sp. (Nematoda: Aporcelaimidae), and SEM observations on three known species from freshwater sources in the Telperion Nature Reserve (Mpumalanga, South Africa) Author Girgan, Chantelle Author Marais, Mariette Author Fourie, Hendrika Author Tiedt, Lourens Author Swart, Antoinette text Zootaxa 2019 2019-08-05 4651 2 201 234 journal article 26092 10.11646/zootaxa.4651.2.1 fff8098d-23d2-45b4-840c-c5aa8539713a 1175-5326 3363080 3EAFEE69-6C8A-454B-A01A-D7E4A23BAD9F Aphanolaimus strilliae n. sp. Figures 2–6 urn:lsid:zoobank.org:act: 6C432C2D-7725-4E79-8EE6-191B5540560E Measurements: Table 2 TABLE 2. Morphometrics of Aphanolaimus strilliae n. sp. females found in freshwater substrates in the Telperion Nature Reserve. All measurements in the form: mean ± standard deviation (range).

Morphological feature	Holotype	Paratypes
n=25
L	1394	1377 ± 86.8 (1240–1613)
a	48.8	40.9 ± 4.1 (36.3–49.5)
b	5.7	5.7 ± 0.3 (5.1–6.6)
c	6.9	7.2 ± 0.3 (7.0–8.0)
c’	10.5	9.5 ± 0.8 (7.7–10.9)
V %	49	49 ± 1.1 (47–52)
G1 %	12	14 ± 2.5 (11–19)
G2 %	13	16 ± 3.3 (12–22)
Nerve ring as percentage of oesophagus	54	55 ± 1.8 (52–59)
Vagina as percentage of corresponding body width	32	36 ± 5.3 (29–48)
Body width at mid–body	29	33 ± 3.9 (28–41)
Body width at vulva	32	34 ± 3.9 (28–43)
Body width at anus	19	20 ± 1.9 (17–26)
Cuticle width at mid–body	3	3 ± 0.5 (2–4)
Annule width at mid–body	2	2 ± 0.3 (1–2)
Number of annules	833	837 ± 24.4 (808–887)
Number of annules from anterior to end of oesophagus	167	156 ± 9.5 (139–179)
Number of annules from anterior to vulva	412	412 ± 18.5 (388–452)
Number of annules between vulva and anus	280	287 ± 11.6 (253–313)
Number annules from anterior to anus	692	699 ± 21.7 (674–746)
Number of annules from anus to tail terminus	141	138 ± 6.5 (120–152)
Start of lateral field (annule)	41	41 ± 3.3 (34–46)
Start of lateral field (μm)	65	64 ± 3.4 (56–70)
End of lateral field from tail tip (μm)	26	29 ± 9.0 (15–55)
Lateral field width at mid–body	2	3 ± 0.7 (2–5)
Lateral body pores (in pairs)	22	23 ± 1.4 (21–26)
Lateral body pores (single)	1	1a
Lateral body pores (total)	45	48 ± 2.9 (43–53)
Lateral body pore pairs (oesophageal region)	5	5a
Lateral body pore pairs (anterior to vulva)	7	10 ± 2.8 (7–14)
Lateral body pore pairs vulva to anus)	9	8 ± 0.8 (7–9)
Lateral body pore pairs (tail)	1	1a
Lateral body pore single (tail)	1	1a
First body pore from anterior (annules)	35	30 ± 2.4 (25–35)
First body pore from anterior (μm)	51	49 ± 4.9 (30–55)
Second body pore from anterior (annules)	71	64 ± 4.2 (56–71)
Second body pore (μm)	101	98 ± 4.5 (89–108)

......continued on the next page TABLE 2. (Continued)

Morphological feature	Holotype	Paratypes
n=25
Head length	9	9 ± 0.8 (7–10)
Head width	8	8 ± 0.5 (7–10)
Cephalic setae length	6	7 ± 1.4 (6–9)
Amphid length	6	6 ± 0.6 (4–7)
Amphid width	4	5 ± 0.5 (4–6)
Stoma length	7	7 ± 0.8 (6–10)
Oesophagus length	243	237 ± 7.7 (217–251)
Oesophagus width at widest point	6	6 ± 1.0 (4–7)
Ventral gland length	56	57 ± 5.9 (48–67)
Ventral gland width at nucleus	7	7 ± 1.0 (6–9)
Nerve ring from anterior	131	131 ± 5.2 (122–140)
Nerve ring length	9	9 ± 0.9 (8–11)
Cardia length	13	10 ± 2.6 (6–16)
Vulva from anterior	689	664 ± 50.0 (560–759)
Vagina length	21	20 ± 2.1 (16–22)
Anterior genital tract length	160	190 ± 42.9 (130–274)
Posterior genital tract length	185	222 ± 54.5 (150–337)
Anterior uterus length	70	79 ± 9.6 (70–96)
Posterior uterus length	92	88 ± 8.5 (72–99)
Rectum length	36	35 ± 4.1 (25–43)
Rectum / anal body width	1.9	1.7 ± 0.2 (1.3–2.1)
Tail length	201	188 ± 11.7 (165–207)
Spinneret length	2	3 ± 0.4 (2–4)

a Value same for all specimens Description: Female (n=26): Body 1240–1613 µm long, tapering towards both extremities, habitus mostly ventral curvature of whole body, some specimens curved dorsally. Body with 808–887 well defined smooth annules, mean width 2 µm . Cuticle 2–4 µm thick at mid-body, lateral field a single ridge with two lines, originating on 34 th to 46 th annule ( 56–70 µm from the anterior end), mean width of 3 µm at mid body. Lateral field ends 15–55 µm anterior to tail tip. Lateral body pores paired, 21–26 pairs on each side, arranged randomly on both ventral and dorsal side of lateral field; five pairs in region of oesophagus, one pair and one single pore on tail. First lateral body pore situated on 25 th to 35 th annule ( 30–55 µm from anterior end); second pore situated on 56 th to 71 st annule ( 89–108 µm from anterior end). Each pore contains a small, hollow projection (observed with SEM) that varies in structure ( Figure 5 ). Each pore connected to an epidermal gland. Labial region not annulated, 7–10 µm high and 7–10 µm wide with cephalic setae measuring 7 µm long, situated at the anterior end of the amphids. Amphid located at base of cephalic region, 4–7 µm high and 4–6 µm wide, circular with no central elevation. Stoma a narrow tube, not sclerotized, 6–10 µm long. Oesophagus a narrow tube, 217–251 µm long, 4–7 µm wide at widest point, ending in a 6–16 µm long cardia. Ventral gland 48–67 µm long, 6–9 µm wide, situated at base of oesophagus, gland opening / excretory pore not observed (excretory pore described as opening in stoma in this genus). Nerve ring situated at 52–59% of oesophagus length. Reproductive system didelphic-amphidelphic, genital tracts almost equal in length, often posterior tract (mean length 222 µm ) slightly longer than anterior tract (mean length 190 µm ). Simple sphincter muscles separate ovijector from both uteri. Anterior uterine branch 70–96 µm long, posterior branch 72–99 µm in length (measured in females without eggs or developing embryos in uterus). Eggs and fully-grown juveniles observed in more than half of specimens, indicating the species is ovoviviparous. Vulva a transverse slit occupying about half corresponding body width, situated at 47–52 % of total body length. Vagina bent anteriorly in V-shape, 16–22 µm long and occupies less than half of corresponding body width. No sperm cells observed in reproductive system of females, indicating that species might be parthenogenetic. Rectum 1.2–2.0 times anal body width. Tail 165–207 µm long ending in an offset 2–4 µm spinneret. Three distinct caudal glands present in anterior half of tail. Male: Unknown. FIGURE 2. Aphanolaimus strilliae n. sp. female A) Oesophageal region with ventral gland; B–D) Anterior region with oesophagus, stoma, position of amphid and position of first lateral body pore; E) Lateral field at mid-body; F) Vulval region; G) Reproductive system; H) Tail region; I) Genital tract with developing juveniles. Diagnosis and relationships: Aphanolaimus strilliae n. sp. is characterised by body length of 1240–1613 µm , more than 800 annules, 8 µm long cephalic setae, lateral field originating between the first and second lateral body pore at the 34 th– 46 th annule, first body pore located at the 25 th– 36 th annule, vulva equatorial, vagina V-shaped and bent anteriorly, 142–195 µm long uterus and 165–207 µm long tail with one paired and one single lateral body pore, spinneret offset. Species is ovoviviparous. Males absent. FIGURE 3. Scanning electron micrographs of Aphanolaimus strilliae n. sp. female A) Head region in ventral view with cephalic setae ( cs ) and amphid ( a ); B + C) Head region in lateral view with cephalic setae ( cs ) and amphid ( a ); D) Lip region in e n face view of lip region with amphid opening ( a ) and stoma opening ( s ). Aphanolaimus strilliae n. sp. belongs to the ovoviviparous group of larger species with more than 700 body annules. This group includes A. aymarae De Waele & Coomans, 1993 , A. spiriferus Cobb, 1914 and A. viviparus Plotnikov, 1899 , which are all ovoviviparous species. Aphanolaimus aquaticus Daday, 1894 and A. tudoranceai Zullini, 1988 belong to the group of large species, but is not ovoviviparous. Aphanolaimus strilliae n. sp. is morphologically closest to A. spiriferus . The new species was compared to the type that was described from the Potomac river, Unit- ed States of America ( U.S.A. ) and other descriptions (Lake Tahoe and Mendocino, U.S.A. ) of A. spiriferus ( Cobb 1914 ; Raski & Coomans 1990 ). Aphanolaimus strilliae n. sp. compare well to A. spiriferus in body length ( 1240– 1613 µm vs 1200–1700 µm ), total number of annules (more than 800) and ovoviviparous development of offspring. It differs from the type population only in the shape of the amphid (circular with no elevation vs spiral), morphology of the vagina (V-shaped vs inverted V-shape) and the presence of males (absent in Aphanolaimus strilliae n. sp. ). Aphanolaimus strilliae n. sp. is however separated from the A. spiriferus from Lake Tahoe and Mendocino based on: lower number of lateral body pores (21–26 pairs vs 26–30 pairs) and the lateral body pores on the tail (one pair and one single pore vs two pairs); presence of males (absent in Aphanolaimus strilliae n. sp. ). Based on SEM observations of both A. strilliae n. sp. and A. spiriferus the following differences were observed: different morphology of annules (rounded, smooth annules vs plate-like annules folding over each other) and different morphology of setae in lateral body pores (various shapes found in all lateral body pores vs simple setae with no setae in first three pores ( Raski & Coomans 1990 ). Aphanolaimus strilliae n. sp. was also compared to various descriptions available of A. aquaticus namely the type description by Daday (1894) and other descriptions by Jägerskiöld (1909) , Höfmanner & Menzel (1914) , Micoletzky (1922) , Coomans & De Waele (1983) , Traunspurger (1989) and Andrássy (2005) . The new species differs from the type population of A. aquaticus described from Lake Balaton, Hungary , in the presence of males. Unfortunately, the original description of A. aquaticus is vague and does not include characteristics currently used for species identification. Morphologically Aphanolaimus strilliae n. sp. was therefore compared to reported populations of A. aquaticus and not the original one. The new species differs from all other populations of A. aquaticus in higher number of body annules {808–887 vs 665 (calculated from Traunspurger (1989) which only stated that “more than 600 annules” are present in A. aquaticus }, vaginal morphology (bent vagina vs straight), structure of amphid (no central elevation vs central elevation), longer stoma ( 6–10 µm vs 2 µm ) ( Coomans & De Waele 1983 ), longer oesophagus ( 215–251 µm vs 190–200 µm ) ( Coomans & De Waele 1983 ), the absence of males (present in A. aquaticus ) (except population of ( Coomans & De Waele (1983) where males were absent) and the development of offspring within the female (oviparous in A. aquaticus ). Aphanolaimus strilliae n. sp. differs from type specimens of A. aymarae described from mud in Bolivia in: lower body width ( 25–46 µm vs to 57–75 µm ), number of annules ( 600–700 in A. aymarae ), number of lateral body pores (26–37 pairs in A. aymarae ), higher head ( 7–10 µm vs 10–12 µm ), longer stoma ( 2–3 µm in A. aymarae ), morphology of the vagina (inverted V-shape in A. aymarae ) and spinneret morphology (offset vs not offset) ( De Waele & Coomans 1993 ). Aphanolaimus aymarae was not found after its description. Aphanolaimus strilliae n. sp. is morphologically close to the type description of A. tudoranceai reported from Lake Zway, Ethiopia , as both are of the larger species with over 700 annules ( Zullini 1988 ). The new species differs in: vaginal morphology (straight vagina in A. tudoranceai ), amphid shape (circular vs kidney shaped), longer tail ( 165–207 µm vs 145–165 µm ) and spinneret morphology (spinneret not offset in A. tudoranceai ). Aphanolaimus tudoranceai was never found after its original description ( Zullini 1988 ). Aphanolaimus strilliae n. sp. is closely related to A. viviparus however, the validity of this species is in question. According to Goodey (1963) A. viviparus may be a synonym of A. aquaticus but unfortunately he did not stipulate the reasons for this. In the literature available on A. viviparus , no distinct differences between A. viviparus and A. aquaticus can be determined apart from one described male of A. viviparus having 13 pre-cloacal supplements compared to the 7–12 supplements reported for A. aquaticus ( Schneider 1916 ; 1922 ). The description of the type female by Plotnikov (1899) could not be located in the present study and apart from the species name being mentioned in literature, no original morphological data could be found for this species. As the validity of A. viviparus is in question, Aphanolaimus strilliae n. sp. therefore is not compared to it. Type material: Holotype female on slide 50587 (first female from the left side) and paratypes on slides 50577– 50578, 50580, 50582–50584, 50586–50590 ( 18 females ) deposited in the National Collection of Nematodes (ARC—Plant Health and Protection, Biosystematics Division, Pretoria). Type locality: Specimens were found in sediment and water samples collected at the wetland site. This wetland is situated along the 11_26 road on the border of the Blaauwpoort 1 and Blaauwpoort 2 sections of the reserve. The site is described in Table 1 . Locality number MP00013 in the National Collection of Nematodes (ARC—Plant Health and Protection, Biosystematics, Pretoria) . Etymology: Aphanolaimus strilliae n. sp. is named after Mrs. Strilli Oppenheimer, owner of Telperion Nature Reserve and well known for her work in conservation.